a 1 1 1 b 3 Mn,b racterization of Air Particulate

Material for Polycyclic Aromatic

Compounds

U.S. DEPARTMENT OF COMMERCE

National Bureau of Standards

Center for Analytical Chemistry

Organic Analytical Research Division

Washington, DC 20234

June 1 982

Final Report

Issued December 1982

“QC100

. U56

02-2595

1982

Prepared for: Environmental Monitoring Division

i ronmental Monitoring Systems Laboratory

, Environmental Protection Agency

3arch Triangle Park, NC 2771

1

NBSIR 82-2595

CHARACTERIZATION OF AIR PARTICULATE

MATERIAL FOR POLYCYCLIC AROMATIC

COMPOUNDS

WATior OF S'

roL. BUrO-Atf, :k;daiuJ6 UBRAB*

JAN IB 1983

S. A. Wise, C. F. Allen, S. N. Chesler, H. S. Hertz, L. R. Hilpert,

W. E. May, R. E. Rebbert, and C. R. Vogt

U.S. DEPARTMENT OF COMMERCE

National Bureau of Standards

Center for Analytical Chemistry

Organic Analytical Research Division

Washington, DC 20234

June 1 982

Final Report

Issued December 1982

Prepared for: Environmental Monitoring Division

Environmental Monitoring Systems Laboratory

U.S. Environmental Protection Agency

Research Triangle Park, NC 27711

U.S. DEPARTMENT OF COMMERCE, Malcolm Baldrige, Secretary

NATIONAL BUREAU OF STANDARDS. Ernest Ambler. Director
'
ABSTRACT

In studies to evaluate the potential health and ecological effects of

atmospheric emissions, bioassays have been employed in conjunction with

chemical characterization to correlate mutagenic and/or carcinogenic

activity with chemical composition. The complexity of an air particulate

extract necessitates the prefractionation of the mixture into suitable

subfractions or chemical classes prior to chemical characterization and/or

biological testing. The goal of this project was to evaluate such a fractionation scheme for air particulate material with respect to chemical

characterization of the various fractions with particular emphasis on the

identification of polycyclic aromatic hydrocarbons (PAH) . In this study we

have used three chromatographic approaches to separate, identify, and

quantitate the complex mixture of PAH extracted from SRM 1649 (Urban Dust/

Organics): (1) capillary gas chromatography (GC) , (2) liquid chromatography

(LC) with selective fluorescence detection, and (3) multidimensional

chromatographic techniques. The analytical methods used for the certification

of several PAH in SRM 1649 are described in this report. In addition, over

80 PAH, including many alkyl-substituted PAH, are tentatively identified

using GC, LC, and gas chromatography-mass spectrometry (GC-MS) data.

Preliminary results of biological testing on selected fractions isolated

from the urban dust sample are reported.

Disclaimer

Identification of any commercial product does not imply

or endorsement by the National Bureau of Standards, nor does

the material or equipment identified is necessarily the best

the purpose.

recommendation

it imply that

available for

iii
CONTENTS

Page

Abstract iii

Disclaimer iii

Figures v Tables vii

1. Introduction 1

Air Particulate Fractionation Schemes 2 Characterization of Polycyclic Aromatic Hydrocarbons

(PAH) from Air Particulate Matter 3

2. Experimental 4

Sample Preparation and Analyses for Certification

of SRM 1649 4

Sample Preparation for Characterization by

Multidimensionsl Chromatography 5 Gas Chromatography /Mass Spectrometry (GC/MS) Analyses. . 5

3. Results and Discussion 7 Quantitation of Major PAH by GC and LC 7 Multidimensional Chromatography for Characterization

of PAH Mixtures 14

Fractionation of Washington Air Particulate Material

for Chemical Analysis and Biological Testing 25

Conclusions 27

References 29

IV
FIGURES

Number Page

1 General scheme for extraction and fractionation of air

particulate material for chemical and biological

characterization 31

2 Gas chromatographic separation of PAH fraction isolated from

Washington urban dust (SRM 1649) 32

3 Reversed-phase liquid chromatograms of total PAH fraction

from Washington urban dust (SRM 1649) 33

4 Reversed-phase LC analysis of PAH from Washington urban dust

(SRM 1649) 34

5 Normal-phase LC separation of PAH isolated from Washington

urban dust for reversed-phase LC analysis 35

6 Reversed-phase LC separations of total PAH fraction and

fractions 2 and 3 obtained from normal-phase fractionation

of PAH from Washington urban dust 36

7 Reversed-phase LC separation of fractions 4-6 obtained from

normal-phase LC fractionation of PAH from Washington

urban dust 37

8 Normal-phase LC separation of PAH isolated from Washington

urban dust for GC-MS analysis 38

9 Gas chromatographic analysis of fraction 1 from Figure 8 39

10 Gas chromatographic analysis of fraction 2 from Figure 8 40

11 Gas chromatographic analysis of fraction 2b from Figure 8 . . . . 41

12 Gas chromatographic analysis of fraction 3 from Figure 8 42

13 Gas chromatographic analysis of fraction 4 from Figure 8 43

14 Gas chromatographic analysis of fraction 5 from Figure 8 44

15 Gas chromatographic analysis of fraction 6 from Figure 8 45

v
Number Page

16 Gas chromatographic analysis of fraction 7 from Figure 8 46

17 Gas chromatographic analysis of fraction 8 from Figure 8 47

18 Extraction and fractionation scheme used by Battelle Columbus

Laboratories (BCL) for the Washington urban dust sample .... 48

19 Normal-phase LC separation of the hexane/benzene (aromatic)

fraction isolated from Washington urban dust using BCL

procedure 49

V20

Gas chromatographic analysis of the basic fraction from

Washington urban dust 50

vi
TABLES

Number Page

1 Fluorescence Spectral Characteristics of PAH 8

2 Fluorescence Conditions for LC Determination of PAH in Air

Particulate Samples 10

3 Fluorescence Conditions for the LC Determination of Selected

PAH in Washington Particulate Matter 11

4 Summary of Results by the Various Analytical Methods for the

Washington Urban Dust (SRM 1649) 12

4A Certified Values for Selected Polycyclic Aromatic Hydrocarbons

in SRM 1649 13

5 Determination of Selected PAH in St. Louis Urban Air

Particulate Matter (SRM 1648) (yg/g) 14

6 Liquid Chromatographic Retention of Polycyclic Aromatic

Hydrocarbons 15

7 Liquid Chromatographic Retention of Polycyclic Aromatic Sulfur

Heterocycles 18

8 Polycyclic Aromatic Hydrocarbons Identified in Washington

Urban Particulate Matter 20

9 Results of Biological Testing on Fractions from Washington

Air Particulate Material 26

10

Aza-Arenes Identified in Basic Fraction of Washington Urban

Dust 28

vii
.
SECTION 1

INTRODUCTION

In studies to evaluate the potential health and ecological effects of

atmospheric emissions (e.g., air particulate matter), bioassays such as the

Ames test have been employed in conjunction with chemical characterization to

correlate mutagenic and/or carcinogenic activity with chemical composition.

The complexity of an air particulate extract necessitates prefractionation of

the mixture into suitable subfractions or chemical classes prior to chemical

characterization and/or biological testing. Classically, air particulate

extracts have been divided into acidic, basic, and neutral fractions; the

neutral fraction is then generally further divided into non-polar neutral,

aromatic, and polar neutral subgroups. The goal of this project was to

evaluate such a fractionation scheme for air particulate material with respect

to the chemical characterization of the various fractions with particular

emphasis on the determination of polycyclic aromatic hydrocarbons.

Polycyclic aromatic compounds (PAC) are widespread environmental pollutants produced by incomplete combustion and pyrolysis of fossil fuels and

other organic materials. A recent book by Lee et_ al_. (1) and a review by

Bartle et_ al. (2) provide excellent discussions concerning the determination

of these compounds. During the past thirty years, many studies have been

undertaken to characterize the polycyclic aromatic hydrocarbon (PAH) content

of airborne particulate matter (2, and references 36-68 therein). Previous

studies have focused on the PAH constituents mainly owing to the known

carcinogenic activity of many of these compounds. Other subgroups of PAC

reported in air particulate samples include polycyclic aromatic sulfur

heterocycles (PASH) , polycyclic aromatic nitrogen heterocycles (PANH) also

known as aza-arenes, nitropolycyclic aromatic hydrocarbons (NPAH) , polycyclic

aromatic oxygen heterocycles (PAOH) , and polycyclic aromatic quinones (PAQ)

.

[These acronyms are assigned according to the format defined by Bartle e_t al . (2)]. In this work we have focused on methods for the determination of PAH,

PASH, and PANH in the air particulate material.

Compound structure, position of substitution, and presence and position

of a heteroatom in the ring have all been found to affect the carcinogenic

and/or mutagenic properties of these aromatic compounds (1), and therefore,

the exact structural elucidation of individual components of a mixture is

necessary to determine which compounds are responsible for the carcinogenic

hazard of the sample. PAC mixtures from air particulate samples are extremely complex because of the presence of alkyl-substituted PAC as well as the numerous isomeric parent compounds. As a result, the determination of

PAC components in such mixtures requires the use of high efficiency

1
chromatographic separation techniques and selective detection systems for

accurate identification and quantitation.

Urban air particulate material collected in Washington, D.C. (see Experimental section) was used for the evaluation of the various fractionation

schemes. Using analytical methods developed at NBS for the determination of

PAH, this urban particulate sample has been certified for the concentrations

of selected PAH and has been issued recently as Standard Reference Material

(SRM) 1649 "Urban Dust/Organics". The analytical methods used for certification of this SRM are described in this report.

AIR PARTICULATE FRACTIONATION SCHEMES

A number of organic solvents including acetone, benzene, toluene,

methylene chloride, methanol, and cyclohexane have been recommended for the

extraction of air particulate samples (3-7). Acetone, benzene, and cyclohexane have all been reported to be nearly 100% efficient in the extraction

of benzo [a.] pyrene (3). Cyclohexane has often been employed for extraction of

air particulate matter for the determination of PAH. However, more polar

solvents such as benzene and methanol are required to remove the more polar

organic constituents. Recently, Grimmer eL al. (7) compared the extraction

efficiency of several solvents for removing PAH from air particulate filters

and found toluene to be the most effective. Dong et^ a^L. (6) used benzene:

methanol (4:1) to extract air filters in the determination of aza-arenes.

