International University, Vietnam National University - HCMC

PRACTICE IN BIOCHEMISTRY

REPORT

EXPERIMENT 3: SOLUBLE CARBOHYDRATE

QUANTIFICATION APPLYING ANTHRONE ASSAY

Date of submission: 25/05/2023

Course: Practice in Biochemistry (Thursday Afternoon)

Instructor: Ms. Nguyen Hong Lan

Group: Campuchia

Group members:

Sq Full name Student ID Contribution Score

1 Hồ Vũ Hoàng Khoa BTBTIU21065 25%

2 Trần Ngọc Hân BTFTUN21021 25%

3 Phạm Đỗ Thùy Linh BTBTIU21217 25%

4 Tống Phước Minh Khang BTBTIU21208 25%

Total score:____/100

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TABLE OF CONTENTS

I. Introduction 3
II. Materials and methods 3
A. Materials and chemicals 3
III. Results and data analysis 6
A. Results 6
B. Data analysis: 10
IV. Discussion: 12
V. Conclusion 15
VI. References 16

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I. Introduction
Fiber, sugars, and starches all fall under the category of carbohydrates, a crucial group of
macronutrients present in many different foods and beverages. Carbohydrates are a crucial part
of our diet and are frequently present in meals and drinks together with proteins and fats. When
analyzing nutritional content and determining dietary consumption, the determination of
carbohydrate concentrations is crucial. In this experiment, we use the anthrone test, a widely
used colorimetric technique to assess the levels of carbohydrates in biological fluids.
Anthrone, a tricyclic aromatic ketone, is used in the anthrone test as a reagent to identify
carbohydrates. Anthrone is created by partially reducing anthraquinone with substances like
sodium hydrogen sulfite or tin chloride. The anthrone assay has been converted to the microtiter
plate format, providing a rapid and practical method of quantifying carbohydrates through
precise mixing, dispensing, and measurement methods.
Carbohydrates are degraded into monosaccharides and dehydrated during the anthrone test while
being exposed to a concentrated sulfuric acid. Furfural is produced as a result of this action, and
it then interacts with anthrone to create a bluish-green complex. At a wavelength of 620–630 nm,
the resultant complex can be identified and colorimetrically measured using a spectrophotometer.
The anthrone test is a useful method for carbohydrate analysis since it can identify hexoses,
aldopentose, and hexuronic acid in both free and polysaccharide forms.
The anthrone assay will be used to measure the amount of carbs in this particular experiment's
banana sample. The banana sample's carbohydrate content is used as an archetype depicting the
procedure of an assay. Along with that, the nutritional makeup of this nutritious fruit is also
valuable, since it is one of the most widely consumed products in the market.

II. Materials and methods

A. Materials and chemicals

Equipment Chemicals

Volumetric flasks (50mL and 100mL) Alcohol 90o

Beakers (50mL and 100mL) Alcohol 80o
Stone mortar Anthrone reagent
Filter paper (11mm) 0.01% Glucose solution
Test tubes
Pipettes (1mL and 10mL)
Spectrophotometer
Condenser
Table 1: List of Equipments and Chemicals
Chemical preparation:
+Anthrone reagent: Prepare a solution of Anthrone in concentrated H2SO4 with a concentration
of 0.002 g/ml. 0.01%
+Glucose solution: Dissolve 0.01 g of glucose (previously dried in a desiccator) in 100 mL of
distilled water to create a 0.01% glucose solution.

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B. Procedure

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Figure 1: The diagram of the procedure.

III. Results and data analysis
A. Results
Step 1 to 6: Preparation of sample
Banana is used in this experiment because it is a rich source of carbohydrates. Because it
dehydrates, Alcohol 90o is employed in this approach to assist get rid of any water that may be in
the sample. This is crucial because water might impede the Anthrone reagent's ability to react
with the carbohydrate, producing unreliable findings. It was then followed by the use of ethanol
80o, which is less harsh than alcohol 90o and less likely to denature or damage the sample's
carbohydrates. This can make sure that the carbohydrates are available for the reaction with the
Anthrone reagent and are in a stable state.

