Question

With reference to the principles of thin-layer chromatography (TLC), explain how silica flash chromatography allows for the purification of organic compounds. What was the purpose of performing TLC analysis to optimise the eluent, and how does changing eluent affect this?

Answer 1

Chromatography is a technique to separate mixture of compounds into its individual compounds.

Thin Layer Chromatography works on the same principal by separating non volatile mixtures into individual components.It consist of Mobile Phase and stationary phase. Mobile phase is silica coated sheet or glass and stationary phase is solvent.

PRINCIPAL : Components present in the mixture separates depending upon their affinity towards mobile phase and stationary phase.

if one component has much more affinity towards stationary phase then it travels slowly and other component having more affinity towards the mobile phase it travels faster and eluate out first .

SILICA FLASH CHROMATOGRAPHY

it also works on the same principle as that of TLC,but it is a rapid column chromatography. sample used for this type of chromatography is less than 10 gm. here Mobile phase is the solvent or eluent and the stationary phase is the silica in the glass column.

relative affinity of the sample towards the mobile and stationary phase, separates the mixture .

PURPOSE OF PERFORMING THE TLC ANALYSIS FOR ELUENT SELECTION.

if the target spot travels 1/3 of the total eluent path or in other words if the Rf value of the target spot is between 0.25 to 0.35 then the eluent is best for the separation.

different eluents or the mixture of eluents are used and analyzed on TLC plates . target spot having Rf value between 0.23 to 0.35 will be selected as the best eluent for separating the mixtures.

if the sample is non polar and we use eluent or mobile phase a non polar one , then it has much more affinity towards each other separates out first with the eluent.

but if we use polar solvent with non polar sample then it travels slowly and separates out later.

mixture of eluent is prepared , analysis on TLC is done, best eluent is selected based on the target spot and Rf value .

By changing the eluent the Rf value of the spots will change which also depends on the affinity of the sample towards different eluents.

let us take an Example,

sample has 2 spots, one is appearing at the top and other at the bottom. we have to collect the top spot. firstly, we use the hexane and checks the Rf value of the top spot . or if the spot travels 1/3 ^rd or more than 1/3 ^rd of the total eluet traveled then it will be good eluent . If not then mixture of eluents is prepared by varying the ratios , example hexane: ethyl acetate 9:1 or 7:3 . by changing the ratios of the eluent mixtures or by taking different eluents , the Rf value will change .depending on the sample affinity with eluent and best one will be chosen and then try on the column, on large scale.

In column, silica is added after that sample is loaded , two types of loading is done dry loading and wet loading depending upon the solubility of the sample with eluent. then cotton is added for the uniformity of the column . and solvent will be added. eluate is collected in test tubes and TLC of eluate present in test tubes is analyzed for the identification of target spot or if spot is appearing or not. when all of the spot completely eluated out and is not appearing in the further TLCs then collect the sample and evaporate the solvent using Rotavapour. TLC are checked under UV chamber.