"Attenuated Total Reflection Fourier Transform Infrared Spectroscopy" In

Attenuated Total Reﬂection Acknowledgments 24 Abbreviations and Acronyms 24 Fourier Transform Infrared Related Articles 24 Spectroscopy References 24

Georg Ramer and Bernhard Lendl Vienna University of Technology, Vienna, Austria Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy is now the most widespread implementation of mid-infrared (MIR) spectroscopy. 1 Introduction 1 While the FTIR technique allows the fast and stable 1.1 Introduction to Attenuated Total collection of MIR spectra, the ATR technique allows Reflection Spectroscopy 1 mechanically stable, robust, and quick sampling. ATR- FTIR spectroscopy is routinely used in industrial and 1.2 History of ATR Spectroscopy 2 research laboratories. The ATR-FTIR technique finds 1.3 A Short Introduction to Mid-infrared application in, e.g. biology, medicine, forensics, process Fourier Transform Spectroscopy 3 analytical chemistry and organic chemistry. Even though 2 Theoretical Aspects of Attenuated Total ATR spectroscopy is often treated as a routine technique, Reflection Spectroscopy 5 it has several intricacies that users should be aware of to 2.1 Reflection, Total Reflection, and the avoid measurement errors and artifacts. Attenuated Total Reflection Signal 5 In this article, starting from the theoretical underpinnings 2.2 Influence of the Measurement of ATR spectroscopy, we aim to give novices in ATR- Parameters on the Attenuated Total FTIR spectroscopy the knowledge to successfully use this Reflection Signal 9 technique and to avoid common errors. The theoretical 3 Practical Aspects of Attenuated Total treatment of ATR spectroscopy is complemented by Reflection Spectroscopy 13 practical information about the routine and advanced uses 3.1 Materials 13 of ATR spectroscopy. Furthermore, the reader will find descriptions of future trends in ATR-FTIR and evanescent 3.2 Number of Reflections 13 wave spectroscopy. Finally, a list of literature for further 3.3 Sample Preparation and Measurement reading and a list of vendors of ATR accessories and Protocols 14 their product lines are given to facilitate using the ATR 3.4 Angle of Incidence 16 technique. 3.5 Surface Modification 17 4 Differences Between Attenuated Total Reflection Spectroscopy and Transmission 1 INTRODUCTION Spectroscopy 18 4.1 Comparison of Attenuated Total 1.1 Introduction to Attenuated Total Reflection Reflection Spectra and Transmission Spectroscopy Spectra 18 4.2 Sample Preparation 19 The attenuated total reflection (ATR) technique is the most frequently used sampling technique for infrared (IR) 5 Applications 19 spectroscopy.(1) IR light traveling in an optically denser 5.1 Biological and Medical Samples 19 medium is totally reflected at the interface to an optically 5.2 Process Analytical Chemistry 20 rarer medium. Although the light rays as such do not 5.3 Microscopy 20 propagate into the optically rarer medium, an evanescent 5.4 Depth Profiling 21 field forms there. Through this evanescent field, the light 6 Vendors 22 can interact with samples placed at the interface, making absorption measurements possible. 7 Further Reading 22 The ATR technique usually needs hardly any sample 8 Future Trends 23 preparation. It allows quick and robust measurements 8.1 Hand-held Attenuated Total Reflection of solid as well as of liquid samples, including pastes Instruments 23 and samples otherwise difficult to handle. Therefore, 8.2 Waveguide Sensors 23 since its invention in the 1960s, the ATR technique has

Encyclopedia of Analytical Chemistry, Online © 2006–2013 John Wiley & Sons, Ltd. This article is © 2013 John Wiley & Sons, Ltd. This article was published in the Encyclopedia of Analytical Chemistry in 2013 by John Wiley & Sons, Ltd. DOI: 10.1002/9780470027318.a9287 2 INFRARED SPECTROSCOPY gradually replaced other sampling methods for solid and Several great minds have investigated total reflection over liquid samples for many applications of IR spectroscopy. the course of the following centuries. E. Hall notes, at the In principle, ATR spectroscopy can also be applied to beginning of The penetration of totally reflected light into the measurement of gases; however, due to the short the rarer medium(3): interaction length of the evanescent field with the sample, gas measurements are of low sensitivity. The problem [attenuated total reflection] was both This article aims at giving a practical and theoret- experimentally and theoretically studied by Fresnel, and more or less treated by Verdet, Young, Huygens, Biot, ical introduction to ATR spectroscopy. An overview of Babinet, Billet, Stokes, and other. The first reliable common applications and designs of ATR accessories quantitative work was published by G. Quincke in 1886. for MIR spectroscopy is given. Practical aspects of ATR spectroscopy, including sample preparation, sampling, and common errors are explained. The current applica- Total reflection was, however, first used in the twentieth tions of the ATR technique in a wide range of fields are century for absorption measurements.(4) In 1926, C.V. reviewed. To make it easy for the reader to start using the Raman,(5) who went on to discover the Raman effect ATR technique, a list of vendors of ATR accessories and a few years later, reexamined the principles of total their respective products is given at the end of the article. reflection. In the early 1930s, the principles of ATR As this article is introductory in nature, several sources absorption measurements were worked out by Taylor recommended for further reading are provided. and Glover,(6) Taylor and Durfee,(7) and Taylor and King(8) and employed to determine the birefringence of layers of organic acids. In their work, the principles of 1.2 History of ATR Spectroscopy ATR spectroscopy are laid out correctly and employed to Isaac Newton famously remarked: ‘If I have seen farther measure absorption spectra, to study surface layers, and (4) it is by standing on the shoulders of giants’. It is therefore to determine refractive indices. fitting that we start this short overview over the history of The next major steps in the development of the the ATR technique and its giants with his work entitled ATR technique happened in quick succession in the late Opticks: Or, A Treatise of the Reflections, Refractions, 1950s and the early 1960s. At the Fourth International Inflections and Colours of Light.(2) Starting with Opticks Conference on Molecular Spectroscopy (Bologna, 1959), has another significance as well: N.J. Harrick, one of the Jacques Fahrenfort of the Royal Dutch Shell Laboratories (4) inventors of the ATR technique, begins his fundamental presented a paper on the ATR technique. In December work on ATR spectroscopy entitled Internal Reflection of the same year, at the Second International Conference Spectroscopy by quoting a passage from Opticks: Or, A on Semiconductor Surfaces (Maryland), Nicolas James Treatise of the Reflections, Refractions, Inflections and Harrick of the Philips Laboratories suggested in a Colours of Light (book III, part 1, query 29), where comment on a talk about ‘Infrared Methods Applied Newton describes total reflection of light at the interface to Surface Phenomena’ (R.P. Eischens) to use ATR between a glass prism and air: measurements to study molecules adsorbed on the surface.(4) Harrick had been studying internal reflection The Rays of Light in going out of the Glass into a Vacuum, and total internal reflection in semiconductors before.(9) are bent towards the Glass; and if they fall too obliquely At this point, however, he presumably did know neither on the Vacuum, they are bent backwards into the Glass, of the works of Taylor et al. nor of Fahrenfort’s and totally reflected;[...] talk.(4) In 1961, ‘Attenuated total reflection’(84) by Fahren- fort was published in Spectrochimica Acta.Inthis and also notes, that light that should be totally reflected work, Fahrenfort introduced the theoretical underpin- will pass into a second optically dense material when the nings of ATR spectroscopy. He showed two different interfaces of both objects are placed close enough to each experimental setups using either a hemicylindric KRS-5 other, even though they do not touch: (Thallium Bromoiodide) or AgCl element to collect ATR And this is still more evident by laying together two Prisms spectra using a focused or a collimated beam. In the of Glass, or two Object-glasses of very long Telescopes, same paper, Fahrenfort also showed the possibility for the one plane, the other a little convex, and so compressing quantification in ATR spectra using Beer’s law and its them that they do not fully touch, nor are too far asunder. limitations. Finally, he suggested using this new technique For the Light which falls upon the farther Surface of the to determine refractive index spectra of samples. first Glass where the Interval between the Glasses is not above the ten hundred thousandth Part of an Inch, will In 1967, Harrick published Internal Reflection Spectro- (10) go through that Surface, and through the Air or Vacuum scopy, which included a thorough treatment of the between the Glasses, and enter into the second Glass,[...] theoretical foundations and practical considerations

400 to determine depth proﬁles of thin layers and to determine the orientation of molecules adsorbed on a surface. 300

200 1.3 A Short Introduction to Mid-infrared Fourier Transform Spectroscopy 100 1.3.1 The Mid-infrared Region of the Electromagnetic

