Chromatography Forum

Hello,

I new to HPLC and still not very familiar with its usage. Thus, I hope you guys can help me out:

Intro: The objective of my work is to achive separation of proteins by RP-HPLC. Hopefully, I will be able to purify, identify and isolate the peptides responsible for the tested bioactivity.
Note: proteins were precipitated with ammonium sulphate and dialyzed to remove the excess salts.

Column: Econosil C18

Solvent system: (i) Water (ii) acetonitrile (without TFA)

1. In almost all RP-HPLC protocols in books/journal papers, people usually use gradient elution with increasing percentage of acetonitrile (let say0-100%).

So, what if I reversed the gradient i.e. i run HPLC with decreasing acetonitrile percentage of 100%-0% and I still obtain decent peaks? Is my result valid? Based on separation mechanism of RP-HPLC, I don't think it works.

2. How should I flush the column after each run and right before I end the run for the day? Different references give me different methods including flushing with methanol, methanol:water etc.

3. The column that was passed to me was an old one. I'm not sure when it was purchased or manufactured. Is there any way for me to check if the column is still in good condition?

4. Since the protein content is so low (25 ug/mL), I might have to collect a large volume of the fractions. These fractions contain acetonitrile. Considering the extremely low yield of proteins, what are the recommended methods other than freeze-drying?

I welcome any suggestions on the approach I should have used in my research. I will be glad if people who did similar work share their experiences with me.

Thank you in advance.


Regards
Joe

Hi Joe,

Please forgive me, but I’m just curious: Why would you want to reverse the gradient (i.e. from high to low ACN concentration)? As you emphasize, you’re new to chromatography, so wouldn’t it be smarter for you to learn the basics before inventing new techniques?
Anyway here is the answer for you, so that you won’t need to try the above perception: It won’t work – just trust me!
Another doubtful notion is to use pure water/ACN gradient (i.e. without at least TFA in the mobile phase). So, my advice is; don’t try that either, unless you have no other plans for the rest of the week – other than cleaning the HPLC system and especially the column for precipitated protein.

Best Regards

Do you have to separate peptides or proteins (it is not clear from your message)?

Dear Danko,

I know this sounds stupid but forgive me for my ignorance. Initially I used the correct (increasing) gradient but I have alot problems i.e. negative peaks, inconsistent results etc (though there may be other reasons for it). Out of curiosity, I did the other way round and I did get some "decent" results (with "nice" peaks). Of course, I doubt the method I used but then someone commented this might work. Now, I am confirm someone might have mislead me.

Would you be able to give me a simple explanation regarding the peaks (obtained from using thewrong gradient)?

As far as I know, TFA's role is to improve the resolution. Why would the absence of TFA causes problems like precipitation of protein in the column? Besides, will the removal of TFA be another problem. I remember I read it somewhere in the forum.

It will be very helpful if you could suggest some modifications which I should apply on my approach.


Dear Kostas Petritis,

Sorry, I think separation of proteins will be more accurate. I've made necessary changes to my first post.

Do you have any comments, apart from what Danko has mentioned?


Thank you.

Hi Joe,

OK, regarding the proposed reversed gradient: According to the reversed phase mechanism the ACN is your strong (strongly eluting) part of the mobile phase. So it’s obvious that if you start the gradient with a strongly eltuting mixture (or even 100 ACN) you’ll elute your analyte immediately (it won’t be retained) – anyway in principal. In most cases however, when protein is the analyte (or just present in the sample) the high concentration of ACN will precipitate it (the protein). So, as you can see; you’re fighting a windmill.

With regards to TFA in the mobile phase; it has several roles in reversed phase chromatography. One of them – in protein context – is to facilitate the solubility of the analytes/proteins. It’s partly due to the low pH, but there are other mechanisms involved (long story).
Whether TFA is difficult to remove or not, there are different opinions on the subject, but if you want to avoid it, you’ll need to utilize some sort of buffer - typically phosphate at low pH (perhaps 2.2 – 2.5).

Finally, I can’t comment on the â€

almost all is said already so

"3. The column that was passed to me was an old one. I'm not sure when it was purchased or manufactured. Is there any way for me to check if the column is still in good condition? " - check the SST solution of other method that is validated or use solution of analytes that is designed to check performance of RP columns (column manufacturers sell this kind of solutions)

"Is my result valid?" - as has been said many times, results are valid if obtained using validated method, as I can see your method is yet to be developed and validated