**2. Solid-phase extraction (SPE)**

Solid-phase extraction (SPE) is the most popular sample preparation technique for environmental and food samples. Due to its high versatility, the SPE procedure is used for many purposes, such as purification, trace enrichment, desalting, derivatization and class fractionation. The principle of SPE is similar to that of liquid-liquid extraction (LLE). It involves partitioning between a liquid (sample matrix or solvent with analytes) and a solid sorbent phase. Anyway, many of the problems associated with LLE, such as incomplete phase separations (emulsion), less-than-quantitative recoveries, use of expensive, breakable specialty glassware, disposal of large quantities of organic solvents, can be prevented by using SPE procedure. In addition, SPE resulted more efficient than LLE because yields quantitative extractions that are easy to perform, is rapid, and can be automated [16,17].

The general SPE procedure has to provide sample extracts that are free of interfering matrix components and concentrated enough for detection. The SPE process basically consists in four different steps: conditioning, sample addition, washing and elution (**Figure 1**).

First, the most suitable solid sorbent will be selected and conditioned using an appropriate solvent. During the conditioning the functional groups of the sorbent bed are solvated in order to make them able to interact with the sample. The sample addition consists in the percolation of the samples through the solid sorbent. During this step, the analytes as well as some matrix components are retained and thus concentrated on the SPE packing material. Successively, the analytes and interferents separation could be realized by the three following ways: selective extraction, selective washing or selective elution. Selective extraction is performed when the SPE procedure is used to remove the interfering components (trace enrichment). In this way, only selected components are retained. Selective washing is accomplished when the target analytes and the impurities are retained on the sorbent bed: the impurities will be rinsed through with wash solutions that are strong enough to remove them, but weak enough to leave the analytes behind. Differently, selective elution consists in the elution of the adsorbed compounds of interest by a solvent that leaves the strongly retained impurities behind. The elution of target analytes could require different solvents, when SPE is applied in order to perform the class compound fractionation.

**Figure 1.** Schematic representation of SPE clean-up procedure.

To achieve optimal SPE extraction conditions, the choice of sorbent is a key factor because this can control parameters of primary importance such as selectivity, affinity and capacity [18]. This choice depends strongly on the nature of the analytes and their physical and chemical properties, which should define the interaction with the chosen sorbent. However, results can also depend heavily on the sample matrix and its interactions with both sorbent and analytes.

After the sorbent choice, the eluotropic strength of adsorption on silica and the polarity index will be helpful in order to select a suitable solvent. The polarity index is an accurate measure of solvent's ability to interact as proton donator, proton acceptor or dipole whereas eluotropic series arranges solvents in order of decreasing elution strength for solutes from a particular sorbent.

## **2.1. SPE sorbents**

128 Chromatography – The Most Versatile Method of Chemical Analysis

reduced or without any sample manipulation [13-15].

recently published.

**2. Solid-phase extraction (SPE)** 

Solid-phase extraction (SPE) based on MIPs is a highly attractive and promising approach for matrix clean-up, enrichment and selective extraction of analytes in such kind of complex samples. Another modern trend in sample preparation for multi-residue applications is the use of the QuEChERS (*Quick, Easy, Cheap, Effective, Rugged and Safe*) method. The QuEChERS method is a recent and fascinating alternative procedure that has become particularly popular for the multi-residue analysis of pesticides in various food matrices [9,10], although this methodology is also being successfully employed for the extraction of other families of compounds [11,12]. Recently, the use of turbulent-flow chromatography (TFC) has also been reported for direct analysis of complex matrices such as honey, milk and animal tissues with

The aim of this chapter is to discuss new trends in sample preparation techniques applied into food and environmental analysis. It includes a selection of the most interesting and promising sample treatment procedures such as on-line SPE methods, MIPs, QuEChERS, and turbulent flow chromatography. The applicability of each technique in food and environmental analysis will be discussed through the analysis of the most relevant papers

Solid-phase extraction (SPE) is the most popular sample preparation technique for environmental and food samples. Due to its high versatility, the SPE procedure is used for many purposes, such as purification, trace enrichment, desalting, derivatization and class fractionation. The principle of SPE is similar to that of liquid-liquid extraction (LLE). It involves partitioning between a liquid (sample matrix or solvent with analytes) and a solid sorbent phase. Anyway, many of the problems associated with LLE, such as incomplete phase separations (emulsion), less-than-quantitative recoveries, use of expensive, breakable specialty glassware, disposal of large quantities of organic solvents, can be prevented by using SPE procedure. In addition, SPE resulted more efficient than LLE because yields quantitative extractions that are easy to perform, is rapid, and can be automated [16,17].