Number of publications 0 1960 1980 2000 Spectrum Year ATR spectroscopy is most often used in the MIR region Figure 1 Plot of the number of publications on ATR of the electromagnetic spectrum. The MIR region is spectroscopy over the past 50 years. Data were collected usually defined as the wavenumber ν˜ region from 4000 to from Scopus. Plotted are all publications listed in Scopus as 400 cm−1 (from 2.5 to 25 μm). The predominant features document type ‘article’, ‘book’, ‘conference paper’, ‘review’, or in this spectral region are primarily due to molecular ‘article in press’ that contain ‘attenuated total reflection’ in title, abstract, or keywords. vibrations. These vibrations give information about the functional groups present in the molecule, as well as the structure of the molecule. regarding ATR spectroscopy. At that time, ATR While it is often a challenge to derive the structure spectroscopy had already taken off with over 600 of complex molecules solely from their IR spectra, papers published.(11) Since then, the interest in ATR these molecules can still be identified by their IR spectroscopy has steadily increased (Figure 1). spectra, in a similar manner in which fingerprints are The ATR technique is sometimes also referred to as used for the identification of humans. This identifi- internal reflection spectroscopy (IRS), especially in works cation of molecules is either performed by compar- by Harrick, Milosevic, and Mirabella. It seems, however, ison with reference spectra determined in the lab that today the generally used term for the technique is or by using IR libraries. Such libraries are available ATR. As reflection measurements at subcritical angles commercially or for free. Examples for free libraries of incidence will lead to spectra that are very different are the Spectral Database for Organic Compounds to those measured at supercritical angles, it makes sense (http://riodb01.ibase.aist.go.jp/sdbs/cgi-bin/cre index.cgi? lang=en) or the National Institute of Standards and Tech- to make a distinction between general internal reflection nology (NIST) Chemistry Webbook (http://webbook.nist. and ATR. Furthermore, the acronym IRS is also used gov/chemistry/). for IR spectroscopy, which no doubt can be a source of According to the harmonic oscillator model, the mix-ups. Finally, most citizens of the United States will energies of the vibrational states of the molecule are have mixed feelings about the IRS at best, as this acronym is also used for the Internal Revenue Service. V = hν (υ + 1 )(1) Other major steps in the general acceptance of the iν i i 2 ATR technique were the broader availability of fast υ ν i is the vibrational quantum number and i the IR instruments, made possible by the invention of the fundamental frequency of the ith mode.(12) The selection FTIR technique and the development of horizontally rules for IR spectroscopy only allow transitions of mounted ATR elements and cylindrical ATR elements υ =±1, where the dipole moment μ changes during (4) i for liquids. the vibration along the normal coordinate Q : Today, the ATR technique is used in broad range i of experimental settings, including routine measurement μ ∂ = protocols as well as advanced research applications. 0 (2) ∂Qi ATR-FTIR spectroscopy is a standard analytical tool for identification of chemicals in many industries, e.g. 1.3.2 Quantitative Mid-infrared Spectroscopy the pharmaceutical and the petroleum industries. Fiber optic or conduit-type ATR probes are used in process IR spectroscopy does not only provide qualitative analytical applications, for in-line measurements in information of a sample but can also be used for laboratory as well as in industrial-scale settings. The quantification. The latter is accomplished using the well- ATR-FTIR technique is used to investigate biological known Beer’s law. This law establishes a relation between systems, especially membranes. ATR-FTIR microscopy the path length l, the concentration c, the molar decadic can be used to image human and animal tissues and has absorption coefficient ε, and the absorption A : A = lεc. also found broad application in forensics and restoration The absorption is the negative decadic logarithm of the =− science. Angle-resolved ATR-FTIR spectroscopy is used transmittance (T )ofthesample:A log10(T ). T is the

3λ Δl = nλ+ λ The largest A is measured, when the light is polarized 4 4 parallel to the dipole moment. ε For general quantitation, neither l nor has to λ be known. Instead, a calibration curve is recorded 2 by measuring the absorption of samples with known concentrations of analyte at a specific wavenumber, e.g. in a flow cell with fixed l in the case of transmission Figure 3 Interference of two sine waves depending on the measurements. As the optical path length and the phase difference. Constructive interference happens when the concentration are independent of the wavenumber, path difference l in the two arms of the interferometer is a Beer’s law also holds for integrated spectral features. whole multiple of the wavelength. Often, integrated bands are used instead of the absorption at a single wavenumber to reduce the influence of noise In contrast, an FTIR instrument generates wavelength in the spectra on the measurement results. Multivariate information in an indirect way by first collecting an inter- methods can also be used, which combine information ferogram I0(l) followed by I (l). These interferograms from different parts of the spectrum to determine the are transformed mathematically into the desired single concentrations of the analytes. A good overview of beam spectra I0 and I using the Fourier transform. While quantitation in MIR spectroscopy can be found in the many different types of interferometers exist, we will use book titled Fourier Transform Infrared Spectrometry by the Michelson interferometer (also used in the famous (12) P. R. Griffiths and J. De Haseth. Michelson–Morley experiment(14)) to explain the general principles. In this interferometer (Figure 2), light emitted 1.3.3 Fourier Transform Infrared Spectroscopy by the source is split into two parts by a beam splitter. One part is directed onto a fixed mirror, the other At of the time of writing this article, the predominant one is directed onto a moving mirror. Both parts are technique in MIR spectroscopy was Fourier transform then reflected back onto the beam splitter. Again being infrared spectroscopy. It has replaced dispersive tech- partially reflected and transmitted at the beam splitter, niques for IR spectroscopy in research, as well as in the light is then recombined and can interfere with itself. industrial applications. The offset of the moving mirror l leads to a phase In dispersive spectrometers, a dispersive element such difference between the wave reflected off the fixed mirror as a grating or prism is used to split the light into its wave- and that reflected off the moving mirror. Depending on lengths. Recording of I0 and I can be performed simul- the wavelength of the light, this causes constructive or taneously, measuring one wavelength after the other. destructive interference (Figure 3). The sample is then

Encyclopedia of Analytical Chemistry, Online © 2006–2013 John Wiley & Sons, Ltd. This article is © 2013 John Wiley & Sons, Ltd. This article was published in the Encyclopedia of Analytical Chemistry in 2013 by John Wiley & Sons, Ltd. DOI: 10.1002/9780470027318.a9287 ATTENUATED TOTAL REFLECTION FOURIER TRANSFORM INFRARED SPECTROSCOPY 5 probed by a beam that contains all wavelengths whose The length of the wave vector of a plane wave | |= π λ intensities will, however, change during the displacement propagating in a medium is given by k 2 n/ 0, where λ of the moving mirror. 0 is the vacuum wavenumber and n is the refractive index. Fourier transform instruments have two fundamental Hence, for a wave propagating in a dielectric medium of advantages when compared with similar dispersive refractive index n in the direction of the x-axis, Equation instruments.(12) The first advantage concerns the signal- (4) can be transformed to to-noise ratio (SNR). When spectra of M datapoints π λ −ω = i(2 xn/ 0 t) are measured with a Fourier transform and a dispersive E(x,t) E0e (5) instrument with equal throughput, resolution, and efficiency, the measurement time of the dispersive The attenuation of the electric field in the medium instrument must be M times that of the FT instrument to can be described by using the complex refractive achieve the same SNR. For equal measurement√ times, the index nˆ = n + ik instead of the real refractive index in SNR of the dispersive instrument will be M times that Equation (5): of the FT instrument (multiplex or Fellgett advantage). π − 2 k The second advantage of FTIR instruments is their i(2πxn/λ −ωt) λ x E(x,t) = E e 0 e 0 (6) higher optical throughput in comparison with dispersive 0 instruments (throughput advantage or Jacquinot advan- From (6) the physical quantity measured in IR tage), as a circular beam shape is accepted by FTIR spectroscopy, the light intensity I, can be calculated as instruments and such slits are no longer required. π 2 Due to the short analysis times made possible by the ε − 2 k cn 0 λ x FTIR technique and the possibility for molecular finger I(x) = E e 0 (7) 2 0 printing, FT MIR spectrometers are now found in almost all industries as tools for routine analysis to identify ε 0 is the vacuum permittivity, c the speed of light, and n incoming or outgoing chemicals and for quantification of the refractive index of the medium. Of practical interest main components of solids, as well as liquids and gases. for most measurements is the determination of the ratio of the intensity of the light before and after traversing an absorbing medium of a given length. This ratio is also = × 2 THEORETICAL ASPECTS OF frequently expressed in percentage T(%) I0 100% ATTENUATED TOTAL REFLECTION and called transmission. It only depends on the path SPECTROSCOPY length x through the medium and the absorbance index k of the medium: π − 4 k I(d) λ d = e 0 (8) 2.1 Reflection, Total Reflection, and the Attenuated I(0) Total Reflection Signal By applying the decadic logarithm to both sides of Total reflection is a special case of reflection of an Equation (8), we get the familiar expression for the electromagnetic wave at an interface between two media. absorption A: The absorbance spectra measured in ATR spectroscopy are dependent on the intensity of the reflected light I(d) 4π log (e)k A =−log = 10 d = αd(9) relative to that of the incident light. The relative intensity 10 I(0) λ of the reflected light is given by the Fresnel equations. 0 In the following section, these equations are derived. α is called the linear decadic absorption coefficient. In More in-depth explanations can be found in introductory spectroscopy, especially when measuring analytes diluted text books about electromagnetism, such as the one by in a matrix, α is replaced by a product of the molar ( ) Pollack and Stump. 15 In this work, vectors such as the decadic absorption coefficient ε and the concentration to electric field (E), the amplitude of the electric field of get Beer’s law: an electromagnetic wave (E0), the wave vector (k), and A = εcd (10) the position (x) are provided in bold font. Scalar values, e.g. time (t) and angular frequency (ω), are provided in It allows the use of spectroscopic measurements for italic font. quantitative analysis of analytes in a given sample. Generally, an electromagnetic plane wave can be Our aim in explaining how ATR spectroscopy works described by the following equation: on a theoretical level will now be to find a connection between the absorption measured in transmission setups = i(kx−ωt) E(x,t) E0e (4) (see Equation 9) and the intensity ratio between the

