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 hydrocarbon solvent 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.

Chromatographic tube. 450 millimeters in length (packing section), inside diameter 19 millimeters ±1 millimeter, equipped with a wad of clean Pyrex brand filtering wool (Corning Glass Works Catalog No. 3950 or equivalent). The tube shall contain a 250-milliliter reservoir and a 2-millimeter tetrafluoroethylene polymer stopcock at the opposite end. Overall length of the tube is 670 millimeters.

Stainless steel rod. 2 feet in length, 2 to 4 millimeters in diameter.

Vacuum oven. Similar to Labline No. 3610 but modified as follows: A copper tube one-fourth inch in diameter and 13 inches in length is bent to a right angle at the 4-inch point and plugged at the opposite end; eight copper tubes one-eighth inch in diameter and 5 inches in length are silver soldered in drilled holes (one-eighth inch in diameter) to the one-fourth-inch tube, one on each side at the 5-, 7.5-, 10- and 12.5-inch points; the one-eighth-inch copper tubes are bent to conform with the inner periphery of the oven.

Beakers. 250-milliliter and 500-milliliter capacity.

Graduated cylinders. 25-milliliter, 50-milliliter, and 150-milliliter capacity.

Tuberculin syringe. 1-milliliter capacity, with 3-inch, 22-gauge needle.

Volumetric flask. 5-milliliter capacity.

Spectrophotometric cells. Fused quartz ground glass stoppered cells, optical path length in the range of 1.000 centimeter ±0.005 centimeter. With distilled water in the cells, determine any absorbance difference.

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

Absorbance repeatability, ±0.01 at 0.4 absorbance.

Absorbance accuracy, 1 ±0.05 at 0.4 absorbance.

Wavelength repeatability, ±0.2 millimicron.

Wavelength accuracy, ±1.0 millimicron.

Nitrogen cylinder. Water-pumped or equivalent purity nitrogen in cylinder equipped with regulator and valve to control flow at 5 p.s.i.g.

Organic solvents. All solvents used throughout the procedure shall meet the specifications and tests described in this specification. The isooctane, benzene, hexane, and 1,2-dichloroethane designated in the list following this paragraph shall pass the following test:

To the specified quantity of solvent in a 250-milliliter beaker, add 1 milliliter of purified n-hexadecane and evaporate in the vacuum oven under a stream of nitrogen. Discontinue evaporation when not over 1 milliliter of residue remains. (To the residue from benzene add a 5-milliliter portion of purified isooctane, reevaporate, and repeat once to insure complete removal of benzene.)

Dissolve the 1 milliliter of hexadecane residue in isooctane and make to 5 milliliters volume. Determine the absorbance in the 1-centimeter path length cells compared to isooctane as reference. The absorbance of the solution of the solvent residue shall not exceed 0.02 per centimeter path length between 280 and 300 mµ and shall not exceed 0.01 per centimeter path length between 300 and 400 mµ.

Isooctane (2,2,4-trimethylpentane). Use 10 milliliters for the test described in the preceding paragraph. If necessary, isooctane may be purified by passage through a column of activated silica gel (Grade 12, Davison Chemical Co., Baltimore, Md., or equivalent).

Benzene, spectro grade (Burdick and Jackson Laboratories, Inc., Muskegon, Mich., or equivalent). Use 80 milliliters for the test. If necessary, benzene may be purified by distillation or otherwise.

Hexane, spectro grade (Burdick and Jackson Laboratories, Inc., Muskegon, Mich., or equivalent). Use 650 milliliters for the test. If necessary, hexane may be purified by distillation or otherwise.

1,2-Dichloroethane, spectro grade (Matheson, Coleman, and Bell, East Rutherford, N.J., or equivalent). Use 20 milliliters for test. If necessary, 1,2-dichloroethane may be purified by distillation.

Eluting mixtures:

1. 10 percent 1,2-dichloroethane in hexane. Pipet 100 milliliters of 1,2-dichloroethane into a 1-liter glass-stoppered volumetric flask and adjust to volume with hexane, with mixing.

