Thin Layer Chromatography

Introduction
(Adapted from Mohrig, 1st ed., pp. 151-162.) Chromatography is a sophisticated method of separating mixtures of two or more compounds. The separation is accomplished by the distribution of the mixture between two phases: one that is stationary and one that is moving. Chromatography works on the principle that different compounds will have different solubilities and adsorption to the two phases between which they are to be partitioned. Thin Layer Chromatography (TLC) is a solid-liquid technique in which the two phases are a solid (stationary phase) and a liquid (moving phase). Solids most commonly used in chromatography are silica gel (SiO2 x H2O) and alumina (Al2O3 x H2O). Both of these adsorbents are polar, but alumina is more so. Silica is also acidic. Alumina is available in neutral, basic, or acidic forms. Thin Layer Chromatography (TLC) is a sensitive, fast, simple and inexpensive analytical technique. It is a micro technique; as little as 10-9g of material can be detected, although the sample size is from 1 to 100x10-6 g. TLC involves spotting the sample to be analyzed near one end of a sheet of glass or plastic that is coated with a thin layer of an adsorbent. The sheet, which can be the size of a microscope slide, is placed on end in a covered jar containing a shallow layer of solvent. As the solvent rises by capillary action up through the adsorbent, differential partitioning occurs between the components of the mixture dissolved in the solvent the stationary adsorbent phase. The more strongly a given component of a mixture is adsorbed onto the stationary phase, the less time it will spend in the mobile phase and the more slowly it will migrate up the plate. The following are some common uses of Thin-Layer Chromatography:

1. 2. 3. 4. 5. 6.

To determine the number of components in a mixture. To determine the identity of two substances. To monitor the progress of a reaction. To determine the effectiveness of a purification. To determine the appropriate conditions for a column chromatographic separation. To monitor column chromatography.

In this experiment, you will use TLC to identify unknown analgesic painkillers using the table of analgesics and their components in the experimental section of this experiment.

Apparatus

Experimental Procedure for TLC Analysis of Analgesic Drugs

(adapted from Fieser & Williamson, pp. 128-129) Obtain 2 TLC plates. Draw a light pencil line about 1 cm from the end of each chromatographic plate. Spot one plate with your 4 known standards (Acetaminophen, Aspirin, Caffeine, and Ibuprofen) and the other plate with the 5 unknown commercial painkillers. Both plates should also have a Reference spot that contains all 4 standards. Use a separate capillary tube for each standard and unknown solution. Make each spot as small as possible, preferably no more than 2-3 mm in diameter. Examine the plate under the ultraviolet (UV) light to see that enough of each compound has been applied; if not, add more. The standards and commercial painkillers will be dissolved in a 50/50 Ethanol/Ethyl Acetate solution.

Prepare a developing chamber as indicated in the picture using a large beaker as the chamber, a halfpiece of filter paper inside, and foil or plastic wrap to cover. Pour the eluting solvent, a 99/1 mixture of Ethyl Acetate/Glacial Acetic Acid, into the beaker to a depth of approximately 1 cm. Place the prepared TLC plates in the developing chamber. After the solvent has risen to near the top of the plate (about 1 cm from the top), remove the plate and mark the solvent front with a pencil. Keep the plates in the hood until the majority of the eluting solvent has evaporated from the plates. Examine the plate under UV light to see the components as dark spots against a bright green-blue background.

the free encyclopedia Thin layer chromatography . The iodine chamber is pre-made and contains a few crystals of iodine in the bottom of a capped jar.wellesley. fluorescence in UV light. The spots should also be visualized by putting the plate in an iodine chamber. Remove the plates when a definite change in appearence takes place on your plates. the reference spot http://www. Wrap your TLC plates in plastic wrap and scan them into your e-lab.html Thin layer chromatography From Wikipedia. changes due to iodine exposure. Unknowns can be identified using Rf values. Note which compounds stained with iodine and to what intensity.edu/Chemistry/chem211lab/Orgo_Lab_Manual/Appendix/Techniques/TLC/thin_l ayer_chrom.Outline the spots with a pencil and note anything distinctive about any of the compounds. Calculate the Rf values for each spot. The iodine stains will dissipate over time. More than 2 plates can be placed in the iodine chamber at one time.

or cellulose (blotter paper). or assaying the radiochemical purity of radiopharmaceuticals identification of medicinal plants and their constituents [3] . which is coated with a thin layer of adsorbent material. After the sample has been applied on the plate. aluminium oxide. separation is achieved.[2] Thin layer chromatography can be used to:    Monitor the progress of a reaction Identify compounds present in a given mixture Determine the purity of a substance Specific examples of these applications include:      analyzing ceramides and fatty acids detection of pesticides or insecticides in food and water analyzing the dye composition of fibers in forensics. This layer of adsorbent is known as the stationary phase. Because different analytes ascend the TLC plate at different rates. plastic.[1] Thin layer chromatography is performed on a sheet of glass.Separation of black ink on a TLC plate Acronym TLC Classification Chromatography Other techniques Related Agarose gel electrophoresis SDS-PAGE Thin layer chromatography (TLC) is a chromatography technique used to separate mixtures. usually silica gel. a solvent or solvent mixture (known as themobile phase) is drawn up the plate via capillary action. or aluminum foil.

