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CHROMATOGRAPHY Definition : Chromatography is process of separation of a mixture into individual components using stationary phase and mobile phase. In Analytical Terms, Chromatography is a non- destructive procedure for resolving a multi-component mixture of trace, minor, or major constituents into individual fractions. IUPAC DEFINITION: It is a method used primarily for the separation of the components of a sample, in which the components are distributed between two phases, one of which is stationary while the other moves . The stationary phase may be solid or a liquid supported on a solid or a gel, and may be packed in a column, spread as a layer or distributed as a film. The mobile phase may be gaseous or liquid.


CLASSIFICATION Principle of Separation Modes of Chromatography Elution technique Type of Analysis Chromatography Nature of Stationary phase and Mobile phase

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Nature of stationary and mobile phase Gas - Solid chromatography Gas - Liquid chromatography Solid - Liquid chromatography E.g. : Column chromatography, thin layer chromatography, HPLC (High performance liquid chromatography) LIQUID CHROMATOGRAPHY E.g. : (Paper chromatography, column partition Chromatography)

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CHROMATOGRAPHY Adsorption completion between solid and Partition completion between liquid and Ion exchange Molecular sieve Gas Gas-Solid Chromatography (GSC) Liquid Liquid Ion Exchange Chromatography Gel Chromatography Column Chromatography Thin Layer Chromatography Column Chromatography Paper Chromatography Thin Layer Chromatography Gas Gas-Liquid Chromatography (GLC) HPTLC HPLC

2. Principle of Separation:

2. Principle of Separation A] Adsorption chromatography When mixture of compounds are dissolve in mobile phase (eluent ), it moves through a column of stationary phase (adsorbent), they travel with relative affinity towards the stationary phase. The compounds have more affinity towards the stationary phase travels slower and compounds have a lesser affinity to ward the stationary phase travel faster. Hence the compound are separated. No two component have the same affinity for combination of stationary phase, mobile phase, and other conditions. E.g., : Thin layer chromatography, HPLC, Column Chromatography,Gas-Solid Chromatography.

B] Partition Chromatography :

B] Partition Chromatography When two immiscible liquid presents , a mixture of solute will be distributed according to their partition co-efficient. When mixture of compounds dissolve in mobile phase and passed through a column of stationary phase. The component which is more soluble in the stationary phase travels slower. The component which are less soluble in stationary phase travels faster. Thus component are separated because the difference in their partition co-efficient. The stationary phase as such cannot be a liquid, hence a solid support is used over which a thin film or coating of liquid is made which acts as a stationary phase. E.g.,(Gas-liquid chromatography, Paper chromatography , column partition chromatography)

Other types of chromatography:

Other types of chromatography Ion exchange Chromatography The principle of separation is ion exchange, which is reversible exchange of functional groups. In ion exchange Chromatography, an ion exchange resin is used to separate a mixture of similar charged ions. A cation exchange resin is used for separation of cation and anion exchange resin is used to separate a mixture of anions .

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Chiral phase Chromatography In this Chromatography, optical isomer (levo and dextro form) can separated by using chiral stationary phase. The stationary phases used for this type of chromatography are mostly chemically bonded silica gels.

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Gel permeation Chromatography A gel is used to separate the components of a mixture according to their molecular sizes. Different gels are used for different molecules. The gels act as molecular sieve and hence mixtures of substance are separated according to their molecular size. The solvent used can be aqueous or non- aqueous type . The stationary phase is a porous matrix . The mechanism involved in separation process is the Diffusion effects in the pores of different gels .

3] Modes of Chromatography:

3] Modes of Chromatography It consists of two modes : Normal phase mode and Reverse phase mode. This modes are basically based on the polarity of stationary phase and mobile phase. Normal phase mode In normal phase mode, the stationary phase is polar in nature and mobile phase is non- polar. In this technique, non polar compound are travels faster and eluted first. This is because less affinity between solute and stationary phase. Polar compounds are retained for longer time in the column because of more affinity towards the stationary phase and take more time to be eluted from the column. This method is not advantageous in the pharmaceutical application since, most of drug mole are polar in nature and required more time to be eluted. E.g., (silica gel) Reverse phase mode Stationary phase - non-polar in nature Mobile phase- polar in nature Hence, polar compound are eluted first and non-polar compounds are retained for longer time. E.g., ODS (octadecyl silane )

4] Elution technique :

4] Elution technique Elution analysis :- In this method small volume of mixture to be separated is added on the top of column and mobile phase is allowed to flow through column. As mobile phase move down the column, the mixture introduced on the column gets separated into zones as the component of mixture are absorbed to the column material to different extent. E ach components of mixture is eluted out as separate component. Isocratic separation :- In this technique, the same mobile phase combination is used throughout the process of separation. The same polarity or elution strength is maintained throughout the process .

