Developing Chamber Thin Layer Chromatography

Thin Layer Chromatography (TLC)

TLC is a simple, quick, and inexpensive procedure that gives the chemist a quick respond as to how many components are in a mixture. TLC is also used to support the identity of a compound in a mixture when the R_f of a chemical compound is compared with the R_f of a known chemical compound (preferably both run on the same TLC plate).

A TLC plate is a sheet of glass, metal, or plastic which is coated with a thin layer of a solid adsorbent (usually silica or alumina). A pocket-sized amount of the mixture to be analyzed is spotted most the bottom of this plate. The TLC plate is then placed in a shallow pool of a solvent in a developing sleeping accommodation so that just the very bottom of the plate is in the liquid. This liquid, or the eluent, is the mobile stage, and it slowly rises up the TLC plate by capillary action.

As the solvent moves by the spot that was applied, an equilibrium is established for each component of the mixture between the molecules of that component which are adsorbed on the solid and the molecules which are in solution. In principle, the components will differ in solubility and in the strength of their adsorption to the adsorbent and some components will be carried farther up the plate than others. When the solvent has reached the top of the plate, the plate is removed from the developing sleeping room, stale, and the separated components of the mixture are visualized. If the compounds are colored, visualization is straightforward. Usually the compounds are not colored, then a UV lamp is used to visualize the plates. (The plate itself contains a fluorescent dye which glows everywhere except where an organic chemical compound is on the plate.)

How To Run a TLC Plate

	Step 1: Fix the developing container The developing container for TLC can be a specially designed chamber, a jar with a lid, or a beaker with a spotter glass on the height (the latter is used in the undergrad labs at CU). Pour solvent into the chamber to a depth of but less than 0.5 cm. To help in the saturation of the TLC chamber with solvent vapors, y'all tin can line role of the inside of the beaker with filter newspaper. Cover the beaker with a watch glass, swirl it gently, and let it to stand while you fix your TLC plate.
	Stride two: Prepare the TLC plate TLC plates used in the organic chem pedagogy labs are purchased as 5 cm x 20 cm sheets. Each large sheet is cutting horizontally into plates which are 5 cm tall by various widths; the more samples you plan to run on a plate, the wider it needs to be. Shown in the photo to the left is a box of TLC plates, a large un-cut TLC sheet, and a small TLC plate which has been cut to a convenient size. Handle the plates advisedly and so that you do not disturb the coating of adsorbent or get them dirty.
	Mensurate 0.5 cm from the lesser of the plate. Using a pencil, draw a line beyond the plate at the 0.v cm marking. This is the origin: the line on which you will spot the plate. Take care not to press and so hard with the pencil that you disturb the adsorbent. Nether the line, marker lightly the name of the samples yous will spot on the plate, or marker numbers for time points. Leave plenty space between the samples so that they exercise non run together; near four samples on a five cm broad plate is advised.
	Step iii: Spot the TLC plate If the sample is not already in solution, dissolve about i mg in 1 mL of a volatile solvent such as hexanes, ethyl acetate, or methylene chloride. As a dominion of pollex, a concentration of 1% normally works well for TLC analysis. If the sample is too concentrated, information technology will run as a smear or streak (see troubleshooting section below); if it is not concentrated enough, you will see null on the plate. Sometimes you lot will demand to use trial and error to get well-sized, like shooting fish in a barrel to read spots.
	Obtain a a microcapillary. In the organic education labs, we use 10µL microcaps - they are easier to handle than the smaller ones used in research labs. Dip the microcap into the solution and then gently bear upon the end of it onto the proper location on the TLC plate. Don't allow the spot to become as well large - if necessary, you tin can touch it to the plate, elevator it off and blow on the spot. If yous repeat these steps, the wet area on the plate will stay pocket-size.
	This example plate has been spotted with 3 dissimilar quantities of the same solution and is ready to develop. If you are unsure of how much sample to spot, you can always spot multiple quantities and run across which looks all-time.
	Step 4: Develop the plate Place the prepared TLC plate in the developing beaker, cover the chalice with the watch glass, and get out it undisturbed on your bench elevation. The solvent will rise upwardly the TLC plate by capillary action. Make sure the solvent does not cover the spot.
	Allow the plate to develop until the solvent is about half a centimeter beneath the summit of the plate. Remove the plate from the chalice and immediately marker the solvent front with a pencil. Allow the plate to dry.
	Stride five: Visualize the spots If there are any colored spots, circle them lightly with a pencil. Most samples are not colored and need to exist visualized with a UV lamp. Hold a UV lamp over the plate and circle any spots you run across. Beware! UV light is dissentious both to your eyes and to your skin! Make sure you are wearing your goggles and practise not expect directly into the lamp. Protect your skin by wearing gloves.
	If the TLC plate runs samples which are too concentrated, the spots will be streaked and/or run together. If this happens, you will have to beginning over with a more dilute sample to spot and run on a TLC plate.
	Here's what overloaded plates look similar compared to well-spotted plates. The plate on the left has a large yellow smear; this smear contains the same two compounds which are nicely resolved on the plate next to it.

