Internal standard

Christopher_D_Southan@SBPHRD.com
Mon, 17 May 1999 17:41:38 +0100

Debora, when I had the same problem many moons ago I used free tryptophan
amino acid for my internal standard. This has the advantage of being about
the right hydrophobicity and a high absorbtion. Solubility is low but you
only need a smidge anyway so I just made a ml or so of saturated solution
in water, filtered to 0.22um, and diluted this out as needed. To get good
early-eluting peak shape for the Trp you could try a gradient "jump start"
i.e. set your program to jump 0-20% in 2 seconds before running the 20-50%
segment (but remember to finish with 50% to 0% as well). This may sharpen
your (assumed) peptide product peaks anyway. I would also suggest that
todays auto-injectors, or manual syringes, are so reproducable you may find
you realy don't need the internal standard. You can use an accurate
substrate peptide stock to calibrate the system and you could probably
achieve linear precursor-product conversion without needing injection
correction factors.

Yours, Chris Southan, SB, U.K.

---------------------- Forwarded by Christopher D Southan/RES/PHRD/SB_PLC
on 17/05/99 17:15 ---------------------------

dfontani@facstaff.wisc.edu on 16-May-1999 03:59

To: abrf
cc: (bcc: Christopher D Southan/RES/PHRD/SB_PLC)
Subject: Internal standard

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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