The general SPE procedure has to provide sample extracts that are free of interfering matrix components and concentrated enough for detection. The SPE process basically consists in

First, the most suitable solid sorbent will be selected and conditioned using an appropriate solvent. During the conditioning the functional groups of the sorbent bed are solvated in order to make them able to interact with the sample. The sample addition consists in the percolation of the samples through the solid sorbent. During this step, the analytes as well as some matrix components are retained and thus concentrated on the SPE packing material. Successively, the analytes and interferents separation could be realized by the three following ways: selective extraction, selective washing or selective elution. Selective extraction is performed when the SPE procedure is used to remove the interfering components (trace enrichment). In this way, only selected components are retained. Selective washing is accomplished when the target analytes and the impurities are retained

four different steps: conditioning, sample addition, washing and elution (**Figure 1**).

The sorbent selectivity depends on the attractive forces between the analytes and the functional groups on the sorbent surface. The sorbent can interact with analytes by hydrophobic (non polar-non polar, van der Waals), hydrophilic (polar-polar, hydrogen bonding, dipole-dipole, dipole-induced dipole), cationic-anionic and selective antigenantibody interactions.

Each sorbent offers a unique mix of these types of interactions.

The sorbent widely used for SPE packing can be classified into polar phases (normal phase), non-polar phases (reversed phase), ion exchange and immunoaffinity adsorbents.

Current Trends in Sample Treatment Techniques for Environmental and Food Analysis 131

analytes is severely reduced. For instance, low recovery of analytes can be observed when

Nevertheless, the narrow pH stability range of all modified silica reversed phase must be taken into account when SPE is carried in extremely acidic or basic media. For this purpose, a reversed phase polymerically bonded, such as copolymers of styrene-divinylbenzene (SPE-PS-DVB) resulted more resistant to pH extremes, and thus is more suitable for environmental applications for trapping organic compounds from acidified aqueous samples. Moreover, PS-DVB resin copolymer is a hydrophobic resin which has greater analyte retention, mainly for polar compounds, than their hydrophobic surface containing a relatively large number of active aromatic sites which allow π-π interactions with unsaturated analytes [20]. The higher potential of PS-DVB over SPE-18 for trapping aromatic compound, especially phenols, is largely demonstrated [21,22]. Anyway, PS-DVB has some drawbacks, such as lack of selectivity and low breakthrough volumes for highly polar compounds, which leads to their incomplete extraction from predominantly aqueous matrices. Over the years, the performance of SPE-PS-DVB has been enhanced by attaching polar groups (i.e. acetyl, hydroxmethyl, benzoyl, o-carboxybenzoil, sulfonate, trimethylammonium) to the aromatic ring on the polymer DVB [23] or by changing the copolymer composition. The SPE-DVB phase modified with *o*-carboxybenzoyl was useful applied for the determination of pesticides and phenolic compounds in environmental waters. The HLB sorbent, a macroporous copolymer prepared from a balance ratio of two monomers the lipophilic divinylbenzene and the hydrophilic Nvinylpyrrolidone has been formulated. It can absorb a wide range of polar and no-polar compounds [24] and its performance is unaffected by sorbent dry. It represents the most

SPE-18 sorbent is accidentally dried down before sample application.

common hydrophilic sorbent used in the herbicides extraction [25,26].

*Ion Exchange phases* are comprised of positively (aliphatic quaternary amine, aminopropyl) or negatively (aliphatic sulfonic acid, aliphatic carboxylic acid) charged groups that are bonded to the silica surface. These sorbents are really suitable for extraction of charged analytes, such as acidic and basic compounds, from aqueous or non-polar organic samples. They exert a retention mechanism based mainly on the electrostatic attraction of the charged functional group of the analytes to the charged groups that are bonded to the silica surface. In order to retain a compound by ion exchange from an aqueous solution, the pH of the sample matrix must be one at which both the compound of interest and the functional group on the bonded silica are charged. Also, there should be few, if any, other species of the same charge as the compound in the matrix that may interfere with the adsorption of the compound of interest. A solution having a pH that neutralizes either the compound's functional group or the functional group on the sorbent surface is used to elute the compound of interest. When one of these functional groups is neutralized, the electrostatic force that binds the two together is disrupted and the compound is eluted. Alternatively, a solution that has a high ionic strength, or that contains ionic species that displaces the adsorbed compound, is used to elute the compound. Positively charged compounds are isolated under cation exchange conditions by using SPE sorbent containing silica linked with aliphatic sulfonic acid (SPE-SCX) or aliphatic carboxylic acid (SPE-WCX). The sulfonic acid group is strongly acidic and attracts or exchanges cationic

*Polar phases* are used under normal phase chromatography conditions. These phases include polar adsorption media (LC Florisil, ENVI-Florisil, and LC-Alumina) and polarfunctionalized bonded silica materials. The retention mechanism of an analyte is primarily due to interactions between polar functional groups of the analyte and polar groups on the sorbent surface. These include hydrogen bonding, π-π interactions, dipole-dipole interactions, and dipole-induced dipole interactions, among others. A compound adsorbed by these mechanisms is eluted by passing a solvent that disrupts the binding mechanism; usually a solvent that is more polar than the sample's original matrix.

