@ G. Balaji - did the fractions come out in the methanol, before adding the dichloromethane, as a mixture? If so, you may wish to start at 80% methanol in water, go to 100% methanol, then step into your dichloromethane or chloroform gradient. I call this techniqu "wide polarity range chromatography" because I can run from very polar to very non-polar solvents in a run. The link at the bottom of this message describes some work I've done.

As for using acetonitrile with dichloromethane- That might work. Acetonitrileis less polar than methanol, so you will need less of dichloromethane to reduce the polarity. Tetrahydrofuran is less polar than acetonitrile, and can be considered as well. Tetrahydrofuran runs fine in C18 columns with water, but I find high back pressure with THF/water systems and so this solvent mixture will be slow on your open column.

If you can get some C18 TLC plates, you can develop methods on those. I see you mentioned TLC, but I didn't see what kind of plates.

Here the Wide Polarity Range Chromatography link:

http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN77_Wide_Polarity_Range_Chromatography.pdf

Do you mean a C18 HPLC column?. In such a case, with a new column you should equilibrate the column with the initial conditions (flow-rate and mobile phase composition) of your analytical method. Record the pressure at these conditions, since this value will give you an idea of some problemn that may arise during the working life of the column.

No sir I would like to do C18 open column. Not a HPLC column

Weight the resin powder, then add 3 or 4 volumes of methanol 100% and stir. You should be able to have for 1 g of resin about 2 mL of liquid resin. Then pack the column and use a peristaltic pump do pull the liquid and let the resin pack in the column. After that, let the column equilibrate with your buffer A. Usually the pressures are pretty high, because the beads of the resin are very small and the volume among the particles is very close (I'm talking about 6KPsi).

If you solve the resin in the buffer A and not methanol you cold have some problems, because organic solvents are not so affine.

is it a HPLC column

No. Open column only. It seems I cant proceed with reverse phase open column. Flow rate is very slow. Need high pressure. Any suggestions? Without high pressure?

The packing of silica gel column should be with low polarity solvent based on your extracts nature. Suppose you are trying to isolate nonpolar constituents like monoterpenes or fattyacids the packing must be with hexane or like solvents. Similarly, if midpolar constituents to be isolate, the solvent should be chloroform or like solvent. but if you want to trying highpolar constituents like glycosides then methanol is the option. For a good separation and avoid the possibility of chocking, the extract must be pre-absorbed with silicagel (approx. 1:1). It does not need any high pressure but with slow flow rate and by increasing polarity of the solvent you can isolate a range of constituents one by one. refer the attached file.

I'd pack the column in methanol (or whatever you will use for the orgainc solvent since acetonitrile and tetrahydrofuran are also used). This will condition the column and get the C18 chains interacting with the solvent. I'd adsorb the compound on Celite, much as one would silica gel. I've had good results using Celite. I'd also start at no lower than 10% organic solvent.

I used air to help move the solvent through the column; a peristaltic pump works too if you are using a column with fittings.

Mr Deepak Semwal thanks for your document. But I am trying to find out a way to perform the open column with reverse phase rather than normal phase silica

Mr.Jack Silver thanks for your suggestion. You mentioned that you used air to move the solvent. Can you explain it in bit detail?

All right you should read a article Preparative Reversed-Phase Flash

Chromatography, a Convenient Method for the Workup of Reaction Mixtures as attached with this reply.

Regards

Dr DK Semwal

@ Dr. Balaji; Please note the attached JPG image. I run air through the fitting with a rubber hose connected to the jag. Depending on your column/fitting, it may have hooks for little springs to hold the gas fitting to the top of the column and also provide for some pressure relief. for this fitting, the gas flow is controlled by a volve at the source, although I've seen these fittings with valves built into them.

If using an Ace column, or something similar, they use a fitting (generally 1/4x28) that you can attach to the gas line. Be careful using this because it it easy to break the column from over pressure.

