mass balance of chromatography columns

[latkan]

Hey guys im doing a project and we have a design of a process which we have to mass balance. I am in charge of the mass balance of the protein a chromatography step.Thing is i have no idea where to start...so far i have worked out the column volume via the dbc and the mass of product attainted from the feremntation and centrifugation (mass is 5kg and dbc is 30g/l) i have also worked out the times for the load, wash, elution and cip...but like how do i go about doing the actual mass balnces of the prod in/out contaminats i/n etc any help on a general method or lay out would be great, im literally stuck on this and im beginning to panic
thanks

[tom jupille]

First of all, provide a bit more detail. Most of the Forum members are involved in analytical-scale chromatography of small molecules, not prep of proteins. Speaking for myself, I don't know what "dbc" "cip" and "i/n" stand for.

That said, the most general way to do a mass balance on a prep separation is to collect fractions, analyze each fraction, and sum up the analyses. The trick lies in how you do the analyses! If you only need to account for the proteins, something as simple as the old-fashioned ninhydrin reaction can work. If you need to account for a wide range of compounds, it gets more complicated. In that situation, the most common technique is analytical scale chromatography, but the catch is that chromatography is only a *relative* quantitation technique, not an absolute technique. In other words, all you can ever do is to compare the response from your sample to the response from known calibrators -- and that implies that you have standards for all of the compounds involved.