Paper chromatography

Paper Chromatography: Principles, Types, and Applications
Introduction:
Paper chromatography is one of the simplest and oldest forms of chromatographic techniques.
It was developed in the early 20th century and has since become a valuable method for
separating and identifying compounds, particularly in organic chemistry and biology. It is widely
used due to its simplicity, low cost, and ability to separate small quantities of substances in a
mixture.
Principles of Paper Chromatography
Paper chromatography operates on the principle of partition chromatography, where the

separation of compounds is based on their di erential partitioning between two immiscible
phases: the mobile phase and the stationary phase.
 Stationary Phase: The stationary phase in paper chromatography is the paper itself.
More specifically, it is the water molecules bound to the cellulose fibers of the paper.
Cellulose paper acts as a porous matrix that holds the water and creates the stationary
phase.
 Mobile Phase: The mobile phase is typically a solvent or a mixture of solvents (liquid)
that moves through the paper by capillary action. Common mobile phases include
alcohol, water, or organic solvents like chloroform or acetone.
Mechanism of Separation:
As the mobile phase moves up the paper, it carries the dissolved components of the sample
along with it. The components in the mixture are separated based on their relative a inities for
the stationary and mobile phases:
 A inity for the Mobile Phase: Components that are more soluble in the mobile phase
will travel further along the paper.
 A inity for the Stationary Phase: Components with a higher a inity for the stationary
phase will adsorb onto the paper and move more slowly.
The result is a series of spots on the paper, each representing a di erent component of the
mixture. The distance each component travels is related to its solubility in the mobile phase and
its interaction with the stationary phase.
Types of Paper Chromatography
Paper chromatography can be performed in several di erent configurations, depending on the

orientation of the paper and the flow of the solvent. Each type has unique characteristics suited
for specific applications.
Type of Paper
Description Application
Chromatography
The mobile phase moves upward through the

Separation of amino
Ascending paper by capillary action. The sample is
acids, plant pigments,
Chromatography applied near the bottom, and as the solvent
sugars
rises, it separates the components.
The mobile phase moves downward with the

Complex mixture
Descending help of gravity. The solvent reservoir is placed
separation, such as in
Chromatography at the top, and the solvent moves through the
plant extracts
paper by gravity-assisted capillary action.
The sample is applied to the center of a

Radial/ Circular circular piece of paper. The solvent flows from Small-scale separations
Chromatography the center outward, forming rings of separated and qualitative analysis
components.
A complex form where the sample is separated

Enhanced separation of
in one direction, and then the paper is rotated
Two-dimensional complex mixtures, like
90 degrees, and a di erent solvent is used to
Chromatography proteins, peptides, and
separate the sample again in the
nucleic acids
perpendicular direction.
Procedure for Paper Chromatography
The general procedure for paper chromatography involves the following steps:
1. Sample Application: A small spot of the sample mixture is placed near the bottom of a
strip of chromatography paper.
2. Solvent Preparation: A suitable solvent or solvent mixture is prepared as the mobile

phase. The choice of solvent depends on the nature of the compounds being separated.
3. Development Chamber: The paper is placed in a chamber with a small amount of

solvent, ensuring that the spot is above the solvent level. The chamber is sealed to
maintain a saturated environment, which helps solvent movement.
4. Chromatogram Development: As the solvent rises through the paper by capillary

action, it carries the sample with it. The di erent components of the sample separate
based on their interactions with the stationary and mobile phases.
5. Drying: Once the solvent front reaches a designated point, the paper is removed from
the chamber and allowed to dry.
6. Visualization: The separated components are visualized by using a variety of methods

such as UV light, iodine vapor, or chemical stains, depending on the nature of the
compounds.
Applications of Paper Chromatography
Paper chromatography has a wide range of applications in various fields such as chemistry,
biology, and environmental science. Below are some of the key areas where this technique is
utilized.
Field Application
Separation and identification of amino acids, peptides, nucleic acids,

