Thin Layer Chromatography Manual

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A manual on how to perform Thin Layer Chromatography (TLC).

Transcript of Thin Layer Chromatography Manual

(CHE 276) Organic Chemistry Lab Experiment 1

Thin Layer Chromatography

"Adapted from: N. I. Totah, CHE 276 Organic Chemistry Laboratory Course Reader, Syracuse University, Fall 2011"Principle of Thin Layer Chromatography : As the solvent containing a mixture of organic molecules (polar and less polar) moves up the silica gel due to capillary action, the polar organic molecules will interact stronger with the silica gel than the less polar organic molecules, and thus the polar organic molecules will move slower than the less polar organic molecule on the silica gel.

This almost magical difference in the interactions between different molecules with the silica gel gives separation of a mixture of organic molecules, ranks the relative polarities of the organic molecules, and to some extent, helps in the identification of organic molecules.


Chromatography is one of the most ubiquitous methods of analyzing and purifying organic compounds. While originally used to separate plant pigments, today this process includes a variety of sophisticated techniques which allow for the separation, isolation, and identification of the components of a mixture. While there are many types of chromatography, the fundamental basis for this technique concerns the distribution of the individual components of a mixture between two phases: the stationary phase and the mobile phase. For any given compound (A), an equilibrium is established such that the compound is either adsorbed on the stationary phase or dissolved in the mobile phase. In thin layer and column chromatography this equilibrium is determined by the polarity of the stationary and mobile phases, and by the polarity of the compound itself. In gas chromatography, however, the equilibrium is determined primarily by temperature.

As the name implies, the stationary phase is a non-moving substance (often SiO2 or Al2O3) to which the components of a mixture may adsorb. It can exist in a variety of forms, but is commonly contained in a column or spread in a thin layer over a glass or plastic backing. The mobile phase percolates over the stationary phase and may be either a gas, as in gas chromatography, or a liquid as in column or thin layer chromatography. A compound dissolved in the mobile phase is carried along in the direction of the flow. A compound adsorbed on the stationary phase does not move.

The individual components of a mixture also exist in equilibrium between stationary and mobile phases. In many cases, the mixture is first loaded on the stationary phase, then the mobile phase is added. As the mobile phase flows over the stationary phase it carries with it all components of the mixture in the direction of flow. Since each component has a different affinity for the stationary phase, each is adsorbed to a greater or lesser extent relative to the other components of that mixture. Compounds which favor the stationary phase are held longer, and as a result move more slowly than do compounds which favor the mobile phase. It is these differences in equilibrium which allow for the separation of compounds in multi- component mixtures.Figure 1: Chromatographic separation of a two component mixture. As the mobile phase moves, it carries blue molecule faster than red molecules because silica gel interacts stronger with red molecules than with blue molecules.

In the example shown here (Figure 1), compound A interacts more strongly with the stationary phase than does compound B. As a result, more of compound A is adsorbed on the stationary phase at any given time. It follows that compound B thus spends more time in the mobile phase than does compound A, and is carried more quickly in the direction of the flow. As such, these two compounds will eventually separate, and the separation will increase the longer the mobile phase travels.

Science of Thin layer chromotography. (This section perhaps requires the most thought-intense reading for an understanding, and is central to many questions in the quiz and final examination.) The plate for a TLC is made of silica gel, which is a very polar material. For organic solvents used for running a TLC, they are not considered as polar as silica gel.(1) Otherwise, if the solvent is too polar, such as water, the silica gel will be disolved. In addition, the silica gel binds to polar molecular stronger than non-polar molecules through noncovalent interactions. These two facts form the basis for many logics explaining how TLC (and column chromotography, LAB 5) works.Consider two cases. First, for a given eluent solvent, how would a polar and nonpolar molecule (to be analyzed) behave on a TLC plate. Second, for a given molecule (to be analyzed), how would a polar and a nonpolar eluent solvent impact and compare the movement of the molecule on a TLC plate.For a given eluent solvent and comparing the behavior of a polar and nonpolar molecule, the interaction between the silica and the molecule dominate over the interaction between the solvent and the molecule. Why? see (1) above. For this reason, it is more important to consider that the silica gel interacts with the the polar molecule stronger than with the nonpolar molecule. Thus, for any eluent solvent (polar or nonpolar), the polar molecule will always move slower than the nonpolar molecules. For a given molecule and comparing the effect of a polar and a nonpolar solvent, the interaction between the silica gel and the molecule is the same for both solvents. Thus, one only needs to consider that the molecule will interact stronger with the polar solvent than with nonpolar solvent. As a result, the molecule will always move faster when polar solvent is used than when nonpolar solvent is used. This fact stays true for both polar and non-polar molecules, because nonpolar molecules still interact stronger with a polar solvent than with a nonpolar solvent.

