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Different profile of the same sample in different IEC column


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5 replies to this topic

#1 Prep!

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Posted 09 May 2011 - 03:01 AM

Hey people... my head is spinning 360 degrees with the unique problem i have got here. I am attaching a pdf file which have the details... essentially i analyzed three different samples (same product) on two different column manufacturers. Its a cation exchange chromatography. Two samples behave similarly while one of the sample is bizzare... All i can think of is there is some problem with the sample and is interacting with the column chemistry and so the bizzare profile. Please suggest.. sorry for the long explanation to the problem and please be patient enough to go thru the word file :-) (I have tried to make it as concise and simple as possible) :D

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#2 mdfenko

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Posted 09 May 2011 - 08:04 AM

have you repeated the runs?

did you run sample 3 after samples 1 and 2?

maybe the column requires cleaning after 1 or 2 samples or sample 3 was too "dirty" for that column?

Edited by mdfenko, 09 May 2011 - 08:05 AM.

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#3 rick112

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Posted 09 May 2011 - 07:34 PM

what is the sequence of the running of sample ?? did u run sample 1 first and then others latter??

jst wondering if you do multiple injections?? do you see a periodic repeatation of this profile ??

#4 Prep!

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Posted 09 May 2011 - 07:47 PM

sequence of runnig is sample 3, sample 2 followed by sample 1 and the run has been repeated more than three times on both the columns (even using the same mobile phase).... :blink:
all samples are purified proteins so sample being dirty is less likely
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#5 mdfenko

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Posted 10 May 2011 - 10:02 AM

all samples are purified proteins so sample being dirty is less likely

what i meant by "dirty" was aggregated, denatured, decomposed.

have you tried making a fresh sample 3?
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#6 Prep!

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Posted 10 May 2011 - 07:25 PM

yeah even i meant it is a purified protein so no impurities related or unrelated... and i prepared the sample fresh each time i run!!!! :( i have mailed to the column manufactureres too.. will post their replys once i get them!!! even they are zonked!!! :lol:
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