ω −iωt and 3 are all equal, the e term can be factored out of Equation (12). From the resulting equation again using the continuity condition x k2 i|k | sin(θ )y i|k | sin(θ )y i|k | sin(θ )y q C e 1 2 + C e 3 3 = C e 2 2 (14) y 2 1 3 2 z it follows that the components of k , k , and k in the n2 x > 0 1 2 3 n1 x < 0 yz-plane have to be equal:

|k | sin(θ ) =|k | sin(θ ) =|k | sin(θ )(15) k q q 1 1 2 2 3 3 1 1 3 k3 | |= π λ λ As kj 2 nj / 0 ( 0 being the vacuum wavelength), θ the angle of incidence 1 is equal to the angle of the θ reflected wave 3. The angle of incidence and the angle of the transmitted wave are related by Snell’s law: Figure 4 Reflection at an interface between two infrared θ = θ transparent, dielectric media (n1 >n2). n1 sin( 1) n2 sin( 2)(16) incident and the reflected light measured in ATR The x component kx of the wave vector can be spectroscopy in a form similar to Beer’s law. calculated once ky is known: Consider two IR transmissive dielectric media with = | |2 − 2 =| | θ refractive indices n1 and n2 with a common interface. A kx k ky k cos (17) plane wave traveling toward this interface at an angle θ of incidence 1 will be partially reflected and partially When the light passes from the optically denser into transmitted (Figure 4). We expect an electric field of the the optically rarer medium (n1 >n2), there are angles of following form: θ θ incidence 1 at which there are no real 2 to fulfill Snell’s law. The smallest angle for which this is true is the critical i(k2x−ω2t) E02e x>0 angle θ : E(x,t) = −ω −ω (11) c E ei(k1x 1t) + E ei(k3x 3t) x ≤ 0 n 01 03 θ = arc sin 2 (18) c n Maxwell’s equations tell us that the components of the 1 electric field (E) and the magnetic H-field tangential to the θ For angles of incidence of c or greater, no light will interface are continuous across the boundary. The same be transmitted into the lower refractive index medium, is true for the components of electric displacement field instead all the incident light is reflected. This is called (D) and the magnetic B-field orthogonal to the interface: total reflection. The amplitudes of the electric field of the reflected + = E: E1,yz E3,yz E2,yz (12a) wave can be calculated using the continuity conditions (15) H: H + H = H (12b) derived from Maxwell’s equations. We have shown 1,yz 3,yz 2,yz in the previous paragraphs that the ω are equal and ε + ε = ε D: 1E1,x 3E3,x 2E2,x (12c) the y components of the wave vectors are equal, too. B: μ H + μ H = μ H (12d) The continuity conditions can therefore be simplified to 1 1,x 3 3,x 2 2,x conditions for the amplitudes of the electric and magnetic fields: If the electric fields are viewed on one point on the interface, the continuity condition will be fulfilled for + = E: E0,1,yz E0,3,yz E0,2,yz (19a) − ω − ω − ω i 1t + i 3t = i 2t + = C1e C3e C2e (13) H: H0,1,yz H0,3yz H0,2,yz (19b) ε + ε = ε D: 1E0,1,x 3E0,3,x 2E0,2,x (19c) and appropriate C1,C2, and C3. This condition can only ω ω ω μ + μ = μ be fulfilled when 1, 2, and 3 are equal. B: 1H0,1,x 3H0,3,x 2H0,2,x (19d) When we set the plane of incidence to the xy-plane, the z components of the wave vector have to be zero. The First, we consider the case of parallel polarized light. projections of the wave vectors onto the y-vector are then This case is often indicated by the symbol ||, the index p, or | | θ ω ω of the form k1 sin( 1). As we already know, that 1, 2, the acronym TM (transverse magnetic). In this case, the

μ = magnetic field vector of the incident light is orthogonal Employing Snell’s law and j 1 for dielectric media, = = θ to the plane of incidence, H0,j,y 0 and H0,j,x 0. To 2 can be eliminated: apply the continuity conditions for the electrical field, the electrical field has to be split up into its component n cos(θ ) − n2 − n2 sin2(θ ) parallel to the interface and its component perpendicular 1 1 2 1 1 r⊥ = (26) to the interface. When the electrical field is written as a θ + 2 − 2 2 θ ˆ n1 cos( 1) n2 n1 sin ( 1) product of a directional vector (E0,j ) and its length (E0j ) = ˆ as E0,j E0,j E0,j , geometrical considerations lead to Equations (21), (22), (25), and (26) are called the Fresnel equations. From them, the ratio of the intensities (E + E )ε (θ ) = E ε (θ )() 0,1 0,3 1 sin 1 0,2 2 sin 2 20a of the incident and the reflected beam can be calculated as 2 2 R⊥ =|r⊥| and R|| =|r||| . Similarly to Equation (7), the for the components perpendicular to the interface and intensity of the light is proportional to the square of the amplitude of the electric field. For nonabsorbing media, (E − E ) cos(θ ) = E cos(θ )(20b) 0,1 0,3 1 0,2 2 light will be totally reflected for angles of incidence above the critical angle R = R = 1 (Figure 5). To calculate for the components parallel to the interface. Next, we ⊥ || the reflection at an interface to an absorbing medium, want to calculate the amplitude reflection coefficient the refractive index n in the Fresnel equations simply r = E /E of parallel polarized light as the ratio of 2 || 0,3 0,1 has to be replaced by its complex counterpart nˆ .(16) the electric field of the reflected and incident light. This 2 The relations between the intensity of the incident and can be done by solving (20a) and (20b) which results in the reflected light, expressed as reflection coefficient R, ε sin(θ ) cos(θ ) − ε sin(θ ) cos(θ ) therefore are r = 2 2 1 1 1 2 (21) || ε θ θ + ε θ θ 2 sin( 2) cos( 1) 1 sin( 1) cos( 2) 2 ˆ2 θ − ˆ2 − 2 2 θ n2 cos( 1) n1 n2 n1 sin ( 1) ε = 2 θ R = (27a) Using Snell’s law and j nj for dielectric media, 2 || ˆ2 θ + ˆ2 − 2 2 θ can be eliminated: n2 cos( 1) n1 n2 n1 sin ( 1) 2 θ − 2 − 2 2 θ n2 cos( 1) n1 n2 n1 sin ( 1) r|| = (22) 2 θ + 2 − 2 2 θ q n2 cos( 1) n1 n2 n1 sin ( 1) 1.00 c

Light polarized perpendicular to the plane of incidence is often indicated by the symbol ⊥, with the index s (German: ‘senkrecht’, orthogonal) or the acronym TE 0.75 (transverse electric). In this case, the continuity conditions for the electric ﬁeld

+ = R 0.50 E0,1 E0,3 E0,2 (23) will not be enough to determine the reflection coefficient. Instead, we have to use the continuity condition for the 0.25 magnetic field: R⊥

− θ = θ (H0,1 H0,3) cos( 1) H0,2 cos( 2)(24a) a = 0.0 cm–1 2 –1 R|| a = 10 cm + μ θ = μ θ 0.000 3 –1 (H0,1 H0,3) 1 sin( 1) H0,2 2 sin( 2)(24b) a = 10 cm a = 104 cm–1 Owing to the interdependence of the electric and 01530 45 60 75 90 = q ° the magnetic fields in electromagnetic waves, H0,j c/ (2πn /c λ μ )E , the ratio of the magnetic fields of j 0 0 j 0,j Figure 5 Reflection of light at an interface between an opti- the incident and the reflected wave equals that of their cally denser (n1 = 2.4) and optically rarer medium (n2 = 1.3). electric fields: The line styles indicate different values for the linear decadic absorption coefficient α of the optically rarer medium. Grey μ cos(θ ) sin(θ ) − μ sin(θ ) cos(θ ) r = 2 1 2 1 1 2 (25) lines denote orthogonal polarization and black lines denote ⊥ μ θ θ + μ θ θ parallel polarization. 2 cos( 1) sin( 2) 1 sin( 1) cos( 2)

in the following way: ‘The thickness de is defined as the As can be seen in the example in Figure 5, with thickness of a film of the sample material that would give increasing absorption coefficient of the sample, there is a the same absorbance for transmission at normal incidence strong change in the intensity of the reflected light. The as that obtained in the IRS experiment.’ This definition shape of the dependence of the reflection coefficient R on leads to the expression the angle of incidence also changes in case of absorbing samples. As Harrick(17) notes, ‘there is no longer a sharp A =−log (R) = αd (31) critical angle for the absorbing case as there was for 10 e the nonabsorbing one and the reflectivity curves become less steep in this region’. As can be seen from Figure 5, in analogy to transmission measurements to describe the sensitivity for changes in the absorption coefficient is the intensity of the light before and after reflection largest close to the critical angle.(17) at the interface for ATR spectra. When measurement The reason for the absorption dependence of the parameters, such as the real part of the refractive index reflection at the interface for angles above the critical of the sample and the coverage of the ATR surface are angle is the evanescent field, which stretches into the kept constant between measurements, the ratio of the rarer medium. If the angle of incidence is increased to reflectance with and without the analyte can be used to θ determine the analyte’s concentration: values greater than c, then k2,y will be an imaginary number. This means, for angles of incidence greater than I × R the critical angle, the electric field (see Equation 11) in =− 0 analyte = ε Aanalyte log10 analytecanalytede (32) the second medium takes the following form: I0 π 2 n1 π To be able to use Equations (31) and (32) practically, i y sin(θ1)−ωt − 2 2 2 θ − 2 λ0 x n sin ( 1) n = λ0 1 2 α E(x,t) E02e e (28) de has to be independent of and canalyte, respec- Hence, the electric field will follow an exponential tively, for all measured samples. For weakly absorbing = decay in the second medium (see Figure 6). samples, an approximate de can be calculated from A − π λ = A common measure for how far the field extends into [ log10(e)4 k]/ 0de log10(R) using first-order approximations in k(1,3): the optically rarer medium is the depth of penetration dp, defined as the depth at which the electrical field falls to λ θ /e 0 n21 cos( ) 1 of the electrical field at the interface. d ⊥ = (33a) e n π − 2 2 θ − 2 1/2 1 (1 n21)[sin ( ) n21] E(dp) 1 λ = (29) = 0 E(0) e de|| n1 λ d = 0 ( ) n cos(θ)[2 sin2(θ) − n2 ] p 30 × 21 21 π 2 2 θ − 2 2 2 2 n sin ( 1) n π − 2 + 2 θ − 2 θ − 2 1/2 1 2 (1 n21)[(1 n21) sin ( ) n21][sin ( ) n21] (33b)

