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Partition Chromatography BY: ARTINA C. AQUITANIA

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Page 1: Partition chromatography & partition paper chromatography

Partition ChromatographyBY: ARTINA C. AQUITANIA

Page 2: Partition chromatography & partition paper chromatography

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

substance. 

Page 3: Partition chromatography & partition paper chromatography

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

Page 4: Partition chromatography & partition paper chromatography

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.

Page 5: Partition chromatography & partition paper chromatography
Page 6: Partition chromatography & partition paper 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.

Page 7: Partition chromatography & partition paper 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.

Page 8: Partition chromatography & partition paper chromatography
Page 9: Partition chromatography & partition paper chromatography

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.

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Page 11: Partition chromatography & partition paper chromatography

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

Page 12: Partition chromatography & partition paper 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

Page 13: Partition chromatography & partition paper chromatography

Types of Partition Chromatography

Partition Chromatography

Liquid - LiquidChromatography

Gas – LiquidChromatography

Page 14: Partition chromatography & partition paper chromatography

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

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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)

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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

Page 17: Partition chromatography & partition paper chromatography
Page 18: Partition chromatography & partition paper chromatography

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.

Page 19: Partition chromatography & partition paper chromatography

Paper Partition Chromatography

Page 20: Partition chromatography & partition paper 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.

Page 21: Partition chromatography & partition paper chromatography

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

Page 22: Partition chromatography & partition 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

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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

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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

Page 25: Partition chromatography & partition paper 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

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• 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

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• 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

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• 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.

Procedures

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• 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

Page 30: Partition chromatography & partition paper chromatography

• 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

Factors affecting Rf Value