EXPERIMENT 2 DETERMINATION OF REDUCING SUGAR USING THE DINITROSALICYCLIC (DNS) COLOURIMETRIC METHOD OBJECTIVE: 1. To determine the concentration of reducing sugar by using the Dinitrosalicyclic (DNS) colourimetric method 2. To studies the application of UV-Visible Spectrophotometry 3. To use the standard curved to determine the concentration of samples INTRODUCTION According
to
Dubois
et.
al
(2000),
“Dinitrosalicyclic
(DNS)
colourimetric method is a tests for the presence of free carbonyl group (C=O), which is reducing sugars. This involves the oxidation of the aldehyde functional group present. 3, 5-dinitrosalicylic acid (DNS) is reduced to 3-amino, 5-nitrosalicylic acid under alkaline conditions.” 3, 5 Dinitrosalicyclic acid + Glucose (Reducing sugar)
3-amino-5-
nitrosalicyclic acid + Gluconic acid The above reaction scheme shows that one mole of sugar will react with one mole of 3, 5-dinitrosalicylic acid and reduced to 3-amino-5nitrosalicyclic acid On
the
other
hand,
the
application
of
spectrophotometric
(colorimetric) method is by using light absorption to determine the concentration of a molecule which absorbs light where this device known as uv-vis spectrophotometer (double beam spectrophotometer) measures the transmittance by calculating the ratio of the two intensities. These obey the Beer-Lambert Law where it relates the absorbance of light by a sample to the concentration of the absorbing species. In the Beer-Lambert Law, the absorbance is directly proportional to the concentration of the absorbance and also to the path length. In this experiment, the reducing
sugar concentration of two samples which are apple juice and strawberry jam will be determined by using colourimetric (spectrophotometric) method
MATERIALS 1. Double-beam spectrophotometer
10. Spatulas
2. Analytical balance
7. Wash bottle
3. Plastic/Glass cuvettes
8. Stop-watch
4. Beakers
9. Ice water
5. Vortex mixer
10. Glucose standard
6. Pipettes
11. 3, 5-dinitrosalicylic acid (DNS)
7. Volumetric flasks 8. Test-tubes and racks tetrahydrate 9. Water-bath (boiling)
12. 2 Molar NaOH 13.
Sodium
potassium
tartrate
14. Distilled water
METHODS A. Preparation of DNS reagent 1. Solution A was prepared first by dissolving 10 g of DNS in 200 mL 2 M NaOH with warming and vigorous stirring 2. Solution B was then prepared by dissolving 300 g sodium potassium tartrate tetrahydrate in 500 mL distilled water 3. Both solution A and B was then mixed together before make up to 1 L distilled water B. Preparation of the Sample a) Solid sample
1. 3 g of solid sample was weighed accurately into a beaker and 50 mL of distilled water was added. The solution was stirred until thoroughly dissolved 2. The solution was then filtered into a 100 mL volumetric flask. The residue was then washed into the volumetric flask with a small amount of distilled water and the volume was making up to 100 mL. The solution was then mixed well by repeated inversion of the volumetric flask b) Liquid sample 1. 5 mL of the liquid sample was pipetted 2. The liquid sample was filtered into a 100 mL volumetric flask. The residue was washed into the volumetric flask with a small amount of distilled water and make up to 100 mL volume. The liquid sample was then mixed well with repeated inversion of the flask 3. 10 mL of sample solution was diluted to 100 mL distilled water in a volumetric flask. The solution was then mixed well by inversion of the volumetric flask C. Preparation of Glucose Standard Solutions 1. 1.0 mL of distilled water was pipetted into a test tube and labelled as blank 2. 1.0 mL of each concentration of glucose standard solution was pipetted in order into appropriately labelled test tubes 3. 1.0 mL of sample solution prepared was pipetted into a test tube and labelled as sample (The sample, standard and blank was prepared in triplicate before testing was conducted) 1. 1.0 mL of DNS reagent and 3 mL of distilled water was then added into prepared test tubes. The solution was then mixed well with a vortex mixer 2. The test tubes was arranged in a test tubes rack before it was placed in the boiling water bath for exactly 5 minutes to allow the reaction between the DNS reagent and glucose to occur 3. The test tubes was then transferred immediately into a container of iced water after 5 minutes in water bath 4. 10 mL of distilled water was then added to each test tube and mixed well by using vortex mixer
5. The maximum wavelength was then determined by measuring the absorbance spectrum of 4 mg/mL of glucose standard solution by using a double-beam spectrophotometer. 6. The spectrum was measured within the wavelength range of 400 to 600 nm. The spectrophotometer wavelength was set by using the highest absorbance obtained 7. The absorbance for each standard was measured for each standard solution and the sample solutions 8. RESULT Absorbance data for DNS colorimetric method Standard/
ʎ
Absorbance reading at
: 477 nm
Sample
Absorban
Absorbanc
Absorbance
Absorbance
Concentration
ce 1
e2
3
Average ±
(mg/mL) 0 (Blank) 2 4 6 8 10 15 Liquid Sample
0 0.456 0.826 1.099 1.312 1.315 1.060 0.068
0 0.443 0.825 1.097 1.223 1.319 1.067 0.039
0 0.434 0.831 1.096 1.310 1.373 1.152 0.038
SD 0 0.444 0.827 1.097 1.282 1.336 1.093 0.048
(Apple Juice) Solid Sample
0.072
0.067
0.061
0.067
(Strawberry Jam)
2. Standard curve graph a) State the glucose concentration range for linear curve mg/mL – 6 mg/mL b) Write the equation derived from the linear curve 0.1925 x c) Write the linear regression obtained
:
0
: y = : 0.9852
CALCULATIONS 1. Show
one
example
of
calculation
for
preparing
a
desired
