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Twelve Twelve
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12 years ago
I'm doing chromatography with leaf extracts to separate pigments. I was wondering about the following:

1. Does the concentration of the sample used affect the Rf values?

I thought that seeing as the Rf value, or how far the pigment travelled depended on the intermolecular forces between the stationary phase and mobile phase, if you increase the amount of mobile phase, there would be a larger force, Hence the pigments will travel less (i.e. the separated pigments will become closer to each other). Is that correct?

2.Secondly, does the darkness of the pigment and the thickness of the line it produces on paper reflect upon the concentration of the pigment in the mixture used to dab the paper?

I know that different pigments are different coloured, but what I mean is the intensity of the colour. The thickness of the line each colour produces is also a bit different. I was wondering what the cause of that was? Is it due to the high concentration? Would it be related to question 1?

Thanks in advance,

Twelve.
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#1
12 years ago
Hi Twelve,

1. The mobile phase is your solvent(s) not your spot. As for the spot, the concentration of your spot should not affect the Rf value, unless your dot is pitch black. Then you might want to dilute it with some organic solvent (dichloromethane, or ethyl acetate). On the other hand, If you only have 1 solvent for your mobile phase, the amount of solvent used should not affect the Rf value, as long as the solvent level is below your spot. However, if you use a mixture of solvents as the mobile phase, your Rf values will change as you change the ratios of the solvents used.

2. Yes, the darkness and the thickness of the spot is depends on the concentration of your substance. If it is too dark, you will need to dilute it a little but for better separation.

Hope this helps,
Laser
Twelve Author
wrote...
#2
12 years ago
@Laser_3,

Thank you for your reply. For question one, I was wondering because I realised that when more leaf extract was used, the separation between pigments became less distinct, thus the thickness of the 'pigments' on the paper decreased.

For question two, I may have interpreted your answer wrong, however I was referring to the 'darkness of the pigment' on the paper strip as opposed to the spot of leaf extract at the bottom. Like you said, I realised that more concentrated 'spots' yielded worse separations.

Once again, thanks for your reply,

Twelve.
wrote...
#3
12 years ago
Hi Twelve,

Yea, I misread your question. In that case, I would presume that the darkness of the spot can be some what of an indication of concentration, but it is not very accurate to go off of the TLC alone. Some compound absorb light better than others; therefore it might be darker. For example, for some compound, you need to look at under UV light in order to see, for some you can see it on the TLC with your eyes alone.

Best,
Laser
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