This method is mainly used in quantitative amino acid analysis (AAA). The determination of the blind value ( = racemisation after hydrolysis) is necessary because for AAA the enantiomers are added as an multi-internal standard.Therefore the concentration of the enantiomers of the sample itself after hydrolysis must be taken into account.

The method involves hydrolysis with 6N hydrochloric acid, derivatization of the free amino acids and gas chromatographic separation of the enantiomers on a chiral column. The results for the enantiomeric purity however have one weakness: under the condition of hydrolysis, racemization occurs and the amount of determined racemate represents the sum of the amount originally presented in the peptide plus the amount generated during hydrolysis. The standard deviation of the racemisation during sample preparation is high due to matrix and sequence influences.

• The racemisation during hydrolysis depends on the amino acid and is between 0.2% and 10%,
• The standard deviation is 0.3-1% and >2% for cysteine and the amino acids which are linked at cysteine.

So this method is not applicable for exact determination of the enantiomeric purity of amino acids in peptides but for half-quantitative determination.

Results: Optical purity after hydrolysis

• Information about choice of method download PDF.