Pressurized solvent extraction or »accelerated solvent extraction," employs temperatures that are higher than those used in other methods of extraction, and requires high pressures to maintain the solvent in a liquid state at high temperatures. It is best suited for the rapid and reproducible initial extraction of a number of samples. The solid biomass sample is loaded into an extraction cell, which is placed in an oven. The solvent is then pumped from a reservoir to fill the cell, which is heated and pressurized at programmed levels for a set period of time. The cell is flushed with nitrogen gas, and the extract, which is automatically filtered, is collected in a flask. Fresh solvent is used to rinse the cell and to solubilize the remaining components. A final purge with nitrogen gas is performed to dry the material. High temperatures and pressures increase the penetration of solvent into the material and improve metabolite solubilization, enhancing extraction speed and yield. Moreover, with low solvent requirements, pressurized solvent extraction offers a more economical and environment-friendly alternative to conventional approaches

As the material is dried thoroughly after extraction, it is possible to perform repeated extractions with the same solvent or successive extractions with solvents of increasing polarity. An additional advantage is that the technique can be programmable, which will offer increased reproducibility. However, variable factors, e. g., the optimal extraction temperature, extraction time, and most suitable solvent, have to be determined for each sample (Kaufmann, 2002; Tsubaki, 2010; Sarker, 2006).

Microwave-assisted extraction (MAE) or simply microwave extraction is a relatively new extraction technique that combines microwave and traditional solvent extraction. The microwave energy has been investigated and widely applied in analytical chemistry to accelerate sample digestion, to extract analytes from matrices and in chemical reactions. Application of microwaves for heating the solvents and plant tissues in extraction process, which increases the kinetic of extraction, is called microwave-assisted extraction. Microwave energy is a non-ionizing radiation that causes molecular motion by migration of ions and rotation of dipoles, without changing the molecular structures if the temperature is not too high. Nonpolar solvents, such as hexane and toluene, are not affected by microwave energy and, therefore, it is necessary to add polar additives. Microwave-assisted extraction (MAE) is an efficient extraction technique for solid samples which is applicable to thermally stable compounds accepted as a potential and powerful alternative to conventional extraction techniques in the extraction of organic compounds from materials. The microwave-assisted extraction technique offers some advantages over conventional extraction methods.

Compared to conventional solvent extraction methods, the microwave-assisted extraction (MAE) technique offers advantages such as improved stability of products and marker compounds, increased purity of crude extracts, the possibility to use less toxic solvents, reduced processing costs, reduced energy and solvent consumption, increased recovery and purity of marker compounds, and very rapid extraction rates.

The use of MAE in natural products extraction started in the late 1980s, and through the technological developments, it has now become one of the popular and cost-effective extraction methods available today, and several advanced MAE instrumentations and methodologies have become available, e. g., pressurized microwave-assisted extraction (PMAE) and solvent-free microwave-assisted extraction (SFMAE).

Comparison between conventional and MAE extraction method

This technique has been used successfully for separation of phenolic compounds from types of biomass, polyphenols derivates, pyrimidine glycosides, alkaloids, terpenes, and so.

In most cases, the results obtained suggested that the microwave assisted method was more convenient even compared to the ultrasound extraction method.

Pyrimidine glycosides

The studies regarding the microwave extraction of vicine and convicine (toxic pyrimidine glycosides) from Vicia faba using a methanol: water mixture (1:1 v/v) involves two successive microwave irradiations (30 s each) with a cooling step in between. No degradation could be observed under these conditions, but further irradiation was found to decrease the yield of vicine and convicine. The yield obtained was 20% higher than with the conventional Soxhlet extraction method.

Alkaloids Sparteine, a lupine alkaloid, was extracted from Lupinus mutabilis, with methanol: acetic acid (99:1, v/v) in a common microwave oven and the microwave irradiation program used one to five cycles of 30 s with a cooling step in between and conduct to 20% more sparteine than was obtained with a shaken-flask extraction using the same solvent mixture for 20 min.

Terpenes Five terpenic compounds: linalool, terpineol, citronellol, nerol and geraniol, associated with grape (Vitis vinifera) aroma was extracted from must samples by MAE (Carro et al.,1997). Was investigated the influence of the parameters: extracting solvent volume, extraction temperature, and amount of sample and extraction time. Several conditions were fixed, such as the extraction time (10 min) and the applied power (475 W). The solvent volume appeared to be the only statistically significant factor, but was limited to 15 mL by the cell size. The highest extraction yield was obtained with both the solvent volume and the temperature at their maximum tested values. In contrast, the sample amount had to be minimized in order to obtain the best recoveries. The final optimized extraction conditions were as follows: 5 mL sample amounts extracted with 10 mL of dichloromethane at a temperature of 90°C for 10 min with the microwave power set at 50% (475 W).

