modern state of high-performance liquid chromatography of polycyclic aromatic hydrocarbons

ISSN 0027�1314, Moscow University Chemistry Bulletin, 2011, Vol. 66, No. 3, pp. 133–143. © Allerton Press, Inc., 2011.Original Russian Text © E.M. Basova, V.M. Ivanov, 2011, published in Vestnik Moskovskogo Universiteta. Khimiya, 2011, No. 3, pp. 163–174.

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Global environmental pollution and the unfavor�able ecological situation in industrial regions requireconstant analytical control (monitoring) of the state ofthe air, natural and drinking water, soil, and vegetationto be carried out. Polycyclic aromatic hydrocarbons(PAHs) belong to supertoxicants, class I hazardoussubstances [1]. PAHs have been added to the list of themost dangerous contaminating agents of water, air,and soil by the Environmental Protection Agencies(EPA) of the United States and the European Unioncountries [2, 3]. Benzo[a]pyrene (3,4�benzopyrene) issubject to obligatory certification in Russia [3].

PAHs are not industrially produced; they areformed upon incomplete combustion of organic com�pounds, e.g., during forest fires and volcanic pro�cesses. The anthropogenic sources of PAH supply intothe environment include bitumen, resins, oil and itsproducts, products of combustion of coal and other fuelsin furnaces of industrial and home incinerators and atthermal power plants, automobile exhaust emissions,and cigarette smoke. Thus, PAH are present in air and getinto soils and underground waters through domestic andindustrial waste storage. The anthropogenic factor is themajor source of contamination of the environment withPAHs. Approximately 5000 t of benzo[a]pyrene is annu�ally released into the environment; 61% of this amount isdue to coal combustion; 20%, to coke production; 8%, toopen burning of forests and crops; 4%, to burning ofwood; 1%, to transport emissions; 0.09%, to oil combus�tion; and 0.06%, to gas combustion [1].

The pollution has a regional character. Near largeindustrial centers, it is particularly strong and can betransferred to other regions with atmospheric fronts

and precipitation. PAHs can pass from soil into plants,which serve as food to animals. Then, they enter thehuman body with meat and dairy products. In waterbasins, PAHs can accumulate in plants, bottom sedi�ments, and fish. The average amount of benzo[a]pyrenethat annually is supplied to the human body with foodproducts in Russia is 1–2 mg [1]. The inhabitants ofmegalopolises annually inhale up to 200 mg ofbenzo[a]pyrene [2].

Supertoxicants exhibit mutagenic and carcinogeniceffects. The carcinogeneity of PAHs depends on theirstructure. Benzo[a]pyrene and dibenzo[a,h]anthraceneexhibit the highest carcinogeneity [1, 3], whereas thestructural isomer of benzo[a]pyrene (benzo[e]pyrene)is not carcinogenic. In Russia, the maximum allow�able concentration (MACs) of PAHs for atmosphericair is 0.1 μg/100 m3; for the air in working areas at industrialplants, it is 1.5 × 10–4 mg/m3; for water, it is 5 × 10–6 mg/l;and for soil, it is 0.02 mg/kg [3]. In the EuropeanUnion countries, the MAC of PAHs in drinking wateris 0.2 μg/l [2].

The composition of PAHs varies for different sources;therefore, each of them is characterized by its own set ofpriority substances; the degree of contamination can beassessed on the basis of the content of these substances.Since not all substances exhibit carcinogenic ormutagenic activity, in order to determine the toxicity ofthe PAH mixture, the content of each component has tobe determined in addition to their total content. Thus, theEPA recommends controlling the content of 16 PAHs inwater: acenaphthene, acenaphthylene, anthracene,benzo[a]anthracene, benzo[a]pyrene, benzo[b]fluoran�thene, benzo[g,h,i]perylene, benzo[k]fluoranthene,

Modern State of High�Performance Liquid Chromatographyof Polycyclic Aromatic Hydrocarbons

E. M. Basovaa and V. M. Ivanovb

a Dubna International University for Nature, Society, and Man, Dubna, Moscow oblast, Russiab Division of Analytical Chemistry, Department of Chemistry, Moscow State University, Moscow, Russia

e�mail: [email protected] September 20, 2010

Abstract—The review is devoted to the recent studies in the field of high�performance liquid chromatogra�phy (HPLC) of polycyclic aromatic hydrocarbons (PAHs). The existing certified techniques, developmentsof synthesis of new sorbents for separating PAH isomers, and study of the retention mechanism are discussed.It also considers the modern methods of extraction and concentration of PAHs, mainly from environmentalobjects, which are compatible with subsequent identification by HPLC, as well as the necessity of analyticalcontrol of contamination of the environment, food sources, and food supplies with PAHs.

