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130 〈171〉 Vitamin B12 Activity Assay / Biological Tests USP 35

cautions to maintain uniformity of sterilizing and cooling

conditions throughout the assay, since packing tubes too
closely in the autoclave, or overloading it, may cause varia-
Chemical Tests and
tion in the heating rate.
Aseptically add 0.5 mL of Inoculum to each tube so pre-
Assays
pared, except two of the four containing no Standard Cya-
nocobalamin Solution (the uninoculated blanks). Incubate the
tubes at a temperature between 30° and 40° held constant
to within ±0.5°, for 16 to 24 hours.
Terminate growth by heating to a temperature not lower
than 80° for 5 minutes. Cool to room temperature. After
agitating its contents, place the container in a spectropho-
tometer that has been set at a wavelength of 530 nm, and
IDENTIFICATION TESTS
read the transmittance when a steady state is reached. This
steady state is observed a few seconds after agitation when
the reading remains constant for 30 seconds or more. Allow
approximately the same time interval for the reading on
each tube.
With the transmittance set at 100% for the uninoculated
blank, read the transmittance of the inoculated blank. If the
difference is greater than 5% or if there is evidence of con- 〈181〉 IDENTIFICATION—
tamination with a foreign microorganism, disregard the re-
sults of the assay. ORGANIC NITROGENOUS BASES
With the transmittance set at 100% for the uninoculated
blank, read the transmittance of each of the remaining
tubes. Disregard the results of the assay if the slope of the This test is for the identification of tertiary amine
standard curve indicates a problem with sensitivity. compounds.
Calculation—Prepare a standard concentration-response Dissolve 50 mg of the substance under test, if in bulk, in
curve by the following procedure. Test for and replace any 25 mL of 0.01 N hydrochloric acid, or shake a quantity of
aberrant individual transmittances. For each level of the powdered tablets or the contents of capsules equivalent to
standard, calculate the response from the sum of the dupli- 50 mg of the substance with 25 mL of 0.01 N hydrochloric
cate values of the transmittances (Σ) as the difference, y = acid for 10 minutes. Transfer the liquid to a separator, if
2.00 – Σ. Plot this response on the ordinate of cross-section necessary filtering it and washing the filter and the residue
paper against the logarithm of the mL of Standard Cyanoco- with several small portions of water. In a second separator
balamin Solution per tube on the abscissa, using for the ordi- dissolve 50 mg of the corresponding USP Reference Stan-
nate either an arithmetic or a logarithmic scale, whichever dard in 25 mL of 0.01 N hydrochloric acid. Treat each solu-
gives the better approximation to a straight line. Draw the tion as follows. Add 2 mL of 1 N sodium hydroxide and 4
straight line or smooth curve that best fits the plotted mL of carbon disulfide, and shake for 2 minutes. Centrifuge
points. if necessary to clarify the lower phase, and filter it through a
Calculate the response, y, adding together the two trans- dry filter, collecting the filtrate in a small flask provided with
mittances for each level of the Assay Preparation. Read from a glass stopper.
the standard curve the logarithm of the volume of the Stan- Determine the absorption spectra of the filtered solutions
dard Preparation corresponding to each of those values of y of both standard and sample without delay, in 1-mm cells
that falls within the range of the lowest and highest points between 7 µm and 15 µm, with a suitable IR spectropho-
plotted for the standard. Subtract from each logarithm so tometer, using carbon disulfide in a matched cell as the
obtained the logarithm of the volume, in mL, of the Assay blank. The spectrum of the solution prepared from the sam-
Preparation to obtain the difference, x, for each dosage ple shows all of the significant absorption bands present in
level. Average the values of x for each of three or more the spectrum of the solution prepared from the Reference
dosage levels to obtain x = M′, the log-relative potency of Standard.
the Assay Preparation. Determine the quantity, in µg, of USP
Cyanocobalamin RS corresponding to the cyanocobalamin
in the portion of material taken for assay by the equation
antilog M = antilog (M′ + log R), in which R is the number
of µg of cyanocobalamin that was assumed to be present in
each mg (or capsule or tablet) of the material taken for
assay.
