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University of Santo Tomas Faculty of Pharmacy Organic Chemistry Laboratory
COLUMN AND THIN LAYER CHROMATOGRAPHY OF MALUNGGAY LEAVES ( Moringa Oleifera ) Eunice Aurelle T. Basco !an Lin"ley C. Cabral Aira #ina A. Cayago $ar"ine #ariel L. Ching Leomariss M. Chua an" Fil%osh &. Cucueco 'rou( ) )A*#e"ical Technology Technology Organic Chemistry Chemistry Laboratory
As!ra"! Chromatogra(hy is a +ay of se(arating an" analy,ing the constituents of a mi-ture by (assing them through an inert stationary environment using a mobile solvent that carries them. The t+o "ifferent ty(es of Chromatogra(hy use" to achieve the ob%ectives of the e-(eriment +ere the Column Chromatogra(hy +hich is a (re(arative techniue +here the eluates +ere ta/en an" the Thin Layer Chromatogra(hy +hich is a fast techniue that reuire" only a small amount of the material. The ob%ectives of this e-(eriment +ere to illustrate the (rinci(les of chroma chromatog togra ra(hi (hic c se(ara se(aratio tions ns an" to "emons "emonstra trate te ho+ Thin Thin Layer Layer Chroma Chromatog togra ra(hy (hy an" Column Chromatogra(hy are use" in Organic Chemistry. !n the e-(eriment the 0 eluates collecte" from the e-tract of #alunggay leaves using the (rocess of Column Chromatogra(hy +ere the yello+*green colore" com(onent 12 st eluate3 +hich ha" 45 "ro(s the blue*green colore" com(onent 1)n" eluate3 +hich ha" 6) "ro(s an" the green colore" com(onent 10 r" eluate3 eluate3 +hich ha" 75 "ro(s. After s(otting s(otting these eluates eluates on the Thin Layer Chromatogr Chromatogra(hy a(hy (late an" (lacing the (late in a "evelo(ing chamber chamber it +as evi"ently seen that from the origin the yello+*green blue*green an" green eluates travelle" a "istance of 4.85 cm ).65 cm an" ).56 cm res(ectively. The U9 lam( +as then use" to visuali,e the TLC (late an" &f values +ere com(ute" giving the yello+*green eluate a 5.:; &f value the blue*green eluate a 5.04 &f value an" the green eluate e luate a 5.45 &f value.
INTRODUCTION Chromatogra(hy first intro"uce" by the &ussian botanist #icharl !s+ett is a se(aration metho" that relies on "ifferences in (artitioning behavior bet+een a flo+ing mobile (hase +hich is a liui" solvent 1or mi-ture of solvents3 that use to carry the sam(le solutes un"er analysis along the (a(er an" a stationary (hase +hich is the a"sorbent to se(arate the com(on com(onent ents s in a mi-tur mi-ture. e. <2= Chroma Chromatog togra ra(hy (hy is a very very s(ecia s(eciall se(ara se(aratio tion n (roces (rocess s for a multitu"e of reasons> 1a3 se(arates com(le- mi-tures +ith great (recision? 1b3 can (urify basically any soluble or volatile substance if the right a"sorbent material carrier flui" an" o(erating con"itions are em(loye"? 1c3 se(arates "elicate (ro"ucts since the con"itions un"er +hich it is (erforme" are
not ty(ically severe? an" 1"3 use" to se(arate the colore" (igments in (lants. <)= !n Column Chromatogra Chromatogra(hy (hy the stationary stationary (hase a soli" a"sorbent a"sorbent is (lace" in a vertical vertical glass 1usually3 column. The mobile (hase a liui" is a""e" to the to( an" flo+s "o+n through the column by either gravity or e-ternal (ressure. Column chromatogra(hy is generally use" as a (urification techniue> it isolates "esire" com(oun"s from a mi-ture. <0=
Thin Layer Chromatogra(hy is a sim(le uic/ an" ine-(ensive (roce"ure that gives the chemist a uic/ ans+er as to ho+ many com(onents are in a mi-ture. TLC is also use" to su((ort the i"entity of a com(oun" in a mi-ture +hen the &f of a com(oun" is com(are" +ith the &f of a /no+n com(oun" 1(referably both run on the same TLC (late3. A TLC (late is a sheet of glass metal or (lastic +hich is coate" +ith a thin layer of a soli" a"sorbent 1usually silica or alumina3. <7= !n or"er to ma/e the techniue more scientific rather than a mere inter(retation by sight &etention 9alue 1&f value3 +as a((lie" in chromatogra(hy. A (articular com(oun" +ill travel the same "istance along the stationary (hase by a s(ecific solvent 1or solvent mi-ture3 given that other e-(erimental con"itions are /e(t constant. !n other +or"s every com(oun" 1"ye (igment organic substance etc.3 have a s(ecific &f value for every s(ecific solvent an" solvent concentration. &f values come very han"y for i"entification because one can com(are &f values of the un/no+n sam(le 1or its constituents3 +ith &f values of /no+n com(oun"s. <6= !n this e-(eriment the grou( shoul" be able to attain the follo+ing ob%ectives> 123 to be able to se(arate the colore" com(onents of malunggay leaves using column chromatogra(hy 1)3 to "etermine the (urity of the com(onents using thin/ layer chromatogra(hy 1TLC3 an" 103 to measure the &f values of the colore" com(onents in TLC.
