question on compound behaviour on thin layer chromatography

[wky]

i have a hexane fraction over here after solevent-solvent partitioning. hexane is supposed to extract non polar compound and so i asssume that the hexane fraction contains non-polar compounds. however, when i use TLC to analyse the hexane fraction with 100% chloroform as the mobile phase, the compounds didn't move far. when chloroform/methanol 9:1 was employed as the mobile phase, the compounds moved high up, which indicates that the compounds have affinity for methanol. methanol is a polar solvent, and base on the principle of like has the affinity of like, this indicates that the compounds are polar. any explanation of this phenomena? or i must have done something wrong in the process?

[tom jupille]

like has the affinity of like

Which is why methanol will stick to the silica gel and displace less polar compounds.

[HW Mueller]

In other words, there is a huge variability in polarity of substances.

[wky]

sorry, but can u all explain more? is it methanol displaced the less poalr compound to stick to the silica and thus the less polar compound had to move further up together with chloroform? how does this correlate with the huge variability in polarity?

and, does that mean that it make sense to get such result? sorry for being slow in understanding. thanks

[chromatographer1]

Retardation Factors Rf of analytes on a chromatography system :

1. analytes won't move unless they are soluble in the mobile phase.

2. Methanol is an excellent solvent for many analytes.

3. Separation of analytes is performed by having mobile phase soluble analytes move through a media that retains the two different analytes at a different rate.

4. Changing the mobile phase composition OR changing the stationary phase will change the manner the analytes move through the system.

Your experience with chloroform and methanol are quite typical.

Try using Hexane/chloroform 80/19 with 1% methanol and keep increasing the methanol content. You could also use 1% acetic acid.
Ethyl Ether is another solvent that might be useful in developing a TLC system that separates the analytes you have. You might review the literature for examples of analytes and the solvent systems that work well for them.

Try Camag web site.

best wishes,

Rod

[wky]

thanks, rod, for the explanation. appreciated =) i dun actually know what my analytes are. another thing i would like to ask is, though methanol is a good solvent for a lot of compounds, the solvent partitioning done before this was between hexane and aqueous methanol. shouldn't the compounds having affinity for aqueous methanol went into the aqueous methanol phase rather than the hexane phase?

[wky]

anybody can help? tq

[tom jupille]

Adsorption chromatography is a complex phenomenon (i.e., you can get an accurate answer to your question, or you can get a simple answer to your question, but you are unlikely to get both ).

But I'll try.

The dominant force in the process is adsorption (a molecule sticking to the surface of the stationary phase). Solubility in the mobile phase is a secondary effect. Consider two compounds (call them "A" and "B").
- If A sticks more tightly to silica than does B, then A will be more strongly retained (move more slowly), even if A is more soluble in the mobile phase.
- If A and B stick about equally, then the less soluble of the two will move more slowly.
- If either A or B stick very tightly, then they will move slowly even if they are very soluble in the mobile phase.
- If you add something to the mobile phase which sticks more tightly to the silica than does your analyte (e.g., methanol), then that additive will "bump" your analyte from the silica and make it move more quickly. This will be true regardless of the solubility of your analyte (unless solubility is very, very low).

As Rod said, your experience is quite typical.

If you want an accurate answer, try to find a copy of the classic reference, a book called Principles of Adsorption Chromatography by Lloyd Snyder (published by Marcel Dekker in 1968; now out of print).

[chromatographer1]

Analytes that have an affinity for aqueous methanol will tend not to be present in the hexane solvent, but don't forget that hexane phase in your extracton is not pure hexane, it actually contains methanol AND water if my understanding of your description of the extraction is accurate.

for example, a simple free fatty acid might not migrate well or far in a TLC system of pure hexane although it would most likely be extracted into hexane from an aqueous methanol solution.

It wouild require some methanol to make this analyte migrate, and even better would be some acetic acid or ethyl acetate to compete for active sites on the TLC plate and to improve solubility in the mobile phase.

If you have nitrogen containing analytes, methanol addition is probably required for some TLC migration. Another solvent to improve migration might be Ethylamine or Triethylamine.

You can sometimes characterize the active structures in your analytes by careful and thoughtful addition of trace solvents to your TLC system.

I hope I have been helpful. Several years of TLC experience would be helpful here. It is not as easy to perform well as many might suppose. But it is an relatively uncomplicated technique that can provide a great deal of information at a very low cost for ID and large scale separation requirements.

[tom jupille]

It is not as easy to perform well as many might suppose. But it is an relatively uncomplicated technique that can provide a great deal of information at a very low cost for ID and large scale separation requirements.

I'll second that comment and add that despite the apparent simplicity, TLC is chromatographically complicated. It's roughly equivalent to doing gradient HPLC with an unequilibrated column. Once you learn how to control it, it can be extraordinarily powerful.

[wky]

i sincerely thank you guys for the info-sharing =)

[Grinberg]

TLC on silica is one of the first technique used in chromatography. It has to be mentioned that the retention on silicagel occurs through H-bonds. Hydrogen bonding has the has the maximum strenght in non polar medium (i.e. non polar mobile phases). Increasing the polarity of the mobile phase disturbs the hydrogen bonding between the analyte and the silica stationary phase. Therefore, in order to elute polar compounds off the start line of a TLC plate requires polare mobile phase. In your case addition of 10% of methanol lead to a total elution of your analyte from the stationary phase. This means that your analyte has groups which are forming relative weak H-bonding with silica stationary phase. It is possible that your analyte may have an ester group or an amide group in its structure?

Nelu Grinberg

[wky]

hmm, this is about column chromatography. i performed thin layer chromatography using silica gel plate. the mobile phase showing the better separation among all was chosen as the mobile phase in the subsequent column chromatograpy separation. However, the compounds seem not to separate well, where around 5-6 of them turned out to be in 1 tube. my question here is: what is the difference between column chromatography and TLC when the same mobile phase is employed?

[tom jupille]

what is the difference between column chromatography and TLC when the same mobile phase is employed?

In column chromatography, the stationary and mobile phases are (usually) in equilibrium at the start of the process and remain in equilibrium unless you change the solvent. In TLC, the phases equilibrate during the run. One side effect is "demixing", in which the more polar solvent is depleted from the leading edge of the solvent, often to the point of forming a secondar solvent front. A second effect is adsorption of polar solvent from the vapor phase in the tank, with the result that the activity of the silica can change as a function of time unless the plate has been extensively pre-equilibrated.

Trust me, TLC is nowhere near as simple as it looks!

I'm assuming that you are using "identical" silica on the TLC plate and in the column. If not, further complications can result from differences in porosity, surface area, and surface chemistry.

[wky]

grinberg and tom, thanks for the replies. truly grateful. haha, i m really not good in all these techniques and m very lacking in knowledge.