1 ± 0. They are prepared by mixing the adsorbent.0 mm for preparative TLC. a purple spot separates into a red and blue spot. or plastic. thick aluminum foil. The resultant plate is dried and activated by heating in an oven for thirty minutes at 110 °C. with a small amount of inert binder like calcium sulfate (gypsum) and water.25 mm for analytical purposes and around 0. to increase the resolution achieved with TLC and to allow more accurate quantization. .5 ± 2. with standard particle size ranges to improve reproducibility. or "high performance TLC". This mixture is spread as a thick slurry on an unreactive carrier sheet. Contents [hide] 1 Plate preparation 2 Technique 3 Preparative TLC 4 Analysis 5 Applications 6 References [edit]Plate preparation TLC plates are usually commercially available. such as silica gel. usually glass. The thickness of the adsorbent layer is typically around 0. This method is referred to as HPTLC.[4] [edit]Technique Development of a TLC plate.A number of enhancements can be made to the original method to automate the different steps.

the separation achieved with a TLC plate can be used to estimate the separation of a flash chromatography column. Also. the separation of components (measured by the Rf value) can be adjusted. about 1. so that its bottom touches the solvent.5 centimeters from the bottom edge. and the lid is closed. or perhaps using a mixture. To run a TLC.[6] Separation of compounds is based on the competition of the solute and the mobile phase for binding places on the stationary phase. By changing the solvent. and the paper lies on the chamber wall and reaches almost to the top of the container. The solvent moves up the plate bycapillary action. otherwise a very poor or no separation will be achieved. A strip of filter paper is put into the chamber. if normal phase silica gel is used as the stationary phase it can be considered polar. The solvent is allowed to completely evaporate off. Given two compounds which differ in . When the solvent front reaches no higher than the top of the filter paper in the chamber. (Failure to saturate the chamber will result in poor separation and nonreproducible results). 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. Because of its simplicity and speed TLC is often used for monitoring chemical reactions and for the qualitative analysis of reaction products. the plate needs to be dried in a vacuum chamber.Chromatogram of 10 essential oilscoloured with vanillin reagent.   Different compounds in the sample mixture travel at different rates due to the differences in their attraction to the stationary phase. For instance. 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. and the choice between different stationary phases. and because of differences in solubility in the solvent. If a non-volatile solvent was used to apply the sample. better separations. The process is similar to paper chromatography with the advantage of faster runs. 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. meets the sample mixture and carries it up the plate (elutes the sample). the following procedure is carried out: [5]  A small spot of solution containing the sample is applied to a plate. the plate should be removed (continuation of the elution will give a misleading result) and dried.

or masking most of the plate and exposing a small section to a chemical developer like iodine. Hexane. Obviously. The backing material is then extracted with a suitable solvent (e. while "weak" elutants barely move them. Dichloromethane. Pentane.polarity. Alternatively. adding more ethyl acetate results in higher Rf values for all compounds on the TLC plate. the less polar compound moves higher up the plate (resulting in a higher Rf value). Consequently. 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. Changing the polarity of the mobile phase will normally not result in reversed order of running of the compounds on the TLC plate. Practically this means that if you use a mixture of ethyl acetate and hexane as the mobile phase. Water. Diethyl ether. 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. 2-Propanol/nButanol. If a reversed order of running of the compounds is desired. Ethylacetate. When developed with solvent the compounds separate in horizontal bands rather than horizontally separated spots. or visible under UV light. Methanol. Aneluotropic series can be used as a guide in selecting a mobile phase. the elutant strength increases in the following order: Perfluoroalkane (weakest).g. a small section of the plate can be chemically developed e. Thus this technique is best used with compounds that are coloured. For C18 coated plates the order is reverse. . For silica gel coated TLC plates.g. 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. preparative TLC can be a far more efficient in terms of time and cost than doing column chromatography.Benzene/Toluene. Formic acid (strongest). cutting a section out and chemically developing it. the whole plate can not be chemically developed or the product will be chemically destroyed. Acetone. It is commonly said that "strong" solvents (elutants) push the analyzed compounds up the plate. DCM) and filtered to give the isolated material upon removal of the solvent. Carbon tetrachloride. For small-scale reactions with easily separated products. Each band (or a desired band) is scraped off the backing material. [edit]Preparative TLC TLC can also be used on a small semi-preparative scale to separate mixtures of up to a few hundred milligrams. If the mobile phase is changed to a more polar solvent or mixture of solvents. Acetonitrile. such as C18-functionalized silica.Triethylamine. The order of strength/weakness depends on the coating (stationary phase) of the TLC plate. an apolar stationary phase should be used. Acetic acid.

