Thin Layer Chromatography

TLC is a simple, quick, and inexpensive procedure that gives the chemist a quick answer 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 Rf of a compound is compared with the Rf of a known compound (preferrably 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 small amount of the mixture to be analyzed is spotted near the bottom of this plate. The TLC plate is then placed in a shallow pool of a solvent in a developing chamber so that only the very bottom of the plate is in the liquid. This liquid, or the eluent, is the mobile phase, and it slowly rises up the TLC plate by capillary action.

As the solvent moves past 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 chamber, dried, and the separated components of the mixture are visualized. If the compounds are colored, visualization is straightforward. Usually the compounds are not colored, so a UV lamp is used to visualize the plates. (The plate itself contains a fluor which fluoresces everywhere except where an organic compound is on the plate.)

The procedure for TLC, explained in words in the above paragraphs, is illustrated with photographs on the TLC Procedure .

TLC Adsorbent

In the teaching labs at CU Boulder, we use silica gel plates (SiO2) almost exclusively. (Alumina (Al2O3) can also be used as a TLC adsorbent.) The plates are aluminum-backed and you can cut them to size with scissors. Our plates are purchased ready-made from EM Sciences or from Scientific Adsorbents. The adsorbent is impregnated with a fluor, zinc sulfide. The fluor enables most organic compounds to be visualized when the plate is held under a UV lamp. In some circumstances, other visualization methods are used, such as charring or staining.

TLC Solvents or Solvent Systems

Choosing a solvent is covered on the Chromatography Overview page. The charts at the bottom of that page are particularly useful.

Interactions of the Compound and the Adsorbent

The strength with which an organic compound binds to an adsorbent depends on the strength of the following types of interactions: ion-dipole, dipole-dipole, hydrogen bonding, dipole induced dipole, and van der Waals forces. With silica gel, the dominant interactive forces between the adsorbent and the materials to be separated are of the dipole-dipole type. Highly polar molecules interact fairly strongly with the polar Si—O bonds of these adsorbents and will tend to stick or adsorb onto the fine particles of the adsorbent while weakly polar molecules are held less tightly. Weakly polar molecules thus generally tend to move through the adsorbent more rapidly than the polar species. Roughly, the compounds follow the elution order given on the Chromatography Overview page.

The Rf value

Rf is the retention factor, or how far up a plate the compound travels. See the Rf page for more details:

Rf's on this Orgchem site

Visualizing the Spots

If the compounds are colored, they are easy to see with the naked eye. If not, a UV lamp is used (see the Procedure page).

Troubleshooting TLC

All of the above (including the procedure page) might sound like TLC is quite an easy procedure. But what about the first time you run a TLC, and see spots everywhere and blurred, streaked spots? As with any technique, with practice you get better. One thing you have to be careful Examples of common problems encountered in TLC:

The compound runs as 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 appear as a streak. Perhaps, the experiment did not go as well as expected.

The sample runs as a smear or a upward crescent.

Compounds which possess strongly acidic or basic groups (amines or carboxylic acids) sometimes show up on a TLC plate with this behavior. Add 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 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. Crookedly run plates make it harder to measure Rf values accurately.

Many, random spots are seen on the plate.

Make sure that you do not accidentally drop any organic compound on the plate. If get a TLC plate and leave it laying on your workbench as you do the experiment, you might drop or splash an organic compound on the plate.

No spots are seen on the plate.

You might not have spotted enough compound, perhaps because the solution of the compound is too dilute. Try concentrating the solution, or, spot it several times in one place, allowing the solvent to dry between applications. Some compounds do not show up under UV light; try another method of visualizing the plate. Or, perhaps you do not have any compound because your experiment did not go as well as 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 allowing them to move up the plate by capillary action. Thus, you will not see spots after the plate is developed.

You see a blur of blue spots on the plate as it develops.

Perhaps, you used an ink pen instead of a pencil to mark the origin?

Thin layer

chromatography

Thin layer chromatography (TLC) is a chromatography technique used to separate mixtures.[1] Thin layer chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose (blotter paper). This layer of adsorbent is known as the stationary phase.

After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved.[2].

Thin layer chromatography can be used to:

Monitor the progress of a reaction Identify compounds present in a given substance Determine the purity of a substance

Specific examples of these applications include:

determination of the components a plant contains analyzing ceramides and fatty acids

Thin layer chromatography

Separation of black ink on a TLC plate

Acronym TLC

Classification Chromatography

Other techniques

Related Agarose gel electrophoresis

SDS-PAGE

detection of pesticides or insecticides in food and water analyzing the dye composition of fibers in forensics, or assaying the radiochemical purity of radiopharmaceuticals

A number of enhancements can be made to the original method to automate the different steps, to increase the resolution achieved with TLC and to allow more accurate quantitation. This method is referred to as HPTLC, or "high performance TLC".

Plate preparation

TLC plates are usually commercially available, with standard particle size ranges to improve reproducibility. They are prepared by mixing the adsorbent, such as silica gel, with a small amount of inert binder like calcium sulfate (gypsum) and water. This mixture is spread as a thick slurry on an unreactive carrier sheet, usually glass, thick aluminum foil, or plastic. The resultant plate is dried and activated by heating in an oven for thirty minutes at 110 °C. The thickness of the adsorbent layer is typically around 0.1 – 0.25 mm for analytical purposes and around 0.5 – 2.0 mm for preparative TLC.[3]

Technique

Development of a TLC plate, a purple spot separates into a red and blue .

