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当前位置:文档库 > 美国药典191一般鉴别试验 0130-0133 [191] IDENTIFICATION TESTS—GENERAL

美国药典191一般鉴别试验 0130-0133 [191] IDENTIFICATION TESTS—GENERAL

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 Chemical Tests and closely in the autoclave, or overloading it, may cause varia-

tion in the heating rate.Assays

Aseptically add 0.5 mL of Inoculum to each tube so pre-

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-IDENTIFICATION TESTS tometer that has been set at a wavelength of 530 nm, and

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?181? IDENTIFICATION—difference is greater than 5% or if there is evidence of con-

tamination with a foreign microorganism, disregard the re-ORGANIC NITROGENOUS BASES sults of the assay.

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 http://www.wendangku.net/doc/f861b3d7dd36a32d727581d1.htmlpounds.

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 any25 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 1N 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.?191? IDENTIFICATION TESTS—Replication—Repeat the entire determination at least

once, using separately prepared Assay Preparations. If the GENERAL

difference between the two log potencies M is not greater

than 0.08, their mean, M, is the assayed log-potency of the

test material (see Vitamin B12 Activity Assay under Design and

Under this heading are placed tests that are frequently Analysis of Biological Assays ?111?). If the two determinations

referred to in the Pharmacopeia for the identification of offi-differ by more than 0.08, conduct one or more additional

cial articles. Before using any acid or base to modify the pH determinations. From the mean of two or more values of M

of the sample solution, make sure that the added substance that do not differ by more than 0.15, compute the mean

will not interfere with the results of the test. [N OTE—The potency of the preparation under assay.

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

USP 35Chemical Tests / ?191? Identification Tests—General131

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 6N ammonium hydroxide, solutions of acid, but is soluble in 6N 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 6N ammonium hydroxide. 1N 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 6N 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

nitric acid solution (1 in 100), and discard the washings.

Antimony—With hydrogen sulfide, solutions of antimony

Add ammonia TS dropwise to this precipitate. It dissolves (III) compounds, strongly acidified with hydrochloric acid,

readily. When a monograph specifies that an article re-yield an orange precipitate of antimony sulfide that is insol-

sponds to the test for dry chlorides, mix the solid to be uble in 6N ammonium hydroxide, but is soluble in ammo-

tested with an equal weight of manganese dioxide, moisten nium sulfide TS.

with sulfuric acid, and gently heat the mixture: chlorine, Barium—Solutions of barium salts yield a white precipi-which is recognizable by the production of a blue color with

tate with 2N sulfuric acid. This precipitate is insoluble in moistened starch iodide paper, is evolved.

hydrochloric acid and in nitric acid. Barium salts impart a

Citrate—To 15 mL of pyridine add a few mg of a citrate yellowish-green color to a nonluminous flame that appears

salt, dissolved or suspended in 1 mL of water, and shake. To blue when viewed through green glass.

this mixture add 5 mL of acetic anhydride, and shake: a Benzoate—In neutral solutions, benzoates yield a light red color is produced.

salmon-colored precipitate with ferric chloride TS. In moder-

Cobalt—Solutions of cobalt salts (1 in 20) in 3N hydro-ately concentrated solutions, benzoates yield a precipitate of

chloric acid yield a red precipitate when heated on a steam benzoic acid upon acidification with 2N sulfuric acid. This

bath with an equal volume of a hot, freshly prepared solu-precipitate is readily soluble in ethyl ether.

tion of 1-nitroso-2-naphthol (1 in 10) in 9N acetic acid.

Bicarbonate—See Carbonate.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 Copper—Solutions of cupric compounds, acidified with by hydrogen sulfide, and the resulting compound dissolves hydrochloric acid, deposit a red film of metallic copper

in a warm mixture of equal parts of nitric acid and water.upon a bright, untarnished surface of metallic iron. An ex-Bisulfite—See Sulfite.cess of 6N 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 6N 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 6N ammonium hydroxide. Add 3N 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 6N 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 6N acetic acid but black precipitate with ammonium sulfide TS. This precipitate dissolves in hydrochloric acid. Calcium salts moistened with is dissolved by cold 3N 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 1N 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

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

and by sodium bisulfite TS, in the cold, and by oxalic acid Ferrous Salts—Solutions of ferrous salts yield a dark blue

TS, in hot solution.

precipitate with potassium ferricyanide TS. This precipitate is

insoluble in 3N hydrochloric acid but is decomposed by Peroxide—Solutions of peroxides slightly acidified with

1N sodium hydroxide. With 1N 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—[N OTE—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 2N nitric acid and in 6N ammonium hydrox-

ide. With ammonium molybdate TS, acidified solutions of Lead—With 2N sulfuric acid, solutions of lead salts yield

orthophosphates yield a yellow precipitate that is soluble in a white precipitate that is insoluble in 3N hydrochloric or

6N ammonium hydroxide. This precipitate may be slow to 2N nitric acid, but is soluble in warm 1N sodium hydrox-

form. With silver nitrate TS, pyrophosphates obtained by ig-ide and in ammonium acetate TS. With potassium chromate

nition yield a white precipitate that is soluble in 2N nitric TS, solutions of lead salts, free or nearly free from mineral

acid and in 6N ammonium hydroxide. With ammonium acids, yield a yellow precipitate that is insoluble in 6N ace-

molybdate TS, a yellow precipitate that is soluble in 6N

tic acid but is soluble in 1N sodium hydroxide.

ammonium hydroxide is formed.

