Republ

icoft
hePhi
l
ippines
LEYTENORMALUNI VERSITY
Tacl
obanCit
y
Sci
enceUnit

Labor
ator
yWor
k__
___
Name:__
_____
_ _
_____
______
_____
_ Pr
ogr
am/Year&Section:_
____
____
____
__
Dat
eAccomplished:___
______
_____
_ Dat
eSubmitted:___
____
____
____
____
___
I
nst
ruct
or:Jef
fersonE.Flor
es,RN,LPT Rat
ing:_
___
____ _
____
____
____
____
___
_

Col
umnChr
omat
ogr
aphy

I
ntr
oduct
ion
Chromatogr aphyisawi del yusedexper iment altechni
quef orthesepar ati
onofa
mixtureofcompoundsi ntoit
sindi v i
dualcomponent s.Achr omat ographictechniquewi l
l
beexpl oredi nthisexperimentwhi chisthecol umnchr omatography .Howev er,another
chromat ographict echnique,paperchr omat ographyi salso usedwi del
yi ndi ff
erent
procedur es.Inpaperchr omatogr aphy ,
thepaperadsor bswat erfr
om t heatmospher eof
thedev elopingchr omatogram.( Thewat erispr esentint heairasv apor,andi tmaybe
suppliedasonecomponenti nt heel uti
ngsol uti
on.)Thewat eristhest at
ionaryphase.
The( other)componentoft heelut ingsolventist hemobi lephaseandcar r
ieswi thitthe
component soft hemi xt
ure.Thisi sal i
quid–l
iquidsy stem.

I
ncolumnchr omatography,ami xtur
eofcomponent sdissol
vedinasolventis
pouredov eracol umnofsol idadsor bentandi selutedwi t
ht hesameoradi ffer
ent
solvent.Thi
sisasol i
d–li
qui
dsy stem;t hestationaryphase( theadsor
bent)i
ssoli
dand
themobi lephase( t
heeluent)isliquid.Columnchr omat ogr
aphyisgeneral
l
yusedf or
preparati
vepurposes,whenonedeal swi thar elat
ivelylar
geamountoft hemixt
ure,and
thecomponent sneedtobei sol
atedi nmi l
l
igram orgr am quanti
ti
es.

Mat
eri
als:
 TomatoPast e  AluminaOxide
 Erl
enmey erFl
ask  Aluminum Foil
 Analyt
icalBal
ance  Petrol
eum Ether
 Buret(25ml–50ml )  Spatul
a
 Glasswool  Ethanol
 Beakers  Funnel
Pr
ocedur
e:
1.Pr
epar
eal
lthenecessar
ymat
eri
alsandchemi
cal
sfort
hisexper
iment
.

2.Pi
gment
sint
het
omat
opast
ewi
l
lbeext
ract
edi
ntwost
eps:

a.Inext r
actingthepigmentsoft hetomatopaste,weighabout10goft omat o
pastei n a 50-mL beaker .Add 15mL of95% et hanol .St i
rt he mi xt
ure
vigorouslywithaspat ul
auntilthepastewil
lnotsticktot hest ir
rer.Placea
smal lamountofgl asswool(thesizeofapea)inasmal lfunnel,blocki
ngt he
funnelexi t.Pl
acet hefunnelintoa50- mLEr l
enmey erf l
askandpourt he
tomat o paste–ethanolmi xt
urei ntothe funnel
.When t he fil
trat
ion is
compl eted,squeezetheglasswooll i
ghtl
ywit
hy ourspat ula.Inthisstep,we
remov edt hewaterfrom t
hetomat opasteandtheaqueouscomponent sare
i
nt hef i
ltr
ate,whichwediscard.Theresi
dueinthegl asswoolwi l
lbeusedt o
extractthepigments.

b.Placetheresi
dueandt heglasswool i
na50- mLbeaker.Add10mLpet r
oleum
etherandstirthemixtur
eforabout2mi n.toext
ractthepigments.Fi
lterthe
extractasbefore,
thr
oughanewf unnelwithgl
asswoolblockingtheexit,i
nto
anew andcl ean50-mLbeaker .Pl
acet hebeakerunderthehoodonahot
plate(orwaterbath)
.Evaporatethesolventtoabout1mLv olume.Usel ow
heatandt akecarenott oevaporateallthesol
vent.Aft
erevaporati
oncov er
thebeakerwithaluminum f
oil.

3.Use a 25- mL buret.(You may use a

chromat ogr
ahi
c column,ifavail
abl
e,of
1.6cm di amet
erandabout13cm l ong;
seeFi gure23.
8.Ify ouuset hecolumn
i
nsteadoft heburet,al
lquanti
ti
esbelow
shouldbedoubled.
)

4.Addasmal lpieceofgl asswool( about

thesizeofapea)andwi t
ht heaidofa
glassrod,pushi tdownneart hestopcock.
Add15–16mLofpet r
oleum ethertot he
buret.Open t he st opcock slowly and
all
owt hesolventtof i
l
lthetipofthebur et
.
Close the stopcock.You shoul d hav e
12–13 mL ofsol ventabov et he glass
wool.

5.Weigh20gofal uminum oxi

de( al
umina)ina100- mLbeaker .Placeasmal l
funnelontopofy ourburet
.Pourthealuminaint
otheburet.All
owi ttosett
leto
form a20-cm column.Drai
nthesolventbutdonotall
owt hecolumnt orundr y
.
Alwayshav eatleast0.
5mLofcl earsolv
entabovetheal
umi nainthecolumn.If
thealuminaadherestothewal
lsoftheburet,
washitdownwi thmoresolvent.

6.Transferbypi pet0.5–1mLoft heextractyoustor

edi nyourdrawerontot he
column.Thepi petcontai
ningt
heextr
actshoul
dbeplacednearthesurfaceofthe
solventont opofthecolumn.Touchi
ngt hewal
lsoftheburetwit
ht hetipofthe
pi
pet ,all
owt heextr
acttodrai
nslowl
yont opofthecolumn.Opent hestopcock
sl
ightly.All
ow thesamplet oent
erthecolumn,butmakesur ethereisasmal l
 amountofsol
ventabovetheal
uminainthecolumn.(Thecolumnshoul
dnever
rundr
y.)Add10ormor emLofpet r
oleum et
herandwasht hesamplei
ntothe
col
umnbyopeningthest
opcockandcoll
ect
ingtheel
utedsol
venti
nabeaker
.

7.Ast hesolventelut
esthesampl e,
youobserv
et hemigrat
ionofthepigment sand
thei
rseparationint
oatl eastt
wobands.Whent hef
astest-movi
ngpigmentband
reachesneart hebott
om ofthecolumn,cl
osethestopcockandobserv et
hecol or
ofthepi gmentbandsandhow f artheymigratedfr
om t hetopoft hecolumn.
Recordy ourobser vati
on on the Repor
tSheet .This concl
udes the column
chromatographicpart.

Resul
ts:

Col
umnChr
omat
ogr
aphy

NumberofBands Di
stanceMigr
atedf
rom Col
or
t
hetopoftheColumn(cm)

Concl
usi
on:

_
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
_
_
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
_
_
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
_
_
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
_
_
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
_
_
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
_
_
___
___
___
___
___
___
___
___
___
__
_
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
__

Ref
lect
ion:

_
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
_
_
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
_
_
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
_
_
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
_
_
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
_
_
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
_
_
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
_
_
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
_
_
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
_
_
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
_
_
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
_
_
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
_
_
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
_
_
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
_
_
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
_
_
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
___
_
_
___
___
___
___
___
___
___
___
___