# Structural Biochemistry/Chromatography/Thin Layer

Thin layer chromatography (TLC) is an extremely valuable technique in the organic lab. It is used to separate mixtures, to check the purity of a mixture, or to monitor the progress of a reaction. The polarity of the solute, polarity of solvent, and polarity of adsorbent are crucial factors that determine the mobility rate of a compound along a TLC plate. This technique helps separate different mixtures of compounds based on their mobility differences. TLC can also be used to identify compounds by comparing it to a known compound
Thin layer chromatography (TLC): this technique was used to separate dried liquids with using liquid solvent (mobile phase) and a glass plate covered with silica gel (stationary phase). Basically, we can use any organic substance (cellulose polyamide, polyethylene, etc.) or inorganic substance (silica gel, aluminum oxide, etc.) in TLC. These substances must be able to divide and form uniform layers. On the surface of the plate, will be a very thin layer of silica which is considered the stationary phase. Then, add a small amount of solvent into a wide-mouth container (i.e. beaker or developing jar) just enough to cover the bottom of the container. Place the prepared TLC plate into the sealed container which has small amount of a solvent (moving phase). Due to capillary action, the solvent moves up to the plate and now we can remove the plate and analyze the R$_f$ values.
Usually TLC is done on a glass, plastic, or aluminum plate coated with silica gel, aluminum oxide, or cellulose. This coating is called the stationary phase. The sample is then applied to the bottom of the plate and the plate placed in a solvent, or the mobile phase. Capillary action pushes the sample up the plate. The rate the samples move up the plate depends on how tightly the sample binds to the stationary phase. This is determined by polarity. The R$_f$ values or the Retention Factors are then compared for analysis. The retardation factor of a solute is defined as the ratio between the distance traveled by a compound to that of the solvent in a given amount of time. For this reason, R$_f$ values will vary from a minimum of 0.0 to a maximum of 1.0. However, this retardation factor for a given protein compound will vary widely with changes in the adsorbents and/or solvents utilized. In addition, the retardation factor can vary greatly with the content of moisture in the adsorbent. The R$_f$ values or the Retention Factors are then compared for analysis. This R$_f$ value can be quantified as such:
R$_f$ = (Distance that compound has traveled)/ (distance that the solvent has traveled)
A light pencil line is drawn approximately 7 mm from the bottom of the plate and a small drop of a solution of the dye mixture is placed along the line. To show the original position of the drop, the line must be drawn in pencil. If it was drawn in ink, dyes from the ink would move up the TLC plate along with the dye mixture and the results would not be accurate. In order to get more accurate results, dot the TLC paper with the dye mixture a few times trying to build up material without widening the spots. A spot with a diameter of 1 mm will give good results. While dotting the TLC plate, be sure to not dot mixtures too close to one another because when the dye mixture rises up the TLC plate, it will clash with the other spots and the R$_f$ values will be difficult to calculate.
When the spots are dry, the TLC plate is placed in a beaker, with the solvent level below the pencil line. Cover the beaker to ensure that the atmosphere in the beaker is saturated with solvent vapor. Line the beaker with some filter paper soaked in solvent because this will help in the process of separating the mixture. Saturating the atmosphere in the beaker with solvent vapor stops the solvent from evaporating as it rises up the plate.
As the solvent slowly travels up the plate, the different components of the dye mixture travel at different rates and the mixture is separated into different colored spots. The solvent is allowed to rise until it approximately 1-1.5 cm from the top of the plate. This gives the maximum separation of the dye components for this particular combination of solvent and stationary phase.
Once the maximum separation of the dye components for this particular solvent and stationary phase solvent is induced, the TLC plate is removed from the beaker and allowed to dry. Immediately after removing the TLC plate, use a pencil to mark the solvent front before the solvent begins to evaporate. The solvent front is the line where the solvent rose up to on the TLC plate. Then, let the solvent evaporate from the TLC plate. The separated compounds are circled/marked to indicate their position on the plate. In some cases, the compounds that have traveled up the TLC plate do not give off any noticeable appearance with the naked eye. In such cases, the TLC plate can be dipped briefly in a visualizing solution containing certain reagents that will react with the separated compounds to form a colored compound upon heating. Another way to visualize colorless organic compounds separated on a TLC plate is by placing them in iodide (I$_2$) vapor to test their absorption of iodide vapor. These TLC plates with colorless marks are placed in a bath of iodine vapor prepared by placing a small amount of iodine crystals in a tightly capped jar. Colorless spots gradually gain a dark brown color after placing the TLC plates in the bath for approximately 10 minutes. For the reason that the colored spots usually disappear in a short period of time, they are outlined immediately with a pencil after the TLC plate is taken out of the iodine bath.
In addition to the visualization technique of an iodine bath, a fluorescent indicator can also aid in helping to determine the distance in which the separated compounds had traveled. A short- wave ultraviolet lamp is used to illuminate the adsorbent side of the plate in a darkened room/ area. Many compounds will decrease the intensity of the fluorescent. Using this UV light visualization technique, the separated compounds appear as dark spots on the fluorescent TLC plates. It is often easier to visualize the darkened spots with 365-nm light. These dark spots are outlined with a pencil while the plate is under the UV light source to give a permanent record of the location in which the analyzed compounds had traveled.
Some examples of interpretation of TLC plates under UV light:
1. TLC gives useful qualitative results and interpretations. For example, if an individual wants to compare the components in an unknown mixture to standard compound A and B, TLC can be ran and if the dark spots for unknown under UV light aligns with those of compound A and B, the unknown contains both A and B.
2. If there is only one dark spot for the unknown and it is uncertain whether the spot for compound A is at the same level as the spot for the unknown, one can co-spot both compounds on the TLC plate for a quick check. Co-spot means to spot compound A on one area of the TLC plate and spot the unknown on the same area as the spot of compound A. If there is only one dark spot under the UV light for the co-spotting lane, the identity of unknown is A.
The co-spot result for example 1 should contain only 2 spots where one spot represents compound A + one component in the unknown, and the other spot represents another component in the unknown mixture. Extra spots may indicate that one of the components in the unknown does not match with the standards.
3. How can someone tell the reaction between A+B actually occurs to give a new product C? TLC can be used to check. Compound A and B are spotted on a TLC plate separately. The mixture of A+B (C ) is then spotted on the TLC plate and after each time period a new sample can be spotted (C2, C3, and so on).
Two spots on C1 align with A and B suggests just a mixture of A+B, not a new product. C2 and C3 still have one spot aligning with reactant A, but C4 has both spots that do not match with either reactant, where C5 only has one dark spot. There are two possible interpretations:
1) C2 to C4 are intermediates to the new product in C5,
2) the desired product is actually C4 and it degrades to just having one component on the plate.
1) A capillary tube is used to transfer solution onto the TLC plate. Smaller origin spots will give smaller area and better separation of dark spots under UV light and this will make calculation of Rf easier and more accurate.
2) The container with TLC plate and solvent should always be on a flat surface in order to get a "straight lane" for the run.

