Michael Dominique B. Allag, Eryll Joy H. Agojo, Camille A. Baetiong, Greanne P. Danica Ballesta, Rachel Anne S.

Barlao Group !C Me"ical #echnology $rganic Chemistry %a&oratory

COLUMN AND THIN LAYER CHROMATOGRAPHY

Abstract
Chromatography is a technique 'or separating mi(tures into their components in or"er to analy)e, i"enti'y, puri'y, an" quanti'y the mi(ture or components. #here are "i''erent types o' chromatography an" each has its o*n a"+antages an" "isa"+antages. ,n this e(periment, DCM-he(ane *as use" to e(tract the "i''erent pigments o' the siling la&uyo. E(tract *as intro"uce" into the column an" eluate *as collecte", this process is the column chromatography .CC/ metho". #he purity o' the components *as "etermine" &y using thin later chromatography .#%C/. 01 lamp *as use" to +isuali)e the "e+elope" #%C plate an" the 2etention or 2etar"ation 3actor *as measure".

I.

Intro !ct"on
sur'ace. All types o' chromatography are use'ul 'or analytical purposes. 0n"er appropriate c o n " itio , n s all types o' chromatography can &e use" 'or preparati+e s c a l e s e p a r a t i o n s . ,n e + e r y type o' chromatography there are three elements to &e consi"ere"7 the si)e o' the sample .%oa"/, relati+e se par atio n o ' co mpo ne nts .2esolution/, an" the 5pee".

Chromatography can &e "e'ine" as a la&oratory technique that separates components *ithin a mi(ture &y using the "i''erential a''inities o' the components 'or a mo&ile me"ium an" 'or a stationary me"ium through *hich they pass. #he un"erlying principle o' chromatography is that "i''erent su&stances ha+e "i''erent partition coe''icients &et*een the stationary an" mo&ile phases. A compoun" that interacts *ea4ly *ith the stationary phase *ill spen" most o' its time in the mo&ile phase an" mo+e rapi"ly through the chromatographic system. Compoun"s that interact strongly *ith the stationary phase *ill mo+e slo*ly. All 'orms o' chromatography *or4 on the same principle. Di+erse types o ' Chromatography are possi&le, "epen"ing on the physical states o' the phases. Employing a gas the mo&ile phase is terme" gas chromatography .gc/ or +apor phase chromatography .+pc/. 5eparations using gas chromatography in+ol+e +apor phase +ersus a"sorption an"6or equili&ria. %iqui" Chromatography .lc/ re'ers to any chromatographic process that employs a mo&ile liqui" phase. Chromatographic separations can also &e carrie" out using thin layer chromatography .tlc/ an" column chromatography *hich a +ariety o' supports, inclu"ing immo&ili)e" silica on glass plates.

,t *oul" &e i"eal i' all three elements coul" &e ma(imi)e" so that complete separation o' samples o' any "esire" si)e coul" &e quic4ly achie+e". ,n practice, generally t*o o' these elements can &e ma(imi)e" at the e(pense o' the thir". 3or routine analytical *or4, resolution an" spee" are ma(imi)e" at the e(pense o' the loa". ,n preparati+e scale separations, loa", an" spee" can &e ma(imi)e" &ut then separations are usually incomplete. Complete separations o' large samples can &e achie+e" &ut the o+erall operation is li4ely to &e slo* an" te"ious, an" may in+ol+e the use o' large quantities o' sol+ent that must &e "istille" 'or reuse, or "iscar"e".

Chromatography separates a su&stance into its component parts, *hich is +ery use'ul, as su&stances are o'ten unique in their composition. ,t can i"enti'y a su&stance an" sho* ho* it "i''ers 'rom others that may loo4 ali4e on the

,n the e(periment, Chromatography an" Chromatography *ere use".

Column #hin

#%C has a num&er o' a"+antages7 ,t is simple, 'ast, e''icient to use an" it requires only small amounts o' sample. #%C is generally use" a qualitati+e analytic technique, such as chec4ing the purity o' a compoun" or "etermining the num&er o' components in a mi(ture or column chromatographic 'unction. ,n a""ition, #%C is use'ul 'or "etermining the &est sol+ents 'or a column chromatographic separation. ,t can &e use" 'or an initial chec4 on the i"entity o' an un4no*n sample. Preparati+e plates can &e carrie" out *ith special thic4-layere" #%C plates.

