AAS Method for Chloride

AAS Method for Chloride determination

A procedure is outlined for the determination of body fluid chloride levels from as little as 0.2-mi. of fluid. The results are highly reproducible and compare very favorably with the widely used method of Schales and Schales. The color produced is quite stable, and the interference of extraneous compounds has been found to have no effect on the accuracy of the method.


This colorimetric method is based on a procedure used in automated systems. It is extremely simple, quite accurate, and gives highly reproducible results even in the hands of student technicians.


Those laboratories not having access to a chloridometer or automated systems, or even those not wanting to burden their automated systems with more work, may find this method suitable for their needs.


Basically, the method consists of the addition of chloride ions to a solution of mercuric thiocyanate and ferric nitrate. The chloride ions upset an equilibrium established between the latter two salts, thereby allowing the formation of a brown ferric thiocyanate complex which is quantitatively proportional to the amount of chloride added.


Method


Reagents


1. Saturated solution of Hg(SCN)5 Add 2.0 gIn, of Hg(SCN)2., to 1 L. of distilled water. Leave the solution at room temperature for 48 hr. or longer, with occasional shaking. Decant and filter the supernatant solution before using. If Hg(SCN)2 is not readily available, it can easily be prepared by dissolving 33 gm. Hg(N03)2H20 in 100 ml. of distilled H20 containing 5.0 ml. concentrated HN03. In a separate 1000-ml Erlenmeyer flask, dissolve 20 gm. of KCNS in 100 ml. of distilled H20. With constant stirring, mix the two solutions together. Allow the precipitated Hg(SCN)2 to settle to the bottom of the flask and decant the supernatant solution. Wash the precipitate 4 times with 900-1000 ml. of distilled water, decanting the supernatant each time. (The Hg(SCN)2., is of sufficiently low solubility that any excess Hg(N03)2 .H20 or KCNS will be washed out while losing very little Hg(SCN)2.) At this point the wet Hg(SCN)2 can be used to prepare a saturated solution of the salt.


2. 6% (w/v) Mercuric nitrate solution: Place 6.0 gIn, of Hg(N03)2 in a 100-mi. volumetric flask. Add 80.0 ml. distilled water and 1.0 ml. of concentrated HNOa. Mix until dissolved, dilute to volume.


3. Standard chloride solution: (0.1 N) Dry reagent grade sodium chloride overnight in a 1150 oven and allow to cool to room temperature in a desiccator. To prepare 0.1000N NaCl, weigh exactly 5.845 gm. of the salt and transfer it quantitatively to a 1000-mi. volumetric flask. Dissolve and dilute to the mark with distilled water. Under the conditions of this test, 0.5 ml. of 0.1N NaC1 is equivalent to 100 mEq. of chloride per liter of serum.


4. Reagent blank solution: Place 13.0 gm. of Fe(N03)3 . 911..0 in a 1-L. volumetric flask. Add approximately 500-600 ml. of distilled water and 1.5 ml. of concentrated nitric acid. Shake until the salt is dissolved and dilute to volume.


Prepare the color reagent by dissolving 13.0 gm. of Fe(NO3)3.9H20 in approximately 400 ml. of distilled water in a 1-L. volumetric flask. Add 1.5 ml. of concentrated nitric acid and 500 ml. of saturated mercuric thiocyanate, and dilute to volume. Next, add 6% mercuric nitrate (approximately 5.0 to 6.0 ml.) until the absorbance of an 80.0 mEq./L. standard (0.4 ml. of the standard chloride solution plus 15.0 ml. of this color reagent) is between 0.07 and 0.10. If desired, 4 or 5 L. of reagent may be prepared at one time to save time in standardization.


Standardization Procedure


Either a precalibrated card or a standard curve should be prepared for this procedure. The absorption-concentration curve does not pass through zero absorbance for a zero chloride concentration, and therefore calculations of chloride values, using absorbance values of the standard and the unknown, are not possible. A standard curve can be constructed by transferring 0.40, 0.45, 0.50, 0.55, and 0.60 ml. of the 0.1N NaC1 standard chloride solution to each of five separate spectrophotometer tubes. These tubes then contain the equivalent of 80, 90, 100, 110, and 120 mEq. chloride per liter of serum, respectively. Bring the volume of each tube to 0.60 ml. and add 15.0 ml. of color reagent to each tube. Prepare a reagent blank by transferring 0.60 ml. of 0.1N NaC1 to a spectrophotometer tube and adding 15.0 ml. of reagent blank solution. Using this blank, set the spectrophotometer to zero absorbance at 480 mjz and obtain the absorbance values for each of the standards. A plot of the absorbance against milliequivalents Cl per liter should yield a straight line on regular graph paper. The range of absorbance for the 80- to 120 mEq./L. standards will usually fall from 0.07 to 0.70, respectively, when using spectrophotometer tubes of 19 mm. diameter. It will be noticed that there is a discrepancy of 0.1 ml. in the total volume between the standards and the unknown, but this amount is negligible in relation to the total volume and contributes no detectable error in the procedure.

Procedure

This entire procedure is best carried out directly in the spectrophotometer tubes.

Transfer 0.5 ml. of serum to each of two spectrophotometer tubes.
To one tube add 15.0 ml. of color reagent; to the second tube add 15.0 ml. of reagent blank. Shake the tubes as the reagents are added. A slight precipitation will form, but this will disappear on standing.
Allow 10 mm. for the color to develop and also for the precipitate to dissolve. Set the spectrophotometer for zero absorhance at 480 mı with the blank and then obtain the absorbance of the unknown. The concentration of the unknown can be obtained from the precalibrated card or standard curve, whichever is preferred.


References
1. Schales, 0., and Sehales, 5. S., J. Biol. Chem. 140, 879 (1941).
2. ZaJI, D. M., Fisher, D., and Garner, M. Q., Anın. Chem. 28, 1665 (1956).
3. Skeggs, L. T., Jr., Am. J. Clin. Path. 28, 311 (1957).