Thin layer chromatography (TLC) is a technique used to separate and identify components of a mixture. It works by distributing the mixture between a stationary phase coated on a plate and a mobile phase solvent that travels up the plate. Components separate based on how much they adsorb to the stationary phase versus dissolve in the mobile phase. To perform TLC, a small amount of the mixture is spotted on the plate and the plate is developed in a solvent chamber. This allows the mobile phase solvent to carry components up the plate at different rates, separating them into visible spots that can then be analyzed.

1. CHCHROMROMATOATOGRAGRAPHYPHY
NAME:SONIKANAME:SONIKA
CLASS: MSC.SEM 2CLASS: MSC.SEM 2
ROLL NO: 06ROLL NO: 06

2. HistoryHistory
Mikhail Tswett, Russian, 1872-Mikhail Tswett, Russian, 1872-
19191919
BotanistBotanist
In 1906 Tswett used to chromatography toIn 1906 Tswett used to chromatography to
separate plant pigmentsseparate plant pigments
He called the new techniqueHe called the new technique
chromatography because the result of thechromatography because the result of the
analysis was 'written in color' along theanalysis was 'written in color' along the
length of the adsorbent columnlength of the adsorbent column
Chroma meansChroma means ““colorcolor”” and graphein means toand graphein means to
““writewrite””

3. ImportanceImportance
Chromatography has application in every branch of theChromatography has application in every branch of the
physical and biological sciencesphysical and biological sciences
1212Nobel prizes were awarded between 1937 and 1972 aloneNobel prizes were awarded between 1937 and 1972 alone
for work in which chromatography played a vital rolefor work in which chromatography played a vital role

4. ChChromromatoatogragraphyphy
Chromatography is a physical method ofChromatography is a physical method of
separation in which the components to beseparation in which the components to be
separated are distributed between twoseparated are distributed between two
phasesphases
one of which is stationary (one of which is stationary (stationary phasestationary phase))
while the other (thewhile the other (the mobile phasemobile phase) moves) moves
through it in a definite directionthrough it in a definite direction..
The chromatographic process occurs due toThe chromatographic process occurs due to
differences in thedifferences in the distribution constantdistribution constant ofof
the individual sample componentsthe individual sample components..

5. ChChromromatoatogragraphyphy
Is a technique used to separate
and identify the components of a
mixture.
Works by allowing the molecules present in the
mixture to distribute themselves between a
stationary and a mobile medium.
Molecules that spend most of their
time in the mobile phase are carried
along faster.

6. Classification of chromatographyClassification of chromatography
according to mobile phaseaccording to mobile phase::
11--Liquid chromatography: mobileLiquid chromatography: mobile
phase is a liquid. (LLCphase is a liquid. (LLC(.(.
22--Gas chromatography : mobile phase isGas chromatography : mobile phase is
a gas. (GLCa gas. (GLC(.(.

7. Classification according to the packing of theClassification according to the packing of the
stationary phasestationary phase::
11--Thin layer chromatography (TLC): the stationaryThin layer chromatography (TLC): the stationary
phase is a thin layer supported on glass, plastic orphase is a thin layer supported on glass, plastic or
aluminium platesaluminium plates..
22--Paper chromatography (PC): the stationary phasePaper chromatography (PC): the stationary phase
is a thin film of liquid supported on an inertis a thin film of liquid supported on an inert
supportsupport..
33--Column chromatography (CC): stationary phaseColumn chromatography (CC): stationary phase
is packed in a glass columnis packed in a glass column..

8. Classification according to the force ofClassification according to the force of
separationseparation::
11--Adsorption chromatographyAdsorption chromatography..
22--Partition chromatographyPartition chromatography..
33--Ion exchange chromatographyIon exchange chromatography..
44--Gel filtration chromatographyGel filtration chromatography..
55--Affinity chromatographyAffinity chromatography..

10. Paper ChromatographyPaper Chromatography
A method of partition chromatography using filterA method of partition chromatography using filter
paper strips as carrier or inert supportpaper strips as carrier or inert support..
The factor governing separation of mixtures of solutesThe factor governing separation of mixtures of solutes
on filter paper is theon filter paper is the partition between twopartition between two
immiscible phasesimmiscible phases..
One is usually water adsorbed on cellulose fibres inOne is usually water adsorbed on cellulose fibres in
the paper (stationary phasethe paper (stationary phase(.(.
The second is the organic solvent flows past theThe second is the organic solvent flows past the
sample on the paper (stationary phasesample on the paper (stationary phase(.(.

