Paper Chromatography

Objectives

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

Principle of Paper 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

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

•       R_fvalue is the ration of distance travelled by the solute to the distance travelled by solvent front

•       R_f = distance travelled by solute /distance travelled by solvent front

•       R_f 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

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

•       R_f value differs, compound may be different from its reference standard  

R_x value

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

•       R_x value is always closer to 1

R_m value

•       To find whether compounds belongs to a homologous series

•       If they belong, R_m values are constant

•       Can be determined by R_m = 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 

•       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

Summary

•       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  

FAQs

1. What is the main principle behind paper chromatography?

The main principle of paper chromatography is the differential migration of compounds through a stationary phase and a mobile phase, leading to their separation based on their affinities to each phase.

2. How is paper chromatography used in the field of forensics?

In forensics, paper chromatography is used to analyze crime scene evidence, such as ink samples and drug residues, helping forensic scientists identify and compare substances.

3. Can you explain the difference between stationary and mobile phases?

The stationary phase is the substrate where compounds are separated based on their affinities, while the mobile phase carries compounds along the paper, facilitating their migration.

4. What are the common visualizing agents in paper chromatography?

Common visualizing agents include iodine vapor, UV light, and specific chemical reagents that react with the separated compounds to make them visible.

5. Are there any safety precautions to consider when performing paper chromatography?

Yes, safety precautions should be followed, such as working in a well-ventilated area and handling solvents with care. Protective equipment like gloves and goggles may also be necessary.

 For Paper Chromatography PDF Notes Click on Download Button