Polar adsorption media comprises underivatized silica material (SPE-Si), magnesium silicate (SPE- Florisil) and aluminum oxide materials (SPE-alumina).

SPE-Si is suitable to adsorb polar compounds from non polar matrices. All samples used with this material must be relatively water-free since the functional group involved in the adsorption of compounds are the free hydroxyl groups on the surface of silica particles.

Polar-functionalized bonded silica sorbent consists of a silica material modified by bonding functional groups, such as ciano (SPE-CN), aminopropyl (SPE-NH2), diol (SPE-Diol) to the surface of the SPE material. These phases are less retentive than SPE-Si toward very polar analytes and therefore permit extractions impossible to achieve with unmodified silica gel [19]. They result useful to adsorb and selectively elute compounds of very similar structure (e.g. isomers), or complex mixtures or classes of compounds such as drugs and lipids. Moreover, SPE-CN, can be used also under reversed phase conditions (with aqueous samples) to extract moderately polar compounds. The SPE-NH2 can also be applied under ion exchange conditions in order to separate charged compounds.

*Non polar phases* are used under reversed phase chromatography conditions. These sorbents comprise alkyl silica and polymer based materials. Alkyl silica sorbents are manufactured by bonding alkyl or aryl functional groups, such as cyano (SPE-CN), octyl (SPE-8), octadecyl (SPE-18) and phenyl (SPE-Ph) to the silica surface. These phases are suitable for the extraction of hydrophobic or polar organic analytes from aqueous matrices. The retention of analytes is due primarily to the non polar-non polar attractive forces between the carbon-hydrogen bonds in the analytes and the functional groups on the silica surface. The elution of adsorbed compounds is generally made by using a non polar solvent to disrupt the forces that bind the compound to the packing. Since all silica based bonded phases contain not-uncapped silanols, which can cause the strongly binding (sometime irreversibly, i.e tetracyclines) of some group of compounds, the addition of a more polar solvent may be often necessary. The main drawback of alkyl silica sorbent, especially of SPE-8 and SPE-18, is their poor water wettability. These cartridges require an initial conditioning step with a water-miscible organic solvent. When the internal surface of sorbent fails to be wetted because of the omission of the conditioning step or if the sorbent runs dry, the accessibility of sorbent surface for adsorbing analytes is severely reduced. For instance, low recovery of analytes can be observed when SPE-18 sorbent is accidentally dried down before sample application.

130 Chromatography – The Most Versatile Method of Chemical Analysis

Each sorbent offers a unique mix of these types of interactions.

usually a solvent that is more polar than the sample's original matrix.

(SPE- Florisil) and aluminum oxide materials (SPE-alumina).

ion exchange conditions in order to separate charged compounds.

The sorbent widely used for SPE packing can be classified into polar phases (normal phase),

*Polar phases* are used under normal phase chromatography conditions. These phases include polar adsorption media (LC Florisil, ENVI-Florisil, and LC-Alumina) and polarfunctionalized bonded silica materials. The retention mechanism of an analyte is primarily due to interactions between polar functional groups of the analyte and polar groups on the sorbent surface. These include hydrogen bonding, π-π interactions, dipole-dipole interactions, and dipole-induced dipole interactions, among others. A compound adsorbed by these mechanisms is eluted by passing a solvent that disrupts the binding mechanism;

Polar adsorption media comprises underivatized silica material (SPE-Si), magnesium silicate

SPE-Si is suitable to adsorb polar compounds from non polar matrices. All samples used with this material must be relatively water-free since the functional group involved in the adsorption of compounds are the free hydroxyl groups on the surface of silica particles.

