I have to start optimising a wet lab metod to separate amino acids, 2-3 amino acid peptide and protein digest on HPLC. We have C-18 column. We should use gradient or isocratic run and which UV wavelength will be right for detection. I have just started and I also want to know about precuations to prevent column. I want to optimise prptide separation by Dry Lab afterwards.

There are many ways to do protein and peptide separations. We will need to know more information about your method before we can recommend anything.

For example, do you have an existing method for the separation? If you do, then please provide that information. If you do not have a method, then there are many decisions to make.

First, what kind of equipment do you have? Binary or quaternary pump? Autosampler? Diode array or variable wavelength or fluorescence detector?

What kind of sample preparation will you be doing, or do you need help with that also?

Please write back with these details and then we can get started.

Thanks. I am starting with Insulin. I want to perform HPLC for active and denatured states of Insulin. We have quaternary pump and this is a Shimazdu machine. We have earlier performed run of insulin in HPLC and obtained one peak. That was bfore I joined here. I think isocratic runs will be sufficient for this experiment.

If you have a method that provides good retention with acceptrable peak shape, then, yes, you can use an isocratic separation. However, make sure that you have adjust your conditions so that you are getting enough retention to see a separation. You do not want your insulin peak eluting near the void time, since you will probably not be able to separate the other peak.

The only thing to worry about with an isocratic method is the presence of other components that are highly retained, such as larger proteins or polymers. These materials will stick to the column under isocratic conditions, and over many injections they will change the nature of the surface. You will often see a change in retention, peak shape and resolution. To prevent this problem, program a "cleaning" gradient, using more acetonitrile, and do this at least once a week, depending on the number of injections.

you are right Dr. Analytical,
i think if some one else has also gone it before then i dont think u need a new method, but i would like to suggest first repeat what the other person has done and see if you are getting same peak at same RT under same conditions. Some times after the excessive usage of column the Rt of peaks shifts, and that may confuse the results.