Keywords: high�performance liquid chromatography, polycyclic aromatic hydrocarbons, environmentalobjects.

DOI: 10.3103/S0027131411030035

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chrysene, dibenzo[a,h]anthracene, fluoranthene, fluo�rene, indeno[1,2,3�c,d]pyrene, naphthalene, phenan�threne, and pyrene [4]. Anthracene, anthanthrene,benzo[a]anthracene, benzo[a]pyrene, benzo[e]pyrene,benzo[k]fluoranthene, benzo[g,h,i]perylene, phenan�threne, fluoranthene, pyrene, chrysene, perylene, andcoronene are typically determined in air [3]. Coal heatersmainly emit benzo[b]naphtha[2,1�d]thiophene into theatmosphere; Otto engines emit cyclopenta[c,d]pyrene.Both substances are carcinogenic. The main componentof automobile exhausts is the noncarcinogenic coronene.

At low PAH content in environmental objects, inorder to determine PAHs simultaneously, highly selec�tive and sensitive methods need to be used, such ascapillary gas chromatography and HPLC with variousdetection methods.

This review summarizes the modern state of deter�mination of PAHs by HPLC based on published worksstarting from 2000.

Official (certified) techniques. Any procedure com�prises three stages: extraction and concentration of ananalyte, separation, and detection. In method EPA550 [4], PAH was extracted from a water matrix withdichloromethane; the solvent was replaced by acetoni�trile. Then, the solution was concentrated by evapora�tion; the concentrate was diluted with water and analyzedby reversed phase HPLC on a column (250 × 4.6 mm;Vydac 201 TP C18; 5 μm) in the gradient elution modewith acetonitrile–water mixtures (flow rate 2 ml/min).Detection was carried out by a detector with a diodematrix at 254 nm. The duration of separation was45 min. The concentration coefficient was 250 (1 l ofwater sample, 1 ml of concentrate + 4 ml of water).p�Terphenyl was used as an internal standard. Thethreshold of detectability of benzo[a]pyrene is 0.02 μg/l(the sample volume in the column was 500 μl). Theprocedure is suitable for analyzing samples of drink�ing, surface, and underground water.

In EPA method 550.1 [5], the extraction stage wasmodified: the stage of liquid–liquid extraction in aseparating funnel was replaced by solid�phase extrac�tion (SPE) with an ENVI�18 disk made of a denseglassy matrix filled with silica gel with grafted C18groups. Prior to analysis, the sample was acidified topH 2; some methanol was added. The advantage ofthis procedure is the high throughput rate of the solu�tion sample (100 ml/min); the extraction occurs fasterthan when using the SPE cartridge. PAHs were elutedfrom the disk with dichloromethane.

In analyzing wastewater samples, PAH microim�purities were extracted with dichloromethane andpurified from disturbing impurities (paraffin andnaphthene hydrocarbons, and other impurities) on acolumn with silica gel; the eluate was evaporated untildry; the residuum was dissolved in an acetonitrile–water mixture (1 : 1) and analyzed by HPLC with twodetectors (diode matrix and fluorescent detector) thatwere connected in series in order to enhance the valid�ity of identification of a complex mixture of contami�

nants [6]. The concentration coefficient was 1250(250 ml of water sample, 200 μl of extract).

EPA method 8310 was designed for analyzing soilsamples [2]. PAHs from a soil sample was extractedwith chloromethane in a Sokslet apparatus or in anultrasound bath; the solvent was replaced with aceto�nitrile. Then, the extract was thickened by means of aKudern–Danish apparatus and purified by SPE. Theseparation was carried out on an HC�ODS Sil�X C18column (250 × 4.6 mm) (5 μm) in the gradient elutionmode with an acetonitrile–water mixture, with theflow rate of 0.5 ml/min. The separation duration was40 min. For naphthaline, acenaphthylene, acenaph�thene, and fluorene, we used a detector with a diodematrix at 220 nm, detection limits being 0.21–2.3 μg/l. Afluorescence detector was used to detect other PAHs.The threshold of detectability was 0.013–0.66 μg/l,including that of benzo[a]pyrene, 0.023 μg/l.

In Germany, two techniques for determining PAHsin soil and solid waste samples were developed [7].According to the first technique, analytes wereextracted with acetonitrile in an ultrasound bath, puri�fied by SPE on a CHROMAFIX 400�SA cartridge(containing a strongly acidic anion exchanger based onsilica gel with benzenesulfonic acid groups), eluted withacetonitrile, concentrated by evaporation (or, on thecontrary diluted with acetonitrile), and analyzed on aNucleosil 100�5 C18 PAH column (150 × 4 mm) with aUV or fluorescent detector. According to the secondtechnique, analytes were extracted with petroleum etherin a Sokslet apparatus, thickened on a rotary evaporator,and purified by SPE on a CHROMABOND CN/SiOHcartridge (containing nonmodified silica gel and silicagel modified with cyanopropyl groups); the mixturewas eluted with an acetonitrile–toluene (3 : 1) mix�ture; the eluate was evaporated (or diluted) and ana�lyzed on a Nucleosil 5 C18 PAH column (250 × 3 mm)with a UV or fluorescent detector.

