Chromatography Forum

i am trying to purify a protein from a biological source. after a series of purification steps the last step i have employed is rp hplc. i get a single peak at this stage. on mass analysis it analysed as two proteins with a mass difference of around 100 daltons. on careful analysis, this protein does have two peaks but the retention times are with a difference of less than 0.5 minutes. how do i seperate these two more accurately? plz help.

There are only a limited number of ways to improve resolution in reversed phase:
- a longer column
- decrease the flow rate
- change the steepness of the gradient
- change the temperature
- change the pH
- change the column

Pick one, try it, and see what happens. If no effect, try the next one, and so on.

Without more details about your exact conditions and results, it's hard to be more specific.

thanx tom. i did try changing the gradient but without any success. can u enlighten me about the system using change of pH? currently i am using the TFAH2O/CH3CN system.do u think changing the flow rate will help? what column do think should i use? currently i am using the rp hplc column C18.

In a TFA/water system, change the concentration of TFA (either double it or cut it in half). TFA does a number of things (controls pH, acts as a ion-pair reagent, and keeps your proteins denatured), but all you want to know is if its concentration affects your selectivity. If selectivity doesn't change, then go back to the original TFA concentration and try something else. If selectivity improves, then change further in the same direction. If selectivity gets worse, then go the other way.

As for changing the column, try a different brand of C18 or C8. For proteins, you will probably get best results with a large-pore material (300A pore size or thereabouts). C18 columns can vary tremendously in selectivity.

Another thing that might be useful is to change from TFA to hexafluorobutyric or formic acid.

Getting rid of fluoroacids, using formic as suggested, + using an alcohol (1- or iso-butanol are even used) might help. It will be very instructive to read one of MTW Hearn´s many reviews on RP of proteins.
Almost forgot, it may be prudent to use a column which has not seen TFA if you should do without it (I am not sure that RP columns are adversely effected, there is at least one SEC column which changed adsorption properties, partially irreversibly, after being exposed to fluoro acids).

Mixed mode chromatography offers an alternative approach to the separation of peptides and proteins. You have two mechanisms which you can use for your benefits: reverse phase and ion-exchange, by modifying amount of organic, buffer nature and concentration, pH of the mobile phase you might have a chance to separate your mixture using our new line of columns Promix. Please check the following link. It describes separation of three insulins. Two of them have exact molecular weight and differ on l in position of two amino acids Pro-Lys vs. Lys-Pro. We believe that this is the first successful separation of human and synthetic insulins (Humulin and Humalog). The application might give you idea on the ability of mixed mode chromatography to separate closely eluted peaks.

http://hplcmethods.com/application_101.php

100 Daltons is a significant difference for this particular type of columns