Amino Acids:
Chromatographic Separation
and Enantioresolution

Ravi Bhushan and Juergen Martens

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March 2010
322 pp.

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ISBN: 978-0-9728061-3-8



Amino acids are important biological compounds that are associated with peptides and proteins, and occupy an important position in the food and pharmaceutical industries. With their simple structures and the ready availability of both enantiomers, amino acids not only serve as a chiral pool for synthesis but also provide an inexpensive pool for resolution studies as either the racemate for resolution or the chiral selector for resolution of racemic mixtures of several other compounds. There has been increasing interest in stereoselective synthesis based on chiral auxiliaries, the production of which requires enantiomerically pure substances, particularly natural substances.

Separation and identification of amino acids have always been important in various analytical situations, including determination of primary structure of peptides and proteins. The methods used for separation and identification have included classic paper chromatography, using butanol档etic acid淡ter as the solvent, and two-dimensional paper chromatography with phenol-water as the solvent for the second run, followed by ninhydrin reaction and application of 2,4-DNFB as the derivatizing reagent. Identification using DNP derivatives in the planar mode was achieved by Sanger to establish the amino acid sequence of insulin (leading to his receiving the Nobel Prize in chemistry). Application of ion-exchange columns for the separation of amino acids, followed by postcolumn derivatization with ninhydrin and recording the absorbance, formed the basis for early amino acid analysis. Automated Edman degradation methods were useful specifically for se quencing. The enantiomeric resolution of amino acids has been as challenging as that of any other chiral compound important in pharmaceutics or the synthesis or examination of chiral purity or racemization in immune response studies穮 particular, of peptides.

The development of analytical methods that can be applied for separation and identification and resolution of enantiomers of chiral compounds, including the control of optical purity, remains an area of interest and importance. Direct enantiomeric resolution by HPLC is hindered by the very high cost of CSPs, less durability, greater dependence on temperature, and other related problems. Therefore, indirect HPLC methods get preference. Indirect enantioseparation in the form of pairs of diastereomers by LC is generally simpler to perform and may offer better results than direct separation because chromatographic conditions can be optimized more easily and chiral derivatizing reagents (CDRs) can provide a highly sensitive detector response, making them suitable for the analysis of biological samples.

Chromatography has progressed from the introduction of the classical column in 1906 to capillary electrochromatography (CEC) in 2000. HPLC has passed through many developmental phases, experiencing decreased separation time due to reduction in particle size from >100 mm to 3 mm and improvements in instrumentation and column materials, including fused-core particles. Although HPLC and TLC use similar stationary and mobile phases, which have closely related separation mechanisms, and can provide equally reliable quantitative data, the latter method is still less frequently utilized for enantioseparations. Nevertheless, a greater variety of stationary and mobile phases is available in TLC, and detection methods for analytes are more flexible and varied. The relative simplicity and low cost of TLC should make it ideal, but it is known for having generally lower separation efficiency than fully instrumental methods (e.g., GC, HPLC, and CE).

This book reflects our research activities over the past 30 years in the area of separation and synthesis related to amino acids. Its significant features are as follows

  1. A unified perspective of both TLC and HPLC of amino acids for their separation and enantiomeric resolution
  2. A unified approach as a handbook as well as a source book for separation of amino acids
  3. Relevant, current information on the procedures for direct and indirect enantioseparation of amino acids
  4. A chapter exclusively on separation and enantioseparation of nonprotein amino acids
  5. Application of several CDRs, including methods of synthesis of both the CDRs and the diastereomers
  6. Impregnation of thin-layer plates by different methods and their use for enantiomeric resolution
  7. A chapter on penicillamine, which is a trifunctional nonproteinogenic amino acid containing a thiol group
  8. Details of various TLC and HPLC experiments that show how to select an optimal method
  9. Discussion and methods for determination of enantiomeric purity without resorting to specific rotation measurements
10. Use of enantiomerically pure amino acids as chiral auxiliaries for synthesis of various CDRs as well as chiral impregnating reagents
11. Discussion on the scope of the CDRs for their application in the enantioseparation of compounds other than amino acids based on identical functional reactivity

This is the first such comprehensive book, and we believe that it will be useful as a complete guide to understanding the current and potential applications of amino acid separations.

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