1.0 n1 n2 = where nij ni /nj and n2 is real. The error between the linear approximations using Equations (33a) and ) y 0.5 (33b) and the exact values calculated from the Fresnel E ( E (0) equations is <10% for reflection losses smaller than 10% per reflection.(17) To measure highly absorbing samples, 0.0 larger angles of incidence can be used. Similarly, to 0.0 0.5 1.0 increase the sensitivity without sacrificing the linearity y of the measurement, multiple reflections can be used.(17) λ0 In such multireflection ATR measurements, the light Figure 6 Magnitude of the evanescent electric field in the optically rarer medium for different angles of incidence. The is reflected several times off the interface between angle of incidence increases from 35° at the nock of the arrow the optical denser and the optically rarer medium. ° to 85 at the arrowhead. n1 = 2.4andn2 = 1.3. The absorption increases linearly with the number of

a Table 1 Critical angles for common solvent (n2) –ATR material (n1) combinations

α-Al2O3 ZrO2 ZnSe KRS-5 ZnSe Cdiamond AMTIR CdTe Ge n 1.74 2.15 2.20 2.37 2.40 2.40 2.50 2.65 4.00

Methanol 1.33 50° 39° 38° 35° 34° 34° 33° 31° 20° Water 1.33 50° 39° 38° 35° 34° 34° 33° 31° 20° Ethanol 1.36 52° 40° 39° 35° 35° 35° 33° 31° 20° EtOAc 1.37 52° 40° 39° 36° 35° 35° 34° 32° 21° Hexane 1.37 53° 40° 39° 36° 35° 35° 34° 32° 21° Cyclohexane 1.42 55° 42° 41° 37° 37° 37° 35° 33° 21° DMF 1.43 56° 42° 41° 38° 37° 37° 35° 33° 21° Glycol 1.43 56° 42° 41° 38° 37° 37° 35° 33° 21° CCl4 1.46 57° 43° 42° 38° 38° 38° 36° 34° 22° DMSO 1.48 59° 44° 43° 39° 38° 38° 37° 34° 22° Toluene 1.49 60° 45° 43° 40° 39° 39° 37° 35° 22° ° ° ° ° ° ° ° ° ° C6H5Cl 1.52 62 46 44 40 40 40 38 36 23 a (20) nD values for the solvents and refractive indices for the ATR materials were taken from Coates.

x parallel to Ex and A2 measured for y parallel to Ex and K1 2 θ + 2 θ + 2 θ = x using cos ( x) cos ( y ) cos ( z) 1, the angles q θ (1,21) x i can be calculated from E q y y |E |2 cos2(θ ) + cos2(θ ) x m A z x |E |2 1 = z (38) A |E |2 y 2 cos2(θ ) + cos2(θ ) x z y |E |2 Figure 7 Sketch of the orientation of the dipole moment of a z diatomic molecule and the electric field of the incident wave for ⊥ polarization. The sample–ATR element interface is parallel The electrical fields at the interface are(1) to the paper plane. θ 2 2 θ − 2 2n1 cos( ) n1 sin ( ) n2 E = E ( ) For ⊥ polarized light (Figure 7), the electric field only x 0,|| 39a n2 − n2 (n1 + n2) 2(θ) − n2 has a component parallel to the interface; therefore, only 1 2 1 2 sin 2 μ vibrations where ∂ /∂Qi is not orthogonal to the interface 2n2 cos(θ) sin(θ) = 1 will interact with the incident radiation. If the absorption Ez E0,|| (39b) 2 − 2 1 + 2 2 θ − 2 of a sample with a marked direction (x ) is placed on the n1 n2 (n1 n2) sin ( ) n2 μ ATR and measured twice the orientation of ∂ /∂Qi in the xy-plane can be determined. The first measurement (21) Mirabella explains the experimental application is done with x parallel to the Ey field vector (Ax ),the ° of these theoretical considerations for solid samples. second one after rotating the sample by 90 (Ay ).The An in-depth introduction into the application of ATR average orientation can then be calculated from(1) spectroscopy to study the orientation of biological membranes has been written by Goormaghtigh et al.(22) 2 A cos θ x = x (37) 2 A cos θ y y 2.2.4 Refractive Indices of Sample and Attenuated Total Reflection Element The angle brackets · denote the average. Light of parallel polarization has an electric field The refractive indices of the sample and the ATR crystal parallel to the interface (Ex ) as well as perpendicular also influence the ATR spectrum. Usually, instead of to the interface (Ez) (Figure 8). Accordingly, spectra = both refractive indices only their ratio n21 n2/n1 (index collected with this polarization can give information about matching) is considered. Higher index matching will lead the orientation of dipole moment in the xz plane. When to a higher effective thickness.(17) the sample is rotated between measurements, information In general, the refractive index of the materials, sample, in three dimensions is accessible. From A1 measured for and ATR element alike is not constant, but instead shows wavelength dependence. In wavenumber regions z far from absorption bands, the refractive index changes very slowly, with a positive slope ∂n/∂ν˜ > 0 (normal dispersion). However, close to absorption bands, that is, m q close to a peak in the imaginary part of the refractive z index nˆ, there will be a much more rapid change in the q x x real part of nˆ. On the low wavenumber flank of the absorption band, the real part of the refractive index will increase significantly above the values farther from E z the band, then drop to values below those farther from the absorption band at the high wavenumber flank and E x finally increase again to values close to the starting value k1 (Figure 9). ∂n/∂ν˜ < 0 is called anomalous dispersion. The interdependence of the real and the imaginary part of nˆ (23) Figure 8 Sketch of the orientation of the dipole moment of a is described by the Kramers–Kronig relations. With diatomic molecule and the electric field of the incident wave for P denoting the principal value of the integral and n∞ polarization. denoting the refractive index at the upper integration

1.31 10 Wavenumber / 103 cm −1 51 0.5 4.5 –3 )/1 /10 v ~ 1.30 5.0 ) ( v ~ ( n 4 Ge k

1.29 0.0 3.5 2000 1800 1600 1400 1200 1000 Si GaAs v~/cm–1 Figure 9 Anomalous dispersion in the vicinity of an absorp- 3 tion band. n and k were calculated using the method of Huang (24) and Urban. / 1 n 2.5 AMTIR-1 KRS-5 Diamond ZnSe ZnS limit, these relations are 2 AgCl ∞ 2 ν˜k(ν˜) CsI n(ν˜ ) = n∞ + P dν˜ (40a) MgO a π ν˜2 − ν˜2 SiO Al O 0 a 1.5 2 2 3 ∞ BaF2 2ν˜ n(ν˜) − n∞ CaF k(ν˜ ) =− a P dν˜ (40b) MgF 2 a π ν˜2 − ν˜2 2 0 a LiF 1 0203010 The change in the real part of the refractive index λ / μm across an absorption band leads to an increase in the Figure 10 Plots of the dispersion relation of common effective thickness on the low wavenumber side of the materials for infrared optics and ATR elements (see band and a decrease in the effective thickness on the high Table 2). wavenumber side of the band. When ATR spectra are compared with spectra measured in transmission mode, The dispersion equations in Table 2 are empirical this leads to a perceived increase in the absorption on relations based on the Sellmeier equation the low wavenumber side of a band and a perceived decrease in the absorption on the high wavenumber A λ2 A λ2 n2(λ) = 1 + 1 + 2 +··· (41) side of a band. The band maximum therefore appears λ2 − λ2 λ2 − λ2 1 2 to be shifted toward lower wavenumbers. The severity of this effect also depends on the refractive index of or Sellmeier–Cauchy-like equations. In these empirical the ATR crystal n1, because in the expressions for the relations between the wavenumber and the refractive A λ effective thicknesses in Equation (33), n2 only shows index, the coefficients j and j are determined by fitting (45) up in the ratio n2/n1. A higher n1 will lead to a experimental data. lower influence of anomalous dispersion. Furthermore, In short, factors that lead to an increased effective the effects are more pronounced for steeper than for thickness are flatter angles and for parallel than for perpendicular • better (closer to unity) index matching; polarization.(17) • steeper angle of incidence; The refractive index of materials used in IR optics, • parallel polarization of the incident light; and such as ATR elements, is also wavelength dependent. • lower wavenumber. A plot of the dependence of the refractive index on the wavelength of several materials commonly The effects of anomalous dispersion on the refractive used for IR optics can be found in Figure 10. In index spectrum are bigger for addition, the dispersion equations for these materials are provided in Table 2. Optical constants for a much • better index matching; wider range of optical materials have been compiled by • a steeper angle of incidence; and Weber.(25) • parallel polarization of the incident light.