The process is similar to paper chromatography with the advantage of faster runs, better separations, and the choice between different stationary phases. Because of its simplicity and speed TLC is often used for monitoring chemical reactions and for the qualitative analysis of reaction products.

To run a TLC, the following procedure is carried out: [4]

A small spot of solution containing the sample is applied to a plate, about 1.5 centimeters from the bottom edge. The solvent is allowed to completely evaporate off, otherwise a very poor or no separation will be achieved. If a non-volatile solvent was used to apply the sample, the plate needs to be dried in a vacuum chamber.

A small amount of an appropriate solvent (elutant) is poured in to a glass beaker or any other suitable transparent container (separation chamber) to a depth of less than 1 centimeter. A strip of filter paper is put into the chamber, so that its bottom touches the solvent, and the paper lies on the chamber wall and reaches almost to the top of the container. The container is closed with a cover glass or any

other lid and is left for a few minutes to let the solvent vapors ascend the filter paper and saturate the air in the chamber. (Failure to saturate the chamber will result in poor separation and non-reproducible results).

The TLC plate is then placed in the chamber so that the spot(s) of the sample DO NOT TOUCH the surface of the elutant in the chamber, and the lid is closed. The solvent moves up the plate by capillary action, meets the sample mixture and carries it up the plate (elutes the sample). When the solvent front reaches no higher than the top of the filter paper in the chamber, the plate should be removed (continuation of the elution will give a misleading result) and dried.

Different compounds in the sample mixture travel at different rates due to the differences in their attraction to the stationary phase, and because of differences in solubility in the solvent. By changing the solvent, or perhaps using a mixture, the separation of components (measured by the Rf value) can be adjusted. Also, the separation achieved with a TLC plate can be used to estimate the separation of a flash chromatography column.[5]

Separation of compounds is based on the competition of the solute and the mobile phase for binding places on the stationary phase. For instance, if normal phase silica gel is used as the stationary phase it can be considered polar. Given two compounds which differ in polarity, the more polar compound has a stronger interaction with the silica and is therefore more capable to dispel the mobile phase from the binding places. Consequently, the less polar compound moves higher up the plate (resulting in a higher Rf value). If the mobile phase is changed to a more polar solvent or mixture of solvents, it is more capable of dispelling solutes from the silica binding places and all compounds on the TLC plate will move higher up the plate. It is commonly said that "strong" solvents (elutants) push the analyzed compounds up the plate, while "weak" elutants barely move them. The order of strength/weakness depends on the coating (stationary phase) of the TLC plate. For silica gel coated TLC plates, the elutant strength increases in the following order: Perfluoroalkane (weakest), Hexane, Pentane, Carbon tetrachloride, Benzene/Toluene, Dichloromethane, Diethyl ether, Ethylacetate, Acetonitrile, Acetone, 2-Propanol/n-Butanol, Water, Methanol, Triethylamine, Acetic acid, Formic acid (strongest). For C18 coated plates the order is reverse. Practically this means that if you use a mixture of ethyl acetate and heptane as the mobile phase, adding more ethyl acetate results in higher Rf values for all compounds on the TLC plate. Changing the polarity of the mobile phase will normally not result in reversed order of running of the compounds on the TLC plate. An eluotropic series can be used as a guide in selecting a mobile phase. If a reversed order of running of the compounds is desired, an apolar stationary phase should be used, such as C18-functionalized silica.

Preparative TLC

TLC can also be used on a small semi-preparative scale to separate mixtures of up to a few hundred milligrams. The mixture is not "spotted" on the TLC plate as dots, but rather is applied to the plate as a thin even layer horizontally to and just above the solvent level. When developed with solvent the compounds separate in horizontal bands rather than horizontally separated spots. Each band (or a desired band) is scraped off the backing material. The backing material is then extracted with a suitable solvent (e.g. DCM) and filtered to give the isolated material upon removal of the solvent. For small-scale reactions with easily separated products, preparative TLC can be a far more efficient in terms of time and cost than doing column chromatography. Obviously, the whole plate can not be chemically developed or the product will be chemically destroyed. Thus this technique is best used with compounds that are coloured, or visible under UV light. Alternatively, a small section of the plate can be chemically developed e.g. cutting a section out and chemically developing it, or masking most of the plate and exposing a small section to a chemical developer like iodine.

Analysis

As the chemicals being separated may be colorless, several methods exist to visualize the spots:

Often a small amount of a fluorescent compound, usually manganese-activated zinc silicate, is added to the adsorbent that allows the visualization of spots under a blacklight (UV254). The adsorbent layer will thus fluoresce light green by itself, but spots of analyte quench this fluorescence.

Iodine vapors are a general unspecific color reagent Specific color reagents exist into which the TLC plate is dipped or

which are sprayed onto the plate[6] o Potassium permanganate - oxidationo Iodine

In the case of lipids, the chromatogram may be transferred to a PVDF membrane and then subjected to further analysis, for example mass spectrometry, a technique known as Far-Eastern blotting.

