Hydrophilic Interaction Liquid Chromatography (HILIC) is a variation of normal-phase chromatography which can be performed with partially aqueous mobile phases. This permits normal phase separation of peptides, carbohydrates, nucleic acids, and many proteins. The elution order is least polar elutes first, the opposite of that in reversed-phase HPLC. The stationary phase in HILIC must be extremely polar. PolyLC has developed bonded phase specifically for this purpose.
PolyHydroxyethyl A retains solutes almost solely on the basis of hydrophilic interaction. Volatile mobile phases can be used. HILIC exhibits retention proportional to the amount of organic solvent in the mobile phase. Typical HILIC mobile phases contain 65 - 90% organic in the initial conditions with the sample in the same. Gradient elution may be performed either by a decreasing organic level, or increasing salt gradient or both.
Each PolyHYDROXYETHYL A SEC column may be used in two different fractionation ranges, merely by changing the mobile phase. With conventional salt buffers, the fraction range is determined by the pore diameter of the packing. Nonspecific interaction with polypeptides are generally lower than with other SEC columns. If the mobile phase contains a denaturing agent (i.e. 50 mM formic acid, or hexafluoro-2 Propanol (HFIP), then the sieving occurs between the polymer chains of the coating. This results in a dramatic shift of the fractionation range to lower values; solutes as small as formic acid can be separated by size. Moreover, these separations can be effected with volatile mobile phases for use with Mass specific and Light scatter detectors.
With our 60 A pore size column, the fractionation range is 20-600 Daltons. This permits SEC of small solutes not possible heretofore. Examples include desalting a dipeptide, or separation of small solutes from a large excess of an even smaller derivatizing agent, and coupling reactions.When to use HILIC:
Need a volatile mobile phase and Reversed Phase does not suffice
Solutes too weakly or too strongly retained in the Reversed-Phase mode.
Solutes which aggregate or are not soluble in aqueous mobile phase (i.e. Amyloid peptides)
Solute differing in hydrophilic residues (i.e. Ser-)
Complementary orthogonal mode of chromatography
Removing electroeluted proteins from SDS, Coomassie blue, and salts.
Elute molecules into Mass Spec detector.Use the PolyHYDROXYETHYL A SEC Column for:
- Routine SEC of proteins.
- SEC of Polypeptides which exhibit nonspecific interaction or poor recovery from other SEC columns.
- Resolution of the smallest peptides, Amino Acids and other solutes by size.
- SEC in a volatile mobile phase for Mass Detection and Light Scatter Detection.
- Desalting , including removal of derivatizing reagents.
- Analysis of residual monomer content of a polymer.
For routine SEC applications, we recommend the 200 x 9.4 mm columns which offer optimal separations at 0.5 ml/min. Smaller columns can be used if the HPLC System can deliver low flow rates accurately (i.e. 120 uL/min for the 4.6 x 200 mm columns.)