The adsorbent layer will thus fluoresce light green by itself. several methods exist to visualize the spots:  Often a small amount of a fluorescent compound. because the sample is warmed to room temperature in the capillary. but spots of analyte quench this fluorescence. the chromatogram may be transferred to a PVDF membrane and then subjected to further analysis. the Rf value . and are not physical constants. if any product has appeared. and it will show if the starting material has disappeared. for examplemass spectrometry. Spots sampled with a capillary tube are placed on the plate: a spot of starting material. which can alter the reaction²the warmed sample analyzed by TLC is not the same as what is in the low-temperature flask. Carotene elutes quickly and is only visible until step . As an example the chromatography of an extract of green leaves (for example spinach) in 7 stages of development. Unfortunately. TLCs from low-temperature reactions may give misleading results. is added to the adsorbent that allows the visualization of spots under a blacklight (UV254). usually manganese-activated zinc silicate. the reaction is complete. a spot from the reaction mixture. Eluent on the thin layer is put on top of the plate [edit]Applications In organic chemistry. The analysis is qualitative. A small (3 by 7 cm) TLC plate takes a couple of minutes to run.[edit]Analysis As the chemicals being separated may be colorless.oxidation Iodine  In the case of lipids.e. and a "co-spot" with both. Once visible. 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. i. or retention factor. and the type of TLC plate. reactions are qualitatively monitored with TLC. and how many products are generated (although this might be under-estimated due to co-elution). 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[7]     Potassium permanganate . a technique known as Far-Eastern blotting. One such reaction is the DIBALH reduction of ester to aldehyde.

2.  Step 1  Step 2  Step 3  . Chlorophyll A and B are halfway in the final step and lutein the first compound staining yellow.

org/wiki/Thin_layer_chromatography .wikipedia. then reacted and then instantly analyzed. [edit] http://en. In this method the alcohol and catalyst solution (for instance iron(III) chloride) are placed separately on the base line.Step 4  Step 5  Step 6  Step 7 In one study TLC has been applied in the screening of organic reactions[8] for example in the fine-tuning of BINAP synthesis from 2-naphthol.

alumina. a fluorescent powder is mixed into the stationary phase to simplify the visualization later on (e. In addition a binder like gypsum is mixed into the stationary phase to make it stick better to the slide.5-1 cm from the bottom of the plate. It also permits the optimization of the solvent system for a given separation problem. Do not use gloves when you pull capillaries. Stationary Phase As stationary phase.g. Why? The start line should be 0. If you pull the capillary inside the flame. In comparison with column chromatography. bright green when you expose it to 254 nm UV light).last updated Friday. Quickly remove the capillary from the flame and pull on both ends to about 2-3 times its original length. you will have a "piece of art". Capillary spotters Place a melting point capillary and in the dark blue part of the Bunsen burner flame. Preparing the Plate Do not touch the TLC plate on the side with the white surface. and then break it in the middle. Allow the capillary to cool down. but not a good spotter. Make sure that you break off the closed end on one of them. it only requires small quantities of the compound (~ng) and is much faster as well. You will have much better control without them! . August 14.25 mm). In order to obtain an imaginary start line. 2009 Introduction Thin-layer chromatography (TLC) is a very commonly used technique in synthetic chemistry for identifying compounds. a metal or a plastic film as a thin layer (~0. Do not use pen. You can also draw a thin line with pencil. determining their purity and following the progress of a reaction. Hold it there until it softens and starts to sag. In many cases. a special finely ground matrix (silica gel. or similar material) is coated on a glass plate. make two notches on each side of the TLC plate.

because this will deteriorate the quality of the separation considerably (µtailing¶). Place a small amount of solvent (= mobile phase) in the container. you should spot the compound or mixture together with the starting materials and possible intermediates on the plate. moving the components of the samples at various rates because of their different degrees of interaction with . Try to avoid spotting too much material. The solvent (eluent) travels up the matrix by capillarity. They will serve as internal reference since every TLC plate is slightly different. The lower edge of the plate is then dipped in a solvent. Developing a Plate A TLC plate can be developed in a beaker or closed jar (see picture below). The spots should be far enough away from the edges and from each other as well. This way you will get a concentrated and small spot. The solvent level has to be below the starting line of the TLC. the solution will rise up in the capillary (capillary forces). otherwise the spots will dissolve away. Allow the solvent to evaporate and spot at the same place again.Watch movie how to pull capillaries here here Spotting the plate The thin end of the spotter is placed in the dilute solution. Touch the plate briefly at the start line. If possible.

