Paper Chromatography

Paper chromatography (PC) is a type of a planar chromatography whereby chromatography

procedures are run on a specialized paper.
It is considered to be the simplest and most widely used of the chromatographic techniques
because of its applicability to isolation, identification and quantitative determination of organic
and inorganic compounds.
It was first introduced by German scientist Christian Friedrich Schonbein (1865).

Types of Paper chromatography:

a. Paper Adsorption Chromatography: Paper impregnated with silica or alumina acts as
adsorbent (stationary phase) and solvent as mobile phase.
b. 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 In general paper chromatography mostly refers to paper partition
chromatography.

Principle of Separation
The principle of separation is mainly partition rather than adsorption. Substances are distributed
between a stationary phase and mobile phase. Cellulose layers in filter paper contain moisture
which acts as stationary phase. Organic solvents/buffers are used as mobile phase. The
developing solution travels up the stationary phase carrying the sample with it. Components of
the sample will separate readily according to how strongly they adsorb onto the stationary phase
versus how readily they dissolve in the mobile phase.
Instrumentation of Paper chromatography
1. Stationary Phase And Papers
Whatmann filter papers of different grades like No.1, No.2, No.3, No.4, No.17, No.20 etc are
used. In general the paper contains 98-99% of α-cellulose, 0.3 – 1% β -cellulose. These papers
differ in sizes, shapes, porosities and thickness.
Other modified papers like 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. Silicon pretreatment and organic non-polar polymers
can also be impregnated to give reverse phase chromatographic mode. Impregnation of silica,
alumna, or ion exchange resins can also be made. Size of the paper used: Paper of any size can
be used. Paper should be kept in a chamber of suitable size.
Factors that governs the choice of paper: (selection of stationary phase)
i. Nature of Sample and solvents used.
ii. Based on Quantitative or Qualitative analysis.
iii. Based on thickness of the paper.
Preparation of Paper
 Cut the paper into desired shape and size.
 The starting line is marked 5cm from the bottom edge.
 On the starting line marks are made 2cm apart from each other.

2. Application of sample
The sample to be applied is dissolved in the mobile phase and applied using capillary tube or
using micropipette. Very low concentration is used to avoid larger zone.
3. Paper Chromatography Mobile Phase
Choice depends on the nature of the substance to be separated.
 Pure solvents, buffer solutions are used.
 If pure solvents do not give satisfactory separation, a mixture of solvents of suitable
polarity may be applied.
 Pure solvents, buffer solutions or mixture of solvents can be used. Some of the Examples
of
Hydrophilic mobile phases
 Isopropanol: ammonia:water 9:1:2
 Methanol: water 4:1 or 3:1
 n-Butanol: glacial acetic acid: water 4:1:5
Hydrophobic mobile phases
 kerosene: 70% isopropanol
 Dimethyl ether: cyclohexane
 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.

4. Chromatographic Chamber
The chromatographic chambers are made up of many materials like glass, plastic or stainless
steel. Glass tanks are preferred most. They are available in various dimensional sizes depending
upon paper length and development type. The chamber atmosphere should be saturated with
solvent vapor.
5. Development technique
Sample loaded filter paper is dipped carefully into the solvent not more than a height of 1 cm and
waited until the solvent front reaches near the edge of the paper.
Different types of development techniques can be used:
a. Ascending Development: Like conventional type, the solvent flows against gravity. The
spots are kept at the bottom portion of paper and kept in a chamber with mobile phase
solvent at the bottom. (Same as in TLC).

b. Descending Type: 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.
c. Ascending – Descending Development: A hybrid of above two techniques is called
ascending-descending chromatography.Only length of separation increased, first ascending
takes place followed by descending.

d. 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. Hence the individual
spots after development look like concentric circles. By making perforations radially, number
of quadrants can be created allowing more number of samples to be spotted.

e. Two dimensional developments: This technique is very similar to 2-Dimensional TLC.

Here the chromatogram development occurs in two directions at right angles.
 In this mode, the samples are spotted to one corner of rectangular paper and allowed for
first development. Then the paper is again immersed in the mobile phase at a right angle
to the previous development for the second chromatogram. In the second direction, either
the same solvent system or different solvent system can be used for development.
f. Drying of Chromatogram: After the development, the solvent front is marked and the left
to dry in a dry cabinet or oven.

