2.4. Amino acid analyses
For analysing the amino acid composition, carotenoprotein
complexes were prepared according
to the methods described by Zagalsky et al.
(1967). The samples were hydrolysed for 36 h at
110 8C. Amino acids were separated in a twocolumn
system using a JEOL JLC-6 AH automatic
amino acid analyser under standard conditions
(JEOL Instruction, Tokyo).
The columns were filled with ICR-2 resin and
the separation temperature was maintained at 52
8C. For the analyses, 0.8 ml samples were injected
and the flow rate of the solvent (buffer solutions)
was programmed to 25.2 mlyh and for the ninhydrine
dye at 12.6 mlyh as outlined by Czeczuga
and Czeczuga-Semeniuk (1998). Alkaline amino
acids (Lys, His, Arg) were separated in an 8=150
mm column using 0.35 N sodium–citrate buffer
solution (pH 5.28) as mobile phase under a pressure
of approximately 8 atm.
Acidic and neutral amino acids were separated
on a 8=500 mm column in buffer solution No. 2:
0.2 N sodium–citrate at pH 4.2 under a pressure
of approximately 20 atm. Standard amino acid
solutions were obtained from Pierce Biotechnology
Inc., USA. By comparing the retention time (Rt)
with the standard amino acid run at identical
conditions, the amino acids present in the sample
were identified and total amino acid content was