Determination of serum chloride concentration using the calibration curve

PRINCIPLE

Chloride ions substitute thiocianate ions in mercury thiocianate. Thiocianate ions bind with Fe³⁺ ions and form a red product - iron thiocianate (Fe(SCN)₃).

2 Cl^– + Hg(SCN)₂ → HgCl₂ + 2 SCN^–

3 SCN^– + Fe³⁺ → Fe(SCN)₃

REAGENTS AND EQUIPMENT

iron(III) nitrate , 20 mmol/L
mercury(II) chloride, 0.8 mmol/L
mercury(II) thiocianate, 2 mmol/L
nitrogen(V) acid, 45 mmol/L
serum sample: control serum for biochemistry. Ordered by Sanolabor, manufactured by Randox
sodium chloride - fundamental solution (120 mmol/L)
spectrometer Spektra^TM
blisters
reagent bottles

Reagent preparation: According to directions of manufacturer, ordered by IRIS d.o.o., manufacturer DIALAB.

HAZARDS

	iron(III) nitrate: may intensify fire, oxidiser, causes skin irritation, causes serious eye irritation R: 8, 36/38 S: 26

	mercury(II) chloride: suspected of causing genetic defects, suspected of damaging fertility, fatal if swallowed, causes damage to organs through prolonged or repeated exposure, causes severe skin burns and eye damage,very toxic to aquatic life with long lasting effects R: 28, 34, 48/24/25, 50/53,62, 68 S: 26, 36/37/39, 45, 60, 61



	mercury(II) thiocianate: fatal if inhaled, fatal in contact with skin, fatal if swallowed, may cause damage to organs through prolonged or repeated exposure, very toxic to aquatic life with long lasting effects, contact with acids liberates very toxic gas R: 26/27/28, 32, 33, 50/53 S: 13, 28, 45, 60, 61


	nitrogen(V) acid: may intensify fire; oxidiser, causes severe skin burns and eye damage,may be corrosive to metals R: 35 S: 23, 26, 36/37/39, 45

PROCEDURE

Concentration of fundamental NaCl solution has to be 120 mmol/L because of linearity of method. The linearity of method is up to 130 mmol/L.

Solutions from 2 to 7 are prepared in 50 mL measuring flasks.

The formulae needed for the preparation of solutions from 2 to 7 is:

c₁ · V₁ = c₂ · V₂

V₁ = (c₂ · V₂) / c₁

V₁ - volume of fundamental solution
V₂ - volume of solution, which has to be prepared
c₁- concentration of solution fundamental solution
c₂ - concentration of solution, which has to be prepared

The following procedure is presented in the table 1. Adequate number of drops is added (with reagent bottles) in blister. The compound is mixed and after 10 minutes the transmittance is measured. The wavelength of measuring transmittance is 480 nm (or. at blue light). Sample blank is adjusted on 100 % transmittance. Then absorbance of all solutions is calculated.

Table 1: Diluted standards

	sample blank	sol.1	sol.2	sol.3	sol.4	sol.5	sol.6	sol.7	sample
Conc. of Cl^– (mmol/L)	/	120	100	80	60	40	20	10	?
Purified water (no. of drops)	1	/	/	/	/	/	/	/	/
Sol. Cl^–(no. of drops)	/	1	1	1	1	1	1	1	1
Working reagent (no. of drops)	11	11	11	11	11	11	11	11	11

Values of measured dilutions absorbance are entered in the diagram and a calibration curve is drawn. The calibration curve indicates that absorbance is proportional with concentration. Then the transmittance of the sample is measured, absorbance calculated and concentration of the sample is read from calibration curve.

Measurements

	sample blank	sol.1	sol.2	sol.3	sol.4	sol.5	sol.6	sol.7	sample
Conc. of Cl^– (mmol/L)	/	120	100	80	60	40	20	10	?
Measured transmittance (%)
Calculated absorbance

RESULTS

Measurements of students:

	sample blank	sol.1	sol.2	sol.3	sol.4	sol.5	sol.6	sol.7	sample
Conc. of Cl^– (mmol/L)	/	120	100	80	60	40	20	10	?
Measured transmittance (%)	100	65.8	66.8	72.1	80.5	89.5	94.4	96.8	70.6
Calculated absorbance	0	0.18	0.17	0.14	0.09	0.05	0.02	0.01	0.15

Calibration curve : Correlation of absorbance (A) with molar concentration.

Comment: straight red line was calculated statistically with LINEST function (linear regression).

Result:
Value of concentration of the unknown serum sample read from calibration curve is 95 mmol/L. The accurate value of control serum offered by manufacturer is 98.7 mmol/L.

Prepared by: Sabina Povhe Lenart, Secondary school for pharmacy, cosmetics and health care, Ljubljana