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- Posts: 10
- Joined: Thu Oct 07, 2010 1:52 pm
- Location: Switzerland
I am Project Chemist and develop UPLC methods for purity and assay for peptides with approx. 1000 - 6000 dalton on Waters Acquity UPLCs. One basic requirement for our methods is that the mobile phases must be suitable for MS. This means we are using mainly TFA containing eluents with 0.02 or 0.05% TFA in water and acetonitril (alternatively also formic acid, ammonium acetate or ammonium formate buffers). Since 1 year (we started more and more with method development on UPLC) we are confronted with a problem when using new Acquity columns for our analyses. After an equillibration procedure (see attachments) we do not obtain sufficient resolution with new columns. We see these problems with BEH PST columns, as well as with BEH Shield or BEH phenyl columns. Further we loose resolution, when we flush the columns afterwards with ACN/H2O 7:3 before storage. This leeds to a enormous efforts for several days to gain again resolution of the column.
We do not fully understand the problem. This problem is also present in other departments and at our customers, when the methods are transfered to them. One approach is, to flush the column with ACN/H2O 7:3 + 0.5% TFA at higher temperatures (60 - 80°C) for one day or over weekend. And we start to store our columns in ACN/H2O 7:3 + 0.05% TFA to "save" resolution (not to loose resolution again). We think that the problem might have something to do with ionparing/TFA/column/ligand/peptide.
Maybe someone of you has similar problems? I would like to know:
* Which equilibration procedure is adequate to gain resolution with new UPLC Acquity columns (using TFA-containing eluents afterwards)
* What happens exactly on the column with TFA?
* What is the secret behind ionparing/TFA/column/ligand/peptide?
* Why do we loose resolution after flushing the columns with ACN/H2O 7:3 before storage?
Here you can find more details (methods and overlay chromatograms). You can easily see my problems (not only including methods and pictures into this thread)
Equilibration procedure for new Acquity UPLC columns:
http://fotos.mtb-news.de/p/757789
Analytical method for one peptide:
http://fotos.mtb-news.de/p/757790
Overlay chromatograms of the peptide (stressed and unstressed) after the equilibration procedure:
http://fotos.mtb-news.de/p/757791
Overlay chromatograms of the peptide (stressed and unstressed) 2 days after:
http://fotos.mtb-news.de/p/757792
Method for the "TFA-flush":
http://fotos.mtb-news.de/p/757793
Overlay chromatograms of the peptide (stressed and unstressed) after the TFA-flush:
http://fotos.mtb-news.de/p/757794
Overlay chromatograms of the peptide (unstressed) after flushing the column for 30 min with ACN/H2O 7:3 and trials for reequilibration:
http://fotos.mtb-news.de/p/757788
I am looking forward to your feedback.
Nicki-Nitro