Packings for High-Pressure Liquid Chromatography
See packings for high-pressure liquid chromatography in the Chromatographic Reagents section under Chromatography
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Palladium Catalyst
Use a suitable grade.
[NoteA suitable grade is available commercially as Palladium Catalyst, Type I (5% Palladium on Calcium Carbonate), from Engelhard Industries, Inc., fax number (864) 885-1375. ]
Palladium Chloride,
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Assay
Dissolve 80 mg, accurately weighed, in 10 mL of diluted hydrochloric acid, dilute with water to 50 mL, and add 25 mL of a 1 in 100 solution of dimethylglyoxime in alcohol. Allow to stand for 1 hour, and filter. Check for complete precipitation with the dimethylglyoxime solution. Ignite the precipitate in a tared platinum crucible at 850
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Change to read:
Pancreatic Digest of Casein
(a bacteriological peptone; Tryptone)A grayish-yellow powder, having a characteristic, but not putrescent, odor. Freely soluble in water; insoluble in alcohol and in ether.
Residue on Ignition
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Analysis:
Ignite 500 mg with 1 mL of sulfuric acid.
Acceptance criteria:
NMT 75 mg (15%)
Microbial Content:
NMT 104 cfu/g
Nitrogen Content (Reagent test)
Analysis:
Determine by the Kjeldahl method.
Acceptance criteria:
9.0%14.0% is found
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Pancreatin
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Change to read:
Papaic Digest of Soybean Meal
A soluble nutrient material prepared by the action of the enzyme papain on soybean meal followed by suitable purification and concentration. It contains fermentable carbohydrates.
Residue on Ignition (Reagent test)
Analysis:
Ignite 500 mg with 1 mL of sulfuric acid.
Acceptance criteria:
NMT 75 mg (15.0%)
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Microbial Content:
NMT 104 cfu/g
Nitrogen Content (Reagent test)
Analysis:
Determine by the Kjeldahl method, using a test specimen previously dried at 105
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Acceptance criteria:
NLT 8.5% is found.
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Para-aminobenzoic Acid
(p-Aminobenzoic Acid),
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Assay
Accurately weigh about 300 mg, previously dried at 105
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Melting range
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Loss on drying
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Residue on ignition (Reagent test):
not more than 0.1%.
Paraformaldehyde,
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Assay
Transfer about 1 g, accurately weighed, to a 250-mL conical flask containing 50.0 mL of 1 N sodium hydroxide VS, and mix by swirling. Immediately, and slowly, add 50 mL of hydrogen peroxide TS, previously neutralized to bromothymol blue, through a small funnel placed in the neck of the flask. After the reaction moderates, rinse the funnel and inner wall of the flask with water, allow the solution to stand for 30 minutes, add bromothymol blue TS, and titrate the excess alkali with 1 N sulfuric acid VS. Each mL of 1 N sodium hydroxide is equivalent to 30.03 mg of HCHO: not less than 95% is found.
Residue on ignition:
not more than 0.1%.
Solubility in ammonia
Dissolve 5 g in 50 mL of ammonia TS: a practically clear, colorless solution results.
Reaction
Shake 1 g with 20 mL of water for about 1 minute, and filter: the filtrate is neutral to litmus.
Pectate Lyase
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Activity
Pectin solution
Transfer a quantity of Pectin, equivalent to 0.05 g on the dried basis, to a 100-mL volumetric flask. [NotePectin has a molecular weight of 103,000 Da; its degree of esterification (percentage of galacturonic acid groups substituted with methyl) is 12. ] Moisten with 0.1 mL of 2-propanol. Add 50 mL of water to the flask, and mix the solution with a magnetic stirrer. Use 0.5 N sodium hydroxide to adjust the solution to a pH of 12. Stop the stirrer, and allow the solution to stand undisturbed at room temperature for 15 minutes. Adjust the solution with 0.5 N hydrochloric acid to a pH of 8.0. Dilute with water to volume.
Tris Buffer solution
Transfer 6.055 g of Tris(hydroxymethyl)aminomethane and 0.147 g of calcium chloride to a 1000-mL volumetric flask containing 950 mL of water, and mix. Adjust the solution with 1 N hydrochloric acid to a pH of 8.0. Dilute with water to volume.
Diluted pectate lyase
Transfer 0.5 mL of Pectate Lyase to a 50-mL volumetric flask, dilute with Tris buffer solution to volume, and mix.
