Chromatography Forum

Hello everybody,
I am trying to purify the small oligopeptide (consists of 6-10 amino acids). Its size should be about 1 kD. As the size is very small, I could not detect this through common protein detection methods like SDS PAGE. I am following the protocol which has been applied for purifying of the similar oligopeptide. As the concentration of my peptide is very low, the molecule was not detectable in HPLC with C18.
One trial experiment was carried out:
I have got the cell free filtrate supernatant (50ml) of recombinant E. coli which contains the overproduced peptide. First technique of purification with Sep Pak plus column, 360 mg (Waters) was carried out with step gradient (10-30-60% Acetonitrile+ 0.1%TFA). The 60% eluate which should have the peptide in, was collected. After this semi-purification procedure which causes diluting the amount of desirable peptide in the purified solution, I would like to detect the molecule in gradient HPLC with C18.
So I checked the standard (synthesized peptide which should be similar to my peptide) with this column in HPLC (Dionex 3000, Thermo) initially. I haven't got any peak in the lower concentrations of standard (less than 1 µg/ml). But sharp peaks were presented in the higher concentrations (600, 60, 4, 2,1 µg/ml).
According to the protocol, for further purifications, they used HPLC in three times to purify the peptide. They work with 5 litter supernatant and have got 0.2mg/5L= 0.04mg/L purified oligopeptide. According to my trail run on standard, this concentration is not detectable through HPLC.
I would like to know how I can detect this low concentration of peptide through HPLC? And then, how I can collect this low amount during HPLC to verify with NMR.

If you have any experience with this type of purification, please give me an advice.

Thanks a lot,
Elham

What is the UV detector---diode array or variable wavelength ? and which wavelength ?

One possible "trick" might be to make multiple injections (n)* of your oligopeptide in a diluent that is weaker than the initial starting condition of the mobile phase (column must be well equilibrated !!). This should concentrate the analyte as a sharp band at the head of the column.

(n) will depend on estimated concn. & volume per injection. I would try to use a total amount of analyte at least 10x greater than the amount in an injection that gives no peak.

Then run the HPLC gradient. This should elute the analyte as a single peak.

First of all, I think the description of your problem is a bit convoluted and makes it hard to follow what is really going on.

However from what I gathered, you have a very low concentration of Analyte, which lies below the LOD.

It appears that there is no chance for your to detect it as it is.

It may be a good idea to try an increase the injection volume to the max and see if it helps.

May be you need to think of another kind of a detector (ECD) which is more sensitive to you substance or a completely different technique ?

Do keep us posted.

Any other way to detect your peptide? (ELISA, cell-based assay, etc?)

Do you have access to a lyophilizer to concentrate your samples? Most laboratories doing anything related to peptides have them...