I don’t know if anyone out there has performed this
protocol, but I am looking for advice. We are trying to determine the
concentration of
polyamines in some plant tissue samples using a
benzoyl-labelling process and HPLC. Unfortunately, we have run into some
problems and
not even gotten beyond the first step of labeling standards.
Analyzing the benzoylated standards on a C18 column using
an isocratic flow of 52% acetonitrile at 1 ml/min, we found no retention
time
differences between labelled putrescine, spermidine and spermine. We found the
same result using a 50% methanol buffer system.
It is unclear to us whether we are
having problems with the HPLC or whether it is an issue with the labelling
process.
I was worried that we were not
getting any labelling at all so we ran a sample of the benzoyl chloride on the
HPLC just to see what its retention
time may be—and we found multiple large
peaks. It’s entirely unclear to me why a reagent like benzoyl chloride should
have such multiple peaks.
Sigma-Aldrich would not admit there was anything wrong with the product and has not yet answered my grad student's inquiries after a week's
time.
We would appreciate any help or advice that anyone can give.
Jim Campanella
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James J. Campanella, PhD
Associate Professor,
Department of Biology
Montclair State University
1 Normal Avenue
Montclair, NJ 07043
Alternate email address:
Ph: 973-655-4097
Fax: 973-655-7047