2. 40 percent benzene in hexane. Pipet 400 milliliters of benzene into a 1-liter glass-stoppered volumetric flask and adjust to volume with hexane, with mixing.

n-Hexadecane, 99 percent olefin-free. Dilute 1.0 milliliter of n-hexadecane to 5 milliliters with isooctane and determine the absorbance in a 1-centimeter cell compared to isooctane as reference between 280 mµ-400mµ. The absorbance per centimeter path length shall not exceed 0.00 in this range. If necessary, n-hexadecane may be purified by percolation through activated silica gel or by distillation.

Silica gel, 28-200 mesh (Grade 12, Davison Chemical Co., Baltimore, Md., or equivalent). Activate as follows: Weigh about 900 grams into a 1-gallon bottle, add 100 milliliters of de-ionized water, seal the bottle and shake and roll at intervals for 1 hour. Allow to equilibrate overnight in the sealed bottle. Activate the gel at 150 °C for 16 hours, in a 2-inch × 7-inch × 12-inch porcelain pan loosely covered with aluminum foil, cool in a dessicator, transfer to a bottle and seal.

Determination of ultraviolet absorbance. Before proceeding with the analysis of a sample determine the absorbance in a 1-centimeter path cell for the reagent blank by carrying out the procedure without a sample. Record the absorbance in the wavelength range of 280 to 400 millimicrons. Typical reagent blank absorbance in this range should not exceed 0.04 in the 280 to 299 millimicron range, 0.02 in the 300 to 359 millimicron range, and 0.01 in the 360 to 400 millimicron range. If the characteristic benzene peaks in the 250 to 260 millimicron region are present, remove the benzene by the procedure described above under “Reagents and Materials,” “Organic Solvents,” and record absorbance again.

Transfer 50 grams of silica gel to the chromatographic tube for sample analysis. Raise and drop the column on a semisoft, clean surface for about 1 minute to settle the gel. Pour 100 milliliters of hexane into the column with the stopcock open and allow to drain to about one-half inch above the gel. Turn off the stopcock and allow the column to cool for 30 minutes. After cooling, vibrate the column to eliminate air and stir the top 1 to 2 inches with a small diameter stainless steel rod. Take care not to get the gel above the liquid and onto the sides of the column.

Weigh out 40 grams ±0.1 gram of the hydrocarbon solvent sample into a 250-milliliter beaker, add 50 milliliters of hexane, and pour the solution into the column. Rinse the beaker with 50 milliliters of hexane and add this to the column. Allow the hexane sample solution to elute into a 500-milliliter beaker until the solution is about one-half inch above the gel. Rinse the column three times with 50-milliliter portions of hexane. Allow each hexane rinse to separately elute to about one-half inch above the gel. Replace the eluate beaker (discard the hexane eluate) with a 250-milliliter beaker. Add two separate 25-milliliter portions of 10 percent 1,2-dichloroethane and allow each to separately elute as before. Finally, add 150 milliliters of 10 percent 1,2-dichloroethane for a total of 200 milliliters. When the final 10 percent 1,2-dichloroethane fraction is about one-half inch above the top of the gel bed, replace the receiving beaker (discard the 1,2-dichloroethane eluate) with a 250-milliliter beaker containing 1 milliliter of hexadecane. Adjust the elution rate to 2 to 3 milliliters per minute, add two 25-milliliter portions of 40 percent benzene and allow each to separately elute as before to within about one-half inch of the gel bed. Finally, add 150 milliliters of 40 percent benzene for a total of 200 milliliters. Evaporate the benzene in the oven with vacuum and sufficient nitrogen flow to just ripple the top of the benzene solution. When the benzene is removed (as determined by a constant volume of hexadecane) add 5 milliliters of isooctane and evaporate. Repeat once to insure complete removal of benzene. Remove the beaker and cover with aluminum foil (previously rinsed with hexane) until cool.

Quantitatively transfer the hexadecane residue to a 5-milliliter volumetric flask and dilute to volume with isooctane. Determine the absorbance of the solution in 1-centimeter path length cells between 280 and 400 millimicrons using isooctane as a reference. Correct the absorbance values for any absorbance derived from reagents as determined by carrying out the procedure without a sample. If the corrected absorbance does not exceed the limits prescribed in paragraph (b)(1)(ii) of this section, the sample meets the ultraviolet absorbance specifications for hydrocarbon solvent.