On Sat, 15 May 1999, Debora Fontanini wrote:
> 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
>