THIN-LAYER CHROMATOGRAPHY AND QUANTITATIVE ANALYSIS OF CARBOHYDRATES

Chryss Yancee G. Oliva, Joel C. Piansay, Magnolia Grac G! Q"in#o, Marian Angelu C. Ramos, Rose Ann M. Refuela Group 7, 2E Pharmacy, Faculty of Pharmacy, University of Santo Tomas

ABSTRACT
Starch obtaine from !otatoes "as the source of carbohy rate use in this e#!eriment. $he ob%ectives of this e#!eriment are &'( to !erform thin)layer chromatogra!hy on the carbohy rate hy rolysates, &*( to correlate the ata obtaine from the thin)layer chromatogra!hy of the carbohy rate hy rolysates an i entify the monosacchari e !resent in the !olysacchari e sam!le, an &+( to etermine the amount of re ucing sugars using ,elson-s test an e#!lain the !rinci!le involve . $he thin)layer chromatogra!hy se!arates the monosacchari es !resent in a given !olysacchari e sam!le. $he ,elson-s Metho , ho"ever, etects the concentration of glucose !ro uce by the !olysacchari e sam!le by measuring the absorbance of the value follo"ing the !rinci!le behin the ,elson-s metho . After !erforming the mentione tests, the thin)layer chromatogra!hy sho"e that the starch sam!le contains e#trin, maltose, an glucose. .hile in the ,elson)Somogyi Metho , the concentration of the un/no"n, "hich is the starch hy rolysate, is 0.1* mg2m3.

INTRODUCTION
Accor ing to the American 4ietetic Association, carbohy rates are one of the three main buil ing bloc/s of foo , along "ith !rotein an fat. 5oo s "ith carbohy rate hel! fuel your bo y. 6t is also /no"n as sacchari es or sugars. Carbohy rate is the most abun ant organic molecule on earth. 6t functions as one of the /ey interme iates of metabolism as sugar, a structural com!onent in !lants as cellulose, com!onent in the in ustrial !ro uction of !a!er, lumber an fibers, an a /ey com!onent of foo sources li/e in sugar, flour, an vegetable fibers &McMurry-s Organic Chemistry 7th(. $he buil ing bloc/s

of all carbohy rates are the sim!le sugras calle monosacchari es. A monosacchari e can be !olyhy ro#y al ehy e &al ose( or a !olyh ro#y /etone &/etose( an oes not un ergo hy rolysis unli/e oligo) an !olysacchari e. $he sim!lest monosacchari es contain three carbon atoms an are calle trioses. 6t has the general formula Cn&8*O(n &Cam!bell 9 5arrell, *00:(. Carbohy rates can also occur as isacchari e or t"o monosacchari es lin/e together. Oligosacchari es are forme "hen many monosacchari es are bon e together &Cam!bell 9 5arrell, *00:(, usually u! to *0 monosacchari es, "hile !olysacchari es

are com!ose of *0 or more monosacchari es bon e to each other. ,elson-s metho "as a!!lie to etermine the concentration of glucose liberate from hy rolysis. ,elson-s metho , also calle ,elson)Somogyi Metho , is one of the classical an "i ely use metho s for the ;uantitative etermination of sugars li/e glucose an galactose.

!lace insi e the evelo!ing chamber an "as evelo!e until the solvent front "as a!!ro#imately ' cm from the to!. $he $3C "as remove an the solvent front "as mar/e "ith a !encil. $he $3C !late "as air) rie an "as s!raye "ith panisal ehy e. After s!raying, the !late "as oven rie until s!ots "ere visible. $he Rf values of the s!ots "ere com!ute using the formula<

Rf = MATERIALS AND METHODS

Materials A. $hin)3ayer Chromatogra!hy ,)butyl alcohol<acetic aci <ether<"ater &:<7<+<'(= Stan ar s< '> e#trin, '> maltose, '> glucose= p) anisal ehy e, ca!illary tubes, ?#'0 cm $3C !late, evelo!ing chamber @. ,elson-s Metho ,elson-s reagent A, ,elson-s reagent @, arsenomolyb ate, glucose stan ar , s!ectro!hotometer, an cuvettes.

distance traveled by substance distance traveled by solvent front

@. ,elson-s Metho ,elson- reagent "as !re!are by m#ing '*.? m3 ,elson-s A "ith 0.? m3 ,elson-s @. $hen, B test tubes "ere !re!are as follo"s< $able '. Glucose stan ar , "ater, an un/no"n concentrations use in the e#!eriment
T"$ No! Gl" S#%! Di&#! H'O Un(no)n

