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Preparative HPLC, running out of column

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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Im in a natural products total synthesis group here in grad school, and we have been starting to use more preparative HPLC in our syntheses. For the prep system we are using C18 and silica colums that are 21.2 mmID x 25 cm with the separations we are seeing the most we can load onto the columns is 50-100mg at a time. For the final steps in the syntheses this isnt a problem, but for the middle intermediates sometimes gram quantities need to be purified. Can you suggest some brands/manufacturers to look at for larger columns, and are they going to be prohibitively expensive? The system we are using is a waters LC 2000 which can push up to 300 ml/min with a max pressure of 2kpsi if that helps any.

Have you talked to Waters? I just looked at an older catalogue, and the largest column in that catalogue is about 7 times larger than what you have.
Also, are you familar with all the tricks that are available to maximize load? Depending on the quality of the separation needed, you might still be able to further increase the load. My guess is that you should be able to load 10 times more on your column. I can send you two publications how to do that...
nk1124 - my lab is responsible for HPLC purifications of many of my company's catalog products and I also observe limited loading capacity when the separations are very difficult. We typically purify anywhere from 200mg-10g quantities of material on this dimension of column. Depending upon the crude sample purity, the difficulty of the purification, the quantity of material, etc we use a variety of methods to yield our pure products with highest possible recoveries:
- when the crude material is fairly clean and run times are fast but the impurities are very closely coeluting we will often choose the option of injecting small volumes to maximize resolution and performing a large number of repeat injections. Automated fraction collectors connected in series are invaluable for these purifications. Also be aware of sample loss at the injector here - you will observe particularly low recoveries when performing larege numbers of injections on a system with a bad injector port, bad injector rotor seal, etc.
- when we are dealing with small quantities of product in very crude extract samples we will often perform a crude purification in which we overload the sample and cut the product out with its coeluters to yield 80-90% pure material (hopefully). We then will resubject this smaller quantity to the purification conditions a second time with lower injection volumes to hopefully yield very high purity material. This 2-step approach will reduce the total number of injections dramatically when dealing with very crude samples. Of course, be sure to use a guard column when dealing with very crude samples.
- when dealing with clean samples in which the impurities are well separated from the main product you can simply continue to overload the column until either you start to get too much coelution or until peak shapes are compromised due to solubility problems. It is surprising sometimes how much you can overload the column - in some cases we have loaded as much as 500-600mg of sample onto a 21.1x250mm column. Under these conditions it is not difficult to purify 10-15g of material in a few days.

If you do need larger columns phenomenex does offer 30mm i.d. x 250mm columns for many phases. These allow for 50% higher loading compared to the 21.2mm column and operate at around 45mL/min typically therefore the purifications are still manageable on a lab scale. If you go to the larger i.d. columns such as the 50mm i.d. columns you need to be sure your pump can handle the very large flow rates (>100mL/min) and you need to consider the large rate of solvent consumption (you will need very large vessels to premix your mobile phases and you will need the appropriate waste handling system. Outsourcing your purifications may be a better option at this scale as the cost of the columns can become prohibitive. (this is where I insert a plug for purification services offered by my company - see link below) ;)

Hope the info helps.

Paul K.
www.caymanchem.com

Wow... You must be the chap who purifies our standards of sulforaphane!
Nice to meet the other end of the supply chain.
nk1124,

I work with a start-up company that is developing a novel monolithic-type of column, and we would be interested in speaking with you about some of the problems you're having and how we might be able to help. Could we possibly contact you privately in this matter?
Thanks!
~blaise5669

What is the run-time of each purification? Chromatographic conditions? Mobile phases?
We are now producing a media capable of separating macromolecules and even virus by LC at about 1 bar. The media uses nanofibrous AlOOH attached to a microglass scaffold. The zeta potential is +50 mv at pH 7. Separation is much like an IX resin, except that the surface of the nano alumina is fully exposed to the moving phase and there are no steric hindrances as compared to HPLC.

Because these columns can operate at or near ambient pressure, then one can envision a "column" even a meter in diameter.
Fred Tepper
President
Argonide Corp
291 Power Court
Sanford, FL 32771
Phone 407-322-2500 x 102
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