Formal Report Exp 5

1  Column and Thin Layer Chromatography Using Red Siling Labuyo (Capsicum frutescens) Extract Rada, R.A.P., Rojo, K.A.A., Roue, !.".#., $antos, R.$.$., $ee, K.A.A.%, $ilang, &.!. Group 7, 2A Pharmacy Department of Pharmacy, Faculty of Pharmacy University of Santo Tomas A'stract Chromatoraphy is a techni!ue use" to separate an" to i"entify components in a mi#tur mi#ture$ e$ %n this this e#per e#perime iment, nt, colum column n chroma chromato tora raphy phy an" thin thin layer layer chromatoraphy &T'C( )ere performe"$ The e#periment )as con"ucte" to sepa separa rate te the the "i*e "i*errent ent pim pimen ents ts in the the pur pure e#tr e#trac actt of re" sili siling ng labuyo labuyo (Capsicum frutescens) throuh throuh column chromato chromatorap raphy, hy, to "etermine "etermine the purity of the "i*erent piments o+taine" throuh thin layer chromatoraphy, an" to calculate the retention factor & f ( of each piment o+taine", inclu"in the pure pure e#trac e#tractt usin usin the "ata "ata o+tai o+taine" ne" from from T'C$ T'C$ The color colore" e" eluate eluates s collec collecte" te" from from column column chro chromat mator oraph aphy y are are yello) yello) &1-. &1-. "rops "rops(, (, re" re" &-7 "rops(, an" re" orane &1/ "rops($ %n T'C, the solvent front travelle" .$2 cm$  The  f  values   values compute" )ere0 )ere0 re" &pure &pure e#tract(0 $/., yello) yello)00 , orane0 , an" re" orane0 $1$ (ntroduction

compon component ents s of the mi#tur mi#ture e +ein +ein

Chroma Chromato torap raphy hy is "e3ne" "e3ne"

separa separate" te"$$ A6n A6nity ity is "eter "etermin mine" e"

as a metho etho" " use" to separ eparat ate e

+y

components

A"sorption

of

a

mi#ture

+y

a"s "so orpti ption is

an" "e3ne"

solu olu+ili +ility ty$ as

the

"i*erenti "i*erential al a+sorpti a+sorption on +et)een +et)een a

+in"in of molecules on a surface$

stat statio iona nary ry phas phase e

mo+i mo+ile le

%n chr chromat omato ora raph phy, y, ho) ho) )ell )ell a

phase$ The stationary phase is the

compon component ent of a mi#tur mi#ture e +in"s +in"s to

non4movin

phase

the stationary phase$ Solu+ility, on

chroma chromato torap raphy, hy,

throu throuh h

an" an"

a

in )hich )hich

the other han", is the amount of 

the mo+ile phase 5o)s, an" )here

su+st su+stanc ance e that that )ill )ill "is "issol solve ve in a

in the "istri+u "istri+ution tion of components components

ive iven n amou amount nt of solv solven ent$ t$ %n this this

+et)e +et)een en the t)o phase phases s occurs occurs$$

case, it is ho) )ell a component of 

 The

mo+ile

phase

carries

the

a

mi#ture

"issolves

in

the

components throuh the stationary

statio stationar nary y phase$ phase$

phase$

a+sorpti a+sorptivity vity are closely closely relate" relate" to the the

 The principle of separation separation in

Solu+ Solu+ili ility ty an" an"

pola polari rity ty of su su+s +sta tanc nces es$$

instance,

a

separation

of  

chromato chromatorap raphy hy is the "i*erentia "i*erentiall

components

a6nities

chromatoraphy is +ein "one, the

of

the

"i*erent

throuh

For

column

2 column +ein compose" of a polar

Desee"e" an" choppe" re"

stationary phase, an" the solvent

siling labuyo  )as rin"e" in a

+ein use" is increasin in polarity$

mortar an" pestle$ After )hich,

Follo)in the

of li8e

the e#tractin solvent, DC<4

eluates

he#ane &101( )as a""e" to the

"issolves

principle

li8e9,

the

collecte" )ill also +e increasin in

rin"e"

sample$

The

pure

polarity, since the most non4polar

e#tract )as collecte" usin a

component of the mi#ture )ill o

Pasteur pipette$

)ith the most non4polar solvent, an" so on an" so forth$ Thin layer chromatoraphy also follo)s the same

principle

as

column

chromatoraphy &T'C($ etention factor &f ( value is the !uantitative measure of a component:s a6nity to the mo+ile phase$ %n normal chromatoraphy,

in

)hich

the

stationary phase is polar an" the mo+ile phase

Figure 1: Extracting the sample

is non4polar, the

B. Column Chromatography  Fiure

2

sho)s

lesser the polarity of a component,

chromatoraphy

the hiher the  f value$

column

to

e#periment  The e#periment

o+;ective is

to

of

separate

+e )as

the

column

set4up$ utili=e"

The in

the

prepare"

+y

this

placin a small cotton +all insi"e a

the

Pasteur pipette, an"

3llin the

piments of the re" siling labuyo

pipette )ith silica el up to its

usin column chromatoraphy, to

nec8$ The column )as clampe" to

use the eluates o+taine" for thin

the iron stan"$

layer

chromatoraphy,

an"

to

compute for the retention factors of each piment from the values o+taine" in T'C$

Experimental  A. Extracting the Sample

 one of the lines$ The pure e#tract an" each of the colore" eluate collecte" )ere spotte" on the thin layer

chromatoraphy

times$

Then,

the

plate

1

"evelopin

cham+er )as prepare" +y 3llin a > m' +ea8er )ith  m' of DC<4 he#ane

&101($

The

+ea8er

)as

covere" )ith a )atch lass an" a Figure 2: Column

tissue, an" )as left to rest for 1.

