Hello,
I am using size exclusion to purify a protein. Before size exclusion a ran my sample through cation exchange.
Where I typically see my protein come off during SEC, there was a drop in conductance and the peak for my protein did not look good. What does this indicate?
What did you dissolve your sample in? It should be dissolved in the mobile phase for injection. If the solution you used is different, then the changes in properties will result in a signal change plus peak distortion. Avoid this types of basic problems by following good chromatography fundamentals. Get help from some local who has practical experience in using your HPLC system. These types of problems are best solved on-site, in-person.
Change of conductance during SEC analysis of proteins might be expected due to separating mechanism of this technique retaining in reversed appearance of molecular size, volume, weight...
Usually some quantities of NaCl is added for stabilisation of
protein structure and avoiding any interaction of analyte and gel in SEC, usually 0.15-0.2% w/v. If more appropriate, buffering salts may be used, which stabilise protein structure.
During travelling the protein molecule across the columns stationary phase, protein can change the ionic strength of his vicinity and close hydrodynamic volume area, as a result of dynamic equilibrium shift with an ions. It will be smart choice to consult experienced colleague or internet data and literature.
Protein Peak distortion in SEC might be of many reason, starting from damaged column inlet surface, channeling of gell, inappropriate dissolving medium and others
Pharmacia Amersham had perfect booklet for Gel Separation or Size Exclusion Chromatography. Short, illustrative and very useful.
Try to purchase, about 20$
Very nice Nick, thanks for uploading...is there others like IEX chrom, Afinity Chrom, RP Chrom, Electrophoresis...?
Is Pharmacia Amersham now part of Cytiva? I haven't use their productd a long time
Hi Nathan,
Do you use the AKTA system for protein purification? Maybe you can consult GE engineers, they have enough experience.
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