12. Techniques of development with various flowTechniques of development with various flow
directionsdirections
Ascending development Descending development

13. B- two-dimensional chromatographyB- two-dimensional chromatography::
When large numbers of substances are to be separatedWhen large numbers of substances are to be separated
on a single chromatogramon a single chromatogram..
Development in a direction perpendicular to the first,Development in a direction perpendicular to the first,
and with a solvent system different from that usedand with a solvent system different from that used
initially is often necessaryinitially is often necessary..
The sample is applied on one corner of a square piece ofThe sample is applied on one corner of a square piece of
paper and after development with the first solvent,paper and after development with the first solvent,
the paper is dried , rotated 90the paper is dried , rotated 90oo
and developed in theand developed in the
second directionsecond direction..
Usually, different types of solvents systems are used inUsually, different types of solvents systems are used in
each direction. It is essential that the first solvent beeach direction. It is essential that the first solvent be
completely volatilecompletely volatile..

16. Thin layer chromatography (TLCThin layer chromatography (TLC((
is a method foris a method for identifyingidentifying substances andsubstances and
testing the puritytesting the purity of compoundsof compounds..
TLC is a useful technique because it isTLC is a useful technique because it is
relativelyrelatively quickquick and requiresand requires smallsmall
quantitiesquantities of materialof material..

17. Separations in TLC involve distributing a mixture of twoSeparations in TLC involve distributing a mixture of two
or more substances between aor more substances between a stationary phasestationary phase and aand a
mobile phasemobile phase..
The stationary phaseThe stationary phase::
is a thin layer of adsorbent (usually silica gel or alumina)is a thin layer of adsorbent (usually silica gel or alumina)
coated on a platecoated on a plate..
The mobile phaseThe mobile phase::
is a developing liquid which travels up the stationaryis a developing liquid which travels up the stationary
phase, carrying the samples with itphase, carrying the samples with it..
Components of the samples will separate on theComponents of the samples will separate on the
stationary phase according tostationary phase according to
how much they adsorb on the stationary phase versushow much they adsorb on the stationary phase versus
how much they dissolve in the mobile phasehow much they dissolve in the mobile phase..

18. Thin Layer Chromatography (TLCThin Layer Chromatography (TLC((

20. TLCTLC

21. Visualizing AgentsVisualizing Agents
Alkaloids: DragendorffAlkaloids: Dragendorff’’s reagents reagent
Cardiac glycosides: Antimony trichlorideCardiac glycosides: Antimony trichloride
Sugar: Aniline phthalateSugar: Aniline phthalate
Amino acids: NinhydrinAmino acids: Ninhydrin

22. Interpreting the DataInterpreting the Data
The RThe Rff (retention factor) value for each spot(retention factor) value for each spot
should be calculatedshould be calculated..
It is characteristic for any given compoundIt is characteristic for any given compound
on the same stationary phase using the sameon the same stationary phase using the same
mobile phase for development of the platesmobile phase for development of the plates..
Hence, known RHence, known Rff values can be compared tovalues can be compared to
those of unknown substances to aid in theirthose of unknown substances to aid in their
identificationsidentifications..

25. ))Note: RNote: Rff values often depend on the temperature andvalues often depend on the temperature and
the solvent used in the TLC experimentthe solvent used in the TLC experiment..
the most effective way to identify a compound is to spotthe most effective way to identify a compound is to spot
known substancesknown substances –– authentic - next to unknownauthentic - next to unknown
substances on the same platesubstances on the same plate.(.(
In addition, the purity of a sample may be estimatedIn addition, the purity of a sample may be estimated
from the chromatogramfrom the chromatogram..
An impure sample will often develop as two or moreAn impure sample will often develop as two or more
spots, while aspots, while a
pure sample will show only one spotpure sample will show only one spot

