30 1.

08 Nitrogen Determination (Semimicro-Kjeldahl Method) / General Tests JP XVI

dium sulfide TS in acidic solution, as their quantity is ex- (95) (1 in 10), fire the ethanol to burn, and carbonize by
pressed in terms of the quantity of lead (Pb). gradual heating. Cool, add 1 mL of sulfuric acid, heat care-
In each monograph, the permissible limit for heavy metals fully, and incinerate by ignition between 5009 C and 6009C.
(as Pb) is described in terms of ppm in parentheses. If a carbonized substance remains, moisten with a small
amount of sulfuric acid, and incinerate by ignition. Cool,
1. Preparation of test solutions and control solutions
dissolve the residue in 3 mL of hydrochloric acid, evaporate
Unless otherwise specified, test solutions and control solu-
on a water bath to dryness, wet the residue with 3 drops of
tions are prepared as directed in the following:
hydrochloric acid, add 10 mL of water, and dissolve by
1.1. Method 1
warming. Add 1 drop of phenolphthalein TS, add ammonia
Place an amount of the sample, directed in the mono-
TS dropwise until a pale red color develops, then add 2 mL
graph, in a Nessler tube. Dissolve in water to make 40 mL.
of dilute acetic acid, filter if necessary, wash with 10 mL of
Add 2 mL of dilute acetic acid and water to make 50 mL,
water, transfer the filtrate and the washing to a Nessler tube,
and designate it as the test solution.
add water to make 50 mL, and use this solution as the test
The control solution is prepared by placing the volume of
solution.
Standard Lead Solution directed in the monograph in a
The control solution is prepared as follows: Take 10 mL
Nessler tube, and adding 2 mL of dilute acetic acid and
of a solution of magnesium nitrate hexahydrate in ethanol
water to make 50 mL.
(95) (1 in 10), and fire the ethanol to burn. Cool, add 1 mL
1.2. Method 2
of sulfuric acid, heat carefully, and ignite between 5009C
Place an amount of the sample, directed in the mono-
and 6009C. Cool, and add 3 mL of hydrochloric acid. Here-
graph, in a quartz or porcelain crucible, cover loosely with a
inafter, proceed as directed in the test solution, then add the
lid, and carbonize by gentle ignition. After cooling, add 2
volume of Standard Lead Solution directed in the mono-
mL of nitric acid and 5 drops of sulfuric acid, heat cau-
graph and water to make 50 mL.
tiously until white fumes are no longer evolved, and inciner-
ate by ignition between 5009C and 6009C. Cool, add 2 mL 2. Procedure
of hydrochloric acid, evaporate to dryness on a water bath, Add 1 drop of sodium sulfide TS to each of the test solu-
moisten the residue with 3 drops of hydrochloric acid, add tion and the control solution, mix thoroughly, and allow to
10 mL of hot water, and warm for 2 minutes. Then add 1 stand for 5 minutes. Then compare the colors of both solu-
drop of phenolphthalein TS, add ammonia TS dropwise tions by viewing the tubes downward or transversely against
until the solution develops a pale red color, add 2 mL of a white background. The test solution has no more color
dilute acetic acid, filter if necessary, and wash with 10 mL of than the control solution.
water. Transfer the filtrate and washings to a Nessler tube,
and add water to make 50 mL. Designate it as the test solu-
tion.
The control solution is prepared as follows: Evaporate a 1.08 Nitrogen Determination
mixture of 2 mL of nitric acid, 5 drops of sulfuric acid and 2 (Semimicro-Kjeldahl Method)
mL of hydrochloric acid on a water bath, further evaporate
to dryness on a sand bath, and moisten the residue with 3 Nitrogen Determination is a method to determine nitrogen
drops of hydrochloric acid. Hereinafter, proceed as directed in an organic substance in which the nitrogen is converted
in the test solution, then add the volume of Standard Lead into ammonia nitrogen by thermal decomposition of the or-
Solution directed in the monograph and water to make 50 ganic substance with sulfuric acid, and the ammonia liber-
mL. ated by alkali and trapped by distillation with steam is deter-
1.3. Method 3 mined by titration.
Place an amount of the sample, directed in the mono-
1. Apparatus
graph, in a quartz or porcelain crucible, heat cautiously,
Use the apparatus illustrated in Fig. 1.08-1. It is thor-
gently at first, and then incinerate by ignition between 5009C
oughly constructed of hard glass, and ground glass surfaces
and 6009C. After cooling, add 1 mL of aqua regia, evapo-
may be used for joints. All rubber parts used in the appa-
rate to dryness on a water bath, moisten the residue with 3
ratus should be boiled for 10 to 30 minutes in sodium hy-
drops of hydrochloric acid, add 10 mL of hot water, and
droxide TS and for 30 to 60 minutes in water, and finally
warm for 2 minutes. Add 1 drop of phenolphthalein TS, add
washed thoroughly with water before use.
ammonia TS dropwise until the solution develops a pale red
Alternatively, apparatus can be used in which some of the
color, add 2 mL of dilute acetic acid, filter if necessary, wash
procedures, such as digestion of organic substances, distilla-
with 10 mL of water, transfer the filtrate and washings to a
tion of the liberated ammonia, and endpoint detection
Nessler tube, and add water to make 50 mL. Designate it as
methods in titrimetry (e.g., potentiometric titration or titra-
the test solution.
tion by colorimeter) are automated.
The control solution is prepared as follows: Evaporate 1
mL of aqua regia to dryness on a water bath. Hereinafter, 2. System suitability
proceed as directed for the test solution, and add the volume If an automated apparatus is used, it is necessary to con-
of Standard Lead Solution directed in the monograph and firm periodically the suitability of the apparatus according to
water to make 50 mL. the following method:
1.4. Method 4 Weigh accurately about 1.7 g of amidosulfuric acid (stand-
Place an amount of the sample, directed in the mono- ard reagent), previously dried in a desiccator (in vacuum,
graph, in a platinum or porcelain crucible, mix with 10 mL silica gel) for about 48 hours, dissolve in water to make ex-
of a solution of magnesium nitrate hexahydrate in ethanol actly 200 mL. Pipet 2 mL of this solution, and transfer to a
JP XVI General Tests / 1.09 Qualitative Tests 31
the vapor of sulfuric acid is condensed at the neck of the
flask, until the solution changes through a blue and clear to
a vivid green and clear, and the inside wall of the flask is free
from a carbonaceous material. If necessary, add a small
quantity of hydrogen peroxide (30) after cooling, and heat
again. After cooling, add cautiously 20 mL of water, cool
the solution, and connect the flask to the distillation appa-
ratus (Fig. 1.08-1) washed beforehand by passing stream
through it. To the absorption flask K add 15 mL of boric
acid solution (1 in 25), 3 drops of bromocresol green-methyl
red TS and sufficient water to immerse the lower end of the
condenser tube J. Add 30 mL of sodium hydroxide solution
(2 in 5) through the funnel F, rinse cautiously the funnel with
10 mL of water, lose the clamp attached to the rubber tubing
G, then begin the distillation with stream, and continue until
the distillate measures 80 to 100 mL. Remove the absorption
flask from the lower end of the condenser tube J, rinsing the
end part with a small quantity of water, and titrate <2.50> the
distillate with 0.005 mol/L sulfuric acid VS until the color of
the solution changes from green through pale grayish blue to
pale grayish red-purple. Perform a blank determination in
the same manner, and make any necessary correction.
Each mL of 0.005 mol/L sulfuric acid VS
＝ 0.1401 mg of N
If an automated apparatus is used, proceed as directed in
the instrumental procedure.

