Chromatography Forum

is there a need to pre-elute the silica gel column chromatography with methanol before using? if yes, what is the purpose of doing so and will the methanol stay in the column and thus affect separation? another thing is: what is the pupose of eluting the column with the mobile phase? is it to ensure a more uniform flow through the column when separation is on? thanks

anybody knows bout this? thanks

This is not always necessary but one benefit of eluting a silica gel column before use is that the methanol may clean out some column impurities. If this is done, however, it is important to flush the column afterwards with less polar solvent to remove the traces of methanol which could affect the separation. Dichloromethane followed by hexane is a good combination. The dichloromethane is necessary because methanol and hexane are not miscible but both are miscible with dichloromethane. You must then flush sufficient mobile phase through the column so that the column is equilibrated. Ten column volumes is usually sufficient.

thanks, skunked_once.

i have heard of flushing silica column with methanol actually helps to "activate" the silica's -OH group since they are initially in the collapsed form. how true is that and how does it actually do the activation? what is meant by the "collapse" form?

the reason of methanol being used: is it bcos it is a "universal solvent "which disslove almost everything? thanks

In following this thread I believe that a lot of misguided information is being shared. Just my opinion but let me explain why.

Concerning 'activating' the silica gel:

I suspect the reason for the methanol wash primarily is to remove excess water from the silica gel so chromatography performance will be more consistent. It is, shall we say, impossible to maintain a dry silica gel column when exposed to the atmosphere. By washing with methanol (which may have a small percentage of water present) the water content may be reduced to a small but reproducible level on the column and thus allow a solvent system to perform reproducibly in separating the matrix placed upon it.

It also 'cleans' the silica gel of much of the contamination that may be present. Washing then with two non-polar solvents brings the column to a state where a consistent selectivity may be attained.

The same thing happens in TLC and one can visibly see the band of 'crud' at the top of the solvent front as the methanol moves up the plate.

Many times in TLC the plate is then placed in an oven and dried to remove water and residual methanol.

I hope this does not offend and is helpful.

best wishes,

Rod

Rod,

Thanks for the additional details. I had forgotten about the water factor on silica columns. You explained it better than I did.

skunked_once

skunked_once and rod, help appreciated. heartfelt thanks =)

regarding the mobile phase used in column chromatography, if i dun know the nature of compounds to be separated, how do i choose the mobile phase? is it through means of trial and error? does the solvent selectivity triangle apply to this? is it the normal practice to choose a mixture of 1 more polar solvent and another not-so-polar 1?

Chromatography is simplest when you can find examples of solvent/column conditions that someone else has worked out for your compound(s) or class of compounds. Keep in mind that these may not be the optimum conditions but they are condidtions that worked for that particular analyst. When you are working with unknown compounds, you have to develop some solvent scheme of polar/non-polar solvents to test with your compounds and sample impurities. Fortunately, many or all of the vendors of chromatography and solid-phase extraction columns have flow charts available that can guide you through the process. There is also a large volume of literature on column chromatography which one can wade through. Good luck!

o, thanks, skunked_once. i wonder if the solvent selectivity triangle applies to all kinds of chromatography? cos when i try to fing info regarding this, it mostly talk about HPLC and other types of chromatography.

wky,

All chromatography involving liquid solvents depends on the interactions of a sample between a fixed adsorbent phase and a mobile phase, whether it is HPLC, low-pressure column chromatography, or TLC. The solvent selectivity triangle is a guide to selecting a solvent mix for the mobile phase. I believe it was developed by HPLC experts but it should apply to all types of chromatography. Anyone else more knowledgable care to chime in?

thanks for the replies. well, i had tried out in separating my sample using the silica gel column, with chloroform/methanol 98:2 as the mobile phase (which was the mixture showing the best separation after trying out with different ratios of chloroform:methanol using TLC). However, there appeared to be many compounds in one fraction, indicating bad separation. erm, a mixture of chloroform and DCM had been previously tried using TLC but the separation was not as good as chloroform/methanol. How can i solve this problem besides collecting eluates of a larger number and smaller volume, which will be tedious and probably may not work as well? By the way, how does the diameter, height, weight and volume of the column affect separation?

How large (volume) are the fractions you are collecting now, and what size is your column?

You have just asked a very complicated question!

First off, as discussed in the other thread, extrapolation from TLC to column chromatography is, at best, an approximation.

Second, the resolving power of liquid chromatography (the maximum number of compounds that can be separated) increases with the square root of the column length (doubling column length gets you 40% more compounds). The resolving power is approximately independent of column diameter (but you can collect larger fractions!). The resolving power increases approximately as the inverse square root of the particle size. That last bit is what HPLC is all about: the use of very small particles to improve resolution. The catch is that the improvement comes about by making the sample bands narrower (hence, you have to collect many small fractions).

TLC plates are typically coated with fairly small particle size material (sub-10 micron, if memory serves -- it's been a long time since I actually worked with TLC!). If you're using "classical" (gravity or low-pressure) column configuration, your silica is probably around 100 micron particle size. To put this in perspective, HPLC columns are typically packed with particles of 5-, 3-, or sub-2 microns. Bottom line here is that it is quite possible to have better "resolution" on a TLC plate than on a silica-packed column, even if the chemistry is similar, and it is quite possible that your compounds are actually separating on the column, but that multiple bands are being collected in a single large fraction.

the column i was using is the 20 g/ 70 ml pre-packed silica gel column.

That has a fairly big particle size. I would not be at all surprised to find that the width of your sample bands was much greater on the column than on the plate. That's why HPLC was developed (to keep sample zone widths narrow).