PRACTICAL 2: COLORIMETRIC DETERMINATION OF REDUCING SUGAR CONCETRATION

ABSTRACT

The aim of this experiment is to determination of reducing sugar (simple sugar) concentration. Beverages and soft drinks produced from different manufactures differ in their sugar concentration .Spectrphotometric (calorimetric) method was used in determination of sugar concentration. As concentration of sucrose increases also absorbance increases thus obeys beer’s Lambert law. The color and concentration of a sample solution affect absorbance, the proportion of the light observed will depend on how many molecules interact with sample solution. Strong colored sample solution (concentrated) it will have high absorbance because there are lots of molecules to interact with light. Dilute solution and less colored experience less interaction with light therefore absorbance is very low. As the graph obtained gives linear relationship between concentration and absorbance and thus obeys Beer’s Lambert law.

INTRODUCTION 

            Beverages and soft drinks produced from different manufactures differ in their sugar concentrations. Calorimetric analysis is a method of determining the concentration of chemical element or chemical compound or amount of sugar or carbohydrate in different solutions for example fruit juices or soft drinks but with the aid of color reagent. it is applicable to both organic and inorganic compound and also may be used with or without an enzymatic stage. The method is useful in industrial process and medical laboratories, for example determination of sugar concentration in soft drinks in industrial process. (Calometric analysis-Wikipedia)

          In this experiment we required to determine amount of sugar or carbohydrate in a soft drinks by spectrophotometric method. The determination of concentration is based upon the color that is formed when sugar reduce 3,5-dinitrosalicylic acid (DSNA) to 3-amino-5-nitrosalicylic acid as shown in the equation. M            

                     3,5-dinitrosalicylic          Gluconic acid       3-amino-5nitrosalicylic

                          acid (yellow)                                             acid (red brown)

        Sucrose cannot be detected directly using the 3,5-dinitrosalicylic acid .Therefore to facilitate this sucrose and other complex carbohydrates must be treated with hydrochloric acid this will digest sucrose to glucose and fructose. The pH of the sample has to be adjusted after the sample have been broken to its corresponding monosaccharide, it has to be kept under basic medium a condition which makes the simple sugar good reducing agents. is a disaccharide formed by condensation of two monosaccharide, glucose and fructose. Sucrose, commonly termed as table sugar is present in natural fruits and vegetables. (Miller, 2001)

Sucrose undergoes hydrolysis under the presence of HCl to yield glucose and fructose as the above reaction shows.

         After the DNSA has been reduced the resulting sample is tested for its light absorbance property (spectroscopy) by using spectrophotometer. Spectrophotometer can measure the amount of light absorbed by the solution, and the quantity of light absorbed is proportional to the intensity of color or pigment in the solution. Therefore this device measures the absorbance and displays its values. Absorbance values themselves cannot describe the concentration of the sample. However, we can determine the concentration of the reducing sugar sample by using a standard curve. The standard curve will translate the absorbance values into concentrations. The sugar in a soft drink is high concentrated for this method, so dilution must be carried out before caring out analysis. . Beer Lambert law can be used to determine sugar concentration, according to beer’s law, the absorbance of a solution should be zero (100%T) if there is none of the absorbing substance is present. The Beer’s Lambert law” state that” the absorbance of a solution is directly proportion to the concentration of the absorbing species in the solution and the path length. (Berberan.Santos, J.chem.Educ 67, 1990, 757.)

MATERIALS AND METHODS

Material used:

   Cuvettes, Test tubes,Test tube rack,  Test tube clamp,  Two 400 ml beaker,Mohr pipettes, Bulb (5 and 10 ml),  25ml volumetric pipette,  Five 100 ml volumetric flask, Pasteur pipette and bulb,  Tissue paper

Chemical used:

       6M HCl

       2.5 M NaOH

       0.05 M 3,5-Dinitrosacyclic acid

       1000 mg/L standard sucrose solution

       Soft drink to test (non-diet, not dark colored)

EXPERIMENTAL PROCEDURES

           1000mg/L of standard solution was prepared by suitable dilution of the stock solution.2:10 dilutions was made as follows, 2.0 ml of the stock solution was pipette into a clean 10 ml test tube and distilled water was added to calibrate mark of 10 ml. The tube was covered and shakes well to mix. In a similar fashion, 4:10, 6:10, and 8:10 dilutions were made

           Five standards were prepared (the original stock solution and the four dilutions) as follows:

2.0 ml of each sucrose standard solution were pipette into test tubes.2.0 ml of 6 M HCl was added into each test tube and placed in a boiling water bath for 10 minutes. The timer was set when the tubes were placed in the hot water so that each tube to stay in the water for the same amount of time. The test tubes were removed from the boiling water bath at the proper time and quickly placed in ice-water bath for 10minutes.Some of the blank (distilled water) solution was poured into a clean, dry corvette (filled half way) and then placed into the spectrophotometer.

The measurement of the standard solution was taken and the absorbance in triplicate in each dilution was recorded.