Procedure
Add the solutions set forth in the table below to quartz cuvettes.
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() 50(103)[(A30
in which 50 is the volume, in mL, of Diluted pectate lyase; 103 is the unit conversion factor; 30 is the time, in minutes, of the reaction; ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]()
Pentadecane,
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Assay
Inject an appropriate specimen into a gas chromatograph (see Chromatography
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Refractive index
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Add the following:
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Pentafluoropropanoic Acid
(Pentafluoropropionic Acid),
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Pentane
(n-Pentane),
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Boiling range (Reagent test)
Not less than 95% distils between 34
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2-Pentanone,
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Pepsin
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Pepsin, Purified
A white or yellowish-white powder, spongy mass, or translucent scales or granules. Freely soluble in water, producing more or less opalescence; practically insoluble in alcohol, in chloroform, and in ether. Purified Pepsin used in the second tier of the Dissolution test has an activity that is determined by the following method.
Activity
Pepsin solution
Transfer about 2.5 mg of Purified Pepsin, accurately measured, to a 100-mL volumetric flask, dilute with 10 mM hydrochloric acid to volume, and mix. [NotePrepare immediately before use. ]
2.0% hemoglobin solutionDissolve and dilute 2.5 g of bovine hemoglobin with water to 100 mL. Dilute 80 mL of this solution with 0.3 M hydrochloric acid to a volume of 100 mL.
Trichloroacetic acid solution
Dilute 5 g of trichloroacetic acid with water to 100 mL.
Test Solution
Transfer 5.0 mL of 2.0% Hemoglobin solution to a suitable container equilibrated at 37
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Control solution
Transfer 5.0 mL of 2.0% Hemoglobin solution to a suitable container equilibrated at 37
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Procedure
Determine the absorbances of the Test solution and Control solution, in 1-cm cells, at a wavelength of about 280 nm, using water as the reference. Calculate the activity of the portion of Purified Pepsin taken by the formula:
10,000(AU
in which AU and AC are the absorbances of the Test solution and the Control solution, respectively.
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Change to read:
Peptic Digest of Animal Tissue
(a bacteriological peptone)Tan powder, having a characteristic, but not putrescent, odor. Soluble in water; insoluble in alcohol and in ether. An autoclaved solution (2 in 100) is clear and is neutral or nearly so in its reaction.
Residue on Ignition
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Analysis:
Ignite 500 mg with 1 mL of sulfuric acid.
Acceptance criteria:
NMT 75 mg (15%)
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Microbial Content:
NMT 104 cfu/g
Nitrogen Content
Analysis:
Determine by the Kjeldahl method, using a test specimen previously dried at 105
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Acceptance criteria:
Between 9.0% and 14.0%
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Peptone, Dried
(Meat Peptone)
Reddish-yellow to brown powder, having a characteristic, but not putrescent, odor. Soluble in water, forming a yellowish-brown solution having a slight acid reaction; insoluble in alcohol and in ether.
Nitrogen compounds (Reagent test)
Determine by the Kjeldahl method, using a test specimen previously dried at 105
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Residue on ignition (Reagent test)
Ignite 500 mg with 1 mL of sulfuric acid: the residue weighs NMT 75 mg (15.0%).
Loss on drying
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Coagulable protein
Heat a filtered solution (1 in 20) to boiling: no precipitate forms.
Microbial content
NMT 104 cfu/g
Perchloric Acid
(70 Percent Perchloric Acid),
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Periodic Acid,
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Phases for Gas Chromatography
See phases for gas chromatography in the Chromatographic Columns section under Chromatography
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Phenacetin
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1,10-Phenanthroline
(Orthophenanthroline),
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o-Phenanthroline Monohydrochloride Monohydrate,
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Phenol
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Phenol Red, Sodium,
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Phenoxybenzamine Hydrochloride
[N-(2-Chloroethyl)-N-(1-methyl-2-phenoxyethyl)benzylamine Hydrochloride],
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Melting range
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Absorptivity
Its absorptivity, 1%, 1 cm, in the range of 272 nm to 290 nm, in chloroform solution is about 178.
