Table Of ContentUniversity of Tennessee, Knoxville
Trace: Tennessee Research and Creative
Exchange
Doctoral Dissertations Graduate School
8-2010
Multidimensional Liquid Chromatography
Separations
Jacob N. Fairchild
University of Tennessee - Knoxville, [email protected]
Recommended Citation
Fairchild, Jacob N., "Multidimensional Liquid Chromatography Separations. " PhD diss., University of Tennessee, 2010.
http://trace.tennessee.edu/utk_graddiss/796
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To the Graduate Council:
I am submitting herewith a dissertation written by Jacob N. Fairchild entitled "Multidimensional Liquid
Chromatography Separations." I have examined the final electronic copy of this dissertation for form and
content and recommend that it be accepted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy, with a major in Chemistry.
Georges A. Guiochon, Major Professor
We have read this dissertation and recommend its acceptance:
Shawn Campagna, Michael Sepaniak, Douglas Hayes
Accepted for the Council:
Carolyn R. Hodges
Vice Provost and Dean of the Graduate School
(Original signatures are on file with official student records.)
To the Graduate Council:
I am submitting herewith a dissertation written by Jacob Nathan Fairchild entitled
“Multidimensional Liquid Chromatography Separations.” I have examined the final
electronic copy of this dissertation for form and content and recommend that it
be accepted in partial fulfillment of the requirements for the degree of Doctor of
Philosophy, with a major in Chemistry.
Georges Guiochon, Major Professor
We have read this dissertation
and recommend its acceptance:
Shawn Campagna
Michael Sepaniak
Douglas Hayes
Accepted for the Council:
Carolyn R. Hodges
Vice Provost and Dean of the Graduate School
(Original signatures are on file with official student records.)
Multidimensional Liquid
Chromatography Separations
A Dissertation Presented for the
Doctor of Philosophy Degree
The University of Tennessee, Knoxville
Jacob Nathan Fairchild
August 2010
Copyright (cid:13)c 2010 by Jacob Nathan Fairchild
All Rights Reserved.
iii
Acknowledgments
The best acknowledgment I can convey is that I was never alone in my work. I
can honestly state that earning this degree has been extremely challenging at points,
but I’ve never regreted the journey. This is due in part to the tremendous support
system I have been gifted from God. To my wife and family, everyone has been very
encouraging and I am grateful to have all of you in my life. Telana, thank you so
much for being a wonderful companion and putting up with me for so long. I’m
glad that our families have been so helpful in getting our life together started. Mom,
Dad, Pat and Roy, I love you all very much. I would like to thank Prof. Guiochon
for his guidance during my time at the University of Tennessee. I feel lucky to have
had the chance to work with him and learn so much about the extraordinary field
of chromatography. It has been a pleasure to conduct the preceding research. The
many researchers that have been in the group since I joined have all helped me in
some way to achieve my goal. To my doctoral committee, I thank each of you for
taking the time to serve on my committee and review this dissertation. I greatly
value your opinions and suggestions.
iv
Abstract
Many mixtures important to research consist of hundreds or even thousands of
individual components of interest. These types of mixtures are far too complex
to separate by a single chromatographic dimension in any reasonable amount of
time. However, if a multidimensional approach is used, where a complex mixture
is separated by an initial dimension, simpler fractions of that separation are collected
and each of those fractions are analyzed individually, highly complex mixtures
can be resolved in relatively short amounts of time. This dissertation serves as
a guide to multidimensional chromatography, in particular, two-dimension liquid
chromatography. There are many aspects of multidimensional separations that have
been investigated to show its aspects, drawbacks and potential ability to separate
highly complex mixtures. Measurements for the performance of multidimensional
chromatography,theeffectsofthefirstandsubsequentdimensionsandtheapproaches
to pairing dimensions are shown with experimental examples. Fundamental and
practical features of multidimensional chromatography are explained as well as
theoretical discussions on current and future multidimensional chromatography
performance. Experimentally, very high peak capacities were obtained (ca. 7000)
and an algorithm to predict how to best optimize a two-dimensional separation based
on the time used and performance was created for designing experiments.