Since aza-arenes are frequently weak bases, salt formation with inorganic and

organic acids is a possibility which necessitates the use of the more polar

solvent (6)

The extraction of air particulate matter with an organic solvent results

in a complex mixture of organic and inorganic constituents. This mixture must

be fractionated into subgroups prior to chemical characterization. A number

of fractionation schemes have been used for air particulate extracts,

primarily for the isolation of PAH components (1-2, 8-10). These procedures

involved chromatographic isolation on silica and/or lipophilic gels and

liquid-liquid partition with nitromethane

A general fractionation scheme for the characterization of the organic

constituents extracted from air particulate material is illustrated in

Figure 1. In general, most fractionation schemes designed for characterization of all groups of organic compounds involve partition with dilute alkali

and acid to separate, respectively, acidic (e.g., phenols) and basic components

(aza-arenes), from the neutral components as shown in Figure 1. The neutral

fraction is further separated into nonpolar neutrals (aliphatics) , aromatic

(PAH and PASH) , and polar neutral fractions by silica column chromatography or liquid-liquid partition.

2
CHARACTERIZATION OF POLYCYCLIC AROMATIC HYDROCARBONS (PAH) FROM AIR

PARTICULATE MATTER

In recent years a number of papers have reported the determination of

PAH in air particulate samples (1,2). The majority of these papers have

reported only qualitative information on the PAH content of particulate

samples; however, several studies illustrate the use of gas chromatography

(GC) (7,11-13) and liquid chromatography (LC) (13-15) for the quantitation of

selected PAH extracted from air particulate matter.

Solvent extraction of air particulate matter provides a complex mixture

of organic constituents from which the PAH must be isolated prior to identification and quantitation. The PAH mixtures isolated from air particulate

extracts are extremely complex because of the presence of numerous alkylated

PAH as well as the numerous isomeric parent PAH. The complexity of these PAH

mixtures necessitates the use of high resolution gas and/or liquid chromatographic techniques with selective detection [e.g., fluorescence or mass

spectrometry (MS)] to achieve separation, identification, and quantitation of

individual components. Recent reviews on the use of capillary gas chromatography (16) and high performance liquid chromatography (17) for the

determination of PAH have appeared. We have used three chromatographic

approaches to separate, identify, and quantitate the complex PAH mixture

isolated from the Washington dust sample: [1] high resolution capillary GC

with flame ionization detection (FID); [2] LC with selective fluorescence

detection; [3] multidimensional chromatographic techniques (i.e., normalphase LC to isolate specific PAH fractions followed by GC , GC-MS , and/or

reversed-phase LC)

.

The first and second approaches were used for quantitation of selected major PAH components in the extract after appropriate

sample cleanup. These first two approaches were used for the certification

of Standard Reference Material (SRM) 1649 "Urban Dust/Organics" . This

material is issued in 10-g quantities with certified and information values

for the concentrations of selected PAH. SRM 1649 is a companion material to

SRM 1648 "Urban Particulate Matter" which is available in 2-g quantities and

certified for inorganic constituents. The third approach was used primarily

for a detailed characterization of both the major and minor PAH components.

The third approach requires isolation and analysis of various fractions and

thus is more time-consuming than the first two approaches.

3
SECTION 2

EXPERIMENTAL

SAMPLE PREPARATION AND ANALYSES FOR CERTIFICATION OF SRM 1649

The air particulate sample was collected in the Washington, D.C. area

using baghouses specially designed for this purpose. This material was collected over a period in excess of 12 months and, therefore, represents a time-integrated sample. While this sample is not intended to be representative of air particulate samples from the area in which it was collected, it

should typify the analytical problems associated with the chemical analysis

of atmospheric particulate matter collected in an urban area. The particulate material was removed from the filter bags by a specially designed vacuum

cleaner and combined into a single lot. This material was subsequently

screened through a fine mesh sieve (120 mesh) to remove bag fibers and other

extraneous material. The sieved material was then thoroughly mixed in a V-blender, bottled, and the bottles sequentially numbered. Randomly selected

bottles were used for the analytical measurements. Sample aliquots of 1 g

were extracted in a Soxhlet extractor for 48 h with a cycle time of about 20

min. Samples prepared for GC analysis were extracted with 450 mL of a 1:1

mixture of benzene/methanol, whereas samples for LC analysis were extracted

with a similar volume of methylene chloride. An internal standard solution

of 1-methylpyrene (for GC analysis) or 7 -methy If luoranthene and/or

perylene-di 2 (for LC analysis) was added to the particulate samples prior to

extraction. The perylene-d^j (^.98%) was obtained from MSD Isotopes (Merck & Co., Rahway, NJ) ; 7-methylf luoranthene was obtained from the National Cancer

Institute Chemical Carcinogen Repository (IIT Research Institute, Chicago,

IL) ; 1-methylpyrene was obtained from ICN K&K Laboratories (Plainview, NY).

The internal standard solutions were prepared in methylene chloride and 1 mL

of the solution was added onto the dust samples in the Soxhlet extraction

thimble. Prior to GC analysis the extract was concentrated in a rotary

evaporator, redissolved in cyclohexane, and partitioned between N,N-dimethylformamide (DMF) and water as described by Bj?Srseth (12). After the liquidliquid partition the total PAH fraction was isolated by normal-phase LC on an aminosilane column (30 cm x 9 mm i.d. , yBondapak NH2 , Waters Associates,

Milford, MA) using 3% methylene chloride in hexane as the mobile phase. The

PAH fraction was collected, concentrated, solvent changed to toluene, and

analyzed by GC (Model 3700 Gas Chromatograph, Varian Associates, Palo Alto,

CA) on a fused silica column (30 m x 0.25 mm i.d.) coated with a 0.25 ym film

thickness of SE-52. The sample (1 yL sample plus 1 yL toluene solvent flush) was injected using an on-column injector (J & W Scientific, Rancho Cordova,

CA)

The carrier gas for the GC analyses was hydrogen. Flame ionization

detection was used in all GC analyses.

4
Sample clean-up for the LC analyses consisted of concentration, solvent

exchange to cyclohexane, followed by a liquid-liquid partition between cyclohexane and nitromethane (4) . The nitromethane solution was concentrated to

approximately 1 mL and diluted with 1 mL tetrahydrofuran. The LC analyses

[Varian Model 5000 Liquid Chromatograph with Vista CDS 401 (Varian Associates,

Palo Alto, CA) and a Model 440 UV absorbance detector at 254 nm (Waters

Assoc., Milford, MA) ] were performed on a 5 pm Ciq column (Vydac 201TP, The

Separations Group, Hesperia, CA) with a solvent gradient from 40% acetonitrile

in water to 100% acetonitrile in 45 min at 1.5 mL/min. A variable wavelength

fluorescence detector (Model 3000, Perkin Elmer Corp. , Norwalk, CT) was used

which is capable of changing excitation and emission wavelength conditions

during the chromatographic run (three sets of excitation and emission wavelength conditions are stored) . Detector response factors for the cuantitation of the selected PAH were obtained by analysis of SRM 1647 — Pixority

Pollutant Polynuclear Aromatic Hydrocarbons in Acetonitrile (18) — in

conjunction with the air particulate extract. The standard compounds which

were not included in SRM 1647 (i.e. , triphenylene (>97%) , perylene (>_99%) and

benzo [e] pyrene (>99%)] were obtained from Fluka/Tridom Chemical Inc.,

(Hauppauge, NY) and the Community Bureau of Reference (Brussels, Belgium).

SAMPLE PREPARATION FOR CHARACTERIZATION BY MULTIDIMENSIONAL CHROMATOGRAPHY

The two PAH fractions which were used for normal-phase LC fractionation

to obtain samples for subsequent analyses by reversed-phase LC and GC-MS were

isolated as described below. The sample (^15 g) which was used for reversedphase LC analysis was Soxhlet extracted for 24 h with benzene/methanol (1:1)

and fractionated by acid-base partition (see Figure 1) to obtain a neutral

fraction which was then partitioned with cyclohexane/DMF/^O to obtain an enriched PAH fraction. This fraction was then separated on the amino column

to obtain the desired fractions. The sample (^21 g) which was used for GC-MS

analysis was Soxhlet extracted for 96 h with cyclohexane. Cyclohexane was

used for extraction of this particular sample to avoid removing significant

quantities of more polar constituents. The extract was then partitioned with

DMF/H2 O followed by a silica gel cleanup on a short column (SepPak silica

cartridge Waters Associates, Milford, MA) . The enriched PAH fraction was

then separated on the amino column to obtain the desired fractions.

GAS CHROMATOGRAPHY /MASS SPECTROMETRY (GC/MS) ANALYSES

GC/MS analyses were performed on a Hewlett-Packard 5985 system equipped

with an HP 1000 computer (Hewlett Packard Co., Palo Alto, CA) . Chromatographic

separations were carried out on a 30 m x 0.25 mm i.d. fused silica capillary

column coated with a 0.25 pm film of SE-52 (J & W Scientific, Inc., Rancho

Cordova, CA) . The column was interfaced directly to the ion source of the

mass spectrometer. Helium was used as the carrier gas. All GC/MS runs were

carried out with a 2 yL injection and 1:10 split. The initial oven temperature was 200 °C for 2 min, programmed at 4 °C/min to a final temperature of

275 °C and then held at this temperature for 10 to 60 min depending on the

fraction being analyzed. The mass spectrometer was operated in the electron

5
tron

ctra were s run.

70 eV and an ion source temperature of

etitively at 200 amu/s over the entire

jmatograpnic run.

V

*

*

'
SECTION 3

RESULTS AND DISCUSSION

QUANTITATION OF MAJOR PAH BY GC AND LC

V

The certification of an SRM requires the use of two independent and

reliable analytical methods (19) . The results from these two independent

methods must agree within a specified uncertainty in order for the values to

be certified. In the case where agreement is not within the specified limits

or where only one method of quantitation was employed, the values are reported only as informational values. In order to provide two independent

analytical methods in the determination of selected PAH in the Washington air

particulate material, different sample extraction solvents, internal standards, and cleanup steps were used prior to the chromatographic separation

and quantitation. GC and LC were used as the two different chromatographic

techniques for separation and quantitation.

GC Analysis

Prior to analysis by GC, the PAH fraction was isolated from the complex

mixture using a liquid-liquid partition with cyclohexane/DMF/water followed

by a normal-phase LC separation to isolate the total PAH fraction (see discussion to follow) . A gas chromatogram of the PAH fraction isolated in this

manner from the Washington urban dust (SRM 1649) is shown in Figure 2. The

major PAH were quantified using 1-methylpyrene as an internal standard. As

shown in Figure 2, the major peaks were the unsubstituted parent PAH. The

majority of the smaller peaks are alkyl-substituted PAH as will be discussed

later.

LC Analysis

In the LC analysis of the Washington dust extract, fluorescence detection

was used to achieve the selectivity necessary to quantify the individual PAH

components in the complex mixture without extensive preseparation or cleanup.