Column chromatography is a"+antageous o+er most other chromatographic techniques &ecause it can &e use" in &oth analytical an" preparati+e applications. 8ot only can column chromatography &e use" to "etermine the num&er o' components o' a mi(ture, &ut it can also &e use" to separate an" puri'y su&stantial quantities o' those components 'or su&sequent analysis. #his is in contrast to paper chromatography, *hich is solely an analytical metho". #he "isa"+antage o' a column chromatography is that it is time-consuming an" te"ious, especially 'or large samples. ,' it is unnecessary to preparati+e separate large quantities o' sample, analytical metho"s such as paper chromatography may &e more suita&le an" easier to per'orm. #hin-%ayer Chromatography .#%C/ in+ol+es the same principles as column chromatography9 it is also a 'orm o' soli" liqui" a"sorption chromatography. ,n this case, ho*e+er, the soli" a"sor&ent is sprea" as a thin layer on a plate o' glass or rigi" plastic. #he sol+ent tra+els up &y plate through capillary action. A "rop o' the solution to &e separate" is place" near one e"ge o' the plate, an" the plate is place" in a container, calle" a "e+eloping cham&er, *ith enough o' the eluting sol+ent to come to a le+el just &elo* the point o' origin. #he sol+ent migrates up the plate, carrying *ith it the components o' the mi(ture at "i''erent rates. #he result then, is a series o' spots on the plate, 'alling on a line perpen"icular to the sol+ent le+el in the container.

DCM he(ane or Dichloromethane he(ane is the sol+ent system use" to elute through a chromatography column. #his means that the mo&ile phase .sol+ent system/ consists o' 7 .ratio o' +olume/ mi(ture o' "ichloromethane .DCM9 CH!Cl!/, an" he(ane .C:H ;/. #he soli" phase .silica gel/ is elute" *ith this sol+ent system until 'ully sol+ate", the compoun" to &e puri'ie" is then loa"e" onto the sol+ate" soli" phase, an" the column is elute" *ith the same sol+ent system until your "esire" compoun" has come o'' the column # he 2etention or 2etar"ation 3actor .2< +alue/ is the ratio o' the "istance that the spot tra+elle" relati+e to the "istance mo+e" &y the sol+ent *hich in this case is the DCMhe(ane.

#he o & j e c t i + e s o ' t h e e ( p e r i m e n t are the 'ollo*ing7 separate the colore" components o' re" siling la&uyo using column chromatography, t o pre"ict the purity o' components using column an" thin layer chromatography .#%C/ an" lastly, to measure the 2etention62etar"ation 3actor .2< +alues/ o' colore" components in #%C.

II.

E#$er"%ental
#he e l u a t e s * e r e a p p l i e " o n t h e >cm ? @cm pre-coate" #%C plate &y equi"istantly spotting each spot = times. #he spot *as allo*e" to "ry 'irst &e'ore applying the succee"ing spots. ,t *as ensure" that the spots ma"e *ere small as possi&le so that *hen the plate "e+elops, the colors *oul" not &e "isarray. De+eloping Cham&er *as prepare" &y placing the appro(imate amount o' DCM he(ane. #he inner *all o' the cham&er *as line" *ith 'ilter paper to allo* the #%C plate to stan". #he "e+eloping cham&er *as co+ere" *ith *atch glass an" *as allo*e" to equili&rate. #he #%C plate *as care'ully intro"uce" in the "e+eloping cham&er. #he sol+ent system *as allo*e" to rise up until it reaches just cm 'rom the upper en". #he #%C plate *as then remo+e" care'ully 'rom the cham&er. #he sol+ent 'ront *as imme"iately mar4e" an" the plate *as allo*e" to "ry. #he components *ere +isuali)e" using the ultra+iolet lamp a'ter the plate has "e+elope" a'ter elution process an" this causes su&stances to appear as colore" spots. #he 2< +alues *ere measure" an" chromatographic plates *ere "ocumente".

