Chromatography Forum

Hello Everybody,

I have a C18 Gemini-NX column of 150x2mm, 3um and 110A. I am trying to use this column (to high pH, around 10) to fractionate a peptide digest. I use these buffers: Buffer A: 20mM TEA + water, pH 10; Buffer B: 20 mM TEA + 80% CH3CN + 20% water, pH 10.
The main problem is that I have a very high pressure values along the run and in several cases I cannot inject the sample for this problem.

Maybe, Could the column be degrading oneself?? Could anybody advise me how to improve my pressure values???

Anyway, in my last run, I don't understand what happened but I had an overpressure problem in the moment of the sample injection. I think that the sample is precipitated for basic buffer. And now I have too high pressure values. I want to clean or regenerate my column. Which solvent is better to clean the column and reduce the pressure values?? DMSO or THF???

Could anybody help me???

Thank you very much.

Wardiam

What is the flow rate with your 2mmID column? Flush your column with ACN in reversed flow without connection to your detector at 0,1ml per minute for minimum 10 column volumes.
Why TEA? Tailing problems? What injector do you have?

By the way: for applications at high pH there are several RP18 columns on the market, based on polymer. There performance is better than there image.
What ever silica based column is used at high pH, it is still silica!!

Try same column, same particle size, but larger ID and shorter column length. Try to replace TEA by a volatile buffer.

From the problem you described I don't really see it is to do with column degradation itself - column voiding due to use of high pH mobile phase typically results in decreased efficiency, resolution, peak broadening, tailing and/or splitting.

High pressure value is usually caused by blockage of column inlet frit - in this case I suspect the culprit is gunk in your sample that deposited on your inlet frit or even the head of the packing bed. My recommendations would be to reverse-flush the column, however consult your column manufacturer (Phenomenex) first, as some manufacturers used frits of different porosity for column inlets and outlets.

If you truly suspect your chromatography problem is due to column degradation, you can try more robust columns, for example XBridge columns from Waters.

If you suspect the column to be fouled with sample (peptide) then a wash first with 1% acetic acid in water for 20 column volumes (~10 mL - to be sure the LC system is purged) followed by 10mL 50/49/1 DMSO/H2O/acetic acid is a good start. Be sure to reduce the flow rate when running the DMSO solution to avoid over pressurizing the column. For good measure you could follow the DMSO/H2O/acetic acid with 10mL 50/49/1 DMF/H2O/acetic acid also at low flow rate. Finish up with 10mL 1% acetic acid then equilibrate with mobile phase.

Is the sample soluble at high pH?

What is the sample dissolved in?

The source of the problem (precipitation?) should be determined in order to determine the most robust anlaysis conditions.

What is your flow rate and inital %ACN?