Because of the selectivity for PAH of LC fluorescence detection compared to

the universal FID for GC, a less rigorous PAH isolation procedure was used for

the LC analyses than for the GC analyses (i.e., cyclohexane/nitromethane

partition vs. cyclohexane/DMF/water partition and normal-phase LC)

.

The fluorescence spectral characteristics of a number of PAH are summarized in Table 1. By selection of the appropriate excitation and emission

wavelengths, a high degree of specificity can be achieved. This spectral

selectivity often permits the determination of individual PAH in a mixture

7
TABLE

1.

FLUORESCENCE

SPECTRAL

CHARACTERISTICS

OF

PAH

00

co 00 CO

O

m r>»

u

O rH ON rH tH

4J 3 3 CO *3 co *3 a

0)a.

CO CM 00 rH 00 f". in r** 1 O >3 rs. CM NO

00 00 ON 3 00 ON sO 1 VO CM CM CO o c CO CO CO 3 CO co CO 1 3 >3 >3 3 *3 oHX

m

x E w •H C CM 1 r^l O 1 CO CM 00 1 CM rH CO 00 >3 1 00 e NO vO i CM 00 m i CO O O O ON 1 ON m w co| co| col 3 CO CO 1 3 3 "3 3 CO 1 3 3

03 r< u

m

O o c CM NO

03 co 00 3 1 ' »H| NO 1 ao 1 Ol 1 ml

m -3 CO I Ol

U

m ON 1 m

O 1 l 00 1 GO 1 ao 1 l 1 NO 1 CO 1 ® CO co m co co| **3 1 col co| CO 1 co | o m 3 1 3 l 03 CM ON »H

)-i 3 >3 >3 O v-/ 3

rH

m i O 1 col nO| col

O 3 1 3 -3 1 Ol CM 1 00 1 3 l r^l col ml r*| 3 I mi m r**. NO >3 00 1 nO NO 1 NO 1 O CO 1 co ol

co i CO 1 co 1 co | >3 | co co | CO CO| co| CO I CO col

m 3 | 3 | 3 |

NO

O

m 3

co co |

co

Ui -uU

0)Ou COcooXWVuc

<DC

CO

<uo3 o3

co

m i cm i co i mm 3 CM CO 1 CM 3 00 00 1 rH NO

CO CO I rH CO CM CM CM 1 00 r-v co CO co| CO CO CO CO CO 1 CO co

CD 00 r» m 00

m 3 ao

O NO

m CM oo NO

ON ONm

O rH CM rH rH rH rH nO 3 o m

CM CM co co CO CM CO CO CO CO CO CO CO 3co £C

r< CM NO NO r>- 3 1 m 3 »H ml co ao CM CM oo no| CO 3|

ON 00 00 co 00 I O NO CO 00 o ON o o CM ON 00

O

CM CM CM co CM | co CM CM CM 1 CO CM CO CO CO CM | CO co|

/•—

CD 'w' v-'

O 00 r^ o o o 3 ON

m i CM no|m i ON | CO 3 00 r>* CM 00 ON m VO I 00 00 oo I O 1 00 1 NO ON

CM CM CM CO CM CM CM CM CM 1 CM CM CM | co 1 CM 1 CO CM

CO 00m 3 CM 3 m col oo I CM 1 NO 1 CM 1 3 CO nO

m | o 3 m | nO NO NO r>» 1 rH NO NO 1 nj | CO CM CM CM CM CM CM | CM 1 CM | CM 1 CO I CM CM CM

on I 3 I CM

0)c

0)

J-4 —uc

ccc

03 S

00 1

CD 'w' r- I o o rHCD s-/ CO

m

m 3 1 3 1 m | NO NO CO 3 m

CM 1 CM I CM | CM CM CM CM CM

<UaVU

<D SI C u

0) c u CO Si c u Q) 0) c a) C

CO Cl c 0) c rH a) u

<u So

u CO -c 0) So 0) u

c- 4J c a c si

rH 0) 0) 0) rH a>u

So E C J= SO rH c

SI *H <V •u SI So CO u Q u c 4J c r—i

0) 1 CO CO <u a; Q) CO | X NO u u c X SI 1 r» r* o 0) i CL N

rH CO

U 3 u rH •H c C rH So U Q) < &L (X H ca

m o m vo

CM CM

vo o m rHm

no on

m

m 3 CM CM CM CM CM 8
Fluorescence

Excitation

Spectra

Fluorescence

Emission

Spectra

w

.J<H

O

<r <r

ON 0

ON ON ON n* 1 nO o o cn ^d* CN 1 <r 00

-a* <r | <r 'd- m

rH

cn 00 ^dn

H r* l m "d- 00 o CN

m ON

H <r CN r—1 j l ON rH CN NO <r cn 'd* >d- <r i m ^d- m ^d" <r

NO

CO co 'w' v«^ r^ 00 CN r^ 1 00 cn i o vOl ON ON o 00 o

CN oo

O rH 1 ON rH 1 N2 ON

m o 00 o m

<r cn >d“ <r l cn <r i <r -d* | <r ^d* <r <r >d*

CO V

<T 1 r** 1 CN | <r 1 rH <r 1 in CN 1 CN I ml

H I rH <r 1 O I ON ON I © m 1 cn I ON m 1 ON cn I <r cn m| cn | cn <r | -3- 1 | cn | cn <T |

CN

Ocn

cn <r

cn

r^cn

vO CN m cn cn <r

vD

m r- m

cn cn EC<

cn l HI oo CO CN

00

m i H 1 O CNm cn

cn cn i cn i "d- cn cn

cn 1 r>* cn 00 1 H cn CN CN NO

NO 1 •'d- <r ON 1 Om CN <rH

cn l cn cn CN 1

cn 'W

<r cn <r cn cn

00 CN NO 00 o ON o

'd* cn CN 00 00 <r ON cn ON

cn cn cn CN cn cn cn cn CN

rH ON CN oo ml cn 00 cn CN 1 ml

m

ON 00 I H o CN ON r-

©©

H

© 00

CN CN | cn cn cn CN | CN cn cn | cn| cn | CN

<r i on

co I r^

CN I CN

in I <r I

on co I CN ] CN

-3* <r i O oo i cn cn l

r^. nt

CN

>d* o

>dvO

ON

CN

CN

ON

CN orHcn

cnon

CN

<r

r^. CN

m on

n£> no

CN CN

vo i n

CO I f"v CN I CN rH cn oo r^. CN I CN

ON I O cn oo

cn I cn

OO vo co

CN CN

CN vO

CN CN I m

<r on

cn cn

420C0

COpH X) u

X 0 0 42 0 0) C 42 C G 42 c c 0 0 0 r—i 0 0 0 0 u d cn U-l 1 C o O £ >N 0 0 0

03 03 0

CL 03

u T3\ 42

U Li 03 r—» 42 1 , CL

42 42

C “X •Ol

uG <r| 0 0 u 44 03 G 42 r—« 4J C C C cn 42 G •4-4 1 cnJ c 0 03 03 Ph w cn| 0 (—1 0 0 u r—i

r—« u C-L

C CLj T3| CNj CL

>.G 1xl\ XL 1 cnJ 0 r> f—i

CL o •H 1 u -o|

H | AJ 1 r—i

03 1

0J| 1—1

XL

H | XL cn 0| 42 1 42 ori XJ o o Q - N N 03 1 —* O C N N 42 ' i O c e o C o c 0 c c LJ o N 03 03 N 03 N 0 XL 0 0 42 N c X 42 c G c -o u 42 42 CL c 0 H H 03 *H 0 d C *H *H 0 0

CO

QQ ca P~ P2

H < Q C2 z 02 0

COC0 U u c 0 c H 44 H 0 0 3 u 00 0 c 0 O 0 H e Li *Hu 0 42 U

42

u

u 00

C

C o

IH 0 •u O •—i 0 0 G

5^ > 0 O 0 O

CN

C >N 0 42 o X oo U 030 c u 5 •H 0 O u H 03 0 H 0 0 O c u IM

00 0 0 U

>NH 42

u 0 O H0 0 0 c0 0 0

4J 0 3 C CL

*H 0

03 Li 0 0 44 > O G 0 U 0

42 CQ Q02

42G • •H /^s • 42 0 adB 0 -H

4*i 42

X 0 rt 0 03 £

CL 0

-U 4)

r* 0 JZ 0 G u C -H

rl T3 H C

O

"O ^8

•H

O

O

H P CO O CQ 0 >> 0 rH 42 Li 0 U 0 44 H 03 0 H B 3 •H

'TOO

X 0 42 o c cr Li •H CL H CL

Li • 0 0 0

T3 c e C •H 0 3 H 42

44 0 c

•H 0 Li

4*i 0

4^O 44 • 0 Li 0 03 0 42 0 0

CL Li 4-1 42 00 G 0 44 0 0 •H 0 Li C Li 0 O

4J 03

G c 0 44 C H C *H 3 H 0

4-J c 0 0 Li 0 Li o 0 44 0 E 'O c c H 03 0 3 C

42 0 0

E-* 0 42 H 44 0 0 Li • • 42 3 0 4* G 44

4-4 0 H

X o 0 42 •H 2 CL 3 B

CO XI 9

Data

from

Reference

20,

spectra

were

obtained

in

cyclohexane.
even when complete liquid chromatographic resolution of the components is not

achieved. The selectivity of LC analysis with fluorescence detection is

illustrated in Figure 3 for the Washington dust sample. In Figure 3 the

upper chromatogram is the reversed-phase LC analysis of a total PAH fraction

(obtained from normal-phase LC isolation procedure as described later) with

UV detection. UV detection provides a nearly "universal" LC detector for

PAH. The major PAH are discernable in this chromatogram; however, accurate

quantitation of the majority of these components would be difficult due to

incomplete chromatographic resolution. The middle and lower liquid chromatograms in Figure 3 are the LC analyses of the same Washington dust total PAH

fraction using fluorescence detection at the wavelength conditions described

in Table 2. These chromatograms illustrate the specificity achievable with

fluorescence detection optimized for various PAH. . N TABLE 2. FLUORESCENCE CONDITIONS FOR LC DETERMINATION OF PAH

IN AIR PARTICULATE SAMPLES

Wavelengths (nm)

(see Figure 3)

excitation emission PAH Quantitated

X 1 250 360 phenanthrene

X 2 285 450 fluoranthene , 7-methylfluoranthene

(I.S.) , benzo [b ] fluoranthene

X 3 295 400 benzo [k] fluoranthene

,

benzo [a] pyrene , benzo [ ghi]perylene

X 4 335 385 pyrene

X

5 285 390 benz [ a] anthracene

,

dibenz [a,h] anthracene

X 6 270 360 chrysene

X

7 400 440 perylene, benzo [k] fluoranthene

£

I.S. = internal standard.

