Protein chacterization using variants of HPLC: Four questions

Philip J. Wyatt phil at
Fri Jan 6 15:51:33 EST 1995

We are a small company that manufactures multi-angle laser light  
scattering detectors (MALLS) used in combination with chromatographically  
separated samples to determine their absolute molecular weights and sizes  
and the distributions of these quantities.  During the past few years, a  
great number of our colleagues seem to be increasingly interested in  
proteins and their characterizations.  (E. g. aggregation phenomena, self  
assembly, stability and just plain molecular weights of various  
preparation.)  We get a lot of complaints, not so much about the  
instruments, but about the chromatography itself and what the separation  
process is doing to the samples.  We're not experts in protein chemistry,  
but obviously feel we have a lot to learn both to help our customers and  
to interact better with the protein chemistry community.  Right now we  
have the following four questions and would be most grateful for any  
comments or suggestions a protein or biochemist might have:

Please be assured that this is NOT a commercial pitch, but it seems to  
touch on some pretty important measurements now going on in the protein  
	1)  What do you think of the chromatographic separation process as  
a tool for characterizing protein samples (assuming that an absolute  
determination may result)? 
	2)  If you use such techniques, what type do you use?  (E. g.  
reverse phase, size exclusion, ion exchange, capillary electrophoresis)

	3)  Do you think the buffer and/or the mobile phase can (will)  
affect  the composition of your sample?  (E. g. might you detect  
aggregates that were not present in your sample?  Could some of the sample  
stick to the columns during the separation process itself?)

	4)  If a column structure could be developed  that would permit  
the use of any type of mobile phase without affecting the separation  
process itself (i. e. no sticking and no false aggregation), would that  
make such chromatographic techniques more attractive?  (We do NOT  
manufacture columns!)

Phil Wyatt
wyatt at

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