Materials Included In Kit

Additional Materials Required

Safety Precautions

Do not allow students to ingest any of the milk samples during this laboratory. The samples have been stored with non–food grade laboratory chemicals and are for lab use only. Sodium hydroxide solution is severely corrosive to eyes and skin. Avoid contact of all chemicals with eyes and body tissues. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for both general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. CaCl₂, EDTA, and HNB solutions can be disposed of according to Flinn Suggested Disposal Method #26b. NaOH solution can be disposed of according to Flinn Suggested Disposal Method #10.

Teacher Tips

• Enough materials are provided in this kit for 30 students working in pairs, or for 15 groups of students. The procedure can reasonably be completed in one 50-minute class period. The prelaboratory assignment should be completed prior to lab, and the calculations and post-lab questions can be assigned as a post-lab activity or performed as a class activity the day following the lab.
• Distilled or deionized water must be used for the control solutions. The microscale titration reaction is sensitive to trace amounts of magnesium and calcium ions found in tap water. All glassware and the microscale reaction plate should be rinsed with distilled water and dried before use.
• The amount of distilled water in the control sample is 2 mL so that the final volume of all solutions after EDTA titration is approximately equal. This allows better comparison of the final indicator colors to determine the endpoint of the reaction.
• Students may notice that the milk sample turns from cloudy (opaque) to translucent when NaOH is added. Casein, the principal milk protein, is virtually insoluble in water under neutral or acidic conditions. It is present in milk as a stable emulsion. Addition of base, however, changes the ionic state of the protein and causes it to dissolve.
• The HNB indicator color depends on the amount of NaOH added to each well. It is important that students add the precise amount of NaOH specified in step 7. If too much base is added, the indicator color changes will not be distinct.
• The accuracy and precision of the results may be improved by carrying out the microscale titration as an optional gravimetric exercise, that is, by determining the mass of EDTA solution needed to titrate a known mass of milk. This can be done by weighing the appropriate Beral-type pipets before and after the solutions have been dispensed.
• Having the students determine the average drop volume for the EDTA solution is a valuable optional exercise. Ask the students to fill a 10-mL graduated cylinder to the 1 mL mark and then add EDTA dropwise to the graduated cylinder. The students should record the volume of EDTA in the graduated cylinder after 10, 20, 30 and 40 drops of EDTA have been added and then calculate the average volume in mL of 10 drops of solution. This may also stimulate the students to develop good lab technique so that the size of a drop is more reproducible and precise.
• Do different types of milk contain different amounts of calcium? Construct a collaborative classroom activity to compare the amount of calcium present in whole milk, 2%, skim, powdered dry milk, buttermilk, etc.
• Engage the students in optional inquiry-based lab experiments to determine the amount of calcium in a variety of calciumfortified foods or in calcium supplements. Problems that may need to be addressed include whether the indicator color change is visible, what pH is needed to dissolve the samples and whether the samples should be diluted first.

Answers to Prelab Questions

1. Name three reasons why calcium is an essential element for good nutrition and good health.
   Calcium serves numerous critical roles in maintaining health. It strengthens our bones and teeth and is vital for the normal clotting of blood. It facilitates the transmission of nerve impulses, contraction of muscles including the heart, and the activation and regulation of enzymes and hormones.
2. What features of the EDTA molecule allow it to form stable complexes with metal ions?
   In basic solution EDTA exists as a tetraanion. It contains four negatively charged oxygen atoms and two nitrogen atoms (with lone pairs of electrons) that can act as electron-donor sites in binding to positively charged metal cations. Coordination of the metal ion to the six sites of the EDTA molecule stabilizes the metal cation and esentially traps it so that it cannot react with other chemicals.
3. Describe in general terms how a microscale titration reaction is carried out.
   In a microscale titration, a known small volume of the sample to be analyzed is added to a microscale plate and the titrating reagent (titrant) is added dropwise until the endpoint is reached.

