logging in or signing up 14-Thin Layer Chromatography Deokate Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 2290 Category: Education License: All Rights Reserved Like it (2) Dislike it (0) Added: September 25, 2010 This Presentation is Public Favorites: 2 Presentation Description No description available. Comments Posting comment... By: vinayjain7013 (9 month(s) ago) HI FRIENDS CAN U PLEASE SEND ME PPT FOR TLC TO THIS ID krishnasai24365@gmail.com Saving..... Post Reply Close Saving..... Edit Comment Close By: Shyam03 (10 month(s) ago) HI...could you please send me the ppt about TLC to 'meghashyam.3@gmail.com' Thank you. Saving..... Post Reply Close Saving..... Edit Comment Close By: prakashchem (10 month(s) ago) hello sir/madam i like this contents please allow meto download this presentation Saving..... Post Reply Close Saving..... Edit Comment Close By: mannemkiranbabu (13 month(s) ago) hello sir...your presentation is excellent.. please allow me download...im b.pharm graduate Saving..... Post Reply Close Saving..... Edit Comment Close By: nishishailesh (17 month(s) ago) can u allow me to download. I wish to use it to teach my PG medical students Saving..... Post Reply Close Saving..... Edit Comment Close loading.... See all Premium member Presentation Transcript Thin Layer Chromatography : 1 Thin Layer Chromatography U.A. Deokate Copyright © by U. A. Deokate, all rights reserved. Thin Layer Chromatography : 14-Dec-06 2 U.A. Deokate Thin Layer Chromatography Thin layer chromatography (TLC) is a widely-used chromatography technique used to separate chemical compounds. It involves a stationary phase consisting of a thin layer of adsorbent material, usually silica gel, alumina, or cellulose immobilised onto a flat, inert carrier sheet. A liquid phase consisting of the solution to be separated dissolved in an appropriate solvent is drawn through the plate via capillary action, separating the experimental solution. It can be used to determine the pigments a plant contains, to detect pesticides or insecticides in food, in forensics to analyze the dye composition of fibers, or to identify compounds present in a given substance, among other uses. Introduction : 14-Dec-06 3 U.A. Deokate 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. The S.P. may be solid or liquid hold as a layer on solid support. 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. Advanced TLC : 14-Dec-06 4 U.A. Deokate Advanced TLC More sensitive, very less sample required Spraying with corrosive agent for identification is possible. TLC can be automated using forced solvent flow, running the plate in an vacuum-capable chamber to dry the plate, and recording the finished chromatogram by absorption or fluorescence spectroscopy with a light source. The ability to program the solvent delivery makes it convenient to do multiple developments in which the solvent flows for a short period of time, the TLC plate is dried, and the process is repeated. This method refocuses the spots to acheive higher resolution than in a single run TLC Theory : 14-Dec-06 5 U.A. Deokate TLC Theory Stationary phase (TLC plate) TLC paper is coated with silica gel Hydrogen bonding is main intermolecular force involved in a TLC separation Polar molecules “stick” to plate Non-polar molecules do not “stick” to plate Non-polar molecules will spend a greater amount of time dissolved in the eluent Separation of compounds occurs due to differences in partitioning between liquid and stationary phases Technique (Operation involved) : 14-Dec-06 6 U.A. Deokate Technique (Operation involved) Choice of adsorbent Preparation of plate Preparation & application of sample Choice of solvent Development of chromatogram Drying of chromatogram Location of spot Quantitative estimation. Choice of adsorbent : 14-Dec-06 7 U.A. Deokate Choice of adsorbent Two general properties decides the selection are Particle size and homogeniscity. (1-25μm preferred). Factor affecting selection: There should not be any reaction with sub to be separated. It should be insoluble with mobile phase and solvent used for elution. It should not catalyses or decompose off substance. It should be colour less. Should have grater mechanical strength. Classification of adsorbents used : 14-Dec-06 8 U.A. Deokate Classification of adsorbents