Shpol'skii spectroscopy as a tool in environmental analysis for amino- and nitro-substituted...

ELSEVIER Analytica Chimica Acta 333 (1996) 193-204

AlfUUXTICA CHIMICA ACTA

Shpol’skii spectroscopy as a tool in environmental analysis for amino- and nitro-substituted polycyclic aromatic hydrocarbons:

A critical evaluation

Igor S. Kozin *, Cees Gooijer, Nel H. Velthorst

Department of General and Analytical Chemistry, Free University, De Boelelaan 1083, 1081 HR Amsterdam, Netherlands

Received 14 February 1996; revised 14 May 1996; accepted 20 May 1996

Abstract

Aiming at the development of an alternative isomer-specific spectral method favourable for identification of mutagenic

amino- and nitro-substituted polycyclic aromatic hydrocarbons (PAHs) in environmental samples, the possibility to utilize Shpol’skii spectroscopy, a high-resolution cryogenic molecular luminescence technique, is critically investigated. New data representing specific, vibrationally resolved cryogenic fluorescence and phosphorescence spectra of a number of individual

amino-substituted naphthalenes, anthracenes and Buorenes are presented in addition to spectra of four- and five-membered ring amino-PAHs reported earlier. Room temperature fluorescence, photostability of the amino-PAHs and the influence of the cooling rate on spectral quality are considered. For determination of the nitro-PAHs, an off-line chemical reduction with zinc powder was optimized to convert these non-fluorescent compounds to the corresponding aromatic amines. Furthermore, the Shpol’skii technique was successfully applied to a mixture of certified standard reference materials (SRM 1587 and SRM 1647), containing a range of parent and nitro-PAHs. Finally, in combination with normal-phase liquid chromatographic separation, the methods developed enabled the determination of 1-nitropyrene in a diesel particulate SRM 1650.

Keywords: Polycyclic aromatic hydrocarbons; Amino-PAHs; Nitro-PAHs; High resolution low temperature molecular luminescence spectroscopy; Shpol’skii spectroscopy; Environmental analysis

1. Introduction

Amino- and nitro-substituted polycyclic aromatic hydrocarbons (PAHs) constitute two important classes

of compounds ubiquitous in the human environment. These chemical species may originate from different sources; for example, the nitro-substituted PAHs are

* Corresponding author. Tel.: +31 20 4447532; fax: +31 20

4447543; e-mail: kozin@chem. vu.nl.

easily formed from the parent compounds through

reactions with nitric acid and nitrogen oxides. Furthermore, under anaerobic conditions, chemical reduction may lead to conversion of nitro-PAHs to

amino-PAHs. A variety of materials such as coal- derived products, shale oil and synthetic fuels, diesel

and gasoline exhausts aerosol particles have been shown to contain either nitro- or amino-PAH derivatives [l-7]. These groups of compounds are known to exhibit strong mutagenic effects [&lo],

which can be enhanced in the presence of the parent

0003-2670/96/$15.00 Q 1996 Elsevier Science B.V. All rights reserved

PII SOOO3-2670(96)00253-X

194 I.S. Kozin et al./Analytica Chimica Acta 333 (1996) 193-204

PAHs. In this respect, correct qualitative and quantitative determination of individual amino- and

nitro-PAHs is strongly needed to be able to estimate

their impact on the ecosystems as well as to advance

our understanding of the environmental fates of PAHs.

By now, several analytical techniques based on chromatographic separation have been developed to detect amino- and nitro-PAHs. High-resolution capil-

lary gas chromatography (GC) coupled to mass-

spectroscopic (MS) [ 1 l-131, nitrogen-selective [14,15], thermionic nitrogen-phosphorus [16], elec-

tron capture [17,18] or flame ionization [12,14] detection has been successfully applied to the

analysis of complex environmental samples. The advantages of liquid chromatography (LC) with on-

line chemical reduction followed by UV/diode array

[ 191, electrochemical [20] or fluorescence detection [20,21] have also been demonstrated. Extremely high

sensitivity in determination of amino-PAHs has been achieved by implementing LC with chemilumines-

cence detection [4,22]

Evidently, the development of an alternative approach, that would enable identification of indivi-

dual compounds at low concentrations, is highly

interesting. Hence, it is worthwhile to investigate the applicability of Shpol’skii spectroscopy (SS) in this field, a cryogenic luminescence method utilizing n-

alkane solutions. The fundamentals of this technique

have been well documented [23-261. The high

isomer-specific selectivity and sensitivity of SS in

the analysis of complex environmental matrices for various parent and alkyl-substituted PAHs have been

demonstrated by several research groups [26-301. Reference Shpol’skii fluorescence spectra of individual isomeric monoaminopyrenes have also been

published [31,32]. Most recently, the applicability of the technique to the detection of nitro-PAHs con-

verted into the amino-substituted analogues has been studied, and the reduction products of 14 nitro-PAHs have been shown to exhibit cryogenic high-resolution fluorescence spectra [33].