4. 1. What is the purpose of adding sodium hydroxide to milk for the EDTA titration of calcium ion?

      NaOH is added to milk to increase the pH of the solution above 10. Basic conditions are needed to convert the EDTA to the correct ionic state so that it will bind efficiently and strongly to the calcium ions present in solution.

   2. Describe the safety hazards and precautions associated with the use of NaOH solution.

      NaOH solution is toxic and corrosive. Wear chemical splash goggles and chemical-resistant gloves and apron. Avoid contact with skin and eyes.

5. What method is used to determine when all of the calcium in milk has been used up in its reaction with added EDTA?
   A metal-ion indicator is added to the test samples prior to titration. The indicator exists in two different colored forms, depending on whether calcium ion is bound to it or not. At the endpoint of the titration, the last drop of EDTA added displaces the last calcium ions from their bound state with the indicator. This causes an abrupt color change that signals the end of the reaction.

Sample Data

Answers to Questions

1. Based on the results for the number of milligrams of calcium in 1 mL of each of the test solutions A, B, C, calculate the amount of calcium in milligrams that would be present in 1 cup (240 mL) of skim milk. Report both the individual values for the three samples and the mean (average value).
   
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2. Calculate the individual deviations from the mean amount of calcium for each test sample: A, B, C. Average the individual deviations to determine the average deviation also.
   
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3. Report the amount of calcium in 1 cup of milk in the following form:
   (mean) ± (average deviation). Don’t forget the units and the number of significant figures.
   Ca content per cup = 330 ±23 mg
4. The average deviation is an indicator of the precision of an experimental procedure. Comment on the precision of your experimental results.
   The precision can be estimated by dividing the average deviation by the mean and calculating a “percent uncertainty.” The percent uncertainty is thus (23/330) × 100 = 7%.
   
   The precision of the results is good. In one case, the difference in the number of drops added is only 1 drop—the smallest difference that can be measured. One important factor that limits the precision of the method is the reproducibility of the drops being added. The small size of the sample being titrated and the resulting minimal differences in the number of drops of EDTA required are also important.
5. 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 calcium requirement would one cup of milk provide? Compare this result with the value reported on the nutritional label for the carton of skim milk.
   (330 mg/1200 mg) × 100 = 28%. The nutritional label on the carton of skim milk states that 1 serving size of milk (1 cup) provides 30% of the RDA for calcium (but does not specify whether that is for an adult or a child or a pregnant woman).
6. The accuracy of an experimental procedure is determined by comparing the results obtained versus the actual or known value for a reference sample. The concentration of calcium in the reference solution (0.050 M CaCl₂) is 2.0 mg per mL. Comment on the accuracy of this method for determining the amount of calcium in a sample.
   The amount of calcium measured in the reference sample was 1.7 mg per mL. This represents a 15% error in the accuracy of the measurement.

References

Special thanks to Robert Lewis, Downers Grove North High School in Downers Grove, IL, and John Little, St. Mary’s High School in Stockton, CA, for the lab idea and sample procedure.

Introduction

Healthy body, good teeth, strong bones—the 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.

Concepts

• Calcium in nutrition
• Titration
• EDTA–complex ion formation
• Accuracy and precision

Background

Calcium is the most abundant mineral element 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, however, 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 carefully balanced to prevent an excessive loss of calcium 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 in the blood, where it controls muscle action, regulates heart function, and assists in blood clotting. Although less than 1 g of the calcium in the body is present as Ca²⁺ ions in the blood, calcium nevertheless plays an essential role in mediating 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 one of the most important elements 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. The equation for the reaction of EDTA with calcium ion is shown in Equation 1.

In the microscale titration analysis of calcium in milk, the number of drops of EDTA (of known molarity) needed for complete reaction with a measured volume of milk are counted. Sodium hydroxide is added to the milk sample to keep the solution basic, and a metal-ion indicator is added to show when all of the calcium in the milk sample has reacted with the EDTA. Parallel titrations are carried out using both a control solution (a solution that does not contain any calcium) and a reference solution (a solution that contains a known quantity of calcium ion). Because the time necessary to do one titration is quite short, it is possible to carry out several experimental trials during one lab period and to average the results that are obtained. Averaging the measurements improves both the accuracy and precision of the results.

