Peptide dimers don't work on HILIC?

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

6 posts Page 1 of 1
Hi,

HILIC is becoming a "big" technique in our lab (mostly peptides) since you get so nice peak symmetry and good separation.

Most of our peptides have disulfide bonds and can therefore form dimers (parallel and anti-parallel dimers). But HILIC doesn't work for these peaks, not for one single peptide.

The dimers are very hard to elute and comes out as ugly "blobs". I do not understand why, since they are more hydrophobic and should elute earlier. Anyone that has a good idea why?
It would help if you let us know what column and mobile phase you're using, as well as the solvent that the peptides are dissolved in when you inject.

Regarding retention time:
Retention in HILIC is promoted by the number of hydrophilic residues; the column tends to ignore the hydrophobic ones. Crosslinking two peptides results in a peptide with approximately twice as many hydrophilic residues as either one would have alone. That's why retention is greater with the crosslinked peptides.

Regarding peak shape:
Peptides that associate with a stationary phase in a well-defined orientation generally elute in the sharpest peaks (example: tryptic peptides on an ion-exchange column) . Crosslinked peptides are the opposite; they can associate in a variety of orientations. Some of these associations are stronger than others, so peptides in the various orientations elute at different points in the gradient, i.e., over a wide range. Result: A wide peak. Adding electrostatic effects to the hydrophilic interaction can constrain the peptide into certain orientations. That should sharpen up your peaks. Using a column of the opposite charge (e.g., an SCX column at pH < 3) would make it necessary to add salt for elution that you probably don't want in your mobile phase. Accordingly, try a column of the same charge, i.e., an anion-exchange column at pH < 3. This combination is called ERLIC: Electrostatic Repulsion-Hydrophilic Interaction Chromatography.
PolyLC Inc.
(410) 992-5400
aalpert@polylc.com
Our peptides are quite similar in structure and we actually use the same method for all:

Mobile phase A: 10 mM NH4OAc pH 5.1 in 90% acetonitrile
Mobile phase B: 10 mM NH4OAC pH 5.1 in 75% acetonitrile
Column: Kinetex HILIC, 2.1x150 mm, 2.6 µm
Gradient from 100% A to 100% B in 20 minutes
Sample is dissolved in mobile phase A

All peptides contain at least two basic amino acids. The dimers of course contains twice the amount, and I did not know that the column "ignores" the hydrophobic parts of the molecule. That can explain the very high retention of the dimers.

I am afraid that I will not get any retention in ERLIC, but it could be worth a try. The dimers are quite important since they are stability indicating impurities
Why do you need 90% ACN to get retention of peptides this size in HILIC? We get perfectly adequate retention of tryptic peptides with 80-85%. At 90% you're liable to start seeing some solubility problems. Better get a different HILIC column!

ERLIC: Your crosslinked peptides should be retained well enough starting with 85% ACN if you use an appropriate column.
PolyLC Inc.

(410) 992-5400

aalpert@polylc.com
Sorry for the late reply!

In isocratic conditions, 80-85% acetonitrile in the mobile phase is fine.

I have tried another column called iHILIC Fusion (+) from a company close to ours, and on this column the dimers elute as nice Gaussian peaks. Quite a difference to the Kinetex column. The drawback is slightly less retention overall.
This is Wen Jiang's column from his new company, Hilicon. He can supply them with various ratios of + and - charges on the surface. It sounds like you got one with a predominance of + charges, meaning that you are operating in the ERLIC mode that you were apprehensive about. The electrostatic repulsion accounts for the decrease in retention time that you've observed. If that bothers you, then just increase the % ACN a little.
PolyLC Inc.

(410) 992-5400

aalpert@polylc.com
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