Biochemistry
sugars, and enzymes.
Detection of additives, preservatives, and contaminants in food

Food Chemistry
products. Analysis of sugars and colorants in fruits and beverages.
Environmental Analysis of pollutants, such as pesticides, herbicides, and other

Science contaminants in water and soil samples.
Pharmaceutical Identification of active ingredients, quality control, detection of

Industry impurities, and drug stability testing.
Analysis of toxic substances, drugs, inks, dyes, and other chemicals in

Forensic Science
forensic investigations.
Botany and Plant Separation of plant pigments, such as chlorophyll, carotenoids, and
Sciences anthocyanins, for botanical studies.
1. Biochemistry and Life Sciences:
 Amino Acid Separation: Paper chromatography is commonly used to separate amino

acids derived from protein hydrolysates. The di erent amino acids exhibit varying
a inities for the mobile and stationary phases, resulting in their separation. Ninhydrin
reagent is often used to visualize the amino acids as colored spots on the paper.
 Sugars and Carbohydrates: Monosaccharides and oligosaccharides can be separated

and identified using paper chromatography. This is particularly useful in carbohydrate
metabolism studies and food chemistry.
2. Food Chemistry:
 Analysis of Additives and Contaminants: Paper chromatography is employed to detect

artificial additives, such as food colorings and preservatives, in various food products. It
also helps in identifying natural sugars and colorants in fruits and beverages.
 Monitoring Fermentation: The progress of fermentation, particularly in the beverage

industry, can be tracked by identifying sugars and their breakdown products.
3. Environmental Science:
 Pesticide Residue Analysis: Paper chromatography can be used to detect trace

amounts of pesticides and herbicides in environmental samples such as water and soil.
These compounds can be extracted from the samples, separated, and identified based
on their migration patterns.
 Pollutant Detection: Various organic pollutants, including hydrocarbons and phenolic
compounds, can be analyzed in environmental matrices, aiding in pollution control and
regulatory compliance.
4. Pharmaceutical Industry:
 Drug Analysis: Paper chromatography is applied for testing the purity and composition
of pharmaceuticals. It is particularly useful in separating di erent alkaloids, glycosides,
and other pharmaceutical compounds. Paper chromatography helps monitor the
stability and shelf life of drugs by detecting degradation products.
 Quality Control: During drug formulation, paper chromatography can be used to check
for impurities and ensure that the correct amount of active ingredients is present.
5. Forensic Science:
 Ink and Dye Analysis: In forensic science, paper chromatography can be applied to
analyze inks, dyes, and other pigments in criminal investigations, such as in questioned
document analysis. It can help link a suspect to a crime scene by identifying ink used in
forgeries or altered documents.
 Drug and Poison Identification: Forensic toxicology uses paper chromatography to

separate and identify drugs, poisons, and other chemicals in biological samples (e.g.,
blood, urine, or tissue extracts).
6. Botany and Plant Sciences:
 Separation of Plant Pigments: One of the classic uses of paper chromatography is in

the separation and identification of plant pigments, such as chlorophyll, xanthophylls,
and carotenoids. This helps in the study of photosynthesis, plant physiology, and
taxonomy.
 Phenolic Compound Analysis: Paper chromatography helps identify various phenolic

compounds in plants, which play important roles in plant defense mechanisms and
antioxidant activity.
Advantages and Disadvantages of Paper Chromatography
Advantages Disadvantages
Simple, inexpensive, and easy to perform Limited resolution for complex mixtures
Requires only small amounts of sample Time-consuming compared to other methods
Limited to small molecules and non-volatile

No need for sophisticated equipment
compounds
Can handle both polar and non-polar

Quantification is less precise than HPLC or GC
compounds
Advantages Disadvantages
Visualizing some compounds may require chemical

