You may find caffeine, uric acid, inosine, hypoxanthine, guanine,
or guanosine to be good choices for your standard. Although I do not know
where they will elute in this system, one or another base or nucleoside
will probably have the right polarity. You might check Rossomando's book
on HPLC in Enzymatic Analysis for further ideas.
Always nice to hear from a fellow Madisonian.
Chris Halkides
>Dear ABRFs,
>
>I am not sure this is the best place to post my question, but many of you
>seem very familiar with HPLC techniques, adn I hope somebody can help me;
>here is my question:
>I am analyzing the activity of an enzyme in the process of being purified,
>using an HPLC assays; the reaction products are analyzed by separation on a
>Zorbax C-18 column, using the following gradient: 0.1% TFA (A) in water
>with 20% of 0.1% TFA (B) in acetonitrile, going up to 50% in 14 minutes.
>The substrate and the products elutes at about 35% of B.
>To accurately quantify the amount of digestion, in presence and without a
>particular inhibitor, I need to use an internal standard. I have tried
>various compounds, included benzene which would work out just fine if it
>was not so volatile that by the time is injected (after a 2 hrs reaction),
>is all gone.
>Can any of you think of something that I could use in my reaction and
>inject as a standard, which possibly elutes before my substrate (I would
>like not to use a longer gradient, if avoidable) and absorbs nicely at 254nm?
>
>Any help is greatly appreciated,
>
>
>Debora Fontanini-Sella
>University of Wisconsin-Madison
>e-mail: dfontani@facstaff.wisc.edu
>phone: (608) 262-4478
Christopher Halkides
Dept. of Chemistry, UNCW
601 S. College Road
Wilmington, NC 28403-3297
(910) 962-7427