Methods for Flavour Enhancers
Methods for glutamates
Methods for nucleosides
Methods for Sweeteners
Purity test for Saccharine
Purity tests for Cyclamates
Methods for Hydrocarbons, Waxes and Oils
Methods for Polyols
Identification tests
Proceed as directed under Chromatography (Thin-layer chromatography) under following condition:
Sample: 1 µl of 1 in 100 solution of the sample. Add a few drops of ammonium hydroxide TS if required to dissolve.
Reference: 1 µl of a 1 in 100 solution of monosodium L-glutamate
Solvent: A mixture of 2 volumes of n-butanol, 1 volume of glacial acetic acid and 1 volume of water
Adsorbent: Silica gel
Stop the development when the solvent front has advanced about 10 cm from the point of application. Dry the plate at 80o for 30 min. Spray ninhydrin TS on the plate, heat at 80o for 10 min and observe the plate under natural light. The Rf value of the sample and that of the reference standard are identical.
Proceed as directed under Chromatography (Thin-layer chromatography) under following condition:
Sample: 2 µl of a 0.5 in 100 solution of the sample
Reference: 2 µl of a 0.5 in 100 solution of monosodium L-glutamate containing 2.5 of pyrrolidone carboxylic acid
Solvent: A mixture of 2 volumes of n-butanol, 1 volume of glacial acetic acid and 1 volume of water.
Adsorbent: Silica gel
Potassium iodide-starch TS: Dissolve 0.5 g of starch in about 50 ml of water by heating, after cooling add 0.5 g of potassium iodide and water to make up to 100 ml.
Stop the development when the solvent front has advanced about 10 cm from the point of the application, dry the plate for 30 min in air.
At the same time, prepare a similar chamber as that for developing, placing in the chamber a 50-ml beaker containing about 3 g of sodium hypochlorite; pour slowly 1 ml of hydrochloric acid into the beaker in order to generate chlorine gas, put on the lid and allow to stand for 30 sec to fill the chamber with the gas. Place the dried plate in this chamber, put on the lid and allow to stand for 20 min. Take out the plate, keep for 10 min in air and spray with ethanol. After drying, spray potassium iodide-starch TS and observe the plate under natural light immediately after the standard spot has appeared.
No spot corresponding to pyrrolidone carboxylic acid standard is detected in the sample (sensitivity = 0.2%).
To 3 ml of a 3 in 10,000 solution of the sample in water, add 0.2 ml of a 1 in 10 solution of orcinol in ethanol and subsequently 3 ml of a 1 in 1,000 hydrochloric acid solution of ferric ammonium sulfate. Heat in a water bath for 10 min. A green colour is produced.
To the solution given in the monograph add 2 ml of magnesia mixture TS. No precipitate is formed. Add 5 ml of nitric acid, boil for 10 min, neutralize with strong ammonia TS, add water to make to 100 ml, add ammonium molybdate TS, and warm. A yellow precipitate, which dissolves in sodium hydroxide TS or ammonia TS.
Proceed as directed under "Thin-layer chromatography" using the solution described in the monograph as the sample solution. Use a mixture of 80 volumes of a saturated solution of ammonium sulfate, 18 volumes of a 13.6% w/v solution of sodium acetate and 2 volumes of isopropanol as the developing solvent. Use microcrystalline cellulose as the absorbent. Stop the development when the solvent front has advanced about 10 cm from the point of the application, dry the plate in air, and observe under ultraviolet light (about 254 nm) in a dark place. Only a spot of 5'-guanylic acid or 5'-inosinic acid is detected.
Purity test for Saccharine
Standard and test solutions
Methylene chloride: Use a suitable pure grade, equivalent to the product obtained by distillation in all-glass apparatus.
Internal standard stock solution: Transfer 100 mg of 95% n-tricosane into a 10 ml volumetric flask. Dissolve in n-heptane, dilute to volume with the same solvent, and mix.
Stock standard preparation: Transfer 20 mg each of reagent grade o-toluenesulfonamide and p-toluene- sulfonamide into a 10 ml volumetric flask. Dissolve in methylene chloride, dilute to volume with the same solvent, and mix.
Dilute standard preparations: Pipet into five 10-ml volumetric flasks 0.1, 0.25, 1.0, 2.5 and 5 ml, respectively, of the "Stock standard preparation". Pipet 0.25 ml of the "Internal standard stock solution" into each flask, dilute each to volume with methylene chloride, and mix. These solutions contain 250 µg of n-tricosane, plus respectively, 20, 50, 200, 500 and 1000 µg per ml of each toluenesulfonamide.
Test preparation: Dissolve 2 g of the sample in 8.0 ml of sodium carbonate TS. Mix the solution thoroughly with 10 g of chromatographic siliceous earth (Celite 545 or equivalent). Transfer the mix into a 25 x 250-mm chromatographic tube having a fritted glass disk and a Teflon stopcock at the bottom, and a reservoir at the top. Pack the contents of the tube by tapping the column on a padded surface, and then by tamping firmly from the top. Place 100 ml of methylene chloride in the reservoir, and adjust the stopcock so that 50 ml of eluate is collected in 20-30 min. To the eluate add 25 µl of "Internal standard stock solution". Mix, and then concentrate the solution to a volume of 1 ml in a suitable concentrator tube fitted with a modified Snyder column, using a Kontes tube heater maintained at 90o.
