EXPERIMENT 5 COLUMN AND THIN LAYER CHROMATOGRAPHY

*Juanico, R., Ibale I., Ibaez E., Jao E. and Joson E.

2D-Pharmacy, Faculty of Pharmacy, Uni ersity of !anto "omas ABSTRACT #hromato$ra%hy is a set of laboratory techni&ues for the se%aration of mi'tures (hich in ol es %assin$ a mi'ture dissol ed in a mobile %hase throu$h a stationary %hase. "his e'%eriment aims to se%arate the colored com%onents of red !ilin$ )abuyo and *alun$$ay lea es usin$ column chromato$ra%hy+ to determine the %urity of the com%onents usin$ thin layer chromato$ra%hy+ and measure the ,f alues of the colored com%onents in thin layer chromato$ra%hy. #olumn #hromato$ra%hy and "hin )ayer #hromato$ra%hy (as used to se%arate the different colored %i$ments. #olored eluate (as collected throu$h #olumn #hromato$ra%hy and the %urity of these eluates (as tested throu$h "hin )ayer #hromato$ra%hy. U- lam% (as used as the isualization method for the determination of colorless substrates and the ,etention or ,etardation Factor (as measured. INTRODUCTION #hromato$ra%hy in chemistry, analytical techni&ue used for the chemical se%aration of mi'tures and substances. "he techni&ue de%ends on the %rinci%le of selecti e adsor%tion, a ty%e of adhesion. #hromato$ra%hy (as disco ered in ./01 by the Italian-born ,ussian botanist *i2hail "s(ett, but (as not (idely used until the ./30s. #olumn chromato$ra%hy is the most $eneral method for the %urification of or$anic com%ounds. #olumn chromato$ra%hy no( uses a (ide ran$e of adsorbent solids, includin$ silica, alumina, and silica $el. It in ol es the se%aration of indi idual com%ounds from a mi'ture by %assin$ it throu$h a column %ac2ed (ith silica $el. 4fter the mi'ture is a%%lied to the column, an a%%ro%riate sol ent is %assed throu$h the column. !ilica $el (as used because com%ounds (ill usually adhere to the silica to different e'tents, they tra el at different s%eeds do(n the column. Each chromato$ra%hic zone re%resents a natural constituent of the or$anism from (hich the mi'ture (as e'tracted. 4 %um% is re&uired to dri e a mi'ture throu$h the column. "hin-layer chromato$ra%hy is another form of column chromato$ra%hy in (hich the adsorbent. "hin layer chromato$ra%hy is ery useful for ra%id analysis of or$anic mi'tures. Plastic, metal, or $lass %lates are %re%ared (ith a thin layer of silica $el. "he %a%er is called the stationary %hase (hile the sol ent is referred to as the mobile %hase. "he or$anic com%ound that (as noticeable (as the chloro%hyll. "hrou$h these %rocesses the follo(in$ ob5ecti es (ere met, mainly6 .. !e%arate the colored com%onents of silin$ labuyo and malun$$ay lea es usin$ column chromato$ra%hy. 2. Determine the %urity of the com%onents usin$ thin layer chromato$ra%hy 7")#8. 3. *easure the ,f alues of the colored com%onents in ")#. EXPERIMENTAL A. Co !oun"# $%#$%" &o' #a !(%# u#%") .0-.9 %ieces of silin$ labuyo, D#*6:e'ane7.6. ratio8, 4cetone, 4cetone *ethanol. B. P'oc%"u'% For the first %rocedure of this e'%eriment, .0-.9 %ieces of silin$ labuyo (ere selected and $rinded usin$ a mortar and %estle. Usin$ D#*6:e'ane7.6.8, %i$ments (ere e'tracted and later on, a %ortion of the e'tracts (ere set aside for ")#. 4fter that, the column (as %lu$$ed (ith cotton at the bottom and uniformly %ac2ed (ith silica $el. "he silica $el

$ot u% to the intended %art acted as the stationary %hase in the set-u%. >ello( ?ran$e )i$ht ?ran$e Dar2 ?ran$e

Usin$ a Pastuer %i%ette, 0.9 m) of the e'tract (as %laced on to% of the column. "he %i$ments (ere then eluted (ith the use of .0 m) of D#*6:e'ane ha in$ a ratio of . is to . 7.6.8. "he column (as %ro%erly obser ed u%on e ery no( and then to a oid it to run dry. "he sol ent system (as then introduced into %ortions (hilst discardin$ the colorless eluate.

