Thin Layer Chromatography

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THIN LAYER CHROMATOGRAPHY I/II , I st Semester M.Pharmacy Dept. of Pharmaceutical Analysis JNTUH, Hyderabad. Ravi Pratap Pulla M.Pharm ., Ph.D Associate Professor SSJ College of Pharmacy, V.N.Pally , Gandipet , Hyderabad-75 Andhra Pradesh.

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 TLC is widely used to separate chemical compounds  It involves S.P – Consists of thin layer adsorbent material  S.P usually consists of – silica gel , alumina, cellulose immobilized onto a flat inert carrier sheet.  M.P consists of the solution to be separated dissolved in appropriate solvent.  It is drawn through the plate via capillary action  Used to determine and detect pigments in plant, pesticides or insecticides in food, in forensic dept. to analyze dye composition of fiber & identification of various compounds.

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 TLC is universal analytical technique in chemical analysis for separation of organic and inorganic matter.  In 1938 , Izmailov and Shraiber describe basic principle used it for separation of plant extract.  In 1944 , Consden , Gorden & Martin used filter papers for separating the Amino acids.  In 1950 , Kirchner identified terpenes on filter paper.  In 1958 , Stahl mainly created with bringing out the work on preparing plates and separation of wide variety of compounds.

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 TLC – common used technique in synthetic chemistry for identifying compounds  Determining their purity  Analyzing the progress of the reaction and also mechanism involved.  S.P may be solid or liquid & hold as a layer on solid support.  It permits optimization of the solvent system for given separation problem.  Compared b/w C.C & TLC – this requires less quantity of compounds & also much faster in progress.

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PRINCIPLE  The basic Principle is based on ADSORPTION Chromatography  The component with more affinity travel slower towards the S.P  The component with lesser affinity travel faster towards the S.P.  TLC is simple and rapid method - carried out by using thin layer of adsorbent on plates.

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 TLC is included under both adsorption and partition chromatographs.  Separation of component may result due to adsorption or partition  both phenomenon depend upon nature of adsorbent used on plate and solvent system used for the development.  S.P – TLC plate, TLC paper is coated with silica gel  In TLC separation – hydrogen bonding is main intermolecular forces involved

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 Polar molecules stick to plate  Non- polar molecules do not stick to plate  Non-polar molecules will spend a great amount of time dissolved in eluent  Separation of compounds occur due to differences in partitioning b/w liquid and S.P  More sensitive & less sample required  Spraying with corrosive agents for identification possible

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 TLC can be automated using forced solvent flow  Running the plate in vacuum  Capable chamber to dry the plate  Recording the finished chromatograph – absorption / fluorescence spectroscopy with light source  Ability to program the solvent delivery makes it convenient to do multiple developments in which the solvent flows for a short period of time and TLC dried and process repeated. This method refocuses the spots to achieve higher resolution than in single run.

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ADVANTAGES  Low cost  Short analysis time  All spots can be visualized  Adaptable to most pharmaceuticals  Uses small quantities of solvents  Requires minimal training  Reliable and quick  Minimal amount of equipment is needed  Densitometers can be used to increase accuracy of spot concentration

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TLC SUPERIOR OVER OTHER METHODS  It requires little equipment  Require little time for separation  It is more sensitive  Very small quantity of sample require for analysis  The method use for adsorption, partition, ion exchange chromatography  Component which are separated can be recovered easily .  Quantative separation of spot and zone are possible  For identification is permitted  S praying of corrosive agent

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OPERATIONAL TECHNIQUE INVOLVED  Choice of adsorbent  Preparation of plate  Preparation and application of sample  Choice of solvent  Development of chromatogram  Drying of chromatogram  Location of spot  Quantitative estimation

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CHOICE OF ADSORBENT  Two properties decide the selection: 1. particle size 2. homogeniscity  Factors affecting selection: 1. Colorless 2. should have great mechanical strength 3. should not catalyze or decompose of substance 4. should be insoluble with mobile phase & the solvent used for elution 5. no reaction at time of separation

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6. Adsorbent do not adhere to glass plate 7. Adsorbent particle size 8. To see whether compound is liable to react chemically with adsorbent. 9. Nature of substance to be separated 10. Characteristic of compound to be separated 11. Solubility of compound

