Tlc

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Submitted To- Submitted By-Dr. Sudipta Saha Smita choudhary M.pharm 1st yr (Pharmacology)

Department of Pharmaceutical Sciences,Babasaheb Bhimrao Ambedkar University,Lucknow

CHROMATOGRAPHY

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CONTENT CHROMATOGRAPHY THIN LAYER CHROMATOGRAPHY TYPES INSTRUMENTATION ADVANTAGE AND APPLICATION PAPER CHROMATOGRAPHY PRACTICAL PREPARATION COLUMN CHROMATOGRAPHY PHASE INSTRUMENTATION ADVANTAGE AND DISADVANTAGE APPLICATION

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CHROMATOGRAPHY• Very useful technique in organic chemistry based on differential

adsorption.

• Used to separate components in a mixture (solid or liquid).

• It depends on the polarity of the ingredients involved intermolecular forces.

• Thin layer chromatography (TLC) is used to analyze components and purity of a mixture.

• Thin layer chromatography (TLC) is also used to monitor the progress of a reaction.

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Conti..

What do we need to perform a chromatographic separation?

• Adsorbent: Silica gel (silicon dioxide), also called “stationary phase”

• Eluent: solvent used to move your compound through the silica gel, also called the mobile phase

• Your compound mixture to be separated

• Patience and chemical intuition

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Conti..• More polar solvent move the molecules more efficiently• Less polar move the molecules less efficiently• Chromatography can be divided in two types Planer and

Column TLC is one type planer chromatof ography

ChromatographyPlanerPaper

TLC

Column

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THIN LAYER CHROMATOGRAPHY

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1.THIN LAYER CHROMATOGRAPHY

• Thin layer chromatography, or TLC is a method for analyzing mixtures by separating the compounds in the mixture.

• TLC can be used to help determine the number of components in a mixture, the identity of compounds, and the purity of a compound by observing the appearance of a product or the disappearance of a reactant, it can also be used to monitor the progress of a reaction.

• TLC is a sensitive technique - microgram (0.000001 g) quantities can be analyzed by TLC- and it takes little time for an analysis (about 5-10 minutes)

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Conti..• TLC is performed using thin sheets of glass, aluminum or

plastic coated with a layer of stationary phase, usually silica gel.

• However, other coatings such as alumina, polyamide, cellulose, ion exchangers, and chemically bonded amino or phenol layers can also be used.

• Using polar group like Silica provide chance of separation of polar and non-polar material by using non-polar solvent.

• Readymade TLC sheets are commercially available but mobile phase is chosen according to the type of sample being used

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Conti..TLC consists of three steps – 1)spotting, 2)development and 3)visualization.SPOTTING: In TLC, the sample must be carefully applied to the plate to

minimize spreading. Sample sizes from 0.1 mg to 50 mg are the best for TLC. Smaller amounts are difficult to visualize, while larger spots result in overloading and variable results. Samples should be dissolved in relatively volatile solvents (0.5 to 5mL) so that the spots do not spread excessively(3-4 mm diameter )

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Conti..

Application of sample

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Conti.. DEVELOPMENT- The spotted plate is placed in a sealed development tank filled

with vapor of mobile phase its lower side immersed in solvent to a level below the applied sample spots.

The solvent rises due to capillary flow in a process called development.Development times can range from 3-60 minutes.

Basic model of TLC chromatography chamber

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Conti..VISUALIZATION:

1) Visible - the bands or spots can be seen immediately. 2) Fluorescence - observed under UV light. 3) Absorb UV - plate has an indicator that when irradiated will show

analytes as dark spots on a bright background. 4) Reaction with a chromogenic reagent - These reagents may be

general (reacting with many different compounds) or selected (reacting only with certain functional groups.

5) Organic Materials - treat plates with concentrated sulfuric acid and then heat at 200oC. Analytes show up as dark spots.

6)The plate is sprayed with ninhydrin and heated at hot air oven that produce colour of specimen.

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Conti…

Visualization by UV light

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Conti..

Rf of A=7.5/10=.75

Rf of B=2.5/10=.25

Relative Rf of A on B=7.5/2.5=3

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ADVANTAGE OF TLC

The Thin layer chromatography advantages include: It is simple process with short development time It helps in visualization of separated compound spots easily The method helps to identify the individual compounds It helps in isolation of most of the compounds The separation process is faster and the selectivity for

compounds is higher even small differences in chemistry is enough for clear separation

The purity standards of the given sample can be assessed easily It is a cheaper chromatographic technique

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APPLICATION OF TLC

1. To check purity of given samples2. Identification of compounds like acids, alcohols, proteins,

alkaloids, amines,antibiotics etc3. To evaluate reaction process by assessment of intermediates,

reaction course etc4. To purify samples i.e. for purification process5. To keep a check on the performance of other separation

processes

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2.PAPER CHROMATOGRAPHY

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PAPER CHROMATOGRAPHY• Paper Chromatography (PC) was first introduced by German

scientist Christian Friedrich Schonbein (1865).• PC is considered to be the simplest and most widely used of

the chromatographic techniques because of its applicability to isolation, identification and quantitative determination of organic and inorganic compounds.

