MIAMI UNIVERSITY the Graduate School Certificate for Approving The

MIAMI UNIVERSITY

The Graduate School

Certificate for Approving the Dissertation

We hereby approve the Dissertation

Craig Anthony Damin

Candidate for the Degree:

Doctor of Philosophy

______André J. Sommer, Advisor

______Neil D. Danielson, Committee Chair

______Jonathan P. Scaffidi, Reader

______David C. Oertel, Reader

______Lei L. Kerr, Graduate School Representative

ABSTRACT

INSTRUMENT DEVELOPMENT AND APPLICATION FOR QUALITATIVE AND QUANTITATIVE SAMPLE ANALYSES USING INFRARED AND RAMAN SPECTROSCOPIES

by Craig Anthony Damin

This dissertation describes the development and application of methods and instrumentation for qualitative and quantitative sample analyses by infrared and Raman spectroscopies. An introduction to the concepts and methods utilized is provided in Chapter 1. A comparative evaluation of solid-core silver halide fiber optics and hollow silica waveguides was performed on the basis of the transmission of mid-infrared radiation using a fiber optic coupling accessory and an infrared microscope is presented in Chapter 2. Increased transmission was reproducibly observed between two identical hollow waveguides due to minimization of insertion and scattering losses resulting from the hollow core. Chapter 3 presents an evaluation of a mid-infrared, attenuated total (internal) reflection (ATR) probe accessory utilizing hollow waveguides based on transmission and signal-to-noise. Quantitative analyses of aqueous succinylcholine chloride and ethanol solutions were also performed. An in situ Raman study of nitrogen incorporation in thin films of zinc oxide using a temperature-controlled reaction cell is discussed in Chapter 4. Monitoring nitrogen incorporation in thin films of zinc oxide at elevated temperatures in the presence of nitrogen-containing precursor reagents proved inconclusive using the proposed method. Chapter 5 presents an evaluation of dispersive and Fourier transform (FT-) Raman spectroscopies for on-line process control in the bottling industry. FT-Raman was determined to be more applicable for on-line determinations of poly(ethylene terephthalate) bottle thickness due to the availabilities of such benefits as increased laser power and fluorescence rejection. Preliminary data from the development of an inverted ATR imaging microscope are discussed in Chapter 6. The inverted optical design of the microscope permits simultaneous viewing of the sample with white light and the collection of infrared spectral images. Summaries of the presented research are provided in Chapter 7.

INSTRUMENT DEVELOPMENT AND APPLICATION FOR QUALITATIVE AND QUANTITATIVE SAMPLE ANALYSES USING INFRARED AND RAMAN SPECTROSCOPIES

A DISSERTATION

Submitted to the Faculty of

Miami University in partial

fulfillment of the requirements

for the degree of

Doctor of Philosophy

Department of Chemistry and Biochemistry

Craig Anthony Damin

Miami University

Oxford, Ohio

2013

Dissertation Director: André J. Sommer

Craig A. Damin

2013

Table of Contents

Chapter 1. Introduction

1.1 Infrared Spectroscopy ...... 2 1.1.1 History of Infrared Spectroscopy ...... 2 1.2 Fourier Transform Infrared (FT-IR) Spectroscopy ...... 3 1.2.1 Instrumentation ...... 3 1.2.2 Advantages of FT-IR Spectroscopy ...... 5 1.3 Infrared Sampling Methods ...... 6 1.3.1 Transmission ...... 7 1.3.2 Reflectance ...... 7 1.3.3 Attenuated Total Internal Reflection ...... 8 1.4 Quantitative Analysis by Infrared Spectroscopy ...... 9 1.5 Infrared Fiber Optics and Waveguides ...... 10 1.5.1 Crystalline Infrared Fibers ...... 10 1.5.1.1 Polycrystalline Infrared Fibers ...... 11 1.5.2 Hollow Waveguides ...... 12 1.5.2.1 Hollow Glass Waveguide ...... 13 1.6 Near-Infrared Spectroscopy ...... 14 1.7 Raman Spectroscopy ...... 14 1.7.1 Dispersive Raman Spectroscopy...... 15 1.7.2 Fourier Transform (FT-) Raman Spectroscopy ...... 16 1.8 Raman Microspectroscopy ...... 17 1.9 Thermogravimetric Analysis ...... 19 1.10 Infrared Microspectroscopy ...... 20 1.10.1 History of Infrared Microspectroscopy ...... 20 1.10.2 Instrumentation ...... 21 1.11 Infrared Microspectroscopic Imaging ...... 24 1.11.1 History of Infrared Imaging ...... 24 1.11.2 Advantages of Infrared Imaging Using a Focal Plane Array ...... 26

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1.11.3 ATR-FT-IR Microspectroscopic Imaging ...... 26 1.12 Dissertation Goals and Specific Aims ...... 28 References ...... 30

Chapter 2. Characterization of Silver Halide Fiber Optics and Hollow Silica Waveguides for Use in the Construction of a Mid-Infrared Attenuated Total Reflection-Fourier Transform Infrared (ATR-FT-IR) Spectroscopy Probe 2.1 Abstract ...... 45 2.2 Introduction ...... 46 2.2.1 Infrared Fiber Optics ...... 46 2.2.2 Hollow Waveguides ...... 48 2.3 Goals and Specific Aims ...... 49 2.4 Experimental ...... 49 2.4.1 Materials ...... 49 2.4.2 Instrumentation ...... 50 2.4.3 Methods...... 52 2.5 Results and Discussion ...... 53 2.5.1 Harrick FiberMate2 ...... 53 2.5.2 Spectra-Tech IR-PLAN Infrared Microscope ...... 55 2.5.3 Single-beam Spectra ...... 61 2.5.4 Effect of Bending ...... 64 2.6 Conclusions ...... 68 2.7 Acknowledgements ...... 68 References ...... 69

Chapter 3. Evaluation of a Mid-Infrared Hollow Waveguide Accessory for Sample Analysis by Attenuated Total Reflection-Fourier Transform Infrared (ATR-FT-IR) Spectroscopy 3.1 Abstract ...... 75 3.1.1 Evaluation of a Hollow Waveguide ATR Probe Accessory ...... 75 3.1.2 Analysis of Hard Samples ...... 76 3.1.3 Quantitative Analysis of Ethanol in Alcoholic Beverages ...... 76 3.2 Introduction ...... 77 3.2.1 Infrared Fiber Optics and Hollow Waveguides ...... 77 3.2.2 Quantitative Analysis of Succinylcholine Chloride ...... 78 3.2.3 Economic Regulations of Alcoholic Beverages ...... 79 3.2.4 Current Methods of Determining Alcohol Content ...... 79 3.2.5 Vibrational Spectroscopy ...... 80 3.2.6 Infrared Fiber-Coupled Probes for Alcohol Content Determinations ...... 81 3.3 Goals and Specific Aims ...... 82 3.4 Experimental ...... 83 3.4.1 Instrumentation ...... 83 3.4.1.1 Evaluation of a Hollow Waveguide ATR Probe Accessory ...... 85 3.4.1.2 Analysis of Hard Samples ...... 85 3.4.1.3 Quantitative Analysis of Ethanol in Alcoholic Beverages ...... 85 3.4.2 Methods and Materials ...... 86 3.4.2.1 Evaluation of a Hollow Waveguide ATR Probe Accessory ...... 86 3.4.2.2 Analysis of Hard Samples ...... 88 3.4.2.3 Quantitative Analysis of Ethanol in Alcoholic Beverages ...... 88 3.5 Results and Discussion ...... 90 3.5.1 Evaluation of a Hollow Waveguide ATR Probe Accessory ...... 90 3.5.1.1 Single-beam Spectra ...... 90 3.5.1.2 Bending Analysis ...... 93 3.5.1.3 Transmission Efficiency ...... 96 3.5.1.4 Quantitative Analysis of Succinylcholine Chloride Solutions...... 98 3.5.1.5 Infrared Spot Size ...... 100

3.5.2 Analysis of Hard Samples ...... 104 3.5.2.1 Polycarbonate Sheet and Tube ...... 104 3.5.2.2 Rubber Eraser...... 106 3.5.2.3 Calcium Oxalate Monohydrate ...... 107 3.5.2.4 Practical Considerations...... 108 3.5.3 Quantitative Analysis of Ethanol in Alcoholic Beverages ...... 109 3.5.3.1 Infrared Spectra of Ethanol and Water ...... 109 3.5.3.2 Ethanol Calibration Curves ...... 110 3.5.3.3 Error Analysis ...... 111 3.5.3.4 Alcohol Content Determination in Beer ...... 112 3.5.3.5 Alcohol Content Determination in Wine ...... 115 3.5.3.6 Alcohol Content Determination in Complex Matrices ...... 115 3.5.3.7 Alcohol Content Determination in Liquor ...... 116 3.5.3.8 Effect of Carbohydrate Sugars on Alcohol Content Determinations ...... 117 3.5.3.9 Sample Volume ...... 118 3.6 Conclusions ...... 119 3.6.1 Evaluation of a Hollow Waveguide ATR Probe Accessory ...... 119 3.6.2 Analysis of Hard Samples ...... 119 3.6.3 Quantitative Analysis of Ethanol in Alcoholic Beverages ...... 120 3.7 Acknowledgements ...... 120 References ...... 121

Chapter 4. Design of a Reaction Cell for Studying Nitrogen Doping in Thin Zinc Oxide Films Using In situ Raman Spectroscopy 4.1 Abstract ...... 130 4.2 Introduction ...... 131 4.2.1 Reaction Cells ...... 131 4.2.2 Thermal Decomposition of Calcium Oxalate Monohydrate ...... 132 4.2.3 Analysis of Nitrogen Incorporation in Zinc Oxide Films ...... 133 4.3 Goals and Specific Aims ...... 135 4.4 Experimental ...... 136

4.4.1 Stainless Steel Reactor Cell for Raman Process Monitoring ...... 136 4.4.2 Thermal Decomposition of Calcium Oxalate Monohydrate ...... 138 4.4.2.1 Materials and Methods ...... 138 4.4.2.2 Instrumentation ...... 139 4.4.3 Analysis of Nitrogen Incorporation in Zinc Oxide Films ...... 140 4.4.3.1 Materials and Methods ...... 140 4.4.3.2 Instrumentation ...... 142 4.5 Results and Discussion ...... 145 4.5.1 Thermal Decomposition of Calcium Oxalate Monohydrate ...... 145 4.5.1.1 Thermogravimetric Analysis of Calcium Oxalate Monohydrate...... 145 4.5.1.2 Process-Raman Analysis of the Thermal Decomposition of Calcium Oxalate Monohydrate ...... 147 4.5.2 Analysis of Nitrogen Incorporation in Zinc Oxide Films ...... 154 4.5.2.1 Process-Raman Analysis of Nitrogen Incorporation in Zinc Oxide Films ...... 154 4.5.3 Background Variation ...... 165 4.6 Conclusions ...... 168 4.6.1 Thermal Decomposition of Calcium Oxalate Monohydrate ...... 168 4.6.2 Analysis of Nitrogen Incorporation in Zinc Oxide Films ...... 168 4.7 Acknowledgements ...... 168 References ...... 169

Chapter 5. Raman Spectroscopic Detection for Process Control in the Bottling Industry 5.1 Abstract ...... 178 5.1.1 Dispersive Raman Spectroscopy...... 178 5.1.2 Fourier Transform (FT-) Raman Spectroscopy ...... 178 5.2 Introduction ...... 179 5.2.1 Production of Plastic Bottles ...... 179 5.2.2 Current Methods of Thickness Determination ...... 179 5.3 Goals and Specific Aims ...... 180 5.4 Raman Spectroscopic Monitoring of Poly(ethylene terephthalate) ...... 181

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5.5 FT-Raman Spectroscopy of Poly(ethylene terephthalate) ...... 182 5.6 Experimental ...... 183 5.6.1 Dispersive Raman Spectroscopy...... 183 5.6.1.1 Materials ...... 183 5.6.1.2 Instrumentation ...... 183 5.6.1.3 Methods...... 185 5.6.2 Fourier Transform Raman Spectroscopy ...... 185 5.6.2.1 Materials ...... 185 5.6.2.2 Instrumentation ...... 185 5.6.2.3 Methods...... 189 5.7 Results and Discussion ...... 190 5.7.1 Raman Spectrum of Poly(ethylene terephthalate) ...... 190 5.7.2 Dispersive Raman Spectroscopy...... 191 5.7.2.1 Preliminary Considerations ...... 191 5.7.2.2 Dispersive Raman Spectra of PET at Short-range Distances ...... 194 5.7.2.3 Optical Considerations ...... 196 5.7.2.4 Sample Displacement Profiles ...... 197 5.7.2.5 Raman Intensity Models ...... 198 5.7.3 FT-Raman Spectroscopy ...... 201 5.7.3.1 Preliminary Considerations ...... 201 5.7.3.2 Optical and Sample Considerations ...... 202 5.7.3.3 Near-Infrared Characterization of PET ...... 205 5.7.3.4 PET Thickness Calibration ...... 208 5.7.3.5 Minimum Thickness Variation ...... 210 5.7.3.6 Sampling Precision ...... 211 5.7.3.7 Effect of Laser Power on Thickness Determinations ...... 212 5.7.3.8 Effect of Number of Scans on Thickness Determinations ...... 215 5.7.3.9 Effect of Spectral Resolution on Thickness Determinations ...... 218 5.7.3.10 Sample Displacement Profiles ...... 222 5.7.3.11 Comparison of Spectrograph Slit and Interferometer J-Stop ...... 227 5.8 Conclusions ...... 228

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5.9 Acknowledgements ...... 228 References ...... 229

Chapter 6. Development of an Inverted Microscope Designed for Attenuated Total Reflection-Fourier Transform Infrared (ATR-FT-IR) Spectroscopic Imaging 6.1 Abstract ...... 235 6.2 Introduction ...... 235 6.2.1 History of Infrared Microspectroscopy ...... 236 6.2.2 FT-IR Microspectroscopic Imaging ...... 237 6.2.3 ATR-FT-IR Microspectroscopic Imaging ...... 238 6.2.4 History of ATR-FT-IR Imaging...... 239 6.2.5 Sample Size and Pixel Resolution ...... 240 6.2.6 Conventional and Aperture-splitting Beamsplitters...... 241 6.2.7 Infinity-corrected Optics in FT-IR Microspectroscopy ...... 242 6.3 Goals and Specific Aims ...... 243 6.4 Experimental ...... 244 6.4.1 Materials and Methods ...... 244 6.4.2 Instrumentation ...... 245 6.5 Results and Discussion ...... 247 6.5.1 Optical Considerations ...... 247 6.5.2 Signal-to-Noise ...... 250 6.5.3 Spatial Resolution ...... 252 6.5.4 Sample Analysis...... 255 6.6 Conclusions ...... 256 6.7 Acknowledgements ...... 256 References ...... 257

Chapter 7. Conclusion 7.1 Summary ...... 263 7.1.1 Characterization of Infrared-Transmitting Silver Halide Fiber Optics and Hollow Silica Waveguides ...... 263 7.1.2 Evaluation of a Mid-Infrared Hollow Waveguide ATR Probe Accessory ...... 263 7.1.3 Design of a Reaction Cell for Studying Nitrogen Doping in Thin Zinc Oxide Films Using In situ Raman Spectroscopy ...... 264 7.1.4 Raman Spectroscopic Detection for Process Control in the Bottling Industry ...... 265 7.2 Future Work ...... 266 7.2.1 Application of an Inverted Microscope Designed for ATR-FT-IR Spectroscopic Imaging ...... 266

List of Tables

Table 2.1: Infrared Transmission by Fibers/Waveguides Using Harrick FiberMate2 ...... 54 Table 2.2: Transmission of Infrared Light Measured by Coupling Fiber Optics and Hollow Waveguides to an Infrared Microscope ...... 58 Table 2.3: Summary of Bending Analysis for Silver Halide Fibers and Hollow Waveguides ... 66

Table 3.1: Summary of Bending Analysis for Hollow Waveguide ATR Probe ...... 95 Table 3.2: Hollow Waveguide ATR Probe Accessory Compared to Open FT-IR Sample Compartment ...... 97 Table 3.3: Comparison of Single-Reflection and Multi-bounce ATR Detection of Succinylcholine Chloride ...... 102 Table 3.4: Infrared Absorptions and Assignments for Polycarbonate ...... 105 Table 3.5 Analytical Figures of Merit for Ethanol Calibration Curves ...... 111 Table 3.6: Summary of Quantitative Analyses of Alcoholic Samples ...... 114 Table 3.7: Analysis of Aqueous Sugar Solutions ...... 118

Table 4.1: Thermogravimetric Analysis Results for Calcium Oxalate Monohydrate ...... 146

Table 5.1: Disperisve Raman Collection Parameters ...... 193 Table 5.2: Near-Infrared Sample Characteristics ...... 206 Table 5.3: Summary of FT-Raman Results for PET: Sample Thickness ...... 209 Table 5.4: Summary of FT-Raman Results for PET: Laser Power ...... 214 Table 5.5: Summary of FT-Raman Results for PET: Number of Scans ...... 217 Table 5.6: Summary of FT-Raman Results for PET: Spectral Resolution ...... 221 Table 5.7: Summary of FT-Raman Results for PET: Sample Displacement (Thickness: 0.0254 cm) ...... 224 Table 5.8: Summary of FT-Raman Results for PET: Sample Displacement (Thickness: 0.1016 cm) ...... 225 Table 5.9: Summary of FT-Raman Results for PET: Sample Displacement (Thickness: 0.1500 cm) ...... 226

Table 6.1: Results of Signal-to-Noise Study with Changes in Number of Scans ...... 250 Table 6.2: Results of Signal-to-Noise Study with Changes in Spectral Resolution ...... 251

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List of Figures

Figure 1.1: Diagram of an FT-IR spectrometer incorporating a Michelson interferometer ...... 3 Figure 1.2: Principal infrared sampling methods. Transmission. Reflectance. Attenuated total (internal) reflection (ATR)...... 6 Figure 1.3: Diagram of a silver halide PC infrared fiber optic ...... 11 Figure 1.4: Diagram of a hollow glass waveguide ...... 13 Figure 1.5: Diagram of x-y discrimination caused by lateral displacement of the sample and z- discrimination caused by axial displacement of the sample...... 18 Figure 1.6: Schematic diagrams of a visible wavelength optical microscope with refractive optics and an infrared microscope with reflective optics...... 23 Figure 1.7: Schematic representation of a rapid-scan FT-IR imaging data set collected using an array detector with n × m pixels ...... 25

Figure 2.1: Diagram of fiber optic evanescent wave spectroscopy (FEWS)...... 47 Figure 2.2: Harrick FiberMate2 fiber optic coupling accessory ...... 50 Figure 2.3: Instrument diagram of fiber optics and waveguides analyzed using the Spectra-Tech IR-PLAN infrared microscope accessory coupled to a PerkinElmer Spectrum 2000 FT-IR spectrometer ...... 51 Figure 2.4: Microscope objectives used to focus infrared radiation into the fibers/waveguides...... 53 Figure 2.5: Optical diagrams of an infrared fiber optic depicting launch angles of 14.5o and 30.0o...... 56

Figure 2.6: Fresnel reflection plots of Rp and Rs for the solid-core silver halide PC infrared fibers with a core refractive index 2.150 ...... 59 Figure 2.7: Single-beam infrared spectra of silver halide fiber optics and hollow silica waveguides ...... 61 Figure 2.8: Photographs of silver halide PIR fiber construction ...... 61 Figure 2.9: Infrared spectra of Parylene and the interior polymer jacket of a silver halide fiber; Single-beam spectrum of a silver halide fiber optic ...... 62 Figure 2.10: Single-beam spectrum acquired for an open-beam FT-IR sample compartment; Single-beam spectrum acquired for a hollow waveguide ...... 63

Figure 2.11: Illustration of bending radius (RB) versus bending angle (θB) ...... 64

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Figure 2.12: Bending angle instrument responses for PIR fiber optics ...... 65 Figure 2.13: Bending angle instrument responses for hollow silica waveguides ...... 67

Figure 3.1: Photographs of the Pike Technologies Mid-IR FlexIR hollow waveguide accessory and ATR probe ...... 83 Figure 3.2: Optical diagram of the Pike Technologies FlexIR hollow waveguide accessory ..... 84 Figure 3.3: Schematic illustration of the lateral bending of the HWG probe from + 60o to -60o ...... 86 Figure 3.4: Single-beam infrared spectrum obtained using the HWG ATR probe accessory; Single-beam infrared spectrum obtained for an open-beam FT-IR sample compartment; Ratio of the single-beam accessory spectrum to the single-beam open sample compartment spectrum ...... 90 Figure 3.5: Comparison of the single-beam ratio spectrum to the infrared spectrum of an ethyl cyanoacrylate adhesive ...... 92 Figure 3.6: Instrument responses for the hollow waveguide ATR probe in a straight configuration and bent at + 60o...... 93 Figure 3.7: Infrared spectrum of a 100 hundred parts per thousand succinylcholine chloride solution ...... 98 Figure 3.8: Calibration curve for the 1167 cm-1 succinylcholine chloride absorption...... 103 Figure 3.9: Infrared spectra of a polycarbonate sheet and a polycarbonate tube ...... 104 Figure 3.10: Infrared spectrum of a pink, rubber eraser; Reference spectrum of calcium carbonate; Reference spectrum of talc...... 106 Figure 3.11: Infrared spectrum of calcium oxalate monohydrate...... 107 Figure 3.12: Infrared absorption spectra of pure, 200 proof anhydrous ethanol and distilled water ...... 109 Figure 3.13: Calibration curves for the 1046 cm-1 and 1088 cm-1 absorptions of ethanol ...... 110 Figure 3.14: Infrared absorption spectra of alcoholic beverages ...... 112 Figure 3.15: Extended calibration curve for the 1046 cm-1 ethanol absorption ...... 116 Figure 3.16: Infrared absorption spectra of 5 wt. % solutions of ethanol, glucose, lactose, and sucrose...... 117

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Figure 4.1: Photographs of stainless steel reaction cell ...... 136 Figure 4.2: Schematic diagrams of stainless steel reaction cell ...... 137 Figure 4.3: Stainless steel sample compartment ...... 138 Figure 4.4: Remote, in situ thermo-Raman sampling configuration using a Renishaw inVia Raman microscope ...... 140 Figure 4.5: Optical diagram of the Renishaw System 100 Raman Analyzer; Optical diagram of the Renishaw RP20 fiber optic Raman microprobe...... 142 Figure 4.6: Close-up photograph of the fiber optic Raman microprobe and the reaction cell ...... 143 Figure 4.7: Instrument setup for the in situ study of nitrogen incorporation in thin films of ZnO ...... 144 Figure 4.8: Thermogravimetric analysis curve for COM...... 146 Figure 4.9: Raman spectra of standard samples...... 148 Figure 4.10: Raman spectra of COM at 25, 100, 200, and 350oC ...... 149 Figure 4.11: Raman spectra of COM over the range of 1400-1700 cm-1 shift at 25, 100, 200, and 350oC...... 150 Figure 4.12: Raman spectra of the thermal decomposition of COM at 500, 600, 750, and 900oC; o Post-reaction scan at 25 C; Raman spectrum of CaCO3 ...... 151 Figure 4.13: Raman spectra of COM at 500, 600, 750, and 900oC over the range of 1000-1160 cm-1 shift ...... 152 o Figure 4.14: Raman spectra of ZnO sample 1 annealed in 5 wt. % NO/N2 at 300 C ...... 155 o Figure 4.15: Raman spectra of ZnO sample 3 annealed in 5 wt. % NO/N2 at 400 C ...... 156 Figure 4.16: Raman spectra over the range of 250-300 cm-1 shift for ZnO samples 1-4 cooled for o 60 minutes after annealing in 5 wt. % NO/N2 at 300, 350, 400, and 450 C ...... 158 o Figure 4.17: Raman spectra of ZnO sample A annealed in 5 wt. % NO/N2 at 350 C ...... 160 o Figure 4.18: Raman spectra of ZnO sample B annealed in 5 wt. % NO/N2 at 400 C ...... 161 o Figure 4.19: Raman spectra of ZnO sample 8 annealed in PPN2 at 450 C ...... 163 Figure 4.20: Raman spectra over the range of 220-400 cm-1 shift for ZnO samples 5-8 cooled for o 60 minutes after annealing in PPN2 at 300, 350, 400, and 450 C...... 164

Figure 5.1: Optical illustration of the Renishaw inVia Raman microspectrometer ...... 183 Figure 5.2: Photograph of the remote, dispersive Raman configuration ...... 184 Figure 5.3: Optical drawing of the FT-Raman sample compartment interfaced to the System 2000 FT-IR; Diagram of the FT-Raman optical system ...... 186 Figure 5.4: Photograph of the PerkinElmer System 2000 NIR FT-Raman sample compartment ...... 187 Figure 5.5: Illustration of the displacement of the PET sample from the FT-Raman collection lens ...... 188 Figure 5.6: Raman spectrum of poly(ethylene terephthalate) ...... 190 Figure 5.7: Intensity of the PET 1614 cm-1 transition as a function of focal length ...... 191 Figure 5.8: Throughput as a function of focal length ...... 192 Figure 5.9: Raman spectra of PET collected using 100, 150, and 200 mm focal length lenses ...... 194 Figure 5.10: Plots of intensity versus scan number for ten, static spectral acquisitions ...... 195 Figure 5.11: Displacement profiles of intensity versus distance from the focal plane ...... 197 Figure 5.12: Intensity versus distance models for a conventional Raman microscope ...... 198 Figure 5.13: Intensity versus distance models for a confocal Raman microscope ...... 199 Figure 5.14: Theoretical model of normalized Raman intensity versus distance for objective lenses possessing focal lengths of 100, 150, and 200 mm ...... 200 Figure 5.15: Near-infrared absorption spectra of PET and water ...... 201 Figure 5.16: Raman scattering intensity versus sample thickness for various scattering coefficients ranging from r = 0 to r = 1000 cm-1 with α = 3 cm-1 and s = 1 cm-1; Raman scattering intensity versus sample thickness for various absorption coefficients ranging from α = 0.1 cm-1 to α = 100 cm-1 with r = 0 and s = 1 cm-1 ...... 204 Figure 5.17: Apparent absorption coefficient (a) versus sample thickness ...... 207 Figure 5.18: Calibration curve of intensity versus thickness for PET ...... 208 Figure 5.19: Plot of intensity versus scan number for ten, repetitive scans ...... 211 Figure 5.20: FT-Raman spectra of PET using different laser powers; Plot of 1614 cm-1 intensity versus laser power ...... 212 Figure 5.21: FT-Raman spectra of PET using different numbers of scans; Plot of 1614 cm-1 intensity versus number of scans ...... 216 Figure 5.22: FT-Raman spectra of PET using different spectral resolutions; Plot of 1614 cm-1 intensity versus log (spectral resolution) ...... 219

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Figure 5.23: FT-Raman displacement profiles for PET samples with thicknesses of 0.0254, 0.1016, and 0.1500 cm generated using the 1614 cm-1 intensity ...... 222 Figure 5.24: Illustration of the magnified image of the illuminated sample focused over a slit and a J-stop...... 227

Figure 6.1: Illustration of an inverted ATR-FT-IR imaging microscope ...... 243 Figure 6.2: Diagram of an ATR cartridge slide ...... 244 Figure 6.3: Photographs of the inverted ATR imaging microscope ...... 245 Figure 6.4: Photograph of the imaging optics; Optical drawing of the imaging optics ...... 246 Figure 6.5: Optical illustration of an inverted ATR-FT-IR imaging microscope ...... 248 Figure 6.6: Infrared reflectance image and response of a 1951 USAF resolution target group 5 element 1 ...... 249 Figure 6.7: Plot of signal-to-noise as a function of the square root of the number of scans ..... 251 Figure 6.8: Plot of signal-to-noise as a function of spectral resolution ...... 252 Figure 6.9: 1951 USAF resolution target group 7 ...... 253 Figure 6.10: 1951 USAF resolution target group 6 elements 6 to 4 and 3 to 2 ...... 253 Figure 6.11: Single-beam background image of bare, germanium IRE at 1000 cm-1; Single-beam background spectrum extracted at pixel 32 × 32; Sample image of adhesive tape at 1732 cm-1; Sample spectrum extracted at pixel 32 × 32 ...... 255

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List of Acronyms

% ABV Percent alcohol by volume % RSD Percent relative standard deviation % T Percent transmission

A/R Antireflective ACS American Chemical Society ATR Attenuated total internal reflection; Attenuated total (internal) reflection ATR-FT-IR Attenuated total internal reflection-Fourier transform infrared; Attenuated total (internal) reflection-Fourier transform infrared

CCD Charge-coupled device CE Capillary electrophoresis CIRCLE Cylindrical internal reflection cell for liquid evaluation COM Calcium oxalate monohydrate CVD Chemical vapor deposition CW Continuous wave

DOF Depth of focus DRIFTS Diffuse reflectance infrared Fourier transform spectroscopy DTA Differential thermal analysis DTGS Deuterated triglycine sulfate

EFL Effective focal length

FEWS Fiber optic evanescent wave spectroscopy FFT Fast Fourier transform FIA Flow injection analysis FPA Focal plane array FT Fourier transform

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FT-IR Fourier transform-infrared FT-Raman Fourier transform Raman FWHM Full-width-half-maximum

HGW Hollow glass waveguide HPLC High performance liquid chromatography HWG Hollow waveguide

IR Infrared IRE Internal reflection element

LOD Limit of detection LOQ Limit of quantitation LVM Local vibrational mode mabs milliabsorbance MCT Mercury-cadmium-telluride MOCVD Metalorganic chemical vapor deposition

NA Numerical aperture NEP Noise equivalent power NIR Near infrared NMR Nuclear magnetic resonance NT Normalized transmittance

OPD Optical path difference OPL Optical path length p-p Peak-to-peak PC Polycarbonate

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PC Polycrystalline PCA Principal component analysis PET Poly(ethylene terephthalate) PIR Polycrystalline infrared PLS Partial least squares PTFE Polytetrafluoroethylene rms root-mean-square RMS Royal Microscopical Society RSD Relative standard deviation

SC Single-crystal SNR Signal-to-noise ratio SQRT Square root

TGA Thermogravimetric analysis TTB Alcohol and Tobacco Tax and Trade Bureau

UV Ultraviolet

WiRE Windows-based Raman Environment wt. % Weight percent

Dedication

To my parents, Tom and Janet-thank you for your love, support and guidance.

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Acknowledgements

The academic achievements I have made would not have been possible without the assistance of several individuals. I would first like to sincerely thank my graduate research advisor, Dr. Andy Sommer, for his guidance and knowledge during my time in the Molecular Microspectroscopy Laboratory. I would like to thank Dr. Patricia Lang, Chairperson of the Department of Chemistry at Ball State University, for her positive influence as my undergraduate chemistry professor and for her advice to attend graduate school. Also, I would like to thank Kathy Dittman for her advice and assistance. To Adam Lanzarotta, thank you for your help and assistance when I first joined the lab. To Willie Tran, Chen Ling, and Taryn Winner, thank you for your added insights and discussions. Thank you to my friends and colleagues for your professional help and friendship; you have made graduate school something to remember. I would not be who I am, nor would I have achieved all that I have, without the love and support of my Mom and Dad. You have always encouraged me to do the best I can in all my endeavors. Cindy Luxford, thank you for your continued love, patience, and understanding. You have made graduate school a memorable experience. I am very thankful to have you in my life and look forward to what the future may bring.

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Chapter 1

Introduction

1.1 Infrared Spectroscopy

1.1.1 History of Infrared Spectroscopy The foundations of infrared spectroscopy date back to over three hundred years ago. Mariotte, in 1686, noticed that heat radiating from a fireplace could be focused using a mirror but did not pass through a glass plate.1 Scheele later confirmed this observation in 1781.2 Infrared spectroscopy was further investigated by Sir William Herschel in 1800 using a prism to disperse solar radiation onto mercury thermometers with blackened bulbs.3-6 Herschel was able to show the presence of invisible, heat-transporting radiation below the red end of the solar spectrum, which he called the infrared. Melloni constructed the first mid-infrared spectrometer after discovering the transparency of sodium chloride for infrared wavelengths.7-9 Abney and Festing first reported spectra of molecular vibrations in 1881.10 An atlas containing absorption, as well as emission and reflectance, spectra of organic compounds was published by Coblentz in 1905.11 Advancements in electronics during World War II resulted in the construction of automatic- recording infrared spectrometers. The development and availability of the PerkinElmer Model 21 spectrometer stimulated worldwide interest in infrared spectroscopy. Over one hundred Model 21 spectrometers were sold with approximately three hundred of the low-cost Model 137 Infracord, in 1957. Further improvements in mid-infrared instrumentation during the 1960s resulted in grating-based spectrometers replacing prism-based spectrometers due to increased optical conductance.

1.2 Fourier Transform Infrared (FT-IR) Spectroscopy

1.2.1 Instrumentation The basis of modern Fourier transform infrared (FT-IR) spectroscopy began during the latter part of the 19th century with the development of a two-beam interferometer by Michelson.12-14 A Michelson interferometer is illustrated in Fig. 1.1. The interferometer utilized a combination of a fixed mirror and a moving mirror with a beamsplitter positioned such that it bisected the planes of the two mirrors.

Source Fixed mirror

Moving mirror

Beamsplitter Sample

Detector

Figure 1.1: Diagram of an FT-IR spectrometer incorporating a Michelson interferometer.

Energy from an infrared source is collimated and directed towards the beamsplitter, usually a thin film of germanium on a potassium bromide substrate. Ideally, the beamsplitter will reflect 50% of the incident radiation and transmit the remaining 50%, resulting in two optical paths. In one path, infrared radiation is reflected by a fixed-position mirror back to the beamsplitter where it is partially reflected back to the source and partially transmitted to the detector. Infrared radiation in the second path is reflected by a moving mirror back to the

3 beamsplitter where it is also partially reflected and transmitted. Translation of the moving mirror results in a difference between the two optical pathlengths, known as the optical path difference (OPD). Additional information regarding the generation of an interferogram and the computation of an infrared spectrum can be found in a publication by Perkins.15 Recognition that the interferogram was related to the spectrum by a mathematical operation known as the Fourier transform occurred as early as 1892 by Lord Rayleigh.16 Fourier transformations require extensive calculation time, so the potential of this technique was not realized until the 1950s with the development of computers capable of performing these transformations quickly. Fellgett, in 1949, is credited as being the first individual to successfully transform an interferogram into is corresponding infrared spectrum.17 The first commercial FT- IR spectrometer designed for laboratory use, the Digilab (now Agilent) FTS-14, became available in 1969.18 The instrument featured a frictionless air bearing system developed by Block Engineering19, a laser reference system for wavelength calibration, and interchangeable beamsplitters, sources, and detectors. Significant advances in FT-IR instrumentation have been achieved since 1969, but the basic optical designs of these spectrometers remain unchanged. Infrared studies discussed in this dissertation were primarily performed using the PerkinElmer Spectrum 2000 and System 2000 (more recently known as the Spectrum GX) FT-IR spectrometers. Additional work was performed using the PerkinElmer Spectrum ONE FT-IR spectrometer. The interferometers incorporated in these instruments operate on a similar principle first described by Sternberg and James in 1964.20 A modification of the conventional Michelson interferometer was performed by mounting two or more mirrors on a common baseplate which was rotated in order to create an OPD. In the PerkinElmer instruments, this optical design resulted in the construction of the Dynascan interferometer.21 In the Spectrum and System 2000 FT-IR spectrometers, four plane mirrors were mounted on a tilt table. The benefit of this design was that any misalignment would result in each beam being affected in the same way; therefore, the interferometer was tilt- compensated. A similar interferometer design, known as the periscope interferometer, was incorporated into the PerkinElmer Paragon 1000, Spectrum 1000, and Spectrum ONE FT-IR spectrometers.22

1.2.2 Advantages of FT-IR Spectroscopy FT-IR spectrometers possess several advantages over dispersive instruments. A discussion of the various advantages associated with FT-IR was presented by Perkins.23 The three fundamental advantages of FT-IR instruments are the multiplex (Fellgett)24, throughput (Jacquinot)25, and wavenumber precision (Connes). A comparison of FT- and grating-based infrared spectrophotometers in terms of the multiplex advantage, relative optical throughputs, and performance was presented by Griffiths et al.26 Fellgett realized that the resolution elements of a dispersive instrument were examined individually as the dispersed spectrum was scanned past the exit slit of the monochromator.27-28 The Fellgett, or multiplex, advantage can be stated as: For spectra measured in the same time at the same resolution, optical throughput, and efficiency, the signal-to-noise ratio of a spectrum measured on an FT-IR spectrometer exceeds the signal-to-noise ratio of an identical spectrum measured on a grating spectrometer by the square root of the number of resolution elements in the spectrum.24 This advantage results from the spectrometer detecting all wavelengths simultaneously during a scan. The multiplex advantage is only realized when the system is detector-noise limited, that is, the detector noise remains constant as the signal increases. The second fundamental advantage of an FT-IR spectrometer over a dispersive spectrometer is the Jacquinot advantage.29 At the same spectral resolution, the optical throughput of an FT spectrometer with circular apertures is generally greater than that of a dispersive instrument equipped with entrance and exit slits. The optical throughput of a beam passing through an FT-IR spectrometer is limited by two apertures. An aperture stop, located at any position in the instrument other than an image plane, limits the intensity of the beam passing through the spectrometer. Alternatively, a field stop located at an image plane, determines the size and shape of the image. FT-IR spectrometers with resolutions better than 1 cm-1 possess a field stop located at a focus between the source and the interferometer. This field stop is referred to as the Jacquinot, or J-, stop. Characterization of an optical system is based on its limiting apertures and energy throughput, or étendue, calculated as the product of the area of the limiting aperture and the solid angle of the beam subtended at that aperture. In an FT-IR spectrometer, calculations of optical throughput are performed using the solid angle and diameter at the J-stop. The third advantage of modern FT-IR spectrometers is associated with increased wavenumber accuracy. The benefit of using a helium-neon laser to trigger data collection in an

FT-IR spectrometer was first realized by Connes.30 The wavenumber stability afforded by the Connes advantage results from the frequency scale of the FT instrument being linked to the helium-neon laser, which provides an internal reference for every interferogram.

1.3 Infrared Sampling Methods The three principal sampling methods employed in infrared spectroscopy are: transmission, reflectance, and attenuated total (internal) reflection (ATR). Illustrations of the principal sampling methods are shown in Fig. 1.2. The following sections will discuss the pertinent sampling methods employed in this dissertation.

Transmission Reflectance ATR

Figure 1.2: Principal infrared sampling methods. (Left) Transmission. (Middle) Reflectance. (Right) Attenuated total (internal) reflection (ATR).

1.3.1 Transmission Transmission is debatably the oldest and most widely-used method of infrared spectroscopy.31 Transmission infrared spectroscopy is the focus of a chapter by Duerst et al.32 Transmission requires the sample be thin enough to allow infrared radiation to pass through and reach the detector. Traditionally, a powdered sample is ground with an infrared transparent material, such as potassium bromide (KBr), and pelletized prior to analysis. Samples possessing thicknesses on the order of 10 µm can be mounted on an infrared-inactive substrate such as KBr, potassium chloride (KCl), or barium fluoride (BaF2) prior to analysis.

1.3.2 Reflectance Reflectance infrared spectroscopy can be divided into either specular33 or diffuse34 reflectance. Specular reflectance involves the analysis of reflective, or mirror-like, samples in which the angle of incidence is equal to the angle of reflection with respect to the sample surface. Diffuse reflectance involves the collection of infrared light reflected in all directions by the rough surfaces of a sample. Infrared specular reflectance is principally used to characterize thin films on reflective substrates; diffuse reflectance (DRIFT or DRIFTS) is used to obtain infrared spectra from finely ground powders. Mid-infrared diffuse reflectance spectroscopy is often performed by diluting the sample in a non-absorbing matrix, such as KBr or KCl. Reflectance measurements and discussions in this dissertation will focus on specular reflectance.

1.3.3 Attenuated Total Internal Reflection Attenuated total (internal) reflection (ATR) spectroscopy, sometimes referred to as internal reflection spectroscopy, is a commonly-used method of infrared spectroscopy due to the limited or nonexistent sample preparation associated with the technique. Development of the modern ATR methodology began in the early 1960s with the works of Harrick35-38 and Fahrenfort39-40. Attenuated total (internal) reflection spectroscopy is performed through the immersion of a sample in an optically dense material possessing a high refractive index, such as diamond, zinc selenide, silicon or germanium. Infrared light passing through the internal reflection element (IRE) will undergo total internal reflection at the interface between the IRE and the sample for angles of incidence greater than or equal to the critical angle, resulting in the formation of an evanescent wave capable of penetrating into the sample and being absorbed.

The critical angle (θc) required for total internal reflection can be determined using Eq. 1.1:

( ) (1.1)

where ns is the refractive index of the sample, and nIRE is the refractive index of the IRE. The depth to which the evanescent wave penetrates into the sample, referred to as the depth of penetration, dp, can be determined using Eq. 1.2:

(1.2)

[ ( ) ]

in which λ is the wavelength and θ is the angle of incidence with respect to the surface normal of the IRE. Additional information regarding ATR can be found in literature.41-43

1.4 Quantitative Analysis by Infrared Spectroscopy Quantitative analyses in the mid-infrared are performed using the Bouguer-Lambert-Beer law, simply referred to as Beer’s law. Bouguer was the first to realize that absorbance is proportional to pathlength44; however, it was not until the mid-1750s that Lambert45 was able to express this relationship in mathematical terms. Infrared spectroscopy is based on the measurement of light transmitted by a sample. Transmittance is defined as the ratio of the radiant power emerging from the rear face of the sample to the radiant power at the front face of the sample. The transmittance, T, of a pure sample of thickness b at wavenumber ̅ is given as:

( ̅) ( ̅) (1.3) ( ̅)

The absorbance of the sample at the selected wavenumber is given as the negative base 10 logarithm of the transmittance.

( ̅) ( ̅) (1.4)

Beer published a paper a century later showing that absorbance was proportional to concentration.46 In the case of a sample being either a pure substance or a mixture, the absorbance of each component, I, at concentration, ci, is given as:

( ̅) ( ̅) (1.5)

In Eq. 1.5, ( ̅) is the absorptivity at wavenumber ̅. In the case of a mixture containing N components where more than one component absorbs at ̅, the total absorbance is given in Eq. 1.6.

( ̅) ∑[ ( ̅) ] (1.6)

Quantitative analyses in the mid-infrared are often performed using either peak height or peak area, commonly referred to as integrated band intensity. When using peak height, small shifts in band position can result in significant error; therefore, quantitative analyses should be performed using integrated absorbance rather than the absorbance at a single wavenumber.