Polar-functionalized bonded silica sorbent consists of a silica material modified by bonding functional groups, such as ciano (SPE-CN), aminopropyl (SPE-NH2), diol (SPE-Diol) to the surface of the SPE material. These phases are less retentive than SPE-Si toward very polar analytes and therefore permit extractions impossible to achieve with unmodified silica gel [19]. They result useful to adsorb and selectively elute compounds of very similar structure (e.g. isomers), or complex mixtures or classes of compounds such as drugs and lipids. Moreover, SPE-CN, can be used also under reversed phase conditions (with aqueous samples) to extract moderately polar compounds. The SPE-NH2 can also be applied under

*Non polar phases* are used under reversed phase chromatography conditions. These sorbents comprise alkyl silica and polymer based materials. Alkyl silica sorbents are manufactured by bonding alkyl or aryl functional groups, such as cyano (SPE-CN), octyl (SPE-8), octadecyl (SPE-18) and phenyl (SPE-Ph) to the silica surface. These phases are suitable for the extraction of hydrophobic or polar organic analytes from aqueous matrices. The retention of analytes is due primarily to the non polar-non polar attractive forces between the carbon-hydrogen bonds in the analytes and the functional groups on the silica surface. The elution of adsorbed compounds is generally made by using a non polar solvent to disrupt the forces that bind the compound to the packing. Since all silica based bonded phases contain not-uncapped silanols, which can cause the strongly binding (sometime irreversibly, i.e tetracyclines) of some group of compounds, the addition of a more polar solvent may be often necessary. The main drawback of alkyl silica sorbent, especially of SPE-8 and SPE-18, is their poor water wettability. These cartridges require an initial conditioning step with a water-miscible organic solvent. When the internal surface of sorbent fails to be wetted because of the omission of the conditioning step or if the sorbent runs dry, the accessibility of sorbent surface for adsorbing

non-polar phases (reversed phase), ion exchange and immunoaffinity adsorbents.

Nevertheless, the narrow pH stability range of all modified silica reversed phase must be taken into account when SPE is carried in extremely acidic or basic media. For this purpose, a reversed phase polymerically bonded, such as copolymers of styrene-divinylbenzene (SPE-PS-DVB) resulted more resistant to pH extremes, and thus is more suitable for environmental applications for trapping organic compounds from acidified aqueous samples. Moreover, PS-DVB resin copolymer is a hydrophobic resin which has greater analyte retention, mainly for polar compounds, than their hydrophobic surface containing a relatively large number of active aromatic sites which allow π-π interactions with unsaturated analytes [20]. The higher potential of PS-DVB over SPE-18 for trapping aromatic compound, especially phenols, is largely demonstrated [21,22]. Anyway, PS-DVB has some drawbacks, such as lack of selectivity and low breakthrough volumes for highly polar compounds, which leads to their incomplete extraction from predominantly aqueous matrices. Over the years, the performance of SPE-PS-DVB has been enhanced by attaching polar groups (i.e. acetyl, hydroxmethyl, benzoyl, o-carboxybenzoil, sulfonate, trimethylammonium) to the aromatic ring on the polymer DVB [23] or by changing the copolymer composition. The SPE-DVB phase modified with *o*-carboxybenzoyl was useful applied for the determination of pesticides and phenolic compounds in environmental waters. The HLB sorbent, a macroporous copolymer prepared from a balance ratio of two monomers the lipophilic divinylbenzene and the hydrophilic Nvinylpyrrolidone has been formulated. It can absorb a wide range of polar and no-polar compounds [24] and its performance is unaffected by sorbent dry. It represents the most common hydrophilic sorbent used in the herbicides extraction [25,26].

*Ion Exchange phases* are comprised of positively (aliphatic quaternary amine, aminopropyl) or negatively (aliphatic sulfonic acid, aliphatic carboxylic acid) charged groups that are bonded to the silica surface. These sorbents are really suitable for extraction of charged analytes, such as acidic and basic compounds, from aqueous or non-polar organic samples. They exert a retention mechanism based mainly on the electrostatic attraction of the charged functional group of the analytes to the charged groups that are bonded to the silica surface. In order to retain a compound by ion exchange from an aqueous solution, the pH of the sample matrix must be one at which both the compound of interest and the functional group on the bonded silica are charged. Also, there should be few, if any, other species of the same charge as the compound in the matrix that may interfere with the adsorption of the compound of interest. A solution having a pH that neutralizes either the compound's functional group or the functional group on the sorbent surface is used to elute the compound of interest. When one of these functional groups is neutralized, the electrostatic force that binds the two together is disrupted and the compound is eluted. Alternatively, a solution that has a high ionic strength, or that contains ionic species that displaces the adsorbed compound, is used to elute the compound.

