COIT- V)

TIHINLAyER_CHROMAOGRAPHY K

(Thin koyer ehroriatography)

separattan baßed on , the
one o fthe
TAC
'AC.
îs
Chromátdgrapny
Chrbmatdgrapny echnquc
eenaue
pheremena. T 1s a tt uid
tauid
adsorptbH
and mobile phasa-is
is a Solid
phase
in., which statiordy siLt c a Je Cora
u m i n a tor) celllo
ue
Ltquid. I n thisedhn Penene
chloroform

Phas N-hezon
'

suged as .statfonony
as mobtPhase.
etc qre ufed

Prtncipie.
basecd on the adsorpttan.
Phepomcna
-En t I g echriqui
PlaceS Om Be
Compounda &re a d s o r b e d at di£f er ent
diernt
phase ofF he" TLe plata ntth, ailterenr strengths.
Stabionary
the mo bYle pha se basin g
a thekr polarity.
these ar desorbed by
b
Erpertmental Procedure
www

AdsorbentS: Stationary Phas¢ StliCa el Aumin0 2ellukose etc

Solvents Moblle Phasc n-hexonc, benzenc, Water » choroform ete.

In thbis techriquc, thln layer ot stlica gel cor alumina is

Coat ed on the glass plate .A di luted Solutiom of he Samplc

to chloroform or acetone Or methanol,
's a p p lied as a
5pot on
end of the plate.
t TLC Plate just 1 cm above froro th
The Spotted TLC Plate is devetoped by Keepln9 VerHcoally tn
the
TLC chambcr ntaining feto ml of Organte olvent such as

ch lorofom or acetunne or
Methonol It must be sught
belo the Spot of the TLe Ploate. The Solventmoves p the

Plote thro ugh the Stationar phasc y the Capiliary acHon.

The 90tvent Pushes the in the Sample uLpuoards
Com pouneds
up o d s . As a result, ne Compound o the
moving
With lfferent 8peed s
somple moves Sotvem

diffevent
and ets ad Sor bed a Ptace S
asirg on th ev

th ro is
Potaity as bright spoFS Atte Solyen aeq

each edi the 's taken out o the cham ber and
TL Plot
markn s dome the 6o lvent Arom i th a pen . hen
 Eolvent is evaporaed.

unt
the plote 1s dried
G lasschombes

-elunt solve

yem
TLC Plote mith pevetopmentof+
Suta gel coated
Sample Spo ) TLCPlat.
TLe plote)

G o e n tf r o m t compoud I

Compound I .

Seperaton ot thbe

on u e TLCPate
Con poundS
TLe Plotes gives
on
the
number of Spots produced
t h e Sample
compownds
im
he
umber ot Compounds in The mirture.
wrh tHe Rf
ot the Spots
Coparing the R^ factor
are knoo by on the plates
Kno n compouds. he Spots
facto of the Seperation
for the
ad leached by using Orgon ic Sotvents
9rapped
of conpounds
APPLicotios in
a míxturc.

urm ber o f Compoun ds

TO detemmine the
in a mixture
Compound
1dentity an unknown
r To
a
mixture.

COmpounds
în
TO Seperate
TO Ceperate pids
vitamins.(Algeno. o ita Ankibio tcs g foodprodug
Seperake have b een separgted 4y
process areaction . wwng TLL
TO moni tor

TO moni tor column chromatography Seperatton.

Equipment is ample

chromatoqram development timg i s Short

Choice is hide
Stationary phase
tast 'and easyy.
ComPonsnts is
Recovery ot 6eparated
Se para Hom eiHeiency iS high.

Tdeni H cation of Sepaateb Componcnts s easy.

Sensitivity s Very high

St ationary Phabc is ehemcally
inert
 Thin Layer chroatpgrpnY
22 ° JeNsr Sla
a t i o n of
Rlates
outthe Coatir
be Consis t e n t trough
The layer musr

he layer
Spension or Slurry
.

moterial is used Sa Su

Pre par ed by the tolowtng me thods

C uring Method
he glass plate is placed on alevel
Surfa.ce d n d
Slurry
Shur
S Put he is titled back ond fprthto Spread
On i t Dloatt
P Iate

the slumy
2 DPeing ethod
The adsorbent s dissolved in CHcl co) cHCI3t ChaoH
The
and are dipped into This
ar joined back
mixtur TWo Plates
is Then
and for Some time The Solv n t
SoLution Kept
.

olatiized.
Rr Methood
is Sprayed on to the glaas Plate nu th a

the srry

SPpayer
Seporahgo mehopl
in
This is idely used. be slurry is Placd

appicator is moved over he glass

app i c a t o r an th
a p pli cator S hetd slattorary and x
Plate. Some time s The
he Suurry . The surry laye are ded
pbte iS puued
throug
and Octivoted at uo'c .

