Separation Examples -- Insights Into the "minds" of chromatographers

Amino Acids (Column, ionic modifier)
a) Underivatised amino acids (C8, citrate -SDS)
b) underivatised amino acids (amine, phosphate)
c) iodoamino acids (c8, acetic acid)
d) 3-methylhistidine (c18, C6SO3Na)
e,f) PTH-amino acids (C18, ammonium acetate)
g,h) dansyl-amino acids (C18, phosphate)
i) dansylated protein hydrolysate (C18, phosphate)
j) dinitrophenyl-amio acids derivative (silica, isoctane-iPrOH-CH2Cl2)
k) Dansyl-amino acids (C8, C12-dien-Zn(II))
l) dansyl-amino acid enantiomers (C8, L-2-isopropyl-dien-Zn(II))
m) resolution of D, L-Valine (C18, Cu (L-Pro)2)
n, o) fluorescent O-phthalaldehyde-amino acid derivatives (C18, phosphate)

The effective separation of amino acids and their derivatives has been readily achieved by reversed phase HPLC. The examples A to O in this section show a representative selection of separations of these solutes. In addition, the following sections of teh text give useful information.

Separation of free amino acids.
The polar amino acids are insufficiently retained on non-polar, reversed phase columns for adequate resolution. Example A shows that the use of a mobile phase which contains sodium dodecylsulphate (SDS) allows adequate retention and thus separation of these solute. Alternatively a polar column (Example B) can be used to achieve this separation.
Example C shows that a buffered aqueous mobile phase is adequate to achieve the separation of the relatively hydrophobic, iodeamino acids. Similarly reversed phase HPLC has been particularly successful in the separation of tryphtophan and other aromatic amino acids from a variety of extracts. A recent example is the analysis of tryphtophan and phenylalanine metabolites in urine by M. Ghebregzabher, et al. Alternatively HPLC techniques can be used to monitor enzymatic activities, such as tryptophan 5-mono-oxygenase activity in brain preparations.
The analysis of polar amino acids in the complex mixtures present in tissue preparations can be achieved with the use of hydrophobic ion pairing reagents. Example D shows a typical separation that was achieved with an alkysulphonate and allowed the analysis of 3-methylhistidine in urine. Baker et al used hexane sulphonate in the analysis of guanidino compounds such as arginine and taurocyamine.