The emphasis of the present paper is placed on the luminescence properties of individual amino-PAHs ranging from two to five ring systems. They were studied in n-alkane solvents both at room and at low (helium) temperatures. To our knowledge, Shpol’skii fluorescence and phosphorescence spectra of several

compounds, including mono- and disubstituted amino-naphthalenes, monoaminoanthracenes, and ami-

notluorenes, are reported for the first time.

Photostability of the compounds, the linearity of emission response under Shpol’skii conditions, and

the influence of the solidification rate on the spectral

features were investigated as well. For determination of nitro-PAHs, an analytical

procedure, comprising the normal-phase separation, off line chemical reduction with zinc powder, and

Shpol’skii luminescence detection, was optimized by using a mixture of standard reference materials

containing parent and nitro-PAHs (SRM 1647 and

SRM 1587, respectively) to resemble a real-world sample. Finally, determination of nitro-PAHs was

attempted in a diesel particulate material (SRM 1650).

2. Experimental

2.1. Chemicals

The amino-substituted PAHs examined in this

study were supplied by several manufacturers, i.e.

Acres-Janssen Chimica (1-aminopyrene, 6-aminochrysene, 3_aminofluoranthene, 2-aminofluorene, 9-

aminofluorene hydrochloride, 9aminophenanthrene), ICN Biomedicals (9-aminoanthracene hydrochlor-

ide), Sigma, Aldrich (1-aminonaphthalene, 2-aminonaphthalene 1-aminofluorene, 1-aminoanthracene, 2-

aminoanthracene, 1,5diaminonaphthalene, 1,8-diaminonaphthalene, 2,3_diaminonaphthalene). 1,2-Dia-

minonaphthalene was a gift from Dr. Carola Heeremans (The Institute for Environmental Re- search (TNO), Delft, Netherlands).

Standard Reference Materials SRM 1647 (acetoni-

trile solution of the 16 EPA PAHs), SRM 1587 (seven nitro-PAHs dissolved in methanol), and SRM 1650 (diesel particulate matter) were supplied by the

National Institute of Standards and Technology (NIST, Gaithersburg, MD).

Deuterated perylene, Per-dl2, used as an internal standard was obtained from Merck, Sharp & Dohme (Montreal, Canada).

n-Octane was supplied by Acres-Janssen Chimica. Methanol, n-pentane and n-hexane were Baker HPLC reagents. Dichloromethane and diethylether (analy-

IS. Kozin et al./AnaEyfica Chimica Acta 333 (1996) 193-204 195

tical grades) were purchased from Riedel-de HaEn

(Seelze, Germany). For chemical reduction reactions, zinc powder (less than 60 pm particle size) was obtained from Merck (Darmstadt, Germany), ammonium acetate was “Baker analysed” grade. The purity

of the solvents was regularly checked by recording fluorescence emission-excitation spectra. Since no significant response was observed, all solvents were used without further purification. To avoid photo-

decomposition, the experiments were conducted under yellow light, and the glassware used was

wrapped in aluminium foil.

2.2. LC fractionation

A normal-phase chromatographic step has been optimized to be able to collect separate fractions of

the parent, nitro- and/or amino-PAHs, depending on analytical purposes. The hardware consisted of two

Applied Biosystems Model 400 Solvent Delivery Systems, and a multi-wavelength Applied Biosystems

Model ABllOOOS Diode Array Detector. For the separation, a Chrompack Lichrosorb aminosilane

column (10 pm particle size, 250x4.6 mm i.d.) equipped with a 200 c1_1 injection loop was employed. For elution, a mixture of n-hexane and dichloro-

methane (DCM) with a flow rate of 1 ml min-’ was used. The DCM level was increased stepwise

gradually from 10% in the beginning of separation

up to 100% in the end of each run. The effluent was usually monitored at 254nm. Fractions contain-

ing the nitro-PAHs were dried in a gentle flow of nitrogen, and redissolved in methanol for chemical

reduction.

