THIN LAYER CHROMATOGRAPHYPresented By

Mr. K.Sudheerkumar M Pharmacy., (PhD)Faculty, Sri Shivani College of PharmacyMulugu road,Warangal, Telangana State

India, E. mail : ksum2810@gmail.com

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.

TLC is universal analytical technique in chemical analysis for TLC is universal analytical technique in chemical analysis for separation of organic and inorganic matter.separation of organic and inorganic matter.

In In 19381938, Izmailov and Shraiber describe basic principle used , Izmailov and Shraiber describe basic principle used it for separation of plant extract.it for separation of plant extract.

In In 19441944, Consden, Gorden & Martin used filter papers for , Consden, Gorden & Martin used filter papers for separating the Amino acids.separating the Amino acids.

In In 19501950, Kirchner identified terpenes on filter paper., Kirchner identified terpenes on filter paper.

In In 19581958, Stahl mainly created with bringing out the work on , Stahl mainly created with bringing out the work on preparing plates and separation of wide variety of preparing plates and separation of wide variety of compounds.compounds.

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.

PRINCIPLEPRINCIPLE

The basic Principle is based on ADSORPTION The basic Principle is based on ADSORPTION ChromatographyChromatography

The component with more affinity towards the S.P- travel The component with more affinity towards the S.P- travel slower slower

The component with lesser affinity towards the S.P - travel The component with lesser affinity towards the S.P - travel faster faster

TLC is simple and rapid method - carried out by using thin TLC is simple and rapid method - carried out by using thin layer of adsorbent on plates.layer of adsorbent on plates.

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

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

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.

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 neededDensitometers 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 analysisThe 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

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 size2. homogeniscity

Factors affecting selection:1. Colorless2. should have great mechanical strength3. should not catalyze or decompose of

substance4. should be insoluble with mobile phase

& the solvent used for elution 5. no reaction at time of separation

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

CLASSIFICATION OF ADSORBENTS USED1.Classification according to binding strength:A.Weak adsorbent: sucrose, starch, talc, celluloseB. Intermediate adsorbent: silica gel, calcium carbonate, calcium phosphate, magnesiaC.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 units2. 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

Reverse Phase ChromatographyReverse Phase Chromatography In this the S.P is Non-polar & M.P is polar & it is widely In this the S.P is Non-polar & M.P is polar & it is widely used used in pharmaceutical analysis.in pharmaceutical analysis.

1. Polar compounds get eluted first 1. Polar compounds get eluted first 2. Non-polar compounds are retained for long time2. Non-polar compounds are retained for long timeComparison of Normal Phase & Reverse Phase :Comparison of Normal Phase & Reverse Phase :

PARAMETER NORMAL PHASENORMAL PHASE REVERSE PHASEREVERSE PHASE

Stationary phaseStationary phase PolarPolar Non-polarNon-polar

Mobile phaseMobile phase Non-polarNon-polar Polar Polar

Compound eluted Compound eluted firstfirst

Non-polarNon-polar PolarPolar

Compound eluted Compound eluted lastlast

PolarPolar Non-polarNon-polar

Example of stationary Example of stationary phasephase

Silica gelSilica gel CC44 ,c ,c88 –bonded phase –bonded phase

STATIONARY PHASENAMENAME COMPOSITIONCOMPOSITION

Silica gel HSilica gel H Silica gel without binderSilica gel without binder

Silica gel GSilica gel G Silica gel + CaSOSilica gel + CaSO44

Silica gel GFSilica gel GF Silica gel + Binder + fluorescent Silica gel + Binder + fluorescent indicatorindicator

AluminaAlumina AlAl22003 3 Without Binder Without Binder

AlAl22003 3 G G AlAl22003 3 + Binder + Binder

Cellulose powderCellulose powder Cellulose Without BinderCellulose Without Binder

Cellulose powderCellulose powder Cellulose With BinderCellulose With Binder

Kieselguhr GKieselguhr G Diatomaceous earth + binderDiatomaceous earth + binder

Polyamide powderPolyamide powder PolyamidePolyamide

Fuller’s earthFuller’s earth Hydrous magnesium aluminaHydrous magnesium alumina

Magnesium SilicateMagnesium Silicate magnesolmagnesol

MOBILE PHASE1)1)Nature of the substance to be separated i.e whether it is Nature of the substance to be separated i.e whether it is polar or non-polar.polar or non-polar.

2)2)Mode of ChromatographyMode of Chromatography

3)3)Nature of Stationary phaseNature of Stationary phase

4)4)Mode Separation i.e Analytical or Preparative techniqueMode Separation i.e Analytical or Preparative technique

Examples: 1) Petroleum ether 2) CyclohexaneExamples: 1) Petroleum ether 2) Cyclohexane 3) Acetone 4) Toluene3) Acetone 4) Toluene 5) Ethyl acetate 6) Benzene5) Ethyl acetate 6) Benzene 7) Alcohols 8) Water7) Alcohols 8) Water 9) Chloroform 10) Pyridine9) Chloroform 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 : Three types :

1)1)Full plate : 20cm × 20 cm.Full plate : 20cm × 20 cm.

