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THIN LAYER CHROMATOGRAPHY

IN 1958, STAHL DEVELOPED STANDARD EQUIPMENT FOR ANALYZING BY THIN LAYER CHROMATOGRAPHY .

DEFINITION Thin layer chromatography (TLC) is a technique used tseparate the components of a mixture using a thin stationary phase ed by an inert backing. Separation depends on competition between adsorption of solute onto the solid surface and its desorption by the solvent needed to elute (wash off) it . Stationary phase: Solid Mobile phase: Liquid

PRINCIPLE ADSORPTION Chromatography  The component with more affinity travel slower towards the S.P The component with lesser affinity travel faster towards the S.P. In TLC separation – hydrogen bonding is main intermolecular forces involved 

 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

REVERSE PHASE TLC BASED ON THE NATURE OF MOBILE AND STATIONARY PHASE USED NORMAL PHASE TLC THIN LAYER CHROMATOGRAPHY

BASED ON PURPOSE OF USE

ANALYTICAL

PREPARATIVE TLC

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

C4 ,c8 –bonded phase

INSTRUMENTATION COMPONENT

PURPOSE

Developing chamber

Create and maintain environment for chromatography

Solid (chromoplates)

s thin film of stationary phase

Stationary phase

Adsorption of material

Mobile phase

Solvent system

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

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 . Adsorbent do not adhere to glass plate

CLASSIFICATION OF ADSORBENTS USED 1. Classification according to binding strength: A. Weak adsorbent: sucrose, starch, talc, cellulose B. Intermediate adsorbent: silica gel, calcium carbonate, calcium phosphate, magnesia C. 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

 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 (C18) attached to silica gel then polar components elute first hence names as RPC.  Synthetic nature of silica gel enables careful control of pore size.

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

ALUMINIUM OXIDE  It is a chemical compound of aluminum and oxygen with chemical formula – Al2O3  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

STATIONARY PHASE NAME

COMPOSITION

Silica gel H

Silica gel without binder

Silica gel G

Silica gel + CaSO4

Silica gel GF

Silica gel + Binder + fluorescent indicator

Alumina

Al203 Without Binder

Al203 G

Al203 + 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

MOBILE PHASE

1) Nature of the substance to be separated i.e whether it is polar or non-polar. 2) Mode of Chromatography

3) Nature of Stationary phase 4) Mode Separation i.e Analytical or Preparative technique  Examples: 1) Petroleum ether 3) Acetone 5) Ethyl acetate 7) Alcohols 9) Chloroform

2) Cyclohexane 4) Toluene 6) Benzene 8) Water 10) Pyridine

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< CCl4 < benzene < toluene < CHCl3 < diethyl ether < ethyl acetate < acetone < ethanol < Methanol < water

GLASS PLATES  Three types : 1) Full plate

: 20cm × 20 cm.

2) Half plate

: 20cm × 10 cm.

3) Quarter plate : 20cm × 5 cm.  Microscopic slides can also be used for monitoring the progress of a chemical reaction.

DEVELOPING A PLATE  TLC plate prepared , P 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

 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.

PREPARATION AND ACTIVATION OF PLATES  The T L C plates can be prepared by following techniques : 1) Pouring 2) Dipping 3) Spraying 4) Spreading

 Activation :It is nothing but removing of water/ moisture & other adsorbed substance from the surface of any adsorbent by heating.

METHOD FOR APPLICATION OF ADSORBENT ON THE PLATE 1. POURING- adsorbent of homogeneous particle size made in slurry and pour on plate. 2. DIPPING- it used for small plate by dipping two plate back to back in slurry of adsorbent in chloroform or other volatile solvent. 3. SPRAYING- simply by spraying slurry on plate 4. SPREADING- slurry spread by using spatula or glass rod

ACTIVATION OF PLATE 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

APPLICATION OF SAMPLE The concentration of the sample should be 2--5µl of a 1% solution

Sample is spotted using a capillary tube or micropipette

Spots can be placed at random process

Spots should be kept atleast 2cm above the base of the plate

Spotting area should not be immersed in the Mobile phase

Go for development

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

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  Spraying of corrosive agent

Development tank The development tank should be lined Inside with filter paper moistened with mobile phase to

saturate the atmosphere & also prevent the “ EDGE EFFECT ” .

•  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 Rf value. •  Development should be carried out at room temperature by covering chamber with glass plate.

DEVELOPMENT TECHNIQUE Different development techniques are : 1) One dimensional development.

2) Two dimensional development. 3) Horizontal development. 4) Multiple development.

DETECTING AGENTS Detecting agents are two types: (A)Non-Specific method 1) 2) 3) 4)

Iodine chamber method. Sulphuric acid spray method. UV chamber for fluorescent compounds. Using fluorescent stationary phase.

(B) Specific method 1) 2) 3) 4) 5)

Ferric chloride. Ninhydrine in acetone. Dregendroff reagent. 3,5 – Dinitro benzoic acid. 2,4 - Dinitro phenyl hydrazine.

DETECTION The Rf 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 Rf =

Distance travelled by solvent front

 Rf value is constant for each component only under identical experimental condition. Polar compounds have low Rf 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

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

VARIOUS TECHNIQUES TO VISUALIZE THE COMPOUNDS:

1. Sulfuric acid/ heat: destructive, leaves charred blots behind 2. ceric stain: destructive, leaves a dark blue blot behind polar compounds 3. Iodine: semi- destructive , iodine absorbs onto the spots , not permanent 4. UV light: non – destructive, long wavelength, (background plate green, spots dark) short wavelength (background plate dark, spots glow)

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.

Zf Zs Zo

 The value of Rf is related to the capacity factor (k) of the solute by the following equation: k = (1- Rf)/ Rf  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 of plates and plate height. N = (Zs / s)2

N = 16*(Zs / Wb)2 Where, 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)

H = Zs /n

 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.  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:

Zf = (xt)1/2 Where,

t = time required by the mobile phase to migrate Zf = distance x = the system constant

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

APPLICATIONS / USES 1) Separation of mixture of chemical,biological,plant origin.

drug

of

2) Separation of Carbohydrates, vitamin, antibiotics, proteins, etc. 3) Identification of drug. Ex :Amoxicillin, Levodopa 4) Detection of foreign substances.

5) To detect the decomposition products of drug.

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

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.

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 Rf 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.

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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