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CLASSIFICATION OF ADSORBENTS USED Classification according to binding strength: Weak adsorbent: sucrose, starch, talc, cellulose Intermediate adsorbent: silica gel, calcium carbonate, calcium phosphate, magnesia Strong adsorbent: alumina, charcoal 2. Classification according to nature: A. Inorganic adsorbent: Silica, Silica gel, Alumina, Calcium phosphate, Glass powder, Kieselguhr ,Magnesium silicate, Calcium silicate, Phosphate , Ferric & Chromic oxides, Zinc carbonate & zinc ferro cyanides, Bentonites B. Organic adsorbent: Normal cellulose powder, Charcoal & activated carbon, Starch, Sucrose, Manitol , Dextran gel

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 SILICA GEL is granular porous form of silica  Made synthetically from sodium silicate  Silica gel is solid and used in chromatography as S.P  Due to silica gel polarity – non polar components tend to elute before polar ones hence named as NPC  Hydrophobic groups (C 18 ) attached to silica gel then polar components elute first hence names as RPC.  Synthetic nature of silica gel enables careful control of pore size.

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CELLULOSE  Cellulose (C6H10O5)n is a long chain polymeric polysaccharide carbohydrate of β – glucose  Adsorbed water or alcohol can be retained by interaction with hydroxyl groups  Two types of cellulose are used in planar chromatography: 1.Polymerization b/w 400-500 glucopyranose units 2. 40 – 200 glucopyranose units

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ALUMINIUM OXIDE  It is a chemical compound of aluminum and oxygen with chemical formula – Al 2 O 3  Commonly referred to as alumina  Manufactured in 3 pH ranges – acidic, basic and neutral  Acidic compounds – phenols, sulphonic , carboxylic & Amino acids are separated on acidic alumina  Basic compounds – amines , dyes separated  Neutral compounds – aldehydes , ketones & lactones

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Reverse Phase Chromatography  In this the S.P is Non-polar & M.P is polar & it is widely used in pharmaceutical analysis. 1. Polar compounds get eluted first 2. Non-polar compounds are retained for long time Comparison of Normal Phase & Reverse Phase : PARAMETER NORMAL PHASE REVERSE PHASE Stationary phase Polar Non-polar Mobile phase Non-polar Polar Compound eluted first Non-polar Polar Compound eluted last Polar Non-polar Example of stationary phase Silica gel C 4 ,c 8 –bonded phase

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STATIONARY PHASE NAME COMPOSITION Silica gel H Silica gel without binder Silica gel G Silica gel + CaSO 4 Silica gel GF Silica gel + Binder + fluorescent indicator Alumina Al 2 0 3 Without Binder Al 2 0 3 G Al 2 0 3 + Binder Cellulose powder Cellulose Without Binder Cellulose powder Cellulose With Binder Kieselguhr G Diatomaceous earth + binder Polyamide powder Polyamide Fuller’s earth Hydrous magnesium alumina Magnesium Silicate magnesol

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MOBILE PHASE Nature of the substance to be separated i.e whether it is polar or non-polar. Mode of Chromatography Nature of Stationary phase Mode Separation i.e Analytical or Preparative technique  Examples: 1) Petroleum ether 2) Cyclohexane 3) Acetone 4) Toluene 5) Ethyl acetate 6) Benzene 7) Alcohols 8) Water 9) Chloroform 10) Pyridine

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CHOICE OF SOLVENT Selection of M.P depends upon nature of substance to be separated Viscosity and polarity of S.P Solvent used may be single or double phase system e.g : n-hexane < cyclohexane < CCl 4 < benzene < toluene < CHCl 3 < diethyl ether < ethyl acetate < acetone < ethanol < Methanol < water

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GLASS PLATES  Three types : Full plate : 20cm × 20 cm. Half plate : 20cm × 10 cm. Quarter plate : 20cm × 5 cm.  Microscopic slides can also be used for monitoring the progress of a chemical reaction.