In the measurement of selected PAH for the certification of SRM 1649,

three LC runs, each with three or four sets of wavelength conditions (one in

each run specific for the internal standard) were used for the quantitation of

11 PAH. The reversed-phase liquid chromatograms for these three runs with

fluorescence detection and a single run with UV detection are shown in Figure

4. The fluorescence detection conditions are given in Table 3. Even with

only minimal sample cleanup (no normal-phase LC as compared to Figure 3), the

selectivity of the fluorescence detection allows quantitation of individual

PAH.

10
TABLE 3. FLUORESCENCE CONDITIONS FOR THE LC DETERMINATION OF SELECTED

PAH IN WASHINGTON PARTICULATE MATTER

Wavelengths (nm)

(see Figure 4)

excitation emission PAH Quantitated

A 1 285 450 fluoranthene and 7-methylf luoranthene

(i.s.) a

X 2 400 440 perylene-di 2 (I.S.) and perylene

X

3 295 405 benzo [k] fluoranthene , benzo[a]-

pyrene, and benzo [ ghi] perylene

X 4 330 385 pyrene

X 5 285 385 benz [a] anthracene , dibenz[a,h]-

anthracene, and benzo [ghi] perylene

X 6 290 360 phenanthrene

X

7 270 360 chrysene

X 8 300 500 indeno [1,2, 3-cd ] pyrene

a

I .S. internal standard.

The results of the GC and LC determinations are summarized in Table 4.

The LC results are presented separately for the measurements using 7-methylfluoranthene and perylene-di 2 as internal standards. It was observed that

after a number of analyses, the LC column would lose some resolution between

the internal standard peak (7-methylf luoranthene) and a minor peak in the

sample. This resolution loss resulted in greater variability in the results.

Thus, a different internal standard, perylene-d^ 2 > was used for the remaining

analyses. In general, the results in Table 4 illustrate the improved

analytical precision for the results from LC method II compared to LC method

I. Data for some PAH are not reported in Table 4 for several reasons. For

example, GC values are not reported for triphenylene, benzo [b_] fluoranthene

,

benzo [k] fluoranthene , and dibenz [a_,h] anthracene because of co-elution with

other compounds (e.g., chrysene and triphenylene). LC values for triphenylene

and benzo [e_] pyrene were not reported owing to the lack of sensitivity and/or

selectivity for these two compounds using fluorescence detection. Triphenylene was measured in three samples only after a normal-phase isolation

of the four condensed ring PAH fraction. Results for phenanthrene and

dibenz [a_, h.] anthracene were reported only for method LC-II which used more

selective fluorescence conditions than were used in method LC-I.

11
TABLE 4. SUMMARY OF RESULTS BY THE VARIOUS ANALYTICAL METHODS FOR THE

WASHINGTON URBAN DUST (SRM 1649)

Compound

Concentration (yg/g)

GC [4] a LC-I [18] LC-II [9]

Phenanthrene —

*Fluoranthene 7.3 ± CNI o •

Pyrene 7.2 ± 0.2

Chrysene 4.6 ± 0.2

d

*Benz [ a] anthracene 2.4 ± 0.1

Triphenylene —

Perylene 0 . 84± 0.09

Benzo[ ejpyrene 3.3 ± 0.2

*Benzo[a] pyrene 3.0 ± 0.3

Benzo [b_] fluoranthene

Benzo [k.j fluoranthene

*Benzo[ ghi jperylene 4.7 ± 0.2

Dibenz [ a_,hj anthracene

*Indeno[ 1,2, 3-cd] pyrene 3.3 ± 0.3

— 4.5 ± 0.3

b

(9)

(4) 7.0 ± 0 .

5

b (24) 6.8 ± 0.4 (9)

(4) 6.3 ± 0.4 (17) 6.2 ± 0.2 {V

(4) 3.5 ± 0.1 (5) 3.7 ± 0.2 (9)

(4) 2.8 ± 0.3 (18) 2.4 ± 0.1 (3) — 1.7 ± o.i e

(3)

(4) o +i oo o 0.04 (17) 0 . 65± 0.02 (9)

(4) — —

(4) 2.6 ± 0.4 (18) 2.6 ± 0.1 (9)

6.2 ± 0.3 (18) —

2.0 ± 0.1 (18) 2.1 ± 0.1 (9)

(4) 3.9 ± 0.8 (12) 5.2 ± 0.6 (9) — 0.41± 0.07 (9)

(4) 3.4 ± 0.4 (16) 3.6 ± 0.2 (9)

0Number in [ ] indicates number of samples extracted.

kUncertainty is one standard deviation of the mean.

QNumber in ( ) indicates number of measurements.

d

GC determination includes triphenylene which coelutes with chrysene.

0Triphenylene determined by LC analysis of four ring PAH fraction isolated

by normal-phase LC.

* Indicates compounds with certified values in Table 4A.

Note: LC-I used 7-methylf luoranthene as the internal standard and LC-II

used perylene-di 2 as the internal standard.

12
The certified values for five PAH are given in Table 4A. Certified

values are provided only when the results of the two independent methods agree

within specified uncertainty. Pyrene and perylene were not certified because

of lack of suitable agreement between the methods. The values for the

remaining PAH were determined by only one method and were, therefore, not

certified but are provided only for information.

TABLE 4A. CERTIFIED VALUES FOR SELECTED POLYCYCLIC AROMATIC HYDROCARBONS

IN SRM 1649

Compound Concentration (yg/g)

a

Fluoranthene 7.1 ± 0.5

Benz [a] anthracene 2.6 ± 0.3

Benzo [a] pyrene 2.9 ± 0.5

Benzo [ghi] perylene 4.5 ± 1.1

Indeno [1,2, 3-cd ] pyrene 3.3 ± 0.5

cl The estimated uncertainty listed for a constituent is the union of 95%

confidence intervals computed separately for each analytical method and

represents an evaluation of the combined efforts of method imprecision,

possible systematic errors among methods, and material homogeneity. The

estimated uncertainty is intended to correspond to approximately 95%

confidence limits.

SRM 1648, "Urban Particulate Matter", which was collected in the

St. Louis area, was also analyzed by LC for the determination of 9 PAH. The

results are summarized in Table 5. The concentrations of most of the major

PAH were slightly higher in the St. Louis sample compared to the Washington

sample. The St. Louis collection site was selected as representative of an industrial area, whereas the Washington site was selected as representative

of an area with less industrial input. Qualitatively, the results for the

PAH in the St. Louis and Washington sample were very similar indicating that

automobile exhaust may be the principle source of PAH in the particulate

samples

.

13
TABLE 5. DETERMINATION OF SELECTED PAH IN ST. LOUIS URBAN AIR

PARTICULATE MATTER (SRM 1648) (yg/g)

a

Compound £ Concentration (yg/g)

Fluoranthene 7.9 + 0.6b

Pyrene 7.4 + 0.2

Chrysene 6 .

6

+ 0.1

Benz [a] anthracene 2.8 + 0.1

Perylene 0.65 + 0.02

Benzo [k] fluoranthene 3.3 + 0.1

Benzo [a] pyrene 2.6 + 0.2

Benzo [ ghi ] perylene 5.5 + 0.8

Indeno [ 1 , 2 , 3-cd ] pyrene 4.8 + 0.2

a Three 1-g samples of particulate matter were extracted.

^Uncertainties are ±la from the mean.

MULTIDIMENSIONAL CHROMATOGRAPHY FOR CHARACTERIZATION OF PAH MIXTURES

For the detailed qualitative characterization of the complex PAH mixture

from the Washington particulate samples, a multidimensional chromatographic

approach was employed. Multidimensional chromatography is the sequential

combination of at least two different chromatographic modes of separation to

analyze complex mixtures, e.g., LC/GC, LC/LC, TLC/GC, etc. The major PAH

constituents in the air particulate extract were found to be the unsubstituted PAH with smaller amounts (< 10 percent of the parent PAH) of the

alkyl-substituted PAH. In order to isolate and identify the numerous minor

components in the PAH mixture, a normal-phase LC procedure on an aminosilane

column was used to separate the PAH according to the number of aromatic

carbons (21-22) . The normal-phase retention characteristics of a number of

PAH and PASH on an aminosilane column are listed in Tables 6 and 7. In

normal-phase LC, isomeric PAH have similar retention characteristics e.g.,

benz [a] anthracene , chrysene, and triphenylene have retention indices of 4.00,

4.01, and 4.07 (A difference of 0.1 retention index units is required for

complete resolution of two compounds. See reference 21 for a complete

discussion of LC retention indices for PAH) . Alkyl-substituted PAH elute in

the same region as the parent PAH as shown in Table 6. This normal-phase LC

procedure provides PAH fractions which contain isomeric PAH and their alkyl

substituted homologs. These fractions were then analyzed by GC-MS and

reversed-phase LC. Reversed-phase LC provides excellent selectivity for the

separation of isomeric PAH and alkyl substituted PAH (17,22-23) e.g., see the

methylchrysene isomers in Table 6. Reversed-phase LC retention characteristics for over 90 PAH have been reported and discussed by Wise et_ al_. (22-23) .

14
TABLE

6.

LIQUID

CHROMATOGRAPHIC

RETENTION

OF

POLYCYCLIC

AROMATIC

HYDROCARBONS

(Logarlthim

of

the

Retention

Index)

3

cn3

-3

P-i

1

1—

1

o o O T—

1

CTM CO o CO CM CM rH CTM CM MO 00 O o

33 o o CM CO MO o co co CO CO 1—

1

ON co

0) CJ

cn CM CM CO CO CO CO co co co co CO co co CO CO co CO <r CO

Sj co

at r- > a

at

-3

at

c

n

3

a.

i i—

i

a)E

S-i o2

3

s

3

pHoo

CM 2

U

uh a

O -H

4-1 a!Eouc

o2

o2

o in rH CM 00 o CM o CM CM rH rH <r o mO rH o m OM o o o o o o rH o rH m m rH co m

CM CM CM CO CO CO co CO CO CO co CO co CO CO CO CO CO co

CM mO mO M3 MO mO

rH MO

CO

CM mOmO

00 CMOM

CMCMmOO

CM

00

rCMOM

CM

CTM CMOM

CM mO MO MO o Csl mO o

o 1—

1

rH CM o i—

!