Once visible, the Rf value , or retention factor, of each spot can be determined by dividing the distance traveled by the product by the total distance traveled by the solvent (the solvent front). These values depend on the solvent used, and the type of TLC plate, and are not physical constants. Eluent on the thin layer is put on top of the plate

Applications

In organic chemistry, reactions are qualitatively monitored with TLC. Spots sampled with a capillary tube are placed on the plate: a spot of starting material, a spot from the reaction mixture, and a "co-spot" with both. A small (3 by 7 cm) TLC plate takes a couple of minutes to run. The analysis

is qualitative, and it will show if the starting material has disappeared, i.e. the reaction is complete, if any product has appeared, and how many products are generated (although this might be under-estimated due to co-elution). Unfortunately, TLCs from low-temperature reactions may give misleading results, because the sample is warmed to room temperature in the capillary, which can alter the reaction—the warmed sample analyzed by TLC is not the same as what is in the low-temperature flask. One such reaction is the DIBALH reduction of ester to aldehyde.

As an example the chromatography of an extract of green leaves (for example spinach) in 7 stages of development. Carotene elutes quickly and is only visible until step 2. Chlorophyll A and B are halfway in the final step and lutein the first compound staining yellow.

Step 1 Step 2 Step 3 Step 4

Step 5 Step 6 Step 7

In one study TLC has been applied in the screening of organic reactions [7] for example in the fine-tuning of BINAP synthesis from 2-naphthol. In this method the alcohol and catalyst solution (for instance iron(III) chloride) are place separately on the base line, then reacted and then instantly analyzed.

Carrying out thin layer chromatography

Background

Chromatography is used to separate mixtures of substances into their components. All forms of chromatography work on the same principle.

They all have a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a gas). The mobile phase flows through the stationary phase

and carries the components of the mixture with it. Different components travel at different rates. We'll look at the reasons for this further down the page.

Thin layer chromatography is done exactly as it says - using a thin, uniform layer of silica gel or alumina coated onto a piece of glass, metal or rigid plastic.

The silica gel (or the alumina) is the stationary phase. The stationary phase for thin layer chromatography also often contains a substance which fluoresces in UV light - for reasons you will see later. The mobile phase is a suitable liquid solvent or mixture of solvents.

Producing the chromatogram

We'll start with a very simple case - just trying to show that a particular dye is in fact a mixture of simpler dyes.

Note:  The chromatography plate will in fact be pure white - not pale grey.

A pencil line is drawn near the bottom of the plate and a small drop of a solution of the dye mixture is placed on it. Any labelling on the plate to show the original position of the drop must also be in pencil. If any of this was done in ink, dyes from the ink would also move as the

chromatogram developed.

When the spot of mixture is dry, the plate is stood in a shallow layer of solvent in a covered beaker. It is important that the solvent level is below the line with the spot on it.

The reason for covering the beaker is to make sure that the atmosphere in the beaker is saturated with solvent vapour. To help this, the beaker is often lined with some filter paper soaked in solvent. Saturating the atmosphere in the beaker with vapour 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 coloured spots.

The diagram shows the plate after the solvent has moved about half way up it.

The solvent is allowed to rise until it almost reaches the top of the plate. That will give the maximum separation of the dye components for this particular combination of solvent and stationary phase.

Measuring Rf values

If all you wanted to know is how many different dyes made up the mixture, you could just stop there. However, measurements are often taken from the plate in order to help identify the compounds present. These measurements are the distance travelled by the solvent,

and the distance travelled by individual spots.

When the solvent front gets close to the top of the plate, the plate is removed from the beaker and the position of the solvent is marked with another line before it has a chance to evaporate.

These measurements are then taken:

The Rf value for each dye is then worked out using the formula:

For example, if the red component travelled 1.7 cm from the base line while the solvent had travelled 5.0 cm, then the Rf value for the red dye is:

If you could repeat this experiment under exactly the same conditions, then the Rf values for each dye would always be the same. For example, the Rf value for the red dye would always be 0.34. However, if anything changes (the temperature, the exact composition of the solvent, and so on), that is no longer true. You have to bear this in mind if

you want to use this technique to identify a particular dye. We'll look at how you can use thin layer chromatography for analysis further down the page.

What if the substances you are interested in are colourless?

There are two simple ways of getting around this problem.

Using fluorescence

You may remember that I mentioned that the stationary phase on a thin layer plate often has a substance added to it which will fluoresce when exposed to UV light. That means that if you shine UV light on it, it will glow.

That glow is masked at the position where the spots are on the final chromatogram - even if those spots are invisible to the eye. That means that if you shine UV light on the plate, it will all glow apart from where the spots are. The spots show up as darker patches.

While the UV is still shining on the plate, you obviously have to mark the positions of the spots by drawing a pencil circle around them. As soon as you switch off the UV source, the spots will disappear again.

Showing the spots up chemically

In some cases, it may be possible to make the spots visible

by reacting them with something which produces a coloured product. A good example of this is in chromatograms produced from amino acid mixtures.

The chromatogram is allowed to dry and is then sprayed with a solution of ninhydrin. Ninhydrin reacts with amino acids to give coloured compounds, mainly brown or purple.

In another method, the chromatogram is again allowed to dry and then placed in an enclosed container (such as another beaker covered with a watch glass) along with a few iodine crystals.

