by
lmh » Thu Aug 08, 2019 8:45 am
The question has been answered well by James and others, so these are just a few loose-ends and irrelevant side-lines that are sort of fun to know about!
James mentioned particle size. Here you may find something counter-intuitive going on in proteomic world: you will probably find people using very narrow, very long, 5u particle columns. Why aren't they embracing UHPLC and running small particles? Such sophisticated LC with such last-decade particle size! The reason is that they're running very complex samples, so they need the highest possible resolution, and therefore the highest possible plate number. Very roughly, if you halve the diameter of the particle, you double the plate number, but you quadruple the resistance to flow, which means you can only run a column a quarter the length (for the same back-pressure), so you've only got half as many plates... so for the very highest resolution with pump-pressure as the limiting factor, you need very loooong columns of fairly large particles.
The other thing relates to "conservation of sample": yes, proteomic people often have very small amounts of very precious sample for their nanoflow operations. As a completely irrelevant aside, this is why they tend to use loop injection autosamplers with low-pressure needles, which can be made of Peek, which can be lowered right to the very bottom of an insert so it just touches the bottom and bends slightly, allowing removal down to the last uL. Peek, of course, is also less inclined to bind things (bioinert and all that...). Those of us who worry more about carry-over and have plenty of sample tend to favour high-pressure needle systems (it's demanding on the autosampler to wash the inside and outside of a loop-injection needle thoroughly).