Replication—Repeat the entire determination at least
〈191〉 IDENTIFICATION TESTS—
once, using separately prepared Assay Preparations. If the
difference between the two log potencies M is not greater
GENERAL
than 0.08, their mean, M, is the assayed log-potency of the
test material (see Vitamin B12 Activity Assay under Design and
Analysis of Biological Assays 〈111〉). If the two determinations Under this heading are placed tests that are frequently
differ by more than 0.08, conduct one or more additional referred to in the Pharmacopeia for the identification of offi-
determinations. From the mean of two or more values of M cial articles. Before using any acid or base to modify the pH
that do not differ by more than 0.15, compute the mean of the sample solution, make sure that the added substance
potency of the preparation under assay. will not interfere with the results of the test. [NOTE—The
tests are not intended to be applicable to mixtures of sub-
stances unless so specified.]
Acetate—Dissolve about 30 mg of the substance to be
examined in 3 mL of water, or use 3 mL of the prescribed
solution. Adjust the pH of the solution with sodium hydrox-
ide to slightly alkaline. Add 0.25 mL of lanthanum nitrate
TS. If a white precipitate is formed, filter the solution. Add
successively 0.1 mL of iodine and potassium iodide TS 3

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USP 35 Chemical Tests / 〈191〉 Identification Tests—General 131

and 0.1 mL of ammonia TS 2 to the solution. If no blue colored red by phenolphthalein TS, while a similar solution
color is observed, heat carefully to boiling. In the presence of a bicarbonate remains unchanged or is only slightly
of acetates, a dark color develops or a blue precipitate is colored.
formed. With neutral solutions of acetates, ferric chloride TS Chlorate—Solutions of chlorates yield no precipitate with
produces a red color that is destroyed by the addition of silver nitrate TS. The addition of sulfurous acid to this mix-
mineral acids. ture produces a white precipitate that is insoluble in nitric
Aluminum—With 6 N ammonium hydroxide, solutions of acid, but is soluble in 6 N ammonium hydroxide. Upon igni-
aluminum salts yield a gelatinous, white precipitate that is tion, chlorates yield chlorides, recognizable by appropriate
insoluble in an excess of 6 N ammonium hydroxide. 1 N tests. When sulfuric acid is added to a dry chlorate, decrepi-
sodium hydroxide or sodium sulfide TS produces the same tation occurs, and a greenish yellow-gas is evolved. [Cau-
precipitate, which dissolves in an excess of either of these tion—Use only a small amount of chlorate for this test, and
reagents. exercise extreme caution in performing it.]
Ammonium—Add 0.2 g of magnesium oxide to the so- Chloride—With silver nitrate TS, solutions of chlorides
lution under test. Pass a current of air through the mixture, yield a white, curdy precipitate that is insoluble in nitric acid
and direct the gas that escapes to just beneath the surface but is soluble in a slight excess of 6 N ammonium hydrox-
of an indicator solution, prepared by mixing 1 mL of 0.1 M ide. When testing amine (including alkaloidal) hydrochlo-
hydrochloric acid and 0.05 mL of methyl red TS 2. In the rides that do not respond to the above test, add one drop
presence of ammonium, the color of the indicator solution of diluted nitric acid and 0.5 mL of silver nitrate TS to a
is changed to yellow. After directing the gas into the indica- solution of the substance being examined containing, unless
tor solution for a sufficient period of time, add 1 mL of otherwise directed in the monograph, about 2 mg of chlo-
freshly prepared sodium cobaltinitrite TS to the indicator so- ride ion in 2 mL: a white, curdy precipitate is formed. Cen-
lution. Upon the addition of the sodium cobaltinitrite TS, a trifuge the mixture without delay, and decant the superna-
yellow precipitate is formed when ammonium is present. tant layer. Wash the precipitate with three 1-mL portions of
Antimony—With hydrogen sulfide, solutions of antimony nitric acid solution (1 in 100), and discard the washings.
