Separation of amino acids by ion mobility spectrometry

The mobilities of the 20 common amino acids were determined by electrospray ionization ion mobility spectrometry. It was found that each amino acid had a different drift time and hence a different reduced mobility constant K0. This difference in drift time was less than 0.1 ms in many cases. With the instrument used in this study it would not be possible to resolve mixtures of some of the amino acids. It would however be possible to determine any single amino acid. In addition, the detection limits were determined for the 20 amino acids. They ranged from 50 to 700 pg. This indicates that the detection limits were less than 3 pmol for all of the amino acids and that many amino acids had detection limits less than 1 pmol.     

Separation of phenylthiohydantoin amino acids by capillary electrochromatography

Capillary electrochromatography (CEC) was employed as a rapid and high-efficiency method for the isocratic separation of all 20 important phenylthiohydantoin (PTH) amino acids, the end products of Edman degradation during N-terminal protein sequencing. For this purpose, 75 microm ID fused-silica capillaries were packed with standard 3 microm Hypersil octadecyl silica (ODS) particles using a two-step column fabrication process, which represents a fast, reliable and efficient means of producing long-term stable columns. The influence of solvent composition, pH, type of buffer cation, buffer concentration, and temperature on retention behavior of PTH amino acids was investigated. Same-day and day-to-day reproducibility of the retention times (over a period of two months) were found to be better than 3%. When comparing this new technique with traditional reversed phase-high performance liquid chromatography (RP-HPLC) methods applied in automated protein sequenators, CEC shows essentially shorter separation times and superior resolution.     

Separation of Di-dansyl amino acids by MEKC with a co-surfactant

Summary A method has been developed for qualitative determination, in some natural products, of those amino acids which form di-dansyl derivatives. The capillary electrophoresis methods so far developed have not completely solved the problem of separation of di-dansyl amino acids. These amino acids are highly non-polar, because they contain two dansyl groups linked to the amino acid part of the molecule, and are poorly separated from each other, or not separated at all, by micellar electrokinetic chromatography. A new separation mechanism has been tested using a different co-surfactant. Preliminary data have led to 2-methyl-1-propanol being chosen for further investigation of the capabilities of the method of separation.     

Separation of amino acids with simulated moving bed chromatography*

Authors have constructed an automatized four-column large laboratory scale (I.D. = 50 mm, L = 500 mm) simulated moving bed (SMB) equipment. The applied model system for separation of biomolecules is glycine, L-phenylalanine, water and Sepabeads SP825 adsorbent. The authors determined the adsorption equilibrium data and the packing characteristics. The operating conditions of SMB equipment were calculated with the help of the Morbidelli variables. During the SMB experiments, glycine and L-phenylalanine were separated in water on Sepabeads SP825 with an average particle size 0.3 mm at temperatures 20 degrees C and 60 degrees C. The measurement series were carried out on a four-column three-zone open loop SMB. Both L-phenylalanine and glycine were produced with more than 99.9% (m/m) purity and 99% yield at productivity 1.7-3.7, with productivity 3.7-8.1 mg/(g adsorbent h) in case of 2-1-1-0 column configuration. The measured and the calculated data agreed well.     

Separation of native amino acids at low pH by capillary electrophoresis

Amino Acids are cations at low PH and can be readily separated by capillarty electrophoresis provided an alkanesulfonic acid is added to the elecrolyte carrier. Formation of a Positive net charge on the bare fused-silica surface at low PH was confirmed by measurement of an anodic electroosmotic flow. The addition of ethanesulfonic acid or octanesulfonic acid to the electrolyte carrier causes a reversal of the EOF. A mechanism is proposed in which the alkanesulfonic acid adsorbs to the positively-charged capillary wall through electrostatci attraction. Adsorption of a second molecule of alkanesulfonate by hudrophobic attraction to the carbon chain forms a negatively-charge coating on the capillary wall. The alkanesuflfonate also imparts selectivety to the system by participation in ionpairing interactions with the native amino acids to improve resolution. The CE separation of a mixture of the twenty common amino acids at PH 2.8 with direct absorabance detection at 185 nm resulted in 17 amino acid peals in 20 minutes with a 30 KV applied voltage. The effect of several variables was studied including electrolyte carrieres containing different alkanesulfonic acids, the influence of PH, applied voltage, and concentration of electrolyte carrier.     

Separation of amino acids, their derivatives and enantiomers by impregnated TLC

The present state of TLC with respect to separation of amino acids, their different derivatives and their enantiomers by the technique of impregnation is discussed. The main approaches to impregnation viz. mixing of a suitable reagent with the adsorbent prior to plate-making, immersion of the untreated plate in the solution of impregnating reagent prior to development, and modification of the adsorbent, have been identified and discussed for each class of these compounds. The role of impregnation in resolving enantiomers or in improving the separation of mixtures of amino acids or their derivatives in terms of ion pairing, complex formation, ligand exchange or other steric interactions has been elaborated in each category.