g. leaves charred blots behind 2. UV light: non-destructive. because the compounds dissolve well and do not interact with the polar stationary phase. Ceric stain: destructive. 1. Measure the distance of the start line to the . let the solvent evaporate completely. Take the plate out and mark the solvent front immediately.the matrix (=stationary phase) and solubility in the developing solvent. Iodine: semi-destructive. Sulfuric acid/heat: destructive. after ~5 beacher min with a lid or a closed jar after ~10 min after drying Visualization There are various techniques to visualize the compounds. Next. Analysis The components. iodine absorbs onto the spots. short wavelength (plate dark. leaves a dark blue blot behind for polar compounds 3. compounds glow). are identified by comparing the distances they have traveled with those of the known reference materials. Non-polar solvents will force non-polar compounds to the top of the plate. long wavelength (background green. Also draw a sketch of the developed plate in your lab notebook. not permanent 4. Allow the solvent to travel up the plate until ~1 cm from the top. Do not allow the solvent to run over the edge of the plate. TLC chamber for development e. visible as separated spots. Do not look into the UV lamp!!! Circle the spots on the TLC plate to have a permanent record how far the compound traveled on the plate. spots dark).

The Rf (=retardation factor) depends on the following parameters: y y y y solvent system absorbent (grain size.0 (spot did not moved from starting line) and 1. a reference compound is usually applied to the plate as well. Then measure the distance of center of the spot to the start line (=a). thickness) amount of material spotted temperature Due to the fact that all those variables are difficult to keep constant. Useful links . water content. The resulting ratio is called Rf-value. Divide the distance the solvent moved by the distance the individual spot moved.0 (spot moved with solvent front) and is unitless.solvent front (=d). The value should be between 0.

chem.htm Acetaminophen Acetaminophen is an analgesic and an antipyretic. 5. The mixture is spotted on the plate and solvent is allowed to run up the plate and separate the compounds. Chemically.24% nitrogen .edu/~bacher/General/30BL/tips/TLC1.43% hydrogen.16 and elemental analysis shows it contains 67. 9.http://www. acetaminophen is p-hydroxy acetanilide. to analyse mixtures or to establish conditions for a preparative separation of compounds using column chromatography. but the exact mechanism of the drug action is not fully understood. It appears to relieve pain by raising the pain threshold so that a greater amount of pain is necessary before it can be felt.html The technique of Thin Layer Chromatography (TLC) is normally used as an analytical method to follow the progress of a reaction.net/labTechniques/TLC. http://chem-ilp. it has a molecular weight of 151.ucla.38% carbon. The stationary phase (often silica) is coated on plastic or aluminium plates. Its antipyretic action results from its effect on the heat-regulating centers of the brain.

TLC is generally very sensitive to small differences in the chemical structure of the sample. It is important to note that no solvent is more polar than the silica gel . caffeine.95% oxygen. and Tylenol) were determined by thin layer chromatography in this experiment using ethyl acetate as both the solvent and the elluent.6 mm in diameter using a strongly polar. Nuprin. For a given sample. Excedrin. Only the edge of the plate nearest the samples is in contact with the eluent. This is because the solvent begins to compete more and more with the silica gel for the polar parts of the molecule. The structure affects the strength and type of interactions between the sample and the gel. The Rf value is the ratio of distance traveled by the spot divided by the distance traveled by the solvent. the less the distance from the origin it migrates. By employing a C18 stationary phase strong dispersive interactions are exploited in the stationary phase whereas predominantly polar interactions are active in the mobile phase. A line is drawn 1 cm from the bottom of the gel and a small volume of sample (dissolved in a solvent. Separations can be easily obtained in 5 to 10 minutes Analysis of Analgesics by Thin Layer Chromatography Abstract: The chemical compositions of four analgesics (Anacin. Nonpolar samples tend to migrate the furthest away from the origin because it is unable to interact readily with the silicon gel (see chart below). These Rf factors were then compared to the Rf factors of the pure standard samples: acetaminophen. Anacin. the Rf value will always be less than 1.and 17. Introduction: Thin layer chromatography (TLC) is a method used in chemistry and biochemistry for the separation and analysis of a wide variety of inorganic ions and organic molecules. and so the sample is eluted up the plate more effectively. Excedrin contains acetaminophen. The plate is then placed in a chamber containing a small volume of the appropriate solvent designated the eluent (in this experiment. The eluent is drawn into the silicon by capillary action and travels up the plate through the samples. Each sample has its own distinct Rf value for a particular solvent. Nuprin contains only ibuprofen. The TLC plate typically consists of a thin layer of gel (fluorescent silicon gel was used in this experiment) bonded to a glass or plastic backing. The migration rate of the sample components over the silicon depends on their chemical structure. the general rule of thumb is the more polar the compound. in this case ethyl acetate) is applied to the silicon with a micropipette and allowed to dry. different samples have different solubilities in a given solvent which is also dependent upon chemical structure. the Rf value (and the distance moved by the spot) will increase as the polarity of the solvent is increased (see chart below). it is mainly used to help reduce temperature and as a mild analgesic. aspirin. and Tylenol were pulverized. Nuprin. Since the solvent front will always be ahead of the sample spots. and ibuprofen. and run plated on a silicon gel to determine their Rf factors. Acetaminophen tablets have been successfully analyzed using reversed phase (C18) columns 20-25 cm long 3-4. In addition. Excedrin. The TLC plates indicated that Anacin contains both aspirin and caffeine. aspirin. ethyl acetate). water-methanolacetonitrile mixture with a tetramethylammonium hydroxide buffer as the mobile phase. Thus. dissolved in ethyl acetate. Acetaminophen is prescribed for arthritis but is not an antiinflammatory. and Tylenol contains only acetaminophen. and caffeine.