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DEVELOPING A PLATE  TLC plate can be prepared in beaker or closed jar  Place a small amount of solvent in container.  Solvent level below the starting line of TLC, else spots dissolve  Low edge of plate dipped in solvent  Solvent travels up the matrix by capillarity  Moving components of samples at various rates because of their different degrees of interaction with matrix & solubility in the developing solvent

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 Non polar solvents force non polar compounds to top of plate because the compounds dissolve well & do not interact with polar S.P  Allow the solvent to travel up the plate until 1 cm from top  Take the plate out and mark the solvent front immediately.  Do not run the solvent over edge of plate  Let solvent evaporate completely.

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PREPARATION AND ACTIVATION OF PLATES  The T L C plates can be prepared by following techniques : Pouring Dipping Spraying Spreading  Activation :It is nothing but removing of water/ moisture & other adsorbed substance from the surface of any adsorbent by heating.

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METHOD FOR APPLICATION OF ADSORBENT ON THE PLATE POURING- adsorbent of homogeneous particle size made in slurry and pour on plate. DIPPING- it used for small plate by dipping two plate back to back in slurry of adsorbent in chloroform or other volatile solvent. SPRAYING- simply by spraying slurry on plate SPREADING- slurry spread by using spatula or glass rod

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ACTIVATION OF PLATE  TLC plates made by mixing adsorbent – silica gel + inert binder calcium sulphate (gypsum) + water  Mixer spread as thick slurry on un-reactive carrier sheet – glass, thick aluminum foil, plastic etc  Resultant plate dried and activated by heating in oven for 30 minutes at 110° C  Thickness of adsorbent layer: A. 0.1 – 0.25 mm for analytical purpose B. 1- 2 mm for preparative TLC

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APPLICATION OF SAMPLE

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DEVELOPMENT CHAMBER / TANK

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 TLC plates are placed vertically in rectangular chromatography tank or chamber .  Glass and stainless steel are suitable chambers.  If tank is not saturated, solvent will evaporate and affect the R f value.  Development should be carried out at room temperature by covering chamber with glass plate.

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The development tank should be lined Inside with filter paper moistened with mobile phase to saturate the atmosphere & also prevent the “ EDGE EFFECT ” .

Development technique:

Different development techniques are : One dimensional development. Two dimensional development. Horizontal development. Multiple development. Development technique

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DEVELOPMENT OF CHROMATOGRAMS  Ascending development- plate after spotting placed in chamber flow of solvent from bottom to top.  Descending development- in this flow of solvent from reservoir to plate by means of filter paper strip. solvent move from top to bottom.  Place spotted plate in developing chamber  Developing solution is drawn up the plate by capillary action  Compounds in the original spots are pulled by silica gel.

Detecting agents:

Detecting agents are two types: Non-Specific method 1) Iodine chamber method. 2) Sulphuric acid spray method. 3) UV chamber for fluorescent compounds. 4) Using fluorescent stationary phase. (B) Specific method 1) Ferric chloride. 2) Ninhydrine in acetone. 3) Dregendroff reagent. 4) 3,5 – Dinitro benzoic acid. 5) 2,4 - Dinitro phenyl hydrazine. Detecting agents

detection:

The R f value is calculated for identification "Rf value is the ratio of distance travelled by The solute to the distance travelled by the solvent front” Distance travelled by solute R f = Distance travelled by solvent front detection

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R f value is constant for each component only under identical experimental condition. Polar compounds have low R f value  It depend on following factors-  Nature of adsorbent  Mobile phase  Activity  Thickness of layer  The temperature  Equilibration  Loading  Dipping zone  Chromatographic technique

Development of t l c:

Development of t l c

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VISUALIZATION METHOD  Previous slide shows colored spots. Most of the time spots wont show unless visualized.  Visualization is a method used to render TLC spots visible  A visualization method can be:  UV light  iodine vapors to stain spots  colored reagents to stain spots  reagents that selectively stain spots leaving others unaffected

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VARIOUS TECHNIQUES TO VISUALIZE THE COMPOUNDS: Sulfuric acid/ heat: destructive, leaves charred blots behind ceric stain: destructive, leaves a dark blue blot behind polar compounds Iodine: semi- destructive , iodine absorbs onto the spots , not permanent UV light: non – destructive, long wavelength, (background plate green, spots dark) short wavelength (background plate dark, spots glow)