To make it more concise, the following Tables are used to compare different scenarios described above.

Sample for analysisPolar solvent

Polar molecule Slow(a)

Nonpolar molecule Fast

Sample for analysisNonpolar solvent

Polar molecule Slow(b)

Nonpolar molecule Fast

Case (a) will be faster than case (b), see the two Tables below.

Sample for analysisPolar solventNon polar solvent

Polar moleculeFast(c)Slow

Sample for analysisPolar solventNon polar solvent

Nonpolar moleculeFast(d)Slow

Case (c) will be slower than case (d), see the previous two Tables.

Note that, of course, the relative polarity (i.e. which is more polar) of two molecules does NOT change in different solvents. The relative polarity of the common organic molecules and solvents are shown in Table 1 and Table 2.

How to compare and determine relative polarity between two different organic molecules? The polarity of a small organic molecule is determined by the functional groups the molecule possesses. The symmetry of the molecule also plays a small role in influencing the polarity of the molecule. Below is a list of general guidelines for ranking the polarity from high to low. 1. Charged groups such as R4N+ or COO- are the most polar functional groups. 2. Functional groups with hydrogen bonding capability are the second category of polar functional groups. 3. Atoms in functional groups with high electronegativity introduce more polarized bonds (and thus more polar molecules) than those with low electronegativity. 4. The more polar functional groups a molecule possess, the more polar the molecule will be. 5. In general, a polar functional group has a more dominating effect over less polar functional groups on a molecule. For example, a carboxylate (RCOO) is likely more polar than a sugar-based surfactant that has five hydroxyl (OH) groups. 6. All else being the same, an asymmetric molecule is more polar than a symmetric molecule. The eluting power of various organic solvents parallels the order shown in Table 2. Thus, the greater the polarity of the solvent, the greater its ability to dislodge and displace a compound from the stationary surface, and the faster the compound will move along. This property of solvents is quantified somewhat by listing them in order of their ability to displace solutes from adsorbents. This listing is known as an "eluotropic series" and will vary somewhat from adsorbent to adsorbent. An eluotropic series shown in Table 2 is for silica gel.

Operation of Thin Layer Chromatography:

Thin layer chromatography (TLC) is used primarily as an analytical technique to determine the purity of a compound, the status of an ongoing reaction, or as a preliminary means of identification. The sample is spotted near the bottom of a glass or plastic plate which is coated with a thin layer (hence the name) of dry adsorbent (Figure 2). The plate is then placed in a covered beaker or jar that contains a small amount of the appropriate solvent. The level of the solvent in the jar must be below the level of the sample spots, and the atmosphere in the jar should be saturated with solvent vapors. A filter paper is used to help with the saturation. Capillary action draws the solvent up the plate (If the jar is not saturated with solvent vapors, the solvent will not run all the way up the plate). When the solvent front is near the top of the plate, it is removed from the beaker and the location of the solvent front is marked with a pencil. If the compounds are colored, and the plate can be read easily, no other method of visualization is needed. If the compounds are not colored then they can be visualized using an ultraviolet lamp or a chemical stain, such as iodine.Figure 2: TLC developing chamber. Note that the spots are all above the level of solvent. This figure shows the TLC plate immediately after it is put in to the chamber.

For each spot on