Paper Chromatography

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I/II , I st Semester M.Pharmacy Dept. of Pharmaceutical Analysis JNTUH, Hyderabad. Ravi Pratap Pulla M.Pharm., Ph.D Associate Professor SSJ College of Pharmacy, V.N.Pally, Gandipet, Hyderabad-75 Andhra Pradesh.

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 Paper Chromatography (PC) was first introduced by German scientist Christian Friedrich Schonbein (1865 A.D).  The discovery of paper chromatography in 1943 by Martin and Synge  PC is considered to be the simplest and most widely used the chromatographic techniques  because of its applicability to isolation, identification and quantitative determination of organic and inorganic compounds.

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 PC is meant for analysis of unknown substances  It is carried out mainly by the flow of solvents on a specially designed filter paper.

What is Chromatography?:

What is Chromatography?  Chromatography is a laboratory technique for separating components within mixtures in order to analyze, identify, purify and /or quantify the mixture or its components.  MIXTURE: a material composed of two or more elements or parts.  COMPONENT: a constituent, element, or part

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Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components. Separate Analyze Identify Purify Quantify Components Mixture Illustration of Separating a Mixture  Analyze – examine a mixture, its components, and their relations to one another Identify – determine the identity of a mixture or components based on known components Purify – separate components in order to isolate one of interest for further study  Quantify – determine the amount of the a mixture and/or the components present in the sample

Uses for Chromatography:

Uses for Chromatography Real-life examples of uses for chromatography:  Pharmaceutical Company – determine amount of each chemical found in new product  Hospital – detect blood or alcohol levels in a patient’s blood stream  Law Enforcement – to compare a sample found at a crime scene to samples from suspects  Environmental Agency – determine the level of pollutants in the water supply  Manufacturing Plant – to purify a chemical needed to make a product

Definition of Chromatography:

Definition of Chromatography Detailed Definition: Chromatography is a laboratory technique that separates components within a mixture by using the differential affinities of the components for a mobile medium and for a stationary adsorbing medium through which they pass. Terminology :  Differential – showing a difference, distinctive  Affinity – natural attraction or force between things  Mobile Medium – gas or liquid that carries the components ( mobile phase)  Stationary Medium – the part of the apparatus that does not move with the sample ( stationary phase )

Definition of Chromatography:

Simplified Definition : Chromatography separates the components of a mixture by their distinctive attraction to the mobile phase and the stationary phase. Explanation :  Compound is placed on stationary phase  Mobile phase passes through the stationary phase  Mobile phase solubilizes the components  Mobile phase carries the individual components a certain distance through the stationary phase, depending on their attraction to both of the phases Definition of Chromatography

Illustration of Chromatography:

Illustration of Chromatography Components Affinity to Stationary Phase Affinity to Mobile Phase Blue ---------------- Insoluble in Mobile Phase Black         Red        Yellow           Mixture Components Separation Stationary Phase Mobile Phase

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LIQUID CHROMATOGRAPHY – separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase) GAS CHROMATOGRAPHY – separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads (stationary phase) PAPER CHROMATOGRAPHY – separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase) THIN-LAYER CHROMATOGRAPHY – separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase) Types of Chromatography

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(A) uses charge, (B) uses pores, and (C) uses covalent bonds to create the differential affinities among the mixture components for the stationary phase.

Principles of Paper Chromatography:

Principles of Paper Chromatography  CAPILLARY ACTION – the movement of liquid within the spaces of a porous material due to the forces of adhesion, cohesion, and surface tension. The liquid is able to move up the filter paper because its attraction to itself is stronger than the force of gravity.  SOLUBILITY – the degree to which a material (solute) dissolves into a solvent. Solutes dissolve into solvents that have similar properties. (Like dissolves like) This allows different solutes to be separated by different combinations of solvents.  SEPARATION- of components depends on both their solubility in the mobile phase and their differential affinity to the mobile phase and the stationary phase.

