What info do you need to know when analysing Protein on HPLC

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

5 posts Page 1 of 1
I provide a HPLC service, but I normally work on synthetic compounds. The chemists fill out a form I have made-up, with the compounds information and I analyse/purify.

However, recently I did some Protein/Peptide work. And I found it was a whole different ballgame. A Protein standard (in this case) wasn't just one peak, the protein precipitated in high organic (which I have had with synthetics) but this was inconsistent. Protein aggregation!

I was finding things out as I was going along, I had not asked enough information on my form. I have now decided to prepare a separate form for proteins/peptides analysis.

The recent job was to quantitate a protein and peptide in the supernatant.

I wanted to know (not being a protein chemist) what is the important information I need to know about the protein or peptides to make my life easier. That is, what questions should I ask on the form?

I am thinking something along the lines of...
Size kD
Sample concentration
What buffer and pH is sample in?
Solubility in Acetonitrile
Sample Storage Conditions
Temperature stability
pH sample stability
Does sample aggregate? If so what can prevent this?
What other possible inclusions is there in the sample solution

Can you think of anything else I should add, or have I asked some ridiculous questions? It probably depends on what they want me to do?

BTW this work is not using the MS.

Thank you very much for reading my waffle, and any help would be appreciated.
I would also add;

1. Sample matrix
2. Excipients and their concentrations

These can 'kill' a column rapidly even though they are unseen.
Thankyou HPLC chemist.

I will do. Brilliant.
If you're going to be doing more protein work, I'd recommend getting a copy of "Basic HPLC and CE of Biomolecules" by Cunico, Gooding, and Wehr. It's out of print, but used copies are available for $25 or so on Amazon:
https://www.amazon.com/exec/obidos/tg/d ... 966322908/
-- Tom Jupille
LC Resources / Separation Science Associates
+ 1 (925) 297-5374
Thankyou Tom,

I have ordered it. Am quite excited. :)
5 posts Page 1 of 1

Who is online

In total there are 7 users online :: 0 registered, 0 hidden and 7 guests (based on users active over the past 5 minutes)
Most users ever online was 234 on Tue Feb 06, 2018 7:33 am

Users browsing this forum: No registered users and 7 guests