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Zinc Sulfate

ZnSO4xH2O.

Sulfuric acid, zinc salt (1:1), hydrate.

Zinc sulfate (1:1) monohydrate 179.46

Zinc sulfate (1:1) heptahydrate 287.56 [7446-20-0].

Anhydrous 161.46 [7733-02-0].

Zinc Sulfate contains one or seven molecules of water of hydration. The

monohydrate contains not less than 89.0 percent and not more than 90.4 percent

of ZnSO4, corresponding to not less than 99.0 percent and not more than 100.5

percent of ZnSO4H2O, and the heptahydrate contains not less than 55.6

percent and not more than 61.0 percent of ZnSO4, corresponding to not less

than 99.0 percent and not more than 108.7 percent of ZnSO47H2O.

Packaging and storage Preserve in tight containers.

Labeling The label indicates whether it is the monohydrate or the

heptahydrate. Label any oral or parenteral preparations containing Zinc Sulfate to

state the content of elemental zinc.

Identification A solution of it responds to the tests for Zinc 191 and for

Sulfate 191 .

Acidity A solution containing the equivalent of 28 mg of ZnSO4 per mL is not

colored pink by methyl orange TS.
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Arsenic, Method I 211 Prepare a Test Preparation by dissolving a portion

equivalent to 215 mg of ZnSO4 in 35 mL of water: the limit is 14 ppm.

Lead 251 Dissolve a portion equivalent to 0.25 g of ZnSO4 in 5 mL of

water, and transfer the solution to a color-comparison tube (A). Add 10 mL of

Potassium Cyanide Solution (1 in 10), mix, and allow the mixture to become

clear. In a similar, matched color-comparison tube (B) place 5 mL of water, and

add 0.50 mL of Standard Lead Solution (see Heavy Metals 231 ) and 10 mL of

Potassium Cyanide Solution (1 in 10). Add to the solution in each tube 0.1 mL of

sodium sulfide TS. Mix the contents of each tube, and allow to stand for 5

minutes: viewed downward over a white surface, the solution in tube A is no

darker than that in tube B. The lead limit is 0.002%.

Alkalies and alkaline earths Dissolve the equivalent of 1.12 g of ZnSO4 in

about 150 mL of water contained in a 200-mL volumetric flask. Precipitate the

zinc completely by means ofammonium sulfide TS, and dilute with water to

volume. Mix, and filter through a dry filter, rejecting the first portion of the filtrate.

To 100 mL of the subsequent filtrate add a few drops of sulfuric acid, evaporate

to dryness in a tared dish, and ignite: the weight of the residue does not exceed 5

mg (0.9%).

Residual solvents 467 : meets the requirements.

(Official January 1, 2007)

Assay Dissolve an accurately weighed quantity of Zinc Sulfate, equivalent to

about 170 mg of ZnSO4, in 100 mL of water. Add 5 mL of ammoniaammonium

chloride buffer TS and 0.1 mL oferiochrome black TS, and titrate with 0.05 M

edetate disodium VS until the solution is deep blue in color. Each mL of 0.05 M

edetate disodium is equivalent to 8.072 mg of ZnSO4.

Auxiliary Information Staff Liaison : Feiwen Mao, M.S., Senior Scientific

Associate
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Expert Committee : (MDOOD05) Monograph Development-Ophthalmics

Oncologics and Dermatologicals

USP29NF24 Page 2292

Phone Number : 1-301-816-8320

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211 ARSENIC

This procedure is designed to determine the presence of trace amounts of

arsenic (As) by converting the arsenic in a substance under test to arsine, which

is then passed through a solution of silver diethyldithiocarbamate to form a red

complex. The red color so produced is compared, either visually or

spectrophotometrically, to the color produced similarly in a control containing an

amount of arsenic equivalent to the limit given in the individual monograph. Limits

are stated in terms of arsenic (As). The content of arsenic does not exceed the

limit given in the individual monograph.

Two methods are provided, the methods differing only in the preliminary

treatment of the test substance and the standard. Generally, Method I is used for

inorganic materials, while Method II is used for organic materials.

Apparatus

The apparatus (see illustration) consists of an arsine generator (a) fitted with a

scrubber unit (c) and an absorber tube (e) with standard-taper or ground glass

ball-and-socket joints (b and d) between the units. However, any other suitable
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apparatus, embodying the principle of the assembly described and illustrated,

may be used.

Arsenic Test Apparatus

Arsenic Trioxide Stock Solution Dissolve 132.0 mg of arsenic trioxide,
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previously dried at 105 for 1 hour and accurately weighed, in 5 mL of sodium

hydroxide solution (1 in 5) in a 1000-mL volumetric flask. Neutralize the solution

with 2 N sulfuric acid, add 10 mL more of 2 N sulfuric acid, then add recently

boiled and cooled water to volume, and mix.

Standard Arsenic Solution Transfer 10.0 mL of Arsenic Trioxide Stock Solution

to a 1000-mL volumetric flask, add 10 mL of 2 N sulfuric acid, then add recently

boiled and cooled water to volume, and mix. Each mL of Standard Arsenic

Solution contains the equivalent of 1 g of arsenic (As). Keep this solution in an

all-glass container, and use within 3 days.

METHOD I

Standard Preparation Pipet 3.0 mL of Standard Arsenic Solution into a

generator flask, and dilute with water to 35 mL.

Test Preparation Unless otherwise directed in the individual monograph,

transfer to the generator flask the quantity, in g, of the test substance calculated

by the formula:

3.0/L,

in which L is the arsenic limit in ppm, dissolve in water, and dilute with water to

35 mL.

Procedure Treat the Standard Preparation and the Test Preparation similarly

as follows. Add 20 mL of 7 N sulfuric acid, 2 mL ofpotassium iodide TS, 0.5 mL

of stronger acid stannous chloride TS, and 1 mL of isopropyl alcohol, and mix.

Allow to stand at room temperature for 30 minutes. Pack the scrubber tube (c)

with two pledgets of cotton that have been soaked in saturated lead acetate

solution, freed from excess solution by expression, and dried in vacuum at room

temperature, leaving a 2-mm space between the two pledgets. Lubricate the

joints (b and d) with a suitable stopcock grease designed for use with organic
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solvents, and connect the scrubber unit to the absorber tube (e). Transfer 3.0 mL

ofsilver diethyldithiocarbamate TS to the absorber tube. Add 3.0 g of granular

zinc (No. 20 mesh) to the mixture in the flask, immediately connect the

assembled scrubber unit, and allow the evolution of hydrogen and the color

development to proceed at room temperature for 45 minutes, swirling the flask

gently at 10-minute intervals. Disconnect the absorber tube from the generator

and scrubber units, and transfer the absorbing solution to a 1-cm absorption cell.

Any red color produced by the Test Preparation does not exceed that produced

by the Standard Preparation. If necessary or desirable, determine the

absorbance at the wavelength of maximum absorbance between 535 and 540

nm, with a suitable spectrophotometer or colorimeter, using silver

diethyldithiocarbamate TS as the blank.

Interfering Chemicals Metals or salts of metals, such as chromium, cobalt,

copper, mercury, molybdenum, nickel, palladium, and silver, may interfere with

the evolution of arsine. Antimony, which forms stibine, produces a positive

interference in the color development with silver diethyldithiocarbamate TS; when

the presence of antimony is suspected, the red colors produced in the two silver

diethyldithiocarbamate solutions may be compared at the wavelength of

maximum absorbance between 535 and 540 nm, with a suitable colorimeter,

since at this wavelength the interference due to stibine is negligible.

METHOD II

NOTES

(1) CautionSome substances may react with explosive violence when digested

with hydrogen peroxide. Exercise safety precautions at all times.

(2) If halogen-containing compounds are present, use a lower temperature while

heating the test specimen with sulfuric acid, avoid boiling the mixture, and add

the hydrogen peroxide with caution, before charring begins, to prevent loss of

trivalent arsenic.
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(3) If the test substance reacts too rapidly and begins charring with 5 mL of

sulfuric acid before heating, use instead 10 mL of cooled dilute sulfuric acid (1 in

2), and add a few drops of the hydrogen peroxide before heating.

Standard Preparation Pipet 3.0 mL of Standard Arsenic Solution into a

generator flask, add 2 mL of sulfuric acid, mix, and add the total amount of 30

percent hydrogen peroxide used in preparing the Test Preparation. Heat the

mixture to strong fuming, cool, add cautiously 10 mL of water, and again heat to

strong fumes. Repeat this procedure with another 10 mL of water to remove any

traces of hydrogen peroxide. Cool, and dilute with water to 35 mL.

Test Preparation Unless otherwise directed in the individual monograph,

transfer to a generator flask the quantity, in g, of the test substance calculated by

the formula:

3.0 / L,

in which L is the arsenic limit in ppm. Add 5 mL of sulfuric acid and a few glass

beads, and digest in a fume hood, preferably on a hot plate and at a temperature

not exceeding 120 , until charring begins. (Additional sulfuric acid may be

necessary to wet some specimens completely, but the total volume added shouldnot exceed 10 mL.) Cautiously add, dropwise, 30 percent hydrogen peroxide,

allowing the reaction to subside and again heating between drops. Add the first

few drops very slowly with sufficient mixing, in order to prevent a rapid reaction.

