Suitability for the Qualitative Determination of Calcium with Phthalein Purple (O-Cresophthalein Complexone) using Stannous Chloride

1. OBJECTIVE
To standardize a procedure to determine the suitability for the qualitative determination of calcium with phthalein purple at Sigma-Aldrich St. Louis.

2. SCOPE
This procedure applies to all products that have a specification for the qualitative determination of calcium with phthalein purple.

3. DEFINITIONS
N/A

4. DISCUSSION
Phthalein purple is a useful indicator in differentiating magnesium from calcium. Phthalein purple reacts with calcium ions to produce a violet color, and this color can be measured photometrically at a pH of greater than 9. Thus, this indicator is useful in measuring the hardness of water. It can be used in biological systems that are free of chelating complexes such as ethylenediametetraacetic acid, citric acid, etc. Magnesium is detectable in neutral solution, pH of approximately 7.5.

5. RESPONSIBILITIES
It is the responsibility of trained Analytical Services laboratory personnel to follow this procedure as written.

6. SAFETY
Refer to Material Safety Data Sheets (MSDS) for hazards and appropriate handling precautions.

7.1 CONDITIONS:
T = 23-25°C, pH > 11.5, A575nm, Light path = 1 cm

7.2 METHOD:
Spectrophotometric Stop Determination

7.3 REAGENTS:

7.3.1    3.6 mM Stannous Chloride/ 15%(v/v) Diethylamine (SnCl2/DEA)
            Prepare 200 ml by dissolving 136.7 mg to 152 mg of Stannous Chloride, Anhydrous, Sigma-Aldrich Product Number S2752, in 40 ml of purified water. After dissolution, add 30.0 ml of Diethylamine, Free Base, Aldrich Product Number 471216 with continuous stirring. Dilute to 200 ml with purified water with continuous stirring. The solution may be hazy in appearance.

7.3.1.1    Use reverse pipetting for delivering Deithylamine, Free Base.

7.3.1.2    Store the solution in a sealed vessel with continuous stirring throughout the assay due to stench.

7.3.1.3    Using pH strips, Sigma-Aldrich Product Number P4411 and a digital thermometer, record pH and temperature. The pH of this reagent must be greater then 11.5 at 23 - 25°C. If not, prepare the reagent again, and check the pH. If pH is below 11.5 again, prepare the reagent using a new unopened bottle of Diethylamine, Free Base, following the same steps.

7.3.2    1 N Hydrochloric Acid Solution (HCl)
            Use Hydrochloric Acid Solution, 1.0 N, Sigma Product Number H3162. Use neat.

7.3.3    10ug/ml or 10 ppm Calcium Standard (Ca)
            Use a 1000 ug Calcium per ml atomic absorption, ICP standard P/N 4400-100091. Dilute 1.0 ml to 100 ml with purified water using a Class A, volumetric flask.

7.3.4    0.12 M Sodium Acetate, 0.062 mM Phthalein Purple, 0.42 N Hydrochloric Acid, 8.7 % (v/v) Diethylamine Solution (Control)
            Immediately before use, prepare a solution containing 2.375 mg to 2.625 mg of Phthalein Purple, Sigma-Aldrich Product Number P5631, 0.95 g to 1.05 g of Sodium Acetate, Trihydrate, Sigma-Aldrich Product Number S8625, 25 mls of Reagent 7.3.2 (HCL), and 34.5 mls of reagent 7.3.1 (SnCl2/DEA)

7.3.4.1    Mix with continuous stirring in a ventilation hood.

7.3.4.2    Prepare immediately and proceed with the spectrophotometric determination.

7.3.5    0.12 M Sodium Acetate, 0.062 mM Phthalein Purple, 0.42 N Hydrochloric Acid, 8.7 % (v/v) Diethylamine Solution (Sample)
            Immediately before use, prepare a solution containing 2.375 mg to 2.625 mg of Phthalein Purple, Sigma-Aldrich Product Number P5631, 0.95 g to 1.05 g of Sodium Acetate, Trihydrate, Sigma-Aldrich Product Number S8625, 25 ml of Reagent 7.3.2 (HCL), and 34.5 ml of reagent 7.3.1 (SnCl2/DEA)

7.3.5.1    Mix with continuous stirring in a ventilation hood.

7.3.5.2    Prepare immediately and proceed with the spectrophotometric determination.

7.4 TEST METHOD

7.4.1    Control Reaction:

7.4.1.1    Pipette the following reagents into suitable glass vials:

  Blank Test-1 Test-2 Test-3
Purified Water 1.00 0.90 0.50 -----
Reagent 7.3.3 (Ca) ----- 0.10 0.50 1.00
Reagent 7.3.4 (Control) 5.00 5.00 5.00 5.00

7.4.1.2    Mix by inversion and immediately filter each through Millex – HV Durapore PVDF25 mm diameter acrodisc, 0.45μm pore size, syringe filter, Sigma-Aldrich Stock Number Z227366.

7.4.1.3    Using a suitable spectrophotometer, record the A575nm against purified water.

7.4.2    Sample Reaction:

7.4.2.1    Pipette the following reagents into suitable glass vials:

  Blank Test-1 Test-2 Test-3
Purified Water 1.00 0.90 0.50 -----
Reagent 7.3.3 (Ca) ----- 0.10 0.50 1.00
Reagent 7.3.5 (Sample) 5.00 5.00 5.00 5.00

7.4.2.2    Mix by inversion and immediately filter each through Millex – HV Durapore PVDF25 mm diameter acrodisc, 0.45μm pore size, syringe filter, Sigma-Aldrich Stock Number Z227366.

7.4.2.3    Using a suitable spectrophotometer, record the A575nm against purified water.

7.5 SUITABILITY CRITERIA

    The A575nm for both control and sample must increase with increased calcium concentration. The difference between the absorbance with the reaction containing zero micrograms calcium and the reaction containing one microgram of calcium must be >=0.005.

8. REFERENCES & ATTACHMENTS

NA

9. APPROVAL

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