LABORATORY REPORT

COURSE CODE MIC548 - INDUSTRIAL APPLICATIONS OF MICROORGANISM

GROUP AS257 3A (SESSION 2)

EXPERIMENT EXPERIMENT 1 : REDUCING SUGAR ANALYSIS

LECTURER’S NAME DR MUHAMMAD NAZIZ SAAT

STUDENT’S NAME SITI NURMAISARAH BINTI AHMAD SHUKRI (2022607976)

GROUP MEMBER’S 1. NUR ZUR AIN BINTI ROZANI (2022699942)

NAME 2. TIHANI SOFEA BINTI ABDUL LATIF (2022494228)
3. NOORSHAWATI ASIKIN BINTI AHMAD MAHADI (2022830324)
4. NUR FATIN MAISARAH BINTI FAIRUL HISYAM (2022461456)

DATE OF 27 OCTOBER 2023

EXPERIMENT

DATE OF 11 NOVEMBER 2023

SUBMISSION
ABSTRACT

Spectrophotometry is a technique that measures light intensity as a light beam travels

through a sample solution to determine how much a chemical compound absorbs light. The

fundamental idea is that, within a specific wavelength range, every substance either transmits or

absorbs light. Measurements of recognised chemical substances, such glucose, can also be made

using this method. A spectrophotometer is a device that counts photons, or the amount of light

that is absorbed, after the light has passed through a sample solution. By detecting light intensity,

the spectrophotometer can also be used to calculate concentrations, or the amount of a known

chemical substance. This experiment applies Beer Lambert’s Law where it states that there is a

linear relationship between the absorbance and the concentration of a sample. From this law, we

could assume that the higher the glucose concentration, the higher the absorbance and we could

simply obtain a linear graph.

INTRODUCTION

Reducing sugar divided into two groups which are monosaccharides such as glucose,

fructose and disaccharides except sucrose due to lack of free -CHO group. All these reducing

sugars consist of a free aldehyde group (-CHO) or a ketone group that makes it different from

non reducing sugar. All reducing sugar will undergo oxidase reaction and it is known as a

reducing agent towards the reactive reagent and form carboxylic acid (Biology Online. (2023,

August 16).

The DNS (3,5-dinitrosalicylic acid) assay was chosen in this study due to cost-friendly,

its simplicity and flexibility. The reaction is known as the quantifying method and works

depending on the reducing sugars present in the sample and DNS reagent that produce

colorimetric signals as it detects the carbonyl group (C=O) in reducing sugar. This signal was

proportional to the concentration of reducing sugar present. The precision of the graph relies on a

standard calibration curve that indicates the concentration of the sample. So, accurate

measurements by spectrophotometer are an essential part in this study (Biocyclopedia.com, n.d.).

The spectrophotometer works by measuring the color intensities of the samples with the

specific wavelength which is 575 nm. This instrument will strike the light from the lamp to the

sample like a prism and be absorbed by the matter based on the Beer-Lambert law. As a result,

the absorbance is going to be linear in relation to the concentration (How Does a

Spectrophotometer Work?, n.d.)

OBJECTIVES

● To create a standard calibration plot of different glucose concentrations.

MATERIALS

Reagent

● DNS Reagent (3,5-Dinitirosalicylic acid 5.0 g + Sodium hydroxide, NaOH 8.0 g + 100

mL distilled water).

● Rochelle Salt Solution or Sodium Potassium Tartrate Solution, (40.0 g dissolved

separately in 100 mL distilled water)

● Glucose stock (0.1 g dissolve separately in 100 mL distilled water).

Apparatus

● Test tubes

● Test tube rack

● Beaker

● Distilled water

Equipment

● Water bath 90 °C

● Visible spectrophotometer (575nm)

● Cuvette
PROCEDURES

1. 1 g/L of glucose stock was diluted with distilled water to prepare 0.1 g/L of glucose

sample to be used as a sample to measure their concentration. We calculated the volume

of glucose that needed to be mixed into distilled water by using the 𝑀1𝑉1 = 𝑀2𝑉2

formula.

2. 0.1 g/L of glucose samples were prepared by adding 1 mL of glucose solution into 9 mL

of distilled water.

3. Steps 1 to 2 were repeated to prepare 0.2 g/L, 0.3 g/L, until 1 g/L of glucose sample.

4. 1 mL of DNSA reagent was added into each test tube containing 3 mL of each reducing

sugar sample with different concentrations. (sodium sulfate solution already added into

DNSA reagent to obtain a stable color).

