Poor recovery with hydrophobic peptide

[rtho5616]

Hi,
I am working with a relatively small glycine rich peptide (around 1300 amu) that is very insoluble in all solvents except DMSO.

My attempts to purify the molecule have been hampered by low recoveries (20%) even though the chromatogram indicates good resolution and separation when i use at-column-dilution to load my compound. Im using a C18 column eluting with water/ACN/TFA.

I check the solvent front and the wash to see if my compound is there but it seems like it is vanishing into thin air.

If anyone has experienced this problem, or could redirect me to another post, it would be much appreciated!
Cheers

[danko]

Does the backpressure increase underway?

Best Regards

[rtho5616]

yes it increases quite substantially while loading but decreases after elution of the DMSO and then again after the product peak. I assumed it was due to the viscosity of DMSO, but couldn't rationalize the higher-than-usual pressure after the injection peak

Is there any way irreversible aggregation can occur within the column without a permanent increase in back-pressure?

In was thinking of flushing the column with 3 injection loops of DMSO, would this be harmful to a C18 column?

[danko]

Is there any way irreversible aggregation can occur within the column without a permanent increase in back-pressure?

That was my idea too and I think that’s what you’re dealing with. The peptide (or some of it) aggregates due to the low pH and high ACN content in the mobile phase. The aggregates stick on the stationary phase and slowly dissociate and elute over time.
To test this thesis, try and inject a blank (DMSO containing solvent) right after a normal sample elution/run. Maybe you’ll see a peak representing some of the peptide that you didn’t recover in the previous run.

If you’re comfortable with the conclusion you can go on and try to optimize the conditions in order to avoid the sample loss you’re experiencing at present.

Here are two suggestions I’d explore if I were in your situation:

1. Choose less hydrophobic stationary phase (column) e.g. C4 or even Phenyl.
2. Change mobile phase e.g. some diluted buffer approx. 0.01 M and alkali pH e.g. 8 – 9 if the column material allows for it.

Remember to test the solubility of your peptide in the modified/new mobile phase, if you choose to follow that path. The way to do it is, mixing some of your sample solution with the mobile phase in a test tube and observing the mixture for cloudiness. Actually it’s also a relevant test to carry out with your current mobile phase.

Best Regards

[HW Mueller]

It seems to me such problems have long since been worked out with highly hydrophobic membrane proteins?

[Uwe Neue]

Do the blank DMSO injection with the exact same protocol as you are currently using, including the at-column dilution. This will tell us if there is precipitated sample somewhere...