PARTITION CHROMATOGRAPHY IS A SEPARATION TECHNIQUE ON THE BASIS OF PARTITION COEFFICIENT

1. BY
Dr.RachanaCHOUDHARY
Asstt.Prof.
Departmentofmicrobiology
ShrISHANKARACHARYAMAHAVIDYALAYAJUNWANI,
BHILAI
Partition
Chromat ography

2. SYNOPSIS
INTRODUCTION
TERMS RELATED TO CHROMATOGRAPHY
HISTORY OF CHROMATOGRAPHY
TYPES OF CHROMATOGRAPHY
PARTITION CHROMATOGRAPHY
 TYPES
 PRINCIPLE
 PROCEDURE
APPLICATIONS
REFERENCES

3. Introduction
Chromatography is an analytical technique used for separation, identification, and analysis of
various components of a mixture. The sample components often vary in physical and/ or
chemical properties.
The compound that is separated during chromatography is called analyte.
Russian botanist - M. S. Tswett. He developed this useful technique in 1906 to separate plant
pigments under gravity using a calcium carbonate column. Tswett also coined the term
‘chromatography’, which comes from the words chroma (Greek) i.e. colour and graphein,
which means "to write". “Towrite with colors”
In fact, this technique can be used to distinguish between two
compounds that are quite similar in molecular mass or
charge; however, this requires an appropriate combination of
materials and operating conditions.
A wide range of soluble or volatile, organic or inorganic
compounds can be thus separated using chromatographic technique.

4. TERMS RELATED TO Chromatography
StationaryPhase - phasethat stays in placeinside the column
usually viscous liquid chemically bonded to the inside of acapillary
column
Mobilephase- solvent moving through the column and is either
liquid orgas.
Elution- processof passing liquid or gasthrough the column.
Eluent -fluid entering the column
Eluate-fluid leaving the column

5. History of Chromatography
• Chromatography was first employed in Russia by the Italian-born
scientist Mikhail Tswett in 1906.
• of Archer John Porter Martin and Richard Laurence Millington
Synge during the 1940s and 1950s, for which they won the 1952 Nobel
Prize in Chemistry.
• They established the principles and basic techniques of partition
chromatography, and their work encouraged the rapid development of
several chromatographic methods.
• Researchers found that the main principles of Tswett's chromatography
could be applied in many different ways.

6. TYPES OF CHROMATOGRAPHY

7. PartitionChromatography
basedon athin film formed on the surfaceof a
solidsupport by aliquid stationary phase.
Solute equilibrates between the mobile phase&
the stationary liquid.
method of separation in which the components
present in the mixture get distributed more likely
into two liquid phasesbecauseof differences in partition coefficients during the
flow of mobile phasein the chromatographycolumn.

8. 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 acrossthe interface between them.
TYPESOFPARTITIONCHROMATOGRAPHY
 Liquid –Liquid Chromatography
 Gas- Liquid Chromatography

9. Liquid - LiquidChromatography
• employsliquid mobile andstationary phases
• usessmall particles with moleculesbonded to their surfaceto give a
thin filmthat hasliquid like properties
• PAPER CHROMATOGRAPHY

10. 1.PAPERADSORPTIONCHROMATOGRAPHY
Paperimpregnatedwith silicaor aluminaactsasadsorbent(stationary phase)and solvent
asmobilephase.
2. PAPER PARTITIONCHROMATOGRAPHY
Moisture / Water present in the pores of cellulose fibers present in filter paperactsas
stationary phase& another mobilephaseisused assolvent
Ingeneral,PaperChromatography=PaperPartitionChromatography
Typesof PaperChromatography

11. PaperPartitionChromatography
 Instandardmethod of analysis,wherein the paperisutilized asa support with
one solvent asmobile phaseand the other is the stationaryphase
 Themigration of substancesisdueto the partition coefficients
 separationof similar substancesby repeated divisionsbetween two
immiscible liquids,sothat the substances,in effect, crossthe partition
betweenthe liquidsin oppositedirections;whereone of the liquidsis
boundasafilm onfilter paper.
• Ascending,
• Descending
• Circular

13. Celluloselayers in filter paper contains moisture which acts as
stationary phase& organicsolvents/buffersareusedasmobile
phase
STATIONARY PHASEAND 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 PaperPartitionChromatography

14. Pure solutions can be applied direct on the paper but solids are always dissolved in small
quantity of asuitablesolvent.
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 exchangeresin.
APPLICATIONOF SAMPLE
The sample to be applied is dissolved in the mobile phaseand applied asasmall spot on
the origin line,usingcapillarytube or micropipette.
verylow concentrationisusedto avoidlargerzone
Thespot isdried onthe filter paperandisplacedin developingchamber.
Preparation of thesolution

16. • Glasstanks are preferredmost.
They are available in various
dimensional sizedepending upon
paper length and development
type.
• The chamberatmosphere
should be saturated with
solventvapor.
Chromatographic
Chamber

17. • Paper is flexible when compared to glass plate used in TLC, several types of
development are possible which increasesthe easeof 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
andefficient result.
Procedures

19. • Inpaperchromatographythe resultsarerepresentedby Rf value.
RfValue

20.  Temperature
 Purity of the solventsused
 Quality of the paper,adsorbents& impurities presentnthe
adsorbents
 Chambersaturation techniques,method of drying & development
 Distancetravelled bythe solute& solvent
 Chemicalreaction between the substancesbeingpartitioned.
 pH of thesolution
Factors affecting RfValue

21. Gas- LiquidChromatography
• mobilephaseisagasandthe stationary phaseisaliquid, usually on smallbeads
packedin along column
Pointstoremember:
Samplehasto beableto bevaporizedwithout
decomposition
Basedonboiling point/vapor pressure
• Mobilephase
o Inert carrier gaslike Helium or Nitrogen
• Stationaryphase
o Layerof liquidor polymeroninert solidsupport
o Insideaglassor metal tubing(COLUMN)

22. Procedures
• Compoundisinjectedwith syringeinto sampleinjector
• Compoundiscarriedbycarriergasandvaporized
• Vaporizedsampleinteractswith wallsof column
o Somesamplesinteract more someless
• Dueto interaction sampleseluteat differenttimes
o Retentiontimes
o Comparisonof retentionstimes iswhat isuseful
• Adetector monitors the outlet streamfrom the column

24. GasPressure Regulator
• Helium - It hasan excellent thermal conductivity, low density,
inertness andit permits greater flow rates. It ishighly expensive
• Nitrogen - Itoffers reduced sensitivity and is inexpensive
• Hydrogen - It hasadistinctly better thermal conductivity and lower
density. Demerits areits reactivity with unsaturated compoundsand
hazardous explosivenature
• Air - It isemployedonly when the atmosphericO2isbeneficial to
the detectorseparation.

25. Applications
usedfor final purification natural extracts, synthetic mixtures and
biological matrices.
It is also usedfor fractionization of complex crude extracts eg. Petroleum
fractions
Determination ofwater quality
Separation of aroma moleculesof wine
Determination of pesticide residue

26. REFERENCES
• BIOPHYSICAL CHEMISTRY BY UPADHYAY & UPADHYAY NATH
• INSTRUMENTAL METHODS OF CHEMICAL ANALYSIS BY DR.B.K.SHARMA
• A TEXTBOOK OF MICROBIOLOGY BY R.C. DUBEY & D.K. MAHESHWARI
• GOOGLE SEARCH
• SLIDESHARE