This week I am at an intensive chromatography bootcamp taught by legacy Dionex experts Nick Glogowski and Daniel Kutscher. Of the hundreds of interesting things I've picked up so far, one of the coolest things is this explanation of smaller particle sizes and why we keep migrating to smaller and smaller particles.
This chart shows the optimal parameters for separation of different particle size solid phase materials. (Sorry about the colors, original document available here.) For the most striking difference look a the 10um particles. There is only a very specific flow rate where the binding is optimal. Look at that in comparison with the 2um and 3um beads where we can handle a much larger variation in flow rates and maintain a nice straight line. While I'm not going to pretend I understand all the math that has passed by on the projector this morning (sorry, guys!) I think it is still pretty clear that consistency in our chromatography sure makes a big difference (especially if we want to load our columns faster than we elute off them!)
Monolithic columns do not adhere (fully, at least) to the Van-deemter equation due to lesser impact of mass transfer (termed C). One thing to remember is that pore size also affect this constant, so smaller particles will take you so far when you're running big proteins. I don't know if you've tried the pepswift, but for applications involving proteins, or a mixture of several sizes you're bound to get better performance with it then the other C4 and C18 300A columns...
ReplyDeleteThanks for the comments! I haven't tried the monolithic columns personally but I was around when Craig Dufresne was experimenting with some and the results did look very nice. I plan to give one a run the next time I go to help someone perform intact or top-down experiments.
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