Procedure
Inject 2.5 µl of the "Test preparation" into a suitable gas chromatograph equipped with a flame-ionization detector. The column is of glass, approximately 3 m in length and 2 mm in inside diameter, and it is packed with 3% methyl phenyl silicone in 100 to 120 mesh silanized calcined diatomaceous silica (Caution: The glass column should extend into the injector for on-column injection and into the detector base to avoid contact with metal). The carrier is helium flowing at a rate of 30 ml per min. The injection port, column, and detector are maintained at 225o, 180o, and 250o, respectively. The instrument attenuation setting should be such that 2.5 µl of the "Dilute standard preparation" containing 200 µg per ml of each toluene sulfonamide gives a response of 40-80% of full-scale deflection. Record the chromatogram, note the peaks for o-toluene sulfonamide, p-toluene sulfonamide, and the n-tricosane internal standard, and calculate the areas of each peak by suitable means. The retention times for o-toluene sulfonamide, p-toluene sulfonamide, and n-tricosane are about 5, 6, and 15 min, respectively.
In a similar manner, obtain the chromatograms for 2.5-µl portions of each of the five "Dilute standard preparations", and for each solution determine the areas of the o-toluene sulfonamide, p-toluene sulfonamide, and n-tricosane peaks. From the values thus obtained, prepare standard curves by plotting concentration of each toluene sulfonamide, in µg per ml, vs. the ratio of the respective toluene sulfonamide peak area to that of n-tricosane. From the standard curve determine the concentration, in µg per ml, of each toluene sulfonamide in the "Test preparation". Divide each value by 2 to convert the result mg/kg of the toluene sulfonamide in 2 g sample taken for analysis. [Note: If the toluene sulfonamide content of the sample is greater than about 500 mg/kg, the impurity may crystallize out of the methylene chloride concentrate (see "Test preparation"). Although this level of impurity exceeds that permitted by the specifications, the analysis may be completed by diluting the concentrate (usually 1:10 is satisfactory) with methylene chloride containing 250 µg of n-tricosane per ml, and by applying appropriate dilution factors in the calculation. Care must be taken to redissolve completely any crystalline toluene sulfonamide to give a homogeneous solution.]
Methyl orange-boric acid solution: Dissolve 200 mg of methyl orange and 3.5 g of boric acid in 100 ml of water, heating on a steam bath to effect solution. Allow to stand for at least 24 h, and filter before use.
Standard solution: Transfer 100 mg of cyclohexylamine into a 100-ml volumetric flask, dissolve in 50 ml of water and 0.5 ml of hydrochloric acid TS, dilute to volume with water, and mix. Transfer 5 ml of the solution into a second 100-ml volumetric flask, dilute to volume with water, and mix. Transfer 5 ml of the solution into a third 100-ml volumetric flask, dilute to volume with water, and mix. Each ml of this solution contains 2.5 µg of cyclohexylamine.
Test preparation: Prepare the test preparation as directed in the individual monograph.
Procedure: Transfer 10 ml each of the Standard preparation and of the Test preparation into two separate 50-ml glass-stoppered centrifuge tubes, and transfer 10 ml of water to a third tube to serve as a blank. To each tube add 3.0 ml of disodium ethylenediamine tetraacetate solution (prepared by dissolving 10 g of disodium ethylenediamine- tetraacetate and 3.4 g of sodium hydroxide in 100 ml of water) and 15 ml of a 20:1 mixture of chloroform and n-butanol, shake the tubes for 2 min, and centrifuge. Remove and discard the aqueous layer in each tube, and then transfer 10 ml of the chloroform solution from each tube into separate centrifuge tubes. To each tube add 2 ml of Methyl orange-boric acid solution, shake the tubes for 2 min, and centrifuge. Remove and discard the aqueous layer in each tube, then add to each tube 1 g of anhydrous sodium sulfate, shake well, and allow to settle. Transfer 5 ml of each clear chloroform solution into separate test tubes, add 0.5 ml of 50:1 mixture of methanol and sulfuric acid TS, and mix. Successively determine the absorbance of the solutions in 1 cm cells at 520 nm with a suitable spectrophotometer, using the blank to set the instrument at zero. The absorbance of the solution from the Test preparation does not exceed that from the Standard preparation.
Dissolve 50 g of the sample in 300 ml of water, add 3 ml of sodium hydroxide TS, and extract with 50 ml and 30 ml of chloroform. Combine the extracts, add 2 g of anhydrous potassium carbonate and filter. Wash the container and the residue on the filter paper several times with 5 ml chloroform, combine the washings to the filtrate and concentrate to 1 ml at 30º. To this solution add 1 ml of nitrobenzene standard solution (100 mg in 500 ml chloroform) as an internal standard and examine for dicyclohexylamine by Gas chromatography using a flame ionization detector under the conditions described below.
Chromatographic column: Stainless steel, 1.5 m x 3-4 mm of inside diameter, packed with 60-80 mesh diatomaceous earth (gas chromatographic grade) in a solution of methanolic potassium hydroxide. The final potassium hydroxide concentration should be about 3% of the diatomaceous support. Evaporate off the methanol, add a chloroform solution of polyethyleneglycol 6000, and evaporate the chloroform. The content of polyethyleneglycol 6000 should be about 10% of the diatomaceous support.
Column temp.: 130-140º
Carrier gas: Nitrogen or helium, flow rate should be set so that the retention time of nitrobenzene is about 7 min
Calculate the dicyclohexylamine content from a standard curve.
Dicyclohexylamine (C12H23N) for the standard curve:
Refractive index:n (25, D): 1.480-1.488
Specific gravity: d (25, 25): 0.905-0.915
Boiling point: 254-256º
Oxidative Microcoulometry
NOTE: All reagents used in this test should be reagent grade: water should be of high purity, and gases must be high-purity grade.
Apparatus: The Dohrmann Microcoulometric Titrating System (MCTS-30), or its equivalent as shown in the Figure should be used. It consists of a constant rate injector (A), a pyrolysis furnace (B), a quartz pyrolysis tube (C), a granular tin scrubber (D), a titration cell (E), and a microcoulometer with a digital readout (F).
Granular-Tin Scrubber: Place 5 g of 20/30 mesh granular reagent grade tin between quartz-wool plugs in an elongated 18/8-12/5 standard-taper adaptor which connects the pyrolysis tube and the titration cell.