The iodine vapour in the container may either react with the spots on the chromatogram, or simply stick more to the spots than to the rest of the plate. Either way, the substances you are interested in may show up as brownish spots.

Using thin layer chromatography to identify compounds

Suppose you had a mixture of amino acids and wanted to find out which particular amino acids the mixture contained. For simplicity we'll assume that you know the mixture can only possibly contain five of the common amino acids.

A small drop of the mixture is placed on the base line of the thin layer plate, and similar small spots of the known amino acids are placed alongside it. The plate is then

stood in a suitable solvent and left to develop as before. In the diagram, the mixture is M, and the known amino acids are labelled 1 to 5.

The left-hand diagram shows the plate after the solvent front has almost reached the top. The spots are still invisible. The second diagram shows what it might look like after spraying with ninhydrin.

There is no need to measure the Rf values because you can easily compare the spots in the mixture with those of the known amino acids - both from their positions and their colours.

In this example, the mixture contains the amino acids labelled as 1, 4 and 5.

And what if the mixture contained amino acids other than the ones we have used for comparison? There would be spots in the mixture which didn't match those from the known amino acids. You would have to re-run the experiment using other amino acids for comparison.

How does thin layer chromatography work?

The stationary phase - silica gel

Silica gel is a form of silicon dioxide (silica). The silicon atoms are joined via oxygen atoms in a giant covalent structure. However, at the surface of the silica gel, the silicon atoms are attached to -OH groups.

Note:  If you aren't sure about it, you will find one possible structure of silicon dioxide towards the bottom of the page you will get to by following this link.

Use the BACK button on your browser to return quickly to this page.

So, at the surface of the silica gel you have Si-O-H bonds instead of Si-O-Si bonds. The diagram shows a small part of the silica surface.

The surface of the silica gel is very polar and, because of the -OH groups, can form hydrogen bonds with suitable compounds around it as well as van der Waals dispersion forces and dipole-dipole attractions.The other commonly used stationary phase is alumina - aluminium oxide. The aluminium atoms on the surface of this also have -OH groups attached. Anything we say about silica gel therefore applies equally to alumina.

What separates the compounds as a chromatogram develops?

As the solvent begins to soak up the plate, it first dissolves the compounds in the spot that you have put on the base line. The compounds present will then tend to get carried

up the chromatography plate as the solvent continues to move upwards.

How fast the compounds get carried up the plate depends on two things:

How soluble the compound is in the solvent. This will depend on how much attraction there is between the molecules of the compound and those of the solvent.

How much the compound sticks to the stationary phase - the silica get, for example. This will depend on how much attraction there is between the molecules of the compound and the silica gel.

Suppose the original spot contained two compounds - one of which can form hydrogen bonds, and one of which can only take part in weaker van der Waals interactions.

The one which can hydrogen bond will stick to the surface of the silica gel more firmly than the other one. We say that one is adsorbed more strongly than the other. Adsorption is the name given to one substance forming some sort of bonds to the surface of another one.

Adsorption isn't permanent - there is a constant movement of a molecule between being adsorbed onto the silica gel surface and going back into solution in the solvent.

Obviously the compound can only travel up the plate during the time that it is dissolved in the solvent. While it is adsorbed on the silica gel, it is temporarily stopped - the solvent is moving on without it. That means that the more strongly a compound is adsorbed, the less distance it can travel up the plate.

In the example we started with, the compound which can hydrogen bond will adsorb more strongly than the one dependent on van der Waals interactions, and so won't travel so far up the plate.

What if both components of the mixture can hydrogen bond?

It is very unlikely that both will hydrogen bond to exactly the same extent, and be soluble in the solvent to exactly the same extent. It isn't just the attraction of the compound for the silica gel which matters. Attractions between the compound and the solvent are also important

- they will affect how easily the compound is pulled back into solution away from the surface of the silica.

However, it may be that the compounds don't separate out very well when you make the chromatogram. In that case, changing the solvent may well help - including perhaps changing the pH of the solvent.

This is to some extent just a matter of trial and error - if one solvent or solvent mixture doesn't work very well, you try another one. (Or, more likely, given the level you are probably working at, someone else has already done all the hard work for you, and you just use the solvent mixture you are given and everything will work perfectly!)

Procedure for TLC

1. Prepare the developing container.

The developing container for TLC can be a specially designed chamber, a jar with a lid, or a beaker with a watch glass on the top:

In the teaching labs, we use a beaker with a watch glass on top.

Pour solvent into the beaker to a

depth of just less than 0.5 cm.

To aid in the saturation of the TLC chamber with solvent vapors, line part of the inside of the beaker with filter paper.

Cover the beaker with a watch glass, swirl it gently, and allow it to stand while you prepare your TLC plate.

2. Prepare the TLC plate.

TLC plates used in the organic chem teaching labs are purchased as 5 cm x 20 cm sheets. Each large sheet is cut 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.

Plates will usually be cut and ready for you when you come to lab.

Handle the plates carefully so that you do not disturb the coating of adsorbent or get them dirty.

Measure 0.5 cm from the bottom of the plate. Take care not to press so hard with the pencil that you disturb the adsorbent.