## Effect of Solvent in TLC plate

As you might already know, the TLC plates are made of silica gel, which is a polar compound, and is the reason why non-polar compounds tend to have a great separation on TLC plates.
As shown in the diagram, initially, the solvent used consisted of a 7:3 ratio of hexane to hexyl acetate. This means that a majority of the solvent reacting with the TLC plate will be nonpolar. Due to the lack of polarity of the solvent, there is less competition between the spotted samples and the TLC plate, thus, the polar parts of the sample will readily react with the silica gel leading to less of a separation. Because there is nothing 'hindering' the sample from reacting with the silica gel, it reacts right away and its separation is 'bogged down.' Think of a dog walking down a pathway, if the dog stops to sniff at every tree on the way, its distance separated from the beginning is less than if it had just kept walking without being distracted by the surroundings. This is the same with these samples, if they are constantly reacting with the silica gel as they are moving they will not move as far.
Now when the ratios are switched, and there is more of the hexyl acetate(more polar), then all of a sudden there is competition for the reacting with the TLC plate. The sample wants to react with the TLC plate, but so does the solvent(since it is now more polar), thus there will be less reaction of the sample with the TLC plate. Obviously the solvent is trying to react with the TLC plate, leading to the sample not getting as much of a chance to "stop and sniff" so it is separated further. The sample reacts less with the TLC plate because now there is the solvent reacting with the same TLC plate, and this explains why there is a greater separation.
Now the 3d TLC plate in the diagram is a bit tricky. One might think that petroleum ether would be semi-polar due to the name(it has ether in it), but actually petroleum ether is a non polar compound which consists of many hydrocarbon molecules. This will not lead to any different separation.