Paper chromatography

• Paper chromatography is defined as technique in

which the analysis of unknown substance is carried
out mainly by the flow of solvents on specially
designed filter paper (Whatman FP).
• In 1961 paper chromatography was first discovered
by SEHON BEN.
 Basic Principle
• The principle of separation is mainly partition rather
then adsorption.
• Cellulose layers in filter paper contains moisture which
acts as stationary phase. organic solvents or buffers are
used as mobile phases. instead of water as stationary
phase other organic solvents can be used by suitable
modification.
• Paper is made of cellulose fibers, and cellulose is a
polymer of the simple sugar, glucose. The key point
about cellulose is that the polymer chains have -OH
groups sticking out all around them.
• The cellulose fibers attract water vapour from the
atmosphere as well as any water that was present
when the paper was made. You can therefore think
of paper as being cellulose fibers with a very thin
layer of water molecules bound to the surface.
• It is the interaction with this water which is the most
important effect during paper chromatography.
• Suppose you use a non-polar solvent such as hexane
to develop your chromatogram.
• Non-polar molecules in the mixture that you are
trying to separate will have little attraction for the
water molecules attached to the cellulose, and so will
spend most of their time dissolved in the moving
solvent. Molecules like this will therefore travel a
long way up the paper carried by the solvent. They
will have relatively high Rf values.
• On the other hand, polar molecules will have a high
attraction for the water molecules and much less for
the non-polar solvent.
• They will therefore tend to dissolve in the thin layer
of water around the cellulose fibers much more than
in the moving solvent.
• Because they spend more time dissolved in the
stationary phase and less time in the mobile phase,
they aren't going to travel very fast up the paper.
• The tendency for a compound to divide its time
between two immiscible solvents (solvents such as
hexane and water which won't mix) is known as
partition. Paper chromatography using a non-polar
solvent is therefore a type of partition
chromatography.
• PRACTICAL REQUIREMENTS
1)Stationary phase & papers used
2)Application of sample
3)Mobile phase
4)Development technique
5)Detecting or Visualizing agents
• STATIONARY PHASE AND PAPERS USED
• Whatman filter papers of different grades like No.1, No.2,
No.3, No.4, No.20, No.40, No.42 etc are used. In general this
paper contains 98-99% of α-cellulose, 0.3 – 1% β -cellulose
• Factors that governs the choice of paper:
• » Nature of Sample and solvents used.
• » Based on Quantitative or Qualitative analysis.
• » Based on thickness of the paper
• Modified Papers – 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.
• Impregnation of silica, alumna, or ion exchange
resins can also be made.
• PREPARATION OF PAPER

