INVESTIGATORY PROJECT

ON
TEST TO FIND THE PRESENCE OF VITAMIN C IN
GUAVA FRUIT

SUBMITTED TO
CENTRAL BOARD OF SECONDARY EDUCATION
NEW DELHI

ACADEMIC YEAR 2020-2021

BY

UNDER THE GUIDANCE OF

Msr.ARIFA SULTANA
(BIOLOGY FACULTY)

DEPARTMENT OF BIOLOGY
SENTHIL PUBLIC SCHOOL
(Affiliated to CBSE, New Delhi, Affiliation No:1930323)
Adhiyamankottai,Dharmapuri-636807

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ACKNOWLEDGEMENT

I am here overwhelmed in all humbleness and gratefulness to acknowledge

my depth to all those who have helped me to put these ideas well above the
level of simplicity.

I would like to express my special note of gratitude to my teacher

Mrs.Arifa sultana for spending many precious hours in helping me for
the quality of the this, kindly providing valuable guidance, motivation and
his constant encouragement.

I would like to thank our senior principal Mr. C.Srinivasan and our
principal Mr.senthil murugan and our vice principal Mr.Raj Kumar and our
academic co-ordinator Mr. N.Soundar Rajan who gave me this golden
opportunity to do this wonderful project. It also helped me in doing lot of
research and I came to know about many new things.

I am really thankful to them. Any attempt at any level can’t be satisfactorily

completed without the support and guidance of my parents my friends and
my lab in charge Mrs.sumathe saravanan

I would like to thank my parents who helped me a lot in gathering

information, collecting data and guiding me from time to time in making
this project, despite of their busy schedule. They gave me different ideas
in making this project unique.

NAME:
GiriDharan.M

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Validation and Vitamin C Testing in Crystal Guava (Psidium guajava L.)

With Variations of
Origin With the HPLC
Method (High
Performance Liquid Chromatography)

Abstract
Crystal guava contains high vitamin C. Vitamin C is contained in different fruits, one of the factors is the altitude of the
fruit plant growing areas. This study aims to determine the level of vitamin C in the slurry of crystal guava flesh with
variations of origin using the HPLC method. Samples of crystal guava are obtained from the city of Bogor, Malang, and
Gunung Kidul. The samples to be analyzed are prepared as a slurry. Qualitative analysis is done by identification using
KMnO4 p.a, FeCl3 p.a, AgNO3 p.a as well as comparing the retention time of the sample with vitamin C. Method validation
for system suitability, linearity and precision provide results that are eligible according to the applicable regulations.
Quantitative analysis using HPLC with C 18 stationary phase, water: methanol mobile phase (95: 5) v/v, flow rate of 1
mL/min, and run time of 7.5 minutes. Qualitative analysis by using KMnO 4, FeCl3, AgNO3, and retention time (tR) shows
positive results of the vitamin C existence. The average levels of vitamin C from Bogor, Malang, and Gunung Kidul, equal
to (0.4139 ± 0.004) mg/mL; (0.6746 ± 0.03) mg/mL; and 0.8608 ± 0.002 mg/mL respectively. Keywords: guava crystal,
Gunungkidul, HPLC, origin, vitamin C
1. Introduction
People become aware of health and starting back to nature lifestyle. One attempt to maintain health is by eating fresh
fruits. This has an impact on increasing the consumption of fruit in society. Indonesia is known as a country that has a
variety of fruits especially tropical fruits. An example of fruit which is often found in the tropics is the guava fruit. Guava
fruit has the scientific name of Psidium guajava. There are many variaties of guava fruit and one of them is the crystal
guava. Crystal guava is a mutation of Bangkok guava found in 1991 in Taiwan. Crystal guava has a crunchy texture, sweet
taste, and few seeds that become the society’s favorite as a fresh-eaten fruit (Hadiati and Apriyanti, 2015).
Crystal guava contains vitamins (A, B1, and C), minerals, carbohydrates, water, proteins, lipids, and fiber (Lubis et al.,
2017). Crystal guava can be used to increase antioxidants. Antioxidants are compounds that play an important role in
protection from free radicals that come from the metabolic processes in the body or that enters the body from outside. One
of the antioxidants contained in crystal guava fruit is vitamin C (Andarwulan et al., 2012).
Vitamin C is the most simple vitamin, easy to change due to oxidation, but very useful for humans. Vitamin C is easily
damaged when it’s in solution form due to oxidation by oxygen. Factors that can accelerate the oxidation are high
temperature (Paul & Ghosh, 2012; El-Ishaq & Ebirinakem, 2015), acidity (Hacisevki, 20009), and storage time (Steskova
et. al., 2006; Cvetkovic & Jokanovic, 2009; Zhang et. al., 2016). Vitamin C found in fruits may be influenced by the type
of fruit (Tareen et. al., 2015), site conditions (Abanto-Rodriguez et. al., 2016), climatic conditions before harvest time
(Kaleem et. al., 2016), fruit maturity level (Lee & Kader, 2000).
The amount of vitamin C content using High Performance Liquid Chromatography (HPLC). HPLC is the fastest
developing analytical method. A good method needs a method validation test. Validation test includes system suitability,
linearity, precision. This study aims to test the validity of the HPLC method and the determination of vitamin C levels
based on the difference in origin of growing regions. The origin areas of guava crystals used are the city of Bogor, Malang
and Gunung Kidul.