Steroids Recently, was demonstrated that only 30-40 s were sufficient to extract ergosterol quantitatively by MAE using 2 mL methanol and 0.5 mL 2 M sodium hydroxide. Microwave irradiation was applied at 375W for 35 s and the samples were cooled for 15 min before neutralization with 1 M hydrochloric acid followed by pentane extraction. The yield was similar to or even higher than that obtained with the traditional methanolic extraction followed by alkaline saponification and pentane extraction.

Alkaloids The extraction of two alkaloids cocaine and benzoylecgonine by focused MAE was optimized by taking into account several parameters such as the nature of the extracting solvent, particle size distribution, sample moisture, applied microwave power and radiation time. MAE was found to generate similar extracts to those obtained by conventional SLE but in a more efficient manner. Indeed, 30s were sufficient to extract cocaine quantitatively from leaves, using methanol as solvent and a microwave power of 125 W. (Kaufmann, 2002).

Phenolic compounds

In recent years, synthetic antioxidants were reported to have the adverse effects such as toxicity and carcinogenicity and this situation has forced scientists to search for new natural antioxidants from herbs or the other materials. Phenolic compounds, the most important bioactive compounds from plant sources, are among the most potent and therapeutically useful bioactive substances, providing health benefits associated with reduced risk of chronic and degenerative disease (Luthria, 2006; Tsubaki et al., 2010; Proestos, 2008).

Extraction is one of the most imperative steps in the evaluation of phenolic compounds from plant. Often is done a saponification prior to the extraction step because is necessary to cleave the ester linkage to the cell walls (Robbins, 2003).

The capability of a number of extraction techniques have been investigated, such as solvent extraction and enzyme-assisted extraction. However, these extraction methods have drawbacks to some degree. For example, solvent extraction is time consuming and enzyme in enzyme assisted extraction is easy to denature. In the case of Soxhlet extraction, the extraction time vary from 1 minute to 6 h. Ultrasonic is one of the most industrially used methods to enhance mass transfer phenomena (Japon-Lujan et. al. 2006; Luthria, 2006; Perez-Serradilla, 2007). Meanwhile, microwave assisted extraction heats the extracts quickly and significantly accelerates the extraction process (Martinez, 1996; Kojima, 2004; Patsias, 2009). Simultaneous ultrasonic/microwave assisted extraction (UMAE) coupled the advantage of microwave and ultrasonic, presenting many advantages (Kojima, 2004).

Extraction of phenolic compounds from solid samples is usually carried out by stirring (Luthria, 2006; Nepote, 2005), although the use of auxiliary energies has demonstrated to accelerate the process (Japon-Lujan, et. al.2006; Perez-Serradilla, 2007). Microwave-assisted extraction (MAE) is the process by which microwave energy is used to heat polar solvents in contact with solid samples and to partition compounds of interest between the sample and the solvent, reducing both extraction time and solvent consumption.

The conventional liquid-solid extraction techniques, such as heat reflux extraction (HRE), ultrasonic extraction (UE) and maceration extraction (ME), are discommodious, laborious, time-consuming and require large volumes of toxic organic solvents. So increasing attention is paid to the development of more efficient extraction methods for the rapid extraction of active compounds from materials.

The current analytical methods used to extract phenolic compounds from liquid samples are based on liquid-liquid extraction (LLE). Although this technique offers efficient and precise results, it is relatively time-consuming, possibly harmful due the use of large volume of organic solvents (frequently toxic) and highly expensive. For these reasons, there is an increasing tendency to replace LLE by solid-phase extraction (SPE) for liquid samples. SPE was developed in the 1980s, and has emerged as a powerful tool for chemical isolation and purification. This methodology is an alternative extraction to LLE due to it reduces organic solvents consumption, the length of analysis and it can be automated (Martinez, et. al., 1996; Kojima, 2004; Patsias, et. al., 2009).

Although most attention has been focused on the determination of phenolic compounds in aqueous samples, more substituted phenols, such as pentachlorophenol, show limited transport in water and they are more likely absorbed in sediments and soils. This fact contributes to the persistent of these compounds in the environment and it results in high concentrations of them that could affect aquatic and earth organism. For extraction, Soxhlet extraction is one of the most popular techniques for isolating phenolic compounds from solid samples, due to its simplicity, inexpensive extraction apparatus. Despite the good results obtained with this methodology, Soxhlet extraction makes the analysis procedure excessive time consuming. Moreover, it requires large amount of hazardous organic solvents.

Ultrasonic extraction is another conventional technique to extract analytes from solid samples. Although sonication is faster than Soxhlet extraction, it also requires large volumes of toxic and expensive organic solvents.

The studies show that the compounds are extracted more effectively when the energy provided by microwave is employed (Perez-Serradilla, 2011).