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Gradient separation :- In this technique, lower polarity or elution strength is used followed by gradually increasing the polarity or elution strength of the mobile phased . Frontal analysis :- It consists in passing the sample solution continuously through the absorbent column. No mobile phase is used for development of column. A mixture of A, B, C is added on the column. If the component A is least absorbed, component B intermediate extent and component C most strongly to column material, as the mixture flows through the column , the least absorbed A runs down the column fast, component B to intermediate extent while component C is retained at the top of the column. Displacement analysis:- This method is encountered in absorption column chromatography. In this method, a small volume of mixture is added to the column and elution is carried out by solvent containing a solute which has high absorptivity for column material. The technique is also known as “ displacement analysis”

5) Type of Analysis:- :

5) Type of Analysis :- Qualitative analysis :- A chromatogram provides only single piece of qualitative information about each piece in a sample, namely its retention time or its portion on the stationary phase after certain elution period. It is used to identify the compound, detect the presence of impurities, to find out the number of compounds, etc. this is done by using retention time values. Quantitative analysis :- Chromatography owes its precipitous growth during the past two decade in a part to measure the perpendicular distance from the particular line to the peak. This measurement can ordinarily be made with reasonably high precision and yielding accurate results. It is Used to determine the quantity of the individual or several components in the mixture


PAPER CHROMATOGRAPHY It may be defined as “ the technique in which the analysis of the unknown substance is carried out mainly by the flow of solvents on specially designed filter paper. T he separation of a mixture into its components by filtering it through a strip of special paper known as “WHATMANN FILTER PAPER” available in different grades.

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PRINCIPLE: 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.


OPERATION METHOD Choice of filter paper: Whatman filter papers are used as chromatography paper. In general this paper contains 98-99% of alpha-cellulose . There are various grade and types of paper available for separation of a sample. 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 .


PREPARATION OF PAPER: 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 starting line marks are made 2cm apart from each other.


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.


SOLVENTS: A number of solvents can be used in the paper chromatography . The solvent selection depends upon nature of substance to be separated. Some E.g. of solvents , in ratio n-Butanol: Gl.acetic acid: Water [4:1:5] Isopropanol : Ammonia : Water [9:1:2] Methanol : Water [3:1 or 4:1] t-Butanol : Water : Formic acid [40:20:5]


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 vapour.


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 completed the paper is taken out of the chamber carefully.

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DRYING OF CHROMATOGRAM : 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. LOCATION OF SPOT: If the substances are coloured they are visually detected easily. But for colourless substance. Physical and chemical methods are used to detect the spot. Physical method: In this method observation are done under UV light, detection of fluorescence and radioisotope measurements. Chemical method: In this method chemical reagent is used to develop the colour. >Amino Acids-Ninhydrin Reagent >Alkaloids-Dragendroff’s Reagent


Rf VALUE: In paper chromatography the results are represented by Rf value which represent the movement or migration of solute relative to the solvent front. The Rf value is calculated as : Distance travelled by the solute Distance travelled by the solvent front


DEVELOPMENT TECHNIQUE: There are various methods of development of paper chromatography… > DESCENDING CROMATOGRAPHY: In this method solvent moves from top to bottom so it is called descending chromatography. > ASCENDING CROMATOGRAPHY: In this case the solvent migrates upward by capillary action.

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ASCENDING-DESCENDING CHROMATOGRAPHY: A hybrid of above two technique is called ascending-descending chromatography. Initial ascending chromatography is performed, often crossing the glass rod changes to descending. RADIAL CHROMATOGRAPHY: This is rarely used method, in this case a circular piece of paper is taken which has a wick cut parallel to the radius from the edge to centre. The sample is applied at the centre of the paper. The paper is then laid on the edge of circular disk with wick dipped into the solvent at the bottom of the dish.

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TWO DIMENSIONAL CHROMATOGRAPHY: In this method a square paper is taken the sample is applied to the one of the corner. Using solvent system the first development is carried as ascending method. The paper is taken out dried and second development is performed at right angels to the first dimensional Development.


APPLICATION OF PAPER CROMATOGRAPHY: Used in the separation of various organic mixtures. Used in almost all area to solve complicated problems in chemistry, biology, biochemistry. Used for both qualitative and quantitative analysis. Used for identification of foreign substances in drugs. Used for identification of decomposition products. Used for Analysis of metabolites of drugs in blood, urine.

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REFERENCE : Pharmaceutical Analysis Volume II Nirali Prakashan . Dr. A.V Kasture Dr. K.R Mahadik Dr. S.G Wadodkar Dr. H.N More Textbook of Pharmaceutical Analysis 3 rd Edition Dr. S. Ravi Shankar INTERNET SOURCE

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