used Classification according to bonding strength Weak adsorbent; eg. Sucrose, starch, talc ,cellulose. Intermediate adsorbent eg. Silica gel, calcium carbonate, calcium phosphate, magnesia Strong adsorbent: alumina charcoals Classification according to nature Inorganic adsorbent: Silica gel, aluminum oxide, Kiselghur, Magnesia, Magnesium silicate. Organic adsorbents: cellulose, charcoal. Silica gel : 14-Dec-06 9 U.A. Deokate Silica gel Silica gel is a granular, porous form of silica made synthetically from sodium silicate. Despite the name, silica gel is a solid. In chemistry, silica gel is used in chromatography as a stationary phase. In this application, due to silica gel's polarity, non-polar components tend to elute before more polar ones, hence the name normal phase chromatography. However, when hydrophobic groups (such as C18 groups) are attached to the silica gel then polar components elute first and the method is referred to as reverse phase chromatography. Silica gel is also applied to aluminum or plastic sheets for thin layer chromatography The synthetic nature of silica gel enables the careful control of pore size. Thin Layer Chromatography : 14-Dec-06 10 U.A. Deokate Thin Layer Chromatography TLC plate silica gel - silicon dioxide (SiO2)x (a common, inexpensive stationary phase) bulk (SiO2)x 5 x 10 cm. TLC slide 250 mm silica gel layer impregnated with a fluorescent indicator, on a plastic backing Cellulose : 14-Dec-06 11 U.A. Deokate Cellulose Cellulose (C6H10O5)n is a long-chain polymeric polysaccharide carbohydrate, of beta-glucose Adsorbed water or alcohols can be retained by the interaction with hydroxyl groups. Two types of cellulose are used in planer chromatography. 1. with a polymerization between 400-500 glucopyranose unit and 2. 40-200 glucopyranose unit. Aluminium oxide : 14-Dec-06 12 U.A. Deokate Aluminium oxide Aluminium oxide is a chemical compound of aluminium and oxygen with the chemical formula Al2O3. It is also commonly referred to as alumina. It is manufactured in three pH ranges acidic, basic and neutral for different types of samples. Acidic comp like phenols, sulphoinc carboxylic and amno acids are separated on the acidic alumina. Basic comp as amines, dyes are separated on basic alumina Neutral comp. such as aldehyeds ketones and lactones are separated on neutral alumina. Stationary Phase : 14-Dec-06 13 U.A. Deokate 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). Plate preparation : 14-Dec-06 14 U.A. Deokate Plate preparation TLC plates are made 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, and the resultant plate is dried and activated by heating in an oven for thirty minutes at 110 OC. The thickness of the adsorbent layer is typically around 0.1–0.25 mm for analytical purposes and around 1–2 mm for preparative TLC. Slide 15: 14-Dec-06 15 U.A. Deokate CAMAG –Vario chamber parallel test of 6 neat solvents or solvent mixtures Applicators Sample Application (spotting) : 14-Dec-06 16 U.A. Deokate TLC plate B. Dissolve solid sample in MeOH C. Use TLC capillary to transfer and spot dissolved sample ACE ASP CAF ACE ASP CAF #5 #5 #5 Ref. Ref. Ref. Sample Application (spotting) Choice of solvent : 14-Dec-06 17 U.A. Deokate Choice of solvent Selection of M.P. depends on nature of substance to be separated. Viscosity & Polarity of S.P. The solvent used may be a single or double phase system. N-hexane, cyclohexane, carbon tetra chloride, benzene, toluene, trichloro ethylene, chloroform, diethyl ether, ethyl acetate, n-butanol, acetone, ethanol, methanol and water Polarity Developing a Plate : 14-Dec-06 18 U.A. Deokate Developing a Plate A TLC plate can be developed in a beaker or closed jar Place a small amount of solvent (m.p.) 