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- Posts: 85
- Joined: Fri Sep 10, 2004 3:10 pm
I'm purifying a natural ligand. I expect it to be ~35-55 amino acids.
Following a preliminary bulk C4 solid-phase extraction step, the first HPLC step is a C4 10 mmID column. The gradient is 27-50% CH3CN over 60 minutes (0.1% TFA). Ambient temperature. I loaded 15 mg extract (Vydac catalog claims the column can handle 50 mg). The active fraction range is Gaussian, though has some tailing. It spans t=14 min to t=40 minutes. In other words, it spans over 1/3 the chromatogram.
At first I would suspect something may be wrong with the column, however, a separate ligand we are screening for elutes AFTER the broad peaks and spans only 3-4 fractions (minutes) according to our assay.
What is the possibility that, in spite of the broad active fraction range for the first, there is somehow only one active species?
Following a preliminary bulk C4 solid-phase extraction step, the first HPLC step is a C4 10 mmID column. The gradient is 27-50% CH3CN over 60 minutes (0.1% TFA). Ambient temperature. I loaded 15 mg extract (Vydac catalog claims the column can handle 50 mg). The active fraction range is Gaussian, though has some tailing. It spans t=14 min to t=40 minutes. In other words, it spans over 1/3 the chromatogram.
At first I would suspect something may be wrong with the column, however, a separate ligand we are screening for elutes AFTER the broad peaks and spans only 3-4 fractions (minutes) according to our assay.
What is the possibility that, in spite of the broad active fraction range for the first, there is somehow only one active species?