TLC Solvents Choice

When y'all need to decide the best solvent or mixture of solvents (a "solvent system") to develop a TLC plate or chromatography column loaded with an unknown mixture, vary the polarity of the solvent in several trial runs: a process of trial and error. Carefully observe and record the results of the chromatography in each solvent system. You will observe that as you increment the polarity of the solvent system, all the components of the mixture move faster (and vice versa with lowering the polarity). The platonic solvent system is but the organisation that gives the best separation.

TLC elution patterns normally acquit over to cavalcade chromatography elution patterns. Since TLC is a much faster procedure than column chromatography, TLC is frequently used to determine the best solvent organisation for column chromatography. For example, in determining the solvent system for a flash chromatography process, the ideal system is the one that moves the desired component of the mixture to a TLC R_f of 0.25-0.35 and will separate this component from its nearest neighbour past difference in TLC R_f values of at least 0.20. Therefore a mixture is analyzed by TLC to determine the ideal solvent(due south) for a flash chromatography procedure.

Beginners oftentimes do non know where to start: What solvents should they pull off the shelf to use to elute a TLC plate? Because of toxicity, cost, and flammability concerns, the common solvents are hexanes (or petroleum ethers/ligroin) and ethyl acetate (an ester). Diethyl ether can exist used, only it is very flammable and volatile. Alcohols (methanol, ethanol) can be used. Acetic acid (a carboxylic acid) can be used, usually as a small percentage component of the arrangement, since it is corrosive, non-volatile, very polar, and has irritating vapors. Acetone (a ketone) can exist used. Methylene chloride or and chloroform (halogenated hydrocarbons) are expert solvents, but are toxic and should be avoided whenever possible. If two solvents are equal in performance and toxicity, the more volatile solvent is preferred in chromatography because information technology will be easier to remove from the desired compound after isolation from a column chromatography procedure.

Inquire the lab instructor what solvents are available and advisable. Then, mix a not-polar solvent (hexanes, a mixture of 6-carbon alkanes) with a polar solvent (ethyl acetate or acetone) in varying percent combinations to brand solvent systems of greater and lesser polarity. The charts below should help y'all in your solvent pick. You can also download this pdf nautical chart of elution order.

Interactions Between the Chemical compound and the Adsorbent

The strength with which an organic compound binds to an adsorbent depends on the force of the following types of interactions: ion-dipole, dipole-dipole, hydrogen bonding, dipole induced dipole, and van der Waals forces. With silica gel, the ascendant interactive forces betwixt the adsorbent and the materials to exist separated are of the dipole-dipole type. Highly polar molecules collaborate fairly strongly with the polar SiOH groups at the surface of these adsorbents, and volition tend to stick or adsorb onto the fine particles of the adsorbent while weakly polar molecules are held less tightly. Weakly polar molecules generally tend to move through the adsorbent more rapidly than the polar species. Roughly, the compounds follow the elution gild given above.

