Chromatography Forum

I am still trying to get a grip on HILIC...

Discovered that I am probably is mostly doing ion-exchange with my positively charged peptides on the Kinetex HILIC column

I need at least 20 mM of salt to get anything out of the column (regardless of water content of mobile phase)

I ended up doing a salt gradient instead, and got razor sharp peaks. So this will be my method now. But can I even call this HILIC?
Is HILIC only for neutral compounds?

Your observations are not surprising. Electrostatic effects don't disappear just because you are superimposing hydrophilic interaction on top of them. Phenomenex' Kinetex material is a core-shell version of uncoated silica. Uncoated silica is covered with silanol groups. Silanols have a pKa around 4, which means that uncoated silica is a cation-exchange material at any pH above 3. In order to elute positively charged analytes (cf. your peptides) from a cation-exchange material, you need salt in the mobile phase. How much salt? Read my paper that introduced ERLIC (Electrostatic Repulsion-Hydrophilic Interaction Chromatography), cf. the following link: http://pubs.acs.org/doi/pdf/10.1021/ac070997p

This paper goes into considerable detail on the effect of salt when HILIC of charged solutes is performed on charged columns. Briefly, though, you can suppress most electrostatic effects with a minimum of 20 mM salt in the mobile phase (cf. your own observations). That's 20 mM overall, not just in the aqueous portion of the mobile phase. That amount suffices to form an electrical double layer of counterions on top of the charged surface, shielding electrostatic effects. For particularly highly charged solutes, use 40 mM. For extreme cases, such as aminoglycoside antibiotics, you may need as much as 120 mM salt in the mobile phase in order to get symmetrical peaks.

HILIC materials with a neutral coating have less of a problem with charged analytes than does uncoated silica. I recommend one such material for an application like yours. However, you will still need some salt in the mobile phase. What mobile phase did you initially try using, and what are the details of that gradient that you're using now (i.e., composition of mobile phases A and B and gradient schedule)?

Thanks Andy for your reply and the reference!

I am currently running two different HILIC (or "mixed-mode") separations on the Kinetex column.

The first application is using 20 mM of ammonium acetate pH 5.2 in a shallow gradient between 80 - 70% acetonitrile. I still think I have quite a lot of ion-exchange left, since this peptide is actually a bit hydrophobic and still elutes after everything else in the sample. This is not a bad thing per see, my peak is clean and well separated. The reason for using HILIC at all here is the sample matrix that is only soluble in high amounts of acetonitrile.

The second application is still in development and is quite different. Here I have very low concentration of the peptide (about 20 ng/ml) in a complex sample matrix (gelatin and salts) in water solution. Using ordinary reversed-phase doesn't work, the gelatin peaks are everywhere. My approach so far is to inject 900 µl on a C18 precolumn (using 5% acetonitrile in water as mobile phase). Then I switch the column switch and elute the sample on the HILIC column with about 80% acetonitrile in water.

In the second application I have great use of the ion-exchange properties, since the sample is eluted on the HILIC column with quite a lot of water (in tubings and precolumn), which should ruin the separation. But it doesn't, since there are no ions present. Then I increase both the water and salt content by a gradient and elute the peptide as a very sharp peak. The gelatin elutes in the front and does not disturb.

I know this is an unorthodox way of using a HILIC column, and I am not sure if I should use a dedicated cation-exchange column instead. Due to the very low amount of peptide I need a low wavelength, so I use sodium sulfate instead of ammonium acetate. I still do not know how robust this is, I need to make more injections. Or is this too crazy?

Do you mean 20 mM in the portion that's aqueous or 20 mM in the overall solution? If the former, then your overall concentration of ammonium acetate is more like 4 mM; far too low for the purpose.

Regarding your second application: In effect the uncoated silica is a cation-exchange column. If it's working satisfactorily, then by all means keep using it that way. I am apprehensive about the solubility of sodium sulfate in the presence of a large amount of acetonitrile, though. Try either triethylammonium sulfate or triethylammonium phosphate (the latter having some buffering capacity below pH 3.5). If you do want to switch to cation-exchange, though, then let me recommend a WCX (weak cation-exchange) material such as our PolyCAT A. It will lose its (-) charge in the same pH range as silanols. An SCX material is a lot harder to uncharge and so you'd have to use a gradient to a higher concentration of salt than you'd like. Here's a link to a poster on the use of a decreasing pH gradient to uncharge a WCX column and elute basic peptides: http://www.polylc.com/downloads/ISPPP_1 ... poster.pdf

In your case you'd have to use some acid other than acetic or formic acid, since you need the mobile phase to be transparent at low wavelengths. You could use a solution of a phosphate salt with a gradient from pH 4.0 to 2.5 or so.