OMNISEC for Protein Characterization – Demo at your desk Q&A

The column used in these measurements was s single 300 A column with a particle size of 3 um.  It was an analytical column 300 mm long x 15 mm ID.

­I’ll assume here that you are asking about detecting fragments in the presence of the IgG.  This is certainly possible but more dependent on the column resolution than anything else.  I’ve certainly studied IgG fragments but don’t have an example exactly as you described that I could show.  If you are interested in such samples, though, I’d be happy to measure some and see what information we could get.

The recoveries for both of the IgG samples (native and heated) was just over 50%.  The most likely explanation for this was simply that I dissolved the lyophilized powder for a short period and that it was not fully dissolved and some sample was subsequently lost, either when I filtered it before injection, or on the column.  Better practice would have been to use a UV spec. to measure the sample concentration before injection.  Then I would have a better idea if any was lost on the column.  In that case, either a different column or mobile phase would improve the sample recovery.

The software integrates the full area of the selected peaks so the results cover that full range.The only limitation here is when one of the detector signals returns to baseline before the edge of the peak.  In this case, the software will extrapolate the molecular weight trend in that region.  The main body of the peak is using the real calculated data though.

These measurements were performed on a 300 A silica column with a 3 um particle size.  We supply these columns and call them PLS3030.

Of course, a lower molecular weight protein like this will scatter less light so we might need a bit more sample but we can see the signals here are very strong.  From other measurements we’ve made with peptides, I think we could make a measurement with somewhere around 5 µg of sample, possibly a little bit less.