EPA APTD-1515
Final Report
(February, 1969 - December, 1971)
Rapid Methods of Analysis for Trace Quantities of
Polynuclear Aromatic Hydrocarbons and Phenols in
Automobile Exhaust, Gasoline, and Crankcase Oil
CRC-APRAC Project CAPE-12-68
R. A. Brown, T. D. Searl, W. H. King, Jr.,
W. A. Dietz and J. M. Kelliher
Esso Research and Engineering Company
Linden, New Jersey
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CRC-APRAC PROJECT CAPE-12-68
PH-22-68-Neg. 38
Panel
F. P. Hochgesang, Project Leader (Mobil Research & Development Corp.,
Paulsboro, N. J.)
C. R. Begeman (General Motors Research, Warren, Michigan)
K. Habibi (E. I. duPont de Nemours & Co., Penns Grove, N. J.)
L. W. Mixon (American Oil Research & Development, Whiting, Indiana)
E. Sawicki (Environmental Protection Agency/Air Pollution Control Office,
Research Triangle Park, North Carolina)
CRC-APRAC Headquarters
A. E. Zengel, Project Manager
T. C. Belian, Assistant Project Manager
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Table o£ Contents
Page
Objective 1
Summary 1
Introduction 3
Method for Benz(a)Anthracene, Benzo(a)Pyrene and Other 4
Polynuclear Aromatic Hydrocarbons in Automobile Exhaust,
Gasoline and Crankcase Oil
Preparation of Samples with Carbon-14 Labeled BaA and BaP 4
Caustic Extraction 4
Preparation of a PNA Concentrate by Adsorption Chrotnatography 6
Gas Chromatography and Trapping of Peaks 6
Ultraviolet Absorption Spectrophotometry 8
Radioactivity Measurements 11
Calculation of PNA's in GC Peaks 11
Test for Accuracy 14
Calculation to a Sample Basis 15
Application of the Method 16
Measurement of Total Polynuclear Aromatic Hydrocarbons 19
By Low Voltage Mass Spectrometery
Procedure for Mass Spectrometer Analysis 21
Calibration 21
Calculation 25
Application of the Method 26
Method for Phenols in Automobile Exhaust Gas 28
Description of Method 28
Preparation of Sample for Gas Chromatograph 28
Gas Chromatographic Separation of Phenols 29
Application of the Method 30
Appendix A - Method for Benz(a)anthracene, Benzo(a)pyrene, and
Other Polynuclear Aromatic Hydrocarbons in Automobile
Exhaust, Gasoline and Crankcase Oil 34
Appendix B - Measurement of Total Polynuclear Aromatic Hydro- 45
carbons by Low Voltage Mass Spectrometry
Appendix C - Method for Measurement of Phenols in Aqueous 49
Condensate of Automobile Exhaust
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Final Report
Rapid Methods of Analysis for Trace Quantities of
Polynuclear Aromatic Hydrocarbons and Phenols in
Automobile Exhaust, Gasoline, and Crankcase Oil
OBJECTIVE
Objectives of CAPE-12-68 project were:
Develop and put to practice a rapid method to measure benz-
(a)anthracene and benzo(a)pyrene in tar recovered from
automobile exhaust, in gasoline, and in crankcase oil.
Extend this method to additional polynuclear aromatic (PNA)
hydrocarbons.
Develop a method to measure total PNA's in auto exhaust tar.
Develop a method to measure individual phenols in auto
exhaust tar.
SUMMARY
A. A rapid method was developed to measure benz(a)anthracene (BaA) and
benzo(a)pyrene (BaP) in auto exhaust tar, gasoline, and crankcase oil. Nine
other polynuclear aromatic hydrocarbons were also included in the measurement.
These included: pyrene, chrysene, triphenylene, methyl BaA, dimethyl BaA,
benzo(e)pyrene (BeP), methyl BaP, methyl BeP, and benzo(g,h,i)perylene. Each
of the 11 compounds occurred in concentrations of 10-2000 ppm and were measured
as 0.1 micrograms and higher. The method can also be extended to other PNA
hydrocarbons, such as benzofluoranthenes, dibenzanthracenes, anthanthrene, and
coronene.
A cyclohexane solution of the sample was extracted with aqueous caustic,
then eluted through a column of partially deactivated alumina and all fractions
containing PNA's were combined. A portion of the PNA concentrate was fractionated
by gas chromatography and individual chromatographic peaks trapped. Concentra-
tions of individual PNA's in the trapped fractions were measured by UV absorption
spectrophotometry. Analytical losses were accounted for by isotope dilution.
Twenty technician-hours were required to measure the 11 individual
PNA's .
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The precision (ICT) for nine of the compounds was in the range 4-12
relative percent. The preceision for benzo(g,h,i)perylene was 18 relative per-
center, and methyl BaP 45 relative percent. Absolute accuracy for BaA and BaP
was within 10 percent.
B. Total PNA's were measured by low voltage mass spectrometry, using the
same concentrates that were prepared for injection into the gas chromatograph in
the GC/UV procedure. PNA's are defined by class and molecular weight. Quantita-
tion is achieved by using tripheylbenzene as an internal standard. Compound classes
range from tricyclic to heptacyclic aromatics. Of the total four and higher ring
PNA hydrocarbons in one sample, BaA represented only 0.95% and BaP only 0.4%.
£. A solvent extraction-gas chromatographic method was developed for the
quantitative determination of individual phenols in the aqueous condensate of
auto exhaust. An internal standard, chlorophenol, was added and neutral compounds
were removed by extraction of the alkaline sample. After acidification, phenols
were removed by extraction with ethyl ether. After removing organic acids from
this extract, evaporation to a residue is carried out. An aliquot of the residue
was then analyzed by capillary column gas chromatography. In two samples of ex-
haust tar, phenol was 65 and 100 ppm, individual cresols ranged from 7 to 15 ppm,
and xylenols were less than 1 ppm.
These methods were developed principally for the CAPE-6-68 Project.
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INTRODUCTION
The initial and most important objective in the CAPE-12-68 Project was
to develop a rapid method to measure BaA and BaP in tar recovered from automobile
exhaust, in gasoline, and in crankcase oil. In,selecting an approach, we were in
position to learn from fifteen years of experience in the assay of petroleum fractions
for PNA's. Our choice of technique was based on a recent method which was developed
at Esso Research and Engineering to measure several PNA compounds in coke oven ef-
fluents (1). Based on combined use of gas chromatography and ultraviolet absorption
spectrophotometry, the new method (GC/UV) appeared to be more advantageous compared
with the other most promising approach in which thin layer chromatography would
provide the final fraction(s) for each PNA measurement. This approach was in
agreement with a conclusion reached earlier in 1967 by McKee and McMahon (2). They
surveyed existing methods for analyzing PNA and concluded that "gas chromatography
appears to show the greatest promise of providing a rapid yet reliable and accurate
method to determine a number of PNA compounds."
The coke oven effluent method was extensively modified in achieving a
satisfactory procedure for the CAPE-12-68 project.
Another objective of CAPE-12-68 was to provide a means of measuring all
of the PNA's in a sample. Some of the promising approaches included: ultraviolet
absorption spectrophotometry, summation of GC peaks, and low voltage mass spec-
trometry. The mass spectrometric capability offered the best potential for com-
prehensiveness and specificity. The value of low voltage ionization measurements
was first demonstrated by Field and Hastings (3). They showed that olefins and
aromatic hydrocarbons can be selectively measured in the presence of other com-
pounds. This capability arises from the fact that ionization potentials of olefins
and aromatics are one or two volts lower than those of aliphatic and cycloparaffins.
By using a low ionization potential, selective ionization occurs. This principle
has been extensively used in the petroleum industry to analyze complex hydrocarbon
mixtures.
In achieving another objective of CAPE-12, a method was developed to
measure individual phenols at the ppm level. This development was based primarily
on the use of an extraction procedure evaluated by Hoffman and Wynder (4) to pre-
pare a sample for analysis by capillary gas chromatography (5, 6).
(1) T. D. Searl, F. J. Cassidy, W. H. King, Jr., R. A. Brown, Anal. Chem. 42,
954 (1970).
(2) H. P. McKee, W- A. McMahon, Technical Report No. 1, Project No. 21-2139,
Committee for Air and Water Conservation, American Petroleum Institute.
(3) F. H. Field, S. H. Hastings, Anal. Chem. 28, 1248 (1956).
(4) Dietrich Hoffman, Ernest L. Wynder, Beitrage zur Tobakforschung ^, 101 (1961).
(5) Jan Hrivnak, J. Chromatog. Sci. 8, 602 (1970).
(6) D. S. Payn, Chemistry & Industry, 1090 (August 20, 1960).
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METHOD FOR BENZ(a)ANTHRACENE, BENZQ(a)PYRENE
AND OTHER POLYNUCLEAR AROMATIC HYDROCARBONS IN
AUTOMOBILE EXHAUST. GASOLINE AND CRANKCASE OIL
The method used carbon-14 labeled BaA and BaP as internal standards,
starting from the first step in the analysis. Some polar compounds were removed
by caustic and then a preliminary separation of PNA's was made by liquid-solid
adsorption column chromatography. Specific compounds were next measured by a
combined gas chromatography/ultraviolet absorption spectrophotometry procedure.
In the following sections, details of the method are discussed, as well as its
applicability to tar from automobile exhaust, gasoline, and crankcase oil.
The method was developed and tested during 1969-70. From mid-1970 on,
it was in routine use in CAPE-6-68. Figure 1 shows the method of analysis for
PNA's in automobile exhaust tar, and complete details of the method are given
in Appendix A.
Preparation of Samples With
Carbon-14 Labeled BaA and BaP
In this work, the starting sample of exhaust tar was in solution as
several gallons of cyclohexane/acetone solvent containing the tar (7). It was
necessary to distill off solvent to a residual volume of one liter. Prior to
starting the distillation, C BaA and C BaP were added to the sample.
For gasoline, a starting sample of 50-1000 ml was spiked with ^C BaA
and ^C BaP and evaporated on a steam bath over nitrogen. As evaporation proceeded
the sample was twice replenished with cyclohexane and finally reduced to 25 ml. The
sample was then ready for charging to the alumina column.
In the case of used crankcase oil, carbon-14 standards were added to a
weighed amount of sample (^500 mg), and the sample charged to the deactivated
alumina column.
Caustic Extraction
Caustic extraction was needed only for tar samples. 500 ml of the one-
liter sample was extracted three times with 0.5 N aqueous sodium hydroxide and
then washed successively with 0.1 N HCl and water. This extraction step reduced
the background of the gas chromatogram. 250 ml of the caustic treated sample
(one-quarter of the original sample) was charged to the alumina column. This
volume usually contained 100-400 mg of tar.