Positively charged compounds are isolated under cation exchange conditions by using SPE sorbent containing silica linked with aliphatic sulfonic acid (SPE-SCX) or aliphatic carboxylic acid (SPE-WCX). The sulfonic acid group is strongly acidic and attracts or exchanges cationic

species in a contacting solution. It is charged over the whole pH range, and therefore can be used to isolate strong cationic (very high pKa >14) or weak cationic (moderately high pKa <12) compounds, as long as the pH of the solution is one at which the compound of interest is charged. Anyway, SPE-SCX cartridges should be used to isolate strong cations only when their recovery or elution is not desired. Weak cations can be isolated and eluted from SPE-SCX; elution is done with a solution at 2 pH units above the cation's pKa (neutralizing the analytes), or by adding a different cation that displaces the analytes. If recovery of a strongly cationic species is desired, SPE-WCX is more suitable. The carboxylic acid group, present in SPE-WCX material, is a weak anion, and is thus considered a weak cation exchanger (WCX). It has a pKa of about 4.8, will be negatively charged in solutions of at least 2 pH units above this value, and will isolate cations if the pH is one at which they are both charged. SPE-WCX can be used to isolate and recover both strong and weak cations because the carboxylic acid functional group on the silica surface can be neutralized (2 pH units below its pKa) in order to elute the strong or weak cations. Weak cations also can be eluted from LC-WCX with a solution that neutralizes the adsorbed cations (2 pH units above its pKa), or by adding a different cation that displaces the analytes.

Current Trends in Sample Treatment Techniques for Environmental and Food Analysis 133

Due to the drawbacks of the commonly used SPE phases (previously discussed), the main current trends is the study and development of new sorbents materials. These new materials try to fulfill the requirements according to present needs, such higher specific surface area, selectively towards the target analytes, easy manipulation allowing coupling on-line configurations and higher biocompatibility, with the overall objective of the enhancement of the efficiency of the extraction process. Among them, molecularly imprinted polymers (MIPs), restricted access material (RAM), porous graphite carbon (PGC) and mixed-mode

*MIP*, which has become more and more popular in recent years, is a technology where recognition sites are created by copolymerization of a target molecule in a macromolecular

*RAM* materials possess a pore size that restricts big molecules from entering the interior extraction phase based on size. They have a bimodal surface topochemistry and enable the

1. size exclusion chromatography (SEC), i.e. macromolecular sample components (>15,000

2. adsorption chromatography (e.g. reversed-phase chromatography), i.e. low-molecularweight sample components are bound adsorptively on the internal pore surface.

Because the ability of these phases to exclude proteins, RAMs are the most suitable choice for clean-up biological and food samples. In a recent work, Chico et al. [30], evaluated the SPE-RAM clean- up for tetracyclines analysis in milk and water samples. The RAM clean-up removed large peaks that otherwise appeared in the initial time window of the chromatograms, attributed to proteins in milk samples and humic substances in water samples. Thus, quantification of analytes in real samples, especially of the most polar

*Porous graphite carbon (PGC)* material is manufactured by impregnating a high porosity LC

PGC behaves as a strong reversed-phase stationary phase, even stronger than SPE-18 silica phase which represents the most hydrophobic of the commonly used alkyl substituted silica phases [31]. The retention mechanism of PGC is different from that observed of reversedphase silicas. The retention mechanism of polar analytes on PGC is a charged-induced interaction of the polar analyte with the polarizable surface of graphite [32]. The strength of interaction between a hydrophobic analyte molecule and the PGC surface largely depends on how well the molecule fit onto the flat graphite surface. PGC has been found to be particularly selective with respect to geometrical isomers and closely related substances. It

*Mixed mode polymeric sorbents* combine the polymeric skeleton with ion-exchange group. It can be divided into cationic (SPE-MCX) or anionic (SPE-MAX) and as weak or strong ion exchange, depending on the ionic group linked to the resin. The retention mechanism of mixed-mode ion exchange chromatography combines the use of reversed-phase and ionexchange modes into a single protocol on a single SPE cartridge. The mixed mode sorbents

polymeric sorbent are attracting much interest [3,27-28].

Dalton) are directly eluted to waste;

matrix. This technique will be discussed in detail in section 3.

simultaneous performance of two different chromatographic processes [29]:

compounds such as oxytetracycline and tetracycline, was clearly improved.

silica gel (to provide the desired pore size) with a phenol-formaldehyde resin.

was found that non-polar analytes were strongly retained on PGC.

Negatively charged compounds can be isolated under anion exchange condition by using SPE sorbent containing silica functionalized with positively charged groups, such SPE-SAX and SPE- NH2.

