[Code of Federal Regulations]
[Title 21, Volume 3]
[Revised as of April 1, 2001]
From the U.S. Government Printing Office via GPO Access
[CITE: 21CFR178.3620]
[Page 400-408]
TITLE 21--FOOD AND DRUGS
CHAPTER I--FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN
SERVICES (CONTINUED)
PART 178--INDIRECT FOOD ADDITIVES: ADJUVANTS, PRODUCTION AIDS, AND SANITIZERS--Table of Contents
Subpart D--Certain Adjuvants and Production Aids
Sec. 178.3620 Mineral oil.
Mineral oil may be safely used as a component of nonfood articles
intended for use in contact with food, subject to the provisions of this
section:
(a) White mineral oil meeting the specifications prescribed in
Sec. 172.878 of this chapter may be used as a component of nonfood
articles provided such use complies with any applicable limitations in
parts 170 through 189 of this chapter. The use of white mineral oil in
or on food itself, including the use of white mineral oil as a
protective coating or release agent for food, is subject to the
provisions of Sec. 172.878 of this chapter.
(b) Technical white mineral oil identified in paragraph (b)(1) of
this section may be used as provided in paragraph (b)(2) of this
section.
(1) Technical white mineral oil consists of specially refined
distillates of virgin petroleum or of specially refined distillates that
are produced synthetically from petroleum gases. Technical white mineral
oil meets the following specifications:
(i) Saybolt color 20 minimum as determined by ASTM method D156-82,
``Standard Test Method for Saybolt Color of Petroleum Products (Saybolt
Chromometer Method),'' which is incorporated by reference. Copies may be
obtained from the American Society for Testing Materials, 1916 Race St.,
Philadelphia, PA 19103, or may be examined at the Office of the Federal
Register, 800 North Capitol Street, NW., suite 700, Washington, DC
20408.
(ii) Ultraviolet absorbance limits as follows:
------------------------------------------------------------------------
Maximum
absorbance
per
Wavelength (m) centimeter
optical
pathlength
------------------------------------------------------------------------
280 to 289.................................................. 4.0
290 to 299.................................................. 3.3
300 to 329.................................................. 2.3
330 to 350.................................................. 0.8
------------------------------------------------------------------------
Technical white mineral oil containing antioxidants shall meet the
specified ultraviolet absorbance limits after correction for any
absorbance due to the antioxidants. The ultraviolet absorbance shall be
determined by the procedure described for application to mineral oil
under ``Specification'' on page 66 of the ``Journal of the Association
of Official Agricultural Chemists,'' Volume 45 (February 1962) (which is
incorporated by reference; copies are available from the Center for Food
Safety and Applied Nutrition (HFS-200), Food and Drug Administration,
200 C St. SW., Washington, DC 20204, or available for inspection at the
Office of the Federal Register, 800 North Capitol Street, NW., suite
700, Washington, DC 20408), disregarding the last two sentences of that
procedure and substituting therefor the following: Determine the
absorbance of the mineral oil extract in a 10-millimeter cell in the
range from 260-350 m, inclusive, compared to the solvent
control. If the absorbance so measured exceeds 2.0 at any point in range
280-350 m, inclusive, dilute the extract and the solvent
control, respectively, to twice their volume with dimethyl sulfoxide and
remeasure the absorbance. Multiply the remeasured absorbance values by 2
to determine the absorbance of the mineral oil extract per centimeter
optical pathlength.
(2) Technical white mineral oil may be used wherever mineral oil is
permitted for use as a component of nonfood articles complying with
Secs. 175.105, 176.200, 176.210, 177.2260, 177.2600, and 177.2800 of
this chapter and Secs. 178.3570 and 178.3910.
(3) Technical white mineral oil may contain any antioxidant
permitted in food by regulations issued in accordance with section 409
of the Act, in an amount not greater than that required to produce its
intended effect.
(c) Mineral oil identified in paragraph (c)(1) of this section may
be used as provided in paragraph (c)(2) of this section.
(1) The mineral oil consists of virgin petroleum distillates refined
to meet the following specifications:
(i) Initial boiling point of 450 deg.F minimum.
(ii) Color 5.5 maximum as determined by ASTM method D1500-82,
``Standard
[[Page 401]]
Test Method for ASTM Color of Petroleum Products (ASTM Color Scale),''
which is incorporated by reference. The availability of this
incorporation by reference is given in paragraph (b)(1)(i) of this
section.
(iii) Ultraviolet absorbance limits as follows as determined by the
analytical method described in paragraph (c)(3) of this section:
------------------------------------------------------------------------
Maximum
absorbance
per
Wavelength (m) centimeter
optical
pathlength
------------------------------------------------------------------------
280 to 289.................................................. 0.7
290 to 299.................................................. 0.6
300 to 359.................................................. 0.4
360 to 400.................................................. .09
------------------------------------------------------------------------
(2) The mineral oil may be used wherever mineral oil is permitted
for use as a component of nonfood articles complying with Secs. 175.105
and 176.210 of this chapter and Sec. 178.3910 (for use only in rolling
of metallic foil and sheet stock), Secs. 176.200, 177.2260, 177.2600,
and 177.2800 of this chapter.