1.09 Qualitative Tests

Qualitative Tests are applied to the identification of drugs
and are done generally with quantities of 2 to 5 mL of the
test solution.
Acetate
(1) When warmed with diluted sulfric acid (1 in 2), ace-
Fig. 1.08-1 tates evolve the odor of acetic acid.
(2) When an acetate is warmed with sulfuric acid and a
small quantity of ethanol (95), the odor of ethyl acetate is
digestion flask. When the test is performed as directed in the evolved.
instrumental manual the nitrogen content (z) in amidosul- (3) Neutral solutions of acetates produce a red-brown
furic acid should be determined between 14.2z and 14.6z. color with iron (III) chloride TS, and a red-brown precipitate
when boiled. The precipitate dissolves and the color of the
3. Reagents, Test Solutions
solution changes to yellow upon addition of hydrochloric
Decomposition accelerator: Unless otherwise specified,
acid.
use 1 g of a powdered mixture of 10 g of potassium sulfate
and 1 g of cupper (II) sulfate pentahydrate. The composition Aluminum salt
and amount of the digestion accelerator may be modified if (1) Solutions of aluminum salts, when treated with am-
it is confirmed that the modified one give almost the same monium chloride TS and ammonia TS, yield a gelatinous,
results using the sample as those obtained from the conven- white precipitate which does not dissolve in an excess of am-
tional catalyst. monia TS.
(2) Solutions of aluminum salts, when treated with so-
4. Procedure
dium hydroxide TS, yield a gelatinous, white precipitate
Unless otherwise specified, proceed by the following
which dissolves in an excess of the reagent.
method. Weigh accurately or pipet a quantity of the sample
(3) Solutions of aluminum salts, when treated with so-
corresponding to 2 to 3 mg of nitrogen (N:14.01), and place
dium sulfide TS, yield a gelatinous, white precipitate which
in the Kjeldahl flask A. Add the decomposition accelerator
dissolves in an excess of the reagent.
and wash down any adhering sample from the neck of the
(4) Add ammonia TS to solutions of aluminum salts
flask with a small quantity of water. Add 7 mL of sulfuric
until a gelatinous, white precipitate is produced. The color
acid, allowing it to flow down the inside wall of the flask.
of the precipitate changes to red upon addition of 5 drops of
Then, while shaking the flask, add cautiously 1 mL of
alizarin red S TS.
hydrogen peroxide (30) drop by drop along the inside wall of
the flask. Heat the flask gradually, then heat so strong that