Quantitative analyses of aqueous solutions of succinylcholine chloride and ethanol using integrated absorbance are performed in Chapter 3 using a hollow waveguide ATR probe.

1.5 Infrared Fiber Optics and Waveguides Infrared fiber optics are commonly defined as fibers capable of transmitting light from 2 to approximately 20 µm. This special type of fiber optic can be divided into three broad categories based on the chemical and physical characteristics of the fibers/waveguides: glass, crystalline, and hollow waveguide (HWG). A review of the different types of infrared fibers/waveguides was published by Harrington.47 Additional sources of general information on infrared fiber types may be found in literature.48-49 The introductory material presented in this chapter will discuss the two most common types of infrared fiber optics currently employed, crystalline and hollow waveguide.

1.5.1 Crystalline Infrared Fibers When compared to infrared glass fibers, such as arsenic trisulfide, crystalline fibers are capable of transmitting longer wavelength radiation. Crystalline infrared fibers can be further divided into single-crystal (SC) and polycrystalline (PC). The most common and viable SC fiber developed is sapphire. Sapphire is an insoluble uniaxial crystal that is extremely hard, chemically inert, and can be conveniently melted and grown in air. Despite these benefits, the usable fiber transmission range is limited to 0.5 to 3.2 µm. Although this spectral range is of little use for mid-infrared spectroscopic sensing, this fiber has been shown to be ideal for the transmission of 2.94-µm light from an Er:YAG laser.50 Less work has been performed using SC fibers compared to PC fibers due to the difficulty associated with fabrication.

1.5.1.1 Polycrystalline Infrared Fibers Polycrystalline fibers are often preferred for use in mid-infrared spectroscopic applications due to their ability to transmit infrared light at long wavelengths. Historically, the first PC fibers were fabricated through the hot extrusion of thallium bromoiodide (TlBrI), commonly referred to as KRS-5, by Pinnow et al. in 1978.51 Selection of KRS-5 as the core material of the fibers was based on appreciable ductility and the availability of an extended transmission range beyond 20 µm. This type of fiber optic is no longer used due to the toxicity of thallium and the minimal flexibility of the fiber. Due to the requirements of the hot extrusion process, such as ductility and a low melting point, only PC fibers consisting of thallium and silver halides have been successfully fabricated. Thallium halides are highly toxic and are optically unstable. Silver halide PC fibers are currently preferred for the transmission of broadband infrared radiation. Figure 1.3 illustrates the construction of a PC silver halide fiber optic.

Silver halide core

Silver halide cladding

Polymer jacket

Figure 1.3: Diagram of a silver halide PC infrared fiber optic.

Silver halide fibers are non-toxic, flexible, and optically transparent over the range of 3- 18 µm. Several disadvantages associated with silver halide fibers include decreased transmission resulting from aging effects and photosensitivity to ultraviolet (UV) or visible light.52 The silver halide core material is also corrosive to many metals; therefore, PC fibers of this type must be connected with inert materials such as titanium, gold, or ceramic materials.

1.5.2 Hollow Waveguides Hollow waveguides offer an attractive alternative to solid-core infrared fibers for the transmission of broadband infrared wavelengths due to the capabilities of high power thresholds, low insertion losses resulting from no end-surface reflections, ruggedness, and small beam divergence.53-55 The theory describing transmission losses in HWGs was first thoroughly studied by Marcatili and Schmeltzer.56 HWGs possess inherent disadvantages associated with their construction and use.57-58 Specifically, attenuation of light transmitted by HWGs as a result of bending varies as 1/R, where R is the bending radius. In addition, attenuation losses in HWGs are also depended upon the bore radius, a, according to 1/a3. Therefore, increased flexibility gained through the use of a small diameter waveguide is offset by increased transmission loss. Attenuation associated with bore size and bending radius of HWGs are characteristics not shared by solid-core fiber optics. Hollow waveguides can be divided into two categories: those with inner core materials with refractive indices greater than 1 (leaky waveguides) and those with an inner wall material with a refractive index less than 1 in the wavelength region of interest (ATR waveguides). Leaky, or n > 1, waveguides have metallic and dielectric films deposited on the inside of metallic59, plastic60, or glass54 tubes. In contrast, ATR waveguides are fabricated using dielectric materials such as sapphire, which possesses a refractive index of 0.67 at 10.6 µm.61 The attenuation of incoherent mid-infrared radiation was investigated for hollow sapphire and silica waveguides by Saggese et al. for applications in spectroscopy and thermometry.62 Additional information on HWGs can be found in reviews by Harrington.47,63-64

1.5.2.1 Hollow Glass Waveguide Hollow-core waveguides have been fabricated using a variety of techniques, but the most popular HWG is the hollow glass waveguide (HGW) developed by Harrington17 at Rutgers University. A diagram of a hollow glass waveguide is shown in Fig. 1.4.

Air core

Silver iodide film

Silver film

Silica substrate

Polymer coating

Figure 1.4: Diagram of a hollow glass waveguide

The hollow glass structure is advantageous due to its simple design, flexibility, and smooth inner surface. Fabrication of these waveguides begins with a hollow silica capillary, which has been coated with a polymer on the exterior surface. A liquid-phase chemistry method, similar to that used by Croitoru et al.65 to deposit metal and dielectric layers on the inside of plastic tubes, is used in depositing silver and silver iodide (AgI) on the inner surface of the waveguide.66 The spectral response of the waveguide has been shown to be dependent upon the thickness of the AgI dielectric layer.54,63 A thin dielectric layer is preferred for the transmission of broadband infrared light.

1.6 Near-Infrared Spectroscopy The near-infrared (NIR) spectral region covers the range of 0.77 to 2.5 µm (12,900 to 4000 cm-1).67 Absorptions located in this region are assigned to overtones and combinations of fundamental C-H, N-H, and O-H stretching vibrations occurring in the mid-infrared between 3000 and 1700 cm-1. Although NIR spectroscopy is comparatively less useful than mid-infrared for qualitative analyses, the greatest utility of the method is the detection and subsequent quantitation of functional groups containing unique hydrogen atoms. Quantitative analyses of water, proteins, low-molecular-weight hydrocarbons, and fats have been performed using NIR spectroscopy in the agricultural, food, petroleum, and chemical industries.68 Additional information regarding the concepts and applications of NIR spectroscopy can be found in recent publications.69-70 Preliminary analysis of the absorption characteristics of poly(ethylene terephthalate) (PET) will be quantitatively performed in Chapter 5 using NIR absorption spectroscopy. The resulting optical characteristics will be applied to the NIR Fourier transform (FT-) Raman study of the polymer using 1064 nm laser excitation.

1.7 Raman Spectroscopy The first reported experimental observation of the Raman scattering effect was published by Raman and Krishnan in 1928.71 Raman spectra are acquired by irradiating a sample with monochromatic light from a visible or NIR laser. The incident radiation causes excitation of the sample molecule to a virtual state and subsequent re-emission of a photon of lower or higher energy. Stokes scattering occurs when the scattered radiation is of a lower frequency than the excitation radiation. Molecules in a vibrational excited state are also capable of producing a Raman signal with a higher frequency than the radiation source. This process is known as anti- Stokes scattering. Rayleigh scattering refers to elastically scattered light possessing the same frequency, or energy, as the excitation source. Although the Raman shift frequencies are independent of the excitation wavelength, the observed intensity of the Raman signal is dependent on several factors, as shown in Eq. 1.7:72

S = NVIoσ (1.7)

14 where N is the number of particles per unit volume (density), V is the volume of uniformly irradiated sample, Io is the excitation laser irradiance, and σ is the differential Raman scattering cross-section. Equation 1.7 can be further expanded to illustrate the dependence of the Raman signal upon several instrument parameters.

1/2 -1 -4 S = NVIoσΔνt Ωξ(NEP) λ (1.8)

These parameters include the spectral resolution, Δν, and the measurement time of each spectral element, t, the limiting aperture in the optical system, Ω, instrument efficiency, ξ, the noise equivalent power of the detector (NEP), and the excitation wavelength, λ.

1.7.1 Dispersive Raman Spectroscopy Conventional, dispersive Raman spectroscopy is generally performed using visible laser excitation wavelengths, such as the 488 or 514 nm lines of an argon-ion laser or the 633 nm line of a helium-neon laser. Equation 1.8 indicated that the intensity of the observed Raman signal is inversely proportional to the fourth power of the excitation wavelength. Although increased Raman signal is observed using short-wavelength laser excitation, sample fluorescence can become increasingly significant as excitation wavelength decreases. NIR laser excitation is often preferred in the collection of Raman spectra from samples exhibiting appreciable fluorescence. Despite the benefit of decreased fluorescence, the disadvantage to using NIR laser excitation is a diminution of Raman intensity. Coupling of Raman scattered light into a spectrograph is accomplished by matching the f/# of the pre-slit lens to that of the spectrograph. Dispersive Raman spectra are obtained by illuminating a prism or diffraction grating with collimated light entering through the entrance slit. After interacting with the dispersing optic, the light is incident upon a detector. The most common detector currently employed for visible Raman spectroscopy is the charge-coupled device (CCD). Silicon-based CCDs have a long-wavelength cutoff close to 1100 nm. Assuming an equivalent wavelength coverage to that of an FT-Raman spectrometer (200-3600 cm-1), the maximum laser wavelength is approximately 800 nm. Raman spectra collected using 785 nm, or even 830 nm, laser excitation provide appreciable fluorescence rejection while maintaining signal intensity.

1.7.2 Fourier Transform (FT-) Raman Spectroscopy The development of FT-Raman spectroscopy was fueled by the desire to reduce sample fluorescence by shifting the excitation wavelength to the NIR. NIR FT-Raman spectroscopy was first demonstrated by Chantry et al. in 196473; however, since little technology required to produce a viable, commercial instrument existed at that time, the study was primarily of academic interest. Several potential problems associated with the method, including Rayleigh line rejection, detector noise, and the “multiplex disadvantage” were addressed by Hirschfeld.74 FT-Raman remained essentially undeveloped until the mid-1980s when Hirschfeld and Chase demonstrated its capability using a NIR 1.064-µm Nd:YAG laser excitation source in the collection of Raman spectra from samples such as cyclohexane and phenol.75 Due to increased popularity, special issues of Spectrochimica Acta were devoted to applications and studies using FT-Raman.76-79 A thorough review of FT-Raman theory and a discussion of the process used in obtaining a spectrum was published by Parker.80 Fundamentally, an FT-Raman spectrometer is similar to a conventional grating-based instrument in that inelastically scattered light must be efficiently collected and passed through the spectrometer. Rabolt et al. showed that either reflective or refractive optics could be successfully used to collect Raman scattered light.81 In the late 1980s, a prototype FT-Raman system was developed and constructed at Southampton University by Hendra et al. in collaboration with PerkinElmer.82-84 Single-purpose and dual-purpose instruments capable of performing FT-Raman spectroscopy have been developed by major instrument manufacturers, including Bruker, PerkinElmer, and Thermo Scientific. For example, the PerkinElmer System 2000 FT-IR/FT-NIR spectrometer was designed to accommodate an FT-Raman sample compartment. Near-infrared and FT-Raman spectra were collected using a quartz beamsplitter; however, mid-infrared spectra could also be obtained by selecting an appropriate beamsplitter. Additional information regarding instrumentation associated with FT-Raman can be found in a publication by Cutler.85

1.8 Raman Microspectroscopy The interrogation of micrometer-sized samples using Raman spectroscopy can be accomplished through the coupling of a Raman spectrometer and a microscope. A review of Raman microspectroscopy is presented by Sommer.72 Raman microspectroscopy possesses an inherent advantage over its infrared counterpart due to the use of visible laser excitation. For a constant numerical aperture objective, the diffraction-limited spot sizes obtained using visible wavelengths are smaller than those obtained in the infrared. Although smaller focused spot sizes can be obtained, this capability does not control the spatial resolution of the measurement. In actuality, the sample dictates the spatial resolution that can be achieved since it is an optical component in the system; the exception is the analysis of optically thin or smooth films. The spatial resolution of Raman microspectroscopy is an order of magnitude better than that of infrared microspectroscopy. Improvements in spatial resolution can be obtained through spatial filtering using a confocal aperture. In confocal Raman microspectroscopy86-88, lateral and axial filtering can be achieved by placing a limiting aperture at an image plane of the illuminated sample. Schematic illustrations of the lateral and axial discriminations offered by confocal Raman microspectroscopy are shown in Fig. 1.5. Confocal Raman microspectroscopy was performed using the entrance slit of the monochromator and the detector elements on an array detector by Williams et al.89 Depth profiling can be performed by axially scanning a sampling through the focal plane of the objective. When applied to the analysis of planar, homogeneous samples, Fig. 1.5 indicates that the observed Raman signal will be the same for each position on the sample. Figure 1.5 also illustrates that axial displacement of the sample from the focal plane of the objective lens in a conjugate lens system results in a corresponding shift in the position of the resulting image. Increased image size at the confocal aperture, or entrance slit, caused from changes in sample position results in a diminution in Raman signal. The effect of sample displacement is studied with respect to the Raman signal of poly(ethylene terephthalate) (PET) in Chapter 5 using both dispersive and FT-Raman spectroscopy.

Figure 1.5: Diagram of (left) x-y discrimination caused by lateral displacement of the sample and (right) z-discrimination caused by axial displacement of the sample.

1.9 Thermogravimetric Analysis Thermogravimetric analysis (TGA) is the process by which the mass of a sample in a controlled atmosphere is recorded continuously as a function of either temperature or time as the temperature of the sample is increased.90 Commercial TGA instruments consists of: (1) a sensitive microbalance, (2) a furnace, (3) a purge-gas system for providing either an inert or reactive environment, and (4) a computer for instrument control and data acquisition and processing. Sample analysis by TGA can be performed using either a gradient temperature program where the sample temperature changes with time or isothermally where the temperature is held constant. The resulting thermogram, or thermal decomposition curve, is capable of providing quantitative information about the sample; however, the method is limited to studies of decomposition, oxidation, vaporization, sublimation, and desorption. The thermal decomposition of calcium oxalate monohydrate (CaC2O4∙H2O) is commonly used as a 91 quantitative example of TGA. The thermal decomposition of CaC2O4∙H2O is employed in Chapter 5 as a preliminary test of the capabilities associated with a custom-designed reaction cell for process-Raman analysis.

1.10 Infrared Microspectroscopy Infrared microspectroscopy represents the coupling of an infrared spectrometer and an optical microscope. Since its inception, infrared microspectroscopy has become a well- established method for sample analysis. Information regarding concepts and applications of infrared microspectroscopy can be found in books and reviews.92-100

1.10.1 History of Infrared Microspectroscopy Infrared microspectroscopy began with the development of a reflecting microscope by Burch.101-102 The microscope consisted of a condenser and an objective with aspherical surfaces, or Schwarzschild aplanats, possessing numerical apertures greater than 0.50. Barer, Cole, and Thompson, in 1949, successfully integrated a microscope of the Burch design to an infrared spectrophotometer.103 The performance of the instrument was demonstrated through the collection of infrared spectra from particles as small as 20 µm in diameter. Several detailed reports concerning infrared microspectroscopy, specifically factors affecting the integration of a microscope and a spectrophotometer, were published by Blout, Bird, and Grey.104-105 The authors demonstrated that objectives employing spherical surfaces could be employed in collecting infrared spectra of microsamples. The first commercial infrared microscope was developed in 1953 by the Perkin-Elmer Corporation. Details of this microscope were described in a publication by Coates, Offner, and Siegler.106 The infrared microscope, designated Model 85, was interfaced to Perkin-Elmer’s Model 12, 112, and 13 spectrophotometers. Revitalization of infrared microspectroscopy occurred with the development of the FT-IR spectrometer during the 1970s and 80s. The first commercial infrared microscope to combine high-quality visual imaging with infrared spectroscopy, known as the IR-PLAN, was introduced by Spectra-Tech, Inc. at the 1986 Pittsburgh Conference. The microscope accessory featured a dual confocal aperture system, also known as Redundant Aperturing, in which the infrared beam was focused at apertures (field stops) positioned at image planes before and after the sample.107 This optical configuration restricted the focused infrared radiation to the sample region of interest while minimizing interference from surrounding areas.108 Infrared microspectroscopy has evolved into a powerful method for the analysis of micrometer-sized samples.

1.10.2 Instrumentation An infrared microscope is a high-magnification beam condenser capable of introducing infrared radiation on a small transmitting or reflecting sample for the purpose of obtaining an infrared spectrum.109 Prior to the development of modern infrared microscopes, infrared microsampling was performed using beam condensing accessories located in the sample compartment of the infrared spectrometer. An optical microscope was used to view the sample prior to the collection of a spectrum. Ensuring that the same location on a sample was analyzed posed a challenge using this method. Infrared microscopes eliminate the problem of preliminary sample viewing by incorporating a white-light microscope in the optical path for viewing and manipulation of the sample at high magnification. The components of an infrared microscope are comparable to those of an optical microscope. A comparison of the optical designs of an optical microscope and an infrared microscope is shown in Fig. 1.6. Infrared microscopes (1) employ infrared radiation from the spectrometer as the source, (2) use reflecting optics, (3) use an aperture at the primary image plane for sample definition, and (4) use an infrared-sensitive detector. Refracting optics, such as those employed in optical microscopes, are not used in IR microscopes due to chromatic aberration. An aperture located at the primary image plane restricts the interaction of infrared light to the sample area of interest and prevents contamination from surrounding areas. A liquid- nitrogen-cooled mercury-cadmium-telluride (HgCdTe, MCT) detector is the most common detector used for infrared microspectroscopy. The function of an infrared microscope is also comparable to that of an optical microscope. An infrared microscope must illuminate the sample with the spectrometers infrared source, collect light that has interacted with the sample and image this light onto a suitable detector, and allow the user to view and select specific sample areas. The incorporation of a white-light microscope in an infrared microscope requires both optical paths be collinear with images formed at the same position along the z-axis, i.e., parfocal.

The smallest sample size capable of being analyzed with minimal contributions from surrounding areas is defined by the diffraction-limited diameter of the focused beam as determined by:

(1.9)

where λ is the wavelength, and NA is the numerical aperture. The NA is defined as the product of the refractive index in which the sample is immersed (n1) and the sine of the maximum collection angle of the optic, θ.

(1.10)

Most conventional infrared microscopes employ objectives and condensers with 0.6 NA. Therefore, the maximum angle of light collected by theses optics is 36.9o.

Figure 1.6: Schematic diagrams of a visible wavelength optical microscope with refractive optics (left) and an infrared microscope with reflective optics (right).

1.11 Infrared Microspectroscopic Imaging

1.11.1 History of Infrared Imaging Fourier transform infrared (FT-IR) spectroscopic imaging has become a well-established technique for the acquisition of spatially-resolved spectroscopic data. The development of FT- IR imaging in which an interferometer was coupled to an infrared microscope and a two- dimensional array detector began during the 1990s. During this time, multichannel infrared array detectors originally designed for military use became commercially available. These detectors often possessed defects, such as dead pixels, that did not meet military specifications. Additional information on the instrumentation used for FT-IR imaging can be found in reviews by Kidder et al110 and Griffiths111. Initial applications of infrared spectroscopic imaging were performed using step-scan FT- IR spectrometers. In step-scan interferometers, the retardation of the moving mirror is changed to the desired value and then held constant. Image frame collection by a focal plane array (FPA) is triggered after the interferometer mirror has been stepped to a new position. The first report of this method of data acquisition was published by Lewis et al. in 1995.112 The authors employed a 128 × 128 indium antimonide (InSb) FPA detector coupled to a step-scan interferometer. Using this system, 16,384 spectra were obtained at 16 cm-1 resolution for a twelve second acquisition time; however, due to the limited spectral range of the InSb array, spectral data was not available in the mid-infrared fingerprint region. Kidder et al. later coupled a mercury- cadmium-telluride (MCT) FPA to a step-scan interferometer and an infrared microscope.113 Mid-infrared spectroscopic imaging was performed over the spectral range of 3500-900 cm-1 at various spectral resolutions. Infrared spectrometers possessing continuous, or rapid-scan, interferometers have also been employed for FT-IR imaging. The first implementation of a rapid-scan FT-IR imaging spectrometer was presented by Bennett et al. in 1993.114 Snively et al. later employed a rapid- scan system consisting of a first-generation, 64 × 64 MCT FPA in the collection of infrared spectra at a slow mirror velocity.115 Compared to step-scan approaches, data acquisition can be performed faster using a rapid-scan approach since there is no requirement for stabilization of the moving mirror. Although this temporal advantage is beneficial, the primary advantage of continuous-scan imaging systems is the reduced instrumentation cost compared to step-scan

24 systems. It is for these reasons that the majority of infrared spectroscopic imaging currently performed is done using FT-IR spectrometers with continuous-scan interferometers. The concept of infrared imaging is shown in Fig. 1.7. Data are first acquired as an interferogram, with each pixel on the array possessing a response determined by it corresponding spatial location on the sample. A fast Fourier transform (FFT) results in each pixel on the array containing a complete infrared spectrum that is representative of the chemical composition at that location on the sample. Reviews regarding concepts and applications of infrared imaging and spectrochemical analyses can be found in publications by Bhargava and Levin.116-117

(n) FFT and Conversion (n) to Absorbance (m) (m)

(Y) (Y)

(X) (X)

Figure 1.7: Schematic representation of a rapid-scan FT-IR imaging data set collected using an array detector with n × m pixels.

1.11.2 Advantages of Infrared Imaging Using a Focal Plane Array The combination of a FPA detector and an interferometer provides both multichannel and multiplex advantages.112 The multichannel advantage results from the simultaneous collection of infrared spectra from the multiple detector elements, pixels, on the array. As with conventional FT-IR spectroscopy, the multiplex advantage is the result of all spectral frequencies across the wavelength region of interest being measured concurrently. Depending on the array and the collection parameters, thousands of infrared spectral images at near-diffraction-limited spatial resolution can be obtained in minutes.118

1.11.3 ATR-FT-IR Microspectroscopic Imaging ATR-FT-IR microscopic imaging refers to the collection of spatially-resolved infrared images using an infrared microscope possessing a FPA detector. ATR imaging utilizes the concept of conventional ATR spectroscopy developed by Harrick119 and Fahrenfort120 in which a sample is placed in direct, intimate contact with a high-refractive-index material, such as germanium. Two approaches are generally used when performing ATR imaging: (1) on-axis imaging and (2) off-axis imaging. In on-axis imaging, the hemispherical IRE and the sample are centered at the focus of the microscope objective and are illuminated globally. Infrared radiation that has undergone total internal reflection at the IRE-sample interface is imaged onto a two- dimensional array detector. Off-axis imaging is performed by centering the hemispherical IRE and the sample are centered in the focus of the microscope with imaging being performed by moving the IRE and sample off-axis. The off-axis imaging technique is typically employed using either a single-element detector or a linear array detector. The development and application of a prototype inverted ATR imaging microscope discussed in Chapter 6 employs the on-axis imaging approach. The optical configuration of an imaging system does not require area defining apertures since the sample area is imaged directly onto the array detector. The size of the FPA detector defines the sample area that can be imaged, and the pixel size defines the spatial element on the sample, a parameter known as pixel resolution. For example, Sommer et al. employed a 64 × 64 MCT FPA possessing 61 × 61 µm pixels for the ATR-FT-IR imaging of several polymers and biological samples.121 For a magnification factor of 13× from the detector to the sample, Sommer et al. determined the FPA detector was capable of imaging a 300 × 300 µm area on the

26 sample at a 4.7 µm pixel resolution. ATR images were collected using a germanium hemisphere with a refractive index of 4.0. An improvement in spatial resolution, as well as an increase in magnification, equal to the refractive index of the hemispherical IRE was obtained. Using the ATR accessory, a 75 × 75 µm area of the sample was capable of being imaged onto the array detector at a pixel resolution of 1.2 × 1.2 µm. The capability of this imaging method was demonstrated through the collection of an infrared image from a single red blood cell. Additional information regarding micro- and macro-ATR-FT-IR spectroscopic imaging can be found in a review by Kazarian and Chan122 as well as a book chapter by Gulley-Stahl et al.123

1.12 Dissertation Goals and Specific Aims The goal of the research presented in this dissertation is to develop and expand upon modern infrared and Raman instrumentation and techniques for qualitative and quantitative sample analyses. The studies discussed will focus on the characterization and evaluation of conventional instrumentation and methods currently used in infrared and Raman spectroscopy. In some cases, however, further development of either instrumentation or techniques is demonstrated in order to provide alternative, and potentially improved, methods of sample analysis. The goals and specific aims of each chapter are:  Chapter 2 presents a comparison of infrared-transmitting polycrystalline silver halide fiber optics and hollow silica waveguides for the purpose of constructing a fiber/waveguide-coupled ATR probe. Characterization of the fibers/waveguides is performed with respect to infrared transmission using a fiber optic coupling accessory and an infrared microscope.  Chapter 3 evaluates a mid-infrared remote sampling accessory employing a hollow waveguide ATR probe on the basis of infrared transmission and signal-to-noise. Practical sampling applications of the accessory include quantitative analyses of aqueous solutions of succinylcholine chloride and ethanol. Determinations of alcohol content in commercial beer, wine, and liquor are performed as a practical application of the proposed quantitative method.  Chapter 4 demonstrates the use of a specially-designed, high-temperature reaction cell for process-Raman analysis. The thermal decomposition of calcium oxalate monohydrate is monitored by coupling the reaction cell to a Raman microspectrometer. An in situ study of nitrogen incorporation in thin films of zinc oxide for the purpose of generating a p- type semiconductor is performed using a Raman microprobe.  Chapter 5 investigates the application of dispersive and Fourier transform Raman spectroscopy to on-line, quantitative determinations of poly(ethylene terephthalate) (PET) bottle thickness. A determination of the optimum sampling configuration applicable for use in on-line process control in the bottling industry is discussed. Impacts on Raman intensity-thickness calibrations resulting from changes in instrument parameters are addressed.

 Chapter 6 discusses the preliminary development of a prototype inverted ATR-FT-IR imaging microscope. Initial testing of the instrument includes an evaluation of image quality and spatial resolution obtained in reflectance mode. Continued assessment of the microscope includes a determination of the response obtained while in ATR imaging mode.

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Chapter 2

Characterization of Silver Halide Fiber Optics and Hollow Silica Waveguides for Use in the Construction of a Mid-Infrared Attenuated Total Reflection-Fourier Transform Infrared (ATR-FT- IR) Spectroscopy Probe†

2.1 Abstract Advances in fiber optic materials have allowed for the construction of fibers and waveguides capable of transmitting infrared radiation. An investigation of the transmission characteristics associated with two commonly used types of infrared-transmitting fibers/waveguides for prospective use in a fiber/waveguide-coupled attenuated total (internal) reflection (ATR) probe was performed. Characterization of silver halide polycrystalline fiber optics and hollow silica waveguides was done on the basis of the transmission of infrared light using a conventional fiber optic coupling accessory and an infrared microscope. Using the fiber coupling accessory, the average percent transmission for three silver halide fibers was 18.1 ± 6.1% relative to a benchtop reflectance accessory. The average transmission for two hollow waveguide (HWGs) using the coupling accessory was 8.0 ± 0.3%. (Uncertainties in relative percent transmission represent standard deviations.) Reduced transmission observed for the HWGs was attributed to the high numerical aperture of the coupling accessory. Characterization of the fibers/waveguides using a zinc selenide lens objective on an infrared microscope indicated 24.1 ± 7.2% of the initial light input into the silver halide fibers was transmitted. Percent transmission obtained for the HWGs was 98.7 ± 0.1%. Increased transmission using the HWGs resulted from the absence or minimization of insertion and scattering losses due to the hollow air core and a better matched numerical aperture. The effect of bending on the transmission characteristics of the fibers/waveguides was also investigated. Significant deviations in the transmission of infrared light by the solid-core silver halide fibers were observed for various bending angles. Percent transmission greater than 98% was consistently observed for the HWGs at the bending angles. The combined benefits of high percent transmission, reproducible

† Reproduced with permission of Society for Applied Spectroscopy via Copyright Clearance Center. Reference: C. A. Damin, A. J. Sommer. Appl. Spectrosc. 2013. 67(11): 1252-1263.

45 instrument responses, and increased bending tolerance indicated HWGs would be preferred in the construction of a fiber/waveguide-coupled ATR probe.

2.2 Introduction

2.2.1 Infrared Fiber Optics Recent advances in mid-infrared-transmitting fibers provide alternatives to the use of benchtop accessories for remote sample analyses. Several materials capable of transmitting infrared light have been developed and used as optical conduits.1-2 The materials used to fabricate infrared-transmitting fiber optics are generally divided into three broad categories: glass, crystalline, and hollow waveguides (HWGs).3 Crystalline fibers include both polycrystalline (PC) fibers, such as silver halides, and single-crystal (SC) fibers, such as sapphire. The most common type of HWG is the hollow glass waveguide constructed using a glass capillary possessing a metallic-dielectric inner surface. Pinnow et al. reported that infrared fiber optics had been prepared using PC cores of thallium bromoiodide (TlBrI), commonly known as KRS-5.4 These fibers are generally no longer used due to the high toxicity of the core materials. Silver halide fiber optics offer an attractive alternative due to such benefits as increased flexibility, non-toxicity, and wide spectral range. Silver halide fibers also possess several disadvantages, such as the capability of undergoing photodegradation when exposed to ultraviolet (UV) and visible light. The silver halide core is also corrosive to many metals, so these fibers must be connectorized using titanium, gold, or ceramic materials. Increased transmission losses are typically observed along the length of the fibers due to aging effects.5 Fiber optic attenuated total (internal) reflection (ATR) probes designed for use the mid- infrared have been previously constructed using stripped, or core-only, silver halide fibers as internal reflection elements (IREs). These probes directly use the evanescent waves formed from total internal reflection of infrared radiation propagating through the core for sample analysis.6-7 This method is known as fiber optic evanescent wave spectroscopy (FEWS). A diagram of the concept and optical arrangements associated with FEWS is shown in Fig. 2.1.

Figure 2.1: Diagram of fiber optic evanescent wave spectroscopy (FEWS). A bare, core-only fiber is used as both the optical conduit and the sensing element. The insert depicts total internal reflection of the infrared radiation at the interface between the sample and the fiber.

The core-only fiber serves as both the IRE and the optical conduit for transmitting infrared radiation. The concept of FEWS can be expanded to include the use of ATR probes possessing high refractive index IREs. In a publication by Artyushenko et al., PC infrared (PIR) fiber probes using different IREs were examined.8 The selected IREs included cones made of zinc selenide (ZnSe), diamond, and germanium and unclad fiber loops of different shapes. Probes and sensors using these configurations have been used in a variety of applications, including the authentication of extra virgin olive oils9 and the examination of medical, pharmaceutical, and bioanalytical samples.10-13 Attenuated total (internal) reflection-Fourier transform infrared (ATR-FT-IR) spectroscopy is commonly used in the analysis of biological or physiological samples, such as urinary calculi (kidney stones).14-15 Urine and urinary calculi have been analyzed using a flattened segment of the infrared-transparent silver halide fiber as the IRE.16 This optical configuration is similar to that of the CIRCLE cell ATR accessory. Raichlin et al. previously used a flattened silver halide fiber to investigate eight of the most common minerals responsible for the formation of kidney stones, including: hydroxyapatite, L-cystine, brushite, and uric acid.17 Fiber optic evanescent wave spectroscopy is often applied to the examination of skin and

47 tissue samples. Research published in 2003 by Heise et al. demonstrated the use of a U-shaped segment of a silver halide fiber for dermatological examinations of the epidermal layer(s) of human skin.18 Infrared fiber optic probes have also been used in diagnosing living and cancerous tissues.19 Li et al., in 2005, used an infrared fiber optic probe with a zinc sulfide IRE for the identification and evaluation of human cartilage degradation.20

2.2.2 Hollow Waveguides Hollow waveguides have emerged as modern alternatives to conventional solid-core fibers. Hollow-core fibers permit the transmission of infrared radiation without significant attenuation associated with insertion. Hollow waveguides can be categorized into two groups depending on their physical construction: (1) waveguides with inner wall materials possessing refractive indices greater than 1 (leaky waveguides) and (2) waveguides with inner wall materials less than 1 (ATR waveguides).2-3 Leaky waveguides possess metallic-dielectric films deposited on the interior surfaces of metallic21, plastic22, or glass23 tubes. In contrast, ATR waveguides are constructed using dielectric materials such as sapphire, which possesses a refractive index of 0.67 at 10.6 µm.24-25 The HWGs employed in the present evaluation were constructed by depositing a dielectric layer of silver-silver iodide on the inner surfaces of silica capillaries. A 2000 publication by Harrington provided a comprehensive review of infrared- transmitting HWGs.26 Hollow glass waveguides are among the most popular HWGs currently used. Waveguides of this form, initially developed by Abel et al., are constructed through the deposition of a silver film on the inner surface of a silica tube.27 A thin layer of the metallic surface is then converted to silver iodide, resulting in the formation of a dielectric layer. A polymer coating of UV acrylate or polyimide is applied to the exterior surface of the waveguide for protection and preservation. The theory describing attenuation losses associated with circular HWGs was first explored by Marcatili and Schmeltzer in 1964.28 Subsequent research by Miyagi and Karasawa provided a measurement of attenuation as a result of bending.29 The resulting work from these groups provided the relationships that govern attenuation losses for HWGs. Current applications of HWGs include laser power delivery and the construction of remote sensors. Matsuura et al. discussed the optical properties of small-bore hollow glass waveguides with respect to

48 fabrication, attenuation, and the capability to transmit laser radiation.23 The thickness of the inner silver iodide layer can be optimized to produce high reflectivity for a specific wavelength or for broadband transmission. Currently, the thickness of the inner dielectric layer can be 30 modified to accommodate either CO2 (10.6 µm) or Er:YAG (2.9 µm) laser systems. Reflectance and ATR probes employing HWGs have been applied in FT-IR spectroscopy.31-33 Matsuura et al. investigated multiple prismatic IRE configurations for use in a HWG ATR probe. Accessories incorporating infrared-transmitting waveguides are advantageous for the analysis of samples that either are too larger for the FT-IR sample compartment or cannot be easily analyzed using conventional accessories.

2.3 Goals and Specific Aims A side-by-side comparison of the infrared transmission capabilities of silver halide fiber optics and hollow silica waveguides will be performed using both a conventional fiber optic coupling accessory and an infrared microscope. The goal of the characterization studies for these fibers/waveguides is the development of a fiber- or waveguide-coupled ATR probe for remote sample analysis. The effect of bending on the instrument responses obtained using the infrared fibers/waveguides will also be performed.

2.4 Experimental

2.4.1 Materials Silver halide fiber optics with core and cladding diameters of 900 and 1000 µm, respectively, were purchased (JT Ingram Technologies, Oviendo, FL); they were 1 m in length and possessed a transmission range of 4-18 µm. According to the listed specifications, the silver halide fibers possessed a numerical aperture (NA) of 0.25, and the silver halide cores possessed a refractive index of 2.150. Hollow silica waveguides were purchased (Polymicro Technologies, LLC, Phoenix, AZ) that possessed an inner diameter of 1000 µm and an outer diameter of 1600 µm. The selected HWGs, optimized for the transmission of 2.9 µm radiation from an Er:YAG laser, exhibited low

49 attenuation over the range of 2.9-12.0 µm. The waveguides were 1 m in length and had a durable acrylate buffer on the exterior surface. The spectral characteristics of the HWGs indicated that smaller attenuations would be obtained using an Er:YAG-optimized waveguide than using a similar waveguide optimized for the 10.6 µm output of a CO2 laser.

2.4.2 Instrumentation Infrared spectra of the silver halide fiber optics and the hollow silica waveguides were collected using a Harrick Scientific FiberMate2 fiber optic coupling accessory interfaced to a PerkinElmer Spectrum 2000 FT-IR spectrometer. A photograph of the FiberMate2 accessory is shown in Fig. 2.2.

Figure 2.2: Harrick FiberMate2 fiber optic coupling accessory.

This accessory uses two 90o off-axis ellipsoidal mirrors capable of focusing infrared radiation to a 1 mm diameter spot. The standard deuterated triglycine sulfate (DTGS) detector located in the Spectrum 2000 macrobench was used in conjunction with the fiber optic coupling accessory. Spectra collected for the fibers/waveguides represent the average of 64 individual scans at 4 cm-1 resolution. Additional analyses of the fibers/waveguides were performed using a Spectra-Tech IR- PLAN infrared microscope accessory coupled to the PerkinElmer Spectrum 2000 FT-IR

50 spectrometer. Figure 2.3 depicts the instrument setup using the infrared microscope to focus infrared radiation into the fibers/waveguides using two microscope objectives: (1) an Ealing 15× Cassegrain reflecting objective and (2) a biconvex ZnSe lens objective. The lens objective was fabricated using a 33.3 mm long aluminum tube with an inner diameter of 22.8 mm and an outer diameter of 28.5 mm. A standard 20 mm microscope objective extension was used to attach the optic to the microscope. A 1-inch diameter ZnSe biconvex lens with an effective focal length of 25 mm at 6 µm was purchased (Smiths Detection, Hopewell Junction, NY); the lens was coated with an antireflective (A/R) material at the specified wavelength. The infrared microscope utilized a Graseby Infrared (Newmarket, UK) liquid-nitrogen-cooled mercury cadmium telluride (MCT) detector (model no. NO170186). Single-beam spectra collected with each fiber/waveguide connected to the microscope represent the average of 64 individual scans at 4 cm-1 resolution. Instrument control of the PerkinElmer Spectrum 2000 FT-IR spectrometer was done using PerkinElmer Spectrum software (version 3.02.00 [2000]).

Figure 2.3: Instrument diagram of fiber optics and waveguides analyzed using the Spectra-Tech IR-PLAN infrared microscope accessory coupled to a PerkinElmer Spectrum 2000 FT-IR spectrometer.

Infrared spectra of the interior polymer jacket of a silver halide fiber and Parylene were collected using a Harrick SplitPea ATR accessory coupled to a PerkinElmer Spectrum ONE FT-

IR spectrometer. The accessory employed a silicon IRE and the standard DTGS detector in the macrobench. Spectra collected using this accessory represent the average of 32 individual scans at 4 cm-1 resolution. Samples were brought into direct contact with the IRE using a loading of 0.5 kg. Instrument control of the Spectrum ONE FT-IR spectrometer was done using PerkinElmer Spectrum software (version 5.3.1 [2005]).

2.4.3 Methods The energy monitoring function of the Spectrum software (version 3.02.00) was used to measure light transmission for the fibers/waveguides. Investigation of the transmission characteristics was performed using the fiber optic coupling accessory with the fibers/waveguides looped between the two SMA (SubMiniature version A) connector ports on the accessory. Optimization of the accessory for each individual fiber/waveguide was performed using the method outlined by Harrick Scientific. The Harrick SplitPea is a horizontal reflection accessory capable of being configured for either ATR or reflectance measurements.34 The SplitPea was converted to its specular reflectance configuration by replacing the IRE plate with an identical plate from which the IRE had been removed and that had a flat, first-surface mirror at the sampling position. The reflectance accessory was used in obtaining a relative energy throughput for the FiberMate2 accessory since the optical configurations of the two accessories are essentially the same. The accessories differ with respect to the orientations of the ellipsoidal mirrors. In the SplitPea, these mirrors are oriented to provide a 45o angle of reflection at the surface of the IRE. The ellipsoidal mirrors in the FiberMate2 are positioned to focus and collect infrared radiation entering and exiting fibers positioned 90o directly above the mirrors. Prior to analysis, the IR-PLAN infrared microscope was aligned in reflectance mode. The fibers/waveguides were mounted to the microscope stage using a custom-designed mounting plate possessing an SMA 905 barrel connector. Infrared radiation was launched into the fibers/waveguides using a 15× reflecting objective (0.28 NA) and a ZnSe objective lens (0.25 NA). Photographs of the two microscope objective are shown in Fig. 2.4.

Figure 2.4: Microscope objectives used to focus infrared radiation into the fibers/waveguides. (Left) The 15× reflecting objective (0.28 NA). (Right) The ZnSe objective lens (0.25 NA).

A flat, front-surface mirror was placed at the output of the fibers/waveguides to direct light back to the detector. The relative amount of infrared radiation passing through the fibers/waveguides was evaluated by removing the reflecting mirror. The energy difference observed between the presence and absence of the mirror was used in determining the overall amount of transmitted infrared radiation. A remote aperture was used to minimize specular reflections at the input end of the fibers/waveguides and to ensure that the radiation was incident only on the cores of the fibers/waveguides.

2.5 Results and Discussion

2.5.1 Harrick FiberMate2 A comparative evaluation of the transmission capabilities of the silver halide fibers and hollow silica waveguides was performed using a fiber optic coupling accessory. Since the optical design of the accessory was identical to that of the reflectance accessory, the transmission values measured for the fibers/waveguides were compared to those obtained using the reflectance accessory. Results from this initial study are shown in Table I; the uncertainties associated with the values listed in this table are standard deviations.

Table 2.1: Infrared Transmission by Fibers/Waveguides Using Harrick FiberMate2

TABLE I. Infrared Transmission by Fibers/Waveguides Using Harrick FiberMate2

Identification Energy Transmission a Percentage Relative to SplitPeab counts

Silver Halide 1 374 ± 6 24.9 Silver Halide 2 196 ± 1 13.0 Silver Halide 3 250 ± 1 16.6

Average 271 ± 89 18.1 ± 6.1

Hollow Waveguide 1 124 ± 2 8.2 Hollow Waveguide 2 117 ± 2 7.8

Average 121 ± 5 8.0 ± 0.3

a Average of two collections

b Energy of SplitPea ATR/reflectance accessory in absence of IRE 1504

c Uncertainties represent standard deviation

Increased transmission was observed for the solid-core silver halide fibers compared to the HWGs. Significant deviations in transmission were also observed among the three silver halide fibers. In contrast, the transmission of infrared radiation by the two HWGs was comparable. The optical mechanisms of coupling light into solid-core fibers and HWGs differ. The effect of coupling light into HWGs in contrast to silver halide fibers has been explained by Harrington.26 The large effective NA associated with the silver halide fibers permits coupling at relatively large angles, such as those employed in the fiber optic coupling accessory. Since the effective NA of the HWGs is very small by comparison, the coupling of light into HWGs is typically done using optics possessing long focal lengths. The optical design specifications of the Harrick FiberMate2 indicate that coupling of infrared radiation into the hollow silica waveguides is not preferred. Data obtained for the fibers/waveguides indicate a low transmission of infrared radiation by the HWGs as a result of high NA coupling. The FiberMate2 represents the most popular fiber coupling accessory currently available. Other available coupling accessories include 90o off-axis parabolic mirrors; however, the NAs associated with these optics are also large, typically on the order of 0.6. The current study presents a side-by-side comparison of the transmission capabilities of solid-core silver halide fibers and hollow silica waveguides using the technology currently available. Infrared sampling accessories employing HWGs as optical conduits require coupling optics optimized for use with low NAs.