3-Phenoxybenzoic Acid,
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Melting range
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2-Phenoxyethanol,
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Assay
To 2 g, accurately weighed, add 10 mL of a freshly prepared solution made by dissolving 25 g of acetic anhydride in 100 g of anhydrous pyridine. Swirl to mix the liquids, heat on a steam bath for 45 minutes, add 10 mL of water, heat for 2 additional minutes, and cool. Add 10 mL of normal butyl alcohol, shake vigorously, add phenolphthalein TS, and titrate with 1 N sodium hydroxide VS. Perform a blank test using the same quantities of the same reagents, and in the same manner, and make any necessary correction. Each mL of 1 N sodium hydroxide is equivalent to 138.2 mg of C8H10O2. Not less than 99% is found.
Phenol
Add 0.2 mL of it to 20 mL of water, mix, and to 5 mL of the mixture add 0.2 mL of Millon's reagent. Warm the solution at 60
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Phenyl Isocyanate,
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Assay
Transfer 250 mg, accurately weighed, to a glass-stoppered, 250-mL flask. Exercise care to avoid loss by volatilization, and avoid breathing the vapor. Add 20 mL of butylamine solution (25 g of butylamine diluted to 1000 mL with dioxane previously dried over potassium hydroxide pellets), insert the stopper in the flask, and allow to stand for 15 minutes. Add a few drops of methyl red TS and 25 mL of water, and titrate the excess amine with 0.1 N sulfuric acid VS. Perform a blank titration on 20 mL of the butylamine solution (see Residual Titrations
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2-Phenylacetamide
(
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Melting range
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dl-Phenylalanine,
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o-Phenylenediamine Dihydrochloride,
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Assay
When tested by thin-layer chromatography, with the use of plates coated with chromatographic silica gel mixture and a developing system consisting of a mixture of butyl alcohol, water, and acetic acid (12:5:3), and examined under short-wavelength UV light, a single spot is exhibited, with trace impurities.
p-Phenylenediamine Hydrochloride
(1,4-Diaminobenzene Dihydrochloride),
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Insoluble matter
Dissolve 1 g in 10 mL of water: the solution is clear and complete.
Molar absorptivity (see Spectrophotometry and Light-scattering
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Phenylglycine
(d(
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Phenylhydrazine,
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Congealing temperature
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Insoluble matter
Shake 1 mL with 20 mL of diluted acetic acid: the resulting solution is clear or practically so.
Residue on ignition (Reagent test)
Ignite 1 mL with 0.5 mL of sulfuric acid: the residue weighs not more than 1 mg (0.1%).
Phenylhydrazine Hydrochloride,
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Phenylmethylsulfonyl Fluoride,
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[NoteA suitable grade is available from Sigma-Aldrich, www.sigma-aldrich.com. ]
3-Phenylphenol
(m-Phenylphenol),
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Assay
Inject an appropriate specimen into a suitable gas chromatograph (see Chromatography
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Melting range
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Phloroglucinol,
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Insoluble in alcohol
Dissolve 1 g in 20 mL of alcohol: a clear and complete solution results.
Melting range, Class Ia
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Residue on ignition (Reagent test)
Ignite 1 g with 0.5 mL of sulfuric acid: the residue weighs not more than 1 mg (0.1%).
Diresorcinol
Heat to boiling a solution of 100 mg in 10 mL of acetic anhydride, cool the solution, and superimpose it upon 10 mL of sulfuric acid: no violet color appears at the zone of contact of the liquids.
Phloxine B
(Acid red 92; Eosin 10B; 2',4',5',7'-tetrabromo-4,5,6,7-tetrachlorofluorescein disodium salt)
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Phosphatase Enzyme, Alkaline
Use a suitable grade from intestinal origin.
[NoteA suitable grade is available from Worthington Biochemical Corp., www.worthington-biochem.com. ]
Phosphatic Enzyme
An enzyme preparation of microbial origin, high in both phosphatase and amylase activity, the former being the property that renders it suitable for use in the liberation of thiamine from its orthophosphate and pyrophosphate esters. Light cream-colored or slightly gray powder. Freely soluble in water. It hydrolyzes 300 times its weight of starch in 30 minutes.