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Figure 2: Final extraction

Step 7 to 9: Dilution

To create the 100-diluted solution, dilute the sample solution 100 times in the volumetric flask
with distilled water. The 10,000-diluted solution is then created by diluting the 100-diluted
solution 100 times with distilled water. These procedures work to lower the carbohydrate
concentration, which aids in keeping the sample below acceptable limits when using a
spectrophotometer.
Step 10 to 11:
Prepared 10 test tubes following the given table to perform a color-forming reaction.

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Step 12:
Put every tube in the ice-water. Each test tube will get an addition of anthrone solution, which
includes concentrated H2SO4 solution; the reaction will produce a significant quantity of heat. It
might be possible to prevent test tubes from shattering while adding Anthrone reagent by first
placing them in ice water.

Figure 3: All the tubes in ice water

Step 13 to 15:
Each tube add 10 mL of Anthrone reagent, which should be added gentlely. This will allow the
reagent to flow into the inside surface of the tube and create a color that indicates the amount of
carbohydrate present. Due to the extreme toxicity of the Anthrone reagent, which contains the
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dangerous acid H2SO4, this step needs to be done in a fume hood. Using a glass stick, carefully
stir the solution. Then, boil all tube for 7.5 minutes in hot water. After that, quickly place all of
the tubes in ice water.

Figure 4: Boiling test tubes in water bath

Figure 5: All the tubes after being put in ice water. Left photo: Tubes 1 to 8 (left to right). Right
photo: tubes 10 and 9

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B. Data analysis:
Tube number 1 2 3 4 5 6 7 8 9 10

1.6979 2.0071 0.4226 0.4239

OD 0.3540 0.7768 0.8622 1.5752 1.4768 1.8323
1.8525 0.4233

ΔOD 0 0.4228 0.5082 1.2212 1.1228 1.4783 1.4985 0.06925

Carbohydrate
X Y
concentration 0 0.02 0.04 0.06 0.08 0.1
(mg/mL)
Table 2: Optical density results

From the spectrophotometer results, data of the 100-diluted pair (7 and 8) and 10,000-diluted
pair (9 and 10) were in the cover range of the standard cuvette (1-6). The final OD value in each
diluted pair was computed by taking the average of replicates (2 in this case). For standard
cuvettes, the OD data are final OD values. The ΔOD values (4 significant figures) of all cuvettes
were calculated by continually subtracting each value with the OD1 (OD of the 1 cuvette). Based
on the standard cuvettes of carbohydrate, the standard curve (linear regression model) was
obtained and depicted below. X and Y represent the unknown concentration in solutions that
were diluted 100 and 10,000 times, respectively.

Figure 6: The standard curve of carbohydrate concentration

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The linear equation was y = 14.578x + 0.0633, with R2 value of 0.9247. Based on this, we
determined the concentration corresponding to values X (ΔOD =1.499) and Y (ΔOD =0.06925).
Solving for X:
1.4985 =14.578x + 0.0633 → X = 0.09845 (mg/mL)

Solving for Y:
0.06925 =14.578x + 0.0633→ Y= 0.000408 (mg/mL)

So the predicted concentration of carbohydrate in 100-fold and 10,000-fold diluted solutions are
0.09845 mg/mL and 0.000408 mg/mL, respectively. Since all values are positive, they can be
evaluated by downstream calculations.
Carbohydrate content using X value:
The solution of X value was diluted 100 times from the stock solution. Hence we use the dilution
factor 100 to predict the carbohydrate concentration within the stock solution (2g banana + 30
mL of alcohol (90° and 80°):

0.09845 (mg/mL) x 10-3 (g/mg) x 102 (dilution factor) = 0.009845 (g/mL)

The stock solution was obtained by extracting 2g banana with 30 mL of alcohol. Thus, the
carbohydrate content within 2g banana is:

0.009845 (𝑔/𝑚𝐿) 𝑥 30 (𝑚𝐿)

2 (𝑔)
(g)

The carbohydrate content in 100 grams of banana:

0.009845 (𝑔/𝑚𝐿) 𝑥 30 (𝑚𝐿) 𝑥 100 (𝑔)

2 (𝑔)
= 14.767 (g)

Carbohydrate content using Y value:

All other calculations are the same with the X value except for the dilution factor of 10,000 or
104. So, the protein concentration within the stock solution (2g banana + 30 mL of alcohol) is:

0.000408 (mg/mL) x 10-3 (g/mg) x 104 (dilution factor) = 0.00408 (g/mL)

Carbohydrate content within 2g of banana, (30 mL of total ethanol solutions is only an

estimation without considering their vaporization):

0.00408 𝑥10 𝑥 30 (𝑚𝐿)

2 (𝑔)

Protein content within 100 g of bananas

0.00408 (𝑔/𝑚𝐿) 𝑥 30 (𝑚𝐿) 𝑥 100 (𝑔)

2 (𝑔)
= 6.12 (g)
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IV. Discussion:
Results discussion:
Our calculation shows that there is a statistically significant variation in concentration of
100-fold and 10,000-fold diluted samples (14.767 versus 6.12). The reasons for this may be
inadequate shaking. The experimenter perhaps did not shake the flask thoroughly so that the
carbohydrates can be evenly distributed in the homogeneous mixture. It should be noted that
Anthrone assay has a bias towards ketose than aldose (Jermyn, 1975) and bananas contain
various types of carbohydrates. Consequently, when we pipette the sample (either in dilution or
in test tube preparation steps), the nonuniform distribution of carbohydrates will strongly affect
the final ΔOD results. So we hypothesize that the 10,000-diluted tubes were unintentionally
pipetted at the low-carbohydrate regions, leading to a diminish in final optical density compared
to 100-diluted tubes. Thus, we chose results from the 100-diluted tubes for further comparison.
Data comparison
In general, our quantification experiment estimated 14.767 g of carbohydrate that underestimates
the actual carbohydrate content in 100 g of bananas. According to a conducted analysis from
Food Data Central of the United States Department of Agriculture (USDA), 100 g of bananas is
expected to contain 22.8 g of carbohydrate on average, which is 8.033 g higher than our results.
We strived to search for another article that uses a similar Anthrone assay to quantify
carbohydrate content in banana pulp, but it yields only 2 potential outcomes. One from Nutrition
Facts DataBase of The Titi Tudorancea Bulletin yields similar results to USDA (22.840 g per
100 g), but is again just actual carbohydrate content on average, perhaps not from Anthrone
method. We found another study from Aquino et al. (2016) that implemented Anthrone assay in
quantification of Musa acuminata (a predominant banana species in South East Asia). But they
do not specify their assay procedure. Unfortunately, we are unable to interpret from their
statistical analysis to obtain raw data, due to limitations in knowledge and time. As a result, our
group has to stick with data from USDA and The Titi Tudorancea. Below we explain the reasons
why our data differs from that of 2 databases.

Table 3 : The average carbohydrate content in 100g of banana (USDA)

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Table 4 : The average carbohydrate content in 100g of banana (Nutrition Facts DataBase of The
Titi Tudorancea Bulletin)

Table 5 : The result table from Aquino et al. (2016)