2.5.2 Spectra-Tech IR-PLAN Infrared Microscope The coupling of light into a fiber optic/waveguide is based on NA, which can be determined using Eq. 1:

NA = n sin θ (2.1) where n is the refractive index of the medium in which the optic is being used, and θ is the acceptance angle of the most divergent ray entering the optic. When applied to fibers/waveguides, NA is a measure of the most extreme angle of light capable of being accepted for transmission. In the case of solid-core fiber optics, the NA can be determined using the refractive indices of the core (ncore) and cladding (ncladding) according to Eq. 2.

√( ) ( ) (2.2)

For a fiber optic possessing a 0.25 NA, the angle of the most extreme ray entering the fiber is 14.5o. Using Eq. 2 and the refractive index of the silver halide core, the resulting refractive index of the cladding material is 2.135. The cladding of a silver halide fiber is usually silver bromochloride (AgBrCl) with a different composition ratio than that of the core. Successful coupling of light into a fiber optic/waveguide requires the NA of the coupling optic be identical to or less than that of the fiber. Figure 2.5 represents the coupling of light into the solid-core silver halide fibers with input angles of 14.5 and 30.0o.

Figure 2.5: Optical diagrams of an infrared fiber optic depicting launch angles of (a) 14.5o and (b) 30.0o. The refractive indices of the silver halide core, the cladding, and the surrounding jacket are 2.150, 2.135, and 1.70, respectively.

Infrared radiation propagates along the length of the fiber by total internal reflection (TIR) at the interface between the core and the cladding for incident angles equal to or greater than the critical angle of 83.2o. The coupling configuration using a focusing optic with the same NA as the silver halide fibers is illustrated in Fig. 2.5a. The coupling of light into the silver halide core at 30.0o (Fig. 2.5b) represents an improper optical matching between the NAs of the coupling optic and the fiber optic/waveguide. A 30.0o coupling angle is improper for both the silver halide fiber optics and the hollow silica waveguides. According to Snell’s law, light enters the core of the fiber optic at a refracted angle of 13.5o; therefore, the resulting angle of incidence

56 at the core-cladding interface is 76.6o. This angle is well below the critical angle required for TIR. Improper coupling of light into the solid-core fiber optics can result in light entering the cladding material and being absorbed by the surrounding polymer jacket, resulting in decreased transmission. Unlike the solid-core silver halide fiber optics, infrared radiation coupled into the HWGs propagates through a series of reflections from the metallic-dielectric surface. NAs are not generally listed for HWGs since they are considered hollow, reflective tubes. Kriesel et al. indicated the effective NA of similar hollow glass waveguides with different bore sizes as being 0.01 to 0.05.35 An article by Polymicro Technologies discusses several of the recommended parameters for coupling light into a HWG.36 Minimization of coupling losses can be accomplished using a low-NA optic. Results obtained for the transmission of infrared radiation by the silver halide fibers and the HWGs using the infrared microscope are shown in Table II. Data listed in this table indicate significant differences in the amount of light transmitted by the fibers/waveguides; uncertainties in the percentages of light transmitted represent standard deviations. Despite the similar NAs between the reflecting and refracting optics, the physical constructions of the two objectives are very different. The reflecting Cassegrain objective consists of a primary and a secondary mirror. The presence of the secondary mirror produces a central obscuration that reduces the amount of infrared radiation coupled into the fibers/waveguides. The large energy values observed for the silver halide fibers in the absence of the output-reflecting mirror can be attributed to two factors: (1) scattering within the solid core, and (2) Fresnel reflection at the front surface of the solid-core fibers. Several publications have shown that a major source of attenuation in PC fibers is the scattering of light by microscopic pores or microvoids formed during the extrusion process.37-38 Increased attenuation can also occur due to the formation of microcracks and microfractures as a result of repeat bending. The formation of reduced silver inside the solid core also decreases the amount of light transmitted.

Table 2.2: Transmission of Infrared Light Measured by Coupling Fiber Optics and Hollow Waveguides to an Infrared Microscope

TABLE II. Transmission of Infrared Light Measured by Coupling Fiber Optics and Hollow Waveguides to an Infrared Microscopea

Identification Microscope Objective Energy with Mirror Energy Without Mirror % of Light Transmitted b counts counts

Silver Halide 1 Reflecting Cassegrain 1216 1004 17.1 Silver Halide 2 Reflecting Cassegrain 1028 872 15.0 Silver Halide 3 Reflecting Cassegrain 1146 943 17.7

Average 16.6 ± 1.4

Silver Halide 1 ZnSe Lens 2566 1749 31.8 Silver Halide 2 ZnSe Lens 2088 1723 17.5 Silver Halide 3 ZnSe Lens 2525 1946 22.9

Average 24.1 ± 7.2

Hollow Waveguide 1 Reflecting Cassegrain 193 24 87.6 Hollow Waveguide 2 Reflecting Cassegrain 199 24 87.9

Average 87.8 ± 0.2

Hollow Waveguide 1 ZnSe Lens 1110 16 98.6 Hollow Waveguide 2 ZnSe Lens 1113 15 98.7

Average 98.7 ± 0.1

a Spectra-Tech IR-PLAN infrared microscope in reflectance mode

b Uncertainties represent standard deviation

Reflections occur at any interface where the refractive index changes from an optically rare material to an optically dense material. The reflectance from the surface of any flat material is governed by the Fresnel equations shown in Eqs. 2.3 and 2.4:39

( ) (2.3) ( )

( ) (2.4)

( )

where Rp is the radiation having an electric field polarized parallel to the plane of incidence and

Rs is radiation having an electric field perpendicular to the plane of incidence. Theta, θ, is the angle of incidence at the surface, and θ’ is the angle of refractive light inside the high-refractive- index material according to Snell’s law. Fresnel plots of Rp and Rs generated using various incident angles with respect to the surface of the silver halide core are shown in Fig. 2.6.

1.00

0.90 nAgBrCl(10.6 µm)= 2.150 0.80 nAir= 1.000 0.70 0.60 Rs 0.50 Rp

0.40 Power reflection Power 0.30 0.20 0.10 0.00 0 10 20 30 40 50 60 70 80 90 Incident angle (deg)

Figure 2.6: Fresnel reflection plots of Rp (solid line) and Rs (dashed line) for the solid-core silver halide PC infrared fibers with a core refractive index 2.150.

For an average angle of 15.4o between the two objectives, approximately 13% of the incident light is lost due to reflections at the front surfaces of the fiber optics. Under ideal conditions, this results in 44% of the initial light being lost after cycling through the fiber optic.

The observed attenuation losses were greater than initially predicted from the Fresnel equations. The higher than expected attenuation results from a combination of scattering within the silver halide cores and absorption of the infrared radiation. The combined effects of absorption, reflection, and scattering reduce the amount of usable infrared radiation transmitted by the fibers/waveguides. Fresnel reflection reduces the amount of usable radiation when using the fiber coupling accessory. When coupled with the microscope, Fresnel reflection also reduces the amount of usable radiation, but the reflected light is detected (false energy) due to the use of an aperture-splitting beamsplitter. As such, fiber probes employing one HWG fiber/waveguide to transmit the radiation to the probe head and another similar fiber to output the radiation may be preferable to using a single fiber/waveguide to perform both tasks. In 1984, Miyagi and Kawakami published a solution for the theoretical attenuation of dielectric-coated HWGs for the infrared.40 Silver is often used as a reflective coating for optical components in infrared instruments due to its high reflectivity at these wavelengths. Fresnel calculations indicated that 99% of the initial infrared radiation transmitted along the HWG is reflected at each point. The high reflectivity of the HWGs supported the transmission values listed in Table II for the 0.25 NA ZnSe objective lens since essentially all infrared radiation reflected back to the microscope is detected.

2.5.3 Single-beam Spectra The single-beam spectra of the silver halide fibers obtained using the fiber optic coupling accessory are shown in Fig. 2.7.

10 Silver halide 1 Silver halide 2 8 Silver halide 3 Hollow waveguide 1

6 Hollow waveguide 2 Energy

0 4000 3600 3200 2800 2400 2000 1600 1200 800 400 Wavenumber (cm-1) Figure 2.7: Single-beam infrared spectra of silver halide fiber optics and hollow silica waveguides.

The spectra illustrate that the amount of energy transmitted by the silver halide fiber optics gradually increased before sharply dropping off at wavenumbers less than 750 cm-1 (13.3 µm). Absorptions below 2000 cm-1 were attributed to the materials employed in the fabrication of the fibers. Photographs of the side and cross-section of a silver halide fiber are shown in Fig. 2.8.

Figure 2.8: Photographs of silver halide PIR fiber construction.

The interior polymer jacket is in direct contact with the silver halide core. As infrared radiation propagates through the core, evanescent waves formed as a result of TIR are capable of penetrating into the surrounding jacket and being absorbed. Infrared spectra of the interior fiber jacket and Parylene are shown in Fig. 2.9.

Parylene

Interior polymer jacket

* %T * * * * *

Silver halide single-beam

4000 3600 3200 2800 2400 2000 1600 1200 800 400 Wavenumber (cm-1)

Figure 2.9: (Top) Infrared spectra of Parylene and the interior polymer jacket of a silver halide fiber. (Bottom) Single-beam spectrum of a silver halide fiber optic. The asterisks denote absorptions characteristic for an epoxy material filled with silica.

Parylene is employed as a protective coating on spectrometer windows. Absorptions observed in the spectrum of the fiber resulting from the interior fiber jacket were observed at 1508, 1243, 1182, 1099, 1037, and 828 cm-1. The positions of these absorptions, indicated by asterisks in Fig. 2.9, are characteristic of an epoxy material filled with silica. Absorptions assigned to Parylene were observed at 2924, 2854, and 1048 cm-1. Absorptions corresponding to a metal carboxylate at 1590 and 1398 cm-1 could not be explained. The single-beam spectra of the HWGs obtained using the fiber optic coupling accessory are shown in Fig. 2.7. Unlike the solid-core silver halide fiber optics, the HWG single-beam spectra exhibited spectral features associated with absorptions by atmospheric CO2 and water vapor trapped inside the waveguides. A comparison of the HWG single-beam spectrum to a

62 single-beam spectrum acquired for an open FT-IR sample compartment under identical conditions is shown in Fig. 2.10.

Open-beam sample compartment Energy

Hollow waveguide single-beam

4000 3600 3200 2800 2400 2000 1600 1200 800 400 Wavenumber (cm-1)

Figure 2.10: (Top) Single-beam spectrum acquired for an open-beam FT-IR sample compartment. (Bottom) Single-beam spectrum acquired for a hollow waveguide.

2.5.4 Effect of Bending The effect of bending on the transmission capabilities of the fibers/waveguides was investigated using the infrared microscope. The ZnSe objective lens was employed due to the increased transmission of infrared radiation using this optic. Bending was performed relative to a central point along the lengths of the fibers/waveguides in a clockwise direction at angles of 0, 30, 45, 60, 90, and 135o with respect to the horizontal axis. Although traditional bending analyses are performed in terms of bend radius, it is more applicable to use bending angles due to the manner in which the fibers/waveguides would be used when incorporated into a probe. Figure 2.11 illustrates the difference between bending measurements based on bending radius,

RB, and bending angle, θB.

Bending position

Fiber optic or waveguide

θB

Figure 2.11: Illustration of bending radius (RB) versus bending angle (θB).

Instrument responses were collected for the fibers/waveguides by taking the ratio of a single-beam spectrum acquired at the designated bending angle to a background single-beam spectrum acquired with the fiber/waveguide in a straight orientation. The resulting responses for the silver halide fiber optics are shown in Fig. 2.12.

(a) 45o (b) 135o

30o

90o

% T % % T %

o 0 60o

4000 3600 3200 2800 2400 2000 1600 1200 800 4000 3600 3200 2800 2400 2000 1600 1200 800 Wavenumber (cm-1) Wavenumber (cm-1)

30o

% T % % T % 60o 0o

4000 3600 3200 2800 2400 2000 1600 1200 800 4000 3600 3200 2800 2400 2000 1600 1200 800 Wavenumber (cm-1) Wavenumber (cm-1)

(e) (f) 45o 135o

90o

30o

% T % % T %

0o 60o

4000 3600 3200 2800 2400 2000 1600 1200 800 4000 3600 3200 2800 2400 2000 1600 1200 800 -1 -1 Wavenumber (cm ) Wavenumber (cm ) Figure 2.12: Bending angle instrument responses for PIR fiber optics. (Top) Silver halide fiber 1. (Middle) Silver halide fiber 2. (Bottom) Silver halide fiber 3.

Peak-to-peak (p-p) variations were determined over the range of 4000-800 cm-1. A summary of the bending evaluation for the silver halide fiber optics is shown in Table III. Data presented in this table illustrate three general trends: (1) a diminution of transmitted radiation as the bending angle increases, (2) an increase in p-p noise at low wavenumbers with increased bending angle, and (3) variability in the production of the fiber optics.

Table 2.3: Summary of Bending Analysis for Silver Halide Fibers and Hollow Waveguides

TABLE III. Summary of Bending Analysis for Silver Halide Fibers and Hollow Waveguides

Identification Bending Angle % of Light Transmitted p-p Variation b degrees %T

Silver Halide 1 0 34.1 - 30 31.9 8.0 45 32.3 6.4 60 33.2 9.0 90 32.3 10.1 135 30.5 10.5

Silver Halide 2 0 20.8 - 30 20.7 5.1 45 21.3 8.7 60 19.8 4.4 90 19.4 8.2 135 17.8 12.5

Silver Halide 3 0 21.7 - 30 21.7 7.1 45 21.4 5.6 60 21.2 4.8 90 21.2 6.9 135 20.2 6.1

Hollow Waveguide 1 0 98.4 - 30 98.2 33.6 45 98.2 36.1 60 98.2 40.0 90 98.2 41.8 135 98.0 63.8

Hollow Waveguide 2 0 98.5 - 30 98.3 27.8 45 98.3 49.0 60 98.3 36.8 90 98.3 36.9 135 98.1 77.0

a Spectra-Tech IR-PLAN Infrared Microscope in reflectance mode using the ZnSe objective lens

b Spectral Range: 4000-800 cm -1

Additional spectral features observed in the silver halide instrument responses include a series of oscillations at various locations in the infrared spectra obtained for the bending of silver halide fiber 1. The most prominent example of this artifact was observed between 1600 and 1100 cm-1. These oscillations were attributed to scattering from large PC grains in the silver halide core. Uncompensated absorptions were observed in the instrument responses for silver halide fiber 3 at 1736, 1460, and 1377 cm-1. The 1736 cm-1 absorption corresponded to the carbonyl stretch of an ester. The 1460 and 1377 cm-1 absorptions were assigned to a methylene bending mode and a methyl deformation mode of a hydrocarbon, respectively. The presence of these absorptions, which were only observed in fiber 3, indicated possible contamination of the silver halide core during manufacturing. Instrument responses generated for the bending of the two HWGs are shown in Fig. 13.

45o (a) (b) 135o

90o

30o

% T % % T %

0o 60o

4000 3600 3200 2800 2400 2000 1600 1200 800 4000 3600 3200 2800 2400 2000 1600 1200 800 Wavenumber (cm-1) Wavenumber (cm-1)

45o (c) (d) 135o

30o

90o

%T %T 0o 60o

4000 3600 3200 2800 2400 2000 1600 1200 800 4000 3600 3200 2800 2400 2000 1600 1200 800 -1 -1 Wavenumber (cm ) Wavenumber (cm ) Figure 2.13: Bending angle instrument responses for hollow silica waveguides. (Top) Hollow waveguide 1. (Bottom) Hollow waveguide 2.

The spectra exhibited a high degree of uniformity when compared to those of the silver halide fiber optics. It is apparent that the instrument responses of waveguides are not significantly affected by bending, except at wavelengths longer than 6.3 µm (1600 cm-1). According to the theory by Marcatili and Schmeltzer, increased attenuation of light propagating

67 through a waveguide will occur at longer wavelengths.28 A summary of the bending analysis for the HWGs is shown in Table III. Percent transmission remains constant at 98% at each bending angle. Peak-to-peak variations increased as the bending angle increases or, alternatively, as the bend radius decreases. Baseline variations observed in the instrument responses of the silver halide fiber optics and HWGs resulted from background and sample scans not being collected with the fiber/waveguide in similar configurations. This effect becomes apparent at longer wavelengths. Correction for the wavelength-dependent baseline variation could be accomplished through the collection of background and sample scans with the fiber/waveguide in similar, if not identical, configurations.

2.6 Conclusions Polycrystalline silver halide fiber optics and hollow silica waveguides were evaluated on the basis of the transmission of mid-infrared radiation using a fiber optic coupling accessory and an infrared microscope. Results have shown that the transmission capabilities of the silver halide fiber optics are appreciably less than those of the hollow waveguides. Reduced transmission and manufacturing inconsistency were observed among the three silver halide fiber optics. The transmission characteristics of two identical HWGs were found to be reproducible. Hollow waveguides do not suffer the same insertion losses as those of the solid-core silver halide fibers due to the hollow core. Transmission of infrared radiation at large bending angles for the hollow waveguides indicated increased tolerance to bending. Reproducible instrument responses and high percent transmission indicated HWGs would be preferred as optical conduits in the construction of an ATR probe.

2.7 Acknowledgements The author thanks Joe Lucania and Harrick Scientific for providing a FiberMate2 fiber optic coupling accessory for the purpose of this research. Financial support for this study was provided by Kodak, PerkinElmer, and Procter & Gamble.

References:

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31. Y. Matsuura, S. Kino, E. Yokoyama, T. Katagiri, H. Sato, H. Tashiro. “Flexible Fiber-Optics Probes for Raman and FT-IR Remote Spectroscopy”. IEEE J. Sel. Top. Quant. 2007. 13(6): 1704-1708.

32. S. Kino, Y. Matsuura. “Nontoxic and Chemically Stable Hollow Optical Fiber Probe for Fourier Transform Infrared Spectroscopy”. Appl. Spectrosc. 2007. 61(12): 1334-1337.

33. Y. Matsuura, S. Kino, T. Katagiri. “Hollow-fiber-based Flexible Probe for Remote Measurement of Infrared Attenuated Total Reflection”. Appl. Opt. 2009. 48(28): 5396-5400.

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Chapter 3

Evaluation of a Mid-Infrared Hollow Waveguide Accessory for Sample Analysis by Attenuated Total Reflection-Fourier Transform Infrared (ATR-FT-IR) Spectroscopy

3.1 Abstract

3.1.1 Evaluation of a Hollow Waveguide ATR Probe Accessory A Pike Technologies, Inc. mid-infrared remote sampling accessory employing a hollow waveguide (HWG) attenuated total (internal) reflection (ATR) probe was evaluated on the basis of infrared transmission and signal-to-noise. The HWG accessory is applicable to remote analyses of a wide variety of samples, such as visible surface contamination, small area material identification, and samples too large to fit into the sample compartment of an FT-IR spectrometer. A bending analysis indicated that baseline variations were minimized by collecting background and sample scans with the probe in similar configurations. Aqueous solutions of succinylcholine chloride were analyzed to determine the quantitative capabilities and limitations of the accessory. Integrated absorbance (peak area) of the 1167 cm-1 C-O-C stretch of succinylcholine chloride was selected for quantitation. A calibration of integrated absorbance versus concentration possessed a linear R2 correlation of 0.9993. Limits of detection and quantitation for succinylcholine chloride in water were 0.03 ± 0.01 and 0.10 ± 0.02 parts per thousand, respectively. (Uncertainties in the limits of detection and quantitation represent standard deviations associated with five replicate determinations of the background noise in distilled water.) Quantitative results obtained using the ATR probe were comparable to those of an earlier study using an ATR-FT-IR microscope with a newer-generation detector.

3.1.2 Analysis of Hard Samples Samples possessing hard surfaces were analyzed using a HWG ATR probe. The capabilities and limitations of the accessory for sample analysis were evaluated using hard samples of polycarbonate (PC) and pliable samples, including a rubber eraser and a pelletized sample of calcium oxalate monohydrate (COM). Data indicated the presence of a slight curvature to the probe internal reflection element (IRE). Although this defect did not facilitate analyses of hard, flat samples, curved and pliable samples possessing appreciable conformability were capable of being analyzed using the ATR probe.

3.1.3 Quantitative Analysis of Ethanol in Alcoholic Beverages Quantitative analyses of aqueous ethanol solutions were performed using a hollow waveguide (HWG) attenuated total (internal) reflection (ATR) probe. Calibration curves constructed using integrated absorbance at 1046 and 1088 cm-1 versus concentration possessed linear R2 correlation coefficients of 0.9994 and 0.9986, respectively. Limits of detection and quantitation of 0.001 ± 0.001 and 0.004 ± 0.001 percent alcohol by volume (% ABV) were determined using the 1046 cm-1 ethanol absorption; limits of detection and quantitation of 0.006 ± 0.001 % ABV and 0.021 ± 0.001 % ABV were determined using the 1088 cm-1 ethanol absorption. (Uncertainties in the limits of detection and quantitation represent standard deviations associated with four replicate determinations of the background noise in distilled water.) Quantitative determinations of alcohol content in beer, wine, and liquor were performed using the ATR probe. Accurate determinations of alcohol content in alcoholic beverages were achieved in the absence of statistical modeling using the proposed quantitative method.

3.2 Introduction

3.2.1 Infrared Fiber Optics and Hollow Waveguides Infrared fiber optics, generally classified as fiber optics capable of transmitting wavelengths greater than 2 µm, can be categorized into three groups: glass, crystalline, and hollow waveguide (HWG). Information regarding the optical and physical properties of infrared fibers/waveguides, as well as their potential applications, can be found in several reviews.1-3

Infrared-transmitting fibers/waveguides have transitioned from glass fibers, such as As2S3-based chalcogenide fibers, to polycrystalline (PC) fibers consisting of solid solutions of silver bromide and silver chloride due to such benefits as increased flexibility, non-toxicity, and wide spectral range. Despite the benefits associated with PC silver halide fibers/waveguides, HWGs have emerged as modern alternatives to solid-core fibers/waveguides for infrared transmission due to a minimization of insertion losses resulting from a hollow air core.4-17 Hollow waveguides are often very durable and are capable of high-efficiency transmission of mid-infrared light over a wider spectral range than that accessible using PC core fibers. Furthermore, HWGs do not suffer from fracture problems typically associated with solid-core fibers/waveguides. Three PC silver halide fiber optics and two hollow silica waveguides possessing an internal metal-dielectric layer of silver/silver iodide were recently characterized by Damin and Sommer using infrared microspectroscopy.18 In the study by Damin and Sommer, each set of fibers/waveguides respectively possessed identical physical characteristics and were obtained from the same locations. In contrast to the PC silver halide fiber optics, comparable transmission efficiencies were obtained using the two HWGs. An increased bending tolerance was also observed for the two hollow silica waveguides. Hollow waveguides have been successfully applied to sample analysis using infrared spectroscopy. Early applications of HWGs included use as infrared absorption cells for the analysis of gaseous samples.19-25 Hollow waveguides have recently been incorporated in spectroscopic probe accessories.26 Infrared spectra of biological samples, including aorta, blood, fatty tissue, and muscle, were collected by Hooper et al. using a catheter constructed using two hollow glass waveguides (HGWs) coupled to diamond and zinc sulfide internal reflection elements (IREs).27 Wang et al. successfully applied multivariate data analysis and classification techniques to infrared spectral data of atherosclerotic rabbit aorta collected using a HWG

77 attenuated total (internal) reflection (ATR) probe.28 The study by Wang represented an in vitro investigation using the accessory with a future goal of in vivo applications. A probe constructed using two HWGs possessing interior silver coatings was later employed by Kino and Matsuura in acquiring infrared spectra of teeth, skin, and oral mucosa.29 The development of a hollow-fiber- based probe for the remote acquisition of infrared spectra in clinical applications was discussed by Matsuura et al.30-32

3.2.2 Quantitative Analysis of Succinylcholine Chloride Quantitative analyses of aqueous succinylcholine chloride solutions have been performed using a succession of ATR accessories. Studies performed by Miller et al. and McKittrick et al. employed multi-bounce ATR accessories for quantitative studies of succinylcholine chloride by flow injection analysis (FIA) with infrared detection. Using the micro-CIRCLE (cylindrical internal reflection cell for liquid evaluation) cell, Miller et al. reported a detection limit of 0.2 parts per thousand using peak height for the 1165 cm-1 succinylcholine chloride absorption.33 An ultramicro-CIRCLE cell was used by McKittrick et al. to obtain a detection limit of 2 parts per thousand using peak area of the 1165 cm-1 succinylcholine chloride absorption.34 Although beneficial for the detection of trace analytes in dilute solutions, multi-bounce ATR can be problematic for strongly-absorbing solvents due to increased pathlength. In contrast, single- bounce ATR permits sample analyses in the presence of strongly-absorbing solvents due to a reduction in pathlength. Patterson et al. reported a detection limit of 0.7 parts per thousand succinylcholine chloride using single-bounce ATR-FT-IR microspectroscopy.35 Quantitative analyses were performed using integrated absorbance (peak area) of the 1167 cm-1 succinylcholine chloride absorption. A detection limit of 0.02 parts per thousand was obtained using a newer-generation infrared microscope in a subsequent study by Patterson.36

3.2.3 Economic Regulations of Alcoholic Beverages Alcohol content is an important quality control parameter used in determining the correct taxes to be levied on alcoholic beverages. In the United States, the Alcohol and Tobacco Tax and Trade Bureau (TTB), a division of the U.S. Department of the Treasury, is responsible for the regulation of alcoholic beverages, including the applied taxes.37 Wine, for example, possesses various tax rates depending on the alcohol content. Normal alcohol content in wine varies from 7% v/v upward to 24% v/v in dessert wines. According to the TTB, wine is categorized in the following groups: 14% alcohol or less, over 14% to 21%, over 21% to 24%, natural sparkling, and artificially carbonated. Taxes ranging from $1.07 to $3.40 per gallons are imposed on these beverages, with higher concentrated beverages possessing higher taxes. In terms of wine regulation, the TTB has tolerances on the ethanol content in the beverages. A ± 1.5% tolerance exits for wines between 7 and 14%. For wines greater than 14%, the tolerance drops to ± 1.0%, and for wines less than 7%, the tolerance is ± 0.75%. In addition to federal taxes, state taxes are also applied to the sale of alcoholic beverages. In the state of Indiana38, beer, flavored malt beverages, and hard ciders carry a tax of $0.115 per gallon. Wine containing less than 21% alcohol is taxed at a rate of $0.47 per gallon while wine and liquor containing 21% or more alcohol have a tax of $2.68 per gallon. Therefore, accurate determinations of alcohol content in alcoholic beverages are economically important for both the producer and the consumer.

3.2.4 Current Methods of Determining Alcohol Content Several analytical methods are employed in the determination of quality control parameters, such as alcohol content, in alcoholic beverages. Jacobson lists several methods of determining alcohol content, including those accepted by the TTB.39 Densitometry is the industry-accepted standard for alcohol content determinations in beverages. A densitometer uses harmonic motion to determine the density of a liquid in a U-shaped tube vibrated electromagnetically to its natural frequency. Although accurate density measurements of ± 0.001-0.000005 g/cm3 are possible depending on the density meter selected, the financial costs associated with these instruments are often high, and direct determinations of alcohol content are not achieved. Instead, alcohol content is measured using both a densitometer and an alcohol analyzer. Anton Paar (Ashland, VA) manufactures a series of alcohol analyzers, known as the

Alcolyzers, for determinations of alcohol content in beer, wine, and spirits. The Alcolyzers employ a near-infrared (NIR) light-emitting diode in an optical configuration similar to that of a UV-visible spectrophotometer. The analyzers use a highly specific range of the NIR spectrum between 1150 and 1200 nm for quantitative determinations. Although accuracies were not provided in the specifications of the beer and spirit analyzers, the listed accuracy of the wine analyzer is ± 0.1% v/v.

3.2.5 Vibrational Spectroscopy Mid-infrared spectroscopy has become a proven technique for the analysis of solutes in aqueous solutions. One of the most popular mid-infrared sampling methods is attenuated total (internal) reflection-Fourier transform infrared (ATR-FT-IR) spectroscopy. ATR-FT-IR spectroscopy is beneficial since limited or no sample preparation is generally required. The only principal requirement of the technique is that the sample must be placed in direct, intimate contact with the internal reflection element (IRE). Additional information regarding total internal reflection spectroscopy and ATR can be found in publications by Harrick.40-42 As early as 1936, infrared absorption spectra of mixtures of ethyl alcohol and water were studied in the region from 1.5-5 µm by Williams et al.43 A quantitative study of ethanol in water using ATR was performed by Malone and Flournoy using the 1080 cm-1 absorption of ethanol.44 Although linear calibrations were obtained, the publication by Malone and Flournoy required further research to prove the validity of the method. ATR-FT-IR spectroscopy has been quantitatively applied in determining characteristics of beer and alcoholic beverages.45-47 A multivariate investigation of proton nuclear magnetic resonance (1H NMR) and ATR-FT-IR as potential tools for the quality control of beer was presented by Duarte et al.48 The authors concluded that an accurate classification of beer types based on ethanol concentration could be obtained using ATR, whereas 1H NMR was more useful in providing information about the sugar content. Polshin et al. used horizontal- and micro-ATR in the analysis of Belgian beer, including lager and white beers, dark and blonde ales, and Lambic and Trappist beers.49 A comparison of the results revealed a similar prediction performance with respect to the sugar and alcohol content in beer. Single-bounce ATR was employed by Cocciardi et al. for the analysis of distilled liquors and wines.50 A partial least squares (PLS) calibration model yielded a sensitivity of 0.061/% v/v with a correlation of 0.993. Cocciardi et al. also reported that the alcohol content of distilled

80 liquor products determined using FT-NIR and single-bounce ATR-FT-IR could be predicted to within 0.17% v/v of the alcohol content determined using a density meter. Despite the results presented by Cocciardi, this method still required significant analysis and computational time and does not permit remote sample analyses.

3.2.6 Infrared Fiber-Coupled Probes for Alcohol Content Determinations In situ analyses using ATR-FT-IR spectroscopy are capable of being remotely performed through advancement in infrared-transmitting fiber optics/waveguides. Fiber optic ATR-FT-IR spectroscopy has been employed for qualitative and quantitative analyses of alcoholic beverages. Küpper et al. used a flexible, fiber optic ATR probe with a diamond prism in the analysis of beer with different alcohol contents directly inside the bottles.51 Infrared spectra of ethanol-free beer and beers possessing 2.7 and 4.5% alcohol were obtained by Küpper; however, no quantitative analyses were performed. Counterfeit samples of Scotch whiskey were detected by McIntyre et al. using a silver halide fiber-coupled ATR immersion probe.52 The alcohol content of seventeen whiskey samples were determined using in situ ATR-FT-IR spectroscopy in conjunction with univariate and multivariate PLS calibrations. The average univariate and multivariate errors reported by McIntyre was ± 0.11% v/v for liquid samples ranging from 31.0-43.1% v/v. Successful demonstrations of fiber optic ATR prove have been performed using polycrystalline silver halide fibers; however, several problems, such as increased attenuation resulting from a combination of front-surface reflections at the ends of the solid-core fibers and scattering within the fiber core, are commonly associated with this type of infrared fiber/waveguide.18 These effects become increasingly significant when the fibers/waveguides are bent.

3.3 Goals and Specific Aims Infrared-transmitting, fiber- or waveguide-coupled probes can be used for sample characterization in laboratory and industrial situations. In contrast to large, portable ATR sampling accessories, such as the Agilent ExoScan, a 1-meter waveguide probe coupled to a small FT-IR spectrometer offers the capability of remote analyses of samples in tight places, such as inside processing equipment, or samples too large for conventional sampling accessories. In 2010, Pike Technologies, Inc. introduced the FlexIR hollow waveguide accessory, a commercial mid-infrared accessory featuring a series of specialized probes for ATR, specular reflectance, and diffuse reflectance measurements.53 The capabilities and limitations of the HWG ATR probe will be evaluated through the analysis of aqueous succinylcholine chloride solutions. Aqueous solutions were chosen as a measure of the accessory’s quantitative utility in order to eliminate problems associated with solid samples, such as particle size. In 2009, quantitative ATR-FT-IR spectroscopy was employed by Gulley-Stahl et al. in the analysis of urinary (kidney) stone components.54 It was concluded that similar sized particles produced linear calibration curves, whereas mixtures of small and large particle produced nonlinear curves, especially at higher concentrations. Succinylcholine chloride was selected as a target analyte due to its high solubility in water and the success of previous quantitative ATR-FT-IR studies employing the compound. Quantitative results obtained using the HWG ATR probe will be compared to those obtained in earlier studies. A qualitative study using hard and pliable samples of rubber, polycarbonate (PC), and calcium oxalate monohydrate (COM) will be performed as an evaluation of the surface quality of the ATR probe IRE. The application of mid-infrared spectroscopy to the characterization and determination of quality control parameters in alcoholic beverages remains underdeveloped. Earlier mid-infrared studies have shown the method to be capable through the application of statistical modeling software; however, a method of providing fast, accurate results that are easily interpretable in the field is still lacking. Analyses of alcoholic solutions with respect to alcohol content were performed as a quantitative application of a hollow waveguide (HWG) ATR probe. Calibration curves of integrated absorbance (peak area) versus alcohol content were constructed and used in verifying alcohol content in commercial beer, wine, and liquor.

3.4 Experimental

3.4.1 Instrumentation Infrared spectra were collected using a Pike Technologies, Inc. (Madison, WI) mid- infrared FlexIR hollow waveguide accessory coupled to a PerkinElmer System 2000 FT-IR spectrometer. The accessory featured an integrated 1.0 mm × 1.0 mm liquid-nitrogen-cooled, wide-band mercury-cadmium-telluride (MCT) detector (model no. 660-106800 00, Infrared Associates, Inc., Stuart, FL) and pre-amplifier. Photographs of the accessory are shown in Fig. 3.1.

Figure 3.1: Photographs of the Pike Technologies Mid-IR FlexIR hollow waveguide accessory and ATR probe.

The ATR probe was constructed using a set of 1-m hollow silica waveguides with inner and outer diameters of 1.0 and 1.6 mm, respectively. An optical diagram of the accessory is shown in Fig. 3.2. A diamond-zinc selenide (ZnSe) IRE was selected for the ATR probe. When coupled to the MCT detector, the spectral range available with the probe was 4000-700 cm-1. The ATR probe was sealed up to 60 mm beyond the tip for liquid analyses. Instrument control of the System 2000 FT-IR spectrometer was done using PerkinElmer Spectrum software (version 5.3.1 [2005]). Spectral analyses were performed using Spectrum software (versions 6.3.1.0132 [2007] and 10.03.06 [2011]).

Integrated LN2-cooled MCT detector

Incoming beam

Reflected beam from probe

HWG probe

Figure 3.2: Optical diagram of the Pike Technologies FlexIR hollow waveguide accessory.

3.4.1.1 Evaluation of a Hollow Waveguide ATR Probe Accessory Infrared spectra of an open FT-IR sample compartment were collected using a separate wide-band MCT detector coupled to the System 2000 macrobench. Transmission efficiencies for both the ATR probe and the open FT-IR sample compartment were performed with both MCT detectors registering an initial energy of 5000 counts when optimized. The diameter of the aperture (B-) stop on the FT-IR was then reduced to obtain energies of 4200 counts. Instrument responses representing ratio scans of 4, 8, 16, 32, and 64 individual scans in the absence of a sample were collected at 4 cm-1 resolution. A 4 cm-1 spectral resolution was selected based on the time (1 minute) required to collect a spectrum representing the average of 64 individual scans. Signal-to-noise ratios were determined over the range of 2600-2400 cm-1; noise was calculated as one-fifth the peak-to-peak (p-p) noise over this spectral range. (Uncertainties in p- p noise and signal-to-noise represent standard deviations associated with five replicate collections for each number of individual scans.)

3.4.1.2 Analysis of Hard Samples

Infrared spectra of calcium carbonate (CaCO3) and talc were collected using a Harrick Scientific SplitPea ATR accessory interfaced to a PerkinElmer Spectrum ONE FT-IR spectrometer. The accessory employed a silicon IRE and the standard deuterated triglycine sulfate (DTGS) detector on the Spectrum ONE macrobench. Spectra collected using the SplitPea accessory represent the average of 32 individual scans at 4 cm-1 resolution. Samples were brought into direct contact with the IRE using a loading of 0.5 kg. Instrument control of the Spectrum ONE FT-IR spectrometer was done using the PerkinElmer Spectrum software (version 5.3.1 [2005]). Analysis of the collected infrared spectra was performed using Spectrum software (version 10.03.06 [2011]).

3.4.1.3 Quantitative Analysis of Ethanol in Alcoholic Beverages Infrared spectra were collected using a Pike Technologies, Inc. (Madison, WI) mid- infrared FlexIR hollow waveguide accessory coupled to a PerkinElmer System 2000 FT-IR spectrometer. The accessory featured a 1.0 mm × 1.0 mm liquid-nitrogen-cooled, wide-band mercury-cadmium-telluride (MCT) detector (model no. 660-1-6800 00, Infrared Associates, Inc., Stuart, FL) and pre-amplifier. The ATR probe was constructed using a set of 1-meter hollow

85 silica waveguides with inner and outer diameters of 1.0 and 1.6 mm, respectively. A diamond- zinc selenide (ZnSe) IRE was selected for the ATR probe. When coupled to the MCT detector, the spectral range available with the probe was 4000-700 cm-1. The ATR probe was sealed up to 60 mm beyond the tip for liquid analyses. Infrared spectra of aqueous ethanol solutions and the alcoholic beverages represent the average of 64 individual scans at 8 cm-1 resolution. Instrument control of the System 2000 FT-IR spectrometer was done using PerkinElmer Spectrum software (version 5.3.1 [2005]). Spectral analyses were performed using Spectrum software (versions 6.3.1.0132 [2007] and 10.03.06 [2011]).

3.4.2 Methods and Materials

3.4.2.1 Evaluation of a Hollow Waveguide ATR Probe Accessory The premise of the bending evaluation of the ATR probe is illustrated in Fig. 3.3.

FlexIR

o +60o -60

o +45o -45 o +30o -30 0o

Figure 3.3: Schematic illustration of the lateral bending of the HWG probe from + 60o to -60o.

Bending was performed at angles of 0o, ± 10o, ± 20o, ± 30o, ± 45o, and ± 60o with respect to a straight orientation. The effects of bending on the transmission of mid-infrared radiation by the HWG probe were evaluated using two situations: (1) single-beam spectra collected at each angle were ratioed to a single-beam spectrum collected with the ATR probe in a straight configuration (0o), and (2) standard ratio spectra consisting of background and sample scans were collected with the ATR probe manually held at each angle. Instrument responses generated in the two situations were analyzed in terms of p-p variations in absorbance and % T over the ranges of 2600-2400 and 4000-1000 cm-1. The spectral range below 1000 cm-1 was excluded due to its close proximity to the MCT detector cutoff. The transmission efficiency of the HWG ATR probe accessory was compared to that obtained using a Harrick Scientific (Pleasantville, NY) SplitPea ATR accessory. The SplitPea accessory was coupled to a PerkinElmer System 2000 FT-IR spectrometer and utilized a wide- band MCT detector coupled to the macrobench. The accessory was equipped with a ZnSe hemisphere IRE. Prior to placing the accessory in the sample compartment, the energy reaching the MCT detector was optimized to 5000 counts. Without changing the settings of the FT-IR spectrometer, the SplitPea accessory was placed in the sample compartment and optimized. Succinylcholine chloride dihydrate was purchased (Sigma-Aldrich, St. Louis, MO). Aqueous solutions of succinylcholine chloride with concentrations of 5-100 parts per thousand were prepared. Infrared spectra collected in absorbance mode using the ATR probe represent the average of 64 individual scans at 8 cm-1 resolution with distilled water used as a background. Integrated absorbance of the 1167 cm-1 absorption of succinylcholine was determined over the range of 1193-1133 cm-1. Limits of detection (LOD) and quantitation (LOQ) were calculated as three and ten times the noise (root-mean-square, rms) in absorbance over the range of 2100-1900 cm-1 for a series of blanks in distilled water divided by the slope of the calibration curve.37 (Uncertainties in the LOD and LOQ represent standard deviations associated with five replicate determinations of the background noise in distilled water.) An empirical determination of the infrared spot size at the surface of the probe IRE was done by laterally scanning a strip of adhesive tape across the surface. The active area was identified through visual observation of the infrared spectrum of the acrylic adhesive on the tape and measured using a Brown & Sharpe (Hexagon Metrology, Inc., North Kingstown, RI) Digit- Cal MK IV digital caliper.

3.4.2.2 Analysis of Hard Samples Sheet and tube polycarbonate were selected as hard samples. The flat PC sheet was obtained from the stripping of a compact disc. A pink, rubber eraser was selected as a pliable sample. Calcium oxalate monohydrate was purchased (ACROS Organics, Morris Plains, NJ); a pellet was pressed at 2.0 tons using a SPEX Industries, Inc. (Metuchen, NJ) press and a 7 mm pellet die. ACS reagent grade CaCO3 was purchased (MP Biomedicals, Inc., Solon, OH). Talc was obtained from an unidentified source. Direct, intimate contact between the samples and the probe IRE was accomplished using a manual laboratory jack. Control of the applied loading force was qualitatively based on visual inspection of the resulting spectra.

