Pre-Lab Exercise

The importance of biochemical analysis in bioprocess technology is to understand the mechanism of

organism which they operate in molecular level. Reducing sugar is one of the common compounds in
bioprocess and routinely analysed due to its presence in almost all lifeforms. Better understanding of
the mechanism can lead to new discoveries that may contribute to science & technology.

Part A: Somogyi-Nelson’s Method Calibration Curve

Wavelength: 660 nm Conversion Factor: 1 absorbance = 180 µg/ml

Glucose concentration Absorbance

µg/ml
0 0
20 0.0728
40 0.2236
60 0.3292
80 0.4429
100 0.5637

Part B: DNS (3,5-dinitrosalicyclic acid) Method Calibration Curve

Wavelength: 750 nm Conversion Factor: - µg/ml

Glucose concentration Absorbance

µg/ml
0 0
200 0.0060
400 0.0022
600 0.0028
800 0.0040
1000 0.0029

Glucose concentration from the manufacturing standard

Sample Glucose
Concentration
(mg/ml)
Coffee 77.08
Soy Bean 82.00
Revive 68.92
Somogyi-Nelson Method Conversion factor: 1 absorbance = 180 µg/ml
BOLD FOR SELECTED DATA

Tube Sample Dilution Sample Absorbance Glucose Glu. Conc. per Glu. Conc. per Manufacturing
volume concentration 1 ml sample 1 ml sample Standard
(ratio) (µg/ml) X
(ml) (µg/ml) Dilution Factor (mg/ml)
(mg/ml)
1 Blank - 0 0 0 0 0 0

2 Coffee 1:25 0.1 1.1457 206.23 2062.30 51.56

3 1:50 0.1 0.0898 16.16 161.60 8.08

77.08
4 1:25 0.5 2.3966 431.39 862.78 21.57

5 1:50 0.5 1.2464 224.35 448.7 22.44

6 Soy 1:25 0.1 0.1172 21.096 210.96 5.274

7 1:50 0.1 0.0834 15.01 150.10 7.51

82.00
8 1:25 0.5 0.4941 88.94 177.88 4.44

9 1:50 0.5 0.3980 71.64 143.28 7.164

10 Revive 1:25 0.1 0.1261 22.70 227.00 5.68

11 1:50 0.1 0.0941 16.94 169.40 8.47

68.92
12 1:25 0.5 0.5700 102.60 205.2 5.13

13 1:50 0.5 0.4500 81.00 162.00 8.10

DNS Method Conversion factor: -- µg/ml
Wavelength: 750 nm

Tube Sample Dilution Sample volume Sample distilled Absorbance Glucose concentration
water (ml)
(ratio) (ml) (µg/ml)

1 Blank - 0 1.0 0 0

2 Coffee 1:25 0.1 0.9 0.0020 -

3 1:50 0.1 0.9 0.0001 -

4 1:25 0.5 0.5 0.0175 -

5 1:50 0.5 0.5 0.0084 -

6 Soy 1:25 0.1 0.9 0.0160 -

7 1:50 0.1 0.9 0.0290 -

8 1:25 0.5 0.5 0.0455 -

9 1:50 0.5 0.5 0.0010 -

10 Revive 1:25 0.1 0.9 0.0007 -

11 1:50 0.1 0.9 0.0006 -

12 1:25 0.5 0.5 0.0038 -

13 1:50 0.5 0.5 0.0047 -

DISCUSSIONS
The purpose of the experiment is determining the reducing sugar using colorimetric analysis.
Two methods in analysis used in the experiment were Somogyi-Nelson method and DNS
method. The sample used in the experiment were coffee, soy bean, and isotonic revive.

Somogyi-Nelson’s Method
Preparing the calibration curve for Somogyi-Nelson, the conversion factor for determining
amount of glucose in micrograms per ml was; 1 absorbance = 180 µg/ml. From the conversion
factor, the amount of glucose in sample can be calculated. The data were carefully selected
(highlighted in bold) based on consistency and used for further analysis and discussion.
A. Coffee
Selected data with dilution ratio 1:25. The 0.1 ml sample shows absorbance of 1.1457
and glucose concentration 206.23 µg/ml. The 0.5 ml sample shows absorbance of 2.3966 and
glucose concentration of 431.39 µg/ml. The 0.5 ml sample should be 5 times higher than the
0.1 ml sample but the data shows only about 2 times higher. The actual glucose concentration
from the experiment (best pick) is 51.56 mg/ml which differ greatly from the manufacturing
standard which is 77.08 mg/ml . The inaccuracy in the data may due to mistakes in preparing
the sample or lies on damaged apparatus
B. Soy Bean
Selected data with dilution ratio 1:25. The 0.1 ml sample shows absorbance of 0.1172
and glucose concentration 21.096 µg/ml. The 0.5 ml sample shows absorbance of 0.4941 and
glucose concentration of 88.94 µg/ml. The data is almost consistent but not precise. The actual
glucose concentration (best pick) measured was 5.27 mg/ml which is incomparable with
manufacturing standard which is 82 mg/ml. The measurement shows a major error and will be
clarified in the recommendations.
C. Isotonic Revive
Selected data with dilution ratio 1:25. The 0.1 ml sample shows absorbance of 0.1261
and glucose concentration 22.70 µg/ml. The 0.5 ml sample shows absorbance of 0.5700 and
glucose concentration of 102.60 µg/ml. The data is consistent but not precise. The actual
glucose concentration (best pick) from the samples was 5.68 mg/ml differed greatly from the
manufacturing values which is 68.92 mg/ml. The measurement shows a major error and will
be clarified in the recommendations.

Among the three type of samples, none of them shows a close value to the manufacturing
standard. Therefore, comparison of amount of glucose between the three samples cannot be
determined. DNS method shows an invalid result maybe due to expired materials, therefore
further discussion cannot be resume. Comparison of method cannot be made.