Chromatography Forum

Hi all. I'm a biology guy. So my knowledge about chemistry is poor, especially chromatography. I want to analyse my recombinant protein by using RP-HPLC. I want to know the retention time of my protein on C8 and C18 column. I had run HPLC for the first time using C18 column but it seem that most of proteins ran out of the column immediately so I can not see any peak after that. My program is equilibration by H2O:0.1% TFA and gradient from 0% acetonitrile:0.1%TFA to 100% acetonitrile:0.1%TFA for 100mins, flow rate is 0.8ml/m. What should I do???

Hi all. I'm a biology guy. So my knowledge about chemistry is poor, especially chromatography. I want to analyse my recombinant protein by using RP-HPLC. What should I do???

You should gain some HPLC experience first. No one starts with a climb up Everest.

Are you following an established, validated method, or are you starting fresh?

If starting fresh, then KM's advice is sound. Get hold of a good text on HPLC of proteins or take a course.

If you are trying to follow an established method, follow it EXACTLY. Specifically, us the same brand of column specified in the method. All "C18" columns are *not* equivalent.

I'm starting fresh. Please tell me know where I can find KM's advices? Thank u so much.

http://www.blogsua.com/pdf/hplc-of-pept ... tocols.pdf

http://www.interscience.be/promotiesite ... ecules.pdf

http://wolfson.huji.ac.il/purification/ ... ookRPC.pdf

Etcetera. Google is your friend. If you have a specific protein, try to find a published method in Journal of Chromatography A or Journal of Chromatography B.

And for a good solid introduction, I usually recommend "Basic HPLC and CE of Biomolecules" by Robert Cunico, Karen Gooding, and Tim Wehr.
You can pick up used copies for around $40 on Amazon:
http://tinyurl.com/8boeoxk

Chromacademy might be a help and also pick the brains of any colleagues who might have some experience. I am guessing you are not the only one there running an HPLC system?

http://www.chromacademy.com/

Le Duy:
Some neophytes believe that RP-HPLC is synonymous with HPLC. It is not. In general, intact proteins are best analyzed by ion-exchange or hydrophobic interaction chromatography. These are modes that do not denature the tertiary structure of proteins. They are frequently more sensitive to single-residue variations in a protein's composition than is RP-HPLC, even if the protein of interest survives passage through a RP column. You can also get up to 100% of the protein to pass through the column and recover it successfully using these alternative modes. Many proteins don't elute from RP-HPLC columns at all or else do so in peaks 15' wide. Accordingly:
1) Are you attempting to follow a protocol for analysis that someone else has implemented successfully with this protein and which involves RP-HPLC (per Tom's earlier response)?
2) If not, then what exactly are you looking for with this analysis? Does this protein occur in variants (presence or absence of sialic acid in a glycan sidechain; deamidation of an asparagine residue; truncation at either the N- or C-terminus; etc.) or something like the presence or absence of unrelated proteins? Some methods are better than others for certain such applications.

I don't disagree with the others, learning more will help you, although I understand the limit on time to do that. We can't all be experts in everything. So for quick and dirty assistance:
1. Not all C18's are created equal. Some will work when others don't. I use a lot of Phenomenex, Grace/Vydac and Thermo C18's. All are different.
2. In column selection pay attention to pore size (angstroms). If you have a higher molecular weight protein, you'll want a higher pore size. But if you're getting blow through, a lower angstrom column may help. Smaller pore size will effect your pressure though.
3. If you have a UPLC or capability to run at higher pressures, try shell-silica columns or smaller micron particles for better resolution.
4. 0.1% TFA H20/ACN is just a starting point in peptide/protein resolution. Not the be all end all. TEAP is phenomenal, especially in the shell-silica columns. Or other phosphate or acetate buffer systems. When TFA is insufficient, I tend to go for TEAP next, you can even buy pre-made stock for cheap.
5. If you are evaluating a native-type protein, the folding may be too complex for RP-HPLC. Denaturing may be required first. Not ideal, but if other, easy options don't work...

Not much of a tutorial, but if you're starting from scratch, there's a few options.