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 method development : 14-Dec-06 19 U.A. Deokate TLC method development Solution for plate conditioning with vapor phase: twin through chambers ( + filter paper ) © Camag 1997 Development of TLC Plate : 14-Dec-06 20 U.A. Deokate Development of TLC Plate TLC Developing Chamber (just a glass jar with solvent in it!) A. Place spotted TLC plate in developing chamber B. Developing solution is drawn up the plate by capillary action C. Remove TLC plate when solvent reaches top line Developing solution (mobile phase) } {keep capped} NOTE: During this ~20 min. developing stage, compounds in the original spots are being pulled through the silica gel. Visualization Method : 14-Dec-06 21 U.A. Deokate Visualization Method The previous slide shows colored spots. Most of the time, the spots won’t show unless they are visualized! Vizualization is a method that is used to render the TLC spots visible. A visualization method can be: Ultraviolet light Iodine vapors to stain spots Colored reagents to stain spots Reagents that selectively stain spots while leaving others unaffected. Visualization : 14-Dec-06 22 U.A. Deokate Visualization There are various techniques to visualize the compounds. Sulfuric acid/heat: destructive, leaves charred blots behind Ceric stain: destructive, leaves a dark blue blot behind for polar compounds Iodine: semi-destructive, iodine absorbs onto the spots, not permanent UV light: non-destructive, long wavelength (background green, spots dark), short wavelength (plate dark, compounds glow) Visualization of TLC Results : 14-Dec-06 23 U.A. Deokate Visualization of TLC Results A. Allow solvent to evaporate from surface of TLC plate. C. Mark spots with a pencil while viewing under UV. UV TLC Experiment : 14-Dec-06 24 U.A. Deokate TLC Experiment First: Mark the origin Origin TLC Experiment : 14-Dec-06 25 U.A. Deokate TLC Experiment First: Mark the origin Second: Apply sample Origin TLC Experiment : 14-Dec-06 26 U.A. Deokate TLC Experiment First: Mark the origin Second: Apply sample Third: Place into chamber Eleunt Origin TLC Experiment : 14-Dec-06 27 U.A. Deokate TLC Experiment Forth: Eluent ascends plate Direction Solvent Travels TLC Experiment : 14-Dec-06 28 U.A. Deokate TLC Experiment Forth: Eluent ascends plate Direction Solvent Travels Fifth: Remove plate & label solvent front Front Determine the Rf for Spots : 14-Dec-06 29 U.A. Deokate Determine the Rf for Spots Origin Solvent Front Spots 2 cm Determine the Rf for Spots : 14-Dec-06 30 U.A. Deokate Determine the Rf for Spots Origin Solvent Front Spots Rf = distance spot traveled distance solvent front traveled 2 cm Determine the Rf for Spots : 14-Dec-06 31 U.A. Deokate Determine the Rf for Spots Origin Solvent Front Spots Rf = distance spot traveled distance solvent front traveled 2 cm 10 cm Determine the Rf for Spots : 14-Dec-06 32 U.A. Deokate Determine the Rf for Spots Origin Solvent Front Spots Rf = distance spot traveled distance solvent front traveled 2 cm 10 cm Rf = 2 cm 10 cm 0.2 Determine the Rf for Spots : 14-Dec-06 33 U.A. Deokate Determine the Rf for Spots Origin Solvent Front Spots Rf = distance spot traveled distance solvent front traveled 2 cm 10 cm Rf = 2 cm 10 cm 0.2 Rule of thumb: polar compounds have low Rf values Use of TLC for comparisons : 14-Dec-06 34 U.A. Deokate Use of TLC for comparisons Determine purity and corroborate identity of sample Pure Sample Recrystallized Sample For Successful TLC Experiment : 14-Dec-06 35 U.A. Deokate For Successful TLC Experiment Do not apply too much test compound to plate Streaking Disproportionately Large Spots Interpretation of TLC Results : 14-Dec-06 36 U.A. Deokate Interpretation of TLC Results A. Determine retention factors (Rf) for each spot detected. B. Use Rf’s of reference spots to identify the other components. How do you interpret any other spots? 4 1 3 2 ? ? How do you identify the unknown tablets? Finally: Uses of TLC : 14-Dec-06 37 U.A. Deokate Uses of TLC To determine how many components there are in a mixture (is it really pure?) To determine the best solvent conditions for separation on a column To identify the substances being studied To monitor the composition of fractions collected from column chromatography To monitor the progress of a reaction uses of Thin-Layer Chromatography : 14-Dec-06 38 U.A. Deokate uses of Thin-Layer Chromatography 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. Troubleshooting TLC : 14-Dec-06 39 U.A. Deokate Troubleshooting TLC All of the above (including the procedure page) might sound like TLC is quite an easy procedure. But what about the first time you run a TLC, and see spots everywhere and blurred, streaked spots? As with any technique, with practice you get better The compound runs as a streak rather than a spot The sample was overloaded. Run the TLC again after diluting your