SEPARATING AND DETERMINING COMPONENTS OF MALUNGGAY LEAVES USING COLUMN AND

THIN LAYER CHROMATOGRAPHY

Marian Erica Rosimo, Carissa Patricia Santos, Miguel Silvestre,
Ellysa Joviel Singzon, Rouville Sosa, and Chrizanne Irah Tagle
Group !C Pharmacy "rganic Chemistry #a$oratory
ABSTRACT
Chromatography is a separating techni%ue used &or solid'li%uid, li%uid'li%uid, and gas'li%uid mi(tures) In
this e(periment, colored pigments o& malunggay leaves *ere e(tracted *ith the use o& he(ane'acetone
and eluates *ere collected through the Column Chromatography) Then, thin layer chromatography *as
per&ormed to measure the purity o& the components) The developed thin layer chromatography plate
*as seen through the +, lamp and the Retention or Retardation &actor *as measured)
INTRODUCTION
Chromatography is a proven method &or separating
comple( samples into their constituent parts, and it is
undou$tedly the most important procedure &or isolating
and puri&ying chemicals) Chromatography relies on the
di&&erential solu$ility and adsorptive o& the components to
$e separated *ith respect to t*o phases- stationary
phase and mo$ile phase) Stationary phase is the part o&
the chromatographic system though *hich the mo$ile
phase &lo*s *here distri$ution o& the solutes $et*een the
phases occurs) Mo$ile phase, on the other hand, is the
part o& the chromatographic system *hich carries the
solutes through the stationary phase) The t*o types o&
chromatography that *ill $e used in this e(periment is
Column Chromatography and Thin #ayer
Chromatography)
,arious types o& chromatography are possi$le,
depending o& *hich t*o phases are used) These
are solid' li%uid .column, thin layer/, li%uid'li%uid
.paper, high per&ormance li%uid/, and gas'li%uid
.vapor'phase/ chromatographic methods)
The stationary phase, a solid adsor$ent, is
placed in a vertical glass, usually columnand the
mo$ile phase, a li%uid, is added tothe top and
&lo*s do*n through the column.$y either gravity
or e(ternal pressure/ in column chromatography)
Column chromatography is generally usedas a
puri&ication techni%ue0 it isolates desired
compounds &rom a mi(ture)
Thin layer chromatography is per&ormed on a
sheet o& glass, plastic, or aluminum &oil, *hich is
coated *ith a thin layer o& adsor$ent material,
usually silica gel, aluminum o(ide, or cellulose
.$lotter paper/) The T#C plate is then placed in a
shallo* pool o& a solvent in a developing cham$er
so that only the very $ottom o& the plate is in the
li%uid) The li%uid or the mo$ile phase slo*ly rises
up the T#C plate $y capillary action) 1hen the
solvent has reached the top o& the plate, the
plate is removed &rom the developing cham$er,
dried, and the separated components o& the
mi(ture are visualized) "nce visi$le, the
Rf value, or retention &actor, o& each spot can $e
determined to compute the purity o& each
component $y dividing the distance the
su$stance travelled $y the distance the solvent
&ront travelled using the initial spotting site as
re&erence) These values depend on the solvent
used and the type o& T#C plate and are
not physical constants)
Through these processes, the o$2ectives o& this
e(periment *ere0 .3/ to separate the color
components in malunggay leaves using column
chromatography, .!/ to determine the purity o&
components using Thin #ayer chromatography
and, .4/ to measure the R& values o& the colored
components in Thin layer chromatography)
EXPERIMENTAL
A. Co!ou"#s $es$e#
Plant used0 Malunggay leaves .Moringa
oleifera)
Solvent'system used0 5e(ane 6cetone
B. P%o&e#u%e
'. E($%a&$io"
E(tract the pigments o& the Malunggay leaves
$y using he(ane0 acetone .704/ a&ter macerating
it) Set aside a portion o& the e(tracts &or T#C)
2. Colu" C)oa$o*%a!)+
Prepare the Pasteur pipette $y plugging cotton
on the inside o& the pipette and add silica gel up
to the indented part o& it) Place 8)9 m# on the
e(tracnt on top o& the the column using Pasteur
pipette) Introduce in succession 9)8 m# each
he(ane'acetone .704/, acetone- and acetone'
methanol .303/) :ever allo* the column to dry,
discard the colorless eluate, and collect the
colored eluates in separate test tu$es) Then, note
the num$er o& drops o& eluate collected in each
vial)
3. T)i" La+e% C)%oa$o*%a!)+
6pply the eluates on a precoated T#C plate $y
spotting 38 times) 6llo* each spot to dry $e&ore
da$$ing another and ma;e sure that the spots
are as small as possi$le and that you da$$ed it
on the same spot as the &irst application o& your
eluate) Prepare the developing cham$er $y
placing an appro(imate amount o& the solvent
system0 <CM' he(ane'acetone .704/ &or
malunggay leaves) #ine the inner *all o& the
cham$er *ith &ilter paper then cover *ith a *atch
glass = allo* e%uili$rating) 6&ter, care&ully place
the plates in the developing cham$er and allo*
the solvent system to rise up to 3 cm &rom the
upper end) Remove the plates &rom the cham$er,
immediately mar; the solvent &ront- air'dry)
>inally, visualize the componets using the +,
lamp, measure the R& values and document the
chromatoplates)
RESULTS AND DISCUSSION
6&ter e(tracting the pigments o& Malunggay
leaves $y using he(ane'acetone .704/ and
per&orming Columnn Chromatography and Thin
layer Chromatography, here is the list o& data
that *as gathered throughout the e(perimenting
process0
Ta$le 3) Column Chromatography Results

Ta$le !) Thin #ayer Chromatography Results
Color o&
Component
<istance o&
Component
&rom origin
.(/ in cm
R& value
3 Invisi$le !)9
! Invisi$le !)?
4 @ello* 4)!
A @ello* Green A
9 <ar; green A)4
B <ull yello* 9)3
7 Cright yello* 7
Color o&
Component
,ol) o& Eluate
<rops
3 #ight green 38
! <ar; green 39
@ello*ish
green
39
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