(7) G. P. Gross, "Gasoline Composition and Vehicle Exhaust Gas Polynuclear
Aromatic Content," U.S. Clearinghouse Federal Science Technology Information,
PB Rep. Issue No. 200266 (1971) 124 pp.
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Figure 1
Outline of GC/UV Method for CAPE-12-68
Prepare Sample Solution
Containing L*C Labeled BaA, BaP
Remove Polars by
Caustic Extraction
Separate on Partially Deactivated Alumina,
Elute by Cyclohexane, Cyclohexane/Benzene,
Benzene, Benzene/Methanol
Cut 1
First 140 ml -
Discard first 100 ml
Cut 2 (PNA Fraction)
Select Front Cut Point
by UV, Evaporate,
Add Acetone
Cut 3
Select Front Cut
Point by Appearance
of Water and Color -
Discard
Run Cut 2 by GC
Chart -
Chromatogram
15%
85%
Trap Peaks
Measure UV Spectra of
Pyrene, BaA, Chrysene, etc
Measure C Activity of
BaA and BaP Peaks
Calc. pg of Pyrene, BaA,
Chrysene, etc. Based on UV and
^C Labeled Internal Standards
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Preparation of a PNA Concentrate
By Adsorption Chromatography
A PNA concentrate was prepared using the materials and conditions
listed in Table I.
Table I
Conditions For Stepwise Elution
Of PNA's From Auto Exhaust Tar
Column Dimensions: 50 cm length and 1.1 cm i.d.
Alumina: Woelm neutral alumina dried at 160°C and then
deactivated with 2 wt.70 water.
Elution Schedule: 100 ml of cyclohexane
100 ml of cyclohexane/benzene (4:1)
100 ml of benzene
100 ml of benzene/methanol (1:1)
The first 100 ml of cyclohexane eluted was discarded. Then, 10 ml fractions were
collected and inspected by UV for the appearance of pyrenes (absorption band at
340 nm). The first fraction containing pyrene and all subsequent fractions to
the appearance of a two liquid phase boundary were combined as the PNA concen-
trate. The appearance of pyrene has varied from the second to the seventh 10 ml
fraction of the cyclohexane/benzene elution step. Selection of the first fraction
containing pyrene is illustrated in Figure 2. In this example, cut B2-3 was the
initial fraction of the PNA concentrate.
The alumina column fraction containing the PNA's will normally consist
of -v160 ml of a cyclohexane/benzene solution. To prepare a sample for injection
into the gas chromatograph, the solution is evaporated to ~s 40 ul.
Evaporate solvent, under a small jet of nitrogen, using a 150 ml beaker
on a steam bath. Add portions of the solution and reduce to 2 ml.
Transfer the 2 ml residue to a one-dram vial and continue the evaporation
on a steam table to constant volume. Add acetone to bring residual volume
up to <~40 jil.
Gas Chromatography and Trapping of Peaks
The operating conditions for the gas chromatographic separation are
shown in Table II.
Table II
Operating Conditions for GC Separation of PNA Fractions
Chromatograph - Perkin-Elmer Model 900.
Gas Flow - Helium at 30 ml/min.
Column - 300 cm-0.22 cm i.d., packed with 2% SE 30 (GC grade) on
Chromosorb "G" 80/100 mesh (acid washed and DMCS treated).
Injection Port - 305°C
Detector, F.I.D. - 345°C
Program - 175° to 300°C at 4°/min., then hold
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Figure 2
SELECTION BY UV OF FRONT CUT PT.
OF PNA FRACTION FROM Al COLUMN
I
CUT B2-2
This fraction discarded.
Cd
O
PQ
06
O
w
M
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The recovery of individual PNA's, as influenced by operating conditions,
indicated that the temperature at the sample injection port was important. This
is shown by the recovery data in Table III.
Table III
Recovery vs. Sample Injector Temperature
Temp, programmed to 300°C
Trap line at 345°C
Damp j.e
Injector (°C)
260
275
305
345
400
no.
Runs
2
6
4
3
1
Pyrene
87
87
91
85
79
nean /«,
BaA
82
81
83
75
71
Kecover)
BaP
84
81
84
70
63
B(g,h,i)P
58
73
77
62
59
Optimum recovery occurred at 305°C injection port temperature. Even for
these conditions, the recovery of B(g,h,i)P is ~ 10 relative 70 lower than
BaA and Bap. For this reason, a correction of 1.10 is applied to the
measurement of B(g,h,i)P.
Figure 3 shows the gas chromatograph trapping assembly. To trap a
fraction the stainless steel tube was slid into position as shown.
As the sample was run on the chromatograph, selected peaks were trapped
and the trap content rinsed out with cyclohexane for UV measurement. Selection
of peaks coincided with the retention times listed near the top of Table IV. In
some instances retention times were virtually identical for two or three different
PNA's. For example, BaA, chrysene and triphenylene elute together and are taken
into one trap. BaP and BeP also elute together. Methyl BaP and methyl BeP elute
together but in three different peaks; each peak is trapped separately. A typical
chrotnatogram is illustrated in Figure 4. All of the PNA's currently measured
plus others are shown.
Ultraviolet Absorption Spectrophotometry
Absorptivity coefficients were measured for those PNA's of interest which
could be obtained. These included: pyrene, BaA, chrysene, triphenylene, some methyl
and dimethylbenz(a)anthracenes, BaP, BeP, and benzo(g,h,i)perylene. These calibrations
were corrected for the observed purity of each compound (purity was established by
GC and MS measurements). Coefficients for methyl BaP and methyl BeP were estimated
as being 0.815 of the parent compounds.
To prepare the trapped fraction for UV spectroscopy, the trap containing
the GC fraction was rinsed with cyclohexane and made up to 3.8 ml. UV absorbance
was measured with 1 cm path cell. For low absorbing solutions a 5-cm cell was used.
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Radioactivity Measurements
The most accurate and finally used radioactive count was obtained in a
long count using a Packard Tri-Carb Counter. Counting was conducted for fifty
minutes or 50 K, whichever came first. Background and counting efficiency was
measured on each sample. On occasion, a waiting period of one week was encoun-
tered in getting this count. To avoid this delay in reporting an analysis, a
tentative analysis was based on a short count. This short count was obtained for
one ml of cyclohexane solution using an Intertechnique SL 20. This count was
always in close agreement with the long count.
Calculation of PNA's in GC Peaks
Concentrations of the individual PNA's were calculated from UV spectra
of GC peaks based on absorptivity coefficients listed in Table IV. Slant base
line measurements were used and the table lists the anchor points; and the ana-
lytical peaks for each compound.
BaA, Chrysene. Triphenylene - Fig. 5 is an UV absorption spectrum of the GC
peak containing BaA, chrysene and triphenylene. To calculate the concentration
of each compound, a set of three simultaneous equations is solved. The array
of calibration coefficients from Table IV is shown below.
Table V
Calibration Matrix for Calculation
of BaA, Chrysene, and Triphenylene
BaA
0.332
0.039
0.015
Chrysene
0
0.464
0.107
Triphenylene
0
0
0.480
(1) 0.332 0 0 A289
(2) 0.039 0.464 0 A269
(3) 0.015 0.107 0.480 A259
A289, A269, A259 = UV absorbance of GC peak in a 1 cm cell.
The equations can be solved directly by substitution.
BaA = A289
0.332
Chrysene = A269 - BaA x 0.039
0.464
Triphenylene = A259 - BaA x .015 - Chrysene x 0.107
0.480
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TABLEF?
GC AND UV CALIBRATION DATA FOR SOME PNA HYDROCARBONS
Dl- Benzo-
Trl- methyl (g,h,i)
Chry- phenyl Methyl /ethyl Methyl Methyl peryl-
Pyrene BaA sene ene BaA BaA BaP BeP BaP BeP ene
27.5
GC Retention 13.6 19.4 19.6 19.4 21.4 23.3 25.8 25.7 28.0 31.4
Times (min) 29.0
Wavelength nm
Peak Base Line
_ ..- . »»-,. Absorptivity (ml/pg cm)
336 327-343 0.240
289 283-295 0.332
269 263-277 0.039 0.464
259 252-263 0.015 0.107 0.480
293a 285-300 0-27
293a 285-300 0-27
383 373-390 0.092
333 325-338 °-135
384b 373-390 °-°75
335 325-340 °-110
382 370-390 °-C7A
a Occurs in range, 290-293 nm
b Occurs in range, 383-385 nm
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Radioactivity Measurements
The most accurate and finally used radioactive count was obtained in a
long count using a Packard Tri-Carb Counter. Counting was conducted for fifty
minutes or 50 K, whichever came first. Background and counting efficiency was
measured on each sample. On occasion, a waiting period of one week was encoun-
tered in getting this count. To avoid this delay in reporting an analysis, a
tentative analysis was based on a short count. This short count was obtained for
one ml of cyclohexane solution using an Intertechnique SL 20. This count was
always in close agreement with the long count.
Calculation of PNA's in GC Peaks
Concentrations of the individual PNA's were calculated from UV spectra
of GC peaks based on absorptivity coefficients listed in Table IV. Slant base
line measurements were used and the table lists the anchor points^ and the ana-
lytical peaks for each compound.
BaA, Chrysene, Triphenylene - Fig. 5 is an UV absorption spectrum of the GC
peak containing BaA, chrysene and triphenylene. To calculate the concentration
of each compound, a set of three simultaneous equations is solved. The array
of calibration coefficients from Table IV is shown below.
Table V
Calibration Matrix for Calculation
of BaA, Chrysene, and Triphenylene
BaA
0.332
0.039
0.015
Chrysene
0
0.464
0.107
Triphenylene
0
0
0.480
(1) 0.332 0 0 A289
(2) 0.039 0.464 0 A269
(3) 0.015 0.107 0.480 A259
A289, A269, A259 = UV absorbance of GC peak in a 1 cm cell.
The equations can be solved directly by substitution.
BaA = A289
0.332
Chrysene = A269 - BaA x 0.039
0.464
Triphenylene = A259 - BaA x .015 - Chrysene x 0.107
0.480
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TABLEF?
GC AND UV CALIBRATION DATA FOR SOME PNA HYDROCARBONS
Dl- Benzo-
Tri- methyl (g»h,i)
Chry- phenyl Methyl /ethyl Methyl Methyl peryl-
Pyrene BaA sene__ ene BaA BaA BaP BeP BaP BeP ene
-----27.5
GC Retention 13.6 19.4 19.6 19.4 21.4 23.3 25.8 25.7 28.0 31.4
Times (min) 29.0
Wavelength nm
Peak Base Line
-....--...-..-. ^-,. Absorptivity (ml/pg cm)
336 327-343 0.240
289 283-295 0.332
269 263-277 0.039 0.464
259 252-263 0.015 0.107 0.480
293a 285-300 0-27
293a 285-300 0-27
383 373-390 P-092
333 325-338 0.135
384b 373-390
335 325-340 °-110
382 370-390 P-iP-Zl
a Occurs in range, 290-293 nm
b Occurs in range, 383-385 nm
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Test for Accuracy
The accuracy of the UV analysis of these three component mixtures was
found to be satisfactory as indicated by the results in Table VI for some known
blends.
