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Thanks- guess I'm old-fashioned, using low-pressure columns. So apparently theoretical plates are still calculated, and have improved a lot- 25000/m is HETP .04 mm, way better than the figure I mentioned. (TP per dollar not so much.) No more sour grapes from me- 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: [log in to unmask] > 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 >