(3) The analytical method for determining ultraviolet absorbance
limit is as follows:
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
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 oil 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-milliliter, 500-milliliter, 1,000-
milliliter, and preferably 2,000-milliliter capacity, equipped with
tetrafluoroethylene polymer stopcocks.
Reservoir. 500-milliliter capacity, equipped with a 24/40 standard
taper male fitting at the bottom and a suitable ball-joint at the top
for connecting to the nitrogen supply. The male fitting should be
equipped with glass hooks.
Chromatographic tube. 180 millimeters in length, inside diameter to
be 15.7 millimeters plus-minus0.1 millimeter, equipped with a
coarse, fritted-glass disc, a tetrafluoroethylene polymer stopcock, and
a female 24/40 standard tapered fitting at the opposite end. (Overall
length of the column with the female joint is 235 millimeters.) The
female fitting should be equipped with glass hooks.
Disc. Tetrafluoroethylene polymer 2-inch diameter disk approximately
\3/16\-inch thick with a hole bored in the center to closely fit the
stem of the chromatographic tube.
Suction flask. 250-milliliter or 500-milliliter filter flask.
Condenser. 24/40 joints, fitted with a drying tube, length optional.
Evaporation flask (optional). 250-milliliter or 500-milliliter
capacity all-glass flask equipped with standard taper stopper having
inlet and outlet tubes to permit passage of nitrogen across the surface
of contained liquid to be evaporated.
Spectrophotometric cells. Fused quartz cells, optical path length in
the range of 5,000 centimeter plus-minus0.005 centimeter;
also for checking spectrophotometer performance only, optical path
length in the range 1,000 centimeter plus-minus0.005
centimeter. With distilled water in the cells, determine any absorbance
differences.
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, plus-minus0.01 at 0.4
absorbance.
Absorbance accuracy \1\ plus-minus0.05 at 0.4 absorbance.
---------------------------------------------------------------------------
\1\ As determined by procedure using potassium chromate for
reference standard and described in National Bureau of Standards
Circular 484, Spectrophotometry, U.S. Department of Commerce (1949). The
accuracy is to be determined by comparison with the standard values at
290, 345, and 400 millimicrons. Circular 484 is incorporated by
reference. Copies are available from the Center for Food Safety and
Applied Nutrition (HFS-200), Food and Drug Administration, 200 C St.
SW., Washington, DC 20204, or available for inspection at the Office of
the Federal Register, 800 North Capitol Street, NW., suite 700,
Washington, DC 20408.
---------------------------------------------------------------------------
Wavelength accuracy, plus-minus1.0 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.
[[Page 402]]
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-milliliter Erlenmeyer
flask, add 1 milliliter of purified n-hexadecane and evaporate on the
steam bath under a stream of nitrogen (a loose aluminum foil jacket
around the flask will speed evaporation). Discontinue evaporation when
not over 1 milliliter of residue remains. (To the residue from benzene
add a 10-milliliter 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 vacuum line in such a
way as to prevent any flow-back of condensate into the flask.
Dissolve the 1 milliliter of hexadecane residue in isooctane and
make to 25 milliliters volume. Determine the absorbance in the 5-
centimeter path length cells compared to isooctane as reference. The
absorbance of the solution of the solvent residue (except for methyl
alcohol) shall not exceed 0.01 per centimeter path length between 280
and 400 m. For methyl alcohol this absorbance value shall be
0.00.
Isooctane (2,2,4-trimethylpentane). Use 180 milliliters 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 centimeters in
length and 5 centimeters to 8 centimeters in diameter.
Benzene, A.C.S. reagent grade. Use 150 milliliters for the test.
Purify, if necessary, by distillation or otherwise.
Acetone, A.C.S. reagent grade. Use 200 milliliters for the test.
Purify, if necessary, by distillation.
Eluting mixtures:
1. 10 percent benzene in isooctane. Pipet 50 milliliters of benzene
into a 250-milliliter glass-stoppered volumetric flask and adjust to
volume with isooctane, with mixing.
2. 20 percent benzene in isooctane. Pipet 50 milliliters of benzene
into a 250-milliliter glass-stoppered volumetric flask and adjust to
volume with isooctane, with mixing.
3. Acetone-benzene-water mixture. Add 20 milliliters of water to 380
milliliters of acetone and 200 milliliters of benzene, and mix.
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 point between
280 m-400 m. 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, A.C.S. reagent grade. Use 10.0 milliliters of methyl
alcohol. Purify, if necessary, by distillation.
Dimethyl sulfoxide. Spectrophotometric grade (Crown Zellerbach
Corporation, Camas, Washington, or equivalent). Absorbance (1-centimeter
cell, distilled water reference, sample completely saturated with
nitrogen).