Discontinue heating if foaming becomes excessive. When the reaction has

abated, heat cautiously, rotating the flask occasionally to prevent the specimen

from caking on glass exposed to the heating unit. Maintain oxidizing conditions at

all times during the digestion by adding small quantities of the hydrogen peroxide

solution whenever the mixture turns brown or darkens. Continue the digestion

until the organic matter is destroyed, gradually raising the temperature of the hot

plate until fumes of sulfur trioxide are copiously evolved, and the solution

becomes colorless or retains only a light straw color. Cool, add cautiously 10 mL

of water, mix, and again evaporate to strong fuming, repeating this procedure to


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remove any trace of hydrogen peroxide. Cool, add cautiously 10 mL of water,

wash the sides of the flask with a few mL of water, and dilute with water to 35

mL.

Procedure Proceed as directed for Procedure under Method I.

Interfering Chemicals See Interfering Chemicals underMethod I.

Auxiliary Information Staff Liaison : Kahkashan Zaidi, Ph.D., Senior Scientific

Associate

Expert Committee : (GC05) General Chapters 05

USP29NF24 Page 2554

Phone Number : 1-301-816-8269

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251 LEAD

The imposition of stringent limits on the amounts of lead that may be present in

pharmaceutical products has resulted in the use of two methods, of which the

one set forth following depends upon extraction of lead by solutions of dithizone.

For determination of the content of heavy metals generally, expressed as a lead

equivalent, see Heavy Metals 231 .

Select all reagents for this test to have as low a content of lead as practicable,

and store all reagent solutions in containers of borosilicate glass. Rinse

thoroughly all glassware with warm dilute nitric acid (1 in 2), followed by water.
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Special Reagents

AMMONIA-CYANIDE SOLUTION Dissolve 2 g of potassium cyanide in 15 mL

of ammonium hydroxide, and dilute with water to 100 mL.

AMMONIUM CITRATE SOLUTION Dissolve 40 g of citric acid in 90 mL of

water. Add 2 or 3 drops of phenol red TS, then cautiously add ammonium

hydroxide until the solution acquires a reddish color. Remove any lead that may

be present by extracting the solution with 20-mL portions of Dithizone Extraction

Solution (see below), until the dithizone solution retains its orange-green color.

DILUTED STANDARD LEAD SOLUTION Dilute an accurately measured

volume of Standard Lead Solution (see Heavy Metals 231 ) [containing 10 g

of lead per mL], with 9 volumes of dilute nitric acid (1 in 100) to obtain a solution

that contains 1 g of lead per mL.

DITHIZONE EXTRACTION SOLUTION Dissolve 30 mg of dithizone in 1000

mL of chloroform, and add 5 mL of alcohol. Store the solution in a refrigerator.

Before use, shake a suitable volume of the dithizone extraction solution with

about half its volume of dilute nitric acid (1 in 100), discarding the nitric acid.

HYDROXYLAMINE HYDROCHLORIDE SOLUTION Dissolve 20 g of

hydroxylamine hydrochloride in sufficient water to make approximately 65 mL.

Transfer to a separator, add 5 drops ofthymol blue TS, then add ammonium

hydroxide until the solution assumes a yellow color. Add 10 mL of sodium

diethyldithiocarbamate solution (1 in 25), mix, and allow to stand for 5 minutes.

Extract this solution with successive 10- to 15-mL portions of chloroform until a 5-

mL portion of the chloroform extract does not assume a yellow color when

shaken with cupric sulfate TS. Add 3 N hydrochloric acid until the solution is pink

(if necessary, add 1 or 2 drops more ofthymol blue TS), and then dilute with

water to 100 mL.
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POTASSIUM CYANIDE SOLUTION Dissolve 50 g of potassium cyanide in

sufficient water to make 100 mL. Remove the lead from this solution by

extraction with successive portions of Dithizone Extraction Solution, as described

under Ammonium Citrate Solution above, then extract any dithizone remaining in

the cyanide solution by shaking with chloroform. Finally dilute the cyanide

solution with sufficient water so that each 100 mL contains 10 g of potassium

cyanide.

STANDARD DITHIZONE SOLUTION Dissolve 10 mg of dithizone in 1000 mL

of chloroform. Keep the solution in a glass-stoppered, lead-free bottle, suitably

wrapped to protect it from light, and store in a refrigerator.

Test Preparation [NOTEIf, in the following preparation, the substance under

test reacts too rapidly and begins charring with 5 mL of sulfuric acid before

heating, use instead 10 mL of cooled dilute sulfuric acid (1 in 2), and add a few

drops of the hydrogen peroxide before heating.] Where the monograph does not

specify preparation of a solution, prepare a Test Preparation as follows. [Caution

Exercise safety precautions in this procedure, as some substances may react

with explosive violence when digested with hydrogen peroxide. ] Transfer 1.0 g of

the substance under test to a suitable flask, add 5 mL of sulfuric acid and a few

glass beads, and digest on a hot plate in a hood until charring begins. Other

suitable means of heating may be substituted. (Add additional sulfuric acid, if

necessary, to wet the substance completely, but do not add more than a total of

10 mL.) Add, dropwise and with caution, 30 percent hydrogen peroxide, allowing

the reaction to subside and again heating between drops. Add the first few drops

very slowly, mix carefully to prevent a rapid reaction, and discontinue heating if

foaming becomes excessive. Swirl the solution in the flask to prevent unreacted

substance from caking on the walls of the flask. [NOTEAdd peroxide whenever

the mixture turns brown or darkens.] Continue the digestion until the substance is

completely destroyed, copious fumes of sulfur trioxide are evolved, and the

solution is colorless. Cool, cautiously add 10 mL of water, evaporate until sulfur


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trioxide again is evolved, and cool. Repeat this procedure with another 10 mL of

water to remove any traces of hydrogen peroxide. Cautiously dilute with 10 mL of

water, and cool.

Procedure Transfer the Test Preparation, rinsing with 10 mL of water, or the

volume of the prepared sample specified in the monograph to a separator, and,

unless otherwise directed in the monograph, add 6 mL of Ammonium Citrate

Solution and 2 mL of Hydroxylamine Hydrochloride Solution. (For the

determination of lead in iron salts use 10 mL of Ammonium Citrate Solution.) Add

2 drops of phenol red TS, and make the solution just alkaline (red in color) by the

addition of ammonium hydroxide. Cool the solution if necessary, and add 2 mL of

Potassium Cyanide Solution. Immediately extract the solution with 5-mL portions

of Dithizone Extraction Solution, draining off each extract into another separator,

until the dithizone solution retains its green color. Shake the combined dithizone

solutions for 30 seconds with 20 mL of dilute nitric acid (1 in 100), and discard

the chloroform layer. Add to the acid solution 5.0 mL of Standard Dithizone

Solution and 4 mL of Ammonia-Cyanide Solution, and shake for 30 seconds: the

color of the chloroform layer is of no deeper shade of violet than that of a control

made with a volume of Diluted Standard Lead Solution equivalent to the amount

of lead permitted in the sample under examination, and the same quantities of

the same reagents and in the same manner as in the test with the sample.


Associate


USP29NF24 Page 2558

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231 HEAVY METALS

This test is provided to demonstrate that the content of metallic impurities that

are colored by sulfide ion, under the specified test conditions, does not exceed

the Heavy metals limit specified in the individual monograph in percentage (by

weight) of lead in the test substance, as determined by concomitant visual

comparison (see Visual Comparison in the section Procedure under

Spectrophotometry and Light-Scattering 851 ) with a control prepared from a

Standard Lead Solution. [NOTESubstances that typically will respond to this

test are lead, mercury, bismuth, arsenic, antimony, tin, cadmium, silver, copper,

and molybdenum.]

Determine the amount of heavy metals by Method I, unless otherwise specified in

the individual monograph. Method I is used for substances that yield clear,

colorless preparations under the specified test conditions. Method II is used for

substances that do not yield clear, colorless preparations under the test

conditions specified for Method I, or for substances that, by virtue of their

complex nature, interfere with the precipitation of metals by sulfide ion, or for

fixed and volatile oils. Method III, a wet-digestion method, is used only in those

cases where neither Method I nor Method II can be used.

Special Reagents

Lead Nitrate Stock Solution Dissolve 159.8 mg of lead nitrate in 100 mL of

water to which has been added 1 mL of nitric acid, then dilute with water to 1000

mL. Prepare and store this solution in glass containers free from soluble lead

salts.
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Standard Lead Solution On the day of use, dilute 10.0 mL of Lead Nitrate

Stock Solution with water to 100.0 mL. Each mL of Standard Lead Solution

contains the equivalent of 10 g of lead. A comparison solution prepared on the

basis of 100 L of Standard Lead Solution per g of substance being tested

contains the equivalent of 1 part of lead per million parts of substance being

tested.