.%$$in. $/% !i. %n$ 30 $tts .0 $tts @2 $tts ..$tts

"he collected colored eluates (ere %laced in se%arate test tubes. ;ote ta2in$ of the number of dro%s of eluate that are collected in each tube became an im%ortant tas2 and (as done cautiously. 4 9 ' < ")# %late (as %re%ared. =ith this ")# %late, eluates (ere a%%lied by s%ottin$ fi e times. !%ots (ere made sure to be as small as %ossible. Each s%ot (ere dried first before a%%lyin$ the ne't eluate.

"he mobile %hase slo(ly flo(s do(n throu$h the silica $el column by $ra ity lea in$ behind zones of color A the chromato$ram. "he theory of column chromato$ra%hy is analo$ous to that of thinlayer chromato$ra%hy. "he different com%onents in the sam%le mi'ture %ass throu$h the column at different rates due to differences in their 2ortionin$ beha iour bet(een the mobile li&uid %hase and the stationary %hase. Ta*(% 0. "hin )ayer #hromato$ra%hy6 #olored Pi$ments and Distance tra elled by the s%otted %i$ment from the ori$in Co(o'%" Pi. %n$# >ello( ?ran$e )i$ht oran$e Dar2 ?ran$e Di#$anc% T'a1%(%" &c ) 2.2 cm .@ cm 0 .9 cm+ 2.@ cm+ 3.9 cm+ 9.2 cm

4 de elo%in$ chamber (as then %re%ared by addin$ .0 m) of the D#*6:e'ane7.6.8 solution and lined the inner (all (ith filter %a%er, co ered (ith a (atch $lass and allo(ed to e&uilibrate.

"he distance tra elled by the sol ent of the Bsol ent frontC. In the e'%eriment, (e obtained D.9 cm as the sol ent front. In addition, to &ualitati e results, ")# can also %ro ide a chromato$ra%hic %arameter 2no(n as ,f -alue. "he ,f alue is the retardation factor or the ratio-to-front alue e'%ressed as a decimal fraction. "he , f alue is the ratio of the distance tra elled by the s%ot from the %oint of ori$in to the distance the sol ent tra els. "he $eneral formula for com%utin$ for the ,f alue is

")# %late (as %laced carefully in the de elo%in$ chamber to allo( the sol ent system to rise u% to . cm from the u%%er end before the %late (as remo ed from the chamber. "hen, the sol ent front and air dry (ere mar2ed immediately. 4fter many %rocedures had been %erformed, com%onents (ere isualized usin$ an ultra iolet lam% in order to measure ,etardation factor alues and to document the chromato%late. RESULTS AND DISCUSSION Ta*(% +. #olored Pi$ments and number of dro%s obtained to $et the %i$ment in #olumn #hromato$ra%hy Co(o' , o- "'o!# o*$ain%" *%-o'%

Co !u$a$ion#2 )i$ht >ello(6 2.2E9.2 F @.2 >ello(6

.@E9.2F0.0DD ?ran$e6 0E9.2F0 Dar2 ?ran$e6 .9E9.2F 0.0/+ 2.@E9.2F0.@1+ 3.9E9.2F0.1D+ 9.2E9.2F. "he ,f alues obtained (ill hel% us determine the identity of a substance. If t(o substances on the same ")# %late $i e s%ots in identical location, they may be the same. Gut still, com%arisons of ,f alues to be alid, ")# %lates must be run under the e'act same conditions for stationary %hase, mobile %hase, and tem%erature. RE3ERENCE4S2 Goo2s6 Pa ia, D., )am%man, H., Iriz, H., J En$el, ,. 7.//<8. Introduction to organic laboratory techniques: a microscale approach.3rd Ed. U!46 !aunders Publishin$ #oma%any. =illiamson, I+ *inard, ,+ *asters I. 7200D8. Macroscale and microscale organic experiments. 9th Ed. Goston, U!46 :ou$hton *ifflin #om%any. Harner, #*. 7.//D8. "echni&ues J e'%eriments for ad anced or$anic laboratory. U!46 John =iley J !ons. Inc. *4#4U)4>, DG. 7200D8. Heneral, or$anic J biolo$ical chemistry an inte$rated a%%roach. U!46 John =iley J !ons. Inc.