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Topic: Detection of enzymes using HPLC  (Read 4883 times)

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Offline welsh_Ajo

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Detection of enzymes using HPLC
« on: August 02, 2007, 11:47:18 AM »
I need to determine the amount of horseradish peroxidase in a plant sample. Is it possible to do this with HPLC ? I have an Agilent HP 1100 series HPLC fitted with a C18 column and UV - ELSD detectors at my disposal. I am willing to buy new equipment within reason. If it is possible can anyone recommend any good literature that I can use to develop a method. Thanks. 

Offline invisiblegs

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Re: Detection of enzymes using HPLC
« Reply #1 on: October 10, 2007, 04:52:48 PM »
While I am admitedly not a biochemist, I don't think that separation of this kind of analytical sample will be very simple.  While the 1100 is an excellent instrument, you need to think about the types and magnitudes of interactions between your analyte and the stationary phase of your column.  The second thing to think about is how great the differences are between your analyte and other species present after extraction.  It just seems to me that enzyme separation on a nonpolar column will be difficult to achieve.  I have to imagine that there is some other sort of enzyme assay technique (titration maybe?) that would be more accurate.  If you could isolate the enzyme first, you could quantify it with the HPLC, but that can also be done using spectroscopic methods.

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