Method I

pH 3.5 Acetate Buffer Dissolve 25.0 g of ammonium acetate in 25 mL of water,

and add 38.0 mL of 6 N hydrochloric acid. Adjust, if necessary, with 6 N

ammonium hydroxide or 6 N hydrochloric acid to a pH of 3.5, dilute with water to

100 mL, and mix.

Standard Preparation Into a 50-mL color-comparison tube pipet 2 mL of

Standard Lead Solution (20 g of Pb), and dilute with water to 25 mL. Using a pH

meter or short-range pH indicator paper as external indicator, adjust with 1 N

acetic acid or 6 N ammonium hydroxide to a pH between 3.0 and 4.0, dilute with

water to 40 mL, and mix.

Test Preparation Into a 50-mL color-comparison tube place 25 mL of the

solution prepared for the test as directed in the individual monograph; or, using

the designated volume of acid where specified in the individual monograph,

dissolve in and dilute with water to 25 mL the quantity, in g, of the substance to

be tested, as calculated by the formula:

2.0/(1000L),

in which L is the Heavy metals limit, as a percentage. Using a pH meter or short-

range pH indicator paper as external indicator, adjust with 1 N acetic acid or 6 N

ammonium hydroxide to a pH between 3.0 and 4.0, dilute with water to 40 mL,

and mix.


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Monitor Preparation Into a third 50-mL color-comparison tube place 25 mL of a

solution prepared as directed for Test Preparation, and add 2.0 mL of Standard

Lead Solution. Using a pH meter or short-range pH indicator paper as external

indicator, adjust with 1 N acetic acid or 6 N ammonium hydroxide to a pH

between 3.0 and 4.0, dilute with water to 40 mL, and mix.

Procedure To each of the three tubes containing the Standard Preparation, the

Test Preparation, and the Monitor Preparation, add 2 mL of pH 3.5 Acetate

Buffer, then add 1.2 mL of thioacetamideglycerin base TS, dilute with water to

50 mL, mix, allow to stand for 2 minutes, and view downward over a white

surface *: the color of the solution from the Test Preparation is not darker than

that of the solution from the Standard Preparation, and the color of the solution

from the Monitor Preparation is equal to or darker than that of the solution from

the Standard Preparation. [NOTEIf the color of the Monitor Preparation is

lighter than that of the Standard Preparation, use Method II instead of Method I

for the substance being tested.]

Method II

NOTEThis method does not recover mercury.

pH 3.5 Acetate Buffer Prepare as directed under Method I.

Standard Preparation Pipet 4 mL of the Standard Lead Solution into a suitable

test tube, and add 10 mL of 6 N hydrochloric acid.

Test Preparation Use a quantity, in g, of the substance to be tested as

calculated by the formula:

4.0/(1000L),

in which L is the Heavy metals limit, as a percentage. Transfer the weighed

quantity of the substance to a suitable crucible, add sufficient sulfuric acid to wet

the substance, and carefully ignite at a low temperature until thoroughly charred.

(The crucible may be loosely covered with a suitable lid during the charring.) Add
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to the carbonized mass 2 mL of nitric acid and 5 drops of sulfuric acid, and heat

cautiously until white fumes no longer are evolved. Ignite, preferably in a muffle

furnace, at 500 to 600 , until the carbon is completely burned off (no longer than

2 hours). If carbon remains, allow the residue to cool, add a few drops of sulfuricacid, evaporate, and ignite again. Cool, add 5 mL of 6 N hydrochloric acid, cover,

and digest on a steam bath for 10 minutes. Cool, and quantitatively transfer the

solution to a test tube. Rinse the crucible with a second 5-mL portion of 6 N

hydrochloric acid, and transfer the rinsing to the test tube.

Monitor Preparation Pipet 4 mL of the Standard Lead Solution into a crucible

identical to that used for the Test Preparation and containing a quantity of the

substance under test that is equal to 10% of the amount required for the TestPreparation. Evaporate on a steam bath to dryness. Ignite at the same time, in

the same muffle furnace, and under the same conditions used for the Test

Preparation. Cool, add 5 mL of 6 N hydrochloric acid, cover, and digest on a

steam bath for 10 minutes. Cool, and quantitatively transfer to a test tube. Rinse

the crucible with a second 5-mL portion of 6 N hydrochloric acid, and transfer the

rinsing to the test tube.

Procedure Adjust the solution in each of the tubes containing the Standard

Preparation, the Test Preparation, and the Monitor Preparation with ammonium

hydroxide, added cautiously and dropwise, to a pH of 9. Cool, and adjust with

glacial acetic acid, added dropwise, to a pH of 8, then add 0.5 mL in excess.

Using a pH meter or short-range pH indicator paper as external indicator, check

the pH, and adjust, if necessary, with 1 N acetic acid or 6 N ammonium

hydroxide to a pH between 3.0 and 4.0. Filter, if necessary, washing the filter with

a few mL of water, into a 50-mL color-comparison tube, and then dilute with

water to 40 mL. Add 2 mL of pH 3.5 Acetate Buffer, then add 1.2 mL of

thioacetamideglycerin base TS, dilute with water to 50 mL, mix, allow to stand

for 2 minutes, and view downward over a white surface*: the color of the solution

from the Test Preparation is not darker than that of the solution from the

Standard Preparation, and the color of the solution from the Monitor Preparation


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is equal to or darker than that of the solution from the Standard Preparation.

[NOTEIf the color of the solution from the Monitor Preparation is lighter than

that of the solution from the Standard Preparation, proceed as directed for

Method III for the substance being tested.]

Method III

pH 3.5 Acetate Buffer Prepare as directed under Method I.

Standard Preparation Transfer a mixture of 8 mL of sulfuric acid and 10 mL of

nitric acid to a clean, dry, 100-mL Kjeldahl flask, and add a further volume of

nitric acid equal to the incremental volume of nitric acid added to the Test

Preparation. Heat the solution to the production of dense, white fumes; cool;

cautiously add 10 mL of water; and, if hydrogen peroxide was used in treating the

Test Preparation, add a volume of 30 percent hydrogen peroxide equal to that

used for the substance being tested. Boil gently to the production of dense, white

fumes. Again cool, cautiously add 5 mL of water, mix, and boil gently to the

production of dense, white fumes and to a volume of 2 to 3 mL. Cool, dilute

cautiously with a few mL of water, add 2.0 mL of Standard Lead Solution (20 g

of Pb), and mix. Transfer to a 50-mL color-comparison tube, rinse the flask with

water, adding the rinsing to the tube until the volume is 25 mL, and mix.

Test Preparation Unless otherwise indicated in the individual monograph, use

a quantity, in g, of the substance to be tested as calculated by the formula:

2.0/(1000L),

in which L is the Heavy metals limit, as a percentage.

If the substance is a solid Transfer the weighed quantity of the test substance

to a clean, dry, 100-mL Kjeldahl flask. [NOTEA 300-mL flask may be used if

the reaction foams excessively.] Clamp the flask at an angle of 45 , and add a

sufficient quantity of a mixture of 8 mL of sulfuric acid and 10 mL of nitric acid to

moisten the substance thoroughly. Warm gently until the reaction commences,


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allow the reaction to subside, and add portions of the same acid mixture, heating

after each addition, until a total of 18 mL of the acid mixture has been added.

Increase the amount of heat, and boil gently until the solution darkens. Cool, add

2 mL of nitric acid, and heat again until the solution darkens. Continue the

heating, followed by addition of nitric acid until no further darkening occurs, then

heat strongly to the production of dense, white fumes. Cool, cautiously add 5 mL

of water, boil gently to the production of dense, white fumes, and continue

heating until the volume is reduced to a few mL. Cool, cautiously add 5 mL of

water, and examine the color of the solution. If the color is yellow, cautiously add

1 mL of 30 percent hydrogen peroxide, and again evaporate to the production of

dense, white fumes and a volume of 2 to 3 mL. If the solution is still yellow,

repeat the addition of 5 mL of water and the peroxide treatment. Cool, dilute

cautiously with a few mL of water, and rinse into a 50-mL color-comparison tube,

taking care that the combined volume does not exceed 25 mL.

If the substance is a liquid Transfer the weighed quantity of the test substance

to a clean, dry, 100-mL Kjeldahl flask. [NOTEA 300-mL flask may be used if

the reaction foams excessively.] Clamp the flask at an angle of 45 , and

cautiously add a few mL of a mixture of 8 mL of sulfuric acid and 10 mL of nitric

acid. Warm gently until the reaction commences, allow the reaction to subside,

and proceed as directed for If the substance is a solid, beginning with add

portions of the same acid mixture.

Monitor Preparation Proceed with the digestion, using the same amount of

sample and the same procedure as directed in the subsection If the substance is

a solid in the section Test Preparation, until the step Cool, dilute cautiously with

a few mL of water. Add 2.0 mL of Lead Standard Solution (20 g of lead), and

mix. Transfer to a 50-mL color comparison tube, rinse the flask with water,

adding the rinsing to the tube until the volume is 25 mL, and mix.

