Chromatography Forum

I need to quantitate a ~3.5 kD peptide with 2xC16 chains attached, and a 55 kD protein in the supernatant. The compounds are in a DPBS pH7.2 buffer.
It needs to be a very sensitive method (<<50ug/mL).

I have had issues in the past and had developed two separate HPLC methods (using SEC for the protein) and a C8 100A for the peptide.

I only had a C8 column on hand at the time.

But, I was wondering if I could analyse them both on a C4 column, possibly in the one method.
However, would I need a 300A pore size column because of the protein? Would this mean the peptide would take ages to elute? I thought a C4 column would be good because I wouldn't require as much acetonitrile to elute the protein, and hence it would not crash out.

I have also learnt in order to keep the peptide in solution (because of the attached lipids) to heat the auto-sampler and the oven compartment to 37C.

I was wondering if there was an ideal ~2um; 100x2.1; ??Angstroms; perhaps coreshell technology UPLC column available. A require a very sensitive column. I have attached a high sensitive flow cell to the Shimadzu DAD.

I would very much appreciate any suggestions. Protein and peptide analysis is relatively new to me.

Edited:

Hi Faith,

I have only a small amount of experience with protein separations, and these are new products; however, they deserve consideration in my opinion:

http://www.mac-mod.com/products-halo-bioclass.php

The HALO 400 columns may serve you well.

Does anyone even make C4 columns anymore? Brownlee used to make them. I would try your C8 column particularly if it is short (10 - 15 cm long and can perform gradient). There is newer technology out there!

Thanks MattM

I clicked your link. Read all about the columns.

Thought the HALO 400 Protein C4 column looked perfect. As the two compounds I want to analyse in the one run are ~3.5kD and ~55kD. And..Included in the info. on the website was a chromatogram with 13.7KD (Ribonuclease) and 82KD (enolase) in the one run on this particular HALO column. The Rib A came out just after 2 mins, but the gradient started at 25% acetonitrile, so there is a possibility the 3.5 kD may be retained.

I wrote to the distributors of the column in Australia with a few questions, and am waiting for them to respond. But the column seems ideal. Very sensitive.

Thankyou so much.

HPLC Chemist,

Yes they still make C4 columns. But your right, perhaps I should just ring up some companies and ask them to suggest something with all the new technology now.

I will also try the protein on the C8, but I am worried that I will have to use too much acetonitrile, as I think I went up to 52% to get the peptide off the column, and I don't want the protein to crash out having to use a lot more.

Hi Faith,

Just curious, which way did you decide to go for the stationary phase?

I have ended up ordering
Halo 400 Protein C4, 2.1 x 100 mm, 3.4 um column.
Am very excited, coreshell technology and the range is from 2 kD to 500kD. They have a chromatograph with a number of compounds in it on the site (from 13.8 kD (Rib A) to 88kD (enolase) ) if I remember correctly. But, that gradient started at 24% acetonitrile, and RT was just after 2 mins for the 13.8 kD so feel confident I can retain a 4kD peptide. Then I later read, the range of the column starts at 2kD. I have to wait three weeks for the column to arrive.

Best Wishes Faith!