Paper Chromatography
Contents
• Paper
chromatography
• Principle
involved
• Applications
of Paper chromatography
Practical requirements
• Stationary
phase
• Application
of sample
• Mobile
phase
• Development
technique
• Detecting
or visualizing agents
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
• 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
• Rfvalue
(retardation factor) is calculated for identifying the spots i.e., qualitative
analysis
• Rfvalue
is the ration of distance travelled by the solute to the distance travelled by
solvent front
• Rf
= distance travelled by solute /distance travelled by solvent front
• Rf
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
• Rf
value of a sample and reference compound is same, compound is identified
by its standard
• Rf
value differs, compound may be different from its reference standard
Rx value
• Ratio
of distance travelled by the sample and distance travelled by the standard
• Rx
value is always closer to 1
Rm value
• To
find whether compounds belongs to a homologous series
• If
they belong, Rm values are constant
• Can
be determined by Rm = 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