3.4.2.3 Quantitative Analysis of Ethanol in Alcoholic Beverages Two hundred proof (100%) anhydrous ethanol was purchased (Decon Laboratories, Inc., King of Prussia, PA). Four commercially available beers possessing alcohol contents of 4.2, 5.6, and 7.5% ABV were randomly purchased from a local supermarket. Two lite, or low carbohydrate, beers listed as 4.2% ABV were selected on the basis of similar nutritional information but were produced by different brewing companies. White and red wines containing 9.5 and 11.0% ABV, respectively, were randomly purchased from a local supermarket. Honey whiskey containing 35.5% ABV was obtained (Wild Turkey, Lawrenceburg, KY). Anhydrous ACS reagent-grade α-lactose and sucrose were purchased (Fisher Scientific, Fair Lawn, NJ). Aqueous ethanol solutions with concentrations of 1-30% ABV were prepared and analyzed in absorbance mode using the HWG ATR probe with pure, distilled water being used as a background. Analysis of the liquor required the preparation of aqueous ethanol solutions of 10-90% ABV. The selected limits of integration for the 1046 cm-1 ethanol absorption were 1067-1024 cm-1; the selected limits of integration for the 1088 cm-1 ethanol absorption were 1098-1067 cm-1. Limits of detection (LOD) and quantitation (LOQ) were respectively calculated as three and ten times the noise (root-mean-square, rms) over the range of 2600-2400 cm-1 for a series of blank measurements in distilled water divided by the slope of the calibration curve. (Uncertainties in the limits of detection and quantitation represent standard deviations associated with four replicate determinations of the background noise in distilled water.)

Evaluations of errors associated with standard preparation and instrument drift were done using the corrected integrated absorbance of the 1046 cm-1 absorption for ethanol solutions containing 8% ABV. Standard preparation error was evaluated using five 8% solutions prepared in the same manner. Instrument drift was evaluated by collecting five spectra for a single 8% solution over a period of five hours. Pure, distilled water was used as a background during these collections. Verification of alcohol content in beer, wine, and liquor was performed using the integrated absorbance of the 1046 cm-1 ethanol absorption. Integrated absorbance values were converted to alcohol content using the constructed calibration curves. Alcohol content determinations for these beverages represent the average of five replicate measurements with uncertainties representing absolute error at 95% confidence.

3.5 Results and Discussion

3.5.1 Evaluation of a Hollow Waveguide ATR Probe Accessory

3.5.1.1 Single-beam Spectra Single-beam spectra acquired using the HWG ATR probe and the open-beam FT-IR sample compartment are shown in Fig. 3.4.

Single-beam hollow waveguide accessory

Single-beam open sample compartment %T

Single-beam ratio

4000 3700 3400 3100 2800 2500 2200 1900 1600 1300 1000 700 Wavenumber (cm-1)

Figure 3.4: (Top) Single-beam infrared spectrum obtained using the HWG ATR probe accessory. (Middle) Single-beam infrared spectrum obtained for an open-beam FT-IR sample compartment. (Bottom) Ratio of the single-beam accessory spectrum to the single-beam open sample compartment spectrum.

The single-beam spectra were collected using two, separate wide-band MCT detectors. The MCT detector attached to the HWG accessory was integrated into the optical design of the accessory and was not compatible for direct mounting in the System 2000 FT-IR spectrometer. A separate wide-band MCT detector designed by PerkinElmer for use in the System 2000 FT-IR macrobench was employed on the basis of comparable optical characteristics to those of the accessory detector. The single-beam spectra (Fig. 3.4) of the HWG accessory and the open- beam FT-IR sample compartment possessed maximum energy values of 117 and 115 counts, respectively. The maximum energy transmitted by the probe occurred at 2397 cm-1 (4.2 µm), whereas the maximum energy transmitted through the open FT-IR sample compartment occurred at 988 cm-1 (10.1 µm). The single-beam spectrum of the HWG accessory exhibited increased absorptions indicative of C-H stretches between 2963 and 2853 cm-1 and an absorption at 1750 cm-1 characteristic for an ester carbonyl stretch. A baseline-corrected ratio of the accessory single-beam spectrum to that of the open sample compartment is also shown in Fig. 3.4. Absorptions assigned to C-H stretches were observed, and an ester carbonyl stretch was identified at 1750 cm-1. Absorptions at 1263 and 1108 cm-1 were respectively assigned to the C-C-O and O-C-C stretches of an ester. The absorptions at 1750, 1263, and 1108 cm-1 are characteristic for an acrylate. According to Pike Technologies, the IRE was sealed in the probe head using a modified acrylic adhesive, and the two-piece probe was assembled using an ethyl cyanoacrylate adhesive containing a photo initiator. A comparison of the single-beam ratio spectrum (Fig. 3.4) to a reference spectrum of an ethyl cyanoacrylate adhesive produced by Loctite® (Henkel Corporation) is shown in Fig. 3.5. The acrylate absorptions observed in the single-beam spectrum are compensated for when a sample scan is ratioed to a background. (An example of a ratio scan collected using the ATR probe in the absence of a sample is presented as the 0o straight/straight spectrum in Fig. 3.6.)

Single-beam ratio

% T % Ethyl cyanoacrylate adhesive

4000 3700 3400 3100 2800 2500 2200 1900 1600 1300 1000 700 Wavenumber (cm-1)

Figure 3.5: Comparison of the single-beam ratio spectrum (top) to the infrared spectrum of an ethyl cyanoacrylate adhesive (bottom).

3.5.1.2 Bending Analysis Instrument responses obtained for the two bending situations are shown in Fig. 3.6.

60o Bent/bent

60o Bent/straight %T

0o Straight/straight

4000 3700 3400 3100 2800 2500 2200 1900 1600 1300 1000 700 Wavenumber (cm-1)

Figure 3.6: Instrument responses for the hollow waveguide ATR probe in a straight configuration and bent at + 60o. The bent/bent and straight/straight spectra represent the ratio of sample and background scans with the probe maintained in bent and straight configurations, respectively. The bent/straight response represents a ratio of a sample scan collected with the probe bent to + 60o to a background scan collected with the probe in a straight configuration.

The response representing the ratio of sample and background spectra collected with the probe maintained in a straight configuration is also shown. The response observed for the ratio of a bent sample scan to a straight background exhibited increased baseline variation at longer wavelengths as a result of the two scans being collected with the probe in different positions. A similar effect was previously observed during a comparative study of solid-core silver halide fiber optics and hollow silica waveguides by Damin and Sommer.18 Baseline variations in instrument responses generated by ratioing single-beam spectra collected at specified bending angles to a straight single-beam background spectrum were observed at wavelengths longer than 6.3 µm for two hollow silica waveguides. Data obtained during the bending investigation are summarized in Table I. In contrast to the values obtained in the bent sample scan/straight background situation, p-p variations obtained

93 using the bent sample scan/bent background approach were appreciably smaller and more consistent at each bending angle over the selected spectral ranges. Bending becomes apparent over the range of 4000-1000 cm-1 for the bent sample scan/straight background scan due to increased baseline variation at longer wavelengths. Appreciably greater signal-to-noise was observed over the range of 4000-1000 cm-1 using the bent sample scan/bent background method. The combined benefits of smaller p-p variations, increased signal-to-noise, and minimal baseline variations indicate that maintaining the probe in the desired sampling configuration between background and sample scans would be preferred. The theory describing attenuation losses associated with circular HWGs was first explored by Marcatili and Schmeltzer in 1964.55 Using a wave optic approach, the theory presented by Marcatili and Schmeltzer predicted for either metallic or dielectric waveguides that α ~ 1/a3, where α is the attenuation coefficient and a is the bore radius of the waveguide. In order to minimize attenuation in spectroscopic applications, large-diameter HWGs are preferred. In addition to the bore radius of the waveguide, bending also increases attenuation. Miyagi et al. have shown that the additional bending loss varies as 1/R, where R is the bending radius.56 In the present study, bending angle was selected as a measure of attenuation due to the manner in which the HWG ATR probe would be utilized during practical sampling applications; increases in bending angle correspond to decreases in the bending radius. Based on the aforementioned parameters, attenuation of light transmitted by HWGs is strongly dependent upon both the core diameter and the bending radius of the waveguide. With respect to the FlexIR HWG probe, the core diameters of the waveguides (1 mm) used in its construction are sufficiently large so as to minimize attenuation based on bore radius. Miyagi and Kawakami have shown that for dielectric coatings deposited over a metallic layer, the attenuation coefficient is also proportional to λ2, where λ is the wavelength.57 Baseline variation, such as those observed in the bent/straight instrument response, result from background and sample scans being collected with the waveguides in different positions; minimization of this effect can be achieved by maintaining the probe in similar configurations between background and sample scans.

Table 3.1: Summary of Bending Analysis for Hollow Waveguide ATR Probe

TABLE I. Summary of Bending Analysis for Hollow Waveguide ATR Probe

Bent Sample Scan/Straight Background

Bending Angle p-p Variationa p-p Variationa SNRa p-p Variationb p-p Variationb SNRb degrees mabs %T ×10 3 mabs %T ×10 2

-60 7.5 1.64 0.3 215.9 38.18 0.1 -45 5.5 1.23 0.4 147.8 28.69 0.2 -30 2.8 0.64 0.8 82.3 17.43 0.3 -20 1.9 0.44 1.1 33.6 7.49 0.7 -10 1.1 0.26 2.0 17.7 4.02 1.2 0 ------10 1.3 0.30 1.7 24.7 5.57 0.9 20 2.0 0.46 1.1 63.0 13.36 0.4 30 3.6 0.79 0.6 114.5 22.73 0.2 45 4.5 1.00 0.5 137.3 26.57 0.2 60 5.7 1.22 0.4 187.4 33.47 0.2

Bent Sample Scan/Bent Background

Bending Angle p-p Variationa p-p Variationa SNRa p-p Variationb p-p Variationb SNRb degrees mabs %T ×10 3 mabs %T ×10 2

-60 0.4 0.10 5.3 6.7 1.54 3.2 -45 0.6 0.13 3.8 5.9 1.35 3.7 -30 0.6 0.14 3.6 3.5 0.79 6.3 -20 0.6 0.13 3.8 4.3 1.00 5.0 -10 0.5 0.11 4.5 2.1 0.49 10.2 0 0.3 0.06 8.3 1.4 0.32 15.6 10 0.5 0.11 4.5 2.7 0.62 8.1 20 0.6 0.13 3.8 4.8 1.10 4.5 30 0.5 0.10 5.0 2.0 0.46 10.9 45 0.3 0.08 6.3 3.9 0.90 5.6 60 0.7 0.16 3.1 7.2 1.64 3.0

a Range: 2600-2400 cm-1

b Range: 4000-1000 cm-1

3.5.1.3 Transmission Efficiency Transmission efficiency of the ATR probe was determined on the basis of SNRs collected under identical conditions with the probe in a straight configuration compared to those of an open-beam FT-IR sample compartment. In both situations, the MCT detectors coupled to the HWG accessory and the FT-IR spectrometer registered the same signal strength. The resulting data in Table II indicate that p-p variations in baseline absorbance using the ATR probe were an order of magnitude greater than those obtained for the open-beam sample compartment. Based on a comparison of the resulting SNRs for the HWG ATR probe accessory and the open- beam sample compartment at the each selected number of scans, the average transmission efficiency of the HWG probe was determined to be 8.7 ± 0.4% of the open-beam value. (Uncertainty represents standard deviation for five ratios of the accessory SNR to that of the open-beam sample compartment.) A diminution in signal-to-noise observed for the HWG probe was attributed to increased noise resulting from a combination of the coupling of infrared light into the 1-mm diameter HWGs and increased attenuation resulting from the presence of a high refractive index IRE. The transmission efficiency of the HWG ATR probe accessory was compared to that of a Harrick Scientific (Pleasantville, NY) SplitPea ATR accessory equipped with a ZnSe IRE. In the absence of the accessory, the open-beam energy reaching the MCT detector was 5000 counts; with the ATR accessory installed and optimized, the energy reaching the detector was 1296 ± 30 counts. This value represents a 25.9 ± 0.6% transmission efficiency compared to the open-beam energy. (Uncertainties represent standard deviations for two separate energy determinations.) Under comparable conditions, the FlexIR HWG probe accessory possessed a transmission efficiency 34% of that obtained using the SplitPea ATR accessory. The smaller transmission efficiency observed using the ATR probe results from the coupling of infrared light into the 1- mm diameter HWGs.

Table 3.2: Hollow Waveguide ATR Probe Accessory Compared to Open FT-IR Sample Compartment TABLE II. Hollow Waveguide ATR Probe Accessory Compared to Open FT-IR Sample Compartmenta

Pike Technologies Mid-IR FlexIR Hollow Waveguide ATR Accessory

Number of Scans p-p Variationb p-p Variationb SNRb,c Normalized SNRd Normalized SQRT of Scans milliabsorbance (mabs) %T ×10 3

4 1.2 ± 0.1 0.27 ± 0.02 1.8 ± 0.2 1.0 1.0 8 1.0 ± 0.1 0.22 ± 0.03 2.3 ± 0.3 1.2 1.4 16 0.6 ± 0.1 0.14 ± 0.02 3.5 ± 0.4 1.9 2.0 32 0.4 ± 0.1 0.09 ± 0.01 5.5 ± 0.8 3.0 2.9 64 0.3 ± 0.1 0.07 ± 0.01 7.0 ± 1.4 3.8 4.0

Perkin Elmer System 2000 FT-IR Open Sample Compartment

Number of Scans p-p Variationb p-p Variationb SNRb,c Normalized SNRd Normalized SQRT of Scans milliabsorbance (mabs) %T ×10 3

4 0.10 ± 0.01 0.02 ± 0.01 22.6 ± 1.7 1.0 1.0 8 0.08 ± 0.01 0.02 ± 0.01 26.5 ± 3.7 1.4 1.4 16 0.06 ± 0.01 0.01 ± 0.01 40.7 ± 6.8 2.0 2.0 32 0.04 ± 0.01 0.01 ± 0.01 60.1 ± 1.6 2.9 2.9 64 0.03 ± 0.01 0.01 ± 0.01 79.7 ± 8.6 4.0 4.0

a 4 cm-1 resolution

b Average and standard deviation of five collections for each number of scans over the range of 2600-2400 cm-1

c Noise calculated as one-fifth peak-to-peak variation in %T

d Based on signal-to-noise ratio (SNR) in %T

3.5.1.4 Quantitative Analysis of Succinylcholine Chloride Solutions The infrared spectrum of succinylcholine chloride has been previously reported.35-38 The infrared spectrum of an aqueous 100 parts per thousand solution of succinylcholine chloride collected using the ATR probe is shown in Fig. 3.7.

0.025

0.020

0.015

-1

0.010 1193 cm Absorbance 0.005 1133 cm-1

0.000

-0.005 2000 1800 1600 1400 1200 1000 800 Wavenumber (cm-1)

Figure 3.7: Infrared spectrum of a 100 hundred parts per thousand succinylcholine chloride solution. The area of the 1167 cm-1 C-O-C stretch selected for quantitation is contained within the indicated region.

Principal absorptions of succinylcholine chloride, as identified by Miller33, include the -1 -1 ester carbonyl stretch (1735 cm ), the CH2 and CH3 deformation absorptions (1480-1490 cm ), and the C-O-C stretch (1167 cm-1). The 953 cm-1 absorption corresponds to the trimethyl quaternary ammonium. Early ATR analyses of liquid samples were often performed using the CIRCLE cell multi-bounce ATR accessory. Aqueous pharmaceutical solutions containing two choline compounds, including succinylcholine chloride, were performed by Miller et al. using a micro- CIRCLE cell accessory.33 Peak height determinations for both the 952 cm-1 trimethyl quaternary ammonium absorption and the 1165 cm-1 C-O-C stretch of succinylcholine chloride were performed. An ultramicro-CIRCLE cell was characterized by McKittrick et al. using a Spectra- Tech IR-PLAN infrared microscope.34 The utility of the cell for FIA was tested using aqueous

98 solutions of succinylcholine. Quantitative analyses of succinylcholine by McKittrick et al. were based on integrated absorbance of the 1165 cm-1 absorption. The FIA studies performed by both Miller et al. and McKittrick et al. using the CIRCLE cells were designed for liquid chromatography with infrared detection. The internal volumes of the micro- and ultramicro- CIRCLE cells were 24 and 1.75 µL, respectively. Injection volumes used by Miller and McKittrick were significantly larger than the cell volumes. Patterson et al. demonstrated the utility of single-reflection ATR-FT-IR microspectroscopy as an alternative detection method for capillary-column high-performance liquid chromatography (HPLC).35 Static measurements of aqueous succinylcholine chloride solutions possessing concentrations of 22-328 parts per thousand were performed using a drop-down germanium ATR accessory. Since only a few drops of each solution were placed on a dimpled slide, sample volumes employed by Patterson were less than 1 mL. A subsequent publication by Patterson et al. further extended the method of single-bounce ATR-FT-IR microspectroscopic detection to capillary electrophoresis (CE).58 CE separations of succinylcholine chloride with sodium salicylate, sodium citrate, and sodium nitrate using acetone as a neutral marker were demonstrated.

3.5.1.5 Infrared Spot Size The diameter of the active infrared spot at the surface of the probe IRE was determined to be 2.3 ± 0.3 mm. (The precision of this measurement represents the distances of the maximum and minimum spot diameters from the average value as determined using the tape scanning method.) The small active area was due to the small beam divergence of the HWGs. Design specifications of the HWG ATR probe listed the ATR sampling diameter as 4.4 mm. Infrared sampling volume can be approximated as the volume of a cone possessing a diameter equal to the active spot size at the surface of the IRE and a height equal to the depth of penetration (dp), or optical pathlength (OPL), of the evanescent wave into the sample. The depth of penetration can be determined using Eq. 3.1:

(3.1)

[ ( ) ]

where λ represents the wavelength, θ is the angle of incidence at the IRE-sample interface, and nIRE and ns are the refractive indices of the IRE and sample, respectively. A 1.29 µm dp was obtained for a wavelength of 8.6 µm ( using the diamond-ZnSe IRE with an average angle of incidence of 45o. (The refractive indices of the IRE and the samples were 2.40 and 1.33, respectively.) The resulting sampling volume for the aqueous succinylcholine chloride solutions was 2.0×10-3 µL. Quantitative analysis of the aqueous succinylcholine chloride solutions using the HWG ATR probe were based on the integrated absorbance of the 1167 cm-1 C-O-C absorption of succinylcholine over the range of 1193-1133 cm-1. The selected limits of integration are the same as those used in earlier studies.34-36,58 A comparative summary of the analytical figures of merit pertinent to past and present quantitative studies of succinylcholine chloride is shown in Table III. The studies conducted by Miller et al. and McKittrick et al. were done using a PerkinElmer Model 1800 FT-IR spectrometer. Research presented by McKittrick et al. employed a Spectra-Tech IR-PLAN infrared microscope coupled to the Model 1800 FT-IR spectrometer.34 Signal-to-noise ratios for infrared microscopes during the late-1980s to early- 1990s were on the order of 1000:1.36 According to McKittrick, the SNR for the ultramicro-

100

CIRCLE cell (1.75-µL internal volume) was poorer than that of the micro-CIRCLE cell (24-µL internal volume) due to a lower throughput. Whereas the ultramicro-CIRCLE cell was coupled to the infrared microscope, the micro-CIRCLE cell accessory was capable of being placed directly in the macro sample compartment of the FT-IR spectrometer. Improvements in optical design and electronics have resulted in the development of infrared microscopes with appreciably high SNRs. Patterson, using a PerkinElmer AutoIMAGE infrared microscope, reported a SNR of 5000:1 during a liquid chromatographic study with ATR-FT-IR detection.35 Since the optical designs of the PerkinElmer AutoIMAGE and Spotlight microscopes are identical, comparable throughput would be expected for the same aperture settings; however, higher SNRs would be expected using the Spotlight due to the incorporation of a newer- generation MCT detector. Data obtained using the HWG ATR probe will be compared to those previously obtained by Patterson using Spotlight ATR-FT-IR microscope since they represent a higher standard for comparison. Despite the contrast in optical design between the infrared microscope and the FlexIR ATR probe, comparable results would be expected based on the use of current technology, comparable throughput, and the correspondence between sampling volume and the amount of sample. Data presented in Table III indicate that reductions in sampling volume and OPL relative to those reported by Miller were obtained in successive studies by McKittrick and Patterson. The large sampling volumes and OPLs reported by Miller and McKittrick are the result of multiple reflections within the IRE; the average numbers of reflections through the cylindrical ZnSe IREs for the micro- and ultramicro-CIRCLE cells were 7.54 and 5.63, respectively.34 Patterson reported a sampling volume of 180 femtoliters using an FT-IR microscope equipped 2 36 with a hemispherical germanium (nIRE = 4.0) IRE and a 100 × 100 µm aperture. The sample volume reported by Patterson was four orders of magnitude smaller than that obtained using the ATR probe due to reductions in OPL and spot size resulting from the use of a germanium hemisphere.

101

Table 3.3: Comparison of Single-Reflection and Multi-bounce ATR Detection of Succinylcholine Chloride

TABLE III. Comparison of Single-Reflection and Multi-bounce ATR Detection of Succinylcholine Chloride

Study Accessory Sampling Volume OPL Sensitivity Y-intercept R2 LODa µ L µ m mabs/ppt a mabs ppt a

Miller33 micro-CIRCLE 0.221 6.0 1.21 0.445 0.9998 0.2

McKittrick34 ultramicro-CIRCLE 0.025 3.4 3.97 8.12 0.9999 2

Patterson36 IR µ-scope w/Ge hemisphere 1.8 × 10-7 1.1 2.30 11.29 0.999 0.02

Daminpresent FlexIR HWG ATR probe 2.0 × 10-3 1.3 3.28 2.86 0.9993 0.03

Miller calculated detection limit as (S/N = 2) using peak height. According to McKittrick, the detection limit was calculated by peak area and was the same for 20-, 50-, or 100-µL injection. Patterson reported a detection limit calculated as (S/N = 3) for peak area using limits of integration of 1193-1133 cm-1. The detection limit reported in the present study was calculated as three times the noise (rms) of a blank over the range of 2100-1900 cm-1 for five replicate measurements divided by the slope of the calibration curve.

a parts per thousand

A calibration curve of integrated absorbance versus concentration in parts per thousand constructed using data collected with the ATR probe is shown in Fig. 3.8. (The error bars associated with each data point represent the absolute error at 95% confidence for series of five replicate collections at each concentration.)

0.40

0.35 y = 0.0033x + 0.0029 0.30 R² = 0.9993

0.25

0.20

Absorbance 0.15

0.10

0.05

0.00 0 20 40 60 80 100 120 Concentration (parts per thousand)

Figure 3.8: Calibration curve for the 1167 cm-1 succinylcholine chloride absorption.

A sensitivity of 3.28 milliabsorbance units (mabs) per parts per thousand represented a 1.4× improvement in sensitivity over that reported by Patterson using the Spotlight ATR-FT-IR microscope. Increased sensitivity was attributed to the combination of an increased OPL for the diamond-ZnSe probe IRE and the use of a newer MCT detector on the HWG accessory. Limits of detection (LOD) and quantitation (LOQ) for succinylcholine chloride in water using the ATR probe were determined to be 0.03 ± 0.01 and 0.10 ± 0.02 parts per thousand, respectively. Despite the increased sensitivity obtained using the ATR probe, the LOD remained comparable to that reported by Patterson. The underlying goal of the quantitative studies performed after Miller was to reduce the sample volume while maintaining, if not improving, detection limits. The quantitative study of succinylcholine chloride using the ATR probe represents a compromise between sample volume and LOD in that the LOD remains comparable to that previously determined using an ATR-FT- IR microscope despite significantly larger sampling volumes being used. Whereas sample

103 volumes less than 1 mL were used by Patterson for quantitation of succinylcholine chloride, sample volumes on the order of several milliliters were analyzed using the ATR probe.

3.5.2 Analysis of Hard Samples

3.5.2.1 Polycarbonate Sheet and Tube The infrared spectrum of polycarbonate has been previously reported and is shown in Fig. 3.9.59-60

Polycarbonate sheet

Polycarbonate tube %T

4000 3700 3400 3100 2800 2500 2200 1900 1600 1300 1000 700 Wavenumber (cm-1)

Figure 3.9: Infrared spectra of a polycarbonate sheet (top) and a polycarbonate tube (bottom).

A summary interpretation of the PC infrared absorptions is listed in Table IV. The infrared spectra of the PC samples (Fig. 3.9) were collected using the HWG ATR probe. The spectrum of the PC sheet was characteristic of an instrument response, or 100% line, indicating that ideal contact between the sample and the IRE was not achieved. The absence of sample- related absorptions corresponded to a curvature of the sampling surface of the IRE. Testing of this hypothesis involved the collection of an infrared spectrum from a hard, round tube of PC (Fig. 3.9). Contrast between the infrared spectra of the PC sheet and tube samples further supported the existence of curvature to the IRE surface.

104

Table 3.4: Infrared Absorptions and Assignments for Polycarbonate

TABLE IV. Infrared Absorptions and Assignments for Polycarbonate

Absorption Assignment cm -1

1769 ν (C=O)

1504 Ring ν (C-C)

1219 1186 νas (O-C-O) 1157

1080 γ (C-C-C)

1014 νs (O-C-O)

829 out-of-plane ring C-H deformation

Polycarbonate band assignments are based on the publications by 59 60 Schnell and Kraus et al.

105

3.5.2.2 Rubber Eraser The infrared spectrum of a pink, rubber eraser collected using the ATR probe is shown in Fig. 3.10.

Talc

Calcium carbonate %T

Rubber eraser

4000 3700 3400 3100 2800 2500 2200 1900 1600 1300 1000 700 Wavenumber (cm-1)

Figure 3.10: (Bottom) Infrared spectrum of a pink, rubber eraser. (Middle) Reference spectrum of calcium carbonate. (Top) Reference spectrum of talc.

Principal absorptions include asymmetric and symmetric methylene C-H stretches and an ester carbonyl stretch at 1742 cm-1. Absorptions attributed to the presence of a carbonate are observed at 1427, 875, and 712 cm-1. Absorptions corresponding to talc are located at 3676 and -1 1015 cm . Reference spectra of CaCO3 and talc collected using the SplitPea ATR accessory are shown in Fig. 3.10. The rubber eraser represents a sample capable of conforming to the surface of the IRE when pressure is applied; the surface of the rubber returned to its initial state following the collection of an infrared spectrum.

106

3.5.2.3 Calcium Oxalate Monohydrate Application of the HWG ATR probe for the analysis of a conformable sample was performed using a pellet of COM. The infrared spectrum of COM has been reported, and the infrared spectrum of COM obtained using the ATR probe is shown in Fig. 3.11.54,61-62

100

80 %T 70

50 4000 3700 3400 3100 2800 2500 2200 1900 1600 1300 1000 700 Wavenumber (cm-1)

Figure 3.11: Infrared spectrum of calcium oxalate monohydrate.

Absorptions at 1600 and 1311 cm-1 correspond to asymmetric and symmetric C=O stretches, respectively. The 778 cm-1 absorption is assigned to a C-O bend. The collection of infrared spectra from pelletized samples, such as COM, is facilitated by the small degree of deformation that occurs when the ATR probe is brought into direct, intimate contact with the sample. The COM pellet was capable of conforming to the surface of the IRE, resulting in the collection of a quality infrared spectrum.

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3.5.2.4 Practical Considerations The combined benefits of small size, flexibility, and portability associated with the ATR probe permit sample analyses in locations where neither benchtop accessories nor large, hand- held ATR accessories are feasible. For example, the HWG ATR probe could be applied in an industrial setting for the remote collection of infrared spectra from films on rollers in paper mills or other industrial processing equipment. Infrared spectra can be collected within a few minutes using the ATR probe accessory, thereby reducing production downtime and preventing significant loss of product. The analysis of thin films or stains on hard surfaces is among the potential uses of the HWG probe. Although the remote sampling configuration inherent with the use of the ATR probe is beneficial, the method is still limited by the requirement that direct, intimate contact between the sample and the probe IRE must be achieved. Data have shown that this requirement can be easily satisfied for samples possessing appreciable pliability or conformability; however, analyses of samples on hard, rigid surfaces are difficult if the surface of the IRE is not flat. Even though contact between the sample and the ATR probe may be apparent, direct interaction of the sample with the active sampling area on the IRE may not be achieved, resulting in the absence of characteristic sample absorptions.

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3.5.3 Quantitative Analysis of Ethanol in Alcoholic Beverages

3.5.3.1 Infrared Spectra of Ethanol and Water The infrared spectrum of ethanol has been previously reported63-65 and is shown in Fig. 3.12 along with that of water. The absorption at 1046 cm-1 is assigned to the C-C-O stretch, and the 1088 cm-1 absorption is assigned to the C-O stretch. The two principal absorptions of water at 3326 and 1637 cm-1 correspond to the O-H stretch and H-O-H bend vibrations, respectively. Selection of the 1046 or 1088 cm-1 ethanol absorptions for use in the quantitative study was based on appreciable absorbance and lack of overlap with water absorptions.

0.50 0.45 Water 0.40 Ethanol 0.35 0.30 0.25

0.20 Absorbance 0.15 0.10 0.05 0.00 4000 3700 3400 3100 2800 2500 2200 1900 1600 1300 1000 700 -1 Wavenumber (cm ) Figure 3.12: Infrared absorption spectra of pure, 200 proof anhydrous ethanol and distilled water.

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3.5.3.2 Ethanol Calibration Curves Calibration curves constructed using the integrated absorbance of the ethanol 1046 and 1088 cm-1 absorptions versus % ABV are shown in Fig. 3.13.

2.00 1.80 a 1.60

1.40 y = 0.061x - 0.017 1.20 R² = 0.9994 1.00

0.80 Absorbance 0.60 0.40 0.20 0.00 0 4 8 12 16 20 24 28 32 % ABV

0.45 0.40 b 0.35

0.30 y = 0.013x - 0.007 0.25 R² = 0.9986

0.20 Absorbance 0.15

0.10

0.05

0.00 0 4 8 12 16 20 24 28 32 % ABV

Figure 3.13: Calibration curves for the (a) 1046 cm-1 and (b) 1088 cm-1 absorptions of ethanol.

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The resulting figures of merit are shown in Table V. The concentration range used to construct these calibrations was applicable to beer and wine possessing low to moderate alcohol contents. A 4.7× increase in sensitivity was observed using the 1046 cm-1 absorption over that of the 1088 cm-1 absorption; therefore, quantitative analyses of alcoholic beverages will be performed using the 1046 cm-1 ethanol absorption.

TABLE V. Analytical Figures of Merit for Ethanol Calibration Curves.

Absorption Slope (abs/% ABV) Y-intercept (abs) R 2 LODa,b LOQa,b

1046 cm-1 0.061 -0.017 0.9994 0.001 ± 0.001 0.004 ± 0.001

1088 cm-1 0.013 -0.007 0.9986 0.006 ± 0.001 0.021 ± 0.001

a % ABV

b Uncertainty represents standard deviation of four replicate determinations of background noise in distilled water. Table 3.5 Analytical Figures of Merit for Ethanol Calibration Curves 3.5.3.3 Error Analysis Errors associated with standard preparation and instrument drift on the quantitative analysis of aqueous ethanol solutions were calculated on the basis of percent relative standard deviation (% RSD) using 8% ABV solutions representative of high-end beers and low-end wines. Results of the standard preparation error evaluation indicated an average alcohol content of 8.0 ± 0.1% ABV with a 1.6% RSD. Instrument drift represents the capability of the instrument to ratio two single-beam spectra collected at different times. The average alcohol content obtained was 8.1 ± 0.1% ABV with a 1.1% RSD. The % RSD determined for the standard preparation error indicated that this aspect is the limiting factor between successive measurements of alcohol content; however, the results of the error analyses also showed that accurate and reproducible determinations of alcohol content were achieved using the ATR probe.

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3.5.3.4 Alcohol Content Determination in Beer Infrared spectra of beers containing 4.2 and 7.5% ABV are shown in Figs. 3.14a and 3.14b, respectively.

0.021 0.05 a b 0.018 0.04 0.015

0.012 0.03 0.009

0.02 Absorbance Absorbance 0.006

0.003 0.01 0.000

-0.003 0.00 1400 1300 1200 1100 1000 900 800 700 1400 1300 1200 1100 1000 900 800 700 Wavenumber (cm-1) Wavenumber (cm-1)

0.08 0.08 c d

0.06 0.06

0.04 0.04

Absorbance Absorbance

0.02 0.02

0.00 0.00 1400 1300 1200 1100 1000 900 800 700 1400 1300 1200 1100 1000 900 800 700 Wavenumber (cm-1) Wavenumber (cm-1)

0.30 e 0.25

0.20

0.15

0.10 Absorbance 0.05

0.00

-0.05 1400 1300 1200 1100 1000 900 800 700 Wavenumber (cm-1)

Figure 3.14: Infrared absorption spectra of alcoholic beverages. (a) Spectrum of beer with 4.2% ABV. (b) Spectrum of beer with 7.5% ABV. (c) Spectrum of white wine with 9.5% ABV. (d) Spectrum of red wine with 11.0% ABV. (e) Spectrum of whiskey possessing 35.5% ABV.

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The spectrum of the 4.2% beer possesses a narrow asymmetric C-C-O stretch since band broadening due to carbohydrates is minimal. The infrared spectrum of the 7.5% beer (Fig. 3.14b) exhibited an increased asymmetric band shape due to a higher carbohydrate concentration. The impact of carbohydrates on the quantitative determination of alcohol content in alcoholic beverages will be addressed later in this study. Data in Table VI shows that the alcohol contents of the beers determined using the ATR probe were comparable to the listed contents. The % RSDs indicated that reproducible verification of alcohol content was achieved using the proposed method. Analysis of the beers was complicated by the adsorption of CO2 bubbles on the surface of the IRE after the beer was first opened. Infrared spectra either did not exhibit ethanol absorptions, or the absorbance intensities were significantly reduced. Gases within the beverages were permitted to naturally evolve at room temperature for 30 minutes prior to the collection of infrared spectra; degassing the beverages with an inert gas could expedite sample analysis.

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Table 3.6: Summary of Quantitative Analyses of Alcoholic Samples

TABLE VI. Summary of Quantitative Analyses of Alcoholic Samples.

Beer

Listed % ABV Calculated % ABV a,b % RSD

4.2 4.2 ± 0.1 1.0 4.2 4.2 ± 0.1 0.4 5.6 5.6 ± 0.1 1.2 7.5 7.5 ± 0.1 0.7

Wine

Sample Listed % ABV Calculated % ABV a,b % RSD

White 9.5 9.5 ± 0.1 0.2 Red 11.0 11.1 ± 0.1 0.3

Liquor

Sample Listed % ABV Calculated % ABV a,b % RSD

Whiskey 35.5 35.8 ± 0.1 0.2

a Average of 5 individual collections of 64 scans at 8 cm -1 resolution.

b Uncertainty represents absolute error at 95% confidence.

3.5.3.5 Alcohol Content Determination in Wine Infrared spectra of white and red wines are shown in Figs. 3.14c and 3.14d, respectively. In contrast to that of the white wine, the C-C-O stretch of the red wine possessed an increased absorbance due to increased alcohol content. Band broadening resulting from increased carbohydrate concentrations in the beverages was also observed; however, quantitative analyses remain possible since the 1046 and 1088 cm-1 were distinctively separate. A summary of the quantitative analyses of the wines is shown in Table VI. Data indicate that the alcohol contents of the wines determined empirically were comparable to the listed values. The % RSDs determined for the wines indicate that reproducible verifications of alcohol content were achieved.

3.5.3.6 Alcohol Content Determination in Complex Matrices Quantitative determinations of alcohol content in complex matrices in which significant overlap exits between two components are often performed using computational processes, such as principal component analysis (PCA) or PLS. Accurate determinations of analyte characteristics, such as alcohol content, can be obtained; however, computational calculations and results may not be easily be performed or interpreted by a novice in the field. Correction for a principal C-O absorption of most carbohydrates was achieved by shifting the lower limit of integration used for quantitative analysis; the proposed correction shortens computational time while maintaining accurate determinations of alcohol content using a constructed calibration curve. The proposed correction was applied to a determination of alcohol content in a liquor sample possessing a high concentration of carbohydrate sugars. Characteristics of the whiskey indicated that the beverage was flavored with a complex blend of honey, caramel, and oranges. Honey, for example, is a complex mixture of sugars, including fructose, glucose, and sucrose.66 Infrared spectra of honey have been previously reported.67-68

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3.5.3.7 Alcohol Content Determination in Liquor The infrared spectrum of the whiskey containing 35.5% ABV is shown in Fig. 3.14e. Broadening of the ethanol 1046 cm-1 absorption was observed as a result of an increased carbohydrate concentration. Correction for the increased carbohydrate concentration was accomplished by shifting the lower limit of integration to 1029 cm-1 while leaving the upper limit unchanged. Quantitative analysis of the whiskey required extension of the 1046 cm-1 calibration curve to accommodate increased alcohol content. A plot of integrated absorbance versus alcohol content over the range of 10-90% ABV is shown in Fig. 3.15. A summary of the quantitative analysis of the whiskey is shown in Table VI. The alcohol content determined using the ATR probe represents a 0.8% error when compared to the listed value. A 0.2% RSD indicates that a reproducible measure of alcohol content was obtained.

7.00

6.00

5.00 y = 0.069x - 0.210 R² = 0.9991 4.00

3.00 Absorbance

2.00

1.00

0.00 0 10 20 30 40 50 60 70 80 90 100 % ABV

Figure 3.15: Extended calibration curve for the 1046 cm-1 ethanol absorption.

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3.5.3.8 Effect of Carbohydrate Sugars on Alcohol Content Determinations The molecular structures of monosaccharides and disaccharides consist of cyclic rings possessing different C-C and C-O bonds. Band broadening results from the complexity of the molecular structures giving rise to vibrations at different frequencies. Data have shown that distortion of the ethanol absorption can render quantitative analysis based on integrated absorbance difficult. The potential interference of carbohydrate sugars on the infrared spectrum of ethanol was investigated using 5 wt. % aqueous solutions of ethanol, glucose, and sucrose. The infrared spectra of these solutions are shown in Fig. 3.16; the locations of the C-C and C-O stretching absorptions are listed in Table VII.

0.030 Ethanol 0.025 Glucose

0.020 Lactose Sucrose 0.015

0.010 Absorbance

0.005

0.000

-0.005 1300 1200 1100 1000 900 800 Wavenumber (cm-1)

Figure 3.16: Infrared absorption spectra of 5 wt. % solutions of ethanol, glucose, lactose, and sucrose.

Lactose, a non-fermentable sugar added to beer during the production of cream stouts, was selected as a replacement for maltose due to its structural similarity. Maltose, a common fermentable brewing sugar, exhibits a strong infrared absorption at 1032 cm-1.69 The complexity of quantitative analyses performed on the basis of integrated absorbance when appreciable overlap exists between the analyte absorption(s) of interest and those of the matrix components is illustrated in Fig. 3.16. Absorptions assigned to the sugar C-C and C-O stretches were comparable to the C-C-O absorbance of ethanol at the same concentration. Overestimations of

117 alcohol content based on integrated absorbance can occur as a result of carbohydrate band broadening.

TABLE VII. Analysis of Aqueous Sugar Solutions.

Sample ν(C-C + C-O) Location(s) Absorbance a,b % of Ethanol Absorbance cm-1

Ethanol 1046 0.03 ± 0.01 - Glucose 1033 0.02 ± 0.01 67 1053 0.02 ± 0.01 67 Lactose 1035 0.01 ± 0.01 33 1049 0.01 ± 0.01 33 Sucrose 1056 0.02 ± 0.01 67

a Average of 3 individual collections of 64 scans at 8 cm -1 resolution.

b Uncertainty represents absolute error at 95% confidence.

Table 3.7: Analysis of Aqueous Sugar Solutions 3.5.3.9 Sample Volume Accurate determinations of alcohol content in beer, wine, and liquor performed using the HWG ATR probe required appreciably smaller sample volumes than those employed in commercial analyzers. The minimum volume of a liquid sample required to conduct either qualitative or quantitative studies using the ATR probe is on the order of 1 mL.

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3.6 Conclusions

3.6.1 Evaluation of a Hollow Waveguide ATR Probe Accessory A remote sampling accessory employing a HWG ATR probe was evaluated on the basis of transmission efficiency and signal-to-noise. It has been shown that the transmission efficiency of the HWG ATR probe is an order of magnitude less than that of an open-beam FT-IR sample compartment. Minor attenuation in the transmission of infrared radiation by the probe was observed as a result of bending. Minimization of spectral distortion, including baseline variation, was achieved through the collection of background and sample scans with the probe in similar configurations. The capability of the ATR probe for liquid sample analyses was demonstrated through the quantitative study of aqueous succinylcholine chloride solutions. A calibration of integrated absorbance versus concentration possessed a high linear R2 correlation. Quantitative results obtained for succinylcholine chloride using the ATR probe were comparable to those of an earlier study using an ATR-FT-IR microscope with a newer-generation detector.

3.6.2 Analysis of Hard Samples Continued evaluation of the Pike Technologies Mid-IR FlexIR HWG ATR probe was performed using hard and pliable samples. Infrared spectra collected from hard polycarbonate samples indicated the presence of curvature to the diamond-ZnSe IRE. Spectral analyses of rubber and pelletized samples have shown that samples possessing small to moderate degrees of conformation can be analyzed using the ATR probe. Future applications of the HWG ATR probe for the analyses of thin films on hard surfaces require an IRE possessing a flat sampling surface.

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3.6.3 Quantitative Analysis of Ethanol in Alcoholic Beverages Quantitative analyses of aqueous ethanol solutions were performed using a mid-infrared HWG ATR probe. Calibration curves constructed using integrated absorbance versus alcohol content possessed linear R2 correlations greater than 0.99. The alcohol contents of commercial beer, wine, and liquor were successfully verified using the proposed quantitative method. Overestimations of alcohol content based on integrated absorbance occurred for beverages possessing high concentrations of carbohydrate sugars. Correction for carbohydrate absorptions located in close proximity to the 1046 cm-1 ethanol absorption was achieved by adjusting the limits of integration. Accurate determinations of alcohol content were demonstrated in the absence of statistical modeling.

3.7 Acknowledgements The author thanks Dr. Jenni Briggs and Z Stanek of Pike Technologies for allowing use of the Mid-IR FlexIR hollow waveguide accessory and ATR probe. Financial support of this research was provided by PerkinElmer, Kodak, and Procter & Gamble.

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63. E. K. Plyler. “Infrared Spectra of Methanol, Ethanol, and Propanol”. J. Res. Nat. Bur. Stand. 1952. 48: 281-286.

64. Y. Mikawa, J. W. Brasch, R. J. Jakobsen. “Polarized Infrared Spectra of Single Crystals of Ethyl Alcohol”. Spectrochim. Acta A. 1971. 27(4): 529-539.

65. J. Gnado, P. Dhamelincourt, C. Pelegris, M. Traisnel, A. Le Mageur Mayot. “’Raman Spectra of Oligomeric Species Obtained by Tetraethoxysilane Hydrolysis-Polycondensation Process”. 1996. 208(3): 247-258.

66. E. Anklam. “A Review of the Analytical Methods to Determine the Geographical and Botanical Origin of Honey”. Food Chem. 1998. 63(4): 549-562.

