02/10

02/10
Actual Topic
	Analysis of polycyclic aromatic hydrocarbons (PAHs)
	Analysis of polycyclic aromatic hydrocarbons with chromatographic methods is very important for analytical investigation of water, soil, waste oil and food, due to carcinogenic and mutagenic properties of these pollutants. Below, diverse chromatographic methods of TLC, HPLC and GC, as well as for sample preparation by SPE are presented with newly developed and well established products.
	Polycyclic aromatic hydrocarbons
	Polycyclic aromatic hydrocarbons (PAHs) are chemical substances consisting of fused aromatic rings without heteroatoms or substituents. As pollutant, they are of concern because some compounds have been identified as carcinogenic, mutagenic and teratogenic. PAHs are natural components of coal or crude oil. They are delivered to our environment by pyrolysis (incomplete burning) of organic materials like e.g. coal, oil, fuel, wood or tobacco and hence can be found globally. Today most PAHs accrue from anthropogenic processes – but also natural origins (e.g. forest fire) are possible. Regarding to past pollutions an important impact had production of coke and gas from black coal. Waste products (e.g. tar) from coking or gas plants are often origin of serious ground water pollutions.
	Since a number of PAHs (e.g. benzo[a]pyrene, benz[a]anthracene) have been proven to be carcinogenic, control of PAH content in food, water and soil is an important task for routine analysis. For selection and limiting values of polycyclics we refer to governmental regulations, which exist in many countries (e.g. EPA method 610 of the United States Environmental Protection Agency, Regulation of German Drinking Water (TVO)). Listed PAHs are representatively determined for general contamination by PAHs.
	PAHs can be analysed by different chromatographic techniques (TLC, HPLC, GC). Thus the 6 PAHs according to German drinking water specification (TVO) can e.g. be analysed by TLC, while much larger numbers of polycyclic aromatics can be determined by HPLC or GC. Due to complex sample matrix (e.g. soil, waste oil, humic acid containing water, smoked food) thorough sample preparation is necessary. Here SPE (solid phase extraction) has established itself as efficient method due to availability of specially developed phases and procedures for PAH analysis.
	TLC analysis
	Special TLC layers impregnated with electron acceptor reagent are qualified for thin layer chromatographic separation of PAHs. Acting as electron donator PAHs form charge-transfer complexes, which can be separated chromatographically. Funk et al. [1, 2] developed a method, in which TLC plates are dipped in a solution of electron acceptor caffeine. 6 PAHs of the German drinking water specification (TVO) can be separated by TLC after this procedure. In order to avoid individual laborious preparation of TLC plates by dipping and drying, MACHEREY-NAGEL developed Nano-SIL-PAH, a ready-to-use HPTLC plate, which is already impregnated with caffeine. It can be used directly for the method stated in German standard TVO, DIN 38407, part 7.
	HPLC analysis
	Besides the 6 TVO PAHs, further PAHs have been found in environmental samples, which show often interferences at TLC separation. Thus, HPLC methods have been developed in the 1980s (e.g. a method with the additional determination of perylene). By introduction of US American EPA method 610 in many countries the determination by HPLC has been finally established. Special modified C18 phases separate reliably the 16 EPA PAHs. Detection of the separated PAHs can be achieved by UV (250 – 280 nm), with diode array or with fluorescence detection at different wavelengths for excitation and emission. However, acenaphthylene cannot be analysed with fluorescence detection. Older fluorescence detectors still take comparatively long times to switch frequency range, limiting acceleration of the separation run. Modern detectors can switch in much shorter periods – shortness of analysis time is necessary for many environmental laboratories to counteract the increasing quantity of samples and the growing cost pressure. For reduction of analysis time, MACHEREY-NAGEL has introduced NUCLEODUR® C18 PAH columns into the market at the beginning of the year. This C18 phase offers a polymer coating with a specially balanced and highly sterical selectivity for a rapid PAH analysis. Thus, the 16 EPA PAHs can be separated in less than 3 min on a 100 mm length column with UV and fluorescence detection.
	Fast separation of 16 EPA PAHs
	To avoid acetonitrile or buffer, NUCLEODUR® C18 PAH can also be successfully applied with a gradient of water / methanol (see application no. 123830). As new regulations require the determination of 2 additional PAHs (1- and 2-methylnaphthalene), a method with short analysis time has also been developed for the separation of these 18 PAHs (see application no. 123840). Committees of European Food Safety Authority (EFSA) have evaluated around 10,000 analytical investigations about PAHs in food from 18 Member States of the European Union and published a scientific expertise for contaminants in the food chain [3]. PAH content of 16 PAHs - partial different from EPA method - has been reviewed. A separation of these “EFSA PAHs” can be successfully operated with NUCLEODUR® C18 PAH.
