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