2)2)Half plate : 20cm × 10 cm.Half plate : 20cm × 10 cm.

3)3)Quarter plate : 20cm × 5 cm.Quarter plate : 20cm × 5 cm.

Microscopic slides can also be used for monitoring Microscopic slides can also be used for monitoring

the progress of a chemical reaction.the progress of a chemical reaction.

DEVELOPING A PLATE TLC plate can be developed in beaker or closed jar.Draw the base line on TLC plate which is 1cm above from lower edge of plate and apply the spots on base line. Place a small amount of solvent in container. Solvent level below the starting line(Base line)of TLC because it its above the base line spots dissolve. Low edge of plate dipped in solvent. Solvent travels up the matrix or adsorbent(S.P) by capillarity action. Moving components of samples at various rates because of their different degrees of interaction with matrix & solubility in the developing solvent (M.P).

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 :The T L C plates can be prepared by following techniques :

1)1)PouringPouring

2)2)DippingDipping

3)3)SprayingSpraying

4)4)SpreadingSpreading

Activation :It is nothing but removing of water/ moisture & other Activation :It is nothing but removing of water/ moisture & other

adsorbed substance from the surface of any adsorbent by heating.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 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

APPLICATION OF SAMPLE

DEVELOPMENT CHAMBER / TANK

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.

The development tank The development tank

should be lined Insideshould be lined Inside

with filter paper moistenedwith filter paper moistened

with mobile phase to with mobile phase to

saturate the atmospheresaturate the atmosphere

& also prevent the & also prevent the

“ “ EDGE EFFECT ” .EDGE EFFECT ” .

Different development techniques are :Different development techniques are :

1)1)One dimensional development.One dimensional development.

2)2)Two dimensional development.Two dimensional development.

3)3)Horizontal development.Horizontal development.

4)4)Multiple development.Multiple development.

DEVELOPMENT TECHNIQUEDEVELOPMENT TECHNIQUE

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.

DEVELOPMENT OF T L CDEVELOPMENT 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

Detecting agents are two types:Detecting agents are two types:(A)(A)Non-Specific methodNon-Specific method 1) Iodine chamber method.1) Iodine chamber method. 2) Sulphuric acid spray method.2) Sulphuric acid spray method. 3) UV chamber for fluorescent compounds.3) UV chamber for fluorescent compounds. 4) Using fluorescent stationary phase.4) Using fluorescent stationary phase.

(B) Specific method(B) Specific method 1) Ferric chloride – for Phenolic compounds and Tannins.1) Ferric chloride – for Phenolic compounds and Tannins. 2) Ninhydrine in acetone – For Amino acids.2) Ninhydrine in acetone – For Amino acids. 3) Dregendroff reagent – For Alkaloids.3) Dregendroff reagent – For Alkaloids. 4) 3,5 – Dinitro benzoic acid – For Cardiac glycosides. 4) 3,5 – Dinitro benzoic acid – For Cardiac glycosides. 5) 2,4 - Dinitro phenyl hydrazines – For Aldehyd and Ketones. 5) 2,4 - Dinitro phenyl hydrazines – For Aldehyd and Ketones.

DETECTING AGENTSDETECTING AGENTS

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)

The RThe Rff value is calculated for value is calculated for

identification "Rfidentification "Rf value is the value is the

ratio of distance travelled byratio of distance travelled by

The solute to the distanceThe solute to the distance

travelled by the solvent front”travelled by the solvent front”

Distance travelled by soluteDistance travelled by solute

RRf f = =

Distance travelled by solvent frontDistance travelled by solvent front

DETECTIONDETECTION

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

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

1)1) Separation of mixture of drug of Separation of mixture of drug of chemical,biological,plant origin.chemical,biological,plant origin.

2)2) Separation of Carbohydrates, vitamin, antibiotics, Separation of Carbohydrates, vitamin, antibiotics, proteins, etc.proteins, etc.

3)3) Identification of drug. Ex :Amoxicillin, LevodopaIdentification of drug. Ex :Amoxicillin, Levodopa

4)4) Detection of foreign substances.Detection of foreign substances.

5)5) To detect the decomposition products of drug. To detect the decomposition products of drug.

APPLICATIONS / USESAPPLICATIONS / USES

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 column8). To identify the substances being studied9). To monitor the compositions & appropriate conditions of the fractions collected from Column Chromatography10). To monitor the progress of the reaction11). To determine identity of two substances12). To determine effectiveness of purification

TLC TROUBLESHOOTING1. 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 caused4. 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.

THANK YOUTHANK YOU