purification of proteins on rp hplc with close ret times

[dhanashreedj]

i am trying to purify a protein from the biological sample by rp hplc. during the elution of this protein there is another protein with a retention time of 0.5 minutes difference which comes as a contamitant. how can i seperate these two effectively? plaease help!

[HW Mueller]

How are you doing this (isoratic, gradient, what is the biological source, what is the mobile phase....)? You seem to be doing a splendid jop already if you really have only two proteins left from a biological matrix, must be a very simple biological system.

[dhanashreedj]

i am using a gradient system of TFA/H2O and 50% CH3CN & the biological source is human semen. literature reports point out that these 2 proteins are probably similar but one could be with a single amino acid missing but this needs to be confirmed.

[ivanvins]

You can try to improve the resolution of your RP separation by using shallow gradient or even isocratic step for the area, where your protein is eluting. The elution of proteins is extremely sensitive to eluent composition and the difference between no elution and no retention can be just a few % of the acetonitrile concetration. If you have 0,5 min diference for a 0-100% ACN gradient, this will probably work.

Other option is to isolate your protein together with the contaminant and try to separate them by another technique (ion exchange)

Ivan Vins