HPLC Problems

[Genepro]

Hi,

I am trying purify a protein using C-18 column (Adsorbosphere XL 250mm x 4.6mm from Alltech). I am facing with a stange problem in the my chromatograms as can be seen from the following url.




http://www2.latech.edu/~ksg015/hplc-chrom.jpg

I was using water and acetonitrile as the mobile phase. There are regular peaks in my chromatogram when the Acetonitrile gradient reaches around 60 %. This make me hard to identify my protein and collect it as fractions. This has been really frustrating me for quite sometime. If anyone has any idea about the cause of the problem and how to resolve it, please do reply me.

Thanks in advance.

Genepro

[tom jupille]

It looks like you are overloading your detector. The pulsations come from fluctuations in your ACN content. The sketch below may illustrate what's going on (apologies for the quality; it's a freehand sketch and my artistic skills are limited, to say the least):

The red trace is a magnified view of a small part of your gradient. The blue trace is the protein output from your column. In effect what happens is that every time the ACN composition drops off a bit, protein stops coming off. Protein doesn't start to come off again until the ACN concentration gets back above its previous high.

This isn't uncommon, because retention of proteins varies very quickly with small changes in ACN concentration.

One work-around is to narrow your gradient range (to 40-70%, for example) and then use 40% ACN in the A reservoir and 70% ACN in the B reservoir. That will reduce the absolute size of the ACN fluctuations.

Also, if you haven't already done it, I would go back and do a loading study on the column (start with small "analytical scale" injections and gradually increase the mass on column).

[Genepro]

Hi Tom,

Thanks for your kind reply. One thing I forgot to mention was there were always air bubbles coming off the column, when the ACN gradient reaches 50%. They continue until I stop the run. I made every effort to degass the sample as well the solvents. I also primed the pump and the lines of my HPLC instument as per the instructions. Do you think the air bubbles might have contributed to the signal?

Thanks,

Genepro

[Mark Tracy]

Looks like air in the detector to me. You should take Tom's advice because it will also help with outgassing. I had a similar problem a while back; turned out my vacuum degasser was defective. Are you seeing any similar waviness in the pump pressure? One other thing you can do is to add some back-pressure after the detector; 100 psi is about right.