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FPLC
Posted: Wed Apr 25, 2007 2:12 pm
by saraswathi
Dear all,
I am running samples on Amersham's Superdex-200 and everytime I get a peak at around the void volume no matter what protein I run...
I ahve tried giving 0.5M NaOH wash to no avail...any ideas on what could be the problem?
Saraswathi.
Posted: Wed Apr 25, 2007 8:12 pm
by danko
Hi Saraswathi,
What kind of column is this â€
Posted: Thu May 03, 2007 1:24 am
by Rande
I assume what you are seeing is what I call "the buffer peak" .
This is totally normal and it is from the buffer your sample is disolved in.
For my SEC-HPLC , I always include a PBS blank as a control sample, or whatever dilution buffer the sample is diluted in.
This is partly as a system suitability test to show the system is running OK, and partially to show to anyone (such as an auditor with questions) that this is normal and not a strange peak from my sample.
This is not really from UV absorbance but from a refraction of light at the interface of 2 different solutions .
I have always known this as the "schlearing effect" I tried to confirm this term and could not find any reference to it. Can anyone help ???
Posted: Thu May 03, 2007 1:37 am
by Uwe Neue
Rande: try "schlieren"
Posted: Thu May 03, 2007 10:03 pm
by Rande
Uwe,
Thanks alot!
"schlieren effect" is what I was refering to.
It was really bugging me. that I was not able to find that term.
I guess I have been mis-pronouncing it for years.
I was starting to question my memory and/or mental health.
(then again ... my sanity - or lack there of , is unrelated to this question)
Posted: Thu May 03, 2007 10:29 pm
by Uwe Neue
Rande, Actually, your pronounciation is fine
which was why I understood you... (it is a German word).