In Russia, several techniques with the use of Rus�sian liquid chromatographs have been developed andcertified. During the analysis of soil samples, PAHswere extracted with an acetonitrile–water mixture(84 : 16); the extract was purified and thickened bySPE sequentially in three cartridges (Diapak A�3,Diapak P�3, and Diapak C); the last eluate was evap�orated until dry; the residuum was dissolved in an ace�tonitrile–water mixture (7 : 3) and analyzed using aMilikhrom�5 chromatograph on a Diasfer�110�S16column in the gradient elution mode with a fluores�cent detector [8]. The degree of extraction was 85%.The lower boundary of detectable content ofbenzo[a]pyrene in soil was 0.01 mg/kg. This techniquecan also be applied for analyzing food sources andfood products. The technique for determiningbenzo[a]pyrene in soil was developed and certified,combining liquid–liquid extraction, HPLC, and fluo�rescence detection using a Fluorat�02 analyzer with anHPLC�3 attachment [9]. The range of detectable con�


MODERN STATE OF HIGH�PERFORMANCE LIQUID CHROMATOGRAPHY 135

centrations of benzo[a]pyrene, pyrene, and chryseneis 0.005–2 mg/kg.

The technique for determining benzo[a]pyrene insamples of natural and drinking water using a Luma�khrom liquid detector and a Fluorat�02�02 fluorimet�ric detector was developed [10]. The lower boundaryof detectable content is 0.5 ng/l. The technique wascertified and included in the register of EnvironmentalProtection Regulation (2006).

There was a report [11] on certification of the tech�nique for controlling benzo[a]pyrene in aerosols,snow cover, and surface waters using short columns ofsmall diameter.

Although the major techniques for determiningPAHs were designed at the end of the 20th century, theflow of publications devoted to using HPLC for PAHdetermination has not subsided. Meanwhile, onlyreversed�phase HPLC is used for separation. Severaldirections of studies can be singled out: developmentof new sorbents; designing new separation configura�tions; using other detectors; searching for new meth�ods or more optimum conditions of separation andthickening; and ecological and analytical monitoring.

Sorbents. The separation is carried out on thereversed phase, usually, C18 or C8. Synthesis of newsorbents is one of the major developmental directionsof chromatography. Thus, retention of PAHs on a newphase that was cross�linked using an agent with threefunctional groups 1,3,5�triacryloyl�1,3,5�triazine wasstudied and compared with octadecyl grafted silica(ODS) [12]. The new phase was shown to be suitablefor PAH separation; moreover, it has enhanced abilityof recognizing planar molecules as compared with thestandard polymeric ODS phase.

A comb�shaped polymer was synthesized using thetelomerization reaction between octadecylacrylateand 3�mercaptopropyltrimethoxysilane, which wasnext immobilized on YMC porous silica gel (15 μm,295 m2/g) [13]. The higher selectivity with respect toPAH was revealed when studying the retention ofPAHs on a new modified silica gel. The retention ofPAHs depends on their structure and is determined byπ–π interactions.

Copper phthalocyanine�modified silica gel withamino propyl groups was prepared to study the mech�anism of PAH retention [14]. The modified immobilephase appeared to be more efficient for separatingPAHs with three and four rings. The retention is basedon π–π interactions between π�electron clouds ofPAHs and the π electron system of the modified silicagel (phthalocyanine).

The interactions between the π�electron system ofPAHs and anion exchangers based on silica gel(Nucleosil 55B) modified with anionic metal com�plexes with porphyrin and phthalocyanine were stud�ied in [15]. The separating power of the column con�taining a copper complex with phthalocyanine as amodifying agent is comparable with that of silica gel,

in which the copper complex with phthalocyanine isbound via the formation of sulfonamide bonds. How�ever, synthesis of the latter sorbents is a much moresophisticated procedure. In nonpolar solvents, theretention occurs because of π–π electron interactions.New immobile phases can be used in preparativeHPLC.