(III) compounds, strongly acidified with hydrochloric acid, Add ammonia TS dropwise to this precipitate. It dissolves
yield an orange precipitate of antimony sulfide that is insol- readily. When a monograph specifies that an article re-
uble in 6 N ammonium hydroxide, but is soluble in ammo- sponds to the test for dry chlorides, mix the solid to be
nium sulfide TS. tested with an equal weight of manganese dioxide, moisten
Barium—Solutions of barium salts yield a white precipi- with sulfuric acid, and gently heat the mixture: chlorine,
tate with 2 N sulfuric acid. This precipitate is insoluble in which is recognizable by the production of a blue color with
hydrochloric acid and in nitric acid. Barium salts impart a moistened starch iodide paper, is evolved.
yellowish-green color to a nonluminous flame that appears Citrate—To 15 mL of pyridine add a few mg of a citrate
blue when viewed through green glass. salt, dissolved or suspended in 1 mL of water, and shake. To
Benzoate—In neutral solutions, benzoates yield a this mixture add 5 mL of acetic anhydride, and shake: a
salmon-colored precipitate with ferric chloride TS. In moder- light red color is produced.
ately concentrated solutions, benzoates yield a precipitate of Cobalt—Solutions of cobalt salts (1 in 20) in 3 N hydro-
benzoic acid upon acidification with 2 N sulfuric acid. This chloric acid yield a red precipitate when heated on a steam
precipitate is readily soluble in ethyl ether. bath with an equal volume of a hot, freshly prepared solu-
Bicarbonate—See Carbonate. tion of 1-nitroso-2-naphthol (1 in 10) in 9 N acetic acid.
Solutions of cobalt salts, when saturated with potassium
Bismuth—When dissolved in a slight excess of nitric acid chloride and treated with potassium nitrite and acetic acid,
or hydrochloric acid, bismuth salts yield a white precipitate yield a yellow precipitate.
upon dilution with water. This precipitate is colored brown
by hydrogen sulfide, and the resulting compound dissolves Copper—Solutions of cupric compounds, acidified with
in a warm mixture of equal parts of nitric acid and water. hydrochloric acid, deposit a red film of metallic copper
upon a bright, untarnished surface of metallic iron. An ex-
Bisulfite—See Sulfite. cess of 6 N ammonium hydroxide, added to a solution of a
Borate—To 1 mL of a borate solution, acidified with hy- cupric salt, produces first a bluish precipitate and then a
drochloric acid to litmus, add 3 or 4 drops of iodine TS and deep blue-colored solution. With potassium ferrocyanide TS,
3 or 4 drops of polyvinyl alcohol solution (1 in 50): an in- solutions of cupric salts yield a reddish-brown precipitate,
tense blue color is produced. When a borate is treated with insoluble in diluted acids.
sulfuric acid, methanol is added, and the mixture is ignited, Hypophosphite—When strongly heated, hypophosphites
it burns with a green-bordered flame. evolve spontaneously flammable phosphine. Hypophosphites
Bromide—Solutions of bromides, upon the addition of in solution yield a white precipitate with mercuric chloride
chlorine TS, dropwise, liberate bromine, which is dissolved TS. This precipitate becomes gray when an excess of hypo-
by shaking with chloroform, coloring the chloroform red to phosphite is present. Solutions of hypophosphites, acidified
reddish brown. Silver nitrate TS produces in solutions of with sulfuric acid, and warmed with cupric sulfate TS yield a
bromides a yellowish-white precipitate that is insoluble in red precipitate.