SPE-SAX material presents an aliphatic quaternary amine as functional group. This is a strong base that exchanges or attracts anionic species in the contacting solution. Its pKa is very high (greater than 14), which makes the bonded functional group charged at all pHs in aqueous solution. As a result, LC-SAX is used to isolate strong anionic (very low, pKa <1) or weak anionic (moderately low, pKa >2) compounds, as long as the pH of the sample is one at which the compound of interest is charged. For an acidic compound of interest, the pH of the matrix must be 2 pH units above its pKa for it to be charged. In most cases, the compounds of interest are strong or weak acids. Because it binds so strongly, LC-SAX is used to extract strong anions only when recovery or elution of the strong anion is not desired (the compound is isolated and discarded). Weak anions can be isolated and eluted from LC-SAX because they can be either displaced by an alternative anion or eluted with an acidic solution at a pH that neutralizes the weak anion (2 pH units below its pKa). If recovery of a strongly anionic species is desired, the use SPE-NH2 is recommended. Generally, SPE-NH2 is used for normal phase separations but it is also considered to be a weak anion exchanger (WAX) when used with aqueous solutions. It has an aliphatic aminopropyl group bonded to the silica surface. The pKa of this primary amine is around 9.8. For it to be used as an anion exchanger, the sample must be applied at a pH at least 2 units below 9.8. SPE-NH2 is used to recover both strong and weak anions because the amine group can be neutralized (2 pH units above its pKa) in order to elute the strong or weak anions.

*Immunoaffinity SPE* phases, also called immunosorbents (ISs), are very interesting materials because of their high selectivity. ISs cartridges are filled with antibody materials bonded onto silica gel support. They allow extraction, concentration and clean up from complex matrices in a single step, and from large sample volumes. The retention mechanism of these sorbents involves reversible and selective antigen-antibody interactions.

Due to the drawbacks of the commonly used SPE phases (previously discussed), the main current trends is the study and development of new sorbents materials. These new materials try to fulfill the requirements according to present needs, such higher specific surface area, selectively towards the target analytes, easy manipulation allowing coupling on-line configurations and higher biocompatibility, with the overall objective of the enhancement of the efficiency of the extraction process. Among them, molecularly imprinted polymers (MIPs), restricted access material (RAM), porous graphite carbon (PGC) and mixed-mode polymeric sorbent are attracting much interest [3,27-28].

132 Chromatography – The Most Versatile Method of Chemical Analysis

displaces the analytes.

and SPE- NH2.

weak anions.

species in a contacting solution. It is charged over the whole pH range, and therefore can be used to isolate strong cationic (very high pKa >14) or weak cationic (moderately high pKa <12) compounds, as long as the pH of the solution is one at which the compound of interest is charged. Anyway, SPE-SCX cartridges should be used to isolate strong cations only when their recovery or elution is not desired. Weak cations can be isolated and eluted from SPE-SCX; elution is done with a solution at 2 pH units above the cation's pKa (neutralizing the analytes), or by adding a different cation that displaces the analytes. If recovery of a strongly cationic species is desired, SPE-WCX is more suitable. The carboxylic acid group, present in SPE-WCX material, is a weak anion, and is thus considered a weak cation exchanger (WCX). It has a pKa of about 4.8, will be negatively charged in solutions of at least 2 pH units above this value, and will isolate cations if the pH is one at which they are both charged. SPE-WCX can be used to isolate and recover both strong and weak cations because the carboxylic acid functional group on the silica surface can be neutralized (2 pH units below its pKa) in order to elute the strong or weak cations. Weak cations also can be eluted from LC-WCX with a solution that neutralizes the adsorbed cations (2 pH units above its pKa), or by adding a different cation that

Negatively charged compounds can be isolated under anion exchange condition by using SPE sorbent containing silica functionalized with positively charged groups, such SPE-SAX

SPE-SAX material presents an aliphatic quaternary amine as functional group. This is a strong base that exchanges or attracts anionic species in the contacting solution. Its pKa is very high (greater than 14), which makes the bonded functional group charged at all pHs in aqueous solution. As a result, LC-SAX is used to isolate strong anionic (very low, pKa <1) or weak anionic (moderately low, pKa >2) compounds, as long as the pH of the sample is one at which the compound of interest is charged. For an acidic compound of interest, the pH of the matrix must be 2 pH units above its pKa for it to be charged. In most cases, the compounds of interest are strong or weak acids. Because it binds so strongly, LC-SAX is used to extract strong anions only when recovery or elution of the strong anion is not desired (the compound is isolated and discarded). Weak anions can be isolated and eluted from LC-SAX because they can be either displaced by an alternative anion or eluted with an acidic solution at a pH that neutralizes the weak anion (2 pH units below its pKa). If recovery of a strongly anionic species is desired, the use SPE-NH2 is recommended. Generally, SPE-NH2 is used for normal phase separations but it is also considered to be a weak anion exchanger (WAX) when used with aqueous solutions. It has an aliphatic aminopropyl group bonded to the silica surface. The pKa of this primary amine is around 9.8. For it to be used as an anion exchanger, the sample must be applied at a pH at least 2 units below 9.8. SPE-NH2 is used to recover both strong and weak anions because the amine group can be neutralized (2 pH units above its pKa) in order to elute the strong or

*Immunoaffinity SPE* phases, also called immunosorbents (ISs), are very interesting materials because of their high selectivity. ISs cartridges are filled with antibody materials bonded onto silica gel support. They allow extraction, concentration and clean up from complex matrices in a single step, and from large sample volumes. The retention mechanism of these

sorbents involves reversible and selective antigen-antibody interactions.