67. J. C. Tewari, J. M. K. Irudayaraj. “Floral Classification of Honey Using Mid-Infrared Spectroscopy and Surface Acoustic Wave Based z-Nose Sensor”. J. Agric. Food. Chem. 2005. 53(18): 6955-6966.

68. S. Hennessy, G. Downey, C. O’Donnell. “Multivariate Analysis of Attenuated Total Reflection-Fourier Transform Infrared Spectroscopic Data to Confirm the Origin of Honeys”. Appl. Spectrosc. 2008. 62(10): 1115-1123.

69. J. Wang, M. M. Kliks, S. Jun, M. Jackson, Q. X. Li. “Rapid Analysis of Glucose, Fructose, Sucrose, and Maltose in Honeys from Different Geographic Regions Using Fourier Transform Infrared Spectroscopy and Multivariate Analysis”. J. Food Sci. 2010. 75(2): C208-C214.

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Chapter 4

Design of a Reaction Cell for Studying Nitrogen Doping in Thin Zinc Oxide Films Using In situ Raman Spectroscopy

4.1 Abstract Applications of process monitoring using remote Raman spectroscopy are demonstrated using a high-temperature reaction cell. The thermal decomposition of calcium oxalate monohydrate (CaC2O4·H2O) was studied as a preliminary test of cell design by coupling the reaction cell to a Raman microscope. A comparison of the process-Raman spectral data to the thermogravimetric analysis (TGA) data indicated that reaction intermediates and products formed during thermal decomposition could be identified. A fiber optic Raman microprobe was utilized in the in situ monitoring of nitrogen incorporation in thin films of zinc oxide (ZnO). ZnO samples were heated in the presence of nitrogen-containing precursor gases of a 5 wt. %

NO/N2 mixture and pre-purified nitrogen (PPN2) within the reaction cell. Results of the nitrogen doping analyses were determined to be inconclusive with respect to the formation of nitrogen- doped ZnO (ZnO:N). Changes in the Raman spectrum of ZnO indicating nitrogen incorporation were not observed using PPN2 as the precursor; however, a low-intensity transition commonly associated with the formation of N-related defects in the ZnO was observed at 275 cm-1 using the

5 wt. % NO/N2 mixture. Repetitive spectral measurements performed under identical reaction conditions indicated that the presence of this peak was not constant.

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4.2 Introduction

4.2.1 Reaction Cells One of the first reported variable temperature reactions cells designed for use with Raman spectroscopy was described by Antion and Durig.1 Antion and Durig presented a new sampling device for observing Raman scattering from condensed phase samples of carbon tetrachloride (CCl4), polycrystalline ammonium iodide (NH4I), and oxalyl bromide (C2Br2O2) over a temperature range of 23 to -178oC. In 1975, Hirschfeld et al. discussed the development and use of a high-temperature cell capable of operating from ambient temperatures to 900oC.2 Hirschfeld et al. demonstrated the capability of the cell by monitoring changes in the Raman intensities of CO2 with changes in temperature. In addition to the use of temperature-controlled Raman cells for monitoring samples under extremely cold or hot conditions, cells have been constructed to examine samples under vacuum3 and in a controlled atmosphere4. A publication by Domenech-Ferrer et al. discussed the use of a high-pressure, high-temperature cell in monitoring the decomposition of sodium aluminum hydride (NaAlH4) with added titanium (III) chloride (TiCl3) at different hydrogen pressures and the decomposition and rehydrogenation of 5 magnesium hydride (MgH2) and lithium amide (LiNH2). Many different cells have been designed and constructed since the first reported use of a Raman cell in 1964.6 Studies of metal oxides and catalysts were performed by Wachs and associates7-12 using either a modified version of a reaction cell developed by Wang et al.13 or a commercially available high-temperature, heated stage. Raman spectroscopy was performed by exciting the samples through a quartz window and collecting the resulting Raman scattered light. In situ Raman spectroscopy was employed by Lee and Wachs while investigating the molecular 14 and electronic structures of SiO2-supported surface metal oxides. Catalyst samples were heated in the presence of oxidizing and reducing environments within a reactor cell. Kim et al. characterized hydrothermally-prepared titanate nanotube powders by in situ Raman spectroscopy using a heated stage for reaction monitoring.15 A 1977 publication by Brown et al. illustrated the use of a vacuum cell for the Raman spectroscopic analysis of various metal oxide catalysts.3 A reaction cell possessing easy sample alignment, vacuum control, temperature treatment capabilities to at least 600oC, and maximum optical transparency to minimize signal loss was constructed to monitor phenomena occurring on

131 sample surfaces. Raman spectroscopy was used in characterizing molybdenum on alumina catalysts using a controlled-atmosphere reaction cell by Cheng et al. in 1980.4 Many, if not most, catalysts are susceptible to thermal degradation resulting from local heating of the sample by the excitation laser. Rotating reaction cells, such as those employed by Brown et al. and Cheng et al., assist in preserving samples that may be susceptible to decomposition or damage from localized heating when using a laser excitation source. A publication by Xie et al. discussed the effect of laser heating on the local temperature and concentration of barium nitrate 16 (Ba(NO3)2 ) and barium peroxide (BaO2) using a stationary in situ Raman cell.

4.2.2 Thermal Decomposition of Calcium Oxalate Monohydrate The thermal decompositions of carbonates, carboxylates, oxalates, acetates, formates, and hydroxides were studied by Mu and Perlmutter.17 The authors described the decomposition processes of the various sample types and provide temperature ranges over which the samples, and intermediates, thermally decompose. Calcium oxalate monohydrate (CaC2O4·H2O, COM) was among the samples included in the analysis by Mu and Perlmutter. COM is commonly used as a calibration standard for TGA and differential thermal analysis (DTA). The thermal decomposition of COM occurs via a stepwise process shown in Eqs. 4.1-4.3.

CaC2O4 ∙ H2O → CaC2O4 + H2O (4.1)

CaC2O4 → CaCO3 + CO (4.2)

CaCO3 → CaO + CO2 (4.3)

Anhydrous calcium oxalate (CaC2O4), calcium carbonate (CaCO3), and calcium oxide (CaO) result from the thermal decomposition of COM. One of the earliest reported Raman 18 spectra of COM and CaC2O4 was published by Shippey. Duval and Condrate reported on the dehydration of COM and the structural information obtained at higher temperatures for 19 anhydrous CaC2O4. Raman spectra of the monohydrate powder were collected over the range of 295-483 K (22-210oC) using a high-temperature reaction cell designed to hold an open capillary tube packed with sample.

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4.2.3 Analysis of Nitrogen Incorporation in Zinc Oxide Films 4 Wurtzite ZnO belongs to the C6v symmetry group with two formula units in the primitive cell. According to group theory, the irreducible representation for this symmetry group is: 20

Γirr = A1 + 2B1 + E1 + 2E2 (4.4)

The A1 and E1 modes are polar and split into transverse optical (TO) and longitudinal optical (LO) phonons. The A1 (TO), A1 (LO), E1 (TO), and E1 (LO) modes are infrared and

Raman active. The two E2 modes, (E2 (high) and E2 (low)), are only Raman active. The B1 modes are silent modes, so they are infrared and Raman inactive. ZnO is a wide direct band gap class II-IV semiconductor that has drawn increasing interest for applications in the construction of high efficiency solar cells21-23, ultraviolet (UV) light-emitting diodes (LEDs)24, UV photodetectors, and displays. Modern concerns surrounding non-renewable energy sources have led to increased interest in renewable energy sources, such as wind, water, and solar. The development of high efficiency solar cells is one of the priorities addressed in the U.S. Photovoltaic Industry Roadmap.25 A tandem solar cell constructed using reliable, stable p-type ZnO would provide a method for achieving this goal. Emerging white light illumination technology is based on gallium nitride (GaN), a semiconductor used in LEDs since the 1990s. ZnO possesses a fundamental advantage over GaN in that the exciton binding energy of ZnO is 60 meV while that of GaN is only 24 meV. The higher exciton binding energy permits efficient excitonic emissions at room temperature, so a p-n homojunction composed of ZnO would be expected to produce an ideal blue/UV solid-state emitter. ZnO is relatively better than GaN in several other areas, including low material costs, radiation hardness, the possibility of low-temperature growth, and the availability of large-area ZnO substrates. Realization of the optoelectronic potential of ZnO requires high-quality p-type ZnO and a reliable and reproducible method of controlling the p-type doping.26-27 A publication by Klingshirn et al. in 2010 provided and in-depth account of results covering 65 years of ZnO research.28 Due to the effect of self-compensation (the formation of donor-type native defects when acceptor doping is attempted), a reproducible method of forming p-type ZnO has not yet been accomplished. Nitrogen has been predicted to be the best dopant candidate for the formation of ZnO:N. Since nitrogen is approximately the same size as oxygen, substitution should be easily

133 accomplished without adding significant strain29-30; however, if growth conditions are not precisely controlled, ZnO:N will exhibit n-type conductivity.31 Raman spectroscopy is a useful method for monitoring spectral changes resulting from controlled-reaction conditions. An ex situ study performed by Kerr et al. demonstrated the use of Raman spectroscopy for the characterization of ZnO films annealed in the presence of a nitrogen-containing dopant for the purpose of studying the mechanism of nitrogen doping.32 Kerr et al. observed additional Raman transitions that were attributed to the presence of nitrogen- related defects in the samples; however, since these studies were performed ex situ, real-time evolution of the defects was not observed. Currently, no in situ Raman study has been performed to investigate the kinetic aspects of nitrogen doping in ZnO. In actuality, ex situ Raman spectroscopic results obtained in earlier studies have not been correlated with the electrical properties of the films. A direct comparison of the existing ex situ studies is complicated because the ZnO films studied were grown using different gas precursors and deposition techniques. Kaschner et al. identified five nitrogen-related local vibrational modes (LVMs) while studying the influence of nitrogen as a potential acceptor on the lattice dynamics of ZnO.33 Similarly, Haboeck et al. also observed the five nitrogen-related LVMs in 2005.34 In the studies by Kaschner et al. and Haboeck et al., ZnO was grown on GaN/sapphire substrates by chemical vapor deposition (CVD) using ammonia (NH3) as the nitrogen source and nitrogen dioxide

(NO2) as the oxidant. Ma et al. examined thin films of ZnO:N obtained using thermal processing of zinc oxynitride alloy films deposited on crystalline silicon (100) by radio frequency reactive 35 magnetron sputtering using an Ar, N2, and O2 gas mixture. Results of the Raman spectroscopic analysis performed by Ma et al. indicated the presence of two additional transitions at 273 and 579 cm-1 not belonging to the Raman spectrum of undoped ZnO. Nickel et al. studied ZnO:N deposited by metalorganic chemical vapor deposition (MOCVD) using diallylamine as the nitrogen source, hydrogen, and oxygen.36 The authors concluded that carbon could be readily incorporated in ZnO grown by MOCVD, resulting in the presence of the disordered (D-) and graphitic (G-) transitions in the Raman spectrum at 1360 and 1565 cm-1, respectively. Nitrogen incorporation can give rise to the formation of NO and NNO complexes that can reduce doping efficiency.

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Park et al. investigated the difficulty associated with p-type doping in ZnO using first principles total-energy calculations.37 The dopants investigated included the group I elements of Li, Na, and K and the group V elements of N, P, and As. Results indicated that group I elements are shallow acceptors and substitutional group V elements, such as P and As are deep acceptors. Nitrogen was ultimately concluded to be a better candidate for forming p-type ZnO. Polarized micro-Raman measurements were performed by Bundesmann et al. in studying the phonon modes of Fe-, Sb-, Al-, Ga-, and Li-doped ZnO thin films grown by pulsed-laser deposition on sapphire substrates.38 Additional modes previously assigned to nitrogen incorporation by Kaschner et al.39 were observed in the doped films. These modes were determined to be unrelated to nitrogen incorporation in the films but rather to host lattice defects since the films were grown in the absence of nitrogen.

4.3 Goals and Specific Aims The capability of a reaction cell designed for process-monitoring using Raman spectroscopy will be demonstrated. Initial evaluation of the reaction cell will be performed by studying the thermal decomposition of calcium oxalate monohydrate via remote, in situ Raman spectroscopy. The resulting Raman spectral data will be compared to data collected using a thermogravimetric analyzer. The reaction cell will be applied to the in situ monitoring of nitrogen incorporation in thin films of ZnO. The goal of the present study is to monitor the formation of ZnO:N at elevated temperatures in the presence of nitrogen-containing precursors.

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4.4 Experimental

4.4.1 Stainless Steel Reactor Cell for Raman Process Monitoring A stainless steel 304 reactor cell for Raman process monitoring was designed by the Molecular Microspectroscopy Laboratory at Miami University and manufactured by the Kurt J. Lesker Company (Pittsburgh, PA). Photographs of the reactor cell are shown in Fig. 4.1, and schematic diagrams of the cell are shown in Fig. 4.2. The Raman reactor cell employed a 0.5 in. (1.27 cm) diameter aluminum oxide resistance button heater (Model No. 101275-28) purchased from HeatWave Labs (Watsonville, CA). Samples were held in place on the surface of the heater using three sample clips located on the front of the heat shield assembly. Temperature control of the reactor cell was done using a variable-output voltage transformer. The temperature of the reaction cell was monitored using a Fluke Model 54 II digital thermometer with chromel/alumel thermocouple leads built into the reaction cell.

Figure 4.1: Photographs of stainless steel reaction cell. (Top left) Front of the reactor. (Top right) Side of the reactor cell showing two thermocouple leads and the power connection. (Bottom) Internal heater assembly with the resistance button heater removed.

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The Raman reaction cell possessed six inlets to allow reagent gases to enter the chamber during a reaction or purging of the cell by an inert gas or vacuum. Four stainless steel 316 toggle valves were used to control the flow of gases into and out of the cell and for evacuation. The inlets and outlets of the toggle valves possessed 1/8 in. Let-Lok tube fittings. The reaction cell was connected to a manifold containing the four toggle valves using polytetrafluoroethylene (PTFE) capillary tubes and stainless steel 316 plugs for 1/8 in. compression tube fittings. The two remaining outlets on the reaction cell were sealed using stainless steel 316 compression caps.

Figure 4.2: Schematic diagrams of stainless steel reaction cell.

The front window of the reaction cell was constructed of a 12.5 mm-thick stainless steel 304 CF (ConFlat) flange. The reaction cell possessed a 3 mm-thick quartz window with an aperture of 38 mm. A stainless steel 316 sample compartment was mounted on the button heater assembly using the standard sample clips. A diagram of the stainless steel sample compartment is shown in Fig. 4.3.

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Figure 4.3: Stainless steel sample compartment.

4.4.2 Thermal Decomposition of Calcium Oxalate Monohydrate

4.4.2.1 Materials and Methods Calcium oxalate monohydrate was purchased from Alfa Aesar (Danvers, MA). Calcium carbonate was purchased from EM Science (Gibbstown, NJ). Calcium hydroxide was purchased from Sigma Aldrich (St. Louis, MO). Calcium oxide possessing a 99.998% purity was purchased from Alfa Aesar (Ward Hill, MA). Thermogravimetric analysis was performed as a preliminary investigation of the thermal decomposition of COM. Ten milligrams of COM was heated from 25 to 900oC at a rate of 40oC per minute in PPN2 with a flow rate of 80 stnd. mL/min. The TGA data was used in identifying intermediate and final products of decomposition, as well as over what temperature ranges these species were present. The thermal decomposition of COM was investigated using Raman spectroscopy and the high-temperature, stainless-steel reaction cell. The thermal decomposition of COM was investigated by packing approximately 10 mg of sample into the sample compartment; the sample compartment was subsequently mounted to the button heater using existing hardware. Packing the sample into the sample compartment increases the Raman scattering intensity while maintaining a mass comparable to that used during the TGA analysis.

Thermal decomposition of COM within the reaction cell was performed under a PPN2 flow rate of 80 stnd. mL/min. The beginning of each sample trial included collection of the Raman spectrum at room temperature (~20-25oC) in air. The sample was then heated to the

138 temperatures identified using the TGA plot under a PPN2 purge. Raman spectra were collected at temperatures at or close to the centers of plateaus and curves along the TGA curve of COM. Samples were held at the designated temperatures for 30 minutes to allow the sample to equalize to the temperature of the reaction cell. The samples were cooled to room temperature at the conclusion of each trial prior to collecting post-reaction Raman spectra.

4.4.2.2 Instrumentation Thermogravimetric analysis data for COM was collected using a PerkinElmer TGA 7 Thermogravimetric Analyzer operated using Pyris series thermal analysis software (version 3.52). Instrument control of the analyzer was through a PerkinElmer TAC 7/DX Thermal Analysis Controller. Raman spectra for the thermal decomposition of COM were collected using a Leica Microsystems (Buffalo Grove, IL) DM 2500M Ren (RL/TL) microscope coupled to a Renishaw (Hoffman Estates, IL) inVia Raman spectrometer. Instrument control of the inVia system was done using the Renishaw Windows-based Raman Environment (WiRE) software (version 3.0 [2007]). Excitation of the samples was done using the 514 nm line of a Lexel Laser model 95 argon ion laser with a 10 mW laser power incident on the sample. A Newport (Irvine, CA) model 840-C power meter equipped with a model 818-ST silicon sensor wand was used to measure laser power. In situ monitoring of the thermal decomposition of COM was performed using the remote sampling configuration shown in Fig. 4.14. Collimated laser light exiting the microscope was directed to a 1-inch (2.54 cm) diameter plano-convex lens possessing an effective focal length (EFL) of 50 mm using a flat, front-surface mirror mounted at 45o on the microscope stage.

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Reactor cell Folding mirror

Lens

Figure 4.4: Remote, in situ thermo-Raman sampling configuration using a Renishaw inVia Raman microscope. The laser exiting the microscope was directed towards the reactor cell using a flat, first-surface mirror oriented at 45o on the microscope stage. The laser was focused on the sample contained within the reactor cell using a plano-convex lens. The lens served to both excite the sample and collect the resulting Raman scattered light.

4.4.3 Analysis of Nitrogen Incorporation in Zinc Oxide Films

4.4.3.1 Materials and Methods Zinc oxide nanopowder with particle sizes less than 100 nm was purchased (Sigma- Aldrich, St. Louis, MO). The undoped ZnO samples studied using in situ Raman spectroscopy were grown on glass substrates using low-pressure CVD by members of Dr. Lei Kerr’s research group in the Department of Paper and Chemical Engineering at Miami University. The growth temperatures of the ZnO films ranged from 300-550oC. Additional ZnO samples were also o annealed in the presence of O2 at 350 C. Zinc nitrate was used in identifying spectral features present in multiple ZnO samples following the doping procedure with a 5 wt.% NO/N2 gas mixture purchased from Airgas (Cincinnati, OH). Potassium hydroxide (KOH) was purchased (Fisher Scientific, Fair Lawn, NY). During the investigation of nitrogen incorporation in the ZnO films using the 5 wt. %

140

NO/N2 gas mixture, a 1.0 M solution of KOH was used to scrub out excess nitric oxide (NO) exiting the reaction cell. The ZnO samples deposited on glass substrates were cut into small, square fragments with dimensions of 0.5 in. × 0.5 in. (1.27 cm × 1.27 cm) prior to the spectroscopic investigations; the sample fragments were mounted directly to the resistance heating element inside the reaction cell. The Raman signal collected using the RP20 fiber optic Raman probe was optimized by laterally adjusting the location of the focal plane for the achromatic lens objective. Initial investigation of nitrogen-doping in the ZnO films was performed using the 5 wt. %

NO/N2 gas mixture. After collecting the initial Raman spectrum of the sample in air at room temperature, the reaction cell was placed under vacuum for no less than 60 minutes. The vacuum pressure obtained using a reciprocal pump was 14 psi (pounds per square inch). The sample was slowly heated under vacuum for 60 minutes to allow the reaction cell and the sample to reach a stable temperature of approximately 300oC. Once a stable temperature was reached, the vacuum line to the cell was closed, and the 5 wt. % NO/N2 gas mixture was flowed through the cell at a rate of 40 stnd. mL/min for 60 minutes. After this time, the flow of the dopant gas to the cell was discontinued, and the vacuum line reopened to purge the cell during a 60 minute cool-down to room temperature. Raman spectra of the ZnO sample at the various sampling conditions were collected over a spectral range of 100-2000 cm-1 shift using a 10 second exposure with 20 accumulated scans. The procedure above was repeated for reaction o temperatures of 350, 400, and 450 C using the 5 wt. % NO/N2 mixture as the dopant. Pre- purified nitrogen (PPN2) was also investigated as a nitrogen-containing dopant at the four annealing temperatures.

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4.4.3.2 Instrumentation In situ Raman spectra of the thin film ZnO samples were collected using a Renishaw RP20 fiber optic Raman microprobe coupled to a Renishaw System 100 Raman Analyzer (RA100; Model No. MK30LC). The System 100 is a compact Raman spectrometer designed for process monitoring, bulk materials analysis, and in situ Raman monitoring in laboratory or industrial applications. The fiber optic Raman microprobe was selected for use in investigating nitrogen doping in ZnO due to the capability of monitoring samples quickly and remotely in the presence of potentially hazardous materials. Optical diagrams of the System 100 Raman spectrometer and the RP20 fiber optic Raman microprobe are shown in Fig. 4.5.

Flat mirror Input/collimating lens

Fiber optic input

Neon lamp

CCD camera CCD lens 1800 lines/mm grating

Flat mirror Laser line filter Input/collimating lens

Sample Laser input

To spectrometer Lens Objective Beamsplitter Rayleigh filter Focusing lens Figure 4.5: (Top) Optical diagram of the Renishaw System 100 Raman Analyzer. (Bottom) Optical diagram of the Renishaw RP20 fiber optic Raman microprobe.

The RA100 spectrometer utilized a set of 75 mm focal length lenses for collimating Raman scattered light entering the spectrometer and for focusing diffracted light from an 1800 lines per millimeter grating onto the CCD camera. The CCD used in the System 100 Raman Analyzer was a Renishaw RenCam EEV 600 × 400 deep depletion/UV camera (Part No. A- 9803-0177) thermoelectrically-cooled to -70oC. The active area of the RenCam CCD was 576 × 400 pixels with each pixel being 22 µm × 22 µm. Control of the System 100 Raman Analyzer

142 was done using the Renishaw WiRE software (version 1.3.15 [1993-1999]). This software was operated in conjunction with the GRAMS/32 Spectral Notebase software (version 4.14 Level II) from the Galactic Industries Corporation (acquired by Thermo Scientific). The RP20 fiber optic Raman microprobe is a high-performance probe designed for general purpose spectroscopic applications and can be configured for different excitation wavelengths. The RP20 microprobe was configured for 488 nm excitation from a Lexel Laser model 95 argon ion laser during the analyses of nitrogen incorporation in ZnO films. A photograph of the sampling configuration is shown in Fig. 4.6.

Figure 4.6: Close-up photograph of the fiber optic Raman microprobe and the reaction cell. The 488 nm laser was focused onto the sample using an 18 mm diameter achromatic lens objective with an effective focal length of 27 mm.

The incident laser power at the sample was measured as 20 mW using a Newport model 840-C power meter a model 818-ST silicon sensor wand. The excitation and collection fiber optics for the RP20 probe possessed core diameters of 50 and 62.5 µm, respectively. A suitable working distance between the probe and the sample was obtained using a custom-designed objective constructed using an 18 mm diameter magnesium fluoride-coated achromatic doublet lens (item no. 45-412) purchased from Edmund Optics (Barrington, NJ). The achromatic lens was mounted in a standard 20 mm microscope objective extension tube. The effective focal length of the lens objective was 27 mm.

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A photograph of the instrument setup for the in situ study of nitrogen incorporation in thin films of ZnO is shown in Fig. 4.7.

Figure 4.7: Instrument setup for the in situ study of nitrogen incorporation in thin films of ZnO. With the exception of the Raman spectrometer, the complete reaction-monitoring system was placed inside a sealed glove box. The glove box was designed to allow the user control of the reagents involved in the reaction while simultaneously providing an exhaust purge of the box.

A polycarbonate glove box was designed by the Molecular Microspectroscopy Laboratory and constructed by the Instrumentation Lab at Miami University. The glove box was designed to contain the entire instrument setup, with the exceptions of the Raman spectrometer and the voltage control of the button heater in the reaction cell. A sealed side port equipped with an industrial, chemical-resistant glove permitted access to the reaction values on the manifold. An industrial exhaust fan coupled to the glove box was used to remove any potentially hazardous gas remaining inside. Ex situ analyses of the ZnO films following the nitrogen doping experiments were performed using a Leica Microsystems DM 2500M Ren (RL/TL) microscope coupled to a Renishaw inVia Raman spectrometer equipped with an 1800 lines per millimeter diffraction grating. Raman spectra were collected using 488 nm excitation from a Lexel Laser model 95 argon ion laser. A Leica 20× (0.40 NA) objective was used on the microscope. Control of the inVia Raman microspectrometer was done using Renishaw WiRE software (version 3.0 [2007]).

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4.5 Results and Discussion

4.5.1 Thermal Decomposition of Calcium Oxalate Monohydrate

4.5.1.1 Thermogravimetric Analysis of Calcium Oxalate Monohydrate The thermogravimetric curve for COM is shown in Fig. 4.8. During the first stage of decomposition, the sample underwent dehydration of the monohydrate to anhydrous CaC2O4 over a temperature range of 150-240oC. The second stage of thermal decomposition represented o the transformation of the anhydrous oxalate to CaCO3 between 410 and 540 C with the release of CO. The final decomposition step between 675 and 850oC was assigned to the transformation of

CaCO3 to CaO with the loss of CO2. The theoretical and experimental weight percent changes during each stage of decomposition are shown in Table I.

145

105.0

95.0

85.0

75.0

65.0

Weight percent Weight 55.0

45.0

35.0 0 100 200 300 400 500 600 700 800 900 Sample temperature (oC)

Figure 4.8: Thermogravimetric analysis curve for COM. The temperature of the sample was o increased at a rate of 40 C per minute in the presence of PPN2.

Table 4.1: Thermogravimetric Analysis Results for Calcium Oxalate Monohydrate

TABLE I. Thermogravimetric Analysis Results for Calcium Oxalate Monohydrate

Transition Molecule(s) Lost Weight Percent Change Theoretical Actual

CaC O ∙H O → CaC O H O 2 4 2 2 4 2 12.3 11.7

CaC O → CaCO 2 4 3 CO 19.2 19.3

CaCO → CaO CO 3 2 30.1 30.0

a Initial sample mass = 9.4120 mg

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4.5.1.2 Process-Raman Analysis of the Thermal Decomposition of Calcium Oxalate Monohydrate Unlike earlier thermo-Raman investigations in which spectra were continuously collected as the sample temperature was increased, the thermal decomposition of COM was monitored in the present study using a step-scan approach in which the temperature inside the reaction cell was increased to the desired value and maintained to allow the sample to equilibrate prior to the collection of a spectrum. The temperatures selected for investigating the decomposition of COM were 25, 100, 200, 350, 500, 600, 750, and 900oC. Complete decomposition of COM to CaO occurred by 900oC. The Raman spectrum of COM is presented in Fig. 4.9c. The Raman transition at 504 cm- 1 was assigned to the O-C-O in-plane bending mode, and the transition at 897 cm-1 was assigned to a C-C stretching vibration. Transitions corresponding to the symmetric C-O stretch were located at 1464 and 1491 cm-1. COM contains both planar and non-planar oxalate units; the non- planar oxalate units possess a shorter C-C bond than the planar configuration.41 Due to the non- equivalence of the symmetric C-O stretching vibrations associated with differences in planarity of the oxalate, two transitions were observed. The 1630 cm-1 Raman transition corresponded to the C-O asymmetric stretch.18,19,40

147

(a) (b)

intensity intensity

Normalized Normalized Normalized Normalized 100 500 900 1300 1700 2100 100 500 900 1300 1700 2100 Raman shift (cm-1) Raman shift (cm-1)

intensity intensity

Normalized Normalized Normalized Normalized 100 500 900 1300 1700 2100 100 500 900 1300 1700 2100 Raman shift (cm-1) Raman shift (cm-1)

(e) (f)

intensity

Normalized Normalized Normalized Normalized 100 500 900 1300 1700 2100 100 500 900 1300 1700 2100 Raman shift (cm-1) Raman shift (cm-1)

Figure 4.9: Raman spectra of standard samples. (a) Calcium carbonate. (b) Calcium hydroxide. (c) Calcium oxalate monohydrate. (d) Calcium oxide. (e) Zinc nitrate. (f) Zinc oxide.

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Raman spectra collected during the thermal decomposition of COM between 25 and 350oC are presented in Fig. 4.10.

350oC

200oC intensity

100oC Normalized Normalized

25oC

100 500 900 1300 1700 2100 Raman shift (cm-1)

Figure 4.10: Raman spectra of COM at 25, 100, 200, and 350oC. Heating of the sample was performed in PPN2. (The area enclosed by the box is expanded in Figure 4.11.)

The Raman spectra shown in Fig. 4.10 correspond to the dehydration of the monohydrate to anhydrous CaC2O4. Changes associated with this transformation were observed over the spectral range of 1400-1700 cm-1. (This region contains symmetric and asymmetric C-O stretching vibrations.) Spectral interpretation and assignment of COM and anhydrous CaC2O4 transitions were based on the works of Shippey18 and Duval and Condrate19. At 25oC, the two transitions assigned to the symmetric C-O stretch of COM were observed at 1464 and 1491 cm-1. These two transitions shifted to 1464 and 1480 cm-1 when the sample was heated to 100oC. At 200oC, the symmetric C-O stretches were located at 1467 and 1478 cm-1. The positions of these Raman transitions at 200oC corresponded to the symmetric stretching modes of anhydrous -1 CaC2O4. The symmetric stretches coalesced into a single transition centered at 1474 cm when the sample was heated to 350oC. The symmetric C-O stretches became equivalent at this temperature, resulting in the formation of a single, broad transition.

149

350oC intensity 200oC

Normalized Normalized 100 C

25oC

1400 1450 1500 1550 1600 1650 1700 Raman shift (cm-1)

Figure 4.11: Raman spectra of COM over the range of 1400-1700 cm-1 shift at 25, 100, 200, and 350oC. The transitions located between 1450 and 1500 cm-1 are assigned to the symmetric C-O stretch of COM, and the transitions at 1640 cm-1 is assigned to the asymmetric C-O stretch.

Changes to the asymmetric C-O stretch at 1630 cm-1 were also observed in the Raman spectra of COM over the range of 25-350oC. A single transition was observed at 1630 cm-1 at 25oC, but this transition split into two separate transitions at 1629 and 1647 cm-1 when the sample was heated to 100oC. Above 100oC, the asymmetric C-O stretch was observed at 1644 cm-1. Further increasing the temperature to 200 and 350oC shifted this transition to 1640 cm-1.

The spectroscopic analysis of the dehydration of COM to anhydrous CaC2O4 was in agreement with previously published data by Frost and Weier.41-42

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Raman spectra collected during the decompositions of anhydrous CaC2O4 to CaCO3 and o CaCO3 to CaO over the temperature range of 500-900 C are shown in Fig. 4.12a.

25oC post-reaction (a) 900oC

750oC intensity

600oC

Normalized Normalized 500oC

100 500 900 1300 1700 2100 Raman shift (cm-1)

(b)

intensity Normalized Normalized

100 500 900 1300 1700 2100 Raman shift (cm-1)

Figure 4.12: (a) Raman spectra of the thermal decomposition of COM at 500, 600, 750, and 900oC. A post-reaction scan at 25oC is also shown. (The area enclosed by the box is expanded in

Figure 4.13.) (b) Raman spectrum of CaCO3.

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Spectral features associated with the oxalate were not observed when the anhydrous o oxalate sample was heated to 500 C. Anhydrous CaC2O4 undergoes thermal conversion to o CaCO3 between 450 and 510 C. A low-intensity transition indicating the formation of a carbonate was observed at 1082 cm-1 upon heating of the sample to 600oC. The Raman spectrum -1 of CaCO3 is shown in Figs. 4.9a and 4.12b. The 1082 cm transition for CaCO3 was assigned to the ν1 symmetric stretching mode of the carbonate. The intensity of this transition increased as more oxalate was converted to carbonate. (The spectral region indicated by the box in Fig. 4.12a is expanded in Fig. 4.13.)

25oC post-reaction

900oC

o intensity 750 C

600oC Normalized Normalized

500oC

1000 1020 1040 1060 1080 1100 1120 1140 1160 Raman shift (cm-1)

Figure 4.13: Raman spectra of COM at 500, 600, 750, and 900oC over the range of 1000-1160 cm-1 shift. A post-reaction scan at 25oC is also shown. The spectra show an increase in the -1 normalized intensity of the ν1 symmetric stretch of calcium carbonate at 1082 cm shift from 600 to 750oC. This transition disappears when the temperature increased to 900oC.

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The intensity of the 1082 cm-1 transition increased as the sample was heated from 600 to 750oC. The 1082 cm-1 carbonate transition was no longer observed once the sample temperature reached 900oC. The TGA curve indicated that COM has thermally decomposed to CaO by 900oC. Controversy has arisen in recent years with regards to the spectral identification of CaO. Chaix-Pluchery et al. used Raman spectroscopy to investigate the thermal treatment of calcium 43 hydroxide, Ca(OH)2. The authors concluded that CaO was Raman-inactive because no spectral features were observed above 200oC. In a follow-up article by Seehra, the author commented on the spectral interpretation of CaO performed by Chaix-Pluchery et al. and indicated that CaO has no first-order Raman effect; however, a second-order Raman effect involving the scattering of two phonons should be observed.44 A study of second-order Raman spectra of alkaline-earth oxides possessing a NaCl-like structure, like CaO, was performed by Reider et al. in 1973.45 Using 514.5 nm laser excitation with a triple monochromator, Reider observed transitions at 533, 655, and 945 cm-1 in the second-order Raman spectrum of high-purity single crystals of CaO. The Raman spectrum of CaO is shown in Fig. 4.9d. Principal Raman transitions were observed at 254, 358, 664, and 1080 cm-1 shift. A low-intensity transition was also observed at 1123 cm-1. The CaO sample selected for use in the present study possessed a purity of 99.998%. At this purity, the Raman spectrum presented in Fig. 4.9d would theoretically be the true Raman spectrum of CaO; however, upon further review, many of the transitions present in the Raman spectrum of CaO were also observed in the Raman spectrum of Ca(OH)2 (Fig. 4.9b). The -1 Raman spectrum of Ca(OH)2 included transitions at 254, 282, 358, 684, 1086, and 1123 cm . -1 The only differences between the Raman spectra of CaO and Ca(OH)2 were a 20 cm difference -1 -1 between the 644 cm band of CaO and the 684 cm band of Ca(OH)2 and the presence of a low- -1 intensity transition at 282 cm in the spectrum of Ca(OH)2. Calcium oxide is capable of readily reacting with water or moisture in the air resulting in the formation of Ca(OH)2. In the present study, the interpretation of CaO by Chaix-Pluchery was utilized in that CaO was identified as being Raman-inactive and the final product of the thermal decomposition of COM due to the absence of Raman transitions at 900oC. No transitions were observed in the Raman spectrum upon cooling of the sample to 25oC.

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4.5.2 Analysis of Nitrogen Incorporation in Zinc Oxide Films

4.5.2.1 Process-Raman Analysis of Nitrogen Incorporation in Zinc Oxide Films

ZnO in 5 wt. % NO/N2. As-grown samples of ZnO grown using CVD were analyzed using remote, in situ Raman spectroscopy. The Raman spectrum of ZnO was previously reported by Damen et al. in 1966.46 The Raman spectrum of ZnO acquired experimentally is shown in Fig. 4.9f. The spectrum of pure ZnO powder possessed fundamental vibrations at 332, -1 -1 383, 438, 581, and 1156 cm . The 332 and 383 cm transitions were identified as the 2E2(M) -1 and A1(TO) modes, respectively. The 2E2(M) mode at 332 cm was due to possible multiple- phonon-scattering processes33 and may have been associated with the Zn-O vibration47. The -1 transition of strongest intensity was observed at 438 cm and was assigned to the E2 (high) vibrational mode. The E2 (high) transition involves an oxygen atom and is often paired with the -1 383 cm transition for the identification of ZnO. A shoulder representing the LO2 mode was -1 -1 observed at 538 cm . The 581 cm transition was attributed to the E1 (LO) mode. The broad band centered at 1156 cm-1 has been associated with C-O bonding between naturally-occurring carbon and oxygen in the air within the sample due to the reactive characteristic of ZnO.32 Monitoring nitrogen incorporation in the ZnO films was performed by annealing the o samples in a 5 wt. % NO/N2 gas mixture at 300, 350, 400, and 450 C. A comparison of the spectra collected at these temperatures indicated that the Raman spectra of the as-grown ZnO samples were essentially the same at the four selected temperatures both pre- and post-reaction with the nitrogen-containing dopant gas. Raman spectra of samples annealed in the 5 wt. % o NO/N2 at 300 and 400 C are shown in Figs. 4.14 and 4.15. Significant changes in the Raman spectra were not observed for the samples annealed with 5 wt. % NO/N2.

154

(a) Initial scan under ambient conditions (b) Under vacuum for 60 min

intensity intensity

Normalized Normalized Normalized 100 500 900 1300 1700 2100 100 500 900 1300 1700 2100 Raman shift (cm-1) Raman shift (cm-1)

Temp = 300oC Temp = 300oC

intensity intensity

Normalized Normalized Normalized 100 500 900 1300 1700 2100 100 500 900 1300 1700 2100 Raman shift (cm-1) Raman shift (cm-1)

(e) Cooled in 5 wt.% NO/N2 for 60 min (f) Post-reaction scan on Renishaw inVia

intensity intensity

Normalized Normalized Normalized 100 500 900 1300 1700 2100 100 500 900 1300 1700 2100 Raman shift (cm-1) Raman shift (cm-1)

o Figure 4.14: Raman spectra of ZnO sample 1 annealed in 5 wt. % NO/N2 at 300 C.

155

(a) Initial scan under ambient conditions (b) Under vacuum for 60 min

intensity intensity

Normalized Normalized Normalized 100 500 900 1300 1700 2100 100 500 900 1300 1700 2100 Raman shift (cm-1) Raman shift (cm-1)

Temp = 400oC Temp = 400oC

intensity intensity

Normalized Normalized Normalized 100 500 900 1300 1700 2100 100 500 900 1300 1700 2100 Raman shift (cm-1) Raman shift (cm-1)

(e) Cooled under vacuum for 60 min (f) Post-reaction scan on Renishaw inVia

intensity intensity

Normalized Normalized Normalized 100 500 900 1300 1700 2100 100 500 900 1300 1700 2100 Raman shift (cm-1) Raman shift (cm-1)

o Figure 4.15: Raman spectra of ZnO sample 3 annealed in 5 wt. % NO/N2 at 400 C.

156

Large, sloping baselines characteristic of fluorescence-related impurities or defects in the samples were observed during initial analyses of many ZnO samples. The background intensity decreased as the temperature within the reaction chamber was increased. This phenomenon was attributed to the removal of sample impurities at elevated temperatures; however, the sloping baseline returned when the sample cooled to room temperature.

The post-annealing reaction spectrum of ZnO annealed in the presence of the NO/N2 dopant at 300oC shown in Fig. 4.14f represents a potential problem if the annealing reaction conditions are not readily controlled. The spectrum collected using the Raman microscope indicated the presence of a low-intensity transition at 730 cm-1 and a moderate-intensity -1 transition at 1055 cm . These transitions correspond to the formation of Zn(NO3)2 resulting o from cooling of the sample from 300 C in the presence of the NO/N2 dopant. Remaining samples, including the sample shown in Fig. 4.15f, were cooled following annealing under vacuum. In doing so, the nitrate peak was no longer observed in the post-reaction Raman spectra. The Raman spectrum of Zn(NO3)2 is shown in Fig. 4.9e. Yan et al. previously discussed theoretical predictions for four different nitrogen- containing gases that have been the focus of many research projects dealing with the incorporation of nitrogen in ZnO for the purpose of creating p-type semiconductors.48 The four gases investigated were nitrogen (N2), nitrogen monoxide (NO), nitrogen dioxide (NO2), and nitrous oxide (N2O). The authors predicted that the use of NO or NO2 would lead to the formation of p-type ZnO and that N2 and N2O would incorporate substitutional nitrogen in the samples, making the ZnO n-type. The underlying principle for their prediction was based on the single nitrogen atoms that could be supplied by NO or NO2. These single atoms could easily be incorporated in the films resulting in the formation of N-related defects in the samples.

Alternatively, use of N2 and N2O complicates the annealing process since pairs of nitrogen atoms that must be broken apart by supplying more energy. In ZnO, nitrogen can act as a donor as well as an acceptor. If a single nitrogen atom substitutes for a single oxygen atom (NO), it will act as a single acceptor. However, if diatomic N2 substitutes for an O atom, forming (NN)O, it is expected to act as a double donor. It is believed that the formation of NO is favored when using single nitrogen-containing dopant gases, such as NO. Based on this prediction, initial investigations of nitrogen incorporation in ZnO were performed using the 5 wt. % NO/N2 gas mixture as a nitrogen-containing precursor.

157

The formation of p-type ZnO is predicted to occur at annealing temperatures around 350- 400oC. A weak-intensity transition at 275 cm-1 was observed in the Raman spectra acquired for the ZnO films annealed in the presence of the NO/N2 mixture after cooling the samples from 350 and 400oC. The Raman spectra presented in Fig. 4.16 represent the 275 cm-1 transition after the samples were cooled to room temperature post annealing.

450oC

o intensity 400 C

350oC Normalized Normalized

300oC

250 260 270 280 290 300 Raman shift (cm-1)

Figure 4.16: Raman spectra over the range of 250-300 cm-1 shift for ZnO samples 1-4 cooled for o 60 minutes after annealing in 5 wt. % NO/N2 at 300, 350, 400, and 450 C. Samples annealed at 350 and 400oC exhibit a transition at 275 cm-1 shift that could be assigned to a local vibrational mode of nitrogen-doped ZnO.