16 1.surface. cyclohexane.8945 -83. ketones.75 75-77 C4H8O2 88.293 169 C8H10N4O2 194.106 0. esters Amines Alcohols Carboxylic acids Common TLC developing solvents: Hexane. thin layer chromatography is principally a competition between the solvent and the silica gel for the polar parts of the molecule.6 . for this reason non-polar samples prefer the polar solvent over the surface of the silica gel causing it to migrate the furthest from the origin.284 0.19 1.23 238 C13H18O2 206.35 135 C8H9NO2 151.oC C9H8O4 180.16 1. g/mol Density g/cm3 Melting pt. Thus. Relative polarity of organic samples: Alkanes Alkenes INCREASING Aromatic hydrocarbons POLARITY Ethers.5 ± 0. alkyl halides Aldehydes. petroleum ether Toluene Dichloromethane Diethyl ether INCREASING Chloroform POLARITY Ethyl acetate Isopropyl alcohol Acetone Ethanol Methanol Acetonitrile Water Table of Physical Properties: Compound Aspirin Acetaminophe Caffeine Ibuprofen Ethyl s and (compoun n (compoun (compoun acetate Solvents d) (compound) d) d) (solvent ) Structural Formula Molecular Formula Molec. Using a common eluent while cmparing and contrasting the Rf values of known pure samples with that of an unknown is the way to identify the unknown by TLC and the main purpose of this experiment. Wt.

Boiling pt.1 .oC 140 77.

The four known solutions used were of caffeine. Nuprin.106 0.016 0.474 cm cm cm Nuprin 5 cm 2.54 2.53 2. After running the TLC plates for the full 5 cm. While being viewed in ultraviolet light. acetaminophen and ibuprofen. the unkowns were compared with the knowns to determine their composition.35 0. they were allowed to dry and were placed under ultraviolet light. TLC plates were cut and marked so that the elluent would have 5 cm to run with 1 extra cm at the top and 1 cm at the bottom.304 cm Composition of Unknowns: Acetaminophen Aspirin Caffeine Ibuprofen Anacin No Yes Yes No Excedrin Yes Yes Yes No Nuprin No No No Yes Tylenol Yes No No No Anacin = Aspirin + Caffeine Excedrin = Acetaminophen + Aspirin + Caffeine Nuprin = Ibuprofen Tylenol = Acetaminophen .11 cm Ibuprofen 5 cm 2.85 0.35 0. Tylenol. and Excedrin.57 cm Anacin 5 cm 0. This elluent was placed in a small jar lined with filter paper to keep the vapors high up in the vessel. and thusly the Rf values for each spot could be determined. Once the Rf values had been determined.308 0.37 0.86 0. The elluent used for the TLCs run was ethyl acetate. aspirin. the movement of the spots placed on the TLC plates could be determined. It was given that the unkown analgesics contained one or more of the known analgesics.57 cm Tylenol 5 cm 1. Ethyl acetate was used as the elluent to run the TLC plates in.53 1.52 0.47 cm Caffeine 5 cm 0. Results: Compound Dist of Dist Dist Dist Rf Rf Rf Solvent (a) (b) (c) (a) (b) (c) Acetaminophen 5 cm 1. The unknown solutions used were of Anacin.55 0.31 cm Aspirin 5 cm 2.Experiment: This experiment involved the use of the technique: thin-layer chromatography.55 0.47 cm cm Excedrin 5 cm 0. Each TLC plate was spotted with two known analgesics in an ethyl acetate solution and one unknown analgesic in an ethyl acetate solution.