2.3. Chemical reduction

An off-line reduction reaction utilized in this study

was conducted in a principally similar manner as it was implemented in LC analysis with on-line reduction of the nitro-PAHs on a packed zinc column

[20]. After optimization, the reaction was carried out in all examined solutions, as follows: (1) A 0.4ml aliquot of a test solution, containing the

nitro-PAHs in methanol, was mixed with the same amount of ammonium acetate buffer solution (0.04 M) in a 6 ml screw cap vial, 0.2 g of Zn powder was added, and the mixture was

(2)

sonicated for 15 min in an ultrasonic bath

(increasing the time of ultrasonic agitation up to 40min did not result in higher yields of amino- PAHs).

After cooling the vial down to room temperature, amino-PAHs were extracted with four 2ml portions of diethylether under sonication for 2min.

The completeness of extractions was checked by the absence of visual luminescence under a UV

lamp (a 370nm cut-off filter was installed) in a last portion of the extract. Diethylether was

evaporated in a gentle stream of nitrogen and replaced by 0.4ml of n-hexane spiked with the

internal standard Per-d12 (0.1 PM). Half of this

volume was taken to replace n-hexane by n-

octane, a more suitable Shpol’skii solvent for

several compounds. In the experiments with simulated samples con-

taining both parent and nitro-PAHs, two solutions in

n-hexane and methanol were prepared by mixing

SRM 1647 and SRM 1587 (IO-fold dilution for both). The n-hexane solution was subjected to LC separa-

tion and, subsequently, to off-line reduction with Zn

as described above to obtain the fraction of amino- PAHs for low temperature fluorescence detection.

The methanol solution was first directly submitted to chemical reduction (following the same reaction

scheme). Secondly, the parent and amino-substituted compounds were extracted with diethylether as

outlined above, and the solvent was exchanged by

n-hexane for LC separation to isolate amino-PAHs. Finally, the collected fraction of amino-PAHs was

dried and redissolved in n-hexane or n-octane spiked with the internal standard for Shpol’skii lumines-

cence detection.

2.4. SRM 1650 extraction

Previously, it has been demonstrated that nitro-

PAHs can be effectively extracted from particulate samples at room temperature by applying (DCM)

with ultrasonic agitation [20]. In our study, following

this simplified approach, two separate subsamples of SRM 1650 (0.1 g each) were extracted with several portions of either DCM or methanol (for comparison

reasons) under sonication for 40min in total. After centrifuging and filtering the extracts through octa-

decyl Bakerbond SPE cartridges (Baker) the solvent

196 I.S. Kozin et al./Analytica Chimica Acta 333 (1996) 193-204

was exchanged by n-hexane mixed with DCM (10%)

for a normal-phase separation (see Section 2.2).

2.5. Luminescence measurements

Room temperature fluorescence emission-excitation spectra of individual amino-PAHs in either n-

hexane or n-octane were recorded with a Perkin-

Elmer LS50 luminescence spectrometer. To provide sufficient resolution, a narrow (2.5 nm) spectral

bandpass was set for both the excitation and the emission monochromator.

In the present study, Shpol’skii spectroscopy was exclusively applied in the conventional model, i.e.

lamp excitation was utilized. To gain additional selectivity, optimal excitation wavelengths were chosen for detection of each compound by dispersing

the light of a 450 W xenon arc lamp through a Bausch & Lomb (Roechester, NY) monochromator (bandpass

5 nm). A closed-cycle helium CT1 Cryogenics (Waltham,

MA) refrigerator was employed to simultaneously

cool down to 15-25 K four 10 pl samples previously

immersed in liquid nitrogen (fast cooling). The

influence of the cooling rate on the spectral shape was studied by placing liquid samples

directly into the vacuum chamber of the refrigerator and decreasing the temperature gradually

from 300 to 15 K in 40 min (slow cooling). A good thermal contact between sample solutions and the

cold tip of a cryogenerator, necessary for rapid

solidification, was provided by using a gilded copper sample holder covered with sapphire windows

[29,30,34]. For luminescence detection, the emission signal

from a front-face illuminated samples was focused on

the entrance slit of a Jobin-Yvon (Longjumeau, France) HRlOOO monochromator (0.08 nm spectral

resolution), and measured by an EG&G (Princeton, NJ) Model 1421B blue-enhanced intensified linear photodiode array enabling simultaneous data acquisition from about 20nm wide spectral regions. The spectral data were collected and processed by employing a Model 1463 detector controller and a Model 1460 OMA III control system. Photo-decomposition of the analytes during luminescence measurements was prevented as much as possible by using very short sample illumination times

(l-2Os, depending on the emission intensity) and

a low intensity green light beam for optical align- ment.

