Peptide aggregation by SEC?

[Mattias]

Hi,

We have a peptide that has a tendency to aggregate, and I am thinking about using SEC to monitor the early signs of aggregation.

I have tested a solution that I know contains aggregates, but nothing shows on the SEC chromatogram.

Column: Superdex peptide
Mobile phase: 0.1% TFA in 30% ACN (default mobile phase for this column)

I guess the problem is that the peptide deaggregates when it is injected (and diluted) in the mobile phase. Do you have any tricks to use SEC for this application?

[Gerhard Kratz]

I would use a SEC column based on silica, with small particles and a 4,6mm ID or smaller with a micro flow cell in the detector.

[Mattias]

I would use a SEC column based on silica, with small particles and a 4,6mm ID or smaller with a micro flow cell in the detector.

Thank you for your reply!

Do you know why a silica column would be better than a polysaccharide column in this case?

[Gerhard Kratz]

Higher resolution due to higher number of theoretical plates! Much higher!

[Andy Alpert]

The composition of your mobile phase is also a problem. As one adds more and more ACN to the mobile phase, you gradually strengthen hydrophilic interactions. Analytes which elute earliest in SEC (cf. aggregates) tend to elute later in HILIC, so there is a reversal of elution order. 30% ACN is not high enough to promote full-bore HILIC, butr it might be high enough to ruin the separation of the aggregate from the monomer. Also, chaotropes such as TFA tend to cause the fractionation range of an SEC material to shift toward lower mol. wts. Read my book chapter on the subject:
http://www.polylc.com/downloads/SEC_boo ... r_ver1.pdf

[dorota]

Hi Mattias
Have you found a solution for your problem? I am interested in this topic.