Procedure Treat the Test Preparation, the Standard Preparation, and the

Monitor Preparation as follows. Using a pH meter or short-range pH indicator


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paper as external indicator, adjust the solution to a pH between 3.0 and 4.0 with

ammonium hydroxide (a dilute ammonia solution may be used, if desired, as the

specified range is approached), dilute with water to 40 mL, and mix.

To each tube add 2 mL of pH 3.5 Acetate Buffer, then add 1.2 mL of

thioacetamideglycerin base TS, dilute with water to 50 mL, mix, allow to stand

for 2 minutes, and view downward over a white surface *: the color of the Test

Preparation is not darker than that of the Standard Preparation, and the color of

the Monitor Preparation is equal to or darker than that of the Standard

Preparation.

* In those countries or jurisdictions where thioacetamide cannot be used, add 10mL of freshly prepared hydrogen sulfide TS to each of the tubes, mix, allow to

stand for 5 minutes, and view downward over a white surface.


Associate


USP29NF24 Page 2556

Pharmacopeial Forum : Volume No. 31(5) Page 1435

Phone Number : 1-301-816-8269

Search USP29Go

TEST SOLUTIONS (TS)
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Certain of the following test solutions are intended for use as acid-base indicators

in volumetric analyses. Such solutions should be so adjusted that when 0.15 mL

of the indicator solution is added to 25 mL of carbon dioxide-free water, 0.25 mL

of 0.02 N acid or alkali, respectively, will produce the characteristic color change.

Similar solutions are intended for use in pH measurement. Where no special

directions for their preparation are given, the same solution is suitable for both

purposes.

Where it is directed that a volumetric solution be used as the test solution,

standardization of the solution used as TS is not required.

In general, the directive to prepare a solution fresh indicates that the solution is

of limited stability and must be prepared on the day of use.

For the preparation of Test Solutions, use reagents of the quality described under

Reagents.

Acetaldehyde TS Mix 4 mL of acetaldehyde, 3 mL of alcohol, and 1 mL of

water. Prepare this solution fresh.

Acetate Buffer TS Dissolve 320 g of ammonium acetate in 500 mL of water,

add 5 mL of glacial acetic acid, dilute with water to 1000.0 mL, and mix. This

solution has a pH between 5.9 and 6.0.

Acetic Acid, Glacial, TS Determine the water content of a specimen of glacial

acetic acid by the Titrimetric Method (see Water Determination 921 ). If the

acid contains more than 0.05% of water, add a few mL of acetic anhydride, mix,

allow to stand overnight, and again determine the water content. If the acid

contains less than 0.02% of water, add sufficient water to make the final

concentration between 0.02% and 0.05%, mix, allow to stand overnight, and

again determine the water content. Repeat the adjustment with acetic anhydride
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or water, as necessary, until the resulting solution shows a water content

between 0.02% and 0.05%.

Acetic AcidAmmonium Acetate Buffer TS Dissolve 77.1 g of ammonium

acetate in water, add 57 mL of glacial acetic acid, and dilute with water to 1000

mL.

Acetone, Buffered, TS Dissolve 8.15 g of sodium acetate and 42 g of sodium

chloride in about 100 mL of water, and add 68 mL of 0.1 N hydrochloric acid and

150 mL of acetone. Mix, and dilute with water to 500 mL.

Acid Ferric Chloride TS Mix 60 mL of glacial acetic acid with 5 mL of sulfuric

acid, add 1 mL offerric chloride TS, mix, and cool.

Acid Ferrous Sulfate TS See Ferrous Sulfate, Acid, TS.

Acid Stannous Chloride TS See Stannous Chloride, Acid, TS.

Acid Stannous Chloride TS, Stronger See Stannous Chloride, Acid, Stronger,

TS.

Albumen TS Carefully separate the white from the yolk of a strictly fresh hen's

egg. Shake the white with 100 mL of water until mixed and all but the chalazahas undergone solution; then filter. Prepare the solution fresh.

AlcoholPhenol TS Dissolve 780 mg of phenol in alcohol to make 100 mL.
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Alcoholic Ammonia TS See Ammonia TS, Alcoholic.

Alcoholic Mercuric Bromide TS See Mercuric Bromide TS, Alcoholic.

Alcoholic Potassium Hydroxide TS See Potassium Hydroxide TS, Alcoholic.

Alkaline Cupric Citrate TS See Cupric Citrate TS, Alkaline.

Alkaline Cupric Iodide TS See Cupric Iodide TS, Alkaline.

Alkaline Cupric Tartrate TS (Fehling's Solution)See Cupric Tartrate TS,

Alkaline.

Alkaline MercuricPotassium Iodide TS See MercuricPotassium Iodide TS,

Alkaline.

Alkaline Picrate TS See Picrate TS, Alkaline.

Alkaline Sodium Hydrosulfite TS See Sodium Hydrosulfite TS, Alkaline.

Amaranth TS Dissolve 20 mg of amaranth in 10 mL of water.

Aminonaphtholsulfonic Acid TS Accurately weigh 5 g of sodium sulfite, 94.3 g

of sodium bisulfite, and 700 mg of 1,2,4-aminonaphtholsulfonic acid, and mix.
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Prepare aminonaphtholsulfonic acid TS fresh on the day of use by dissolving 1.5

g of the dry mixture in 10 mL of water.

AmmoniaAmmonium Chloride Buffer TS Dissolve 67.5 g of ammonium

chloride in water, add 570 mL of ammonium hydroxide, and dilute with water to

1000 mL.

AmmoniaCyanide TS Dissolve 2 g of potassium cyanide in 15 mL of

ammonium hydroxide, and dilute with water to 100 mL.

Ammonia TS It contains between 9.5% and 10.5% of NH3. Prepare by

diluting 350 mL ofAmmonia Water, Stronger(see in the section, Reagents) with

water to make 1000 mL.

Ammonia TS, Alcoholic A solution of ammonia gas in alcohol. Clear, colorless

liquid having a strong odor of ammonia. Specific gravity: about 0.80. It contains

between 9% and 11% of NH3. Store it in alkali-resistant containers, in a cold

place.

Ammonia TS, Stronger Use Ammonia Water, Stronger(see in the section

Reagents).

Ammoniacal Potassium Ferricyanide TS Dissolve 2 g of potassium

ferricyanide in 75 mL of water, add 25 mL of ammonium hydroxide, and mix.

Ammoniated Cupric Oxide TS See Cupric Oxide, Ammoniated, TS.
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Ammonium Acetate TS Dissolve 10 g of ammonium acetate in water to make

100 mL.

Ammonium Carbonate TS Dissolve 20 g of ammonium carbonate and 20 mL

ofammonia TS in water to make 100 mL.

Ammonium Chloride TS Dissolve 10.5 g of ammonium chloride in water to

make 100 mL.

Ammonium ChlorideAmmonium Hydroxide TS Mix equal volumes of water

and ammonium hydroxide, and saturate with ammonium chloride.

Ammonium Molybdate TS Dissolve 6.5 g of finely powdered molybdic acid in

a mixture of 14 mL of water and 14.5 mL of ammonium hydroxide. Cool the

solution, and add it slowly, with stirring, to a well-cooled mixture of 32 mL of nitric

acid and 40 mL of water. Allow to stand for 48 hours, and filter through a fine-

porosity, sintered-glass crucible. This solution deteriorates upon standing and is

unsuitable for use if, upon the addition of 2 mL ofdibasic sodium phosphate TS

to 5 mL of the solution, an abundant yellow precipitate does not form at once or

after slight warming. Store it in the dark. If a precipitate forms during storage, use

only the clear supernatant.

Ammonium Oxalate TS Dissolve 3.5 g of ammonium oxalate in water to make100 mL.

Ammonium Phosphate, Dibasic, TS (Ammonium Phosphate TS) Dissolve 13

g of dibasic ammonium phosphate in water to make 100 mL.
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Ammonium Polysulfide TS Yellow liquid, made by saturating ammonium

sulfide TS with sulfur.

Ammonium Reineckate TS Shake about 500 mg of ammonium reineckate

with 20 mL of water frequently during 1 hour, and filter. Use within 2 days.

Ammonium Sulfide TS Saturate ammonia TS with hydrogen sulfide by

bubbling hydrogen sulfide gas through the solution for 1 minute. This solution

must be freshly prepared. The solution is not rendered turbid either by

magnesium sulfate TS or by calcium chloride TS (carbonate). This solution is

unstable for use if an abundant precipitate of sulfur is present.

Residue on ignition: not more than 0.05%.

Ammonium Thiocyanate TS Dissolve 8 g of ammonium thiocyanate in water

to make 100 mL.

Ammonium Vanadate TS Dissolve 2.5 g of ammonium vanadate in 500 mL of

boiling water, cool, and add 20 mL of nitric acid. Mix, cool, and add water to

make 1 L. Store in polyethylene containers.

Anthrone TS Within 12 hours of use, rapidly dissolve 35 mg of anthrone in a

hot mixture of 35 mL of water and 65 mL of sulfuric acid. Immediately cool in an

ice bath to room temperature, and filter through glass wool. Allow the solution to

stand at room temperature for 30 minutes before use.

