Peter Hsu wrote:
Hi all,
I've generally always thought as long as the peak was symmetrical and not too
broad would suggest a good sample. However, looking at my previous runs in the
past, I've had peaks as narrow as 1.5-2mL on a 24mL SD200, or slightly broader
peaks with about 3mL (all symmetrical peaks, roughly similar amounts loaded on
the columns). I'm curious to see what people's views are as far as what
constitutes a broad peak and how much that can end up affecting crystallization
of the sample.
Thanks for any responses.
Peter
The width itself may not be a good indicator unless its always the same protein- in general a molecule that elutes later
will have a broader peak.
Supposing that each time a molecule diffuses into the stationary phase it resides there for a certain time on the
average, then the extra retention time is proportional to that time, times the number of times it enters stationary
phase (N, "theoretical plates"). The variance in elution time is proportional to the square root of N (like standard
error of the mean) and the dwell time. This gives sigma/(retention time) = 1/sqrt(N). If N is the same for all
molecules, the criterion to look at is peak width divided by retention time. If it varies (the reason some molecules
elute slower is not just that they stay in the stationary phase longer, but also they enter more often; k-on as well as
k-off) that would still be better than just peak width. People don't talk about theoretical plats and HTEP much any
more, perhaps because the driving force in chromatography is HPLC and FPLC, and fast chromatography is antithetical to
good resolution?
However I'm not familiar with this column and can't advise. You can calculate N more exactly (see wikipedia "van Deemter
equation") as 8*ln(2)*square of (elution volume over width at half height), divide length of column by that to get HETP,
and compare with values like .7 mm reported for resins like ultragel A at optimum (very slow) flow rate.
eab