nitric acid and is slightly soluble in 6 N ammonium Iodide—Solutions of iodides, upon the addition of chlo-
hydroxide. rine TS, dropwise, liberate iodine, which colors the solution
Calcium—Solutions of calcium salts form insoluble oxa- yellow to red. When the solution is shaken with chloroform,
lates when treated as follows. To a solution of the calcium the latter is colored violet. The iodine thus liberated gives a
salt (1 in 20) add 2 drops of methyl red TS, and neutralize blue color with starch TS. Silver nitrate TS produces, in solu-
with 6 N ammonium hydroxide. Add 3 N hydrochloric acid, tions of iodides, a yellow, curdy precipitate that is insoluble
dropwise, until the solution is acid to the indicator. Upon in nitric acid and in 6 N ammonium hydroxide.
the addition of ammonium oxalate TS, a white precipitate is Iron—Ferrous and ferric compounds in solution yield a
formed. This precipitate is insoluble in 6 N acetic acid but black precipitate with ammonium sulfide TS. This precipitate
dissolves in hydrochloric acid. Calcium salts moistened with is dissolved by cold 3 N hydrochloric acid with the evolution
hydrochloric acid impart a transient yellowish-red color to a of hydrogen sulfide.
nonluminous flame.
Ferric Salts—Acid solutions of ferric salts yield a dark blue
Carbonate—Carbonates and bicarbonates effervesce with precipitate with potassium ferrocyanide TS. With an excess
acids, evolving a colorless gas that, when passed into cal- of 1 N sodium hydroxide, a reddish-brown precipitate is
cium hydroxide TS, produces a white precipitate immedi- formed. With ammonium thiocyanate TS, solutions of ferric
ately. A cold solution (1 in 20) of a soluble carbonate is

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132 〈191〉 Identification Tests—General / Chemical Tests USP 35

salts produce a deep red color that is not destroyed by di- Permanganate—Solutions of permanganates acidified
lute mineral acids. with sulfuric acid are decolorized by hydrogen peroxide TS
Ferrous Salts—Solutions of ferrous salts yield a dark blue and by sodium bisulfite TS, in the cold, and by oxalic acid
precipitate with potassium ferricyanide TS. This precipitate is TS, in hot solution.
insoluble in 3 N hydrochloric acid but is decomposed by Peroxide—Solutions of peroxides slightly acidified with
1 N sodium hydroxide. With 1 N sodium hydroxide, solu- sulfuric acid yield a deep blue color upon the addition of
tions of ferrous salts yield a greenish-white precipitate, the potassium dichromate TS. On shaking the mixture with an
color rapidly changing to green and then to brown when equal volume of ethyl ether and allowing the liquids to sep-
shaken. arate, the blue color is found in the ethyl ether layer.
Lactate—When solutions of lactates are acidified with Phosphate—[NOTE—Where the monograph specifies the
sulfuric acid, potassium permanganate TS is added, and the identification test for Phosphate, use the tests for orthophos-
mixture is heated, acetaldehyde is evolved. This can be de- phates, unless the instructions specify the use of the pyro-
tected by allowing the vapor to come into contact with a phosphate tests or indicate that the product is to be ignited
filter paper that has been moistened with a freshly prepared before performing the test.] With silver nitrate TS, neutral
mixture of equal volumes of 20% aqueous morpholine and solutions of orthophosphates yield a yellow precipitate that
sodium nitroferricyanide TS: a blue color is produced. is soluble in 2 N nitric acid and in 6 N ammonium hydrox-
Lead—With 2 N sulfuric acid, solutions of lead salts yield ide. With ammonium molybdate TS, acidified solutions of
a white precipitate that is insoluble in 3 N hydrochloric or orthophosphates yield a yellow precipitate that is soluble in
2 N nitric acid, but is soluble in warm 1 N sodium hydrox- 6 N ammonium hydroxide. This precipitate may be slow to
ide and in ammonium acetate TS. With potassium chromate form. With silver nitrate TS, pyrophosphates obtained by ig-
TS, solutions of lead salts, free or nearly free from mineral nition yield a white precipitate that is soluble in 2 N nitric
acids, yield a yellow precipitate that is insoluble in 6 N ace- acid and in 6 N ammonium hydroxide. With ammonium
tic acid but is soluble in 1 N sodium hydroxide. molybdate TS, a yellow precipitate that is soluble in 6 N
Lithium—With sodium carbonate TS, moderately concen- ammonium hydroxide is formed.
