Research Question:

Does the temperature at which spinach is cooked at (25.0C, 40.0C, 55.0C, 70.0C, 85.0C)
affect the calcium content (in mg), determined by EDTA Titration, using Eriochrome Black T
Indicator?

Introduction:
Spinach also known as a superfood is a type of a vegetable. This leafy vegetable is known for its
high calcium content. There are different methods of cooking the leave such as stir frying,
steaming etc. but the most common way of doing so is by boiling the leaves in water.

Other day, my mother came through a WhatsApp message in which it stated that the vitamin and
mineral content of vegetables are affected by the different methods of cooking. One of the
factors influencing this is the temperature at which the food is cooked at. This intrigued me and I
decided to verify it experimentally. Then I came across this method by which I could possibly
prove it. This curiosity led me to develop this research question for my Chemistry Internal
Assessment.

Background Information:

Hypothesis:
At higher temperatures, leaves have greater chances of releasing greater amount of calcium
because of leaching. At higher temperatures, the bonds between the cell walls also breaks
thereby releasing greater amount of calcium.

Therefore, I predict that as the temperature rises the amount of calcium in the Spinach leaf
decreases, but after a certain temperature the amount of calcium content in the Spinach leaf
stabilizes as there would be some calcium which would have made very strong bonds and would
might not get affected because of the change in temperature and would remain in the leaves after
cooking.

Variables:
Table 1: Independent Variable
Independent Values which I will take How it would be varied
Variable (C)
The temperatures would be varied
using a water bath. The water bath
Temperature 25.0, 40.0, 55.0, 70.0, 85.0 would be set to the required
temperatures and a thermometer
would be used to confirm the
temperature.
Table 2: Dependent Variable
Dependent Variable How it would be measured
Using complexometric titration mass would be measured.
Spinach is cooked in water. The sample of water is taken and
EDTA, ammonia buffer and EBT indicator are added to it.
Mass of calcium in spinach Titrate the solution against magnesium chloride till it turns
(mg) from dark blue to permanent pink color. Calculations are
carried out to find the calcium content in the water; which is
then subtracted from the original calcium content before
cooking to find the calcium content that remains in the
spinach after cooking.
Table 3: Controlled Variables (Sample Preparations)
Control Variable How it would be controlled Importance of controlling
All the leaf samples would be The rate of loss of calcium would
Surface area of the leaf cut into 1.50 cm square using be higher for greater surface area
a ruler and a pair of scissors. affecting the accuracy.
Table 3: Continued
Control Variable How it would be controlled Importance of controlling
A timer for five minutes would More calcium is lost from the leaf
Duration of cooking be set for each test tube so that if it is cooked for a longer time
the spinach the spinach only cooks for five because there is more time for the
minutes and not more than minerals to leach away affecting
that. the accuracy.
There is a possibility that there
Mass of 7.00 g would be would higher levels of calcium in
Mass of spinach measured using an analytic greater masses of spinach
balance. therefore it is very important to
keep the mass constant.
If greater amount of water is used
A 20 cm3 volumetric pipette is for cooking spinach the solution
Volume of water used used to measure out 20 cm3 of would be more dilute and if very
for cooking the spinach deionized water and added to a less water is used, then the
test tube. solution would be very
concentrated affecting the readings
of calcium present in the leaf.
Table 4: Controlled Variables (For Titration)
Control Variable How it would be controlled Importance of Controlling
Concentration of A solution of 0.05 mol dm-3
EDTA solution EDTA solution is used and is
(0.05 mol dm-3) taken from the same
volumetric flask throughout
the experiments.
Volume of EDTA Using volumetric pipette to
solution (20.0 cm3) pipette out 20.0 cm3 of EDTA
solution.
 Concentration of
magnesium chloride
solution (0.025 mol
dm-3)
Volume of deionized
water added to the
sample solution prior to
the experiment
Volume of sample
(10.0 cm3)
Volume of ammonia
buffer solution added
to the mixture (10.0
cm3)

Apparatus:
S. No. Name of Apparatus Quantity Uncertainty
1 Test tubes
2 Clamp stand 1
3 Burette 0.05cm3
4 Graduate Pipette 0.1cm3
5 Volumetric Pipette 0.03cm3
6 Volumetric Flask with Stopper 3 0.25cm3
7 Water Bath 1
8 Spinach 1 Big Leaf
9 Deionized Water
10 Chronometer 1 0.01s
11 Conical Flask
12 Measuring Cylinder 0.5cm3
13 Pasteur Pipette 1
14 Tweezers 1
15 Thermometer 1 0.5C
16 Spatula
17 Top Pan Scale 1 0.01g
18 Funnel 2
19 Weighing Boat 4
20 Ruler 1 0.05cm
22 Scissors 1 pair
23 EBT Indicator
24 Ammonia Buffer
25 Magnesium Chloride Hexahydrate
Crystals
26 EDTA Powder
27 Sieve 1
 28 Glass Rod
29 Heat Resistance Gloves 1 pair

Calculations for standard solutions:

Before the preparation of any standard solution it is always necessary to know the amount of
solids to be added to solvents. In order to obtain the correct concentrations of solutions
calculations are done which are shown below.
Preparation of a standard solution of EDTA of 0.05mol dm-3
Molar mass of EDTA (C10H16N2O8) = 10(12.01) + 16(1.01) + 2(14.01) + 8(16.00) = 372.24 g
mol-1
Mass required = moles  Molar mass (g mol-1)  volume (dm3)
Mass required = (0.05  372.24  1) g dm-3 = 18.612 g dm-3  18.61 g
Therefore, 14.61 g of EDTA is added to 1.000 dm-3 of water to prepare a solution of 0.05 mol
dm-1
Preparation of a standard solution of Magnesium Chloride of 0.025mol dm-3
Molar mass of Magnesium Chloride (MgCl26H2O) = 24.31 + 2(35.5) + 6[2(1.01) + 16] =
203.43 g mol-1
Mass required = moles  Molar mass (g mol-1)  volume (dm3)
Mass required = (0.025  203.43  1) g dm-3 = 5.08575 g dm-3  5.09 g
Therefore, 5.09 g of Magnesium Chloride is added to 1.000 dm-3 of water to prepare a solution of
0.025 mol dm-1

Method:
Preparation of a standard solution of EDTA of 0.05mol dm-3
1)Take a weighing plate and weigh out 14.61 g of EDTA powder using an analytic balance.
2)Using a spatula add the powder to a 1.00 dm3 volumetric flask.
3)Add some deionized water to the weighing plate and pour this into the volumetric flask as
well.
4)Add some water to the volumetric flask and swirl it vigorously until the powder is completely
dissolved.
5)Add more deionized water to the volumetric flask till it reaches the marked line of 1.00 dm3.
Make sure that it is the actually reading of the solution. As the liquid would be colorless
therefore, we would consider the lower meniscus.
Preparation of a standard solution of Magnesium Chloride of 0.025mol dm-3
6)Repeat steps one to six for 5.09 g of Magnesium Chloride using a fresh weighing plate.
Preparation of a standard solution of Eriochrome Black T Indicator
7)Take a fresh weighing plate and weigh out 0.2 g of Eriochrome Black T indicator and add it to
a 40 ml beaker.
8)Place a 15 ml measuring cylinder inside a fume box and measure out 15 ml of concentrated
ammonia.
9)Place the beaker with the indicator in the fume box and add 15 ml of concentrated ammonia to
it.
10)Take a 5 ml measuring cylinder and add ethanol to it.
11)Add the ethanol to the solution placed in the fume box.
Preparation of a standard solution of Ammonia Buffer
12)Take a fresh weighing plate and weigh out 7.0 g of ammonium chloride and add it to a 100 ml
volumetric flask.
13)Place a 100 ml measuring cylinder in a fume box and measure out 57 ml of concentrated
ammonia.
14)Place the volumetric flask in the fume box and add the concentrated ammonia to it.
15)Add deionized water to it until it reaches the 100 ml marked line. Make sure it is the actual
reading of the solution. As the liquid would be colorless therefore, we would consider the lower
meniscus.
Preparation of the spinach sample solution
16)Set the water bath at ___C. (___C higher than the wanted temperature)
17)Using a measuring measure out 20 cm3 of deionized water and pour it into a test tube.
18)Place the test tube in the water bath.
19)Measure out a 1.5 cm square sample of spinach using a ruler and a scissor and measure out 6
g of it using an analytic balance.
20)Check the temperature of the water in the test tube using a thermometer. When it reaches
___C add the spinach into the test tube and submerge it completely using a glass rod.
21)Start the timer for five minutes as soon as the leaf is submerged completely.
22)When the time is up, remove the test tube from the water bath and measure out 10.0 cm3 of
the spinach water using a 10.0 cm3 volumetric pipette and let the solution cool.
23)Repeat steps 16-22 for four other test tubes as well.
Complexometric Titration
24)Add the sample from step 22 to a 250 cm3 conical flask.
25)Add 20 cm3 of EDTA solution to the conical flask using a volumetric pipette.
26)Place the conical flask in the fume box and add 10.0 cm3 of ammonia buffer using a 10 cm3
volumetric pipette.
27)Add 1 cm3 of Eriochrome Black T indicator using a 5 ml measuring cylinder. The color of the
solution will now turn dark blue.
28)Prepare the setup for titration.
29)Fill the burette till the 0.00 cm3 mark with the magnesium chloride solution.
30)Titrate the solution until the solution turns into a permanent pink color and record the end
point of the titration.
31)Repeat the whole experiment for the other temperatures as well. (___C, ___C, ___C,
___C)

Raw Data:
Temperature(C) Volume of 0.025moldm-3 MgCl2 Solution Added (cm3) ( 0.5cm3)
( 0.5C) Trial 1 Trial 2 Trial 3 Trial 4 Trial 5
Start Stop Start Stop Start Stop Start Stop Start Stop
25 0.00 28.80 0.00 31.35 0.00 29.70 0.00 29.70 0.00 29.70
40 0.00 29.50 0.00 28.60 0.00 28.60 0.00 28.60 0.00 26.25
55 0.00 27.80 0.00 29.35 0.00 27.80 0.00 26.90 0.00 27.80
70 0.00 27.20 0.00 26.20 0.00 23.10 0.00 27.20 0.00 27.20
85 0.00 25.90 0.00 25.90 0.00 25.40 0.00 26.10 0.00 25.90
Qualitative Observation:
-While preparing the EDTA solution, not all the EDTA powder was dissolved despite vigorous
shaking and swirling.
-Each piece of spinach had a different stem structure, where some were leafier, and some had
more stems.