------------------------------------------------------------------------
Absorbance
Wavelength (maximum)
------------------------------------------------------------------------
261.5....................................................... 1.00
270......................................................... .20
275......................................................... .09
280......................................................... .06
300......................................................... .015
------------------------------------------------------------------------
There shall be no irregularities in the absorbance curve within these
wavelengths.
Phosphoric acid. 85 percent A.C.S. reagent grade.
Sodium borohydride. 98 percent.
Magnesium oxide (Sea Sorb 43, Food Machinery Company, Westvaco
Division, distributed by chemical supply firms, or equivalent). Place
100 grams of the magnesium oxide in a large beaker, add 700 milliliters
of distilled water to make a thin slurry, and heat on a steam bath for
30 minutes with intermittent stirring. Stir well initially to insure
that all the adsorbent 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 adsorbent to a glass trough lined
with aluminum foil (free from rolling oil). Break up the magnesia with a
clean spatula and spread out the adsorbent on the aluminum foil in a
layer about 1 centimeter to 2 centimeters thick. Dry for 24 hours at 160
deg.C plus-minus1 deg.C. Pulverize the magnesia with mortar
and pestle. Sieve the pulverized adsorbent between 60-180 mesh. Use the
magnesia retained on the 180-mesh sieve.
Celite 545. Johns Mansville 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 minutes. Transfer the mixture
to a glass trough lined with aluminum foil (free from rolling oil)
[[Page 403]]
and spread it out on a layer about 1 centimeter to 2 centimeters thick.
Reheat the mixture at 160 deg.C plus-minus1 deg.C for 2
hours, and store in a tightly closed flask.
Sodium sulfate, anhydrous, A.C.S. 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 grams of anhydrous sodium
sulfate in a 30-milliliter course, fritted-glass funnel or in a 65-
millimeter filter funnel with glass wool plug; wash with successive 15-
milliliter portions of the indicated solvent until a 15-milliliter
portion of the wash shows 0.00 absorbance per centimeter path length
between 280 m and 400 m when tested as prescribed
under ``Organic solvents.'' Usually three portions of wash solvent are
sufficient.
Before proceeding with analysis of a sample, determine the
absorbance in a 5-centimeter path cell between 250 millimicrons and 400
millimicrons for the reagent blank by carrying out the procedure,
without an oil sample, recording the spectra after the extraction stage
and after the complete procedure as prescribed. The absorbance per
centimeter pathlength following the extraction stage should not exceed
0.02 in the wavelength range from 280 m to 400 m; the
absorbance per centimeter pathlength following the complete procedure
should not exceed 0.02 in the wavelength range from 280 m to
400 m. If in either spectrum the characteristic benzene peaks
in the 250 m-260 m region are present, remove the
benzene by the procedure under ``Organic solvents'' and record
absorbance again.
Place 300 milliliters of dimethyl sulfoxide in a 1-liter separatory
funnel and add 75 milliliters of phosphoric acid. Mix the contents of
the funnel and allow to stand for 10 minutes. (The reaction between the
sulfoxide and the acid is exothermic. Release pressure after mixing,
then keep funnel stoppered.) Add 150 milliliters of isooctane and shake
to pre-equilibrate the solvents. Draw off the individual layers and
store in glass-stoppered flasks.
Weigh a 20-gram sample of the oil and transfer to a 500-milliliter
separatory funnel containing 100 milliliters of pre-equilibrated
sulfoxide-phosphoric acid mixture. Complete the transfer of the sample
with small portions of preequilibrated isooctane to give a total volume
of the oil and solvent of 75 milliliters. Shake the funnel vigorously
for 2 minutes. Set up three 250-milliliter separatory funnels with each
containing 30 milliliters of pre-equilibrated isooctane. After
separation of liquid phases, carefully draw off lower layer into the
first 250-milliliter separatory funnel and wash in tandem with the 30-
milliliter portions of isooctane contained in the 250-milliliter
separatory funnels. Shaking time for each wash is 1 minute. Repeat the
extraction operation with two additional portions of the sulfoxide-acid
mixture and wash each extractive in tandem through the same three
portions of isooctane.
Collect the successive extractives (300 milliliters total) in a
separatory funnel (preferably 2-liter) containing 480 milliliters of
distilled water; mix, and allow to cool for a few minutes after the last
extractive has been added. Add 80 milliliters of isooctane to the
solution and extract by shaking the funnel vigorously for 2 minutes.