Antimony Trichloride TS Dissolve 20 g of antimony trichloride in chloroform to

make 100 mL. Filter if necessary.


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Barium Chloride TS Dissolve 12 g of barium chloride in water to make 100

mL.

Barium Hydroxide TS A saturated solution of barium hydroxide in recently

boiled water. Prepare the solution fresh.

Barium Nitrate TS Dissolve 6.5 g of barium nitrate in water to make 100 mL.

Betanaphthol TS See 2-Naphthol TS.

Biuret Reagent TS Dissolve 1.5 g of cupric sulfate and 6.0 g of potassium

sodium tartrate in 500 mL of water in a 1000-mL volumetric flask. Add 300 mL of

carbonate-free sodium hydroxide solution (1 in 10), dilute with carbonate-free

sodium hydroxide solution (1 in 10) to 1000 mL, and mix.

Blue Tetrazolium TS Dissolve 500 mg of blue tetrazolium in alcohol to make

100 mL.

Brilliant Blue G TS Transfer 25 mg of brilliant blue G to a 100-mL volumetric

flask, add 12.5 mL of alcohol and 25 mL of phosphoric acid, dilute with water to

volume, and mix.

Bromine TS (Bromine Water) A saturated solution of bromine, prepared by

agitating 2 to 3 mL of bromine with 100 mL of cold water in a glass-stoppered

bottle, the stopper of which should be lubricated with petrolatum. Store it in a cold

place, protected from light.


26/103

BromineSodium Acetate TS Dissolve 100 g of sodium acetate in 1000 mL of

glacial acetic acid, add 50 mL of bromine, and mix.

p-Bromoaniline TS Add 8 g of p-bromoaniline to a mixture of 380 mL of

thiourea-saturated glacial acetic acid, 10 mL of sodium chloride solution (1 in 5),

5 mL of oxalic acid solution (1 in 20), and 5 mL of dibasic sodium phosphate

solution (1 in 10) in a low-actinic glass bottle. Mix, and allow to stand overnight

before using. Protect from light, and use within 7 days.

Bromocresol Blue TS Use Bromocresol Green TS.

Bromocresol Green TS Dissolve 50 mg of bromocresol green in 100 mL of

alcohol, and filter if necessary.

Bromocresol GreenMethyl Red TS Dissolve 0.15 g of bromocresol green

and 0.1 g of methyl red in 180 mL of alcohol, and dilute with water to 200 mL.

Bromocresol Purple TS Dissolve 250 mg of bromocresol purple in 20 mL of

0.05 N sodium hydroxide, and dilute with water to 250 mL.

Bromophenol Blue TS Dissolve 100 mg of bromophenol blue in 100 mL of

diluted alcohol, and filter if necessary.

Bromothymol Blue TS Dissolve 100 mg of bromothymol blue in 100 mL of

diluted alcohol, and filter if necessary.


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Buffered Acetone TS See Acetone, Buffered, TS.

Calcium Chloride TS Dissolve 7.5 g of calcium chloride in water to make 100

mL.

Calcium Hydroxide TS Use Calcium Hydroxide Topical Solution (USP

monograph).

Calcium Sulfate TS A saturated solution of calcium sulfate in water.

Ceric Ammonium Nitrate TS Dissolve 6.25 g of ceric ammonium nitrate in 10

mL of 0.25 N nitric acid. Use within 3 days.

Chloral Hydrate TS Dissolve 50 g of chloral hydrate in a mixture of 15 mL of

water and 10 mL of glycerin.

Chlorine TS (Chlorine Water) A saturated solution of chlorine in water. Place

the solution in small, completely filled, light-resistant containers. Chlorine TS,

even when kept from light and air, is apt to deteriorate. Store it in a cold, dark

place. For full strength, prepare this solution fresh.

Chromotropic Acid TS Dissolve 50 mg of chromotropic acid or its disodiumsalt in 100 mL of 75% sulfuric acid, which may be made by cautiously adding 75

mL of sulfuric acid to 33.3 mL of water.

CobaltUranyl Acetate TS Dissolve, with warming, 40 g of uranyl acetate in a


28/103

mixture of 30 g of glacial acetic acid and sufficient water to make 500 mL.

Similarly, prepare a solution containing 200 g of cobaltous acetate in a mixture of

30 g of glacial acetic acid and sufficient water to make 500 mL. Mix the two

solutions while still warm, and cool to 20 . Maintain the temperature at 20 forabout 2 hours to separate the excess salts from solution, and then pass through

a dry filter.

Cobaltous Chloride TS Dissolve 2 g of cobaltous chloride in 1 mL of

hydrochloric acid and sufficient water to make 100 mL.

Congo Red TS Dissolve 500 mg of congo red in a mixture of 10 mL of alcohol

and 90 mL of water.

m-Cresol Purple TS Dissolve 0.10 g of metacresol purple in 13 mL of 0.01 N

sodium hydroxide, dilute with water to 100 mL, and mix.

Cresol Red TS Triturate 100 mg of cresol red in a mortar with 26.2 mL of 0.01N sodium hydroxide until solution is complete, then dilute the solution with water

to 250 mL.

Cresol RedThymol Blue TS Add 15 mL ofthymol blue TSto 5 mL ofcresol

red TS, and mix.

Crystal Violet TS Dissolve 100 mg of crystal violet in 10 mL of glacial acetic

acid.

Cupric Acetate TS Dissolve 100 mg of cupric acetate in about 5 mL of water
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to which a few drops of acetic acid have been added. Dilute to 100 mL, and filter,

if necessary.

Cupric Acetate TS, Stronger (Barfoed's Reagent) Dissolve 13.3 g of cupric

acetate in a mixture of 195 mL of water and 5 mL of acetic acid.

Cupric-Ammonium Sulfate TS To cupric sulfate TS add ammonia TS,

dropwise, until the precipitate initially formed is nearly but not completely

dissolved. Allow to settle, and decant the clear solution. Prepare this solution

fresh.

Cupric Citrate TS Dissolve 25 g of cupric sulfate, 50 g of citric acid, and 144 g

of anhydrous sodium carbonate in water, and dilute with water to 1000 mL.

Cupric Citrate TS, Alkaline With the aid of heat, dissolve 173 g of dihydrated

sodium citrate and 117 g of monohydrated sodium carbonate in about 700 mL of

water, and filter through paper, if necessary, to obtain a clear solution. In a

separate container dissolve 17.3 g of cupric sulfate in about 100 mL of water,

and slowly add this solution, with constant stirring, to the first solution. Cool the

mixture, add water to make 1000 mL, and mix.

Cupric Iodide TS, Alkaline Dissolve 7.5 g of cupric sulfate (CuSO45H2O) in

about 100 mL of water. In a separate container dissolve 25 g of anhydrous

sodium carbonate, 20 g of sodium bicarbonate, and 25 g of potassium sodiumtartrate in about 600 mL of water. With constant stirring, add the cupric sulfate

solution to the bottom of the alkaline tartrate solution by means of a funnel that

touches the bottom of the container. Add 1.5 g of potassium iodide, 200 g of

anhydrous sodium sulfate, 50 to 150 mL of 0.02 M potassium iodate, and

sufficient water to make 1000 mL.


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Cupric Oxide, Ammoniated, TS (Schweitzer's Reagent)Dissolve 10 g of cupric

sulfate in 100 mL of water, add sufficient sodium hydroxide solution (1 in 5) to

precipitate the copper hydroxide, collect the latter on a filter, and wash free from

sulfate with cold water. Dissolve the precipitate, which must be kept wet during

the entire process, in the minimum quantity ofammonia TS necessary for

complete solution.

Cupric Sulfate TS Dissolve 12.5 g of cupric sulfate in water to make 100 mL.

Cupric Tartrate TS, Alkaline (Fehling's Solution)

The Copper Solution (A) Dissolve 34.66 g of carefully selected, small crystals

of cupric sulfate, showing no trace of efflorescence of adhering moisture, in water

to make 500 mL. Store this solution in small, tight containers.

The Alkaline Tartrate Solution (B) Dissolve 173 g of crystallized potassium

sodium tartrate and 50 g of sodium hydroxide in water to make 500 mL. Store

this solution in small, alkali-resistant containers.

For use, mix exactly equal volumes of Solutions A and B at the time required.

Delafield's Hematoxylin TS Prepare 400 mL of a saturated solution of

ammonium alum (Solution A). Dissolve 4 g of hematoxylin in 25 mL of alcohol,

mix it with Solution A, and allow it to stand for 4 days in a flask closed with a

pledget of purified cotton and exposed to light and air (Solution B). Then filter

Solution B, and add to it a Solution C consisting of a mixture of 100 mL of

glycerin and 100 mL of methanol. Mix, and allow the mixture to stand in a warm

place, exposed to light, for 6 weeks until it becomes dark-colored. Store in tightly

stoppered bottles.


31/103

For use in staining endocrine tissue, dilute this test solution with an equal volume

of water.

Denigs' Reagent See Mercuric Sulfate TS.