Microcoulometer: Must have variable attenuation, gain control, and be capable of measuring the potential of the sensing-reference electrode pair, and comparing this potential with a bias potential, amplifying the potential difference, and applying the amplified difference to the working-auxiliary electrode pair so as to generate a titrant. Also the microcoulometer output voltage signal must be proportional to the generating current.
Pyrolysis Furnace: The sample should be pyrolyzed in an electric furnace having at least two separate and independently controlled temperature zones, the first being an inlet section that can maintain a temperature sufficient to volatilize all the organic sample. The second zone shall be a pyrolysis section that can maintain a temperature sufficient to pyrolyze the organic matrix and oxidize all the organically bound sulfur. A third outlet temperature zone is optional.
Pyrolysis Tube: Must be fabricated from quartz and constructed in such a way that a sample, which is vaporized completely in the inlet section, is swept into the pyrolysis zone by an inert gas where it mixes with oxygen and is burned. The inlet end of the tube shall hold a septum for syringe entry of the sample and side arms for the introduction of oxygen and inert gases. The center, or pyrolysis section, should be of sufficient volume to ensure complete pyrolysis of the sample.
Sampling Syringe: A microlitre syringe of 10-µl capacity capable of accurately delivering 1 to 10 µl of sample into the pyrolysis tube. Three-inch x 24-gauge needles are recommended to reach the inlet zone of the pyrolysis furnace.
Titration Cell: Must contain a sensor-reference pair of electrodes to detect changes in triiodide ion concentration, a generator anode-cathode pair of electrodes to maintain constant triiodide ion concentration, and an inlet for a gaseous sample from the pyrolysis tube. The sensor electrode shall be platinum foil and the reference electrode platinum wire in saturated triiodide half cell. The generator anode and cathode half-cell shall also be placed on a magnetic stirrer.
Preparation of Apparatus: Carefully insert the quartz pyrolysis tube into the furnace, attach the tin scrubber, and connect the reactant and carrier-gas lines. Add the Cell Electrolyte Solution (see below) to the titration cell and flush the cell several times. Maintain an electrolyte level of 3.8 cm (1.5 in.) above the platinum electrodes. Place the titration cell on a magnetic stirrer and connect the cell inlet to the tin scrubber outlet. Position the platinum foil electrodes (mounted on the movable cell head) so that the gas-inlet flow is parallel to the electrodes with the generator anode adjacent to the generator cathode. Assemble and connect the coulometer in accordance with the manufacturer's instructions. Double-wrap the adaptor containing the tin scrubnber with heating tape and turn the heating tape on. Adjust the flow of the gases, the pyrolysis furnace temperature, the titration cell, and the coulometer to the desired operating conditions. Typical operating conditions are as follows:
Reagent gas flow (oxygen): 200 cm3/min
Carrier-gas flow (Ar, He): 400 cm3/min
Furnace temperatures
Inlet zone: 700o (maximum)
Pyrolysis zone: 800 - 1000o
Outlet zone: 800º (maximum)
Tin-Scrubber flow rate: 200 cm3/min
Titration cell: Stirrer speed set to produce slight vortex
Coulometer
Bias voltage: 160 mV
Gain: 50
Constant Rate Injector: 0.25 µl/sec
The tin scrubber must be conditioned to sulfur, nitrogen, and chlorine before quantitative analysis can be achieved. A solution containing 10 mg/kg butyl sulfide, 100 mg/kg pyridine, and 200 mg/kg chlorobenzene in isoctane has proven an effective conditioning agent. With a fresh scrubber installed and heated, two 30-µl samples of this conditioning agent injected at a flow rate of 0.5 µl/sec produces a steady increasing response, with final conditioning indicated by a constant reading from the offset during the second injection.
Reagents
- Argon or Helium, (Argon preferred) High-purity grade: two-stage regulators must be used.
- Cell Electrolyte Solution: dissolve 0.5 g of potassium iodide and 0.6 g of sodium azide in 500 ml of high-purity water, add 5 ml of glacial acetic acid and dilute to 1 L. Store in a dark bottle or in a dark place and prepare fresh at least every 3 months.
- Oxygen: high-purity grade.
- Iodine: resublimed, 20 mesh or less.
- Sulfur Standard (approximately 100 mg/kg): weigh accurately 0.1569 g of n-butyl sulfide, into a tared 500-ml volumetric flask. Dilute to the mark with isooctane and reweigh. Calculate the sulfur concentration (S), in percent, by the formula:
where
Ws = weight of n-butyl sulfide
Wc = weight of the solution.
Calibration: Prepare a calibration standard (approximately 5 mg/kg) by pipetting 5 ml of Sulfur Standard into a 10-ml volumetric flask and diluting to volume with isooctane. Fill and clamp the syringe onto the constant rate injector, push the sliding carriage forward to penetrate the septum with the needle, and zero the meter in case of long-term drift in the automatic baseline zero circuitry. Switch S1 automatically starts the stepper-motor syringe drive and initiates the analysis cycle. At 2.5 min (after setting switch S1) set the digital meter with the scan potentiometer to correspond to the sulfur content of the known standard to the nearest 0.01 mg/kg. At the 3-min point, the number displayed on the meter stops, the plunger drive block is retracted to its original position, as preset by switch S2 and a baseline re-equilibration period equal to the injection period must be allowed before a new sample may be injected. Repeat the Calibration step a total of at least four times.
Procedure: Rinse the syringe several times with sample: then fill it, clamp it onto the constant-rate injector push the sliding carriage forward to penetrate the septum with the needle, and zero the meter. Turn on switch S1 to start the stepper-motor syringe drive automatically and initiate the analysis cycle. After the 3-min hold point, the number displayed on the meter corresponds to the sulfur content of the injected sample.
