Quality Control 2

1. Partition
Chromatography
BY: ARTINA C. AQUITANIA

2. 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.

3. 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

4. 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.

6. 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.

7. 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.

9. 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.

11. 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

12. 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

13. Types of Partition Chromatography
Partition Chromatography
Liquid - Liquid
Chromatography
Gas – Liquid
Chromatography

14. 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

15. Gas - Liquid Chromatography
• mobile phase is a gas and the stationary phase is a liquid, usually on
small beads packed in a long column
Points to remember:
Sample has to be able to be vaporized without
decomposition
Based on boiling point/vapor pressure
• Mobile phase
o Inert carrier gas like Helium or Nitrogen
• Stationary phase
o Layer of liquid or polymer on inert solid support
o Inside a glass or metal tubing (COLUMN)

16. Procedures
• Compound is injected with syringe into sample injector
• Compound is carried by carrier gas and vaporized
• Vaporized sample interacts with walls of column
o Some samples interact more some less
• Due to interaction samples elute at different times
o Retention times
o Comparison of retentions times is what is useful
• A detector monitors the outlet stream from the column

18. 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.

19. Paper Partition
Chromatography

20. 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.

21. 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

22. 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

23. 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

24. 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

25. • 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

26. • 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

27. • 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

28. • Paper is flexible when compared to glass plate used inTLC, 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

29. • 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

30. • 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