A new immobile phase for reversed�phased HPLC,an alkylphosphonate�modified magnesia�zirconiacomposite, was studied in [16]. It was shown that, onthe new immobile phase, the capacity coefficient ofcertain PAHs correlates with their logarithms of distri�bution coefficients in the immobile phase octanol–water system. Methanol was proposed as an organicmodifying agent of the mobile phase in PAH separation.The new immobile phase separates isomers of phenan�threne, anthracene, and terphenyl. It is characterizedby a better selectivity as compared with Zorbax ODSsorbent, although analytes are more weakly retained onit. Another new sorbent for reversed�phase HPLC wasobtained—porous cerium–zirconium oxide beadsmodified with alkylphosphorous acid [17]. The elu�tion order of PAHs on this immobile phase coincideswith that on silica�gel�based reversed phases.

A number of immobile phases with arylalkyl sub�stituents, which are stable in a wide pH range, weresynthesized; their selectivity to PAHs was studied [18].The retention of various sorbates (including PAHs) onfluorophenyl and fluoroalkyl immobile phases wasstudied and discussed [19].

The investigation of retention on the conventionalreversed phases is in progress. The results of compara�tive study of the selectivity of PAH retention on immo�bile phases of C18 and phenyl types are of interest [20].The column with grafted phenyl radicals was shown topossess a better separating power with respect to linearPAHs, whereas the structural isomers can be betterseparated on the C18 phase. PAH molecules exhibit thestrongest interaction with the propylphenyl immobilephase, the carbon content on its surface being the low�est among the sorbents investigated (3%). This factalso attests to the predominance of contribution of π–π�electron interactions to the total retention. Theinteraction with the most hydrophobic immobilephase Aqua C18 (surface content of carbon being equalto 15%) appeared to be a factor of two lower. Theinvestigated synergetic reversed phase was also of phe�nyl type; however, in terms of its properties, itappeared to be closer to C18 than to the phenyl phase.

The comparative study of selectivity of various col�umns—(200 × 4.6 mm) Hypersil ODS, (150 × 4.6 mm)Vydac 2017 P 5415, and (250 × 3.0 mm) Lichrospher®PAH—with water–methanol and water–acetonitrilemobile phases for determining 16 PAHs was describedin [21]. Under optimum conditions, the threshold ofdetectability varied from 1 μg/l (the sample volume inthe column was 20 μl) to <1 mg/l.

The selectivity of monomeric and polymeric C18phases to the shape of PAHs and their methyl deriva�

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tives was studied by means of a chemometric methodof fractional least squares [22]. The polymeric phasewas characterized by a higher selectivity to distinguishfine structures of planar and nonplanar isomers. Spe�cific and topological descriptors were introduced tothe models, which had been preliminarily used forranging structural differences between PAHs (theshape of condensed rings, length and width of themolecules) that are responsible for controlling theselectivity of chromatographic separation with respectto the shape. The limited space between alkyl chainsmay serve for recognizing the differences in shapebetween 3D and nonplanar sorbates in the case of ahigh�density polymeric immobile phase. Immobilephases with polymeric alkyl chains contain “holes”that are selective with respect to the size and shape ofmolecules, which facilitates the separation of structur�ally related sorbates. The monomeric immobile phasehas limited possibilities for separating PAH isomers, aswell as PAHs different in their nonplanar form.

Thermodynamics and kinetics of PAH distributionin the reversed�phase HPLC [23, 24] was studied. Itwas shown by the example of six PAHs (phenanthrene,chrysene, picene, benzo[a]pyrene, tetrabenzonaph�thalene, and phenanthro[3,4�c]phenanthrene that thecoefficient of retention on the polymeric phase ishigher than that of retention on the monomeric phase[23]. Increasing temperature reduces the retention,while pressure has little effect on it. The changes inmolar enthalpy values increase with growing numberof rings in a molecule, these values being lower fornonplanar molecules as compared with those for pla�nar molecules. The existence of very rapid sorbatetransfers between the mobile and immobile phases wasshown. Among tetranuclear PAHs, the change inmolar enthalpy and molar volume is lower for pyreneas compared with chrysene [24]. Pyrene transfer ischaracterized by a higher rate as compared with thetransfer rate of chrysene, which has a more linearstructure.

Thus, the synthesized new immobile phases are ofinterest for theoretical chromatography and studyingthe retention mechanism; however, they are poorlysuitable for analytical use. For analytical purposes, it iseasier to choose any commercial column (C8–C18)and modify the conditions of separation of the ana�lyzed PAH mixture. It should be noted that the type ofinteraction specific for PAHs (π–π interactions) asso�ciated with electron structure of these molecules wasused at the sample preparation stage for selectiveextraction of PAHs with silica gel with grafted cyano�propyl groups [7].

