practice in biochemistry report soluble carbohydrate quantification applying anthrone assay

INTRODUCTION 1.1 Objectives - To know how to apply Anthrone assay for quantification of carbohydrate in a source, particularly, banana.. In this assay, anthrone is immediately dissolved

PRACTICE IN BIOCHEMISTRY REPORT

Experiment 3

SOLUBLE CARBOHYDRATE QUANTIFICATION APPLYING

ANTHRONE ASSAY Group 1

CONTENTS

1 INTRODUCTION

1.1 Objectives

- To know how to apply Anthrone assay for quantification of carbohydrate in a source, particularly, banana

- To practice handling hazardous chemicals in the fume hood

- To know how to use Microsoft Excel’s standard curve to analyze data

1.2 General principles

Hexoses, aldopentose, and hexuronic acid may all be measured quickly and easily using the Anthrone assay, whether they are free-from or in the form of polysaccharides In this assay, anthrone is immediately dissolved at a 2% concentration in concentrated sulfuric acid without the need for distilled water to be added to the reagent preparation Furfural is created when the monosaccharides are dehydrated with the help of the concentrated acid A

spectrophotometer can measure the blue-green complex that is created when the furfural condenses with two molecules of naphthol from the Anthrone reagent at a wavelength of 620-630 nm

O

Sugar + conc H,SO, —m— [ 0 I HO+ C —— Blue-green

(Anthrone)

2 MATERIALS AND METHOD

2.1, Materials

¢ Volumetric flasks (50mL and 100 mL) ® Alcohol 900

¢ Beakers (SOmL and 100mL) ¢ Alcohol 800

¢ Filter paper (11mm)

¢ Test tubes

¢ Pipettes (ImL and 10 mL)

2.2, Procedure and Method

2.2.1, Preparation of sample

Step 1: Take 5g of banana sample into the beaker

Step 2: Add 10mL of 90% ethanol into the beaker

Step 3: Put the beaker in the water bath at 80° C Then, using the stirring rod to stir the solution well during the heating process

Step 4 Filter the extract into an E flask Return the grains of the banana back to the beaker and get the solutes in alcohol

Step 5 Add 10mL of 80% ethanol into the beaker of grain and do the same at step 3 and 4

Do step 5 twice to get the total of 30mL final volume of banana extract

Step 6: Put 30mL of banana extract into the falcon and put it into the centrifuge

Step 7: After centrifugation, dissolve 1mL banana extracted solution in 99mL distilled water

to make 100-diluted solution, put 5mL of the solution into test tube 8 and 9

Step 8: Dissolve mL of a 100-diluted solution in 99mL distilled water to make a

10 000-diluted solution, put 5mL of the solution into test tube 10 and 11

2.2.2 Color-forming reaction

(100)(100)| (10,000)| (10,000)

(mL)

Distllwater(mL) |5 |5 |4 |3 |2 | 1 4] 0

Anthrone (ml) 10[ 10 10 | 10 |10 | 10 |10 x Vụ

Step 1: Put all tubes in the ice-water

Step 2: Put slowly 10 mL of Anthrone reagent into each tube Let the reagent flow

along the inside-surface of the tube

Step 3: Stir the solution very slowly with a glass stick Then boil all tubes in hot

water from 7 to 10 minutes After that, put all the tubes in cool water immediately

Step 4: Let all test tubes cool down and measure the Aeon

3 RESULT AND DISCUSSION

3.1 Cell number determination

JH a

Wt ache

l

After adding 0.01% Glucose and Distilled water

Before adding Anthrone reagent

After adding Anthrone reagent and boiling in 10 minutes (test tube 1->11) before measuring colormetrically at the wavelength of 630nm by a spectrophotometer

number

OD at 0.1919 |0.1990 10.4694 |0.7223 10.9348 |1.2287 |1.4916 10.2219 10.2629 0.2188 0.2190

As30nm

AOD at 0 0.2739 |0.5268 |9.7393 11.0332 |1.2961 | 0.0469 0.0234

As30nm

Carbohydrate 0 0 0.1 | 02 | 03 | 04 | 05 x Y

concentration

(ug/mL)

3.2 Data analysis

Carbohydrate

(ug/mL)

Corretlation between the Carbohydrate concentration versus AOD

we

1.2 FO) = 2.56 Ot

1

074v

a 08Ƒ————T—————T————— ‘

3 06 053 vàn

we ° `

wl `

0

oe"