Amylase activity
Place in a test tube 5 mL of a 1 in 50 solution of soluble starch in 0.2 M, pH 5 sodium acetate buffer (containing 1.6 g of anhydrous sodium acetate in each L and sufficient glacial acetic acid to adjust to a pH of 5), and add 4 mL of water. Mix, and place in a water bath at 40
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Phosphomolybdic Acid,
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Phosphoric Acid,
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Phosphorous Acid
(Phosphonic Acid),
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Phosphorus, Red,
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Yellow phosphorus
Shake 20 g with 75 mL of carbon disulfide in a glass-stoppered vessel, and allow to stand in the dark overnight. Filter, and wash the residue with carbon disulfide until the filtrate, collected in a graduated cylinder, measures 100 mL. Evaporate the solvent to 10 mL by immersing the cylinder in hot water. Dip a strip of cupric sulfate test paper in the remaining solvent: no more color is produced than in a similar strip dipped into 10 mL of solution in carbon disulfide containing 3 mg of yellow phosphorus (0.015% as P).
Soluble substances
Digest 2 g with 30 mL of acetic acid on a steam bath for 15 minutes. Cool, dilute with water to 40 mL, and filter. Evaporate 20 mL of the filtrate on a steam bath, and dry at 105
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Phosphorus Pentoxide
(Phosphoric Anhydride),
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Phosphotungstic Acid,
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Insoluble matter (Reagent test):
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Chloride (Reagent test)
One g shows not more than 0.3 mg of Cl (0.03%).
Nitrate
Dissolve 500 mg in 10 mL of water, and add about 10 mg of sodium chloride, 0.1 mL of indigo carmine TS, and 10 mL of sulfuric acid: the blue color does not disappear within 1 minute (about 0.01%).
Sulfate (Reagent test, Method I )
A 500-mg portion shows not more than 0.1 mg of SO4 (0.02%).
o-Phthalaldehyde
(Phthalic Dicarboxaldehyde),
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Phthalic Acid,
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Phthalic Anhydride,
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Phthalimide,
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Assay
Mobile phase
Prepare a mixture of isooctane and methyl-tert-butyl ether (88:12).
Procedure
Inject about 20 µL into a suitable liquid chromatograph (see Chromatography
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Melting range
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2-Picoline,
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Assay
Inject an appropriate specimen into a suitable gas chromatograph (see Chromatography
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Refractive index
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Picric Acid
(2,4,6-Trinitrophenol; Trinitrophenol),
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Picrolonic Acid
(3-Methyl-4-nitro-1-(p-nitrophenyl)-5-pyrazolone),
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Melting range
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Residue on ignition (Reagent test):
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Sensitiveness
Dissolve 25 mg in 10 mL of warm water containing 0.1 mL of glacial acetic acid, and filter the solution, if necessary. Dissolve 100 mg of calcium chloride in 250 mL of water, and mix. Heat 1 mL of the calcium chloride solution in a test tube to about 60
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Pipemidic Acid
(8-Ethyl-3,8-dihydro-5-oxo-2-(1-piperazinyl)pyrido[2,3-d]-pyrimidine-6-carboxylic acid),
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Piperazine
(Diethylenediamine),
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Piperidine,
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Congealing range
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Boiling range (Reagent test)
Not less than 95% distills between 104
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Refractive index:
about 1.454.
Platinic Chloride
(Chloroplatinic Acid),
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Polydimethylsiloxane, viscosity 0.65 centistokes
(Hexamethyldisiloxane),
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Refractive index
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Specific gravity
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Polyethylene Glycol 200,
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Refractive index
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Density:
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Viscosity:
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Polyethylene Glycol 600
![]() ![]() ![]() It meets the requirements of all of the tests under Polyethylene Glycol (NF monograph) except Limit of ethylene glycol and diethylene glycol.
Change to read:
Polyethylene Glycol 20,000
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pH
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Residue on ignition
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Polyoxyethylene 10 Lauryl Ether (Decaethylene Glycol Monododecyl Ether),
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Polyoxyethylene (23) Lauryl Ether (Brij-35)
Use a suitable grade.
[NoteA suitable grade is available commercially as Brij-35. ]
Polyoxyethylene (20) Sorbitan Monolaurate
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Polysaccharide Molecular Weight Standards
Polymaltotriose polymers of different weight-average molecular weight, MW, values ranging from 5,000 to 400,000 Da.
[NoteA suitable set is available from Shodex (www.shodex.com) as Kit P-82. ]
Polytef
Use Poly(tetrafluoroethylene).
Polyvinyl Alcohol,
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pH
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Loss on drying
Dry it at 110
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Residue on ignition:
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[NoteSuitable grades are available as catalog number U 232, from J.T. Baker Chemical Co., www.jtbaker.com. ]
Potassium Acetate,
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Potassium Alum
Use Potassium Alum [see Potassium Alum (USP monograph)].