Table VI
UV Analyses of Known Blends Containing
Benz(a)anthracene, Chrysene and Triphenylene
BaA
Chrysene
Triphenylene
Known
0.4
1.2
2.4
Found
0.5
1.0
2.3
Known Found
/ i
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1.5 1.5
1.5 1.3
1.5 1.5
Known
0.5
1.5
1.0
Found
0.5
1.4
1.0
BaP, BeP - Spectrum of a GC peak containing BaP and BeP is shown in
Figure 6. Base line constructions are shown for these compounds.
Figure 6
UV Spectrum of GC Fraction Containing BaP and BeP
- 5 cm cell -
2.0
1.5
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350
400
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Methyl BaP, Methyl BeP - Of the other PNA's measured, the calculation
of pyrene, methyl BaA, and dimethyl BaA is straightforward. Benzo(g,h,i)perylene
is corrected upward by 1.10 as previously noted (p. 8). Methyl substituted BaA
and BeP isomers require special handling as they occur in three separate GC peaks.
In practice, each of the three peaks is separately trapped and measured by UV.
Summed values are then used to calculate the quantity of each methyl isomer. A
more efficient approach would be to blend the solutions of the three fractions
prior to the UV measurement. This is not done because there are variations in
the wave length position of the maximum absorption and the UV absorption of the
blend would therefore be non-additive. For methyl BaP the band position was ob-
served to vary from 384 to 386 nm, and for methyl BeP it was 334 to 337 nm (see
Table VII) .
Composition information was obtained for one CAPE-6 sample in which
the GC peaks were examined by UV and mass spectrometer. Results are summarized
in Table VII. Peak 20 contained BaP and BeP.
Table VII
Identification of GC Fractions Containing Alkyl Substituted BaP and BeP
BaP BeP
Series Series
Peak No. nm nm Mol. Wt. Identification
20 383 332 252 BaP, BeP
22 384 334 266 - 100% Methyl BaP, Methyl BeP
23 384 to 334,337 266 - 58% Methyl BaP, Methyl BeP
3883 280 - 427, (Dimethyl/Ethyl BaP,
(Dimethyl/Ethyl BeP
24 385 335 266 15% Same as Fraction No. 23
above
280 - 85%
a = Broad peak.
Peak 22 showed only 266 as a molecular weight (MW) ion which indicates the presence
of only methyl BaP and methyl BeP. In peak 23, however, only 587= of MW 266 was found
and there was 42% of MW 280. MW 280 is probably due to dimethyl and/or ethyl BeP
and BeP. Peak 24 is primarily MW 280. An estimate of the overall distribution of
the methyl and dimethyl/ethyl species in the three peaks was:
methyl (BaP + BeP) = 0.8
dimethyl/ethyl (BaP + BeP) = 0.2
Calculation to a Sample Basis
Based on the measurements of the previous steps, the micrograms of each
PNA in the sample were calculated from the aliquot factor (activity ratio), the
PNA concentrations as measured for each GC peak, and a correction for the weights
of added 14C BaA and l^C BaP.
Details are in Appendix A.
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Application of the Method
Time Requirement - During the past two years, this method has been
routinely used to analyze all of the sample types for which it was intended plus
others. Experience with the method indicates that a technician can be trained
in all of the steps during a 1-2 week period. Trained technicians can analyze
two or three samples in parallel and at this level of proficiency, each sample
requires 20 man hours of effort.
Expansion of the Method - In addition to the eleven PNA's currently
being measured, the gas chromatographic separation provides numerous other well-
defin ed peaks that can be trapped and analyzed. Based upon our experience other
4+ ring PNA's can be added, including: fluoranthene, benzo(b)fluoranthene, benzo-
(j)fluoranthene, benzo(k)fluoranthene, perylene, anthanthrene, and coronene.
Analysis of Tar - A principal test of the method was to compare results
on 11 samples with measurements by an independent procedure. In the latter method,
which also employed carbon-14 labeled BaA and BaP as internal standards, a pre-
liminary separation was carried out in a deactivated alumina column. A composite
of these fractions was next separated on a thin layer chromatographic (TLC) plate
containing 20% acetylated cellulose. A fraction from the plate, rich in BaA (or
BaP) was then examined by UV and counted for radioactivity to obtain a quantitative
measurement.
Comparison between the two methods is shown graphically in Figure 7.
Shown are the ratios of GC to TLC. Measurements of BaP by GC/UV are consistently
higher, particularly for small quantities. We have no explanation of this, al-
though only small differences in absolute amounts are involved. An opposite re-
lationship exists for BaA in that the measurement by TLC is higher than by GC/UV.
High values by TLC can be attributed to the presence of methyl BaA in TLC frac-
tions (8).
Precision of the method is indicated by several analyses of a tar sample
as shown in Table VIII.
Table VIII
Precision3 of GC/UV
Compound
Pyrene
BaA
Chrysene
Triphenylene
Methyl BaA
Dimethyl/ethyl BaA
BaP
BeP
Benzo(g,h, i) perylene
Methyl BaP
Methyl BeP
a = six analyses from
Method as Applied to a
Amount
(ug)
676
117
111
25
58
14
15
153
137
5
63
March-October,
cr
(ug)
25.4
5.7
13.1
1.4
4.0
1.1
1.4
8.5
24.9
2.2
6.2
1971.
Tar
cr
q)
3.8
4.9
11.8
5.6
6.9
7.9
10.0
5.6
18.2
45.2
9.8
(8) R. A. Brown, J. M. Kelliher, Amer. Petrol. Inst. Proc., Div. Ref. 51,
349 (1971).
-------
-17-
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-18-
Analysis of Gasoline - Repetitive analysis of a gasoline was obtained
at a time when only 2-5 compounds were being measured. These results are in
Table IX.
Table IX
Analysis of a Gasoline
No. of Ug per Gallon CT
Compound Measurements Amount ff (%)
Pyrene 4
BaA 5
Chrysene 4
BaP 5
BeP 4
Analysis of Crankcase Oil - A sample of unused multi-grade oil was
analyzed. Two grams of oil were eluted through a deactivated alumina column as
described in the procedure. Both the "PNA" fraction and the more polar fraction
which elutes afterwards were inspected by GC/UV. BaP and BaA were below the
detectable limit. Three known blends of BaA and BaP in this oil were then an-
alyzed with accuracy as shown in Table X.
TableX
Measurement of BaA and Bap
As Added to Unused Multi-Grade oil
Sample BaA BaP
- - - mlcrograms in 250 ml - - -
Addod Found Added Found
A 12 12 11 11
B 23 23 17 19
C 48 47 44 43
A 250 mg sample of crankcase oil, estimated to have been used for
3,000 miles, was found to contain 6 pg BaA and 7 ug BaP, equivalent to 24 and
28 ppm, respectively. These concentrations are comparable to those reported
by Begeman and Colucci (9).
(9) Charles R. Begeman, Joseph M. Colucci, SAE Trans., _79, 1782 (1970).
-------
-19-
KEASUREMENT OF TOTAL POLYNUCLEAR AROMATIC
HYDROCARBONS BY LOW VOLTAGE MASS SPECTROMETRY
Low voltage mass spectrometry appears to be an Ideal tool for measuring
PNA's in a mixture. It is common practice in the petroleum industry to use this
technique for olefins and aromatics. Lumpkin and Aczel (10) observed aromatics
in gas oil and numerous others have reported on its use. The low voltage tech-
nique can be used with either high or low resolution spectrometers. High resolu-
tion offers the advantage of unequivocal measurement by empirical formula. A
shortcoming of this approach is that is requires expensive, complex equipment.
For general utility, a low resolution (1/600) mass spectrometric method is
attractive.
A partial spectrum (Figure 8) shows the nature of a measurement for a
polynuclear aromatic mixture. Monoisotopic peaks are shown for the mass number
ranges 188-190 and 220-228. Individual peaks are identified by molecular weight.
Compound(s) in a given series start at the lowest number consistent
with the molecular weight of the parent compound(s) and occur at intervals of
fourteen mass units in ascending value. Successive substitution of methyl,
dimethyl or ethyl, and higher alkyls account for the regular increase of fourteen
mass units. Phenanthrenes occur as the series 178, 192, 206, etc., and pyrenes
and fluoranthenes at 202, 216, 230, etc. Based on this behavior, a spectrum
provides additional insight into identifications. For example, the spectrum in
Figure 8 shows an intense peak at 202 and a weak 14 units lower, which identifies
the 202 as the parent peak of pyrene and/or fluoranthene. Another series, benzo-
(g,h,i)fluoranthenes (mol wt 226), starts at 226, and the much lower peak at 212
indicates that the first member of this series does indeed start at MW 226.
Another series starts at MW 228 (benzanthracenes, etc.) although a much smaller
peak at 214 indicates a small amount of another class of aromatics.
Cyclopentaphenanthrenes are handled as if their lowest molecular weight
is 190 and this is consistent with a small 176 peak that is observed but which
is not shown in Figure 8.
The sample to be analyzed by mass spectrometer is the aromatic fraction
from the chromatographic separation in the alumina column. Oxygenated compounds
are present in the original sample, and even though they largely are separated
into a polar fraction, there is a likelihood that some would also occur in the
aromatic fraction. There was concern that such oxy's might be present so as
to subsequently be measured along with PNA's.
In order to evaluate this source of error, a typical PNA concentrate
from a CAPE-6 run was analyzed by high resolution mass spectrometry3. The
analysis showed that oxygenates were absent in the spectral region of interest
for this method.
(10) H. E. Lumpkin, Thomas Aczel, Anal. Chem. 36, 181 (1964).
a = Analysis was done by H. E. Lumpkin, Esso Research and Engineering Company,
Baytown, Texas.
-------
-20-
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-21-
Procedure for Mass Spectrometer Analysis
A sample to be analyzed is spiked with a known quantity (40 micrograms)
of triphenylbenzene as the internal standard. The sample is then charged to the
alumina column and handled the same way as for preparing the PNA concentrate for
GC. Three pi of this sample is charged to the mass spectrometer.
A Model 21-102 mass spectrometer manufactured by Consolidated Electro-
dynamics Corporation was used. This instrument is equipped with an all-glass
sample inlet system that is maintained at a temperature of 316°C. It was im-
portant to demonstrate that typical compounds could be quantitatively handled
by the spectrometer. This implies the ability to quantitatively introduce a
sample to the inlet system, measure its spectrum, and pump out without decom-
position occurring.