CO

CM CM CM rH CNj CM CN

atHA4-1 3

3

3 3 3 3 3 32 3

3 3 3 3 HI 3

3 3 S-I 3 3 3 3 3 -3 3

03 3 3 3= S-i S-i S-i S-i 3 Cl 3

3 3 3 4J r3 oa r3 JC 3 i—i 3

3 a CJ 3 4-J 4-J 4-J 4-1 3 IH S-4 O 3 3 3 3 3 3 3 -3 3 3 3 3 to a H U <—

1

3 3 3 3 Cl 3 3 33 3 Cu E JC -3 to 3 3 3 3 rH 3 3 ! 1 3 rH o 4-J 4-1 r3 3 3 3 3 U S-i 3 S-i to u 3 3 4J 3 -3 -3 r- 3= O o 4-J to -3 3

at 3 3 3 3 CL a. "5- 3. u 3 3 3 a 4H 3

3 at i—

i

rH E 3 rH i—

i

rH rH 0) rH <—

1

3 rH 3 3

at 3 to to •H S-4 to to to B 14—

1

<4H rr to E -3

i—

i

at at r3 r a r1 -3 -c r— r3 •H i 1 .

1 o r3 •H 4-J 3 3 CJ 4H H i 4-4 4J 4-J 4-J AJ o 3 | -o| I—

1

4-1 Q 3

j= at 3 3 3 o 3 3 3 3 3 1 o 3 3 1 3

4-1 H H 2 2 rH 3 2 2 2 2 00 O o to 3 2 S-i JC O r3 1 l 3 1 l 1 1 * N N CJ 3 1 •r O

a 3 4—1 CM <T\ OM 3 rH CM CO OM rH 3 3 1 S-4 pH CM 3

at i—

1

3 -3 3 3 T* to rH 2 pH C PU pa ca or Ou PH

15

202
No.

of

Normal-Phase

Reversed-Phase

Compound

Mol.

wt.

Aromatic

C

NH2

Column

Ci

q

Column

o

0 >3

* CO 0 c O' CO 0 CO CM O' 00 m sf 0 ^a- m O' ^d* CM 00 00 0 *-H CN r—i r-H CO «-H f-H uo CM r-H CO »“H SO O' CN CN SO CN

^a- •a- >3

“ •a

- •a- a1 •a- CO CO CO "d* >d* >3* "d*

0 0 <r CO CO 00 r-H t-H co CM m >d" 0 <r 00 SO m 00 sO 0 O' 0 0 0 0 O' 0 0 0 l"H O' O' *—

0 sO CO m co -a

'd* CO ^d- <r ^d' CO <r 'd* CO co <r CO CO >a- ^a- «a- sf <r

cooooooocooooooooooooooooooooooooo o o

CM CN CM000CM CM

CO CM CM CM CN CN CM 00 CM CM CN CN CN CN 00 00 vO CM CM CN CM CM CM <r M3" Mj- <r <r CM <r <r <r <r CM CM CN m m LO m m

CM CM CM CM CN CN CM CM CM CM CN CN CN CN CM CM CN CM CM CN CM CM

03 <u (U 03 03 <13 d

d d d d d 03

a) <u <u 03 03 O

0 0 0 O C3 03

03 03 CO 03 03 3h

3-i >-1 3-i >-1 X

X X X x X 4-1

4-1 4-1 4J 4-1 4-1 d

d £ d d d 03

03 03 03 03 03 1—03 03 03 03 03 03 3 .—1 1 r—

1—1 1—1 03

d d d d d d

C jaj 03

jo| 03

J0|

03 03 03 03 03 03 03 U-—1 CM CO CO CO CO CO CO O CM CM CM CM CM d >» >s >s >s Ss Sh

03 d d d d d 03 3-i 34 3-( u 34 )-i U a) <u (U (U 03 X X X X x X X

x x X X -O X i-H 0 a CJ CJ O O

4-1 r-H 1—t-H 1—

»—< >, i-H 1—

r-H i-H 1—i-H d fo So So So S>s X >H S>1 S>s S>s 03 JC r x X X 4-1 03 X X r- X X X 1—1 4-1 4-1 4-1 4-> 4-1 03 d 4-1 4-1 4J 4—1 4-1 4-1 03

<u 0) a> 03 03 2 03 03 03 0) 03 03 03 2 2 2 2 2 1 CO 2 2 S 2 2 2

CM 1 1 1 1 1 rH S*s 1 1 1 l 1 1 d rH uo sO 00 O' r—

!-i i-H CM cn ~T m sO

a) i— CO CJ

03c

03 03 03 03 d X d d

03 4—1 03 03

3-i d X r- X d 4-1 4J

4-1 3-i d c

d O d Cd d 0 S-i u

03 d i-H 0 0

d 03 U-l 3 3

0) X 1—1 1—1 H r-H CU •pH U-l U-H Sh X 1—1 r—

1 d

JjJ

to -Q| •H

03 '

1 —4

r- O 0 O 0

'a. CM CM CM CM •H d O d d

d 03 03 03 03 H ea ca sa sa

<uc

<u

c

d 03 03 u c c

O 03 0) 3 l-i iH S^

U-l C- 51 5 000 CM CM CM c c c

0) 0) 0) ca ca ca

16

.lAd

[o]
33

to 3

rC e

Pm 3 I 3

HP

O

0)

U

CO3 00 3> u

a) pi

cocococoocmoo mr^cricO'd-r^OiH

X5 3

Pm rH rH cn o o CO o CO 1 o MO CO 00 a> ON OM o o

rH

u 3 <r <r <r cn

m 3 Pd o z 2 u

4Mo

4-J COEo3< O CM CM CM CM N (N IN N N

!N CM

N

N CM CM

CM MO MO MO 00 00 00 00

LO I''- i--. r*-. O'.

CNI CN CM CM CN CM CN CM o2

hp33333Oa

mo3i—i

rQ3H 33 3 3 3 3 3

HP 3 3 3 3 3 3 3 3 3 a 3 3 O 3 r—i 3 3 3 a i—1 Hpl 3 3 3 B >M 3 3 4-1 3 o 3 1 , 3 3 3

CJ 3 3 co | 3 3 ?o

0- 3 i—

p

r

—i 3

r—i 3 CMJ 3 1

3j 3 3 3 * 3 3 XI 3= 1—1 1 J0| jo| i—i 3 aa 4-J L 1 3 | 3 i—i 3 o N N 3

rH o 3 3 3 3 O 3

>» CM] -3 3 3 3 N 3 3 3 •u HP 3 3 3 a 3 3 3 3 •H •H 3 •rH Pm P3 < M Q Q CO PH 3

CM333 • 3 3 3 3 3 3 3 JO 3 Cu 3 3 3 3 3 •H 3 joOE X3 3

HO 3 3

•H 33 3 3 O 3 333 3 3 •H 33 3 3 3

•H 3 3

J3 3 3 33 3 O O 33 B 3

x: 3 3 • H 3 3 3 3 3 3 3 3

00 -3 3 O a 3 3 3 3 a. 3 3 3 •H cn

-3 00

HPO 3 E 3 3 o 3 3 O JO o

3- 3 r-v 33 3 3 3 o 3O 3 3 3 3 3 3 3 3 3 X 3 3 3 3 3 Xi X 3 1 1 3

Pi 3 2 3 iO u

17
TABLE 7. LIQUID CHROMATOGRAPHIC RETENTION OF POLYCYCLIC AROMATIC SULFUR

HETEROCYCLES (Logarithm of the Retention Index)

3

Compound Mol. wt.

No . of

Aromatic C

Normal-Phase^

NH2 Column

Dibenzo thiophene 184 12 2.87

Benzo [b ] naphtho [ 1 , 2—d ] thiophene 234 16 3.65

Benzo [ b ] naphtho [ 2 , 1-d ] thiophene 234 16 3.59

Benzo [b ] naphtho [ 2 , 3-d] thiophene 234 16 3.78

Chryseno [4 , 5-bcd] thiophene 258 18 4.04

Benzo [2,3] phenanthro [ 4 , 5-bcd ]

thiophene 258 18 3.83

Triphenyleno [ 4 , 5-bcd] thiophene 258 18 4.09

Dinaphtho [ 1 , 2-b : 1

' , 2 * -d ] thiophene 284 20 4.33

Dinaphtho [ 1 , 2-b : 2

' , 1 ’ -d ] thiophene 284 20 4.40

3. See reference 21-22 for discussion of retention index system,

^n-hexane as mobile phase.

The normal-phase separation of the PAH mixture isolated from the

Washington dust (see experimental section for details of the isolation) is

shown in Figure 5. The reversed-phase liquid chromatograms of these fractions

(including a total PAH fraction) are shown in Figure 6 and 7 to illustrate the

usefulness of this approach for the analysis of complex mixtures. Using UV

absorption detection at 254 nm (generally considered as a "universal" detector

for PAH), quantitation of even the major PAH in the total PAH fraction would

be difficult owing to the complexity of the mixture. However, the normalphase LC preseparation based on the number of aromatic carbons provides

fractions suitable for LC analysis even with the "universal" UV detector.

Fraction 2 contains the three condensed ring isomers, phenanthrene and

anthracene, and alkyl substituted phenanthrenes /anthracenes ; fraction 3 contains the peri-condensed four ring isomers, fluoranthene and pyrene, and

their alkyl substituted isomers. The four ring cata-condensed isomers of

triphenylene , benz [a] anthracene , and chrysene are found in fraction 4.

Quantitation of triphenylene in the air particulate samples (see Table 4) was achieved by reversed-phase LC analysis of fraction 4. Chrysene and

triphenylene were not resolved in the GC analyses. Fraction 5 contains the

isomers of molecular weight 252, i.e., benzo [b_] fluoranthene , perylene,

benzo [k] fluoranthene , and benzo [aj pyrene. The six condensed ring isomers of

molecular weight 276, benzo

[

ghi ] perylene and indeno [1,2, 3-cd ] pyrene , are the

major peaks in fraction 6. The PAH components in these fractions were

identified based on LC retention times and fluorescence spectra compared with

standard compounds.

All of the chromatograms in Figures 6 and 7 were obtained using UV detection. By using fluorescence detection in the analysis of these fractions,

18
additional selectivity could be achieved. For example, fraction, 3, which

contains pyrene/fluoranthene and alkyl substituted isomers, could be analyzed

with fluorescence conditions selective for only pyrene and alkylpyrenes

(excitation-333 nm, emission-370 nm) or selective for only fluoranthene and

alkylf luoranthenes (excitation-284 nm, emission-463 nm)

.

For detailed characterization of the numerous minor PAH and PASH, normalphase LC isolation of the various fractions followed by GC and GC-MS analysis

of these fractions was used. Lee ejt al_. (24) reported the GC-MS analysis of

LC fractions from gel chromatography on LH-20 for the determination of PAH

from air particulate material. The normal-phase separation on an aminosilane

column of the PAH isolated from the air particulate extract by liquid-liquid

partition and silica cleanup (see experimental section for details) is shown

in Figure 8. The individual fractions were separated by GC and identified

based on GC retention times (compared with standard compounds) and mass

spectra. Information from LC analyses of these same fractions was also used

for identifications. The gas chromatograms of the individual fractions are shown in Figure 9 to 17 and the peak identifications are listed in Table 8.

