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Davidmb Davidmb
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3 years ago
My question relates to plant pigment chromatography.  Reading up high school level literature it can be seen that in paper chromatography xanthophyll has a much higher relative Rf value (second only to carotene and hence above chlorophyll) whilst in thin layer chromatography it has a much lower relative Rf value (below that of chlorophyll).  I am having difficulty understanding this.  The explanations I have seen refer to the polarity of the xanthophyll causing it to have a lower Rf value than chlorophyll but this same explanation does not work for paper chromatography.  Any help would be appreciated.
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wrote...
Educator
3 years ago Edited: 3 years ago, bio_man
Can you provide a source that confirms your speculation? For anyone else not familiar with these methods:

Paper chromatography – uses paper (cellulose) as the stationary bed

Thin layer chromatography – uses a thin layer of adsorbent (e.g. silica gel) which runs faster and has better separation

Here's an illustration of what you should observe:



Generally speaking, you should see this order, where xanthophyll is the most polar while carotene is the least:

xanthophyll > chlorophyll b > chlorophyll a > pheophytin > carotene
Davidmb Author
wrote...
3 years ago
Thanks for this but if xanthophyll is the most polar pigment  it should have the lowest Rf value but as can be seen in your image it shows up in the middle - which appears to be common for paper chromatography but on the other hand if you refer to TLC resources such as "https://www.saps.org.uk/secondary/teaching-resources/1347-a-level-set-practials-tlc"  the xanthophyll does indeed have the lowest Rf value.
Apologies not sure how to include a copy of the Rf values given in this resource but have attached resource.



 Attached file 
You must login or register to gain access to this attachment.
wrote...
Educator
3 years ago Edited: 3 years ago, bio_man
I see your point now. In general, low polarity compounds have higher retention factor values than high polarity compounds, such as xanthophyll. When conducting TLC, we should expect to see xanthophyll at the bottom of the column. In general, the adsorptivity of compounds increases with increased polarity; for example, the more polar the compound, then the stronger it binds to the adsorbent. Therefore, the stronger a compound is bound to the adsorbent , the slower it moves up the TLC plate. This doesn't seem to be the case with paper chromatography. In paper chromatography, we see xanthophyll posses a much high Rf, meaning that it travels a lot higher, as seen in my illustration above. The reason I'm thinking is because xanthophyll doesn't bind to the paper as well as it does to the silica (SiO2) or alumina (Al2O3) powder that coats the TLC plates. That's my guess.
wrote...
Staff Member
3 years ago
Quote
The reason I'm thinking is because xanthophyll doesn't bind to the paper as well as it does to the silica (SiO2) or alumina (Al2O3) powder that coats the TLC plates. That's my guess.

I believe that's it!

Chromatography is based on the principle that different compounds will have different affinity to the two phases (Stationary phase and mobile phase).

Thin Layer Chromatography (TLC) is an adsorption method in which the stationary phase is a solid (ex-silica or alumina) and a moving phase (solvent either non polar or mixture of polar & non polar solvent). The analyte is adsorbed on the stationary phase or desorbed from the stationary phase in to the mobile phase.)

Paper Chromatography is a partition method in which the stationary phase is a liquid (water adsorbed on the cellulose of paper) and a moving phase (solvent either non polar or mixture of polar & non polar solvent). The analyte is partitioned between the stationary phase and the mobile phase.

Basis of difference in movement is due to polarity difference. As the stationary phase (silica gel or adsorbed water) are polar in nature. So more polar compounds will have greater affinity for the stationary phase as compared to mobile phase. So these compounds will move slowly with the mobile phase as compared to non polar compounds.
Ask another question, I may be able to help!
wrote...
Educator
3 years ago
Hi Davidmb

Can we mark this topic solved?
Davidmb Author
wrote...
3 years ago
Who marks it as solved?
Not sure how the system works?
wrote...
Educator
3 years ago
Yes, there's a green "solved" button on your initial post.

Anyway, I've gone ahead and done it for you.

Best of luck
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