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