Analyzing Calcium in Milk

12
Introduction
Healthy body, good teeth, strong bonesthe benefits of calcium in good nutrition are well known.
Milk and dairy products are widely promoted as an important source of calcium in the diet. Explore
the chemistry behind the nutrition with this experiment that measures the amount of calcium in milk. Ca
Dairy

Concepts
Calcium in nutrition Titration
EDTAcomplex ion formation Accuracy and precision

Background
Calcium is the most abundant mineral in the body. An average young adult carries approximately 2% of his body
weight in calcium. That amounts to about 1 kg (1000 g) of calcium in the body! More than 99% of calcium present in
the body is in the form of calcium phosphate, Ca3(PO4)2, a crystalline compound that gives structure and strength to
bones. The calcium in bones also serves as a reservoir of calcium. Bone tissue is continuously being made and broken
down by specialized bone cells. The amount of calcium in the diet must be balanced to prevent an excessive loss of cal-
cium from bones. This is particularly important for children, adolescents, and young adults, where insufficient calcium
intake is a known risk factor for poor growth (and for osteoporosis later in life).
Calcium in the bones is exchanged with body fluids to maintain a constant level of calcium ions in the blood. Calcium
in the blood controls muscle action, regulates heart function, and assists in blood clotting. Although less than one gram
of the calcium in the body is present as Ca2+ ions in the blood, calcium nevertheless plays an essential role in mediat-
ing a variety of crucial body functions. In fact, the amount of calcium in the blood must be precisely regulated within
very narrow limits in order for the body to function normally. Since virtually all of the calcium in the body has to come
from the diet, calcium is a vital element in good nutrition.
How Is Calcium Measured?
One of the most reliable methods for determining the amount of calcium present in a sample involves measuring the
precise amount of a complex-forming reagent called ethylenediaminetetraacetic acid (mercifully, EDTA for short) that
reacts with calcium ions in solution (Equation 1).
Ca2+ + EDTA CaEDTA Equation 1
Complex ion

The microscale titration of calcium in milk involves counting the number of drops of EDTA (of known molarity)
needed for complete reaction with a measured volume of milk. Sodium hydroxide is added to the milk to keep the
solution basic, and a metal-ion indicator is added to show when all of the calcium has reacted with the EDTA. Parallel
titrations are carried out using both a blank (a solution that does not contain any calcium) and a reference solution or
control that contains a known quantity of calcium ions.
What Is EDTA?
As summarized in Equation 1, calcium ions form a stable complex-ion product with EDTA. When EDTA binds to
Ca2+, it is acting as an electron donor molecule. In basic solution (pH >10), the chemical structure of EDTA (Figure 1)
contains six electron-donor sitestwo nitrogen atoms and four negatively charged oxygen atomsthat will bond to
a positively charged metal cation. EDTA forms stable complex ions with calcium and a wide variety of other metal
cations. The EDTA ion effectively surrounds the metal cation and isolates it so it cannot react with anything else in
solution. This type of complex is called a chelate complex, derived from the Greek word chelos, meaning claw. EDTA
can be pictured as a claw that traps the metal ion and keeps it tightly in its grip. The structure of the CaEDTA chelate
complex is shown in Figure 2.

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Analyzing Calcium in Milk continued

O2CCH 2 CH 2 CO 2

N CH 2 CH 2 N

O2CCH 2 CH 2 CO 2

Figure 1. Structure of EDTA Ion (EDTA4)

O

C
H 2C O
CH 2 CH 2
N N CH 2
H 2C
Ca+2
C
C O O
CH 2 O
O
O
C

Figure 2. CaEDTA Complex Ion [CaEDTA]2

Indicators for EDTA Titrations

Both Ca2+ ions and CaEDTA complex ions are colorless. The point at which all uncomplexed calcium ions in milk
have been consumed by reaction with EDTA is called the equivalence point or endpoint of the titration. An indicator
must be used to provide a visible color change or signal showing that the endpoint has been reached. A metal-ion indica-
tor (Ind) is a compound whose color changes when it binds to a metal ion. For an indicator to be useful, it must bind to
the metal cation less strongly than EDTA does. The indicator used in this experiment will be hydroxynaphthol blue
(HNB), a water-soluble dye.

Experiment Overview
The purpose of this experiment is to analyze the calcium content in skim milk by microscale titration with EDTA. At the
start of the titration, a small amount of indicator (Ind) is added to the colorless solution of Ca2+ to form a red complex
(CaInd). As EDTA is added, it will react with free, colorless Ca2+ ions. When all of the free Ca2+ ions have reacted, the
last drop of EDTA added before the endpoint will dislodge the indicator from the red CaInd complex (Equation 2).
The change from the red color of CaInd to the blue color of unbound Ind will signal the endpoint of the titration.
CaInd + EDTA CaEDTA + Ind Equation 2
Red Colorless Colorless Blue

Materials
Calcium chloride solution, CaCl2, 0.050 M, 1 mL Beakers or small test tubes, 5
Ethylenediaminetetraacetic acid, disodium Beral-type pipets, graduated, 5
salt solution (EDTA), 0.040 M, 510 mL Microspatula
Hydroxynaphthol blue (HNB), 0.2 g Reaction plate, 24-well
Skim milk, 5 mL Toothpicks, 5
Sodium hydroxide solution, NaOH, 6 M, 2 mL White paper
Water, distilled

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 Analyzing Calcium in Milk continued

Safety Precautions
 odium hydroxide solution is a corrosive liquid and causes severe skin burns and eye damage. Hydroxynaphthol
S
blue causes skin and eye irritation. Avoid contact of all chemicals with eyes and skin. Wear chemical splash gog-
gles, chemical-resistant gloves, and a lab coat or chemical-resistant apron. Do not drink the milk! All food-grade
items that have been brought into the lab are considered laboratory chemicals and are for lab use only. Do not
taste or ingest any materials in the chemistry laboratory and do not remove any remaining food items after they
have been used in the lab. Wash hands thoroughly with soap and water before leaving the lab.