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PRINCIPLE INVOLVED BEHIND  The principle involved in separation by paper chromatography is largely by partition coefficient phenomenon.  Separation of components depends on both their solubility in the mobile phase and their differential affinity to the mobile phase and stationary phase.

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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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TYPES OF PAPER CHROMATOGRAPHY  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  Filter paper –act as an inert support  Water –stationary phase  Organic solvent –mobile phase  Mechanism –partition between the two phases  PAPER ADSORPTION CHROMATOGRAPHY  Paper impregnated with silica or alumina acts as adsorbent (stationary phase) and solvent as mobile phase.  Impregnated filter paper with alumina or silica stationary phase  Alumina or silica –stationary phase  Organic solvent –mobile phase  Mechanism –adsorption IN GENERAL P.C – PAPER PARTITION CHROMATOGRAPHY

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THEORY  Two types of forces operate when a drop of solution is applied on the filter paper and treated with a solvent: (A) PROPELLING FORCE (B) IT TRIES TO DRAG THE SUBSTANCES IN THE DIRECTION OF THE FLOW OF THE SOLVENT.  This depends upon: (A) THE RATE OF THE SOLVENT FLOW (B) The solubility of the substance in the solvent  The component with higher solubility will move rapidly along the filter paper than the less soluble component

Rf VALUE::

In paper chromatography the results are represented by R f value It represents the movement or migration of solute relative to the solvent front. R f VALUE: The R f value is calculated as :- Distance travelled by the solute Distance travelled by the solvent front Retardation force / FACTOR

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Factors affecting R f values The temperature The solvent used Quality of the paper, adsorbents & impurities present n the adsorbents Technique employed The distance travelled by the solvent and the solute Chemical reactions between the substance Purity of the solution The concentration of the separated substance pH of the solution Chamber saturation technique

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Techniques of paper chromatography  The various operations involved in PC are  Choice of filter paper depends upon:  Thickness  Flow rate  Purity  Net strength of paper. (Generally Whatman filter papers are 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.  Chemical composition of Whatman paper  α -cellulose 99%  β-cellulose 0.3-1%  Pentosans 0.5-0.8%  Ash 0.01-0.07%  Ether soluble substance 0.01-0.1%

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TYPES OF PAPERS  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, alumina, or ion exchange resins can also be made.

PAPER CHROMATOGRAPHY EXPERIMENT PRACTICAL REQUIREMENTS 1)Stationary phase & papers used 2)Application of sample 3)Mobile phase 4)Development technique 5)Detecting or Visualizing agents :

PAPER CHROMATOGRAPHY EXPERIMENT PRACTICAL REQUIREMENTS 1)Stationary phase & papers used 2)Application of sample 3)Mobile phase 4)Development technique 5)Detecting or Visualizing agents

MATERIALS LIST:

 6 beakers or jars  6 covers or lids  Distilled H 2 O  Isopropanol & many more  Graduated cylinder  6 strips of filter paper  Different colors of Sharpie pens  Pencil  Ruler  Scissors  Tape MATERIALS LIST

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PREPARATION OF PAPER  Cut the paper into desired shape and size depending upon work to be carried out.  The most common shape of the filter paper is rectangular. Although square paper can also be used  on the paper with an ordinary pencil 5 cm from the bottom edge.

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PREPARATION OF THE SOLUTION  Choice of suitable solvent for making solution is very important.  Pure solution can be applied directly on the filter paper but solids are always dissolved in small quantity of a suitable solvent  Concentrated solutions are usually applied on the filter paper to avoid diffusion.  Biological tissues are treated with suitable solvents and their extracts obtained.  Proteins can be precipitated with alcohol and salts can be removed by treatment with ion exchange resin.

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APPLICATION OF SAMPLE  The sample mixture to be separated is applied as a small spot on the origin line .  The spot is dried on the Filter paper and is placed in developing chamber .  Micropipette or glass capillary is used for sample application.