Multidimensional Chromatography and Combining Various Chromatographic Methods

HPLC is a multiunit method; however, its resolvingpower is limited, as well. The samples can simulta�neously contain large amount of PAHs, their deriva�

tives, and contaminants belonging to other classes.Frequently, the use of only one method is not suffi�cient to analyze such complex samples. Therefore, thedirection of two�dimensional liquid chromatographyhas been developing recently. Very few works have beenpublished along this direction for PAHs. Thus, two�dimensional HPLC [25] was described in which a col�umn with pentabromobenzyl phase was used duringthe first measurement, and two short monolith col�umns with the C18 phase were used during the secondmeasurement. The two measurements were connectedvia a ten�channel two�position valve; after the firstmeasurement, the eluate was delivered to the second�measurement column every 12 s.

The system for HPLC described in [26] consists ofthree columns with different immobile phases andallows performing both real�time purification of thesample and component separation. An unpurifieddiluted sample containing complex industrial matrices(oil, asphalt, etc.) was introduced to the system. Theduration of one analysis run was 45 min. The resultsobtained are comparable with the results of analysiscarried out in the mode with time separation and con�sisting of individual stages of sample preparation,extraction, and chromatographing. The identificationprocedure of benzo[a]pyrene to carry out routineanalyses was developed.

In order to identify PAHs and their alkyl homo�logues in the heavy oil fraction (Tboil = 287–481°C),the sample in which paraffin hydrocarbons were pre�liminarily deposited in cold acetone (–20°C) wasdivided into five fractions depending on the number ofrings by semipreparative normal�phase HPLC in a sil�ica gel column [27]. Individual fractions were ana�lyzed by normal�phase HPLC on two columns con�nected in series with reversed phase and water–aceto�nitrile mobile phase and UV detection. Within thegroup, the compounds were identified on the basis ofretention times. The separation of nonsubstituted andalkyl�substituted PAHs was achieved.

Combining the systems for HPLC and gas chroma�tography made it possible to simplify the sample prep�aration and reduce the duration of PAH identificationin particles of diesel engine emissions [28]. The systemin which supercritical fluid extraction, liquid chroma�tography, gas chromatography, and mass spectrometrywere combined in real time was used to identifyorganic substances in atmospheric aerosol particles[29]. The extract was transferred into the column of aliquid chromatograph and fractionated into four frac�tions on the basis of the polarity; then, each fractionwas placed into the gas chromatograph for final sepa�ration. PAHs were found in the first two fractions; theremaining fractions containing more polar com�pounds. Thus, the use of two�dimensional chromatog�raphy or a combination of different chromatographicmethods is required only when analyzing samples thathave complex matrices or when performing simulta�neous identification of PAHs and other environmental



contaminants. The tasks of analysis of atmosphericaerosol samples, natural waters, and soil can be suc�cessfully solved using one�dimensional HPLC.

Extraction and Concentration

Direct loading of the sample into the chromato�graph column is possible only in rare cases. The anal�ysis of most samples requires the stage of preliminaryextraction of PAH, which is typically accompanied byabsolute and (or) relative concentrating. A number ofreview articles have been published which weredevoted to the recent developments in methods ofsolid�phase microextraction, single�drop microex�traction and liquid�phase microextraction [30], super�critical fluid extraction of contaminants from soils[31], extraction using microwave heating (radiation)[32], and accelerated liquid extraction of stableorganic contaminants from environmental objectsusing solvents at increased temperature and pressure(PAHs being discussed among these objects) [33].

The table summarizes the data on PAH determina�tion in various objects by means of HPLC. The objectsused were environmental samples, terrestrial plants,biota, vegetable and animal food, and urine. It can beseen that extraction with organic solvents (hexane,dichloromethane, toluene, mixtures with acetone) aretypically used to extract PAHs from solid samples(aerosol particles, soil, and bottom sediments sorbedon the filters).

Since extraction with organic solvents is a long�term process that does not necessarily occur quantita�tively, procedures such as extraction with microwaveheating (radiation) [37, 41] and accelerated liquidextraction under increased pressure [35] and in anultrasound field [36, 53, 62, 65] were developed. Ascompared with regular extraction in the Sokslet appa�ratus, the use of focused microwaves has followingadvantages: reduced duration of the analysis, quanti�tative extraction of analytes from solid samples, andreduced extragent consumption. Indeed, the durationof extraction was 20–30 min [36, 37, 41, 53, 62, 65].Along with the target components, soil extract maycontain accompanying organic impurities; therefore,it is purified prior to chromatographing. The purifica�tion stage allows one to enhance the selectivity of theprocedure, improve the efficiency of the chromato�graphic column, and increase its lifetime. Purificationis usually performed using methods of solid�phaseextraction (SPE) or column chromatography [53, 55,56]. Silica gel [56], a mixture of silica gel and alumi�num oxide [53], and reversed phase C18 [55] served assorbents for purification. In the latter case, the sampleis simultaneously thickened. It is shown [54] that moreefficient separation of PAH from matrix componentsis achieved when using preparative HPLC. Whencomparing the methods of purification of biologicalobjects (biota) using SPE, microwave saponification,

and gel permeation chromatography, it was shown thatthe latter technique yields purer extractants [73].