For quantitative determination, an internal standard procedure utilizing Per-d12 was applied. Experimen-

tal aspects of the measurements were given in more detail elsewhere [29,30].

3. Results and discussion

In this section, first, room and low temperature

fluorescence spectra of individual amino-PAHs ex-

amined will be compared, which will demonstrate the expected strong increase in spectral resolution.

Secondly, critical experimental conditions, necessary to successfully implement Shpol’skii luminescence

measurements, are investigated. Furthermore, for the determination of nitro-PAHs, an analytical procedure

combining preliminary chromatographic separation, off-line chemical reduction reaction, and Shpol’skii spectroscopic detection is described, and the overall

recoveries for these steps are estimated. Finally, the

analysis of a diesel particulate SRM 1650 for the contents of nitro-PAHs is discussed, and the results are compared with the certified values.

3.1. Luminescence spectra of amino-PAHs

In contrast to nitro-PAHs, aromatic amines gen-

erally are good fluorophores exhibiting relatively

high fluorescence quantum yields. It should be

realized, however, that, in comparison to parent PAHs the presence of the NH2 substituent can give

rise to some problems as far as high-resolution luminescence is concerned. Aromatic amines, as is

obvious from their efficiency in peroxyoxalate chemiluminescence [4], are prone to electron-transfer

mechanisms which might negatively influence their

photostability. Their lowest singlet excited electronic state (S1), which together with the ground state (So) largely determines the fluorescence transition and

thus the spectral shape, has a significant charge transfer character. Last but not least, due to the

increased polarity of the analytes concerned, aggregate formation in the electronic ground state So has to be expected, which may deteriorate the Shpol’skii effect. Within this context, as a result of possible

IS. Kozin et al./Analytica Chimica Acta 333 (1996) 193-204

rotation of the NH2 group, the non-planarity of

amino-PAHs may also play a role. In general a charge-transfer Sr state is usually

associated with such spectral features as non-

structured Sc=KSr absorption bands, a large Stokes’ shift (see [36] and references therein), and broad-

banded fluorescence emission. In agreement with these considerations, for most of the amino-substi-

tuted compounds examined in this study, the shape of

room temperature fluorescence spectra is character- ized by a single featureless emission band stretched

over a wide spectral region (see Figs. l-4). Although

emission wavelengths may differ depending upon the position of the substituent and the configuration of

the aromatic system, for some positional isomers (e.g. l- and 2-aminoanthracene) virtually identical room temperature fluorescence spectra were recorded (Fig.

3). As anticipated for amino-PAHs, aggregate forma-

tion has to be accounted for. For this reason, the shape of the fluorescence emission-excitation spectra

is concentration dependent, and the emission response may have a non-linear character. For various

amino-PAHs in the liquid state at ambient temperatures with analyte concentrations exceeding 50 uM

level, changes in the spectral shape of fluorescence

excitation were indeed observed. Thus, analysis of

a NH2 s

A

-- T=25K. Ercitabon at 292 “m. Room tempemture. Exc”a”o” at 290 “m. (n-hcnane. 10 @d wncentratm”,

370 380 390

Wavelength (“m)

2

.% ’ $H*

B

0 6 - T=25K. Excitation at 300 “m.

9 Rwm tcmpmturc. PCS. lkitat~o” at 294 “m. (n-hexane, IO @I concentration,

Fig. 1. Room temperature and Shpol’skii fluorescence spectra of

2-aminonaphthalene (A) and 1,2-diaminonaphthalene (B).

- T=20K. Exc”a,,“” at 270 ““I Room tempemmre. Excitation at 271 “m (n-hexanc, IO pM concentrano”)


amino-substituted three-membered ring PAHs: 9-aminophenan-

threne (A), 2-aminofluorene (B), I-aminofluorene (C).

multi-component samples by room temperature

fluorescence requires sufficiently diluted solutions. Furthermore, the lack of detailed spectral features implies that fluorescence spectrometry is not specific

enough for this purpose, and that chromatographic

separation is needed.

