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J. Biochem. Biophys. Methods 70 (2007) 253 261 www.elsevier.

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Review

Techniques of preparing plant material for chromatographic separation and analysis


G. Romanik a,, E. Gilgenast a,1 , A. Przyjazny b , M. Kamiski a,1
a

Gdask University of Technology, Chemistry Department, Analytical Chemistry Division, 80 952 Gdask, ul. Narutowicza 11/12, Poland b Science and Mathematics Department, Kettering University, 1700 West Third Avenue, Flint MI 48504, USA Received 28 June 2006; accepted 29 September 2006

Abstract This paper discusses preparation techniques of samples of plant material for chromatographic analysis. Individual steps of the procedures used in sample preparation, including sample collection from the environment or from tissue cultures, drying, comminution, homogenization, leaching, extraction, distillation and condensation, analyte enrichment, and obtaining the final extracts for chromatographic analysis are discussed. The techniques most often used for isolation of analytes from homogenized plant material, i.e., Soxhlet extraction, ultrasonic solvent extraction (sonication), accelerated solvent extraction, microwave-assisted extraction, supercritical-fluid extraction, steam distillation, as well as membrane processes are emphasized. Sorptive methods of sample enrichment and removal of interferences, i.e., solid-phase extraction, and solid-phase micro-extraction are also discussed. 2006 Elsevier B.V. All rights reserved.
Keywords: Plant material; Sample preparation; Chromatographic analysis

Contents 1. 2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preliminary sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Collection of plant material and preparation of sample for analysis general principles 2.2. Drying, auxiliary techniques and procedures for volatile compounds . . . . . . . . . . . 2.3. Comminution and homogenization . . . . . . . . . . . . . . . . . . . . . . . . . . . . Techniques of isolation of analytes from plant material . . . . . . . . . . . . . . . . . . . . . 3.1. Extraction/leaching general principles . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Soxhlet extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Ultrasonic extraction (sonication) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Accelerated solvent extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Microwave-assisted extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Steam distillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. Membrane processes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8. Supercritical fluid extraction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9. Solid-phase micro-extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.10. Sample disruption method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.11. Comparison of extraction techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254 254 254 255 256 256 256 257 257 257 258 258 258 258 259 259 259

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Corresponding author. Tel.: +48 58 347 25 58; fax: +48 58 347 26 94. E-mail address: anyzar@wp.pl (G. Romanik). 1 Tel.: +48 58 347 25 58; fax: +48 58 347 26 94. 0165-022X/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jbbm.2006.09.012

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4. Solid-phase extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260 5. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260

1. Introduction Plant metabolites often occur as complex mixtures of many substances of a wide range of polarity and hydrophobicity. The most important groups of substances in plant material are: lowpolar (waxes, terpenoids), semi-polar (lipids, phenolic compounds, low-polar alkaloids), and high-polar (polar glycosides, polar alkaloids, saccharides, peptides, proteins). The sample preparation procedure for the investigation of the composition of plant material includes at least three steps, the first two being always used, while the third one is common, although not required: 1. Preliminary sample preparation, preceded by comminution or homogenization of the examined material. The techniques commonly employed at this stage involve drying, lyophilization, or steam distillation. 2. Extraction/leaching of soluble components of the examined material with suitable solvents or their mixtures, or a supercritical fluid, including desorption, hydrolysis, saponification, etc. When planning extraction/leaching of the metabolites, it should be realized that a fraction of the total amount of the components can be adsorbed or otherwise bound to the cellular wall or organelles. Oftentimes, the metabolites can form adducts with peptides or phospholipids. Alternatively, the metabolites can be present in the form of glycosides. Each step of a sample preparation procedure can result in loss of a fraction of analyte, and this is especially important when the amounts of isolated substances are very small. Consequently, in order to relate the content of metabolites in the final extract to their content in the tissue or organism, it is necessary to determine the socalled recovery, which in turn can be inaccurate for the metabolites adsorbed or otherwise bound to the cellular wall or high-molecular-weight components of the cell. The range of extracted components depends on the kind of extrahent and conditions of the extraction process. First, solvents and conditions that enable isolation of a wide range of metabolites are used. The extracts will contain metabolites and other substances which can interfere with their analysis belonging to such groups as lipids, phospholipids, glycosides, saccharide, peptides, and other products of plant metabolism. To reduce the error in determining the recovery, the standard addition method is used for homogenized plant material. However, even this approach may not eliminate the analytical error, particularly when the standard added does not undergo natural physiological processes in dried or homogenized material. 3. Analyte enrichment with the simultaneous removal of interferences by techniques such as liquid/liquid extraction, solid-phase extraction (SPE), selective adsorption, prepara-