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In our experiment, the underestimation of carbohydrate content might comes from several
reasons listed by the likelihood from the most impactful to the least: (1) incomplete extraction,
(2) variation in carbohydrate composition and Anthrone bias, (3) Anthronol oxidation, and (4)
calibration curve. First, our group might not extract all of the carbohydrate, which is usual in
every extraction procedure (cannot obtain 100% efficiency). To optimize it for better yield, we
suggest that 1 or 2 more times of ethanol extraction is required. Second, the total carbohydrate
content would vary due to influences of seasonal quality, supply chains, post-harvest handling,
and ripeness. In addition, information about the cultivar of banana or species and its condition is
not provided, causing a barrier to compare data from other articles. Below is a graph
demonstrating the change in composition of bananas. Also, studies have shown that the reaction
of coloration in Anthrone test will be more rigorous towards ketoses (like fructose) compared
with aldoses (like glucose). The combination of those factors leads to a consistent result of
banana samples (like tube 7 and 8) and subsequently alters the final prediction of carbohydrate
content. Third, the anthronol, which is responsible for chromomeric properties of Anthrone
assay, is susceptible to oxidative factors and develops a brownish color over time. The principle
of anthrone assay is that the carbohydrate will be (1) dehydrated and (2) broken down under a
strong protonated system of concentrated sulfuric acid. Under these conditions, the active form
of anthrone - anthronol is formed. The oxidation of anthronol can occur, since our group had to
wait for a long time to use the spectrophotometer. Fourth, our calibration curve is strongly
affected by a failed tube (tube 4), which results in an insufficient equation to predict the data
from banana samples. However, we believe that this is just a minor factor, since the gap between
actual and our estimated data is tremendous.

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Figure 7: The change in carbohydrate composition of banana over time

V. Conclusion
Among a myriad of assays for total carbohydrate quantification, Anthrone-sulfuric assay is the
most effective, suitable for novices, and reliable. If carbohydrate exists in the form of
polysaccharides or disaccharides, the concentrated sulfuric acid provides a protonated system to
hydrolyze them into monomers and dehydrate these monosaccharides to form furfural or
hydroxymethyl furfural. Anthrone reagents then react with these compounds to generate the
blueish-green color, which is quantified at A630nm. Our experiment underestimates the total
carbohydrate content in 100 g of bananas with only 14.767 g. Explanation and suggestions are
provided in the discussion section to obtain higher yield content from a single sample.

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VI. References
[1] Biochemistry Lab Manual. (n.d.). N.H.Lan. Practice in Biochemistry. International
University-VNU
[2] Jermyn, M. A. (1975). Increasing the sensitivity of the anthrone method for carbohydrate. Analytical
Biochemistry, 68(1), 332–335. https://doi.org/10.1016/0003-2697(75)90713-7

[3] FoodData Central. (n.d.).

https://fdc.nal.usda.gov/fdc-app.html#/food-details/173944/nutrients

[4] Bananas, raw, Musa acuminata Colla: Nutrition Facts. Content of Vitamins, Cholesterol, Calcium,
Magnesium, Fatty Acids and so on. (n.d.).
https://www.tititudorancea.com/z/nutrition_facts_musa_acuminata_colla_bananas_raw.htm

[5] Aquino, C. F., Salomão, L. C. C., Ribeiro, S. M. R., De Siqueira, D. L., & Cecon, P. R. (2016).
CARBOHYDRATES, PHENOLIC COMPOUNDS AND ANTIOXIDANT ACTIVITY IN PULP AND PEEL OF 15
BANANA CULTIVARS. Revista Brasileira De Fruticultura, 38(4).
https://doi.org/10.1590/0100-29452016090

[6] Haldar, D., Sen, D., & Gayen, K. (2017). Development of Spectrophotometric Method for the Analysis
of Multi-component Carbohydrate Mixture of Different Moieties. Applied Biochemistry and
Biotechnology, 181(4), 1416–1434. https://doi.org/10.1007/s12010-016-2293-3

[7] Kashyap, A. (2022). Comparative study of total sugar content among selected fruits using
standard protocol. Helix - The Scientific Explorer | Peer Reviewed Bimonthly International Journal,
12(4), 15-18. Retrieved from https://helixscientific.pub/index.php/home/article/view/406
[8] Phillips, K. W., McGinty, R. C., Couture, G., Pehrsson, P. R., McKillop, K., & Fukagawa, N. K. (2021).
Dietary fiber, starch, and sugars in bananas at different stages of ripeness in the retail market. PLOS ONE,
16(7), e0253366. https://doi.org/10.1371/journal.pone.0253366

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