In Shpol’skii spectroscopy, the analytes are in-

corporated into a (poly)crystalline matrix formed

upon rapid solidification of room temperature solutions. Consequently, under cryogenic sampling con-

ditions the inhomogeneous spectral broadening is largely reduced, so that instead of featureless emission, concealed vibrational structure of lumines-

cence spectra can be revealed and relatively narrow (2-20cm-’ F-width at half maximum) fluorescence and (in some instances) phosphorescence bands can be observed. In our study, Shpol’skii fluorescence spectra, favourable for unambiguous identification

198 I.S. Kozin et al./Anulytica Chimica Acta 333 (1996) 193-204

A 7 I I T=25K Excitation at 283 nm.

c Room temperature. Excwion at 252 nm. (n-octane, 10 phi conccntmtion)

430 440 450 480

Wavelength (nm)

- T=25K. Excitation at 269 nm. ~~~.~~~~. Room tcmpretum. Exatatmn a 250 nm.

(n-cct&ne, IO ph4 concentition)

I

4& 4do 470

Wavelength (nm)

I

480

- T=25K. Excitation at 261 nm. Room temperature. Exc~tauon at 2HI nm. (n-cdne. IO JIbi concalt?ahon)

400 410 420 430 440

Wavelength (nm)


monoaminoanthracenes.

and quantitation, have been recorded for a number of amino-substituted two- to four-membered ring PAHs.

A dramatic increase in spectral resolution is demon-

strated in Figs. 14, where ambient and low tempera-

ture fluorescence spectra are overlaid. Phospho- rescence spectra, which can be used as complemen-

tary for qualitative and quantitative determination, are depicted in Fig. 5.

The linearity of the fluorescence and phosphores-

cence emission responses under Shpol’skii conditions was examined by using several multi-component solutions with equimolar concentrations of amino- PAHs varied in the range from 10uM down to 0.1 uM. For these purposes, 2arninonapthalene, l- and 2+uninofluorene, 9-aminophenanthrene and 3-

aminofluoranthene were dissolved in n-hexane, while the solutions of the aminoanthracenes, &uninochry- sene, and I-aminopyrene were prepared in n-octane.

- A 21 - T=ZSK. Excitation at 364 nm. m Room temperature. Excitation at 3.55 nm.

(n-octane, 10 pM concentration)

I I I I 400 410 420 430

WaveIengths (nm)

- T=ZSK. Excitation at 282 nm.

3 Rwm tempcmture. Excitation at 278 nm.

J (n-cctanc. 1opM conctntration)

E : “‘.’ ‘, r., . . . . _ .c

. . ..._..., ,__,. 8 a 2 2

345 1

400 I

405

Wavelength (nm)

7=25K. Excitation at 308 nm. Room rempcratum. Excilation at 305 nm (n-hexane. 10 phi concentration)

4bo Wavelen@h (nm)


amino-substituted four-membered ring PAHs: 1 -aminopyrene (A),

6-aminochrysene (B), 3-aminofluoranthene (C).

Linear calibration graphs were obtained for most

compounds in the concentration range measured. 3- aminofluoranthene, and l- and 9-aminoanthracene, exhibiting comparatively bad spectral resolution, i.e.

sharp features together with intense “phonon wings” (Fig. 3(B) and (C), and Fig. 4(C)), and relatively low

fluorescence quantum yields, could not be quantified in the mixed solutions. In the separately prepared single solutions of the examined amino-PAHs, the

absolute limits of detection achieved varied from 0.05 ng for I-aminopyrene to 2 ng for 3-aminotluor- anthene,

Highly resolved spectra were obtained not for all the amino-PAHs studied. In fact, under the present experimental conditions, utilizing available normal

and iso-alkane solvents, the Shpol’skii effect was not observed at all for 1 -aminonapthalene, 9-aminoff uor- ene, and several isomers of 1,2diaminonapthaIene.

IS. Kozin et al./Analytica Chimica Acta 333 (1996) 193-204 199

Fig. 5. Shpol’skii phosphorescence spectra of amino-PAHs: 1,2-

diaminonanphthalene (A), 1-aminofluorene (B), 2-aminofluorene

(C). Xenon emission bands are indicated with stars.

The data obtained indicate that the Shpol’skii

method, although successfully implemented for a number of amino-PAHs, cannot be generally applied.

Furthermore, the limits of detection achieved are distinctly less favourable than those obtained for the

parent PAHs. To discuss these results, possible

influences of photostability, sample solidification rate and analyte-matrix interactions will be considered consecutively.