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2.Research Methods
Ingredients: crystal guava (Psidium guajava L.) varieties, standard vitamin C with 99% purity (E-Merck), aquabidest of
HPLC grade (Ika Farma), methanol of HPLC grade (E-Merck), KMnO4 pro analysis (E-Merck) , FeCl3 pro analysis.
(EMerck), AgNO3 pro analysis (E-Merck).
Equipments: glassware, a set of HPLC (LC-20AT with SPD-20A SHIMADZU detector), analytical scale (Ohaus of
PA214 type), centrifuged (Eickmeyer), sonicator (Elmasonic), juicer (Sapporo), micropipette (Socorex), blue tip, 1 mL
syringe, millipore PVDF of 0.22 μm filter (Sartoriusstedim), filter paper no. 1 and no. 42 (Whatman). Study course:
Slurry making: Crystal guava is washed with running water and drained. Crystal guava is separated between the skin,
pulp, and seeds, then the flesh of fruit is smoothed using juicer (Safqatullah, et al, 2013).
Qualitative Analysis: performed with FeCl3 reagent, KMnO4 reagent, AgNO3 reagent, sample retention time.
Validation Methods include: System suitability test, Linearity, and Precision (Alvi and Hammami, 2011).
Quantitative analysis include:
Sample Preparation: Crystal guava of each region are taken in 5 pieces for analysis. The flesh of fruit that has been
cleaned then is smoothed (slurry) using a juicer. Once the fruit is made into a slurry then weighed 10 grams of it carefully,
then put it in a 10 mL flask and add aquabidest up to the mark. Next, it’s centrifuged at a speed of 5000 rpm for 5 minutes.
The obtained supernatant is then filtered using a filter paper (Whatman no. 42) (Teepoo et al., 2012).
Determination of vitamin C: Clear samples are drawn as many as 100 uL then put in a 10 mL flask, aquabidest is then
added up to the mark. Millipore paper of 0.22 μm filter is used to filter the samples. Samples of 20 mL are injected into
the HPLC system (Kumar, et al., 2011).
Data Analysis: Levels of vitamin C in each sample of guava crystals of Margajaya (Bogor), Junrejo (Malang), and Nglipar
(Gunung Kidul) is calculated by entering the AUC (Area Under Curva) values of vitamin C in the sample as a function of
"y" of the regression line of y = bx + a, where x is the standard concentration on making the standard curve and y is the
absorbance of the standard solution of vitamin C.
2. Results and Discussion Qualitative analysis
Results of the qualitative analysis with FeCl3 reagent, KMnO4 reagent, and AgNO3 reagent shows positive results
containing vitamin C for all the crystal guavas of Bogor, Malang and Gunung Kidul. Test with Ferric chloride reagent
(FeCl3) is one of the compounds that can be reduced. In this reaction, vitamin C serves as a reductant. Ferric chloride is
reduced by vitamin C so that the yellow color of the ferric ions (Fe 3+) turn into colorless (Fe2+). Vitamin C is oxidized by
ferric ions (Fe3+) into dehydroascorbic acid (Erina, et al. 2015).
The qualitative test results of slurry with FeCl3 reagent formed a yellow color that quickly disappears. It can be concluded
that the slurry of crystal guavas contain vitamin C (Shishir et. al., 2014).
Potassium permanganate will oxidize vitamin C, the reaction results will lead to the purple color of the permanganate ion
(MnO4-) to colorless (Mn2+). Meanwhile, vitamin C is oxidized by permanganate ion into dehydroascorbic acid (Azmat et
al., 2012). The reaction with KMnO4 solution can be seen in Figure 1.