26. SummarySummary
A TLC plate is a sheet of glass, metal, or plastic which is coatedA TLC plate is a sheet of glass, metal, or plastic which is coated
with a thin layer of a solid adsorbent (usually silica orwith a thin layer of a solid adsorbent (usually silica or
aluminaalumina(.(.
A small amount of the mixture to be analyzed is spotted near theA small amount of the mixture to be analyzed is spotted near the
bottom of this platebottom of this plate..
The TLC plate is then placed in a shallow pool of a solvent in aThe TLC plate is then placed in a shallow pool of a solvent in a
developing chamber so that only the very bottom of the plate isdeveloping chamber so that only the very bottom of the plate is
in the liquidin the liquid..
This liquid, or the eluent, is the mobile phase, and it slowly risesThis liquid, or the eluent, is the mobile phase, and it slowly rises
up the TLC plate by capillary actionup the TLC plate by capillary action..
As the solvent moves past the spot that was applied, anAs the solvent moves past the spot that was applied, an
equilibrium is established for each component of the mixtureequilibrium is established for each component of the mixture
between the molecules of that component which are adsorbedbetween the molecules of that component which are adsorbed
on the solid and the molecules which are in solutionon the solid and the molecules which are in solution..

27. In principle, the components will differ in solubility and inIn principle, the components will differ in solubility and in
the strength of their adsorption to the adsorbent and somethe strength of their adsorption to the adsorbent and some
components will be carried farther up the plate thancomponents will be carried farther up the plate than
othersothers..
When the solvent has reached the top of the plate, the plate isWhen the solvent has reached the top of the plate, the plate is
removed from the developing chamber, dried, and theremoved from the developing chamber, dried, and the
separated components of the mixture are visualizedseparated components of the mixture are visualized..
If the compounds are colored, visualization isIf the compounds are colored, visualization is
straightforward. Usually the compounds are not colored,straightforward. Usually the compounds are not colored,
so a UV lamp is used to visualize the platesso a UV lamp is used to visualize the plates..

28. Columnar Chromatography (CCColumnar Chromatography (CC((
This includes chromatographic methods inThis includes chromatographic methods in
whichwhich::
The stationary phase is packed into a columnThe stationary phase is packed into a column..
The mobile phase is a moving liquid or gasThe mobile phase is a moving liquid or gas..
According to the mechanism of separation ofAccording to the mechanism of separation of
solutes, five major types of CC aresolutes, five major types of CC are
ditinguished. Usually, one mechanismditinguished. Usually, one mechanism
predominates but does not exclude the otherspredominates but does not exclude the others

29. Different Types of chromatography
Mode or type Stationary phase Mobile phase Mechanism
Adsorption
Chromatography
Solid that attracts
the solutes
Liquid or gas Solutes move at different rates
according to the forces of attraction
to the stationary phase.
Partition
Chromatography
Thin film of liquid
formed on the
surface of a solid
inert support
Liquid or gas Solutes equilibrate between the 2
phases according to their partition
coefficients
Ion Exchange
Chromatography
Solid resin that
carries fixed ions
& mobile
couterions of
opposite charge
attached by
covalent bonds
Liquid
containing
electrolytes
Solute ions of charge opposite to the
fixed ions are attracted to the resin
by electrostatic forces & replace the
mobile counterions.
Molecular Exclusion
Chromatography
Porous gel with no
attractive action
on solute
molecules
Liquid Molecules separate according to
their size:
1.Smaller molecules enter the pores
of the gel, and need a larger volume
of eluent.
2.Larger molecules pass through the
column at a faster rate.
Affinity
Chromatography
Solid on which
specific molecules
Liquid or gas Special kind of solute molecules
interact with those immobilized on

30. Column ChromatographyColumn Chromatography
ColumnColumn
chromatographychromatography
Stationary phase isStationary phase is
held in a narrowheld in a narrow
tube throughtube through
which the mobilewhich the mobile
phase is forcedphase is forced
under pressure orunder pressure or
under the effectunder the effect
of gravityof gravity

31. Term Definition
Solvent
Mobile liquid phase with no affinity to the stationary phase
(i.e. inert towards it) & no effect on solutes.
Developer
Any liquid with more affinity to the stationary phase than
the solvent but less than solutes and just capable to move
them through the column.
Effluent Any liquid that passes out of the column.
Eluent
Any liquid that has lesser affinity to the stationary phase
than solutes but is capable to move them out of the column.
Eluate Fraction of eluent containing a required specific substance.
Retention
volume (VR
)
(or retardation volume): Volume of mobile phase that
passes out of the column, before elution of a specific
substance.

32. Number of Theoretical PlatesNumber of Theoretical Plates
(N(N((
H = Theoretical Plate Height
L = Length of the Column.
N = L / H
As HETP decreases efficiency
of the column increases.

33. REFRENCESREFRENCES

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