Potassium Arsenate Monobasic,
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Potassium Bicarbonate,
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Potassium Biphthalate
(Acid Potassium Phthalate; Phthalic Acid Monopotassium Salt; Potassium Hydrogen Phthalate Acidimetric Standard),
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Potassium Bisulfate,
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Acidity
Dissolve 4 g, accurately weighed, in 50 mL of water, add phenolphthalein TS, and titrate with 1 N alkali: it contains between 34% and 36%, calculated as H2SO4.
Insoluble matter and ammonium hydroxide precipitate
Dissolve 10 g in 100 mL of water, add methyl red TS, render slightly alkaline with ammonia TS, boil for 1 minute, and digest on a steam bath for 1 hour. Pass through a tared filtering crucible, wash thoroughly, and dry at 105
![]() For the following tests, prepare a Test solution as follows. Dissolve 6 g in 45 mL of water, add 2 mL of hydrochloric acid, boil gently for 10 minutes, cool, and dilute with water to 60 mL.
Heavy metals (Reagent test)
To 30 mL of Test solution add phenolphthalein TS, and neutralize with ammonia TS. Add 0.5 mL of glacial acetic acid, dilute with water to 40 mL, and add 10 mL of hydrogen sulfide TS: any brown color produced is not darker than that of a control containing 10 mL of Test solution and 0.02 mg of added Pb (0.001%).
Iron
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Potassium Bromate,
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Potassium Bromide,
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Potassium Carbonate, Anhydrous,
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Potassium Chlorate,
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Potassium Chloride,
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Potassium Chloroplatinate,
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Assay
Accurately weigh about 300 mg, transfer to a 600-mL beaker, add 20 mL of hydrochloric acid, and heat gently if necessary to achieve complete solution. Add zinc granules, slowly, until no more dissolves, then add 2 mL of hydrochloric acid, and digest for 1 hour on a steam bath to coagulate the reduced platinum. Add more acid, if necessary, to ensure that all of the zinc has dissolved. Filter through paper, rinsing the beaker with diluted hydrochloric acid until all of the precipitate is transferred to the filter, then wash with several small portions of water. Ignite the filter in a tared crucible at 800 ± 25
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Potassium Chromate,
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Potassium Cyanide,
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Potassium Dichromate,
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[NotePotassium dichromate of a quality suitable as a primary standard is available from the National Institute of Standards and Technology, Washington, DC, www.nist.gov, as standard sample No. 136. ]
Potassium Ferricyanide,
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Potassium Ferrocyanide,
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Potassium Hyaluronate
White to cream-colored powder. Freely soluble in water. Store in a tight container, in a refrigerator.
Inhibitor content
Prepare as directed in the Assay under Hyaluronidase for Injection (USP monograph) a quantity of Standard solution containing 1 USP Hyaluronidase Unit in each mL, and a similar quantity of acetate-buffered Standard solution using as the solvent 0.1 M, pH 6 sodium acetate buffer (prepared by diluting the 0.2 M buffer prepared as directed below with an equal volume of water). Prepare from the potassium hyaluronate under test 10 mL of Potassium hyaluronate stock solution, and dilute 2 mL of it with the specified Phosphate buffer solution to make a Hyaluronate solution. In the same way, and concurrently, dilute a second 2-mL portion of the stock solution with 0.2 M, pH 6 sodium acetate buffer (containing 16.4 g of anhydrous sodium acetate and 0.45 mL of glacial acetic acid in each 1000 mL).
Place 0.50-mL portions of the Hyaluronate solution in each of four 16- × 100-mm test tubes, and place 0.50-mL portions of the acetate-buffered Hyaluronate solution in two similar tubes. To two of the four tubes containing Hyaluronate solution add 0.50 mL of Diluent for hyaluronidase solutions, prepared as directed in the Assay under Hyaluronidase for Injection (USP monograph). To the remaining two tubes, on a rigid schedule, at 30-second intervals, add 0.50 mL of Standard solution. Similarly, to the two tubes containing acetate-buffered Hyaluronate solution add at 30-second intervals 0.50-mL portions of acetate-buffered Standard solution. Then proceed as directed in the second paragraph for Procedure, beginning with Mix the contents, as far as Plot the average. The reduction in absorbance of acetate-buffered Hyaluronate solution is not less than 25% of that observed in the Hyaluronate solution.
