Detailed Overview of Thin Layer and Paper Chromatography Techniques
- 1. A Constituent College of Yenepoya (Deemed to be University) Naringana, Mangaluru Thin layer Chromatography & Paper chromatography Ms . Sajini Assistant professor Dept of Pharmaceutical chemistry
- 2. Table of content • Introduction • Principle • Methodology • Advantages • Disadvantages • Application
- 3. Specific learning outcomes At the end of the lecture students should know • What is TLC & Paper chromatography • Practical aspect of TLC & Paper chromatography • Purpose and application of TLC & Paper chromatography in the pharmaceutical field
- 4. Introduction Thin Layer Chromatography can be defined as a method of separation or identification of a mixture of components into individual components by using finely divided adsorbent coated or spread over a chromatographic plate. TLC is commonly used for • Monitoring the progress of reactions • Identifying compounds • Checking the purity of substances
- 5. Principle • Thin Layer Chromatography (TLC) is a laboratory technique used for the separation of mixtures into their individual components. It involves applying a small sample of the mixture to a thin layer of adsorbent material (usually silica gel, alumina, or cellulose) coated on a flat, inert substrate like glass, plastic, or aluminum. • The mobile phase solvent flows through because of capillary action (against gravitational force).
- 6. • The components move according to their affinities towards the adsorbent. • The component with more affinity towards the stationary phase travels slower. • The component with lesser affinity towards the stationary phase travels faster. • Thus the components are separated on a thin layer chromatographic plate based on the affinity of the components towards the stationary phase.
- 7. Methodology • Choice of adsorbent • Methods for the production of a thin layer on a plate • Application of the sample on the chromatoplate • Choice of solvent • Detecting reagent • Developing a reagent • Development and detection
- 8. Stationary Phase(Adsorbent ) Inorganic Organic Silica gel Cellulose & its derivatives Alumina Charcoal & activated carbon Kieselguhr Magnesia Magnesium silicate calcium silicate
- 9. • Stationary phases, their composition and the ratio in which they have to be mixed with water or other solvents to form a slurry for preparing thin layer chromatographic plates
- 10. Preparation of the Plates • Glass plates used for TLC are typically 20 x 20 cm, 20 x 10 cm, or 20 x 5 cm in size, matching the sizes of commercially available spreaders • Smaller plates, like microscope slides, can also be used for specific tasks, such as checking the progress of a chemical reaction • The glass should be of high quality and able to withstand the temperatures needed for drying
- 11. General method: • To prepare TLC plates, mix 30 grams of the adsorbent (like silica gel) with the correct amount of water or solvent until smooth. Quickly pour this mixture into a spreader and evenly coat 4 to 5 glass plates (each 20 x 20 cm in size). Let the coating set for about 4 minutes, especially if it contains calcium sulfate. After setting, place the plates in a holder, dry them at 100-120°C for 1 hour to activate the adsorbent, then cool and store them in a desiccator to keep them moisture-free. The coating should be about 0.25 mm thick.