Supercritical fluid extraction (SFE) is an efficientmethod for extracting organic compounds from solidsamples [31]. It ensures a larger degree of extraction ofPAHs from soil as compared with regular liquidextraction and SPE [74]. In [75], the dependence ofPAH extraction with hexane as a function of tempera�ture and pressure was studied, and the results wereshown to be described by the Peng–Robinson stateequation. The use of CO2 modified with a dichlo�romethane–n�hexane (5 : 1) mixture allows extractingPAHs from solid samples in only 15 min, the degree ofextraction being 70–90% [66]. PAHs were extractedfrom roasted bread with CO2 modified by acetonitrile[67]. The technique makes it possible to perform anexpress determination of 11 PAHs over the concentra�tion range from 0.323 to 9.40 μg/kg. Thus, in order toenhance the efficiency of PAH extraction, a modifyingagent—organic solvent that was used for liquid extrac�tion of PAHs from soils (table)—was added to theCO2. SFE combined with HPLC has not been widelyused for PAH determination yet. The procedure com�bining extraction by SFE with PAH retention on thecolumn with polytetrafluoroethylene and elutiondirectly into the fluorescence detector cell was described[76]. The optimization of PAH extraction from complexmatrices (earthworms) was described in [77].

In general, it should be noted that regardless of theconsiderable savings in consumption of organic solvents,SFE is rarely used at present for ecological analysis.

A drawback of liquid extraction is that toxic sol�vents are used. Micellar solutions were proposed as analternative. Extraction of PAHs from marine depositswith a micellar medium was described [62]. PAHs areextracted by a solution of polyethoxylene dodecylether [78]. The degree of conversion of PAHs uponextraction with micelles of sodium dodecyl sulfonateat the acid�induced cloud point was 67–93% [79].

Matrix solid�phase dispersion is a new method forpreparation of soil samples [57]. Quantitative extrac�tion of 16 PAHs was attained using an alkaline solu�tion. The results are comparable with those obtainedby extraction using microwave heating. The newapproach to direct extraction of PAHs from soils [80]consists in dynamic extraction in a rotating spiral col�umn. The competitiveness of this approach is not yetclear.

The main method for extracting PAHs from watersamples is SPE (table) on commercial cartridges,mostly those with reversed�phase C18. New sorbentsfor extracting and concentrating PAHs are beingsought: foamed polyurethanes [46], immunosorbentsfor extraction of pyrene and fluorine [61], fluoroplastsand sorbents based on cross�linked polystyrene [81],and multiwalled carbon nanotubes [47] have been pro�posed.

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Determination of polycyclic hydrocarbons by high�performance liquid chromatography

Object Method of PAH extraction Detector cmin (cM) References

Oil Treatment with acetone MDM (UV) – [27]

Oil, asphalt – – billion–1 [26]

Crude oil – UV – [34]

Aerosols accelerated liquid extraction FLD, tandem MS – [35]

Atmospheric particles extraction in ultrasound field with acetonitrile

FLD – [36]

Same extraction under microwave heat�ing with hexane–acetone mixture (1 : 1)

FLD 0.1 ng/ml (anthracene) – 5.9 ng/ml (pyrene)

[37]

Atmospheric aerosol – FLD – [38]

Aerosol extraction under microwave heat�ing with hexane–acetone mixture (1 : 1), SPE

DMD (254 nm), FLD 0.3–9.5; 0.035–0.86 pg/m3

[39]

Cigarette smoke SPE – – [40]

Wastewater extraction under microwave heating DMD 4–12 ng/l [41]

Drinking water – FLD – [42]

Same solid�phase nanoextraction FLD 0.9 ng/l (anthracene) 0.58 ng/l (fluorene)

[43]

Natural waters SPE – – [44]

Underground water – tandem MS with elec�trosputtering ionization

0.1 µg/l (naphtha�lene derivatives)

[45]

Water sorption with foamed polyure�thanes

FLD – [46]

River and tap water SPE – – [47]

Water capillary solid�phase microextrac�tion

– 0.9 µg/l (anthracene and 1,2�benzoan�thracene)

[48]

Environmental water stir rod extraction FLD (0.1–1.2 ng/l) [49]

Water, olive oil SPE FLD – [50]

Seawater stir rod extraction DMD – [51]

Seawater, bottom sedi�ments

SPE, extraction with dichlo�romethane

FLD, MS with chem�ical ionization

– [52]