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CHOICE OF THE SOLVENT  The commonly employed solvents are the polar solvents, but the choice depends on the nature of the substance to be separated.  If pure solvents do not give satisfactory separation, a mixture of solvents of suitable polarity may be applied.  The solvent selection depends upon nature of substance to be separated.

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 A number of solvents can be used in the paper chromatography.  Some examples of solvents are:  Ethyl alcohol  n-Butanol  N-Hexane  water  Benzene  methanol  Toluene  chloroform  These solvents are used in different ratio with different mixtures….

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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

CHROMATOGRAPHIC CHAMBER:

 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. CHROMATOGRAPHIC CHAMBER

DEVELOPMENT OF CHROMATOGRAM:

 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 by capillary action. It is allowed to run 2/3 rd of paper height for better and efficient result.  After development is complete paper is taken out of the chamber carefully .  Paper is flexible when compared to glass plate used in TLC, several types of development are possible which increases the ease of operation. DEVELOPMENT OF CHROMATOGRAM

DEVELOPING THE CHROMATOGRAM:

DEVELOPING THE CHROMATOGRAM  Place the strips in the beakers  Make sure the solution does not come above your start line  Keep the beakers covered  Let strips develop until the ascending solution front is about 2 cm from the top of the strip  Remove the strips and let them dry

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DRYING OF CHROMATOGRAM  After the solvent has moved a certain distance for certain time the chromatogram is taken out from the tank & position of the solvent front is marked with a pencil.  The chromatogram is dried after its development  They are dried by cold or hot air depending on volatility of solvents.  A simple hair dryer is a convenient device to dry chromatograms.

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LOCATION OF SPOT  If the substance are colored they are visually detected easily. But for colorless substance.  Physical and chemical methods are used to detect the spot.  Physical method: In this method observation are done under UV light, iodine chamber, detection of fluorescence and radioisotope measurements.  Chemical method / spraying method: colorless compound can be detected by converting them into colored compound by reaction with some reagents.  Amino Acids- Ninhydrin Reagent  Alkaloids- Dragendroff’s Reagent  Ferric Chloride – phenolic compounds and tannins  3, 5 dinitro benzoic acid – cardiac glycosides

OBSERVING THE CHROMATOGRAM:

OBSERVING THE CHROMATOGRAM CONCENTRATION OF ISOPROPANOL 0% 20% 50% 70% 100%

BLACK DYE:

BLACK DYE CONCENTRATION OF ISOPROPANOL 0% 20% 50% 70% 100% 1. Dyes separated – purple and black 2. Not soluble in low concentrations of isopropanol 3. Partially soluble in concentrations of isopropanol >20%

BLUE DYE:

BLUE DYE CONCENTRATION OF ISOPROPANOL 0% 20% 50% 70% 100% 1. Dye separated – blue 2. Not very soluble in low concentrations of isopropanol 3. Completely soluble in high concentrations of isopropanol

GREEN DYE:

GREEN DYE CONCENTRATION OF ISOPROPANOL 0% 20% 50% 70% 100% 1 . Dye separated – blue and yellow 2. Blue – Soluble in concentrations of isopropanol >20% 3. Yellow – Soluble in concentrations of isopropanol >0%

RED DYE:

RED DYE 1. Dyes separated – red and yellow 2. Yellow –soluble in low concentrations of isopropanol and less soluble in high concentrations of isopropanol CONCENTRATION OF ISOPROPANOL 0% 20% 50% 70% 100% 3. Red – slightly soluble in low concentrations of isopropanol , and more soluble in concentrations of isopropanol >20%

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DEVELOPMENT TECHNIQUES 1) ASCENDING DEVELOPMENT (go up)  Like conventional type, the solvent flows against gravity & migrates upward by capillary action.  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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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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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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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.

