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Specialty Chemistry Forums => Biochemistry and Chemical Biology Forum => Topic started by: DeltaForce on January 22, 2007, 12:15:19 PM

Title: Peptide disappears/rendered inactive following HPLC
Post by: DeltaForce on January 22, 2007, 12:15:19 PM

Hi  :)

I'm trying to isolate a peptide thought to be around 3kDa in size, via HPLC so I can pop it on a MALDI-TOF for structural analysis.  I'm running it through the HPLC "C18 - Hypersil Gold" column with the solvents below on the timescale shown.

Time (min)   0.1% TFA   ACN
0   95%   5%
5   95%   5%
50   5%   95%
60   5%   95%

This table shows that we're starting with 95% 0.1%TFA and 5% ACN at the start, and at 50mins we reverse that to 5% 0.1%TFA and 95% ACN to elute the peptide.

However, when placed on MALDI it doesn't show up anything significant.  There was a slight peak at 6kDa but it was shallow and wide.

I can't think why this peptide is showing up in a bioassay prior to HPLC but after HPLC it is nowhere to be found.  Am I doing something wrong with the solvents? 

I'm thinking whether the peptide solution is concentrated enough, or maybe it's sticking to the column? 

Hope someone can help an undergrad in distress, thanks in advance!!  8)

DeltaForce  ;D

Title: Re: Peptide disappears/rendered inactive following HPLC
Post by: chiralic on January 23, 2007, 09:00:58 AM

Did you read thishttp://www.vydac.com/vydacpubs/NL97SPR/NL97SPR.html#Art2?

Chiralic