What is EDTA?

As shown in Equation 1, calcium ion in solution is able to form a stable complex-ion product with EDTA. When EDTA binds to Ca²⁺ in this manner, it is acting as an electron donor molecule. In basic solution (pH >10), the chemical structure of EDTA (Figure 1) contains six electron-donor sites—two nitrogen atoms and four negatively charged oxygen atoms—that are able to interact with the positively charged metal cation and stabilize it. Under these conditions EDTA forms exceptionally stable complex ions with calcium (as well as a wide variety of other metal cations). The EDTA molecule 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. The picture analogy is that EDTA is like a claw—it traps the metal ion and keeps it tightly in its grip. The structure of the Ca–EDTA chelate complex is shown in Figure 2.

Indicators for EDTA Titrations

Both Ca²⁺ ion and the Ca–EDTA complex ion are colorless. The point at which all uncomplexed calcium ions in milk have been completely consumed by reaction with EDTA is called the endpoint of the titration. A chemical indicator must be used to provide a visible color change that can be taken as a sign that the endpoint has been reached. A metal-ion indicator (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 is hydroxynaphthol blue (HNB), a water-soluble dye molecule.

At the start of the titration reaction, a small amount of indicator (Ind) is added to the colorless solution of Ca²⁺ to form a red complex (Ca–Ind). As EDTA is added, it reacts with free, colorless Ca²⁺. When all of the free Ca²⁺ has been used up, the last drop of EDTA added before the endpoint dislodges the indicator from the red Ca–Ind complex (Equation 2). The change from the red color of Ca–Ind to the blue color of unbound Ind signals the endpoint of the titration.

Materials

Safety Precautions

Sodium hydroxide solution is severely corrosive to eye and skin tissue. Wear chemical splash goggles, chemical-resistant gloves, and a chemical-resistant apron when handling this chemical. Do not drink the milk! Any food product brought into the lab is thus considered a chemical. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Preparation

1. Obtain the required reagents (CaCl₂, EDTA, HNB, milk and NaOH) in labeled beakers or small test tubes.
2. Label five Beral-type pipets to use with the corresponding solutions.
3. Place the 24-well microscale plate on a piece of plain white paper so that the color comparisons will be more distinct. Use the appropriate graduated Beral pipet to fill a 24-well reaction plate (steps 4–8):
4. To the first well in the first row; add 2 mL of distilled water. This is the control solution.
5. Add 1 mL of skim milk to wells 2, 3, and 4 in the first row. These are the sample 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 add 3 drops of 6 M NaOH solution. Stir thoroughly with a wooden toothpick.
8. Use a microspatula to add a small amount of HNB indicator to each filled well. Stir to dissolve. Note the initial color of each solution in the Data Table.

Microscale Titration

9. Add 0.04 M EDTA solution slowly, one drop at a time, to the control solution in well 1 until it just turns blue (turquoise). There should be no trace of purple left in the solution color. Record the number of drops of EDTA added in the Data Table.
10. Add EDTA solution one drop at a time to the milk solution in well 2 until the solution turns the same shade of blue as the control solution. Record the number of drops of EDTA added in the Data Table.
11. Repeat steps 11 and 12 for each of the other two milk samples in wells 3 and 4, respectively.
12. Record the number of drops of EDTA needed for each solution in the Data Table.
13. 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.
14. Add EDTA solution one drop at a time to the reference CaCl₂ solution in well 5 until the solution turns the same shade of blue as the control solution. Record the number of drops added in the Data Table.
15. Consult the teacher for proper disposal of the contents of the well plate and the laboratory solutions.

Student Worksheet PDF

11940_Student1.pdf