the activity of the adsorbent. caffeine. the more strongly it will be adsorbed on the surface of the solid phase. The sample is applied in a small drop a short distance from one end of the plate. The separation of the components in a mixture is dependent on the relative values of the adsorption-desorption equilibrium constants for each of the components in the mixture. the presence of an ultra-violet-absorbing compound on the adsorbent will result in a dark spot on exposure to ultra-violet light. Another possibility is to impregnate the plate in advance with a fluorescent dye. thin layer chromatography (TLC) has found many applications in medical. The activity of the adsorbent (adsorptive power) depends on the type of material and on the mode of its preparation. their Rf values can be calculated from the equation: . The separation of the components of a mixture depends on adsorption-desorption equilibria between compounds adsorbed on the solid stationary phase and in the moving liquid phase. instead of a column of adsorbent. The extent of adsorption of a single component depends upon the polarity of the molecule. they can be located visibly. Find structures for these products in the Pharmacopedic. If these approaches still do not make the TLC components visible.2). and codeine. and the polarity of the mobile liquid phase. In general. Illumination with ultra-violet light will excite many compounds to fluoresce. The choice of the proper adsorbent will depend on the types of compounds to be chromatographed (1. Once the spots are located. Elution. Separation is stopped by removal of the plate when the solvent front approaches the top edge. chemical and pharmaceutical sciences (1. is accomplished by capillary movement of the solvent up the thin layer of adsorbent.2). as used in column chromatography. Some of the more common components found in over-the-counter analgesic products include aspirin. The plate is then placed vertically into a closed jar with its lower edge dipping into a pool of eluting solvent. phenacetin (withdrawn). in which a thin layer of adsorbent coated onto a flat surface is utilized. a color. Thin layer chromatography is a special application of adsorption chromatography. biological. If the components are colored. paracetamol (acetaminophen). to render the spots visible. the more polar a functional group in the compound. as the compound quenches the fluorescence of the dye. but more often they are invisible and must be located by other means. Introduction: Because of its simplicity and speed. (methylmorphine) (3). or development of the chromatogram.or fluorescence-producing reagent can be sprayed onto the dried plate.Determination of analgesics by thin layer chromatography (TLC) 1. and the solvent evaporated off. The most commonly used adsorbent in TLC is silica gel and the flat surface is a plain rectangular or square glass plate.

(d) Set the gap of the TLC applicator to 0. Confirmatory tests can then be used to positively identify the analyte (4. (g) Wash and drain the applicator. TLC analysis of an extract of a forensic sample. With a single constant motion. It is important that the surface of the glass be kept free of grease and dirt. to give a smooth slurry (25g in about 60-70mL water). Wash plates throughly with detergent and water. allows the analyte in the sample to be tentatively identified. with the 20 x 5cm plates on either end and clamp the plates to provide an even spreading surface. For example. The position of the bands on the developed TLC plate can alternately be located under ultra-violet light. 2. Place applicator on an end-plate. Separate the plates by means of a spatula. TLC plates will be prepared and used.25mm. Once the bands are located. using the feeler guage provided. they are scraped from the plates and the material leached from the adsorbent using an appropriate solvent. employing thicker layers of adsorbent and applying the sample as a band instead of individual spots. to ensure complete spreading of the adsorbent. The dissolved components of the bands can then be analyzed by other techniques. with UV spectroscopy. The chromatogram can be developed. remove both end-plates and discard. then release the pressure on the plates. TLC may also be used as a means of identification of an analyte. for use by the next group. with the gap away from the analytical 20 x 20cm plates. and the edges of the plate treated with visualizing agents to locate the bands of interest. Wipe plates free of grease and dirt with acetone-soaked tissue. (f) Tap the sides of the plate holder to smoothen the surface of the slurry layer.Rf = Distance moved by compound Distance moved by solvent system TLC can be used for small-scale preparative separations.5). draw the slurry along the plates. (c) Mix the recommended weight of silica gel adsorbent with distilled water. (e) Pour the slurry and distribute evenly in the reservoir. including UV spectroscopy. (b) Mount plates on the plate spreader. rinse with distilled water and allow to drain. stopping only when the applicator is on the other end-plate. for coating with silica gel. . In this experiment. . for the semiquantitative determination of selected analgesics in pharmaceutical preparations. alongside standards of analytes suspected to be present in the sample. Experimental procedure: (A) Preparation of TLC plates: (a) Obtain two 20cm x 20cm glass plates and two 5cm x 20cm plates.

Take care not to make craters or holes on the adsorbent layer. in the volume ratio of 120:30:30:02. (b) Filter the mixture through filter paper (Whatman No.5g of the finely powdered analgesic mixture provided and extract by swirling with 4mL analytical grade methanol/actone (1:1 v/v).(h) Allow the analytical plates to air-dry. Thus.1) into a 10mL volumetric flask. (B) Sample preparation: (a) Weigh out accurately 1. (c) Sample application and development of chromatogram: (i) On each of the activated TLC plates. Begin at least 1.5 cm apart. gently mark the intended positions of samples and standards with a clean pointed glass rod. N. (i) Place plates horizontally in a plate rack provided. (vi) Remove the plates from the tank and allow the solvent to evaporate. (C) Detection of sample spots: (a) Some of the constituents in the mixture may not be very stable on the TLC plate. and activate in an oven at 110° to 120°C overnight and cool in a desiccator before use. (v) Develop the plates in tanks pre-equilibrated for one hour with a toluene:acetic acid:diethyl ether:methanol mixture. starting from one edge of the plate. starting from at least 2cm from one edge of the plate. Repeat the extraction procedure as described in (i) and make up to the mark with methanol. once the plates are dried. using a clean 10uL micropipette (iv) Score a horizontal line 15 cm from the origin line. in a small beaker for about 5 minutes. Ensure that the level of the solvent is below that of the applied spots. then remove from plate holder. until the watery sheen has disappeared. to provide a stop mark for the developing solvent. (ii) Apply the standards. they must be examined immediately under short wavelength UV (254nm) .B. using a separate graduated 10uL micropipette for each standard. (iii) Apply the sample spots to the other marked positions on the origin line.5 cm from either vertical edge and keep samples at least 1.