Table 2 Dispersion equations of common infrared optical materialsa Material Dispersion formula Range (μm) Source 1.4313496λ2 0.65054713λ2 5.3414021λ2 Sapphire n2 = 1 + + 0.22–5.0 26 o λ2 − (0.0726631)2 λ2 − (0.1193242)2 λ2 − (18.028251)2 1.5039759λ2 0.55069141λ2 6.59273791λ2 n2 = 1 + + + e λ2 − (0.0740288)2 λ2 − (0.1216529)2 λ2 − (20.072248)2 5.298λ2 0.6039λ2 AMTIR-1 n2 = 1 + + 1–14 27 λ2 − (0.29007)2 λ2 − (32.022)2 0.643356λ2 0.506762λ2 3.8261λ2 BaF n2 = 1 + + + 0.27–10.3 28 2 o λ2 − (0.057789)2 λ2 − (0.10968)2 λ2 − (14.3864)2 1.03961212λ2 0.231792344λ2 1.01046945λ2 N-BK7 n2 = 1 + + + 0.3–2.5 29 λ2 − 0.00600069867 λ2 − 0.0200179144 λ2 − 103.560653 0.5675888λ2 0.4710914λ2 3.8484723λ2 CaF n2 = 1 + + + 0.23–9.7 30 2 λ2 − (0.050263605)2 λ2 − (0.1003909)2 λ2 − (34.649040)2 4.3356λ2 0.3306λ2 Diamond n2 = 1 + + 0.225-∞ 31 λ2 − (0.1060)2 λ2 − (0.1750)2 0.34617251λ2 1.0080886λ2 0.28551800λ2 0.39743178λ2 CsI n2 = 1 + + + + + 0.29–50 32 λ2 − (0.0229567)2 λ2 − (0.1466)2 λ2 − (0.1830)2 λ2 − (0.2120)2 3.3605359λ2 λ2 − (161.0)2 7.4969λ2 1.9347λ2 GaAs n2 = 3.5 + + 1.4–11 33 λ2 − (0.4082)2 λ2 − (37.17)2 6.72880λ2 0.21307λ2 Ge n2 = 9.28156 + + 2–12 34, 35 λ2 − 0.44105 λ2 − 3870.1 0.92549λ2 6.96747λ2 LiF n2 = 1 + + 0.1–10 30 λ2 − (0.07376)2 λ2 − (32.79)2 0.48755108λ2 0.39875031λ2 2.3120353λ2 MgF n2 = 1 + + + 2 o λ2 − (0.04338408)2 λ2 − (0.09461442)2 λ2 − (23.793604)2 0.41344023λ2 0.50497499λ2 2.4904862λ2 n2 = 1 + + + 0.27–7 36 e λ2 − (0.03684262)2 λ2 − (0.09076162)2 λ2 − (23.793604)2 1.111033λ2 0.8460085λ2 7.808527λ2 MgO n2 = 1 + + + 0.36–5.4 37 λ2(0.0712465)2 λ2(0.1375204)2 λ2(26.89302)2 0.663044λ2 0.517852λ2 0.175912λ2 0.565380λ2 1.675299λ2 SiO α− n2 = 1 + + + + + 2( quartz) o λ2 − (0.060)2 λ2 − (0.106)2 λ2 − (0.119)2 λ2 − (8.844)2 λ2 − (20.742)2 0.665721λ2 0.503511λ2 0.214792λ2 0.539173λ2 1.807613λ2 n2 = 1 + + + + + 0.18–0.71 38 e λ2 − (0.060)2 λ2 − (0.106)2 λ2 − (0.119)2 λ2 − (8.792)2 λ2 − (197.709)2 10.66842933λ2 0.003043475λ2 1.54133408λ2 Si n2 = 1 + + + 1.36–11 39, 40 λ2 − (0.3015116485)2 λ2 − (1.13475115)2 λ2 − (1104.0)2 2.062508λ2 0.9461465λ2 4.300785λ2 AgCl n2 = 1 + + + 0.54–21.0 41 λ2 − (0.1039054)2 λ2 − (0.2438691)2 λ2 − (70.85723)2 1.8293958λ2 1.6675593λ2 1.1210424λ2 0.04513366λ2 12.380234λ2 T1[BrI] n2 = 1 + + + + + 0.58–39.4 42 λ2 − (0.150)2 λ2 − (0.250)2 λ2 − (0.350)2 λ2 − (0.450)2 λ2 − (164.59)2 0.3390426λ2 3.7606868λ2 2.7312353λ2 β-ZnS n2 = 1 + + + 0.55–10.5 43 λ2 − (0.31423026)2 λ2 − (0.1759417)2 λ2 − (33.886560)2 4.2980149λ2 0.62776557λ2 2.8955633λ2 ZnSe n2 = 1 + + + 0.55–18 43 λ2 − (1920630)2 λ2 − (0.37878260)2 λ2 − (46.994595)2 1.347091λ2 2.117788λ2 9.452943λ2 ZrO Y O n2 = 1 + + + 0.36–5.1 44 2:12% 2 3 λ2 − (0.062543)2 λ2 − (0.166739)2 λ2 − (24.320570)2 a Excerpt from Weber.(25)

3 PRACTICAL ASPECTS OF ATTENUATED beam width can lead to parts of the beam undergoing TOTAL REFLECTION SPECTROSCOPY more reflections at the interface than others,(10) which in turn leads to nonlinearities in the measurement 3.1 Materials (Figure 11). Owing to the high surface sensitivity of the ATR The material of an ATR element has a profound effect on technique, the contact between the sample and the ATR the experiment that can be performed with this element. element is very important. For solid samples, this contact Apart from a good transmission in the spectral range is often assured by pressing the sample onto the ATR of interest as well as a higher refractive index than element mechanically. In a single bounce ATR, it is the sample, ATR elements also have to be chemically easier to fully cover the smaller ‘hot’ zone of the ATR inert in the measurement environment and insoluble in element, where light can interact with the sample than the intended sample. Usable wavelength regions and in a multibounce ATR, which has N such ‘hot’ zones. solubilities of common materials for ATR elements can The Harrick SplitPea™ is one example of an ATR setup be found in Table 3. that is specifically designed to have a small ‘hot’ spot and Mechanical properties of the ATR element also thereby increase the sensitivity for the measurement of influence sampling. A hard ATR element allows to use solid samples.(16) more force to press the sample against the surface of the In multiple reflection ATR setups, either one or both element to ensure a better contact. Softer materials will sides of the ATR element can be used for measurements. allow a better contact with brittle samples. A common design of ATR setups uses a horizontally mounted ATR (HATR) element, where only the top of 3.2 Number of Reflections the element is covered with the sample. ATR accessories In the simplest ATR setup, light will be reflected off with this design are, for example, the Spectra-Tech ARK the ATR element–sample interface once. This is, for (Thermo Scientific), the Gateway (Specac), the Overhead ™ example, the case in a prism-shaped ATR element with ATR (Bruker), and the HATR and ATRmax II (Pike a triangular cross-section. If an ATR element with a Technologies). trapezoidal cross-section is used instead, the radiation will bounce off the top and bottom surface multiple times. In this case, the effective thickness of the setup increases linearly by the number of reflections. In theory, the absorption measured in a setup using unpolarized (a) (b) light with N reflections is

N N R⊥ + R|| A =−log (42) 10 2 (c) The number of reflections, however, cannot be Figure 11 Cross-section of single and multi reflection ATR increased without limit. The width of the IR beam elements. (a) Triangular, single reflection; (b) trapezoidal, increases along its path through the ATR element. A high multireflection; and (c) fiber waveguide.

Table 3 Optical and chemical properties of common materials for ATR elements

(25,46) −1 (46,47) −1 (47) Material Trivial name Usable range (cm )H2O solubility (g L ) Other solvents AgBr 20 000–300 8.4 × 10−5 NaCl α-Alumina Sapphire 55 000–1800 ≈0 Bases Ge33As12 Se55 AMTIR-1 11 000–750 ≈0 BaF2 50 000–840 1.2 CaF2 50 000–1140 ≈0 Cdiamond Diamond 45 000–2500 and 1650 to < 200 ≈0 Ge 5500–600 ≈0 GaAs 11 000–580 ≈ ® MgO Irtran-5 62 500–1100 0.0062 Acids, NH4 salts SiO2 Quartz 55 000–4000 Si Silicon 8300–660 ≈0HF+ HNO3 TlBr-TlI KRS-5® 20 000–250 0.05 β-ZnS Cleartran 17 000–833 6.5 × 10−4 Acid

Thermocouple

Sample in Sample out

Reflecting cone

Optical rays

ATR element Figure 12 Cylindrical TNL-130H CIR element (Axiom Analytical Inc.) for use with FTIR spectrometers. (Reprinted with permission of Axiom Analytical Inc.)

A special ATR element type designed for multire- the measurement, and by forming the fiber into a loop, flection measurements using a FTIR spectrometer is the number of reflections can further be increased.(49) cylindrical in shape with conical ends. Focusing elements, Selective layers can be used to enrich the analyte close such as parabolic mirrors, are used at the ends of the rods to the surface of the fiber and thereby increase the to introduce light into the ATR element and reshape the sensitivity.(50) Like ATR elements, fiber evanescent wave exiting beam to conform to the expected beam path in a sensors (FEWSs) can be employed in flow cell setups, as spectrometer. These cylindrical internal reflection (CIR) well as in remote setups, where additional fibers guide elements are usually used for the measurement of liquids. the light from the light source to the FEWS and back to For that end, they have to be enveloped in a flow cell. the detector.(49) It is possible to seal the flow cell toward the cylindrical element using o-rings without reducing the light intensity 3.3 Sample Preparation and Measurement Protocols significantly. Axiom Analytical offers a special type of 3.3.1 Liquid Samples CIR element that is specifically designed for reproducible measurements of liquids. This is achieved by restricting Liquid samples can be measured either in a flow cell or the angles of incidence to a small range (Figure 12).(48) by using a trough. A flow cell makes the use of an ATR Thick fiber waveguides are similar to long cylindrical in an automated analytical system, such as a sequential ATR elements. Light travels along the waveguide and is injection analysis (SIA) or flow injection analysis (FIA) totally reflected at the interface between the waveguide system possible. But even when the samples are injected and air or sample as long as the angle of incidence is into the flow cell manually, a flow cell offers the additional larger than the critical angle. They are based on IR benefit of precluding sample evaporation. Flow cells are transparent fibers, such as chalcogenide glasses, fluoride cleaned in between samples by flushing with solvent glasses, silica-based glasses, thallium halides, and silver instead of wiping and applying solvent several times as in halides.(49) Like in an ATR setup, the interaction between trough-type sampling equipment. Another reason to use the sample and the light happens via the evanescent field. a flow cell is when many similar samples are measured Generally, fiber waveguides show a higher sensitivity and only the difference in the absorption between the than ATR sensors as they have more reflections than pure solvent and sample dissolved in the solvent is of an ATR sensor. This sensitivity can be further increased interest. by changing the shape of the fiber: a tapered fiber will To assure a uniform attenuation of all of the incident show more reflections on the same length of the fiber, light, it is important to that flow cells are kept bending the fiber decreases the angle of incidence in completely free of air bubbles. If this is not achieved, the fiber and thereby increases the effective thickness of nonlinearities in the recorded absorption spectra will

