Separation
4.2.5.
Amino acid separation was performed with an Onyx C18 4.6×25 mm monolithic column attached at the periphery of the SIC instrument valve (A). A precolumn filter of 2 µm porosity was used in front of the separation column to prevent clogging. The chromatogram (B) shows that some amino acids (Gly+Arg, Tyr+Ala, Trp+Met) co-elute as one peak. Also, there is little resolution between Gln and His. The remaining amino acids are well-separated. The reason for the incomplete resolution is the limited length of the gradient formed in the holding coil. An extended, less steep gradient would be required for complete resolution.
The stepwise gradient (Rigobello-Masini 2008), also produced using SIChrom instrument, yields a better resolution, since in the mixture of 18 amino acids co-elution occurred only between Val and Met, Ile and Leu and Gln and His.
A
B
