2.4. LIMIT TESTS
2.4.1. AMMONIUM
METHOD A
Reagents: alkaline potassium tetraiodomercurate solution (Nessler´s reagent) in alkaline solution of
NaOH
Dissolve the prescribed quantity of the substance to be examined in 14 mL of water R in a test-tube,
make alkaline if necessary by the addition of dilute sodium hydroxide solution R and dilute to 15 mL
with water R. To the solution add 0.3 mL of alkaline potassium tetraiodomercurate solution R. Prepare
a standard by mixing 10 mL of ammonium standard solution (1 ppm NH4) R with 5 mL of water R and
0.3 mL of alkaline potassium tetraiodomercurate solution R.
After 5 min, any yellow colour in the test solution is not more intense than that in the standard.
NH4
+
+ (x) K2[HgI4] Hg2I3NH2
I
Hg
I
Hg
I
NH2
{[Hg2N]+I-. 2H2O}x
High molecular base, three- dimensional structure
Orange to red- brown colour
2.4.2. ARSENIC
METHOD B
Reagents: hydrochloric acid, potassium iodide, hypophosphorous reagent
Introduce the prescribed quantity of the substance to be examined into a test-tube containing 4 mL of
hydrochloric acid R and about 5 mg of potassium iodide R and add 3 mL of hypophosphorous reagent
R. Heat the mixture on a water-bath for 15 min, shaking occasionally. Prepare a standard in the same
manner, using 0.5 mL of arsenic standard solution (10 ppm As) R.
After heating on the water-bath, any colour in the test solution is not more intense than that in the
standard.
AsO4
3+
2I-
AsO3
3+
I2
2 AsO3
3+
3 H3PO2 2 As + 3 H3PO4
Dark colour to black precipitate
2.4.3. CALCIUM
Reagents: ammonium oxalate, dilute acetic acid
All solutions used for this test should be prepared with distilled water R.
To 0.2 mL of alcoholic calcium standard solution (100 ppm Ca) R, add 1 mL of ammonium oxalate
solution R. After 1 min, add a mixture of 1 mL of dilute acetic acid R and 15 mL of a solution
containing the prescribed quantity of the substance to be examined and shake. Prepare a standard in
the same manner using a mixture of 10 mL of aqueous calcium standard solution (10 ppm Ca) R, 1
mL of dilute acetic acid R and 5 mL of distilled water R.
After 15 min, any opalescence in the test solution is not more intense than that in the standard.
Ca2+
+ (NH4)2(COO)2 Ca(COO)2 + 2NH4
+
White precipitate
2.4.7. MAGNESIUM AND ALKALINE-EARTH METALS
To 200 mL of water R add 0.1 g of hydroxylamine hydrochloride R, 10 mL of ammonium chloride
buffer solution pH 10.0 R, 1 mL of 0.1 M zinc sulfate and about 15 mg of mordant black 11 triturate R.
Heat to about 40 °C. Titrate with 0.1 M sodium edetate until the violet colour changes to full blue.
To the solution add the prescribed quantity of the substance to be examined dissolved in 100 mL
of water R or use the prescribed solution. If the colour of the solution changes to violet, titrate with 0.1
M sodium edetate until the full blue colour is again obtained.
The volume of 0.1 M sodium edetate used in the second titration does not exceed the prescribed
quantity.
2.4.4. CHLORIDES
Reagents: dilute nitric acid, silver nitrate
To 15 mL of the prescribed solution add 1 mL of dilute nitric acid R and pour the mixture as a single
addition into a test-tube containing 1 mL of silver nitrate solution R2. Prepare a standard in the same
manner using 10 mL of chloride standard solution (5 ppm Cl) R and 5 mL of water R. Examine the
tubes laterally against a black background.
After standing for 5 min protected from light, any opalescence in the test solution is not more intense
than that in the standard.
Cl+
Ag+
AgCl
White precipitate (turning grey- violet)
2.4.8. HEAVY METALS
METHOD A
Reagents: buffer solution pH 3.5, thioacetamide reagent
Test solution. 12 mL of the prescribed aqueous solution of the substance to be examined.
Reference solution (standard). A mixture of 10 mL of lead standard solution (1 ppm Pb) R or lead
standard solution (2 ppm Pb) R, as prescribed, and 2 mL of the prescribed aqueous solution of the
substance to be examined.
Blank solution. A mixture of 10 mL of water R and 2 mL of the prescribed aqueous solution of the
substance to be examined.
To each solution, add 2 mL of buffer solution pH 3.5 R. Mix and add to 1.2 mL of thioacetamide
reagent R. Mix immediately. Examine the solutions after 2 min.
System suitability: the reference solution shows a slight brown colour compared to the blank solution.
Result: any brown colour in the test solution is not more intense than that in the reference solution.
H3C-CS-NH2 + 2H2O H3C-COONH4 + H2S
M2+
+ S2-
MS
Dark precipitate
2.4.9. IRON
Reagents: thioglycollic acid R, citric acid, ammonia R
Dissolve the prescribed quantity of the substance to be examined in water R and dilute to 10 mL with
the same solvent or use 10 mL of the prescribed solution. Add 2 mL of a 200 g/L solution of citric acid
R and 0.1 mL of thioglycollic acid R. Mix, make alkaline with ammonia R and dilute to 20 mL with
water R. Prepare a standard in the same manner, using 10 mL of iron standard solution (1 ppm Fe) R.
After 5 min, any pink colour in the test solution is not more intense than that in the standard.
Fe2+
+ 2 HS-CH2COOH [Fe(S-CH2COO)2]2+
4H+
S
S
CH2
CH2
COOH
COOHCH2
COOH -2H+
2 Fe2+ +2 Fe3+ + 2 HS
complex Fe(II)- thioglycolate oxidation to Fe (III)
colourless or light yellow pink or red
2.4.11. PHOSPHATES
Reagents: sulfomolybdic reagent, stannous chloride
To 100 mL of the solution prepared and, if necessary, neutralised as prescribed add 4 mL of
sulfomolybdic reagent R3. Shake and add 0.1 mL of stannous chloride solution R1. Prepare a
standard in the same manner using 2 mL of phosphate standard solution (5 ppm PO4) R and 98 mL of
water R. After 10 min, compare the colours using 20 mL of each solution.
Any colour in the test solution is not more intense than that in the standard.
(NH4)6Mo7O24.4H20 + PO4
3+
H2SO4 H3[P(Mo3O10)4)]
Phosphomolybdic acid
+ SnCl2 reduction to molybdenum blue
Reagents: sodium tetraphenylborate
To 10 mL of the prescribed solution add 2 mL of a freshly prepared 10 g/L solution of sodium
tetraphenylborate R. Prepare a standard in the same manner using a mixture of 5 mL of potassium
standard solution (20 ppm K) R and 5 mL of water R.
After 5 min, any opalescence in the test solution is not more intense than that in the standard.
Na[B(C6H5)4] + K+
K[B(C6H5)4] + Na+
White precipitate
2.4.13. SULFATES
Reagents: barium chloride, acetic acid
All solutions used for this test must be prepared with distilled water R.
Add 3 mL of a 250 g/L solution of barium chloride R to 4.5 mL of sulfate standard solution (10 ppm
SO4) R1. Shake and allow to stand for 1 min. To 2.5 mL of this suspension, add 15 mL of the solution
to be examined and 0.5 mL of acetic acid R. Prepare a standard in the same manner using 15 mL of
sulfate standard solution (10 ppm SO4) R instead of the solution to be examined.
After 5 min, any opalescence in the test solution is not more intense than that in the standard.
SO4
2+
Ba
2+
BaSO4
White precipitate