Investigation of important variables had been done previously in some
other work. This previous work showed that hydrocarbons were stable but that
benzanthrone, an oxygenated aromatic, does decompose at certain conditions.
Even though we were not concerned with this type of compound, we used operating
conditions for its stability. This involved operating at a lowered ion source
temperature. In all of our work the ion source was operated at a n-hexadecane
cracking pattern of m/e 127 _ _ This was obtained by running at a relatively
7 , /v U .o.
m/e 226
low temperature in the ion source. This is in contrast to that associated with
a cracking pattern of 1.40 obtained at higher temperatures.
At these conditions, some partially hydrogenated compounds behaved
normally (9,10-dihydrophenanthrene, dihydropyrene); others were found to par-
tially decompose, as also reported by Shultz (11). Decomposition occurred for
9,10-dihydroanthracene and 3-methylcholanthrene. In agreement with Shultz, we
found that decomposition of these compounds was greatly reduced at sample bottle
temperature of 260°C, as compared with 316°C. The lower temperature was not put
to practice, however, because an actual sample of tar analyzed at 316°C and 260°C
gave similar compositional results. Vulnerable compounds were thereby shown to
be absent or present at an insignificant level.
Calibration
Low voltage measurements are based on the intensities observed for
molecular or parent ions. For quantitative work it is necessary to calibrate
the mass spectrometer to establish peak intensities per unit weight, mol or
volume. We chose to measure peak intensity per microgram (sensitivity) of each
compound. Triphenylbenzene was used as an internal standard with each calibration
blend. In the calibration step, therefore, the sensitivity of individual com-
pounds was measured on an absolute basis, and also in comparison with triphenyl-
benzene. Sensitivities were observed by examining blends of 4-5 compounds, each
at a known concentration of approximately one milligram per milliliter of solution.
(11) J. L. Shultz, Spectroscopy Letters, I (8 & 9), 345 (1968).
-------
-22-
Cotnpounds for each blend were selected to be of different molecular weights so
that each compound could be separately observed. Toluene was the solvent used
for 3-5 ring compounds, but 6+ ring compounds required pyridine solvent.
Samples of 1-3 microliters are introduced with the sintered disk at
40°C. This temperature is then raised to 316°C prior to running.
More than thirty calibration blends were run in duplicate. Results
for a typical blend are tabulated in Table XI.
Table XI
Calibration of a Typical Blend (# LV-21) of PNA's
g
Concentration Mol. Purity Sensitivity
Compound (mg/ml) Wt._ (%) (PkHt/yg)
2-Methylphenanthrene 2.03 192 100 111, 123
2,3-Dihydrp-lH-cyclo-
penta [1] phenanthrene 2.08 218 84 86, 94
BaP 2.00 252 100 62, 67
Dibenz (a, h) anthracene 2.08 278 100 54, 58
Triphenylbenzene 1.96 206 100 42, 46
a = Purity was measured by GC and MS.
Sensitivities were measured for fifty-three compounds as tabulated in Table XII.
Each sensitivity value represents the average of duplicate measurements. Replicate
measurements are based on a given compound being present in different blends.
2-methyl pyrene, for example, was measured in three different blends.
Based on sensitivities from Table XII, average sensitivities were es-
timated for groups of compounds having identical empirical formulae, viz., phen-
anthrenes and anthracenes of the formula, CnH2n-18- Parent compound sensitivities
were first calculated and then estimates were made for the alkyl-substituted
isomers. When available, knowledge of composition was employed to obtain weighted
averages. For example, in auto exhaust and in gasoline, phenanthrenes occur in
greater abundances than anthracene. Thus, the average for this group was weighted
in the direction of phenanthrenes. Values as used from Table XII are:
Phenanthrene = 138
Anthracene = 126
Mean = 132
Instead of the mean of 132, a value of 134 was selected.
-------
-23-
Table XII
Low Voltage Sensitivities of 3-7 Ring PNA Hydrocarbons
Compound Mol Wt Sensitivity
(tnm/^g)
Phenanthrencs, Anthracenes
Phenanthrene 178 138
1-Methyl Phenanthrene 192 106
2-Methyl Phenanthrene 192 133
9,10-Dimethyl Phenanthrene 206 115
Anthracene 178 126, 127
2-Methyl Anthracene 192* 78, 95
j9-Methyl Anthracene 192 136
2,6-Dimethyl Anthracene 206 128, 131
2,7-Dimethyl Anthracene 206 113
Fluorcnes, Others
Fluorene 166 84
2-Methyl Fluorene 180 121
9-Methyl Fluorene 180 97
9,10-Dihydrophenanthrene 180 93
9,10-Dihydroanthracene 180 69
Pyrenes, Fluoranthenes, Benzofluorenes
Pyrene 202 91, 101
1-Methyl Pyrene 216 97
2-Methyl Pyrene 216 85, 79, 74
4-Methyl Pyrene 216 106
1,9-Dimethyl Pyrene 230 95
Fluoranthene 202 80
5H-B«nzo(a)fluorene 216 75
llH-Benzo(b)fluorene 216 58
Cyclopentnphonanthrenos, Other
Dihydropyrene 204 90
2,3-Dihydro-lH-cyclopenta jjjphenanthrene 218 102
Ben7.o(g,h, i) f luornnt hones , Clio Ion thrones
Benzo(g,h,i)fluoranthene 226 61
Cholanthrene 254
3-Methyl Cholanthrene 268 63
4-Mcthyl Cholanthrene 268 52
(continued)
-------
-24-
Table XII (continued)
Compound Mo I WE Sensitivity
(i
Benzanthracenas, Chrysenes, Triphenylcncs
BaA 228 93
7-Methyl BaA 242 73
8-Methyl BaA 242 76
12-Methyl BaA 242 90
7,12-Dimethyl BaA 256 71, 75
Chrysene 228 99
Trlphenylene 228 68
Benzo(e)phenanthrene 228 79
Benzopyrenes, Benzof1uoran thene s
Benzo(a)pyrene 252 81
Benzo(e)pyrene 252 76
Perylene 252 67
Benzo(b)fluoranthene 252 62
Benzo(k)fluoranthene 252
Benzoperylenes, Anthantbrones
Benzo(g,h,i)perylene 276 63
Anthanthrene 276 26
Dlbenzanthracenes
Dibenz(a,h)anthracene 278 63, 62
Dibenz(a,e)anthracene 278 57
3,4,5,6-Dibenzophenthrene 278 58
Picene 278 51
-3,4-Benztetraphene 278 61
Pentacene 278 -a-
Coronenos
Coronene 300 48
ptbcnzopyrcnes
Dibenzofa,e)pyrene 302 42
Dibenzo(a,h)pyrene 302 38
Dibenzo(a,i)pyrenc 302 36
2,3,4,5-Dibcnzopyrene 302 40
l-Methyl-2,3,7,8-dibenzopyrene 316 -a-
5-Methyl-3,4,8,7-dibenzopyrene 316 -a-
-fl- - No or slight vaporization in GC and MS,
-------
-25-
Sensttivities for alkyl-substituted compounds were estimated from the
sensitivity behavior of methyl and dimethyl isomers of anthracene, phenanthrene,
pyrene, and benz(a)anthracene. This is summarized below in terms of relative
values (Table XIII).
Table XIII
Relative Low Voltage Sensitivities
Phenanthrenes
Anthracenes Pyrenes Benz(a)Anthracenes Mean
Parent 1.00 1.00 1.00 1.00
Methyl Subst. 0.93 0.98 0.90 0.94
Dimethyl 0.91 0.98 0.79 0.90
This trend of diminishing sensitivity with increased substitution on the ring is
common to simpler aromatics. Ratios of 1.00, 0.94, 0.90, 0.88, 0.85, and 0.82
were used to represent compounds ranging, successively, from the parent compound
to its alkyl substituted form of up to 5 carbon atoms. Applied to the pyrene,
fluoranthene series this refers to compounds of molecular weights: 202, 216, 230,
244, 258. Both pyrene and fluoranthene have a molecular weight of 202. A methyl-
substituted pyrene has a molecular weight of 216, a dimethyl or ethyl pyrene is
230, etc.
Average sensitivity data for the method are shown in Appendix B.
In the calculation of the mass spectrum, monoisotopic peaks are first
obtained and then, based on the sensitivities supplied in the method, microgram
quantities are calculated. Quantities on an original sample basis are obtained
by proportioning the amount of triphenylbenzene found in the mass spectrometer
to the forty micrograms initially added.
Calculation
Peaks are read from a chart or selected from a digitizer/computer
printout. All peaks in the m/e range, 178-330, are recorded.
Calculate monqisotopic peaks for all even m/e numbers.
Calculate the triphenylbenzene peak at m/e 306 by correcting for
sample background as follows:
m/e Peak Height
292 117.0
306 266.1
320 70.1
Triphenylbenzene peak (M/e 306) = 266.1 - I(117.0 + 70.0)/2 I = 172.6.
- |(117.0 + 70.0)/2 I =
-------
-26-
Divide tnonoisotopic peaks by appropriate sensitivity to
obtain micrograms of each mol wt species in the sample.
Based on the internal standard, triphenylbenzene, convert
all microgram quantities to an original sample basis.
Calculation of a partial spectrum is outlined in Table XIV.
Table
Partial Calculation of a Sample
Type of Mol Wt Peak Micrograms Micrograms
Compound (m/e) Height Sensitivity (Peak T Sensitivity! in Sample_
Pyrenes, 202 4150 88 47.1 540
Fluoranthenes
216 2262 86 26.3 300
80 13.4 153
78 7.6 87
75 4.7 54
This illustrated calculation covers the group, pyrenes and fluoranthenes,
for MW's from 202 to 258. Micrograms of each MW species are shown in column 5;
for example, 4150 T 88 = 47.1 micrograms. The quantity of internal standard
(triphenylbenzene) is calculated.
172.6 (peak height) '- 50 (sensitivity) = 3.5
Since 40 micrograms of triphynylbenzene was originally added to the sample,
the micrograms by MW (column 5) can be converted to a total sample basis by
multiplying each quantity by 40 4 3.5, or 11.4.
Application of the Method
Two samples, X and Y, were analyzed as shown in Table XV. Including
the tricyclics, eleven groups of compounds are reported and several of these
include different molecular structures. For example, one group includes benz-
anthracenes, chrysenes, and tripheylenes. If we count molecular structure
variations, the number is eighteen. This includes 3- to 7-ring structures.
In addition to unsubstituted PNA's, many alky-substituted PNA's are
present in auto tar. The parent compounds represent the principal isomer in
each group, although the sum total of substituted compounds generally exceeds
that of the parent compound itself.