Diazobenzenesulfonic Acid TS Place in a beaker 1.57 g of sulfanilic acid,

previously dried at 105 for 3 hours, add 80 mL of water and 10 mL of diluted

hydrochloric acid, and warm on a steam bath until dissolved. Cool to 15 (some

of the sulfanilic acid may separate but will dissolve later), and add slowly, with

constant stirring, 6.5 mL of sodium nitrite solution (1 in 10). Then dilute with water

to 100 mL.

Dichlorofluorescein TS Dissolve 100 mg of dichlorofluorescein in 60 mL of

alcohol, add 2.5 mL of 0.1 N sodium hydroxide, mix, and dilute with water to 100

mL.

Dicyclohexylamine Acetate TS Dissolve 50 g of dicyclohexylamine in 150 mL

of acetone, cool in an ice bath, and add, with stirring, a solution consisting of 18

mL of glacial acetic acid in 150 mL of acetone. Recrystallize the precipitate that

forms, by heating the mixture to boiling and allowing it to cool in an ice bath, then

collect the crystals on a filtering funnel, wash with a small volume of acetone, and

air-dry. Dissolve 300 mg of the dicyclohexylamine acetate so obtained in 200 mL

of a mixture of 6 volumes of chloroform and 4 volumes of water-saturated ether.

Use immediately.

2,7-Dihydroxynaphthalene TS Dissolve 100 mg of 2,7-dihydroxynaphthalene

in 1000 mL of sulfuric acid, and allow the solution to stand until the yellow color

disappears. If the solution is very dark, discard it and prepare a new solution from


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a different supply of sulfuric acid. This solution is stable for approximately 1

month if stored in a dark bottle.

Diiodofluorescein TS Dissolve 500 mg of diiodofluorescein in a mixture of 75

mL of alcohol and 30 mL of water.

Diluted Lead Subacetate TS See Lead Subacetate TS, Diluted.

p-Dimethylaminobenzaldehyde TS Dissolve 125 mg of p-

dimethylaminobenzaldehyde in a cooled mixture of 65 mL of sulfuric acid and 35

mL of water, and add 0.05 mL offerric chloride TS. Use within 7 days.

Dinitrophenylhydrazine TS Carefully mix 10 mL of water and 10 mL of sulfuric

acid, and cool. To the mixture, contained in a glass-stoppered flask, add 2 g of

2,4-dinitrophenylhydrazine, and shake until dissolved. To the solution add 35 mL

of water, mix, cool, and filter.

Diphenylamine TS Dissolve 1.0 g of diphenylamine in 100 mL of sulfuric acid.

The solution should be colorless.

Diphenylcarbazone TS Dissolve 1 g of crystalline diphenylcarbazone in 75 mL

of alcohol, then add alcohol to make 100 mL. Store in a brown bottle.

Dithizone TS Dissolve 25.6 mg of dithizone in 100 mL of alcohol. Store in a

cold place, and use within 2 months.

Dragendorff's TS Mix 850 mg of bismuth subnitrate with 40 mL of water and


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10 mL of glacial acetic acid (Solution A). Dissolve 8 g of potassium iodide in 20

mL of water (Solution B). Mix equal portions of Solution A and Solution B to

obtain a stock solution, which can be stored for several months in a dark bottle.

Mix 10 mL of the stock solution with 20 mL of glacial acetic acid, and dilute with

water to make 100 mL.

Edetate Disodium TS Dissolve 1 g of edetate disodium in 950 mL of water,

add 50 mL of alcohol, and mix.

Eosin Y TS (adsorption indicator)Dissolve 50 mg of eosin Y in 10 mL of water.

Eriochrome Black TS Dissolve 200 mg of eriochrome black T and 2 g of

hydroxylamine hydrochloride in methanol to make 50 mL.

Eriochrome Cyanine TS Dissolve 750 mg of eriochrome cyanine R in 200 mL

of water, add 25 g of sodium chloride, 25 g of ammonium nitrate, and 2 mL of

nitric acid, and dilute with water to 1000 mL.

Fehling's Solution See Cupric Tartrate TS, Alkaline.

Ferric Ammonium Sulfate TS Dissolve 8 g of ferric ammonium sulfate in water

to make 100 mL.

Ferric Chloride TS Dissolve 9 g of ferric chloride in water to make 100 mL.

Ferroin TS Dissolve 0.7 g of ferrous sulfate and 1.76 g of o-phenanthroline

monohydrochloride monohydrate in water, and dilute with water to 100 mL.


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Ferrous Sulfate TS Dissolve 8 g of clear crystals of ferrous sulfate in about

100 mL of recently boiled and thoroughly cooled water. Prepare this solution

fresh.

Ferrous Sulfate, Acid, TS Dissolve 7 g of ferrous sulfate crystals in 90 mL of

recently boiled and thoroughly cooled water, and add sulfuric acid to make 100

mL. Prepare this solution immediately prior to use.

Folin-Ciocalteu Phenol TS Into a 1500-mL flask introduce 100 g of sodium

tungstate, 25 g of sodium molybdate, 700 mL of water, 50 mL of phosphoric acid,

and 100 mL of hydrochloric acid. Gently reflux the mixture for about 10 hours,

and add 150 g of lithium sulfate, 50 mL of water, and a few drops of bromine. Boil

the mixture, without the condenser, for about 15 minutes, or until the excess

bromine is expelled. Cool, dilute with water to 1 L, and filter: the filtrate has no

greenish tint. Before use, dilute 1 part of the filtrate with 1 part of water. When

used for protein determination (i.e., Lowry assay), this reagent must be further

diluted (1:5) with water. See Method 2 in Total Protein Assay under

Biotechnology-Derived ArticlesTests 1047 .

Formaldehyde TS Use Formaldehyde Solution (see in the section Reagents).

FuchsinPyrogallol TS Dissolve 100 mg of basic fuchsin in 50 mL of water

that previously has been boiled for 15 minutes and allowed to cool slightly. Cool,

add 2 mL of a saturated solution of sodium bisulfite, mix, and allow to stand for

not less than 3 hours. Add 0.9 mL of hydrochloric acid, mix, and allow to stand

overnight. Add 100 mg of pyrogallol, shake until solution is effected, and dilute

with water to 100 mL. Store in an amber-colored glass bottle in a refrigerator.
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FuchsinSulfurous Acid TS Dissolve 200 mg of basic fuchsin in 120 mL of hot

water, and allow the solution to cool. Add a solution of 2 g of anhydrous sodium

sulfite in 20 mL of water, then add 2 mL of hydrochloric acid. Dilute the solution

with water to 200 mL, and allow to stand for at least 1 hour. Prepare this solution

fresh.

Gastric Fluid, Simulated, TS Dissolve 2.0 g of sodium chloride and 3.2 g of

purified pepsin, that is derived from porcine stomach mucosa, with an activity of

800 to 2500 units per mg of protein, in 7.0 mL of hydrochloric acid and sufficient

water to make 1000 mL. [NOTEPepsin activity is described in the Food

Chemicals Codex specifications under General Tests and Assays.] This test

solution has a pH of about 1.2.

Gelatin TS (for the assay of Corticotropin Injection)Dissolve 340 g of acid-

treated precursor gelatin (Type A) in water to make 1000 mL. Heat the solution in

an autoclave at 115 for 30 minutes after the exhaust line temperature has

reached 115 . Cool the solution, and add 10 g of phenol and 1000 mL of water.

Store in tight containers in a refrigerator.

Glacial Acetic Acid TS See Acetic Acid, Glacial, TS.

Glucose OxidaseChromogen TS A solution containing, in each mL, 0.5 mol

of 4-aminoantipyrine, 22.0 mol of sodium p-hydroxybenzoate, not less than 7.0units of glucose oxidase, and not less than 0.5 units of peroxidase, and buffered

to a pH of 7.0 0.1.


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Suitability When used for determining glucose in Inulin, ascertain that no

significant color results by reaction with fructose, and that a suitable absorbance-

versus-concentration slope is obtained with glucose.

[NOTEA suitable grade is available, as a concentrate, from Worthington

Diagnostics, Division of Millipore Corp., www.millipore.com.]

Glycerin Base TS To 200 g of glycerin add water to bring the total weight to

235 g. Add 140 mL of 1 N sodium hydroxide and 50 mL of water.

Gold Chloride TS Dissolve 1 g of gold chloride in 35 mL of water.

Hydrogen Peroxide TS Use Hydrogen Peroxide Topical Solution (USP

monograph).

Hydrogen Sulfide TS A saturated solution of hydrogen sulfide, made by

passing H2S into cold water. Store it in small, dark amber-colored bottles, filled

nearly to the top. It is unsuitable unless it possesses a strong odor of H2S, and

unless it produces at once a copious precipitate of sulfur when added to an equal

volume offerric chloride TS. Store in a cold, dark place.

Hydroxylamine Hydrochloride TS Dissolve 3.5 g of hydroxylamine

hydrochloride in 95 mL of 60% alcohol, and add 0.5 mL of bromophenol blue

solution (1 in 1000 of alcohol) and 0.5 N alcoholic potassium hydroxide until a

greenish tint develops in the solution. Then add 60% alcohol to make 100 mL.

