Chromatography Forum

hi..
i wish to know waht the typical protein loading amounts (for aggregate evaluation) on an analytical sec column

for example on a tsk gel, tosoh column.. 7.8*300 mm, 7 micron, i do a 40 mcg per 100 mcl protein injection where aggregates are observed to be near 0.2-0.5%.
are these appropriate. In order to have higher confidence on the aggregate % i would report i wish to inject protein higher than 40 mcg.. would this be considered appropriate.

up to how much protein amount can be injected for sucha colulmn, such analysis.

thanks..

In any kind of of this type of analysis (similar to impurity profile), the limitations on the injected amount are either overload of the detector for the parent or loss of resolution between parent and impurity (or between impurities). If neither is a problem, you can inject more.

In SEC, you usually will have to increase the injection volume, which will widen the peaks, since there is nothing that concentrates the analyte. AN alternative would be to work from a higher sample concentration, which is most of the time limited by the analyte solubility.

For SEC, typically analytical is around 500ug loading depending on size. For prep, a lot more: http://www.sepax-tech.com/SEC.php