Retention :

Retention  The fundamental parameter in TLC is the retardation factor, R f : R f = Z s / (Z f – Z o )  Z f : Distance traveled by the solvent front from the point of application.  Z s : Distance traveled by the solute front from the point of application.  Z o : Distance between the point of application of solvent and solute. Z f Z s Z o

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 The value of R f is related to the capacity factor (k) of the solute by the following equation: k = (1- R f )/ R f  By using the above equation, planar chromatography can be used to obtain estimates of k for a solute on different stationary phase and mobile phase combinations.  This can be useful in screening a number of columns or mobile phase for use in column liquid chromatography. EFFICIENCY  The efficiency of a separation in planar chromatography is described in terms of plates and plate height. N = (Z s / s ) 2 N = 16*(Z s / W b ) 2 H = Z s /n Where, N: number of theoretical plates; H: plate height s : standard deviation of the solute band (in distance units) W b : baseline width of the solute band (in distance units)

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 Note that the efficiency of a planar system is not constant, but depends on the distance that the solute has traveled, or its retention and R f value.  The change in efficiency of a planar chromatography system with distance and the presence of a third phase have made the derivation of exact plate height equations for planar chromatography difficult.  These concurrently occur with another complicating factor: the flow rate of mobile phase through a system with capillary flow is not constant with time.  For a system with capillary flow, the change in the mobile phase velocity with time is described by the following equation: Z f = ( xt ) 1/2 Where, t = time required by the mobile phase to migrate Z f = distance x = the system constant

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PERFORMING THE TLC ANALYSIS: CALCULATE THE RF VALUES  The R f value is calculated by measuring the distance the sample zone travels divided by the distance the developing solvent travels  Values below 0.1 is considered poor: the spots are too close to origin  Values of 0.1 to 0.8 are good and any other spots (impurities) or other actives are resolved form each other  Above 0.8: poor: spots may be too broad or distorted

Applications / uses:

Separation of mixture of drug of chemical,biological,plant origin. Separation of Carbohydrates, vitamin, antibiotics, proteins, etc. Identification of drug. Ex :Amoxicillin, Levodopa Detection of foreign substances. To detect the decomposition products of drug. Applications / uses

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6). To determine how many compounds are there in a mixture – is it real pure? 7). To determine the best solvent conditions for separation on column 8). To identify the substances being studied 9). To monitor the compositions & appropriate conditions of the fractions collected from Column Chromatography 10). To monitor the progress of the reaction 11). To determine identity of two substances 12). To determine effectiveness of purification

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TLC TROUBLESHOOTING 1 . CAUSE: the compound runs as streak rather than a spot REASON:  the sample was overloaded  Run the TLC again after diluting your sample  Sample might contain many components  It creates many spots which run together & appear as streak 2. CAUSE: the sample runs as a smear or a upward crescent (moon) REASON:  compounds which possess strongly acidic or basic groups (amines or carboxylic acids) show this behavior  Add few drops of ammonium hydroxide(amines) or acetic acid (carboxylic acids) to the eluting solvent to obtain clear plates.

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3. CAUSE: the sample runs as a downward crescent (moon) REASON:  adsorbent was disturbed during spotting caused 4. CAUSE: plate solvent front runs crookedly (curved) REASON:  adsorbent flaked of the sides of plate  Adsorbent moved towards the side of the plate or touching the sides of the container or the paper used to saturate the container as plate develops.  Crookedly run plates makes it harder to measure the R f value accurately. 5. CAUSE: many random spots are seen on the plate REASON:  accidently check not any organic compound on the plate or any new foreign substance touched incidentally.

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6. CAUSE: no spots seen on plate REASON:  you might have not spotted enough compound, perhaps because the solution of the compound is too dilute.  Try concentrating the solution or else spot it several times in one place allowing solvents to dry b/w capillaries  Some compounds do not show under UV light  Try another method of visualization of plate  Perhaps you don’t have any compounds because the experiment did not go as well planned  If solvent level in developing jar is deeper than the origin of the TLC plate  Solvent will dissolve the compounds into the solvent reservoir  It allows them to move up the plate by capillary actions.  Thus you will not see the spots after the plate is developed.

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