Thin Layer Chromatography

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HI...could you please send me the ppt about TLC to 'meghashyam.3@gmail.com' Thank you.

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TECHNIQUES AND APPLICATION THIN LAYER CHROMATOGRAPHY ABDUL RAZZAQM.PHARMA(PHARMACHEMISTRY) DEPARTMENT OF PHARMACHEMISTRYLUQMAN COLLEGE OF PHARMACY GULBARGA

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INTRODUCTION TLC is universal analytical technique in chemical analysis for organic and inorganic matter HISTORY- In 1938 Izmailov and Shraiber describe basic principle used it for seperation of plant extract. In 1958 Stahl mainly created with bringing out the work on preparing plates and seperation of wide varity of compound. TLC is simple and rapid method carring out using thin layer of adsorbent on plates.

ADVENTAGES : 

ADVENTAGES 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

TLC SUPERIOR OVER OTHER METHOD : 

TLC SUPERIOR OVER OTHER METHOD It requires little equipment Require little time for seperation It is more sensitive Very small quantity of sample require for analysis The method use for adsorption,partition,ion exchange chromatography Component which are seperated can be recovered easily . Quantative seperation of spot and zone are possible. For identification is permited.spraying of corrosive agent

PRINCIPLE OF TLC : 

PRINCIPLE OF TLC TLC is included under both adsorption and partition chromatographs. Separation of component may result due to adsorption or partition or both phenomenon depend upon nature of adsorbent used on plate and solvent system used for development.

TLC TECHNIQUE : 

TLC TECHNIQUE TlC is carried out on glass or plastic plate which is coated with thin uniform layer of adsorbent such as silica gel Plate are activated Sample applied by capillary or microsyring at bottom of plate After drying spot plate is placed in sutable tank Solvent rised by capallary action Resolving sample mixture into descrete spot Seperated spot are located and identified by various physical and chemical method

1) ADSORBENT : 

1) ADSORBENT Adsorbent use such as silica gel,alumina,kieselguhr Factor consider for adsorbent- Characteristic of compound to be separated Solubility of compound Nature of substance to be separated To see whether compound is liable to react chemically with adsorbent. Adsorbent particle size Adsorbent do not adhere to glass plate.

INORGANIC ADSORBENT : 

INORGANIC ADSORBENT Silica Silica gel Alumina Calcium phosphate Glass powder Kieselger Magnessium silicate Calcium cilicate Phosphate Ferric & Cromic oxides Zinc carbonate & zinc ferro cyanides Bentonites

ORGANIC ADSORBENT : 

ORGANIC ADSORBENT Normal celluose powder Charcoal & activated carban Starch Sucrose Manitol Dextran gel

2) PREPERATION OF CHROMATOGRAPHIC PLATE : 

2) PREPERATION OF CHROMATOGRAPHIC PLATE Glass plate or plastic plate used to spreyed adsorbent. Standard size of plate is 20 X 5cm ,20 X 10 cm,20 X 20cm. Plate surface is flat and regular. Standard film thickness is 250 um. Thicker layer 0.5to 2 mm used for preperative seperation.

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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

3)Activation of plate : 

3)Activation of plate After spreading plate allowed to dry and activated by heating about 1000 cfor 30 min. Plate made with volatile organic solvent may not require further drying

4) SOLVENT SYSTEM : 

4) SOLVENT SYSTEM Choice of mobile phase depend on nature of substance to be seperated. And also depend on adsorbent material to be used . Polarity of solvent and substance to be seperated playes important role in selection. Purity of solvent also important.

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Factor affecting mobile phase- Nature of the substance to be separated. Nature of the stationary phase used. Mode of chromatography. Nature of separation. Suitable eluents are usually selected by trial and error method, literature review The solvent used should be of high purity. Other factor which are taken into consideration while selecting solvents include polarity, solubility etc. Combination of two solvents gives better separation than with a single solvent

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Solvent used – Petroleum ether Benzene Carbon tetrachloride Chloroform Diethyl ether Ethanol Methanol Acetone Dichloromethane Diethyl form amide

5)APPLICATION OF SAMPLE : 

5)APPLICATION OF SAMPLE The area of application is kept as small as possible for sharper and greater resolution of sample. For preperative work sample applied in narroe band The pipette ,loop or syringe use for applying sample. The spot should be within 2-5 mm diameter. For preperative work sample upto 4 mg is appied on starting line

6)DEVELOPMENT CHAMBER : 

6)DEVELOPMENT CHAMBER TlC plate placed vertically in rectangular chromatography tank or chamber . Glass and stainless steel are sutable chamber. If tank is not saturated,solvent will evoparate and affect the Rf value. Development should be carried out at room temp.by covering chamber wuth glass plate.

DEVELOPING CHAMBER : 

DEVELOPING CHAMBER

7)DEVELOPMENT OF CHROMATOGRAMS : 

7)DEVELOPMENT OF CHROMATOGRAMS Ascending development- plate after spotting placed in chamber . and flow of solvent from bottom to top. b)Descending – in this flow of solvent from reservoir to plate by means of filter paper strip. solvent move from top to bottom.

