EMST

ECO
AATOGRARR
 Seperalion of duembturaMetbyl.orangc and
Metbylenc Bue using TLG
Aim 10 Seperate the mixtuYe ot dyes on silica.gel plate bu
nin laye chromatographic method
Principle hinlayer chromato.graphic technique invalves the
Seperation of Components usinq a qlass slide or plate covered
Loith tbin lauer of adsorbent like alumina or Silica qel
The Seperation of Compound in TLC iS based on differentiall
adsorption as uell as partition of analute between the liquid
staionaru phase and mobileSoluentphasehe mobile phase
flouis thiough the stoationaryphase and carries the Component
- S of the mixture uoith it: Sifferent components trauel a t
different Jates based on thei affinities touwards statianary
phase and mobile phase
Thusthe compounds that aIe more polar will haue a
greateraffinity to polar stationary phase and less polar compo
unds are less attacted to pola stationaxy phases
Ihe movement of SubstanceComponent elafive to the
Solventis expressed in termsof Rf value and is given by
the equation.

Rf
Distance trauelled by the analyte
Oistancetrauelled by the mobilephase cMS
he seperated analyte are identified bycomparinq theiy
RE Values to that of reference standards
lhen silicagelsusEed as the staionaryphase in
Seperation of dye mixture, methulene blue is more sthongly
retained becausemetbulene blue is basic and bas bigh affinitu
to the acidic silica qel than methul orangeSo methyl oange
 N CH3) Methylene blue (A)
(CH)

N =N

cH3
N Methyl Orange le)
CH3
br
Table: Calculation of Rf values of standard Samples

Name of Sistance trauellOistance trauelled

the standard-ed by the solute 'by the solvent
Re dld
from the stayting front from the
line d: Ccm) Starting line d
Ccro

Methyl bue ( 3 Co 4 8 Cm O.62 cm

bollnt
Methyl ora ngel) 45 cm 48 Cm o-93 m
 spend more timein mobile phaseand moves fastex
K Since bothmethulOTangeand methulene blue are telativelu
polar a polar soluent ubill uoik best to seperate them
Materials Requted
ghopparatus TLC chamber TLC plates OY glass slides beakers,
capillary tubes glass 10dHot aiz Oven Drier etc
Chemicals Solution of dyes2:5 methul orange and a5.mq
methyleneblue in 1o.ml of uwater seperatelu, soluent metharol
silica gel
Eper imental prOcedure
4 Preparation of _plates
4 0 mg of Silica gel uas uweighed and toaken into a beaker
containinq 1ooml of dishlled uater, mixed well until the
small qranules of the qel gets wetted uniformluy to become
slurry
The alass plate were cleaned with ethanol and auanged on

the Stand
xPouI the slurry ioto the application and coat the gel along
the qlass plates0y xapicly movingq the application the coatinq
o f the adsorbentshould be of uniform thickness. Or glass

slides can be coated by dipping them in paiisbeld back

to back in the slurry and with drauoing slouly and steadily
temperature to let the solvent diain auwau

Ax drythe plates at i0om femperatuyefoz 5 bours The

coated plates were octivated by keéping them in hot air
Oven at uo'c late t plates were iernoved and allowed
plates
temperature in desicated chamber
to Coo at
Table a-Calculation of R values of onknouon
Samples
Name of Oistance trauelle-Oistance trauelled
the standlar-d by the solute by the sduent
RF d
-d trom the starhing front from the
line (da) cm Starbing line C)cm

O3 Co = O62 Cm

45Cm y&Cm -5 O-93 cm

y.8
 5

2.evelopment of Chromatoqrapm
Spot the dues standoids and test Sample oith the
help of
Seperate capillaxy tube a t anequidistance of 2 to3 cms and
at
a height of 3cms above the of qlass plate
* Place the
the spotted platesinto methanol saturated TLCc
chamber Cae must be taken that tbe
leuel of mobile phase
shouldbe belouthe spotted points
Allou the mobile phase to runascendingly for about 3lsth
length of the plate
* `olvent risesin the adsorbent through capillary action
and carries along uoitb heComponents at different speeds
kRemoNe tbe plcte faom the TLC Cchamber and dry ín a
hot ai Oven for 5-10minutes to deelop the coloured spots
Measuie and calculate the Rf values of each Standard by
uSing the foxmula

Rf=
Oistance txavelled by the solute from origin line
Oistance tuavelled by the solvent tom origin line

Result
*From the obtained results tbat it was found RE value
of thesample a s coin.cidinq with methylene bue and
methyO1ange tHenceit is confirmedhat given mixtuie
containsmethylene blueand methyl orange
Levthn