Using a pencil, draw a line across the plate at the 0.5 cm mark. This is the origin: the line on which you will "spot" the plate.

It's kind of hard to see the pencil line in the above photos, so here is a close-up of how the plate looks after the line has been drawn.

Under the line, mark lightly the name of the samples you will spot on the plate, or mark numbers for time points. Leave enough space between the samples so that they do not run together, about 4 samples on a 5 cm wide plate is advised.

Use a pencil and do not press down so hard that you disturb the surface of the

plate. A close-up of a plate labeled "1 2 3" is shown to the right.

3. Spot the TLC plate

The sample to be analyzed is added to the plate in a process called "spotting".

If the sample is not already in solution, dissolve about 1 mg in a few drops of a volatile solvent such as hexanes, ethyl acetate, or methylene chloride. As a rule of thumb, a concentration of "1%" or "1 gram in 100 mL" usually works well for TLC analysis. If the sample is too concentrated, it will run as a smear or streak; if it is not concentrated enough, you will see nothing on the plate. The "rule of thumb" above is usually a good estimate, however, sometimes only a process trial and error (as in, do it over) will result in well-sized, easy to read spots.

add a few drops of solvent . . .

. . . swirl until dissolved

The solution is applied to the TLC plate with a 1µL microcap.

Not every organic lab uses microcaps to spot plates; some use drawn-out pipets. We have chosen the microcaps from

experience in working with undergraduates; they give consistant sized spots and are convenient. They are a bit expensive, at

about $5 for 100. If you use a new one for each spot, you could go through 10-20 in a single lab period. Environmentally and

financially, it is much better to reuse them by rinsing between spots. Directions for rinsing and reusing microcaps are given

below.

Microcaps come in plastic vials inside red-and-white boxes. If you are opening a new vial, you will need to take off the silver cap, remove the white styrofoam plug, and put the silver cap back on. A small hole in the silver cap allows you to shake out one microcap at a time. Microcaps are very tiny; the arrow points to one, and it is hard to see in the photo.

Take a microcap and dip it into the solution of the sample to be spotted. Then, touch the end of the microcap gently to the adsorbent on the origin in the place which you have marked for the sample. Let all of the contents of the microcap run onto the plate. Be careful not to disturb the coating of adsorbent.

dip the microcap into solution - the arrow points to the microcap, it is tiny and hard to see

make sure it is filled - hold it up to the light if necessary

touch the filled microcap to TLC plate to spot it - make sure you watch to see that all the liquid has drained from the microcap

do this rinse

process 3

times!

rinse the microcap with clean solvent by first filling it . . .

. . . and then draining it by touching it to a paper towel

If the microcap breaks or clogs, you may obtain a new one. The microcaps should be cleaned and re-used whenever possible both

because this is an environmentally sound practice and because they are

relatively expensive.

here's the TLC plate, spotted and ready to be developed

4. Develop the plate.

Place the prepared TLC plate in the developing beaker, cover the beaker with the watch glass, and leave it undisturbed on your bench top. Run until the solvent is about half a centimeter below the top of the plate (see photos below).

place the TLC plate in the developing container - make sure the solvent is not too

deep

The solvent will rise up the TLC plate by capillary action. In this photo, it is not quite

halfway up the plate.

In this photo, it is about 3/4 of the way

up the plate.

The solvent front is about half a cm below the top of the plate - it

is now ready to be removed.

Remove the plate from the beaker.

quickly mark a line across the plate at the

solvent front with a pencil

Allow the solvent to evaporate completely from the plate. If the

spots are colored, simply mark them with

a pencil.

5. Visualize the spots

If your samples are colored, mark them before they fade by circling them lightly with a pencil (see photo above).

Most samples are not colored and need to be visualized with a UV lamp. Hold a UV lamp over the plate and mark any spots which you see lightly with a pencil.

Beware! UV light is damaging both to your eyes and to your skin! Make sure you are wearing your goggles and do not look 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 start over with a more dilute sample to spot and run on a TLC plate.

this is a UV lamp

here are two proper sized spots, viewed under a UV

lamp

(you would circle these while viewing them)

The plate to the left shows three compounds run at three different concentrations. The middle and right plate show reasonable spots; the left plate is run too concentrated and the spots are running together, making it difficult to get a good and accurate Rf reading.

Here's what overloaded plates look like 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. The plate to the far right is a UV visualization of the same

overloaded plate

Thin Layer Chromatography

Chromatography is a word used to encompass a range of techniques in which mixtures of pure substances are separated into the individual substances by using a mobile phase (usually a liquid or gas) to push the mixture along a

stationary phase (usually a solid or liquid coated on a solid). Because the individual substances have different molecular structures, they interact differently with both the stationary and mobile phases, and consequently are "pushed" at different rates by the mobile phase. A number of chromatographic techniques are summarized in Table 1.