Procedure
1. Obtain the required reagents (CaCl2, EDTA, HNB, milk, and NaOH) in labeled beakers or small test tubes.
2. Label four graduated, Beral-type pipets to use with the corresponding solutions in step 1.
3. Place the 24-well reaction plate on a piece of white paper so that the color comparisons will be more distinct. Use
the appropriate graduated pipet to fill the reaction plate (steps 48).
4. To the first well in the first row, add 2 mL of distilled water. This is the blank solution.
5. Add 1 mL of skim milk to wells 2, 3, and 4 in the first row. These are the test solutions.
6. Add 1 mL of CaCl2 solution to well 5 in the first row. This is the reference solution.
7. To each filled well, carefully add 23 drops of 6 M NaOH solution. Stir each with a different toothpick. Do not
add extra base!
8. Using a microspatula, add several grains of HNB indicator to each filled well. Stir to dissolve. Observe and record the
color of each solution.
9. Add EDTA solution slowly, one drop at a time, to the blank solution in well 1 until it just turns blue (turquoise).
There should be no trace of purple left in the solution. Record the number of drops of EDTA added.
10. Add EDTA solution one drop at a time to the milk solution in well 2 until it just turns blue (turquoise). There
should be no trace of red or purple left. Record the number of drops of EDTA added.
11. Repeat step 10 to analyze the milk samples in wells 3 and 4, respectively.
12. If the results obtained for the three milk samples do not show good agreement, run additional trials as needed.
Consult your instructor if consistent results are not obtained after five trials.
13. Add EDTA solution one drop at a time to the reference solution in well 5 until the solution turns the same shade
of blue as the blank. Record the number of drops added.
14. The contents of the reaction plate may be rinsed down the drain using running water.

Laboratory Experiments for General, Organic and Biological Chemistry 3

Pre-Laboratory Assignment 12
Name:_____________________________________________ Section:__________ Date:___________

Analyzing Calcium in Milk

1. Name three reasons why calcium is an essential element for good nutrition and good health.

2. What features of the EDTA molecule allow it to form stable complexes with metal ions?

3. What is the purpose of adding sodium hydroxide to milk for the EDTA titration of calcium ion? Describe the
safety hazards and precautions associated with the use of NaOH solution.

4. What method will be used to determine when all of the calcium in milk has been used up in its reaction with added
EDTA? Describe in detail the role of the indicator.

5. EDTA titration is often used to determine water hardness levels arising from the concentration of Mg2+ and
Ca2+ ions in water. (For simplicity, water hardness is usually expressed as if all the ions are Ca2+.) A 50.0-mL
sample of pool water was titrated using EDTA15.4 mL of 0.010 M EDTA was required to reach the endpoint.
Calculate the concentration (molarity) of Ca2+ ions in the pool water.

4 Laboratory Experiments for General, Organic and Biological Chemistry



Laboratory Report 12
Name:_____________________________________________ Section:__________ Date:___________

Analyzing Calcium in Milk

Reaction Well 1 2 3 4 5

Blank Sample Test Sample A Test Sample B Test Sample C Reference

Solution
Distilled Water Skim Milk Skim Milk Skim Milk CaCl2
Initial color with
HNB indicator
Number of drops
of EDTA added
Corrected volume
of EDTA
Millimoles
of calcium
Milligrams
of calcium

1. For each sample in wells 25, subtract the number of drops of EDTA added to the blank from the number of
drops of EDTA added to each test or reference solution, respectively. This is the corrected volume of EDTA
required to titrate the calcium in each test or reference sample. Record the results in the table.

2. Use the following equation to calculate the number of millimoles of calcium ion present in each test and reference
solution, respectively. Record the results in the table. Note: The equation assumes that 25 drops of EDTA solution
are equal to 1 mL.
Millimoles = Corrected volume 1 mL Molarity of EDTA
of calcium of EDTA added (millimoles/mL)
25 drops
(drops)

3. Use the atomic mass of calcium to calculate the number of milligrams of calcium present in each test and reference
solution, respectively. Record the results in the table. Hint: The units for atomic mass are grams per mole, which
can also be expressed as milligrams per millimole.

4. Based on the number of milligrams of calcium in 1.0 mL of each test solution A, B, C, calculate the amount of cal-
cium in milligrams that would be present in one cup (240mL) of skim milk. Report both the individual values for
the three samples and the mean (average value).

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Analyzing Calcium in Milk continued

5. Calculate the average deviation from the mean for test samples A, B and C.

6. Report the amount of calcium in one cup of milk in the following form: (mean) (average deviation). Dont
forget the units and the number of significant figures.

7. The average deviation is an indicator of the precision of an experimental procedure. Comment on the precision of
your results and sources of experimental error that affect the precision.

8. The federal government has set a Recommended Daily Allowance (RDA) for calcium of 1200 mg per day for
adolescents and young adults. Based on your results, what percent of the daily requirement would one cup of milk
provide? Compare this result with the value reported on the Nutrition Facts label for the carton of skim milk.

9. Accuracy is determined by comparing the results obtained for the reference sample versus its actual or known
value. The amount of calcium in the reference solution (0.050 M CaCl2) is 2.0 mg per mL. Comment on the accu-
racy of this method for determining the amount of calcium in a sample.

6 Laboratory Experiments for General, Organic and Biological Chemistry