tive liquid chromatography (PLC) with normal (NP-PLC) or reversed (RP-PLC) stationary phases, ion chromatography (IC), size-exclusion chromatography (SEC), as well as solidphase micro-extraction (SPME), or solvent micro-extraction. The sample, prepared by the procedures described above, is subjected to separation and final determination, primarily by liquid chromatography, electrophoresis, or gas chromatography. Qualitative analysis, i.e., identification of selected or (less commonly) all components, is performed first. Next, quantitative analysis is carried out, following calibration of the instrument. Simultaneous performance of both qualitative and quantitative analysis is preferred in order to save time. 2. Preliminary sample preparation 2.1. Collection of plant material and preparation of sample for analysis general principles The determination of the composition of plants, fungi, or bacteria is based on consecutive (or, very rarely, simultaneous) application of several techniques for preliminary preparation of the examined material. First, the material is dried or lyophilized, followed by comminution or homogenization. Next, the material is extracted (leached) with a specific solvent or either a series or mixture of solvents. Each extract (leachate) is purified by removing solids via filtration, ultrafiltration, or centrifugation. The majority of these sample preparation techniques have been used for a long time. However, recently some of the classical extraction techniques carried out at elevated temperature, such as several-day maceration, long-term leaching with stirring, agitation in water or a buffer solution, and Soxhlet extraction are being replaced with more modern techniques, which are more effective, require less solvent, and permit more readily automation of the apparatus and procedures [1]. The main objective of sample preparation procedures is the selective isolation of analytes with the simultaneous matrix simplification. Distillation and purification of the extract via removal of solids is followed by the removal of interferences with a concurrent enrichment of the extract in the analytes. The concentration of interferences can substantially exceed that of the analytes [2]. Sample enrichment aims at increasing the concentration of analytes above the detection limit of the instrument used for their determination [3]. An important requirement in analytical chemistry is that the sample analyzed be representative. This means that samples must be collected, treated and stored in such way that their chemical composition is similar to the average composition of the total material. In the analysis of plant material the collection of a representative sample is difficult due to variability of individual plants among a species or variety.

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Consequently, a specific program of sample collection is often required. For example, at a selected site a dozen or so plants are collected from a small area (ca. 5 cm2). Soil and contaminants are removed from the plant material collected. Each plant is rinsed with deionized water, removing particles loosely bound to the plant. In addition, the plant material is rinsed with other liquids or solutions, containing complexing agents such as EDTA, with dilute HCl solutions or, sometimes, with organic solvents [4].

2.2. Drying, auxiliary techniques and procedures for volatile compounds The analysis of natural samples is often carried out on dried materials. This allows the determination of specific components on a dry mass basis and largely reduces the problems associated with high water content in crude samples (living organisms, their fragments, or tissues). It should be realized, however, that drying plant material does not remove water completely and the term

Table 1 Compilation of various operations and steps of the procedure associated with preparation of biological material for chromatographic analysis No. Operation, step of procedure 1 Comminution, homogenization (breaking, cutting, grinding, disintegration or lysis of cellular wall) Division of sample Decomposition of glycosides, adsorbates, aggregates, complexation of metal ions, acidic, basic or enzymatic hydrolysis Leaching, extraction of metabolites Soxhlet extraction Ultrasonic extraction (sonication) Microwave-assisted extraction (MAE) Accelerated solvent extraction (ASE) Supercritical fluid extraction (SFE) Steam distillation Decantation/filtration or centrifugation/ ultracentrifugation Drying extracts Objective Increasing interfacial surface area; obtaining a homogeneous sample Sample storage, saving a spare sample Obtaining free forms of analytes Remarks References

