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.

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

http://www.wellesley.edu/Chemistry/chem211lab/Orgo_Lab_Manual/Appendix/Techniques/TLC/thin_l ayer_chrom.html

Thin layer chromatography

From Wikipedia, the free encyclopedia

Thin layer chromatography

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.[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 themobile 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 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, or assaying the radiochemical purity of radiopharmaceuticals identification of medicinal plants and their constituents [3]

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 quantization. This method is referred to as HPTLC, or "high performance TLC".
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, 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.[4]
[edit]Technique

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

Chromatogram of 10 essential oilscoloured with vanillin reagent.

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: [5]


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 nonreproducible 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 bycapillary 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.[6] 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/nButanol, 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 hexane 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. Aneluotropic 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.
[edit]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.

[edit]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[7]
 

 

Potassium permanganate - oxidation Iodine

In the case of lipids, the chromatogram may be transferred to a PVDF membrane and then subjected to further analysis, for examplemass 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
[edit]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 reactionthe 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[8] 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 placed separately on the base line, then reacted and then instantly analyzed.
[edit]

http://en.wikipedia.org/wiki/Thin_layer_chromatography

last updated Friday, August 14, 2009 Introduction

Thin-layer chromatography (TLC) is a very commonly used technique in synthetic chemistry for identifying compounds, determining their purity and following the progress of a reaction. It also permits the optimization of the solvent system for a given separation problem. In comparison with column chromatography, it only requires small quantities of the compound (~ng) and is much faster as well. Stationary Phase As stationary phase, a special finely ground matrix (silica gel, alumina, or similar material) is coated on a glass plate, a metal or a plastic film as a thin layer (~0.25 mm). In addition a binder like gypsum is mixed into the stationary phase to make it stick better to the slide. In many cases, a fluorescent powder is mixed into the stationary phase to simplify the visualization later on (e.g. bright green when you expose it to 254 nm UV light).

Preparing the Plate Do not touch the TLC plate on the side with the white surface. In order to obtain an imaginary start line, make two notches on each side of the TLC plate. You can also draw a thin line with pencil. Do not use pen. Why? The start line should be 0.5-1 cm from the bottom of the plate.

Capillary spotters Place a melting point capillary and in the dark blue part of the Bunsen burner flame. Hold it there until it softens and starts to sag. Quickly remove the capillary from the flame and pull on both ends to about 2-3 times its original length. If you pull the capillary inside the flame, you will have a "piece of art", but not a good spotter. Allow the capillary to cool down, and then break it in the middle. Make sure that you break off the closed end on one of them. Do not use gloves when you pull capillaries. You will have much better control without them!

Watch movie how to pull capillaries here here

Spotting the plate The thin end of the spotter is placed in the dilute solution; the solution will rise up in the capillary (capillary forces). Touch the plate briefly at the start line. Allow the solvent to evaporate and spot at the same place again. This way you will get a concentrated and small spot. Try to avoid spotting too much material, because this will deteriorate the quality of the separation considerably (tailing). The spots should be far enough away from the edges and from each other as well. If possible, you should spot the compound or mixture together with the starting materials and possible intermediates on the plate. They will serve as internal reference since every TLC plate is slightly different.

Developing a Plate A TLC plate can be developed in a beaker or closed jar (see picture below). Place a small amount of solvent (= mobile phase) in the container. The solvent level has to be below the starting line of the TLC, otherwise the spots will dissolve away. The lower edge of the plate is then dipped in a solvent. The solvent (eluent) travels up the matrix by capillarity, moving the components of the samples at various rates because of their different degrees of interaction with

the matrix (=stationary phase) and solubility in the developing solvent. Non-polar solvents will force non-polar compounds to the top of the plate, because the compounds dissolve well and do not interact with the polar stationary phase. Allow the solvent to travel up the plate until ~1 cm from the top. Take the plate out and mark the solvent front immediately. Do not allow the solvent to run over the edge of the plate. Next, let the solvent evaporate completely.

