INTRODUCTION
In this experiment, the reducing sugar compound in the carbohydrate of the sample was be determined. determined. A sugar is only a reducing sugar if it has an open chain with an aldehyde or a ketone group. Monosaccharides which contain an aldehyde group are known as aldoses, and those with a ketone ketone group are known as ketoses. Reducing monosaccharides monosaccharides include glucose, fructose, glyceraldehyde and galact galactos ose. e. Ma Many ny disacc disacchar haride ides s like like lactos lactose e and malto maltose se also also do have have a reducing form, form, as one of the two units may have an open-chain with an aldehyde group. owever, owever, sucrose and trehalose, in which the anomeri carbons of the two units are linked together, are non-reducing disaccharides. !owa !owaday days s threr threre e are are sever several al method methods s were were estab establis lished hed in order order to determi determine ne the reducin reducing g sugar sugar such as "initrosal "initrosalicylic icylic Acid method, method, #enedict$s #enedict$s reagent, reagent, %ehling&s solution and !elson-'omogyi !elson-'omogyi Method. (he "initrosalicylic "initrosalicylic Acid method was used because it is simple, sensitive and adoptable during handling of a large number of samples at a time. #enedict$s reagent reagent and %e %ehling$s hling$s solution are used to test for the presence of a reducing sugar. (he reducing sugar reduces copper)II* ions in these test solutions to copper)I*, which then forms a brick red copper)I copper)I** oxide oxide precipit precipitate. ate. +hile (he !elson-'omo !elson-'omogyi gyi method method is one of the classical and widely used methods for the uantitative determination of reducing suga sugars rs.. "ie "ierrent ent reduc educin ing g suga sugars rs give give blue blue colo colorred prod produc ucts ts of vary varyin ing g intens intensiti ities es when when estima estimated ted this this metho method, d, there thereby by implyi implying ng that that a mixtur mixture e of reducing sugars should not be estimated using this protocol. roteins present in the reaction medium should be eliminated by /inx hydroxide precipitation as they can interfere with this assay. In this experiment the !elson-'omogyi method was applied. (he principle of this method is (he reducing sugars when heated with alkaline copper tartrate reduce the copper from the cupric to cuprous state and thus cuprous oxide is formed. +hen cuprous oxide is treated with arsenomolybdic acid, the reduction of molybdic acid to molybdenum blue takes place. (he blue color developed is compared with a set of standards in a colorimeter at 012nm.
APPARATUS
Material 3
a* 122m4 beaker b* ipette c* 5olumetric 6ask. d* cuvvete e* spectrophotometer. 7hemicals 3 a* 4ow-alkalinity copper reagent 8 sodium potassium tartrate )92g* and anhydrous )1:g* were dissolved in about ;22 m4 of water. A solution of <2= cupric acid sulphate pentahydrate in water ):2m4* was added in small amounts with stirring. <:2 g anhydrous was added in small amounts and was dilute to < litre. After < day of standing, the clear supernatant solution was used. b* Arsenomolybdate reagent- 1>g of ammonium molybdate in 9>2 m4 water was added 1= sulphuric acid, followed by @.2g of disodium hydrogen arsenate heptahydrate dissolved in 1>m4 water. (he mixed solution was incubated 1> hours at @;7 and stored in a glass- stoppered brown bottle. c* 'tandard glucose solution 8 stock solution was diluted containing
PROCEDURES
A 3 sample preparation
<* (he bottle containining the carbonated drinlCks was shaken. 1* <22m4 of the drink was poured into 1>2Mlbeaker. @* (he drink were transfered from ane beaker to another. (his steps was repeated until nogas remains in the dark. 9* (he degassed sample was pipette out >,2 m4 into 1>2 volumetric 6ask and the volume was diluted. (his is calle dsolution is A. >* <2m4 of solution A was pipette out into a <22m4 beaker and >2m4 of distilled water. (he solution was neutrali/ed. (he solution was transfer into <22ml volumetric 6ask and the volume was diluted. (his is solution is #. # 3 determination of reducing sugar <* A blank and standar solutios containing between 2 and 9>2 ug glucose were prepared as similar to the manual. 1* A sample solution of containing not more than 1m4 of solution # was made. A series of dilution was made and added distilled water until 1m4. @* 'amples, blanks and standard sugar solution were added with 1m4 copper reagent and the content was mix properly. 9* 'amples, blanks and standard sugar solution was heated <2min in avigorously boiling water bath and then was cooled under running water for > min >* < m4 of arsenomolybdate reagent was added to altest tubes and mix well. 0* +hen all the cuprous oxide was dissolved after mixing, the solution was diluted to the <2 m4 using volumertric 6ask and then allowed to stand and at least <> minute, but not more than 92min. ;* Read absorbance was read at >12nm.