Scanned by CamScarner
 DeveloPment chambs:
ApPLimtion of Sarnpie:
Purpo Se C a p i Llar y
MCrO Syringes are used. For te

tubes Can also be used.

Development chamber
he Chomber is made fsorn gla s s and he Platt is

Placed at an angle of 45°c as shocon in the igure.

1mm.
the tan k is coveres upto neaily
The bottomn of
a trd th
Nibh
Tne top iS Cover ed
Welgh by the sovent
Sattrated Nitb Solvent vapour
e n i re ehomber is
tormed
Detecio ofthe Spots W
Components re detected
ot 1he Seperot ed
The Spbts producngreagenE
appropriate celour'
b.Spraying
Or locat ed .

are charagterisH
".tompounds
7hesc reagents molec
to. the Spots. bimolecule
on coT) Organi malc

Sitable for inorganic . Cai on

Amino acidss

Carbornyl
compound 2,4 ONpH

A id
olKMnoy,
T2 Vapou
Amn

Scanned by CamScarnner
 tedwM& applicalisns s (olumn
(ol umn

Alesh priaiple, trpuiualAt o

thMMAoaghy?
Comn Cromatogra
Column omatography
is One of he SeparA tng
technqe
lechn
a liq
id chomatograph.
chromatographby.
Tt S
med on adsorptiom Pheno m e n a .
a Sotid ad he
is
pha s e
in Which is Stationary
tech nique Silca ge el Cor) alu
Jn this tech nique
|mobile Pha is a Alquid.
n-hexane
cor) chono A.
o
enzenc con
s used aS Stationary phase.

Tsused as moile Phase.

Parciple
It is bosed On the adsorptiom pheromena In trs
9';
on
On
technte
the
|diferent compounds are adsorbed ar dîfferent places

6tationary Phas of the blwnn ilt dittertnt Streng th6 Thesu

Ore describd by the mobile pha se s i n g on ther polaritY

Epemental Prooed use

Stottonaryphase: Si lfca Gel, Aluminb etC
Mobile Phase: n-hexane, benzepe, iater, chloroform ete

Sovent

arsure
9e
Pooder

Colun wtth sifca (eo uon uw

gel poodey. mltue and Ccownn wtb
sitcoqe Sepanated
boder). Compourd) Ccolectons o
Coopouod s dA E8)-
In this techni
que ndy poadered Lca gel o r alumlo
1s plaocd in a burette ltke 9lass ube Called/
co tumn. I t 's clan
Vericaly he mlature ot organic compounds hih cn are to be
e parated 1s dissoved o a solvent ad Pou ed from the to ot

the Column. . the Compounds n the nmi x ture

hile o ving dovo
th colum move wi h di Aferent Speeds nd ae a d s o r b e«
ar differ emt places dl4fertnt sbeng s on hc
C6totonar y Pha te) Sili ca
dependtrg On heir
Foarity .th
en the Organe

Scanned by CamScariner
 Solvents mobile phas Such as n-hezane or' benzèhe ov ChHor@form
are added tp flo Over the statonary phage. As a result, Cornpounds
of the mixture and
are deSorbe d from the Statona phac
awe collected Seporately tro the Column.

APPLcattons:
Ca) he SeParation of geometric SomerS.
cb) The SParation ot Compounds in h e miatgre-
c) The CPOration of Compoundsrin. the Racemic mixturC.

d) he Se Paration of amino acids

-pn each otp
paration Techniuesin Colum eromatographiy
s M

Column chromatography techniques a clasifted on the

osis he nature. ot the interaction bettoeen the Solutes and
the 9tationary PhaSe.

CI) Adsorption chromatograpby

) TonExchange chromatography.
3). Gel Flltearation
C4) Atftnit y chromatography
K5. High Performonce Kiquld Chromatography-
6) Gas Chrbmatogra phy
ci) Adsorption Chromatograp
the 1nteraction aSolute and thc SoLid
this techniquesS

bcking in
the Çolumn is bastcally adsorptisn and the packing
materlas. tunetions as the Sblid adSorbent

2 on Erchonge chroatograpby
Th th's techaue the Colump are packed

ExchangerS. Ton Fxchange materials are

materials Called Ion
nature theS Wege not
mostl Synttetic resin of o
Buitable for Separafon .of large moleaue Such as prot
Erchavge ot 1on

RSo Nat + NtH R Rso

Ry cl+ 6o cR' R,N... Oo cRt e l *

Scanned by CamScanner
C8 Ge l Eilteration: thc basis of
YMwiwm on
Separated
be
Mole cules Can
also Contaln
them
the Solu tt
on

in thdr Stzes by passing

difference Swollen parHcals
oF Gel.

down a column. Contai nfng

Cel particalS
S m a l artoals
O- Lage pamaals

CA) wAffinlty
wm
chornabography
w w Mw

is gure filustrate the purt Heat on of ensmes.