2 3

Used only infrequently

Analyte solubilization

[68]

5 6

Derivatization of analytes

Complete or partial solvent evaporation, usually with solvent exchange

Extract enrichment in analytes and removal of interferences using: SPE, liquidliquid extraction, steam distillation, supercritical fluid extraction

10 11

Storage of extracts Calibration

12

Method validation

Separation of solvent from plant material particles, Especially important when grinding a sample separation of extract and raffinate, colloid peptization in the presence of a solvent Removal of water, which can interfere with the Drying can be accomplished by: analysis due to: Change in surface activity of the stationary phase Passing the extract through a column packed with a drying agent Shortening chromatographic column life Adding a drying agent (e.g. anhydrous Na2SO4) to the extract Stopping flow of carrier gas as a result of flooding capillary column in case of gas chromatography of volatile analytes Conversion of analytes into derivatives with properties enabling: Simple determination Higher stability of analytes Increased volatility Higher sensitivity of determination Increasing analyte concentration in the extract; This step makes use of: replacing solvent (through redissolving dry Evaporation in a rotary evaporator (often at residue) with the one more suitable for subsequent a reduced pressure) steps of the analytical procedure Evaporation in a stream of inert gas Increasing concentration of analytes, removal of Can be accomplished by using: interferences 1. Gel chromatography 2. Column chromatography with: silica gel alumina Florisil Proper planning of work in the analytical laboratory Extracts should be stored in tightly capped vials at lowered temperature (below 0 C) Preparation for the final determination Most common calibration methods are: calibration curve internal standard standard addition Investigation whether the procedure is valid for Reference materials and certified reference specific applications material are required; typically carried out together with validation of the entire analytical procedure

[9] [8]

[11]

[6,7]

[6,7]

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dry mass means that the material contains from several to a dozen or so percent water. The drying of natural materials is frequently performed at 70 C in ventilated ovens or in ventilated ovens with a flow of warm air or, sometimes, nitrogen. Convective ovens are often used in small laboratories. Laboratory vacuum ovens with water absorption, adsorption, or freezing-out systems, placed before a vacuum pump, have been found effective for the isolation of nonvolatile and nonsubliming substances. Recent literature reports indicate that effective drying can be accomplished at temperatures as low as 4050 C [4,5]. Low drying temperature is especially important when the analytes are relatively volatile or subliming. In this case, however, vacuum ovens cannot be used. In case of highly volatile substances, drying should be replaced with distillation in a stream of warm gas (typically nitrogen) and freezing out the distillate or, in case of nonpolar compounds of medium volatility, with steam distillation. This refers to substances with boiling points below ca. 250 C as well as the substances subliming under these conditions. Any preparation of natural material for analysis also requires the determination of water content in the primary sample, while realizing that it may be highly variable and depend on the developmental stage of the plant, on the kind of species, cultivation conditions, storage of the material, etc. Following preliminary preparation of a plant sample, various techniques of isolation of specific groups of analytes are used. The operations used to prepare biological material for chromatographic analysis are compiled in Table 1. 2.3. Comminution and homogenization The next operation in preparing raw plant material for analysis proper is comminution and homogenization [10]. The choice of comminution technique depends on the consistency of the material and its hardness. In case of raw materials containing essential oils, any increase in temperature should be avoided. The material should be comminuted in small batches, which prevents the loss of essential oils [11]. Roots, hard stems, fruits