Fraction 1 contains fluorene and dibenzothiophene as the major parent PAH

and PASH. Methylf luorenes and Cj- through Ci+-substituted dibenzothiophenes

are the remaining constituents. Both fluorene and dibenzothiophene have 12

aromatic carbons and elute prior to phenanthrene in the normal-phase LC

separation (retention indices of 2.55 and 2.87, respectively). Phenanthrene

is the major peak in fraction 2. Anthracene is present, but only at about the same level as several of the methylphenanthrenes . 4H-cyclopenta

[

def ] phenanthrene and the sulfur containing analog of molecular weight 208 are also

included in fraction 2 as would be expected by the number of aromatic carbons.

The isomeric pyrene and fluoranthene were isolated into separate LC fractions, 2b and 3, (LC retention indices of 3.37 and 3.51, respectively) for the

GC analysis whereas they were combined in one fraction for the reversed-phase

LC analysis (see Figure 6, fraction 3). Minor components in the pyrene

fraction include alkylsubstituted 4H-cyclopenta

[

def ] phenanthrene isomers and

methylpyrenes . The fluoranthene fraction (no. 3) contains methylf luoranthenes

,

benzof luorenes , benzo[ ghi ] fluoranthene, benzo [cj phenanthrene and three PASH

of molecular weight 234, i.e., benzo [bjnaphtho [_l,2-d] thiophene , benzo [bjnaphtho [2_,_l-d] thiophene, and benzo [bjnaphtho [2_,_3-d] thiophene . All of the PAH

and PASH in this fraction have 16 aromatic carbons except benzo [£] phenanthrene

and benzo

[

ghi ] fluoranthene. These two PAH elute earlier than expected based

on aromatic carbon number because of their compact structure.

The four ring cata-condensed PAH found in fraction 4 include benz[ajanthracene, chrysene, triphenylene , C]_- and Cj-substituted molecular weight

228 isomers, methylbenzo

[

ghi ] fluoranthene , and possible PASH of molecular

weight 258. The major peaks in fraction 5 are the five ring peri-condensed

isomers of molecular weight 252 (i.e., benzo [b_] fluoranthene , benzo[k]-

f luoranthene , benzo [ e_] pyrene, benzo [aj pyrene , and perylene) , three isomers of

molecular weight 276, and a compound of molecular weight 306 (possibly

quaterphenyl) . Benzo

[

ghi ] perylene and anthanthrene (six ring peri-condensed

PAH appear in fraction 5, but could have been fractionated into fraction 6 (as in Figure 6, fraction 6) or a separate fraction based on the retention

19
TABLE 8. POLYCYCLIC AROMATIC HYDROCARBONS IDENTIFIED IN WASHINGTON URBAN

PARTICULATE MATTER

3

Peak No. Mol. wt. Compound

Fraction 1

1 166 Fluorene'3 ’ C

2 (a,b) 180 Methylf luorene'

3

3 184 b c Dibenzothiophene ’

4 (a-c) 198 Me thy ldibenzo thiophene'3

5 (a-i) 212 C^-substituted dibenzothiophene'3

6 (a-g) 226 C^-substituted dibenzothiophene'3

7 240 C^-substituted dibenzothiophene'3

Fraction 2

1 184

b c Dibenzothiophene ’

2 178 Phenanthrene'3 ’ c ’ ^

3 178 Anthracene'3 ’ c ’ ^

4 192

b c 3-Methylphenanthrene ’

5 192

b c 2-Methylphenanthrene ’

6 192

b c 2-Methylanthracene ’

7 190

b c 4H-Cyclopenta[def] phenanthrene ’

8 192

b c 1-Methylphenanthrene ’

9 (a-i) 206 C„-substituted phenanthrene/anthracene'3

10 208

1 be Phenanthro [4 , 5-bcd] thiophene ’

11 218 C ^-substituted 4H-cyclopenta [def] phenanthrene

12 (a,b) 218 C 0 -substituted 4H-cyclopenta [ def ] phenanthrene

13 (a,b) 232 C 0 -substituted 4H-cyclopenta[def ] phenanthrene

20
TABLE 8. (Continued)

Peak No. Mol. wt. Compound

Fraction 2b

1 204 C^-subs tituted 4H-cyclopenta [ def ] phenanthrene^

2 (a-d) 206 C

2 ~substituted phenanthrene/ anthracene'

3

3 (a,b) 218 C 0 -substituted 4H-cyclopenta [ def ] phenanthrene'

3

4 202

" b , c, d

Pyrene

5 204 C ^-substituted 4H-cyclopenta [def ] phenanthrene'3

6 218 C 0 -substituted 4H-cyclopenta [ def ] phenanthrene'

3

7 216

b c 2-Methyl- or 4-methylpyrene ’

8 218 C^-substituted 4H-cyclopenta [def ] phenanthrene'

3

9 216

b c 1-Methylpyrene ’

10 218 C„ -substituted 4H-cyclopent a [def] phenanthrene'

3

11 244 C^-substituted pyrene /fluoranthene'3

12 258 unknown

13 242 unknown

Fraction 3

1 202 Fluoranthene'3 ’ c ’ ^

2 202

b e Acephenanthylene ’

3 202

b c Pyrene (overlap from Fraction 2b) ’

4 230 unknown

5 218 C^ -substituted 4H-cyclop ent a [ def ] phenanthrene'

3

6 230 C 0 -substituted fluoranthene /pyrene'

3

7 216

^ b f

Methylf luoranthene ’

8 216 Methylf luoranthene'

3 ’

9 216

b c Benzo[a] f luorene ’

10 216

b c Benzo [b ] f luorene ’

11 216 Methylf luoranthene'3 ’

12 216 Methylf luoranthene'3 ’

13 216 Methylf luoranthene'

3 ’

14 (a-e) 230 C 0 -substituted fluoranthene /pyrene'

3

15 234

b c Benzo[b]naphtho[2 ,1-d] thiophene ’

21
TABLE 8. (Continued)

Peak No. Mole. wt. Compound

Fraction 3 (Continued)

16 226 Benzo[ ghi] fluoranthene ’

17 228 b c Benzo [c_] phenanthrene *

18 234 Benzo [ bj naphtho [ 1 , 2-d ] thiophene*3

’ C

19 234 b c Benzo [ bj naphtho [ 2 , 3-d ] thiophene ’

20 228

b c Benz [^] anthracene (overlap with Fraction 4) ’

226

b c Cyclopenta[ cd]pyrene ’

21 228 b c Chrysene (overlap with Fraction 4) ’

iction 4

1 234 Benzo [ b ] naphtho [ 1 , 2-d ] thiophene isomer*

3

2 234 Benzo [ b ]naphtho[ 1, 2-d] thiophene isomer*

3

3 228 b c Benz [a] anthracene ’

4 228 b,c Chrysene

228

b c Triphenylene ’

5 242 Methylchrysene/benz [a] anthracene/triphenylene*3

6 242 Methylchrysene/benz [ a ] anthracene/ triphenylene*3

7 242 Methylchrysene/benz [a]anthracene/ triphenylene*3

8 240 Methylbenzo[ ghi] fluoranthene*3

9 242 Methylchrysene/benz [ a ] anthracene/ triphenylene*3

10 256 C -substituted 228 isomer*

3

11 268 unknown (methylbinaphthyl)

12 258 unknown

268 unknown (methylbinaphthyl)*3

13 268 unknown (methylbinaphthyl)*3

14 258 unknown

268 unknown (methylbinaphthyl)*3

22
TABLE 8. (Continued)

Peak No

.

Mol. wt. Compound

Fraction 5

1 242 Methyl-substituted 228 isomer

2 254 Binaphthyl*5

3 254 Binaphthyl*5

4 254 Binaphthyl*5

5 256 C -substituted 228 isomer*

5

6 252

1 be Benzofluoranthenes ’

7 252 unknown

8 252 Benzo[ e] pyrene*

5 ’ ° ,(*

9 252 Benzo [ a ] pyrene*

5 ’ C ’ °*

10 252

_ . b ,c ,d Perylene

11 266 Methyl-substituted 252 isomer or dibenzofluorene isomer*

5

12 306 Quaterphenyl*5

13 266 Methyl-substituted 252 isomer or dibenzofluorene isomer*

5

14 266 Methyl-substituted 252 isomer or dib enzofluorene isomer*

5

15 264 unknown

16 (a ,b) 284 C^-substituted 252 isomer*

5

17 276 unknown

18 276 Benzo [ ghi] perylene*5 ’ C ’

*“*

19 276

b c Anthanthrene 5

Fraction 6

1 300 unknown

2 276

b c Indeno [ 1 , 2 , 3-cd] Fluoranthene ’

3 276 unknown

4 276 Indeno [1,2 , 3-cd] pyrene*

5 ’ c ’ ^

5 276 Benzo [ghi] perylene (overlap from

_ . cN b , c f-raction 5)

23
TABLE 8. (Continued)

Peak No. Mol. wt. Compound

Fraction 7

1 278 Dibenz [_a,j_] anthracene ,C ’

2 278 Dibenz [a., cj anthracene and dibenz [ a, h]

anthracene*3 ’ c ’ ^

3 278 unknown

4 278 Benzo [b ] chrysene*

3 ’ c ’ ^

5 278 n • b,c,d Picene

6 290 unknown

7 292 Methyl substituted 278 isomer*

3

8 304 unknown

9 302 Dibenzopyrene*3

10 302 Dibenzopyrene*3

11 302 Dibenzopyrene*3

12 300 Unknown

13 300 Unknown

Lction 8

1 302 Dibenzopyrene isomer*3

2 302 Dibenzopyrene isomer 0

3 302 Dibenzopyrene isomer*3

4 302 Dibenzopyrene isomer*3

5 302 Dibenzopyrene isomer*

3

6 302 Dibenzopyrene isomer*3

£Identifications are best estimates based on mass spectral data, GC and LC

retention data compared to standard compounds, and fluorescence spectra

compared to standard compounds.

^Identification based on MS.

c Identification based on GC retention.

^Identification based on LC retention and fluorescence spectra.

0Reference [25].

“Identified as methyl fluoranthene rather than methylpyrene based on normalphase LC retention.

24
indices of these compounds, i.e., 4.61 for perylene compared to 4.80 and 4.83

for anthanthrene and benzo[ ghi ] perylene. Minor components identified in

fraction 5 include binaphthyl isomers and methyl substituted 252 isomers (or

dibenzof luorenes)

.