3. I. 1. Photostability

Since many amino-PAHs are known to be light-

sensitive, in order to prevent phototransformation, the

analytes should be exposed to radiation in the UV- visible region only for a short time. Experiments on 2-aminofluorene, 9-aminophenanthrene and 6-aminochrysene demonstrate that already after 30 s of illumination the fluorescence intensity rapidly de-

Fig. 6. The influence of sample illumination time on fluorescence

emission response of amino-PAHs under Shpol’skii conditions: 9.

aminophenanthrene (A), 2aminofluorene (B), 6-aminochtysene

(C).

creases (Fig. 6). On the basis of these observations, it

must be emphasized that for amino-PAHs, the use of a diode array as a system enabling simultaneous fluorescence emission detection over a relatively

wide spectral region is of crucial importance. The duration of exposure and the spectral data acquisition time used for the measurements in this study were, therefore, limited to l-20 s depending on the signal intensity. Of course, it cannot be excluded that even under these conditions for all the amino-PAHs

concerned, photodecomposition should still be taken into consideration.

3.1.2. Solidijkation rate

Since in Shpol’skii spectroscopy the analyte molecules should be trapped in substitutional sites

200 IS. f&in et al./Analytica Chimica Acta 333 (1996) 193-204

of a cryogenic polycrystalline matrix, during the

sample cooling non-equilibrium conditions have to be maintained, i.e. aggregate formation has to be avoided [23,24]. This explains why cooling rate

may influence the shape of the cryogenic spectra, a phenomenon well known for some parent PAHs, but

presumably even more important for amino-PAHs because of their non-planarity and low compatibility

with the host matrix. For this reason, fast and slow temperature decrease procedures (see Section 2.5)

were applied to a sample of 2-aminoanthracene in n- octane (lOpM), and the spectra recorded were compared. No significant difference in fluorescence

spectra was observed. Apparently, in both procedures,

in spite of the different cooling rates applied, the

solidification time (which in fact determines the spectral features) is almost equal, because in the

experimental set-up employed efficient heat ex- change between the sample and the cold finger of the cryostat is guaranteed [29,34]. Under these

conditions, the actual freezing time is shorter than 1 s [14]. With the present experimental set-up, further

accelerating the sample solidification process is not readily achievable.

3.1.3. Andyte-matrix interactions

The Shpol’skii effect is based on the fact that under

cryogenic sampling conditions the interaction between the electrons of the analyte and the solvent

lattice, the so-called electron-phonon coupling (EPC) is small [23]. However, in view of the mesomeric effect of the amino group, for aromatic amines EPC

may be much more important than for parent PAHs, so that the spectral resolution will be drastically

decreased. This is underlined by the experimental results presented in Figs. 1-4. Obviously, the spectra reflect the presence of the photon wings

which can be attributed to significant EPC, especially in the case of I-aminoanthracene (Fig. 3(B)) and 3-

aminofluoranthene (Fig. 4(C)). Comparing low temperature fluorescence spectra of positional isomers as, for example, 1 - and 2-aminonaphthalenes; l-, 2- and 9-aminofluorenes and I -, 2- and 9-aminoanthracenes

(see Fig. 3) it reveals that the importance of EPC is strongly influenced by the position of the substituent. To conclude this section, it has to be stressed that for amino-PAHs the choice of the particular solvent to form a cryogenic matrix is much more sensitive than

for parent PAHs. Moreover, the purity of the solvent is a critical factor. According to our observations, for example, the spectral resolution was severely affected

when only a small portion of n-octane (O.l%, v/v) was added to a 1 pM n-hexane solution of 2- aminofluorene.

3.2. Determination of nitro-PAHs in test solutions

As indicated in Section 2.3, the seven individual

nitro-PAHs contained in IO-fold diluted SRM 1587 were chemically reduced to the corresponding amino- PAHs by treatment with zinc powder. After extracting

and exchanging the solvent either for n-hexane or n-

octane, the solutions of the reduced SRM 1587 were

submitted for low temperature luminescence measurements. Using optimal excitation wavelengths derived for n-octane solutions of individual amino-

PAHs, Shpol’skii fluorescence spectra of the reduction products of 6-nitrochrysene and I-nitropyrene

were successfully recorded (see Fig. 7(C)). These data match perfectly the spectra of corresponding

individual amino-PAHs depicted in Fig. 4. A highly resolved fluorescence spectrum was also

observed for reduced Gnitrobenzo[a]pyrene (Fig.

