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benzoylation of polyamines
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 -- ---------- 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 |
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