and seeds are first cut mechanically or manually and then ground in any of a number of mechanical mills [11]. Comminution by manual cutting is simple and does not require sophisticated tools. It results in fragments of different sizes; thus, sieving of the cut material is recommended. These techniques have been used, for example, for comminution of onion and garlic, which were subsequently extracted without further homogenization of the sample material [12]. Serine was isolated from yam by dicing the roots, followed by homogenization in a medium of three extracting agents [13]. Comminution usually precedes the next stage of sample preparation, i.e., homogenization of the material. In the laboratory, homogenization is often carried out with ceramic or agate mortar-and-pestle sets. A variety of mechanical homogenizers are also employed, although their use can result in local overheating of the material and thermal degradation of thermolabile substances despite cooling. High-energy ultrasonic vibrations, freezing under conditions resulting in the rupture of cellular wall or membrane, enzymatic lysis (usually hydrolysis), and other nonmechanical physicochemical processes can also be employed for sample homogenization [5]. 3. Techniques of isolation of analytes from plant material 3.1. Extraction/leaching general principles In order to isolate the analytes from plant material, extraction/leaching with various solvents is used, as a rule, in order of increasing polarity of the extracting agent [11,14]. The analytical procedure is shown in Fig. 1. Making use of various solvents, extracts containing different analytes can be obtained (Extracts A, B, C, D). The procedure should be carried out in several steps so that particular analytes are present in one extract only, while others are present in different extracts A in Fig. 1. Application of additional operations, for example extract purification, results in obtaining further fractions (Fractions I, II) B in Fig. 1. Each of the fractions can then be

Fig. 1. Separation steps used for isolation of plant metabolites [14].

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chromatographically separated into individual components C in Fig. 1. 3.2. Soxhlet extraction Soxhlet extraction is one of the oldest techniques for isolating metabolites from natural material. The technique is used for the isolation and enrichment of analytes of medium and low volatility and thermal stability. It allows a high recovery, but has a number of shortcomings, including long extraction time and large consumption of solvents, cooling water and electric energy. Another disadvantage of Soxhlet extraction is lowered extraction efficiency due to the fact that the temperature of condensed solvent flowing into the thimble is lower than its boiling point [15,16]. These disadvantages are partially eliminated by the automated Soxhlet extractor recently introduced to the market. As a result, the extraction time is shortened considerably, while reproducibility of the results is comparable with the classical Soxhlet extraction [15]. This extraction is now in common use, being applied to the determination of, among others, lipids and polycyclic aromatic hydrocarbons in natural products (e.g., coffee, soybean and coconut oil, mushrooms, fruits, and vegetables) [15,17]. Soxhlet extraction was also used in investigations of antiinflammatory and antibacterial properties of plant metabolites. Nine African plants were examined and their therapeutical properties determined on the basis of pharmacological properties of the extracts [18]. 3.3. Ultrasonic extraction (sonication) Ultrasounds are waves with frequencies ranging from 16 kHz to 1 GHz, inaudible to humans. Ultrasonic vibrations are the source of energy facilitating the release of some analytes from the sample matrix. The improvement in extraction efficiency due to ultrasound appears at certain values of so-called acoustic pressure [19]. Among the most important phenomena taking place in the acoustic field are: cavitation (generation and collapse of mostly empty cavities), friction at the boundary and interfacial surfaces, and increase in the diffusion rate. Cavitation is the most significant phenomenon, because it has a direct effect on a number of phenomena occurring in a liquid subjected to ultrasound. Cavitation involves the formation of pulsating bubbles as a result of strong stretching forces, originating from abrupt local pressure drops [19]. At constant ultrasound intensity, dynamic equilibrium is established between the forming and collapsing bubbles. The process of generation and collapse of the cavities actively interacts with the liquid/solid boundary surface, thus enhancing the erosion processes of solids [19]. The average time of ultrasonic extraction typically ranges from a few to 30 min, although it can be as long as 70 min (Table 2). The recoveries obtained during this time are comparable to those obtained after a dozen or so hours of Soxhlet extraction, carried out at the same temperature. The extraction conditions can be optimized with respect to time, polarity and amount of solvent, and the mass and kind of sample. The investigations

