This experiment is adapted from an experiment prepared by Dr. Myton, Lake Superior State University, 1993 and the Orion Model 93-20 Calcium Electrode Instruction Manual. In this experiment, the calcium concentration of an unknown will be determined using an ion selective electrode (ISE). A modern ISE instrument will be used to measure calibration standards, generate the calibration curve internally on a point-to-point basis with background correction, and display the unknown sample concentration directly.

The calcium ion selective electrode consists of an electrode body and replaceable sensing module. The sensing module consists of an internal Ag/AgCl reference electrode which is in contact with a calcium sensitive membrane. When the electrode is in a solution containing calcium ions an electrode potential develops across the membrane which is dependent on the activity of free calcium in solution. The potential, E, between the calcium electrode and a separate reference electrode ca be measured directly with a digital voltmeter, a digital pH/mV meter or a specific ion meter. The measured potential in millivolts (mV) can be described by a form of the Nernst equation:

E = E₀ + S log(A)

where E is the measured electrode potential, E₀ is ta constant reference potential, S is the electode slope, and A is the calcium ion activity. The calcium ion activity is related to the free calcium ion concentration, C_A, by the activity coefficient, f:

A = fC_A

If the background ionic strength is high and constant relative to the sensed ion concentration, the activity coefficient is constant and activity is directly proportional to concentration. For solutions containing calcium ion an ionic strength adjustor (ISA) is added to all standards and samples to give a uniformly high and constant ion strength. In this experiment the student should add 2 mL of 4M KCl to 100 mL of the sample of standard.

As with any ion selective electrode the manufacturers specification sheets should be consulted to ensure that the electrode is prepared and used in accordance with its design.

4 M KCl

0 .100 M CaCl₂ standard stock

Orion Calcium specific ion electrode

Accumet AB15 pH/ISE Meter

stir plates

stir bars

100 mL volumetric flasks

The calcium sensing module is expected to last six months during normal operation after which time the slope will decrease and readings begin to drift. The electrode sensing unit should be stored in 10^-2 M calcium standard between short term uses and should be dissembled and stored dry in the vial for periods of more than 3 days. After storage the electrode should be soaked in water 15 minutes and in calcium standard for at least one hour then the electrode operation checked to insure proper operation and response.

1. Place 100 mL of distilled water into a 150 mL beaker and 2 mL of ISA (4M KCl) and stir thoroughly. Set the instrument to the mV setting. Rinse and blot dry the electrode being careful not to damage the sensing membrane.
2. Place the electrodes in the solution and add 1 mL of 0.100 Ca²⁺ standard. Allow the electrode to stabilize, record the potential in millivolts.
3. Carefully pipet 10 mL of the same standard to the solution and record the new potential after stabilitzation. If the potential increase is 25-30 mV at 25^oC then the electrode operation is satisfactory, if not consult the instructor.

2. Determination of an Unknown

1. Prepare a set of calibration standards from the 0.100 M CaCl₂ standard stock Although any concentrations or concentration units are equally acceptable it is suggested that the calibration standards be 100 mL in volume and 0.010, 0.001, 0.0001, and 0.00001 M in calcium. Add 2 mL of ISA to each standard and allow all standards to reach room temperature before measurement.
2. Obtain an unknown from the instructor. The unknown will be analyzed in triplicate. Pipette 3 aliquots of 100 mL of each unknown into 200 mL beakers and add 2 mL of ISA to each.
3. Using the Accumet AB15 pH/ISE Meter:

Press mode button until the meter displays the Relative mV mode.

Immerse the electrode in a blank solution and press the std button. The millivolt value will change to a 0.0 mV reading. Subsequent mV readings will be displayed relative to this 0.00 mV reading.

Rinse the electrode with distilled water being careful not to touch the tip of the electrode.

Place the electrode into the standard with the lowest concentration, allow the reading to equilibrate and then record the mV reading.

Repeat step d. for each standard working from dilute to concentrated.

After the last standard has been run, place the electrode in the first unknown sample and record the mV reading. Repeat the process for the second and third aliquots of the unknown sample.

4. Using semilog graph paper, prepare a calibration curve by plotting the millivolt values on the linear axis and the standard concentrations on the logarithmic axis. Use the method of least squares to determine an equation for the plotted line on your calibration curve.

2. Determination of an Unknown Using the Method of Standard Additions

1. Using the Accumet AB15 meter:

1. Place the electrode in one of the aliquots of the first unknown and record the mV reading after the electrode equilibrates.
2. Pipet 10 mL of the highest calibration standard into the unknown sample and record the mV reading after the electrode equilibrates.

Determine the average concentration and the standard deviation for the measurement of the unknown for both the direct measurement procedure and the method of standard additions.

Write up a laboratory report using the standard format. Report the average concentration of the two unknown samples, and the standard deviations associated with the measurement of each unknown using each method. Compare the concentrations obtained using each method. Did they agree with one another? If not, explain why you think they didn’t.