by Dr C Mahugo Santana, Dr M E Torres Padrón, Dr Z Sosa Ferrera,Dr J J Santana Rodríguez
Heavily used in industry and in agriculture, phenols are some of the most wide-spread contaminants in the environment. Because of their toxicity and presence in the environment, certain phenols have been included in the “priority pollutant” lists of the EU and the US EPA.

Phenolic compounds in the environment
Industrial processes such as the production of drugs, textiles, dyes, pesticides and paper, are the main source of phenolic compounds in the environment. In addition, chlorophenols have been widely used as wood preservative agents and disinfectants for decades, which also results in their release into the environment media. Phenols are also created by natural processes, e.g. by the degradation of humic substances, tannins and lignins. Detection of the presence of some chlorophenols in relatively pristine areas suggests that they may be formed in forest fires.

Phenols that are more hydrophilic, e.g. less chlorinated phenols, can easily enter the aquatic environment, while non polar compounds, e.g. more chlorinated phenols such as pentachlorophenol, usually persist longer in soils and sediments in the environment. As a result of their hydrophobic properties, the compounds tend to accumulate in the lipid stores of animals and humans.
 
Toxic effects of phenolic compounds
The potential of chemicals from the environment to disrupt the endocrine system of animals and humans has attracted a great deal of public concern. Some chlorophenols are known to possess carcinogenic and immunosuppressive properties. In addition, they can affect the taste and odour of drinking water with concentrations as low as a few µg·L-1. As a consequence, both the US Environmental Protection Agency (EPA) and the European Union (EU) have included some phenols, mainly chlorophenols and nitrophenols, in their lists of priority pollutants. EU directive 2455/2001/EC set a maximum total concentration of 0.5 µg·L-1 in drinking water with individual concentrations not exceeding 0.1 µg·L-1 [1].

Chlorophenols
Although the use of pentachlorophenol is prohibited in most countries, it is still widely found in the wood used in pallets, containers, crates and in cardboard, paper, etc. It has been used in the maintenance of boats, trailers, station wagons, fences, outdoor furniture and similar articles and has been detected in the effluents of many industries.  These industries include coal mining, iron and steel manufacturing, pharmaceutical manufacturing, textile, pulp and paperboard mills and steam electric power plants.

Since wooden crates and cardboard boxes are often used to store and transport fresh fruits, chlorophenols such as pentachlorophenol that are present in the packaging may contaminate the stored fruits by migration. Chlorinated phenols can also be generated from non-chlorinated phenols by the chlorination of drinking water. Tri-, tetra- and pentachlorophenol are considered the precursors in the formation of corresponding chloroanisoles, known to be powerful odourants in corks and wine. This is one of the most critical problems in the wine industry.

Nitrophenols
This class of molecules is released into the environment in waste water and as fugitive emissions during production and use as chemical intermediates, primarily in the manufacture of the insecticides parathion, methyl parathion and N-acetyl-p-aminophenol. Agricultural workers using parathion may be exposed to nitrophenols dermally or via inhalation since they are both degradation products and impurities in pesticides. When released on soil, nitrophenols do not adsorb significantly to soil except for some clays. Nitrophenols are also found in suspended particulate matter in the atmosphere, originating mostly from secondary photochemical reactions in the air and partly from emissions of vehicle exhaust gas.

 
Determination of phenols
Analytical techniques commonly used in the determination of phenols are high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) in combination with ultraviolet (UV), electrochemical or mass spectrometry detection (MS). In addition, gas chromatography (GC) has been used, using several detection methods such as flame ionisation detection (FID), electron-capture detection (ECD) or mass spectroscopy.  If GC is used a derivatisation step is needed.

Despite the fact phenolic compounds are widely distributed in the environment, they are present in low concentrations; an enrichment step therefore needs to be carried out prior to their separation and detection. Current official analytical methods for phenolic compounds extraction are liquid-liquid extraction (LLE) for liquid samples, and Soxhlet extraction for solid samples (US EPA Methods 604, 605, 8041 and 3540 B, respectively). These methods require expensive and hazardous organic solvents, which are undesirable for health and disposal reasons; in addition, the analysis takes a long time.

 
Improved methods of determination
The setting up of new analytical methods which improve quality and sensitivity in the determination of environmental pollutants, in both liquid and solid samples, is one of the main lines of research in environmental chemistry. A main focus is to replace traditional sample extraction methods with other approaches that are more sensitive, selective, rapid and environmentally friendly. Furthermore, the detection limits imposed by current environmental quality legislation can only be achieved by using appropriate sample preparation techniques, which provide high enrichment factors for the analytes.