Table 2 Substances isolated using ultrasonic extraction Analyte Cobalamins Tartaric and malic acid Isoflavones Isoflavonoids Flavonoids Polysaccharides Volatile compounds Aroma compounds Steroids and triterpenoids Antioxidants Sample Biological samples Grapes Recovery [%] Time [min] Reference [21] [22] [23] [24] [25] [26] [27] [28] [29] [30]

94.8101.1 1 30 20 60 60 70 10 30 30 60/45

Soybeans 100 Root 83 Plant extract 91.295.6 Buckwheat hulls 147 Citrus flowers and honey Aged brandies 45113 Stems, leaves and flowers Herbs

carried out by Melecchi et al. [20] have demonstrated that solvent polarity and extraction time have the greatest effect on the recovery. Examples of substances isolated by ultrasonic extraction along with extraction time and recoveries are compiled in Table 2. The advantage of this technique is the possibility of extraction of many samples at once in an ultrasonic bath. The extraction is carried out at room temperature, which makes it suitable for the extraction of thermally labile analytes. The need for separation of the extract from the sample following the extraction is a disadvantage of this technique. 3.4. Accelerated solvent extraction Accelerated solvent extraction (ASE) makes use of the same solvents as do other extraction techniques, but at an increased pressure (ca. 100140 atm) and at an elevated temperature (50 200 C). The design of the extractor, capable of withstanding high pressures, allows the extraction temperature to be raised above the boiling point of the solvent used. The high pressure allows maintaining the solvent in a liquid state at a high temperature. Under these conditions, the solvent has properties favoring the extraction process, such as low viscosity, high diffusion coefficients, and high solvent strength. This results in good kinetics of dissolution processes and favors desorption of analytes from the cellular wall or organelles. The sample is placed in an extraction cell, made of stainless steel. Following addition of the solvent, the cell is pressurized, heated to the desired temperature, and the sample is extracted statically for a specific period of time. Next, the extract is removed from the cell and the cell is flushed with fresh solvent. The cycle can be repeated. When the extraction is complete, compressed nitrogen moves all of the solvent from the cell to the vial for analysis. The total extraction time typically ranges from 5 to 15 min, and the volume of the solvent used is about 150% of the volume of the extraction cell. The extract is filtered prior to being collected in the receiver, thus eliminating the need for a separate filtration step. ASE has been used successfully for the extraction of analytes from natural plant products, food, pharmaceuticals, etc. The disadvantage of ASE is the high cost of the equipment.

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3.5. Microwave-assisted extraction Microwave-assisted extraction (MAE) is based on absorption of microwave energy by molecules of polar chemical compounds. The energy absorbed is proportional to the dielectric constant of the medium, resulting in rotation of dipoles in an electric field (usually 2.45 GHz). The extraction is carried out at a temperature from 150 to 190 C. The hot solvent allows rapid isolation of thermally stable analytes. The efficiency of microwave-assisted extraction depends on solvent properties, sample material, the components being extracted, and, specifically, on the respective dielectric constants. The higher the dielectric constant, the more energy is absorbed by the molecules and the faster the solvent reaches the boiling point. In most cases, the extracting solvent has a high dielectric constant and strongly absorbs microwave radiation. The extraction is then carried out in closed containers made of materials resistant to high temperatures (e.g., PTFE). Solvents with a low dielectric constant can also be used. In this case, the sample matrix is heated and the analytes are released into a cooler solvent. This approach is employed for the extraction of thermally labile analytes of low polarity. Major advantages of microwave-assisted extraction include: shortened extraction time, reduced size of extraction apparatus, ease of control of sample heating, reduced amount of solvent used. The limitation of this technique, when applied to extraction of nonpolar analytes from nonpolar materials, is the need for using solvents with dipole moments greater than zero (n-hexane or iso-octane can be replaced by dichloromethane or a mixture of acetone and n-hexane) [3133]. 3.6. Steam distillation Steam distillation is a valuable technique, allowing isolation from plants of volatile components, such as the essential oils, some amines and organic acids, as well as other, relatively volatile compounds, insoluble in water. Among others, steam distillation has been used to isolate the essential-oil fraction from either plant material or a previously prepared extract in a low-boiling solvent (petroleum ether or diethyl ether) [11]. Volatile amines of relatively low polarity can be isolated by steam distilling them from a medium alkalized with calcium carbonate, while volatile acids can be steam-distilled from a medium acidified with orthophosphoric acid [11]. Vitzthym et al. [34] used steam distillation to isolate the flavor components of black tea. Steam distillation was also used for the isolation of antioxidants from herbs and the essential oils having antioxidative properties from caraway, clove, rosemary, sage, and thyme [34]. However, the technique is not free from shortcomings. It involves substantial energy consumption. An elevated temperature (ca. 100 C) may cause thermal decomposition of substances. This can also affect the essential-oil components, resulting in flavor changes [35]. 3.7. Membrane processes Membrane techniques are finding ever-increasing application in the isolation of groups of components from the plant

