ABSTRACT REVERSED PHASE LIQUID CHROMATOGRAPHY USING THE IONIC LIQUID ISOPROPYLAMMONIUM FORMATE AND COMPARISON OF INDIRECT SPECTROPHOTOMETRIC METHODS FOR PHOSPHATE by Matthew P. Collins A new room temperature ionic liquid, isopropylammonium formate, IPAF, has been synthesized. Its application as a mobile phase replacement for traditional organic reversed phase-high performance liquid chromatography solvents is demonstrated. Solvent characteristics of IPAF, such as, polarity index, viscosity, and solvent strength are measured. Various van Deemter plots using different mixing approaches of the mobile phase and different columns are compared. The utility of IPAF has been further shown to be a suitable modifier solvent for the separation of cytochrome c from tryptophan. At 50°C, IPAF was able to maintain cytochrome c in its native form during the course of a chromatography separation, while a similar methanol modifier causes denaturation. Our long term goal is to develop a semi-preparative method to separate proteins in their native conformation. A comparison of atomic absorption and molecular spectrophotometry for the use in determining phosphate in an undergraduate teaching environment is also presented. REVERSED PHASE LIQUID CHROMATOGRAPHY USING THE IONIC LIQUID ISOPROPYLAMMONIUM FORMATE AND COMPARISON OF INDIRECT SPECTROPHOTOMETRIC METHODS FOR PHOSPHATE A Thesis Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Master of Science Department of Chemistry and Biochemistry by Matthew P. Collins Miami University Oxford, OH 2011 Advisor____________________ Dr. Neil D. Danielson Reader____________________ Dr. James A. Cox Reader____________________ Dr. Michael Novak Reader____________________ Dr. C. Scott Hartley TABLE OF CONTENTS Page List of Tables v List of Figures vi List of Abbreviations ix Dedications x Acknowledgements x CHAPTER 1 Introduction 1 Section 1 A: General Chromatography 1 Section 1 B: Reversed Phase High Performance Chromatography 8 Section 1 C: Room Temperature Ionic Liquids 12 Section 1 D: Specific Aims 16 References 17 CHAPTER 2 Isopropylammonium Formate as a Mobile Phase Modifier in Liquid Chromatography Section 2 A: Introduction 19 Section 2 B: Experimental 21 2 B 1: Reagents 22 2 B 2: Ionic Liquid Synthesis 22 2 B 3:Chromatographic Conditions 23 Section 2 C: Results and Discussion 23 2 C 1: Solvent Characterization 23 2 C 2: HETP Study 29 2 C 3: Protein Stability 41 Section 2 D: Conclusion 49 ii CHAPTER 3 Comparison of Atomic Absorption and Molecular Spectrophotometry for the Indirect Determination of Phosphate Compounds in an Undergraduate Teaching Environment Section 3 A: Introduction 52 Section 3 B: Experimental 55 3 B 1: Reagents 56 Section 3 C: Results and Discussion 58 3 C 1: Iron (III) Salicylate Spectrophotometry Method 58 3 C 2: Calcium Atomic Absorption Spectrophotometry Method 61 3 C 3: Comparison of the Two Methods for Sample Analysis 64 Section 3 D: Conclusion 71 Section 3 E: Acknowledgement 71 References: 72 CHAPTER 4 Future Work Future Work 74 iii LIST OF TABLES Table Title Page 1-1 Summary of reversed phase chromatography separation factors that can affect 4 resolution. 1-2 Thermal prope rties of previously reported alkylammonium formate s 14 1-3 Physiochemical prope rties of previously reported alkylammonium formate s 14 2-1 Summary of the solvent strength parameters found for standard organic 25 solvents and for IPAF 2-2 Peak area analysis for tryptophan and cytochrome c 44 2-3 Peak characteristics for cytochrome c and tryptophan during the separations 46 shown in Figure 2-17a for IPAF 2-4 Peak characteristics of cytochrome c and tryptophan during the separations 47 shown in Figure 2-17b for Methanol 3-1 Student sensitivity data for Fall 2010 semester 63 3-2 Summary of phosphate levels in common sodas and mouthwash 68 3-3 Summary of the statistical differences between the two techniques for each sample 70 tested iv LIST OF FIGURES Figure Title Page 1-1 Sample chromatogram illustrating peak width and the retention factor variables 3 1-2 Solvent strength r elationship 5 1-3 The v an Deemter t erms 7 1-4 Representations of solvophobic theory and adsorption theory 9 1-5 Representation of p arti ti oning theory in RP -HPLC 11 1-6 Comparison of HPLC and UHPLC separations of h ydrocortisone cream 12 2-1 UV -VIS photo diode array spectra for pure IPAF 24 2-2a Retention f actors for p-nitroaniline in methanol, acetonitrile, and IPAF 26 2-2b Retention f actors for caffeine in methanol, acetonitrile, and IPAF 26 2-3 Sample c hromatogram is a test mixture of the analytes in 3 5% IPAF / 65 % water 27 2-4 Plot of l og 10 viscosity as a function of volume fraction of IPAF in water 28 2-5 Back pressure dependence on % organic mobile phase composition 29 2-6a The van D eemter plot for p -nitroaniline in 25% of three organic