M
 6

Seperation of Amino acids by

As.cendingpaper
chromatography
him To Seperate
and identify the oamino.ocid presentin a
mixtureby Paper chuomatoqiaphy
Principle Chiomatoqraphy is used to Seperatemiatures of
substances intotheir individual componentsThe basic iequie
-mentso f stationary phasea liquid supported on a solid and
a mobile phasea liquid In paper chiomatoqiaphy the
stationary phaseis a uexy uniform adso1bent paper The
mobilephase is a suitable liquid soluent o mixtuie of solvents
he phase flouws through the stationaru phase and
cairiesthe components of the mixture coith it Oifferent
components tiavel at different _rates based on theixaffinitiés
touardsstationary phaseand mobile phase
Thus compounds that are moré polar uill bave a gxeater
affinity to polar stationary/phase and less pola_compounds
are less attracted to pola stabionau phases
Amino acids a e sepeiated based on theiydistaibubion co-effic-
-ient between the liquids stationaiy phase and liquid mobile

phase
Theseperated analytes axe identihed by_compauinq theix
Rf values to that of reference sBandards

Sistance tiauelled by analyte

Rf Distance trauelled
by mobile phase

Aoporatus Required: Glass beaker, lbatmann filter paper

Measurinq cylinde Oeueloping chambeí and capillazy tubes
driey
Table i: Calculation of Rf values of standard Sample

istance trauelledoistance travelled

Name of by the solutie from 'by the soluent froo
the standard the starbing line from the starting
R
c) m line (8). em

8-8 Cm O.9
Glycine O102 Cm

phenyl alanine 5-8 Crm 8-9 cm 5-3

O-6sO-6S
8-8 Cm
Chemicols required:
Developing soluent nbutanol, acehic acid,disilled water
41:5)
Glycime phenylalanine1omg of each amino acid in 1oml of
1oisopropanal
Nlinhydain reagent Oissalue 200mg of ninbydrin in 100ml
of Acetone
xpeximental procedure.
Take wbatmann No 1 filter papez with appropriaie dimension
Orau the pencil line aaossthepaper leauing3cm iom the.
edge of the paper tbis_denotes the oriqin-
kabel the oamino acids and the unknouon to be spotted at the
oriqin and using
a pencil
* öpot the aminoacid soluion with tbe belp of a capillary tube

and dry the spot using hot ain druer

Hold the filter pape uíthsupport which can stand upríght
Place tbe paperin satuxated chromatoqiapby chbamber containin
-g themobile phase

* The spotted end edqe should be placed in_Such a was thatt

solvent leuel
Sample spots are 2 cm abovethe
Run chromoatoqram and after that. 1un the papei is Iemoved
the soluent front ismatked usinq a pencil and later it is

died at 1oom tempeiatuie

The chromatoq1am is spraued with Dlinhydrin_reaqent and
alloued to dy at oom tenmperaturE
Outline colour spots oith the pencil, measure the distance in
centimeters faom the origin to center of the outline spot and
calculatethe Rf value for the Sepexate aminoacids
 of Rf values of unknouor
Table a Calculation
amples
trauelledetstande travelled
b . istonce
the solute from by the solvent front
Name ot by RE
from the starting
the stondardthe starting line line (d) cm 'n
(da) Cr

cm
O-1Cro

s6
56
om -63 O-63 Cm
 RF Distance trawelled by test
Oistance travelled by mobile phase (cms)
The unknouan aminoacids are identified by compaing the Rf
alue oith that of standard aminoacids
Result:
Fom the obtained iesults it was found that the R Value
ofthe sample was coinciding wifh 3tandard glycine and
phenylalanime Hepee it is confizmed that the qiven sample
mixtuie containsglycine and pbenylalanine-
CCH) NCCH3

N=N

,CH3

CH
 Seperahion of Oue mixtureMethul orange and Methul
Bhue by Using Column chromatogapbu

Aim 1 0 SepeIate the mixtuie of dyeS by Column chiomatogia

phic metbod
Principle Column chromatography is a technique uwhich is
used to seperateCompounds from a mixture thioughequilibriu
-m distibuhon betuoeenn tuuo phoses Tt Seperates Substances
based on differential adsoiption af compounds to the adsoxbent
as the compounds move thiouab the column_at different rates
which allouo them to qet sepeiated in fractions he components
with loer adsorphion to stationary phase tiauel foaster thxough
mobile phase uwhen compored to the qreater adsorphion uwith the
Stabonary phase lhe components that move fast axe eluted first
ubere as the components that move slou aie eluted last

An analyte is in equilíbrium_ betuween_the tuio phases,

A mobile) A (stationay)
K-CSIcM
Alhere the equilibrium constant k is called the Paxtition coefficient
CS molar conentrahion o fanalyte in stationany_phase

CM molar concentiafion of analytein mobile phase

ulhen silico gel is used as the stationary phase in seperation of
the
dye miatures, methylene blue 1S more Staongly retained at
affinits_
top because methylene blue is basic and bas biqh
to the acidic Silica qelthan methyl oranqe Hence methyl
Orange elutes fixst andmethylene blue later witb the polax
elutingsolvent