concentration of glucose solution from the stock solution given To prepare 4 mg/mL glucose standard solution:C1V1 = C2V2 100 mg/mL x V1 = 4 mg/mL x 100 mL V1 = 4 mL 2. Show the calculation for determining sample concentration from the equation derived from the linear standard curve 1. Liquid sample (apple juice) Y = mx 0.048 = 0.1925 x X = 0.2494 Concentration:= X x dilution factor (Final dilution / Initial dilution) = 0.2494 x (100/10) = 2.494 mg/mL 2. Solid sample (Strawberry jam) Y = mx 0.067 = 0.1925 x X = 0.3481
Concentration: = X x dilution factor (Final dilution / Initial dilution) = 0.3481 x (250/10) = 8.7025 mg/mL DISCUSSION Through the experiment, the concentration of reducing sugar which are in apple juice (non-added sugar) and strawberry jam was determined by using the Dinitrosalicyclic (DNS) colourimetric method. Both samples which are solid sample and liquid sample was first prepared by weighing approximately 3 g of solid sample and pipetting 5 mL of the liquid sample respectively. The samples were then undergoing several dilutions after being filtered properly into 100 mL of volumetric flask. The solid sample solution was then diluted again by using ratio 1: 25 while the liquid sample was diluted to 1: 10 dilution ratio. This is to ensure the concentration in the sample is not too high and causing the sample absorbance values is above highest point in the standard curve. After that, a series of glucose standard solutions of 2 mg/mL, 4 mg/mL, 6 mg/mL, 8 mg/mL, 10 mg/mL and 15 mg/mL was prepared by diluting 100 mg/mL glucose stock solution by using distilled water in 100 mL volumetric flask. Volume of glucose stock solution required to make dilution was determined by using this formula:C1V1 = C2V2 Meanwhile, the measurement of absorbance was prepared by pipetting each 1.0 mL of glucose standard solution and samples solution, 1.0 mL of DNS reagent and 3.0 mL of distilled water into the test tubes. DNS reagent is a reagent used to determine sugar content especially glucose. Meanwhile, the sample was prepared triplicate to minimize human error. All the solution was mixed properly by using vortex before it was placed in boiling water bath for 5 minutes to allow the reaction to
occur. In hot solution, the DNS reagent will reacts with the reducing sugars producing the reddish-brown product (3-amino-5-nitosalicil acid). As the concentration of the glucose standard solution increases, the colour of the solution will be darker. As the more sugar there is, the more carbonyl groups there are hence resulting in darker solution. After 5 minutes the test tubes was immediately transferred to container of ice water to stop the reaction from occurring.
The concentration of the
coloured solution was then measured with the uv-vis spectrometer at 477 nm. From the results, the absorbance’s average was calculated and reducing sugar standard curve (linear and non-linear) was constructed to calculate linear regression (R2) and obtained equation derived from standard curve. From the equation, the concentration of samples solution (liquid and solid sample) can be calculated where it was obeying the BeerLambert Law. The calculations show that the concentration of liquid sample (apple juice) is 2.494 mg/mL meanwhile the concentration of solid sample (strawberry jam) is 8.7025 mg/mL. The results show that the concentration of solid sample (strawberry jam) is higher than liquid sample (apple juice) as the strawberry jam contains a lot of glucose. This is because; the strawberry jam was made with higher amount of sugar where it can inhibit the growth of bacteria hence extending the shelf life of the jam. Meanwhile, apple juice that was used is non-sugar added typed where there is no sugar added in their juice’s preparation. The sugars were only obtained from the fruits itself which is from apple. According to Malaysian Food Law and Regulation (1985), the soluble solids for apple juice shall not less than 11.5 g. Meanwhile, for jam the soluble solids should be at the 65 per cent of soluble solids. During the experiment, they are some sources of error that occur which may affect the accuracy of the result. For example, there are parallax error that happen when the eyes does not directly proportional to
the reading of the pipette. Poor pipetting technique may also contribute to the error besides there might be the present of bubbles during pipetting. In addition, enzyme mixture (DNS) might not be prepared correctly or the stability limit had exceeded. To minimize these errors various precautions should be taken such as, make sure our eyes are directly proportional during reading the pipette measurement. The fresh enzyme mixture was prepared and re-assay. Lastly, three consecutive of measuring need to be carried out to eliminate the errors of the handlers. CONCLUSION In conclusion, the concentration of reducing sugar in apple juices and strawberry jam was determined by using the Dinitrosalicyclic (DNS) colourimetric method which are 2.494 mg/mL and 8.7025 mg/mL respectively. REFERENCE C. Michael, (2003), How does Dinitrosalicyclic Acid stops the enzymesubstrate reaction in
enzyme assay, Retrieved October 6, 2003 from
http://www.researchgate.net/post/How_does_Dinitrosalicylic_Acid_stops_th e_enzy
me-substrate_reaction_in_enzyme_assay
M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers and Fred Smith, (2000), Determination of Reducing Sugar by Colourimetric Method and Other Substances, Anal. April
Chem., 1956, 28 (3), 26,
pp
350–356
2000
Retrieved
from
http://pubs.acs.org/doi/pdf/10.1021/ac60111a017 Malaysia. (1985). Food Act 1983 and Food Regulations 1985: Revised 7th October, 1985: Act 281. Kuala Lumpur: MDC