In the GC/UV analysis of sample Y, BaA was found to be 174 ^ug, and
BaP was 73 /ug. Considering that this sample contained 18,294 /ug of 4+ ring
PNA hydrocarbons, BaA represented only 0.95% and BaP was only 0.4% of the
total PNA content.
-------
-27-
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-28-
METHOD FOR PHENOLS IN AUTOMOBILE EXHAUST GAS
The internal standard method described in this report was developed
as the last part of the CAPE-12-68 project. Due to lack of funds, the procedure
was not thoroughly tested. Limited data indicate that it adequately handles
an aqueous sample. Since most of the phenols in automobile testing occur in
the water condensate, the method satisfies most of the needs in this kind of
work. Furthermore, the method can be extended directly to measure phenols in
hydrocabon samples.
For an aqueous condensate sample of 25 ml, sensitivity of the
method is 1 pg (40 ppb). Since CAPE-6 tests generally gave 5 liters of the
condensate, it is possible to measure much lower quantities of a phenol by
using a larger starting sample.
Description of Method
A known amount of an internal standard, o-chlorophenol, is added
to the original sample. Neutral compounds are removed by extraction of the
alkaline sample. The extract is acidified, saturated with sodium sulfate,
and the phenols are extracted by five successive portions of ethyl ether.
Organic acids are then removed by a sodium bicarbonate extraction. The
ether is distilled in a special concentrator leaving a small residue which
is injected into a capillary column gas chromatograph. The recorded peaks
are identified by retention time. Peak areas are measured and multiplied by
the appropriate factors to convert the area to weight.
Preparation of Sample for Gas Chromatograph
To prepare a sample for the gas chromatograph, the phenols are
separated from the aqueous sample and then purified by a classical solvent
extraction technique. A similar separation procedure was evaluated by Hoffman
and Wynder (4) in their study on tobacco smoke. In one step, these workers
separate the phenols from impurities by steam distillation. This causes a
five-fold increase in volume and was found to be unnecessary in the present
work.
After adding 0.8 mgs of o-chlorophenol, the internal standard, to
25 ml of the aqueous condensate and making it alkaline, the neutral compounds
are removed by ethyl ether extraction. The aqueous phase is then acidified,
saturated with sodium sulfate and extracted five times with ether to isolate
the phenols. Any organic acids in the aqueous condensate are extracted along
with the phenols. They are removed from the combined ether solution by
extracting with a small volume of saturated sodium bicarbonate. Finally,
dissolved water is removed by anhydrous sodium sulfate. Ether is distilled
off in a "concentrator" and a phenol-rich residue gathers in a depression of
the glass still pot. Using a medicine dropper the residue is transferred to
a small vial and 10 drops of methanol added. This volume is reduced to 0.2 ml.
An aliquot of this sample is injected to the gas chromatograph for the phenols
measurement.
-------
-29-
Gas Chromatographic Separation of Phenols
As listed in Table XVI, a number of GC columns were evaluated prior
to selecting a capillary column described by Hrivnak (5). Retention times
and response factors of twenty-three phenols were obtained and are presented
in Table XVI.
Table XVI
Analytical Data on Phenols
Source
(a)
E
E
P
P
P
P
A
P
P
P
P
P
P
P
N
A
N
P
A
A
A
K
CS
CS
B.P.-°C Compound
80 Benzene
176 o-Chlorophenol
182 Phenol
192 o-Cresol
203 m-Cresol
207 o-Ethyl phenol
218 m-Ethyl phenol
219 p-Ethyl phenol
218 2,3-Xylenol
212 2,4-Xylenol
242 2,5-Xylenol
212 2,6-Xylenol
219 3,5-Xylenol
225 3,4-Xylenol
to-Isopropylphenol
220 m-n-Propylphen61
229 p-Isopropylphenol.
236 2,3,5-Trimethylphenol
220 2,4,6-
249 3,4,5-
247 2,3,5,6-Tetramethylphenol
242 Indol-4
255 Indol-5
3-tert. Butylphenol
Minutes Past Flame Relative
Injection Sensitivity (t>)
2.
7.
.76
.40
9.5
12.2
15.7
18.6
25.8
24.8
24.2
19.8
20.5
13.0
28.0
29.
35.
29.
42.
41.6
22.0
63.0
51.6
56.
70.
.5
.5
.1
.2
.1
.5
1.12
0.45
0.61
0.66
0.60
0.63
0.61
0.60
0.62
0.60
0.68
0.68
0.69
0.61
0.64
0.70
0.56
0.60
0.62
0.47
0.66
0.59
52.5
(a) E Eastman Kodak Co., Rochester, N.Y.
A Aldrich Chemical Co., Inc., Milwaukee, Wisconsin
N Newton Maine Res. Chem. Co., England
CS Chemical Samples Co., Columbus, Ohio
K K&K Chemical Co., New York, N.Y.
P Polyscience Corporation, Evanston, Illinois
(b) For flame ionization detector, divide area by relative sensitivity
to get corrected area. Normalize corrected areas to get weight
percent.
-------
0.125" o.d. 125
-30-
In brief, relative sensitivities were measured by preparing weighed blends
of selected compounds with a known internal standard (one of the C.. aromatics)
In many cases, the relative sensitivity values are the average of twelve
determinations, involving four or more blends. The values are estimated to
be accurate within + 3 relative ?». These calibration data are also reported
elsewhere (12).
Table XVII
GC Columns for Separation of Phenols
Column Temp.
Column Substrate Type Support Length, ft. Diameter
Didecylphthalate Capillary Chromo G 150
4- 10% H3P04
27, Didecylphthalate Packed Chromo T 6
+ 0.2% H3P04
2% Didecylphthalate " " " " "
+ 2% Bentone 34
10 wt.% FFAP (free " " 10 " 225
fatty acid phase)
Chromosorb 102 " Chromo 102 4 " 150
Chromosorb 104 " Chromo 104 " " 200
Application of the Method
The method was first evaluated with a synthetic blend containing
nine phenols. Results are presented in Table XVIII and the chromatogratn is
reproduced in Figure 9.
Table XVIII
Analysis of Phenol Synthetic
Retention ppm
Time Phenol Known Found
8.0 Chlorophenol Int. Std.
10.2 Phenol 32.5 32.0
13.1 o-Cresol 11.2 12.9
14.0 2,6-Xylenol 10.8 10.8
15.8 p-Cresol 12.4 13.6
16.4 m-Cresol 12.4 13.3
20.8 2,4-Xylenol 10.8 9.2
21.3 2,5-Xylenol 11.2 11.2
25.8 p-Ethyl Phenol 10.8 11.6
20.8 3,4-Xylencl 10.8 12.0
(12) W. A. Dietz, J. Chromatog. Sci. K), 423 (1972)
-------
-31-
A water condensate from a CAPE-6 test was analyzed as shown in
Table XIX. Figure 10 is the chromatogram of the sample. The sum of the
individual phenols is 141 ppm compared with 125 ppm as found by the routine
method for measuring total phenols.
Table XIX
Phenol .Content of Aqueous Condensate, Commercial Fuel
Phenol
Phenol
o-Cresol
P-Cresol
P-Cresol
2,6 Xylenol
2,4 Xyienol
2,5 Xylenol
3,5 Xylenol
3,4 Xylenol
o-Ethyl Phenol
Total By GC
Total by UV
-------
-32-
w
a
w
Q
W
O
o
OS
CM
S3
o
M
H
U
H
X!
W
O
&
§
53
W
g
O
W
W
2
O
I
O
w
525
J.IO IN 1A
X9t
X9I '
10N3TAX
10N31AX 5'
lONSHd TAHia-d
10N31AX f'
H
c
o
*o -H
< 4J
01
-------
-33-
xze iON3HdONOino-°
xet lo.'nud
W
H
W
Q
o
o
o §
-> w
w
Pi
§ o
pLf JV|
1
o
Xb 10N31AX "y'Z
Xt- 10fJ3Hd lAIU3-<<
Xfr 10N31AX
-------
-34-
APPENDIX A
Method for Benz(a)anthracene, Benzo(a)pyrene,
And Other Polynuclear Aromatic Hydrocarbons in
Automobile Exhaust, Gasoline and Crankcase Oil
Introduction
This method was developed to determine polynuclear aromatic
hydrocarbons in gasoline, crankcase oil, and tar from auto exhaust-
The method covers polynuclear_ aromatics (PNA) ranging from pyrene
to benzo(g,h,i)perylene. As described herein, eleven PNA's are measured,
including: pyrene, benz(a)anthracene, chrysene, triphenylene, methylbenz-
(a)anthracene, dimethyl and/or ethylbenz(a)anthracene, benzo(a)pyrene
benzo(e)pyrene, methylbenzo(a)pyrenes, methylbenzo(e)pyrenes and benzo( g,h,i)-
perylene. Additional PNA's can be included. The procedure was successfully
demonstrated for gasoline, exhaust tars and crankcase oils.
Summary
A sample to be analyzed is spiked with known Quantities of carbon-14
labeled benzo(a)anthracene (BaA) and benzo(a)pyrene (BaP). Some polar com-
pounds are removed by a caustic treatb and then a PNA hydrocarbon concentrate is
obtained by solvent elution off a column of partially deactivated alumina.
The solvents are cyclohexane, cyclohexane-benzene, benzene, and benzene-methanol.
The fraction containing the PNA's is reduced to a small volume by evaporation
on a steam bath. An aliquot of this sample is injected into a gas chromatograph
and fractions are collected for measurement by UV and, in the case of BaA and
BaP peaks, also for carbon-14 activity. These activities, compared with known
concentrations originally added, give factors by which to relate the concen-
trations of each PNA to its total weight in the sample.
Apparatus & Isotope Dilution
1. The Gas Chromatograph is equipped with a flame ionization detector,
a linear temperature programmer and a flow controller. The injection port con-
sisted of an aluminum block with a stainless steel tube which slides into the
block. A quartz sleeve fits inside of the stainless steel tube. The chromato-
graph is modified by the addition of a by-pass and trap so that 15% of the
column effluent will go to the detector and 85% to the trap. This arrangement is
shown in Figure 3 (p.9). A Perkin-Elmer 900 chromatograph was used in the de-
velopment of this method, but equivalent instruments should perform satisfactorily.
a = At the time of this report, work is in progress to also include fluoranthene,
benzo(b)fluoranthene, benzo(j)fluoranthene, benzo(k)fluoranthene, perylene,
and coronene.
b = The caustic treat is done on tar samples only.
-------
- 35 -
2. Recorder, 0-1 millivolt, 2 min./inch chart speed.
3. Trapping Tubes, 20 or more stainless steel tubes, 18 cm long by
0.32 cm diameter.
4. Column, 300 cm of .22 cm I.D. stainless steel tubing packed with
2% SE-30 (GC Grade) on Chromosorb G (acid washed and DMCS treated), 80/100 mesh.