Preparation of unknown sample solutions (Beverage)

Dilutions of beverages were prepared as follows: 0.1:100, 0.2:100, 0.4:100, 0.6:100 and 0.7:100 mL

The 2.00 mL aliquots of the diluted samples were treated in the same manner as we have done for the standards. The measurements in duplicate were carried out for each dilution. Care was taken in ensuring the same time intervals were the same after adding the same DNSA used in the standards.

The absorbance values were recorded in triplicates.

RESULTS

            PART A

Table of results for absorbance values of the Sucrose standard solution

Dilution(ml)	Concentration(mg/l)	Absorbance
2:10	200	0.199
4:10	400	0.403
6:10	600	0.687
8:10	800	0.842
10:10	1000	1.110

 The graph of absorbance versus concentration of sucrose

PART B:

Table of results for Absorbance values of the Unknown sample solutions (Beverages)

Dilution	Absorbance 1	Absorbance 2	Absorbance 3	Average absorbance
0.1:100	0.189	0.177	0.173	0.180
0.2:100	0.342	0.324	0.325	0.330
0.4:100	0.664	0.666	0.670	0.667
0.6:100	0.703	0.710	0.713	0.709
0.7:100	0.936	0.928	0.932	0.932

The slope and intercept of that line provide a relationship between absorbance and concentration:

                                       A = slope c + intercept

The unknown solution is then analyzed. The absorbance of the unknown solution, A_u, is then used with the slope and intercept from the calibration curve to calculate the concentration of the unknown solution, c_u.

Cu =	Au - intercept slope

Results of unknown concentration of sucrose from the equation above;

DILLUTION	ABSORBANCE	CONCENTRATION (mg/l)
0.1:100	0.180	201.9
0.2:100	0.330	338.3
0.4:100	0.667	644.6
0.6:100	0.709	682.6
0.7:100	0.932	885.5

The above results were obtained from the equation on the graph from   y=0.0011x-0.0421 ; where by ‘Y’ is an absorbance and ‘X’ is the concentration.

Determinations of concentration of the unknown Sucrose sample solution:

(conc X dilution factor)₁ + (conc X dilution factor)₂ +……….(conc X dilution factor)_n

                                                               N

= {(201.9 X 100/0.1) + (338.3X 100/0.2)+(644.6X 100/0.4)+(682.6X100/0.6)+(885.5X100/0.7)}

                                                                    5

                                   =   772500 mg/l     =154500mg/l

                                               5

                                              =154500mg/l X 1g

                                                         1000mg

                                                       =154.5g/l

The concentration of sucrose in the original beverage is 154.5g/l

DISCUSSION

            According to the result and data obtained above shows that as concentration of sucrose increases also absorbance increases thus obeys beer’s Lambert law. The color and concentration of a sample solution affect absorbance, the proportion of the light observed will depend on how many molecules interact with sample solution. Strong colored sample solution (concentrated) it will have high absorbance because there are lots of molecules to interact with light. Dilute solution and less colored experience less interaction with light therefore absorbance is very low. Graph above gives straight line, the concentration on the x- axis and the absorbance on the y-axis, we saw that there is a linear relationship concentration and absorbance ( Hodge and Hofreiter,1962)

          The concentration of sucrose in the original beverage calculated deviate from the true value due to various errors during the time of conducting an experiment, such errors include failing to take accurate volume measurement, incorrect reading of absorbance in a spectrophotometer. Under a certain conditions Beer-Lambert law fails to maintain linear relationship between attenuation and concentration of analyte. These deviation are classified into three categories include, real fundamental deviations due to the limitations of the law itself, chemical deviations observed due to specific chemical species of the sample which is being analysed and equipment deviations (e.n.m.wikipedia.org/Beer- Lambert law at16:24, 06-12-2014)

CONCLUSION

          As the graph obtained gives linear relationship between concentration and absorbance and thus obeys Beer’s Lambert law. So this help in determination of concentration. Upon the knowledge we obtained from this scientific experiment at industrial level we can employ the method and determine amount of sugar concentration in different drinks.

REFERENCES

 Miller,G.L 1956,use of dinitrosalcylic acid reagent for determination of reducing sugar, Anal chem.,31,426,

Berberan.Santos, J.chem.Educ 67, 1990, 757

J,M Berg, J,L Tymoczko, L Stryer Biochemistry, 5^th  Edition

BN206, practical 4 handouts, calometric determination of reducing sugar concentration    

Chplin,M.Fand Kennedy,J.F, 1994, carbohydrates: A practical approach,Oxford University Press

Hodge,J.E, and Hofreiter, B.T, 1962,Determination of reducing sugars and carbohydrates. Chemistry Vol. 1,PP 380-394,London, Academic press.

Lehinger,A.L, Nelson D.L, Cox, M. 1993, Principle of biochemistry 2^nd Edition.

e.n.m.wikipedia.org/Beer- Lambert law at16:24, 06-12-2014