• Cut the paper into desired shape and size depending upon
work to be carried out. (Rectangular shape)
• The starting line is marked on the paper with an ordinary
pencil 1-2 cm from the bottom edge.
• On the staring line marks are made 1-2 cm apart from each
other.
• 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.
• APPLICATION OF SAMPLE
• The sample to be applied is dissolved in the mobile phase
and applied as a small spot on the origin line, using capillary
tube or micropipette.
• very low concentration is used to avoid larger zone
• The spot is dried on the filter paper and is placed in
developing chamber.
• Sample is placed on paper in such way that sample should not
be dips in to mobile phase. If spot is placed below the place
up to which paper dips in mobile phase, solute will spread in
mobile phase.
• Choice of the Solvent
• 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. MOBILE PHASE
• Pure solvents, buffer solutions or mixture of solvents
• Examples- Hydrophilic mobile phase
• Isopropanol: ammonia: water (9:1:2 v/v/v) ;
• Methanol : water (4:1 v/v) ;
• N-butanol : glacial acetic acid : water (4:1:5 v/v/v)
• Hydrophobic mobile phases: dimethyl ether: cyclohexane;
kerosene: 70% isopropanol
• CHROMATOGRAPHIC CHAMBER
• The chromatographic chamber are made up of many
materials like glass, plastic or stainless steel.
• Glass tanks are preferred most. They are available in
various dimensional size depending upon paper
length and development type.
• The chamber atmosphere should be saturated with
solvent vapor.
• DEVELOPMENT TECHNIQUE
• Paper is flexible when compared to glass plate used
in TLC, several types of development are possible
which increases the ease of operation.
• The paper is dipped in solvent in such a manner that
the spots will not dip completely into the solvent.
• The solvent will rise up and it is allowed to run 2/3rd
of paper height for better and efficient result.
 Types of Paper chromatography
• Four type (Based on development techs)
• 1. Ascending PC
• 2. Descending PC
• 3. Ascending-Descending PC
• 4. Circular (Radial) PC
• 5. Two dimensional PC
• 1) 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.
• Mobile phase rises in upward direction due to capillary
action. This motion is against the gravitational forces.
• When mobile phase rises to ¾ th of paper, it is taken out of
the chamber and mobile phase is allowed to dry.
• If we are doing separation of mixture of colored components
we can see spots of different colors on papers. Thus
identification of such components is easy.
• If mixture being separated becomes colorless then spraying
reagent is required to develop the color on the paper.
• 2) DESCENDING TYPE (a downward slope)
• 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.
• ADVANTAGE IS THAT, DEVELOPMENT IS FASTER
• 3)ASCENDING – DESCENDING DEVELOPMENT
• A hybrid of above two technique is called ascending-
descending chromatography.
• Only length of separation increased, first ascending takes
place followed by descending.
• 4)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.
• 5)TWO DIMENSIONAL DEVELOPMENT
• In this method the paper is developed in one direction and
after development, the paper is developed in the second
direction allowing more compounds to be separated into
individual spots.
• In the second direction, either same solvent/different solvent
system can be used for development.
• 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.
• DETECTING / VISUALISING AGENTS
• If the substance are colored they are visually detected easily.
But for colorless substance, Physical and chemical methods
are used to detect the spot.
• (a) Non specific methods ( Physical methods)
• E.g. iodine chamber method,
• UV chamber for fluorescent compounds – at 254 or at 365nm.
• (b) Specific methods (Chemical methods) or
Spraying method
• For Phenolic mixtures- FeCl3 solution is sprayed
• Starch- Iodine is used
• Amino acids- Ninhydrine reagent
• Carbonyl compounds- 2,4, dinitrophenyl hydrazine
• Primary aromatic amine-NaNO2/HCl and the BMR
reagent to get red color
• Dragendroff’s-Alkaloids
• Identification/ Qualitative analysis
• 1) Relative method:
• This method uses test and standard. Distance travelled by
solute is affected by temperature, pressure and environment
etc.
• Both test and standard are affected by same variables and so
error is minimized.
• 2) Absolute method:
• This method use Rf Value for identification of unknown.
• Rf VALUE (Retardation Factor)
• Rf = Distance travelled by solute from origin
• _______________________________
• Distance travelled by solvent from origin
• Factors affecting Rf VALUE
• i. The temperature
• ii. The purity of the solvents used
• iii. The quality of the paper, adsorbents & impurities present
n the adsorbents
• iv. Chamber saturation techniques, method of drying &
development
• v. The distance travelled by the solute & solvent
• vi. Chemical reaction between the substances being
partitioned.
• vii. pH of the solution
• Rx VALUE
• In many cases it has been observed that the solvent
front is run off the end of the paper.
• It is the ratio of distance travelled by the sample and
the distance travelled by the standard.
• Rx is always closer to 1.
• Assay or Quantitative methods:
• In this technique, the spots are cut into portions and
eluted with solvents. This solution can be filtered
and analyzed by any techniques of analysis like
spectrophotometry, electrochemical methods, etc.
• Sources of Error Sources
• 1. Error during application of the spots
• Apply minimum volume of the concentrated solution in order to
avoid diffusion through the paper which leads to poor separation
• Spots should be approximately of the same diameter.
• 2. Development
• Improper adjustment of the paper in the tank leads to this error
so the paper should be held vertically.
• Do chamber saturation
• 3. Detection
• The spraying methods affect the final result
• APPLICATIONS
• Separation of mixtures of drugs
• Separation of carbohydrates, vitamins, antibiotics, proteins,
etc.
• Identification of drugs
• Identification of impurities
• Analysis of metabolites of drugs in blood , urine ….