Respected sir/madam, for example, I am using two chromatography like ion exchange and gel permeation chromatography to separate my compound
is it any order like first, ion exchange , then gel permeation chromatography. or there is no such specific order we can follow any random steps.
if it there any order, please kindly share the reason.
Not really. It depends on your sample and sample prep. In most cases these are separate. However, in the environmental area you must perform a pH 1st before acidifying the sample for further analysis (like Atomic Absorption) and thus precipitation of cations.
Gel permeation serves either to desalt the sample or to separate the components of interest according to their molecular weight.
In contrast Ion Exchange separates (more or less, depending on the ion exchanger) individual components according to size, charge and polarizability.
Say, you would like to separate carbohydrates by anion exchange chromatography at high pH, and would like to avoid an overload of the anion exchange column with any anions from your sample (say after an acid hydrolysis), then the order would be GP, and then AEX.
Same thing if you would like to separate large from smaller molecules (or vice versa), and then analyze the isolated fraction with AEX to obtain more detailed information about the isolated fraction.
My initial approach - but this might be driven by my background - would be to use the "lower efficient chromatography" first. And in your case this would GP.
What is it that you would like to achieve combining both techniques? May be this would help evaluating your question better.
In the case of enzymes and proteins ,i think ,it will be more easy to make a first separation according to the charge using the chromatography ion exchange then we could separate proteins having same charge depending on their molecular weight. In most articles currently reported, we always start with a FPLC or big beads "mono Q or Mono S" then we use gel filtration chromatography.
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