Figure 1. Vitamin C reaction with KmnO4 (Svehla, 1985)

AgNO3 is a metal compound that can be reduced. In this reaction, vitamin C is as a substance which undergoes oxidation.
AgNO3 is reduced by vitamin C so that the silver ions (Ag +) will lose the charge into Ag that can form a black color
precipitate. Vitamin C which is oxidized by silver ions (Ag +) into dehydroascorbic acid (Chairam, et al. 2011). The
reaction can be observed in Figure 2.

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Figure 2. Vitamin C reaction with AgNO3 (Svehla, 1985)

The qualitative test results on the slurry with silver nitrate forms a black ash color solution which settles as time goes
on.This happens because the oxidation reaction between vitamin C which is a strong reducing agent with Ag + is a metal
so that reduction occurs quickly (Songsasen and Poowanathai, 2002). The potential of silver ions (Ag +) reduced by the
samples are indicated by the change of color into black ash. Table I presents the examples of qualitative reactions of
crystal guavas of Bogor.
Table I. Examples of Vitamin C test results with FeCl3, KMnO4, dan AgNO3 on crystal guavas of Bogor
Figure

Sample Result
Before After

FeCl3

Standard

Bogor
KMnO4 reagent

Standard

Bogor

AgNO3 reagent

Standard

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Figure 3. Chromatogram a) Standard Vitamin C b) Sampels from Bogor c) Sampels from Malang d) Samples from
Gunung Kidul with FG metanol:aquabidest (5:95) v/v, FD C18, λ 265 nm, run time of 7,5 minutes, flow rate of 1
mL/minute.
Based on Figure 3, the obtained average value of tR on a standard vitamin C is 2.621 minutes, an average tR value of fruit
samples coming from Bogor, Malang, Gunung Kidul are 2.622 minutes, 2.673 minutes, and 2.578 minutes respectively.
Based on these data, the value tR owned by the standard vitamin C and the samples are relatively the same. It can be
concluded that the sample of slurry contains vitamin C. HPLC Method Validation System Suitability Test
System suitability test is conducted to determine the start of the used tools, methods, and HPLC systems can give good
results in the analysis process or not. System suitability test results are shown in Table II. Table II. System
Suitability Parameters

1 2.627 0.980 2134.441

2 2.668 1499691 1.031 2240.654

Average of CV 2.590 0.936 3 2.596
(%) 1.94 1.523 0.644 5.74 1533179
References of CV 5 5 15 15 0.940
1491676 2016.839
4 2.563
1467415 0.903 2010.040
5 2.543 1502495 0.885 1942.284

6
1495589 2.543 0.877

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AUC (Area Under Curve)
Based on the table, it can be known that the factors of analytes separation with the HPLC method are the retention time,
AUC, tailing factor, and theoritical plate (N). The obtained results from several parameters indicate that the CV value of
the retention time is 1.94% < 2% and for the CV value of the AUC is 1.523% < 5%. The average value for the tailing
factor is 0.644 < 15% and the value of N is 5.7% <15% (Ganjar and Rohman, 2007). From these results, it can be concluded
that the HPLC method has good system suitability. Linearity
Linearity is parameters of analytical method validation used to obtain the proportional results to the analyte concentration.
Results of the linearity test can be seen in Table III.

Table III. Relation of the standard concentration of vitamin C solution with AUC
Rate AUC
No. (μg/mL) (mV) Linear Regression R count R theory

1 1 201617

2 3 289877
3 5 571615
y = 106053.1x + 431039 0.9911 0.9911
4 7 793137

5 9 1010572

AUC (Area Under Curve)

A correlation coefficient or the R value indicates that the linearity level of the relation between vitamin C levels with
AUC area. From the table, the linear regression equation of y = 106053.1x + 431039 with R values is calculated at 0.9911.
R value is a correlation coefficient that indicates the linearity level of relation between the analyte concentration and the
peak area (Sugihartini et al, 2014). The obtained R value is at 0.9911, whereas according to AOAC (2013), the suggested
correlation coefficient is ≥ 0.99. That result fulfills linearity requirements and demonstrates that the test with the HPLC
method has good linearity, therefore the linear regression equation can be used to determine levels of vitamin C in the
samples (Alvi and Hammami 2011).
Precision
Precision is a value that indicates the closeness of the analysis results that can be accepted. Precision is expressed as
standard deviation or coefficient of variation. Careful analysis methods will provide fixed measurement results at any time
from the same sample. The precision results are presented in Table IV.
Table IV. Standard Vitamin C Precision Test Data
Rate (μg/mL) AUC (mV) Parameter

