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IN 1958, S TAHL DEVELOPED STANDARD EQUIPMENT FOR ANALYZING BY THIN LAYER

CHROMATOGRAPHY .



DEFINITION

Thin layer chromatography (TLC) is a technique usedtseparate the components of a mixture using a thin stationary

phase supported by an inert backing.

Separation depends on competition between adsorption ofsolute onto the solid surface and its desorption by the solventneeded to elute (wash off) it .

Stationary phase: SolidMobile phase: Liquid



PRINCIPLE

ADSORPTION Chromatography

The component with more affinity travel slowertowards the S.P

The component with lesser affinity travel faster towardsthe S.P.

In TLC separation – hydrogen bonding is mainintermolecular forces involved



Polar molecules stick to plate

Non- polar molecules do not stick to plate

Non-polar molecules will spend a great amount of timedissolved 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



THIN LAYERCHROMATOGRAPHY

BASED ON PURPOSE OF USE

BASED ON THE NATURE OF MOBILEAND STATIONARY PHASE USED

NORMAL PHASE TLC

REVERSE PHASE TLC

ANALYTICAL PREPARATIVE TLC



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 stationaryphase

Silica gel C4 ,c8 – bonded phase

COMPARISON OF NORMAL PHASE &REVERSE PHASE



INSTRUMENTATION

COMPONENT PURPOSE

Developing chamber Create and maintainenvironment forchromatography

Solid support(chromoplates)

Supports thin film ofstationary 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 ADSORBENTTwo properties decide the selection:

1. particle size2. homogeniscity

Factors affecting selection:1. Colorless2. should have great mechanical strength3. should not catalyze or decompose ofsubstance4. should be insoluble with mobile phase & the solvent

used for elution5. no reaction at time of separation

. Adsorbent do not adhere to glass plate



CLASSIFICATION OF ADSORBENTS USED1. Classification according to binding strength:A. Weak adsorbent: sucrose, starch, talc, cellulose

B. Intermediate adsorbent: silica gel, calcium carbonate, calciumphosphate, magnesia

C. Strong adsorbent: alumina, charcoal

2. Classification according to nature:A. Inorganic adsorbent: Silica, Silica gel, Alumina, Calciumphosphate, Glass powder, Kieselguhr ,Magnesium silicate, Calciumsilicate, 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 (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.



CELLULOSE

Cellulose (C6H10O5)n is a long chain polymericpolysaccharide carbohydrate of β – glucose

Adsorbed water or alcohol can be retained by interactionwith 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 withchemical formula – Al 2O3

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 + CaSO 4

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 itis polar or non-polar.

2) Mode of Chromatography

3) Nature of Stationary phase

4) Mode Separation i.e Analytical or Preparativetechnique

Examples: 1) Petroleum ether 2) Cyclohexane

3) Acetone 4) Toluene5) Ethyl acetate 6) Benzene7) Alcohols 8) Water

9) Chloroform 10) Pyridine



CHOICE OF SOLVENT

Selection of M.P depends upon nature of substance to beseparated

Viscosity and polarity of S.P

Solvent used may be single or double phase system

e.g: n-hexane < cyclohexane< CCl 4 < 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 PLATETLC 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 theirdifferent degrees of interaction with matrix & solubility in thedeveloping solvent



Non polar solvents force non polar compounds to topof plate because the compounds dissolve well & do notinteract with polar S.P

Allow the solvent to travel up the plate until 1 cmfrom top

Take the plate out and mark the solvent frontimmediately.

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) Pouring2) 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 ONTHE 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 orother volatile solvent.

3. SPRAYING- simply by spraying slurry on plate

4. SPREADING- slurry spread by using spatula or glassrod



ACTIVATION OF PLATE



ACTIVATION OF PLATE

plate dried and activated by heating in oven for 30 minutesat 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



APPLICATION OF SAMPLE

Go for development

Spotting area should not be immersed in the Mobile phase

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

Spots can be placed at random process

Sample is spotted using a capillary tube or micropipette

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





ADVANTAGESLow cost

Short analysis timeAll spots can be visualizedAdaptable to most pharmaceuticals

Uses small quantities of solventsRequires minimal trainingReliable and quickMinimal amount of equipment is neededDensitometers can be used to increase

accuracy of spot concentration



TLC SUPERIOR OVER OTHER METHODSIt requires little equipment

Require little time for separationIt is more sensitiveVery small quantity of sample require for analysis

The method use for adsorption, partition, ionexchange chromatographyComponent which are separated can be recovered

easily .

Quantative separation of spot and zone are possibleFor identification is permittedSpraying of corrosive agent



Development tankThe 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 rectangularchromatography tank or chamber .

