Ion-pair and HILIC chromatography for short and charged peptides

Reverse-phase chromatography retains by hydrophobic interaction. A dipeptide of two polar residues has almost no hydrophobic surface, so it is almost not retained, and a compound that is not retained is not separated from anything. Vilon elutes at 3.94 minutes on the index reference method; carnosine at 3.72; epitalon at 4.62. Those numbers are close to the column dead time, and a purity figure derived from them describes very little.

Ion-pair reverse phase

Adding an amphiphilic counter-ion to the mobile phase forms a neutral ion pair with the charged analyte, which then has enough hydrophobic character to be retained.

HILIC

Hydrophilic-interaction chromatography inverts the problem: a polar stationary phase (bare silica, amide, zwitterionic) with a high-organic mobile phase retains polar analytes strongly and hydrophobic ones weakly. It is the natural technique for the sub-500 Da bioregulators and it is entirely mass-spectrometry compatible, because the high acetonitrile content improves desolvation rather than suppressing it.

The practical costs are a long equilibration time — HILIC columns need 20 or more column volumes and are notoriously sensitive to injection-solvent mismatch — and a smaller literature to draw method conditions from.

What the index does

• Compounds below about 520 Da in the bioregulator class are recorded as method-inappropriate when assayed by plain C18. Twelve of nineteen vilon reports on file fall into that category.
• One laboratory offers ion-pair and HILIC as routine, and it is the source of every bioregulator-class report the index does not mark method-inappropriate.
• The status is METHOD LIMITED rather than rejected. The record is published, because the fact that the wrong method was used is itself the most useful information available about that submission.