TLC chamber for development e.g. 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. 1. Sulfuric acid/heat: destructive, leaves charred blots behind 2. Ceric stain: destructive, leaves a dark blue blot behind for polar compounds 3. Iodine: semi-destructive, iodine absorbs onto the spots, not permanent 4. UV light: non-destructive, long wavelength (background green, spots dark), short wavelength (plate dark, compounds glow), 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. Also draw a sketch of the developed plate in your lab notebook. Analysis The components, visible as separated spots, are identified by comparing the distances they have traveled with those of the known reference materials. Measure the distance of the start line to the

solvent front (=d). Then measure the distance of center of the spot to the start line (=a). Divide the distance the solvent moved by the distance the individual spot moved. The resulting ratio is called Rf-value. The value should be between 0.0 (spot did not moved from starting line) and 1.0 (spot moved with solvent front) and is unitless.

The Rf (=retardation factor) depends on the following parameters:

y y y y

solvent system absorbent (grain size, water content, thickness) amount of material spotted temperature

Due to the fact that all those variables are difficult to keep constant, a reference compound is usually applied to the plate as well. Useful links

http://www.chem.ucla.edu/~bacher/General/30BL/tips/TLC1.html

The technique of Thin Layer Chromatography (TLC) is normally used as an analytical method to follow the progress of a reaction, to analyse mixtures or to establish conditions for a preparative separation of compounds using column chromatography. The stationary phase (often silica) is coated on plastic or aluminium plates. The mixture is spotted on the plate and solvent is allowed to run up the plate and separate the compounds.

http://chem-ilp.net/labTechniques/TLC.htm

Acetaminophen Acetaminophen is an analgesic and an antipyretic, 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. Its antipyretic action results from its effect on the heat-regulating centers of the brain. Chemically, acetaminophen is p-hydroxy acetanilide, it has a molecular weight of 151.16 and elemental analysis shows it contains 67.38% carbon, 9.43% hydrogen, 5.24% nitrogen

and 17.95% oxygen. Acetaminophen is prescribed for arthritis but is not an antiinflammatory, it is mainly used to help reduce temperature and as a mild analgesic. Acetaminophen tablets have been successfully analyzed using reversed phase (C18) columns 20-25 cm long 3-4.6 mm in diameter using a strongly polar, water-methanolacetonitrile mixture with a tetramethylammonium hydroxide buffer as the mobile phase. 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. 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, Excedrin, Nuprin, and Tylenol) were determined by thin layer chromatography in this experiment using ethyl acetate as both the solvent and the elluent. Anacin, Excedrin, Nuprin, and Tylenol were pulverized, dissolved in ethyl acetate, and run plated on a silicon gel to determine their Rf factors. These Rf factors were then compared to the Rf factors of the pure standard samples: acetaminophen, aspirin, caffeine, and ibuprofen. The TLC plates indicated that Anacin contains both aspirin and caffeine; Excedrin contains acetaminophen, aspirin, and caffeine; Nuprin contains only ibuprofen; and Tylenol contains only acetaminophen. 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. 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. A line is drawn 1 cm from the bottom of the gel and a small volume of sample (dissolved in a solvent, in this case ethyl acetate) is applied to the silicon with a micropipette and allowed to dry. The plate is then placed in a chamber containing a small volume of the appropriate solvent designated the eluent (in this experiment, ethyl acetate). Only the edge of the plate nearest the samples is in contact with the eluent. The eluent is drawn into the silicon by capillary action and travels up the plate through the samples. The migration rate of the sample components over the silicon depends on their chemical structure. TLC is generally very sensitive to small differences in the chemical structure of the sample. The structure affects the strength and type of interactions between the sample and the gel. In addition, different samples have different solubilities in a given solvent which is also dependent upon chemical structure. Thus, the general rule of thumb is the more polar the compound, the less the distance from the origin it migrates. 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). The Rf value is the ratio of distance traveled by the spot divided by the distance traveled by the solvent. Since the solvent front will always be ahead of the sample spots, the Rf value will always be less than 1. Each sample has its own distinct Rf value for a particular solvent. For a given sample, the Rf value (and the distance moved by the spot) will increase as the polarity of the solvent is increased (see chart below). This is because the solvent begins to compete more and more with the silica gel for the polar parts of the molecule, and so the sample is eluted up the plate more effectively. It is important to note that no solvent is more polar than the silica gel