*MIP*, which has become more and more popular in recent years, is a technology where recognition sites are created by copolymerization of a target molecule in a macromolecular matrix. This technique will be discussed in detail in section 3.

*RAM* materials possess a pore size that restricts big molecules from entering the interior extraction phase based on size. They have a bimodal surface topochemistry and enable the simultaneous performance of two different chromatographic processes [29]:


Because the ability of these phases to exclude proteins, RAMs are the most suitable choice for clean-up biological and food samples. In a recent work, Chico et al. [30], evaluated the SPE-RAM clean- up for tetracyclines analysis in milk and water samples. The RAM clean-up removed large peaks that otherwise appeared in the initial time window of the chromatograms, attributed to proteins in milk samples and humic substances in water samples. Thus, quantification of analytes in real samples, especially of the most polar compounds such as oxytetracycline and tetracycline, was clearly improved.

*Porous graphite carbon (PGC)* material is manufactured by impregnating a high porosity LC silica gel (to provide the desired pore size) with a phenol-formaldehyde resin.

PGC behaves as a strong reversed-phase stationary phase, even stronger than SPE-18 silica phase which represents the most hydrophobic of the commonly used alkyl substituted silica phases [31]. The retention mechanism of PGC is different from that observed of reversedphase silicas. The retention mechanism of polar analytes on PGC is a charged-induced interaction of the polar analyte with the polarizable surface of graphite [32]. The strength of interaction between a hydrophobic analyte molecule and the PGC surface largely depends on how well the molecule fit onto the flat graphite surface. PGC has been found to be particularly selective with respect to geometrical isomers and closely related substances. It was found that non-polar analytes were strongly retained on PGC.

*Mixed mode polymeric sorbents* combine the polymeric skeleton with ion-exchange group. It can be divided into cationic (SPE-MCX) or anionic (SPE-MAX) and as weak or strong ion exchange, depending on the ionic group linked to the resin. The retention mechanism of mixed-mode ion exchange chromatography combines the use of reversed-phase and ionexchange modes into a single protocol on a single SPE cartridge. The mixed mode sorbents

are useful for fractionation of analytes. It can be used to isolate and separate neutral, acidic, and basic compounds from a single complex matrix. Intermediate washes with organic solvent mixtures of appropriate elution strength may be used to isolate neutral compounds, including ionizable analytes in their neutral state. Selective elution of ionically bound analytes may be attained by manipulating the charge of either the analyte (when bound to strong ion exchangers) or the sorbent (for analytes bound to weak ion exchangers).

Current Trends in Sample Treatment Techniques for Environmental and Food Analysis 135

Vegetable oils GC-FID Silver silica gel [34]

C18

GC-MS Phenothiazine bonded silica

water GC-MS Multi-walled carbonphase [43]

Pesticides Berry fruits GC-MS Envi-Carb + SPE-NH2 [35] Anthocyanin Berry fruits HPLC-MS Amberlite XAD7 [36] Sudan dyes Hot chili powder HPLC-DAD Alumina [37]

HPLC-MS

HPLC– MS/MS

**technique SPE column Reference** 

Chromabond C18ec, Strata-X RP,

LiChrolut RP-18, Supelclean LC-18, Discovery DSC-18,Zorbax

Hydrophilic functional gel and

GC-MS Florisil; Alumina [41]

Sep-Pak Vac C18, Oasis HLB, Bond Elut-ENV, Bond Elut Plexa,LiChrolut EN (500 mg)

cation exchange sorbent [39]

(PTZ-Si) [40]

[38]

[42]

Bakerbond Octadecyl, Bakerbond SDB-1, Bakerbond SPE Phenyl, Oasis HLB,

**Compound Sample Detection** 

Food products (aqueous solution)

Egg, pork, liver, kidney of pig, shrimp, honey, soybean milk, soybean powder, protein powder, milk and other dairy products

Lake water, sanitary

wastewater, and pond water

Wastewater HPLC-MS

**Table 1.** Some recent examples about applications on off-line SPE

Food samples (fish, meat and vegetables) environmental samples (soil or sediments)

Environmental

*Food analysis*  Mineral oil saturated hydrocarbons

High-intensity sweeteners

Melamine and cyanuric acid

Nitrobenzene compounds

Polybrominated diphenyl ethers

Alkylphenol ethoxylates; steroidal hormones; bisphenol-A;

Polycyclic aromatic hydrocarbons

*Environmental analysis* 

## **2.2. Off-line SPE**

SPE is widely used in environmental and food analysis in order to clean-up complex matrices and to isolate or/and concentrate target compounds. Two different SPE approaches are currently available: off-line and on-line procedures. In any case, the method development in SPE is accomplished by investigating pH, ionic strength, polarity and flowrate of the elution solvent and physico-chemicals characteristics of the sorbent bed. Briefly, if the target analytes are polar, normal phase extraction is indicated. When the analytes are less polar, reverse phase separation is advised. While ion exchange SPE extraction is suitable when the analytes are charged.

