Document Detail

Chiral o-phthaldialdehyde reagents for fluorogenic on-column labeling of D- and L-amino acids in micellar electrokinetic chromatography.
MedLine Citation:
PMID:  9221886     Owner:  NLM     Status:  MEDLINE    
Following a recent communication from this laboratory (A. Tivesten et al., J. High Resol. Chromatogr. 1996, 19, 229-233) where on-column chiral derivatization of D- and L-amino acids in micellar electrokinetic chromatography (MEKC) was demonstrated for the first time, we now present further details of the labeling procedure. The basis of the method is the consecutive injection of a sample and the reagent onto the capillary as two discrete plugs. By utilizing their difference in mobility, the zones are mixed by the electrophoretic process in a controllable way. In this way the amino acids are both derivatized within a few seconds and subsequently separated in a single step. Compared with pre-column derivatization, dilution of the original sample is minimized, which is why the method is highly useful for microchemical analytical work, i.e., labeling of nano- to picoliter samples. Four different chiral thiols were compared in this study, 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranose (TATG), N-acetyl-L-cysteine (AC), N-acetyl-D-penicillamine (AP), and N-isobutyryl-L-cysteine (IBC). Together with o-phthaldialdehyde (OPA) these constitute the chiral reagent. The reaction rate as well as the spectroscopic and chromatographic properties of the formed derivatives were examined. It was found that the fastest reaction is obtained with OPA/TATG, as was the case with L-alanine (L-ala), and that the rate is greatly affected by the presence and concentration of acetonitrile or methanol. Moreover, OPA/TATG yields superior resolution of D- and L-amino acids over the other OPA/thiol combinations in a sodium dodecyl sulfate (SDS) micellar buffer, whereas the OPA/AC and OPA/IBC-amino acid derivatives have a higher fluorescence quantum yield. With laser-induced fluorescence detection (He-Cd, 325 nm) the mass limit of detection is at the low amol level.
A Tivesten; S Folestad
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Electrophoresis     Volume:  18     ISSN:  0173-0835     ISO Abbreviation:  Electrophoresis     Publication Date:  1997 Jun 
Date Detail:
Created Date:  1997-09-22     Completed Date:  1997-09-22     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  8204476     Medline TA:  Electrophoresis     Country:  GERMANY    
Other Details:
Languages:  eng     Pagination:  970-7     Citation Subset:  IM    
Department of Analytical and Marine Chemistry, University of Göteborg/Chalmers University of Technology, Sweden.
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MeSH Terms
Amino Acids / chemistry,  isolation & purification*
Chromatography / methods*
Indicators and Reagents
Molecular Conformation
Spectrophotometry, Ultraviolet
Sulfhydryl Compounds / chemistry,  isolation & purification
o-Phthalaldehyde / chemistry*
Reg. No./Substance:
0/Amino Acids; 0/Indicators and Reagents; 0/Micelles; 0/Sulfhydryl Compounds; 643-79-8/o-Phthalaldehyde

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