Fraction 6 contains four molecular weight 276 isomers ( indeno [1,2, 3-cd ]

pyrene is the major peak and traces of benzo

[

ghi ] perylene overlap from

fraction 5) and four unknown constituents of molecular weight 300. Five

isomers of molecular weight 278 are the major components of fraction 7.

Three compounds of molecular weight 300 and four dibenzopyrene isomers (MW

302) are the minor components. Additional dibenzopyrene isomers (5 peaks) are the major components of fraction 8.

A comparison of the gas chromatograms obtained from the normal-phase LC

fractionation and the chromatogram of the total fraction PAH (see Figure 2)

illustrates the advantage of multidimensional LC/GC for detailed characterization of complex PAH mixtures.

FRACTIONATION OF WASHINGTON AIR PARTICULATE MATERIAL FOR CHEMICAL ANALYSIS

AND BIOLOGICAL TESTING

After preliminary investigation of the classical acid/base/neutral

liquid-liquid fractionation scheme for characterization of the organic

constituents on the Washington dust sample, a meeting was held with EPA

personnel to discuss the progress and future approach to validation of an overall fractionation scheme. As a result a plan was proposed to more

effectively and thoroughly evaluate a fractionation scheme in conjunction with

Battelle Columbus Laboratories (BCL) and Research Triangle Institute (RTI)

.

The classical liquid-liquid partition scheme described by Pellizzari et_ al . (26) was modified to include silica gel column chromatography of the neutral

fraction to isolate [1] aliphatic hydrocarbons, [2] aromatic compounds, [3]

moderately polar compounds, and [4] highly polar compounds, in addition to

the initial acidic and basic fractions, for a total of six fractions. The

fractionation scheme utilized by BCL to isolate the fractions is illustrated

in Figure 18. Individual fractions were analyzed by laboratories with

expertise for a particular class of compounds as follows: aromatic fraction

(NBS) , acidic fraction (RTI) , and moderately polar and highly polar fractions

(BCL) . The basic fraction was qualitatively characterized by both RTI and

NBS, and BCL determined nitro-PAH in the aromatic fraction. These fractions

were also tested for biological activity by EPA. The aromatic fraction was further fractionated by NBS on an aminosilane column to isolate and characterize the polycyclic aromatic hydrocarbon content. The resulting fractions

were also tested for biological activity by EPA.

A 130 mg aromatic fraction (obtained from the extraction of ^30 g of

Washington dust) was received from BCL. This sample was fractionated on an aminosilane column to obtain four fractions as shown in Figure 19. Aliquots

of these fractions were sent to EPA for biological testing. The results of

the biological testing are summarized in Table 9 (27). The majority of the

activity was found in fractions 1 and 2. The PAH are contained in fraction 1,

however, other components are also present as indicated by the presence of

25
TABLE

9.

RESULTS

OF

BIOLOGICAL

TESTING

ON

FRACTIONS

FROM

WASHINGTON

AIR

PARTICULATE

MATERIAL

(27)

>> K'

AJ os • 00 CN H 1 H • • O C/5 • m O + C

<uco

CO z <r in

AJ os • 00

m 3 1 H • • • E C/5 • VsO CN o 1 2 in

•H

CJ Os r-H •H 1 H Os m o C C/5 • • • •

OJ +

00

cd aj 2 CM co o so sE

T3 OS

0) I •u c/5 s: i oo

<u 3 Co

cO 00 > m

3- *H • co \ AJ H • • • CO u • i—

00 o aj co C

CO Os

aj 1 2 i"H u c/5 m + >

<Un co

-H

>, AJ (0 aj co • O'

•H > H rH co CM > -H • • • • •H AJ

AJ CJ O co 2 m CN o <

OsI C/0I

05 S'? s-5 CO CN <r o o

cO E

CO CM o

rH

s£5

n CN v£> 00 Os <* o vD • • • m • CN CM rH cn

rH

s£> v£> CN vO o CN <r rH • • • CN • CM r-H O • o o

26

not

tested,

assumed

to

have

no

activity.
biological activity without S-9 activation (PAH require activation for

mutagenicity) . The total mutagenic activity without activation of all the

fractions compares favorably with the activity of two hexane/benzene fractions

prior to fractionation on the aminosilane column (2.11 vs. 2.06 and 1.96).

However, the activity of the hex/benz fractions tested after ^60 days was found to be significantly lower than the initial testing.

The results of the qualitative chemical characterization indicated that

the PAH were contained in fraction 1 as expected. The most characteristic

features of the mass chromatogram was the broad unresolved hump with a number

of superimposed peaks. The spectra for over 100 of these peaks were examined;

however many of the spectra did not provide useful information because of the

complex superimposed background spectra. The majority of the peaks in the

chromatogram gave characteristic PAH spectra. These compounds are not

reported here since the PAH constituents were characterized extensively in

earlier analyses (see Table 8). The additional peaks (several present in

similar quantities as the PAH) gave mass spectra characteristic of methyl

esters of fatty acids and PAH quinones/ketones, (e.g., benzaldehyde

,

fluorenone, methylmyristate, methylpalmitate methylstearate or oleate,

anthraquinone , and benzanthrone) . In addition, other experiments involving

the isolation of the PAH fraction from the Washington dust indicate that the

amount of PAH material should be only about 25 percent of the mass collected

in fraction 1. Modifications to the fractionation scheme such as a cyclohexane/DMF/^O partition of the aromatic fraction prior to normal-phase

LC separation on the amine column may provide a "pure" PAH fraction. GC-MS

analysis of fraction 2 revealed the presence of numerous phthalates, methyl

silicone artifacts (presumably from the aminosilane column) , aza-PAH

(molecular weight 227) carbazole, and two benzanthrone isomers, superimposed

on an unresolved hump. The largest peak was dioctylphthalate . Analysis of

fraction 3 indicated that the only constituents present were probably due to

artifacts from the LC column. The basic fraction was analyzed by GC with

identifications by MS. The gas chromatogram is shown in Figure 20 and the

tentative identifications are listed in Table 10.

CONCLUSIONS

Biological testing can provide valuable information concerning the

potential health hazards associated with various fractions isolated from air

particulate material. Since the polycyclic aromatic hydrocarbon (PAH) fraction has been found to possess mutagenic activity, it is important to develop

methods for complete characterization of the fraction. The analytical

methods described in this work illustrate the capabilities of liquid and gas

chromatographic techniques for the separation, identification, and quantitation of individual components in this complex PAH mixture. Future work will

emphasize the chemical characterization and biological testing of various PAH

fractions to identify which PAH fractions (or compounds) are responsible for

the mutagenic activity in air particulate samples.

27
TABLE 10. AZA-ARENES IDENTIFIED IN BASIC FRACTION OF

WASHINGTON URBAN DUST

Peak # Mol. Wt. Compound

1 121 C^-substituted pyridine

2 129 isoquinoline

3 143 C^-substituted quinoline/ isoquinoline

4 143 C^-substituted quinoline/ isoquinoline

5 143 C^-substituted quinoline/isoquinoline

6 143 C ^-substituted quinoline /isoquinoline

7 157 C

2 ~substituted quinoline /isoquinoline

8 157 C

? -substituted quinoline /isoquinoline

9 179 Benzoquinoline

10 179 Benzoquinoline

11 179 Benzoquinoline

12 194 Caffeine

13 — Unknown

14 Unknown
REFERENCES

1. Lee, M. L., Novotny, M. , and Bartle, K. D. Analytical Chemistry of

Polycyclic Aromatic Compounds, Academic Press, New York, 1981. 462 pp.

2. Bartle, K. D., Lee, M. L., and Wise, S. A. Chem. Soc. Rev., 10:113,

1981.

3. Stanley, T. W. , Meeker, J. E., and Morgan, M. J. Environ. Sci. Technol.

,

1:927, 1967.

4. Hill, H. H. , Chan, K. W. , and Karasek, F. W. J. Chromatogr., 131:245,

1977.

5. Pierce, R. C., and Katz, M. Anal. Chem., 47:1743, 1975.

6. Dong, M. , Locke, D. C. , and Hoffmann, D. J. Chromatogr. Sci., 15:32,

1977.

7. Grimmer

,

311:475,

G. , Naujack, K.-W.

,

1982.

and Schneider, D. Fres. Z. Anal. Chem.,

8. Gr immer

,

G. , and Bohnke, H. Fres. Z. Anal. Chem., 261:310, 1972.

9. Moore, G . E. , Thomas, R. S .

,

and Monkman, J. L. J. Chromatogr., 26:

456, 1976.

10. Novotny, M. , Lee, M. L., and Bartle, K. D. J. Chromatogr. Sci., 12:

606, 1974.

11. Giger, W. , and Schaffner, C. Anal. Chem., 50:243, 1978.

12. Bj^rseth, A. Anal. Chim. Acta, 94:21, 1977.

13. Dong, M. , Locke, D. C., and Ferrand, E. Anal. Chem., 48:368, 1976.

14. Lankmayr, E. P., and Muller, K. J. Chromatogr., 170:139, 1979.

15. Fechner, D. , and Seifert, B. Polynuclear Aromatic Hydrocarbons,

P. W. Jones and P. Leber, eds. Ann Arbor Science Publishers, Inc.,

Ann Arbor, Michigan, 1979. p. 191.

16. Lee, M. L. , and Wright, B. W. J. Chromatogr. Sci., 18:345, 1980.

29
17. Wise, S. A. in: Handbook of Polycyclic Aromatic Hydrocarbons,

A. Bj«5rseth, ed. Marcel Deckker, New York, in press.

18. National Bureau of Standards Certificate of Analysis, SRM 1647,

December 1981.

19. Uriano, G. A., and Gravatt, C. C. CRC Crit. Rev. Anal. Chem. , 6:361,

1977.

20. McKay, J. F., and Latham, D. R. Anal. Chem., 44:2132, 1972.

21. Wise, S. A., Chesler, S. N., Hertz, H. S., Hilpert, L. R. , and

May, W. E. Anal. Chem., 49:2306, 1977.

22. Wise, S. A., Bonnett, W. J., and May, W. E. Polynuclear Aromatic

Hydrocarbons: Chemistry and Biological Effects, A. Bj^rseth and

A. J. Dennis, eds. Battelle Press, Columbus, Ohio, 1980. p. 791.

23. Wise, S. A., Bonnett, W. J., Guenther, F. R. , and May, W. E.

J. Chromatogr. Sci., 19:457, 1981.

24. Lee, M. L. , Novotny, M. , and Bartle, K. D. Anal. Chem., 48:1566, 1976.

25. Krishnan, S., and Hites, R. A., Anal. Chem., 53:342, 1981.

26. Pellizzari, E., Little, L. W. , Sparacino, C., Hughes, T. J. , Claxton, L

and Waters, M. D. Application of Short-Term Bioassays in the Fractiona

tion of Complex Environmental Mixtures, M. D. Waters, S. Nesnow,

J. L. Huisingh, S. S. Sandhu, and L. Claxton, eds. Plenum Publishing

Corp., New York, 1979. p. 333.