Carbohydrate concentration (ug/mL)

Figure Standard curve

3.3 Calculation

From the change of the absorbance based on the carbohydrate concentration, the equation y

=2.5631x + 0.0041 with R? = 0.9986 is established, in which x represents for

carbohydrate concentration and y represents for the change of the absorbance, the unknown

carbohydrate concentration, therefore, can be determined

- The carbohydrate concentration of the stock solution (test tubes 10 and 11) is:

- The amount of carbohydrates in 5g banana is:

- The amount of carbohydrates in 100g banana is:

10

4, DISCUSSION

Based on the standard curve, it shows that R? = 0 converts into R = 0.8143, which means R is in the range of 0.5 to 0.8 (0.5 <R < 0.8); thus, the moderate correlation between

absorbance and concentration could not be seen

The protein concentration in 100 g of soybean is approximately 35.92 g based on our result that calculated, which means the protein concentration takes 35.92% of the total mass of soybean used in this practice

These tubes contain glucose (tube 3 to tube 7) have blue color because it is the commercialized glucose solution

On the other hand, According to the data given from the U.S Department of Agriculture, Food Data Central (2019), the protein quantification in 100 g of soybean is approximately 36.49 g of protein Our group result is 35.92 g which is 0.57 g lower than that data Hence, it

is simply to be seen that the data of our group is slightly high compared with the literature value Therefore, even though it is not different greater than the data from USDA, the result could be still acceptable

11

Bananas, raw

Name Amount Unit

Protein 1.09 &

Ash 0.82 g

Carbohydrate, by difference 22.84 =

Fiber, total dietary 2.6 g |

Sugars, total including NLEA 12.23 ø |

Source: Data from U.S Department of Agriculture (2019)

¢ Several errors could have occurred in the the practices:

+ The protein extraction from soybean could be performed poorly

+ Improper use of pipettes could result in failure to apply the correct volume of reagents + The instruments could have been improperly washed before use, resulting in reagent contamination

+ Failure to combine the right ratios of solution A and B could result in an incorrect solution C

+ Since the Folin — Ciocalteu reagent degrades rapidly when exposed to light, the content of solutions can deteriorate when waiting to use the spectrophotometer, resulting in an inaccurate OD reading

+ The amount of reagents or chemicals were added incorrectly by pipettes

+ The stirring part was not done carefully

+ The equipments were not cleaned carefully, which led to the contamination of reagents

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+ The chemicals are not totally pure

+ The test tubes after adding Anthrone reagent were not well- stirred which result in Anthrone cannot react fully with carbohydrate

¢ Solutions to improve the result:

- Read pipettes more carefully before pouring reagents or chemicals into the test tubes

- Stir the solution gently to make sure all the raw materials become white in the end

- Use washing room to clean the test tubes, to make sure all the contaminations in test tubes were removed Use distilled water to clean the pipettes

- Stir the solution carefully and gently

- Prepare the chemicals carefully so they are pure

5 CONCLUSION

In this experiment, we determine the amount of protein content in soybean samples by using the Hartree-Lowry assay, analyze data, and create a standard curve by using Microsoft Excel However, the results of this experiment differ minimally from the standard data because of a few mistakes that we made during our laboratory work The significant differences can come from some main causes such as inaccurately using the pipette, and incorrectly preserving the sample after adding Folin-Ciocalteu

By means of this practice, we have learned how to perform Hartree — Lowry assay, how the can mechanism works Also, We know that Folin — Ciocalteu reagent is quickly degraded under contact with light, thus, we have to be more careful in performing reactions involve to

Folin — Ciocalteu

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6 REFERENCES

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1 School of Biotechnology (2022), Biochemistry lab manual, HCM International

University, Vietnam National University

U.S Department of Agriculture, Agricultural Research Service, Food Data Central,

Soybeans, mature seeds, raw Published January 1 2019, SR Legacy, 174270, No

16108

Arti Nigam and Archana Ayyagari, Lab Manual in Biochemistry: Immunology and Biotechnology, page 33

David T; Plummer (1990) An Introduction to Practical Biochemistry,179 Third Edition

School of Biotechnology (2020), Biochemistry lab manual, Ho Chi Minh, International University, Vietnam National University