(ASTM D 2502 See TEST for Viscosity for Copyright permission)
Determine the kinematic viscosity of the sample at 37.8 and 98.9° as described in the method for Viscosity, 100°. Read the value of H that corresponds to the measured viscosity at 37.8° by the use of table 1; linear interpolation between adjacent columns may be required. Read a viscosity -molecular weight chart for H and 98.9° viscosity (the chart is available from the American Society for Testing and Materials (ASTM). A simplified version is shown in Figure 1 for illustration purposes only. Interpolate where necessary between adjacent lines of 98.89° viscosity. After locating the point corresponding to the value of H (ordinate) and the 98.89° viscosity (superimposed lines), read the molecular weight along the abscissa.
(ASTM D 2887 See TEST for Viscosity for Copyright permission.)
"Carbon number" is number of carbon atoms in a molecule. Determine the boiling point distribution of the sample by gas chromatography using the following conditions:
The system must have the following performance characteristics:
sensitivity: 1% dodecane must be detected with a peak height of at least 10% of full scale under the conditions prescribed below.
stability: when operated at the required sensitivity level, the baseline drift is not more than 1% of full scale per hour
repeatability of retention times: 6 sec for each component of the calibration mixture.
resolution (R): determined for a solution of 1% of each of hexadecane and octadecane in n-octane is not less than three and not more than eight, using the following formula:
R = 2d / (W1+W2)
where
d is the distance in mm between the peak maxima of hexadecane and octadecane
W1 is the peak width in mm at the baseline of hexdecane
W2 is the peak width in mm at the baseline of octadecane
Typical conditions which may be used:
Column:
length: 1.5m
outside diameter: 3.2 mm
liquid phase: SE - 30.5 %
support material: Chromosorb G, mesh 60/80
Column temperature:
initial: 10°
final: 350°
rate: 6.5°/ min.
Carrier
gas: helium
flow: 30 ml/min.
Detector: FID
Detector temperature: 370°
Injection temperature: 370°
Sample size: 0.3 µl
Calibration mixture:
Prepare a mixture of hydrocarbons of known boiling points covering the range of the sample. At least one compound must have a boiling point lower than the initial boiling point of the sample.
Procedure:
Calibration: Cool the column to the selected starting temperature (the retention time for the initial boiling point must be at least 1 min) and inject the calibration mixture. Record the retention time of each peak maximum and the peak areas for each component. Plot the retention time of each peak versus the corresponding normal boiling point of that component in degrees Celsius to obtain a calibration curve.
Sample analysis:
Using the exact conditions used in the calibration run, inject the sample. Record the area of each time segment at fixed time intervals not greater than 1% of the retention time equivalent to a boiling point of 538° obtained from the calibration curve.
Calculation:
Sum the area segments to obtain the cumulative area at each time interval during the run. At the point of the chromatogram, where the baseline at the end first becomes steady, observe the cumulative area counts. Move back along the record until a cumulative area equal to 99.5% of the total at the steady point appears. Mark this point as the final boiling point. Observe the area counts at the start of the run until the point is reached, where the cumulative area count is equal to 0.5% of the total area. Mark this point as the initial boiling point of the sample. Divide the cumulative area at each interval between the initial and final boiling points by the total cumulative area and multiply by 100. This will give the cumulative percent of the sample recovered at each time interval. Tabulate the cumulative percent recovered at each interval and the retention time at the end of the interval. Using linear interpolation, if necessary, determine the retention time associated with 5% and read the corresponding boiling temperature from the calibration curve.
General Instructions: Because of the sensitivity of the test, the possibility of errors arising from contamination is great. It is of the greatest importance that all glassware be scrupulously cleaned to remove all organic matter such as oil, grease, detergent residues, etc. Examine all glassware, including stoppers and stopcocks, under ultraviolet light to detect any residual fluorescent contamination. As a precautionary measure it is a recommended practice to rinse all glassware with purified isooctane immediately before use. No grease is to be used on stopcocks or joints. Great care to avoid contamination of wax samples in handling and to assure absence of any extraneous material arising from inadequate packaging is essential. Because some of the polynuclear hydrocarbons sought in this test are very susceptible to photo-oxidation, the entire procedure is to be carried out under subdued light.
Apparatus
- Separatory funnels: 250-ml, 500-ml, 1,000-ml, and preferably 2,000-ml capacity, equipped with tetrafluoroethylene polymer stopcocks.
- Reservoir: 500 ml capacity, equipped with a 24/40 standard taper male fitting at the bottom and a suitable balljoint at the top for connecting to the nitrogen supply. The male fitting should be equipped with glass hooks.
- Chromatographic tube: 180 mm in length, inside diameter to be 15.7 mm ± 0.1 mm, equipped with a coarse, fritted-glass disc, a tetrafluoroethylenepolymer stopcock, and a female 24/40 standard tapered fitting at the opposite end. (Overall length of the column with the female joint is 235 mm). The female 24/40 standard tapered fitting at the opposite end.
- Disc: Tetrafluoroethylene polymer 2-inch diameter disc approximately 3/16-inch thick with a hole bored in the center to closely fit the stem of the chromatographic tube.
- Heating jacket: Conical, for 500-ml separatory funnel. (Used with variable transformer heat control).
- Suction flask: 250-ml or 500-ml filter flask.
- Condenser: 24/40 joints, fitted with a drying tube, length optional.
- Evaporation flask (optional): 250-ml or 500-ml capacity all-glass flask equipped with standard taper stopper having inlet and outlet tubes permit passage of nitrogen across the surface of the liquid to be evaporated.
- Vacuum distillation assembly: All glass (for purification of dimethyl sulfoxide); 2-l distillation flask with heating mantle; Vigreaux vacuum-jacketed condenser (or equivalent) about 45 cm in length and distilling head with separable cold finger condenser. Use of tetrafluoroethylene polymer sleeves on the glass joints will prevent freezing. Do not use grease on stopcocks or joints.
