Why do tlc spots have to be small

For the next step co-spotting , an authentic sample of the compound closest in Rf value to the unknown is chosen.

TLC co-spotting of a second plate allows for preliminary identification of your compound. Three spots are applied to the adsorbent on the baseline of the TLC plate: the purified unknown, an authentic sample, and a co-spot of unknown and authentic sample. If the developed TLC plate shows only one row of spots, it can be concluded that the unknown has been purifed, and that the unknown is possibly the same compound as the authentic sample.

However, because Rf values are relative, not absolute, some compounds may have very similar Rf values. A mixed melting point measurement is needed to unambiguously identify the unknown compound. It is primarily used to determine the purity of a compound. A pure solid will show only one spot on a developed TLC plate. In addition, tentative identification of the unknown compound can be made through TLC analysis.

The progress of a reaction can be monitored by Thin Layer Chromatography. At various times during the experiment, a TLC plate is spotted three times, then developed and viewed under UV light: - At position 1 is an authentic sample of the starting compound.

The reaction has gone to completion, when the TLC plate shows that there is no more starting material left in the reaction mixture. Earl N. Meyer wanted to determine the purity of the benzoic acid which he had obtained from Chemical Recycling. He decided to check the purity by chromatographic analysis using a silica gel TLC plate. On the TLC plate he marked the baseline with a pencil, indicating the spotting position with a tick. Earl dissolved about 20 mg of the benzoic acid in 1 ml of methanol.

Then he dipped a micropipette into the solution and lightly touched the TLC plate at the tick marked on the baseline. Next, Earl prepared the developing chamber. He cut a filter paper to line a ml beaker.

He then prepared the eluent: 10 ml of a mixture of dichloromethane and methanol in a ratio of 10 : 1. He added the eluent to the beaker, swirling it onto the filter paper to completely wet it. Earl placed the TLC plate in the developing chamber using tweezers and watched the eluent travel up the plate by capillary action.

When the eluent was near the top of the plate, he used tweezers to remove the plate from the chamber, taking care to immediately mark the solvent front with a pencil.

When the TLC plate was dry, Earl viewed it using long wavelength, ultraviolet light. He observed a single blue spot on a light green background. His TLC indicated that the sample tested contained a single component and that his benzoic acid was pure.

He used a pencil to circle the spot and a ruler to make measurements needed for calculation of the retention factor. First, prepare a TLC plate: dissolve a very small amount of your unknown sample in a volatile solvent, then apply the solution i. Next, prepare the TLC chamber: Cut the portion of the filter paper that rises above the beaker edge.

Prepare the eluent using a mixture of the suggested solvents. Rf or Retention factor is a unique value for each compound under the same conditions. The Rf for a compound is a constant from one experiment to the next only if the chromatography conditions below are also constant: solvent system. By definition, Rf values are always less than 1. An Rf value of 1 or too close to it means that the spot and the solvent front travel close together and is therefore unreliable. This happens when the eluting solvent is too polar for the sample.

Why is a small spot of pigment needed on the TLC plate? Category: science chemistry. What happens if you apply too much compound on the TLC plate? What is Rf value? Is it possible to distinguish two spots that have the same Rf value?

What does TLC tell you about purity? Is silica polar or nonpolar? What will be the appearance of a TLC plate of a solvent of too low polarity?

What will happen if the spots on the TLC plate come in contact with the eluent? What happens if the spots on the TLC plate are made too large when preparing the plate for development? Why should a TLC plate be removed before the solvent reaches the top? Good general stain, very well with polyhydroxylated and carbonyl compounds. Blue or green spot. Upon heating, very sensitive! Good general stain, particularly sensitive towards nucleophiles.

Varying colors on light pink plate upon heating. Does not work with alkenes, alkynes or aromatic system unless functional groups are present. Phosphomolybdic acid PMA. Very sensitive. Dark green spot on light green plate. Sensitivity can be enhanced by use of cobalt II chloride. Indoles, amines. Pink or red-violet. Dragendorff-Munier Stain.

Amines even the ones that are low in reactivity. Various colors. Due to the fact that all those variables are difficult to keep constant, a reference compound is usually applied to the plate as well. TLC University of Colorado. TLC Macherey Nagel. TLC chamber for development e. As with plate selection, keep in mind the chemical properties of the analytes. A common starting solvent is hexane:ethyl acetate. When performing your experiment, you do not want your values to be 0 or 1 because your components that you are separating have different polarities.

If the value is 0, you need to increase your solvent polarity because the sample is not moving and sticking to the stationary phase. If the value is 1, you need to decrease your solvent polarity because the compound was not able to separate. If you know that one component of a mixture is insoluble in a given solvent, but another component is freely soluble in it, it often gives good separations.

How fast the compounds travel up the plate depends on two things:. Acids, bases, and strongly polar compounds often produce streaks rather than spots in neutral solvents. Adding a few percent of acetic or formic acid to the solvent can correct streaking with acids. Similarly for bases, adding a few percent triethylamine can improve results.

For polar compounds adding a few percent methanol can also improve results. The volatility of solvents should also be considered when chemical stains are to be used. Any solvent left on the plate may react with the stain and conceal spots. Many solvents can be removed by allowing them to sit on the bench for a few minutes, but very nonvolatile solvents may require time in a vacuum chamber.

Volatile solvents should only be used once. If the mobile phase is used repeatedly, results will not be consistent or reproducible. Developing a TLC plate requires a developing chamber or vessel. This can be as simple as a wide-mouth jar, but more specialized pieces of glassware to accommodate large plates are available. The chamber should contain enough solvent to just cover the bottom.

It should also contain a piece of filter paper, or other absorbent material to saturate the atmosphere with solvent vapors. Finally, it should have a lid or other covering to minimize evaporation. If fluorescent plates are used, a number of compounds can be seen by illuminating the plate with short-wave UV. Quenching causes dark spots on the surface of the plate. These dark patches should be circled with a pencil. For compounds which are not UV active, a number of chemical stains can be used.

These can be very general, or they can be specific for a particular molecule or functional group. Iodine is among the most common stains.

Plates are placed in a jar containing iodine crystals, or covered in silica gel with iodine dispersed throughout, for approximately one minute. Most organic compounds will be temporarily stained brown.

Some popular general use stains are Permanganate, ceric ammonium molybdate CAM , and p-anisaldehyde. These can be kept in jars which plates are dipped into, or in spray bottles.

To develop a plate with permanganate, spray or dip the plate and heat it with a heat-gun. Hold the plate face up 10 to 20 cm above the heat gun until the bulk water evaporates. Overheating will turn the entire plate brown, obscuring the spots. If glass plates are used it is often easier to see spots through the backing because it is harder to overheat.

CAM and p-anisaldehyde stained plates are developed similarly. Overheating CAM stained plates turns everything blue. There are common problems in TLC that should be avoided.

Normally, these problems can be solved or avoided if taught proper techniques.