Chromatography Forum

Hi friends! i am working with polypeptide having molecular weight of 3800 and net charge of +0.2. I have used PL-Strong Cation exchange column (4.6X150mm, 8 micron, 1000A) with gradient of sod. acetate (pH 5.5) and 1M NaCl (10 to 90% NaCl in 40 min). i did not get retention, please give me suggestions.


Thanks & Regards
Harry

Your peptide is excluded in the void volume, if I understand?

You are starting with pH 5.5 NaOAc buffer + 10 % (of 1 M NaCL)?

You can try lowing the pH of the mobile phase, but I would first try equilibrating the column with 10-15 mM NaOAc (no salt) and running a gradient from 0-90% NaCL. The [buffer], pH, in your sample should match initial conditions.

i am using pH 5.5, because the isoelectric point (pi) of peptide is 9.1. i have tried equilibration of column with sod.acetate initially also, but it didn't work.


Regards
Harry

Hi, Harry -

Several suggestions:
1) Dropping the pH to something like 3.0 will uncharge the carboxyl- groups in your peptide, leaving it with a net (+) charge. It should then stick to a decent SCX material. This is how most groups perform cation-exchange of peptides. You will have to use formate instead of acetate as the buffering salt, though. Better yet, use sodium phosphate, since then you will have a mobile phase that's transparent at 220 nm.
2) Your column is inappropriate. A 1000-Å pore material has 4x less surface area than the corresponding 300-Å material.

If you'd like to discuss this further, you're welcome to contact me offlist.

what is the purpose of the chromatography you are doing? assay? oxidized forms?
do you have other compounds in the sample that you need to separate it from?

Dear sir, i want to purify my crude peptide by ion exchange prep column, for that purpose i am developing analytical ion exchange method and later on i will scale it up to prep column. i have tried reverse phase HPLC method initially but due to very minor difference in polarity i could not able to get resolution ( two much impurities were co-eluting with the desired peak). i have read some articles where people have used orthogonal purification method initial isolation by ion exchange column and then polishing with reverse phase column.

What is your sample dissolved in when loaded on the column?

Do you include organic?

Why(?) start with 100 mM NaCL!?

My sample is dissolved in sodium acetate buffer having pH of 5.5, till now i did"t try organic solvent and i am using 100mM NaCl initially because linear increase in salt concentration will elute peptide at some point of time. if u have any suggestion regarding 100mM initially, please let me know.

i am advising a case of a 7500 da protein, with S=S bonds in the folding.
they team as so far failed to clean the protein, because of HMW and smaller fragments left over from the synthesis. they have tried, both c-18 and mixed mode and failed
i am working on them on trying a SEC column of 3u particle size, 80 or 100A pore size, that should be capable of giving them the solution.
i do think this possibility will be the solution for them because we have an MS-MS MW result for each peak or part of peak in the chromatogram. and the main issue is between the compound of interest and it's dimer.
maybe you need to look as well this way if you can show the MW of each peak in your chromatogram.

100 mM salt is too high a level to start with if a peptide is not being retained. Try 10 mM, and an initial pH of 2.7-3.0. You will have to use some buffer other than acetate in that pH range. Again, your 1000-Å pore column is probably not going to work for this application and you should shop around for another.

You haven't said what variants of this polypeptide you wish to look for. Cation-exchange is good at separation of charge variants. It can also separate oxidation variants and truncation variants in some cases, as well as disulfide-bridged peptides with the bridges arranged the wrong way. I agree that SEC is probably better at separation of aggregates such as dimers, but sometimes ion-exchange can separate those too.

Organic solvent in the mobile phase: PL-SCX columns are based on polystyrene. I'm not sure how much organic solvent they'll tolerate; better look it up if you want to assess the consequences of inclusion of, say, 20% ACN in the mobile phases.