surface; 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. Thus, thin layer chromatography is principally a competition between the solvent and the silica gel for the polar parts of the molecule. 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. Relative polarity of organic samples: Alkanes Alkenes INCREASING Aromatic hydrocarbons POLARITY Ethers, alkyl halides Aldehydes, ketones, esters Amines Alcohols Carboxylic acids Common TLC developing solvents: Hexane, cyclohexane, 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. Wt. g/mol Density g/cm3 Melting pt.oC

C9H8O4 180.16 1.35 135

C8H9NO2 151.16 1.293 169

C8H10N4O2 194.19 1.23 238

C13H18O2 206.284 0.5 0.75 75-77

C4H8O2 88.106 0.8945 -83.6

Boiling pt.oC

140

77.1

Experiment: This experiment involved the use of the technique: thin-layer chromatography. Ethyl acetate was used as the elluent to run the TLC plates in. This elluent was placed in a small jar lined with filter paper to keep the vapors high up in the vessel. 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. Each TLC plate was spotted with two known analgesics in an ethyl acetate solution and one unknown analgesic in an ethyl acetate solution. The four known solutions used were of caffeine, aspirin, acetaminophen and ibuprofen. The unknown solutions used were of Anacin, Nuprin, Tylenol, and Excedrin. It was given that the unkown analgesics contained one or more of the known analgesics. The elluent used for the TLCs run was ethyl acetate. After running the TLC plates for the full 5 cm, they were allowed to dry and were placed under ultraviolet light. While being viewed in ultraviolet light, the movement of the spots placed on the TLC plates could be determined, and thusly the Rf values for each spot could be determined. Once the Rf values had been determined, the unkowns were compared with the knowns to determine their composition. Results: Compound Dist of Dist Dist Dist Rf Rf Rf Solvent (a) (b) (c) (a) (b) (c) Acetaminophen 5 cm 1.55 0.31 cm Aspirin 5 cm 2.35 0.47 cm Caffeine 5 cm 0.55 0.11 cm Ibuprofen 5 cm 2.86 0.57 cm Anacin 5 cm 0.53 2.35 0.106 0.47 cm cm Excedrin 5 cm 0.53 1.54 2.37 0.016 0.308 0.474 cm cm cm Nuprin 5 cm 2.85 0.57 cm Tylenol 5 cm 1.52 0.304 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

Determination of analgesics by thin layer chromatography (TLC) 1. Introduction:

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

Rf = Distance moved by compound Distance moved by solvent system TLC can be used for small-scale preparative separations, employing thicker layers of adsorbent and applying the sample as a band instead of individual spots. The chromatogram can be developed, and the edges of the plate treated with visualizing agents to locate the bands of interest. The position of the bands on the developed TLC plate can alternately be located under ultra-violet light. Once the bands are located, they are scraped from the plates and the material leached from the adsorbent using an appropriate solvent. The dissolved components of the bands can then be analyzed by other techniques, including UV spectroscopy. TLC may also be used as a means of identification of an analyte. For example, TLC analysis of an extract of a forensic sample, alongside standards of analytes suspected to be present in the sample, allows the analyte in the sample to be tentatively identified. Confirmatory tests can then be used to positively identify the analyte (4,5). In this experiment, TLC plates will be prepared and used, , with UV spectroscopy, for the semiquantitative determination of selected analgesics in pharmaceutical preparations.

