Partition chromatography & partition paper chromatography

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Transcript of Partition chromatography & partition paper chromatography

Partition Chromatography

Partition ChromatographyBy: ArtinA c. aQUITANIA

ChromatographyTo write with colors -- literally translated from its Greek rootschromaandgrapheinanalytical 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 substance.

We know that mixture is the combination of more than one substance in a certain ratio-- distillation, crystallization, separating funnel, chromatography2

ChromatographyStationary Phase - phase that stays in place inside the column usually viscous liquid chemically bonded to the inside of a capillary columnMobile 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 ChromatographyPartition 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.

different principles however both of them are efficient analytical techniques for the separation of components4

Partition Chromatographybased 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 Chromatographymethod 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 phasesStationary 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 outThe separation depends on the relative solubility in the stationary liquid layer because of different partition coefficient, different component of sample are separated.

Partition Chromatography Procedurethe 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 Applicationsused for final purification natural extracts, synthetic mixtures and biological matrices. It is also used for fractionization of complex crude extracts eg. Petroleum fractionsDetermination of water qualitySeparation of aroma molecules of wineDetermination of pesticide residue

best chromatographic method to get high performance and pure components12

Types of Partition ChromatographyPartition ChromatographyLiquid - LiquidChromatographyGas LiquidChromatography

Liquid - Liquid Chromatographyemploys liquid mobile and stationary phasesuses small particles with molecules bonded to their surface to give a thin film that has liquidlike properties

Gas - Liquid Chromatographymobile 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 pressureMobile phaseInert carrier gas like Helium or NitrogenStationary phaseLayer of liquid or polymer on inert solid support Inside a glass or metal tubing (COLUMN)

ProceduresCompound is injected with syringe into sample injector

Compound is carried by carrier gas and vaporized

Vaporized sample interacts with walls of columnSome samples interact more some less

Due to interaction samples elute at different timesRetention timesComparison of retentions times is what is useful

A detector monitors the outlet stream from the column

Gas Pressure RegulatorHelium - It has an excellent thermal conductivity, low density, inertness and it permits greater flow rates. It is highly expensiveNitrogen - It offers reduced sensitivity and is inexpensiveHydrogen - 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

Paper 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 phaseThe 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 thepartitionbetween the liquids in opposite directions; where one of the liquids is bound as a film on filterpaper

Cellulose layers in filter paper contains moisture which acts as stationary phase & organic solvents/buffers are used as mobile phaseSTATIONARY 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 -cellulose, 0.3 1% -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 chamber.

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.Chromatographic Chamber

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.

Rf Value

temperaturepurity of the solvents usedquality of the paper, adsorbents & impurities present n the adsorbentsChamber saturation techniques, method of drying & developmentdistance travelled by the solute & solventChemical reaction between the substances being partitioned.pH of the solution

Factors affecting Rf Value