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The spectrum collected at 400oC (Fig. 4.16) possessed the highest normalized intensity at 275 cm-1. Publications by Li et al. and Kerr et al. indicated the formation of p-type ZnO can only be achieved under carefully regulated conditions at temperature between 400 and 450oC.31-32 The ZnO samples employed in the ex situ study by Kerr were thin film polycrystalline samples whereas the ZnO samples characterized during the present in situ Raman study were nanostructured samples. The process of synthesizing these samples was recently reported by Mu et al.49 In the previous study by Kerr et al., the formation of p-type ZnO was determined by the Hall effect.32 In contrast, the conclusion that ZnO:N was formed in the present study was based solely on the occurrence of the low-intensity 275 cm-1 Raman transition. Repetitive measurements performed under identical reaction conditions were unable to reproducibly confirm the presence of the 275 cm-1 transition. Further investigation of the 275 cm-1 transition o resulting from annealing of ZnO in the 5 wt.% NO/N2 mixture at 350 and 400 C was performed using samples of ZnO grown in an oxygen environment. The Raman spectra for samples monitored at 350oC and 400oC are presented in Figs. 4.17 and 4.18, respectively.

159

(a) Initial scan under ambient conditions (b) Under vacuum for 60 min

intensity intensity

Normalized Normalized Normalized 100 500 900 1300 1700 2100 100 500 900 1300 1700 2100 Raman shift (cm-1) Raman shift (cm-1)

Temp = 350oC Temp = 350oC

intensity intensity

Normalized Normalized Normalized 100 500 900 1300 1700 2100 100 500 900 1300 1700 2100 Raman shift (cm-1) Raman shift (cm-1)

(e) Cooled under vacuum for 60 min (f) Post-reaction scan on Renishaw inVia

intensity intensity

Normalized Normalized Normalized 100 500 900 1300 1700 2100 100 500 900 1300 1700 2100 Raman shift (cm-1) Raman shift (cm-1)

o Figure 4.17: Raman spectra of ZnO sample A annealed in 5 wt. % NO/N2 at 350 C. The ZnO sample was grown in the presence of O2.

160

(a) Initial scan under ambient conditions (b) Under vacuum for 60 min

intensity intensity

Normalized Normalized Normalized Normalized 100 500 900 1300 1700 2100 100 500 900 1300 1700 2100 Raman shift (cm-1) Raman shift (cm-1)

Temp = 400oC Temp = 400oC

intensity intensity

Normalized Normalized Normalized Normalized 100 500 900 1300 1700 2100 100 500 900 1300 1700 2100 Raman shift (cm-1) Raman shift (cm-1)

(e) Cooled under vacuum for 60 min (f) Post-reaction scan on Renishaw inVia

intensity intensity

Normalized Normalized Normalized Normalized 100 500 900 1300 1700 2100 100 500 900 1300 1700 2100 Raman shift (cm-1) Raman shift (cm-1)

o Figure 4.18: Raman spectra of ZnO sample B annealed in 5 wt. % NO/N2 at 400 C. The ZnO sample was grown in the presence of O2.

161

Initial comparison of the spectra for the samples grown in the presence of O2 to those of the previously examined as-grown ZnO samples indicated only minor spectral differences. First, the intensity of the sloping baseline was reduced for the samples grown in O2, both before and after the annealing reaction. Next, nitrogen-related transitions, including the 275 cm-1 peak, were not observed in the Raman spectra. The absence of the nitrogen-related vibrational modes was attributed to the increased oxygen content within the ZnO films grown in the presence of O2.

ZnO in PPN2. PPN2 was also investigated as a potential nitrogen-containing dopant for generating p-type ZnO:N. Raman spectra of the ZnO samples were collected at annealing temperatures of 300, 350, 400, and 450oC. The Raman spectra collected for the sample annealed o in PPN2 at 450 C are shown in Fig. 4.19.

162

(a) Initial scan under ambient conditions (b) Under vacuum for 60 min

intensity intensity

Normalized Normalized Normalized Normalized 100 500 900 1300 1700 2100 100 500 900 1300 1700 2100 Raman shift (cm-1) Raman shift (cm-1)

Temp = 450oC Temp = 450oC

intensity intensity

Normalized Normalized Normalized Normalized 100 500 900 1300 1700 2100 100 500 900 1300 1700 2100 Raman shift (cm-1) Raman shift (cm-1)

(e) Cooled under vacuum for 60 min (f) Post-reaction scan on Renishaw inVia

intensity intensity

Normalized Normalized Normalized Normalized 100 500 900 1300 1700 2100 100 500 900 1300 1700 2100 Raman shift (cm-1) Raman shift (cm-1)

o Figure 4.19: Raman spectra of ZnO sample 8 annealed in PPN2 at 450 C.

163

In addition to the spectra presented in Fig. 4.19, Raman spectra collected 300, 350, and o 400 C in the presence of PPN2 were essentially the same as those collected in the presence of the

5 wt. % NO/N2 mixture. Spectral changes indicating the formation of ZnO:N were not observed o for the samples annealed in PPN2 at 300, 350, and 400 C. Raman spectra acquired for the four samples cooled after annealing are shown in Fig. 4.20. The only potential nitrogen-related transition observed in the Raman spectra was located at 280 cm-1 for the sample annealed at o 450 C in PPN2. Nitrogen-containing dopant gases containing two nitrogen atoms, such as N2 or

N2O, are not predicted to be ideal doping reagents for generating ZnO:N because either a N=N or a N≡N bond must be broken in order to supply single nitrogen atoms.

450oC

intensity 400 C

350oC Normalized Normalized

300oC

220 240 260 280 300 320 340 360 380 400 Raman shift (cm-1)

Figure 4.20: Raman spectra over the range of 220-400 cm-1 shift for ZnO samples 5-8 cooled for o 60 minutes after annealing in PPN2 at 300, 350, 400, and 450 C. A low-intensity transition observed at 280 cm-1 could be attributed to the formation of ZnO:N.

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Nitrogen has been regarded as the most likely candidate for p-type doping in ZnO; however, a recent publication by Lyons et al. contradicts this supposition.50 Based on advanced first-principle density functional calculations, Lyons concluded that nitrogen is a deep acceptor in ZnO and would not likely result in p-type conductivity. Alternative methods of synthesizing p-type ZnO films have been investigated. For example, Ryu et al. demonstrated the synthesis of p-type ZnO obtained by arsenic doping of films grown on GaAs substrates.51 Manjon et al. reported that many of the anomalous transitions previously identified as being nitrogen-related actually corresponded to silent Raman modes observed by disorder-activated Raman scattering as a result of a relaxation of the Raman selection rules due to a breakdown of the translational symmetry of the crystal lattice.52 Manjon et al. also showed that the same silent modes could be observed in other wurtzite-type compounds, such as nitride films and alloys. Ultimately, a reliable and reproducible method of generating a p-type semiconductor using ZnO has yet to be accomplished. A consistent agreement with respect to the spectral interpretation of the transitions present in the Raman spectra of doped and undoped ZnO has not been reached. The use of nitrogen-containing precursors remains uncertain with respect to the formation of p-type ZnO:N. Future research may help to clarify the nitrogen-doping process and ultimately determine if nitrogen is or is not the best candidate for use in creating a ZnO p-type semiconductor.

4.5.3 Background Variation Significant changes in the background intensities of the samples were observed at elevated temperatures during the investigations of the thermal decomposition of COM and the nitrogen doping of the ZnO films. Background variations in the Raman spectra observed during the thermal investigations have been previously reported in literature.43,53 Chaix-Pluchery et al. observed a sharp increase in the background intensity during the dehydration of Ca(OH)2 to CaO that was theorized to result from diffusion of the laser light coming from fast and large amplitude motions in the sample.43 Similar shifts in background intensity were reportedly observed during 54-55 studies of TiO2 performed by Chang and Huang. In a 2001 publication, Hendra addressed the issue of hot samples in Raman spectroscopy.56 Effects of blackbody radiation on the resulting Raman spectra of hot samples were discussed, including the presence of a high background signal for samples at elevated

165 temperatures. The presence of the high baseline was apparent in the high-temperature Raman spectra of the ZnO samples; the slope of the baseline decreased as the temperature increased, especially at large Raman shifts. This effect was initially attributed to the minimization of sample fluorescence; however, the slope of the baseline returned to the initial state upon cooling of the samples. Alternatively, a high background was observed in the low wavenumber shift region of the high-temperature ZnO Raman spectra. Although the ZnO Raman transitions were still observed, their intensities were significantly reduced due to the presence of an increased thermal background. A study on the effect of blackbody radiation on the Raman spectrum of diamond up to 1900 K was presented by Zouboulis and Grimsditch.57 When the temperature of the diamond was raised to 1898 K, a strong background signal due to blackbody radiation was observed, resulting in a masking of the 1332 cm-1 phonon band. A similar occurrence was observed in the present study while monitoring the thermal decomposition of COM. Blackbody radiation can significantly influence the intensities of observed Raman transitions, especially at elevated temperatures, since a hot sample is capable of emitting light in the visible region of the spectrum. Increased sample temperature results in a higher portion of this light being in the short-wavelength, or blue region, of the spectrum. When a sample is heated, the total energy emitted increases with the fourth power of the absolute temperature according to the Stefan-Boltzmann law. The intensity of the baseline increased towards higher Raman shifts during the investigation of the COM thermal decomposition. The increasing baseline intensity was attributed to increasing blackbody radiation from the heated sample. A similar occurrence was observed in a publication by Bennett regarding the removal of thermal backgrounds from the FT-Raman spectra of samples excited using the 1064 nm line of a Nd:YAG laser.58 The Raman spectrum of polystyrene (PS) was included as an example of the effects of an increased thermal background. At room temperature, the spectrum of PS possessed a flat baseline with no significant distortions being evident; however, when the PS sample was heated to 140oC, a significant increase in the background was present at longer wavenumber shifts. Bennett observed that the increased thermal background at these frequencies clearly distorted the Raman transitions associated with C-H stretching vibrations. During the present investigation of nitrogen incorporation in thin ZnO films, an increased thermal background was observed at low Raman shifts for the ZnO samples at the elevated annealing temperatures. According to Bennett, the exact form of the thermal background is dependent upon the

166 emissivity of the sample, its temperature, and the transmission characteristics of the spectrometer. It is therefore possible for different samples, such as COM and ZnO, to exhibit different forms of thermal backgrounds in their Raman spectrum. Thermal background at elevated sampling temperatures continues to be an issue in Raman spectroscopy since Raman scattered radiation must be discriminated from the intense blackbody radiation emitted from high-temperature samples. The continuous blackbody radiation emitted by a hot sample is capable of masking the weak Raman signal. The high- temperature Raman spectra collected in the present study represented a combination of the Raman signal and the thermal background. Temporal filtering using pulsed laser excitation and gated detection would offer an efficient method of minimizing the effect of blackbody radiation.59-60 Since the array detector is only active for the duration of the laser pulse, spontaneous Raman scattering can be distinguished from the thermal background. This technique was reported by Exarhos et al. for the collection of Raman spectra of silica up to 2000oC.61 Pulsed-laser Raman spectroscopy was later employed for monitoring the plasma- assisted growth of diamond films in situ.62-64 You et al. reported high-temperature Raman data collected for sodium disilicate up to 1773 K using a pulsed copper vapor laser and a monochannel time-resolved photomultiplier detector.65 Pulsed-laser excitation is advantageous because increased Raman signal can often be observed in contrast to the use of continuous wave (CW) laser excitation; however, prices associated with pulsed lasers are greater than those of CW lasers.

167

4.6 Conclusions Application of a temperature-controlled reactor cell designed for in situ process-Raman spectroscopy was demonstrated by monitoring the thermal decomposition of calcium oxalate monohydrate and the incorporation of nitrogen in thin films of zinc oxide under controlled reaction conditions. The simple design of the reaction cell paired with its capability allows this accessory to be used for a wide range of reaction processes under different conditions. The multiple gas outlets attached to the reaction chamber permit different reagents, purges, or vacuum lines to be attached to the cell. The reactor cell design should prove useful for future analyses of samples under controlled process conditions.

4.6.1 Thermal Decomposition of Calcium Oxalate Monohydrate The reactor cell was successfully employed in verifying the step-wise thermal decomposition products of calcium oxalate monohydrate using remote Raman spectroscopy.

4.6.2 Analysis of Nitrogen Incorporation in Zinc Oxide Films In situ Raman spectroscopy was employed in studying nitrogen incorporation in thin films of ZnO using 5 wt. % NO/N2 and pre-purified nitrogen as dopant gases at 300, 350, 400, and 450oC. Low-intensity, irreproducible transitions, potentially due to nitrogen incorporation in ZnO, were observed for samples annealed at 350 and 400oC in the presence of the 5 wt. % o NO/N2 dopant and at 450 C in the presence of pre-purified nitrogen. The resulting data proved inconclusive with respect to the formation of nitrogen-doped ZnO and indicated that Raman spectroscopy may not be the preferred method for monitoring this process.

4.7 Acknowledgements The author thanks Mr. Barry Landrum and Mr. Lynn Johnson of the Instrumentation Shop at Miami University for their assistance in fabricating the various mechanical and electrical components required for the investigations described in this paper. Financial support of this research was provided by the United States Department of Energy (grant # DE-FG02-07ER) and Pressco Technology.

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34. U. Haboeck, A. Hoffman, C. Thomsen, A. Zeuner, B. K. Meyer. “High-energy Vibrational Modes in Nitrogen-doped ZnO”. Phys. Stat. Sol. B. 2005. 242(3): 21-23.

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36. N. H. Nickel, F. Friedrich, J. F. Rommeluere, P. Galtier. “Vibrational Spectroscopy of Undoped and Nitrogen-doped ZnO Grown by Metalorganic Chemical Vapor Deposition”. Appl. Phys. Lett. 2005. 87(21): 211905-1-211905-3.

37. C. H. Park, S. B. Zhang, S. Wei. “Origin of p-type Doping Difficulty in ZnO: The Impurity Perspective”. Phys. Rev. B. 2002. 073202-1-073202-3.

38. C. Bundesmann, N. Ashkenov, M. Schubert, D. Spemann, T. Butz, E. M. Kaidashev, M. Lorenz, and M. Grundmann. “Raman Scattering in ZnO Thin Films Doped with Fe, Sb, Al, Ga, and Li”. Appl. Phys. Lett. 2003. 83(10): 1974-1976.

39. V. Tazzoli, C. Domeneghetti. “The Crystal Structures of Whewellite and Weddellite: Re-examination and Comparison”. Am. Min. 1980. 65: 327-334.

40. C. G. Kontoyannis, N. CH. Bouropoulos, P. G. Koutsoukos. “Use of Raman Spectroscopy for the Quantitative Analysis of Calcium Oxalate Hydrates: Application for the Analysis of Urinary Stones”. Appl. Spectrosc. 1997. 51(1): 64-67.

41. R. L. Frost, M. L. Weier. “Thermal Treatment of Weddellite—A Raman and Infrared Emission Spectroscopic Study”. Thermochim. Acta. 2003. 406(1-2): 221-232.

42. R. L. Frost, M. L. Weier. “Thermal Treatment of Whewellite—A Thermal Analysis and Raman Spectroscopic Study”. Thermochim. Acta. 2004. 409(1): 79-85.

43. O. Chaix-Pluchery, D. Ciosmak, J. C. Niepce, M. Peyrard. “Raman Study of Prereactional Transformations in Calcium Hydroxide Crystals During a Thermal Treatment Leading to Dehydration”. J. Solid State Chem. 1984. 53(2): 273-276.

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44. M. S. Seehra. “Comment on the Raman Study of the Thermal Transformation of Calcium Hydroxide”. J. Solid State Chem. 1986. 63(2): 344-345.

45. K. H. Reider, B. A. Weinstein, M. Cardona, H. Bilz. “Measurement and Comparative Analysis of the Second-Order Raman Spectra of the Alkaline-Earth Oxides with a NaCl Structure”. Phys. Rev. B. 1973. 8(10): 4780-4786.

46. T. C. Damen, S. P. S. Porto, B. Tell. “Raman Effect in ZnO”. Phys. Rev. 1966. 142(2): 570- 574.

47. U. Ozgur, Y. I. Alivov, C. Liu, A. Teke, M. A. Reshchikov, S. Doganbrevean, V. Avrutin, S. J. Cho, H. Morkoc. “A Comprehensive Review of ZnO Materials and Devices”. J. Appl. Phys. 2005. 98(4): 041301-1-041301-98.

48. Y. Yan. S. T. Pantelides, S. B. Zhang. “Control of Doping by Impurity Chemical Potentials: Predictions for p-Type ZnO”. Phys. Rev. Lett. 2001. 86(25): 5723-5726.

49. W. Mu, L. L. Kerr, D. C. Look. “Enhanced p-type Conductivity of Nitrogen Doped ZnO by Nano/Micro Structured Rods and Zn-Rich Co-Doping Process”. Electron. Mater. Lett. 2011. 7(2): 115-119.

50. J. L. Lyons, A. Janotti, C.G. Van de Walle. “Why Nitrogen Cannot Lead to p-type Conductivity in ZnO”. Appl. Phys. Lett. 2009. 95(25): 252105-1-252105-3.

51. Y. R. Ryu, S. Zhu, D. C. Look, J. M. Wrobel, H. M. Jeong, H. W. White. “Synthesis of p- type ZnO Films”. J. Crystal. Growth. 2000. 216(1-4): 330-334.

52. F. J. Manjon, B. Mari, J. Serrano, A. H. Romero. “Silent Raman Modes in Zinc Oxide and Related Nitrides”. J. Appl. Phys. 2005. 97(5): 053516-1-053516-4.

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53. H. Chang, P. J. Huang, S. C. Hou. “Application of Thermo-Raman Spectroscopy to Study dehydration of CaSO4·2H2O and CaSO4·0.5H2O”. Mater. Chem. Phys. 58(1): 12-19.

54. P. J. Huang, H. Chang, C. T. Yeh, C. W. Tsai. “Phase Transformation of TiO2 Monitored by Thermo-Raman Spectroscopy with TGA/DTA”. Thermochim. Acta. 1997. 297(1-2): 85-92.

55. H. Chang, P. J. Huang. “Thermo-Raman Studies on Anatase and Rutile”. J. Raman Spectrosc. 1998. 29(2): 97-102.

56. P. J. Hendra. “Hot Samples in Raman Spectroscopy”. Internet J. Vib. Spec. 2001. 5(3): 4. http://www.ijvs.com/volume5/edition3/section1.html

57. E. S. Zouboulis, M. Grimsditch. “Raman Scattering in Diamond up to 1900 K”. Phys. Rev. B. 1991. 43(15): 12490-12493.

58. R. Bennett. “Applications of a Modulated Laser for FT-Raman Spectroscopy-1. Removal of a Thermal Background”. Spectrochim. Acta A. 1994. 50(11): 1813-1823.

59. K. F. McCarty. “Investigations of Materials at High Temperatures Using Raman Spectroscopy”. In: J. W. Hastie, editor. Materials at High Temperatures. Vol. 1. Clifton, NJ: Humana Press, 1990. Pp. 19-30.

60. P. Simon, B. Moulin, E. Buixaderas, N. Raimboux, E. Herault, B. Chazallon, H. Cattey, N. Magneron, J. Oswalt, D. Hocrelle. “High Temperatures and Raman Scattering Through Pulsed Spectroscopy and CCD Detection”. J. Raman Spectrosc. 2003. 34(7-8): 497-504.

61. G. J. Exarhos, W. S. Frydrych, G. E. Walrafen, M. Fisher, E. Pugh, S. H. Garofalini. In: R. J. H. Clark, D. A. Long, editors. Proceedings of the 11th International Conference on Raman Spectroscopy. 1988. Wiley. p. 503.

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62. L. Fayette, B. Marcus, M. Mermoux, N. Rosman, L. Abello, G. Lucazeau. “In situ Raman Spectroscopy During Diamond Growth in a Microwave Plasma”. J. Appl. Phys. 1994. 76(3): 1604-1606.

63. N. Rosman, L. Abello, J. P. Chabert, G. Verven, G. Lucazeau. “In situ Raman Characterization of a Diamond Film During its Growth Process in a Plasma Jet Chemical Vapor Deposition Reactor”. J. Appl. Phys. 1995. 78(1): 519-527.

64. M. Mermoux, B. Marcus, L. Abello, N. Rosman, G. Lucazeau. “In situ Raman monitoring of the Growth of CVD Diamond Films”. J. Raman Spectrosc. 2003. 34(7-8): 505-514.

65. J. You, G. Jiang, K. Xu. “High Temperature Raman Spectra of Sodium Disilicate Crystal, Glass and its Liquid”. J. Non-Cryst. Solids. 2001. 282(1): 125-131.

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Chapter 5

Raman Spectroscopic Detection for Process Control in the Bottling Industry

5.1 Abstract

5.1.1 Dispersive Raman Spectroscopy Poly(ethylene terephthalate) (PET) is commercially used in the production of bottles and other plastic containers. Preliminary analyses of PET bottles were performed using dispersive Raman spectroscopy with 633 nm laser excitation. An assessment of Raman intensity as a function of focal length indicated that the observed signal was throughput-dependent. Symmetric displacement profiles were observed as a result of the illumination and collection foci being coincident. Dispersive Raman spectra were collected using visible laser excitation; reduction of background fluorescence requires the use of laser excitation in the near-infrared (NIR).

5.1.2 Fourier Transform (FT-) Raman Spectroscopy Near-infrared (NIR) Fourier transform (FT-) Raman spectroscopy employing 1064 nm Nd:YAG laser excitation was evaluated for use in on-line determinations of PET bottle thickness. Preliminary determinations of the optical characteristics of the PET samples were conducted using NIR absorption spectroscopy. A calibration curve of the PET 1614 cm-1 Raman scattering intensity versus thickness possessed a linear R2 correlation of 0.9810. The effect of changes in laser power, number of scans, and spectral resolution on the uncertainty of the thickness calibration were determined. Displacement profiles representing changes in the position of the PET bottles with respect to the optimum sampling position were generated. The average full width at half maximum for the profiles was 0.4 ± 0.1 cm. This distance is comparable to the predicted displacement distances for PET bottles on a process line. Benefits, such as minimized fluorescence, higher laser powers, and increased throughput, indicated that NIR FT-Raman would be preferred for product control in the bottling industry.

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5.2 Introduction

5.2.1 Production of Plastic Bottles Plastic bottles and containers have superseded glass containers due to their light weight and increased durability. Poly(ethylene terephthalate) (PET) is debatably the most common thermoplastic polymer resin used in the production of plastic bottles and containers. Production methods of PET containers include extrusion blow molding and injection blow molding, but the most common production method is stretch blow molding. Stretch blow molding begins with the automated mixing of new PET pellets with recycled PET flakes reclaimed from previous production cycles. After mixing, the dry, solid mixture enters a plastic injection machine that heats the mixture to a liquid state. The hot, liquid PET is then injected into a mold at high pressure, resulting in the formation of a parison, or preform, capable of being transformed into a wide range of shapes and sizes. When cooled, the preform enters the stretch blow molder where it is reheated and formed to a desire shape. Stretch blow molding is performed through the insertion of a rod, or mandrel, into the preform. The mandrel stretches the preform lengthwise while simultaneously blowing in air at an extremely high pressure. During this process, the preform expands to take the shape of the mold. A thorough, technical review of stretch blow molding was published by Brandau.1

5.2.2 Current Methods of Thickness Determination Quality control is of significant interest during the production of plastic bottles and containers. An industrial manufacturer is capable of producing an average of 60,000 bottles per hour. At this rate of production, a small defect can result in significant losses in a short period of time. The amount of material contained in each section of the container is an important parameter of quality control. Measurements of base or sidewall mass have become accepted indicators of product quality. Current quality control and inspection methods include visual inspection using machine vision and absorption spectroscopy in the mid- and near-infrared. Determinations of sidewall and bottom thicknesses for plastic containers can be done using absorption techniques. Pressco Technology, Inc. currently offers an instrument known as the INTELLIMASS for the process control of PET bottles and containers.2 This electronic bottle sectioning system uses on-line, virtual sectioning to measure the material distribution of PET

179 bottles. Near-infrared absorbance is measured over a 50 mm diameter spot transmitted through both sides of the plastic bottle. The method averages attenuation effects associated with bottle shape and provides a more stable measure of process variability. The transmittance of NIR light through the bottle is divided by the signal obtained in the absence of the bottle. The output normalized transmittance (NT) can be related to mass through calibration of the instrument by sectioning of the bottle into the base, sidewall, and opening sections, each with a height on the order of the NIR beam width. Absorption measurements of the base are performed using a sensor that stares down the throat of the bottle and detects light transmitted from below.3-6 Although absorption and sectioning methods are widely accepted, the techniques possess several inherent disadvantages. Absorption measurements in the NIR, and even the mid- infrared, can be impacted by the presence of water on or in the sample as well as water vapor in the atmosphere. Sectioning is destructive of the sample and requires additional time for sample preparation. Another technique capable of overcoming these limitations is needed for fast, on- line determinations of PET bottle thickness.

5.3 Goals and Specific Aims Raman spectroscopy is a potential technique capable of being employed in the nondestructive determination of PET bottle thickness on a process line. The application of Raman spectroscopy for the qualitative or quantitative analyses of samples requires considerations of:7  resolution  signal-to-noise ratio  fluorescence  time Application of Raman spectroscopic detection as a method of product control in on-line determinations of sample thicknesses in the bottling industry has not been fully investigated. Raman spectroscopy has the potential for fast, reproducible measurements of bottle thickness. Backscattering geometries using dispersive Raman spectroscopy with visible laser excitation and FT-Raman with NIR laser excitation will be investigated. A determination of which sampling method/geometry is more applicable to on-line determinations of bottle thickness will be performed.

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5.4 Raman Spectroscopic Monitoring of Poly(ethylene terephthalate) Applications of Raman spectroscopy to the analysis of PET samples in moving and industrial environments have been performed. Hendra et al. employed Raman spectroscopy in the study of flowing PET melts extruded through a glass die.8 Swierenga et al. developed a universal calibration model for on-line and off-line predictions of PET yarn shrinkage using Raman spectroscopy.9 The calibration method developed by Swierenga et al. was capable of transferring a multivariate calibration model from an off-line to an on-line application without remeasuring the sample subset. Raman spectroscopy has become a popular method for process analysis. Adar, Geiger, and Noonan provided a review of Raman spectroscopy for process/quality control, including the aspects of instrumentation, chemometrics, and sampling applications.10 Process Raman spectroscopy focusing on industrial sampling applications, including studies of polymers, was discussed by Lewis.11 Raman spectroscopy has been employed in the study of polymeric materials.12 The principal disadvantage associated with the use of visible laser excitation in the Raman spectroscopic analysis of polymeric samples arises from fluorescence.13 Minimization of fluorescence inherent in many polymeric samples can be accomplished by shifting the laser excitation to the NIR.

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5.5 FT-Raman Spectroscopy of Poly(ethylene terephthalate) Polymers often contain additives, fillers, contaminants, or other impurities capable of causing fluorescence when exposed to visible wavelengths, such as those commonly used in dispersive Raman spectroscopy. Colorants are often added to PET bottles to provide an aesthetic design or to improve performance, such as extending the shelf life of a product. Absorption of light by the sample can be problematic when performing dispersive Raman with visible laser excitation since both the excitation and the Raman scattered light could be absorbed, resulting in decreased intensity. Colorant impurities in bottles can also cause fluorescence that can appear as a sloping baseline in the Raman spectrum. The development of FT-Raman was fueled by the desire to reduce the effect of sample fluorescence by shifting the excitation wavelength to the NIR. At these wavelengths, the laser excitation lacks sufficient energy to excite electronic transitions responsible for fluorescence. FT-Raman spectroscopy employing NIR laser excitation has been used in the analysis of polymers.14-17 Several major polymers, including PET, were studied by Florestan as an application of NIR FT-Raman to the automatic identification of recyclable plastics.18 FT-Raman was shown to provide a rapid, highly selective method for plastic identification. A study of density and orientation in PET using FT-Raman spectroscopy and multivariate data analysis was performed by Everall et al.19 Everall showed that the 1615 cm-1 ring stretching mode differentiated samples on a basis other than by density or orientation alone. Ellis et al. employed FT-Raman spectroscopy in the study of commercial PET with controlled thermal history as a function of the degree of extension.20 Results presented by Ellis revealed a series of previously unreported transitions associated with the trans- conformers introduced into the non-crystalline material when the polymer was drawn below the glass transition temperature. A review of FT- Raman pertaining to investigations of polymer crystallinity was presented by Stuart.21 PET was among the polymers studied by Brookes et al. at temperatures at, below, and above their melting points using FT-Raman with 1064 nm laser excitation.22 Brookes et al. showed that the PET 1615 cm-1 transition shifted in position as the temperature of the polymer increased, indicating a potential breakage of hydrogen bonds. A more recent publication concerning sampling considerations for the FT-Raman spectroscopic analysis of polymers and solutions was presented by Barrera and Sommer.23 Optimum sampling thicknesses for FT-Raman measurements were reported based on the NIR absorption spectra of the samples, including PET1.

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5.6 Experimental

5.6.1 Dispersive Raman Spectroscopy

5.6.1.1 Materials A clear (colorless), flat-walled PET beverage bottle was purchased at random from a local supermarket. The contents of the bottle were discarded and the bottle rinsed and dried prior to analysis.

5.6.1.2 Instrumentation Dispersive Raman spectra were collected using a Renishaw (Hoffman Estates, IL) inVia Raman microspectrometer. The optical design of the Raman microscope is illustrated in Fig. 5.1.

65 µm Slit 1800 l/mm Grating

CCD Sample Objective Lens 50 mm f.l. 150 mm f.l. 250 mm f.l. Pixel Size: 22 x 22 µm Figure 5.1: Optical illustration of the Renishaw inVia Raman microspectrometer.

The inVia Raman spectrometer was configured with an 1800 lines/mm diffraction grating and utilized a slit width of 65 µm. The instrument featured a high-sensitivity, ultra-low noise RenCam CCD thermoelectrically cooled to -70oC. A Leica Microsystems (Buffalo Grove, IL) DM 2500M Ren (RL/TL) microscope was coupled to the Raman spectrometer. Instrument control of the inVia system was done using Renishaw Windows-based Raman Environment (WiRE) software (version 3.0 [2007]). Excitation of the PET sample was done using the 633 nm line of a 35 mW JDS Uniphase (Santa Rosa, CA) helium-neon laser. The output laser power was measured using a Newport

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(Irvine, CA) Model 840-C hand-held optical power meter equipped with a model 818-ST silicon wand. The laser power at the sample did not exceed 11 mW. The laser excitation was focused on the sample using 25.4 mm-diameter Newport BK7 plano-convex lenses with focal lengths of 50-1000 mm. Raman spectra were collected in static scan mode with a central frequency of 1600 cm-1 shift using an exposure time of 3 seconds for a single accumulation. A photograph of the remote, dispersive Raman sampling geometry is shown in Fig. 5.2. Preliminary testing of the remote sampling configuration was done using a flat, front-surface mirror mounted at 45o on the microscope stage; this reflecting optic was later reconfigured to a custom-designed, slide-on attachment for the microscope. Coupling of the remote sampling accessory to the microscope required an adapter to convert the threading of the Leica microscope to the standard Royal Microscopical Society (RMS) threading.

Figure 5.2: Photograph of the remote, dispersive Raman configuration.

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5.6.1.3 Methods The sidewall thickness of the PET bottle was measured using a Brown & Sharpe (Hexagon Metrology, Inc., North Kingstown, RI) Digit-Cal MK IV digital caliper. The sidewall thickness of the bottle was 0.0439 cm. Displacement of the PET bottle from the optimum sampling positions of the 100, 150, and 200 mm focal length objective lenses was performed by mounting the bottle to a Velmex (Bloomfield, NY) Series A25 UniSlide translational stage. The PET bottle was laterally scanned through the focal plane of each lens in increments of 0.254 cm (0.10 in.).

5.6.2 Fourier Transform Raman Spectroscopy

5.6.2.1 Materials Pre-cut slides of clear, colorless PET were obtained from Pressco Technology, Inc. (Cleveland, OH). The thicknesses of the PET slides were 0.0254, 0.1016, and 0.1500 cm. A clear, flat-walled beverage bottle was purchased at random from a local supermarket. The contents of the bottle were discarded and the bottle rinsed and dried prior to analysis. The sidewall thickness of the bottle was 0.0439 cm.

5.6.2.2 Instrumentation NIR absorption spectra of the 0.0254, 0.0439, and 0.1046 cm thick PET samples were acquired using a PerkinElmer (Waltham, MA) System 2000 Fourier transform infrared (FT-IR) spectrometer configured for use in the NIR. In this configuration, light from a 50W tungsten halogen light source is directed through the sample compartment attached to the right side of the spectrometer. A quartz beamsplitter was used in the interferometer. The NIR optical path is used to align the instrument for FT-Raman measurements by locating the position of zero path difference. Collimation of the NIR light passing through the sample compartment was achieved by setting the aperture (B-) and field (J-) stops to 5.00 mm. Absorption spectra collected using the standard deuterated triglycine sulfate (DTGS) detector located within the macrobench represent the average of 32 individual scans at 4 cm-1 resolution. Three, repetitive collections

185 were obtained for each sample thicknesses. (Uncertainties in the resulting values represent the 95% confidence limit.) FT-Raman spectra were collected using a PerkinElmer NIR FT-Raman sample compartment coupled to the System 2000 FT-IR/FT-NIR spectrometer. An optical illustration of the FT-Raman sample compartment is shown in Fig. 5.3, and a photograph is shown in Fig. 5.4.

1064 nm Nd:YAG Laser Lens

Filters

Sample

Prism PerkinElmer System Collection lens InGaAs 2000 FT-IR detector optics

PerkinElmer System 2000 NIR FT-Raman J-stop

To interferometer

Collection lens MP1 MP2 MP12’ MT23 ME25 J-stop

Sample Detector

38 mm 260 mm 130 mm 140 mm 172 mm 170 mm 245 mm 35 mm

Figure 5.3: (Top) Optical drawing of the FT-Raman sample compartment interfaced to the System 2000 FT-IR. (Bottom) Diagram of the FT-Raman optical system. (MP1, MP2, and MP12’ are parabolic mirrors, MT23 is a toroidal mirror, and ME25 is an ellipsoidal mirror.)

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Figure 5.4: Photograph of the PerkinElmer System 2000 NIR FT-Raman sample compartment.

The optical design of the System 2000 FT-Raman spectrometer results in a magnification of 1 from the sample position to the detector. Excitation of the PET samples was done using the -1 1064 nm (9394.69 cm ) line of a 6 W TEM00 PerkinElmer Diode Pumped Nd:YAG laser (model # SNJ2000PE1.2). The Nd:YAG laser was manufactured by IE Optomech, Ltd. (Newnham, UK). The laser source was focused on the sample using a 100 mm focal length lens. The diameter of the laser at the sample position is on the order of 1 mm. In the System 2000 FT- Raman instrument, the foci of the excitation and collection lenses are not coincident. As a result, the laser beam at the sample is slightly divergent. Laser power at the sample did not exceed 500 mW. Raman spectra were collected using the standard f/0.6 aspheric collection lens in a 180o backscattering geometry. Filtering of the elastically-scattered light was accomplished using a series of longpass dielectric filters. A quartz beamsplitter was employed in the interferometer of the System 2000. The internal optics of the spectrometer were gold-coated to optimize reflection in the NIR. Raman spectra were collected at room temperature using an indium-gallium- arsenide (InGaAs) detector. Unless otherwise stated, FT-Raman spectra represent the average of 32 individual scans at 4 cm-1 resolution using an optical path difference (OPD) velocity of 0.1 cm/sec. Spectra were transformed using magnitude phase correction and a filler apodization function.

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Instrument control of the PerkinElmer System 2000 NIR/FT-Raman spectrometer was done using PerkinElmer Spectrum software (versions 4.07 [2000] and 5.3.1 [2005]). Analysis of the resulting spectra was done using Spectrum software (versions 6.3.1.1032 [2007] and 10.03.06 [2011]). Displacement of the PET samples from the optimum sampling position in the FT-Raman sample compartment was done using a Velmex Series A25 UniSlide translational stage. Positive and negative displacements of the samples were done in increments of 0.127 cm (0.05 in.). The concept of sample displacement is illustrated in Fig. 5.5.

Figure 5.5: Illustration of the displacement of the PET sample from the FT-Raman collection lens.

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5.6.2.3 Methods Poly(ethylene terephthalate) sample thicknesses were measures using a Brown & Sharpe Digit-Cal MK IV digital caliper. The NIR absorption spectrum of pure, distilled water was obtained by placing a drop of water between clear, glass microscope slides. The absorption spectra represent the average of 32 individual scans at 8 cm-1 resolution over the range of 10000-5000 cm-1 using an OPD velocity of 0.1 cm/sec. A calibration curve of intensity versus sample thickness was generated through successive stacking of the PET samples. The PET slides were held in close contact in order to form a continuous sample. Data used in generating the calibration represent the average of five collections at each thickness. The impacts of laser power, number of scans, and spectral resolution on quantitative determinations of PET thickness were performed using the 0.1500 cm-thick PET sample slide. Peak-to-peak variations in the baseline were determined over the range of 2500-2000 cm-1 shift. Five, repetitive collections were performed at the selected laser powers, number of scans, and spectral resolutions. (Uncertainties in the resulting values of p-p variation, 1614 cm-1 intensity, and signal-to-noise represent absolute error at 95% confidence.) The effect of sample displacement was investigated through the repetitive collection of three spectra at each sample position.

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5.7 Results and Discussion

5.7.1 Raman Spectrum of Poly(ethylene terephthalate) The Raman spectrum of PET has been previously reported8,18-19,24-28 and is shown in Fig. 5.6. The 1614 cm-1 transition was assigned to a ring stretching mode29-30, and the 1726 cm-1 transition was assigned to the carbonyl stretch of an ester. The 1614 cm-1 transition was selected for quantitation on the basis of its strong intensity.

Intensity

200 700 1200 1700 2200 2700 3200 Raman Shift (cm-1)

Figure 5.6: Raman spectrum of poly(ethylene terephthalate).

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5.7.2 Dispersive Raman Spectroscopy

5.7.2.1 Preliminary Considerations Raman spectroscopy is inherently a weak scattering process; therefore, it becomes necessary to collected as many Raman scattered photons as efficiently possible. It is for this reason that Raman spectroscopy is often performed using high numerical aperture (NA) refracting or reflecting optics. Although short focal length optics are beneficial in a laboratory setting, such optics may not be physically applicable to the short-range detection of bottle thickness on a process line. The effect of focal length on collection efficiency was studied with the resulting plot of intensity versus focal length is shown in Fig. 5.7. The intensity of the PET 1614 cm-1 transition decreased as the focal length of the objective/collection lens decreased according to a 1/x2 dependency, where x is the focal length of the lens.

10000

8000

6000

Intensity 4000

2000 y = 1E+07x-1.85 R² = 0.9973

0 0 200 400 600 800 1000 1200 Focal length (mm)

Figure 5.7: Intensity of the PET 1614 cm-1 transition as a function of focal length.

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Throughput, Φ, is defined as the product of the solid angle of collection, Ω, in steridians, and the area of the collection optic, A, in cm2. The collection throughput can be represented by Eq. 5.1: Φ = ΩA (5.1) where

Ω = 2π (1-cos θ) (5.2) and theta, θ, is the half-angle of collection. A plot of throughput versus lens focal length is shown in Fig. 5.8. The throughput curve possessed a 1/x2 relationship comparable to that observed in the intensity versus focal length plot (Fig. 5.7), indicating that Raman intensity was directly related to the collection throughput. A summary of the dispersive Raman collection parameters is listed in Table I.

0.35

0.30

) 0.25

∙ 2 0.20

0.15

Throughput (cm Throughput 0.10 y = 615.48x-1.94 0.05 R² = 0.9988

0.00 0 200 400 600 800 1000 1200 Focal length (mm)

Figure 5.8: Throughput as a function of focal length.

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TABLE I. Dispersive Raman Collection Parametersa

Lens Focal Length f/# NA Solid Angle Throughput b Intensity c % RSD d mm sr sr·cm2

100 5.2 0.10 0.029 0.085 3504 ± 51 2.0 150 7.8 0.06 0.013 0.038 1966 ± 54 3.8 200 10.4 0.05 0.007 0.020 1071 ± 24 3.1

a Effective aperture of collection lens: 19.28 mm

b Collection area of lens: 2.92 cm 2

c Average of 10 single acquisitions with 3 second exposure

d With respect to maximum intensities of single acquisitions

Since the lens objectives employed in the dispersive Raman study served to simultaneously illuminate the sample and collect the Raman scattered light, selection of appropriate short-range lenses was based on the criteria of obtaining a strong Raman signal while simultaneously maintaining an appreciable distance between the optic and the sample. A very strong intensity for the PET 1614 cm-1 transition was observed using the 50 mm focal length lens; however, the working distance of this optic was not practical for short-range monitoring in an industrial setting. Plano-convex lenses with focal lengths of 100, 150, and 200 mm were selected for use in the dispersive Raman study because strong transition intensities were observed for the PET bottle at sufficient working distances.

Table 5.1: Disperisve Raman Collection Parameters

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5.7.2.2 Dispersive Raman Spectra of PET at Short-range Distances Dispersive Raman spectra of the PET bottle collected using lenses with focal lengths of 100, 150, and 200 mm are shown in Fig. 5.9. The spectra indicate that signal-to-noise decreased as the focal length of the illumination/collection lens decreased.

3500 100 mm FL 3000

2500

2000

1500 Intensity 1000

500

0 1350 1450 1550 1650 1750 Raman shift (cm-1)

1600 150 mm FL 1400 1200 1000 800

Intensity 600 400 200 0 1350 1450 1550 1650 1750 Raman shift (cm-1)

1200 200 mm FL

1000

800

600 Intensity 400

200

0 1350 1450 1550 1650 1750 Raman shift (cm-1)

Figure 5.9: Raman spectra of PET collected using 100, 150, and 200 mm focal length lenses.

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Reproducible acquisition of Raman spectra from PET bottles on a process line is critical for quality control. The reproducibility associated with the collection of Raman spectra from the PET bottle was evaluated through the collection of ten, static acquisitions using each of the three objective lenses. The resulting plots of intensity versus scan number shown in Fig. 5.10 illustrate a high degree of reproducibility was obtained using the dispersive Raman backscattering geometry. Average intensities and percent relative standard deviations (% RSDs) for the static accumulations are presented in Table I. (The uncertainties in the intensities represent absolute error at 95% confidence for ten acquisitions.)

4000

3500

3000

2500

intensity

1 - 2000

1500

PET 1614cmPET 1000

500 100 mm FL 150 mm FL 200 mm FL

0 0 1 2 3 4 5 6 7 8 9 10 Scan number

Figure 5.10: Plots of intensity versus scan number for ten, static spectral acquisitions.