2. Experimental procedure: (A) Preparation of TLC plates:

(a) Obtain two 20cm x 20cm glass plates and two 5cm x 20cm plates, for coating with silica gel. Wash plates throughly with detergent and water, rinse with distilled water and allow to drain. Wipe plates free of grease and dirt with acetone-soaked tissue. It is important that the surface of the glass be kept free of grease and dirt, to ensure complete spreading of the adsorbent. (b) Mount plates on the plate spreader, with the 20 x 5cm plates on either end and clamp the plates to provide an even spreading surface. (c) Mix the recommended weight of silica gel adsorbent with distilled water, to give a smooth slurry (25g in about 60-70mL water). (d) Set the gap of the TLC applicator to 0.25mm, using the feeler guage provided. Place applicator on an end-plate, with the gap away from the analytical 20 x 20cm plates. (e) Pour the slurry and distribute evenly in the reservoir. With a single constant motion, draw the slurry along the plates, stopping only when the applicator is on the other end-plate. (f) Tap the sides of the plate holder to smoothen the surface of the slurry layer, then release the pressure on the plates. Separate the plates by means of a spatula, remove both end-plates and discard. (g) Wash and drain the applicator, for use by the next group.

(h) Allow the analytical plates to air-dry, until the watery sheen has disappeared, then remove from plate holder. (i) Place plates horizontally in a plate rack provided, and activate in an oven at 110 to 120C overnight and cool in a desiccator before use.

(B) Sample preparation:

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

and long wavelength UV (365nm) light. (b) Using a clean pointed glass rod, mark the outline of the standard and sample spots, and note the color of each visualized spot. (c) Identify the individual components in the mixture from their respective Rf values and colors, by comparison with those of the standards.

(D) UV Spectroscopy of sample extracts:

(a) Carefully scrape all the silica gel outlined for each sample component from the TLC plate, into the centre of a fluted 5cm diam. Whatman No. 1 filter paper. Take care not to contaminate a component with materials from other spots on the plate. (b) Elute each sample component with 3 successive 3mL volumes of analytical grade methanol into a 10mL volumetric flask. Make up to the mark with methanol. (c) Scan each analgesic standard solution provided and note their respective absorbances at the wavelength of maximal absorption. Scan each sample extract similarly and determine their absorbances. (d) Hence calculate the concentration of each analgesic component identified in the sample, using tabulated absorption extinction coefficients of each standard.

3. Exercise:
Explain how it might be possible to analyse a mixture such as Asprin/Phenacetin/Codeine by UV-VIS spectroscopy, without separating the individual components from the mixture.

What is TLC?
Thin Layer Chromatography (TLC) is a commonly used analytical technique that allows for rapid and inexpensive analysis of various mixtures. For organic chemists, TLC is most commonly thought of as being done on silica plates. In reality, many more sorbents in TLC format are available: aluminum, C18 reverse phase, cellulose, ion exchange resins, and many more. Besides speed and low price, there are three more features that make TLC very popular:

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It is easy to run up to 20 samples simultaneously It is possible to use a square plate, run in one direction with one elutant; then rotate plate 90deg and run with another elutant (creating a "twodimensional" TLC). Besides providing better resolution, this experiment determines whether any of the mixture components happen to be unstable under separation conditions. Plates (unlike HPLC columns) are disposable.

Setting up a TLC analysis is very simple. A suitable beaker or a jar with some kind of cover, a piece of filter paper, glass capillaries, and the TLC plate itself constitute all the materials needed. TLC plates differ on backing material. Aluminum, polyester, and glass are the most common materials. Some people insist on glass backing, but, in our experience, aluminum seems the most convenient.