many more sorbents in TLC format are available: aluminum. (d) Hence calculate the concentration of each analgesic component identified in the sample. Besides speed and low price. (D) UV Spectroscopy of sample extracts: (a) Carefully scrape all the silica gel outlined for each sample component from the TLC plate. and note the color of each visualized spot. (b) Using a clean pointed glass rod.and long wavelength UV (365nm) light. Exercise: Explain how it might be possible to analyse a mixture such as Asprin/Phenacetin/Codeine by UV-VIS spectroscopy. by comparison with those of the standards. Make up to the mark with methanol. cellulose. Take care not to contaminate a component with materials from other spots on the plate. mark the outline of the standard and sample spots. Scan each sample extract similarly and determine their absorbances. For organic chemists. TLC is most commonly thought of as being done on silica plates. using tabulated absorption extinction coefficients of each standard. there are three more features that make TLC very popular: . 1 filter paper. (c) Scan each analgesic standard solution provided and note their respective absorbances at the wavelength of maximal absorption. (c) Identify the individual components in the mixture from their respective Rf values and colors. 3. without separating the individual components from the mixture. and many more. ion exchange resins. Whatman No. What is TLC? Thin Layer Chromatography (TLC) is a commonly used analytical technique that allows for rapid and inexpensive analysis of various mixtures. (b) Elute each sample component with 3 successive 3mL volumes of analytical grade methanol into a 10mL volumetric flask. In reality. into the centre of a fluted 5cm diam. C18 reverse phase.

The elutant must not touch the spots. this experiment determines whether any of the mixture components happen to be unstable under separation conditions. TLC plates differ on backing material. Setting up and running a TLC A TLC plate must be cut/divided to size (most of them come as 20cm x 20cm or 20cm x 5cm sheets) and a start line drawn lightly with soft pencil (no inks!) at about 3/4" (17mm) from the bottom edge. run in one direction with one elutant. y Setting up a TLC analysis is very simple. the spotted TLC plate is inserted carefully into the jar. A small amount of elutant is poured into jar (to a depth about 1/2 to 2/3 of the bottom of TLC sheet-to-start line distance). and the jar is tilted temporarily so that all the filter paper gets soaked in elutant. Then. but. in our experience. and glass are the most common materials. a piece of filter paper. Some people insist on glass backing. Besides providing better resolution. Aluminum.y y It is easy to run up to 20 samples simultaneously It is possible to use a square plate. and the TLC plate itself constitute all the materials needed. A suitable beaker or a jar with some kind of cover. aluminum seems the most convenient. replacing the lid afterwards. The solution of material to be analyzed is spotted onto start line carefully (this step discussed in greater detail in the next section). A cover is placed on top of the jar. mostly above the elutant pool. It is a good practice to allow for 1/2" (12mm) between elutant level and spots. and a rectangular piece of filter paper inserted so that it touches the elutant and lies on the jar wall. then rotate plate 90deg and run with another elutant (creating a "twodimensional" TLC). The picture to the . Plates (unlike HPLC columns) are disposable. glass capillaries. The top edge of filter paper should be close to the jar mouth. This step ensures that all inner volume of the jar is saturated with elutant vapors. polyester.

Spotting a TLC To spot a TLC. which always lead to overloading the TLC plate. so results are immediately seen (are colorful and visible to the naked eye). The right dilution is found by trial and error. Due to these. creating a wet circular spot. The spotting solvent must be completely removed from the TLC before developing. Dimethylacetamide. and press the capillary onto the TLC place medium. the plate is removed from chamber. instead of neat spots large streaks appear. are very popular. The initial spot diameter should be 1/8" to 3/16" (3 5mm) immediately after application. The capillary diameter should be around 1/32" (0. the solvent should all drain onto the medium.5 to 5wt% is good enough. Spotting too concentrated or non-homogenous of a solution. DMSO or pyridine. This mark is needed to calculate Rf (Retention factor) values. There is no point in spotting using solutions prepared in DMF. 0. If done correctly. however. After the solvent front reaches within ~1" (25mm) of the top edge of the TLC plate. The solution to be spotted should be free of precipitate and insoluble oils. y Spotting too much of a solution.7 to 1mm). The solvent is then allowed to evaporate. to name a few. and should not be too concentrated. Three common mistakes. so some kind of visualization is required. felt pen inks were subjected to the analysis. and the exact elutant front location is marked with a pencil.left shows what happens next. the spots are colorless. first dissolve the material to be separated in a solvent. A not too volatile solvent is used. it is possible to place the y y . Spotting a TLC plate does require some practice. For DMF and pyridine. Then. Here. before placing the TLC plate into the elutant to "develop". which bear no information. draw some solvent into a capillary. More often. leaving behind only material to be separated.

