Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2011 Innovating Two-Dimensional Liquid Chromatography Bradley J. (Bradley James) Vanmiddlesworth Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] THE FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES INNOVATING TWO-DIMENSIONAL LIQUID CHROMATOGRAPHY By BRADLEY J. VANMIDDLESWORTH A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy Degree Awarded: Fall Semester, 2011 Bradley J. VanMiddlesworth defended this dissertation on November 2, 2011. The members of the supervisory committee were: John G. Dorsey Professor Directing Dissertation Michael Ruse University Representative William T. Cooper Committee Member Michael G. Roper Committee Member Hong Li Committee Member The Graduate School has verified and approved the above-named committee members, and certifies that the dissertation has been approved in accordance with university requirements. ii TABLE OF CONTENTS List of Tables v List of Figures vi Abstract ix 1. Fundamentals of Chromatographic Theory 1 1.1 Introduction to chromatography 1 1.2 The stationary phase 2 1.3 Mobile phase flow 4 1.4 Separation theory 6 1.5 The need for two-dimensional chromatography 8 1.6 Research goals 11 2. Reduction of Reequilibration Time in Gradient Elution Reversed 12 Phase Liquid Chromatography 2.1 Introduction 12 2.2 Experimental 14 2.2.1 Reagents 14 2.2.2 Liquid chromatograph instrumentation 15 2.2.3 Liquid chromatographic method 16 2.2.4 Gas chromatograph conditions 19 2.3 Results and discussion 20 2.3.1 Column reequilibration 20 2.3.2 Offline LC-GC determination of %MeCN 24 2.3.3 Pure organic-highly aqueous interface injections 24 2.4 Conclusions 30 3. Quantifying Injection Solvent Effects in Reversed-Phase Liquid 32 Chromatography 3.1 Introduction 32 3.2 Theory 34 3.2.1 Hydrophobic-subtraction model 34 3.2.2 Acetonitrile excess absorption isotherm 35 3.3 Experimental 37 3.3.1 Reagents 37 3.3.2 Liquid chromatograph instrumentation 37 3.3.3 Liquid chromatography methods 38 iii 3.4 Results and discussion 41 3.4.1 Injection solvent sensitivity, s, of methyl ketones 41 3.4.2 Comparison to the hydrophobic-subtraction model 52 3.4.3 Comparison to the acetonitrile excess absorption isotherm 55 3.4.4 Injection solvent sensitivity of lidocaine 58 3.5 Conclusions 61 4. Simultaneous Two-dimensional Planar Chromatography 62 4.1 Introduction 62 4.2 Instrumentation 65 4.3 Reagents 69 4.4 Results and discussion 70 4.4.1 Serial vs. simultaneous study 70 4.4.2 Simultaneous separation study 72 4.4.3 Amino acid separation 77 4.5 Conclusions 78 5. Summary, Significance, and Beyond 79 References 83 Biographical sketch 90 iv LIST OF TABLES Table 1.1 Maximum peak capacities of a given chromatographic 9 technique. Table 2.1 Measured void volumes of the columns studied. 16 Table 2.2 Calculated capillary pressure to wet stationary phase in 27 bar (psi) from equation 2.2. Table 3.1 Parameters of columns used in this work, as reported by 38 manufacturer. Table 3.2 Measured sensitivities for all columns, conditions, and analytes. 50 Table 3.3 Column and solute parameters measured by the 51 hydrophobic-subtraction model. Table 3.4 Fitting parameters for equation 3.7. 55 Table 4.1 Migration of FD&C blue 1 from initial offline spot to 69 center-of-mass. Table 4.2 Comparison of migration distance of methylene blue between 72 single dimension runs and simultaneous two-dimensional run. Table 4.3 Reproducibility of FD&C blue 1 in three runs by simultaneous 76 method. v LIST OF FIGURES Figure 2.1 Graphic representation of flow path (colored) and control 17 signals (lined arrows) at initial setup. Figure 2.2 Chromatogram comparison of acetonitrile front (black trace 18 at 190nm) after switching from 100% MeCN to 100% H2O at Valve A (tA = 0.50 min) and an acetone injection (cyan trace at 254 nm) at Valve B (tinj = 0.53 min). Figure 2.3 Truncated chromatograms depicting the effect of injecting 19 simultaneously (red trace) with mobile phase switching (tinj = tA) versus injecting at the pure organic:highly aqueous interface (black trace) (tinj = tA + 0.03 min). Figure 2.4 Representation of the run-to-run reproducibility of injection 19 series (red, green, and blue trace). Figure 2.5 Plot of acetone retention time vs. injection number for the 21 100% MeCN to 100% H2O runs. Figure 2.6 Plot of acetone retention time vs. injection number for the 22 100% MeCN to 90% H2O:10% MeCN runs. Figure 2.7 Plot of acetone retention time vs. injection number for the 22 100% MeCN to 97% H2O:3% 1-PrOH runs. Figure 2.8 Equilibration volumes required for each column at each solvent 23 system. Figure 2.9 Equilibration volumes required for each column at each solvent 23 system. Figure 2.10 %MeCN of eluent as determined by GC after Valve A switches 24 the solvent. vi Figure 2.11 Comparison of truncated chromatograms of acetone injections 27 on the Kinetex column with ~14bar priming pressure (red) and ~360bar priming pressure (black). Figure 3.1 Chromatograms showing the effects of injection solvent on 39 peak shape of homologous series of methyl ketones, C3-7. Figure 3.2 Change in retention time with injection solvent strength. 