- 12. Preparation of the Glass Plates • Coating of a glass plate with an adsorbent layer can be achieved by spreading, pouring, spraying, and dipping • Most uniform layers are obtained by the spreading technique • Coating glass plates by spreading:A slurry of adsorbent (like silica gel or alumina) mixed with a binder and solvent is prepared. The slurry is poured onto a glass plate and spread evenly using a glass rod, spatula, or a specialized applicator known as a spreader. • Advantages: Simple, low-cost, and doesn't require specialized equipment • Disadvantages: Can result in uneven coating if not done carefully, leading to inconsistent results
- 13. Coating the glass plate by pouring: • In this method slurry is made by shaking silica gel in a closed flask with ethyl acetate in the ratio of 1:3 • The slurry is evenly distributed on a glass plate by slightly tipping the plate to and fro, without touching its edges • Chromatoplate air dried for 10-20 min Coating microscope slides by dipping: • In this method slurry is prepared by shaking silica gel with chloroform-methanol(2:1) in a stoppered bottle • Two cleaned sandwiched microscopic slides are then dipped into the adsorbent slurry • After 20 seconds slides are carefully withdrawn and allowed to drain by resting them with their lower edges upon the rim of the bottle
- 14. • Two slides separated immediately after evaporation of the solvent • Adsorbent adhering to edges scrapped off and slides are exposed to steam for few minutes • Coating glass plates and slides by spraying: • In this method a slurry of silica gel (15gm) is prepared with water (35ml) and then sprayed onto glass plates with help of ordinary spray gun using an air pressure 1-1.2psi • Precoated plates and sheets • Readymade layers of adsorbent on glass plate are also available commercially • Plastic sheet and aluminium foil coated with silicic acid frequently available
- 15. • Activation of plates: Coated plates are kept in air for 30min and then in a hot air oven at 110 Celsius for another 30min • Purification or washing of plates Silica Gel Impurity: Silica gel, used in chromatography, often contains iron, which can interfere with the results. Purification Process: To remove the iron, the silica gel-coated plates are treated with a mixture of methanol and concentrated hydrochloric acid. Iron Removal: The iron impurities move to the top edge of the plate along with the solvent during this process. Drying and Activation: After this, the plates are dried and reactivated to make them ready for use. Final Step: This washing step ensures that the TLC plates are clean and prepared for accurate testing.
- 16. Application of sample: • To obtain clear spots on a TLC plate, apply 2-5 µl of a 1% solution of the sample or standard using a capillary tube or micropipette. The spots can be placed randomly or spaced evenly using a template with markings. Ensure the spots are at least 2 cm above the bottom of the plate so they don't touch the mobile phase when the plate is placed in the development tank. This helps prevent the spots from smearing or washing away during the chromatography process.
- 17. Choice of solvent Choice of solvent depends on the following 2 factors • Nature of the substance to be separated • Material on which the separation is to be carried out Since more polar solvent produce greater migration, better separation is effected in their presence It also been observed that combination of 2 solvents give better results than obtained with single solvents A typical solvent mixture for adsorption thin layer chromatography is n-hexane-diethyl ether-acetic acid in the ratio 90:10:1
- 18. Detecting reagents • Many compounds separated in tlc are colourless, their position thus located or detected with help of reagents known as locating or detecting reagents • Iodine vapour and sulphuric acid are common locating reagents • Specific methods: In this method particular detecting agents are used to find out the nature of compounds or for identification purposes. • Ferric chloride- for phenolic compounds and tannins. • Ninhydrin in acetone- for amino acids • Dragendroff’s reagent – for alkaloids • 2,4 – Dinitrophenyl hydrazine – for aldehydes and ketones compounds or for identification purposes.
- 19. Nonspecific methods: Where the number of spots can be detected, but not the exact nature or type of compound. • Iodine chamber method: Where brown or amber spots are observed when the TLC plates are kept in a tank with a few iodine crystals at the bottom. • Sulphuric acid spray reagent: 70-80% v/v of sulphuric acid with a few mg of either potassium dichromate or potassium permanganate or a few ml of nitric acid as an oxidizing agent is used. This reagent, after being sprayed on TLC plates, is heated in an oven. Black spots are seen due to the charring of compounds. • Using fluorescent stationary phase: When the compounds are not fluorescent, a fluorescent stationary phase is used. When the plates are viewed under a UV chamber, dark spots are seen on a fluorescent background. • Examples of such a stationary phase are Silica gel GF
- 20. The detecting techniques can be categorized as • Destructive technique: Specific spray reagents, Sulphuric acid spray reagent, etc where the samples are destroyed for detection. • Non-Destructive technique: like UV chamber method, Iodine chamber method, densitometric method, etc where the sample is not destroyed even after detection. These detecting techniques are used in TLC method development and in preparative TLC.