3.3.2 Solid Samples A (ν˜ )/A (ν˜ ) ν˜ Trans 2 Trans 1 = 2 ν˜ ν˜ ν˜ AATR( 2)/AATR( 1) 1 For solid samples, a good contact of the sample and the ATR element’s surface is not as easy to guarantee as When a clamp is used to improve the contact between for liquids. The contact can be improved either by using the ATR element and the sample, background spectra a mechanical clamp to press the sample onto the ATR are still collected from an empty ATR element that is not element, by melting the sample and letting it solidify as touched by the clamp. Brittle samples should be ground a film on top of the ATR element or by spin-coating the before measurement. In any case, the ATR element ATR with the analyte. The contact between the ATR should be fully covered with the sample to obtain best element and the sample can also be improved by adding results. a soft, high index of refraction material (e.g. AgCl and Even if all the steps mentioned earlier are considered, AgBr) between the ATR element and the sample.(19) The the contact between the ATR element and the sample most common method to improve the contact is, however, can vary slightly from measurement to measurement. This will impair the use of the ATR spectra for quantitative using a mechanical clamp that presses the sample onto measurements. A common solution to this problem is the ATR element (Figure 13). to look for a band that should be constant across To check whether a good optical contact has been estab- all samples of the measurement series (e.g. backbone lished, Mirabella(19) suggests to compare the absorptions || ⊥ ° bands in polymers and filler substances in pharmaceutics) at and polarization for a 45 angle of incidence. At and then use the ratio of this band and the band of this angle of incidence, A is twice as large as A⊥. Ekgasit interest for quantification.(19) Application of pressure to (51) and Padermshoke propose to compare the normal- a solid sample, however, can cause additional difficulties. || ⊥ ized absorption spectra collected at and polarization. This can be the case when analyzing crystalline samples When there is no air gap, these spectra should be alike. A which change their crystal structure upon application of (51) second method proposed by Ekgasit and Padermshoke pressure. Therefore, in case of such polymorph samples, is to compare a normalized ATR absorption spectrum of special attention has to be paid to assure proper spectrum the sample recorded at any incidence with a transmis- acquisition. sion spectrum of the sample. For two wavenumbers, the ratio of the absorption in the transmission spectra ν˜ ν˜ 3.3.3 Contaminants ATrans( 2)/ATrans( 1) and the ratio of the absorption in ν˜ ν˜ the ATR spectra AATR( 2)/AATR( 1) should be equal to The surface sensitivity of the ATR technique makes the ratio of the compared wavenumbers: it especially sensitive to measurement errors due to

Laboratory grease

Carbon dioxide Nitrile glove

Finger grease

PTFE

Ethanol Water vapour Absorption/a.u. Absorption/a.u.

2-propanol

Water

4000 3500 3000 2500 2000 1500 1000 4000 3500 3000 2500 2000 1500 1000 –1 Wavenumbers/cm–1 Wavenumbers/cm Figure 14 Spectra of several contaminants in ATR measure- Figure 15 MIR transmission spectra of CO2 and water vapor. ments. The spectra were collected on a MIRacle single (Created using data from Ref. 52.) reflection diamond ATR. (Reprinted with permission of PIKE Technologies.) Depending on the design of the ATR setup, some contamination of the surface of the ATR element. These further restrictions concerning possible cleaning tools contaminations can be caused by insufficient cleaning and protocols that go beyond those imposed by the protocols or inadvertent touching of the ATR element. mechanical and chemical properties of the ATR may Griffiths and De Haseth(12) give as common contaminants apply. For ATR units where a glue used to hold the ATR finger grease, hand lotion, talc, plasticizers, and slip element in place is the fact that such a glue will most agents from laboratory gloves as well as silicon grease. likely be more sensitive to organic solvents than the ATR Furthermore, all fluids that come into contact with the element itself has to be considered. If the materials used ATR element have to be expected as contaminants. Also, for the ATR accessory and their chemical and mechanical a combination of otherwise unsuspicious things, such as resistance are not known, it is best to consult the vendor solvents and cleaning procedures, can cause errors that for recommended cleaning procedures. are hard to trace. Furthermore, when the sample does not fully cover the surface of the ATR element, bands caused by the material of the clamp will also show up in the 3.4 Angle of Incidence spectrum. The spectra of several important contaminants are shown in Figure 14. The plane wave view of the ATR signal generation used Gases in the beam path will also interfere with the in Section 2 does not represent the complete picture. In measurement when their local concentration changes a normal ATR setup, there will be a range of angles between measurements. Two gases whose bands often of incidence at once. However, as different angles of incidence lead to different effective thicknesses, a wider show up in MIR spectra are water vapor and CO2 (Figure 15). range of angles of incidence will decrease the range of concentrations at which the ATR signal is approximately linearly dependent on the concentration of the analyte.(48) 3.3.4 Cleaning Attenuated Total Reflection Elements In ATR setups with a small ‘hot’ spot, as mentioned Although most materials used to fabricate ATR elements, in Section 3.2, a wider range of angles of incidence is such as germanium, zinc selenide, or diamond, are accepted in trade for a higher sensitivity and a lower relatively robust, ATR elements are nonetheless sensitive sample consumption. In these setups, the IR beam is optical instruments and should be treated as such. focused onto the interface between the sample and the Generally, a combination of washing or flushing with ATR element. For more sophisticated applications, such fitting solvents and wiping with optical tissues is a as the determination of the complex refractive index recommended cleaning procedure. However, depending spectra of samples or for depth profiling, ATR setups on the fabrication of the ATR accessory, cleaning using an with a narrow range of angles of incidence are necessary. ultrasound bath might also be possible. For dry powders Finally, many vendors offer variable angle ATR setups that do not stick to the surface of the ATR element, a soft where the angle of incidence can be selected by the user brush can be used as a cleaning implement. (Figures 16 and 17).

ATR element. These modiﬁcations can increase the sensitivity by either increasing the electromagnetic ﬁeld or selectively enriching the analyte in close vicinity of the surface of the ATR element.

3.5.1 Attenuated Total Reﬂection–Surface-enhanced Infrared Spectroscopy

The intensity of the absorption bands in IR measurements can be enhanced by introducing certain metal substrates into the sample or depositing metal island films of a few nanometers on ATR elements before application of the sample.(53,54) This SEIRA effect is caused by two Figure 16 Optical layout of the VeeMAX II variable angle different mechanisms, an electromagnetic and a chemical accessory with ATR. (Reprinted with permission of PIKE mechanism. The electromagnetic mechanism of SEIRA Technologies.) works through the increase in the electric field of the incident light close to the metal substrate. This increase in turn leads to an increase in the absorption.(54) For ATR geometries, only the electric field parallel to the surface will be enhanced.(54) The chemical enhancement μ is in part due to orientational effects that align ∂ /∂Qi with the enhanced electric field vector. At the time of writing, the sensitivity of measurements when using SEIRA can be increased by one to two orders of magnitude. This is clearly smaller than the enhancement factor found in surface-enhanced Raman spectroscopy (SERS). While SEIRA has shown itself to be a decisive advantage in advanced scientific experiments, so far ATR- FTIR/SEIRAS is not developed enough for routine use.

3.5.2 Selective Layers

Owing to the surface sensitivity of the ATR technique, the sensitivity of an ATR measurement for one analyte or a group of analytes in a sample can be increased by selectively enriching them close to the surface of the ATR element. This can be achieved by placing a film on top of the ATR element that shows a higher affinity to the sample than to the matrix components. Several different types of such layers have been reported in the literature. Polymer layers with a thickness of a few micrometers have been shown to increase the sensitivity for the detection of organic analytes, such Figure 17 Photograph of the VeeMAX II with ATR and one (55,56) of the exchangeable ATR elements. (Reprinted with permission as chlorinated hydrocarbons, in aqueous matrices of PIKE Technologies.) by several orders of magnitude. Molecularly imprinted polymers(57) (MIPs) are tailor-made to enrich one specific analyte.(58) Dedicated sol-gels can also be used as 3.5 Surface Modification enrichment layers in ATR spectroscopy.(59) Zeolites have The sensitivity and the selectivity of ATR measurements been shown to allow the detection of gaseous analytes can be enhanced by modifying the surface of the using the ATR technique.(60,61)

4 DIFFERENCES BETWEEN ATTENUATED 300 TOTAL REFLECTION SPECTROSCOPY AND TRANSMISSION SPECTROSCOPY 200 4.1 Comparison of Attenuated Total Reﬂection Spectra and Transmission Spectra 100

As stated in the previous section, Beer’s law is valid Absorption/mAU for ATR spectra, when certain constraints are met. For 0 spectroscopists, it is, however, not only the intensity 1600 1500 1400 of the absorption band that contains information but v~/cm–1 also its shape, position, and relative sensitivity. All these properties can differ between transmission and Figure 19 Calculated ATR spectra for different refractive ATR spectra due to the different mechanisms with indices of the ATR element n1. Along the arrow, n1 increases from 2 to 4. The grey line marks the position of the band which the optical constants of the sample are converted maximum in a transmission setup. When n1 increases, the height into absorption spectra. And while the name ‘effective of the band decreases. Owing to anomalous dispersion, the thickness’ may evoke the image of a transmission cell of maximum of the band is shifted toward lower wavenumbers in a deﬁned thickness, it has been shown in the earlier the ATR spectra. sections to be only a theoretical construct that will change across a spectrum depending on the wavenumber and the refractive index of the sample and the ATR element. A visualization of the differences between the 400 absorptions measured in transmission (when reﬂections at the interfaces are neglected) and ATR spectra can be found in Figure 18. With increasing wavenumber, the 200

intensity of the bands decreases. As mentioned before, Absorption/mAU an increase in the refractive index of the ATR element will lead to a decrease in the magnitude of absorption 0 bands (Figure 19). In ATR spectra, due to anomalous 1600 1500 1400 dispersion, an increase in the absorption at one band v~/cm–1 will lead to a shift of the band maximum (Figure 20). Figure 20 Calculated ATR spectra for different magnitudes This is another good reason to use integrals of spectral of the absorption. Along the arrow, α increases from 0 to − features for quantitative ATR spectroscopy, in addition 0.2 μm 1. The grey line marks the position of the band maximum to the already mentioned improvement of the SNR. The in a transmission setup. While the height of the band in the ATR spectra increases when α increases, the band maximum also shifts due to an increase in the anomalous dispersion. 150 Interactions between molecules have been neglected in this example.