Table 1: Types of Chromatography

TypeStationary phase

Gas chromatography (GC)

Polar or non-polar liquid

High Performance liquid Chromatography (HPLC)

Solid

Gel Permeation Chromatography (GPC)

 

Thin Layer Chromatography (TLC)

Solid on glass or plastic plate

Thin-Layer Chromatography (TLC) is a simple and inexpensive technique that is often used to judge the purity of a synthesized compound or to indicate the extent of progress of a chemical reaction. In this technique, a small quantity of a solution of the mixture to be analyzed is deposited as a small spot on a TLC plate, which consists of a thin layer of silica gel (SiO2) or alumina (Al2O3) coated on a glass or plastic sheet. The plate constitutes the stationary phase. The sheet is then placed in a chamber containing a small amount of solvent, which is the mobile phase. The solvent gradually moves up the plate via capillary action, and it carries the deposited substances along with it at different rates. The desired result is that each component of the deposited mixture is moved a different distance up the plate by the solvent. The components then appear as a series of spots at different

locations up the plate. Substances can be identified from their so-called Rf values. The Rf value for a substance is the ratio of the distance that the substance travels to the distance that the solvent travels up the plate. For example, an Rf value of 0.5 means that the spot corresponding to the substance travels exactly half as far as the solvent travels along the plate.

The Process of TLC. Performing a TLC analysis consists of a number of steps: preparing a spotting capillary; marking the TLC plate; spotting the TLC plate; developing the TLC plate; drying the plate; visualizing the substance spots, and measuring the Rf values. We consider these steps in turn.

Preparing a spotting capillary. Glass capillaries used for spotting TLC plates are commercially available. However, it is occasionally necessary to make your own capillaries. To accomplish this, light a Bunsen burner and adjust for a medium flame. Hold a melting point capillary in the flame until it just begins to soften, then quickly pull the two ends of the capillary in opposite directions. The central, soft part of the glass will elongate and thin down to a capillary with very small diameter. Break the two pieces apart at the center of the thin portion to obtain two TLC spotting capillaries.

Marking the TLC Plate. Obtain a silica gel TLC plate that is approximately 2 cm wide and 5 cm long. Mark the TLC plate as follows using a pencil (pencil must be used rather than pen because inks are moved by many developing solvents). First, LIGHTLY draw a straight line parallel to the short dimension of the plate, about 1 cm from one end of the plate. Don't gouge the silica gel or make a trough with the pencil. Second, LIGHTLY make two small marks perpendicular to this line to divide the line into thirds. This subdivided line will serve as a guide for placing the substance spots, and as a point from which to measure Rf values. Third, LIGHTLY draw a second line parallel to the first line and about 1 cm from the other end of the plate. When you develop the plate, you will allow the solvent front to rise to this

second line.

Activating the TLC plate. Place your marked TLC plate in an oven at 50-60oC for 15-20 minutes to "activate" it. Activation involves driving off water molecules that bond to the polar sites on the plate.

Spotting the TLC plate. Place the narrow end of one of your capillaries into a vial containing a solution of the substance to be analyzed, and allow the solution to rise in the capillary; this will happen spontaneously. Once the capillary is loaded, hold it vertically just above the subdivided pencil line on the plate and centered in one of the three sections. Lower it until the narrow end of the capillary just touches the plate right on the pencil line. You will observe that some of the solution leaves the capillary and deposits on the plate. Leave the capillary in contact with the plate only briefly so that the spot is no larger than 1 mm in diameter, then raise the capillary. Allow the solvent to completely evaporate from the spot (if the solvent is water, evaporation will be slow; you can hasten it by putting the plate in the oven for 5 minutes). Then, if desired, make a second deposit on the same spot with the capillary. Again allow the solvent to completely evaporate.

Developing the TLC plate. Pour the desired developing solvent into a small wide-mouth glass or plastic bottle to a depth of about 4-5 mm. Using tweezers, pick up the TLC plate at the top, which is the end opposite where the subdivided pencil line is drawn. Place it carefully in the developing bottle so that it stands somewhat but not excessively tilted--that is, the bottom of the slide should be somewhat away from the wall of the bottle, whilc the top of the slide rests against the wall of the bottle. It is important that you not allow the TLC slide to tilt too much when in the developing bottle. If the slide is excessively tilted, solvent will not advance uniformly along the plate and development will not take place properly. Similarly, if the bottom of the slide is against the wall of the bottle, solvent will advance more rapidly up the edges of the slide

than in the middle, causing the substances to be pushed toward the center of the slide as they move up. Leave the slide in the chamber until solvent has advanced to the top pencil line on the slide. Development normally requires at least 30 minutes. When the solvent front has advanced to the top pencil line, use the tweezers to withdraw the slide from the chamber.

Drying the Plate. Place the plate flat on a clean dry surface and allow the solvent to completely evaporate. If the solvent is not highly volatile, this can be facilitated by placing the slide on a flat surface in an oven at a temperature of 50-60 oC (higher temperatures will melt the plastic substrate material). When the plate is completely dry, it is ready for visualization.

Visualization of the TLC Plate. If the substances being separated are colored, the spots can be seen without any further effort. Using a pencil, draw a boundary around each spot that matches the shape of the spot. Many substances are colorless (white) and do not show up on the white silica gel unless steps are taken to make them visible. There are a number of techniques for doing this. First is the technique of iodination. The dry plate is placed in a chamber containing a few crystals of iodine. The iodine vapor in the chamber oxidizes the substances in the various spots, making them visible to the eye. Once the spots are visible, they may be outlined with a pencil before the iodine coloration fades. Second is the ninhydrin technique, which is particularly effective for visualizing amino acid spots. In this method, a solution containing 0.2% ninhydrin in ethanol is sprayed on the dry plate. Alternately, the plate can be dipped in ninhydrin solution. In contact with an amino acid, ninhydrin displays a purple coloration that is easily seen. It usually takes a few minutes for this color to develop, so after the plate is sprayed, it is allowed to sit for several minutes. Placing it in a 50 oC oven will hasten the appearance of the purple color. Once this appears, the spots may be circled in pencil to permanently mark their positions. NOTE: NINHYDRIN STAINS THE SKIN PURPLE, TOO, DUE TO THE PRESENCE OF PROTEIN (AMINO ACIDS!).