- 22. Development chamber Container: A chamber or container that can be sealed to create a saturated environment with solvent vapors. It can be a jar, a beaker with a lid, or a specialized TLC development tank. Chamber saturation: • If chamber saturation is not done edge effect occurs • Edge effects: where the solvents front moves faster in middle of the plate than that of the edges • Therefore spots are not regular
- 23. Development Technique "Developing Technique" refers to the process of allowing a solvent (or solvent mixture) to move up a TLC plate, separating the components of a mixture based on their interactions with the stationary phase (the TLC plate) and the mobile phase (the solvent). 1. Ascending or vertical development 2. Horizontal development 3. Multiple development 4. Stepwise development 5. Gradient development 6. Continuous development 7. Two dimension development 8. Chromatography on discontinuous layer 9. Chromatography on gradient layers 10. Chromatography is shaped areas
- 24. Ascending or vertical development • The sample is spotted at one end of the plate and then developed by ascending technique • The plate was placed vertically in a container saturated with developer vapor and solvent as end from bottom to top Horizontal development • In this development, the sample was placed in the center of the plate and developed either by slowly dripping solvent on it from the micropipette or by supplying solvent from the reservoir through the wick • As the solvent spreads out from the center, it moves radially outward, causing the components of the sample to separate in a circular pattern around the center spot. This method is also referred to as Radial Development or Circular TLC
- 26. Multiple development • In this, the development takes place repeatedly with the same solvent and in the same direction, each time after drying Stepwise development • Stepwise development is carried out consecutively with 2 different solvents, but in the same direction • One of the solvents is run to a height of 15-18 cm, and the other to 10-12cm • This method is desirable if the mixture to be separated contains a group of compounds that have wide different properties
- 27. Gradient development • Thin Layer Chromatography (TLC) is a technique where the composition of the solvent (mobile phase) is gradually changed during the development process. This method is particularly useful for separating mixtures of compounds that have a wide range of polarities or affinities for the stationary phase. • Instead of using a single solvent, a gradient of solvents with varying polarities is used. The gradient allows better separation of components with a wide range of polarities.
- 28. Continuous development • Continuous development in Thin Layer Chromatography (TLC) is a technique used to enhance the separation of compounds that are difficult to distinguish due to similar 𝑅𝑓 values. • Extended Migration: Allowing the compounds to migrate further. This extended migration provides a better separation between compounds, especially those with similar 𝑅𝑓 values. • Special Chambers:The BN chamber refers to a special chamber setup using Benzene/Nitromethane as the solvent system.
- 29. Two-dimensional development The sample is spotted in a corner of the plate and then developed consecutively, in 2 directions, either with the same solvent or with different solvents
- 30. Chromatography on discontinuous layer Discontinuous Layer: Instead of having a continuous layer of adsorbent across the entire plate, the adsorbent is applied only in specific spots or areas. Ex TLC plate with three different patches of silica gel. A mixture is applied, and as the solvent moves up the plate, different components of the mixture will separate out depending on which patch they interact with.
- 31. Rf value • The Rf value is calculated for identifying the spots in qualitative analysis. • Rf value is the ratio of distance travelled by the solute to the distance travelled by the solvent front. • Rf = D𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑢𝑡𝑒 𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡h𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛t
- 32. • The 𝑅𝑓 value ranges from 0 to 1. The ideal value is 0.3 to 0.8 • The 𝑅𝑓 value is constant for every compound in a particular combination of stationary and mobile phase. When the Rf value of a sample and reference compound is same, the compound is identified by its standard. When the 𝑅𝑓 value differs, the compound may be different from its reference standard.
- 33. • R𝒙 value is the ratio of distance travelled by the sample and the distance travelled by the standard. • Rx value is always closer to 1
- 34. Rf (Retention factor) value is a critical parameter in Thin Layer Chromatography (TLC) for several reasons: • Compound Identification: The R f value is used to identify compounds by comparing the observed R f values with known standards under the same experimental conditions • Purity Assessment: In TLC, a pure compound typically shows a single spot with a specific R f value. Multiple spots with different R f values indicate the presence of impurities • Monitoring Reaction Progress: By comparing the R f values of reactants and products over time, chemists can determine how far a reaction has proceeded.