Draw off the lower aqueous layer into a second separatory funnel
(preferably 2-liter) and repeat the extraction with 80 milliliters of
isooctane. Draw off and discard the aqueous layer. Wash each of the 80-
milliliter extractives three times with 100-milliliter portions of
distilled water. Shaking time for each wash is 1 minute. 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-milliliter
Erlenmeyer flask (or optionally into the evaporation flask). Wash the
first separatory funnel with the second 80-milliliter isooctane
extractive and pass through the sodium sulfate. Then wash the second and
first separatory funnels successively with a 20-milliliter portion of
isooctane and pass the solvent through the sodium sulfate into the
flask. Add 1 milliliter of n-hexadecane and evaporate the isooctane on
the steam bath under nitrogen. Discontinue evaporation when not over 1
milliliter of residue remains. To the residue, add a 10-milliliter
portion of isooctane, reevaporate to 1 milliliter of hexadecane, and
repeat this operation once.
Quantitatively transfer the residue with isooctane to a 200-
milliliter volumetric flask, make to volume, and mix. Determine the
absorbance of the solution in the 1-centimeter pathlength cells compared
to isooctane as reference between 280 m-400 m (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 an oil sample. If the
corrected absorbance does not exceed the limits prescribed in this
paragraph, the oil meets the ultraviolet absorbance specifications. If
the corrected absorbance per centimeter pathlength exceeds the limits
prescribed in this paragraph, proceed as follows: Quantitatively
transfer the isooctane solution to a 125-milliliter flask equipped with
24/40 joint, and evaporate the isooctane on the steam bath under a
stream of nitrogen to a volume of 1 milliliter of hexadecane. Add 10
milliliters of methyl alcohol and approximately 0.3 gram of sodium
borohydride. (Minimize exposure of the borohydride to the atmosphere. A
measuring dipper may be
[[Page 404]]
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 minutes 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 milliliters of isooctane and evaporate to a volume of about 2-3
milliliters. Again, add 10 milliliters of isooctane and concentrate to a
volume of approximately 5 milliliters. 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 vacuum (approximately 135 millimeters Hg
pressure). Weigh out 14 grams of the 2:1 magnesium oxide-Celite 545
mixture and pour the adsorbent mixture into the chromatographic tube in
approximately 3-centimeter 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 millimeters 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 grams 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 centimeters in depth. Turn off the vacuum and remove
the suction flask. Fit the 500-milliliter reservoir onto the top of the
chromatographic column and prewet the column by passing 100 milliliters
of isooctane through the column. Adjust the nitrogen pressure so that
the rate of descent of the isooctane coming off the column is between 2-
3 milliliters per minute. 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-milliliter 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-milliliter portions of isooctane,
swirling the flask repeatedly each time to assure adequate washing of
the residue. Just before the final 5-milliliter wash reaches the top of
the adsorbent, add 100 milliliters of isooctane to the reservoir and
continue the percolation at the 2-3 milliliters per minute rate. Just
before the last of the isooctane reaches the adsorbent level, add 100
milliliters of 10 percent benzene in isooctane to the reservoir and
continue the percolation at the aforementioned rate. Just before the
solvent mixture reaches adsorbent level, add 25 milliliters of 20
percent benzene in isooctane to the reservoir and continue the
percolation at 2-3 milliliters per minute until all this solvent mixture
has been removed from the column. Discard all the elution solvents
collected up to this point. Add 300 milliliters of the acetone-benzene-
water mixture to the reservoir and percolate through the column to
eluate the polynuclear compounds. Collect the eluate in a clean 1-liter
separatory funnel. Allow the column to drain until most of the solvent
mixture is removed. Wash the eluate three times with 300-milliliter
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 pre-washed with
benzene (see Sodium sulfate under ``Reagents and Materials'' for
preparation of filter) into a 250-milliliter Erlenmeyer flask (or
optionally into the evaporation flask). Wash the separatory funnel with
two additional 20-milliliter portions of benzene which are also filtered
through the sodium sulfate. Add 1 milliliter 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 residue with isooctane
to a 200-milliliter volumetric flask and adjust to volume. Determine the
absorbance of the solution in the 1-centimeter pathlength cells compared
to isooctane as reference between 250 m-400 m. Correct
for any absorbance derived from the reagents as determined by carrying
out the procedure without an oil sample. If either spectrum shows the
characteristic benzene peaks in the 250 m-260 m
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 200-milliliter volumetric flask.
Record the absorbance again. If the corrected absorbance does not exceed
the limits proposed in this paragraph, the oil meets the proposed
ultraviolet absorbance specifications.
(d) Mineral oil identified in paragraph (d)(1) of this section may
be used as provided in paragraph (d)(2) of this section.
(1) The mineral oil consists of virgin petroleum distillates refined
to meet the following specifications:
(i) Distillation endpoint at 760 millimeters pressure not to exceed
371 deg.C, with a maximum residue not to exceed 2 percent, as
determined by ASTM method D86-82, ``Standard Method for
[[Page 405]]
Distillation of Petroleum Products,'' which is incorporated by
reference. The availability of this incorporation by reference is given
in paragraph (b)(1)(i) of this section.
(ii) Ultraviolet absorbance limits as follows as determined by the
method described in paragraph (d)(3) of this section.