5. 1 mL of DNS reagent was added into 3 mL of distilled water as a blank solution.

6. All reaction mixtures in the test tubes were vortex.

7. All the tubes were placed in a boiling water bath for 10 minutes and A red-brown color

developed.

8. After 10 minutes, the test tubes were rinsed using tap water.

9. Each sample was poured into a cuvette to be measured.

10. The absorbance of the tube was measured at 575 nm against a blank.

11. The standard curve was prepared for the determination of reducing sugar concentration of

unknown samples using a glucose stock solution of 1 g/L.

RESULTS

Sample Glucose Distilled Glucose Absorbance (575nm)

concentration water solution
(g/L) (mL) (mL) Trial 1 Trial 2 Trial 3 Average

1 0.1 9 1 0.0870 0.1250 0.1310 0.1143

2 0.2 8 2 0.2220 0.1850 0.1880 0.1983

3 0.3 7 3 0.3000 0.3280 0.3290 0.3190

4 0.4 6 4 0.4450 0.4140 0.4320 0.4300

5 0.5 5 5 0.0700 0.0610 0.0590 0.2463

6 0.6 4 6 0.1590 0.1730 0.1700 0.1673

7 0.7 3 7 0.2460 0.2480 0.2250 0.2396

8 0.8 2 8 0.2830 0.2790 0.2890 0.2836

Table 1: The absorbance (nm) of 8 different glucose concentration

Calculation

Formula used: 𝑀1𝑉1 = 𝑀2𝑉2

Sample 1 Sample 2

𝑀1𝑉1 = 𝑀2𝑉2 𝑀1𝑉1 = 𝑀2𝑉2

(1.0M)𝑉1= (0.1M)(10mL) (1.0M)𝑉1= (0.2M)(10mL)

𝑉1= 1mL 𝑉1= 2mL

10mL - 1mL = 9mL 𝐻2𝑂 10mL - 2mL = 8mL 𝐻2𝑂

Sample 3 Sample 4

𝑀1𝑉1 = 𝑀2𝑉2 𝑀1𝑉1 = 𝑀2𝑉2

(1.0M)𝑉1= (0.3M)(10mL) (1.0M)𝑉1= (0.4M)(10mL)

𝑉1= 3mL 𝑉1= 4mL

10mL - 3mL = 7mL 𝐻2𝑂 10mL - 4mL = 6mL 𝐻2𝑂

Sample 5 Sample 6

𝑀1𝑉1 = 𝑀2𝑉2 𝑀1𝑉1 = 𝑀2𝑉2

(1.0M)𝑉1= (0.5M)(10mL) (1.0M)𝑉1= (0.6M)(10mL)

𝑉1= 5mL 𝑉1= 6mL

10mL - 5mL = 5mL 𝐻2𝑂 10mL - 6mL = 4mL 𝐻2𝑂

Sample 7 Sample 8

𝑀1𝑉1 = 𝑀2𝑉2 𝑀1𝑉1 = 𝑀2𝑉2

(1.0M)𝑉1= (0.7M)(10mL) (1.0M)𝑉1= (0.8M)(10mL)

𝑉1= 7mL 𝑉1= 8mL

10mL - 7mL = 3mL 𝐻2𝑂 10mL - 8mL = 2mL 𝐻2𝑂

 Figure 1 : Linear regression of glucose concentration

Before After

Table 2 : colour changes of the glucose with different concentration before and after water bath
DISCUSSION

This experiment was more focused on constructing a standard calibration curve for

reducing sugar analysis and finding out the sample concentration by using the DNS method. This

method has proven many analyses involving a wide range of sugar including monosaccharides

especially ketoses and aldoses and disaccharides by giving a colorimetric signal which is yellow

color change to orange or red (Whitney, 2011). Since the practical is more flexible, make it a

suitable method to analyze the mixed sugar from a simple fruit juice until the complicated one.

As a result, the additional amount of sugar introduced is equal to the rise in absorbance upon the

color development.

