Multiple Approaches to the Separation of Amino Acids and Biologically-Relevant Compounds

156 Pages·2016·6.48 MB·English

by  Lance Andrew Riddle

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Doctoral Dissertations Graduate School
12-2006
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Lance Andrew Riddle
University of Tennessee - Knoxville
Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss
Part of the Chemistry Commons
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Riddle, Lance Andrew, "Multiple Approaches to the Separation of Amino Acids and Biologically-Relevant
Compounds. " PhD diss., University of Tennessee, 2006.
https://trace.tennessee.edu/utk_graddiss/2011
This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee
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To the Graduate Council:
I am submitting herewith a dissertation written by Lance Andrew Riddle entitled "Multiple
Approaches to the Separation of Amino Acids and Biologically-Relevant Compounds." 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:
Michael J. Sepaniak, Bin Zhao, Eric C. Drumm
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 Lance Andrew Riddle entitled
“Multiple Approaches to the Separation of Amino Acids and Biologically-Relevant
Compounds.” 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:

Michael J. Sepaniak

Bin Zhao

Eric C. Drumm

Accepted for the Council:

Linda Painter
Interim Dean of Graduate Studies

(Original signatures are on file with official student records.)
MULTIPLE APPROACHES TO THE SEPARATION
OF AMINO ACIDS AND BIOLOGICALLY-
RELEVANT COMPOUNDS

A Dissertation
Presented for the
Doctor of Philosophy Degree
The University of Tennessee, Knoxville

Lance Andrew Riddle
December 2006
DEDICATION

This dissertation is dedicated to my Mom and Dad for their constant encouragement,
support, and, most of all, love.
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ACKNOWLEDGEMENTS
There are so many people I would like to thank for helping me through my time
here…both inside and outside of the lab. First of all, I would like to thank God for all
His gracious blessings He has poured out on me. His love is amazing, and I realize more
and more day by day how grateful I am to Him for what He has done.
I would also like to thank my family, especially my Mom and Dad, for everything
they have done to help me through this time as well as make me the man I am now.
Their hopes of my return to North Carolina have definitely come true.
I wish to thank Dr. Georges Guiochon and Dr. Mike Sepaniak for their guidance
and insightful wisdom I have gained as a graduate student in their groups. I have learned
so much from both of them about chemistry and (just as important) life, and I will always
be thankful for that. I would also like to thank Dr. Bin Zhao and Dr. Eric Drumm for
serving on my committee.
Special thanks go to Dr. Don Jackman of Pfeiffer University for his wonderful
teaching and guidance that laid the framework for my chemistry knowledge. I am also
honored by his confidence in me as I replace him as a faculty member, and I hope I am
able to follow in his footsteps at Pfeiffer.
During my time here, there have been so many co-workers that I have had in both
research groups that it is difficult to thank all of them, but there are three who deserve
special mention. I would like to thank Dr. Maggie Connatser, Dr. David Bailey, and Dr.
Nicola Marchetti for their help in the completion of the research presented here. I hope I
was not too annoying when I ran into problems, but I thank all three of them for not only
being great co-workers but also great friends.
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As for others in the research groups as well as in the department, I would like to
thank Chris Tipple and Nate Carrington for being the two closest friends I made in the
department. It is so ironic that Chris’s graduation coincided with Nate’s arrival because
the two of them have always been there for me, and we have shared many fun times
together. For that, I am extremely grateful. I would also like to wish Amber Wellman
nothing but the best as she finishes up and that I hope I was as helpful to her making it
through our first semester here as she was to me.
Outside the department, I would like to especially thank the David Mitchell, Don
Harrell, and Chris Moore families for their support, encouragement, and friendship
during my time here. Special thanks go to Mitch, Drew, and Nancy Ann Mitchell for
allowing me the chance to be their “brother” and making me feel like family to them.
I wish to thank Aaron Matthews and Nick Meals for being the two guys outside
the department I could count on to be there for me during the good and the bad, and I
hope I did the same for them. We shared so many great moments together, and I hope a
suitable replacement for me during the Wednesday morning Bible study can be found. I
also would like to thank Andy Large for being, well, Large.
Finally, my last few months here have been quite enjoyable thanks to the place
that Sorina Dragoi has found in my life. I never dreamed that all the circumstances and
events that have happened would have led us to each other, but with God all things are
possible, and I cling to that promise and assurance as we continue to live our lives and
see where God takes us.
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ABSTRACT
The branch of analytical chemistry known as separations has continued to grow
and expand ever since its beginnings a century ago. With all the technological advances
occurring to make separations a more powerful technique, the main goal continues to be
the desired separation of the compounds of interest in a mixture. Although a desired
separation may be achieved, there can always be room for improvement with regards to
the time necessary for the separation as well as better resolution of closely-related species
as well as enantiomers of the same analyte.
In this work, a variety of separation methods and variations are used to achieve
separations of biologically-related compounds. The technique of high performance liquid
chromatography was used for the separation of the peptides from a protein digest as well
as sterols from synthetic mixtures as well as from naturally occurring fruit juices.
Monolithic column technology was used for the separation of the protein digest, but the
use of different mobile phases as well as mobile phase gradients afforded different
separation orders of the peptides. High temperatures were incorporated for the separation
of the sterols, so this separation included the study of how different stationary phases that
could withstand the high temperatures affected the resolution of the sterols investigated.
Amino acids labeled with a fluorescent tag were also separated in this work. The
investigation of a polyamide column for the separation of amino acids was performed,
and capillary electrophoresis incorporating the use of cyclodextrins as well as mixed
micelles for the separation of amino acids is presented here.
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TABLE OF CONTENTS