------------------------------------------------------------------------
Maximum
absorb-
ance per
Wavelength (m) centimeter
optical
pathlength
------------------------------------------------------------------------
280 to 299.................................................. 2.3
300 to 319.................................................. 1.2
320 to 359.................................................. .8
360 to 400.................................................. .3
------------------------------------------------------------------------
(iii) Pyrene content not to exceed a maximum of 25 parts per million
as determined by the method described in paragraph (d)(3) of this
section.
(2) The mineral oil may be used only in the processing of jute fiber
employed in the production of textile bags intended for use in contact
with the following types of food: Dry grains and dry seeds (for example,
beans, peas, rice, and lentils); whole root crop vegetables of the types
identified in 40 CFR 180.34(f); unshelled and shelled nuts (including
peanuts); and dry animal feed. The finished processed jute fiber shall
contain no more than 6 percent by weight of residual mineral oil.
(3) The analytical method for determining ultraviolet absorbance
limits and pyrene content is as follows:
I. Apparatus. A. Assorted beakers, separatory funnels fitted with
tetrafluoroethylene polymer stopcocks, and graduated cylinders.
B. Volumetric flasks, 200-milliliter.
C. A chromatographic column made from nominal 1.3 centimeters
outside diameter x 75 centimeters glass tubing tapered at one end and
joined to a 2-millimeter-bore tetrafluoroethylene polymer stopcock. The
opposite end is flanged and joined to a female 24/40 standard taper
fitting. This provides for accommodating the 500-milliliter reservoir
described in item I.E below.
D. A chromatographic column made from nominal 1.7 centimeters
outside diameter x 115 centimeters glass tubing tapered at one end and
joined to a 2-millimeter-bore tetrafluoroethylene polymer stopcock. The
opposite end is flanged and joined to a 2.5 centimeters outside diameter
x 9.0 centimeters glass tube having a female 24/40 standard taper
fitting. This provides for accommodating the 500-milliliter reservoir
described in item I. E below.
E. A 500-milliliter reservoir having a 24/40 standard taper male
fitting at bottom and a suitable ball joint at the top for connecting to
the nitrogen supply. The female fitting of the chromatographic columns
described in items I. C and D above and the male fitting of the
reservoir described in this item E should both be equipped with glass
hooks.
(Note: Rubber stoppers are not to be used. Stopcock grease is not to
be used on ground-glass joints in this method.)
F. A spectrophotometer equipped to automatically record absorbance
of liquid samples in 1-centimeter pathlength cells in the spectral
region of 280-400 m with a spectral slit width of 2 m
or less. At an absorbance level of about 0.4, absorbance measurements
shall be repeatable within plus-minus0.01 and accurate within
plus-minus0.05. Wavelength measurements shall be repeatable
with plus-minus0.2 m and accurate within
plus-minus1.0 m. Instrument operating conditions are
selected to realize this performance under dynamic (automatic) recording
operations. Accuracy of absorbance measurements are determined at 290,
345, and 400 m, using potassium chromate as the reference
standard. (National Bureau of Standards Circular 484, Spectrophotometry,
U.S. Department of Commerce, 1949.)
G. Two fused quartz cells having pathlengths of
1.00plus-minus0.005 centimeter or better.
II. Purity of reagents and materials. Reagent-grade chemicals shall
be used in all tests. It is further specified that each chemical shall
be tested for purity in accordance with the instruction given under
``Reagents and Materials'' in III below. In addition, a blank run by the
procedure shall be made on each purified lot of reagents and materials.
Unless otherwise indicated, references to water shall be understood to
mean distilled water.
III. Reagents and materials-- A. Organic solvents. All solvents used
throughout the procedure shall meet the specifications and tests
described in this section III. The isooctane, benzene, cyclohexane,
nitromethane, and n-hexadecane designated shall pass the following test:
To the specified quantity of solvent in a 150-milliliter beaker, add 1
milliliter of purified n-hexadecane and evaporate on the steam bath
under a stream of nitrogen. Discontinue evaporation when not over 1
milliliter of residue remains (to the residue from benzene and
nitromethane add a 10-milliliter portion of purified isooctane, re-
evaporate, and repeat once to insure complete removal of solvent).
Dissolve the 1 milliliter of n-hexadecane residue in isooctane and make
to 10-milliliter volume. Determine the absorbance in 1.0-centimeter
pathlength cells compared to water as reference. The absorbance of the
solution of solvent residue shall not exceed 0.05 between 280 and 400
m.
[[Page 406]]
1. Isooctane (2,2,4-trimethylpentane). Use 240 milliliters for the
above test. Purify, if necessary, by passage through a column of
activated silica gel.
2. Benzene. Use 200 milliliters for the above test. Purify, if
necessary, by distillation or otherwise.
3. Cyclohexane. Use 70 milliliters for the above test. Purify, if
necessary, by distillation, silica gel percolation, or otherwise.
4. Nitromethane. Use 125 milliliters for the above test. Purify, if
necessary, by distillation or otherwise.