Lithium—With sodium carbonate TS, moderately concen-

Potassium—Potassium compounds impart a violet color trated solutions of lithium salts, made alkaline with sodium

to a nonluminous flame, but the presence of small quanti-hydroxide, yield a white precipitate on boiling. The precipi-

ties of sodium masks the color unless the yellow color pro-tate is soluble in ammonium chloride TS. Lithium salts

duced by sodium is screened out by viewing through a blue moistened with hydrochloric acid impart an intense crimson

filter that blocks emission at 589 nm (sodium) but is trans-color to a nonluminous flame. Solutions of lithium salts are

parent to emission at 404 nm (potassium). Traditionally, co-not precipitated by 2N sulfuric acid or soluble sulfates (dis-

balt glass has been used, but other suitable filters are com-tinction from strontium).

mercially available. In neutral, concentrated or moderately Magnesium—Solutions of magnesium salts in the pres-concentrated solutions of potassium salts (depending upon

ence of ammonium chloride yield no more than a slightly the solubility and the potassium content), sodium bitartrate hazy precipitate when neutralized with ammonium carbon-TS produces a white crystalline precipitate that is soluble in ate TS, but on the subsequent addition of dibasic sodium6N ammonium hydroxide and in solutions of alkali hydrox-phosphate TS, a white, crystalline precipitate, which is insol-ides and carbonates. The formation of the precipitate, which uble in 6N ammonium hydroxide, is formed.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 2N 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 1N sodium hydroxide. They yield also, in acid, but is readily soluble in 6N ammonium hydroxide. A neutral solutions with potassium iodide TS, a scarlet precipi-solution of a silver salt to which 6N 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 1N 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 6N 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-

6N 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-

cipitate when added to solutions of sulfates (distinction from Oxalate—Neutral and alkaline solutions of oxalates yield

thiosulfates).

a white precipitate with calcium chloride TS. This precipitate

is insoluble in 6N acetic acid but is dissolved by hydrochlo-Sulfite—When treated with 3N 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.

USP 35Chemical 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 101N 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 yellow fluorescent spots: the R F value of the principal spot Thiocyanate—With ferric chloride TS, solutions of thiocy-obtained from the Test Solution and from the Mixed Test anates yield a red color that is not destroyed by moderately Solution corresponds to that obtained from the Standard concentrated mineral acids.

Solution .

Thiosulfate—With hydrochloric acid, solutions of thiosul-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 Resolution Solution—Unless otherwise directed in the quickly disappears.

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.

3N hydrochloric acid. Ammonium sulfide TS produces a Developing Solvent—Prepare a mixture of 0.5 M oxalic similar precipitate in neutral and in alkaline solutions. With acid, previously adjusted with ammonium hydroxide to a potassium ferrocyanide TS, zinc salts in solution yield a pH of 2.0, acetonitrile, and methanol (80:20:20).

white precipitate that is insoluble in 3N hydrochloric acid.

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.

Procedure—Separately apply 1 μL each of the Standard ?193? IDENTIFICATION—

Solution , the Test Solution , and the Resolution Solution to the Chromatographic Plate . Allow the spots to dry, and develop TETRACYCLINES

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 R F 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

IDENTIFICATION TESTS

METHOD I

pH 3.5 Buffer—Dissolve 13.4 g of anhydrous citric acid Spectrophotometric tests contribute meaningfully toward and 16.3 g of dibasic sodium phosphate in 1000 mL of the identification of many compendial chemical substances.water, and mix.

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 3and 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 evidence of the identity of the substance that can be real-Chromatographic Sheet—Draw a spotting line 2.5 cm ized from any single test. The UV absorption spectrum, on from one edge of a 20-cm × 20-cm sheet of filter paper the other hand, does not exhibit a high degree of specific-(Whatman No. 1, or equivalent). Impregnate the sheet with ity. Conformance with both IR absorption and UV absorp-pH 3.5 Buffer by passing it through a trough filled with pH tion test specifications, as called for in a large proportion of 3.5 Buffer , and remove the excess solvent by firmly pressing compendial monographs, leaves little doubt, if any, regard-the sheet between nonfluorescent blotting papers.

ing the identity of the specimen under examination.

Procedure—To a suitable chromatographic chamber,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-