5. Syringe, 10 microliter.
6. Spectrophotometer, spectral range 225-400 nanometers, 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: absorbance repeatability, +.0.01 at 0.4 absorbance;
absorbance accuracy, + 0.05 at 0.4 absorbance; wavelength repeatability, + 0.2 nm;
wavelength accuracy, +1.0 nm.
7. Spectrophotometric Cells, fused quartz cells having optical path
lengths of 1.000+ 0.005 cm and 5 cm cells. The 5-cm cells are microcells and
contain about 3 ml of solution.
8. Graduate Glass, 5 ml.
9. Vials, one- and three-dram with cap.
10. Steam Bath equipped with nitrogen outlet for purging vials.
11. Medicine Dropper, with tip drawn out.
12. Special Low UV Emission Room Lights, Westinghouse W-40-gold
fluorescent lamps.
13. Chromatographic Column, two feet long 0.5 inch OD and 0.43
inch ID with 300 ml receiving bulb on top and stopcock with Teflon barrel on
the bottom.
14. Radiation Counters used are Intertechnique SL-20 and Packard
Tri-Carb.
15. Counting Vials, 20 ml with cap.
Reagent and Materials
Reagent grade chemicals must be used throughout. Benzo(a)pyrene was
repurified but other polynuclear compounds were used as purchased. Compounds
of comparable purity from other suppliers may be employed.
1. Cyclohexane. As a large amount of cyclohexane is used, it must
be of highest purity. To check purity, evaporate 180 ml down to 5 ml. Run a
UV scan on this residue in a one-cm cell from 280-400 nm. The absorbance should
-------
- 36 -
not exceed 0.01 units. To purify, percolate through activated silica gel,
Grade 12, in a glass column, 90 cm long and 5-8 cm diameter. "Distilled in
Glass Solvents" are generally of suitable purity.
2. Toluene.
3. Acetone.
4. Sodium hydroxide.
5. Hydrochloric Acid.
6. Benzene B&J.
7. Methanol B&J.
8. Benz(a)anthracene C18H12' ^ 228> Eastman 4672.
9. Benzo(a)pyrene C20H12' MW 252, Eastman 4951.
10. Pyrene C^H,., MW 202, Eastman 3627.
* ID 1U
11. Benzo(g, h, i)perylene C22H12' ^ 276' Columbia Organic.
12. Test Blend.
CAUTION
Exercise care when handling these compounds to avoid inhaling
them or getting them on the skin. Wash hands thoroughly after handling.
Weigh exactly 25.0 mgs of each of the four compounds listed as Items 8
through 11. Place in a 25^-ml volumetric flask, add 20 ml of toluene and swirl
until compounds are in solution. Then make up to volume. This will contain
1 microgram of each compound per microliter of solution. Pour into a small,
narrow neck brown bottle and keep in a cool, dark place, preferably a refrigerator,
13. Organic Counting Solution, 8 gm of BBOT in a liter of toluene.
14. C benz (a)anthracene, ^C benzo(a)pyrene, Mallinkrodt Chemical
Works.
-------
- 37 -
Method
Precautionary Note: 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 re-
move all organic matter such as oil, grease, detergent residues, etc.
Examine all glassware, including stoppers and stopcocks, under ultra-
violet light to detect any residual fluorescent contamination. As a pre-
cautionary 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 samples in
handling and to assure absence of any extraneous material arising from
inadequate packaging or storing is essential. Because sone of the poly-
nuclear hydrocarbons sought in this test are very susceptible to photo-
oxidation, the entire procedure is to be carried out under subdued light.
Avoid use of fluorescent lamps. Special yellow lights are available.
Tygon tubing must absolutely be avoided to eliminate di-isooctyl phthalate
(DOP) contamination which interferes in both the GC and UV.
Radioactive Materials must be handled and disposed of by accepted
methods. Glassware should be rinsed with chloroform-acetone solvent and
washed in dichromate-sulfuric acid cleaning solution.
A. Sample Preparation
1- Distillation of Tar Samples
Before commencing the distillation prepare the radioactive
BaA and BaP spikes. Add 50 X of BaA to a 25 ml. volumetric flask
and 50 X of BaP to a second. Make up to volume with cyclohexane and remove
duplicate lOOXaliquots from each for counting. Pour the contents of the
flasks into the still and rinse out well with cyclohexane and acetone.
Conduct the distillation as described elsewhere (1A).
2. Gasoline
The volume of starting sample is varied according to the level of
PNA's. PNA-rich gasolines can be analyzed by using 50 ml and spiking with
50 A each of C labeled BaA and BaP. For gasolines of low PNA content,
1000 ml of sample is spiked with 10 A each of the BaA and BaP internal
standards.
3. Used Crankcase Oil
A weighed amount of sample (-"' 500 mg) is made up to 25 ml of cyclo-
hexane which has been previously spiked with 10X each of carbon-14 labeled
BaA and BaP.
(1A) G. P. Gross, "Gasoline Composition and Vehicle Exhaust Gas Polynuclear
Aromatic Content," U.S. Clearinghouse Federal Science Technology
Information, PB Rep. Issue No. 200266 (1971) 124 pp.
-------
- 38 -
B. Caustic Extraction (Tar Samples)
1. The volume of still bottoms will be about one liter. Take exactly
one-half of this and place in a one-liter separatory funnel.
2. Add 50 ml of 0.5 N aqueous sodium hydroxide solution, shake one
minute, and remove lower aqueous phase.
3. Repeat the extractions with 30 and 20 ml portions of sodium hydroxide.
4. Wash with 50 ml of water, then 50 ml of 1/10 N HC1.
5. Finally, wash with 25 ml of water.
6. Evaporate cyclohexane to about 150 ml and filter, if necessary.
7. Evaporate filtrate to below 50 ml, place in a 50 ml volumetric flask
and make up to volume with cyclohexane.
8. Take 25 ml for placing on the alumina chromatographic column.
C. Column Chromatographic Separation
1. Seventy-five grams of Woeltn Neutral alumina is dried for one hour
at 150°C. The alumina is transferred to a bottle and allowed to reach room
temperature. 1.5 ml of water are added dropwise with shaking. The bottle is
capped and placed on a paint shaker for 15 minutes to obtain a uniform mixture.
2. A glass wool plug is placed in the end of the chromatographic
column and the alumina is poured in along with gentle tapping to pack the alumina.
The column is filled to within one inch of the top and another glass wool plug
is placed on top.
3. Ten ml of cyclohexane are placed on top of the column and allowed
to run into the column under nitrogen pressure of 2 pounds. The sample is then
poured into the 200 ml bulb on top and allowed to run into the column. 90 ml
cyclohexane is added to the bulb.
4. When the cyclohexane has run into the column below the top glass
wool plug, 100 ml of cyclohexane benzene (4:1) are added and run into the column.
The first 100 ml of cyclohexane is collected and set aside. The following solutions
are collected in 3-dram vials and capped. Each should contain 10 ml of solution.
5. After the cyclohexane/benzene (4:1) is below the top of the glass
wool plug, 100 ml of benzene are added to the column. Collection of 10 ml fractions
is continued.
-------
- 37 -
Method
Precautionary Note; 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 re-
move all organic matter such as oil, grease, detergent residues, etc.
Examine all glassware, including stoppers and stopcocks, under ultra-
violet light to detect any residual fluorescent contamination. As a pre-
cautionary measure it is recommended practice to rinse all glassware with
purified iscoctane immediately before use. No grease is to be used on
stopcocks or joints. Great care to avoid contamination of samples in
handling and to assure absence of any extraneous material arising from
inadequate packaging or storing is essential. Because sons of the poly-
nuclear hydrocarbons sought in this test are very susceptible to photo-
oxidation, the entire procedure is to be carried out under subdued light.
Avoid use of fluorescent lamps, bpecial yellow lights are available.
Tygon tubing must absolutely be avoided to eliminate di-isooctyl phthalate
(DOP) contamination which interferes in both the GC and UV.
Radioactive Materials must be handled and disposed of by accepted
methods. Glassware should be rinsed with chloroform-acetone solvent and
washed in dichromate-sulfuric acid cleaning solution.
A. Sample Preparation
1- Distillation of Tar Samples
Before commencing the distillation prepare the radioactive
BaA and BaP spikes. Add 50 X of BaA to a 25 ml. volumetric flask
and 50 X of BaP to a second. Make up to volume with cyclohexane and remove
duplicate 100 Xaliquots from each for counting. Pour the contents of the
flasks into the still and rinse out well with cyclohexane and acetone.
Conduct the distillation as described elsewhere (IA).
2. Gasoline
The volume of starting sample is varied according to the level of
PNA's. PNA-rich gasolines can be analyzed by using 50 ml and spiking with
50 ?>. each of ^C labeled BaA and BaP. For gasolines of low PNA content,
1000 ml of sample is spiked with 10 r<- each of the BaA and BaP internal
standards.
3. Used Crankcase Oil
A weighed amount of sample (~* 500 mg) is made up to 25 ml of cyclo-
hexane which has been previously spiked with 10X each of carbon-14 labeled
BaA and BaP.
(1A) G. P. Gross, "Gasoline Composition and Vehicle Exhaust Gas Polynuclear
Aromatic Content," U.S. Clearinghouse Federal Science Technology
Information, PB Rep. Issue No. 200266 (1971) 124 pp.
-------
- 38 -
B. Caustic Extraction (Tar Samples)
1. The volume of still bottoms will be about one liter. Take exactly
one-half of this and place in a one-liter separatory funnel.
2. Add 50 ml of 0.5 N aqueous sodium hydroxide solution, shake one
minute, and remove lower aqueous phase.
3. Repeat the extractions with 30 and 20 ml portions of sodium hydroxide.
4. Wash with 50 ml of water, then 50 ml of 1/10 N HC1.
5. Finally, wash with 25 ml of water.
6. Evaporate cyclohexane to about 150 ml and filter, if necessary.
7. Evaporate filtrate to below 50 ml, place in a 50 ml volumetric flask
and make up to volume with cyclohexane.
8. Take 25 ml for placing on the alumina chromatographic column.
C. Column Chromatographic Separation
1. Seventy-five grams of Woelm Neutral alumina is dried for one hour
at 150°C. The alumina is transferred to a bottle and allowed to reach room
temperature. 1.5 ml of water are added dropwise with shaking. The bottle is
capped and placed on a paint shaker for 15 minutes to obtain a uniform mixture.
2. A glass wool plug is placed in the end of the chromatographic
column and the alumina is poured in along with gentle tapping to pack the alumina.
The column is filled to within one inch of the top and another glass wool plug
is placed on top.
3. Ten ml of cyclohexane are placed on top of the column and allowed
to run into the column under nitrogen pressure of 2 pounds. The sample is then
poured into the 200 ml bulb on top and allowed to run into the column. 90 ml
cyclohexane is added to the bulb.
