Chromatography Forum

I read somewhere, on the topic of semiprep protein/peptide purification, that since at low eluent strenghts the analytes would more or less adsorb to the front of the column, it was possible to introduce samples by dunking a solvent line in analyte solution.

I was wondering if anyone has actual experience with that, as it sounds really dubious, and I can't really get my head around how you would even do it in practice, without either not getting all the analyte applied or getting huge amounts of air sucked into the system.


source: David Carr: A Guide to the Analysis and Purification of Proteins and Peptides by Reversed-Phase HPLC, p. 42, published by ACE columns


thanks in advance!

--Chris

My boss told me that during PREP HPLC (Columns from 25 mm to 200 mm in ID), they routinely load large sample volumes directly onto the head of the column (usually a precolumn or Column #1) by pumping the solution directly. This is the most common way of loading large samples. The volume of the sample is still relatively small compared to the column volume (Prep columns with volume in liters). He said they have a separate line plumbed into the system before one of the pump heads (or use another prep high pressure pump head which is dedicated to sample loading, a third pump). Taking one of the existing MP lines out of a drum or solvent vessel would indeed add air into the system. A separate injection line tee'd into a MP line or dedicated sample introduction line can be used.

Ah that makes sense. I guess a single channel on a quad pump could also be used, but it might not be desirable to risk contaminating your main system pump.

Thanks for the insight!