With C18, I find maximum back pressure at around 60-70% organic solvent, remainder water. If you are running step gradients through this range, one will need to be patient with the flow.

Is there a reason you aren't using CHP-20? The particle size of this material lends itself well to open column chromatography; I've used it many times for plant extracts. It is a polymeric reverse phase material.

you can pack the column with methanol 100%. you can load the sample by dropper or pre-adsorption on C-18. to eluate your sample, first start with 50% methanol in water, methanol, then decrease the polarit by dichloromethane. finally flash your column by methanol.

good luck

I agree with Donato Angelino and Jack Silver, perhaps only needless to mention stirring time (could be about 20-30 min)

for beginner, I suggest to choose a rubber bulb pump instead of attaching the solvent container to the pressurised air line or an inert gas cylinder. It allows controlled pumping and avoids overpressure -> bursting the glass solvent reservoir.

sample can be loaded in solution, or absorbed on normal phase silica, cellite or other sorbent (cellulose, alumina, whatever). for solution loading, try to choose the eluent mixture as a solvent (or more polar)

column shall be filled/conditioned in pure MeOH or MeCN (the less polar solvent used in your particular eluent mixture) and then equilibrated with the same mixture as planed for the separation itself.

choose properly the particle size. the minimum size for the glass column flash chromatography is ca 40-60 microns, smaller particles can be used only with high pressure systems, comprising high pressure column hardware, loop injector and high pressure pump.

mixtures H2O-MeCN have lower backpressure than those with MeOH, thus MeCN shall be the first choice, but today bit pricey. Mixtures around 50%are the worse.

and do not underestimate the role of additives: most C18 sorbents separate better at lower pH, thus adding 0.1% of AcOH, TFA or formic acid is good idea. If acid can bring some complications, basic additive, such as TEA or NH3 in MeOH can be tried. Even neutral additive, such as ammonium acetate or phosphate buffer will improve the performance, increasing the ionic strenght of the eluent.

Thanks for your all valuable points. I gave a try with C18 gel in an open column with methanol as a starting eluent. Sample was pre adsorbed over C18 gel and loaded. so far so good. I wanted it to be like non aqueous reverse phase column since my fraction is of non polar. So started with methanol. But no success. Fractions collected were mixture of compounds. Then decreased the polarity with chloroform(5-10%) but no success. I found using CHCl3 in RP phase wouldnt be a good idea as my TLC plated showed an additional spot (new spot) on the solvent front which i hope should be C18 gel. So i have to repeat this with some modifications. Will it be worth to give a try with Acetonitrile instead of Methanol? Can Dichlromethane be a good choice to be added with acetonitrile to reduce the polarity?

@ G. Balaji - did the fractions come out in the methanol, before adding the dichloromethane, as a mixture? If so, you may wish to start at 80% methanol in water, go to 100% methanol, then step into your dichloromethane or chloroform gradient. I call this techniqu "wide polarity range chromatography" because I can run from very polar to very non-polar solvents in a run. The link at the bottom of this message describes some work I've done.

As for using acetonitrile with dichloromethane- That might work. Acetonitrileis less polar than methanol, so you will need less of dichloromethane to reduce the polarity. Tetrahydrofuran is less polar than acetonitrile, and can be considered as well. Tetrahydrofuran runs fine in C18 columns with water, but I find high back pressure with THF/water systems and so this solvent mixture will be slow on your open column.

If you can get some C18 TLC plates, you can develop methods on those. I see you mentioned TLC, but I didn't see what kind of plates.

Here the Wide Polarity Range Chromatography link:

http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN77_Wide_Polarity_Range_Chromatography.pdf

Mr.Jack Silver. Yes it came out in methanol itself.

Firstly, flush the column with CH3OH:H2O (1:1) for at least 2 hours by using 1 mL/min. flow. the column will be ready to use. ENJOY with your HPLC instrument. :)