HPLC method development

[sathyanarayanan]

We have product(nutrient supplement) consist of seven enzymes.
We required to develop method for assay analysis.
can you suggest

[skunked_once]

Enzymes are proteins/peptides so you will need to use a column with 300 angstroms pore size of the C-18 particles. Moble phase A should be water with 5% acetonitrile and mobile phase B should be acetonitrile with 5% water. You will need very pure water to avoid background peaks. Add 0.1% trifluoroacetic acid (TFA) to both mobile phases. It must also be of high quality. Run a 0 to 100% gradient over 50 minutes initially to determine the retention time of the enzymes. After that, you can adjust the gradient for faster runs. Be sure to allow equilibration time after the gradient. Monitor at 210-220 nm UV.