Chromatography Forum

For the determination of vitamin E in Plasma , we use HPLC-Fluorescence Detector , with 100% Methanol as a mobile phase. But the pressure of the pump keeps increasing day after day ( from 990 PSI to 2800 PSI)

I can not understand where is the problem?

I would like to ask about the best protocol to follow for flushing the column C18? I tried 1% acetic acid in water (30 min, flow 1ml/min) after that 100 % methanol (30min flow: 1 ml/min)..

Thank You

Most, but not all, proteins are very hydrophobic, resulting in many proteins sticking to C18 columns, not coming off. This is why the majority of protein applications are done using C4, C5, C8, ion-exchange, or SEC.

Proteins, and some peptides, may not be soluble in 100% organic, such as methanol and acetonitrile, plus the fact that 100% organic is one of the methods used to precipitate proteins in biological samples before analysis. That being said, I suspect that you may be experiencing one or both of the above affects from using 100% organic as the mobile phase.

There is no mention of the type of clean up being used to remove proteins and other compounds in biological samples that foul HPLC columns. If doing an extraction method and still getting increased pressure, I suspect that some of the other components in a biological sample is the root cause, but if using straight plasma or diluted plasma, first the proteins are being precipitated by using 100% organic as the mobile phase, and second, the other biological compounds are also contributing to fouling of the column.

If there is a need to use 100% organic as the mobile phase, and there is no sample clean up done, the starting mobile phase needs to be =/< 20% organic to prevent protein precipitation. Once the proteins have been eluted from the column, then the ratio of organic to buffer, water, etc. can be increased. If the sample is being extracted, the method needs to be improved to remove more of the other biological compounds that stick on the inlet frit, such as fatty acids, phospholipids, etc. on the head of the column. In either case, you should be using a guard column, one that matches the column packing, and a disposable filter in front of the guard.

If proteins are sticking to the column, I rather doubt it can be regenerated to its original condition; however, if you wish to try to clean the column of proteins, there is an article that LCGC-Europe Magazine published in their column watch series that contains a section on removing proteins from reversed columns. All that you need to do to find it is to do a search using "protein" + “C18" + "flush" + "reversed". If you find that the column cannot be restored to its original performance, or acceptable performance, I suggest that you find a good SPE method for sample clean up, use a guard column, and possibly a disposable filter in front of the guard.

I hope this information is useful.

Linda Koch

Thank you very much, and i will try to follow the article "The Cleaning and Regeneration of Reversed-Phase HPLC Columns"LCGC-Europe.

I don't know if there's a more recent article on the subject, but the original article was the following: LC-GC 17 (April 1999) 354. The authors stated that injection of some pure trifluoroethanol at the start of a regular organic solvent gradient, followed by injection of more trifluoroethanol at the end of the gradient, was adequate to elute proteins from a C-18 column and restore its performance. LC-GC doesn't have this article in its archives, so I've scanned in my hard copy. Contact me offlist and I'll send it as an attachment.

Be advised that trifluoroethanol is quite toxic.

Andy Alpert
aalpert@polylc.com

I don't know if there's a more recent article on the subject, but the original article was the following: LC-GC 17 (April 1999) 354. The authors stated that injection of some pure trifluoroethanol at the start of a regular organic solvent gradient, followed by injection of more trifluoroethanol at the end of the gradient, was adequate to elute proteins from a C-18 column and restore its performance. LC-GC doesn't have this article in its archives, so I've scanned in my hard copy. Contact me offlist and I'll send it as an attachment.

Be advised that trifluoroethanol is quite toxic.

Andy Alpert
aalpert@polylc.com

Please, how can i contact you (by my e-mail adress )

aalpert@polylc.com

My telephone# is (410) 992-5400

Andy Alpert

You already got two excellent responses. My question is, what kind of sample preparation you use to eliminate proteins for example by SPE from your sample. Some proteins may stick for ever on your column and I'm afraid that you will not get a 100% regenerated column. To extend the lifetime of your column please try to eliminate the proteins during sample preparation. Good luck.

You already got two excellent responses. My question is, what kind of sample preparation you use to eliminate proteins for example by SPE from your sample. Some proteins may stick for ever on your column and I'm afraid that you will not get a 100% regenerated column. To extend the lifetime of your column please try to eliminate the proteins during sample preparation. Good luck.

Thank you for all your responses.
I am trying to devlop an HPLC method for determination of creatinine in human serum using C18column DAD detector and phosphate buffer pH 7,4 and 1% methanol as mobile phase.

For sample preparation,for the elimination of proteins , i add 200µl of methanol to 100µl of plasma (vortex 3 min) and centrifugation for 8 min at 12000 rpm.
I am still searching a better way to get 100% precipitation. any suggestions

Thank you again