Oxalate Ions in Guava Fruit

RAHUL
INTERNATIONAL
SCHOOL
CHEMISTRY PROJECT
Study of Oxalate Ions in

Guava Fruit at Different
Stages of Ripening
Created by: IRFAN HUSSAIN ASAMDI

Introduction:
The study of oxalate ions in guava fruit at different stages

of ripening is crucial for understanding the changes in
nutritional composition during the maturation process.
Oxalate ions play a significant role in human health, and
their levels can vary in fruits based on factors such as
ripening stages. This investigation aims to analyze the
oxalate content in guava fruits at various ripening stages
to gain insights into the nutritional dynamics of this
popular fruit.
Apparatus:
1. Spectrophotometer
2. Weighing balance
3. Pipettes
4. Test tubes
5. Centrifuge
6. Reagents: Sulfuric acid, potassium permanganate,

oxalic acid, distilled water
Theory:
The method involves the oxidation of oxalate ions by

potassium permanganate in an acidic medium. The
resulting manganese ions are then complexed with oxalic
acid, forming a stable color. The intensity of this color is
directly proportional to the oxalate ion concentration and
can be measured spectrophotometrically.
Experimental Procedure:
1. Preparation of Guava Extract:

- Extract guava juice from fruits at different ripening
stages.
- Centrifuge the extract to obtain a clear supernatant.
2. Oxidation Reaction:
- Mix guava extract with sulfuric acid to acidify.
- Titrate with a standardized solution of potassium

permanganate until a faint pink color persists.
3. Color Development:
- Add oxalic acid to complex the manganese ions.
- Allow the solution to stand for a specified time for
color development.
Experiment 1 - Determination of Oxalate

Content:
Sample Collection:
Obtain guava fruits at unripe (green), semi-ripe (yellow),

and fully ripe (orange) stages.
Guava Extract Preparation:
- Peel and pulp guava fruits separately for each
ripening stage.
- Extract juice using a blender and centrifuge for a
clear supernatant.
Oxidation Reaction:
- Mix 5 mL of guava extract with 5 mL of 2 M
sulfuric acid.
- Titrate with 0.02 M potassium permanganate until a
faint pink color persists.
Color Development:
- Add 1 mL of 0.1 M oxalic acid to the titrated
solution.
- Allow the solution to stand for 10 minutes for
color development.
Measurement:
- Measure the absorbance of each solution at a
wavelength of 525 nm using a spectrophotometer.
- Record the volume of potassium permanganate used
for each ripening stage.
Calculation:
Calculate the oxalate concentration for each ripening
stage using the formula:
Procedure:
Oxalate concentration (mg/L)=
Volume of KMnO4 used (mL)×Concentration of
KMnO4 (M)×Molar mass of oxalate (g/mol)
Volume of guava extract (mL)×Volume factor
Experiment 2 - Calibration Curve:
Procedure:
1. Prepare Standard Solutions:

- Prepare a series of standard solutions with known
oxalate concentrations. In this case, we already have the
concentrations from Experiment 1.
2. Perform Oxidation and Color Development:

- Follow the same oxidation and color development steps
as described in Experiment 1 for each standard solution.
3. Spectrophotometric Measurement:
- Measure the absorbance of each standard solution at a
wavelength of 525 nm using a spectrophotometer.
4. Plot Calibration Curve:

- Plot a graph with Oxalate Concentration (mg/L) on the x-
axis and Absorbance at 525 nm on the y-axis.
- Use the measured absorbance values for the standards
to create the calibration curve.
Calculation:
Given that the calibration curve equation is
Oxalate Concentration (mg/L)=2.0×Volume of

KMnO4 Used (mL)
You can use this equation to convert the volume of

KMnO4 used in your standard solutions to oxalate
concentrations.
Experiment 3 - Verification:
To validate the results obtained from Experiment 1