extracts. A membrane is a selective barrier between two phases. The phase from which mass transport takes place is called the donor phase while the receiving phase is called the acceptor (permeation) phase. The general principle of separation of liquid mixture components is depicted in Fig. 2. The main disadvantages of membrane techniques include their slowness, low efficiency, and susceptibility to membrane fouling by solid impurities in the donor phase with sizes comparable to the membrane pores. On the other hand, the method is characterized by low solvent consumption, simplicity, and high selectivity. Membrane separation has been applied, for example, in the isolation and enrichment of polyphenols from grapes [36], where a membrane with 0.22-m pore-size and ethanol as the extracting agent (acceptor phase) were used. The amount of polyphenols extracted was 11.4% of the total mass of grape seeds. The authors [36] suggested that the food and pharmaceutical industries could use this technique for the isolation of polyphenols which are anti-oxidants. Ultrafiltration, and especially nanofiltration, could also be used for the isolation of protein fractions of a specific range of molecular masses. When the concentration of salts is high, proteins and peptides can also be isolated by dialysis [37]. 3.8. Supercritical fluid extraction Supercritical fluids penetrate samples of plant material almost as well as gases, and this results from their high diffusion coefficients and low viscosity. At the same time, their dissolving power is similar to liquids. The most commonly used extracting agent is carbon dioxide, because of its low cost, low toxicity, and favorable critical parameters (Tc = 31.1 C, Pc = 74.8 atm). CO2 as a nonpolar substance is capable of dissolving nonpolar or moderately polar compounds. A mixture of CO2 with modifiers (polar organic solvents) is used for the extraction of polar substances. The modifiers increase the solubility of analytes, preventing them from adsorption on the active sites of sample matrix. The most important advantages of supercritical fluid extraction include: considerable reduction in the volume of solvent used, shortened extraction time, ease of automation, small sample size needed, possibility of on-line

Fig. 2. Separation of mixtures using membrane techniques.