• Glass and stainless steel are suitable chambers.

• If tank is not saturated, solvent will evaporateand affect the R f value.

• Development should be carried out at roomtemperature 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.

DEVELOPMENT TECHNIQUE

DETECTING AGENTS



Detecting agents are two types:(A)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 foridentification "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

DETECTION

R value is constant for each component only under



Rf value is constant for each component only underidentical experimental condition.Polar compounds have low R f value

It depend on following factors-Nature of adsorbentMobile phase

ActivityThickness of layerThe temperature

EquilibrationLoadingDipping zone

Chromatographic technique



DEVELOPMENT OF T L C

VISUALIZATION METHOD



VISUALIZATION METHODPrevious 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 spotscolored reagents to stain spotsreagents that selectively stain spots leaving othersunaffected

VARIOUS TECHNIQUES TO VISUALIZE THE COMPOUNDS:



VARIOUS TECHNIQUES TO VISUALIZE THE COMPOUNDS:

1. Sulfuric acid/ heat: destructive, leaves charred blotsbehind

2. ceric stain: destructive, leaves a dark blue blotbehind polar compounds

3. Iodine: semi- destructive , iodine absorbs onto thespots , not permanent

4. 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 :Rf = Zs / (Z f – Zo)

Zf : Distance traveled by the solvent front from the point ofapplication.

Zs: Distance traveled by the solute front from the point ofapplication.

Zo: Distance between the point of application of solvent and solute.

Z f

Z s

Z o

The value of R f is related to the capacity factor (k) of the solute by



The value of R f is related to the capacity factor (k) of the solute bythe following equation:

k = (1- Rf )/ R f

By using the above equation, planar chromatography can be used toobtain estimates of k for a solute on different stationary phase andmobile phase combinations.

This can be useful in screening a number of columns or mobilephase for use in column liquid chromatography.EFFICIENCY

The efficiency of a separation in planar chromatography isdescribed in terms of plates and plate height.

N = (Zs / ) 2

N = 16*(Z s / W b)2 H = Zs /n

Where,N: number of theoretical plates; H: plate height

: standard deviation of the solute band (in distance units)

Wb: baseline width of the solute band (in distance units)

Note that the efficiency of a planar system is not constant, but



Note that the efficiency of a planar system is not constant, butdepends on the distance that the solute has traveled, or its retentionand R f value.

The change in efficiency of a planar chromatography system withdistance and the presence of a third phase have made the derivationof exact plate height equations for planar chromatography difficult.

These concurrently occur with another complicating factor: the flowrate 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 migrateZf = distancex = the system constant

PERFORMING THE TLC ANALYSIS: CALCULATE THE RF VAL



PERFORMING THE TLC ANALYSIS: CALCULATE THE RF VAL

The R f value is calculated by measuring the distance the

sample zone travels divided by the distance the developingsolvent 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 eachother

Above 0.8: poor: spots may be too broad or distorted

APPLICATIONS USES



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

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.

APPLICATIONS USES



6). To determine how many compounds are there ina mixture – is it real pure?7). To determine the best solvent conditions forseparation on column8). To identify the substances being studied9). To monitor the compositions & appropriateconditions of the fractions collected from ColumnChromatography10). To monitor the progress of the reaction11). To determine identity of two substances

12). To determine effectiveness of purification

TLC TROUBLESHOOTING



TLC TROUBLESHOOTING1. CAUSE:the compound runs as streak rather than a spot

REASON: the sample was overloaded

Run the TLC again after diluting your sampleSample might contain many componentsIt creates many spots which run together & appear asstreak

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 aceticacid (carboxylic acids) to the eluting solvent to obtainclear plates.

3. CAUSE:the sample runs as a downward crescent (moon)



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 plateAdsorbent moved towards the side of the plateor touching the sides of the container or the

paper used to saturate the container as platedevelops.Crookedly run plates makes it harder to measurethe R f value accurately.

5. CAUSE:many random spots are seen on the plateREASON: accidently check not any organic compound on

the plate or any new foreign substance touched

incidentally.

6. CAUSE: no spots seen on plate



p pREASON: you might have not spotted enough compound,

perhaps because the solution of the compound is toodilute.Try concentrating the solution or else spot it severaltimes in one place allowing solvents to dry b/w capillariesSome compounds do not show under UV lightTry another method of visualization of platePerhaps you don’t have any compounds because theexperiment did not go as well planned

If solvent level in developing jar is deeper than the originof the TLC plateSolvent will dissolve the compounds into the solventreservoir

It allows them to move up the plate by capillary actions.

Thus you will not see the spots after the plate is developed.



THANK YOU

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jo tlc.ppt