2. • Paper Chromatography (PC) was first introduced by German
scientist Christian Friedrich Schon bein (1865) and later
Archer Porter Martin and R L M Synge were jointly awarded
the nobel prize for their contribution to paper partition
chromatography in 1952.
• PC 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
• INTRODUCTION
3. Types of Paper chromatography:
i. Paper Adsorption Chromatography: Paper impregnated with silica or alumina
acts as adsorbent (stationary phase) and solvent as mobile phase.
ii. 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.
iii. In general paper chromatography mostly refers to paper partition
chromatography.
Paper Chromatography (PC)
4. 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 and 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
distributed/adsorb onto the stationary phase versus how readily they dissolve in the mobile
phase.
5. Methodology of Paper chromatography
1. Stationary phase & papers used
2. Mobile phase
3. Application of sample
4. Developing Chamber
5. Detecting or Visualizing agents
6. 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 pre-treatment and organic non-polar polymers can also be impregnated to give reverse
phase chromatographic mode.
7. • Paper can be impregnated with alumina, silica or ion exchange resin.
• The size of the paper should be suitable for the size of the chamber and apply the sample
by using capillary or micro pipette.
8. • Cut the paper into desired shape and size depending upon
work to be carried out.
• The starting line is marked on the paper with an ordinary
pencil 2cm from the bottom edge
• On the staring line marks are made 2cm apart from each
other.
PREPARATION OF PAPER
9. • Preparation of the solution
• Choice of suitable solvent for making solution is very important.
• Pure solutions can be applied direct on the paper but solids are
always dissolved in small quantity of a suitable solvent.
• Biological tissues are treated with suitable solvents and their
extracts obtained.
• Proteins can be precipitated with alcohol and salts can be removed
by treatment with ion exchange resin.
10. • 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.
• The spot is dried on the filter paper and is placed in developing chamber.
APPLICATION OF SAMPLE
11. • PAPER CHROMATOGRAPHY MOBILE PHASE :
• 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 4:1:1
• Hydrophobic mobile phases
Hexane: 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.
12. • 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.
CHROMATOGRAPHIC CHAMBER:
13. 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:
• There may be Ascending, Descending, Circular, Two dimensional and Ascending
Descending type different development techniques
14. • Ascending development technique is conventional technique in
which the mobile phase moves against the gravity and spot the
sample at bottom portion.
• In descending development the mobile phase kept at the top and
the solvent flow down the paper. Here the samples applied at the
top and the development is fast due to gravity assisted solvent
flow.
15. Ascending and Descending Chromatography :
• This is the combination of both the above techniques.
• In this paper is folded over a glass rod.
• This allows the first ascending and followed by descending.
16. • In circular or radiant development : The sample applied at the center of the paper and
the mobile phase flown through a wick at the Centre and spread uniformly in all direction.
17. • In two dimensional development techniques: The samples is applied in one corner and
develop the paper on one axis then dry the paper. After drying turn the paper on ninety
degree angle and develop the paper on another axis. This technique is used for more
complex sample
18. • Drying of Chromatogram:
• After the solvent has moved a certain distance for certain time the chromatogram
is taken out from the tank & position of the solvent front is marked with a pencil.
• They are dried by cold or hot air depending on volatility of solvents.
• A simple hair dryer is a convenient device to dry chromatograms
19. • Detection :
• After development of the chromatogram the isolated compound can be visualized by detecting agents.
Detecting agents can be two types:
(a) Destructive type and (b) Non destructive type
• In destructive type the sample cannot be recovered or it will be destroyed due to chemical reaction of
spraying reagent with sample.
• In non destructive method sample can be detected by UV chamber method, densitometric method or
iodine chamber method.
20. • Specific methods: Specific spray reagents or detecting or visualizing agents are
used to find out the nature of compounds or identification purposes.
a. Ferric chloride- For phenolic compounds and tannins
b. Ninhydrin in acetone- for amino acids
c. Dragendroff’s reagent- for alkaloids
d. 3,5-Dinitro benzoic acid- for cardiac glycosides
e. 2,4- Di-nitrophenyl hydrazine- for aldehydes and ketones
21. • Paper chromatography can be use for both qualitative and quantitative purpose. For
qualitative purpose Rf value can be determined.
22. • 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 analysed by any conventional techniques of analysis like spectrophotometry,
electrochemical methods, etc.
23. • Advantages of Paper Chromatography:
1. Simple and Rapid
2. Paper Chromatography requires very less quantitative material.
3. Paper Chromatography is cheaper compared to other chromatography methods.
4. Both inorganic as well as organic compounds can be identified by paper chromatography method.
5. Paper chromatography does not occupy much space compared to other analytical methods or equipment’s.
• Limitations of Paper Chromatography
1. Large quantity of sample cannot be applied on paper chromatography.
2. In quantitative analysis paper chromatography is not effective.
3. Complex mixture cannot be separated by paper chromatography.
4. Less Accurate compared to HPLC or HPTLC
24. Applications :
1. Used for separation of mixture having polar and non polar compounds.
2. Used for separation of sugars , amino acids , lipids and inorganic compounds like salts.
3. Used for analysis of food colours in synthetic drinks and beverages , ice creams , jams
etc
4. Used in forsenic field for crime scene investigation.
5. The presence of metal ions like Ag+ , Fe3+ , Hg2+ can be analysed using paper
chromatography.
25. • Questions :
1. What is paper chromatography? Explain in detail the principle involved in PC.
2. Describe various development methods in PC.
3. Describe application , advantages and disadvantages of PC.