' * +

0 0.' 0.* 0.1 0.7 0.B '.0 0

'.0 0.: 0.B 0.7 0.1 0.* 0 0.7

0 0 0 0 0 0 0 0.1

Methods A. $hin)layer Chromatogra!hy $he solvent system, n)butyl alcohol<acetic aci <ether<"ater, "as measure an !lace insi e the evelo!ing chamber "here it "as covere an e;uilibrate for '0 minutes. $he $3C !late "as then mar/e "ith a !encil, 'cm from one of the e ges. A;ui istant !oints "ere ma e for the s!ots. $he stan ar s, e#trin, maltose an glucose, "ere a!!lie for ? times, an the hy rolysates for '0 times using ca!illary tubes. $he $3C !late "as

1 ? 7 C B

$he '.0 m3 ,elson-s reagent "as then a e to each tube an sha/e "ell. $he tubes "ere heate in a boiling "ater bath for *0 minutes. $he test tubes "ere coole o"n using a bea/er of "ater. $hen, '.0 m3

arsenomolyb ate reagent "as a e to each test tube an mi#e "ell until the !reci!itate issolve . $he solution "ere transferre to cuvettes an rea at 1B0 nm using the s!ectro!hotometer. $he glucose stan ar curve "as !lotte an the concentration of the un/no"n in mg2tube an mg2m3 "as com!ute .

&ol"# R- ,al" 0.'? 0.*'+ 0.BC?

C.B 0.*? 0.CC? 0.:C?

RESULTS AND DISCUSSION

! Thin-layer "hromato#raphy 5igure '. 4evelo!ing the $3C !late $able * sho"s the istance travele by the solvent "hich is B cm an the istances travele by the stan ar s a!!lie "hich are '.* cm for e#trin, '.C for maltose, an C cm for glucose. Mean"hile, the aci hy rolysate se!arate into three com!onents having the measurements * cm, 7.* cm, an C.B cm, res!ectively. $he Rf values "ere com!ute using the formula<

Rf =

distance traveled by substance distance traveled by solvent front

@ase on 5igure ', the Rf values of the stan ar s an the aci hy rolysate "as measure an com!ute . $he follo"ing ata "ere obtaine < $able *. Result of $3C D * Di&#anc #ra, l % $+ &ol, n# Di&#anc #r,al % $+ Bcm Bcm Bcm Bcm Mal Gl" H+%

'.*cm

'.Ccm

C cm

* 7.*

$he Rf values of the stan ar s are 0.'? for e#trin, 0.*'+ for maltose, an 0.BC? for glucose "hile the Rf values of the + com!onents of the aci hy rolysate are 0.*?, 0.CC?, an 0.:C?, res!ectively. @ase on the Rf values com!ute , the Rf value of e#trin &0.'?( an maltose &0.*'+( is close to the 'st com!onent of the hy rolysate "hich is 0.*?. $his means that e#trin an maltose is !resent in the aci hy rolysate of starch. $he Rf value of glucose "hich is 0.BC? is also close to the Rf value of the * n an +r com!onent of the aci hy rolysate "hich are 0.CC? an 0.:C?, res!ectively. 6n conclusion, the aci hy rolysate of the starch is com!ose of the three stan ar s, e#trin, maltose, an glucose. $! %elson&s nalysis Method for 'uantitative

$he re ucing sugars "hen heate "ith al/aline co!!er tartrate re uce the co!!er from the cu!ric to cu!rous state an thus cu!rous o#i e is forme . .hen cu!rous o#i e is treate "ith arsenomolyb ic aci , the re uction of molyb ic aci to molyb enum blue ta/es !lace. $he blue color evelo!e is com!are "ith a set of stan ar s in a colorimeter at 1B0 nm.

5 6 7"n(8

0.++ (!)2

0.' (!)2

0.*0' '.?+?

After rea ing the absrbance at 1B0 nm, the un/no"n "as etermine to have '.?+? nm absorbance value. $he concentration "as com!ute as follo"s<

y = mx + b x= x= y b m 1.535 + 0.0817 3.905

x = 0.42
5igure *. Gra!h of the concentration against the absorbance at 1B0 nm

REFERENCES
$able +. Com!ute concentration of glucose in mg2tube an mg2ml an their absorbance at 1B0 nm Mg.#"$ 0 ' 1 2 3 4 0 0.0+ 0.0C 0.'+ 0.*0 0.*C Mg./l 0 0.0' 0.0* 0.01 0.07 0.0B A$&or$anc 0.000 0.07* 0.00: 0.'': 0.'00 0.0:+ @iochemical Metho . &Accesse 5eb *+, *0'1( htt!<22""".ne"age!ublishers.com2sam! lecha!ter20000:'.! f Cam!bell, M. D. 9 5arrell, S. O. &*00:(. @iochemistry, Si#th A ition. Cengage 3earning Asia Pte 3t . Eoet 4. 9 Eoet J. &*0''(. $iochemistry )th edition. FSA< John .iley 9 Sons 6nc. '11)'1?