trated solutions of lithium salts, made alkaline with sodium Potassium—Potassium compounds impart a violet color
hydroxide, yield a white precipitate on boiling. The precipi- to a nonluminous flame, but the presence of small quanti-
tate is soluble in ammonium chloride TS. Lithium salts ties of sodium masks the color unless the yellow color pro-
moistened with hydrochloric acid impart an intense crimson duced by sodium is screened out by viewing through a blue
color to a nonluminous flame. Solutions of lithium salts are filter that blocks emission at 589 nm (sodium) but is trans-
not precipitated by 2 N sulfuric acid or soluble sulfates (dis- parent to emission at 404 nm (potassium). Traditionally, co-
tinction from strontium). balt glass has been used, but other suitable filters are com-
Magnesium—Solutions of magnesium salts in the pres- mercially available. In neutral, concentrated or moderately
ence of ammonium chloride yield no more than a slightly concentrated solutions of potassium salts (depending upon
hazy precipitate when neutralized with ammonium carbon- the solubility and the potassium content), sodium bitartrate
ate TS, but on the subsequent addition of dibasic sodium TS produces a white crystalline precipitate that is soluble in
phosphate TS, a white, crystalline precipitate, which is insol- 6 N ammonium hydroxide and in solutions of alkali hydrox-
uble in 6 N ammonium hydroxide, is formed. ides and carbonates. The formation of the precipitate, which
is usually slow, is accelerated by stirring or rubbing the in-
Manganese—With ammonium sulfide TS, solutions of side of the test tube with a glass rod. The addition of a
manganous salts yield a salmon-colored precipitate that dis- small amount of glacial acetic acid or alcohol also promotes
solves in acetic acid. the precipitation.
Mercury—When applied to bright copper foil, solutions Salicylate—In moderately dilute solutions of salicylates,
of mercury salts, free from an excess of nitric acid, yield a ferric chloride TS produces a violet color. The addition of
deposit that upon rubbing, becomes bright and silvery in acids to moderately concentrated solutions of salicylates
appearance. With hydrogen sulfide, solutions of mercury produces a white, crystalline precipitate of salicylic acid that
compounds yield a black precipitate that is insoluble in am- melts between 158° and 161°.
monium sulfide TS and in boiling 2 N nitric acid.
Silver—With hydrochloric acid, solutions of silver salts
Mercuric Salts—Solutions of mercuric salts yield a yellow yield a white, curdy precipitate that is insoluble in nitric
precipitate with 1 N sodium hydroxide. They yield also, in acid, but is readily soluble in 6 N ammonium hydroxide. A
neutral solutions with potassium iodide TS, a scarlet precipi- solution of a silver salt to which 6 N ammonium hydroxide
tate that is very soluble in an excess of the reagent. and a small quantity of formaldehyde TS are added depos-
Mercurous Salts—Mercurous compounds are decomposed its, upon warming, a mirror of metallic silver upon the sides
by 1 N sodium hydroxide, producing a black color. With of the container.