The R_f value

The memory factor, or R_f, is defined every bit the distance traveled by the chemical compound divided past the distance traveled by the solvent.

For example, if a compound travels 2.one cm and the solvent front travels 2.8 cm, the R_f is 0.75:

The R_f for a chemical compound is a constant from one experiment to the next only if the chromatography atmospheric condition below are as well constant:

solvent system
adsorbent
thickness of the adsorbent
corporeality of fabric spotted
temperature

Since these factors are difficult to keep constant from experiment to experiment, relative R_f values are generally considered. "Relative R_f" means that the values are reported relative to a standard, or it ways that you lot compare the R_f values of compounds run on the same plate at the same fourth dimension.

The larger an R_f of a compound, the larger the distance it travels on the TLC plate. When comparing two unlike compounds run under identical chromatography weather condition, the compound with the larger R_f is less polar because it interacts less strongly with the polar adsorbent on the TLC plate. Conversely, if you know the structures of the compounds in a mixture, you lot can predict that a chemical compound of low polarity volition have a larger R_f value than a polar chemical compound run on the same plate.

The R_f tin provide corroborative testify as to the identity of a chemical compound. If the identity of a compound is suspected but non still proven, an accurate sample of the compound, or standard, is spotted and run on a TLC plate adjacent (or on peak of each other) with the compound in question. If two substances have the same R_f value, they are likely (but not necessarily) the same compound. If they have different R_f values, they are definitely different compounds. Note that this identity bank check must exist performed on a single plate, because information technology is hard to duplicate all the factors which influence R_f exactly from experiment to experiment.

Troubleshooting TLC

All of the above (including the procedure folio) might sound like TLC is quite an easy procedure. Merely what near the showtime time y'all run a TLC, and see spots everywhere and blurred, streaked spots? As with any technique, with do you become better. Examples of common issues encountered in TLC:

The compound runs every bit a streak rather than a spot: The sample was overloaded. Run the TLC again after diluting your sample. Or, your sample might just contain many components, creating many spots which run together and announced as a streak. Mayhap, the experiment did not go as well equally expected.
The sample runs equally a smear or a upward crescent: Compounds which possess strongly acidic or bones groups (amines or carboxylic acids) sometimes bear witness up on a TLC plate with this behavior. Add together a few drops of ammonium hydroxide (amines) or acetic acid (carboxylic acids) to the eluting solvent to obtain clearer plates.
The sample runs as a downward crescent: Likely, the adsorbent was disturbed during the spotting, causing the crescent shape.
The plate solvent front end runs crookedly: Either the adsorbent has flaked off the sides of the plate or the sides of the plate are touching the sides of the container (or the paper used to saturate the container) as the plate develops. Crooked plates make it harder to mensurate R_f values accurately.
Many random spots are seen on the plate: Make sure that yous do non accidentally drop any organic compound on the plate. If go a TLC plate and leave it laying on your workbench as yous do the experiment, you might drop or splash an organic compound on the plate.
You see a blur of blue spots on the plate as it develops: Perhaps you used an ink pen instead of a pencil to marker the origin?
No spots are seen on the plate: You might not have spotted enough chemical compound, perhaps because the solution of the compound is too dilute. Try concentrating the solution, or spot information technology several times in 1 place, allowing the solvent to dry between applications. Some compounds do not show up under UV light; try another method of visualizing the plate (such every bit staining or exposing to iodine vapor). Or, perhaps y'all do non have any compound because your experiment did not go as well equally planned. If the solvent level in the developing jar is deeper than the origin (spotting line) of the TLC plate, the solvent will dissolve the compounds into the solvent reservoir instead of assuasive them to movement up the plate by capillary action. Thus, yous will not see spots later on the plate is adult. These photos show how the yellow compound is running into the solvent when lifted from the developing jar.

TLC Technique Quiz

See how well you sympathize TLC by taking the online TLC Technique Quiz!