sample. Or, your sample might just contain many components, creating many spots which run together and appear as a streak. The sample runs as a smear or a upward crescent (Moon) Compounds which possess strongly acidic or basic groups (amines or carboxylic acids) sometimes show up on a TLC plate with this behavior. Add a few drops of ammonium hydroxide (amines) or acetic acid (carboxylic acids) to the eluting solvent to obtain clearer plates. Troubleshooting TLC : 14-Dec-06 40 U.A. Deokate Troubleshooting TLC The sample runs as a downward crescent. Likely, the adsorbent was disturbed during the spotting, causing the crescent shape. The plate solvent front runs crookedly. (curved) Either the adsorbent has flaked off the sides of the plate or the sides of the plate are touching the sides of the container (or the paper used to saturate the container) as the plate develops. Crookedly run plates make it harder to measure Rf values accurately. Many, random spots are seen on the plate. Make sure that you do not accidentally drop any organic compound on the plate. If get a TLC plate and leave it laying on your workbench as you do the experiment, you might drop or splash an organic compound on the plate. Troubleshooting TLC : 14-Dec-06 41 U.A. Deokate Troubleshooting TLC No spots are seen on the plate. You might not have spotted enough compound, perhaps because the solution of the compound is too dilute. Try concentrating the solution, or, spot it several times in one place, allowing the solvent to dry between applications. Some compounds do not show up under UV light; try another method of visualizing the plate. Or, perhaps you do not have any compound because your experiment did not go as well as planned. If the solvent level in the developing jar is deeper than the origin (spotting line) of the TLC plate, the solvent will dissolve the compounds into the solvent reservoir instead of allowing them to move up the plate by capillary action. Thus, you will not see spots after the plate is developed. You see a blur of blue spots on the plate as it develops. Perhaps, you used an ink pen instead of a pencil to mark the origin? You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.
14-Thin Layer Chromatography Deokate Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 2290 Category: Education License: All Rights Reserved Like it (2) Dislike it (0) Added: September 25, 2010 This Presentation is Public Favorites: 2 Presentation Description No description available. Comments Posting comment... By: vinayjain7013 (9 month(s) ago) HI FRIENDS CAN U PLEASE SEND ME PPT FOR TLC TO THIS ID krishnasai24365@gmail.com Saving..... Post Reply Close Saving..... Edit Comment Close By: Shyam03 (10 month(s) ago) HI...could you please send me the ppt about TLC to 'meghashyam.3@gmail.com' Thank you. Saving..... Post Reply Close Saving..... Edit Comment Close By: prakashchem (10 month(s) ago) hello sir/madam i like this contents please allow meto download this presentation Saving..... Post Reply Close Saving..... Edit Comment Close By: mannemkiranbabu (13 month(s) ago) hello sir...your presentation is excellent.. please allow me download...im b.pharm graduate Saving..... Post Reply Close Saving..... Edit Comment Close By: nishishailesh (17 month(s) ago) can u allow me to download. I wish to use it to teach my PG medical students Saving..... Post Reply Close Saving..... Edit Comment Close loading.... See all Premium member Presentation Transcript Thin Layer Chromatography : 1 Thin Layer Chromatography U.A. Deokate Copyright © by U. A. Deokate, all rights reserved. Thin Layer Chromatography : 14-Dec-06 2 U.A. Deokate Thin Layer Chromatography Thin layer chromatography (TLC) is a widely-used chromatography technique used to separate chemical compounds. It involves a stationary phase consisting of a thin layer of adsorbent material, usually silica gel, alumina, or cellulose immobilised onto a flat, inert carrier sheet. A liquid phase consisting of the solution to be separated dissolved in an appropriate solvent is drawn through the plate via capillary action, separating the experimental solution. It can be used to determine the pigments a plant contains, to detect pesticides or insecticides in food, in forensics to analyze the dye composition of fibers, or to identify compounds present in a given substance, among other uses. Introduction : 14-Dec-06 3 U.A. Deokate 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. The S.P. may be solid or liquid hold as a layer on solid support. 