hydrochloric acid, solutions of mercurous salts yield a white Sodium—Unless otherwise specified in an individual
precipitate that is blackened by 6 N ammonium hydroxide. monograph, prepare a solution to contain 0.1 g of the so-
With potassium iodide TS, a yellow precipitate, that may dium compound in 2 mL of water. Add 2 mL of 15% potas-
become green upon standing, is formed. sium carbonate, and heat to boiling. No precipitate is
Nitrate—When a solution of a nitrate is mixed with an formed. Add 4 mL of potassium pyroantimonate TS, and
equal volume of sulfuric acid, the mixture is cooled, and a heat to boiling. Allow to cool in ice water and, if necessary,
solution of ferrous sulfate is superimposed, a brown color is rub the inside of the test tube with a glass rod. A dense
produced at the junction of the two liquids. When a nitrate precipitate is formed. Sodium compounds impart an intense
is heated with sulfuric acid and metallic copper, brownish- yellow color to a nonluminous flame.
red fumes are evolved. Nitrates do not decolorize acidified Sulfate—With barium chloride TS, solutions of sulfates
potassium permanganate TS (distinction from nitrites). yield a white precipitate that is insoluble in hydrochloric
Nitrite—When treated with dilute mineral acids or with acid and in nitric acid. With lead acetate TS, neutral solu-
6 N acetic acid, nitrites evolve brownish-red fumes. The so- tions of sulfates yield a white precipitate that is soluble in
lution colors starch-iodide paper blue. ammonium acetate TS. Hydrochloric acid produces no pre-
Oxalate—Neutral and alkaline solutions of oxalates yield cipitate when added to solutions of sulfates (distinction from
a white precipitate with calcium chloride TS. This precipitate thiosulfates).
is insoluble in 6 N acetic acid but is dissolved by hydrochlo- Sulfite—When treated with 3 N hydrochloric acid, sulfites
ric acid. Hot acidified solutions of oxalates decolorize potas- and bisulfites yield sulfur dioxide, which blackens filter paper
sium permanganate TS. moistened with mercurous nitrate TS.

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USP 35 Chemical Tests / 〈197〉 Spectrophotometric Identification Tests 133

Tartrate—Dissolve a few mg of a tartrate salt in 2 drops graphic chamber with the bottom edge touching the Devel-
of sodium metaperiodate solution (1 in 20). Add a drop of oping Solvent. When the solvent front has risen about 10
1 N sulfuric acid, and after 5 minutes add a few drops of cm, remove the sheet from the chamber, and expose the
sulfurous acid followed by a few drops of fuchsin–sulfurous sheet to ammonia vapor. Examine the chromatogram under
acid TS: a reddish-pink color is produced within 15 minutes. long-wavelength UV light. Record the positions of the major
Thiocyanate—With ferric chloride TS, solutions of thiocy- yellow fluorescent spots: the RF value of the principal spot
anates yield a red color that is not destroyed by moderately obtained from the Test Solution and from the Mixed Test
concentrated mineral acids. Solution corresponds to that obtained from the Standard
Thiosulfate—With hydrochloric acid, solutions of thiosul- Solution.
fates yield a white precipitate that soon turns yellow, and
sulfur dioxide, which blackens filter paper moistened with METHOD II
mercurous nitrate TS. The addition of ferric chloride TS to
solutions of thiosulfates produces a dark violet color that
quickly disappears. Resolution Solution—Unless otherwise directed in the
individual monograph, prepare a solution in methanol con-
Zinc—In the presence of sodium acetate, solutions of taining 0.5 mg each of USP Chlortetracycline Hydrochloride
zinc salts yield a white precipitate with hydrogen sulfide. RS, USP Doxycycline Hyclate RS, USP Oxytetracycline RS,
This precipitate is insoluble in acetic acid, but is dissolved by and USP Tetracycline Hydrochloride RS per mL.
3 N hydrochloric acid. Ammonium sulfide TS produces a
similar precipitate in neutral and in alkaline solutions. With Developing Solvent—Prepare a mixture of 0.5 M oxalic
potassium ferrocyanide TS, zinc salts in solution yield a acid, previously adjusted with ammonium hydroxide to a
white precipitate that is insoluble in 3 N hydrochloric acid. pH of 2.0, acetonitrile, and methanol (80:20:20).