The dinitro salicylic acid (DNSA) is sensitive. (DNSA Reagent Instructions for

Preparation and Use, 2016). DNSA technique works on the idea of detecting free carbonyl

groups (C=O) in reducing sugars. (Deshavath et al., 2020). In this process, the aldehyde and

ketone functional groups in glucose and fructose are oxidised. In this process, the yellow

3,5-DNSA undergoes reduction to 3-amino-5-nitrosalicylic acid (ANSA) in an alkaline solution,

producing a brown-red complex with a wavelength absorbance of 575 nm. (Deshavath et al.,

2020). The deeper the brown colours, the more concentrated the glucose. This is because the

absorbance of a substance shows its ability to absorb light at a specific wavelength it prevents

from passing through. The proportion of absorbed light is determined by the number of

molecules that interact with the light. (Clark, 2023). Concentrated solutions have a deeper brown

colour because more molecules interact with the light penetrating and increase its absorption.

The absorbance of the dilute solution is low, as indicated through the lighter yellow-brown

colour, since there are less molecules in the solution to interact with the light.
 The glucose concentration that was used for this standard calibration plot was within the

range of 0.1 to 1 g/L. To produce multiple absorbances, the glucose stock solution (1 g/L) was

diluted in three different batches. After the reading obtained, the average of absorbance is

calculated. With the average of absorbances, the graph was plotted and the mean and standard

deviation were calculated. Based on the graph plotted, the slope, m is calculated by using the

formula of 𝑦 = 𝑚𝑥 + 𝑐 and the slope obtained is 1.075 (based on 0.4 g/L glucose

2
concentration). The coefficient of determination value (𝑅 ) was 0.8017. However, the mean

absorbance of 0.5 g/L was low and caused the graph obtained not in linear relationship. It was

due to inaccuracy during dilution and the reading of absorbances. Therefore, the graph obtained

should be in linear form as the higher the glucose concentration (g/L), the higher the mean of

absorbance (nm) at the wavelength of 575nm.

In this experiment, we use sodium tartrate as a constituent to prepare DNS to increase

stability of DNS reagent. We also stored DNS in a Florence flask covered with aluminium foil to

prevent the DNS from degrading which makes it less effective. The DNS also changes its colour

depending on the presence of reducing sugar. The structure of DNS is altered as it oxidises to

3-amino-5-nitrosalicylic acid, changing the colour with it. When DNS reacts with glucose, its

initial yellow-orange colour changes to a reddish-brown colour.

In practice, there are other sources of error, such as environmental effects on the

photometer and sample, temperature, vibrations, contamination, or heating of the sample. All

these factors may impair the measured result and ways. There are some limitations and errors

that can happen during the reducing sugar analysis using the DNSA method. During the heating

step, all the samples need to be heated at 95–100 °C for 10 min to ensure full coloration.
 Overheating the sample not only limits the overall speed of the assay but potentially

causes inaccuracies by evaporation and interrupts the result (Wood et al., 2012). To minimize the

evaporation process as much as possible, the sample needs to be covered with a plastic cover.

Another limitation of this experiment is the DNS assay has a relatively low specificity, which

means that one must run blanks diligently if the calorimetric result is to be interpreted correctly

and accurately. The DNS assay also cannot be applied in measuring pectinase activity against

pectins because the DNSA reagent is destructive for polysaccharides. The DNS assay is also

associated with over-estimations (Answer in Biochemistry for MONAAMBIGHAI #95812, n.d.).

Lastly, an error can happen if there is the presence of other active carbonyl groups. It can

potentially also react with DNS leading to incorrect yields of reducing sugars. The current study

reveals the limitation involved with the DNS assay, in which 3,5-dinitrosalicylic acid is seen to

react with the unwanted by-products along with the reducing sugars. This leads to incorrect

estimation of sugars due to a parallel reaction going on inside the reaction medium (Deshavath et

al., 2020).
CONCLUSION

In conclusion, we were able to plot the standard curve of different glucose concentration

based on the result that we obtained. However, the standard curve that we obtained was

inaccurate as it was not linear. Hence, we identified various errors that could have occurred

during the conducted experiment, leading to the non-linearity of the standard curve. Besides that,

several precautions should be taken when conducting this experiment in order to obtain an

accurate result which include correct personal skills and technique such as precision in pipetting

the reagent, contamination control by using clean glass tubes and cuvette and calibration using a

blank. Other than that, We also should maintain a consistent temperature during the experiment

since variations of temperature can affect reaction rates which result in inaccurate results.

Furthermore, the standard curve of known different glucose concentration is important due to the

fact that it allows the conversion of the sample absorbance reading to the corresponding

concentration of reducing sugar. Therefore, It would be difficult to accurately determine the

concentration of reducing sugars in an unknown sample without a standard curve.

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