100 shift of the maximum will have a lower impact on the integral of the band than on the absorption measured at one wavenumber. 50 In transmission measurements, reflections at the Absorption/mAU interfaces between different materials, e.g. between 0 the window and a sample solution, will give rise to 2500 2000 1500 1000 interference fringes that depend on the refractive index (17) v~/cm–1 of the materials and the length of the flow cell. While Figure 18 Simulated spectra for a transmission setup these fringes are often used to accurately determine the ° (l = 2.5 μm) and for a n1 = 2.4andθ = 45 ATR setup. The optical path length of the flow cell, they also make the spectrum for a transmission setup is plotted in black, the ATR determination of absolute absorption spectra impossible spectrum for parallel polarization is denoted by a dark grey line, in transmission setups. Usually, liquids with known optical and that for perpendicular polarization is denoted by a lighter constants and low absorption are used as a reference when grey line. The intensities of the band in the ATR spectra visibly increase with decreasing wavenumber. The spectrum collected absolute values are required. These fringes cannot occur with parallel polarization shows more intense bands than that in ATR measurements, even when the sample is a thin collected with perpendicular polarization. layer.

4.2 Sample Preparation soft solid samples, it is often enough to press the samples against the ATR element using a mechanical clamp. As of today the ATR technique has almost completely replaced sampling techniques which required cumber- some sample pre-treatment. This is why many young scientists will learn of these legacy techniques in theory 5 APPLICATIONS only or not at all. For solid samples that are not thin or smooth enough to be used directly in a transmission setup, there are several 5.1 Biological and Medical Samples ways of sample preparation that can be used to bring the samples in an appropriate form. The most common The MIR region of the spectrum gives a wide range ones(62) are dissolution; grinding and mulling with a fluid; of information about biological samples. This informa- grinding and pressing into a disk together with KBr; tion includes the secondary structure, flexibility, and (64) (65) remelting; pressing or casting into films; or pyrolysis. function of proteins, the properties of lipids and (66) When using the mulling method, samples are mixed membranes, and conformational transitions and base (67) with a mulling fluid that has a refractive index similar to pairing in nucleic acids. The combination of strong the sample to reduce scattering and then spread between absorption of water, which is the predominant solvent two windows. The mulling fluid introduces additional for biological samples, and the presence of particles in bands in the spectrum, therefore, when a complete MIR biological samples both are good reasons to prefer ATR spectrum of the sample is needed, mulls in two different measurements to transmission measurements for biolog- fluids have to be prepared. ical samples. As in the preparation for mulls, the sample has to be ATR techniques have been applied to blood and ground for the preparation of KBr disks. The ground plasma samples to determine clinically important param- sample is mixed with KBr. This mixture is then pressed eters. In blood, the hematocrit could be determined; in to a small disk using an evacuated press. This disk is plasma, cholesterol, glucose, total protein, triglycerides, (68) placed in a holder to collect transmission spectra. The urea, and uric acid could be quantified. The concentra- KBr disk technique is quantitative. For hard, inorganic tion of urea, creatinine, uric acid, sulfate, phosphate, and samples, a few drops of ethanol can be added to make pH could be determined in urine samples with accuracies ( ) the grinding easier. Transmission spectra collected with sufficient for clinical use. 68 the KBr disk technique will probably contain water bands It was shown that cells can be classified into benign, and some samples may undergo crystalline modification hyperplasia, and malignant using ATR microscopy. In due to application of pressure. KBr disks can be kept in a contrast to transmission methods, ATR spectroscopy desiccator for later examination. allows to measure tissue sections on IR intransparent If a sample has a low enough melting point, it can be glass slides.(68) smeared or pressed into a thin film on a warmed window. As the ATR element will be in contact with the sample, Soft samples can be pressed between two windows to attention has to be paid to possible interactions between reduce their thickness to an acceptable value. If the the surface and sensitive biosamples. Nevertheless, ATR sample can be dissolved in a solvent that is easily microscopy has been shown to be a viable tool to evaporated, it can be cast into a film on a window. For investigate colonies of microorganisms.(69) very small samples or samples that cannot be adequately ATR spectroscopy can also be employed to determine pressed to a film between two windows, a diamond anvil the orientation of functional groups in membranes.(22) cell(63) can be used. In this type of cell, two diamonds are The angle of the dipole moment can be determined used to compress the sample. These diamonds are then from the ordering parameter, also used as windows for the transmission measurement. Finally, sample pyrolysis can be used as a pretreatment cos2() + 1 S = (43) step. A part of the sample is placed in a glass tube and 2 heated until it is decomposed. The residue, the pyrolyzate, is then scraped out of the tube and analyzed. As the which is related to the dichroic ratio: residue differs from the sample, special reference spectra of pyrolyzates have to be used for identification. The 2 2 A|| E E 3S gasses produced during pyrolyzation can also be collected RATR = = x + z 1 + (44) 2 2 − to measure their IR spectra. A⊥ Ey Ey 1 S In contrast, the sample preparation for ATR spectroscopy is rather straightforward (see Section 3.3). For The coordinate system is oriented as in Figures 7 and 8.

5.2 Process Analytical Chemistry Methods used in process analytical chemistry are different to those of laboratory analysis in many ways. Process Optical rays analytical methods are optimized for speed, avoid manual sample handling and will often rely on sensor type measurements. Process analytical methods need to be robust enough to withstand industrial environments (vibrations, temperature changes, solvents, ...) and deliver Znse art coupling a predictable, constant performance over a long time. element Process analytical methods can be classified by their integration into the process into Spring loaded seal • in-line: the sensor measures in the main process stream; • on-line: the sensor measures in a side stream, sample preparation steps can be included; Diamond element • at-line: the sensor is located close to the process stream, sampling and sample preparations is done manually; and • off-line: measurements are performed at a central Air gap laboratory. DMD-270 diamond atr probe ATR based methods can be used with all four of these Figure 21 DMD-270 diamond ATR probe (Axiom Analytical degrees of integration. For off-line and at-line sampling, Inc.) that can be used as an in-line sensor for process analytical chemistry. (Reprinted with permission of Axiom the common laboratory ATR accessories can be used. Analytical Inc.) This approach is often used for rapid identification of materials. For almost real-time monitoring of chemical processes, on-line sampling ATR flow cells can be depends on the wavenumber of the light.(71) Silver halide employed. All parts of the flow cell – ATR element fibers are fit for broadband MIR transmission. itself, o-rings, glues, the holder of the ATR element – Optical conduits are made out of pipes with a reflective have to be chemically inert toward the chemicals they coating on the inside to guide the light.(72) The light come in contact with. Sensors placed on-line can usually propagating in this pipe is approximately collimated, light be disconnected from the process for cleaning and that deviates too far from a beam path collinear with maintenance without shutting down the process. pipe is reflected back into the pipe by the reflective Specially designed ATR probes can also be placed coating. Corner pieces with off-axis mirrors can be used in-line for real-time process monitoring. There are two to change the direction of the light. Those arrangements common designs for ATR in-line sampling, one uses a allow to bridge a distance of up to 2 m between the cylindrical ATR element at the end of a cylindrical metal spectrometer and the ATR sensor element. Optical rod, the other places a flat ATR element at the end conduits require a more elaborate alignment each time of this rod. Light guides in the metal rod guide the IR they are put in place as compared to fiber optic probes. radiation to the ATR element. In the case of a CIR Furthermore, optical conduits also require a dry air purge sensor, a reflector placed at the opposite end of the CIR for optimal performance, the higher effort in alignment element reflects light back into a second light guide in and maintenance is, however, offset by a broader spectral the metal rod. In the case of a flat element, the beam coverage. path in the ATR element leads from one guiding element to the other with one or several reflections at the ATR element–sample interface (Figure 21). As spectrometers 5.3 Microscopy are usually placed 2 m from the probe’s tip, the light has to transverse a large distance. The Rayleigh criterion describes the resolution (i.e. To guide the light from the spectrometer to the ATR the minimum distance between two points to resolve sensor and back, either optical fibers(71) or hollow optical them as two distinct points) in microscopes: d = (72) λ θ conduits can be employed. Light is confined inside the 1.220 /n1 sin( ). n1 is the refractive index of the medium fiber through total reflection. The attenuation in MIR thesampleisimmersedin,θ is the angle of the most waveguides is in the range of 0.1–10 dB m−1 and strongly extreme ray. Therefore, placing a high refractive index