- Spectrophotometric cells: Fused quartz cells, optical path length in the range of 5,000 ± 0.005 cm; also for checking spectro- photometer performance only, optical path length in the range 1,000 ± 0.005 cm. With distilled water in the cells, determine any absorbance differences.
- Spectrophotometer: Spectral range 250 nm-400 nm with spectral slit width of 2 nm or less, under instrument operating conditions for these absorbance measurements, the spectrophotometer shall, also meet the following performance requirements:
- Nitrogen cylinder: Water-pumped or equivalent purity nitrogen in cylinder equipped with regulator and valve to control flow at 5 p.s.i.g.
Reagents and materials
- Organic solvents: All solvents used throughout the procedure shall meet the specifications and tests described in this specification. The isooctane, benzene, acetone, and methyl alcohol designated in the list following this paragraph shall pass the following test:
To the specified quantity of solvent in a 250-ml Erlenmeyer flask, add 1 ml of purified n-hexadecane and evaporate on the steam bath under a stream of nitrogen (a loose aluminium foil jacket around the flask will speed evaporation). Discontinue evaporation when not over 1 ml of residue remains. (to the residue from benzene add a 10 ml portion of purified isooctane, reevaporate, and repeat once to insure complete removal of benzene).
Alternatively, the evaporation time can be reduced by using the optional evaporation flask. In this case the solvent and n-hexadecane are placed in the flask on the steam bath, the tube assembly is inserted, and a stream of nitrogen is fed through the inlet tube while the outlet tube is connected to a solvent trap and vacuun line in such a way as to prevent any flow-back of condensate into the flask.
Dissolve the 1 ml of hexadecane residue in isooctane and make to 25 ml volume. Determine the absorbance in the 5 cm path length cells compared to isooactane as reference. The absorbance of the solution of the solvent residue (except for methyl alcohol) shall not exceed 0.01 per cm path length between 280 and 400 nm. For methyl alcohol this absorbance value shall be 0.00.
- Isooctane. (2,2,4-trimethylpentane): Use 180 ml for the test described in the preceding paragraph. Purify, if necessary, by passage through a column of activated silica gel (Grade 12, Davison Chemical Company, Baltimore, Maryland, or equivalent) about 90 cm in length and 5 cm to 8 cm in diameter.
- Benzene, reagent grade: Use 150 ml for the test. Purify, if necessary, by distillation or otherwise.
- Acetone, reagent grade: Use 200 ml for the test. Purify, if necessary, by distillation.
- Eluting mixtures:
- n-Hexadecane, 99% olefin-free: Dilute 1.0 ml of n-hexadecane to 25 ml with isooctane and determine the absorbance in a 5-cm cell compared to isooctane as reference point between 280-400 nm. The absorbance per centimeter path length shall not exceed 0.00 in this range. Purify, if necessary, by percolation through activated silica gel or by distillation.
- Methyl alcohol, reagent grade: Use 10.0 ml of methyl alcohol. Purify, if necessary, by distillation.
- Dimethyl sulfoxide: Pure grade, clear, water-white, m.p. 18º minimum. Dilute 120 ml of dimethyl sulfoxide with 240 ml of distilled water in a 500-ml separatory funnel, mix and allow to cool for 5-10 min. Add 40 ml of isooctane to the solution and extract by shaking the funnel vigourously for 2 min. Draw off the lower aqueous layer into a second 500 ml separatory funnel and repeat the extraction with 40 ml of isooctane. Draw off and discard the aqueous layer. Wash each of the 40 ml extractives three times with 50 ml portions of distilled water. Shaking time for each wash is 1 min. Discard the aqueous layers. Filter the first extractive through anhydrous sodium sulfate prewashed with isooctane (see Sodium sulfate under "Reagents and Materials" for preparation of filter), into a 250-ml Erlenmeyer flask, or optionally into the evaporating flask. Wash the first separatory funnel with the second 40 ml isooctane extractive, and pass through the sodium sulfate into the flask. Then wash the second and first separatory funnels successively with a 10 ml portion of isooctane, and pass the solvent through the sodium sulfate into the flask. Add 1 ml of n-hexadecane and evaporate the isooctane on the steam bath under nitrogen. Discontinue evaporation when not over 1 ml of residue remains. To the residue, add a 10 ml portion of isooctane and reevaporate to 1 ml of hexadecane. Again, add 10 ml of isooctane to the residue and evaporate to 1 ml of hexadecane to insure complete removal of all volatile materials. Dissolve the 1 ml of hexadecane in isooctane and make to 25 ml volume. Determine the absorbance in 5 cm path length cells compared to isooctane as reference. The absorbance of the solution should not exceed 0.02 per cm path length in the 280-400 nm range. (Note - Difficulty in meeting this absorbance specification may be due to organic impurities in the distilled water. Repetition of the test omitting the dimethyl sulfoxide will disclose their presence. If necessary to meet the specification, purify the water by redistillation, passage through an ion-exchange resin, or otherwise).
Purify, if necessary, by the following procedure: To 1,500 ml of dimethyl sulfoxide in a 2 l glass-stoppered flask, add 6.0 ml of phosphoric acid and 50 g of Norit A (decolorizing carbon, alkaline) or equivalent. Stopper the flask, and with the use of a magnetic stirrer (tetrafluoro-ethylene polymer coated bar) stir the solvent for 15 min. Filter the dimethyl sulfoxide through four thicknesses of fluted paper (18.5 cm, Schleicher & Schuell, No. 597, or equivalent If the initial filtrate contains carbon fines, refilter through the same filter until a clear filtrate is obtained. Protect the sulfoxide from air and moisture during this operation by covering the solvent in the funnel and collection flask with a layer of isooctane. Transfer the filtrate to a 2-l separatory funnel and draw off the dimethyl sulfoxide into the 2-l distillation flask of the vacuum distillation assembly and distill at approximately 3 mm Hg pressure or less. Discard the first 200 ml fraction of the distillate and replace the distillate collection flask with a clean one. Continue the distillation until approximately 1 l of the sulfoxide has been collected.
