Paper Chromatography - Instrumental Methods of Analysis B. Pharma 7th Semester

Paper Chromatography


       Paper chromatography

       Principle involved

       Applications of Paper chromatography

Practical requirements

       Stationary phase

       Application of sample

       Mobile phase

       Development technique

       Detecting or visualizing agents


By the end of this session, students will be able to:

Ø  Explain the principle involved in Thin layer Chromatography

Ø  Outline the components of Paper chromatography

Ø  Discuss development techniques of Paper chromatography

Ø  Explain various detecting or visualizing agents 

Paper Chromatography

       Analysis of unknown substances carried out mainly by the flow of solvents on specially designed filter paper

       Two types of paper chromatography are there:

Paper adsorption chromatography

       Stationary phase- paper impregnated with silica or alumina acts as adsorbent

       Mobile phase- solvent

Paper partition chromatography

       Stationary phase- moisture/water present in the pores of cellulose fibres present in filter paper

       Mobile phase- solvent

       In general, most separations are based on paper partition chromatography


       Mainly partition rather than adsorption

       Cellulose layers in filter papers contains moisture acts as stationary phase

       Organic solvents or buffers are used as mobile phases

Practical requirements

       Stationary phase and papers used

       Application of sample

       Mobile phase

       Development technique

       Detecting or visualizing agents

Stationary Phase

       Paper of chromatographic grade consists of

       α-cellulose- 98-99%

       β-cellulose- 0.3-1%

       Pentosans- 0.4-0.8%

       Ether soluble matter- 0.015-0.02%

       Ash- 0.01-0.07%

       Whatman filter papers of different grade like No.1, No.2, No.3, No.17, No.20 etc are used

       Papers differ in sizes, shapes, porosity and thickness

       Choice of filter paper depends upon thickness, flow rate, purity, technique, etc

       Modified papers- acid or base washed filter paper, glass fibre type paper

       Hydrophilic papers- papers modified with methanol, formamide, glycol, glycerol, etc

       Hydrophobic papers- acetylation of –OH groups leads to hydrophobic nature

       Can be used for reverse phase chromatography

       Silicon pretreatment and organic non-polar polymers can also be impregnated to give reverse phase chromatographic mode

       Impregnation of silica, alumina or ion exchange resins can also be made

       Paper of any size can be used

       Paper should be kept in a chamber of suitable size

Application of Sample

       Sample to be applied is dissolved in mobile phase

       Applied on paper using capillary tube or micro pipette

       Very low concentration is used to avoid larger zone


Mobile phase

       Pure solvents, buffer solutions, or mixture of solvents are used

Examples of hydrophilic mobile phase:

       Isopropanol:ammonia:water = 9:1:2

       n-butanol:glacial acetic acid:water = 4:1:5

       Methanol:water = 3:1 or 4:1

       t-butanol:water:formic acid = 40:20:5

Mobile Phase

       Examples of hydrophobic mobile phase

       Kerosene:70% isopropanol

       Dimethyl ether:cyclohexane

       Single or two or three phase solvent system can be used

Development technique

       As paper is flexible, several types of development are possible

       For increase of ease and efficiency of separation

Ascending development

       Conventional, solvent flows against gravity

Development Techniques

Descending development

       Carried out in a special chamber

       Solvent holder is at the top

       Spot is kept at the top and solvent flows down the paper

       Advantage- flow of solvent is assisted by gravity

       Development is faster

Ascending-descending development

       Combination of ascending and descending type

       Length of separation is increased by using a combination of techniques

       First ascending takes place followed by descending development 

Circular or radial development

       Spot is kept at the center of circular paper

       Solvent flows through a wick at the center and spreads in all directions uniformly

       Individual spots after development looks like concentric circles

       Number of quadrants can be created allowing more number of samples to be spotted

Two dimensional development

       Similar to two dimensional TLC

       Paper is developed in one direction and after development

       Paper is developed in second direction

       More compounds or complex mixtures to be separated into individual

Detecting or Visualizing Agents

       After development of chromatogram, spots should be visualized

       Detecting colored spots can be done visually

       For detecting colorless spots, following techniques can be used 

Non-specific methods

       Number of spots can be detected

       Exact nature or type of compound is not known

For example

       Iodine chamber method- brown spots are observed

       UV chamber for fluorescent compounds

Specific methods

       Specific spray reagents or detecting reagents or visualizing agents are used

       Nature of compound can be identified

For example

       Ferric chloride- for phenolic compounds and tannins

       Ninhydrin reagent- for amino acids

       Dragendroff’s reagent- for alkaloids

       2,4-dinitrophenyl hydrazine- for aldehydes and ketones

Can also be categorized as

Destructive technique

       Samples are destroyed by spray reagents

       For example- ninhydrin reagent

Non-destructive technique

       Samples are not destroyed

       For example- UV chamber, Iodine chamber, densitometric method 

Qualitative Analysis

       Rfvalue (retardation factor) is calculated for identifying the spots i.e., qualitative analysis

       Rfvalue is the ration of distance travelled by the solute to the distance travelled by solvent front

       Rf = distance travelled by solute /distance travelled by solvent front 

       Rf value ranges from 0 to 1

       Ideal values are from 0.3 to 0.8

       Specific and constant for every compound in a particular combination of stationary and mobile phase

       Rf value of a sample and reference compound is same, compound is identified by its standard

       Rf value differs, compound may be different from its reference standard  

Rx value

       Ratio of distance travelled by the sample and distance travelled by the standard

       Rx value is always closer to 1

Rm value

       To find whether compounds belongs to a homologous series

       If they belong, Rm values are constant

       Can be determined by Rm = log ((1/(R_f  )-1)

Quantitative Analysis

       By Direct and indirect method

       Direct method

Using densitometer

       Quantity of individual spots can be determined by using densitometric method

       Called as in situ method

       Here, optical density of the spots of standard and test solution are measured

       Quantity of substance can be calculated

       Plates are neither destroyed nor eluted with solvents to get the compounds

Indirect method

       Can be done by eluting individual spots with solvent and filtering off the stationary phase

       Filtrate can be concentrated and exact quantity of compound

       Can be determined by conventional methods by colorimetry, UV spectrophotometry, fluorescence method, flame photometry, electrochemical methods, etc.



       Applications are wider

       No limitation to the compounds that can be analysed

       Separation of mixtures of drugs of chemical or biological origin, plant extracts, etc

       Separation of carbohydrates, vitamins, antibiotics, proteins, alkaloids, glycosides, etc

       Identification of drugs

       Identification of related compounds in drugs

       To detect the presence of foreign substances in drugs

       To detect decomposition products in drugs


       Principle is partition

       Mobile phase solvent flows through because of capillary action (against gravitational force)

       Components move according to their affinities towards adsorbent

       Very small particle size can be used to increase the efficiency of separation

       Needs less solvent, stationary phase and time for every separation when compared to column chromatography

       Paper of good quality should be selected  

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