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. Advanced TLC : 14-Dec-06 4 U.A. Deokate Advanced TLC More sensitive, very less sample required Spraying with corrosive agent for identification is possible. TLC can be automated using forced solvent flow, running the plate in an vacuum-capable chamber to dry the plate, and recording the finished chromatogram by absorption or fluorescence spectroscopy with a light source. The ability to program the solvent delivery makes it convenient to do multiple developments in which the solvent flows for a short period of time, the TLC plate is dried, and the process is repeated. This method refocuses the spots to acheive higher resolution than in a single run TLC Theory : 14-Dec-06 5 U.A. Deokate TLC Theory Stationary phase (TLC plate) TLC paper is coated with silica gel Hydrogen bonding is main intermolecular force involved in a TLC separation Polar molecules “stick” to plate Non-polar molecules do not “stick” to plate Non-polar molecules will spend a greater amount of time dissolved in the eluent Separation of compounds occurs due to differences in partitioning between liquid and stationary phases Technique (Operation involved) : 14-Dec-06 6 U.A. Deokate Technique (Operation involved) Choice of adsorbent Preparation of plate Preparation & application of sample Choice of solvent Development of chromatogram Drying of chromatogram Location of spot Quantitative estimation. Choice of adsorbent : 14-Dec-06 7 U.A. Deokate Choice of adsorbent Two general properties decides the selection are Particle size and homogeniscity. (1-25μm preferred). Factor affecting selection: There should not be any reaction with sub to be separated. It should be insoluble with mobile phase and solvent used for elution. It should not catalyses or decompose off substance. It should be colour less. Should have grater mechanical strength. Classification of adsorbents used : 14-Dec-06 8 U.A. Deokate Classification of adsorbents used Classification according to bonding strength Weak adsorbent; eg. Sucrose, starch, talc ,cellulose. Intermediate adsorbent eg. Silica gel, calcium carbonate, calcium phosphate, magnesia Strong adsorbent: alumina charcoals Classification according to nature Inorganic adsorbent: Silica gel, aluminum oxide, Kiselghur, Magnesia, Magnesium silicate. Organic adsorbents: cellulose, charcoal. Silica gel : 14-Dec-06 9 U.A. Deokate Silica gel Silica gel is a granular, porous form of silica made synthetically from sodium silicate. Despite the name, silica gel is a solid. In chemistry, silica gel is used in chromatography as a stationary phase. In this application, due to silica gel's polarity, non-polar components tend to elute before more polar ones, hence the name normal phase chromatography. However, when hydrophobic groups (such as C18 groups) are attached to the silica gel then polar components elute first and the method is referred to as reverse phase chromatography. Silica gel is also applied to aluminum or plastic sheets for thin layer chromatography The synthetic nature of silica gel enables the careful control of pore size. Thin Layer Chromatography : 14-Dec-06 10 U.A. Deokate Thin Layer Chromatography TLC plate silica gel - silicon dioxide (SiO2)x (a common, inexpensive stationary phase) bulk (SiO2)x 5 x 10 cm. TLC slide 250 mm silica gel layer impregnated with a fluorescent indicator, on a plastic backing Cellulose : 14-Dec-06 11 U.A. Deokate Cellulose Cellulose (C6H10O5)n is a long-chain polymeric polysaccharide carbohydrate, of beta-glucose Adsorbed water or alcohols can be retained by the interaction with hydroxyl groups. Two types of cellulose are used in planer chromatography. 1. with a polymerization between 400-500 glucopyranose unit and 2. 