8-Hydroxyquinoline TS Dissolve 5 g of 8-hydroxyquinoline in alcohol to make

100 mL.
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Indigo Carmine TS (Sodium Indigotindisulfonate TS) Dissolve a quantity of

sodium indigotindisulfonate, equivalent to 180 mg of C16H8N2O2(SO3Na)2, in

water to make 100 mL. Use within 60 days.

IndophenolAcetate TS (for the assay of Corticotropin Injection)To 60 mL of

standard dichlorophenol-indophenol solution (see in the section Volumetric

Solutions) add water to make 250 mL. Add to the resulting solution an equal

volume of sodium acetate solution freshly prepared by dissolving 13.66 g of

anhydrous sodium acetate in water to make 500 mL and adjusting with 0.5 N

acetic acid to a pH of 7. Store in a refrigerator, and use within 2 weeks.

Intestinal Fluid, Simulated, TS Dissolve 6.8 g of monobasic potassium

phosphate in 250 mL of water, mix, and add 77 mL of 0.2 N sodium hydroxide

and 500 mL of water. Add 10.0 g of pancreatin, mix, and adjust the resulting

solution with either 0.2 N sodium hydroxide or 0.2 N hydrochloric acid to a pH of

6.8 0.1. Dilute with water to 1000 mL.

Iodine TS Use 0.1 N Iodine (see in the section Volumetric Solutions).

Iodine, Diluted TS Transfer 10.0 mL of 0.1 N iodine VS to a 100-mL

volumetric flask, dilute with water to volume, and mix.

Iodine Monochloride TS Dissolve 10 g of potassium iodide and 6.44 g ofpotassium iodate in 75 mL of water in a glass-stoppered container. Add 75 mL of

hydrochloric acid and 5 mL of chloroform, and adjust to a faint iodine color (in the

chloroform) by adding dilute potassium iodide or potassium iodate solution. If

much iodine is liberated, use a stronger solution of potassium iodate than 0.01 M
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at first, making the final adjustment with the 0.01 M potassium iodate. Store in a

dark place, and readjust to a faint iodine color as necessary.

Iodine and Potassium Iodide TS 1 Dissolve 500 mg of iodine and 1.5 g of

potassium iodide in 25 mL of water.

Iodine and Potassium Iodide TS 2 Dissolve 12.7 g of iodine and 20 g of

potassium iodide in water, and dilute with water to 1000.0 mL. To 10.0 mL of this

solution, add 0.6 g of potassium iodide, and dilute with water to 100.0 mL.

Prepare immediately before use.

Iodobromide TS Dissolve 20 g of iodine monobromide in glacial acetic acid to

make 1000 mL. Store in glass containers, protected from light.

Iodochloride TS Dissolve 16.5 g of iodine monochloride in 1000 mL of glacial

acetic acid.

Iodoplatinate TS Dissolve 300 mg of platinic chloride in 97 mL of water.

Immediately prior to use, add 3.5 mL ofpotassium iodide TS, and mix.

IronPhenol TS (Iron-Kober Reagent) Dissolve 1.054 g of ferrous ammonium

sulfate in 20 mL of water, and add 1 mL of sulfuric acid and 1 mL of 30 percent

hydrogen peroxide. Mix, heat until effervescence ceases, and dilute with water to

50 mL. To 3 volumes of this solution contained in a volumetric flask add sulfuric

acid, with cooling, to make 100 volumes. Purify phenol by distillation, discarding

the first 10% and the last 5%, collecting the distillate, with exclusion of moisture,

in a dry, tared glass-stoppered flask of about twice the volume of the phenol.

Solidify the phenol in an ice bath, breaking the top crust with a glass rod to
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ensure complete crystallization. Weigh the flask and its contents, add to the

phenol 1.13 times its weight of the ironsulfuric acid solution prepared as

directed, insert the stopper in the flask, and allow to stand, without cooling but

with occasional mixing, until the phenol is liquefied. Shake the mixture vigorously

until mixed, allow to stand in the dark for 16 to 24 hours, and again weigh the

flask and its contents. To the mixture add 23.5% of its weight of a solution of 100

volumes of sulfuric acid in 110 volumes of water, mix, transfer to dry glass-

stoppered bottles, and store in the dark, protected from atmospheric moisture.

Use within 6 months. Dispense the reagent from a small-bore buret, arranged to

exclude moisture, capable of delivering 1 mL in 30 seconds or less, and having

no lubricant, other than reagent, on its stopcock. Wipe the buret tip with tissue

before each addition.

Iron Salicylate TS Dissolve 500 mg of ferric ammonium sulfate in 250 mL of

water containing 10 mL of diluted sulfuric acid, and add water to make 500 mL.

To 100 mL of the resulting solution add 50 mL of a 1.15% solution of sodium

salicylate, 20 mL of diluted acetic acid, and 80 mL of a 13.6% solution of sodium

acetate, then add water to make 500 mL. Store in a well-closed container.

Protect from light. Use within 2 weeks.

Lead Acetate TS Dissolve 9.5 g of clear, transparent crystals of lead acetate

in recently boiled water to make 100 mL. Store in well-stoppered bottles.

Lead Acetate TS, Alcoholic Dissolve 2 g of clear, transparent crystals of lead

acetate in alcohol to make 100 mL. Store in tight containers.

Lead Subacetate TS Triturate 14 g of lead monoxide to a smooth paste with

10 mL of water, and transfer the mixture to a bottle, using an additional 10 mL of

water for rinsing. Dissolve 22 g of lead acetate in 70 mL of water, and add the


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solution to the lead oxide mixture. Shake it vigorously for 5 minutes, then set it

aside, shaking it frequently, during 7 days. Finally filter, and add enough recently

boiled water through the filter to make 100 mL.

Lead Subacetate TS, Diluted Dilute 3.25 mL oflead subacetate TS with

water, recently boiled and cooled, to make 100 mL. Store in small, well-filled,

tight containers.

Litmus TS Digest 25 g of powdered litmus with three successive 100-mL

portions of boiling alcohol, continuing each extraction for about 1 hour. Filter,

wash with alcohol, and discard the alcohol filtrate. Macerate the residue with

about 25 mL of cold water for 4 hours, filter, and discard the filtrate. Finally digest

the residue with 125 mL of boiling water for 1 hour, cool, and filter.

Locke-Ringer's Solution See Locke-Ringer's TS.

Locke-Ringer's TS (Locke-Ringer's Solution)

Sodium Chloride 9.0 g

Potassium Chloride 0.42 g

Calcium Chloride 0.24 g

Magnesium Chloride 0.2 g

Sodium Bicarbonate 0.5 g

Dextrose 0.5 g

Water, recently distilled from a hard-glass flask, a sufficient quantity to make 1000 mL

Prepare fresh each day. The constituents (except the dextrose and the sodiumbicarbonate) may be made up in stock solutions and diluted as needed.

Magnesia Mixture TS Dissolve 5.5 g of magnesium chloride and 7 g of

ammonium chloride in 65 mL of water, add 35 mL of ammonia TS, set the


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mixture aside for a few days in a well-stoppered bottle, and filter. If the solution is

not perfectly clear, filter it before using.

Magnesium Sulfate TS Dissolve 12 g of crystals of magnesium sulfate,

selected for freedom from efflorescence, in water to make 100 mL.

Malachite Green TS Dissolve 1 g of malachite green oxalate in 100 mL of

glacial acetic acid.

Mallory's Stain Dissolve 500 mg of water-soluble aniline blue, 2 g of orange

G, and 2 g of oxalic acid in 100 mL of water.

Mayer's Reagent See MercuricPotassium Iodide TS.

Mercuric Acetate TS Dissolve 6.0 g of mercuric acetate in glacial acetic acid

to make 100 mL. Store in tight containers, protected from direct sunlight.

MercuricAmmonium Thiocyanate TS Dissolve 30 g of ammonium

thiocyanate and 27 g of mercuric chloride in water to make 1000 mL.

Mercuric Bromide TS, Alcoholic Dissolve 5 g of mercuric bromide in 100 mL

of alcohol, employing gentle heat to facilitate solution. Store in glass containers,

protected from light.

Mercuric Chloride TS Dissolve 6.5 g of mercuric chloride in water to make

100 mL.


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Mercuric Iodide TS (Valser's Reagent) Slowly add potassium iodide solution

(1 in 10) to red mercuric iodide until almost all of the latter is dissolved, and filter

off the excess. A solution containing 10 g of potassium iodide in 100 mL

dissolves approximately 14 g of HgI2 at 20 .

Mercuric Nitrate TS Dissolve 40 g of mercuric oxide (red or yellow) in a

mixture of 32 mL of nitric acid and 15 mL of water. Store in glass containers,

protected from light.

MercuricPotassium Iodide TS (Mayer's Reagent) Dissolve 1.358 g of

mercuric chloride in 60 mL of water. Dissolve 5 g of potassium iodide in 10 mL of

water. Mix the two solutions, and dilute with water to 100 mL.