v
Contents
List of Tables x
List of Figures xi
1 Introduction 1
1.1 Objectives of this proposal . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Introduction to chromatography . . . . . . . . . . . . . . . . . . . . . 2
1.3 High Performance Liquid Chromatography Basics . . . . . . . . . . . 5
1.4 Main modes of liquid chromatography . . . . . . . . . . . . . . . . . 7
1.5 Chromatographic theory . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.6 Peak Capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
1.7 Multidimensional Peak Capacity . . . . . . . . . . . . . . . . . . . . . 13
1.8 Very high peak capacity separations . . . . . . . . . . . . . . . . . . . 17
1.9 Optimization of two-dimensional liquid chromatography . . . . . . . . 18
2 Peak Capacity 20
2.1 Peak Capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.2 Peak Capacity Equations . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.3 Peak capacity and number of peaks . . . . . . . . . . . . . . . . . . . 24
2.4 Implications of peak capacity . . . . . . . . . . . . . . . . . . . . . . 27
2.5 Peak capacity and protein sequence coverage . . . . . . . . . . . . . . 28
2.6 Generating very high peak capacities . . . . . . . . . . . . . . . . . . 35
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3 Multidimensional chromatography 47
3.1 Origins of multidimensional chromatography . . . . . . . . . . . . . . 47
3.2 Two-dimensional liquid chromatography and optimization . . . . . . 49
3.3 Optimization of the separation of BSA digest . . . . . . . . . . . . . 54
3.4 Two-dimensional optimization outline . . . . . . . . . . . . . . . . . . 58
4 Approaches to multidimensional liquid chromatography (MDLC) 60
4.1 The three basic approaches . . . . . . . . . . . . . . . . . . . . . . . . 60
4.2 Time of analysis in MDLC . . . . . . . . . . . . . . . . . . . . . . . . 61
4.3 Effect of additional time on MDLC . . . . . . . . . . . . . . . . . . . 63
4.4 Calculation of aggregate times in MDLC . . . . . . . . . . . . . . . . 65
4.5 Comparing the approaches . . . . . . . . . . . . . . . . . . . . . . . . 67
4.5.1 Instrumentation and Data Handling . . . . . . . . . . . . . . . 67
4.5.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
4.5.3 Experimental Procedure . . . . . . . . . . . . . . . . . . . . . 69
4.5.4 2D Peak Capacities Generated . . . . . . . . . . . . . . . . . . 71
4.5.5 Comparison of aggregate times . . . . . . . . . . . . . . . . . 73
4.6 Calculations of t vs. n . . . . . . . . . . . . . . . . . . . . . . 77
agg c,total
4.7 Possibilities of decreasing the aggregate time of 2D-LC separations . . 80
5 Parameters that control peak capacity in multidimensional liquid
chromatography 82
5.1 Parameter optimization background . . . . . . . . . . . . . . . . . . . 83
5.2 Achievable peak capacity theory . . . . . . . . . . . . . . . . . . . . . 84
5.2.1 The achievable peak capacity in on-line 2D-LC . . . . . . . . . 84
5.2.2 Calculation of retention times and variances in gradient chro-
matography . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
5.2.3 Separation power of the on-line scheme . . . . . . . . . . . . . 88
5.3 Method of Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . 90
vii
5.3.1 Effect of the operating parameters on the achievable 2D peak
capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
5.3.2 Limitations of on-line 2D-LC . . . . . . . . . . . . . . . . . . 99
6 Practical aspects of detection in 2DLC 101
6.1 Dilution in 2DLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
6.2 Retention time and peak variance in gradient chromatography . . . . 103
6.3 Peak height in 2DLC . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
6.4 Dilution factors in 2D-LC . . . . . . . . . . . . . . . . . . . . . . . . 109
6.5 Peak width and elution strength of the sample diluent . . . . . . . . . 112
6.6 Effect of the frequency of sample collection on the peak heights in the
second dimension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
7 Multidimensional liquid chromatography utilizing multiple-, parallel
second-dimension columns 119
7.1 Multiple-, parallel columns . . . . . . . . . . . . . . . . . . . . . . . . 120
7.2 Calculation of peak capacity . . . . . . . . . . . . . . . . . . . . . . . 122
7.3 Calculated results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
7.4 Practical applications of on-line two-dimensional liquid chromatography128
7.5 Some speculation about three dimensional liquid chromatography . . 130
8 Application of 2DLC to the separation of water-soluble metabolites132
8.1 Metabolite detection background . . . . . . . . . . . . . . . . . . . . 132
8.2 Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
8.2.1 Microbial growth and extraction . . . . . . . . . . . . . . . . . 134
8.2.2 Instrumentation and data handling . . . . . . . . . . . . . . . 135
8.3 Design and results . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
8.4 Improving results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
9 Conclusions and recommendations 144
viii