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5.7.2.3 Optical Considerations The diffraction-limited spot size of light focused to a point using a lens or objective can be determined according to:

(5.3)

where λ is the wavelength of light, θ is the half-angle of the laser at the focusing lens, and n is the refractive index in which the measurement is being performed. The diffraction-limited spot sizes of the 633 nm laser at the foci of the 100, 150, and 200 mm focal length lenses were 38.5, 58.2, and 77.6 µm, respectively. When translated to the position of the spectrometer entrance slit using the focal lengths of the pre-slit and objective lenses, the images sizes of the focused laser spot sizes at the sample were 19.4 µm; therefore, all Raman scattered light incident upon the 65 µm entrance slit will be transferred to the detector for a sample placed at the focal plane of each lens. The distance over which the diameter of the focused laser spot remains unchanged and the Raman intensity essentially constant is known as the depth of focus (DOF). The depth of focus, z, can be determined according to Eq. 5.4.

(5.4)

According to Eq. 5.4, the DOFs obtained using the 100, 150, and 200 mm focal length lenses/objectives were 6.3, 14.4, and 25.6 mm, respectively. A discussion of DOF as it pertains to the sampling conditions of Raman spectroscopy was published by Pelletier.31

196

5.7.2.4 Sample Displacement Profiles Displacement profiles generated by scanning the PET bottle through the focal planes of the selected objective lenses are shown in Fig. 5.11. The intensity of the PET 1614 cm-1 transition was monitored as a function of distance from the lens/objective focal plane.

5000

4000 100 mm FL 150 mm FL 3000 200 mm FL

Intensity 2000

1000

0 -30.00 -20.00 -10.00 0.00 10.00 20.00 30.00 Distance from focal plane (mm)

Figure 5.11: Displacement profiles of intensity versus distance from the focal plane.

The symmetric responses of the displacement profiles (Fig. 5.11) were attributed to the foci of the illumination and collection optic being coincident. The corresponding FWHMs of the 100, 150, and 200 mm profiles were 8.2 21.6, and 38.4 mm. The generated profiles illustrate that the Raman response became increasingly independent of distance as the focal length of the lens/objective increased for a constant laser power. Figure 5.11 also indicates that deviations in sample location relative to the optimum sampling position at the focal plane of the objective lenses resulted in a decrease in Raman intensity. When applied to a calibration of PET bottle thickness based on peak height, a diminution in intensity would make samples appear thinner than they actually are. Thickness errors resulting from sample displacement are expected to be greater when short focal length, or high NA, objective lenses are employed. For example, the intensity of the PET 1614 cm-1 transition decreased rapidly as the PET bottle was translated away from the focal plane of the 100 mm focal length lens. In this situation, small changes in sample position would correspond to increased error in sample thickness.

197

5.7.2.5 Raman Intensity Models The intensity variation along the z-axis located in close proximity to the image plane of a point object positioned at a normalized distance u from the focal plane of the objective is given by:32

( ) ( ) | | (5.5) ( ) where

( ) (5.6)

In Eq. 5.6, λ is the excitation wavelength, z is the displacement distance, and α is the maximum collection angle of the objective. Theoretical plots of intensity versus displacement distance were constructed for the 100, 150, and 200 mm focal length objective lenses using Eq. 5.5. The resulting plots are shown in Fig. 5.12.

1.2

1.0

0.8 100 mm FL 150 mm FL 200 mm FL

0.6 Intensity

0.4

0.2

0.0 -1000 -750 -500 -250 0 250 500 750 1000 Distance (µm)

Figure 5.12: Intensity versus distance models for a conventional Raman microscope.

198

The intensity-distance models (Fig. 5.12) are comparable to the experimental displacement profiles presented in Fig. 5.11. Broadening of the intensity-distance profiles occurs as the focal length of the objective/collection lens increases. When applied to a confocal Raman sampling configuration, the intensity variation is dependent upon both the optical properties of the objective and the pre-slit/aperture lenses. In this configuration, Eq. 5.5 is modified to:32

( ) ( ) | | (5.7) ( )

The resulting confocal models are shown in Fig. 5.13.

1.2

1.0

100 mm FL 0.8 150 mm FL 200 mm FL

0.6 Intensity

0.4

0.2

0.0 -1000 -750 -500 -250 0 250 500 750 1000 Distance (µm)

Figure 5.13: Intensity versus distance models for a confocal Raman microscope.

The flux of inelastically scattered light transmitted through a variable aperture (ФT), i.e., the Raman intensity, can be determined using Eq. 5.8:32

(5.8)

[ ( { √ ( ( ) ) })]

where D is the aperture diameter, or in the present situation, the entrance slit width of 65 µm, Γo is the total magnification of the sample image at the aperture/slit, NA is the numerical aperture of the objective lens, and Do is the diffraction-limited laser spot size at the sample as determined using Eq. 5.3. The Raman intensity transmitted through the aperture/slit was plotted as a

199 function of the displacement distance using Eq. 5.8, and the resulting curves for the three objective lenses are shown in Fig. 5.14.

1.2

1.0 100 mm FL 150 mm FL 200 mm FL 0.8

0.6

0.4 Normalized Raman intensity Raman Normalized 0.2

0.0 -12.5 -10.0 -7.5 -5.0 -2.5 0.0 2.5 5.0 7.5 10.0 12.5 Distance (µm)

Figure 5.14: Theoretical model of normalized Raman intensity versus distance for objective lenses possessing focal lengths of 100, 150, and 200 mm.

The Raman intensity-distance curves presented in Fig. 5.14 were normalized to the maximum intensity determined for the 100 mm focal length lens at zero displacement. The normalized intensities of the 150 and 200 mm FL lens curves were respectively 44% and 25% the theoretical intensity predicted for the 100 mm focal length lens. These values were determined to be similar to the experimental results based on a comparison of the maximum intensities for the displacement profiles in Fig. 5.11. A comparison of the experimental displacement profiles (Fig. 5.11) revealed that the maximum Raman intensity observed using the 150 mm focal length objective lens was 44% of the maximum value obtained using the 100 mm focal length lens. Similarly, the observed maximum intensity of the 200 mm focal length lens profile was 29% of that for the 100 mm focal length lens.

200

5.7.3 FT-Raman Spectroscopy

5.7.3.1 Preliminary Considerations Optimization of the Raman signal requires both the excitation wavelength and the emitted Raman transitions be in regions where the sample itself possesses no absorptions. Application of NIR FT-Raman spectroscopy to the quantitative analysis of bottle thickness requires an investigation of the NIR absorption characteristics of PET. The NIR absorption spectrum of PET has been previously reported33 and is illustrated in Fig. 5.15 along with that of water.34-35

9395 cm-1 7781 cm-1 (1064 nm Nd:YAG) (1614 cm-1 shift)

PET

Water Absorbance

10000 9000 8000 7000 6000 5000 Wavenumber (cm-1)

Figure 5.15: Near-infrared absorption spectra of PET and water. The dotted lines are the absolute wavenumbers of the 1064 nm Nd:YAG laser and the 1614 cm-1 transition of PET.

The 6017 cm-1 (1662 nm) absorption of PET was assigned to the first overtone of the aromatic C-H stretching vibration. A 5798 cm-1 (1725 nm) absorption corresponded to the first overtone of an aliphatic C-H stretch. Absorptions at 5247 (1906 nm) and 5122 cm-1 (1952 nm) were assigned to the second overtones of the ester linkages.33-34 Water poses a potential problem in the acquisition of NIR FT-Raman spectra due to absorptions within the spectral region of interest. Absorption of the NIR laser excitation or the resulting Raman scattered light by water

201 can reduce the intensity of a Raman transition used for quantitative analysis, resulting in significant errors in sample thickness. A 5170 cm-1 (1934 nm) absorption in the spectrum of water results from a combination of the asymmetric stretching and bending modes of water. The broad 6800 cm-1 (1471 nm) absorption was assigned to a combination of symmetric and asymmetric stretching modes. The locations of the 1064 nm (9395 cm-1) Nd:YAG laser wavelength and the resulting 1614 cm-1 shift (7781 cm-1) transition of PET are indicated in Fig. 5.15. The absence of appreciable absorption at these wavelengths indicates that FT-Raman employing 1064 nm NIR laser excitation is applicable to the quantitative analysis of PET bottle thickness.

5.7.3.2 Optical and Sample Considerations The two most common configurations employed in the collection of FT-Raman spectra are: (1) right angle scattering at 90o and (2) backscattering at 180o. Due to the ease of implementation, the backscattering geometry would be preferred for use in on-line monitoring of PET bottle thickness. Fundamentally, two processes are involved in the collection of Raman spectra: (1) excitation of the sample and (2) collection and detection of the resulting Raman scattered light. Under the condition that the FT-Raman sampling geometry is required to be highly efficient due to a reduction in scattering intensity resulting from the use NIR laser excitation, it becomes important to consider the optical properties of the sample, including the characteristics of absorption, inelastic (Raman) scattering, and elastic scattering (Rayleigh). The optical properties of pigments were described according to absorption and Raman scattering parameters by Kubelka and Munk.36-37 Schrader and Bergmann later extended the theory for the purpose of describing the Raman scattering process in crystalline powders.38 The study by Schrader and Bergmann showed that the theory could be applied to liquid and transparent films. The extended Raman scattering theory was applied to sampling geometries in FT-Raman by Schrader.39 The relevant optical properties of the sample include the linear Napierian absorption coefficient (α), the linear Napierian scattering coefficient (r), and the linear Napierian Raman scattering coefficient (s). Equations representing Raman scattering intensities obtained using the different scattering geometries were reported by Schrader as part of the extended theory associated with 39-41 FT-Raman. The Raman backscattering intensity, JR, can be represented by:

202

( ) (5.9) [( ) ] where

√ (5.10)

The absorption coefficient represents the arithmetic mean of the coefficients related to excitation and Raman scattered light. The Raman backscattering intensity expressed in Eq. 5.9 is also a function of sample thickness, d. The intensity of the laser excitation is represented by Io. Figure 5.16, adapted from the works of Schrader and Bergmann38 and Barrera and Sommer23, illustrate plots of the Raman backscattering intensity as a function of sample thickness for various values of r and α.

203

PET bottle thickness (a) r = 0

r =10

ntensity i

r = 100 scattering

r = 1000 Raman Raman

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 Sample thickness (cm)

α = 0.1 PET bottle thickness (b)

α = 1

ntensity

i scattering

Raman Raman α = 10 α = 100

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 Sample thickness (cm)

Figure 5.16: (a) Raman scattering intensity versus sample thickness for various scattering coefficients ranging from r = 0 to r = 1000 cm-1 with α = 3 cm-1 and s = 1 cm-1. (b) Raman scattering intensity versus sample thickness for various absorption coefficients ranging from α = 0.1 cm-1 to α = 100 cm-1 with r = 0 and s = 1 cm-1.

The Raman scattering intensity versus sample thickness plots (Fig. 5.16a) were generated for α = 3 cm-1 and s = 1 cm-1. The linear absorption coefficient (α) selected is representative of the PET samples. Liquids and solutions, including polymers, possess an elastic scattering coefficient of zero. Coarse, medium, and fine powders have elastic scattering coefficients on the order of 10, 100, and 1000 cm-1, respectively. The plots presented in Fig. 5.16a indicate that the

204

Raman backscattering intensity quickly becomes independent of sample thickness for increasing values of r. Plots of Raman scattering intensity versus sample thickness for different absorption coefficients (α) are shown in Fig. 5.16b. The curves shown Fig. 5.16b were generated using r = 0 and s = 1 cm-1. A significant reduction in Raman scattering intensity is observed as the absorption coefficient increases. The r = 0, α = 3 cm-1 plot in Fig. 5.16b is representative of the PET samples studied using FT-Raman. The average sidewall thickness of a PET bottle (0.04 cm) is indicated in the plots shown in Fig. 5.16. Over the range of 0-0.10 cm, the Raman intensity increases linearly as the thickness increases. Increased collection throughput was obtained in the System 2000 FT-Raman as a result of the large-diameter collection optic. The diameter and focal length of the aspheric collection lens are 63 and 38 mm, respectively. The resulting solid angle of collection calculated using Eq. 5.2 is 1.43 sr. The resulting throughput of 43.9 cm2∙sr is 516× greater than that obtained using the 100 mm focal length collection lens in the dispersive Raman study. Although this improvement is substantial, the short focal length collection optic is not practical for use in on- line determinations of bottle thickness. The application of Raman spectroscopic detection in the bottling industry requires the use of collection optics with extended working distances. The use of a large-diameter collection optic with a moderate focal length simultaneously maintains a high throughput while providing working distance between the sample and the lens.

5.7.3.3 Near-Infrared Characterization of PET Near-infrared absorption characteristics for PET samples with thicknesses of 0.0254, 0.0439, and 0.1016 cm are summarized in Table II. The apparent absorption coefficient (a), also referred to as the linear decadic absorption coefficient, was determined using the absorbance values of the PET samples at 7781 cm-1 and their respective thicknesses. Apparent absorption coefficients plotted as function of sample thickness are shown in Fig. 5.17.

205

Table 5.2: Near-Infrared Sample Characteristics

TABLE II. Near-Infrared Sample Characteristicsa,b

Sample Thickness Apparent Absorbance Apparent Absorption Coefficient, a Linear Napierian Absorption Coefficient, αc cm cm-1 cm-1

0.0254 0.05 ± 0.01 1.9 ± 0.1 4.2 ± 0.3 0.0439 0.06 ± 0.01 1.3 ± 0.1 2.9 ± 0.1 0.1016 0.04 ± 0.01 0.4 ± 0.1 0.8 ± 0.1

a Absolute wavenumber of 1614 cm- 1 vibration: 7781 cm-1

b Average of three collections consisting of 32 individual scans at 8 cm -1 resolution for each sample thickness

c α = 2.303 a

2.0

) 1

- 1.6 (cm

y = -1.08ln(x) - 2.10 1.2 R² = 0.9999

0.8 absorption absorption coefficient

0.4 Apparent Apparent

0.0 0.00 0.02 0.04 0.06 0.08 0.10 0.12 Sample thickness (cm)

Figure 5.17: Apparent absorption coefficient (a) versus sample thickness.

The natural logarithmic equation representing the relation between the apparent absorption coefficient and sample thickness was similar to an equation previously presented by Barrera and Sommer relating the apparent absorption coefficient to the optimal sample thickness.23 The Napierian absorption coefficient is related to the apparent absorption coefficient according to Eq. 5.11.

(5.11)

For example, an apparent absorption coefficient of 1.4 cm-1 was determined for a 0.0439 cm thick PET sample possessing an apparent absorbance of 0.06 at 7781 cm-1. A resulting linear Napierian absorption coefficient of 3.2 cm-1 is obtained when this value is input into Eq. 5.11. These considerations indicate that PET does not exhibit any appreciable absorptions within the spectral region of interest and that the correlation between Raman backscattering intensity and sample thickness is linear over the ranges of sample thicknesses to be studied.

207

5.7.3.4 PET Thickness Calibration A calibration of intensity versus sample thickness constructed using the intensity of the PET 1614 cm-1 transition is shown in Fig. 5.18. The calibration possesses a linear correlation up to a thickness of 0.23 cm. The intensity of the Raman transition starts to become independent of sample thickness for thicknesses greater than 0.23 cm. The thickness of a PET beverage bottle (0.04 cm) is indicated in Fig. 5.18. Data obtained for the FT-Raman study of PET thickness are listed in Table III.

PET bottle thickness 30

20 y = 90.76x + 2.63 R² = 0.9810

15 Intensity

0 0.00 0.05 0.10 0.15 0.20 0.25 0.30 Thickness (cm)

Figure 5.18: Calibration curve of intensity versus thickness for PET.

An evaluation of the variance in peak height for five repetitive collections at each thickness was performed in terms of % RSD. The results of these determinations are listed in Table III. The average % RSD in determining the intensity of the PET 1614 cm-1 transition at each thickness was found to be 1.1%.

208

Table 5.3: Summary of FT-Raman Results for PET: Sample Thickness

TABLE III. Summary of FT-Raman Results for PET: Sample Thicknessa

Thickness 1614 cm-1 Intensity % RSD b p-p Variation Signal-to-Noise Ratioc % RSDd cm

0.0254 5.3 ± 0.1 1.6 0.16 ± 0.02 167 ± 29 13.6 0.0381 6.4 ± 0.1 0.9 0.18 ± 0.04 178 ± 29 13.1 0.1016 10.4 ± 0.1 1.1 0.23 ± 0.04 233 ± 42 14.8 0.1270 14.3 ± 0.1 0.6 0.20 ± 0.04 353 ± 50 11.3 0.1500 16.9 ± 0.3 1.3 0.20 ± 0.02 438 ± 63 11.5 0.1754 18.7 ± 0.3 1.2 0.18 ± 0.01 507 ± 38 6.1 0.2516 23.7 ± 0.4 1.2 0.25 ± 0.02 473 ± 61 9.0 0.2770 24.6 ± 0.3 0.9 0.22 ± 0.01 561 ± 29 4.0

a 32 scans at 4 cm-1 resolution using 500 mW laser power; 5 repetitive scans at each thickness

b With respect to intensity

c rms noise calculated as one -fifth p-p variation over 2500 -2000 cm-1 shift

d With respect to signal-to -noise

5.7.3.5 Minimum Thickness Variation A determination of the minimum thickness variation detected for FT-Raman spectra representing the average of 32 individual scans at 4 cm-1 resolution with laser power of 500 mW was done using the intensity-thickness calibration presented in Fig. 5.15. Uncertainties in 42 thickness (sx) were calculated using Eq. 5.12.

( ̅) √ (5.12)

| | ∑( ̅)

In Eq. 5.12, sy is the standard deviation, or error, in y, m is the slope of the calibration curve, k, is the number of replicate measurements of the sample, n is the number of data points for the calibration line, ̅ is the mean intensity value for the points on the calibration line, xi are the individual sample thicknesses for the points on the calibration line, and ̅ is the mean thickness for the points on the calibration line. The average uncertainty in sample thickness determined from the eight calibration thicknesses was 0.012 ± 0.001 cm.

210

5.7.3.6 Sampling Precision The reproducibility associated with the collection of FT-Raman spectra from a PET sample was evaluated by collecting a series of ten spectra, each representing the average of 32 individual scans at 4 cm-1 resolution with a laser power of 500 mW. The 0.1500 cm thick PET slide was selected for this evaluation. The resulting plot of intensity versus scan number is shown in Fig. 5.19.

17.4

17.2

intensity

1 - 17.0

PET 1614cmPET 16.8

16.6 0 1 2 3 4 5 6 7 8 9 10 Scan number

Figure 5.19: Plot of intensity versus scan number for ten, repetitive scans.

The average 1614 cm-1 intensity was 17.0 ± 0.1, and the % RSD of these measurements was 0.5%; the % RSD obtained using the FT-Raman was significantly less than the corresponding values determined using the dispersive Raman instrument. Reproducible determinations of Raman intensity correlates to increased precision in determinations of bottle thickness performed on the basis of peak height.

211

5.7.3.7 Effect of Laser Power on Thickness Determinations Raman spectra collected using laser powers of 50, 100, 150, 200, 300, and 500 mW are shown in Fig. 5.20a. A plot of intensity as a function of laser power is shown in Fig. 5.20b.

(a)

500 mW

Intensity 300 mW

200 mW

150 mW 100 mW 50 mW 200 400 600 800 1000 1200 1400 1600 1800 2000 Raman shift (cm-1)

20 (b) 16 y = 0.04x - 0.32 R² = 0.9998 12

Intensity 8

0 0 50 100 150 200 250 300 350 400 450 500 550 1064 nm Nd:YAG laser power (mW)

Figure 5.20: (a) FT-Raman spectra of PET using different laser powers. (b) Plot of 1614 cm-1 intensity versus laser power.

212

Data obtained from the quantitative analysis of the PET sample at different laser powers are presented in Table IV. Peak-to-peak (p-p) variations were constant at each laser power. Unlike dispersive Raman spectroscopy in which shot noise is dominant, FT-Raman is detector noise limited; therefore, increasing or decreasing the laser power has no appreciably impact on the resulting noise. The effect of changes in laser power on the uncertainty in the minimum detectable thickness variation was performed on the basis of variations (standard deviations) in the PET 1614 cm-1 transition intensity at different laser powers. The corresponding % RSDs are listed in Table IV. With the exception of data collected using a 300 mW laser power, variations in peak height decreased as the laser power increased. A 2.7% RSD observed at 50 mW was representative of significant deviations in intensity for successive determinations. In contrast, a 0.3% RSD determined using a laser power of 500mW represented an appreciable reduction in spectral variance between successive determinations. According to Eq. 5.12, large standard deviations in intensity (sy), such as those observed at low laser powers, would result in increased uncertainty in the minimum detectable thickness variation. Data have shown that intensity variations can be abated through the use of high-power laser excitation. For example, based on the corresponding % RSDs, a 9× improvement in uncertainty would be expected using a 500 mW laser excitation compared to that obtained using a 50 mW laser power.

213

Table 5.4: Summary of FT-Raman Results for PET: Laser Power

TABLE IV. Summary of FT-Raman Results for PET: Laser Powera

Nd:YAG Laser Power 1614 cm-1 Intensity % RSD b p-p Variation Signal-to-Noise Ratioc % RSD d mW

50 1.3 ± 0.1 2.7 0.15 ± 0.01 46 ± 5 8.8 100 3.2 ± 0.1 1.6 0.16 ± 0.02 101 ± 15 11.6 150 5.0 ± 0.1 1.0 0.15 ± 0.01 162 ± 9 4.3 200 6.7 ± 0.1 0.8 0.16 ± 0.01 213 ± 24 9.1 300 10.3 ± 0.2 1.2 0.18 ± 0.01 287 ± 21 5.9 500 17.1 ± 0.1 0.3 0.21 ± 0.04 423 ± 76 14.4

a 32 scans at 4 cm-1 resolution; 5 repetitive scans at each laser power

b With respect to intensity

c rms noise calculated as one -fifth p-p variation over 2500-2000 cm -1 shift

d With respect to signal-to -noise

On-line, quantitative determinations of bottle thickness require spectral acquisitions with high SNRs. The most effective way of accomplishing this would be to increase the incident laser power at the sample. However, consideration must be given to the laser power density at the focus of the illumination optic. If the same objective lenses previously used in the preliminary, dispersive Raman investigation were to be applied to FT-Raman, the diffraction-limited spot size (Eq. 5.3) of the NIR laser at the sample will inherently be greater than that obtained using visible laser excitation. The larger spot size reduces the laser power density and prevents destruction of the sample due to heating. In order to obtain Raman spectra possessing high SNRs, on-line determinations of bottle thickness should be performed using as high of a NIR laser power as the sample can safely tolerate. For comparative purposes, the remaining investigations will be performed with a laser power of 500 mW.

5.7.3.8 Effect of Number of Scans on Thickness Determinations In FT-based instruments, increased signal-to-noise can be gained by averaging greater numbers of scans. FT-Raman spectra representing the averages of 1-128 individual scans at 4 cm-1 resolution are shown in Fig. 5.21a. A plot of the PET 1614 cm-1 intensity versus number of scans is shown in Fig. 5.21b. An evaluation of the resulting data indicated that the observed Raman intensity of the 1614 cm-1 transition was essentially independent of the number of scans averaged. Results from the FT-Raman study of PET with respect to the number of scans are shown in Table V. Data have shown that a reduction in noise was obtained by increasing the number of averaged scans. For constant intensities, increases in SNRs were observed as the number of averaged scans increased, thereby allowing for increased accuracy in thickness determinations.

215

(a) 128 scans

64 scans

32 scans

16 scans

Intensity 8 scans

4 scans

2 scans

1 scan 200 400 600 800 1000 1200 1400 1600 1800 2000 Raman shift (cm-1)

20 (b)

16 y = 0.01x + 16.31

R² = 0.9615 Intensity

12 0 16 32 48 64 80 96 112 128 144 Number of scans

Figure 5.21: (a) FT-Raman spectra of PET using different numbers of scans. (b) Plot of 1614 cm-1 intensity versus number of scans.

216

Table 5.5: Summary of FT-Raman Results for PET: Number of Scans

TABLE V. Summary of FT-Raman Results for PET: Number of Scansa

Number of Scans 1614 cm-1 Intensity % RSD b p-p Variation Signal-to-Noise Ratioc % RSD d

1 16.2 ± 0.3 1.4 0.98 ± 0.16 84 ± 11 12.9 2 16.3 ± 0.3 1.3 0.54 ± 0.02 152 ± 9 4.9 4 16.3 ± 0.1 0.3 0.48 ± 0.05 171 ± 17 8.4 8 16.3 ± 0.1 0.7 0.34 ± 0.09 251 ± 53 17.0 16 16.4 ± 0.2 0.9 0.23 ± 0.04 355 ± 56 12.5 32 16.5 ± 0.1 0.3 0.18 ± 0.01 449 ± 27 5.0 64 16.6 ± 0.1 0.4 0.15 ± 0.01 555 ± 40 5.7 128 16.9 ± 0.3 1.3 0.13 ± 0.01 672 ± 76 9.1

a 500 mW laser power; 4 cm-1 spectral resolution; 5 repetitive scans at each number of scans

b With respect to intensity

c rms noise calculated as one-fifth p-p variation over 2500 -2000 cm-1 shift

d With respect to signal -to-noise

Data presented in Table V indicate that the PET 1614 cm-1 intensity was essentially independent of the number of averaged scans. An assessment of the variation in the intensity of the PET 1614 cm-1 transition with changes in the number of averages scans was performed with respect to % RSD. The corresponding % RSD values listed in Table V indicate that increased variation in intensity occurred for spectra consisting of a single scan or two averaged scans. According to Eq. 5.12, % RSDs of 1.4 and 1.3% would correspond to increased uncertainties in the minimum detectable thickness variation based on a constructed intensity-thickness calibration. A reduction in peak height variation, and by extension, thickness uncertainty, occurred for spectra consisting of a minimum of four averaged scans. Although variations in peak height do not appreciably change between 4 and 64 averaged scans, collection time increases as the number of scans increases.

5.7.3.9 Effect of Spectral Resolution on Thickness Determinations Despite the benefits of decreased spectral variance and increased signal-to-noise, extended collection times, especially at high spectral resolution, are not applicable for on-line determinations of bottle thickness. One potential solution may result from changes in spectral resolution. FT-Raman spectra representing the average of 32 individual scans at spectral resolutions of 4, 8, 16, 32, and 64 cm-1 are shown in Fig. 5.22a. A correlation between Raman intensity and spectral resolution is shown in Figure 5.22b.

218

(a)

4 cm-1

8 cm-1 Intensity

16 cm-1

32 cm-1 64 cm-1 200 400 600 800 1000 1200 1400 1600 1800 2000 Raman shift (cm-1)

16 y = -11.85x + 23.89 R² = 0.9929 12

Intensity 8

4 (b)

0 0.50 0.75 1.00 1.25 1.50 1.75 2.00 Log [spectral resolution (cm-1)]

Figure 5.22: (a) FT-Raman spectra of PET using different spectral resolutions. (b) Plot of 1614 cm-1 intensity versus log (spectral resolution).

Spectra shown in Fig. 5.22a exhibit a decrease in intensity as the spectral resolution decreases. Broadening of the Raman transitions occurred as the spectral resolution decreased. Data presented Fig. 5.22b indicates that the intensity of the PET 1614 cm-1 transition decreased logarithmically as the spectral resolution decreased. The natural linewidth of a sample, typically 4-16 cm-1, often limits the spectral resolution associated with dispersive Raman experiments. An evaluation of the spectral resolutions

219 employed in the FT-Raman study of PET bottle thickness was performed on the basis of the FWHMs of the PET 1614 cm-1 transition for the spectra presented in Fig. 5.22a. The FWHMs of the PET 1614 cm-1 transition observed in the high-resolution 4 and 8 cm-1 Raman spectra were 12 and 15 cm-1, respectively. The similarity between the FWHM values at these spectral resolutions indicates that the natural linewidth of the sample dictates the spectral resolution at 4 and 8 cm-1. In contrast, the FWHM of the 1614 cm-1 transition increases to 23, 42, and 88 cm-1 as the spectral resolution is reduced to 16, 32, and 64 cm-1, respectively. The spectral resolution is dictated by the instrument at 16, 32, and 64 cm-1. A summary of the FT-Raman study of PET with changes in spectral resolution is shown in Table VI. Reductions in collection time were obtained at the expense of intensity. Assessment of variations in intensity was done in terms of % RSD for five, repetitive collections at each spectral resolution. The resulting % RSD values are listed in Table VI. In addition to extended collection times, increased intensity variations were observed at spectral resolutions of 4 and 8 cm-1. The large % RSDs observed at these spectral resolutions would give rise to increased uncertainty in minimum detectable thickness variations based on a constructed intensity-thickness calibration curve. Normalization of the % RSD values to that obtained using a 4 cm-1 spectral resolution indicated a 43% reduction in the uncertainty would be obtained by decreasing the spectral resolution to 16 cm-1. Despite reductions in collection time, increased variations in the intensity of the PET 1614 cm-1 transition were observed at 32 and 64 cm-1 resolutions. Peak-to peak variations listed in Table VI indicate that a reduction in sampled noise occurred as the spectral resolution decreased. Signal-to-noise remained appreciable despite a diminution in intensity as the spectral resolution was decreased. A reduction of the spectral resolution to 16 cm-1 provided a shorter collection time while simultaneously maintaining appreciable signal intensity and signal-to-noise. Quantitative determinations of bottle thickness performed on the basis of peak intensity for Raman spectra collected at 16 cm-1 resolution are possible since the 1614 and 1730 cm-1 transitions remain distinctively separate.

220

Table 5.6: Summary of FT-Raman Results for PET: Spectral Resolution

TABLE VI. Summary of FT-Raman Results for PET: Spectral Resolutiona

Resolution 1614 cm-1 Intensity % RSD b p-p Variation Signal-to-Noise Ratioc % RSD d cm-1

4 16.9 ± 0.1 0.7 0.18 ± 0.01 481 ± 35 5.7 8 13.4 ± 0.1 0.4 0.15 ± 0.01 454 ± 39 8.7 16 9.2 ± 0.1 0.3 0.11 ± 0.02 441 ± 84 15.4 32 5.5 ± 0.1 0.5 0.06 ± 0.01 511 ± 122 19.3 64 3.1 ± 0.1 0.7 0.04 ± 0.01 355 ± 98 22.1

a 32 scans with 500 mW laser power; 5 repetitive scans at each spectral resolution

b With respect to signal-to- noise

c rms noise calculated as one -fifth p-p variation over 2500 -2000 cm-1 shift

d With respect to signal-to- noise

5.7.3.10 Sample Displacement Profiles While on a process line, the positions of the bottles or the location of the focused laser spot on the sample may change. An evaluation of sample displacement and its effect on peak height and uncertainty was performed by scanning three PET samples with thicknesses of 0.0254, 0.1016, and 0.1500 cm through the optimum FT-Raman sampling position. The resulting displacement profiles are shown in Fig. 5.23.

12 0.1500 cm 0.1016 cm 9

0.0254 cm Intensity

0 -1.50 -1.00 -0.50 0.00 0.50 1.00 1.50 Displacement distance (cm)

Figure 5.23: FT-Raman displacement profiles for PET samples with thicknesses of 0.0254, 0.1016, and 0.1500 cm generated using the 1614 cm-1 intensity.

The profiles shown in Fig. 5.23 exhibit a high degree of asymmetry with respect to the optimal sampling position resulting from the foci of the illumination and collection optics not being coincident. The intensity of the PET 1614 cm-1 transition decreased rapidly on either side of the optimum sampling position. This characteristic is comparable to that previously observed in the sample displacement profiles obtained using dispersive Raman with 633 nm excitation. Respective FWHMs of 0.47, 0.39, and 0.42 cm for the 0.0254, 0.1016, and 0.1500 cm thick PET samples indicated that the displacement distance was independent of sample thickness at 50% of the maximum intensity. The determined values also indicate that a PET bottle would be capable of moving ± 2 mm before the scattering intensity decreases to 50% of the maximum value. This tolerance is comparable to the potential displacement of a bottle on a process line.

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Summaries of the FT-Raman results for the three PET samples are listed in Tables VII, VIII, and IX. Data presented in these tables show that the relative uncertainty in the intensity of the PET 1614 cm-1 transition increased as the displacement distance increased. For example, a 14× increase in uncertainty was observed when the 0.0254 cm thick sample was translated -1.016 cm from the optimum sampling position. The increased uncertainty in peak height observed at greater displacement distances would ultimately correspond to increased uncertainties in bottle thickness. Further comparison of the % RSD vales listed in Tables VII, VIII, and IX reveals a decrease in peak height uncertainty occurred as the sample thickness increased. The averages of the % RSDs in the intensity of the PET 1614 cm-1 transition over the distances moved were 4.4, 2.6, and 1.5% for the 0.0254, 0.1016, and 0.1500 cm thick PET samples, respectively. It has been shown that FT-Raman measurements are detector noise limited; therefore, decreases in peak height with constant noise resulted in decreased signal-to-noise as the samples were moved away from the optimum sampling position. The resulting SNRs and the corresponding % RSDs for three, repetitive collections at each distance are shown in Tables VII, VIII, and IX.

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Table 5.7: Summary of FT-Raman Results for PET: Sample Displacement (Thickness: 0.0254 cm)

TABLE VII. Summary of FT-Raman Results for PET: Sample Displacementa,b

Thickness: 0.0254 cm

Distance Moved 1614 cm-1 Intensity % RSD c Signal-to-Noise Ratiod % RSD e cm

-1.016 0.5 ± 0.2 15.2 12 ± 2 7.6 -0.889 0.8 ± 0.2 5.0 19 ± 5 9.3 -0.762 1.2 ± 0.1 1.0 32 ± 10 13.4 -0.635 1.7 ± 0.1 2.0 40 ± 15 14.6 -0.508 2.2 ± 0.1 1.4 63 ± 25 16.5 -0.381 2.1 ± 0.2 2.9 55 ± 20 14.6 -0.254 2.1 ± 0.3 5.6 51 ± 17 12.8 -0.127 2.8 ± 0.1 1.3 68 ± 30 17.9 0.000 4.3 ± 0.1 0.9 104 ± 30 11.5 0.127 4.1 ± 0.3 3.1 107 ± 25 9.7 0.254 1.8 ± 0.2 3.9 44 ± 2 2.8 0.381 0.9 ± 0.1 3.7 20 ± 2 5.9 0.508 0.7 ± 0.1 2.7 15 ± 2 7.5 0.635 0.5 ± 0.1 5.8 13 ± 7 23.0 0.762 0.4 ± 0.1 10.8 10 ± 2 4.3

a 16 scans at 4 cm-1 resolution with 500 mW laser power

b Averages of three collections at each sample displacement

c With respect to intensity

d rms noise calculated as one -fifth p-p variation over 2000-2500 cm -1 shift

e With respect to signal-to- noise

Table 5.8: Summary of FT-Raman Results for PET: Sample Displacement (Thickness: 0.1016 cm)

TABLE VIII. Summary of FT-Raman Results for PET: Sample Displacementa,b

Thickness: 0.1016 cm

Distance Moved 1614 cm-1 Intensity % RSD c Signal-to-Noise Ratiod % RSD e cm

-1.016 1.1 ± 0.1 3.4 29 ± 7 10.0 -0.889 1.9 ± 0.2 1.4 45 ± 18 15.6 -0.762 2.9 ± 0.3 3.6 65 ± 31 19.0 -0.635 4.2 ± 0.3 2.8 102 ± 17 6.9 -0.508 5.1 ± 0.2 1.3 125 ± 21 6.5 -0.381 5.0 ± 0.1 0.5 114 ± 22 7.8 -0.254 4.8 ± 0.1 1.1 114 ± 8 2.6 -0.127 7.3 ± 0.2 1.0 154 ± 65 17.1 0.000 11.0 ± 0.1 0.4 223 ± 44 8.0 0.127 8.1 ± 0.2 1.1 166 ± 62 15.0 0.254 3.2 ± 0.2 2.4 78 ± 9 4.8 0.381 1.8 ± 0.2 4.1 47 ± 12 10.6 0.508 1.4 ± 0.1 2.4 37 ± 14 15.0 0.635 1.2± 0.1 4.9 27 ± 6 9.5 0.762 0.9 ± 0.1 5.3 20 ± 4 9.0 0.889 0.7 ± 0.1 6.4 18 ± 8 17.9 1.016 0.7 ± 0.1 1.5 16 ± 2 5.9

a 16 scans at 4 cm-1 resolution with 500 mW laser power

b Averages of three collections at each sample displacement

c With respect to intensity

d rms noise calculated as one -fifth p-p variation over 2000-2500 cm -1 shift

e With respect to signal-to- noise

Table 5.9: Summary of FT-Raman Results for PET: Sample Displacement (Thickness: 0.1500 cm)

TABLE IX. Summary of FT-Raman Results for PET: Sample Displacementa,b

Thickness: 0.1500 cm

Distance Moved 1614 cm-1 Intensity % RSD c Signal-to-Noise Ratiod % RSD e cm

-1.016 1.9 ± 0.1 0.5 49 ± 1 1.1 -0.889 3.1 ± 0.2 2.1 72 ± 13 7.1 -0.762 4.7 ± 0.1 1.1 118 ± 41 14.2 -0.635 6.8 ± 0.1 0.4 167 ± 61 14.7 -0.508 8.0 ± 0.2 0.8 184 ± 27 6.0 -0.381 7.7 ± 0.2 0.8 174 ± 80 18.4 -0.254 8.0 ± 0.2 0.8 172 ± 20 4.7 -0.127 11.7 ± 0.1 0.4 265 ± 34 5.2 0.000 16.5 ± 0.2 0.4 311 ± 72 9.3 0.127 11.4 ± 0.2 0.6 237 ± 102 17.2 0.254 4.5 ± 0.1 0.3 102 ± 23 9.2 0.381 2.8 ± 0.2 3.2 69 ± 16 9.2 0.508 2.1 ± 0.2 3.6 53 ± 9 7.2 0.635 1.7 ± 0.1 2.7 41 ± 19 18.4 0.762 1.3 ± 0.1 3.1 34 ± 5 5.8 0.889 1.1 ± 0.1 3.4 27 ± 10 15.4 1.016 0.9 ± 0.1 1.1 22 ± 2 10.8

a 16 scans at 4 cm-1 resolution with 500 mW laser power

b Averages of three collections at each sample displacement

c With respect to intensity

d rms noise calculated as one -fifth p-p variation over 2000-2500 cm -1 shift

e With respect to signal-to- noise

5.7.3.11 Comparison of Spectrograph Slit and Interferometer J-Stop The fundamental concepts of FT-Raman spectroscopy are analogous to those of dispersive Raman spectroscopy. In dispersive Raman spectrometers, inelastically scattered light collected from a sample must properly illuminate the diffraction grating, or prism, through an entrance slit, whereas for interferometric-based spectrometers, the beamsplitter must be illuminated through a circular aperture (J-stop). Raman scattered light focused through the entrance slit or J-stop and onto the detector contributes to the detected Raman signal. An optical comparison of a slit and a circular aperture is illustrated in Fig. 5.24. The focused image of the illuminated sample at the entrance slit or aperture will be circular. The use of an entrance slit results in a poor system throughput, or étendue, since the illumination and collection system will inevitably overfill the µm-sized slit. The throughput, or Jacquinot, advantage is realized in interferometric-based spectrometers since the diameter of the entrance aperture may be as large as 8 mm.

Figure 5.24: Illustration of the magnified image of the illuminated sample (shaded area) on (a) a slit and (b) a J-stop.

In a conjugate optical system consisting of two focusing optics, displacement of the sample from the optimum sampling position of the collection lens results in a shift in the position of the resulting image relative to the aperture (J-stop). For a sample located at the optimum sampling position in the System 2000 FT-Raman, the magnification of the sample image on the J-stop was 7×. Since the laser diameter on the sample was on the order of 1 mm, all Raman scattered light collected from the sample will reach the detector for a J-stop diameter of 8.00 mm. Increases in the size of the image at the J-stop resulting from changes in sample position resulted in an overfilling of the circular aperture and a subsequent loss of light.

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5.8 Conclusions Dispersive Raman and FT-Raman sampling arrangements were evaluated as a short- range detection method for the on-line process control of PET bottle thickness. Although dispersive Raman spectroscopy employing visible laser excitation is impractical for the analysis of polymers due to the potential for strong sample fluorescence, the study has shown that a simplification of the optical system can be achieved using a single optic for both sample illumination and collection of the resulting Raman scattered light. Application of Raman spectroscopic detection for the on-line process control of PET bottles requires simultaneous considerations of working distance between the sample and the optic and throughput. Near-infrared FT-Raman spectroscopy successfully fulfills the required criteria for on- line sample analyses of PET bottles. A NIR FT-Raman instrument featuring a single, larger- diameter illumination/collection lens would be the preferred sampling arrangement for on-line process control of thickness in the bottling industry. The use of high-power, NIR laser excitation can be used in collecting Raman spectra with high signal-to-noise ratios while simultaneously minimizing the effect of sample fluorescence. Collection times can be reduced by sacrificing spectral resolution. Data have shown that FT-Raman spectroscopy could be successfully applied to on-line determinations of PET bottle thickness.

5.9 Acknowledgements Financial support of this research was provided by Pressco Technology, Inc. through a grant from the Ohio Third Frontier. Also, thanks to Mr. Jayson Alexander of the Miami University Instrumentation Laboratory for fabrication of the slide-on microscope accessory.

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References:

1. O. Brandau. Stretch Blow Molding. New York, NY: Elsevier, 2012. 2nd ed.

2. Pressco Technology, Inc., “Non-Contact Bottle Sectioning: INTELLIMASS”. 2012. http://www.pressco.com/content.php?page=76 [accessed Nov 9 2012].

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5. S. D. Cech. System and Method for Inspecting the Structural Integrity of Visibly Clear Objects. US Patent 6894775. Filed Apr 27 2000. Issued May 17 2005.

6. D. W. Cochran, S. D. Cech. Apparatus and Method for Inspecting Multi-Layer Plastic Containers. Filed Apr 21 2000. Issued Nov 22 2005.

7. P. J. Hendra. “Sampling Considerations for Raman Spectroscopy”. In: J. M. Chalmers and P. R. Griffiths, editors. Handbook of Vibrational Spectroscopy. Chichester, UK: Wiley, 2002. Pp. 1263-1288.

8. P. J. Hendra, D. B. Morris, R. D. Sang, H. A. Willis. “Raman spectroscopy of Flowing Poly(ethylene terephthalate) Melts”. Polymer. 1982. 23: 9-13.

9. H. Swierenga, A. P. De Weijer, L. M. C. Buydens. “Robust Calibration Model for On-Line and Off-Line Prediction of Poly(ethylene terephthalate) Yarn Shrinkage by Raman Spectroscopy”. J. Chemometrics. 1999. 13(3-4): 237-249.