- 35. • Comparison of Similar Compounds:TLC allows the comparison of structurally similar compounds. Even small changes in structure can lead to different R f values, helping in distinguishing between closely related substances. • Cost-Effective and Rapid Analysis
- 36. Factors affecting the Rf value Solvent Polarity Polar solvents tend to increase the Rf values of polar compounds by helping them move up the stationary phase more effectively. Non-polar solvents, conversely, will favor the movement of non-polar compounds. Stationary Phase Characteristics Polar stationary phases will strongly retain polar compounds, resulting in lower Rf values, while non-polar stationary phases will interact more with non-polar compounds. Sample Polarity More polar compounds tend to have lower R f values on polar stationary phases because they interact more strongly with the stationary phase. Solvent Saturation Proper saturation helps in maintaining consistent conditions and more accurate Rf values.
- 37. Thickness of the Stationary Phase: A thicker layer of stationary phase will generally result in lower Rf values because the compounds will interact more with the stationary phase. Amount of Sample Applied Applying too much of the sample can cause tailing and broaden the spots, potentially altering the Rf values due to overloading the stationary phase. Nature of the Compounds Compounds with similar structures and functional groups tend to have similar Rf values, but small differences in structure can lead to significant changes in Rf .
- 38. Advantages • The instrument required is simple compared to liquid chromatography or gas chromatography. • This method is speedy and cheaper. • This method is flexible because the TLC plate can be treated with a variety of reagents, including corrosive reagents for the detection of the analyte. • Separation of mg of the substance can be achieved.
- 39. Disadvantages • Accurate quantitative analysis may not be performed by TLC • Reproducible results may not be obtained • The number of theoretical plates is less • It has low sensitivity compared to HPLC • It is not suitable for volatile compounds
- 40. Application Test the purity of the sample TLC helps to detect the purity of the sample by direct comparison with standard Any impurity in the sample shows up as an extra spot in chromatography Identification of Active Pharmaceutical Ingredients TLC is used to identify and confirm the presence of active pharmaceutical ingredients (APIs) in drug formulations. By comparing the R f values and spot characteristics with known standards, the presence of the desired API can be confirmed Examination of reaction mixtures During the synthesis of pharmaceutical compounds, TLC is used to monitor the progress of chemical reactions. It helps in determining the completion of a reaction and the formation of the desired product.
- 41. • Biochemical analysis Biochemical metabolites from the body fluids, blood plasma, serum, urine etc. can be isolated using TLC • Qualitative analysis or detect impurities in various medicines like hypnotics, sedatives, anticonvulsants, tranquilizers, antihistamines, analgesics, local anaesthetics, steroids etc • In food and cosmetic industry Any artificial colour, preservatives, sweetening agent, other impurities in food and cosmetic product can be detected and isolated by TLC technique
- 42. Paper chromatography • Paper chromatography is a laboratory technique used to separate and identify the components of a mixture, where the process is carried out on specialized paper • The stationary phase, cellulose in filter paper, holds moisture • The mobile phase can be an organic solvent or buffer
- 43. Types of Paper Chromatography Paper Adsorption Chromatography: Paper impregnated with silica or alumina acts as an adsorbent (stationary phase) and solvent as a mobile phase Paper Partition Chromatography: Moisture / Water present in the pores of cellulose fibers present in filter paper acts as a stationary phase & another mobile phase is used as solvent In general paper chromatography mostly refers to paper partition chromatography
- 44. Principle • The principle of paper chromatography is based on the partitioning of components between two phases: the stationary phase and the mobile phase • Stationary phase, cellulose in filter paper, holds moisture • The mobile phase can be an organic solvent or buffer • When a sample is applied to the paper and the mobile phase is allowed to move through it, the components of the sample will travel at different rates depending on their relative affinities for the stationary phase and the mobile phase • Components that have a higher affinity for the stationary phase will move more slowly, while those with a higher affinity for the mobile phase will move more quickly
- 45. Methodology of Paper chromatography • Stationary phase & papers used • Mobile phase • Application of sample • Developing Chamber • Detecting or Visualizing agents
- 46. Stationary phase and papers: Whatman filter papers of different grades, such as No.1, No.2, No.3, No.4, No.17, No.20, etc., are used. In general, paper contains 98-99% α-cellulose and 0.3 – 1% β —β-cellulose. These papers differ in sizes, shapes, porosities, and thickness. • Other modified papers, like Acid or base-washed filter paper, glass fiber type paper. • Hydrophilic Papers – Papers modified with methanol, formamide, glycol, glycerol, etc. • Hydrophobic papers – acetylation of OH groups leads to a hydrophobic nature, hence can be used for reverse-phase chromatography. Silicon pretreatment and organic non-polar polymers can also be impregnated to give a reverse-phase chromatographic mode. • Impregnation of silica, alumina, or ion exchange resins can also be made. • Size of the paper used: Paper of any size can be used. Paper should be kept in a chamber of suitable size.
