Chromatography Forum

I'm interested in concentrating a low-abundant peptide ligand prior to separation on 1 mm PLRP RP column.

The sample is in a large volume of 50% MeCN 0.1% TFA. The idea is to be able to load the sample without diluting the MeCN.

After adjusting sample pH to 5 with concentrated buffer, I would load on the WCX guard column (disconnected from the PLRP at this point), wash, connect PLRP column and elute with a few column volumes (delivered through the sample loop) 0.2% HCOOH or 0.5 M NaClO4

I'm hoping the sample will concentrate at the head of the PLRP column following elution from the cation exchange guard.

Think this will work?

Considering strong eluent options (NaClO4 for either, dilute acid for WCX), as well as loading capacity, and recovery, which would be better- a WCX or SCX?

In principle a good idea. You need to be able to get the peptide into a charge state that will make it stick to the ion exchanger. You need to know the charge of the peptide at pH 5 for doing what you describe. If you do not know this, I would stick with the TFA solution and use a strong cation exchanger.

I've used a polycat step already in this purification. Ligand binds well in pH 5 NH4OAc. Elution was carried out with a linear gradient to unbuffered acetic acid (40%). The ligand elutes under 20% HOAc.

I did some trials (with synthetic peptide stds) on the 2.1 mm ID Polycat A with increasing MeCN in the mobile phase. Made a big difference as far as resolution, peak shape, and retention time. So, should be no problem loading this hydrophobic, basic, peptide on the polycat at high MeCN.

Any thoughts on how HFIP would effect retention?

why do you want to play with HFIP for a prep separation? It is difficult to get rid of afterwards...

why do you want to play with HFIP for a prep separation? It is difficult to get rid of afterwards...

This peptide binds, horrifically, to surfaces. This is especially true for enriched, low abundant samples. I'm guessing the interaction with a surface denatures the peptide, maximizing irreversible binding. HFIP supposedly has a conformational effect on proteins, which solubilizes them.

If it is tough to get rid of, I will stick to storing HPLC fractions with 50% MeCN and perhaps 50 mM HCOOH

The fact that you can run the peptide on a weak cation exchanger at pH 5 indicates that the peptide has multiple positive charges. Such a peptide is likely to stick to glass. For peptides, I would not think about conformational changes. Consider storing the stuff in something else than glass.

I'm less concerned about the HFIP than about the TFA in your sample. It will form ion pairs tenaciously with the basic residues in your peptides, thereby making them unavailable for interaction with a cation-exchange material. I'd suggest drying down the sample to get rid of the TFA first (NOTE: It may take 3 SpeedVac cycles to dryness in succession to accomplish this, adding back a little water:MeOH 1:1 between cycles). Resolubilize the residue in an aqueous solution containing 15 mM ammonium acetate, pH 5. If the peptide is quite hydrophobic. it may be necessary to include 20-40% organic solvent (e.g., ACN:PrOH 1:1). This will not impair retention in cation-exchange. Use of > 50% organic solvent will lead to hydrophilic interaction superimposed on the electrostatic effects. If your peptide still won't go into solution, then use the minimum amount of pure HFIP to solubilize it, then slowly dilute with the starting solvent.
Incidentally, why bother with a WCX guard cartridge if you just want to concentrate the peptide from a dilute solution? That's what SPE cartridges are for. There are SPE cartridges available with both WCX and SCX materials for peptide applications like this, for samples in volumes from 1-5000 ul.