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12. I. R. Lewis. “Process Raman Spectroscopy”. In: I. R. Lewis and H. G. M. Edwards, editors. Handbook of Raman Spectroscopy: From the Research Laboratory to the Process Line. New York, NY: Marcel Dekker, Inc., 2001. Chap. 23, Pp. 919-973.

12. G. Xue. “Laser Raman Spectroscopy of Polymeric Materials”. Prog. Polym. Sci. 1994. 19: 317-388.

13. N. Everall. “Raman Spectroscopy of Synthetic Polymers”. In: M. J. Pelletier, editor. Analytical Applications of Raman Spectroscopy. Malden, MA: Blackwell Science, Ltd., 1999. Chap. 4, Pp. 127-192.

14. J. K. Agbenyega, G. Ellis, P. J. Hendra, W. F. Maddams, C. Passingham, H. A. Willis. “Applications of Fourier Transform Raman Spectroscopy in the Synthetic Polymer Field”. Spectrochim. Acta. 1990. 46A(2): 197-216.

15. W. F. Maddams. “A Review of Fourier-transform Raman Spectroscopic Studies on Polymers”. Spectrochim. Acta. 1994. 50A(11): 1967-1986.

16. G. Xue. “Fourier Transform Raman Spectroscopy and its Application for the Analysis of Polymeric Materials”. Prog. Polym. Sci. 1997. 22: 313-406.

17. R. Ferwerda, C. Petty. “The Use of FT-Raman Spectroscopy and Chemometric Procedures in the Analysis of Polymers”. Nicolet Application Note AN-97100. Nicolet Instrument Corp. 1997.

18. J. Florestan, A. Lachambre, N. Mermilliod, J. C. Boulou, C. Marfisi. “Recycling of Plastics: Automatic Identification of Polymers by Spectroscopic Methods”. Resour. Conserv. Recy. 1994. 10: 67-74.

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19. N. Everall, P. Tayler, J. M. Chalmers, D. MacKerron, R. Ferwerda, J. H. van der Maas. “Study of Density and Orientation in Poly(ethylene terephthalate) Using Fourier Transform Raman Spectroscopy and Multivariate Data Analysis”. Polymer. 1994. 35(15): 3184-3192.

20. G. Ellis, F. Roman, C. Marco, M. A. Gomez, J. G. Fatou. “FT-Raman Study of Orientation and Crystallization Processes in Poly(ethylene terephthalate)”. Spectrochim. Acta A. 51: 2139- 2145.

21. B. H. Stuart. “Polymer Crystallinity Studied Using Raman Spectroscopy”. Vibr. Spectrosc. 1996. 10(2): 79-87.

22. A. Brookes, J. M. Dyke, P. J. Hendra, S. Meehan, “The FT-Raman Spectroscopic Study of Polymers at Temperatures in Excess of 200oC”. Spectrochim. Acta A. 1997. 53: 2313-2321.

23. B. A. Barrera, A. J. Sommer. “Sampling Considerations for FT-Raman Spectroscopy of Polymers and Solutions”. Appl. Spectrosc. 52(11): 1483-1487.

24. S. K. Bahl, D. D. Cornell, F. J. Boerio, G. E. McGraw. “Interpretation of the Vibrational Spectra of Poly(ethylene terephthalate)”. J. Polym. Sci. Pol. Lett. 1974. 12: 13-19.

25. F. J. Boerio, S. K. Bahl, G. E. McGraw. “Vibrational Analysis of Polyethylene Terephthalate and its Deuterated Derivatives”. J. Polym. Sci. Pol. Phys. 1976. 14: 1029-1046.

26. J. Stokr, B. Schneider, D. Doskocilova, J. Lovy, P. Sedlacek. “Conformational Structure of Poly(ethylene terephthalate). Infra-red, Raman and N.M.R. Spectra”. Polymer. 1982. 23(5): 714-721.

27. F. Adar, H. Noether. “Raman Microprobe Spectra of Spin-Oriented and Drawn Filaments of Poly(ethylene terephthalate)”. Polymer. 1985. 26(13): 1935-1943.

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28. F. J. Boerio, R. A. Bailey. “Polarized Raman Scattering from Polyethylene Terephthalate Fibers”. J. Polym. Sci. Pol. Lett. 1974. 12: 433-437.

29. J. Purvis, D. I. Bower, I. M. Ward. “Molecular Orientation in PET Studied by Polarized Raman Scattering”. Polymer. 1973. 14: 398-400.

30. W. Y. Yeh. Structure-Property Relationships in Engineering Polymer Fibers. [Ph.D. Dissertation]. Manchester, UK: University of Manchester, 1995.

31. M. J. Pelletier. “Raman Instrumentation”. In: M. J. Pelletier, editor. Analytical Applications of Raman Spectroscopy. Malden, MA: Blackwell Science, Ltd., 1999. Chap. 2, Pp. 61-64.

32. P. Dhamelincourt. “Raman Microscopy”. In: J. M. Chalmers and P. R. Griffiths, editors. Handbook of Vibrational Spectroscopy. Vol. 2. Chichester, UK: Wiley, 2002. Pp. 1419-1428.

33. E. J. C. Richardson. Investigating the Characterization and Stability of Polyamide 6,6 in Heritage Artifacts. [Ph.D. Dissertation]. Southampton, UK: University of Southampton, 2009.

34. J. Workman, Jr., L. Weyer. “Water”. In: Practical Guide and Spectral Atlas for Interpretive Near-Infrared Spectroscopy. 2nd ed. Boca Raton, FL, 2012. Chap. 6, Pp. 55-61.

35. H. Yamatera, B. Fitzpatrick, G. Gordon. “Near Infrared Spectra of Water and Aqueous Solutions”. J. Molec. Spectrosc. 1964. 14(1-4): 268-278.

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38. B. Schrader, G. Bergmann. “Die Intensitat des Ramanspektrums Polykristalliner Substanzen (The Intensity of the Raman Spectrum of Polycrystalline Materials)”. Fresen. Z. Anal. Chem. 225(2): 230-247.

39. B. Schrader, A. Hoffmann, S. Keller. “Near-infrared Fourier Transform Raman Spectroscopy: Facing Absorption and Background”. Spectrochim. Acta. 1991. 47A(9-10): 1135-1148.

40. B. Schrader. “Tools for Infrared and Raman Spectroscopy”. In: Infrared and Raman Spectroscopy: Methods and Applications. New York, NY: VCH Publishers, Inc., 1995. Pp. 138-151.

41. B. Schrader. “FT-Raman Spectroscopy”. In: J. M. Chalmers and P. R. Griffiths, editors. Handbook of Vibrational Spectroscopy. Vol. 2. Chichester, UK: Wiley, 2002. Pp. 1289-1301.

42. D. C. Harris. Quantitative Chemical Analysis. New York, NY: W. H. Freeman and Co., 2007. 7th edition.

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Chapter 6

Development of an Inverted Microscope Designed for Attenuated Total Reflection-Fourier Transform Infrared (ATR-FT-IR) Spectroscopic Imaging

6.1 Abstract The development and application of a prototype inverted attenuated total internal reflection-Fourier transform infrared (ATR-FT-IR) imaging microscope is discussed. The inverted infrared microscope functions through a separation of the visible and infrared optical paths. Separation of the optical paths permits the visible observation of the sample using white light illumination while infrared spectral images are simultaneously acquired from below. Preliminary evaluation of the designed microscope was done on the basis of signal-to-noise resulting from generated instrument responses and the quality of the resulting spectral images.

6.2 Introduction Infrared microspectroscopy represents the coupling of a microscope to an infrared spectrometer. In general, an infrared microscope must fulfill three basic functions: (1) illuminate the sample with radiation from the spectrometer’s infrared source, (2) collect the infrared radiation that has interacted with the sample and image this radiation onto an infrared- sensitive detector, and (3) allow the user to view and select specific areas of the sample to be interrogated at high magnification. Since its inception, infrared microspectroscopy has become a well-established method for micro-sample analysis and characterization. Information regarding the design of infrared microscopes, as well as concepts and applications, can be found in various books and reviews.1-5

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6.2.1 History of Infrared Microspectroscopy Early development of infrared microspectroscopic instrumentation and techniques began during the late 1940s, shortly after the ending of World War II. As early as 1943, Burch reported on the construction of a reflecting microscope consisting of a condenser and an objective with aspheric surfaces, or Schwarzschild aplanats, with numerical apertures greater than 0.5.6 In 1949, Barer, Cole, and Thompson successfully integrated a microscope of the Burch design7 to a dispersive infrared spectrophotometer.8 Barer et al. demonstrated the utility of the microscope system through the collection of infrared spectra from particles of biological interest whose size was on the order of 20 µm in diameter. Several reports detailing the concept of infrared microspectroscopy, specifically the factors affecting the integration of a microscope and an infrared spectrophotometer, were published by Blout, Bird, and Grey.9-10 The first commercial infrared microscope, designated the Model 85, was later developed by the Perkin-Elmer Corporation in 1953. Details regarding the design and utility of this microscope were presented in a publication by Coates, Offner, and Siegler.11 Infrared microspectroscopy was revitalized with the development of the Fourier transform infrared (FT-IR) spectrometers during the 1970s and 80s. An increase in the performance of the IR microscope was realized because FT-IR spectrometers were capable of handling IR radiation more efficiently than their dispersive predecessors. In addition to the advantages of the FT technique, such as the multiplex, throughput, and wavenumber precision advantages, the circular aperture(s) of these spectrometers matched those of the microscope, thereby facilitating integration. The first commercial FT-IR microscope accessory was designed by Digilab, LLC. in 1983. At the 1986 Pittsburgh Conference, Spectra-Tech, Inc. introduced the IR-PLAN Infrared Microscope Accessory. This instrument was the first commercial infrared microscope to successfully combine high-quality visual imaging with infrared spectroscopy. The IR-PLAN microscope featured a dual confocal aperture system, also referred to as Redundant Aperturing, in which the infrared beam was focused at apertures (field stops) positioned at image planes before and after the sample.12 Modern infrared microspectrometers possessing both single-element and array detectors are currently offered by several major instrument manufacturers, including Agilent, Bruker, and PerkinElmer.

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6.2.2 FT-IR Microspectroscopic Imaging The combination of a focal plane array (FPA) detector and an interferometer provides both multiplex and multichannel advantages. The multiplex advantage results from all spectral frequencies across the wavelength region of interest being measured concurrently. The multichannel advantage is due to the simultaneous collection of IR spectrum from the multiple detector elements, or pixels, on the array. Depending on the size of the array, thousands, or tens of thousands, of spectral can be collected in minutes. For example, 4096 spectra can be collected in a single image using 64 × 64 FPA. This value increases to 65,536 using a 256 × 256 FPA. Initial applications of infrared spectroscopic imaging were performed using step-scan FT- IR spectrometers. In 1995, Lewis et al. first reported the use of a step-scan interferometer for infrared spectroscopic imaging using an indium antimonide (InSb) FPA.13 Principles of step- scan imaging, details of instrument design, and infrared chemical imaging results were presented. The first commercial instrument to incorporate an InSb array detector on an infrared microscope was introduced by Bio-Rad in 1996. An MCT FPA was later employed for mid- infrared FT-IR spectroscopic imaging by Kidder et al.14 In contrast to an InSb array, which is capable of imaging over the range of 1-5 µm, an MCT array is capable of performing imaging over the range of 2.8-11 µm. The necessity to use step-scan interferometers for infrared imaging resulted from the slow read-out (frame rate) capabilities of the first-generation MCT arrays; however, as electronics improved, faster read-out times permitted the use of continuous, rapid-scan, interferometers for FT-IR imaging. The first implementation of a rapid-scan FT-IR imaging spectrometer was presented by Bennett et al. in 1993.15 Snively et al. later employed a rapid-scan system consisting of a first-generation, 64 × 64 MCT FPA in the collection of infrared spectra using a slow mirror velocity.16 Compared to step-scan imaging, data acquisitions can be performed faster using a rapid-scan approach since stabilization of the moving mirror is not required. In addition to the temporal advantage afforded by the use of a rapid-scan system, reduced instrumentation costs are realized. It is for these reasons that the majority of infrared spectroscopic imaging is currently performed using continuous-scan interferometers. Additional information regarding the instrumentation and methods employed for FT-IR imaging can be found in various books and reviews.17-20

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6.2.3 ATR-FT-IR Microspectroscopic Imaging ATR-FT-IR microspectroscopic imaging utilizes an infrared microscope in which the single-element detector has been replaced with a FPA detector. Attenuated total (internal) reflection imaging utilizes the concept of conventional ATR developed by Harrick21 and Fahrenfort22 where the sample is placed in direct, intimate contact with a material possessing a high refractive index, such as germanium. The focused beam size obtained using an infrared microscope is limited by diffraction. The diffraction-limited diameter (d) of infrared light focused to a point is defined as the bright central maximum of the Airy disk and is given by:

(6.1)

where λ is the wavelength of light and NA is the numerical aperture. Equation 6.1 is also used to determine the diffraction-limited spatial resolution. The NA is defined as the product of the refractive index of the medium in which the sample is immersed (n1) and the sine of the angle (θ) for the most extreme ray entering the optic according to Eq. 6.2.

(6.2)

Conventional infrared microscopes often utilize objectives and condensers with NAs on the order of 0.6. In addition to the benefit of limited or no sample preparation, ATR provides an improvement in spatial resolution equal to the refractive index of the immersion medium. A 4× improvement in spatial resolution over that obtained for reflectance measurements in air (n1 =

1.0) is obtained using a germanium (n1 = 4.0) internal reflection element (IRE). Prismatic and hemisphere IREs are typically employed for infrared spectroscopic imaging; however, when applied to the ATR-FT-IR microspectroscopic imaging, the hemispherical IRE provides a magnification of the sample image equal to the refractive index of the IRE.23

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6.2.4 History of ATR-FT-IR Imaging Two approaches are used when performing ATR imaging: (1) off-axis imaging and (2) on-axis imaging. Off-axis imaging requires the IRE/sample be initially centered in the focus of the microscope with imaging being conducted by moving the IRE/sample off-axis. This imaging method is typically performed using either a single-element or a linear array detector. On-axis ATR-FT-IR imaging is conducted by centering the IRE/sample composite in the focus of the microscope and illuminating the sample globally. Infrared radiation that has interacted with the sample is imaged onto a two-dimension array detector. Improvements in the spatial resolution of an infrared ATR measurement performed using a germanium IRE were first reported by Nakano and Kawata.24-25 Utilizing the off-axis imaging approach on a custom-built evanescent wave microscope, the authors demonstrated that an improvement in spatial resolution equal to the refractive index of the germanium (n = 4.0) could be obtained. A chevron-shaped IRE was later employed by Esaki et al. on conventional microscope.26 Esaki et al. demonstrated the ability to obtain ATR maps as large as 400 × 400 µm; however, since a hemispherical IRE was not used, no improvement in spatial resolution was realized. Lewis and Sommer reported on the off-axis ATR imaging approach of Nakano et al. using a commercial PerkinElmer i-series microscope.27 Lewis and Sommer demonstrated that one-dimensional ATR maps could be obtained with improved spatial resolution over transmission measurements and that spherical aberrations could be compensated for by collecting a background at the exact same off-axis position as the sample. In 1997, Bio-Rad introduced an infrared microscope outfitted with an MCT array detector, the Stingray. In contrast to off-axis ATR imaging, in which either a single-element or a linear array detector is used, on-axis ATR imaging is typically performed using a two- dimensional array detector. Initially proving unsuccessful due to instrument-related problems, Sommer successfully demonstrated ATR-imaging using an MCT array based infrared microscope coupled to a step-scan interferometer in the spring of 2000. Using an on-axis approach, the sample was globally illuminated. The pixels on the array spatially isolated selected points on the sample. An area of approximately 75 × 75 µm could be imaged in minutes with near-diffraction-limited performance. Sommer et al. demonstrated the capabilities of the imaging system by measuring the surface image of a single, human red blood cell. The results of this work were presented at the 2000 Pittsburgh Conference28 and later published in Applied

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Spectroscopy.29 In October of 2000, Bio-Rad (now Agilent) was issued a patent for ATR imaging based on microscopic on-axis measurements done with an array detector.30 The patent obtained by Bio-Rad outline the concept of ATR imaging; however, no supporting research was provided. In 2006, Patterson and Havrilla employed a 12.5 mm diameter germanium hemisphere for off-axis ATR imaging.31 Using a PerkinElmer Spotlight 300 imaging microscope with a 16 × 1 linear MCT detector, Patterson and Havrilla reported a 70× improvement in the available sampling area using the large-diameter hemisphere. Patterson et al. later employed the same hemisphere on a two-dimensional array system with a mapping stage in the off-axis imaging mode.32 Using mosaic tile imaging, the authors reported that the micro-imaging system was capable of collecting sample images over an area of 1.5 × 2.0 mm. Following the initial study by Patterson and Havrilla, PerkinElmer developed and introduced an ATR accessory based on the off-axis imaging concept of Nakano and Kawata24-25 and Lewis and Sommer27. Gulley-Stahl et al., in 2010, performed off-axis ATR imaging of kidney biopsies using this accessory.33 Gulley- Stahl et al. demonstrated that ATR was capable of overcoming many disadvantages associated with transflection and transmission measurements for tissue analysis, including an elimination of spectral artifacts. Further information regarding micro-ATR-FT-IR imaging can be found in a review by Kazarian and Chan.34

6.2.5 Sample Size and Pixel Resolution In a conventional infrared microscope, the principal source of diffraction results from the high-contrast edge of the area-defining aperture located at the primary image plane of the sample. The effect of diffraction-induced stray light on the spatial resolution in infrared microspectroscopy was previously investigated by Katon and Sommer using different aperturing modes, aperture sizes, and wavelengths.35 Infrared radiation diffracted by the aperture could propagate to the detector where it was capable of degrading the theoretical resolution. ATR-FT- IR micro-imaging systems do not typically utilize apertures since the entire sample area is imaged onto a two-dimensional array detector. Removal of this aperture during imaging minimizes the prominent source of diffraction. Diffraction-limited performance remains possible in an array-based system so long as the diffraction-limited beam diameter at the sample

240 is greater than the pixel size when imaged onto the array.36 In an infrared imaging system, the pixels on the detector serve as the area-defining apertures. Infrared radiation that has underdone total internal reflection at the interface between the sample and the IRE is imaged onto the FPA detector. The size of the detector defines the sample area capable of being imaged, and the pixel size defines the spatial element on the sample, a parameter referred to as pixel resolution. For example, Sommer et al. employed a 64 × 64 MCT FPA with a 61 × 61 µm pixel size for ATR-FT-IR mapping using an imaging microscope.29 Based on a magnification of 13× from the sample to the detector, the sample area capable of being imaged was 300 × 300 µm. When the sample was immersed in a germanium hemisphere, the sample area was reduced by 4× to 75 × 75 µm. The pixel resolution obtained in the ATR imaging mode was 1.2 × 1.2 µm. A larger array detector possessing a smaller pixel size can be used to increase the sample area interrogated as well as the pixel resolution.

6.2.6 Conventional and Aperture-splitting Beamsplitters Infrared microspectroscopic imaging is capable of being performed using either a conventional, or full, beamsplitter or an aperture-splitting beamsplitter. Conventional beamsplitters are not often employed for infrared imaging since the overall intensity of the radiation reaching the detector would be 25% at best. Modern FT-IR micro-imaging systems often employ aperture-splitting beamsplitters where 50% of the infrared radiation exiting the interferometer is directed into one-half of the microscope objective (or condenser). In this optical configuration, one half of the optic is used to illuminate the sample, and the second half is used to collect the infrared radiation after it has interacted with the sample. The resulting intensity of the radiation at the detector would be expected to be at least twice as large using an aperture-splitting beamsplitter as that obtained using a conventional beamsplitter. The use of aperture-splitting beamsplitters in FT-IR microspectroscopic imaging systems has a direct impact on the sampling characteristics, more specifically, the diffraction-limited spot size and the spatial resolution. Sommer et al. reported a 50% diminution in spatial resolution resulting from a 2× reduction of the NA due to the use of an aperture-splitting beamsplitter.29 Chan and Kazarian later proposed that the imperfect character of the diffraction pattern obtained from a non-uniformly illuminated aperture would result in the NA also being non-uniform;

241 therefore, the NA along the axis with the fully-illuminated objective would be expected to be higher than that along the axis with the half-filled optic.37 Ultimately, Eq. 6.1 may not necessarily provide a good estimate of the diffraction-limited diameter obtained in reflectance measurements.

6.2.7 Infinity-corrected Optics in FT-IR Microspectroscopy Infrared microscopes employ reflecting objectives with two optical designs: (1) fixed tube length or (2) infinity-corrected. Fixed tube length, or focusing, objectives possess a conjugate focal plane located behind the objective. An adjustable, remote aperture is often placed at this image plane for definition of the sample area. The term infinity-corrected refers to the collimation of both infrared and visible light by the microscope objective, condenser, or both. An infinity-corrected optic is designed to collect and focus light from a source located at “infinity”. The formation of an image at the primary focal plane of an optical microscope using an infinity-corrected objective requires the use of a focusing tele lens. In an infrared microscope, the formation of an image at the aperture requires a focusing mirror. Modern FT-IR imaging systems do not require area-defining apertures since the entire sample area is imaged onto an array detector. Advantages of infinity-corrected optics in FT-IR microspectroscopy have been discussed by Ellis.38 Collimation of the infrared radiation exiting the objective or condenser contributes to improvements in the quality of spectral images and signal-to-noise. Optical aberrations, such as spherical aberrations, associated with fixed-length optics can be minimized using infinity- corrected optics. The incorporation of infinity-corrected optics in infrared microscopes permits a simplification of the optical design because flat, first-surface mirrors can be used to direct the collimated radiation.

242

6.3 Goals and Specific Aims Commercial infrared microscopes capable of ATR imaging employ hemispherical IREs in a downward-facing orientation. In this configuration, the sample is positioned under the IRE using visible illumination. The IRE is then rotated, or dropped, into position in order to obtain direct, intimate contact with the sample. Correspondence between visible viewing and ATR-FT- IR imaging can be problematic, especially with the use of visibly-opaque IREs with high refractive indices, such as silicon or germanium. Viewing of the sample with white light illumination while simultaneously performing ATR imaging will be demonstrated by separating the visible and infrared light paths through inversion of the IRE. Samples will be supported by the IRE, allowing infrared images to be collected from below while visible imaging is conducted from above. An illustration of a prototype inverted ATR-FT-IR imaging microscope capable of simultaneous viewing and collection modes is shown in Fig. 6.1.

Visible objective

ATR cartridge

Infrared condenser

Infrared in Infrared out

Figure 6.1: Illustration of an inverted ATR-FT-IR imaging microscope. Sample viewing using white light illumination is performed from above while the collection of infrared spectra is performed from below.

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6.4 Experimental

6.4.1 Materials and Methods An ATR cartridge slide was designed by the Molecular Microspectroscopy Laboratory and fabricated by the Instrumentation Laboratory at Miami University. A diagram of the slide accessory is illustrated in Fig. 6.2.

3.00 in.

0.12 in.

1.00 in

0.01 in.

120.00 deg 0.19 in. Figure 6.2: Diagram of an ATR cartridge slide.

The cartridge slide was fabricated using aluminum and was designed to contain a 3 mm diameter germanium hemisphere. The bottom of the cartridge was recessed at 120o to accommodate the maximum collection angle for the microscope condenser.

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6.4.2 Instrumentation The inverted ATR imaging microscope was coupled to a Digilab FTS-7000 FT-IR spectrometer. A potassium bromide (KBr) beamsplitter was employed in the interferometer. An aperture-splitting beamsplitter was employed in directing half of the infrared beam exiting the interferometer to an infinity-corrected, variable compensation 15× (0.58 NA) Reflachromat (Thermo Scientific, Waltham, MA) condenser. The condenser optic, originally designed for the Spectra-Tech ImageMax/Insight microimaging system, is currently used in the construction of the Thermo Scientific Continuµm microscope. ATR-FT-IR images were collected using a Digilab Lancer 128 × 128 MCT FPA with a pixel size of 40 × 40 µm. With the FTS-7000 operating in rapid-scan mode, the active area on the detector was apertured down to 64 × 64. Instrument control of the FTS-7000 was done using Digilab Resolutions Pro software (version 4.0.0.030 [2004]). Photographs of the prototype inverted microscope are shown in Fig. 6.3.

Figure 6.3: Photographs of the inverted ATR imaging microscope. (Left) The constructed microscope. (Right) Detector optics.

Visual examination of the samples was performed using a standard Olympus three-way eyepiece coupled to an Olympus BH2-UMA Universal Vertical Illuminator possessing a 50 W tungsten filament bulb. Alignment of the microscope optical system was performed using white

245 light EPI illumination passing through a 100 µm diameter pinhole aperture. A 15× (0.58 NA) Reflachromat objective was used to focus light through the aperture. The detector optics used in the construction of the inverted imaging microscope were obtained from a Spectra-Tech ImageMax/Insight microimaging system. A close-up photograph of the detector optics, as well as an optical diagram, is shown in Fig. 6.4.

MCT focal plane array

Infrared beam from microscope Offner telescope

Figure 6.4: (Left) Photograph of the imaging optics. (Right) Optical drawing of the imaging optics.

Unless stated otherwise, infrared spectral images represented the average of 32 individual scans at 8 cm-1 resolution over the range of 3200-900 cm-1. The interferometer was scanned in rapid-scan mode at a rate of 2.5 kHz. The frame rate was 3773.58 Hz with an integration time of 0.0742 milliseconds. Spectral image data were processed using Digilab Resolutions Pro software (version 4.0.0.030 [2004]) and Thermo Galactic (Thermo Scientific) GRAMS/AI software (version 7.02 [2002]).

246

6.5 Results and Discussion

6.5.1 Optical Considerations The first optical requirement that must be considered when performing ATR imaging is the critical angle, θc. The critical angle required for total internal reflection of the infrared radiation at the interface between the IRE and the sample can be determined using Eq. 6.3:

( ) (6.3)

where ns and nIRE are the refractive indices of the sample and IRE, respectively. For a sample possessing a refractive index of 1.5, the critical angle required using a germanium IRE (nIRE = 4.0) is 22.0o. Using the 0.58 NA condenser, infrared radiation entering the IRE at angles between 22.0o and 35.5o will undergo total internal reflection. A critical angle of 26.2o would be required if the germanium IRE were replaced with a silicon IRE possessing a refractive index of 3.4. The NA of the microscope condenser successfully meets the required critical angles of these two IRE materials; however, a problem occurs when zinc selenide (ZnSe) (nIRE = 2.4) is used as the IRE. In this situation, the maximum collection angle of the condenser (35.5o) is below the required critical angle of 38.7o for total internal reflection at the IRE-sample interface. In order to successfully employ a ZnSe IRE for infrared imaging, the NA of the lower condenser must be increased to at least 0.63.

247

The next optical parameter of interest is the magnification from the sample position to the detector. An optical diagram of the inverted infrared imaging microscope is shown in Fig. 6.5.

Figure 6.5: Optical illustration of an inverted ATR-FT-IR imaging microscope.

The magnification of the infrared microscope from the sample to the detector was determined using a 1951 United States Air Force resolution target. An infrared reflectance image of group 5 element 1 on the resolution target is shown in Fig. 6.6. Specifications of the resolution target indicate that the thicknesses of the lines/spaces for group 5 element 1 were 15.63 µm.

248

(a) (b)

Figure 6.6: 1951 USAF resolution target group 5 element 1. (a) Infrared reflectance image. (b) Detector response.

At the detector position, the resulting separation between the centers of two adjacent lines (2× the line thickness) in group 5 element 1 was determined to be 486.93 µm. Additional reflectance images (not shown) of a 15 µm diameter pinhole and resolution lines in group 5 element 6 (8.77 µm line/space width), group 4 element 4 (22.10 µm line/space width), and group 4 element 6 (8.77 µm line/space width) resulted in a magnification of 15.0 ± 1.2 from the sample to the detector. (Uncertainty represents standard deviation for five determinations of system magnification.) Taking into account a system magnification 15×, the 2560 × 2560 µm FPA would correspond to a sample area of 171 × 171 µm when imaged onto the sample. Similarly, for a 40 × 40 µm, the pixel resolution at the sample is expected to be 2.7 × 2.7 µm. The addition of a germanium hemisphere to the optical system reduces the sample area and pixel resolution by a factor of equal to the refractive index of the material; the refractive index of germanium is 4.0. The resulting sample area capable of being imaged onto the FPA would be 43 × 43 µm at a pixel resolution of 0.7 × 0.7 µm.

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6.5.2 Signal-to-Noise An investigation of the resulting signal-to-noise obtained in ATR imaging mode was performed as a function of both the number of averaged scans and the spectral resolution. Instrument responses (100% T lines) were generated through the ratio of background and sample images of the germanium IRE in the absence of a sample. Extraction of the resulting 100% T lines was done using the center pixel (32 × 32) of the sample image. Peak-to-peak (p-p) variations in % T were determined over the range of 2600-2400 cm-1; root-mean-square (rms) noise was calculated as one-fifth of the p-p variation. Results from the signal-to-noise study with respect to the number of scans are listed in Table I. A plot of signal-to-noise versus the square root of the number of scans is shown in Fig. 6.7.

Table 6.1: Results of Signal-to-Noise Study with Changes in Number of Scans TABLE I. Results of Signal-to-Noise Study with Changes in Number of Scansa Number of Scans SQRT of Number of Scans p-p Variationb,c SNRb,d Normalized SNR % RSD %T

4 2.0 6.2 ± 0.8 82 ± 9 1.0 11.0 8 2.8 3.6 ± 0.5 139 ± 16 1.7 11.5 16 4.0 2.8 ± 0.4 178 ± 20 2.2 11.2 32 5.7 2.1 ± 0.5 245 ± 42 3.0 17.1

a 8 cm-1 resolution.

b Average and standard deviation for three replicate collections at each number of scans.

c Peak-to- peak variation in %T determined over range of 2600-2400 cm-1.

d Noise calculated as one -fifth peak-to- peak variation in %T.

250

300

y = 42.8x + 6.0 250 R² = 0.9826

200

Noise

to -

150 Signal 100

50 1 2 3 4 5 6 SQRT of number of scans

Figure 6.7: Plot of signal-to-noise as a function of the square root of the number of scans. (Error bars represent standard deviation of three replicate measurements at each number of scans.)

A similar study of signal-to-noise in ATR imaging mode was performed through the collection of infrared images at different spectral resolutions. Data obtained during this investigation are presented in Table II. A plot of signal-to-noise versus spectral resolution is shown in Fig. 6.8. Table 6.2: Results of Signal-to-Noise Study with Changes in Spectral Resolution

TABLE II. Results of Signal-to-Noise Study with Changes in Spectral Resolutiona

Spectral Resolution p-p Variationb,c SNRb,d Normalized SNR % RSD cm-1 %T

4 3.0 ± 0.2 169 ± 10 1.0 5.8 8 2.1 ± 0.5 245 ± 51 1.4 20.8 16 1.3 ± 0.1 376 ± 29 2.2 7.7

a Average of 32 individual scans.

b Average and standard deviation for three replicate colle ctions at each spectral resolution.

c Peak-to-peak variation in %T determined over range of 2600- 2400 cm-1.

d Noise calculated as one -fifth peak- to-peak variation in %T.

251

450

400

350

Noise 300

to - 250 y = 17.1x + 103.5 R² = 0.9986

Signal 200

150

100 0 4 8 12 16 20 Spectral resolution (cm-1)

Figure 6.8: Plot of signal-to-noise as a function of spectral resolution. (Error bars represent standard deviation of three replicate measurements at each number of scans.)

6.5.3 Spatial Resolution The diffraction-limited spatial resolution was determined using Eq. 6.1 at a wavelength of 3.1 µm (3200 cm-1). The resulting value was found to be ~2λ, or 6.5 µm, using an NA of 0.58. Since an aperture-splitting beamsplitter was used in directing infrared radiation to the condenser, only half of the optic was filled; therefore, the NA would be reduced by a factor of two to 0.29. The resulting spatial resolution was ~4λ, or 13.0 µm. A 1951 USAF resolution target was utilized in determining the spatial resolution of the inverted infrared microscope in reflectance mode. An empirical determination of the spatial resolution was done using group 7 of the resolution target. A reflectance image of this resolution group is shown in Fig. 6.9

252

Figure 6.9: 1951 USAF resolution target group 7.

As illustrated in Fig. 6.9, the lines in element 3 are the first to possess discernible separation between the individual lines. The resolved lines in element 3 were located along the optical axis in which the condenser was fully-illuminated. The lines (and spaces) in group 7 element 3 possess a width of 3.1 µm, so the separation distance between the centers of the individual lines is twice the width, or 6.22 µm. This value is comparable to the spatial resolution determined using Eq. 6.1 for a wavelength of 3.1 µm (3200 cm-1) and an NA of 0.58. A similar determination of spatial resolution along the half-filled optical axis was done using the elements included in group 6 of the resolution target. Images of elements 6 to 2 are shown in Fig. 6.10.

Figure 6.10: 1951 USAF resolution target group 6. (a) Elements 6 to 4. (b) Elements 3 and 2.

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The vertical resolution lines shown in Fig. 6.10 were oriented along the optical axis of interest. The first lines to be completely resolved were those of element 3. The listed line/space width of this element was 6.2 µm. Therefore, the corresponding separation between two adjacent lines (or spaces) would be 12.4 µm. This value is comparable to the spatial resolution determined using Eq. 6.1 for a wavelength of 3.1 µm (3200 cm-1) and an NA of 0.29.

254

6.5.4 Sample Analysis The capability of the instrument for sample analysis was demonstrated using a strip of adhesive tape mounted directly on the germanium IRE. A background scan of the bare IRE surface was collected prior to the collection of a sample scan. The resulting images are shown in Fig. 6.11. Sample spectra (collected in absorbance) represent the average of 32 individual scans at 8 cm-1 resolution.

a 0.90 b 0.80

0.70

0.60

0.50

0.40 Response 0.30

0.20

0.10

0.00 3100 2900 2700 2500 2300 2100 1900 1700 1500 1300 1100 900 Wavenumber (cm-1)

c 0.30 d

0.25

0.20

0.15 Absorbance 0.10

0.05

0.00 3100 2900 2700 2500 2300 2100 1900 1700 1500 1300 1100 900 Wavenumber (cm-1) Figure 6.11: (a) Single-beam background image of bare, germanium IRE at 1000 cm-1. (b) Single-beam background spectrum extracted at pixel 32 × 32. (c) Sample image of adhesive tape at 1732 cm-1. (d) Sample spectrum extracted at pixel 32 × 32.

The single-beam background image (Fig. 6.11a) represents the response at 1000 cm-1 (10.0 µm). Figure 6.11c represents the absorbance of the acrylate C=O stretch at 1732 cm-1. The resulting spectrum extracted from the central pixel (32 × 32) of this image is shown in Fig. 6.11d. In addition to the 1732 cm-1 C=O stretch, the tape spectrum possessed absorptions corresponding to C-H stretches between 3000 and 2800 cm-1 and a acrylate C-C-O stretch at

255

1160 cm-1. The transparent, adhesive tape used in the present study was manufactured using an acrylic adhesive mounted on an acetate backing. The background and sample images shown in Figs. 6.11a and 6.11c indicate that the detector area was sufficiently filled and that the acquired sample image exhibited a uniform response over most of the detector area.

6.6 Conclusions A prototype inverted attenuated total (internal) reflection-Fourier transform infrared imaging microscope was constructed. Initial testing of the optical design was performed through determinations of magnification and spatial resolution in reflectance mode. The spectral response of the microscope demonstrated in ATR imaging mode was uniform with respect to absorbance by the sample over essentially all of the detector area. Preliminary data showed that the constructed inverted infrared imaging microscope was capable of acquiring high- magnification reflectance images; although, additional testing is required to prove the utility of the ATR imaging aspect of the instrument.

6.7 Acknowledgements The author thanks Gloria Story, Tom Cambron, and Ed Grundner of Procter & Gamble for allowing access to the laboratory and instrumentation during this project, as well as for their assistance and training. Thanks also go to Mr. Jayson Alexander and Mr. Barry Landrum of the Instrumentation Laboratory at Miami University for the fabrication of the various microscope components and accessories.

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33. H. J. Gulley-Stahl, S. Bledsoe, A. P. Evan, A. J. Sommer. “The Advantages of an Attenuated Total Internal Reflection Infrared Microspectroscopic Imaging Approach for Kidney Biopsy Analysis”. Appl. Spectrosc. 2010. 64(1): 15-22.

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Chapter 7

Conclusion

7.1 Summary Research presented in this dissertation has demonstrated the characterization and application of novel instrumentation and methods for the analysis of samples using infrared and Raman spectroscopy.

7.1.1 Characterization of Infrared-Transmitting Silver Halide Fiber Optics and Hollow Silica Waveguides Infrared-transmitting silver halide polycrystalline fiber optics and hollow silica waveguides were evaluated on the basis of the transmission of mid-infrared radiation using a fiber optic coupling accessory and an infrared microscope. Although polycrystalline silver halide fibers have been successfully utilized in the construction of fiber-coupled accessories, this type of solid-core fiber is subject to appreciable attenuation losses as a result of front-surface reflections at the ends of the fibers and scattering within the silver halide core. Hollow waveguides offer an attractive alternative to the use of solid-core fibers due to a minimization of insertion loses resulting from the air core. Increased transmission was obtained using the hollow waveguides as a result of the high reflectivity of the inner dielectric surface. In contrast to the silver halide fiber optics, the hollow silica waveguides exhibited an increased tolerance to bending. Data obtained during the characterization study of the fibers/waveguides indicated that HWGs would be preferred as optical conduits in the construction of a waveguide-coupled ATR probe.

7.1.2 Evaluation of a Mid-Infrared Hollow Waveguide ATR Probe Accessory A remote, ATR-FT-IR sampling accessory utilizing a set of HWGs was evaluated on the basis of transmission efficiency and signal-to-noise. The hollow waveguide ATR probe accessory possessed a diamond-ZnSe IRE and an integrated wide-band MCT detector. A comparison of signal-to-noise ratios revealed that the transmission efficiency of the HWG ATR probe was an order of magnitude less than that of an open-beam FT-IR sample compartment under identical conditions. Data obtained from a bending analysis showed that baseline

263 variations could be minimized by collecting background and sample scans with the probe in similar configurations. The HWG ATR probe was utilized for the quantitative analyses of aqueous solutions of succinylcholine chloride and ethanol. In both quantitative studies, calibration curves possessing high linear R2 correlations were constructed. Despite an increased sampling volume, the analytical figures of merit for the quantitative analysis of aqueous succinylcholine chloride solutions were comparable to those obtained in an earlier study using an infrared microscope. Verification of alcohol content in commercial beer, wine, and liquor was performed using constructed calibration curves of absorbance versus alcohol content. Correction for band broadening resulting from carbohydrate absorptions located in close proximity to the ethanol absorption used for quantitation was accomplished through adjustment of the limits of integration. Accurate determinations of alcohol content in alcoholic beverages can be achieved in the absence of statistical modeling using the proposed method.

7.1.3 Design of a Reaction Cell for Studying Nitrogen Doping in Thin Zinc Oxide Films Using In situ Raman Spectroscopy Remote process monitoring of the thermal decomposition of calcium oxalate monohydrate and the incorporation of nitrogen in thin ZnO films were performed using Raman spectroscopy and a high-temperature reaction cell. The custom-designed reaction cell permits in situ sample analysis at variable temperatures in the presence of gaseous reagents, under purge, or under vacuum. A comparison of the data obtained from the process-Raman and thermogravimetric analyses indicated that the proposed method was capable of identifying reaction intermediates and products formed during the thermal decomposition of COM. In situ Raman spectroscopy using a fiber optic Raman microprobe was utilized in studying nitrogen incorporation in thin films of ZnO. Low-intensity transitions commonly associated with the formation of N-related defects in ZnO were observed at 350 and 400oC in the presence of the 5 o wt. % NO/N2 mixture and at 450 C in the presence of PPN2. Since the presence of these transitions could not be supported in additional trials, process monitoring of nitrogen incorporation in ZnO using Raman spectroscopy was deemed inconclusive. The reactor cell design should prove useful for the future analyses of samples under controlled conditions.

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7.1.4 Raman Spectroscopic Detection for Process Control in the Bottling Industry Dispersive and Fourier transform Raman spectroscopy were investigated as short-range detection methods for PET bottle thickness on a process line. Preliminary assessment of the Raman backscattering configuration using dispersive Raman with 633 nm excitation indicated that optimization of the sampling configuration for on-line, industrial applications required a compromise of Raman intensity and working distance. Although increased Raman signal was observed using a short focal length optic, the small working distance afforded by the lens would not be practical for on-line measurements. The backscattering geometry can be easily implemented in an industrial setting. Correction of background fluorescence in polymeric samples requires shifting of the excitation wavelength to the near-infrared. Near-infrared Fourier transform (FT-) Raman spectroscopy offers a potential alternative to the on-line spectroscopic detection of PET bottle thickness. Minimization of sample fluorescence was observed using 1064 nm Nd:YAG laser excitation. A calibration curve of the PET 1614 cm-1 shift intensity versus sample thickness possessed a linear R2 correlation of 0.9810. Studies of the effects of laser power, number of scans, and spectral resolution on the uncertainty in bottle thickness indicated that on-line determinations should be performed using as high of a laser power as the sample can safely tolerate. In order to expedite sample analysis and prevent any hold-ups in production, FT-Raman spectra should be collected using a small number of scans. Spectral resolution can be sacrificed up to 16 cm-1 in order to minimize uncertainties in intensity and sample thickness while maintaining production rates. Data have shown that an FT- Raman spectrometer possessing a large-diameter illumination/collection lens successfully fulfills the required sampling criteria and could be successfully applied to on-line determinations of PET bottle thickness.

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7.2 Future Work

7.2.1 Application of an Inverted Microscope Designed for ATR-FT-IR Spectroscopic Imaging Preliminary imaging data obtained using the inverted ATR-FT-IR imaging microscope indicated that the system was capable of obtaining quality images in reflectance mode. The ability of the instrument to collect ATR spectra was demonstrated using a strip of adhesive tape. A uniform response in absorption was observed over the full detector area. Additional optimization and testing in ATR imaging mode is required to prove the utility of the instrument. It is anticipated that the inverted optical design of the microscope will facilitate the collection of infrared spectra of samples placed in direct, intimate contact with the internal reflection element while optically viewing the samples from above.

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