Materials Included In Kit

Additional Materials Required

Safety Precautions

Sodium hydroxide solution is a corrosive liquid and causes severe skin burns and eye damage. Hydroxynaphthol blue causes skin and eye irritation. Avoid contact of all chemicals with eyes and skin. Wear chemical splash goggles, chemical-resistant gloves and a lab coat or chemical-resistant apron. 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. Remind students to wash hands thoroughly with soap and water before leaving the lab. Review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Calcium chloride and EDTA solutions may be rinsed down the drain with water according to Flinn Suggested Disposal Method #26b. Sodium hydroxide solution may be neutralized according to Flinn Suggested Disposal Method #10.

Lab Hints

• This experiment can reasonably be completed within a typical 2-hour lab period. For best results, review the Prelaboratory Assignment and the calculations for the Post-Lab Questions during the lab period.
• Distilled or deionized water must be used for the “blank” solutions. The titration reaction is sensitive to trace amounts of magnesium and calcium ions found in tap water. All glassware and the reaction plates should be rinsed with distilled water and dried before use.
• The amount of distilled water in the “blank” is 2 mL so that the final volume of all solutions after EDTA titration will be approximately equal. This allows better comparison of the final indicator colors to determine the endpoint.
• Students may notice that the milk 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 change depends on the amount of NaOH added and on the amount of indicator. If no base is added, the initial color of the indicator is purple, not red. Remember that the NaOH is required to increase the pH to the needed range for the titration of Ca²⁺ ions with EDTA. If too much NaOH is added, however, the color change may go “grayscale” and become less distinct. Individuals who are color blind will have a difficult time recognizing the pink– purple–blue color transition.
• The accuracy of the results may be improved significantly by carrying out the microscale titration as a 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.
• Alternatively, the accuracy of this microscale titration may also be improved by having students analyze the number of drops of EDTA required for a series of reference solutions. Students can then plot a calibration curve of number of drops of EDTA versus Ca²⁺ ion concentration and determine the concentration of Ca²⁺ ions in milk using the calibration curve.
• Having students determine the average drop volume for the EDTA solution will also improve accuracy. Ask the students to fill a 10-mL graduated cylinder to the 1.00 mL mark and then add EDTA dropwise to the graduated cylinder. Count the number of drops that must be added to reach first the 2.00 mL mark and then the 3.00 mL mark. Take the average to determine the number of drops per mL. This exercise may also motivate 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. The amount of fat may interfere with the titration of whole milk or buttermilk.
• This activity is an excellent springboard for optional inquiry-based experiments to determine the amount of calcium in calcium-fortified foods or 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.
• EDTA is used clinically in “chelation therapy” to treat lead poisoning or other types of heavy metal poisoning. Because EDTA forms very stable complex ions with most metal ions, it is used as a “scavenger” to remove toxic heavy metals from the body.

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-ion allow it to form stable complexes with metal ions?

   In basic solution, EDTA exists as a tetra-anion. It contains four negatively charged oxygen atoms and two nitrogen atoms (with lone pairs of electrons) that can bond with a metal ion. Coordination of the metal ion to the six sites of the EDTA molecule stabilizes the metal cation and essentially traps it so that it cannot react with other chemicals.

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.

   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. NaOH solution is corrosive and will cause skin burns and eye damage. Wear chemical splash goggles and chemical-resistant gloves and apron or lab coat. Avoid contact with skin and eyes.

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.

   A metal-ion indicator is added to the test samples prior to titration. The indicator exists in two different colored forms, depending on whether the 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.

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 EDTA—15.4 mL of 0.010 M EDTA was required to reach the endpoint. Calculate the concentration (molarity) of Ca²⁺ ions in the pool water.

   Moles of EDTA = (0.0154 L) x (0.010 moles/L) = 1.5 x 10^–4 moles
   Moles of Ca²⁺ = moles of EDTA = 1.5 x 10^–4 moles
   Concentration of Ca²⁺ = (1.5 x 10^–4)/(0.050 L) = 0.003 M

Sample Data

Answers to Questions

1. For each sample in wells 2–5, 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.

   See the sample data and results 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.
   {14035_Answers_Equation_3}
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.
   {14035_Answers_Equation_4}

   See the sample data and results table for the results of the other calculations.

4. Based on the number of milligrams of calcium in 1.0 mL of each test solution A, B, C, calculate the amount of calcium in milligrams that would be present in one cup (240 mL) of skim milk. Report both the individual values for the three samples and the mean (average value).
   {14035_Answers_Table_2}
5. Calculate the average deviation from the mean for test samples A, B and C.
   {14035_Answers_Table_3}
6. Report the amount of calcium in one 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 = 350 ±10 mg

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.

   The precision can be estimated by dividing the average deviation by the mean and calculating a “percent uncertainty.” The percent uncertainty is

   {14035_Answers_Equation_5}
   The precision of the results is excellent. The difference in the number of drops added was 0–3 drops. Two main factors limiting the precision of the method are the reproducibility in the size/volume of the drops being added and the method used to measure 1.0 mL of milk.
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.
   {14035_Answers_Equation_6}
   The nutritional label on the carton of skim milk states that one serving of milk (1 cup) provides 30% of the RDA for calcium but does not specify whether that is for an adult, child or pregnant woman.
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 accuracy of this method for determining the amount of calcium in a sample.

   The amount of calcium measured in the reference sample was 1.9 mg per mL. This represents a 5% error in the accuracy of the measurement. Better accuracy could be obtained using volumetric glassware, such as serological pipets and burets.

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 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 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 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 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 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).

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 (see Figure 1) contains six electron- donor sites—two nitrogen atoms and four negatively charged oxygen atoms—that 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 Ca–EDTA chelate complex is shown in Figure 2. Indicators for EDTA Titrations
Both Ca²⁺ ions and Ca–EDTA 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 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 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 (Ca–Ind). 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 Ca–Ind complex (Equation 2). The change from the red color of Ca–Ind to the blue color of unbound Ind will signal the endpoint of the titration.

Materials

Prelab Questions

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 EDTA—15.4 mL of 0.010 M EDTA was required to reach the endpoint. Calculate the concentration (molarity) of Ca²⁺ ions in the pool water.

Safety Precautions

Sodium hydroxide solution is a corrosive liquid and causes severe skin burns and eye damage. Hydroxynaphthol blue causes skin and eye irritation. Avoid contact of all chemicals with eyes and skin. Wear chemical splash goggles, 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 4–8).
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 2–3 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.

Student Worksheet PDF

14035_Student1.pdf