At completion of the distillation, the reagent should be stored in glass-stoppered bottles since it is very hygroscopic and will react with some metal containers in the presence of air.
- Magnesium oxide (Sea Sorb 43, Food Machinery Company, Westvaco Division, distributed by chemical supply firms, or equivalent): Place 100 g of the magnesium oxide in a large beaker, add 700 ml of distilled water to make a thin slurry, and heat on a steam bath for 30 min with intermittent stirring. Stir well initially to insure that all the absorbent is completely wetted. Using a Buchner funnel and a filter paper (Schleicher & Schuell No. 597, or equivalent) of suitable diameter, filter with suction. Continue suction until water no longer drips from the funnel. Transfer the absorbent to a glass trough lined with aluminium foil (free from rolling oil). Break up the magnesia with a clean spatula and spread out the absorbent on the aluminium foil in a layer about 1-2 cm thick. Dry for 24 h at 160±1º. Pulverize the magnesia with mortar and pestle. Sieve the pulverized absorbent between 60-180 mesh. Use the magnesia retained on the 180-mesh sieve.
- Celite 545: Johns-Manville Company, diatomaceous earth, or equivalent.
- Magnesium oxide-Celite 545 mixture (2+1) by weight: Place the magnesium oxide (60-180 mesh) and the Celite 545 in 2 to 1 proportions, respectively, by weight in a glass-stoppered flask large enough for adequate mixing. Shake vigorously for 10 min. Transfer the mixture to a glass trough lined with aluminium foil (free from rolling oil) and spread it out on a layer about 1 to 2 cm thick. Reheat the mixture at 160±1o for 2 h, and store in a tightly closed flask.
- Sodium sulfate, anhydrous, reagent grade, preferably in granular form: For each bottle of sodium sulfate reagent used, establish as follows the necessary sodium sulfate prewash to provide such filters required in the method: Place approximately 35 g of anhydrous sodium sulfate in a 30 ml coarse, fritted-glass funnel or in a 65 ml filter funnel with glass wool plug; wash with successive 15 ml portions of the indicated solvent until a 15 ml portion of the wash shows 0.00 absorbance per cm path length between 280 nm and 400 nm when tested as prescribed under "Organic solvents." Usually three portions of wash solvent are sufficient.
Procedure
Before proceeding with the analysis of a sample, determine the absorbance in a 5 cm path cell between 250 nm and 400 nm for the reagent blanck by carrying out the procedure, without a wax sample, at room temperature, recording the spectra after the extraction stage and after the complete procedure as precribed. The absorbance per centimeter path length following the extraction stage should not exceed 0.040 in the wavelength range from 250 to 400 nm; the absorbance per cm path length following the complete procedure should not exceed 0.070 in the wavelength range from 250 to 299 nm, inclusive, nor 0.045 in the wavelength range from 300 nm to 400 nm. If in either spectrum the characteristic benzene peaks in the 250-260 nm region are present, remove the benzene by the procedure under "Organic solvents" and record absorbance again.
Place 300 ml of dimethyl sulfoxide in a 1-l separatory funnel and add 75 ml of phosphoric acid. Mix the contents of the funnel and allow to stand for 10 min. (The reaction between the sulfoxide and the acid is exothermic. Release pressure after mixing, then keep funnel stoppered). Add 150 ml of isooctane and shake to preequilibrate the solvents. Draw off the individual layers and store in glass-stoppered flasks.
Place a representative 1 kg sample of wax, or if this amount is not available, the entire sample, in a beaker of a capacity about three times the volume of the sample and heat with occasional stirring on a steam bath until the wax is completely melted and homogenous. Weigh four 25 ± 0.2 g portions of the melted wax in separate 100 ml beakers. Reserve three of the portions for later replicate analyses as necessary. Pour one weighed portion immediately after remelting (on the steam bath) into a 500 ml separatory funnel containing 100 ml of the preequilibrated sulfoxide-phosphoric acid mixture that has been heated in the heating jacket at a temperature just high enough to keep the wax melted. (Note: In preheating the sulfoxide-acid mixture, remove the stopper of the separatory funnel at intervals to release the pressure).
Promptly complete the transfer of the sample to the funnel in the jacket with portions of the preequilibrated isooctane, warming the beaker, if necessary, and using a total volume of just 50 ml of the solvent. If the wax comes out of solution during these operations, let the stoppered funnel remain in the jacket until the wax redissolves. (Remove stopper from the funnel at intervals to release pressure).
When the sample is in solution, remove the funnel from the jacket and shake it vigorously for 2 min. Set up three 250 ml separatory funnels with each containing 30 ml of preequilibrated isooctane. After separation of the liquid phases, allow to cool until the main portion of the sample-isooctane solution begins to show a precipitate. Gently swirl the funnel when precipitation first occurs on the inside surface of the funnel to accelerate this process. Carefully draw off the lower layer, filter it slowly through a thin layer of glass wool fitted loosely in a filter funnel into the first 250 ml separatory funnel, and wash in tandem with the 30 ml portions of isooctane contained in the 250 ml separatory funnels. Shaking time for each wash is 1 min. Repeat the extraction operation with two additional portions of the sulfoxide-acid mixture, replacing the funnel in the jacket after each extraction to keep the sample in solution and washing each extractive in tandem through the same three portions of isooctane.