6. Detection
After the development of chromatogram, the spots should be visualized. Detecting colored spots
can be done visually. But for detecting colorless spots, any one of the following technique can be
used.
a. Nonspecific methods: where brown or amber of spots can be detected, but not the exact
nature or type of the compound.
Examples
i. Iodine chamber method where brown or amber spots are observed when the developed
papers are kept in atank with few iodine crystals at the bottom.
ii. UV chamber for fluorescent compounds: When compounds are viewed under UV
chamber, at 254nm (short λ) or at 365nm (long λ), fluorescent compounds can be
detected. Bright spots can are seen against a dark background.
b. Specific methods: Specific spray reagents or detecting or visualizing agents are used to find
out the nature of compounds or identification purposes.
i. Ferric chloride- For phenolic compounds and tannins
ii. Ninhydrin in acetone- for amino acids
iii. Dragendroff’s reagent- for alkaloids
iv. 3,5-Dinitro benzoic acid- for cardiac glycosides
v. 2,4- Di-nitrophenyl hydrazine- for aldehydes and ketones.
The detecting techniques can also be categorized as
a. Destructive technique: Specific spray reagents etc where the samples are destroyed
before detection e.g. Ninhydrin reagent.
b. Non-destructive technique: UV chamber method, Iodine chamber method,
densitometric method, etc., where the sample is not destroyed even after detection.

For radioactive materials, detection is by using autoradiographyor Geiger muller counter. For
antibiotics, the chromatogram is layed on nutrient agar inoculated with appropriate strain and the
zone of inhibition is compared.

Quantitative Analysis
Direct technique: Densitometer is an instrument which measures quantitatively the density of
the spots. When the optical densities of the spots for the standard and test solution are
determined, the quantity of the substance can be calculated. The papers are neither destroyed nor
eluted with solvents to get the compounds. The method is also known as in-situ method.
 Indirect techniques: In this technique, the spots are cut into portions and eluted with solvents.
The solution can be analyzedby any conventional techniques of analysis like spectrophotometry,
electrochemical methods, etc.
Qualitative Analysis:
a. Rf value
𝑅𝑓=𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑢𝑡𝑒/𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡
The Rf value ranges from 0 to 1. But the ideal values are from 0.3 to 0.8.
b. Rx value: It is always closure to 1. 𝑅𝑥=𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑢𝑡𝑒/𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒
𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑
c. Rm value: It is mainly used to find out whether the compounds belong to a homologous
series. If they belong to a homologous series, the 𝚫Rm values are constant. The 𝚫Rm
values for a pair of adjacent member of a homologous series are determined by using the
below formula:
𝑅𝑚=log(1/𝑅𝑓−1)
Applications:
i. To check the control of purity of pharmaceuticals,
ii. For detection of impurities

Drug Mobile phase Detecting agent

Hydroxocobalamin Butyl alcohol: acetic acid:potassium Elution and measurement of
cyanide absorbance at 361nm.

iii. Detect the contaminants in foods and drinks,

iv. For the detection of drugs

Drug Mobile phase Detecting agent

Gentamycin Chloroform:Methanol:Ammonia:Water 10:5:3:2) Ninhydrin in pyridine-acetone mixture
Vancomycin t-Amyl alcohol : Acetone: water (2:1:2) Nutrient agar containing Bacilus subtilis

v. In analysis of cosmetics
vi. Analysis of the reaction mixtures in biochemical labs.
vii. Identification of decomposition products
viii. Analysis of metabolites of drugs in blood, urine etc.
ix. In the study of ripening and fermentation

Advantages of Paper Chromatography

i. Simple and Rapid
ii. Paper Chromatography requires very less quantitative material.
iii. Paper Chromatography is cheaper compared to other chromatography methods.
iv. Both unknown inorganic as well as organic compounds can be identified by paper
chromatography method.
v. Paper chromatography does not occupy much space compared to other analytical
methods or equipment’s.

Limitations of Paper Chromatography

i. Large quantity of sample cannot be applied on paper chromatography.
ii. In quantitative analysis paper chromatography is not effective.
iii. Complex mixture cannot be separated by paper chromatography.
iv. Less Accurate compared to HPLC or HPTL.