 
Preparation of liquid samples
Due to the disadvantages of current official analytical methods used to extract phenolic compounds from liquid samples, there is an increasing tendency to replace them by other techniques to reduce both the consumption of organic solvents and the time of analysis. One such alternative technique is solid-phase extraction (SPE) [2]. However, SPE has some limitations, especially for analytes such as phenolic derivatives, which exhibit different behaviour in terms of polarity and acidity. Solid phase micro-extraction (SPME) has been developed to resolve some of the drawbacks that present the SPE technique. As for the solvents used to desorb the phenolic compounds in SPE and SPME, organic solvents such as methanol, acetonitrile or a mixture of both are the most frequently used [3].

Other methods have been developed with a view to eliminate or, at least, to minimise the use of organic solvents. As an alternative to organic solvents, the use of micellar systems to extract and pre-concentrate organic compounds from aqueous or solid samples, offers advantages such as safety and cost, as well as compatibility with the aqueous-organic mobile phase in liquid chromatography (LC) [4]. Aqueous solutions of certain surfactants have been used to extract and pre-concentrate phenols from liquid samples in several techniques, one of which is cloud-point methodology (CPE). This is based on the system’s capacity to establish two different phases, namely a surfactant-rich phase and an aqueous phase as a function of temperature, surfactant concentration, equilibration time, salts addition or acid addition. The small volume of surfactant-rich phase allows the extraction and pre-concentration of the analytes in a single step prior to their determination.

A solid phase microextraction method with a new desorption mode using a micellar medium as desorbing agent has been developed. This approach achieves high efficiencies in the extraction stage and can be combined with HPLC (SPME-MD-HPLC)  [5]. In this procedure, desorption is carried out using a non-ionic surfactant in a 100 µL conical glass vial to avoid the disadvantages that are present in the desorption chamber using organic solvent.

 
Preparation of solid samples
Although most attention has been focused on the determination of phenolic compounds in aqueous samples, more substituted phenols, such as pentachlorophenol, are more likely to be absorbed in sediments and soils. This is the reason for the persistence of these compounds in the environment which results in high concentrations that could affect both earth and aquatic organisms. Probably because of its simplicity and inexpensive extraction apparatus, Soxhlet extraction is one of the most popular techniques for isolating phenolic compounds from solid samples. However Soxhlet extraction suffers from the disadvantage that it is excessively time consuming and requires large amount of hazardous organic solvents.

In this last decade, microwave energy has been widely applied to extract analytes from matrices. Since its development, microwave assisted extraction (MAE) has became a viable alternative to conventional methodologies; it has many substantial improvements over other techniques, such as shorter extraction time, lower amount of solvent and the ability to analyse multiple samples at the same time. For the extraction of phenols, solvent mixtures such as acetone-hexane and acetone-methanol are usually employed [6], but it is also possible to use surfactants in microwave extraction. This technique, known as microwave assisted micellar extraction (MAME), provides an alternative to organic solvents, thereby reducing cost and toxicity, and creating an environmentally friendly extraction method [4].

References
1. IARC, in: IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, IARC, Lyon, France, 1999, p. 769.
2. Rodríguez I, Llompart MP & Cela, R. Solid-phase extraction of phenols. J Chromatogr A 2000; 885: 291.
3. González-Toledo E, Prat MD & Alpendurada MF. Solid-phase microextraction coupled to liquid chromatography for the analysis of phenolic compounds in water. J Chromatog A 2001; 923: 45.
4. Sosa-Ferrera Z, Padrón-Sanz C, Mahugo Santana C & Santana-Rodríguez JJ. The use of micellar systems in the extraction and pre-concentration of organic pollutants in environmental samples. Trends Anal. Chem 2004; 23: 469.
5. Mahugo Santana C, Torres Padrón ME, Sosa Ferrera Z & Santana Rodríguez JJ. Development of a solid-phase microextraction method with micellar desorption for the determination of chlorophenols in water samples: Comparison with conventional solid-phase microextraction method. J. Chromatogr A 2007; 1140: 13.
6. Llompart  MP, Lorenzo RA, Cela R & Jocelyn Paré JR. Optimization of a microwave-assisted extraction method for phenol and methylphenol isomers in soil samples using a central composite design. Analyst 1997; 122: 133.

 The authors
Dr C. Mahugo Santana, Dr M.E. Torres Padrón,
Dr Z. Sosa Ferrera, Dr J.J. Santana Rodríguez
Department of Chemistry
Faculty of Marine Sciences
University of Las Palmas de Gran Canaria
35017 Las Palmas de Gran Canaria, Spain.
E-mail: jsantana@dqui.ulpgc.es