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rpHPLC chromatograph shift

HPLC protein separation

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3 replies to this topic

#1 ionchannelbk



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Posted 23 October 2012 - 01:28 PM


Recently I used reverse phase HPLC with a C18 column to purify a spider toxin, using acetonitrile+0.1% TFA and water+0.1% TFA as mobile phase. The elution protocol worked very steady until recently. The retention time of the spider toxin kept delayed further and further. It used to be eluted at ~ 21% acetonitrile, now it is eluted at 95% acetonitrile.

I tried to regenerate the C18 column by wash with Methanal-Chrloroform-Methanol as instructed by the column manual. It did not solve the problem. Later I change the security guard cartridge of the column because it may get dirty and cause the delay. But it still did not solve the problem.

Do you guys have any solutions for my problem? Is the beads of the column getting bad? Any suggestion will be appreciated.

#2 Adrian K

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Posted 23 October 2012 - 05:45 PM

Check pump calibration and possible loose tubing.
Also, check your system pressure, it might be inconsistent.
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#3 mdfenko


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Posted 24 October 2012 - 07:42 AM

is your mobile phase prepared fresh or used over several days?

how do you degas? are you sparging with helium?

the tfa may be evaporating. try fresh mobile phase (if you haven't already done so).
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#4 ionchannelbk



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Posted 24 October 2012 - 08:14 AM

Thanks for reply, Adrian and mdfenko.

The mobile phase was always fresh. Actually I have to make it fresh. I ran Prep-HPLC, which consumes 4L mobile phase in 2 hours. I degas it by vacuum which looks working well for my many previous runs.

Now I think it is worth checking the pumps and the mixer.

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