solvents in 75% 31 water 2-6b The van D eemter plot for acetophenone in 25% of three organic solvents in 75% 31 water 2-6c Back pressures measured for a YMCBasic column with 25% organic solvent in 75% 32 water 2-7 A sample chromatogram of the v an Deemter plot test mixture separation 32 2-8 The van D ee mter plot for p -nitroaniline in 35% IPAF in water and 25% m ethanol in 33 water 2-9 IPAF v an Deem ter plot comparison for 15%, 25%, and 35% IPAF in water 34 2-10a The van D eemter plot for p -nitroaniline in premixed 25% of IPAF / 75% water and 35 online mixed 25% / 75% water 2-10b The van D eemter plot for acetophenone in premixed 25% of IPAF / 75% water and 36 online mixed 25% / 75% water 2-11 Measured backpressure during both the online mixed IPAF and premixed IPAF runs 36 v 2-12a Overlay of chromatograms showing the differences in retention times with online 38 vs. premix IPAF at 0.5mL/min 2-12b Overlay of chromatograms showing the differences in retention times with online 38 vs. premix IPAF at 3.0mL/min 2-13a Comparison of retent ion factors for p -nitroaniline for 25% online mixed organic 39 and 75% water mobile phase compositions 2-13b Retention times for p-nitroaniline using varying mobile phase flow rates 40 2-13c Peak widths for p -nitroaniline using varying mobile phase flow rates 40 2-14 The v an Deemter plot of p -nitroan iline using a Phenomenex 3μm particle size 41 phenyl column 2-15 Fluorescence emission at 360nm of cytochrome c dissolved in varying amounts of 42 methanol and IPAF 2-16a Sample chromatogram illustrating the separation of tryptophan and cytochrom e c 43 in a 5% methanol / 95% water to 50% methanol / 50% water mobile phase gradient from 0 to 10 min 2-16b Sample chromatogram illustrating the separation of tryptophan and cytochrom e c 44 in a 5% IPAF /95% water to 50% IPAF / 50% Water mobile phase gradient from 0 to 10 min 2-17a Overlay style chromatograms of a tryptophan and cytochrome c separation at 46 different temperatures ranging from 10°C to 50°C. The gradient was programmed from 5% IPAF / 95% Water to 50% IPAF / 50% Water mobile phase gradient from 0 to 10 min. 2-17b Overlay style chromatograms of a tryptophan and cytochrome c separation at 47 different temperatures from 10°C to 50°C. The gradient was programmed from 5% methanol / 95% water to 50% methanol / 50% water mobile phase gradient from 0 to 10 min 2-18 Ratio of fluorescence peak data obtained during the temperature study 48 separations 3-1a Molecular structure of iron (III) salicylate 53 vi 3-1b The structures of sodium trimetaphosphate, sodium fluorophosphates, and 53 sodium hexametaphosphate 3-2 UV -VIS absorbance spectrum taken of the iron (III) salicylate complex showing the 58 λmax 3-3 Iron -salicylate absorbance measurements @525nm as a function of pH 58 3-4 Molecular spectrophotometry sensitivity determinations for four phosphate containing species: Orthophosphate, Trimetaphosphate, Fluorophosphate, and 59 Hexametaphosphate 3-5 The effect of pH on the calibration curve for the indirect determination of NaH 2PO 4 60 3-6 The effect of pH on the calibration curve for the indirect determination of Na 2PO 3F 61 3-7 Student and teaching assistant ( TA ) data showing decreased Ca signal for 62 increasing [NaH 2PO 4] 3-8 Student and TA data showing decreased Ca signal for increasing [Na 2PFO 3] 62 3-9 Student and TA data showing decreased Ca signal for increasing [NaH 5P6O18 ] 63 3-10 Stability of Ca atomic absorption spectrophotometry signal with time 64 3-11 Scott’s Miracle Gro and Scott’s Turfbuilder Phosphate Content Determination 65 3-12 Cola and mouthwash phosphate content determination 68 3-13 Analysis of statistical difference between techniques 69 3-14 Analysis of statistical differences between brands 70 vii LIST OF ABBREVIATIONS Abbreviation Technique LC Liquid Chromatography RPLC Reversed Phase Liquid Chromatography HPLC High Performance Liquid Chromatography UHPLC Ultra High Performance Liquid Chromatography AAS Atomic Absorbance Spectrophotometry UV-VIS Ultraviolet-Visible Spectrophotometry Abbreviation Chemical RTIL Room Temperature Ionic Liquid IPAF Isopropylammonium Formate AAF Alkylammonium Formate MAF Methylammonium Formate EAF Ethylammonium Formate PAF Propylammonium Formate BAF Butylammonium Formate viii Acknowledgement I would like to acknowledge all of the students who were enrolled in Miami University CHM145H/161 during the Spring/Fall 2010 semesters who initiated the work presented in Chapter 3. ix Chapter 1 Introduction The first research study of this thesis focuses on the characterization and analytical application of the room temperature ionic liquid (RTIL) isopropyl ammonium formate (IPAF). It has been synthesized for the first time and applied as a mobile phase modifier for reversed phase liquid chromatography (RPLC).