Dear Ting Wong
The standard mthod for Accqtag requires the sample to be injected in
something like 20% acetonitrile while the eluting gradient actually starts
at a lower value than this. This means that the early-eluting peaks samples
don't bind to the column that easily and it only takes a small increase in
sample volume for those peaks to start splitting, which is what I think you
have observed. The best way to avoid this is to keep your loaded sample
volume small, but that can compromise your sensitvity as it means you
cannot load the whole sample. The way round this, which is what I prefer to
do, is to actually speedvac all the samples after derivatization and then
resuspend in 5% acetonitrile/95% water. Mix well to ensure it's all
dissolved. From this diluent, you can inject the whole sample volume if you
wish and all the peaks are tight. Moreover, there is less variation in the
peak elution position run-to-run and the samples are stable for weeks. The
method takes a little longer with the dry step, but it's worth it for
better results.
Len
>To all those AAA experts out there:
>
>I am trying to run the AAA (amino acid analysis) using the Waters'
>AccQTag method on the HP1100 HPLC system according to a paper Liu et al.
>in Journal of AOAC international, 78 (1995). Unfortunately, the Asp and
>the Glu peaks are eluting with a shoulder and tend to split into two
>peaks. Anybody know how to fix that?
>
>Thanks in advance.
>
>Ting Wong
*********************************************************************
Dr Len C. Packman
Assistant Director of Research
Protein and Nucleic Acid Chemistry Facility
Department of Biochemistry
University of Cambridge
80 Tennis Court Road
Old Addenbrookes Site
Cambridge, CB2 1GA, UK
Tel: +44 (1223) 333639
FAX: +44 (1223) 766002
e-mail: lcp2@mole.bio.cam.ac.uk
Visit my WWW page at http://www.bio.cam.ac.uk/proj/adr/PNAC/pnac.html
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