help with Flash or Column Chromatography (instead of HPLC)

[PureMP]

Hello everyone.

First off let me say thank you for all the support that has been available to me so far. Now it is time for me to stop silently monitoring and ask a question:

My lab partner "Zhensr" and I are purifying meta-Cresol Purple, an indicator dye used in spectrophotometric pH measurements. Through advice from this forum we were able to purify it using a SIELC Primesep B2 column and 70% ACN 30% H20 with TFA. The prep-scale method uses too much ACN (30mL/min for 45 min X five runs for only a few 100 mg isolated product).

I have been researching 'flash or column chromatography' but it is a new topic to me. I am not sure how to choose a proper solvent system or column packing material for prep-scale purification of the dye using flash chromatography. Any comments/input would be very much appreciated.

Thank you.

MP

[Vlad Orlovsky]

I represent SIELC so I will try to advice you on this problem.

Your compound is reteined by combination of reversed-phase and anion-exchange mechanism. We screened this method for your compound (and I can send you file). You can easily reduce amount of ACN from 70% to 30-40%. You will need to increase pH and use ammonium formate or acetate. This will increase ionization of your molecule and make MCP more polar and less retentive by RP, which will allow you to use less ACN. Also it will help you increase loadability of the columns. I am not sure that flash chromatography will work because I think that your impurities are structural isomers and you might nee efficiency of HPLC.
I would reccomend running the following experiments
40% ACN with 50 mmol ammonium formate pH 3
25% ACN with 70 mmol ammonium formate pH 3
40% ACN with 50 mmol ammonium acetate pH 5
25% ACN with 70 mmol ammonium acetate pH 5.
You can alos try to add 10% of THF to replace part of your ACN (going from 70% to 20% of ACN and 10% THF). There is a chance that when you reduce amount of buffer or increase pH your impurity retains longer (double sulfated compound?) and you can trap it on the column and elute it later with higher concentration of buffer. Alternatively you can trap this impurity on the guard column and flash it during analysis similar to this application:
http://www.sielc.com/pdf/SIELC_September_2004.pdf

Do you know structure of the impurity? As far as I know impurity retain longer than you MCP, so there is a chance that you can trap it on corresponding guard.

[mbreslav]

If you want to screen conditions for silica gel flash-chromatography, use TLC. Select solvent mixture that will gives you Rf ~ 0.15 for your product. When using flash-chromatography for maximum performance do not inject liquid sample, but evaporate your solution in a low boiling solvent with silica gel (1:2 - 1:5 ratios), load silica gel on top of the column, or in a separate injection cartridge on a dry flash-chromatography cartridge, and run your mobile phase.