Question

Describe and explain the possible effect on your TLC results of the following experimental errors or variations:

a) The analyte spots are too large

(b) The analyte solution is too dilute.

Answer 1

TLC works by the following principle:

On a polar plate various compounds with different (though possibly very close) polarities are placed. The compounds are then pushed up the plate by a solvent. While being pushed up, the polar plate tries to hold back the polar compounds while the non-polar compounds ignore the plate and head straight to the top. You can alter the interaction between the compounds and the plate by changing the solvent. If you use a very polar solvent, it has the effect of making the polar compounds more attracted to the solvent than they were previously. This means that when the compounds are pushed up the plate by a polar solvent, they spend more time traveling with the solvent and less time being held back by the plate. The non-polar compounds are less attracted to polar stationary phase; therefore they travel further in polar solvents.

More polar compounds = less distance traveled

Less polar compounds = more distance traveled

a) The analyte spots are too large -If the sample spot is too concentrated, the substance will travel up the stationary phase as a streak rather than a single separated spot. You want all the molecules in the plate to be able to interact with the plate effectively. If your spots are too large (or too concentrated), then you have molecules that aren’t interacting with the plate when they should be. This means they will travel further than they normally would. The result is uncertain because some compound in spot is interacting with the plate more than other compound in spot and you'll end up with a large streaking spot.

Spotting sizes of your sample should be not be larger than 1-2 mm in diameter. If you have an over-large spot, this could cause overlapping of other component spots with similar Rf values on your TLC plate. If overlapping occurs, it would prove difficult to resolve the different components.

b) The analyte solution is too dilute - If a solution to be spotted happens to be too diluted, then it is possible to spot same place a few times. Just allow the solvent to dry between spotting’s. This, however, leads to poorer TLC resolution. The specific concentration of the solution is not crucial, however it should not be too dilute. An approximate concentration range for solutions to be analyzed by TLC is approximately 10mg/200microlitre. When you use diluted solution for TLC some minor compounds or impurities will not appear as a separated spot on TLC plate.