41 Figure 3.3 Change in peak width at 10% height with injection solvent 42 strength with (A) constant γ0μg mass, varied volume and (B) constant 1ημδ volume, varied mass. Figure 3.4 Change in asymmetry at 10% height with injection solvent 44 strength with constant γ0μg injection mass for the (A) Poroshell 120 EC-C18, (B) Zorbax Stablebond-C18 (C) Zorbax 300Extend-C18, and (D) Zorbax Bonus-RP. Figure 3.5 As figure 3.4, but with constant 15μδ injection volume for the 47 (A) Poroshell 120 EC-C18, (B) Zorbax Stablebond-C18 (C) Zorbax 300Extend-C18, and (D) Zorbax Bonus-RP. Figure 3.6 Plot of efficiency vectors showing effect of percent gradient 48 on injection solvent sensitivity, s. Figure 3.7 Sensitivity of all columns tested as a function of (A) injection 49 volume, with a constant γ0μg mass or (B) injection mass, with a constant 15uL volume. Figure 3.8 Comparison of sensitivity values for 30μg of 2-heptanone in 54 30uL to (A) bonding density (μmol/m2), number of sorbed acetonitrile layers, and column parameters for the hydrophobic-subtraction model, and (B) to ratios of column parameters, H/A. Figure 3.9 Measured acetonitrile excess isotherms from equation 3.4a 55 and 3.4b. Figure 3.10 Correlation between K(C18)/K(OH) and sensitivity for values 57 of γ0μg of β-heptanone. Figure 3.11 δidocaine peak shape with changes in the 1ημδ of 80% εeCζ 59 injection solvent onto the Poroshell column. Figure 3.12 Lidocaine peak shape with changes in the injection volume 60 of 10% MeCN injection solvent onto the Bonus-RP column. vii Figure 4.1 Instrumentation of the 2D planar chromatography setup. 64 Figure 4.2 Qualification of simultaneous 2D planar chromatography 68 instrumentation. Figure 4.3 Single-dimensional migrations of methylene blue in 75% MeOH 72 and 25% 10mM acetate buffer on reversed phase plates are comparable to the component migrations in simultaneous mode. Figure 4.4 MATLAB graph of pixel data using the described simultaneous 74 2DPC technique. Figure 4.5 Spots remain unresolved using A) electrochromatography and 75 B) conventional TLC. Figure 4.6 Migration distance of blue 1 as a function of run time. 76 Figure 4.7 4.0 minute separation of the four amino acids.1 – histidine, 77 2 – arginine, 3 – lysine, and 4 – alanine. viii ABSTRACT Liquid chromatography is ubiquitous, but two-dimensional liquid chromatography is rare. The difference is in the difficulty of method development, as once floated variables have increased pertinence. Three issues for method development in two-dimensional liquid chromatography have been described previously: 1) To preserve the resolution produced in the first dimension, an eluting analyte peak from the first dimension must be sampled at least four times across the band width. 2) The mobile phase of the first dimension becomes the injection solvent for the second dimension. 3) Each additional dimension adds further dilution of the analyte band. It is the goal of this research to fundamentally describe and mitigate the hurdles of two- dimensional liquid chromatography while concurrently adding to the overall practice of chromatography. Three investigations have been undertaken. The first describes the elucidation of pressure as the limiting factor in column reequilibration post-gradient for the goal of reducing the time of analysis of a second dimension. The second quantifies the effect of band shape distortion due to injection solvent mismatch with the goal of making predictions for column selection and organic modifier choice. The third is a proof-of-concept for a novel approach to separations where two force vectors are applied simultaneously to produce a two dimensional separation in less time and therefore, less dilution. Taken separately, these three are of general use to fundamental separation science, but collected they are of specific use to reduce the difficulty of method development of two-dimensional liquid chromatography. ix CHAPTER ONE FUNDAMENTALS OF CHROMATOGRAPHIC THEORY 1.1 Introduction to chromatography It is of general interest to quantify or qualify a property of a substance of a mixture in the realm of science. Numerous detectors have been designed to target specific properties – mass detectors, spectroscopic detectors, conductivity detectors, etc. When more than one component of a mixture produces a detector response, it is desirable to separate the components to ensure the validity of the measurement.