S OLID –L IQUID E XTRACTION

2.3.1 S OLID –L IQUID E XTRACTION

As mentioned earlier, solid –liquid extraction is probably the most widely used procedure in the analysis of pesticides in solid samples. Solid –liquid extraction includes various extraction techniques based on the contact of a certain amount of sample with an appropriate solvent. Figure 2.1 shows a scheme of the different steps

Organic matter

Solvent

FIGURE 2.1 Scheme of the different steps involved in the extraction of a target analyte A from a solid particle.

Sample Handling of Pesticides in Food and Environmental Samples 39 that take place in a solid –liquid extraction procedure and will influence the final

extraction efficiency. In the first stage (step 1), the solvent must penetrate inside the pores of the sample particulates to achieve desorption of the analytes bound to matrix active sites (step 2). Subsequently, analytes have to diffuse through the matrix (step 3) to be dissolved in the extracting solvent (step 4). Again, the analytes must diffuse through the solvent to leave the sample pores (step 5) and be finally swept away by the external solvent (step 6). Obviously, the proper selection of the solvent to be used is a key factor in a solid –liquid extraction procedure. However, other parameters such as pressure and temperature have an important influence on the extraction efficiency. Working at high pressure facilitates the solvent to penetrate sample pores (step 1) and, in general, increasing temperature increases solubility of the analytes on the solvent. Moreover, high temperatures increase diffusion coeffi- cients (steps 3 and 5) and the capacity of the solvent to disrupt matrix –analyte interactions (step 2). Depending on the strength of the interaction between the analyte and the sample matrix, the extraction will be performed in soft, mild, or aggressive conditions. Table 2.1 shows a summary and a comparison of drawbacks and advantages of the different solid –liquid extraction techniques (which will be described later) most commonly employed in the analysis of pesticides in food and environmental samples.

2.3.1.1 Shaking It is a very simple procedure to extract pesticides weakly bound to the sample and is

very convenient for the extraction of pesticides from fruits and vegetables. It just involves shaking (manually or automatically) the sample in presence of an appro- priate solvent for a certain period of time. The most commonly used solvents are acetone and acetonitrile due to their miscibility with water making ease the diffusion of analytes from the solid sample to the solution, although immiscible solvents such as dichloromethane or hexane can also be used for the extraction depending on the properties of target analytes. In a similar manner, the use of mixtures of solvents is a typical practice when analytes of different polarity are extracted in multiresidue analysis. Once analytes have been extracted, the mixture needs to be filtered before further treatments. Besides, since volume of organic solvents used following this procedure is relatively large, it is usually necessary to evaporate the solvent before final determination.

However, shaking might not be effective enough to extract analytes strongly bound to the sample. In order to achieve a more effective shaking, the use of ultrasound-assisted extraction is recommended. Ultrasound radiation provokes molecules vibration and eases the diffusion of the solvent to the sample, favoring the contact between both phases. Thanks to this improvement, both the time and the amount of solvents of the shaking process are considerable reduced.

An interesting and useful modification for reducing both the amount of sample and organic solvents is the so-called ultrasound-assisted extraction in small columns proposed by Sánchez-Brunete and coworkers [1,2] for the extraction of pesticides from soils. Briefly, this procedure just involves placing the sample (~5 g) in a glass column equipped with a polyethylene frit. Subsequently, samples are extracted with

40 TABLE 2.1

Solid–Liquid Extraction Techniques

Technique

Drawbacks Shaking

Description

Advantages

Samples and solvent are placed in a glass vessel.

Filtration of the extract is necessary Shaking can be done manually or mechanically

Simple

Fast (15 –30 min)

Dependent of kind of matrix

Low cost

Moderate solvent consumption (25 –100 mL)

Analysis

Soxhlet Sample is placed in a porous cartridge and

Time-consuming (12 –48 h) solvent recirculates continuously by

Standard method

High solvent volumes (300 –500 mL) distillation –condensation cycles

No further filtration of the extract necessary

Independent of kind of matrix

Solvent evaporation needed

Low cost

of Pesticides

USE Samples and solvent are placed in a glass vessel and

Filtration of the extract is necessary introduced in an ultrasonic bath

Fast (15 –30 min)

Low solvent consumption (5 –30 mL)

Dependent of kind of matrix

Bath temperature can be adjusted

Low cost

in Food

MAE Sample and solvent are placed in a reaction vessel.

Filtration of the extract is necessary Microwave energy is used to heat the mixture

Fast (~15 min)

Low solvent consumption (15 –40 mL)

Addition of a polar solvent is required

and PSE

Easily programmable

Moderate cost

Sample is placed in a cartridge and pressurized

Fast (20

Initial high cost

Environmental

with a high temperature solvent

Low solvent consumption (30 mL)

–30 min)

Dependent on the kind of matrix

Easy control of extraction parameters (temperature, pressure)

High temperatures achieved

Samples Note:

High sample processing

USE, Ultrasound-assisted extraction; MAE, microwave-assisted extraction; PSE, pressurized solvent extraction.

Sample Handling of Pesticides in Food and Environmental Samples 41 around 5 –10 mL of an appropriate organic solvent in an ultrasonic water bath. After

extraction, columns are placed on a multiport vacuum manifold where the solvent is filtered and collected for further analysis.

2.3.1.2 Soxhlet Extraction As indicated earlier, in some cases shaking is not enough for disrupting interactions

between analytes and matrix components. In this regard, an increase of the tempera- ture of the extraction is recommended. The more simple approach to isolate analytes bound to solid matrices at high temperatures is the Soxhlet extraction, introduced by Soxhlet in 1879, which is still the more used technique and of reference of the new techniques introduced during the last few years.

