Chromatography• “To write with colors” -- literally translated from its
Greek roots chroma and graphein • analytical technique commonly used to separate
mixtures into their components, purify compounds and also to test its purity. • most reliable technique as it gives 99% pure
Chromatography• Stationary Phase - phase that stays in place inside the
column • usually viscous liquid chemically bonded to the inside of
a capillary column• Mobile phase - solvent moving through the column
and is either liquid or gas. • Elution - process of passing liquid or gas through the
column. • Eluent - fluid entering the column • Eluate - fluid leaving the column
Difference Between Adsorption and Partition Chromatography• Partition chromatography - separation on the stationary
phase occurs by partition due to differences in partition coefficients.• Used for liquid-liquid or liquid gas chromatography
• Adsorption Chromatography - relative differences in adsorption of constituents of given sample. Because of differences in their affinity towards stationary phase, the components of the mixture adsorb with different rates.• used only for solid-liquid or solid-gas chromatography.
Partition Chromatography• based on a thin film formed on the surface of a solid support
by a liquid stationary phase. • Solute equilibrates between the mobile phase and the
stationary liquid.• applies to liquid-liquid, liquid-gas chromatography and not
to solid-gas chromatography.
Partition Chromatography• method of separation in which the components present
in the mixture get distributed more likely into two liquid phases because of differences in partition coefficients during the flow of mobile phase in the chromatography column.• • Partition Coefficient - the ratio of the concentrations
of a solute in two immiscible or slightly miscible liquids, or in two solids, when it is in equilibrium across the interface between them.
Partition Chromatography Principle• separation of components of given sample occurs due to
partition of components between two liquid phases• Stationary phase is coated with a liquid which is immiscible in
mobile phase.• stationary phase immobilizes the liquid surface and makes
it stationary phase.• The mobile phase passes over the stationary phase and
separate out• The separation depends on the relative solubility in the
stationary liquid layer because of different partition coefficient, different component of sample are separated.
Partition Chromatography Procedure
• the separation of components of the mixture occurs due to distribution of components between two liquid phases. • The separation of components occurs on the basis of partition coefficients of constituents of mixture. • The flow of components or mobile phase separates all components over stationary phase. • The polar phase attracts polar components of sample and non-polar components are attracted by non-polar phase of the system.• Partition chromatography can be done with different phases like Liquid-liquid and liquid-gas chromatography
Partition Chromatography Applications• used for final purification natural extracts, synthetic
mixtures and biological matrices. • It is also used for fractionization of complex crude extracts
eg. Petroleum fractions• Determination of water quality• Separation of aroma molecules of wine• Determination of pesticide residue
Types of Partition Chromatography
Liquid - LiquidChromatography
Gas – LiquidChromatography
Liquid - Liquid Chromatography• employs liquid mobile and stationary phases• uses small particles with molecules bonded to their surface
to give a thin film that has liquidlike properties
Gas - Liquid Chromatography• mobile phase is a gas and the stationary phase is a liquid,
usually on small beads packed in a long columnPoints to remember:Sample has to be able to be vaporized without
decompositionBased on boiling point/vapor pressure
• Mobile phaseo Inert carrier gas like Helium or Nitrogen
• Stationary phaseo Layer of liquid or polymer on inert solid support o Inside a glass or metal tubing (COLUMN)
Procedures• Compound is injected with syringe into sample injector
• Compound is carried by carrier gas and vaporized
• Vaporized sample interacts with walls of columno Some samples interact more some less
• Due to interaction samples elute at different timeso Retention timeso Comparison of retentions times is what is useful
• A detector monitors the outlet stream from the column
Gas Pressure Regulator• Helium - It has an excellent thermal conductivity, low
density, inertness and it permits greater flow rates. It is highly expensive
• Nitrogen - It offers reduced sensitivity and is inexpensive
• Hydrogen - It has a distinctly better thermal conductivity and lower density. Demerits are its reactivity with unsaturated compounds and hazardous explosive nature
• Air - It is employed only when the atmospheric O2 is beneficial to the detector separation.
Paper Partition Chromatography
• first introduced by German scientist Christian Friedrich Schonbein (1865).• 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.
1.PAPER ADSORPTION CHROMATOGRAPHY Paper impregnated with silica or alumina acts as adsorbent
(stationary phase) and solvent as mobile phase.
2.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 = Paper Partition Chromatography
Types of Paper Chromatography
Paper Partition Chromatography• In standard method of analysis, where in the paper is utilized as a support with one solvent as mobile phase and the other is the stationary phase• The migration of substances is due to the partition coefficients • separation of similar substances by repeated divisions between two immiscible liquids, so that the substances, in effect, cross the partition between the liquids in opposite directions; where one of the liquids is bound as a film on filter paper
Cellulose layers in filter paper contains moisture which acts as stationary phase & organic solvents/buffers are used as mobile phase
• 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
Principle of Paper Partition Chromatography
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.
Paper Partition Chromatography
• 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.
Paper Partition Chromatography
• 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
Preparation of the solution
• 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.
• 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.
• In paper chromatography the results are represented by Rf value which represent the movement or migration of solute relative to the solvent front.
• temperature• purity of the solvents used• quality of the paper, adsorbents & impurities present n the adsorbents• Chamber saturation techniques, method of drying & development• distance travelled by the solute & solvent• Chemical reaction between the substances being partitioned.• pH of the solution