chromatography set-up

DC<4he#ane, solvent,

)as

the

run

e#tractin

throuh

the

column until it )as )et$ Then, $. m' of the pure e#tract )as place" on top of the column$  m' of DC<4 he#ane

)as

intro"uce"

to

the

column, an" colore" eluate )as collecte"$

Throuh

the

same

process, another +atch of colore" eluate )as collecte" in a "i*erent

minutes

to

allo)

the

solvent

system to e!uili+rate$ After 1. minutes, the T'C plate )as place" insi"e the cham+er$ The cham+er )as once aain covere" )ith a )atch lass, an" the solvent system )as left to "evelop$ After reachin the 1$. cm mar8, the T'C plate

)as

remove"

from

the

cham+er to "ry$

test tu+e usin  m' of DC<$ Aain, the process )as repeate", an"

another

+atch

of

colore"

eluate )as collecte", this time usin DC<0 methanol &101( as the solvent$

C. Thin Layer Chromatography   The thin layer chromatoraphy plate )as prepare" for use +y

Figure : !e"elopment 

After

"ryin,

the

"istances

measurin an" linin a 1$. cm

travelle" +y the components )ere

space on +oth en"s of the plate,

visuali=e" un"er U? liht, an" their

an" "ra)in > e!ui"istant spots on

correspon"in places )ere mar8e"

> )ith a pencil for the measurement

)hich is DC<4he#ane, its a6nity to

of

the stationary phase is the lo)est

the

"istance

that

they

ha"

travelle"$

of the all the three components$  Therefore, it is the least polar

D. Computation of the etention

component$ From the same loic

!actors of the "ure Extract an#

applie" in the e#planation a+ove, it

the Colore# Eluates  The retention factor values

is

)ere compute" usin the

=

is

the

Results and )iscussion %n column chromatoraphy, the solvents use" )ere increasin

interme"iate

in

the

most non4polar

eluates collecte" are increasin in polarity$

Color o* 

+olume o* 

component  #ello /range Red orange

eluate in drops1-. "rops -7 "rops 1/ "rops

Ta'le 01 +olume o* the eluates

solvent +ein use" 3rst$ Ta+le 1

collected *rom Column

sho)s the volumes of each eluate

Chromatography

collecte"

orane

the most polar$ %n summary, the

distancetravelled by the pigment  distance travelled by the solvent 

in polarity,

component

that

polarity, an" the re" orane +ein

formula0

 Rf 

inferre"

from

chromatoraphy$

@ase"

column on

the

 The

color

of

eluates

results, the yello) component is

collecte"

the most a+un"ant, )ith 1-. "rops

chromatoraphy can +e e#plaine"

collecte", follo)e" +y the orane

+y the piments present in the

component

)ith -7 "rops, an"

pure e#tract of re" siling labuyo$

lastly, the re" orane component,

 There are three piments present

)ith 1/ "rops$ From the o+taine"

in the e#tract, namely Carotene,

"ata, an" the fact that the solvents

an" Capsanthin, an" Capsor+in$

use" in the e#periment use" )ere

 The yello) component o+taine" is

increasin in polarity, it is inferre"

Carotene, the orane component

that the yello) component is the

Capsanthin, an" the re" orane

most non4polar of all the three$

+ein Capsor+in$

Since the yello) component 5o)e" )ith the most non4polar solvent,

from

the

column

. Fiure > sho)s the T'C plate

the

e#periment

yiel"e"

correct

after the e#periment$ Ta+le 2 sho)s

results, the results o+taine" )oul"

the "istances travelle" +y the pure

have

e#tract an" the yello), orane, an"

component

re"

orane

eluates

"evelopment

sho)n

that

the

ha"

the

yello) hihest

"urin

the

retention factor value, since it is

an"

the

the most non4polar component of 

stae

compute" f  values$

the piment$ The secon" piment )oul" have ha" the interme"iate retention factor value, +ecause in comparison )ith the other t)o piments present, it is interme"iate in non4polarity$ The re" orane piment )oul" have ha" the lo)est retention factor, since it shoul" have ha" the lo)est a6nity to the

Figure #: $LC plate after the

mo+ile phase amon the three components,

experiment 

Color o* 

)istance

compone

o* 

nt

componen

R*  3alue

Chromatoraphy is a metho"

origin 2-

of use" to separate components of 

in cm >$> cm  cm  cm $7 cm

a mi#ture +ase" on "i*erential $/.   $1

)ere

some

errors

"urin the e#periment, +ecause results

"istances

a+sorption$

%n

column

chromatoraphy, the last eluate collecte" )as the least non4polar of  all the components, since it )as

 There

o+taine"$

solvent

Conclusion

orange

the

the

system use" is non4polar$

t *rom

Red  #ello /range Red

since

e#pecte"

%"eally,

)ere

none

travelle"

of +y

not the the

component shoul" +e  cm$ a"

use"

)ith

solvent$

the

least

Amon

non4polar

the

three

components, it )oul" have ha" the stronest

a6nity

to

the

polar

stationary phase, e#plainin the scarce

volume

collecte"$

The

of

the thin

eluate layer

B chromatoraphy &T'C( e#periment

t4prepEmcatEchemical4p

performe"

"i"

e#pecte"

results$

not

yiel"

the

rocessesEseparations4

a"

the

puri3cationsEaEprinciples4of4

e#periment sho)e" the e#pecte" results, )oul"

the have

yello) ha"

component the

hihest

retention factor value compute" +ecause it is the most non4polar of  all

the

components$

%t

a6nity

to

the

polar

stationary phase$

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