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coupling with the separation and determination techniques (SFE/GC, SFE/HPLC), high purity and small volume of the extract, and high selectivity. Supercritical fluid extraction (SFE) is relatively efficient even for materials with compact and hardly accessible structure. It is especially well suited for the isolation of substances of low and medium polarity and high volatility. As a rule, carbon dioxide or carbon dioxide with a volatile polar modifier, such as methyl acetate, diethyl ether, methanol, formic acid, or ammonia, are used as supercritical fluids. A review of recent literature reveals an increasing number of papers on the application of SFE to the extraction of tocopherols, terpenes, fatty acids, steroids, and triglycerides from plant and animal material and from oils [38]. 3.9. Solid-phase micro-extraction Solid-phase micro-extraction (SPME) is a sample preparation technique best suited for gas chromatography, Although SPME has been successfully combined with HPLC, this requires relatively complicated procedures and additional devices. Therefore, it can be anticipated that the technique will be used mostly with gas chromatography. Analyte enrichment by SPME involves two steps. In the first step, a fiber, coated with an adsorbent or stationary liquid, is exposed to a liquid sample or the headspace above a sample and the analytes are sorbed on the fiber. In the second step, the fiber is introduced into the injection chamber of a gas chromatograph, where it is subjected to a high temperature, or it is introduced into the injector of a liquid chromatograph. The released analytes are swept into the chromatographic column [39]. The advantages of SPME include: speed (equilibrium between the sample and the fiber is reached in 2 to 30 min, so the technique is suitable for rapid monitoring), sensitivity (detection limits down to ppt can be achieved), low cost (SPME is solvent-free, a fiber can be used ca. 100 times), general applicability (SPME can be used with any gas chromatograph or liquid chromatograph having a SPME/HPLC sampling accessory), possibility of extraction from a variety of matrices, and ease of automation. Volatile components of medicinal plants and herbs can be determined by SPME/GC/MS. For example, terpenoids can be adsorbed on fibers coated with polydimethylsiloxane (PDMS) [40]. SPME/GC has also been used for the determination of tobacco alkaloids. The equilibrium between the plant extract components and a 100-m PDMS fiber was reached in 12 min [41]. 3.10. Sample disruption method In the last few years, matrix solid-phase dispersion (MSPD) has become an extraction method for naturally occurring compounds. MSPD is primarily used because of its flexibility, selectivity, and the possibility of performing extraction and clean-up in one step (saving analysis time). This results in rapid pre-treatment and low solvent consumption. This technique is based on blending of a viscous, solid, or semi-solid sample with an abrasive solid support material.

The process requires only simple devices and it comprises steps such as: 1. a liquid, viscous, semi-solid, or solid sample is placed in glass mortar and blended together with a solid support, using a glass pestle to obtain complete disruption and dispersion of the sample on the solid support; 2. the sample is packed into an empty column or on top of a solid-phase extraction (SPE) sorbent, the main difference between MSPD and SPE being that the sample is dispersed throughout the column and not retained in just the first few millimeters; 3. elution can be in two ways: either the target analytes are retained on the column and interfering compounds are eluted in the washing step while, the target analytes are subsequently eluted by a different solvent, or interfering matrix components are selectively retained on the column and the target analytes are directly eluted; 4. additional clean-up is performed or the sample is directly analyzed [42]. The selectivity of a MSPD procedure depends on the sorbent/solvent combination used. Often reversed-phase sorbents, like C8- and C18-bonded silica, are used as the solid support [43,44]. 3.11. Comparison of extraction techniques Selection of an appropriate extraction technique entails consideration of not only the recovery but also the cost, time of extraction, and the volume of solvent used. A comparison of the previously described extraction techniques for the isolation of groups of components from plant material is shown in Table 3 [4,45]. Raised temperature and pressure during the extraction profitably influence the process efficiency, because the solvent properties are changed and mass-transfer efficiency is enhanced. Ong and Len [46] performed the extraction of baicalein in a Soxhlet extractor and compared the results with pressurized-liquid extraction. Pressurized-liquid extraction (with 2025 ml methanol at a pressure 1030 atm and at 100 C) over a period of 20 min gave results comparable to Soxhlet extraction (with 100120 ml of methanol/water 70:30) carried out for 34 h. Grigonis et al. [47] compared different extraction techniques: Soxhlet extraction, MAE, and SFE, carried out as onestep and two-step extractions. MAE and SFE were found to be

Table 3 Comparison of various liquidsolid extraction techniques used in the analysis of plant metabolites [45] Extraction Cost Extraction time Solvent use [mL] Soxhlet Low 648 h 200600 USE Low b30 min b50 ASE High b30 min b100 MAE Medium b30 min b40 SFE High b60 min b10

Designation: USE ultrasonic extraction; ASE accelerated solvent extraction; MAE microwave-assisted extraction; SFE supercritical fluid extraction.