CHAPTER PAGE
1
INTRODUCTION TO SEPARATION METHODS

History and General Description....................................................................................1

Fundamentals of High-Performance Liquid Chromatography.......................................2
Elution in Liquid Chromatography.............................................................................2
General Elution Problem.............................................................................................5
Instrumentation...........................................................................................................7

Monolithic Columns.......................................................................................................9
Introduction.................................................................................................................9
Monolithic Silica Columns.......................................................................................11
Advantages, Disadvantages, and Limitations...........................................................11

High Temperature Liquid Chromatography.................................................................12
Introduction...............................................................................................................12
Temperature and Retention.......................................................................................14
Temperature and Selectivity.....................................................................................15
Temperature and Packed Columns...........................................................................16
Isothermal Separations and Temperature Programming...........................................17
Separations in Hot Water..........................................................................................21
Separations with Non-Aqueous Systems..................................................................21
Practical Implications of High-Temperature LC......................................................22

Capillary Electrophoresis..............................................................................................22
Introduction...............................................................................................................22
Theory.......................................................................................................................23
Instrumentation.........................................................................................................25

Modes of Capillary Electrophoresis..............................................................................27
Capillary Zone Electrophoresis.................................................................................27
Cyclodextrin Capillary Electrophoresis....................................................................29
Micellar Electrokinetic Chromatography.................................................................31
Cyclodextrin-Modified Micellar Electrokinetic Chromatography...........................33

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2 INFLUENCE OF MOBILE PHASE GRADIENTS ON THE
SEPARATION OF PEPTIDES FROM A CYTOCHROME-C DIGEST
BY REVERSED-PHASE LIQUID CHROMATOGRAPHY

Introduction...................................................................................................................35

Theory...........................................................................................................................36
Retention in Gradient Elution...................................................................................36
Retention Factor in Gradient Separations.................................................................37
Peak Capacity in Gradient Separations.....................................................................38

Experimental.................................................................................................................39
Chemicals..................................................................................................................39
Equipment.................................................................................................................40
Separation Conditions and Measurements................................................................40
Protein Digestion......................................................................................................41

Results and Discussion.................................................................................................43
Separation of the Cytochrome-C Digest Mixture and Calculations.........................43
Separations Obtained with Acetonitrile....................................................................54
Separations Obtained with Methanol........................................................................54
Separations Obtained with Tetrahydrofuran.............................................................57

Conclusion....................................................................................................................60

3
SEPARATION OF FREE STEROLS BY HIGH
TEMPERATURE LIQUID CHROMATOGRAPHY

Introduction...................................................................................................................61

Experimental.................................................................................................................63
Materials and Reagents.............................................................................................63
HPLC Instrumentation and Columns........................................................................64
Separation Conditions...............................................................................................64
Mass Spectrometer....................................................................................................65
Safety Considerations...............................................................................................65

Results and Discussion.................................................................................................66
Sterols.......................................................................................................................66
Separation on the Graphitic Carbon Column............................................................66
Separation on the Polymeric C Column.................................................................71
18
Separation on the Zirconia Particle Column.............................................................76
Determination of Sterols in Fruit Juices...................................................................80
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