27. Lewtas, J. , Unpublished data, Health Effects Research Laboratory,

Research Triangle Park, NC.
PARTICULATE MATERIAL

INORGANIC RESIDUE FILTRATE

1. Basify to pH 10

2. Extract 3X with CH 2 CL,

1. Base wash sequence

1 1 | 1 1

Figure 1. General scheme for extraction and fractionation of air

particulate material for chemical and biological characterization

(adapted from reference 26)

.

31
</)

CM

J 2 03 s C

X cO • C/3 03 c O c v / r* CO •H

44

—

E

44

X—V

C

A •H

m • CO 03 03

U 3 03 34 C 0 X • O 03 X M 3 34 03 CM c n—/

rH , 03

to <44

a 3 44

X * O r—

<44 cO

>4 at

N

x 1 3 c c O CM

O

03 03 1 , in o C M X m| l o w O u O- /-n CMj C/3 c

oo sO

X c S4 1—1 1 • •H CO X o X N>«/ A O • LJ

03 03 c •rH CO C 0)

X 3 03 03 X E 0) C rH c E £ e 01 Sh -H £ o X C m cO

34 44 03 CM 14

<44 c X o • CO

CO CL i—o o X )4 •H N—/

>4

<13 o >4

X CL

44 3 44

A

cO i"H

—

ai E rs

rH <44 03 c X o C 03 O rH

CO /*N 03 rH

X o

•H CM 03 >.

X 'w*' >s 34 I e 34 03 a o A r- CL c iH

iH 03

u E E

4-1

C r—. 3 o 03 OS rH cn 3 Vj m o

>4

X V4 o

U-( 44

A

u

C

A 0) o

XI CO 03 c • • < e C 03 03 o

Pu 0) 03 >4 c o X O >, o CM

<44

a CO

a •H o (4 r—

44 1 v X CO •H c r—

44

X E o s—

3 O C cO •H C0 N O (4 44 03 i—

c O 00

CO cO cO 03 o

>4

X 34

CO

X

N • a

a. 0)C s 03

03 •H 03X

C 03 CO <44 X 03 34 •H rH 3 o 44 A >, 44

1-1 c <r 0) 34 CO

r- 03 c 03 34 CL X 0) a 0)

CO •H

a 34

r

—

CL

>4 03

H E

CO CO c CX

X 0) o 4s! 0) 1—1 44 44 CO »4 JU

CO 0) 0 • cs £ PH a. o N >3 o rH N c H

34 >> c 03 ctf X • 0) X rH o /-S LJ X rH OS 0) —\ •H*

03 <T E “s rH CcO sO 1 i-i X c3 O i—

rH s ' ' / a

CM z>

~L 3

60

32
o 10 20 30 40 50

Time (min)

Figure 3. Reversed-phase liquid chromatograms of total PAH fraction from

Washington urban dust (SRM 1649) . Sample cleanup included normalphase LC. Upper chromatogram: UV detection at 254 nm; middle and

lower chromatograms : fluorescence detection at conditions

described in Table 2. (See Experimental Section for conditions.)

33
E

Figure 4

,V I ! "•'1,

• "U<1uuvj

> o

m

LU _0

K- w z .52 LU J; o

® Z c

LU = o

c

co m

LU 00 CC CNi o c 3 o

3ENZOU)

fluoranthene

X r*

i'll

! i O o

,

I N Ni Z z a a

I' l!--

— X o Ui

•si a.

Xl

J! X 2 X3

i;

BENZO(k) FLUORANTHENE

I I

^ 5 X 2 Xg

30 40 50

x £

x 7

* z

Z c

-03

g

z ©

— a

TIME (min) Reversed-phase LC analysis of PAH from Washington urban dust

(SRM 1649). No normal-phase LC cleanup. Fluorescence

detection at conditions described in Table 3. (See

Experimental Section for conditions.)

34
0 20 40 60 80

Time (min ) Figure 5. Normal-phase LC separation of PAH isolated from Washington urban

dust for reversed-phase LC analysis. Conditions: semi-prep

yBondapak NH2 column; mobile phase — 1% methylene chloride in

hexane at 5 mL/min; UV detection at 254 run.

35
</> ®

Fraction 3

uv A I . H^W/

>

0 10 20 30 40 50

Time (min)

Figure 6. Reversed-phase LC separations of total PAH fraction and fractions 2 and 3 obtained from normal-phase LC fractionation of PAH from

Washington urban dust (see Figure 5). Conditions: column — Vydac

201TP 5y ; mobile phase — linear gradient from 40-100% acetonitrile

in water at 1%/min at 1 mL/min; UV detection at 254 nm. 36
a) c

o

o

0 10 20 30 40 50

Time (min)

Figure 7. Reversed-phase LC separation of fractions 4-6 obtained from

normal-phase LC fractionation of PAH from Washington urban dust.

Conditions: same as Figure 6.

37
38

Time

(min )

Figure

8.

Normal-phase

LC

separation

of

PAH

isolated

from

Washington

urban

dust

for

GC-MS

analysis

Analysis

conditions:

semi-prep

pBondapak

NH2

,

mobile

phase

—

0.5%

methylene

chlori

e

in

pentane

at 4

mL/min;

UV

detection

at

254

nm.
ll5 i

o iZ

39

Figure

9.

Gas

chromatographic

analysis

of

fraction

1

from

Figure

8.

Conditions:

column

30m

x

0.25mm

i.d.

SE-52

fused

silica

capillary;

temperature

program

—

175

°C

(2

min

hold)

programmed

to

280

°C

at 4

°C/min.
FRACTION 2

40

Figure

10.

Gas

chromatographic

analysis

of

fraction 2

from

Figure

8.

Conditions:

same

as

Figure
FRACTION 2b

ON

_o

u°cE

41 ii

Figure

11.

Gas

chromatographic

analysis

of

fraction

2b

from

Figure

8.

Conditions:

same

as

Figure
FRACTION 3

42

Figure

12.

Gas

chromatographic

analysis

of

fraction 3

from

Figure

8.

Conditions:

same

as

Figure
FRACTION 4

43

Figure

13.

Gas

chromatographic

analysis

of

fraction 4

from

Figure

8.

Conditions:

same

as

Figure
FRACTION 5 I

44

Figure

14.

Gas

chromatographic

analysis

of

fraction 5

from

Figure

8.

Conditions:

same

as

Figure

except

temperature

program:

200

°C

(2

min

hold)

programmed

to

280

°C

at 4

°C/min.
FRACTION 6 o

.00

-3

CN

-8

C«

_

O

_o

-2

45

Figure

15.

Gas

chromatographic

analysis

of

fraction 6

from

Figure

8.

Conditions:

same

as

Figure

14
V

46

Figure

16.

Gas

chromatographic

analysis

of

fraction 7

from

Figure

8.

Conditions:

same

as

Figure

14.
FRACTION 8

47

Figure

17.

Gas

chromatographic

analysis

of

fraction 8

from

Figure

8.

Conditions,

same

as

Figure

1-+
Particulate Sample oX

<U

>-l o

COuo

J2

CO=3

rHoo

>N

-O

-33

CO

"3C3C

48

for

the

Washington

urban

dust

sample.
I

1 1 1 r

0 20 40 60 80

Time (min )

Figure 19. Normal-phase LC separation of the hexane/benzene (aromatic)

fraction isolated from Washington urban dust using BCL

procedure. Conditions: semi-prep yBondapak NH2 column,

mobile phase — 1% methylene chloride at 5 mL/min, UV

detection at 254 run.

49
Figure 20. Gas chromatographic analysis of the basic fraction from

Washington urban dust. Conditions: column — 30m x 0.25 mm

i.d. SE-30 fused silica capillary; temperature program—

50 °C (1 min hold) programmed to 275 °C at 4 °C/min.

50
NBS-114A (REV. 2-8C) U.S. DEPT. OF COMM.

BIBLIOGRAPHIC DATA

1. PUBLICATION OR

REPORT NO.

2. Performing Organ. Report No. 3. Publication Date

SHEET (See instructions) NBSIR 82-2595 January 1983

4. TITLE AND SUBTITLE

Characterization of Air Particulate Material for Polycyclic Aromatic Compounds

5 . author(S) s. A. Wise, C. F. Allen, S. N. Chesler, H. S. Hertz,

L. R. Hilpert, W. E. May, R. E. Rebbert, and C. R. Vogt

6. PERFORMING ORGANIZATION (If joint or other than NBS. see in struction s) 7. Contract/Grant No. NATIONAL BUREAU OF STANDARDS

DEPARTMENT OF COMMERCE 8. Type of Report & Period Covered

WASHINGTON, D.C. 20234

9. SPONSORING ORGANIZATION NAME AND COMPLETE ADDRESS (Street. City. State. ZIP)

NBS and the U.S. Environmental Protection Agency

Research Triangle Park, NC 27711

10.

SUPPLEMENTARY NOTES

J Document describes a computer program; SF-185, FlPS Software Summary, is attached.

11.

ABSTRACT (A 200-word or less factual summary of most significant information. If document includes a significant

bi bliography or literature survey, mention it here)

In studies to evaluate the potential health and ecological effects of

atmospheric emissions, bioassays have been employed in conjunction with chemical

characterization to correlate mutagenic and/or carcinogenic activity with chemical

composition. The complexity of an air particulate extract necessitates the

prefractionation of the mixture into suitable subfractures or chemical classes

prior to chemical characterization and/or biological testing. The goal of this

project was to evaluate such a fractionation scheme for air particulate material

with respect to chemical characterization of the various fractions with particular

emphasis on the identification of polycyclic aromatic hydrocarbons (PAH). In this

study we have used three chromatographic approaches to separate, identify, and

quantify the complex mixture of PAH extracted from SRM 1649 (Urban Dust/Organics):

(1) capillary GC, (2) LC with selective fluorescence detection, and (3) multidimensional chromatographic techniques.

12.

KEY WORDS (Six to twelve entries; alphabetical order; capitalize only proper names; and separate key words by semicolons)

air particulate matter; biological testing; chemical fractionation; fluorescence

detection; gas chromatography (GC); liquid chromatography (LC); mass spectrometry

(MS); polycyclic aromatic hydrocarbons (PAH); SRM's

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