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suitable for extraction of antioxidants from sweet grass. The extraction times were 6 h, 0.25 h, and 2/h for Soxhlet extraction, MAE, and SFE, respectively. A two-step extraction was found to be more efficient than one-step extraction. For two-step SFE, the extraction yields of anti-oxidants were 0.46% and 0.058% of the total sample mass for the first and second step, respectively. Pan et al. [48] compared MAE with other techniques, including classical liquid/liquid extraction, ultrasonic extraction, and heat reflux extraction for the isolation of polyphenols and caffeine from green tea leaves. MAE was found to be superior in terms of the extraction efficiency, yielding 7.1% more polyphenols and 11% more caffeine than the other techniques. D. M. Teixeira et al. [49] compared between disruption methods and solid-liquid extraction (SLE) to extract phenolic compounds (phenolic acid, flavonols and cumarins) from Ficus carica levels. More compounds and higher yields were obtained by these method, using smaller amounts of solvents, and less sample preparation time. Higher extraction yields and smaller RSD values were obtained with SSDM when compared with MSPD. 4. Solid-phase extraction Sample preparation often includes an extract enrichment step, wherein the analyte concentration is increased above the determination limit of the final determination technique. Several enrichment techniques are common, including gas, liquid and solid-phase extraction. One of the primary considerations is the need for reduction of the amount of organic solvent used. This has resulted in extensive use of solid-phase extraction (SPE). SPE involves adsorption of sample components on the surface of a solid sorbent (aminopropyl or octadecyl stationary phases, bonded to silica gel, etc.), followed by elution with a selected solvent. SPE is carried out in glass or polypropylene columns or on extraction disks. SPE has a number of advantages, including the ability to isolate and enrich both volatile and nonvolatile analytes, long storage time of adsorbed analytes, elimination of emulsion formation (common in liquid/liquid extraction) or foaming (common in gas/liquid extraction). A wide selection of sorbents enables substantial selectivity of the enrichment process. An important advantage of SPE compared to liquid/ liquid extraction is a significant reduction in volume of solvent used [50]. A variety of sorbents available on the market allows not only the isolation of analytes but also the removal of interferences. Dry and wet modes of extraction were compared and found to be equivalent. Due to its simplicity, SPE is finding ever-increasing fields of application, including the isolation of proteins and peptides [51,52]. Nonpolar stationary phases are used [52] and the technique has been automated and coupled to HPLC or electrophoresis [53,54]. SPE is not free from shortcomings, including incomplete recoveries. SPE extracts can be introduced into the HPLC system if methanol, acetonitrile, water, or even acetone or 2-propanol are used as elution solvents. However, sometimes the extract cannot be introduced into the HPLC column, because it is insoluble in the mobile phase, requiring solvent replacement. During this operation the analytes could decompose or precipitate.

5. Summary The majority of sample preparation procedures for the determination of plant metabolites are developed in such a way that the final extract introduced into the GC and HPLC columns or CE capillary contains only the analytes with all the interferences removed, although full implementation of this goal may not always be possible or economically justified. Modern extraction/leaching techniques, i.e., MAE, SFE, or ASE are not always available in the average laboratory, due to the high cost of equipment. However, the use of these techniques results in shortened sample preparation time and a reduction in the volume or elimination of organic solvents by employing, e.g., SPME or SFE. This review shows that even relatively simple and inexpensive laboratory equipment can be effective in preparing samples of natural materials for chromatographic analysis. In every sample preparation procedure, especially for complex samples containing a large number of components, analyte enrichment and interference removal are essential. These two steps are often combined into one. The most common analyte isolation/enrichment techniques include SPE, SPME, and, recently, also solvent micro-extraction (the microdrop technique). Development of novel stationary phases for this step is anticipated. Current research effort is focused on automation of analytical procedures. Miniaturization is another recent trend in analytical chemistry. This will result in further reduction in sample size, analysis time, and the amount of solvents used. Miniaturization and novel sample preparation techniques will also be used more often in the control of industrial processes. The techniques discussed in this review can be used not only in the preparation of plant material for analysis but also in practical organic chemistry or in the synthesis of plant or animal metabolites. References
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