(spectrophotometric method) using an alternative method, in
this case, High-Performance Liquid Chromatography
(HPLC).
Procedure:
1. Alternative Method:
- Utilize an HPLC system equipped with a suitable
column for the separation of oxalate ions.
- Set up the HPLC conditions, including mobile phase

composition, flow rate, and detection wavelength.
2. Sample Analysis:
- Take a subset of guava samples from Experiment 1,
including those at different ripening stages (Unripe, Semi-
Ripe, Fully Ripe).
- Prepare guava extracts using a suitable solvent for
HPLC analysis.
3. Instrument Calibration:
- Calibrate the HPLC system using standard solutions
with known oxalate concentrations. This establishes a
relationship between peak area and oxalate concentration.
4. HPLC Analysis:
- Inject the guava extract samples into the HPLC system.
- Record the chromatograms and identify the peaks

corresponding to oxalate ions.
5. Quantification:
- Quantify the oxalate concentration in each guava sample
by comparing the peak areas to the calibration curve
obtained during instrument calibration.
Calculation:
The calculation involves using the calibration curve

obtained from the HPLC standard solutions to determine
the oxalate concentration in the guava samples. The
general equation of the calibration curve is:
Oxalate Concentration (mg/L)=m×Peak Area+b
Here, 'm' is the slope, and 'b' is the y-intercept of the

calibration curve.
Compare the HPLC results with the spectrophotometric

results obtained in Experiment 1. Calculate the correlation
coefficient to assess the agreement between the two
methods.
Note: The values for 'm' and 'b' in the HPLC calibration
curve equation will be obtained during the calibration
process using standard solutions with known oxalate
concentrations. The correlation between the
spectrophotometric and HPLC results will provide
insights into the reliability of the spectrophotometric
method for oxalate determination in guava fruits.
Results:
Volume of Oxalate
Ripenin
KMnO4 Concentratio
g Used n
Stage (mL) (mg/L)
Unripe
2.1 4.2
(Green)
Semi-
Ripe 1.5 3.0
(Yellow)
Fully Ripe
0.8 1.6
(Orange)
Record the observations form the result of Experiment no
1 and plot the calibration curve using the formula
Volume of KMnO4 used (mL) × Concentration of

KMnO4 (M) × Molar mass of oxalate (g/mol)
Volume of guava extract (mL)×Volume factor
And produce a calibration curve for the HPLC system using

the formula
Oxalate Concentration (mg/L) = m × Peak Area + b
Precautions:
- Standardize all reagents before use.
- Ensure accurate pipetting for precise results.
- Handle chemicals with care, especially sulfuric acid
and potassium permanganate.
- Maintain uniform experimental conditions for all samples.

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RAHUL

Study of Oxalate Ions in

Created by: IRFAN HUSSAIN ASAMDI

The study of oxalate ions in guava fruit at different stages

6. Reagents: Sulfuric acid, potassium permanganate,

The method involves the oxidation of oxalate ions by

1. Preparation of Guava Extract:

- Centrifuge the extract to obtain a clear supernatant.

- Titrate with a standardized solution of potassium

Experiment 1 - Determination of Oxalate

Obtain guava fruits at unripe (green), semi-ripe (yellow),

1. Prepare Standard Solutions:

2. Perform Oxidation and Color Development:

4. Plot Calibration Curve:

Given that the calibration curve equation is

Oxalate Concentration (mg/L)=2.0×Volume of

You can use this equation to convert the volume of

To validate the results obtained from Experiment 1

- Set up the HPLC conditions, including mobile phase

- Record the chromatograms and identify the peaks

The calculation involves using the calibration curve

Oxalate Concentration (mg/L)=m×Peak Area+b

Here, 'm' is the slope, and 'b' is the y-intercept of the

Compare the HPLC results with the spectrophotometric

Volume of KMnO4 used (mL) × Concentration of

And produce a calibration curve for the HPLC system using

Oxalate Concentration (mg/L) = m × Peak Area + b

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