Chromatographic Plate—Use a suitable thin-layer chro-
matographic plate (see Thin-layer Chromatography under
Chromatography 〈621〉) coated with a 0.25-mm layer of
octylsilanized chromatographic silica gel mixture. Activate
the plate by heating it at 130° for 20 minutes, allow to
cool, and use while still warm.
〈193〉 IDENTIFICATION— Procedure—Separately apply 1 µL each of the Standard
Solution, the Test Solution, and the Resolution Solution to the
TETRACYCLINES Chromatographic Plate. Allow the spots to dry, and develop
the chromatogram in the Developing Solvent until the sol-
vent front has moved about three-fourths of the length of
the plate. Remove the plate from the developing chamber,
The following chromatographic procedures are provided mark the solvent front, and allow to air-dry. Expose the
to confirm the identity of Pharmacopeial drug substances plate to ammonia vapors for 5 minutes, and promptly lo-
that are of the tetracycline type, such as doxycycline, oxy- cate the spots on the plate by viewing under long-wave-
tetracycline, and tetracycline, and to confirm the identity of length UV light: the chromatogram of the Resolution Solution
such compounds in their respective Pharmacopeial dosage shows clearly separated spots, and the principal spot ob-
forms. Two procedures are provided, one based on paper tained from the Test Solution corresponds in RF value, inten-
chromatography (Method I) and the other on thin-layer sity, and appearance to that obtained from the Standard
chromatography (Method II). Method I is to be used unless Solution.
otherwise directed in the individual monograph.
Standard Solution—Unless otherwise directed in the in-
dividual monograph, dissolve the USP Reference Standard
for the drug substance being identified in the same solvent
and at the same concentration as for the Test Solution.
Test Solution—Prepare as directed in the individual
monograph. 〈197〉 SPECTROPHOTOMETRIC
METHOD I
IDENTIFICATION TESTS
pH 3.5 Buffer—Dissolve 13.4 g of anhydrous citric acid
and 16.3 g of dibasic sodium phosphate in 1000 mL of Spectrophotometric tests contribute meaningfully toward
water, and mix. the identification of many compendial chemical substances.
The test procedures that follow are applicable to substances
Developing Solvent—On the day of use, mix 10 that absorb IR and/or UV radiation (see Spectrophotometry
volumes of chloroform, 20 volumes of nitromethane, and 3 and Light-Scattering 〈851〉).
volumes of pyridine. The IR absorption spectrum of a substance, compared
Mixed Test Solution—Mix equal volumes of the Stan- with that obtained concomitantly for the corresponding USP
dard Solution and the Test Solution. Reference Standard, provides perhaps the most conclusive
Chromatographic Sheet—Draw a spotting line 2.5 cm evidence of the identity of the substance that can be real-
from one edge of a 20-cm × 20-cm sheet of filter paper ized from any single test. The UV absorption spectrum, on
(Whatman No. 1, or equivalent). Impregnate the sheet with the other hand, does not exhibit a high degree of specific-
pH 3.5 Buffer by passing it through a trough filled with pH ity. Conformance with both IR absorption and UV absorp-
3.5 Buffer, and remove the excess solvent by firmly pressing tion test specifications, as called for in a large proportion of
the sheet between nonfluorescent blotting papers. compendial monographs, leaves little doubt, if any, regard-
Procedure—To a suitable chromatographic chamber, ing the identity of the specimen under examination.
prepared for ascending chromatography (see Chromatogra-
phy 〈621〉) add Developing Solvent to a depth of 0.6 cm. INFRARED ABSORPTION
Apply at 1.5-cm intervals 2 µL each of the Standard Solution,
the Test Solution, and the Mixed Test Solution to the spotting Seven methods are indicated for the preparation of previ-
line of the Chromatographic Sheet. Allow the sheet to dry ously dried test specimens and Reference Standards for anal-
partially, and while still damp place it in the chromato-

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