Is there any specific formula to know the exact amount of injection volume and protein content for RP-HPLC column (for column of diameter 4.6 and 5 micron?)
Thank you for your time and concern.
What is the column capacity of the column that you are usig for your particular application. What is your sample concentration? By general rule the sample volume shall not be more than 10% of the total volume of column. Moreover it is appropriate to use a concentrated sample.
Dear …the column volume is around 2.9 ml (so I think 290 microliter will to too high to load as well as the HPLC injection loop is of 100 microliter)..the protein molecule is of 20Kda…the concentration of sample is about 0.5 mg/mL...so if i load max amount is 100microliter i.e 50 microgram...
I hope the amount of your protein of interest in this 50 microgram is sufficient enough else you need to further concentrate the protein sample.
In the HPLC system I have used there was option to load sample gradually along with the injection of buffer so if you also have that option you can overcome 100microlitre limit of loading as well, let the sample purge gradually it will have no effect on resolution. Finally since it's 20kDa protein a better option would be to use c 18 or C 12 column. I hope you are using the same.
All the best
Thank you ...will let u know after trying the same