RESULT
Dlucose solution )<22EgBml* Dlucose solution )@22EgBml* "istilled water )ml* Absorbance reading
(ube < 2.2
(ube 1 2.>
(ube @ <.2
(ube 9 <.>
(ube > 1.2
(ube 0 -
(ube ; -
-
-
-
-
-
<.2
<.>
1.2
<.>
<.2
2.>
2.2
<.2
2.>
2.2>>
2.1?;
2.>2;
2.;;?
<.21?
<.09<
<.?01
Draph of glucose standard curve
Glucose Standard Curve 1.> 1
f)x* C 2x F 2.29 RG C 2.?:
<.> Absorbance !"nm
< 2.> 2 2
>2 <22 <>2 122 1>2 @22 @>2 922 9>2 >22 Amount og glucose µg
Carbonated dr#n$s results%
'ample
Amount )ml*
Absorbance
A< A1 #< #1
2.> 2.> <.2 <.2
%rom standard curve, y = 0.004x + 0.044
T&e amount o' glucose #n t&e sam(le) Sample A1
H
C )2.199 8 2.299* B 2.229 C >2Eg
Sample A2
H
C )2.190 8 2.299* B 2.229 C >2.>Eg
Sample B1
H
C )2.91: 8 2.299* B 2.229 C ?0Eg
Sample B2
H
C )2.920 8 2.299* B 2.229 C ?2.>Eg
Calculat#on o' reduc#ng sugar concentrat#on
2.199 2.190 2.91: 2.920
*or "+ml sam(le A1
C >2 x <2 -0Eg x >2 x <2 x 9 C 2.<22 gBml
A2
C >2.> x <2 -0Eg x >2 x <2 x 9 C 2.<2< gBml
Average C )2.<22 gBml F 2.<2gBml
*or ,+"ml sam(le) B1
C ?0 x <2 -0Eg x >2 x <2 x 1 C 2.2?0 gBml
B2
C ?2.> x <2 -0Eg x >2 x <2 x 1 C 2.2?2> gBml
Average C )2.2?0 gBml F 2.2?2> gBml* B 1 C 2.2?@1> gBml
*#nal concentrat#on o' carbonated dr#n$s reduc#ng sugar #s -.
C 2.<22> gBml F 2.2?@1> gBml* B 1 C 2.2?; gBml
"I'7''I!
%or this experiment, the 'omogyi !elson method have been used in determining the concentration of reducing sugar in the sample which is carbonated drink. (his method is commonly used in determining the reducing sugar rather than the "!' method because of its eectiveness for all types of sample. (hat is because, it does not matter about the color of the sample, it can be easily applied in determination of reducing sugar. %or this experiment, the sample that we used is 7oke drink which is a kind of carbonated drink. (here several steps in doing this method and it started with removing the gas of that carbonated drink. (hen, the sample solution is diluted with the distilled water for neutrali/ation. After the several dilution of the sample, the sample is added with copper reagent and arsenomolybdate reagent. After mixing and incubated at room temperature for several minutes, the absorbance of the sample can be read by spectrophotometer at >12nm wavelength. In principle, during the Jrst step, glucose )or a reducing sugar* is oxidised using a solution of 7u)II* ion which in the process is reduced to 7u)I*. In the second step the 7u)I* ions are then oxidised back to 7u)II* using a colourless hetero-polymolybdate complex, which is, in the process, reduced to give the characteristic blue colour. %inally the absorption of the hetero-poly molybdenum blue is measured using a colorimeter and compared to standards prepared from reacting sugar solutions of known concentration, to determine the amount of reducing-sugar present. +hen the reading of absorbance is got, the concentrations of reducing sugar can be determined by refer to the reducing sugar standard curve that has been produced before. In order to achieve the exact concentration of the reducing sugar, we need to multiply the result with the dilution factor. And after all, the concentration of reducing sugar of coke drink that we got is only 2.2?; gBml. %rom the result we got, the concentration of reducing sugar that we determined is not really high.
CONCLUSION
(he concentration of reducing sugar of the carbonated drink is not really high which is only about 2.2?; gBml. owever, if compared to the other type of drinks, the amount of reducing sugar of carbonated drink is much higher. RE**ERENCES
<* http3BBen.wikipedia.orgBwikiBReducingKsugar 1* http3BBwww.eplantscience.comBindexKJlesBplant =12protocolsB7arbohydratesBreducingKsugarsKbyK!elson'omogyiKmethod.php
