Quantitation of amino acids and amines by chromatographymethods and protocols

669 Pages·2005·26.42 MB·English

by  Ibolya Molnár-Perl (Eds.)

JOURNAL OF CHROMATOGRAPHY — volume 70
quantitation of amino
acids and amines by
chromatography
methods and protocols
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JOURNAL OF CHROMATOGRAPHY — volume 70
quantitation of ami no
acids and amines by
chromatography
methods and protocols
edited by
Ibolya Molnár-Perl, PhD, DSc
Institute of Inorganic and Analytical Chemistry,
L. Eötvös University, Budapest, Hungary
2005
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Dedication and Thanks
To my parents,
Erzsébet and Sándor,
and husband,
Miklós
children
András and Éva,
and grandchildren
AnnMarie, Eszter, Marcel, Daniel and Nóra,
for their love,
and my colleagues and students
for inspiration, encouragement and support of my scientific activity.
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VII
Preface
Quantitation ofAmino Acids and Amines by Chromatography: Methods and Protocols
is intended to serve as a ready-to-use guide for the identification and quantification of
amino acids (AAs) and amines (As) in various matrices.
Quantitative determination of AAs and As is in a similar relationship with the
chemistry of proteins and peptides as elementary analysis with the chemistry of organic
molecules: these methodologies are still relevant, and there is also an urgent need of
increasingly sophisticated systems and approaches to clarify the composition and struc-
ture of proteomes, genomes, etc.
This book is structured in such a way that the main sections are classified by the
chronological order of the development of chromatographic methods: ion exchange
chromatography (IEC), (in the case of AAs only); gas chromatography (GC); high-
performance liquid chromatography (HPLC); and capillary electrophoresis (CE). Sub-
sections are grouped according to the preliminary preparation/derivatization process(es)
as the main ordering concept. This means that first the simple methods will be dis-
cussed - those suitable for the analysis of the selected compound(s) in their ini-
tial/natural forms - followed by their chromatographic analysis subsequent to various
derivatization protocols.
The first approach to the automatic chromatographic analysis of Aas - known
today as IEC - was published by Spackman, Stein and Moore in 1958 and who were
awarded the Nobel prize in 1972. Now, 46 years later, instead of one day it takes (in
special cases) less than five minutes to separate and quantitate the essential protein
AAs.
Over the last decades, in addition to the spectacular improvement of the IEC
analysis of AAs (in terms of both speed and sensitivity), GC, HPLC and CE have of-
fered unlimited possibilities on both the preparative and analytical scale. The wide
choice and sophisticated columns, detectors, derivatization procedures, the development
of modern instrumentation and data handling systems have reduced time and costs, and
give versatility and automation in Good Laboratory practice (GLP)-controlled condi-
tions for selectivity, sensitivity and reproducibility. It is the responsibility of the re-
searcher to choose the most appropriate method for the given task. The most popular
HPLC method for analysis of both AAs and As, (free AAs, present in many natural
VIII
matrices, biological fluids and tissues, feed- and foodstuffs and of those constituents of
protein hydrolyzates), is now reversed-phase (RP) chromatography after pre-column
derivatization.
Numerous protocols for derivatization are available. This book will discuss the advan-
tages and drawbacks of the commonly used procedures.
Analysis of AAs in protein hydrolyzates requires a special paragraph dealing
with the hydrolysis step itself: since, hydrolysis conditions have a definitive and special
influence both on the following anlytical steps and on the reproducibility and recovery
of AAs obtained from hydrolyzates. Consequently, hydrolysis of proteins is discussed
in detail as a subsection of 1.1.2. , i.e., together with the HPLC analysis of the phenyl-
thiocarbamoyl (PTC) derivatives of AAs: this follows form my multi-facetted experi-
ence in the field of protein hydrolysis, and all of this experience is associated with the
determination of the PTC-AAs.
Developing new methods and altering/improving old ones is an ongoing proc-
ess. Despite my more than three decades in the field, and that I follow the literature con-
tinuously, readers may have additional, useful proposals or consider some particularly
important method to be missing: any questions, comments or suggestions for including
other methodologies in a future edition will be gratefully recieved.
I am extremely grateful to the distinguished authors for their time, expertise, and
devotion, and for making this book possible.
IX
Contents
Part 1. Amino Acids
1.1. Gas Chromatography
1.1.1. Quantitation of Amino Acids as Chloroformates - A Return to
Gas Chromatography
PetrHusek 2
1.1.2. Quantitation of Amino Acids by Gas-Liquid Chromatography
Charles W. Gehrke 39
1.1.3. Chiral separations of Amino Acids by Gas Chromatography
RalfPdtzold and Hans Bruckner 98
1.2. High Performance Liquid Chromatography
1.2.1. HPLC of Amino Acids without Derivatization
Claire Elfakir 120
1.2.2. HPLC of Amino Acids as Phenylthiocarbamoyl Derivatives
Ibolya Molndr-Perl 137
1.2.3. HPLC of Amino Acids as o-Phthalaldehyde Derivatives
Ibolya Molndr-Perl 163
1.2.4. HPLC of Amino Acids as Chloroformate Derivatives
Bjorn Josefsson 199
1.2.5. HPLC of Amino Acids as Dansyl and Dabsyl Derivatives
Toyohide Takeuchi 229
1.2.6. Quantitation of Amino Acids as 6-Aminoquinolyl-N-
hydroxysuccinimidyl Carbamate Derivatives
Steven A. Cohen 242
1.3. Capillary Electrophoresis/Capillary Electrochromatography
1.3.1. Determination of Underivatized Amino Acids by Capillary Electrophoresis
and Capillary Electrochromatography
Christian W. Klampfl 269
1.3.2. Quantitation of Amino Acids as o-Phthalaldehyde derivatives
Shigeyuki Oguri 297
1.3.3 Capillary Electrophoresis and Capillary Electrochromatography of
Amino Acids as Dansyl Derivatives
ZilinChen 309