Spotting capillaries are available commercially. the elutant 'strength' ie ability to drag a spot up the plate can be approximately represented by the following sequence (in order of increasing strength): Perfluoroalkanes (weakest). vacuum won't work . Usually a TLC is informative enough if the lower spot has Rf > 0. This. Often it more convenient to use the same mixture of elutants while changing their ratio to adjust the total strength of a mixture. It is also possible to pull a capillary out of a pipette with a good gas burner. For instance ethylacetate : hexane mixture is very popular for eluting TLC of medium polar materials. Methanol.85. . Pentane. 2-Propanol/n-Butanol. Acetone. then try 10:90 or 25:75 mixtures in order to increase Rf values of the spots. Just allow the solvent to dry between spottings. Formic acid (strongest) So basically one needs to use stronger elutant if spots are too low or weaker elutant if spots are too high. Benzene/Toluene.spotted TLC plate for ~5min into vacuum before the run to evaporate the solvent. Dichloromethane.1 while the top spot exhibits Rf < 0. For a silica coated TLC plate. So if say 5:95 ethylacetate : hexane mixture would barely move spots. For DMSO and Dimethylacetamide. leads to poorer TLC resolution.one has to do a mini workup to remove these solvents first. How to choose a solvent (elutant) for TLC Choosing a right elutant (or elutant mixture) to obtain an useful TLC requires some trial and error. Diethyl ether. Hexanes. Acetic acid. If a solution to be spotted happens to be too diluted (like after a column when a too low of Rf fraction was finally pushed out with large amount of elutant). Acetonitrile. Ethylacetate. Do not use melting point determination capillaries . For this approach to work one component of the mixture must be a weak elutant while another must be of excessive strength. Carbon tetrachloride. Water. Triethylamine. however.they are way too thick. then it is possible to spot same place a few times.

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. As the solvent moves past the spot that was applied.) The procedure for TLC. explained in words in the above paragraphs.30 (which means spots are overlapping) for a some mixture then 5 : 95 methanol : dichloromethane or 3 : 97 acetone : toluene mixture likely to produce Rf values in the similar range (0. In principle. (The plate itself contains a fluor which fluoresces everywhere except where an organic compound is on the plate. and it slowly rises up the TLC plate by capillary action. y TLC Procedure on this Orgchem site . metal. and the separated components of the mixture are visualized. When the solvent has reached the top of the plate. For example if 30 : 70 ethylacetate : hexane produces Rf1 = 0. is the mobile phase. the plate is removed from the developing chamber.TLC y Study Questions/Answers from the Handbook for Organic Chemistry Lab TLC is a simple. It is sometimes possible to separate overlapping spots while trying different mixtures of similar strength. visualization is straightforward. This liquid. dried. or plastic which is coated with a thin layer of a solid adsorbent (usually silica or alumina). 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).3 to 0. or the eluent. Thin Layer Chromatography .4) but it well might happen that Rf1 will became different from Rf2.There is one more subtle thing about elutant mixtures. is illustrated with photographs on the TLC Procedure page.30 and Rf2=0. and inexpensive procedure that gives the chemist a quick answer as to how many components are in a mixture. 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. quick. so a UV lamp is used to visualize the plates. A TLC plate is a sheet of glass. Usually the compounds are not colored. If the compounds are colored. A small amount of the mixture to be analyzed is spotted near the bottom of this plate. 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.

dipole-dipole. and van der Waals forces. the dominant interactive forces between the adsorbent and the materials to be separated are of the dipole-dipole type.) The plates are aluminumbacked and you can cut them to size with scissors. hydrogen bonding. Roughly. such as charring or staining. the compounds follow the elution order given on the Chromatography Overview page. With silica gel. The adsorbent is impregnated with a fluor. Weakly polar molecules thus generally tend to move through the adsorbent more rapidly than the polar species. (Alumina (Al2O3) can also be used as a TLC adsorbent. other visualization methods are used. Our plates are purchased readymade from EM Sciences or from Scientific Adsorbents. In some circumstances. we use silica gel plates (SiO2) almost exclusively. dipole induced dipole. zinc sulfide.TLC Adsorbent In the teaching labs at CU Boulder. The fluor enables most organic compounds to be visualized when the plate is held under a UV lamp. . 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. TLC Solvents or Solvent Systems Choosing a solvent is covered on the Chromatography Overview page. Highly polar molecules interact fairly strongly with the polar Si²O 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.

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