Apparatus requiIed Column Glass wool, Fraction collection

tubes beakers, dioppeY, pipette
Chemicals required:
DyesO5mgof methuyl orange and 0-a5 mq of methylene
blue are dissolved in 10.ml of water seperately
Eluting soluentsacetone methanol
a Adsobent silica qel
Sand
Experimental Procedure
Preparahion of Golumm
1 Insert a small plug of alass wool into the neck of a colum0-
The glass wool is packed fixnmiy into the neck by wetting ith
soluent and is covered with a thin layer of Sand
2.Silicagel is added to the calumn until it reaches about half
way up the column depends on size of the columrnhe silicagel
is packed by oentl tapping the outside wall of the columo
with uour finger on pencil
3 Suitable solvent acetone is then added to the column
and allouwed to flow through to "wet the column-1f needed
add mOTe solvent-

Allow the soluent to flou thxough the column until only

about05cmremains above the stathionary phase
Seueloprment of column
5 Add 5 diops of the dye mixtuie to the top ot the column
belouStationay phase and
Allou the mixture to flo jusE
caxefullu add a thin layer of sand
6 Add the eluting soluent methanol and allouo the soluent to
clute thioughthe column to seperate the dye components and
a column. when the Soluent qets close to the top Sand layer
uSe Odropperpipette to addmore soluent Never allow the
coluan to become day:
alhen the first component of the mixture appears ready to
elute fiom the _column place a collecting tube under the stopcock
to collect the eluent
8 Oncethe fiast due component bas been eluted iemove the
flask and label it
9 Addmethanol soluent to the top of the columoand collect
the secand due component in the collecting tube
o Compaie Tesults from collected samples
Result:
Using the silica.qel as the stationary phase and methanolaS
the mobile phase methyl orange elutes first and methylene
blue elutesAater
 CaHs SOg Nat
N-CH2

N-CH-

CaHs SOg na
Green 3 fapoie

drotus

N=N s o mat

Sog nNat

Red 40
 Seperation of Food dues bu Coluon chromatoqapb

Aim Io seperate the miature of foods dyes by column

chromatographic method
Principle Column chromatography is a technique uhich_1s
used toseperate compounds froma mixture through equilib
-tium distribution betudeen two phases ItSeperates Substance
S based o n diffetential adsorption of compounde to the
adsotbent as thecompounds move throughthe column at
different rates which allouo them to qet Seperated in
ftactions The componernt.s with lower adsorption to stationary
tto
o
phase trauel faster thaouah mobile_phase uoben compared
phase
to thegieater adsorption with the stationaryphose The
he
components bat move fast are eluted firstubere as tbe
components tbat move slouo are eluted out last
An analyte is in cquilibaium betuween he tuwophases
a mobile A stationary
k CSs
CM

Albere he cquilibrium constant k is callecd partibion

coefficient

CS concentration of analyte i n stabionary pbase-

Molar
CM Molar concentration of analyte in mobile pbase.
o f the food dyes have relatively compleistructures
and al hayédoublebondsLwhich makes them higbly Colaur
-ed Food dyes also Contain some -COo Or -503 qaoups
These qroups contribute to the polariby of the molecule he
he more polar the dye t h e more
larqer he net charge
larger
affinify Howardshe stationaxy phase
 3

Greenis Some uwbat more polaz molecule thanRed uo

So qeen basmore affinity towards stationaryphase
and r e d bas more affiDtty touwaids mobile phase obich
elutes first
Apparatus Tequlred Golumn, Glass wool, fraction collecting
tubes beakers dropperpipett.e
chemicals required
aMixture of any two dyesg Green and red yo food dyes]
each O:5.m dissolved in 5ml ofwater seperately
eluting solvents Alater Butanolt water t ammonia are in
3:EL
x Adsoibent [Silica gel
xSand
Experimental Procedure
Preparaion of Column
Tnsert a small plug of glass wool into he neck o f
column.Theglassuwaol is packed firmlyioto the neck by
wetting with soluent and is covered Lwith a 1hin layer of
Sand
a.Silica qel is added to the column until it_reaches about
half wau up the columndepends upon_Ihe size of column
The silica gel is packed by qently tapping the outside wall
o f ihe coluron uoiltb youz finger Or apencil.
3. A Suitable sowent uatrr is henadded to the cdumm
and allouwedd t oflou thiough the wet Column, if needed

add moie Solueni

Allou lhe soluert to flou thiougb fhe column until onls

about0'ScmremainS above the stationaryphase.
 4

Deuelopment of Column
5 Add b drops of food dye mixture to Ibe top of the column
Allouo the mixtture to flouw just below stationaiy phase
and Carefullyadd a tbin 1ayer of Sand
6.dd the elutinq Soluent Butanoltwater t ammonia in
ratio in 3 Slouoly and allouw tbe Soluent to elute tbrau-
-gh column to Seperatethe food dye components - hlhen the
soluent gets close to lhe top Sand layes useda_dzopper
pipetteto addSome more sovent Never allou tbe Column
to become dxg
Wlben the fiast component of the minture appeas reads
to elute from he column placea collecting tube_undey
the Stop cock to collect the cluent
8 Once the first dye component bas been eluttdremove
the flask and label i
9.dd eluling solvent toThe top of the column to collect
the Second due component in a collecting tube
1o Comparexesults from ollected samples
Result-
USing the Silica gel as tbe stationaxy phase and
oater

Butonol twater tammonia in rahon3Las te mobile

phase red food dye elutts first and gaeen elutes late