40-200 glucopyranose unit. Aluminium oxide : 14-Dec-06 12 U.A. Deokate Aluminium oxide Aluminium oxide is a chemical compound of aluminium and oxygen with the chemical formula Al2O3. It is also commonly referred to as alumina. It is manufactured in three pH ranges acidic, basic and neutral for different types of samples. Acidic comp like phenols, sulphoinc carboxylic and amno acids are separated on the acidic alumina. Basic comp as amines, dyes are separated on basic alumina Neutral comp. such as aldehyeds ketones and lactones are separated on neutral alumina. Stationary Phase : 14-Dec-06 13 U.A. Deokate 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). Plate preparation : 14-Dec-06 14 U.A. Deokate Plate preparation TLC plates are made 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, and the resultant plate is dried and activated by heating in an oven for thirty minutes at 110 OC. The thickness of the adsorbent layer is typically around 0.1–0.25 mm for analytical purposes and around 1–2 mm for preparative TLC. Slide 15: 14-Dec-06 15 U.A. Deokate CAMAG –Vario chamber parallel test of 6 neat solvents or solvent mixtures Applicators Sample Application (spotting) : 14-Dec-06 16 U.A. Deokate TLC plate B. Dissolve solid sample in MeOH C. Use TLC capillary to transfer and spot dissolved sample ACE ASP CAF ACE ASP CAF #5 #5 #5 Ref. Ref. Ref. Sample Application (spotting) Choice of solvent : 14-Dec-06 17 U.A. Deokate Choice of solvent Selection of M.P. depends on nature of substance to be separated. Viscosity & Polarity of S.P. The solvent used may be a single or double phase system. N-hexane, cyclohexane, carbon tetra chloride, benzene, toluene, trichloro ethylene, chloroform, diethyl ether, ethyl acetate, n-butanol, acetone, ethanol, methanol and water Polarity Developing a Plate : 14-Dec-06 18 U.A. Deokate Developing a Plate A TLC plate can be developed in a beaker or closed jar Place a small amount of solvent (m.p.) 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 method development : 14-Dec-06 19 U.A. Deokate TLC method development Solution for plate conditioning with vapor phase: twin through chambers ( + filter paper ) © Camag 1997 Development of TLC Plate : 14-Dec-06 20 U.A. Deokate Development of TLC Plate TLC Developing Chamber (just a glass jar with solvent in it!) A. Place spotted TLC plate in developing chamber B. Developing solution is drawn up the plate by capillary action C. Remove TLC plate when solvent reaches top line Developing solution (mobile phase) } {keep capped} NOTE: During this ~20 min. developing stage, compounds in the original spots are being pulled through the silica gel. Visualization Method : 14-Dec-06 21 U.A. Deokate Visualization Method The previous slide shows colored spots. Most of the time, the spots won’t show unless they are visualized! Vizualization is a method that is used to render the TLC spots visible. A visualization method can be: Ultraviolet light Iodine vapors to stain spots Colored reagents to stain spots Reagents that selectively stain spots while leaving others unaffected. Visualization : 14-Dec-06 22 U.A. Deokate Visualization There are various techniques to visualize the compounds. Sulfuric acid/heat: destructive, leaves charred blots behind Ceric stain: destructive, leaves a dark blue blot behind for polar compounds Iodine: semi-destructive, iodine absorbs onto the spots, not permanent UV light: non-destructive, long wavelength (background green, spots dark), short wavelength (plate dark, compounds glow) Visualization of TLC Results : 14-Dec-06 23 U.A. Deokate Visualization of TLC Results A. Allow solvent to evaporate from surface of TLC plate. C. Mark spots with a pencil while viewing under UV. UV TLC Experiment : 14-Dec-06 24 U.A. Deokate TLC Experiment First: Mark the origin Origin TLC Experiment : 14-Dec-06 25 U.A. Deokate TLC Experiment First: Mark the origin Second: Apply sample Origin TLC Experiment : 14-Dec-06 26 U.A. Deokate TLC Experiment First: Mark the origin Second: Apply sample Third: Place into chamber Eleunt Origin TLC Experiment : 14-Dec-06 27 U.A. Deokate TLC Experiment Forth: Eluent ascends plate Direction Solvent Travels TLC Experiment : 14-Dec-06 28 U.A. Deokate TLC Experiment Forth: Eluent ascends plate Direction Solvent Travels Fifth: Remove plate & label solvent front Front Determine the Rf for Spots : 14-Dec-06 29 U.A. Deokate Determine the Rf for Spots Origin Solvent Front Spots 2 cm Determine the Rf for Spots : 14-Dec-06 30 U.A. Deokate Determine the Rf for Spots Origin Solvent Front Spots Rf = distance spot traveled distance solvent front traveled 2 cm Determine the Rf for Spots : 14-Dec-06 31 U.A. Deokate