By ritusaini on Tuesday, August 17, 2004 - 03:18 pm:

I am trying to analyse wheat proteins by RP-HPLC. I am using a C-8 column 4.5mm id x 25cm and running gradient elution..the eluents being water and acetonitrile with 0.1% TFA. The actonitrile percentage ranging from 16 to 42% and then back to 16% over a period of 68min. My problem is that at the beginning of the first sample run the pressure seems to be pretty constant but by the end of 1 or 2 runs the pressurs starts fluctuating a lot..say from 2500 to 400 or so..I don't know how to tackle this problem

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By A.Mouse on Tuesday, August 17, 2004 - 03:31 pm:

You may have an air bubble in your pump..

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By ritusaini on Tuesday, August 17, 2004 - 03:48 pm:

What do i do to remove it..i have already tried washing with methanol

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By A.Mouse on Tuesday, August 17, 2004 - 07:10 pm:

You need to carefully degas the mobile phase. Apply vacuum to the solvent bottles for about 30 seconds, and you will be doing fine.

If your HPLC system has an in-line vacuum degaser, it may not work any more.

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By JM on Wednesday, August 18, 2004 - 12:56 am:

Some of the HPLCs Pump in the Market does not do well when gradient is run with the 100 % ACN with TFA in one pump.Verify if the pump seals are OK??? Try using some amount of water added( like 95:5) in place of 100 % ACN. change pump seals

JM

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By Tim on Wednesday, August 18, 2004 - 03:43 am:

Since you are not running with 100% of either Water/TFA or MeCN/TFA, you could have 16% MeCN in Water/TFA in line A and 42% MeCN in Water/TFA in line B then run from 100%A to 100%B and back. That way you have some water constituent in both lines.

If you have got an air bubble and pumping with methanol into an old column/restriction capillary (to provide back pressure) isn't working, try moving up to IPA. If the bubble is really stuck, strip down the pump head, give it a good clean and rinse well, replace the seals and try again.

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By ritusaini on Wednesday, August 18, 2004 - 05:30 am:

Thanks a lot ...I'll tru out these things and will let you know if it works

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By ritusaini on Wednesday, August 18, 2004 - 01:54 pm:

One small question...Is it normal to have pressure dropping down from 1800 to 400psi while the composition of solvent changes say from 14 to 49% acetonitrile

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By HPLC user on Thursday, August 19, 2004 - 10:48 am:

yes

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By ritusaini on Thursday, August 19, 2004 - 02:37 pm:

thanks....how much time does it take for the pressure to come back to the initial value...and how long should I wait befor injecting another sample

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By Uwe Neue on Thursday, August 19, 2004 - 03:32 pm:

I do not think that such a drastic change in pressure is normal for this change in solvent composition. It should change only from 1800 to maybe 1400 psi, not to 400 psi. If indeed it goes to 400, then there is something wrong with your pump.

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By ritusaini on Thursday, August 19, 2004 - 07:54 pm:

any other comments?
pls..I need to solve this thing soon

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By ritusaini on Friday, August 20, 2004 - 11:58 am:

Hi JM

I tried it with 95% ACN...pressure seems to be pretty constant (within acceptable limits)...but somehow the pressure is lower (1400psi) than when I used to run it with 100% ACN (2300). Any thoughts on that?
Thanks a lot

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By JM on Monday, August 23, 2004 - 09:00 am:

Hi ritu,
It is now confirmed that the pressure drop is due to flow problem in 100 % ACN pump. Pls check the flow of your gradient if you are having pressure problem. if any of the pump is not working your actual flow will be different from the set value. do it for both methods and see the difference.

which HPLC u have??

JM

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By ritusaini on Tuesday, August 24, 2004 - 09:12 am:

Hi JM

I am using waters 600 delivery system.I will check the flow and let you know.

I really appreciate your help.Thanks.
Ritu

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By ritusaini on Tuesday, August 24, 2004 - 09:32 am:

JM
I checked the flow rate with 95% ACN..it seems there is some problem there..it's pumping slower than what it's showing....Is there anything that I can do on my own to rectify it.
I am kind of new to this technique so please bear if I ask obvious/simple questions

Ritu

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By JM on Wednesday, August 25, 2004 - 05:21 am:

Hi Ritu,
Change the pump seals and clean the check valves( inlet and outlet) or ask your service engineer to do it and things will be alright.

good luck
JM