- 47. Paper chromatography mobile phase Pure solvents, buffer solutions, or a mixture of solvents can be used. Hydrophilic mobile phases Isopropanol: ammonia: water 9:1:2 Methanol: water 4:1 or 3:1 n-Butanol: glacial acetic acid: water 4:1:5 Hydrophobic mobile phases kerosene: 70% isopropanol Dimethyl ether: cyclohexane • The commonly employed solvents are the polar solvents, but the choice depends on the nature of the substance to be separated. • If pure solvents do not give satisfactory separation, a mixture of solvents of suitable polarity may be applied.
- 48. Application of sample: The sample to be applied is dissolved in the mobile phase and applied using capillary tube or using micropipette. Very low concentration is used to avoid larger zone Chromatographic chamber The chromatographic chambers are made up of many materials like glass, plastic or stainless steel. Glass tanks are preferred most. They are available in various dimensional sizes depending upon paper length and development type. The chamber atmosphere should be saturated with solvent vapor
- 49. • Development technique • Drying of Chromatogram • Detection • Quantitative Analysis Explanation same as TLC
- 50. Development Technique "Developing Technique" refers to the process of allowing a solvent (or solvent mixture) to move up a Paper, separating the components of a mixture based on their interactions with the stationary phase (the paper) and the mobile phase (the solvent). • Ascending or vertical development • Horizontal development • Multiple development • Stepwise development • Gradient development • Continuous development • Two-dimensional development • Chromatography on discontinuous layer • Chromatography on gradient layers • Chromatography shapes areas
- 51. Ascending or vertical development • The sample is spotted at one end of the plate and then developed by the ascending technique • The paper was placed vertically in a container saturated with developer vapor and solvent from the bottom to the top Horizontal development • In this development, the sample was placed in the center of the plate and developed either by slowly dripping solvent on it from the micropipette or by supplying solvent from the reservoir through the wick • As the solvent spreads out from the center, it moves radially outward, causing the components of the sample to separate in a circular pattern around the center spot. This method is also referred to as Radial Development or Circular PC
- 53. Multiple development • In this the development takes place repeatedly with the same solvent and in the same direction, each time after drying Stepwise development • Stepwise development is carried out consecutively with 2 different solvents, but in the same direction • One of the solvents is run to a height of 15-18 cm and the other to 10-12cm • This method desirable if the mixture to be separated contains group of compounds they have wide different properties
- 54. Gradient development • Paper Chromatography (PC) is a technique where the composition of the solvent (mobile phase) is gradually changed during the development process. This method is particularly useful for separating mixtures of compounds that have a wide range of polarities or affinities for the stationary phase. • Instead of using a single solvent, a gradient of solvents with varying polarities is used. The gradient allows better separation of components with a wide range of polarities.