8)LOCATION OF SPOT : 

8)LOCATION OF SPOT The method of location of colourless substance are similler to paper chromatography. Physical method include ultravoilet,fluoroscence or radioactive counting. In chemical method locating agent are applied by spraying and not by dipping Conc. Sulphuric acid use as locating agent to produce coloured spot which visible in dayligjt Iodine vapoure use for organic substance

APPLICATION : 

APPLICATION Purity of sample Examination of reaction Identification of compound Biochemical analysis In chemistry In pharmaceutical industry In seperation of multicomponent pharmaceutical formulation In food and cosmetic industry

Rf VALUE(RETENTION FACTOR) : 

Rf VALUE(RETENTION FACTOR) DISTANCE TRAVLE BY SOLUT FROM STARTING POINT Rf =---------------------------------------------------------- DISTANCE TRAVLE BY SOLVENT FROM STARTING POINT

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Rf value is constant for each component only under identical experimental condition. It depend on following factors- Nature of adsorbent Mobile phase Activity Thickness of layer The temperature Equilibration Loading Dipping zone Chromatographic technique

REFERENCES : 

REFERENCES PHARMACEUTICAL ANALYSIS,VOL-II,DR.V KASTURE,PAGE-18-27. PHARMACEUTICAL ANALYSIS,III EDITION,DR.S .RAVI SANKAR.PAGE-14-1 TO 14-12. INSTRUMENTAL METHOD OF CHEMICAL ANALYSIS,G.R.CHATWAL,PAGE-2.599-2.616. www.google.com.

4. Retention: : 

4. Retention: The fundamental parameter in TLC is the retardation factor Rf: Rf = Zs / (Zf – Zo) Zf: Distance traveled by the solvent front from the point of application. Zs: Distance traveled by the solute front from the point of application. Zo: Distance between the point of application of solvent and solute.

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b. The value of Rf is related to the capacity factor (k) of the solute by the following equation: k = (1- Rf)/ Rf c. 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. 5. Efficiency The efficiency of a separation in planar chromatography is described in terms of plates and plate height. N = (Zs / s)2 N = 16*(Zs / Wb)2 H = Zs /n N: number of theoretical plates; H: plate height. s: standard deviation of the solute band (in distance units) Wb: baseline width of the solute band (in distance units)

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b. 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 Rf value. c. 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. d. For a system with capillary flow, the change in the mobile phase velocity with time is described by the following equation: Zf = (xt)1/2 t : time required by the mobile phase to migrate distance Zf. x : the system constant.

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Rx value : It is ration of the distance travelled by the sample and the distance travelled by the std. Rx value closer to 1. Rm value: Rm = 1-1/Rf this is measured to know wheter the samples are homogenous or not.

TLC : 

TLC The two most common classes of TLC are: Normal phase Reversed phase

Normal Phase : 

Normal Phase Normal phase is the terminology used when the stationary phase is polar; for example silica gel, and the mobile phase is an organic solvent or a mixture of organic solvents which is less polar than the stationary phase.

Reversed Phase : 

Reversed Phase Reversed phase is the terminology used when the stationary phase is a silica bonded with an organic substrate such as a long chain aliphatic acid like C-18 and the mobile phase is a mixture of water and organic solvent which is more polar than the stationary phase.

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ADSORBENT; When choosing the most suitable grain size, the technical preparation and migration times on the chromatogram must be considered and a compromise arrived at. The grain size of most TLC adsorbent lies between 5 and 50 μm. The purity of the adsorbent is often significant for the adsorption properties; occasionally inorganic or organic impurities interfere procedure if were not removed.

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Silica Silica gel Alumina Calcium phosphate Glass powder Kieselger Magnessium silicate Calcium cilicate Phosphate Ferric & Cromic oxides Zinc carbonate & zinc ferro cyanides Bentonites

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Organic adsorbent Normal celluose powder Charcoal & activated carban Starch Sucrose Manitol Dextran gel

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Characteristics of the carrier: It should be stable to all types of solvent used. It must not react with spray reagents Mechanical strength and possibility of repeated use are also desirable . Low cost. Glass plates are used for these reasons.

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Preparation of TLC plate: The adsorbent is usually spread as an aqueous slurry on the carrier plates. The water must be removed by the process of drying. it clearly saves time if it the bulk of the water is first removed in current of air (predrying) and the process then completed by drying the plates at higher temperature.

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Very active plates are obtained by heating silica gel and alumina plates for 3—4hrs at 150c. High activation is worthwhile only if the substances are applied in a correspondingly dry atmosphere and if completely anhydrous solvents are used. This is necessary only in the TLC of hydrocarbon mixtures. There is a grave danger that the substances will decompose on very active layers. In the TLC of polar compounds eg amino acids, no pre and hot air drying is carried out and the plates are allowed to dry overnight at room temperature. Layers of these type show good adhesion and yield more reproducible results than do activated plates.