Turbidity production
The average absorbance of the solutions in the two tubes containing Hyaluronate solution and Diluent for hyaluronidase solutions prepared in the test for Inhibitor content is not less than 0.26 at a wavelength of 640 nm in a suitable spectrophotometer using a 1-cm cell.
Potassium Hydrogen Sulfate,
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Melting point
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Potassium Hydroxide,
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Potassium Iodate,
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Potassium Iodide,
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Potassium Metabisulfite
(Potassium Disulfite; Potassium Pyrosulfite),
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Potassium Nitrate,
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Potassium Nitrite,
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Potassium Perchlorate,
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Potassium Periodate
(Potassium meta-Periodate),
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Potassium Permanganate,
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Potassium Persulfate,
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Potassium Phosphate, Dibasic
(Dipotassium Hydrogen Phosphate; Dipotassium Phosphate),
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Potassium Phosphate, Dibasic, Trihydrate
(Dipotassium Hydrogen Phosphate Trihydrate; Dipotassium Phosphate),
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Potassium Phosphate, Monobasic
(Potassium Biphosphate; Potassium Dihydrogen Phosphate),
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[NoteCertified Potassium Dihydrogen Phosphate is available from the National Institute of Standards and Technology, Washington, DC, www.nist.gov, as standard sample No. 186. ]
Potassium Phosphate, Tribasic,
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Potassium Pyroantimonate
(Potassium hexahydroxyantimonate), KSb(OH)6
262.90
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Potassium Pyrophosphate,
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Potassium Pyrosulfate
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Potassium Sodium Tartrate,
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Potassium Sulfate,
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Potassium Tellurite
(Potassium Tellurate IV),
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Assay
Weigh accurately about 120 mg, transfer to a beaker, and dissolve in a mixture of 10 mL of nitric acid, 10 mL of sulfuric acid, and 25 mL of water. Heat to boiling, and boil until copious fumes of sulfur trioxide are evolved. Cool, cautiously add 100 mL of water, heat to boiling, add 6 g of sodium fluoride, and titrate the hot solution with 0.1 N potassium permanganate VS. Each mL of 0.1 N potassium permanganate is equivalent to 12.69 mg of K2TeO3. Not less than 98% is found.
Chloride (Reagent test)
One g shows not more than 0.1 mg of Cl (0.01%).
Potassium Thiocyanate,
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Pregnenolone Acetate
(3
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Propionaldehyde,
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Propionic Anhydride,
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Assay
Accurately weigh about 350 mg into a tared, glass-stoppered flask containing 50 mL of dimethylformamide previously neutralized to the thymol blue endpoint with 0.1 N sodium methoxide in methanol VS. Titrate with 0.1 N sodium methoxide in methanol VS to the thymol blue endpoint. Perform a blank determination, and make any necessary correction. Each mL of 0.1 N sodium methoxide is equivalent to 13.014 mg of C6H10O3. Not less than 97.0% is found.
Refractive index
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Propiophenone,
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n-Propyl Alcohol
(1-Propanol),
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Propylamine Hydrochloride
(1-Propanamine hydrochloride; n-Propylamine hydrochloride),
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Protein Molecular Weight Standard
Also known as protein molecular weight markers (for SDS-PAGE) and consists of a mixture of several proteins of well-defined molecular weights. The products are generally available in a suitable buffer containing a suitable reducing agent (generally, 100 mM DTT), a preservative (for example, sodium azide), and 50% glycerol to prevent freezing. Use a suitable grade. Store at 20
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Protein Standard Solution (8 g/dL)
A solution containing 5 g of Albumin Human and 3 g of human gamma globulin per dL.
[NoteA suitable grade is available as Protein Standard Solution, catalog number 540-10, from Sigma-Aldrich, www.sigma-aldrich.com. ]
Protocatechuic Acid (3,4-Dihydroxybenzoic acid),
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Pullulanase
(Amylopectin-6-gluconohydrolase)
![]() ![]() ![]() ![]()
Measurement of relative pullulanase activity
Determination of pullulanase activity
Substrate
Dissolve pullulan1
1
A suitable supplier for pullulan is www.hayashibara-intl.com.
in water to make a 1.25% (w/v) solution.