Determine the Rf for Spots Origin Solvent Front Spots Rf = distance spot traveled distance solvent front traveled 2 cm 10 cm Determine the Rf for Spots : 14-Dec-06 32 U.A. Deokate Determine the Rf for Spots Origin Solvent Front Spots Rf = distance spot traveled distance solvent front traveled 2 cm 10 cm Rf = 2 cm 10 cm 0.2 Determine the Rf for Spots : 14-Dec-06 33 U.A. Deokate Determine the Rf for Spots Origin Solvent Front Spots Rf = distance spot traveled distance solvent front traveled 2 cm 10 cm Rf = 2 cm 10 cm 0.2 Rule of thumb: polar compounds have low Rf values Use of TLC for comparisons : 14-Dec-06 34 U.A. Deokate Use of TLC for comparisons Determine purity and corroborate identity of sample Pure Sample Recrystallized Sample For Successful TLC Experiment : 14-Dec-06 35 U.A. Deokate For Successful TLC Experiment Do not apply too much test compound to plate Streaking Disproportionately Large Spots Interpretation of TLC Results : 14-Dec-06 36 U.A. Deokate Interpretation of TLC Results A. Determine retention factors (Rf) for each spot detected. B. Use Rf’s of reference spots to identify the other components. How do you interpret any other spots? 4 1 3 2 ? ? How do you identify the unknown tablets? Finally: Uses of TLC : 14-Dec-06 37 U.A. Deokate Uses of TLC To determine how many components there are in a mixture (is it really pure?) To determine the best solvent conditions for separation on a column To identify the substances being studied To monitor the composition of fractions collected from column chromatography To monitor the progress of a reaction uses of Thin-Layer Chromatography : 14-Dec-06 38 U.A. Deokate uses of Thin-Layer Chromatography 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. Troubleshooting TLC : 14-Dec-06 39 U.A. Deokate Troubleshooting TLC All of the above (including the procedure page) might sound like TLC is quite an easy procedure. But what about the first time you run a TLC, and see spots everywhere and blurred, streaked spots? As with any technique, with practice you get better The compound runs as a streak rather than a spot The sample was overloaded. Run the TLC again after diluting your sample. Or, your sample might just contain many components, creating many spots which run together and appear as a streak. The sample runs as a smear or a upward crescent (Moon) Compounds which possess strongly acidic or basic groups (amines or carboxylic acids) sometimes show up on a TLC plate with this behavior. Add a few drops of ammonium hydroxide (amines) or acetic acid (carboxylic acids) to the eluting solvent to obtain clearer plates. Troubleshooting TLC : 14-Dec-06 40 U.A. Deokate Troubleshooting TLC The sample runs as a downward crescent. Likely, the adsorbent was disturbed during the spotting, causing the crescent shape. The plate solvent front runs crookedly. (curved) Either the adsorbent has flaked off the sides of the plate or the sides of the plate are touching the sides of the container (or the paper used to saturate the container) as the plate develops. Crookedly run plates make it harder to measure Rf values accurately. Many, random spots are seen on the plate. Make sure that you do not accidentally drop any organic compound on the plate. If get a TLC plate and leave it laying on your workbench as you do the experiment, you might drop or splash an organic compound on the plate. Troubleshooting TLC : 14-Dec-06 41 U.A. Deokate Troubleshooting TLC No spots are seen on the plate. You might not have spotted enough compound, perhaps because the solution of the compound is too dilute. Try concentrating the solution, or, spot it several times in one place, allowing the solvent to dry between applications. Some compounds do not show up under UV light; try another method of visualizing the plate. Or, perhaps you do not have any compound because your experiment did not go as well as planned. If the solvent level in the developing jar is deeper than the origin (spotting line) of the TLC plate, the solvent will dissolve the compounds into the solvent reservoir instead of allowing them to move up the plate by capillary action. Thus, you will not see spots after the plate is developed. You see a blur of blue spots on the plate as it develops. Perhaps, you used an ink pen instead of a pencil to mark the origin?