Collect the successive extractives (300 ml total) in a separatory funnel (preferably 2-liter), containing 480 ml of distilled water, mix, and allow to cool for a few min after the last extractive has been added. Add 80 ml of isooctane to the solution and extract by shaking the funnel vigorously for 2 min. Draw off the lower aqueous layer into a second separatory funnel (preferably 2-l) and repeat the extraction with 80 ml of isooctane. Draw off and discard the aqueous layer. Wash each of the 80 ml extractives three times with 100 ml portions of distilled water. Shaking time for each wash is 1 min. Discard the aqueous layers. Filter the first extractive through anhydrous sodium sulfate prewashed with isooctane (see Sodium Sulfate under "Reagents and Materials" for preparation of filter) into a 250-ml Erlenmeyeer flask (or optionally into the evaporation flask). Wash the first separatory funnel with the second 80 ml isooctane extractive and pass through the sodium sulfate. Then wash the second and first separatory funnels successively with a 20 ml portion of isooctane and pass the solvent through the sodium sulfate into the flask. Add 1 ml of n-hexadecane and evaporate the isooctane on the steam bath under nitrogen. Discontinue evaporation when not over 1 ml of residue remains. To the residue, add a 10 ml portions of isooctane, reevaporate to 1 ml of hexadecane, and repeat this operation once more.
Quantitatively transfer the residue with isooctane to a 25 ml volumetric flask, make to volume, and mix. Determine the absorbance of the solution in the 5 cm path length cells compared to isooctane as reference between 280 nm-400 nm (take care to lose none of the solution in filling the sample cell). Correct the absorbance values for any absorbance derived from reagents as determined by carrying out the procedure without the sample. If the corrected absorbance does not exceed the limits prescribed in the Characteristics, the sample meets the ultraviolet absorbance specifications. If the corrected absorbance per centimeter path length exceeds the limits prescribed in the Characteristics, proceed as follows:
Quantitatively transfer the isooctane solution to a 125 ml flask equipped with 24/40 joint and evaporate the isooctane on the steam bath under a stream of nitrogen to a volume of 1 ml of hexadecane. Add 10 ml of methyl alcohol and approximately 0.3 g of sodium borohydride (Minimize exposure of the borohydride to the atmosphere. A measuring dipper may be used). Immediately fit a water-cooled condenser equipped with a 24/40 joint and with a drying tube into the flask, mix until the borohydride is dissolved, and allow to stand for 30 min at room temperature, with intermittent swirling. At the end of this period, disconnect the flask and evaporate the methyl alcohol on the steam bath under nitrogen until the sodium borohydride begins to come out of the solution. Then add 10 ml of isooctane and evaporate to a volume of about 2-3 ml. Again, add 10 ml of isooctane and concentrate to a volume of approximately 5 ml. Swirl the flask repeatedly to assure adequate washing of the sodium borohydride residues.
Fit the tetrafluoroethylene polymer disc on the upper part of the stem of the chromatographic tube, then place the tube with the disc on the suction flask and apply the vaccuum (approximately 135 mm Hg pressure). Weigh out 14 g of the 2:1 magnesium oxide-Celite 545 mixture and pour the adsorbent mixture into the chromatographic tube in approximately 3 cm layers. After the addition of each layer, level off the top of the adsorbent with a flat glass rod or metal plunger by pressing down firmly until the adsorbent is well packed. Loosen the topmost few mm of each adsorbent layer with the end of a metal rod before the addition of the next layer. Continue packing in this manner until all the 14 g of the adsorbent is added to the tube. Level off the top of the adsorbent by pressing down firmly with a flat glass rod or metal plunger to make the depth of the adsorbent bed approximately 12.5 cm in depth. Turn off the vacuum and remove the suction flask. Fit the 500 ml reservoir onto the top of the chromatographic column and prewet the column by passing 100 ml of isooctane through the column. Adjust the nitrogen pressure so that the rate of descent of the isooctane coming off of the column is between 2-3 ml per min. Discontinue pressure just before the last of the isooctane reaches the level of the adsorbent. (Caution:Do not allow the liquid level to recede below the adsorbent level at any time). Remove the reservoir and decant the 5 ml isooctane concentrate solution onto the column and with slight pressure again allow the liquid level to recede to barely above the adsorbent level. Rapidly complete the transfer similarly with two 5 ml portions of isooctane, swirling the flask repeatedly each time to assure adequate washing of the residue. Just before the final 5 ml wash reaches the top of the adsorbent, add 100 ml of isooctane to the reservoir and continue the percolation at the 2-3 ml per min rate. Just before the last of the isooctane reaches the adsorbent level, add 100 ml of 10% benzene in isooctane to the reservoir and continue the percolation at the aforementioned rate. Just before the solvent mixture reaches adsorbent level, add 25 ml of 20% benzene in isooctane to the reservoir and continue the percolation at 2-3 ml per min until all this solvent mixture has been removed from the column. Discard all the elution solvents collected up to this point. Add 300 ml of the acetone-benzene-water mixture to the reservoir and percolate through the column to elute the polynuclear compounds. Collect the eluate in a clean 1-l separatory funnel. Allow the column to drain until most of the solvent mixture is removed. Wash the eluate three times with 300 ml portions of distilled water, shaking well for each wash. (The addition of small amounts of sodium chloride facilitates separation). Discard the aqueous layer after each wash. After the final separation, filter the residual benzene through anhydrous sodium sulfate prewashed with benzene (see Sodium sulfate under "Reagents and Materials" for preparation of filter) into a 250-ml Erlenmeyer flask (or optionally into the evaporation flask). Wash the separatory funnel with two additional 20 ml portions of benzene which are also filtered through the sodium sulfate. Add 1 ml of n-hexadecane and completely remove the benzene by evaporation under nitrogen, using the special procedure to eliminate benzene as previously described under "Organic Solvents". Quantitatively transfer the resiue with isooctane to a 25 ml volumetric flask and adjust the volume. Determine the absorbance of the solution in the 5 cm path length cells compared to isooctane as reference between 250 - 400 nm. Correct for any absorbance derived from the reagents as determined by carrying out the procedure without a was sample. If either spectrum shows the characteristic benzene peaks in the 250 - 260 nm region, evaporate the solution to remove benzene by the procedure under "Organic Solvents". Dissolve the residue, transfer quantitatively, and adjust to volume in isooctane in a 25 ml volumetric flask. Record the absorbance again. If the corrected absorbance does not exceed the limits prescribed in the Characteristics the sample meets the ultraviolet absorbance specifications.