200151 Mean : 197220.3

1 196674 SD : 2699.29
194836 CV : 1.37%
551378 Mean : 544876.7
5 555964 SD : 15403.85
527288 CV : 2.83%

1011207 Mean : 10000526

995184 SD : 9250.31
9
995186 CV : 0.93%

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Based on the calculations in Table IV, CV values obtained at levels of 1 mg/mL of 1.37%; levels of 5 mg/mL of 2.83%; and
levels of 9 mg/mL of 0.93%. The method is stated accurately if the results of CV is <5%. These results indicate that this
method qualifies precision requirements (Anonymous, 2001). Quantitative Analysis
This method is chosen because it has high selectivity and sensitivity, and faster process. (Wardani, 2012). The linear
regression equation obtained y = 106053.1x + 431039 with the value of R 0.9911. The results of the data analysis, the
levels of vitamin C contained in the crystal guavas coming from Bogor amounted to 0.4139 mg/mL; from Malang
amounted to 0.6746 mg/mL; and from South Mountain amounted to 0.8608 mg/mL. Results of the calculation levels of
vitamin C are shown in Table V.

Table V. Calculation Levels of Vitamin C

AUC Rate X±LE CV
Sample
(mV) (mg/mL) (mg/g) (%)
BG1 481469 0.4133
BG2 477000 0.4091
BG3 474265 0.4065 0.4139±0.004 1.52%
BG4 489142 0.4205
BG5 488552 0.4200
ML1 774336 0.6894
ML2 779825 0.6946
ML3 763558 0.6793 0.6746±0.03 5.38%
ML4 784125 0.6987
ML5 744216 0.6109
GK1 954859 0.8597
GK2 951303 0.8563
GK3 956909 0.8616 0.8608±0.002 0.37%
GK4 960754 0.8616
GK5 960712 0.8652
*BG (Bogor); ML (Malang); GK (Gunung Kidul), LE=Limit of Error, CV= Correlation Coefficient
The highest levels of vitamin C is present in crystal guavas of Gunung Kidul. The lowest levels of vitamin C is present in
crystal guavas of Bogor. This can happen because there are differences in the altitude of the crystal guavas planting areas.
The collection sites of crystals guavas in Bogor has the altitude of ± 300m above sea level, the Malang area of ± 700m
above sea level, and Gunung Kidul area of ± 886m above sea level. Therefore, the higher the altitude of the crystal guavas
location planting, the higher the levels of vitamin C in crystal guavas. These results are in line with the research conducted
by Fatchurrozak et al., (2013) which showed that fruit that grows in areas with an altitude of 1400 ± 50 meters above sea
level has vitamin C content of 11.94%, an altitude of 1900 ± 50 meters above sea level is of 13.41%, and an altitude of
2400 ± 50 meters above sea level is of 14.27%. This is due to the higher altitude, the higher the environmental stress, for
example, the lower the temperature, the higher the humidity, the less light intensity, which means the lighting duration is
shorter. When plants are under stress, then the production of vitamin C increased. This is an attempt of plants to combat
environmental stress (Fatchurrozak et al., 2013; Chandra and Sharma, 2013). Figure 4 presented the histogram acquisition
of vitamin C.

0.8608

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0.6746

0.4139

Bogor Malang Gunung Kidul

Figure 4. Vitamin C content (mG/G ) of Bogor, Malang, and Gunung Kidul

3. Conclusion
The results show that the crystal guavas of Bogor, Malang, and Gunung Kidul contain vitamin C. Method validation with
system suitability test, linearity, and precision are in accordance with the provisions. Levels of vitamin C in crystal
varieties of guava fruit from Margajaya (Bogor City), Bumiaji (Malang), and Nglipar (Gunung Kidul) are 0.4139 mg/mL;
0.6746 mg/mL; and 0.8608 mg/mL respectively.
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INFORMANT ABOUT GUAVA FRUIT:

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REASON TO EAT GUAVA FRUIT:

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HISTORY OF GUAVA FRUIT:

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GUAVA-NUTRITIONAL FACTS PER 100 g:

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