UNIVERSITY OF DAR ES SALAAM
DEPARTMENT OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY
BN: 206 BIOCHEMISTRY PRACTICALS
COLORIMETRIC DETERMINATION OF REDUCING SUGAR CONCETRATION
NAME: FOVO Frank P.
REGISTRATION NUMBER: 2009-04-05754
DATE OF SUBMISSION: 06th JANUARY, 2011
SIGNATURE (WRITER) SIGNATURE (RECEPIENT)
The aim of this experiment is to determine the amount of sugar or carbohydrates in a soft drink provided by spectrophotometric (cololorimetric) method.
One useful and often used way of obtaining concentration of chemical in a solution, if it has color is by colorimetric method. If the analyte is not colored an appropriate reagent must be added that reacts with the analyte to produce a colored compound.
In this experiment, the method for determining concentration of sugar is based upon the color that forms when sugar reduce 3,5-dinitrosalicyclic acid(DNSA) to 3-amino-5-nitrosalicyclic acid.
This method tests for the presence of free carbonyl group (C=O), the so-called reducing sugars. This involves the oxidation of the aldehyde functional group present in, for example, glucose and the ketone functional group in fructose. Simultaneously, 3,5-dinitrosalicylic acid (DNS) is reduced to 3-amino,5-nitrosalicylic acid under alkaline conditions:
aldehyde group ----------> carboxyl group
3,5-dinitrosalicylic acid ----------> 3-amino,5-nitrosalicylic acid
In this experiment sucrose is provided which is the non-reducing sugar, does not undergo reaction with 3,5-dinitrosalicyclic acid. Therefore the sucrose and complex carbohydrate must be broken down into simple sugars like glucose first. The hydrolysis can be done by boiling the sample with hydrochloric acid, and then the pH is adjusted to give a basic solution under which conditions are good for reducing sugar.
This method is a straightforward modification of the original DNS method for glucose analysis.
The sugars in the soft drink are at too high concentration for this method. So dilutions must be carried out before carrying out analysis.
The measurement of transmittance (T) is made by determining the ratio of the intensity of incident ( I0) and transmitted (I) light passing through pure solvent and sample solutions as a function of wavelength. [Note: The percent transmittance (%T) is obtained by multiplication of T by 100.]
The logarithm of the reciprocal of the transmittance is called the absorbance (A),
A = log (1 / T)
According to Beer's law, the absorbance of a solution should be zero (100%T) if there is none of the absorbing species present. A blank solution that does not contain analyte being analyzed but have the same composition as the solution can be used to calibrate the machine into zero reading Absorbance (100%T). Then the machine can be used to find concentration of other solution.
The Beer-Lambert Law is only obeyed (the standard curve is linear) for reasonably dilute solutions.
Nature of graphs obeying Beer’s-Lambert law:
· 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
· 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)
Preparation of sucrose standard solutions
· 1000mg/L of standard solution was prepared by suitable dilution of the stock solution.
· 2:10 dilution 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:
o 2.0 ml of each sucrose standard solution were pipette into test tubes.
o 2.0 ml of 6 M HCl was added into each test tube and placed in a boiling water bath for 10 minutes.
o The test tubes were removed and carefully, 0.8 ml of 2.5 M NaOH , 2.0 ml of 0.0050 M 3,5-dinitrosalicyclic acid (DNSA) were pipette into each test tube.
o Then the tubes were placed in a boiling water bath for 5 minutes.
o 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.
o The test tubes were removed from the boiling water bath at the proper time and quickly placed in ice-water bath for 10minutes.
o Some of the blank (distilled water) solution was poured into a clean, dry corvette (filled half way) and then placed into the spectrophotometer.
o The measurement of the standard solution was taken and the abundance was recorded.
Preparation of unknown sample solution (Beverage)
· Dilutions of beverages were prepared as follows:
o 0.1:100, 0.2:100, 0.4:100, 0.6:100, and 0.7:100 ml
o Treated 2.0 ml aliquots of the diluted samples in the same manner that you did for the standards.
o The absorbance for the diluted samples was recorded.
RESULTS AND DISCUSION:
Determination of concentrations is done by using the formula;
Sucrose standard solutions:
Unknown sample solution (Beverage):
Results from the graph:
(Concentration × dilution factor)1 + (concentration × dilution factor)2
160 × (100/0.1) + 780 × (100/0.2) +1060 × (100/0.4) + 1210 × (100/0.6) +450 × (100/0.7) mg/L
Therefore average is 216,190.47mg/L
216,190.47mg/L × 350 ml
75,666.666mg/L × 1g
Therefore the concentration of sucrose in the original beverage is 75.666g/L
The DNS method can be applied twice to measure the individual concentrations of a mixture of glucose and sucrose. First, a small part of the original sample is consumed in measuring the glucose concentration by following the original DNS procedure. Another part of the sample is hydrolyzed and subsequently subjected to the same DNS procedure. The difference in the absorbance between the acid treated sample and the untreated sample is due to the presence of sucrose. The sucrose concentration can then be calculated from a calibration curve based on that difference in the absorbance.
The concentrations of standard solution were used to plot a graph.
The above concentrations were obtained by extrapolation of the standard graph of absorbance against concentration, since the absorbances of the sample were known but the concentrations are the ones which were not known.
The concentration of sucrose in the original beverage calculated deviate from the true value. This is due to various errors taking place in experiment. The mainly errors were due to reading absorbance on the spectrometer or during measurement of volumes. Other sources of error include variation of temperature and humidity.
The nature of the graph is a straight line, hence obeys the Beer’s law, and the concentration of the original beverage was obtained to be 75.666g/L
· Lehinger, A.L, DL Nelson and M. Cox, 1993. Principles of Biochemistry 2nd Edition.
· Practical hand-out
· Biochemistry, 3rd Edition.
· Miller, G.L., Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal. Chem., 31, 426, 1959.
· Chaplin, M.F. and Kennedy, J.F. 1994. Carbohydrates: A practical approach. Oxford University Press, Oxford.
· Hodge, J.E. and Hofreiter, B.T. 1962. Determination of reducing sugars and carbohydrates. In Methods in carbohydrate chemistry, Vol. 1 (R.L.
Whistler, and M.L. Wolfrom, eds.) pp. 380-394. Academic Press, London.