- 55. Continuous development • Continuous development in Paper Chromatography (PC) is a technique used to enhance the separation of compounds that are difficult to distinguish due to similar 𝑅𝑓 values. • Extended Migration: Allowing the compounds to migrate further. This extended migration provides a better separation between compounds, especially those with similar 𝑅𝑓 values. • Special Chambers: The BN chamber refers to a special chamber setup using Benzene/Nitromethane as the solvent system.
- 56. Two-dimensional development The sample is spotted in a corner of the paper and then developed consecutively, in 2 directions, either with the same solvent or with different solvents
- 57. Chromatography on discontinuous layer Discontinuous Layer: Instead of having a continuous layer of adsorbent across the entire plate, the adsorbent is applied only in specific spots or areas. Ex paper with three different patches of silica gel. A mixture is applied, and as the solvent moves up the plate, different components of the mixture will separate out depending on which patch they interact with
- 58. Detecting reagents • Many compounds separated in paper chromatography are colorless; their position is thus located or detected with the help of reagents known as locating or detecting reagents • Iodine vapor and sulphuric acid are common locating reagents • Specific methods: In this method, particular detecting agents are used to find out the nature of compounds or for identification purposes • Ferric chloride- for phenolic compounds and tannins. • Ninhydrin in acetone- for amino acids • Dragendroff’s reagent – for alkaloids • 2,4 – 2,4-Dinitrophenyl hydrazine – for aldehyde and ketones compounds or for identification purposes
- 59. Nonspecific methods: • Where the number of spots can be detected, but not the exact nature or type of compound • Iodine chamber method: Where brown or amber spots are observed when the papers are kept in a tank with few iodine crystals at the bottom • Sulphuric acid spray reagent: 70-80% v/v of sulphuric acid with a few mg of either potassium dichromate or potassium permanganate or a few ml of nitric acid as an oxidizing agent is used. This reagent after spraying on papers is heated in an oven. Black spots are seen due to the charring of compounds • Using fluorescent stationary phase: When the compounds are not fluorescent, a fluorescent stationary phase is used. When the plates are viewed under a UV chamber, dark spots are seen on a fluorescent background • Examples of such stationary phases is Silica gel GF
- 60. The detecting techniques can be categorized as • Destructive technique: Specific spray reagents, Sulphuric acid spray reagents, etc where the samples are destroyed for detection • Non-destructive technique: like UV chamber method, Iodine chamber method, densitometric method, etc where the sample is not destroyed even after detection. These detecting techniques are used in TLC method development and in preparative TLC
- 62. Rf value • The Rf value is calculated to identify the spots in qualitative analysis. • Rf value is the ratio of the distance travelled by the solute to the distance travelled by the solvent front. • Rf = D𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑢𝑡𝑒 𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡h𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛t
- 63. Thin-layer chromatography paper chromatography The stationary phase is usually a thin layer of adsorbent material, such as silica gel or alumina, coated onto a glass, metal, or plastic plat The stationary phase is the cellulose fibers of filter paper, often with the paper's natural moisture acting as the stationary phase Separation is often based on adsorption, where components of the mixture adhere to the adsorbent layer at different rates Separation is primarily based on partitioning between the water retained in the paper fibers and the moving solvent. better resolution and sensitivity compared to paper chromatography. less sensitive with lower resolution but it's simpler and more cost-effective. Faster and more efficient; the separation process usually takes minutes Slower; the process can take hours depending on the paper and solvent used. Widely used for more complex and precise separations, Commonly used for simpler separations
- 64. Advantages • Simple and Rapid • Paper Chromatography requires very less quantitative material. • Paper Chromatography is cheaper compared to other chromatography methods. • Both unknown inorganic as well as organic compounds can be identified by paper chromatography method. • Paper chromatography does not occupy much space compared to other analytical methods or equipment’s.
- 65. Disadvantages • Large quantity of sample cannot be applied on paper chromatography • In quantitative analysis paper chromatography is not effective • Complex mixture cannot be separated by paper chromatography • Less Accurate compared to HPLC or HPTLC