amino acids is not required, and preliminary experiments or routine analyses of special collagen features such as the ,glycine or imino acid contents may thus be performed in only 115 min. Only 3-Hyp, homoserine and the Met-S-oxides are not accounted for by program B.
Fig. shows a typical elution curve of collagen amino acids obtained by using program A. 3-Hyp, the two Met-S-oxides, 4-Hyp and Asp appear as well-separated peaks if the pH of the first elution buffer is lowered to 2.84. Also at the flow rates used (105 ml/h), there are no difficulties in resolving Ser and Thr if the first buffer contains 4% (v/v) of methanol. The other three citrate .buffers are free of methanol. The figure also shows that the conditions used give a good separation of Glu-Pro and Gly-Ala, in spite of the amounts present, as well as Ile-Leu and Tyr-Val, At present the basic amino acids are eluted from a second short column (14 cm; Aminex A-5) at go" in 85 min. The pH of the citrate buffer used is 5.28. Nyl, Lys, His, ammonia and Arg are well separated.
Approximately 0.05 micromole of an amino acid is required for an accurate analysis. This figure has to be multiplied by a factor of 2 and 4, respectively, for the determination of the imino acids Pro and Hyp. The amount of protein necessary for a total analysis is approximately 1.2 mg and may be scaled down to I mg if experienced workers operate the instrument. The reproducibility of the results is very satisfactory.