TWO DIMENSIONAL PAPER CHROMATOGRAPHY :

TWO DIMENSIONAL PAPER CHROMATOGRAPHY  one dimensional chromatography is quite satisfactory for separation for separation of many mixtures.  But for complex mixtures, two dimensional developments (i.e. the development of the chromatography in the other direction at right angle to the first, using another solvent) can be carried out. First solvent flow D B C A D C B A ABCD Second solvent front

Advantages:

Advantages  Simplicity and availability of materials and equipment  High efficiency of separation  The characterization of substances in mixtures without prior separation of individual components  Good separation of homologues  Its applicability to structural analysis  The study the reaction kinetics

Quantitative estimation:

Quantitative estimation There are several methods for quantitative determination of a particular compound in a mixture. The choice of method depends upon properties, composition of the compound and degree of accuracy required. The methods can be divided into four types: 1.Direct measurement methods  Comparison of Visible Spots  Photodensitometry  Fluorimetry  Radiotracer method Polarographic and conductometric methods 2. Indirect measurement method 3. Destructive technique 4. Non destructive technique

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DIRECT MEASUREMENT METHOD (I) COMPARISON OF VISIBLE SPOTS A rough quantitative measurements Component in a mixture can be carried out by comparing the intensity and size of the spot with a standard substance. (II) PHOTO DENSITOMETRY The method is used with the chromatograms of colored compound, instrument which measures quantitatively the density of the spots.

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(iii) Fluorimetry  The compound to be determined by fluorimetry must be fluorescent or convertible into fluorescent derivatives. (iv) Radiotracer Method  The compound containing radioactive element is labeled and treated with locating reagent. Using Geiger Muller counter. (v) Polarographic & Conductometric methods  Used to measure the amount of material in the spot

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3) Destructive techniques  Specific spray reagents, samples destroyed before detection e.g. – ninhydrin reagent 4) Non-destructive techniques  For radio active materials – Geiger Muller counter, UV chamber, iodine chamber R x VALUE  In many cases it has been observed that the solvent front runs off the end of the paper. R x value is used.  It is the ratio of distance travelled by the sample and the distance travelled by the standard. R x value is always closer to 1.

ELUTION METHOD:

ELUTION METHOD S M S S M S S M S X X X X X X X X X Standards Origin line Spot containing known volume of mixture Paper before development Area to be eluted Paper After development

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SOURCES OF ERROR 1. Error during application of the spots  Apply minimum volume of the concentrated solution in order to avoid diffusion through the paper which leads to poor separation  Spots should be approximately of the same diameter. 2. Development  Improper adjustment of the paper in the tank leads to this error so the paper should be held vertically.  Do chamber saturation 3. Detection  The spraying methods affect the final result.

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APPLICATIONS  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 PRECAUTIONS IN P.C  Establishing the vapor solvent equilibrium  Stability of solvent mixture is first ensured

ALTERNATIVE EXPERIMENTS:

ALTERNATIVE EXPERIMENTS  Test different samples:  Other markers, pens, highlighters  Flower pigments  Food Colors  Test different solvents:  Other alcohols: methanol, ethanol, propanol, butanol  Test different papers:  Coffee filters  Paper towels  Cardstock  Typing paper

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Experiment 1-to separate cations (Pd 2+, Ag + , Hg 2+ )  Solvent: Distilled water or ethyl alcohol  Locating agent:0.25 M potassium chromate solution  Colour: Pd-Yellow,0.08, Ag-Orange red,0.16 Hg-Orange,0.85 Experiment 2- To separate mixture of amino acids – arginine, glutamic acid, Lycine and aspartic acid  Solvent: Ethanol water ammonia in the ratio of 20:2.5:2.5  Locating agent:300mg ninhydrin dissolved in 100ml acetone  Color: Blue spots compare the R f value with the literature value Experiment 3- To separate the cations (Hg 2+ .CU 2+ .Cd 2+ .Bi 3+ )  Solvent: n-butanol alcohol and HCl  Locating agent: Conc solution of dithizone in chloroform  Color: Cu-brown. Hg-pink. Cd-purple. Bi-brown

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