ATR element on the sample can significantly improve the the sample and the ATR element, measurement positions spatial resolution of the measurement. on soft materials should be placed far enough apart, so For IR microscopy using the ATR technique, an ATR that the indentations formed by the ATR elements do element is placed on the sample in the beam path of not overlap. In ATR microscopic measurements of wet or an IR microscope. In first experiments by Nakano and sticky samples (e.g. tissue sections), the cleanliness of the Kawata,(73) a germanium hemisphere was placed onto the ATR element should be checked after each measurement, sample. An off-axis mirror was used to direct light coming as traces of the sample can be stuck to the surface from the interferometer toward the interface between the and lead to contamination and errors in the following sample and the ATR element. Cassegrain optics were measurements. employed to collect the reflected light and focus it onto To acquire two-dimensional (2-D) maps without the detector. A piezo stage was used to move the sample moving the ATR across the sample, a focal plane across a range of about 100 μm to collect scans of the array (FPA) detector can be used. FPAs are a type sample. As Nakano and Kawata noted, only scanning the of detector consisting of several single detecting elements sample without adjusting the optics meant that larger scan that are arrayed in a grid and read out separately(12) ranges would lead to optical aberrations. much like the charge coupled device (CCD) in a digital A different approach for IR microscopy is now camera. An image of the sample in contact with the taken by several vendors of commercially available ATR element is formed on the FPA and a spectrum ATR objectives for modern IR microscopes (Figure 22). is collected for each individual pixel of the FPA. These can be used to view the sample through the These spectra then correspond to different sections of optical beam path as well as taking ATR measurements. the sample. When using a germanium ATR element, For viewing the sample between measurements, the a wavelength-dependent spatial resolution of 2–4 μm ATR element can be retracted into the objective. For can be achieved. Alternatively, a single reflection ATR measurements, it is extended and then pressed onto element in combination with a FPA detector can also the sample with a predefined force. To measure several be used for imaging of samples. Using this macro positions, the sample is first shifted vertically away from imaging approach, a spatial resolution of about 18 μm the ATR element, then translated sideways so that has been demonstrated.(74) Samples imaged with this the next measurement spot comes to rest below the macro imaging approach are polymers, biological and ATR objective, finally it is moved upward again for pharmaceutical materials, as well as microfluidic systems the measurement. In these objectives, the position of the placed on top of the ATR element. ATR element relative to the optics is the same for all measurement positions, the scan range is not limited by the optics. Movements of the sample are controlled by a 5.4 Depth Profiling computer controlled stage. The ATR element will leave visible deformations in soft The strong dependence of the evanescent field on the samples. Therefore, to guarantee an even contact between distance from the interface between sample and ATR element already suggests that this technique can be used for depth profiling. By decreasing the angle of incidence, the evanescent field can be extended further into the sample. There are several ways to calculate depth profiles – profiles of either spectra or concentrations with respect to the distance from the ATR element–sample interface. Ekgasit and Ishida(75) explain the use of the inverse Laplace transform and iterative methods using either a multifrequency approach or a multiangle of incidence approach. For the inverse Laplace transform, several ATR spectra at different known angles of incident with either ⊥ or || polarization have to be collected. These spectra can be converted to depth profiles using the inverse Laplace transform. For both iterative methods, the samples are considered to consist of several isotropic horizontal layers. Each of these layers has an unknown spectrum of complex Figure 22 Bruker 20× ATR objective. refractive indices n(ˆ ν˜). These are to be determined.

For the iterative method using a multifrequency light onto the interface could be set. Films of thicknesses approach, an ATR spectrum collected at a known angle between 240 and 400 nm could be resolved. of incidence as well as the optical constant spectra of the analytes in the sample are needed. Several wavelengths are selected by the user. From these wavelengths and the expressions for the evanescent field, a matrix equation 6 VENDORS connecting the molar fractions of the constituents in each of the layers of the sample and the measured absorptions In this section, an overview over vendors of ATR is built. As the mean squared electrical field (MSEF) accessories is given. The information was either supplied needed for this equation depends on the optical constants by the vendors themselves or taken from their respective of the layers, this equation has to be solved iteratively. Web pages. Web addresses and general information about The drawback of this method is that n(ˆ ν˜) has to be the vendors can be found in Table 4. An overview about known for all constituents of the samples. This can the types of ATR equipment available is given in Table 5. be challenging as these optical constants can often not be found in the literature. Furthermore, in mixtures, the interactions between the constituents can cause the optical constants of the mixture to deviate from the sum 7 FURTHER READING of the optical constants of the constituents. This effect can be ameliorated by excluding bands of the analytes, The other articles on IR spectroscopy in the Encyclopedia which are known to shift or broaden in mixtures from the of Analytical Chemistry offer the reader an overview calculations. of other aspects, theoretical as well as practical, of IR For the iterative method using a multiple angle spectroscopy. approach, several ATR spectra collected at different Concise information about ATR-FTIR spectroscopy known angles of incidence are needed. For each of can be found in the chapters ‘Principles, Theory and the measured wavenumbers, a system of equations Practice of Internal Reflection Spectroscopy’,(79) ‘Depth connecting the absorption with the optical constants Profiling by ATR’,(75) and ‘Macro and Micro Internal can be built. Starting from an estimate for the MSEF, Reflection Accessories’(11) of the Handbook of Vibra- the refractive indices are calculated. Then – iteratively – tional Spectroscopy.(80) More extensive information about the values for the refractive indices and the MSEF are the theory and application of ATR spectroscopy can be improved. As this method does not need reference values found in Mirabella’s Internal Reflection Spectroscopy(81) for n(ˆ ν˜) of the components, interactions between the and Harrick’s Internal Reflection Spectroscopy.(17) A very constituents will not cause problems. recent monograph about the theoretical underpinnings Kazarian et al.(76–78) showed a combination of ATR- of ATR spectroscopy is Internal Reflection and ATR FTIR depth profiling and imaging to get the three- Spectroscopy.(1) dimensional chemical information of their sample. They The FTIR technique gets an exhaustive treatment in employed an FPA detector and an ATR of a triangular Fourier Transform Infrared Spectrometry(12) by Griffiths cross-section. A slit was placed in the beam path between and De Haseth. the lens focusing light onto the ATR element–sample A good resource for tables of physical properties of interface. By moving this slit, the angle of incidence of the optical materials is the Handbook of Optical Materials.(25)

Table 4 Overview of vendors of ATR equipmenta Web site Spectrometer Compatible Agilent Technologies www.home.agilent.com Yes Axiom Analytical www.goaxiom.com No a.m.m. Bruker Optics www.brukeroptics.com Yes Harrick Scientific www.harricksci.com No a.m.m. Mettler-Toledo www.mt.com Yes Perkin-Elmer www.perkinelmer.com Yes PIKE Technologies www.piketech.com No a.m.m. Smiths Detections www.smithsdetection.com Specac www.specac.com No a.m.m. Thermo Fisher Scientific www.thermofisher.com Yes

a a.m.m., all major manufacturers.

Table 5 Types of ATR accessories available from vendors at the time of writinga HATR HATR Variable CIR Probe Microscope Portable (single) (multi) CIR angle Agilent ••• Axiom •• Bruker •• • ••• Harrick •• • • Mettler-Toledo • Perkin-Elmer •• • Pike •• • • Smiths Detections • SpecAc •• • Thermo Fisher •• • •••

a Information is either from personal communication or from the vendor’s Web pages.

8 FUTURE TRENDS

8.1 Hand-held Attenuated Total Reflection Instruments The ATR technique can be used to measure almost any sample that can be brought in contact with the ATR element. As the ATR element is usually connected to a rather bulky spectrometer, it is necessary to bring the sample to the ATR element. Movable ATR probes, such as the Axiom Dipper allow a little more flexibility. Recently, portable ATR instruments have become available. The Thermo Scientific TruDefender FT (Figure 23) is a hand-held (1.3 kg, battery powered) FTIR spectrometer with a diamond ATR element for sampling. The instrument is optimized for portable use. Diamond ATR spectra are collected with a carbon film heated to 750°C as a light source and an uncooled DLaTGS detector. The spectral range of the instrument is 4000–650 cm−1 with a resolution of 4cm−1. The TruDefender is used by firemen, military personnel, and law enforcement to identify unknown substances. The users themselves are not expected to know about spectroscopy; instead, the instrument uses spectral databases and algorithms for identification. The instrument is also employed as a tool for quality control Figure 23 Photograph of the TruDefender hand-held in industrial applications. ATR-FTIR spectrometer. (Reprinted with permission of Similar portable ATR-FTIR spectrometers are avail- Analyticon Instruments.) able from Smiths Detection and Bruker Optics. In the future, we expect a further miniaturization of ATR constructed in a dedicated fashion using microfabrication spectrometers, made possible by micro-opto-electro- techniques. mechanical systems (MOEMS), new tunable laser light Waveguides fabricated using microfabrication tech- source and new, smaller detectors. niques are usually planar. This makes it easier to grow them on flat substrates, such as wavers, and allows to 8.2 Waveguide Sensors use established techniques used in the fabrication of inte- An evanescent field-based sensor type that has recently grated devices, such as quantum cascade lasers (QCLs). been investigated by several groups for the use in MIR In the simplest case, these waveguides consist of three sensors are waveguide sensors. These sensors can either layers: a substrate layer, a guiding layer, and a cover be built by modifying fiber waveguides (Section 3.2) or layer. In waveguide sensors, the cover layer is usually the

Infrared Spectroscopy (Volume 12) ACKNOWLEDGMENTS Interpretation of Infrared Spectra, A Practical Approach • Quantitative Analysis, Infrared • Spectral Databases, The authors would like to thank Anette Fey of Analyticon Infrared Instruments, David Pfeiffer of Thermo Fisher Scientific, Infrared Spectroscopy (Suppl 2) Jenni Briggs of Pike Technologies, Steven W. Sharpe Two-dimensional Optical Spectroscopy: Theory and of the Pacific Northwest National Laboratories and Experiment • Liquid Chromatography-infrared and Mike Doyle of Axiom Analytical for their time and Size Exclusion Chromatography-infrared Analysis for assistance. Engelene Chrysostome, Karin Wieland, and Polymer Characterization • Infrared Sensors Andrea Ramer served as proof readers and test audience. Financial support was provided by the Austrian research funding association (FFG) under the scope of the COMET program within the research network ‘‘Process REFERENCES Analytical Chemistry (PAC)’’. 1. M. Milosevic, Internal Reflection and ATR Spectroscopy, John Wiley & Sons, Hoboken, N.J., 264, 2012. ABBREVIATIONS AND ACRONYMS 2. I. Newton, Opticks: or, A Treatise of the Reflections, Refractions, Inflections and Colours of Light, 4th edition, ATR-FTIR Attenuated Total Reflection Fourier W. and J. Innys, London, 1730. Transform Infrared 3. E.E. Hall, ‘The Penetration of Totally Reflected Light into ATR Attenuated Total Reflection the Rarer Medium’, Phys. Rev., 15, 73–106 (1902). CCD Charge Coupled Device 4. F.M. Mirabella, ‘History of Internal Reflection CIR Cylindrical Internal Reflection Spectroscopy’, in Internal Reflection Spectroscopy,ed. FEWSs Fiber Evanescent Wave Sensors F.M. Mirabella, Marcel Dekker, Inc., New York, 1–16, FIA Flow Injection Analysis Chapter 1, 1993.

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