Some reasons for low sample recovery in SPE are: inappropriate cartridge conditioning, toostrong loading and wash solvent, too large volume (mass) of loaded, and too weak or too small volume of elution mobile phase [16].

The off-line SPE equipment is economical and uncomplicated, thus fully applicable to on-site sampling. This consists in a sorbent material, which come in different packaging (syringe barrels, microtubes-tips and discs), in a solvent system (eluent) and in a vacuum manifold. The most popular packaging format for off-line SPE results a syringe barrels which are easy to handle by using vacuum or positive-pressure manifold. However, it is not easy to control the flow-rate, and care should be taken to prevent the column from drying out prior to sample application. As it could be difficult to elute the analytes of interest from SPE syringe using minimal solvent volume unless organic solvent composition rises up to 100%, special SPE discs are typically used for these purposes. This approach is much quicker as evaporation to dryness and reconstitution are no longer necessary because elution can be performed directly by a mobile phase. A major drawback is the small sample capacity of the discs.

The off-line SPE procedure presents the following weaknesses: it is time consuming, it requires large amount of the organic solvent for the elution, and it could cause a possible loss of analytes during the evaporation steps. In addition off-line SPE provide a large manipulation of the samples thus possibility of contamination, less accuracy and precision can be found.

In spite of all disadvantages, off-line SPE approach remains useful for analyzing complex samples, because of its greater flexibility and whenever elution solvent is not compatible with the subsequent method of analysis [33].

Off-line SPE remain the widely used sample preparation technique for environmental and food analysis. Some of the most recent applications of SPE to environmental and food matrices are summarized in **Table 1**.



**2.2. Off-line SPE** 

when the analytes are charged.

small volume of elution mobile phase [16].

with the subsequent method of analysis [33].

matrices are summarized in **Table 1**.

are useful for fractionation of analytes. It can be used to isolate and separate neutral, acidic, and basic compounds from a single complex matrix. Intermediate washes with organic solvent mixtures of appropriate elution strength may be used to isolate neutral compounds, including ionizable analytes in their neutral state. Selective elution of ionically bound analytes may be attained by manipulating the charge of either the analyte (when bound to

SPE is widely used in environmental and food analysis in order to clean-up complex matrices and to isolate or/and concentrate target compounds. Two different SPE approaches are currently available: off-line and on-line procedures. In any case, the method development in SPE is accomplished by investigating pH, ionic strength, polarity and flowrate of the elution solvent and physico-chemicals characteristics of the sorbent bed. Briefly, if the target analytes are polar, normal phase extraction is indicated. When the analytes are less polar, reverse phase separation is advised. While ion exchange SPE extraction is suitable

Some reasons for low sample recovery in SPE are: inappropriate cartridge conditioning, toostrong loading and wash solvent, too large volume (mass) of loaded, and too weak or too

The off-line SPE equipment is economical and uncomplicated, thus fully applicable to on-site sampling. This consists in a sorbent material, which come in different packaging (syringe barrels, microtubes-tips and discs), in a solvent system (eluent) and in a vacuum manifold. The most popular packaging format for off-line SPE results a syringe barrels which are easy to handle by using vacuum or positive-pressure manifold. However, it is not easy to control the flow-rate, and care should be taken to prevent the column from drying out prior to sample application. As it could be difficult to elute the analytes of interest from SPE syringe using minimal solvent volume unless organic solvent composition rises up to 100%, special SPE discs are typically used for these purposes. This approach is much quicker as evaporation to dryness and reconstitution are no longer necessary because elution can be performed directly

The off-line SPE procedure presents the following weaknesses: it is time consuming, it requires large amount of the organic solvent for the elution, and it could cause a possible loss of analytes during the evaporation steps. In addition off-line SPE provide a large manipulation of the samples thus possibility of contamination, less accuracy and precision can be found.

In spite of all disadvantages, off-line SPE approach remains useful for analyzing complex samples, because of its greater flexibility and whenever elution solvent is not compatible

Off-line SPE remain the widely used sample preparation technique for environmental and food analysis. Some of the most recent applications of SPE to environmental and food

by a mobile phase. A major drawback is the small sample capacity of the discs.

strong ion exchangers) or the sorbent (for analytes bound to weak ion exchangers).