7(A)). It is virtually identical with the spectrum recently published by Matsuzawa et al. [33]. The

wavelengths of the most intense fluorescence bands in the Shpol’skii spectrum of the reduced 7- nitrobenzo[a]anthracene, illustrated in Fig. 7(B), also

closely coincide with the data reported earlier [33]. In a n-hexane solution, the reduction product of 2-

nitrofluorene exhibited a specific Shpol’skii fluorescence spectrum, which was very similar to that of 2-

aminofluorene presented in Fig. 2. However, as observed in the experiments on 9-aminoanthracene in n-octane and 3-aminofluoranthene in n-hexane,

after chemical reduction in the examined multi- component solutions of nitro-PAHs, clearly distin-

guished spectra from the reduced 9-nitroanthracene and 3-nitrofluoranthene could not be recorded.

The efficiency of the Zn reduction was estimated qualitatively by applying normal-phase LC to a reduced mixture of SRM 1587 and SRM 1647. The chromatograms depicted in Fig. 8(A) demonstrate the separation of parent, nitro- and amino-PAHs before and after chemical reduction and indicate that the reaction was practically complete. Independently, the

IS. Kozin et al./Analytica Chimica Acta 333 (1996) 193-204 201

Fig. 7. Shpol’skii fluorescence spectra of reduction products of

nitro-PAHs obtained after chemical reduction of SRM 1587 (1110

dilution) with zinc powder: 6aminobenzo[a]pyrene (A), 7-

aminobenzo[a]anthracene (B), 6-aminochtysene (indicated with

asterisks) and 1 -aminopyrene (C). T=25 K, n-octane.

isolated fraction of nitro-PAHs was chemically

reduced and submitted to quantitative Shpol’skii

fluorescence analysis. The concentrations measured for 2-aminofluorene, 6aminochrysene and l-amino-

pyrene indicate that the overall recoveries, estimated for the whole analytical procedure including preliminary fractionation, were in the range of 70-80%, which is high enough to allow the analysis of a real-

world sample. A different analytical scheme, where chemical reduction was followed by LC (to isolate

the aromatic amines) and subsequent Shpol’skii fluorescence detection, yielded approximately the same recoveries. Finally, we stress that the reduction with Zn is a relatively simple and reliable reaction to convert nitro-PAHs into the amino-substituted analo-

B

Fig. 8. Normal-phase LC separation of mixed SRM 1647 and

SRM 1587 (l/10 dilution) before and after chemical reduction (A),

and the extract of a diesel particulate SRM 1650 (B) on a

Chrompack Lichrosorb aminosilane column (250x4.6 min I.D.).

Gradient elution program, IO-100% dichloromethane in hexane at

1 ml min-‘. Peak identities are as follows: (1) 2_nitrofluorene, (2)

9-nitroanthracene, (3) 3-nitrofluoranthene and I-nitropyrene, (4) 7-

nitrobenzo[a]anthracene and 6-nitrochtysene, (5) 6-nitrobenzo[a]-

pyrene.

gues. Moreover, unlike in the case of reduction with

pottasium borohydride (formation of benzo[a]pyrene as a co-product was reported after that reaction [33]),

there are no indications that any co-product is formed in significant amount.

3.3. Determination of nitro-PAHs in SRM 1650

The positive results obtained for the simulated samples outlined above induced us to attempt the

analysis of a diesel particulate material known to contain nitro-PAHs. To reassure the correctness of the

analytical procedure a spiking experiment was carried out. Different amounts of the nitro-PAHs from a SRM 1587 solution were added to two separate samples of the SRM 1650 extract (for extraction details see Section 2), so that the nitro-PAHs spike concentra-

202 IS. Kozin et al./Analytica Chimica Acta 333 (1996) 193-204

tions in the first and in the second subsamples were equal to those in lo-fold and lOO-fold diluted

solutions of the initial SRM 1587, respectively. For example, the level of I-nitropyrene spiked with the

first and the second subsamples was 0.7 and 0.07 ug ml-‘, respectively.

According to literature data, particulate materials are extremely complex for chemical analysis since

they can be composed of a variety of compounds with different chemical nature. SRM 1650, a particulate matter representative of heavy-duty diesel engine

particulate emissions, has been shown to contain various parent and nitro-PAHs as well as oxidized

PAH derivatives [20,35]. With respect to nitro-PAHs present in SRM 1650, a certified value for l-

nitropyrene (19f2 ug g-l), a predominant nitro-

PAH, and non-certified values for 2-nitrofluorene (0.27 pg gg’), 7-nitrobenzo[a]anthracene (2.8 pg gg’)

and &nitrobenzo[a]pyrene (1.6 pg g-‘) have been reported 1351.