MercuricPotassium Iodide TS, Alkaline (Nessler's Reagent) Dissolve 143 g

of sodium hydroxide in 700 mL of water. Dissolve 50 g of red mercuric iodide and

40 g of potassium iodide in 200 mL of water. Pour the iodide solution into the

hydroxide solution, and dilute with water to 1000 mL. Allow to settle, and use the

clear supernatant.

Mercuric Sulfate TS (Denigs' Reagent) Mix 5 g of yellow mercuric oxide with

40 mL of water, and while stirring slowly add 20 mL of sulfuric acid, then add

another 40 mL of water, and stir until completely dissolved.

Mercurous Nitrate TS Dissolve 15 g of mercurous nitrate in a mixture of 90

mL of water and 10 mL of diluted nitric acid. Store in dark, amber-colored bottles

in which a small globule of mercury has been placed.


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Metaphenylenediamine Hydrochloride TS Dissolve 1 g of

metaphenylenediamine hydrochloride in 200 mL of water. The solution must be

colorless when used. If necessary, decolorize by heating with activated charcoal.

MetaphosphoricAcetic Acids TS Dissolve 15 g of metaphosphoric acid in 40

mL of glacial acetic acid and sufficient water to make 500 mL. Store in a cold

place, and use within 2 days.

3-Methyl-2-benzothiazolinone Hydrazone Hydrochloride TS Dissolve 0.1 g of

3-methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate in 10 mL of

water, dilute the resulting solution with methanol to 100 mL, and mix.

Methyl Orange TS Dissolve 100 mg of methyl orange in 100 mL of water, and

filter if necessary.

Methyl Purple TS Use Methyl RedMethylene Blue TS.

Methyl Red TS Dissolve 100 mg of methyl red in 100 mL of alcohol, and filter

if necessary.

Methyl Red TS, Methanolic Dissolve 1 g of methyl red in 100 mL of methanol,

and filter, if necessary. Store protected from light, and use within 21 days.

Methyl RedMethylene Blue TS Add 10 mL ofmethyl red TS to 10 mL of

methylene blue TS, and mix.
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Methyl Violet TS Use Crystal Violet TS.

Methyl Yellow TS Prepare a solution containing 0.10 mg per mL in alcohol.

Methyl YellowMethylene Blue TS Dissolve 1 g of methyl yellow and 100 mg

of methylene blue in 125 mL of methanol.

Methylene Blue TS Dissolve 125 mg of methylene blue in 100 mL of alcohol,

and dilute with alcohol to 250 mL.

Methylthionine Perchlorate TS To 500 mL of potassium perchlorate solution

(1 in 1000) add dropwise, with constant shaking, methylene blue solution (1 in

100) until a slight, permanent turbidity results. Allow the precipitate to settle,

decant the supernatant through paper, and use only the clear solution.

Millon's Reagent To 2 mL of mercury in a conical flask add 20 mL of nitric

acid. Shake the flask under a hood to break up the mercury into small globules.

After about 10 minutes, add 35 mL of water, and, if a precipitate or crystals

appear, add sufficient dilute nitric acid (1 in 5, prepared from nitric acid from

which the oxides have been removed by blowing air through it until it is colorless)

to dissolve the separated solid. Add sodium hydroxide solution (1 in 10)

dropwise, with thorough mixing, until the curdy precipitate that forms after the

addition of each drop no longer redissolves but is dispersed to form asuspension. Add 5 mL more of the dilute nitric acid, and mix. Prepare this

solution fresh.

Molybdo-phosphotungstate TS (Folin-Denis Reagent) To about 350 mL of


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water contained in a round-bottom flask add 50 g of sodium tungstate, 12 g of

phosphomolybdic acid, and 25 mL of phosphoric acid. Boil the mixture under a

reflux condenser for 2 hours, then cool, dilute with water to 500 mL, and mix.

Store in tight containers, protected from light, and in a cold place.

1-Naphthol Reagent Dissolve 1 g of 1-naphthol in 25 mL of methanol.

Prepare this solution fresh.

1-Naphthol TS Use 1-Naphthol Reagent.

2-Naphthol TS (Betanaphthol TS) Dissolve 1 g of 2-naphthol in 100 mL of

sodium hydroxide solution (1 in 100).

p-Naphtholbenzein TS Dissolve 250 mg of p-naphtholbenzein in 100 mL of

glacial acetic acid.

N-(1-Naphthyl)ethylenediamine Dihydrochloride TS Dissolve 100 mg of N-(1-

naphthyl)ethylenediamine dihydrochloride in 100 mL of a mixture of 7 parts of

acetone and 3 parts of water.

Nessler's Reagent See MercuricPotassium Iodide TS, Alkaline.

Neutral Red TS Dissolve 100 mg of neutral red in 100 mL of 50% alcohol.

Nickel Standard Solution TS Dissolve 4.78 g of nickel(II) sulfate heptahydrate

in water, and dilute with water to 1000 mL. Immediately prior to use, dilute 10.0
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mL of the solution so obtained with water to 1000 mL. Suitable nickel standard

solutions are also available commercially.

Ninhydrin TS Use Triketohydrindene Hydrate TS.

p-Nitroaniline TS To 350 mg of p-nitroaniline add 1.5 mL of hydrochloric acid,

and mix. Dilute with water to 50 mL, mix, and allow to settle. Place 5 mL of the

clear supernatant in a 100-mL volumetric flask, and immerse it in an ice bath.

While it is in the ice bath, add 1 mL of hydrochloric acid, then add, in small

portions, 2 mL of sodium nitrite solution (1 in 100), dilute with water to volume,

and mix.

Nitrophenanthroline TS Dissolve 150 mg of 5-nitro-1,10-phenanthroline in 15

mL of freshly prepared ferrous sulfate solution (1 in 140).

Oracet Blue B TS A 1 in 200 solution of oracet blue B in glacial acetic acid.

Orthophenanthroline TS Dissolve 150 mg of orthophenanthroline in 10 mL of

a solution of ferrous sulfate, prepared by dissolving 700 mg of clear crystals of

ferrous sulfate in 100 mL of water. The ferrous sulfate solution must be prepared

immediately before dissolving the orthophenanthroline. Store in well-closed

containers.

Oxalic Acid TS Dissolve 6.3 g of oxalic acid in water to make 100 mL.

Palladium Chloride TS, Buffered Weigh 500 mg of palladium chloride into a

250-mL beaker, add 5 mL of concentrated hydrochloric acid, and warm the

mixture on a steam bath. Add 200 mL of hot water in small increments with


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continued heating until solution is complete. Transfer the solution to a 250-mL

volumetric flask, and dilute with water to volume. Transfer 50 mL to a 100-mL

volumetric flask. Add 10 mL of 1 M sodium acetate and 9.6 mL of 1 N

hydrochloric acid. Dilute with water to volume.

Perchloric Acid TS Dilute 8.5 mL of perchloric acid with water to 100 mL.

Phenol Red TS (Phenolsulfonphthalein TS) Dissolve 100 mg of

phenolsulfonphthalein in 100 mL of alcohol, and filter if necessary.

pH 4.7 Phenol Red TS Dissolve 33 mg of phenolsulfonphthalein in 1.5 mL of

2 N sodium hydroxide solution, dilute with water to 100 mL, and mix (Solution A).

Dissolve 25 mg of ammonium sulfate in 235 mL of water, add 105 mL of 2 N

sodium hydroxide solution and 135 mL of 2 N acetic acid, and mix (Solution B).

Add 25 mL of Solution A to Solution B, and mix. If necessary, adjust the pH of

this solution to 4.7.

Phenoldisulfonic Acid TS Dissolve 2.5 g of phenol in 15 mL of sulfuric acid in

a flask of suitable capacity. Add 7.5 mL of fuming sulfuric acid, stir well, and heat

at 100 for 2 hours. Transfer the product, while still fluid, to a glass-stoppered

bottle, and, when desired for use, warm in a water bath until liquefied.

Phenolphthalein TS Dissolve 1 g of phenolphthalein in 100 mL of alcohol.

Phenylhydrazine Acetate TS Dissolve 10 mL of phenylhydrazine and 5 mL of

glacial acetic acid in water to make 100 mL.


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PhenylhydrazineSulfuric Acid TS Dissolve 65 mg of phenylhydrazine

hydrochloride in 100 mL of a cooled mixture of equal volumes of sulfuric acid and

water.

Phloroglucinol TS Dissolve 500 mg of phloroglucinol in 25 mL of alcohol.

Store in tight containers, protected from light.

Phosphatic Enzyme TS Dissolve 5 g of phosphatic enzyme in water to make

50 mL. Prepare this solution fresh.

Phosphomolybdic Acid TS Dissolve 20 g of phosphomolybdic acid in alcohol

to make 100 mL. Filter the solution, and use only the clear filtrate.

Phosphotungstic Acid TS Dissolve 1 g of phosphotungstic acid in water to

make 100 mL.

Picrate TS, Alkaline Mix 20 mL of trinitrophenol solution (1 in 100) with 10 mL

of

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