4. When the cyclohexane has run into the column below the top glass
wool plug, 100 ml of cyclohexane benzene (4:1) are added and run into the column.
The first 100 ml of cyclohexane is collected and set aside. The following solutions
are collected in 3-dram vials and capped. Each should contain 10 ml of solution.
5. After the cyclohexane/benzene (4:1) is below the top of the glass
wool plug, 100 ml of benzene are added to the column. Collection of 10 ml fractions
is continued.
-------
- 39 -
6. After all the benzene has passed the top glass wool plug, 100 ml
of benzene/methanol (1:1) is added to the column. The benzene/methanol front
can usually be followed by the movement of an orange colored band. The front
can also be detected by a warm front moving down the column which is apparent
to the touch or it can be followed by a UV lamp. When the front reaches the
end of the column, a two-phase system becomes visible in the collecting vial.
At this point, the remaining solution is collected in one large bottle and
allowed to run off the column (cut 3). The column should not be stopped or
allowed to run dry between solvent additions.
7. The solutions in the individual three-dram vials are scanned in
order of elution on a UV spectrophotometer. The peak at 340 nm is used as a
guide in determining the start of elution of tetracyclic PNA compounds. The
solution whose spectrum shows a 340 peak is the start and all remaining solutions
are combined as the PNA fraction (cut #2).
D. Preparation of Concentrated PNA Solution
1. Transfer the contents of the first 4 vials of cut #2 to a
150 ml beaker and rinse each vial twice with 1 to 2 ml of cyclohexane and
combine with the contents of the beaker.
2. Place the 150 ml beaker on the steam bath under a small jet
of nitrogen.
3. As evaporation progresses, add the contents of the remaining
vials with similar rinsing to the beaker. Do not evaporate to less than
two mis and in no case let the contents go to dryness.
4. Transfer the concentrated solution to a one-dram vial.
Wash beaker with cyclohexane and add to vial.
5. Place the vial in a 30 ml beaker for support. The beaker is
placed on a steam bath under a gentle jet of nitrogen.
6. Rinse the 150 ml beaker with 3 or 4 small portions of cyclo-
hexane ( 1 ml) and combine with the contents of the one-dram vial as
evaporation proceeds.
7. If, as is preferable, it is unnecessary to obtain the weight
of the PNA residue, evaporate the solution down to about 50 jil (0.05 ml).
Add about 0.5 ml of acetone and again evaporate to 50 jul.
8. If the weight of the residue must be obtained, exercise
great care as the solution approaches dryness. Keep the vial under constant
observation and the instant all the solvent has been removed, cool and weigh
the vial.
9. Return the vial to the beaker under nitrogen for an additional
minute, cool and reweigh. Repeat this procedure until a constant weight is
reached. For the GC analysis add 10 to 20 microliters of acetone to lower
the viscosity of the sample. Cap and save.
-------
- 40 -
E. Gas Chromatographic Analysis
Parameters
The following parameters are employed in developing the gas
chromatographic separation with the Perkin-Elmer 900. Other instruments
might use different conditions.
Carrier Gas - Helium
Flow Rate - 30 ml./min.
Hydrogen and Air - at manufacturers
recommendation, or optimum rates.
Injection Port - 300°C.
Detector - 340°C.
Program - 175°C to 300°C at 4° per
minute. The temperature is held at
300° until all peaks are eluted, but
in any case, for 20 minutes.
Column Conditioning
1. The Supelco Co. states that only minimum conditioning is
needed on the column specified in (4) of apparatus. Connect the column
to the inlet of the GC but not with the detector.
2. Pass helium through at 30 ml. per minute program at one
degree per minute from 150°C to 275°C. Keep at 275 for 30 minutes. Cool
and connect to the detector.
3. If SE 30 as supplied by the manufacturer is used in place of GC
grade, connect as in (1). Purge with helium for 10 minutes and then put a cap
on the exit end, keeping a helium pressure on the column.
4. Raise the oven to 350"C and bake for 3 hours.
5. Cool to room temperature, remove cap, pass helium through
at 30 ml. per minute and continue conditioning column as shown below
without attaching to the detector.
Temp., 'C. 150 200 250 275
Time, Min. 30 30 30 60
6. Cool to room temperature and attach to detector. The column
is now ready for use.
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- 41 -
Performance Test
This test is used to measure retention times and demonstrate
suitable recovery of individual PNA's. Recoveries of > 837. are required
except for benzo(g,h,i)perylene. For this compound, a recovery of > 757.
is satisfactory. With the column conditioned and the parameters seT as
described in 1, prepare to inject a sample.
7. Flush the 10 pi syringe twice with the test blend.
8. The third time fill the syringe and hold vertically point
up. Stick the tip in a rubber septum and depress the plunger to compress
air bubbles which rise to the top.
9- Now advance the plunger to the desired amount, for instance
2 fill the rubber is removed, and the plunger retracted until the air-liquid
meniscus enters the glass bore.
10. Note the volume from meniscus to plunger.
11. Insert the syringe up to the hilt in the GC injection port,
depress the plunger. Start the recorder.
12. Remove the syringe from the port and retract the plunger.
13. Note the volume remaining (generally about 0.2 jil) and sub-
tract from the previously noted volume to determine the net volume
injected.
14. Have stainless steel trapping tubes and one-drara vials
available. The vials should be supported in small holes drilled in a
board. A plastic cup filled with ice surrounds the stainless steel insert.
15. As a peak approaches, insert a tube. Remove as the trace
returns to the baseline. Mark the peak No. 1 and place the tube in a one-
dram vial labeled 1.
16. Continue taking cuts, putting each tube in a separate vial
labeled 2, 3, etc.
17. Typical retention times obtained in setting up this method
are shown in Table IA.
-------
- 42 -
Table IA
OBSERVED RETENTION TIMES 3
Compound Retention Time, Min.
Pyrene 13.6
Triphenylene 19.4
Benz(a)anthracene 19.4
Chrysene 19.6
Methylbenz(a)anthracenes 21.4
Dimethyl/ethylbenz(a)anthracenes 23.3
Benzo(e)pyrene 25.7
Benzo(a)pyrene 25.8
Methylbenzo(e)pyrenes° ) 27.5, 28.0, ^9,U
Methylbenzo(a)pyrenes )
Benzo(g,h,i)perylene 31.4
a = Conditions given on page 40.
b = Measured in three different peaks.
18. When the run is completed take the first tube and place in
a 5-ml graduace. With an elongated medicine dropper add enough cyclohexane
through the top of the tube to fill the spectrophotometric cell to be
used. In developing this method, 3.8 ml was a convenient volume to em-
ploy, but all cuts must be made up to the same volume.
19- Pour the solution into the original vial, cap and protect
from light. Rinse out the graduate with cyclohexane and use for the next
tube. Save the fractions for UV, and calculation of recoveries.
20- When the run is completed, measure and record the retention
time in minutes from injection point for each compound including the in-
ternal standard.
Analysis of Samples
21. From the vial containing the PNA concentrate, take 5-10 pi of
sample and inject into the GC unit. Trap the peaks at the retention times
obtained on the test blend and also trap any other peak that might be of interest.
Trapped fractions must be made up to the same volume employed for the test blend.
a - At the time of this report, some preliminary data indicated that benzo(g,h,i)
perylene might not be completely dissolved by cyclohexane, resulting in low
recovery. A preferred step would be to use acetone. In this case, the UV
measurement might be done with the cyclohexane/acetone mixed solvent or it
might be necessary to replace the acetone with cyclohexane by careful
evaporation.
-------
- 43 -
F. Ultraviolet Spectrophotometrlc Analysis and^Scintillation Counting
1. Prepare solutions of known concentrations of each PNA in
cyclohexane, e.g., 2.4 yug/ml.
2. With cyclohexane in the reference cell, obtain the spectrum
of each solution from 400 to 225 nm.
3. From the spectra obtain the absorbances of the various compounds
at the wavelength shown in Table IV (p. 12). Calculate absorptivity from
the known concentrations. Typical calibration data are also shown in the
table.
4. Rinse the cell thoroughly with cyclohexane between spectra
and remove the last traces of solvent by vacuum or a gentle jet of air.
5. Obtain the UV spectrum of each selected fraction as directed
in step E-21. Measure the absorbance at the specified wavelength and
reference point as determined by the base line having anchor points as
listed in Table IV.
6. One ml of each fraction containing BaA and BaP is placed in
a counting vial, along with 9 ml of toluene and 10 ml of counting solution.
Counts per minute are measured using a scintillation counter.
-------
- 44 -
G. Calculation to a Sample Basis
1, To calculate the micrograms of the individual PNA's in the sample,
the aliquot factor (activity ratio) must be determined based on radioactivity
per ml of the cyclohexane solutions of GC fractions containing ^C BaA and BaP,
respectively.
Equation 1. M =
M = activity ratio
Q = activity in DPM added to sample
K = average CPM in a measured volume (ml) of the solution
containing the GC fraction
L = background in CPM
N = counting efficiency CPM per DPM
CPM is counts per minute
DPM is disintegrations per minute.
The concentration of each component is determined from the UV absorbance.
A
Equation 2. D =
BC
D = concentration of each PNA (pg/ml)
A = absorbance of each PNA
B = absorptivity of each PNA (ml/^ig cm)
C = cell length (cm)
The weight of added radio tracer is determined.
Equation 3. X = -§-
D
X = weight of radio tracer added, e.g., 1 jig of C BaP.
S = specific activity (DPM/pg)
Q = activity in DPM added to sample.
Calculate weight of PNA in the sample.
Equation 4. T = MVD - X
T = weight of PNA in the sample.
V = volume in ml of the solution containing the GC
fraction taken for counting.
The subtraction of X (Equation 4) is dnne only for BaA and BaP. The weight of benzo
(g,h,i) perylene is corrected upward by 1.10 (p. 8).
Where more than one internal standard is used, all components eluting
in the same peak with a standard are calculated based on that standard's activity
ratio. All other components are calculated using the smallest activity ratio
since this represents the maximum recovery of standard.
-------
- 45 -
APPENDIX B
Measurement of Total Polynuclear Aromatic
Hydrocarbons by Low Voltage Mass Spectrometry
Introduction
This method was developed to measure total polynuclear aromatic
hydrocarbons in gasoline, crankcase oil, and tar from auto exhaust.
The method covers eleven groups of polynuclear aroma tics (PNA)
ranging from 3-ring to 7-rings and including alkyl substituted compounds.
Compound classes include: phenanthrenes; fluorenes and dihydrophenanthrenes;
pyrenes and fluoranthenes; cyclopentaphenanthrenes; benzo(g,h,i)fluoranthenes
and cholanthrenes; benzanthracenes, chrysenes and triphenylenes; benzopyrenes
and benzofluoranthenes; benzoperylenes and anthanthrenes; dibenzanthracenes;
coronenes; and dibenzopyrenes. This procedure was demonstrated for auto
exhaust tar. It should be applicable also to gasoline and crankcase oil.
