I'm interested in performing semi-preparative HPLC upon a mid-range library of compounds (2000-5000 compounds) for purification. I'm currently running a complete Shimadzu system and have everything that I need. I need to translate from an analytical method to a semi-prep method and retain some level of consistency. So far, I get good separation on the analytical column (Supelco C8, 15cm, 4.6mm, 5um particles), but one broad messy peak on the semiprep column (Supelco C8, 15cm, 10mm, 5um). Can someone suggest a translation method from anal to semi? Some calculation, equation, anything to make this run a little better.
Answer:
I'm thinking you're just flooding your column by overloading it. If you go from a 4.6 mm column to a 10 mm column with the same everything else, then your column volume is only four times what you had with your analytical setup. Try shooting ten times as much stuff onto your analytical machine and see if the peak broadens out the same way. That will tell you whether it's a sample loading issue.
Separating so many compounds sounds really cool. The probability of your compounds to have similar retention time is large. This is probably why you have large messy peaks. I would suggest trying gradient HPLC instead of isosyncratic to see if you have any better separation. Increasing the run time a little might also help. Best.
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