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Conti..

ANALYSIS OF UNKNOWN SUSTANCES

It is carried out mainly by the flow of solvents on specially designed filter paper.

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Conti.. TYPES OF PAPER CHROMATOGRAPHY

1.PAPER ADSORPTION CHROMATOGRAPHY Paper impregnated with silica or alumina acts as adsorbent

(stationary phase) and solvent as mobile phase.

2.PAPER PARTITION CHROMATOGRAPHY Moisture / Water present in the pores of cellulose fibers

present in filter paper acts as stationary phase & another mobile phase is used as solvent

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Conti..

PRINCIPLE OF SEPERATION

The principle of separation is mainly partition rather than adsorption

Cellulose layers in filter paper contains moisture which acts

as stationary phase & organic solvents/buffers are used as mobile phase

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Conti..

PRACTICAL REQUIREMENTS 1) Stationary phase & papers used2) Application of sample3) Mobile phase4) Development technique5) Detecting or Visualizing agents

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STATIONARY PHASE AND PAPERS USED whatman filter papers of different grades like no.1, no.2, no.3,

no.4, no.20, no.40, no.42 etc are used. In general this paper contains 98-99% of α-cellulose, 0.3 – 1% β -cellulose

FACTORS THAT GOVERNS THE CHOICE OF PAPER:

» Nature of sample and solvents used. » Based on quantitative or qualitative analysis. » Based on thickness of the paper.

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Conti..

• Modified Papers – acid or base washed filter paper, glass fiber type paper.

• Hydrophilic Papers – Papers modified with methanol, formamide, glycol, glycerol etc.

• Hydrophobic papers – acetylation of OH groups leads to hydrophobic nature, hence can be used for reverse phase chromatography.

• Impregnation of silica, alumna, or ion exchange resins can also be made.

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Conti..• Cut the paper into desired shape and size depending upon

work to be carried out.• The starting line is marked on the paper with an ordinary

pencil 5cm from the bottom edge.• On the staring line marks are made 2cm apart from each

other.

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Conti..

APPLICATION OF SAMPLE The sample to be applied is dissolved in the mobile phase

and applied as a small spot on the origin line, using capillary tube or micropipette.

Very low concentration is used to avoid larger zone The spot is dried on the filter paper and is placed in developing

chamber.

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Conti.. MOBILE PHASE• Pure solvents, buffer solutions or mixture of solvents• Examples- Hydrophilic mobile phase • Isopropanol: ammonia:water 9:1:2• Methanol : water 4:1• N-butanol : glacial acetic acid : water 4:1:5

Hydrophobic mobile phases dimethyl ether: cyclohexane kerosene : 70% isopropanol

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Conti.. The chromatographic chamber are made up of many materials

like glass, plastic or stainless steel. Glass tanks are preferred most. They are available in various dimensional size depending upon paper length and development type

The chamber atmosphere should be saturated with solvent vapor

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Conti..

DEVELOPMENT TECHNIQUE• Paper is flexible when compared to glass plate used in TLC,

several types of development are possible which increases the ease of operation

• The paper is dipped in solvent in such a manner that the spots will not dip completely into the solvent

• The solvent will rise up and it is allowed to run 2/3rd of paper height for better and efficient result

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Conti..Different types of development tech. are

1) ASCENDING DEVELOPMENT (go up)

Like conventional type, the solvent flows against gravity. The spots are kept at the bottom portion of paper and kept in a chamber with mobile phase solvent at the bottom

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Conti..2) DESCENDING TYPE (a downward slope)• This is carried out in a special chamber where the solvent

holder is at the top. The spot is kept at the top and the solvent flows down the paper.

• In this method solvent moves from top to bottom so it is called descending chromatography.

ADVANTAGE IS THAT, DEVELOPMENT IS FASTER

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Conti..

3)ASCENDING – DESCENDING DEVELOPMENT

A hybrid of above two technique is called ascending-descending chromatography.

Only length of separation increased, first ascending takes place followed by descending

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Conti..

4)CIRCULAR / RADIAL DEVELOPMENT Spot is kept at the centre of a circular paper. The solvent

flows through a wick at the centre & spreads in all directions uniformly

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Conti..

5)TWO DIMENSIONAL DEVELOPMENT In this method the paper is developed in one direction and

after development, the paper is developed in the second direction allowing more compounds to be separated into individual spots

In the second direction, either same solvent/different solvent system can be used for development

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Conti..

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APPLICATION

• Separation of mixtures of drugs• Separation of carbohydrates, vitamins, antibiotics, proteins,

etc• Identification of drugs• Identification of impurities• Analysis of metabolites of drugs in blood , urine

ADVANTAGES OF P.C Simple ,rapid ,inexpensive ,excellent resolving power

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COLUMN CHROMATOGRAPHY

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3.COLUMN CHROMATOGRAPHY

Column Chromatography was developed by the American chemist D.T Day in 1900, M.S. Tswett,the Polish botanist, in 1906 used adsorption columns in his investigations of plant pigments

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COLUMN CHROMATOGRAPHY• Column chromatography is one of the most useful methods for

the separation and purification of both solids and liquids. • This is a solid - liquid technique in which the stationary phase

is a solid & mobile phase is a liquid. 