Summary
A sample to be analyzed is spiked with a known quantity (40
micrograms) of triphenylbenzene. PNA hydrocarbons are removed from the
total sample by separation on a column of partially deactivated alumina.
The solvents used are cyclohexane, cyclohexane/benzene, benzene and benzene/
methanol. The fraction containing the PNA's is reduced to about 30 pi by
evaporation on a steam bath. An aliquot of this sample is injected into the
mass spectrometer for a low voltage measurement of its spectrum. Quantitative
measurement of PNA groups are calculated by applying sensitivity calibrations
to monoisotopic peaks. Sample quantity is based on the internal standard,
triphenylbenzene.
Apparatus
1. The mass spectrometer is equipped with an all glass sample
inlet system that is heated to 316°C. Samples are introduced through a glass
frit sealed with gallium. Resolution of one part in six hundred is maintained
over a mass range of 12-600. A Consolidated Electrodynamics Corp. Model 21-102
was used but any comparable instrument is suitable.
2. An Infotronics Digital Readout System, Model CRS-160 is used
for printout of the spectrum. Peaks can also be read manually or acquired
by on line computer.
3. Micropipette, 3 /ul.
-------
- 46 -
Materials
1. 1,3,5-Triphenylbenzene (recrystallized) Pract. MW 306.41
Aldrich Chemical T8200-7.
2. Selected PNA hydrocarbons.
Method
1. Settings £or the Mass Spectrometer
With the mass spectrometer in a high voltage (70 volts) mode of
operation, adjust the ion source temperature so as to give a 127/226 peak
ratio of 0.8 for n-hexadecane. At high voltage, the metastable suppressor
dial reads 89, its maximum setting. The first step in changing to low voltage
is to set this dial at 40, thereby lowering the ionizing voltage to approximately
15 volts. The inner repeller dial is set at its maximum position, which is 97,
and the outer repeller turned to 60. The low voltage switch is moved from the
"N1 to the 'L' position, and the metastable suppressor dial is adjusted to
give a meter value of 11 for the ionization voltage. The ionizing current
is maintained at 20 microamperes, with no change in the inner and outer focus
slits.
2. Sample Introduction
A 3 ^ul volume is charged to the sample inlet with the sintered
disk at approximately 40°C. The disk temperature is then raised to 316°C
(600°F).
3. Scan of Sample
The sample is scanned from m/e 150 to m/e 400.
4. Calculation
Peaks are read from a chart or selected from a digitizer/computer
printout. All peaks in the m/e range, 178-330, are recorded. Steps to be
followed from this point include:
a. Calculate monoisotopic peaks for all even m/e numbers.
b. Calculate the triphenylbenzene peak at m/e 306 by correcting
for sample background as illustrated:
m/e Peak Height
292 117.0
306 266.1
320 70.0
Triphenylbenzene Peak (m/e 306) = 266.1 - ( 117'° * 7°'° )= 172.6
-------
- 47 -
c. Divide monoisotopic peaks by appropriate sensitivity
from Table IB to obtain micrograms of each mol wt species as contained
in the mass spectrometric sample.
d. Based on the internal standard, triphenylbenzene, convert
all microgram quantities to a total sample basis.
-------
- 48 -
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APPENDIX C
Method for Measurement of Phenols in
Aqueous Condensate of Automobile Exhaust
Introduction
This method was devised specifically for the determination of
phenols in the aqueous condensate obtained from automobile exhaust. In-
dividual phenols are measured, including phenol, cresols, xylenols, ethyl
phenols, and a few others. The method is applicable to water samples or
extracts from other sources.
Summary
A known amount of an internal standard, o-chlorophenol, is added to
the sample. Neutral compounds are removed by extraction of the alkaline
sample with cyclohexane. Tne extract is acidified, saturated with sodium
sulfate, and the phenols are extracted by five successive portions of ethyl
ether. Organic acids are then separated by a sodium bicarbonate extraction.
The ether is distilled in a special concentrator leaving a small residue
which is injected into a capillary column gas chromatograph. The recorded
peak areas are identified by the retention time, measured and multiplied
by the appropriate factors which relate the area to weight.
Apparatus
1. The gas chromatograph is equipped with a hydrogen flame
ionization detector, a sample splitter and an oven capable of isothermal
temperature control to at least 120 + 0.2°C. A Perkin-Elmer 900 chromatograph
was used in the development of this method, but equivalent instruments should
perform satisfactorily.
2. Recorder, 0-1 millivolt, 1 second full scale deflection, 2 inches/
minute chart speed.
3. Column, 150' by 0.020" I.D. stainless steel capillary tubing
coated with a mixture of didecyl phthalate and 10 percent phosphoric acid.
4. Syringe, 10 ill.
5. Vials, 1 dram.
6. Concentrator, Cat.# K-283500, Kontes Glass Co., Vineland, New
Jersey, or equivalent apparatus for evaporation.
Reagents
1. Ethyl Ether, anhydrous.
2. Sulfuric Acid, pour 100 ml of water in a beaker and cool the
beaker. Slowly and carefully add 100 ml of concentrated sulfuric acid, H2S04-
-------
- 50 -
3. Sodium Sulfate, anhydrous.
4. Sodium Hydroxide, 50% solution.
5. Sodium Hydroxide, 0.5N.
6. Sodium Bicarbonate, saturated solution.
7. 0-Chlorophenol Solution A. Weigh approximately 400 trigs to
the nearest 0.1 mg. Dissolve in methanol and make up to 100 ml in a
volumetric flask.
8. 0-Chlorophenol Internal Standard. Pipette 10.0 ml of
Solution A into a 100-ml volumetric flask and dilute to volume with water.
9. Standard Phenol Solution. To prepare a standard for checking
retention times, resolution and factors for quantitative analysis, weigh
out from 100 to 300 milligrams of each compound and make up to volume in a
10-tnl volumetric flask. Benzene, chlorophenol, and phenol should be
included in the standard, along with some phenols which will cover the b.p.
range of interest. Table XVII contains data on a number of phenols. It should
not be necessary to calibrate with each phenol. Data in Table XVII, for
example, should be applicable as shown. Retention times, on the other hand,
can be established by cross comparison and interpolation.
Calibration of the Gas Chromatograph
The following operating conditions were employed with the Perkin-
Elmer 900. Other instruments might use different parameters.
Sample Splitter - No. 3 (400-1)
Carrier Gas - 4 ml/tnin
Injection Temp. - 250°C
Detector - 250°C
Column - 125°C
1. Set the instrument controls as described above.
2. Inject 2 /il of toluene and allow helium to flow for 30 minutes
to see if column is clean and base line straight.
3. Inject about 0.5 fil of the synthetic phenol standard (Reagent
9). Determine the retention time and relative sensitivity of each compound.
By comparing retention times with those shown in Table XVII and by interpolation,
the retention time of each compound can be determined. Relative sensitivities
of Table XVII can be used directly although this should be confirmed by satis-
factory agreement between the on-hand measurement and each corresponding
value in Table XVII. Retention times decrease as the column is used so the
standard should be checked frequently.
Method
A. Extraction of Phenol and Separation
of Neutral and Acidic Components
1. Pipette 25.0 ml of the aqueous sample into a 60-ml separatory
funnel and then add by pipette 2.0 ml of the internal standard.
-------
- 51 -
2. Neutralize with 507= sodium hydroxide and add 1 ml in excess.
3. Add 15 ml of ethyl ether (ether) and shake for one minute.
Draw off the aqueous phase into a second separatory funnel.
4. Add 10 ml of ether to the aqueous phase and shake for one
minute. Draw off the aqueous phase into a 50-ml beaker and save for
Step 6.
5. Combine this ether with the first ether extract and add 5 ml
of 0.5N sodium hydroxide solution. Shake for one minute, draw off the
aqueous phase into the 50-ml beaker and discard the ether.
6. Acidify the contents of the 50 ml beaker with (1:1) sulfuric
acid. Add 5 g of anhydrous sodium sulfate to the beaker, stir until the
crystals are dissolved, and pour into a 60-ml separatory funnel.
7. Add 25 ml ethyl ether to the beaker, then transfer combined
ether and water phase to a separatory funnel.
8. Shake gently and vent. Repeat the gentle shaking and venting
several times and then shake vigorously for 2 minutes. Drain the aqueous
phase into a second 60-ml separatory funnel, and pour the ether phase into
a 125-ml separatory funnel.
9. With aqueous phase, repeat steps 3 and 4 four times using
20, 15, 15, 10 ml of ether, combining all of the ether washings in the 125-ml
separatory funnel.
10. Add five ml of saturated sodium bicarbonate solution to the
combined ether extracts and shake for one minute.
11. Draw off the bicarbonate solution into a 60-ml separatory
funnel, extract the ether with another 5 ml of bicarbonate and add the
bicarbonate solution to the first.
12. Extract the bicarbonate solutions successively with two 5-ml
portions of ether, and add the ether to the main ether fraction.
13. Add 4 g of anhydrous sodium sulfate to the combined ether
phase and shake for two minutes to remove dissolved water.
B. Evaporation of Ethe_r
14. Assemble the condenser and receiver of the evaporator, and
decant the ether into the special still pot. Connect and distill off the
ether at a gentle but steady rate.
15. When about 1 ml remains in the pot, and the distilling rate
has slowed down considerably, remove the flask from the heat and cool
immediately with a damp cloth.
-------
-52-
16. With an elongated medicine dropper, transfer the ether from
the still to a one-dram vial. Rinse the still with 10 drops of ether and
add the rinsings to the vial. Repeat with 10 drops of methanol.
17. Place the vial on a steam bath or other controlled heat
source and carefully evaporate off the ether. A gentle stream of nitrogen
may be applied to the vial, but keep the vial warm so that moisture from
the air does not condense on the sides. Remove from the heat when about
0.2 ml remains.
18. Cap the vial and place in a cool location until the GC
analysis can be performed.
C. Gas Chromatographic Analysis
19. Set the instrument controls as described under Calibration.
20. Inject 2 yul of the residue and run at 125°C for at least
70 minutes.
21. Measure the retention times and the area of each peak by
a suitable method.
Calculations
1. Determine the phenols present by comparing the retention
times of the sample with those of the standard or from the table.
2. Calculate the amount of each phenol present by the following
equation:
, C F BC 0.45 0.45 BC
A = BxiX D =^X~D- = ^5
where: A = weight of respective component,
B = weight of internal standard,
C = measured area of respective component,
D = relative sensitivity of component,
E = measured area of internal standard,
F = relative sensitivity for Int. Std. (0-chlorophenol = 0.45).
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