Paper chromatography

PAPER CHROMATOGRAPHY

• Introduction : Developed by AH Gorden AJP Martin. (1941), For separation of amino acids
• AJP Martin & RYL Synge : Shared Nobel prize in 1952
• Paper is used as support/ adsorbent
• Partition plays imp. Role in separation of components than adsorption
• Because: Cellulose fibers – have film of moisture surrounding them in air dried state also

PAPER CHROMATOGRAPHY

• Movement of components on paper- depends on amount & nature of stationary phase compared with the amount of mobile phase in same part of paper & on partition coefficient of components
• It offers means of tentative identification of components
• Factors Responsible : Temp., Stationary phase, Size of spot, Quality of paper, equilibrium times

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PAPER CHROMATOGRAPHY

• To eliminate or to minimize the effect of variables, Rf values are related to std. subst.
• Eg: Rx= Dist. sub. moves from origin / Dist. reference sub. moves from origin
• Where x is reference compound
• Rf values= Dist. travelled by component / Dist. travelled by solvent front

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Methodology

• Basic apparatus required
• Closed chamber: to avoid loss of mobile phase

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Development techniques: �1. Ascending Tech.

• Draw a line with pencil

from one end of paper

• Apply 2.5µl vol. (sample & ref.) at about 2 cm interval on line
• Tank: Mobile phase to a depth of 1 cm,
• Line tank with paper
• Equilibrium time: 2-3 hr
• Suitable ht. 15-20 cm
• Remove the paper, Dry, locate the components

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sample

Components

2. Descending Tech.

3. Radial Paper chromatography

Solvent spreads radially by capillary action

4. 2D PAPER CHROMATOGRAPHY

• 1^st development in one solvent by ascending.
• Then at perpendicular to 1^st in 2^nd solvent
• Done for Complex mixtures

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METHODOLGY (STEPS INVOLVED)

1. Choice of technique: Depends on nature of substance to be separated
2. Choice of paper: Depends on qualitative or quantitative analysis, for analytical or preparative chromatography or substances used is hydrophylic or lipophilic, neutral or charged species

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METHODOLOGY (Choice of paper)

• Whatman chromatographic papers : depends on type of separation
• Coarser or faster papers: Whatman 31ET: used when the components have Rf values wide apart
• Slow paper: rarely used, but facilitate better resolution of subs with close Rf values
• Slow papers: whatman 20, Schleicher & schull 2045, Macherey nogel 261, Edrol 208
• Heavy papers- W 3MM, preparative purpose

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METHODOLOGY (Choice of paper)

• Paper is composed of 99% α-cellulose & mineral content
• Modifications to paper can be done
• For separation of polar subs. efficiently– exchange capacity of paper can be ↑sed by ↑sing carbonyl content (1.4%) by partial oxidation
• By partial hydrolysis- soaking paper for 24 hrs. in 7% HCl & washing with H₂O & ethanol successively- capillarity of paper is ↑sed
• Rigidity of paper: ↑sed by acetylation, esterification & other chemical methods

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3. METHODOLOGY (selection of solvent)

3. Selection of Mobile phase: depends on the fact that the Rf values should be different for different constituents present in a mixture.

Generally solvent mixture which give Rf between 0.2-0.8 for a sample is selected

From the DEC, the polarity of solvent can be obtained

DEVELOPMENT (selection of solvent)

4. DEVELOPMENT (Preparation of samples)

• No standard procedure
• Because of presence of variety of components like fats, salts, proteins etc.
• Sample volume of 10-20µl containing few µg of sample is ideal sample

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5. DEVELOPMENT (Spotting)

• For ascending technique : 24cm x7cm whatman paper
• Graduated micro pipette for sampling the spots
• Drying by stream of hot or cold air

6. Preparing the tank/ chamber

• The chamber is loaded with selected solvent mixture and poured in to the chamber such that the level of solvent is about 1 cm from bottom.

7. Saturation of tank: After placing solvent system, hang a blotting paper by the sides of tank and allow the solvent to rise to top.

8. Placing the paper in to the chamber: With the help of clips/holder hang the paper in to chamber such that the edge of the paper just touches the edge of solvent layer.

8. Allow to run the solvent onto paper till top but before the other edge.

9. Drying the chromatogram: Oven, drying cabinets with temp. control

10. Visualization/Detection: Done by

• Chemical : Chromogenic / visualizing /locating agent: By dipping or fine spray atomizer or
• I₂ vapours used in cupboard

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Detection

• Physical method: colorless spots- under U.V. lamp- fluorescence reveals the presence
• Irradiation with light of 254nm for compounds that absorbs in U.V. range
• 365nm : the fluorescence before and after irradiation with shorter wavelength light

11. Calculation of R_f

12. Estimation : After extraction from paper

• Isolation of separated components from paper
• Cut the spot part of paper, soak in min. qty. of solvent
• Semi micro extractor (soxhlet), Eluation

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��Estimation After extraction from paper� �

• Determine by U.V. spec., colorimetry, Flourimetry, Flame photometry, conductometry etc.
• Factors: Nature of subs., equipment avail., senstivity of method, time etc.

In situ methods for estimation

1. By visual assessment: By human eye- not quantitative

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b. Measurement of areas: Size of spot: Det. Qty. of subs., Linera relation obtained b/w spot area & amt. of subs. Present

Random errors: spot shape, vol. applied, speed of application must be identical

3. By densitometer: Intensity of color of subs. Measured directly on chromatogram

4. Potentiometry: changes in potential of metal electrode in contact with filter paper

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ADVANTAGES

• Low cost
• Requires less quantitative material
• Very efficient for polar substances
• Easy to handle
• Both inorganic and organic compounds can be identified
• Simple
• Various modes are available
• Quantitation is possible

DISADVANTAGES

• Cannot separate complex mixtures
• Quantification is not efficient
• Takes more time- 2-3 hr.
• Accuracy is less
• Results on paper cant be stored for long time
• Separation is not sharp
• Corrosive chemicals destroy paper
• U.V. chamber can be used for florescent compounds only

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APPLICATIONS

• To monitor the reaction
• To check purity of pharmaceuticals
• In fermentation and ripening
• Analysis of reaction mix. in biochemical labs
• Forensic testing
• Beverages and food industry
• Analysis of Cosmetics
• Drug testing
• Separation of carbohydrates, antibiotics, amino acids, vitamins, mix. of drugs etc.

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