(ASTM D 445 Adapted, with permission, from the Annual Book of ASTM Standards, copyright American Society for Testing and Materials, 100 Harbor Drive, West Conshohocken, PA 19428. Copies of the complete ASTM standard may be purchased direct from ASTM, phone: 610-832-9585, fax: 610-832-9555, e-mail: service@astm.org, website: http://www.astm.org)
Use a viscometer of the glass capillary type, calibrated and capable of measuring kinematic viscosity with a repeatability exceeding 0.35 % only in one case in twenty. Immerse the viscometer in a liquid bath at the temperature required for the test ± 0.1° ensuring that at no time of the measurement will any portion of the sample in the viscometer be less than 20 mm below the surface of the bath liquid or less than 20 mm above the bottom of the bath. Charge the viscometer with sample in the manner dictated by the design of the instrument. Allow the sample to remain in the bath for about 30 min. Where the design of the viscometer requires it, adjust the volume of sample to the mark. Use pressure to adjust the head level of the sample to a position in the capillary arm of the instrument about 5 mm ahead of the first mark. With the sample flowing freely, measure, in seconds (±0.2 s), the time required for the meniscus to pass from the first to the second timing mark. If the time is less than 200 s, select a viscometer with a capillary of smaller diameter and repeat the operation. Make a second measurement of the flow time. If two measurements agree within 0.2 %, use the average for calculating the kinematic viscosity. If the measurements do not agree, repeat the determination after thorough cleaning and drying the viscometer.
Viscosity, 100o (mm2/s) = C x t
where
C = calibration constant of the viscometer (mm2/s2)
t = flow time (s)
Examine by thin layer chromatography using silica gel as the coating substance, and using standard and test solutions described in the individual monograph.
4-Aminobenzoic acid reagent: Prepare a solution by dissolving 1 g of 4-aminobenzoic acid in a solvent mixture composed of 18 ml acetic acid, 20 ml water and 1 ml phosphoric acid. Prepare this reagent immediately before use.
Sodium periodate reagent: 0.2% w/v sodium periodate in water
Procedure: Apply 2 µl of each of the standard and test solution to the bottom of the TLC plate. Develop the chromatogram over a path of 17 cm using as the mobile phase a mixture of 70 volumes of propanol, 20 volumes of ethyl acetate and 10 volumes of water. Allow the plate to dry in air and spray with a mixture of 2 volumes of 4-aminobenzoic acid reagent with 3 volumes of acetone. Heat at 100o for 15 min. Spray with the sodium periodate reagent. Heat at 100o for 15 min. The principal spot in the chromatogram obtained from the test solution corresponds in position, colour and size to the principal spot obtained from the standard solution.
Test solution: Dissolve 20.0 g of the sample in a mixture of equal volumes of dilute acetic acid TS and water and dilute to 100 ml with the same mixture of solvents. Add 2.0 ml of a 1% w/v solution of ammonium pyrrolidinedithiocarbamate and 10 ml of methyl isobutyl ketone. Mix and allow the layers to separate and use the methyl isobutyl ketone layer for analysis.
Standard solutions: Prepare three standard solutions in the same manner as the test solution but adding 0.5 ml, 1.0 ml, and 1.5 ml, respectively, of a standard nickel solution containing 10 mg/kg Ni, in addition to the 20.0 g of the sample.
Procedure: Set the instrument to zero using methyl isobutyl ketone prepared as described for the preparation of the test solution but omitting the substance to be examined. Use a nickel hollow-cathode lamp as source of radiation and an air-acetylene flame. The analysis wavelength for all solutions is 232.0 nm.
Dissolve as completely as possible 0.01 g of the sample by shaking with 0.15 ml of 0.1 N sodium hydroxide and add 1 ml of acid ferric sulfate TS. Within 5 min, a cherry-red colour develops that finally becomes deep purple.
To 2 ml of a 2% solution of the sample in water add 2 ml of water, 0.1 g of sodium bicarbonate and about 0.02 g of ferrous sulfate. Shake and allow to stand. A deep violet colour is produced, which disappears on the addition of 5 ml of dilute sulfuric acid TS.
Dissolve a quantity of the sample in water to obtain a solution containing 10 mg/ml, heating in a water bath at 60o, if necessary. Similarly, prepare a standard solution of potassium gluconate in water containing 10 mg/ml.
Apply separate 5-µl portions of the test solution and the standard solution on a suitable thin-layer chromatographic plate coated with 0.25-mm layer of chromatographic silica gel, and allow to dry. Develop the chromatogram in a solvent system consisting of a mixture of ethanol, water, ammonium hydroxide, and ethyl acetate (50:30:10:10) until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the chamber, and dry at 110o for 20 min. Allow to cool, and spray with a reagent, prepared as follows: Dissolve 2.5 g of ammonium molybdate in about 50 ml of 2 N sulfuric acid in a 100-ml volumetric flask, add 1.0 g of ceric sulfate, swirl to dissolve, dilute with 2 N sulfuric acid to volume, and mix. Heat the plate at 110o for about 10 min. The principal spot obtained from the test solution corresponds in colour, size, and retention to that obtained from the standard solution.
Heat a few drops of the sample in a test tube with about 0.5 g of potassium bisulfate; pungent vapours of acrolein are evolved.
Transfer the solution described in the individual monograph into a porcelain dish and add 10 mg of sulfanilic acid. Heat the solution on a water bath for a few min, add 5 ml of a 1 in 5 solution of sodium nitrite and heat slightly. Make alkaline with sodium hydroxide TS. A red colour is produced.