SEPARATION OF AMINO-ACIDS USING ION-PAIRED REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH SPECIAL REFERENCE TO COLLAGEN HYDROLYSATE

The collagen study includes the analysis of its characteristic amino a cids: proline, hydroxyproline, lysine, hydroxylysine. HPLC offers an i nteresting device if associated with on-line radiometric detection for the determination of radiolabelled amino acids in the case of metabol ism studies. To avoid pre or post-column derivatization which may be p oorly quantitative in the case of the hydrolysate of unpurified sample s, we developed an ion-paired reversed-phase chromatography using a C8 column (econosphere C8 5 mum, length: 250 mm, ID: 4.6 mm from Alltech Ass.) and an elution carried out with an acetonitrile gradient in hep tane-sulfonate solution. A direct detection at 210 nm was used. Ninete en amino acids were separated within 40 min. Lag time was 7.3 min betw een hydroxyproline and proline, and 6.9 min between hydroxylysine and lysine. In the case of radiolabelled amino acid, there was a linear co rrelation (r = 0.92) between HPLC and ion-exchange chromatography.