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?

Answer 1

Answer 1) Retardation factor (Rf) is the fraction of an analyte in the mobile phase of a chromatographic paper or sheet. It is defined as the ratio of distance traveled by the center of an analyte spot to the distance traveled by the solvent front. RF are same as R values in column chromatography.

Basically ,in column chromatography, the retention factor (k) is the ratio of time an analyte is retained in the stationary phase to the time it is retained in the mobile phase. Retention factor is inversely proportional to the retardation factor.

If retention factor is (k), retardation factor ,Rf then their relation is given by R=1/K+1

Answer2) Spinach   leaves contain   chlorophyll a, chlorophyll b and carotene, xanthophylls(oxidized carotenes) ,pheophytins ( like chlorophyll but Mg2+ is replaced by two H+).The pigments have different polarity due to their structural difference.This difference in polarity is used to elute the pigments using a suitable non-polar solvent like hexane, through a polar stationary phase like Alumina.The polar pigments would be eluted slowly as they would be strongly held by the polar stationary phase by polar bonds like hydrogen bonds or dipole-dipole interactions.

The order of the elution of spinach pigment based omn their polarity is as follows:

Carotenes (yellow –orange band eluted first,least polar)

Pheophytin a (gray)

Pheophytin a

chlorophyll a(blue green)

chlorophyll b(green)

xanthophylls(yellow, three spots)

3)Two solvent systems one slightly polar (ether), and the other non- polar (hexane) may need to be run through the column to get the less-polar pigments separated initially and more polar one eventually.

In column chromatography , more than one mobile phase can be used to separate the analyte from the mixture, provided the solvents have more affinity for one component of the analyte than the other. A series of increasingly polar solvent systems like ether/hexane can be used to elute a column,while a less-polar solvent elutes a less-polar compound ,the more-polar solvent is added to the column later to elute the more-polar compound.

But in TLC, a single solvent is used for elution.Often a mixture of solvents is used to develop a TLC plate. Highly polar solvent system makes all the components of the mixture to move faster and no clear separation. So it needs a trial of solvent systems to know an ideal solvent system,( which is easier in column chromatography to figure out as solvents are not mixed)

4) At no point should the solvent level dropped below the top of the stationary phase or the column allowed to run dry.

Solvent needs to be replenished regularly, and care must be taken to maintain the solvent level atleast more than 2 cm above the stationary phase.

This is done help to minimize the disturbance to the stationary surface and the resultant disruption of any analyte bands that may be still present near the top of the column .