Sample is placed in an apparatus (Soxhlet extractor) and extraction of analytes is achieved by means of a hot condensate of a solvent distilling in a closed circuit. Distillation in a closed circuit allows the sample to be extracted many times with fresh portions of solvent, and exhaustive extraction can be performed. Its weak points are the long time required for the extraction and the large amount of organic solvents used.

In order to minimize the mentioned drawbacks, several attempts toward auto- mation of the process have been proposed. Among them, Soxtec systems (Foss, Hillerød, Denmark) are the most extensively accepted and used in analytical labora- tories and allow reducing the extraction times about five times compared with the classical Soxhlet extraction.

Table 2.2 shows a comparison of the recoveries obtained for several pesticides in soils after extraction using different techniques. In this case, it is clear that ultrasound-assisted extraction allows the isolation of target analytes, whereas the

TABLE 2.2 Recoveries (%) of Pesticides in Soils Obtained by Different Extraction Techniques

Soxhlet Pesticide

Concentration

Ultrasound-Assisted

Extraction Shaking Atrazine

(mg=mL)

0.05 a -Cypermethrin

0.12 Tetrametrin

0.26 Diflubenzuron

0.02 Source: Reproduced from Babic, S., Petrovic, M., and Kastelan, M., J. Chromatogr. A, 823, 3, 1998.

With permission from Elsevier. Experimental conditions : 10 g of soil sample spiked at indicated concentration level. Ultrasound-assisted

extraction: 20 mL of acetone, 15 min; Soxhlet extraction: 250 mL of acetone, 4 h; Shaking: 20 mL of acetone, 2 h.

42 Analysis of Pesticides in Food and Environmental Samples simple shaking is not effective enough to extract the selected pesticides quantitatively.

It is important to stress that recoveries after Soxhlet extraction were too high, which means that a large amount of matrix components were coextracted with target analytes. At this regard, it is clear that an exhaustive extraction is not always required and a balance between the recoveries obtained of target analytes and the amount of matrix components coextracted needs to be established.

2.3.1.3 Microwave-Assisted Extraction Microwave-assisted extraction (MAE) has appeared during the last few years as a

clear alternative to Soxhlet extraction due to the ability of microwave radiation of heating the sample –solvent mixture in a fast and efficient manner. Besides, the existence of several instruments commercially available able to perform the sequen- tial extraction of several samples (up to 14 samples in some instruments), allowing extraction parameters (pressure, temperature, and power) to be perfectly controlled, has made MAE a very popular technique.

Microwave energy is absorbed by molecules with high dielectric constant. In this regard, hexane, a solvent with a very low dielectric constant, is transparent to microwave radiation whereas acetone will be heated in few seconds due to its high dielectric constant. However, solvents with low dielectric constant can be used if the compounds contained in the sample (i.e., water) absorb microwave energy.

A typical practice is the use of solvent mixtures (especially for the extraction of pesticides of different polarity) combining the ability of heating of one of the components (i.e., acetone) with the solubility of the more hydrophobic compounds in the other solvent of the mixture (i.e., hexane). As an example, a mixture of acetone:hexane (1:1) was used for the MAE of atrazine, parathion-methyl, chlorpy- riphos, fenamiphos, and methidathion in orange peel with quantitative recoveries in

<10 min [3]. As a summary, in general, the recoveries obtained are quite similar to those obtained by Soxhlet extraction but the important decrease of the extraction time (~15 min) and of the volume of organic solvents (25 –50 mL) have made MAE to be extensively used in analytical laboratories.

2.3.1.4 Pressurized Solvent Extraction Pressurized solvent extraction (PSE), also known as accelerated solvent extraction

(ASE), pressurized liquid extraction (PLE), and pressurized fluid extraction (PFE), uses solvents at high temperatures and pressures to accelerate the extraction process. The higher temperature increases the extraction kinetics, whereas the elevated pressure keeps the solvent in liquid phase above its boiling point leading to rapid and safe extractions [4].

Figure 2.2 shows a scheme of the instrumentation and the procedure used in PSE. Experimentally, sample (~10 g) is placed in an extraction cell and filled up with an appropriate solvent (15 –40 mL). Subsequently, the cell is heated in a furnace to the temperatures below 2008C, increasing the pressure of the system (up to a

20 Mpa) to perform the extraction. After a certain period of time (10 –15 min),

Sample Handling of Pesticides in Food and Environmental Samples 43

ASE ® Schematic

Load sample into cell. Pump Fill cell with solvent.

Time (min)

0.5-1

Heat and pressurize cell. 5 Purge valve Oven

Solvent

Hold sample at pressure and temperature.

Extraction Pump clean solvent into

cell sample cell.

Static Purge solvent from cell

valve with N 2 gas.

1-2

Collection Extract ready for analysis. Total 12-14

Nitrogen vial

FIGURE 2.2 Pressurized solvent extraction equipment. (Courtesy of Dionex Corporation. With permission.)

the extract is directly transferred to a vial without the necessity of subsequent filtration of the obtained extract. Then, the sample is rinsed with a portion of pure

solvent and finally, the remaining solvent is transferred to the vial with a stream of nitrogen. The whole process is automated and each step can be programmed, allowing the sequential unattended extraction of up to 24 samples.

This technique is easily applicable for the extraction of pesticides from any kind of sample and the high temperature used allows to perform very efficient extraction in a short time. In addition, the considerable reduction in the amount of organic solvents used makes PSE a very attractive technique for the extrac- tion of pesticides. The main limitations of this technique are the high cost of the apparatus and the unavoidable necessity of purifying obtained extracts, which is common to other efficient extraction techniques based on the use of organic solvents as mentioned earlier.