5. n-Hexadecane. Determine the absorbance on this solvent directly.
Purify, if necessary, by silica gel percolation or otherwise.
B. Other materials--1. Pyrene standard reference. Pyrene, reagent
grade, melting point range 150-152 deg.C. (Organic Chemical 3627,
Eastman Kodak Co., Rochester, N.Y., or equivalent). The standard
reference absorbance is the absorbance at 334 millimicrons of a standard
reference solution of pyrene containing a concentration of 1.0 milligram
per liter in purified isooctane measured against isooctane of the same
spectral purity in 1.0-centimeter cells. (This absorbance will be
approximately 0.28.)
2. Chrysene solution. Prepare a solution at a concentration of 5.0
milligrams per liter by dissolving 5.0 milligrams of chrysene in
purified isooctane in a 1-liter volumetric flask. Adjust to volume with
isooctane.
3. Nitrogen gas. Water pumped or equivalent purity, cylinder with
regulator, and valve control flow at 5 p.s.i.
4. Silica gel. 100-200 mesh (Davison Chemical, Baltimore, Md., Grade
923, or equivalent), purified and activated by the following procedure:
Place about 1 kilogram of silica gel in a large column and wash with
contaminant-free benzene until a 200-milliliter sample of the benzene
coming off the column will pass the ultraviolet absorption test for
benzene. This test is performed as stipulated under ``Organic solvents''
in A under III above. When the silica gel has been sufficiently cleaned,
activate the gel before use by placing the 1-kilogram batch in a shallow
container in a layer no greater than 1 inch in depth and heating in an
oven (Caution! Explosion Hazard) at 130 deg.C. for 16 hours, and store
in a vacuum desiccator. Reheating about once a week is necessary if the
silica gel is repeatedly removed from the desiccator.
5. Aluminum oxide (Aluminum Co. of America, Grade F-20, or
equivalent grade). 80-200 mesh, purified and activated by the following
procedure: Place about 1 kilogram of aluminum oxide in a large column
and wash with contaminant-free benzene until a 200-milliliter sample of
the benzene coming off the column will pass the ultraviolet absorption
test for benzene. This test is performed as stipulated under ``Organic
solvents'' in A under III above. (Caution! Remove Benzene From Adsorbent
Under Vacuum To Minimize Explosion Hazard in Subsequent Heating!) When
the aluminum oxide has been sufficiently cleaned and freed of solvent,
activate it before use by placing the 1-kilogram batch in a shallow
container in a layer no greater than 1 inch in depth. Heat in an oven at
130 deg.C for 16 hours. Upon removal from heat, store at atmospheric
pressure over 80 percent (by weight) sulfuric acid in a desiccator for
at least 36 hours before use. This gives aluminum oxide with between 6
to 9.5 percent volatiles. This is determined by heating a weighed sample
of the prepared aluminum oxide at 2,000 deg.F for 2 hours and then
quickly reweighing. To insure the proper adsorptive properties of the
aluminum oxide, perform the following test:
a. Weigh 50 grams plus-minus1 gram of the activated
aluminum oxide and pack into the chromatographic column (1.3 centimeters
x 75 centimeters) described under ``Apparatus'' in C under I above.
Use glass wool at the column exit to prevent the aluminum oxide from
passing through the column.
b. Place a 250-milliliter graduated cylinder under the column to
measure the amount of eluate coming from the column.
c. Prewet the aluminum oxide by passing 40 milliliters of isooctane
through the column. Adjust the nitrogen pressure so that the rate of
descent of the isooctane coming off the column is between 1.5 to 2.5
milliliters per minute.
d. Just prior to the last of the isooctane reaching the top of the
aluminum oxide bed, add 10 milliliters of the isooctane solution
containing 5.0 milligrams of chrysene per liter.
e. Continue percolation until the isooctane is just above the
aluminum oxide. Then add 200 milliliters of a mixture of benzene and
isooctane (33\1/3\ percent benzene and 66\2/3\ percent isooctane by
volume) to the reservoir and continue percolation.
f. Continue percolation, collecting the eluates (40 milliliters of
the prewet solution, 10 milliliters of the sample solution, and 200
milliliters of the gradient solution) in the 250-milliliter graduated
cylinder until the level of the gradient solution is just above the
aluminum oxide. Add 200 milliliters of the eluting solution of benzene
and isooctane (90 percent benzene and 10 percent isooctane by volume) to
the column and continue collecting until a total of 250 milliliters of
solution has been obtained. This may be discarded. Now begin to collect
the final eluate.
g. Place a 100-milliliter graduated cylinder under the column and
continue the percolation until a 100-milliliter eluate has been
obtained.
h. Measure the amount of chrysene in this 100-milliliter fraction by
ultraviolet analysis. If the aluminum oxide is satisfactory, more than
80 percent of the original amount of chrysene should be found in this
fraction.