A Hrity materias. Enxymc

Tnh

A
A
mturedfenayne

generated Foz
Coluumn) Separab Bund onenzum
en7ym)

Scanned by CamScarnner
 hth ineiple, Cxpurituntaprotedeo &opptitaron d
He ortotogdap
igh Rressure/ Perforroange kiguid chromeDgTP9 CHPLC)
HPLe 1is ba sed on
One of the Separating techntques

distri bution law. Tt is a ehroatography techniquc.

Ltquíd
In
Wheh
Statonary Phase is a soud and mobi te phase is aliquld. I n ths

techntqu Porous s t t c a ton nitrile groups bonded s u c a or) n-akyl

n-bex a n e Cor)n- o ctanc
bond ed siluca etC is oed as sta ionary Pha Se .
corCHcs ete is used as mobîte pha se .

Prirepie
Tt is bo th distH u i om ond adsoTptt on pheromena
based on
compounds ae.
Tt is based Pvinctple
on the tha different

adsor bed at aifferent Paces on the StaHonary

phosc ot h e Coltimnn

wilh diferent strengths. TheSe are desorbed by the mob)le phase

asing m their Polarily.

bperimental Procedure:
Stationary pha St : Poro us Siuca con nitrile groups boe d iuça
Cor) n-alkyl bordcd î c a etc.
Mobile phasc :n-hec a ne coT) n-Octam com CHcig, CH3 OH> Hoj etc.

Saiwent
Reses vo'

i 8ample
ne ceiom part -

Sovent Mioro Hlgh pressurt

ftlte RmP.

-Column
Deteeto d
unte is nected ntd the columnn
thltechtque th
frorn he lnjrctfan int en th
Ifferent fles uith / *rernt
r
treryttn Ced o oft n o ad cands a
desor be. Pacir tolven CHal ht)

Scanned by CamScanner
 OV
desorbed
detector. +he
Gdmpourds

v detector
ro tHe

measUeso
eolumn

res the
montioy
areODHo
absor bae o
the each Com pond. the ot he Compoud in h
Co ncenira.tt o n ombeit -b
Sampie fs determ'ne d frorn ?tS
absorbance using been
kaso
Absorbance = Abe.
C
Con Centra Hon ot the Compound.
b= p a lengm of the b e .

Az Pro porHonality constant

HPLC is
ctass1fted into
Co NOrma phase Hplc.
Cb Reveese phase
HpLc
Ca Nemal phase Hpc
n Normal phase HpLc StatfonaT Phase 1s used.

adsorbed Compounds ae desorbecd from the stati orary phase by

As a result, In this technique tess
Polar Solvents.
Passinga on

Componds are turther Separated. These are folo coed by

Pola
more Polar compounds.

Ch) ReVerse phase HpLa

r e verse pha$e Hplc, Phase s non Polar staHonary

used. he adsorbed cornpoend are desorbed fom De Stahonary

Phas by passing polar Solvents. As aresut, to this teetnique more
Pola con pounds are Ptrst, eparared hese are foltouoed yles
Fblaa compound9.
APpications:
TD Seporale and dgtermine omino actds, itamins, a
lkalojdS
esticides and evronmenta' Þeltutants.
For nve s Hgating crtne lests by forenste labs and
Po lîce
deportment
Tn onalysis of natual and artttical dmips
for determination ot Puf y oConm ounds
TdentifHca tton ot Knoun Kmpaund n
ami uve
Scaned by C'amScaner
 13

Retentionie Com Retavdation Time

in the column cor)
the onpound
The Hme Spent by cpressed in mlnutes.
is cxrcssco
Jt
Stottonary phase s itsretention time? Cotumn
Chromatographey.

fntd the
The tinme of injectom ot Somple
re
ette
ennttio
onn
different
Dierent have
is taken as O' Compounds
identítce
d entiicd by
are

Compounds in the Sample

Unknown ot
of the
he
times. ith the
etenion
etention
t imes
times
retent on times
their
Compeairg
Stondard compounds. Dmin
Omin 1mln

o t Conpo n d
A = 1min
Reentm
of
Compounod =min.
Retention

chvoat e 6
cA
chronatoqra

Aeule
Thin kayt
tt n