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. The solution of material to be analyzed is spotted onto start line carefully (this step discussed in greater detail in the next section). 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 a rectangular piece of filter paper inserted so that it touches the elutant and lies on the jar wall, mostly above the elutant pool. The top edge of filter paper should be close to the jar mouth. A cover is placed on top of the jar, and the jar is tilted temporarily so that all the filter paper gets soaked in elutant. This step ensures that all inner volume of the jar is saturated with elutant vapors. Then, the spotted TLC plate is inserted carefully into the jar, replacing the lid afterwards. The elutant must not touch the spots. It is a good practice to allow for 1/2" (12mm) between elutant level and spots. The picture to the

left shows what happens next. Here, felt pen inks were subjected to the analysis, so results are immediately seen (are colorful and visible to the naked eye). More often, however, the spots are colorless, so some kind of visualization is required. After the solvent front reaches within ~1" (25mm) of the top edge of the TLC plate, the plate is removed from chamber, and the exact elutant front location is marked with a pencil. This mark is needed to calculate Rf (Retention factor) values.

Spotting a TLC
To spot a TLC, first dissolve the material to be separated in a solvent. Then, draw some solvent into a capillary, and press the capillary onto the TLC place medium. If done correctly, the solvent should all drain onto the medium, creating a wet circular spot. The solvent is then allowed to evaporate, leaving behind only material to be separated, before placing the TLC plate into the elutant to "develop". Spotting a TLC plate does require some practice. Three common mistakes, which always lead to overloading the TLC plate, are very popular. Due to these, instead of neat spots large streaks appear, which bear no information.
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Spotting too much of a solution. The capillary diameter should be around 1/32" (0.7 to 1mm). The initial spot diameter should be 1/8" to 3/16" (3 5mm) immediately after application. Spotting too concentrated or non-homogenous of a solution. The solution to be spotted should be free of precipitate and insoluble oils, and should not be too concentrated. 0.5 to 5wt% is good enough. The right dilution is found by trial and error. A not too volatile solvent is used. There is no point in spotting using solutions prepared in DMF, Dimethylacetamide, DMSO or pyridine, to name a few. The spotting solvent must be completely removed from the TLC before developing. For DMF and pyridine, it is possible to place the

spotted TLC plate for ~5min into vacuum before the run to evaporate the solvent. For DMSO and Dimethylacetamide, vacuum won't work - one has to do a mini workup to remove these solvents first.

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), then it is possible to spot same place a few times. Just allow the solvent to dry between spottings. This, however, leads to poorer TLC resolution. Spotting capillaries are available commercially. Do not use melting point determination capillaries - they are way too thick. It is also possible to pull a capillary out of a pipette with a good gas burner.

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. Usually a TLC is informative enough if the lower spot has Rf > 0.1 while the top spot exhibits Rf < 0.85. For a silica coated TLC plate, 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), Hexanes, Pentane, Carbon tetrachloride, Benzene/Toluene, Dichloromethane, Diethyl ether, Ethylacetate, Acetonitrile, Acetone, 2-Propanol/n-Butanol, Water, Methanol, Triethylamine, Acetic acid, Formic acid (strongest)

So basically one needs to use stronger elutant if spots are too low or weaker elutant if spots are too high. Often it more convenient to use the same mixture of elutants while changing their ratio to adjust the total strength of a mixture. For this approach to work one component of the mixture must be a weak elutant while another must be of excessive strength. For instance ethylacetate : hexane mixture is very popular for eluting TLC of medium polar materials. So if say 5:95 ethylacetate : hexane mixture would barely move spots, then try 10:90 or 25:75 mixtures in order to increase Rf values of the spots.

There is one more subtle thing about elutant mixtures. It is sometimes possible to separate overlapping spots while trying different mixtures of similar strength. For example if 30 : 70 ethylacetate : hexane produces Rf1 = 0.30 and Rf2=0.30 (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 (0.3 to 0.4) but it well might happen that Rf1 will became different from Rf2.

Thin Layer Chromatography - TLC

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Study Questions/Answers from the Handbook for Organic Chemistry Lab

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 page.
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TLC Procedure on this Orgchem site

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 aluminumbacked and you can cut them to size with scissors. Our plates are purchased readymade 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 SiO 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.