1. Retention time (tR) is a measure of how long a compound is retained on a stat

Question

1. Retention time (tR) is a measure of how long a compound is retained on a stationaryphase in column in column chromatography. In TLC, Rf was a measure of how long thecompound was retained on the stationary phase. What is the relationship between tR and Rf?

2. What would be the order of elution if one separated the pigments of spinach on columnchromatography?

3. What if any would you have to change in terms of the mobile phase if you wanted toactually perform the column chromatography in order to separate the pigments of spinachcompare to the one used for the TLC Experiment? Explain.

4. Why is it important to keep the solvent level above the stationary phase? What wouldhappen if one lets the solvent get much lower than the alumina stationary phase?

Explanation / Answer

1) the retention is the measure of the speed at which a substance moves in a chromatographic system. In coloum chromatography where the compounds are eluted with eluent,the retention is usually measured as the retention time Rt or tR,

Rt or tR = the time between injection and detection

In Thin layer Chromatography (TLC) the retention is measured as the retention factor Rf,the run length of the compound by the run length of the eluent

Rf= distance of sample migration/distance of solvent migration

2.Spinach is green in color ,However the pigments it contains are green orange and yellow,Spinach leaves contain chlorophyll a and b and -carotene as major pigments and other minor pigments like Phenophytin a gray, Phenophytin b gray and Xanthophylls yellow .These pigments are seperated in coloumn chromatography using their polarity differences

-carotene is a hydrocarbon it is very nonpolar and Chlorophyll A and b are polar compounds.

1. Carotenes (1 spot) (yellow-orange)

Phenophytin a (gray, may be nearly as intense as chlorophyll b)

Phenophytin b (gray, may not be visible)

Chlorophyll a (blue-green, more intense than chlorophyll

Chlorophyll b (green)

Xanthophylls (possibly 3 spots: yellow)

3.in separation of spinach pigments in coloumn the mobile phase to be used should be a non polar solvent like n-hexane. But for TLC mobile phase we use a mixture of polar and non polar solvent mixture

(like 70: 30 hexane:acetone).

4.The solvent level has to be below the starting line of the TLC, otherwise the spots will dissolve away

4.

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