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POURING PROCEDURE Pouring procedure require no special apparatus. The necessary amount of the adsorbent is weighed out and shaken to a homogenous suspension with a suitable solvent. A previously determined volume of this is rapidly poured in one movement on to the middle of the plate to be coated. By gentle tilting and shaking ,the suspension is distributed over the plate which is then dried in a horizontal position. Horhammer and coworkers has suggested that the a uniform layer over the whole plate is obtained when the silica gel used is suspended in pure ethyl acetate or acetone but not in water.

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IMMERSION PROCEDURE: The adsorbent must be evenly suspended in organic solvent Two equally sized, clean glass plates are placed face to face and then immersed in this suspension. Only one surface of the plate is thus coated with this “sandwich” technique. The solvent evaporates rapidly from this layer. The binder, in this case gypsum, is made effective by holding the strip briefly in the steam. The layer is activated ready for use by drying for 1-3 min on a wire rack over a hot plate.

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Spreading procedures: Layers are prepared in laboratory by spreading the suspensions of adsorbents on glass plates. Two types of spreaders-- suspension is run into a rectangular trough which has suitable sized slit on one side. A) fixed spreader (kirchner-type):Has a fixed trough .It is rigidly joined to the base at the side and the plates are pushed through succession. B) movable spreader: (stahl-type):if several carrier plates of the same width and thickness are laid together in a row on a aligning tray or templet,they can be coated by a single passage over them of a spreader.

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Application of the substance mixture for separation: The mixture is brought into solution and applied to the start ( starting point or line) as a spot or band respectively. Strongly polar or in volatile solvents should not be used in TLC for dissolving solid samples or diluting liquid mixtures. These yield large starting spots and ring chromatograms. If necessary, very small volumes must be applied successively and the solvent removed between additions with a current of warm air. Care must be taken to ensure that the substance do not crystalline out at the starting point. Should this happen, chromatograms with long streaks (tails ,breads) are obtained,in the direction from start to front.

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MOBILE PHASES: Selection of mobile phase depend upon different factors: Nature of the substance to be separated. Nature of the stationary phase used. Mode of chromatography. Nature of separation. Suitable eluents are usually selected by trial and error method, literature review The solvent used should be of high purity. Other factor which are taken into consideration while selecting solvents include polarity, solubility etc. Combination of two solvents gives better separation than with a single solvent.

DEVELOPIONG CHAMBERSRactangular chamber : 

DEVELOPIONG CHAMBERSRactangular chamber Rectangular TLC Tanks• Sizes available: 20x20, 10x10,thinline 10x10, 10x20, and5x5cmTLC plates • Unique beveled lip to eliminatesharp edges• Uniform fl at top for max i mumlid sealThis Rectangular TLC Tank is a heavy wall, clear glassblock with flat surface walls. The top has been ground toa uniform flatness for perfect lid fit. Edges are beveledinside and out to remove sharp edges. Lids are polishedglass in sizes to match the tank dimensions.

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E. Elution and Development: 1. Solutes can be eluted in planar chromatography using either isocratic or gradient elution. 2. For gradient elution in TLC a. Mobile phase gradient b. stationary phase gradient c. Vapor phase gradient 3. Development methods Linear development; b. Radial development; c. Extended development d. Over-pressured or forced-flow development; and e. Multi-dimensional

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Gradient techniques in TLC: Gradient elution: When fractionating compounds of widely different properties are present then it is advantageous to change the composition of the solvent continuously during chromatography .this technique is called gradient elution. In this technique the chromatogram is lowered into a jar containing the solvent

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Then a second more polar solvent is allowed to enter the chamber with the help of a burette. The elute is continuously stirred. As a the polarity of the former solvent is modified by the second solvent. •Linear development •Ascending & desscending development •Ascending development •Descending development •Horizontal development •Redial development

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G. Detection a. Direct measurement; b. Chemical derivatization. 1. Direct measurement a. Florescence: emitting light or quenching fluorescence from the substrate. Green light: manganese-activated zinc silicate, which is stimulated to green fluorescence emission by 254 nm

Performing the TLC Analysis: Calculate the Rf Values : 

Performing the TLC Analysis: Calculate the Rf Values The Rf 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

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Quantitative TLC: Two basic principles are applied:----- 1. Analysis of the fraction . 2. Determination of the fraction after elution from the coating material

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Analysis of the fraction: A) Visual comparison with standards B) The precise measurement of spot areas or spot sizes. C) Photo densitometry. Radioactive substances can be assayed by: A) Photo densitometry of X-ray prints B) By liquid scintillation counting in suspension C) Direct radio-scanning of chromatoplates.

Reference: : 

Reference: Instrumental method of chemical analysis by B. K. SHARMA. Instrumental method of chemical analysis by CHATWAL & ANAND http://www.chem.ufl.edu/~cao/CHM6154/Lecture30

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