Pigments o' the siling la&uyo *ere e(tracte" &y cutting it to pieces an" &y pouring DCMhe(ane an" e+entually triturating it &y using a mortar an" pestle *ith the ratio o' 7 . #he e(tracte" pigments *ere set asi"e 'or a *hile. 5ilica Gel Column *as prepare" &y plugging the column *ith cotton 'ollo*e" &y the silica gel *hich *as uni'ormly pac4e" an" containe" no holes or air &u&&les until it reache" the in"ente" part o' the Pasteur pipette. =.> ml o' the e(tract *as place" on top o' the column using Pasteur pipette. #he p i g m e n t m i ( t u r e * a s e l u t e " u s i n g =ml DCM-he(ane. #he s y s t e m s o l + e n t *as intro"uce" in portions. #he column *as not allo*e" to run "ry an" the colorless eluate collecte" *as "iscar"e". #est tu&es *ere change" each time the color o' the eluate +aries. #he num&er o' "rops 'or each color *as note". A'ter collecting the eluates 'rom the column, #hin %ayer Chromatography *as per'orme". #he color o' the eluate +aries. #he num&er o' "rops 'or each color *as note". A'ter collecting the eluates 'rom the column, #hin %ayer Chromatography *as per'orme".

III.

Res!lts an D"sc!ss"on

Plant !se & 5iling %a&uyo Sol'ent S(ste% !se 7 DCM-He(ane

Column Chromatography7 #*o eluates *ere yiel"e" 'rom the e(traction o' the colore" components o' siling la&uyo using column chromatography. #*o "i''erent sha"es o' colors *ere o&taine"7 %ight yello* an" light orange. #he +olume o' the light yello* eluate collecte" 'rom the column *as A> "rops *hile on the other han", the +olume o' the light orange *as @> "rops.

tra+elle" ;.A cm *hile the light orange eluate tra+elle" =.> cm. #he color o' the "e+elope" plate *as not +isi&le &y the na4e" eye. ,t *as place" 01 light 'or +ie*ing. #he general 'ormula 'or computing the 2< +alue is sho*n &elo*7

Color o' Componen light yello* ! light orange

1olume o' eluate .no. o' "rops/ A> @>

Table ) Col!%n Chro%ato*ra$h( +Table o, Res!lts-

A'ter measuring the "istance tra+ele" 'or each spot, #he 2< +alue .also 4no*n as 2etar"ation or 2etention 3actor *as compute"/ 2etar"ation or 2etention 3actor is the ratio o' time spent in the stationary phase relati+e to time spent in the mo&ile phase. Distance o' sol+ent7 >cm

#hin %ayer Chromatography

Table . Th"n La(er Chro%ato*ra$h( +Table o, Res!ltsColor o' Component %ight Cello* %ight $range Cru"e Distance o' Component 'rom origin .(/ in cm ;.A cm =.> cm ;.= cm 2' 1alue =.@: =. =.@

Bith re'erence to 3igure ;, .3rom le't to right/ the 'irst spot is the Cru"e Eluate9 the secon" spot is the 'irst eluate collecte" 'rom the column an" the thir" spot is the secon" eluate collecte" 'rom the Column Chromatography. #he cru"e eluate tra+elle" ;.= cm 'rom the origin9 the light yello* eluate

! A

#he "e+elope" plate *asnDt a&le to sho* completely the separation o' colors. #he possi&le sources o' error are 'rom the spotting o' the #%C plate. Bhen the e(tracte" pigments o' siling la&uyo *ere spotte" on the plate, it *as not le't completely "ry &e'ore placing the succee"ing spots in

a""ition to that9 the spots *erenDt small enough *hich ha+e cause" color the color to "isarray. Another source o' error is not co+ering completely the "e+eloping cham&er "uring the "e+elopment o' #%C plate.

/. Re,erences BOO0S&
3e"essen"en, 2.J., 3e"essen"en, J.5., E 3eist P. .!== /. $rganic %a&oratory #echniques. Cana"a7 Broo4s6 Cole. Pg. F- ;= 2o&ar"s, G., Ha""a",P.2., Jac4son,P.E., . FF;/. Principles an" Practice o' Mo"ern Chromatographic Metho"s. 5an Diego,CA7 Aca"emic Press ,nc. Pg. -A;, A:-!!> Billiams, #. ,., . F;H/. An ,ntro"uction to Chromatography.8e* Cor47 Chemical Pu&lishing Co., ,nc. Pg. -@>

1EBSITES THIN LAYER CHROMATOGRAPHY 2etrie+e"

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COLUMN CHROMATOGRAPHY
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