Regardless of the methods applied to determine nitro-PAHs in that type of environmental matrices,

preliminary fractionation for isolating the nitro-PAHs is usually a prerequisite. This is why the separation scheme optimized by means of a simulated sample,

as described above, was applied to the extracts of SRM 1650 as well. A typical chromatogram of the

unspiked extract is illustrated in Fig. g(3). On the basis of the retention times observed for mixed SRM

1587 and SRM 1647 samples, in the analysis of SRM

1650 the fraction containing nitro-PAHs was collected in the time span between 13 and 24 min. After

chemical reduction Shpol’skii fluorescence spectra

were recorded, and 1-aminopyrene in all examined extracts was positively identified (see Fig. 9). The

analysis of the different subsamples of SRM 1650

revealed the following concentrations of l-aminopyrene (the amounts spiked had been subtracted): for the

methanol extraction - 13.2 pg g-’ (unspiked sample),

13.7 ugg-’ (spiked with lOO-fold diluted SRM 1587)

and 14.4 ug g-’ (spiked with 1 O-fold diluted SRM 1587); for the DCM extraction - 12.5 pgg-‘. Taking

into account that these data are not corrected for the overall recoveries (up to 80%, see above) and comparing the results obtained with the certified

values of 1 -nitropyrene determined by the chromatographic methods (19~t2 pgg-’ [35]), it can be seen

that good agreement between different techniques is observed. For the extract spiked with the larger amounts of nitro-PAHs, cryogenic fluorescence

spectra of the reduced 6-aminochrysene and 6-

aminobenzo[a]pyrene could be recorded as well. In

that sample, however, the other reduction products of nitro-PAHs were not detected probably because of spectral overlap and strong interferences of impu-

rities. Presumably, intermolecular interactions between the corresponding amino-PAHs and other

chemically reactive constituents of the sample (like aromatic phenols and quinones, note the complexity

of the extract chromatogram in Fig. 8 between 20 and 30min) deteriorate the Shpol’skii effect, so that a

more detailed chromatographic separation prior to low temperature luminescence detection might be

required. The analysis of the subsample spiked with

the smaller nitro-PAHs quantities did not result in identification of any amino-PAH except for l-

Fig. 9. Identification of I-arninopyrene by means of Shpol’skii fluorescence detection in fractionated methanol extracts of a diesel particulate

SRM 1650 subjected to chemical reduction. Detector exposure time: 20s - unspiked extract; 10s - extract spiked with lOO-fold diluted SKM

1587; 2s - extract spiked with lo-fold diluted SF&l 1587. Excitation at 364nm, n-octane, T=25 K.

I.S. Kozin et al./Analytica Chimica Actu 333 (1996) 193-204 203

aminopyrene. Thus, the presence of 7-nitrobenzo[a]anthracene, &nitrobenzo[a]pyrene and 2-nitrofluorene (identification was not attempted) in the examined samples of SRM 1650 could not be confirmed in our experiments.

4. Conclusions

Summarizing the results obtained in the present

study, it should be stressed that the Shpol’skii method

can be implemented in the analysis of solutions containing amino- and nitro-PAHs as major constitu-

ents, although the influences of photostability,

solidification rate and the electron-phonon coupling

for amino-PAHs derived from the corresponding nitro-PAHs are stronger than for the parent PAH.

As a consequence of these influences, the Shpol’skii technique is not generally applicable to the detection

of the whole range of amino-PAHs, and the limits of

detection achievable are less favourable than for the parent compounds. In the highly complex diesel

particulate, the reference material SRM 1650, l- nitropyrene could be identified and quantified. This was not possible for the other nitro-PAHs present at

much lower levels.

It can be concluded that Shpol’skii spectroscopy

has some potential as a complementary analytical tool for the determination of a number of individual

amino- and/or nitro-PAHs. Its applicability might be

increased if sample solidification could be signifi- cantly accelerated (by further improving the sample

holder construction), since under these conditions the

negative influence of aggregation and formation of

complexes with sample impurities would be con- siderably reduced. Another possible way out is to involve chemical derivatization of the amino-PAHs as it has also been utilized to improve detectability of

the hydroxy-substituted PAHs [29,34,37].

Acknowledgements

This study was financed in part by the European Environmental Research Organization. We thank Dr. Carola Heeremans and Dr. Mark Houtzager of the Institute for Environmental Research (TNO, Delft,

Netherlands) for providing the amino-PAHs and

reference materials used in experiments. Technical

assistance of Drs. Gerard Stroomberg (IVM VU, Amsterdam, Netherlands) is gratefully acknowl- edged.

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