Soil extraction with dichloromethane in ultrasound field

FLD – [53]

Same extraction with hexane FLD 0.001 mg/kg benzo[a]pyrene

[54]

Same extraction under microwave heating FLD (10 µg/kg) [55]

Same extraction with dichloromethane UV (254 nm) – [56]

Same matrix solid�phase dispersion FLD 0.01–0.6 ng/g [57]



Solid�phase nanoextraction [43] is based on theexistence of a strong affinity between PAH and goldnanoparticles. The method is environmentally safeand inexpensive, with only 500 μl of the samplerequired for the extraction; less than 100 μl of organicsolvent is required to perform extraction from onesample. The degree of extraction is 83.3–95.7%.

Solid�phase microextraction is typically combinedwith the determination by gas chromatography [3]. Theprocedure of PAH extraction into the polymeric film of agas chromatography capillary followed by its desorptioninto an injector of a liquid chromatograph using a smallamount of organic solvent was developed [48].

Stir rod sorptive extraction is one of the methodsfor impurity extraction from liquid samples that hasbeen developed recently. In [49], a polysiloxane rodwith diameter 1 mm and length 10 mm was used. Thedrawback of this method is the duration of extraction

(3 h). A stir rod made of a monolith material obtainedby copolymerization of octyl methacrylate and ethyl�ene dimethacrylate was synthesized. This rod is char�acterized by high sorption and desorption rates andconcentrating coefficients of 150, 134, and 189 forphenanthrene, anthracene, and pyrence, respectively[51]. The rods can be repeatedly used.

Detectors. A fluorescence detector (FLD) was usedin most extractions, its sensitivity being higher thanthat of a diode matrix detector, which operates in theUV region (table). It is possible to use a mass spectro�metric detector. It was used to determine the polarderivative of naphthalene in underground waters neara gas�producing plant [45], oxidized PAHs in aerosolsamples [35], and monohydroxy metabolites of PAHsin urine [71, 72]. HPLC combined with mass spectro�metric detection is required when identifying the new

Table (Contd.)

Object Method of PAH extraction Detector cmin (cM) References

Same liquid extraction FLD 0.12–1.57 µg/kg [58]

Same extraction with acetone–petro�leum�ether mixture (3 : 1)

UV (254 nm) – [59]

Bottom sediments – FLD, UV – [60]

Bottom sediments, waste sludge

SPE DMD – [61]

Marine sediments extraction in ultrasound field with micellar medium

UV – [62]

Same – FLD – [63]

Dust from metallurgical plant

extraction with toluene SFE (365 nm) – [64]

Marine biota extraction in ultrasound field FLD – [65]

Solid samples SFE DMD, FLD – [66]

Roasted bread SFE FLD (0.323 µg/kg) [67]

Meat products SPE – – [68]

Food products preparative exclusion chromatog�raphy

FLD (0.1 µg/g) [69]

Aboveground plant material

– FLD – [70]

Urine – Hybrid quadrupole time�of�flight MS

– [71]

Same – Triple quadrupole tan�dem MS

– [72]

Notation: DMD—diode matrix detector; FLD—fluorescence detector; MS—mass spectrometric detector; SFE—supercritical fluidextraction; and SPE—solid�phase extraction.

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BASOVA, IVANOV

PAH C28H14 as a pyrolysis product of toluene androcket fuel under supercritical conditions [82].

It was proposed that PAHs can be determinedaccording to selective fluorescence quenching [83].After separating PAHs by HPLC, a laser�induced flu�orescence spectrum was recorded. Nitromethane ordiisopropylmethane were added for selective fluores�cence quenching of substituted or nonsubstitutedPAHs, respectively. Selective fluorescence quenchingof PAHs at ambient temperature was observed in anionic liquid, 1�butyl�3�methylimidazolium hexafluo�rophosphate [84].

A surface ionization detector compatible withmicrocolumn chromatography using water–methanoleluents was designed [85]. Methanol content in themobile phase up to 50 vol % does not result in loss ofdetection sensibility. The detector responds to PAHs,among other substances.

Environmental Monitoring

PAH content in the environment is controlled bothin Russia and abroad. The results of investigation ofwater in tributaries of South Baikal [86], atmosphericair in St. Petersburg [38], soil in an industrial district ofSamara [59], bottom sediments in the Belaya River[60, 87], the undercurrent of the Mississippi River andthe Gulf of Mexico [88], atmospheric air in La Coruña(Spain) [39], dust at the Krivoi Rog MetallurgicalPlant (Ukraine) [64], the network of drinking watersupply in Hamburg [89], the environment in Ioannim(Greece) [90], the atmosphere in Ouargla (Algeria)[91], deposits in the Baltic Sea and waters near theGerman coast [63], and soil in Tokushima Prefecture(Japan) [56] were published.