[NoteAdd pullulan to the water. Clumping will occur if water is added to pullulan. ]
Buffer solution A, pH 5.0
Add 0.1 M disodium phosphate (27 g of dibasic sodium phosphate in each L of the solution) to 0.1 M citric acid (21 g of citric acid in each L of the solution) to adjust pH to 5.0.
Buffer solution B, pH 6.0
Add diluted acetic acid to 1 M sodium acetate (136 g of sodium acetate in each L of solution) to adjust the pH to 6.0. Dilute with water to prepare the final buffer solution as 0.01 M acetic acid buffer, pH 6.0.
Somogyi reagent
Add 54 g of disodium phosphate heptahydrate or 28 g of anhydrous disodium hydrogen phosphate and 40 g of potassium sodium tartrate to about 650 mL of water or about 700 mL for anhydrous disodium hydrogen phosphate. Add 100 mL of 1 N sodium hydroxide to this solution and mix. Add 80 mL of 10% cupric sulfate to the solution, and mix. Heat until any solid is completely dissolved. Add 180 g of anhydrous sodium sulfate to the solution and adjust the volume to 1 L. Allow the solution to stand at room temperature for 1 or 2 days to let insoluble matter precipitate. Filter the solution with standard filter paper, and keep the solution in a brown bottle with a ground-glass stopper.
Nelson reagent
Dissolve 50 g of ammonium molybdate in 900 mL of water. Add 42 g of sulfuric acid, and mix. Dissolve 6 g of sodium arsenate or 3.6 g of monobasic potassium arsenate in 50 g of water. Allow the solution to stand in a brown bottle with a ground-glass stopper at 37
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Glucose standard solution
Dry anhydrous glucose crystals under less than 50-mm Hg at 60
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Pullulanase diluent
Dilute pullulanase with Buffer solution B, pH 6.0 to prepare a solution having the enzyme activity of about 0.2 units per mL. [NoteThe measurement range is between 0.1 and 0.4 units per mL. ] Record the dilution factor (FPD). This diluent is used as a diluted enzyme solution.
Procedure
Transfer 4 mL of Substrate to a test tube and add 0.5 mL of Buffer solution A, pH 5.0, mix, and incubate at 30
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Measurement of protein amount (measured as albuminoid amount) for the calculation of specific activity
Reagent A
Prepare a solution having known concentrations of about 0.1 N sodium hydroxide and about 0.2 M sodium carbonate.
Reagent B
Transfer 0.5 g of cupric sulfate and 1.0 g of sodium citrate dihydrate to a 100-mL volumetric flask, dissolve in and dilute with water to volume, and mix.
Lowry solution
Mix Reagent A and Reagent B at the proportion of 50:1.
Diluted Folin-Ciocalteu's phenol reagent (for albuminoid quantification)
Prepare a two-fold dilution of 2 N Folin-Ciocalteu's phenol reagent commercially available or prepare a solution by making an appropriate dilution from Folin-Ciocalteu Phenol TS (see Method 2 in Biotechnology-Derived ArticlesTotal Protein Assay
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Bovine serum albumin standard stock solution
Transfer 0.05 g of bovine serum albumin to a 500-mL volumetric flask, dissolve in and dilute with water to volume, and mix. It contains 100 µg of bovine serum albumin per mL.
Standard solutions
Using appropriate dilutions of Bovine serum albumin standard stock solution in water, prepare five Standard solutions having concentration equally spaced between 5 and 100 µg of bovine serum albumin per mL.
Test solution
Dilute pullulanase with Buffer solution B, pH 6.0 in order to obtain a solution having a concentration between 60 and 70 µg of albuminoid per mL. [NoteWater can be used as diluent. ] Record the dilution factor, FTS.
Blank solution
Use water.
Procedure
To 0.3 mL in separate tubes of the Standard solutions, the Test solution, and the Blank solution, add 3 mL of Lowry solution, and mix. Allow to incubate at room temperature for 10 minutes. Add 0.3 mL of Diluted Folin-Ciocalteu's phenol reagent to each tube, mix immediately, and allow to stand at room temperature for 60 minutes. Determine the absorbances of the Standard solutions and the Test solution at the wavelength of maximum absorbance at about 750 nm, using the Blank solution as the blank.