	GC analysis
	First 6 PAHs from German drinking water specification have been used to determine PAH by gas chromatography. GC columns of polarity 5 (e.g. OPTIMA® 5) were mostly used with FID detectors. Requirements of lower analytical limits for PAH determination could be fulfilled by introduction of highly sensible MS detectors and suitable GC-MS phases with low bleeding characteristics – application 211160 shows analysis of 16 EPA PAHs on OPTIMA® 5 MS. High sensibility of MS columns has been enhanced furthermore by silarylene phases, such like nonpolar OPTIMA® 5 MS Accent. In addition, medium polar silarylene phase OPTIMA® 35 MS with ultra-low bleeding characteristics shows a good separation of all 16 PAHs of EPA 610. Hence, it is often used as column for a confirmation of analytical results in combination with a 1 MS or 5 MS column.
	Separation of PAHs acc. EPA 610 on silarylene phase
	Besides PAHs also toxic and cancer-causing PCBs (polychlorinated biphenyls) can be found in environmental samples. Centre d´Analyses de Recherche [4] has developed an impressive method for separation of PCBs and PAHs in one single run on high selective OPTIMA® XLB in 2007. The critical determination of PCB-28 / PCB-31 can be achieved in less than 10 min with 82 % separation part. A successful application for samples from slag is shown in application note 212930.
	Sample preparation by SPE
	Preparation of PAH samples is necessary for protection of HPLC or GC instruments and columns, as well as to achieve required detection limits. By sample preparation PAHs are enriched and interfering matrix contaminations are removed. Here SPE (solid phase extraction) with its diverse modified silica gels or polymers has been proven as cost-efficient and time-saving method. Selection of a suitable SPE phase is mainly determined by the sample matrix. Thus, for the enrichment from water silica based C18 or polymeric RP phases are applied (see application nos. 302170, 302790). Good recovery rates are achieved with CHROMABOND® C18 PAH, which is especially balanced for PAHs from water. For water samples containing humic acids removal of these interfering compounds is recommend prior to GC/HPLC analytic. Using combination phase CHROMABOND® NH2/C18 humic acids are removed by NH2 adsorbent while PAHs are enriched on the C18 phase during one loading step (see application no. 301260). Also for extraction from soil samples combination phases can be used. The polycyclic aromatics are selectively adsorbed at CN modified silica of CHROMABOND® CN/SiOH via π-π interactions. Then enriched PAHs are eluted with acetonitrile / toluene, whereas polar soil ingredients remain on silica SiOH.
	Extraction of PAHs from soil
	Hartmann [5] used CHROMABOND® CN/SiOH combination phase also for enrichment of benzo[a]pyrene from meat samples, smoked with artificial aroma. Edible fats and oils are analysed on PAHs with different ring sizes after an extraction with polymeric SPE adsorbent CHROMABOND® HR-P by Weißhaar [6]. Further methods describe sample preparation from oil, crude oil, hexane extracts or blood, serum and plasma.
	Conclusion
	Today, chromatographic analysis of toxic and cancer-causing PAHs is one of the most important routine analyses. By continuous enhancements of products and methods, analysis time per sample could be considerably decreased while sample throughput increased. With newly developed HPLC column NUCLEODUR® C18 PAH 16 EPA PAHs can be separated in less than 3 min. OPTIMA® GC columns provide several methods for efficient PAH determination. For sample preparation from different matrices highly efficient and specialised CHROMABOND® SPE phases are available.
	Literature
	[1] Funk, W., Glück, V., Schuch, B., Donnevert, G., J. Planar Chromatogr. 2 (1992) 28-32 [2] Funk, W., Glück, V., Schuch, B., Becker, J., J. Planar Chromatogr. 2 (1992) 317-320 [3] European Food Safety Authority (EFSA), Polycyclic Aromatic Hydrocarbons in Food [1] – Scientific Opinion of the Panel on Contaminants in the Food Chain, EFSA-Q-2007-136 (August 2008) [4] Centre d´Analyses de Recherche, Lab. d´Hydrologie, Illkirch, France (2007), MN-Appl. No. 212920/212930 [5] Hartmann, K., Deutsche Lebensmittel-Rundschau 96, Heft 5 (2000), 163-166 [6] Weißhaar, R., Eur. J. Lipid Sci. Technol. 104 (2002), 282-285
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