Long�term observations and control made it possi�ble to reveal the seasonal and interannual variation inPAH content in the environment. Thus, PAH contentin suspended matter and the soluble phase of theundercurrent of the Mississippi River and the Gulf ofMexico varies and depends on seasonal dischargethroughout the year. In 1999, coastal erosion was themajor source of PAH entry into the gulf [88]. The balancewith respect to three compounds was performed:anthracene, benzo[a]pyrene, and benzo[g,h,i]perylene.The distribution of PAHs in the air in Ionnim wasstudied with allowance for certain meteorologicalparameters (temperature and humidity) [90]. Anincreased content of PAHs in the center of the city, aswell as at the beginning of the week, was found. Theaverage content of benzo[a]pyrene in the air varied inthe range from 0.32 to 2.63 ng/m3.

In bottom sediments of the Baltic Sea (Germany)collected at different depths, 11–1900 ng/g of PAHs wasdetected depending on the sampling site [63]. The corre�lation between PAH concentration and content oforganic carbon was observed. The major PAHs found inthe surface soil layers taken from different sites in

Tokushima Prefecture included fluoranthene, pyrrole,benzo[a]fluoranthene, and benzo[a]anthracene [56].

Water from the network of drinking water supply inthe northern suburbs of Hamburg had a strange flavorand odor [89], which was linked to the presence ofabandoned military units in that area and entry ofdecomposition products of ammunition, cable insula�tion material, etc., into underground water. In thedrinking water at the outlet from the station, phenan�threne, anthracene, and fluoranthene were detected.

In samples of marine water and bottom sediments nearthe coast of Spain, benzo[b]fluoroanthene, benzo[k]fluo�roanthene, benzo[a]pyrene, benzo[g,h,i]perylene, andindeno[1,2,3�c,d]pyrene [52] were detected.

Underground waters near the gas�producing plantturned out to be contaminated with 1� and 2�naph�thoic acids, 1� and 2�naphthylacetic acids, 1�hydroxy�2�naphthoic acid, 2�hydroxy�3�naphthoic acid, andnaphthyl�2�methyl�succinic acid [45]. The concen�tration of benzo[a]pyrene in surface waters of back�ground regions in Russia do not exceed 10–11 ng/l; inbottom sediments, the average concentration is 1–5 ng/gdry weight [1]. Serious contamination of the aquaticecosystem with PAHs was revealed in Sterlitamak[87]. Analysis of bottom sediments of the Belaya River100 and 1000 m below the biological purification facil�ities detected an abrupt increase in concentrations ofmost PAHs (with the exception of anthracene) in thelatter case, which was associated with the partialmigration of the contamination spot downstream inthe river and with the disturbance of the natural riverflow. The values of PAH content (with respect tobenzo[a]pyrene) at the biological purification facili�ties and 100 and 1000 m below them were 0.835 MAC,0.4555 MAC, and 2.685 MAC, respectively.

The map of PAH distribution in soil in Samara wasplotted [59]. On the basis of the results of analysis ofsamples near industrial plants and transportationroutes in the Oktyabr’skii district of the city, contami�nation with acenaphthylene, phenanthrene,anthracene, pyrene, and chrysene was revealed. Back�ground concentrations in the surface soil layers (wherebenzo[a]pyrene is concentrated owing to the highestamount of organic matter) in rural districts of Russiaare lower than 5–8 ng/g [1].

Since PAHs that are contained in the environmentcan enter and be accumulated in agricultural and foodproducts, MACs (1 μg/kg) for grains, smoked meat,fish, and fat�containing product were established[2, 8]. Thermal treatment of raw foods results in anincrease in PAH content. Thus, after thermal treat�ment of meat products (Silesia, Poland) under house�hold conditions, the amount of PAHs in beef, pork, andchicken meat was 2.43–16.10 ng/g, which is higher thanthe MAC [68]. The linear range of the procedure ofdetermining PAH content in roasted bread includes theMAC values for grain and is 0.323–9.40 μg/kg [67]. Ingeneral, the content of benzo[a]pyrene in wheat grain



may vary from 0.68 to 1.44 μg/kg, whereas in ham andbrisket, it may vary from 16.5 to 29.5 μg/kg [1].

Thus, permanent analytical control of PAH con�tent (in particular, of carcinogenic and mutagenic rep�resentatives) in the environment, raw food sources,and prepared food products is required for the sake ofhuman safety. The recent research allows more sensi�tive, reliable, and rapid control procedures to bedesigned.

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modern state of high-performance liquid chromatography of polycyclic aromatic hydrocarbons

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