Chromatography Forum

Hi all,

I am developing an LC method for a peptide with 20 AAs. I've been told there are 5 arg/lys/his

My problem is that the peak is looks jagged and I would say that it must have several coeluting peaks. I have tried different types of chromatography and columns to try to resolve this:

C4/C12/C18 columns with ACN/ 0.1% TFA in various combinations/gradients
SCX pH 2.5 early eluting, poorly retained
IP with hexane sulfonate on C18,

I have tried diluting the samples in water, 6M urea, 0.1% TFA with similar results.

all of the chromatograms suggest several coeluting peaks (4 or more).


Is it possible that this is an artifact? (eg. non-homogeneous charge states? or some kind of isomerism?)

If it is an artifact, what else could I try to resolve this? The chromatographies seem reproducible, so I'm thinking that perhaps the sample prep may be the real issue...

Suggestions?

cheers

Hooky:
It isn't clear why the peptides eluted early in SCX. Normally a peptide with this many basic residues would require about 0.5 M salt for elution. What SCX column and mobile phases were you using? Anyway, try ERLIC. ERLIC has the best selectivity I've seen for peptides with numerous basic residues. Here's a link to the relevant paper: http://pubs.acs.org/doi/pdf/10.1021/ac070997p
Initially, I'd recommend running isocratically with 20 mM sodium methylphosphonate, pH 2.0, with 65% acetonitrile. To get sodium methylphosphonate, dissolve some methylphosphonic acid in water and add NaOH until you get to pH 2.0.

Assuming you have the resources, the first obvious thing to do is LC-MS to see if your peptides have the same mass and fragmentation... What wavelength are you detecting those?

I agree with Andy that the peptide should had been very retained in SCX if it contained so many basic residues...

I agree with Andy too