OK, lets see-
25000 plates/m = 7500 "plates" in a 30 cm column, so variance in elution volume
of the molecules should be elution volume/sqrt(7500) = .0115 Ve.
Full width at half height is 2*sqrt(2ln2), or 2.35, times sigma, so 0.0275
times elution volume.
For elution volumes 10 - 15 ml, FWHH of peak should be 0.27 to 0.4 ml,
if the sample is applied in an infinitely small volume.
For finite samples I guess width of the peak would be the sample volume
increased by .27 to .4 ml due to peak broadening.
eab
Zhijie Li wrote:
Hi Ed,
I guess by "24mL SD200" Peter meant the Superdex 200 10/300 column, which
most of us should be quite familiar with. According to GE healthcare, a new
Superdex 200 10/300 GL column should have TP >25000/m. For comparison, a new
Superdex200 16/600 PG, which uses bigger beads, has TP >13000/m. The TP
difference of the two should be mainly caused by the different resin sizes.
Of course in reality columns change over time and in cases like Peter's, it
might be a good idea to test the performance of the column before drawing a
conclusion. When we are concerned about resolution of a column, we load a
standard sample and calculate the TP based on the peak shape. As I remember,
GE healthcare's SEC manuals has recommended procedures on TP determination.
Zhijie
-----Original Message-----
From: Edward A. Berry
Sent: Saturday, June 29, 2013 7:43 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] off topic: good peak on gel filtration
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