[[Page 407]]
(Note: If the amount of chrysene recovered is less than 80 percent, the
original batch of aluminum oxide should be sieved between 100-160 mesh.
Activation and testing of this sieved batch should indicate a
satisfactory aluminum oxide for use.)
IV. Sampling. Precautions must be taken to insure that an
uncontaminated sample of the mineral oil is obtained since ultraviolet
absorption is very sensitive to small amounts of extraneous material
contaminating the sample through careless handling.
V. Procedure. A. Blank. Before proceeding with the analysis of a
sample, determine the absorbance of the solvent residues by carrying out
the procedure without a sample.
B. Sample. 1. Weigh out 20.0 grams plus-minus0.1 gram of
the mineral oil into a beaker and transfer to a 250-milliliter
separatory funnel fitted with a tetrafluoroethylene polymer stopcock,
using enough cyclohexane (25 milliliters) to give a final total volume
of 50 milliliters (mineral oil plus cyclohexane).
2. Add 25 milliliters of nitromethane saturated with cyclohexane and
shake by hand vigorously for 3 minutes. Recover the lower nitromethane
layer in a 150-milliliter beaker containing 1 milliliter of n-hexadecane
and evaporate on the steam bath under nitrogen. Repeat the extraction
four more times, recovering each extract in the 150-milliliter beaker.
Exercise care not to fill the beaker to such a capacity that solvent
losses may occur. Evaporate the combined nitromethane extracts to 1
milliliter of n-hexadecane residue containing the nitromethane-soluble
mineral oil extractives. (Note: Complete removal of the nitromethane is
essential. This can be assured by two successive additions of 5
milliliters of isooctane and reevaporation.)
3. Remove the beaker from the steam bath and allow to cool.
4. Weigh 50 grams plus-minus1 gram of activated aluminum
oxide and pack into the chromatographic column (1.3 centimeters x 75
centimeters) described under ``Apparatus'' in C under I above. (Note: A
small plug of glass wool is placed at the column exit to prevent the
aluminum oxide from passing through the column. After adding aluminum
oxide, tap the column lightly to remove air voids. All percolations
using aluminum oxide are performed under nitrogen pressure. The 500-
milliliter reservoir described under ``Apparatus'' in E under I above is
to be used to hold the elution solvents.)
5. Prewet the column by adding 40 milliliters of isooctane to the
column. Adjust nitrogen pressure so that rate of descent of the
isooctane coming off the column is 2.0 to 3.0 milliliters per minute. Be
careful to maintain the level of solvent in the reservoir to prevent air
from entering the aluminum oxide bed. New or additional solvent is added
just before the last portion of the previous solvent enters the bed. To
minimize possible photo-oxidation effects, the following procedures
(steps 6 through 18) shall be carried out in subdued light.
6. Before the last of the isooctane reaches the top of the aluminum
oxide bed, release the nitrogen pressure and turn off the stopcock on
the column. Transfer the n-hexadecane residue from the 150-milliliter
beaker from procedure step 3 above onto the column, using several washes
of isooctane (total volume of washes should be no greater than 10-15
milliliters).
7. Open the stopcock and continue percolation until the isooctane is
about 1 centimeter above the top of the aluminum oxide bed. Add 200
milliliters of isooctane to the reservoir, and continue the percolation
at the specified rate.
8. Just before the isooctane surface reaches the top of the aluminum
oxide bed, add 200 milliliters of a mixture of benzene and isooctane
(33\1/3\ percent benzene and 66\2/3\ percent isooctane by volume) to the
reservoir, and continue the percolation.
9. Just before the surface of this mixture reaches the top of the
aluminum oxide bed, release the nitrogen pressure, turn off the
stopcock, and discard all the elution solvents collected up to this
point.
10. Add to the reservoir 300 milliliters of a mixture of benzene and
isooctane (90 percent benzene and 10 percent isooctane by volume), place
a 25-milliliter graduated cylinder under the column, continue the
percolation until 20 milliliters of eluate has been collected, and then
discard the eluate.
11. At this point, place a clean 250-milliliter Erlenmeyer flask
under the column. Continue the percolation and collect all the remaining
eluate.
(Note: Allow the column to drain completely. An increase in the
nitrogen pressure may be necessary as the last of the solvent comes off
the column.)
12. Place 1 milliliter of n-hexadecane into a 150-milliliter beaker.
Place this onto a steam bath under a nitrogen stream and transfer in
small portions the eluate from step 11 above. Wash out the Erlenmeyer
flask with small amounts of benzene and transfer to the evaporation
beaker. Evaporate until only 1 milliliter of hexadecane residue remains.
(Note: Complete removal of the benzene is essential. This can be assured
by two successive additions of 5 milliliters of isooctane and
reevaporation.)