PRINCIPLE• Adsorption Mixture of components dissolved in the M.P is introduced in

to the column. Components moves depending upon their relative affinities

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Conti. Adsorption column chromatography, the adsorbent, packed in

a glass column, and a solvent, the mobile phase, that moves slowly through the packed column. A solvent used as a mobile phase is called an eluent

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Conti..• A compound attracted more strongly by the mobile phase will

move rapidly through the column, and elute from, or come off, the column dissolved in the eluent.

• In contrast, a compound more strongly attracted to the stationary phase will move slowly through the column.

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TYPES OF ADSORBENDS

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Conti..

PREPARATION OF ADSORBENTS; Many adsorbents such as alumina,activated carbon etc.are

available commercially. They often requires activated before use.this can be achieved by

heating,possibley in vaccume,when adsorbent loses water and other adsorbent material.

Generally there is no optimum temp.for activation eg.alumina(about 400 degree C).

The period of heating is also important as long time heating will lose its activity.

Three to four hours is usually sufficient.In most cases heating at 200 for fours hours is safe and desirable for most solids.

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Conti.. If freshly activated solid proves too active for separation,deactivation

may be carried our by controlled addition of water. Powdered sugar,sieved and dried in a desicator has been used for the

separation of colouring matters.DEVELOPERS These are the compound or reagents which are used for the

production of color for colorless substance.These substance should have less affinity for adsorbents than the components to be separated on coloumn.

The general reagent such as hydrogen sulphide ammonium sulphide ,potassium ferrocyanide, potassium thioscynate etc.are used a developers.

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Conti..

SOLVENTS The success of chromatography analysis depends upon the

solid adsorbents and the solvent mobile phase.Solvents plays an important role.Solvents are used-

1-to transfer the mixture to the column2-to effect the process of the process of the development of

chromatogram by which the zones an separated to the highest possible extent and,

3-for the removal of the required content from each zone i.e.for the complete elution

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PACKING TECHNIQUES IN C.C

There are two types of preparing the column, they are:• i. Dry packing / dry filling• Ii. Wet packing / wet filling

DRY PACKING METHOD :

Add dry silica / Alumina to the column and apply to the bottom of

the column. This will compress the silica gel and keep it

compressed for the next steps. Packing can be improved by tapping

the column.

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Conti..

• While applying vacuum; pour solvent in it.

• Allow the solvent to move though the column until reaches to

the bottom. At this stage vacuum is not require.

• Allow 5–6 columns value of solvent to flow through the

column to make sure it is complete packed.

• Drain the solvent till the solvent level is just even with the

surface of the stationary phase

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Conti..

WET PACKING METHOD:

• Fill the column about one third with solvent• In a beaker, measure out the required amount of silica /

alumina.• In another beaker, take solvent approximately one and a half

times the amount of silica / alumina.• Add silica/alumina to the solvent while swirling in small

quantity at a time. Use a glass rod to mix the slurry.

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Conti..

• Pour some of the slurry into column & allow solvent to drain to avoid overflowing.

• Tap the column carefully to encourage bubbles to rise and the silica to settle

• Continue to move the slurry to the column until all the silica or alumina is added.

• Wash the inside of the column by pouring solvent down the inside edge.

• Drain the solvent till the solvent level is just even with the surface of the stationary phase

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DEVELOPMENT TECHNIQUE

(I) ELUTION• By elution technique, the individual components are separated out from

the column. The two techniques are:• (i) Isocratic elution technique : in this elution technique , same solvent

composition or solvent of same polarity is used throughout the process of separation.

• Example: chloroform only(II) GRADIENT ELUTION TECHNIQUES:

( gradient – gradually)• Solvents of gradually ↑ polarity or ↑ elution strength are used during the

process of separation.• E.g. initially benzene, then chloroform, then ethyl acetate then

chloroform

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FACTORS AFFECTING COLUMN EFFICIENCY

1. Dimension of the column: column efficiency has been improved by increasing length/width ratio of the column.

2. Particle size of column packing: separation to be improved by decreasing the particle size of the adsorbent.

3. Activity of the adsorbent4. Temperature of the column: The speed of the elution

increases at higher temperatures.5. Packing of the column6. Quality of solvents: solvents having low viscosities is giving

better results.

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APPLICATIONS

Separation of mixture of compounds Purification process Isolation of active constituents Estimation of drugs in formulation Isolation of active constituents Determination of primary and secondary glycosides in digitalis leaf. Separation of diastereomers

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ADVANTAGES

Any type of mixture can be separated Any quantity of mixture can be separated Wider choice of Mobile Phase Automation is possible

DISADVANTAGE Time consuming more amount of Mobile Phase are required Automation makes the techniques more complicated &

expensive

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