All glassware should 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 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 petroleum naphtha 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.

Separatory funnels. 250-milliliter, and 2,000-milliliter capacity, equipped with tetrafluoroethylene polymer stopcocks.

Erlenmeyer flask. 125-milliliter with 24/40 standard taper neck.

Evaporation flask. 250-milliliter capacity all-glass flask equipped with 24/40 standard taper stopper having inlet and outlet tubes to permit passage of nitrogen across the surface of the container liquid to be evaporated.

Condenser. 24/40 joints, fitted with drying tube, length optional.

Spectrophotometric cells. Fused quartz cells, optical path length in the range of 5,000 centimeters ±0.005 centimeter; also for checking spectrophotometer performance only, optical path length in the range 1,000 centimeter ±0.005 centimeter. With distilled water in the cells, determine any absorbance difference.

Spectrophotometer. Spectral range 250-400 mµ with spectral slit width of 2 mµ or less; under instrument operating conditions for these absorbance measurements, the spectrophotometer shall also meet the following performance requirements:

Ultraviolet lamp. Long wavelength (3400-3800A°).

Isooctane (2,2,4-trimethylpentane). Use 180 milliliters in a 250-milliliter Erlenmeyer flask, add 1 milliliter of purified n-hexadecane, insert the head assembly, allow nitrogen gas to flow into the inlet tube and connect the outlet tube to a solvent trap and vacuum line in such a way as to prevent any back flow of condensate into the flask. The contents of the flask are evaporated on a steam bath until 1 milliliter of residue remains. Dissolve the 1 milliliter of hexadecane residue in isooctane and make up to 25 milliliters. Determine the absorbance in a 5-centimeter path length cell compared to isooctane as reference. The absorbance should not exceed 0.01 per centimeter path length between 280-400 mµ. If necessary, isooctane may be purified by passage through a column of activated silica gel (Grade 12, Davidson Chemical Co., Baltimore, Md., or equivalent) or by distillation.

Methyl alcohol, A.C.S. reagent grade. Use 10 milliliters and proceed as with isooctane. The absorbance per centimeter of path length should be 0.00 between 280-400 mµ. Methyl alcohol may be purified by simple distillation or by refluxing in the presence of potassium hydroxide (10 grams/2 liters) and zinc dust (25 grams/2 liters) for 3 hours followed by distillation.

n-Hexadecane, 99 percent olefin-free. Dilute 1.0 milliliter of n-hexadecane to 25 milliliters with isooctane and determine the absorbance in a 5-centimeter cell compared to isooctane as reference between 280-400 mµ. 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.

Sodium borohydride. 98 percent.

Water. All distilled water must be extracted with isooctane before use. A series of three successive extracts of 1.5 liters of distilled water with 100-milliliter portions of isooctane is satisfactory.

Determination of ultraviolet absorbance. Add a 25-milliliter aliquot of the hydrocarbon solvent together with 1 milliliter of hexadecane to the 125-milliliter Erlenmeyer flask. While flushing with nitrogen, evaporate to 1 milliliter on a steam bath. Nitrogen is admitted through a 8±1-milliliter outer-diameter tube, drawn out into a 2±1-centimeter long and 1±0.5-millimeter inner-diameter capillary tip. This is positioned so that the capillary tip extends 4 centimeters into the flask. The nitrogen flow rate is such that the surface of the liquid is barely disturbed. After the volume is reduced to that of the 1 milliliter of hexadecane, the flask is left on the steam bath for 10 more minutes before removing. Add 10 milliliters of purified isooctane to the flask and reevaporate the solution to a 1-milliliter volume in the same manner as described above, except do not heat for an added 10 minutes. Repeat this operation twice more. Let the flask cool.

Add 10 milliliters of methyl alcohol and about 0.3 gram 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 sodium borohydride is dissolved, and allow to stand for 30 minutes at room temperature, with intermittent swirling. At the end of this time, disconnect the flask and evaporate the methyl alcohol on the steam bath under nitrogen until sodium borohydride begins to drop out of solution. Remove the flask and let it cool.

Add 6 milliliters of isooctane to the flask and swirl to wash the crystalline slurry. Carefully transfer the isooctane extract to a 250-milliliter separatory funnel. Dissolve the crystals in the flask with about 25 milliliters of distilled water and pour this also into the separatory funnel. Adjust the water volume in the separatory funnel to about 100 milliliters and shake for 1 minute. After separation of the layers, draw off the aqueous layer into a second 250-milliliter separatory funnel. Transfer the hydrocarbon layer in the first funnel to a 25-milliliter volumetric flask.

Carefully wash the Erlenmeyer flask with an additional 6 milliliters of isooctane, swirl, and transfer to the second separatory funnel. Shake the funnel for 1 minute. After separation of the layers, draw off the aqueous layer into the first separatory funnel. Transfer the isooctane in the second funnel to the volumetric flask. Again wash the Erlenmeyer flask with an additional 6 milliliters of isooctane, swirl, and transfer to the first separatory funnel. Shake the funnel for 1 minute. After separation of the layers, draw off the aqueous layer and discard. Transfer the isooctane layer to the volumetric flask and adjust the volume to 25 milliliters of isooctane. Mix the contents well, then transfer to the first separatory funnel and wash twice with 50-milliliter portions of distilled water. Discard the aqueous layers after each wash.

Determine the ultraviolet absorbance of the isooctane extract in 5-centimeter path length cells compared to isooctane as reference between 280-400 mµ. Determine a reagent blank concurrently with the sample, using 25 milliliters of purified isooctane instead of a solvent sample and measuring the ultraviolet absorbance of the blank between 280-400mµ.

The reagent blank absorbance should not exceed 0.04 per centimeter path length between 280-289 mµ; 0.020 between 290-359 mµ; and 0.010 between 360-400 mµ.

Determination of boiling-point range. Use ASTM method D86-82, “Standard Method for Distillation of Petroleum Products,” which is incorporated by reference. Copies may be obtained from the American Society for Testing Materials, 100 Barr Harbor Dr., West Conshohocken, Philadelphia, PA 19428-2959, or may be examined at the National Archives and Records Administration (NARA). For information on the availability of this material at NARA, call 202-741-6030, or go to: http://www.archives.gov/federal_register/code_of_federal_regulations/ibr_locations.html.

Determination of nonvolatile residue. For hydrocarbons boiling below 121 °C, determine the nonvolatile residue by ASTM method D1353-78, “Standard Test Method for Nonvolatile Matter in Volatile Solvents for Use in Paint, Varnish, Lacquer, and Related Products;” for those boiling above 121 °C, use ASTM method D381-80, “Standard Test Method for Existent Gum in Fuels by Jet Evaporation,” which methods are incorporated by reference. Copies may be obtained from the American Society for Testing Materials, 100 Barr Harbor Dr., West Conshohocken, Philadelphia, PA 19428-2959, or may be examined at the National Archives and Records Administration (NARA). For information on the availability of this material at NARA, call 202-741-6030, or go to: http://www.archives.gov/federal_register/code_of_federal_regulations/ibr_locations.html.