Ideal for teaching and educational use
reagents
Rf Value Calculation
The Rf value (Retention Factor) is a numerical representation of how far a compound travels
relative to the solvent front. It is used to compare and identify substances based on their
movement along the paper.
Rf=Distance traveled by the soluteDistance traveled by the solventR_f = \frac{\text{Distance

traveled by the solute}}{\text{Distance traveled by the solvent}}Rf
=Distance traveled by the solventDistance traveled by the solute
 Rf values are characteristic of specific compounds and depend on the solvent used, the
type of paper, and the conditions of the experiment.
Example of an Rf value table for di erent amino acids under specific solvent conditions:
Amino Acid Rf Value (in ethanol-water mixture)
Alanine 0.45
Glycine 0.35
Lysine 0.18
Tyrosine 0.52
Conclusion
Paper chromatography remains a fundamental technique in the separation and analysis of

small organic molecules. Despite being overshadowed by more advanced chromatographic
methods like HPLC and GC, it retains its importance in educational, research, and some
applied industrial settings due to its simplicity and a ordability. Its applications, from amino
acid separation to environmental pollutant detection, demonstrate its versatility and enduring
value in scientific investigation.
Citations:
1. Grinberg, N. (2009). Modern Thin Layer Chromatography. Marcel Dekker.
2. Kirchner, J. G. (1953). "Paper Chromatography." Journal of Chemical Education.
3. Poole, C. F. (2003). The Essence of Chromatography. Elsevier.

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Paper Chromatography: Principles, Types, and Applications

Principles of Paper Chromatography

Paper chromatography operates on the principle of partition chromatography, where the

Types of Paper Chromatography

Paper chromatography can be performed in several di erent conﬁgurations, depending on the

The mobile phase moves upward through the

The mobile phase moves downward with the

The sample is applied to the center of a

A complex form where the sample is separated

Procedure for Paper Chromatography

2. Solvent Preparation: A suitable solvent or solvent mixture is prepared as the mobile

3. Development Chamber: The paper is placed in a chamber with a small amount of

4. Chromatogram Development: As the solvent rises through the paper by capillary

6. Visualization: The separated components are visualized by using a variety of methods

Separation and identiﬁcation of amino acids, peptides, nucleic acids,

Detection of additives, preservatives, and contaminants in food

Environmental Analysis of pollutants, such as pesticides, herbicides, and other

Pharmaceutical Identiﬁcation of active ingredients, quality control, detection of

Analysis of toxic substances, drugs, inks, dyes, and other chemicals in

1. Biochemistry and Life Sciences:

 Amino Acid Separation: Paper chromatography is commonly used to separate amino

 Sugars and Carbohydrates: Monosaccharides and oligosaccharides can be separated

 Analysis of Additives and Contaminants: Paper chromatography is employed to detect

 Monitoring Fermentation: The progress of fermentation, particularly in the beverage

 Pesticide Residue Analysis: Paper chromatography can be used to detect trace

 Drug and Poison Identiﬁcation: Forensic toxicology uses paper chromatography to

6. Botany and Plant Sciences:

 Separation of Plant Pigments: One of the classic uses of paper chromatography is in

 Phenolic Compound Analysis: Paper chromatography helps identify various phenolic

Advantages and Disadvantages of Paper Chromatography

Requires only small amounts of sample Time-consuming compared to other methods

Limited to small molecules and non-volatile

Can handle both polar and non-polar

Visualizing some compounds may require chemical

Rf=Distance traveled by the soluteDistance traveled by the solventR_f = \frac{\text{Distance

Amino Acid Rf Value (in ethanol-water mixture)

Paper chromatography remains a fundamental technique in the separation and analysis of

1. Grinberg, N. (2009). Modern Thin Layer Chromatography. Marcel Dekker.

2. Kirchner, J. G. (1953). "Paper Chromatography." Journal of Chemical Education.

3. Poole, C. F. (2003). The Essence of Chromatography. Elsevier.

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