13. Remove the beaker from the steam bath and cool.
14. Place a sample of 113.5 grams activated 100- 200-mesh silica gel
in a 500-milliliter glass-stoppered Erlenmeyer flask. Add to the silica
gel 46.2 grams (41 milliliters) of nitromethane. Stopper and shake the
flask vigorously until no lumps of silica gel are observed and then
shake occasionally during a period of 1 hour. The resultant
nitromethane-treated silica gel is 29 weight-
[[Page 408]]
percent nitro-methane and 71 weight-percent silica gel.
15. Place a small plug of glass wool in the tapered end of the 1.7
centimeters outside diameter x 115 centimeters column, described under
``Apparatus'' in D of I above, adjacent to the stopcock to prevent
silica gel from passing through the stopcock. Pack the nitromethane-
treated silica gel into the column, tapping lightly. The resultant
silica gel bed should be about 95 centimeters in depth. Place into a
flask 170 milliliters of isooctane saturated with nitromethane.
16. Place a 100-milliliter graduated cylinder under the column and
transfer the residue from the beaker in procedure step 13 above with
several washes of the 170 milliliters of isooctane, saturated with
nitromethane, onto the top of the column. (Total volume of washes should
be no greater than 10 to 15 milliliters.) Permit isooctane solution to
enter the silica gel bed until the liquid level is at the top bed level.
Place the remaining amount of the 170 milliliters of isooctane,
saturated with nitromethane, in the reservoir above the bed for
percolation through the silica gel. Apply nitrogen pressure to the top
of the column, adjusting the pressure so that the isooctane is collected
at the rate of 2.5 to 3.5 milliliters per minute, and percolate
isooctane through the bed until a quantity of 75.0 milliliters of eluate
is collected. Discard the 75 milliliters of eluate. Turn off the
stopcock and add 250 milliliters of benzene to the reservoir above the
bed. Use a 400-milliliter beaker to collect the remaining eluate.
17. Open the stopcock, renew the pressure, and percolate the
remaining isooctane and benzene through the column eluting the remaining
aromatics. Transfer the eluate in small portions from the 400 milliliter
beaker to a 150-milliliter beaker containing 1 milliliter of n-
hexadecane and evaporate on the steam bath under nitrogen. Rinse the
400-milliliter beaker well with small portions of isooctane to obtain a
complete transfer.
(Note: Complete removal of the nitromethane and benzene is
essential. This can be assured by successive additions of 5 milliliters
of isooctane and reevaporation.)
18. Transfer the residue with several washes of isooctane into a
200-milliliter volumetric flask. Add isooctane to mark.
19. Record the spectrum of the sample solution in a 1-centimeter
cell compared to isooctane from 270 to 400 m. After making
necessary corrections in the spectrum for cell differences and for the
blank absorbance, record the maximum absorbance in each of the
wavelength intervals (m), 280-299, 300-319, 320-359, 360-400.
a. If the spectrum then shows no discernible peak corresponding to
the absorbance maximum of the pyrene reference standard solution at 334
m, the maximum absorbances in the respective wavelength
intervals recorded shall not exceed those prescribed in paragraph
(d)(1)(ii) of this section.
b. If such a peak is evident in the spectrum of the sample solution,
and the spectrum as a whole is not incompatible with that of a pyrene
contaminant yielding such a peak of the observed absorbance, calculate
the concentration of pyrene that would yield this peak (334 m) by the
base-line technique described in ASTM method E169-63 (Reapproved 1981),
``Standard Recommended Practices for General Techniques of Ultraviolet
Quantitative Analysis,'' which is incorporated by reference. The
availability of this incorporation by reference is given in paragraph
(b)(1)(i) of this section. Correct each of the maximum absorbances in
the respective specified wavelength intervals by subtracting the
absorbance due to pyrene, determined as follows:
[GRAPHIC] [TIFF OMITTED] TR01JA93.407
where:
Cp=Calculated concentration of pyrene in sample solution;
Sp=Concentration of pyrene reference standard solution in same units of
concentration;
Sa=Absorbance of pyrene reference standard solution at wavelength of
maximum absorbance of sample solution in the respective specified
wavelength intervals.
Also calculate the pyrene content of the oil sample in parts per
million as follows:
[GRAPHIC] [TIFF OMITTED] TR01JA93.408
where:
C=Calculated concentration of pyrene in milligrams per liter of sample
solution.
c. The pyrene content so determined shall not exceed 25 p.p.m. The
maximum absorbances corrected for pyrene content as described in this
step 19 for each of the specified wavelength intervals shall not exceed
the limits prescribed in paragraph (d)(1)(ii) of this section.
d. If the spectrum as a whole of the sample solution is in any
respect clearly incompatible with the presence of pyrene as the source
of the peak at 334 m, then the maximum absorbances in the
respective wavelength intervals without correction for any assumed
pyrene content shall not exceed the limits prescribed in paragraph
(d)(1)(ii) of this section.
[42 FR 14609, Mar. 15, 1977, as amended at 47 FR 11847, Mar. 19, 1982;
49 FR 10112, Mar. 19, 1984; 54 FR 24898, June 12, 1989]
[[Page 409]]