TO AVOID THIS, WEAR GLOVES WHEN HANDLING IT. Third, one may use TLC plates that have been loaded with a fluorescent substance that is uniformly distributed in the silica gel. Substances moving up the plate block this fluorescence at their locations. When the dried plate is viewed using a special UV light, the substance spots are visible for their lack of fluorescence on an otherwise uniformly fluorescing field. The spots may be outlined with pencil while being viewed in the light.

Measurement of Rf. The distance between the 2 horizontal pencil lines is the distance of solvent advance. The distance from the bottom pencil line to the center of a substance spot is the distance of advance of the substance. The ratio of substance advance distance to solvent advance distance is the Rf value for the substance. The figure shows a developed plate for a mixture consisting of 4 components with Rf values of about 0.05, 0.2, 0.5, and 0.9

TLC of Inks. Before attempting to apply TLC to the challenging problem of separating and identifying amino acids, it is advisable to learn and practice the technique by applying it to mixtures that are easily visualized and separated. Inks provide an ideal practice vehicle for TLC because they normally contain several colored components that separate nicely in common solvents such as ethanol, acetone, or chloroform. Spotting the plate is also easy: it may be done simply by making a VERY small mark on the plate with the tip of a pen, just above the pencil line drawn across the bottom edge of the plate. Inks are of different types and colors, of

course. Some are washable (water-soluble), others are permanent. Different types require different solvents for development. Common sources of ink are ball point pens, felt-tip markers, and roller ball pens. Bottled ink is still available for people using fountain pens. Particularly good ink sources are bottled inks by Parker, Sheaffer, and Mont Blanc; Sharpie marker inks (black, red, blue, orange, brown, yellow, green); Marks-a-Lot Stay sharp markers; and Sanford calligraphy pens.You should select inks from at least 2 different sources available in the lab and should find a solvent or mixture of solvents that separates each ink into its component colors.

Experimental Procedure. You will be assigned two ink samples to examine. Obtain four TLC plates, four TLC spotting capillaries, and 3 developing tanks (snap-top plastic vials). Mark your TLC plates with a narrow pencil line about 1cm from the bottom. Prepare spotting solutions of your ink samples in 1-dram vials by diluting 1 drop of ink with 9 drops of ethanol (this is called a 1-to-10 dilution). Insert one end of a spotting capillary into the first ink solution. You should see the ink solution rise in the capillary. Withdraw the capillary when the liquid has risen to a height of 1-2 cm. Hold the capillary vertically and briefly touch the filled end to the pencil line, about 5 mm from the left edge of the TLC plate. Liquid should flow from the capillary to the plate to form a spot. THIS HAPPENS RAPIDLY. If possible, lift the capillary before the spot gets any bigger than 1 mm in diameter. Allow the ethanol to evaporate. Touch the capillary to a Kimwipe to empty it of ink solution. Use the other end of the same capillary to draw up your second ink solution, and spot the plate about 5 mm from the right edge. Discard the capillary in the broken glass receptacle. Allow the ethanol to evaporate from the plate.

Into your first developing tank, pour ethanol to a depth of about 5 mm. Using tweezers to hold the top end of the spotted plate, lower the plate into the tank. Allow the bottom of the plate to rest on the bottom of the tank, with the top of the plate

leaning against a tank sidewall. NOTE: IT IS VERY IMPORTANT THAT THE START LINE AND SPOTS BE ABOVE THE LEVEL OF SOLVENT. Close the tank and monitor the movement of solvent up the plate. When solvent has advanced to the top pencil line, remove the plate from the tank, and allow all solvent to evaporate. Use a pencil to outline each observed spot on the plate, preserving the shape of the spot.

If ethanol did not effect a separation of the ink into components, try another solvent. If you found that the ink moved along with or close to the solvent front, try a less polar solvent. If you found that the ink did not move at all with ethanol, try a more polar solvent (e.g., methanol). Your goal is to cleanly separate each of your ink samples into its constituents, each constituent producing a single TLC spot. Be aware that some inks contain only one component!