Calculation
[NoteThe relationship of absorbance to protein concentration is nonlinear; however, if the standard curve concentration range is sufficiently small, it will approach linearity. ] Using linear regression method, plot the absorbances of the Standard solutions versus the protein (bovine serum albumin) concentrations, in µg per mL, and determine the best fit curve. Using the plot, determine the concentration, Calbuminoid, in µg per mL, of protein (albuminoid amount) in the Test solution. Calculate the albuminoid concentration, in mg per mL, in the pullulanase taken by the formula:
Cprotein = (Calbuminoid × FTS)/1000
Calculate the specific activity, SA, in units per mg, of pullulanase using the formula:
SA = PA/Cprotein
Pumice
A substance of volcanic origin consisting chiefly of complex silicates of aluminum and alkali metals. Occurs as very light, hard, rough, porous, gray masses, or as a gray-colored powder. Is insoluble in water and is not attacked by diluted acids.
Acid- and water-soluble substances
Boil 2.0 g of powdered pumice with 50 mL of diluted hydrochloric acid under a reflux condenser for 30 minutes. Cool, and filter. To half of the filtrate add 5 drops of sulfuric acid, evaporate to dryness, ignite, and weigh: the residue weighs not more than 60 mg (6.0%).
Purine,
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Melting range
![]() ![]() ![]() ![]() ![]() A single spot is exhibited when it is examined by thin-layer chromatography, with the use of plates coated with chromatographic silica gel mixture and a developing system consisting of butyl alcohol, water, and glacial acetic acid (60:25:15).
Putrescine Dihydrochloride,
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Pyrazole,
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Melting range
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Pyrene,
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Assay
Transfer about 9 mg, accurately weighed, to a 100-mL volumetric flask, dissolve in methanol, dilute with methanol to volume, and mix. Transfer 2.0 mL of this solution to a 100-mL volumetric flask, dilute with methanol to volume, and mix. Using a suitable spectrophotometer, 1-cm cells, and methanol as the blank, record the absorbance of the solution at the wavelength of maximum absorbance at about 238 nm. From the observed absorbance, calculate the absorptivity (see Spectrophotometry and Light-scattering
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Melting range
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Pyridine,
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Pyridine, Dried
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Pyridoxal Hydrochloride,
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Melting range
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Residue on ignition (Reagent test):
not more than 0.1%.
Loss on drying
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Nitrogen content (Reagent test)
Determine by the Kjeldahl method, using a test specimen previously dried at 105
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Chloride content
Accurately weigh about 500 mg, previously dried at 105
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Pyridoxal 5-Phosphate,
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Pyridoxamine Dihydrochloride,
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Melting range
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Residue on ignition (Reagent test):
not more than 0.15%.
Loss on drying
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Nitrogen content (Reagent test)
Determine by the Kjeldahl method, using a test specimen previously dried at 105
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Chloride content
Determine as directed in the test for Chloride content under Pyridoxal Hydrochloride: between 29.1% and 29.6% of Cl is found.
1-(2-Pyridylazo)-2-naphthol,
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Melting range
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Sensitiveness
Add 0.1 mL of a 1 in 1000 solution of it in alcohol to a mixture of 10 mL of water and 1 mL of a buffer solution prepared by mixing 80 mL of 0.2 M acetic acid and 20 mL of sodium acetate solution (8.2 in 100), and mix. To this solution add 1 mL of a mixture of 1 mL of cupric sulfate TS and 2 mL of water, and mix: the color changes from yellow to red.
4-(2-Pyridylazo)resorcinol (PAR),
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3-(2-Pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-5¢,5¢¢-disulfonic Acid, Disodium Salt
(3-(2-Pyridyl)-5,6-bis(5-sulfo-2-furyl)-1,2,4-triazine, Disodium Salt Hydrate),
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[NoteA suitable grade is available as product number P4272 from Sigma-Aldrich, 1-800-558-9160; www.sigma-aldrich.com. ]
Pyrogallol,
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Pyrrole,
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Boiling range (Reagent test)
Not less than 90% distills between 128
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Pyruvic Acid,
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Refractive index
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Assay
Accurately weigh about 1 g, transfer to a suitable container, and add 100 mL of water. Mix, add phenolphthalein TS, and titrate with 0.5 N sodium hydroxide VS. Each mL of 0.5 N sodium hydroxide is equivalent to 44.03 mg of CH3COCOOH: not less than 98% of CH3COCOOH is found.
Auxiliary Information
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