Proce"ure> 2. Column Chromatogra(hy Pigments of Moringa oleifera +ere e-tracte" by grin"ing the leaves +ith a mortar an" (estle an" a""ing he-ane*acetone 1;>03. A (ortion of the e-tracts +as then
Fi#ure &. Pasteur (i(ette (lugge" +ith cotton an" (ac/e" +ith silica gel an" e-tract on to( set asi"e for Thin Layer Chromatogra(hy. After finishing the set*u( the (igments of Moringa oleifera +ere then elute" +ith the use of 6.5mL of eluents he-ane*acetone 1;>03 acetone*methanol 12>23 an" acetone +hich +ere intro"uce" in succession.
Fi#ure $. 'rin"e" malunggay
A Pasteur (i(ette +as then (lugge" +ith cotton for the (ur(ose of be" su((ort an" +as uniformly (ac/e" +ith silica gel u( to the in"ente" (art. 5.6mL of the e-tract +as then (lace" on to(.
t"e solvent system into t"e eaer+ .CM/"exane (#:#) as poured into t"e red siling labuyo "ile t"e "exane/acetone (:!) as mixed it" malunggay leaves+ '"en, t"e inner all of t"e c"amer as lined it" -lter paper it" a atc" glass on top as covering and as next alloed to e0uilirate+ Afterards, t"e plates ere carefully placed in t"e developing c"amer and as left for a"ile in order for t"e t"e solvent system to rise up to t"e # cm mar from t"e upper end+
Figure 5. .eveloping C"amer '"e plates ere removed after from t"e c"amer and t"e solvent front as immediately mared+ '"e plates t"en ere air/dried+ After t"e results ere otained, t"e components ere visuali1ed t"roug" t"e use of 23 lamp+ '"e data attained as used to measure t"e distance traveled y t"e spot and utili1ed for t"e computation of 4f values+
Figure 6. 5late exposed to 23 lig"t
The column +as not allo+e" to run "ry. The colorless eluate +as "iscar"e" +hile colore" eluates +ere collecte" in se(arate. The number of "ro(s of colore" eluate in each vial +as note".
). Thin Layer Chromatogra(hy $n a % cm x & cm precoated 'LC plate, t"e eluates ere applied as small as possile y sloly spotting #* times it" t"e use of a capillary tue+ '"e careful spotting as to mae sure t"at t"e colors ould not mix+ Eac" spot mared t"en as alloed to dry -rst efore t"e next spot as executed+ After t"e careful application of t"e eluates, t"e developing c"amer as prepared t"roug" placing approximate amount of
9
lue 7reen
9+%*cm
*+!8
!
7reen
9+*%cm
*+9<
Resu!s a)* Dis"ussio) Table ). Com(onents obtaine" an" their "istances from the origin The "istance travelle" by the solvent +as ;.55cm from the origin of the TLC (late to the solvent front? the first com(onent travelle" 4.85 cm the secon" com(onent travelle" ).65cm an" the thir" com(onent travelle" ).56 cm.
Plant Use"> #alunggay Leaves 1Moringa Olifera3 Solvent*System use"> @e-ane*Acetone Column Chromatogra(hy !n the column chromatogra(hy three eluates +ere (ro"uce" mainly yello+ green blue green an" green.
Color of Component
3olume of eluate (drops)
#
6ello 7reen
8* drops
9
lue 7reen
%9 drops
!
7reen
;* drops
Table 2. Color of com(onents (ro"uce" an" their res(ective volumes Table 2 sho+s ho+ many eluates +ere (ro"uces an" their res(ective volumes. The first eluate yello+ green yiel"e" 45 "ro(s. The secon" eluate blue green yiel"e" 6) "ro(s an" the last eluate green yiel"e" 75 "ro(s. Fi#ure $. TLC (late after intro"ucing to the "evelo(ing chamber. The yello+ green com(onent contains -antho(hyll +hich is a non*(olar com(oun". This can be confirme" by the column chromatogra(hy. !t +as the first eluate to come out of the column after using he-ane* acetone a non*(olar com(oun". !t follo+s the conce(t of li/e "issolves li/e. Both chloro(hyll a an" chloro(hyll b contain functional grou(s that ma/e them (olar. Thus they "issolve" more +hen acetone is a((lie" to the column. The yello+ com(onent travelle" the farthest in the TLC (late "ue to it being a non*(olar com(oun". Polar com(oun"s stic/ more to the TLC (late as to com(are" to non*(olar com(oun"s. As time (asse" the yello+ com(onent eventually fa"es /ee(ing only the green com(onents.
!n Thin*Layer Chromatogra(hy the &f values of the cru"e e-tract an" the eluates from column chromatogra(hy +as com(ute" base" on the "istance travelle" by the s(ot "ivi"e" by the s(ot travelle" by the solvent front. Since the &f is a ratio it has no unit of measurement. The Thin*Layer Chromatogra(hy (ro"uce" three "ifferent colors from the cru"e e-tract an" the eluates from Column Chromatogra(hy. These +ere yello+ green blue green an" green. Color of t"e Component
The com(ute" values signify the ratio of the "istance travelle" by the center of a s(ot to the "istance travelle" by the solvent front. !n generally it is the fraction of an analyte in the mobile (hase of a chromatogra(hic system.