TLC of Amino Acids. TLC of amino acids is more difficult than TLC of inks, because amino acids are colorless. Therefore, not only can you not monitor their progress up the plate, but you cannot see the spots with the naked eye once the plate is fully developed and dried. To see the spots, it is necessary to use either the ninhydrin or the black-light visualization techniques. Of course, the latter works only if you use fluorescent TLC plates, which are available in the lab. Until you see the spots, you will not know whether or not a chosen solvent system has been effective in moving an amino acid or in separating a mixture. Therefore the process of finding an effective solvent system can be long and painstaking. For this reason, we will specify the solvent system that you are to use. As points of general information, amino acids are quite polar and tend to move on silica gel plates with polar solvents. They have Rf values close to 1 when water or concentrated ammonia is used as the developing solvent, probably because of their high solubility in water. Diluting a polar solvent with a less polar one results in smaller Rf values, roughly in proportion to the amount of less polar solvent used, Thus, alanine, glycine, threonine,

and proline all have Rf values of around 0.60 when developed with a 50/50 mixture of water and n-propanol, and around 0.40 when developed with a 30/70 mixture of concentrated NH3 and n-propanol. The following procedure assumes the use of 50/50 water/n-propanol as the developing solvent, but you are free to try other polar/non-polar combinations.

Experimental Procedure. In the hood, prepare 10 mL of a mixture consisting of 50% 1-propanol and 50% water by volume, and pour about half of this into a clean developing tank. Make sure that the level of liquid in the tank is no higher than 5 mm, and close the lid. In a 1-dram vial, prepare a solution of about 0.001 g of your amino acid in 0.2 mL of water. Dissolve the acid, then draw some solution up in a spotting capillary and double-spot a properly marked and activated TLC plate. Allow the plate to dry for 5 minutes, then use tweezers to carefully lower the plate into the developing tank so that its bottom is submerged in the developing solvent. NOTE: IT IS VERY IMPORTANT THAT THE START LINE AND SPOTS BE ABOVE THE LEVEL OF SOLVENT. Close the lid, and allow the plate to develop until solvent has risen to the pencil line at the top of the plate. Remove the plate from the tank and place it in an oven at 50 oC to dry. When the plate is dry, visualize it using ninhydrin spray or iodination. Circle the amino acid spots with pencil, and calculate Rf values. Compare the measured Rf values with the values posted for the amino acids.

On this basis, you can narrow down the identity of your amino acid. In combination with other data that you obtain, this information will help you unambiguously identify your amino acid. Suppose that your amino acid has an Rf value similar to that of, say, alanine. You should then prepare a small amount of alanine solution and spot it alongside your amino acid on a new TLC plate. Develop, dry, and visualize the plate to confirm that your amino acid indeed has exactly the same Rf value as alanine, and that the spot is the same shape and color.

Finally, it is very important to be observant of detail in doing TLC. In addition to the Rf value for a substance, the shape of the spot produced by a particular developing solvent and the shade of color produced by iodine or ninhydrin can be characteristic of the substance. Please note all of these things. For example, when alanine, glycine, threonine, and proline are spotted side-by-side on a plate and developed with 70% n-propanol/30% conc NH3, the following observations can be made:

Amino AcidSolvent Spot Color

after Iodination

Spot Color with Ninhydrin

alanine 30/70 conc NH3/n-propanol

white on brown bkgrnd

purple

alanine 50/50 water/n-propanol

white on brown bkgrnd

purple

glycine 30/70 conc NH3/n-propanol

white on brown bkgrnd

pink

glycine 50/50 water/n-propanol

white on brown bkgrnd

pink

threonine 30/70 conc NH3/n-propanol

white on brown bkgrnd

purple

threonine 50/50 water/n-propanol

white on brown bkgrnd

purple

proline 30/70 conc NH3/n-

dark brown on brown

yellow with pink border

propanol bkgrnd

proline 50/50 water/n-propanol

white on brown bkgrnd

yellow with pink border

Problems in TLC.

Over-large Spots. Sample spots made using TLC capillaries should be no larger than 1-2 mm in diameter, because component spots in the developed plate will be no smaller than, and will usually be larger than, the size of the initial spot. If the initial spot is larger than 2 mm in diameter, then components with similar Rf values may not be resolved because their spots will be so large that they will overlap considerably and may appear to be one large spot. Small initial spots, on the other hand, maximize the potential of complete separation of components.

Uneven Advance of Solvent Front. A common problem in TLC is uneven advance of solvent along the plate. Instead of a straight line, the solvent front may appear to bow either up or down in the center. Uneven advance of solvent leads to uneven advance of substance spots, and inaccurate Rf values result. A frequent cause of uneven solvent advance is the use of a developing chamber that does not have a flat bottom. Glass bottles usually have bottoms that curve upward from the edges to the center. If the bottom of the TLC plate is placed on this curved surface, the shape of the solvent advance line may mirror the shape of the container bottom. It is therefore important to use flat-bottomed developing tanks in TLC. A bowed solvent front may also result if too little developing solvent is placed in the chamber; if the plate is cut improperly, so that the sides are not exactly perpendicular to the bottom edge; and if the slide is excessively tilted in the chamber. Care in choosing and using a developing chamber is the best defense against curved solvent fronts.

Water is seldom used as a developing solvent because it has a tendency to produce a dramatically curved front. This may be due to its

unusually high surface tension.

Streaking. Sometimes a substance will move along a TLC plate as a long streak, rather than as a single discrete spot. This is the result of spotting the plate with too much substance, more than the moving solvent can handle. The solvent moves as much substance as it can, but a substantial amount of substance is left behind. The substance is dragged along by the solvent leaving a trail of substance that may sometimes span the entire distance between the starting line and the solvent front. Streaking can be eliminated by systematically diluting the spotting solution until development and visualization show the substances moving as single spots, rather than elongated streaks.