2.7: Chloride Determination (Coulometric Method)

RELATED READINGS: Page 285, See Methods in CD-ROM for Chloride

OBJECTIVES

Upon completion of this exercise, appropriate discussion, and related reading, the student will be able to:

1. Clean a chloridometer and prepare it for operation.
2. Blank the instrument according to manufacturer’s instructions.
3. Check and adjust calibration of the instrument with the chloride (Cl^-) standard.
4. Prepare serum samples for Cl^- determination.
5. Determine the Cl^- concentration of unknown samples with 90% accuracy.
6. List the common causes of error in coulometric Cl^- determinations.

PRINCIPLE

In the coulometric measurement of chloride ions, silver ions (Ag⁺) are generated at a constant rate by an electrode and released into a dilute acid solution. There they combine with Cl^- ions in the test sample to form an insoluble precipitate, AgCl. When all Cl^- is removed from solution, an excess of Ag⁺ ions is detected by a sensor and the titration is stopped. Because the rate of Ag⁺ ion generation is constant, the amount of time required to remove all Cl^- ions is directly proportional to concentration as shown in the following:

\[\frac{[Unknown]}{time_{Unknown}}=\frac{[Standard]}{time_{Standard}}\]

OR

\[[Unknown] = \frac{time_{Unknown} [Standard]}{time_{Standard}}\]

Some instruments report a titration time in seconds from which the Cl^- concentration can be calculated. Others perform an internal calculation and directly show a Cl^- value in mmol/L.

GLOSSARY

Spike: a known amount or volume of a substance that is added to a biological sample.

MATERIALS

• Buchler-Cotlove Chloridometer And/or Corning Chloridometer
• Sample beakers
• Acid Reagent
• Silver polish
• Acid Buffer
• Distilled Water
• Standard
• Controls
• Gelatin Reagent (indicator)
• Serum Samples

PROCEDURE

BUCHLER METHOD

1. Clean electrode with silver polish and rinse well with distilled water. This should be repeated approximately every 10 specimens (or whenever results become erratic) to insure reproducible results.
2. Blanking instrument
   1. Set DIRECT READER to BLANK
   2. Set TITRATION RANGE to HIGH
   3. Set D.C. MICROAMPERES to +10
   4. Set DIGITAL READOUT to 0000
   5. Set ADJUST TITRATE to 2
   6. With a pipet or Oxford Pipettor, dispense 4 mL of Nitric-Acetic Acid into chloride titration cups.
   7. Add 4 drops of gelatin indicator.
   8. Place cup under stirrer and turn ADJUST TITRATE control to 1.
   9. When the MICROAMPERES needle stops falling, set the adjustable microampere needle to 10 Microampere above this value.
   10. Turn ADJUST TITRATE control to 2.
   11. Run duplicate blanks to determine if readout is stable. Run additional blanks, if necessary, until stability is obtained.
   12. Adjust the Time Delay Dial to the number appearing on the digital readout. This interval will be automatically subtracted from the final mmol/L.
3. Titrating Samples
   1. Set DIRECT READER to TITRATE.
   2. Set DIGITAL READOUT to 0000.
   3. With a pipet or Oxford Pipettor dispense 4 mL of Nitric—Acetic Reagent into chloride titration cups.
   4. Transfer 0.05 mL of standard, specimen, or control into reagent with pipettor.
   5. Add 4 drops of indicator.
   6. Place cup under stirrer and turn ADJUST TITRATE to 1.
   7. When MICROAMPERES needle moves to near 0, turn ADJUST TITRATE control to 2.
   8. Multiply the number appearing on the digital read-out after titration by 2 to determine the chloride concentration in mmol/liter.
   9. Repeat steps c through h for each additional sample to be tested.
   10. When all samples have been tested, leave the titration electrode immersed in distilled H₂O.

CORNING METHOD

Make certain all electrodes are clean and have been rinsed with distilled water. Use silver polish if necessary.

Conditioning

1. Place approximately 15 mL (exact volume is not critical) of Acid Buffer into a sample cup.
2. Place cup on platform.
3. Squeeze release clips on the electrode head and gently lower electrodes into buffer solution.
4. Press the “Condition" switch down. The instrument should operate and stop at <200. If the instrument continues beyond a reading of 300, turn the power switch off and consult with your instructor.

Calibration

1. Using the same sample cup and solution that were conditioned, pipet 100 uL of standard into the acid buffer solution.
2. Press the titrate switch down. When titration stops, the value displayed is the Cl- concentration in mmol/L.
3. If the observed reading for the standard is ±2 mmol/L from the expected value, proceed with the titration procedure. If the instrument is out of calibration, consult with your instructor and then proceed as follows.
4. Turn the appropriate calibration screw on the back of the instrument clockwise to increase the value or counter clockwise to decrease the value. One half turn will increase/decrease the value approximately 2 mmol/L.
5. Continue testing and adjusting the standard until stable calibration is achieved.

Titration

1. Pipet 100 uL of sample (patient, control) into the sample cup*.
2. Press the titrate control down.
3. When titration stops, read and record your value on the data sheet.
4. Repeat steps 1, 2, and 3 for each sample to be tested.
5. When all titrations are complete, place the electrodes in a sample cup containing 15-20 ml of distilled water.

*The conditioned acid buffer solution may be used for multiple samples, and should be changed within 3 samples after the change solution indicator comes on. When changing the acid buffer solution repeat steps 1 through 4 of the conditioning procedure. It is not necessary to re- calibrate when changing the buffer solution.

OPTIONAL EXERCISE

1. Add 0.05 mL of a 1 M KBr solution to one of the previously analyzed unknowns or controls.
2. Vortex-mix the sample.
3. Assay the sample for Cl^- as per the above directions.
4. Record Cl^- result on the data sheet.

DATA SHEET, EXERCISE #7

NAME: ___________ DATE: ___________

RESULTS

	Cl^-, (mmol/L)
	Buchler	Corning
Standard
Normal Control
Abnormal Control
Unknown 1
Unknown 2

OPTIONAL EXERCISE

\[\text{Unspiked}\; [Cl^{-}] = \text{original result for sample} \times (0.95) = \_\_\_\_\_\_\; mEq/L\]

\[\text{Sample spiked with}\; Br^{-} = \_\_\_\_\_\_\_\; mEq/L\]

\[\% \; \text{reactivity of Br} = \frac{\text{Spiked result} - \text{unspiked result}}{50\; mEq / L} \times 100 \%\]

Discussion Questions

1. If Br^- reacts with Ag⁺ in the same manner as Cl^-, what error would be introduced to a sample if it contained unusually high levels of Br^- ions?
2. Do you think that iodide (I^-) will interfere in this assay?
3. Why is it necessary to blank or condition only the first titration?