Liquid Chromatography: Applications

China

Chapter 1

Sample preparation for liquid chromatography

Hiroyuki Kataoka    Shujitsu University, Okayama Japan

Abstract

Among various analytical processes from sampling to data analysis, sample preparation is the most important step in the extraction, isolation, and concentration of desired analytes in complex matrices, and greatly influences a reliable and accurate analysis. Recent trends in sample preparation include miniaturization, automation, high-throughput performance, on-line coupling with analytical instruments, and reductions in solvent consumption and operation time. Various efficient sample preparation techniques, such as liquid-phase microextraction, solid-phase extraction, solid-phase microextraction, and related microextraction techniques, have been used as pretreatment for liquid chromatography. Extraction devices and coating materials developed for these techniques include restricted access media, molecularly imprinted polymer, and monolithic materials. Column switching is also useful for automation of sample preparation. This chapter provides an overview of recent convenient sample preparation methods for liquid chromatography, and provides a guide for selecting the most effective sample preparation techniques for sample analysis.

Keywords

Sample preparation; High-performance liquid chromatography; Liquid-phase microextraction; Solid-phase extraction; Fiber solid-phase microextraction; In-tube solid-phase microextraction; Column switching technique; Automated analysis; On-line analysis; Extraction device

Chapter Outline

1.1 Introduction

1.2 Overview

1.2.1 Objectives of Sample Preparation

1.2.2 Classification of Sample Preparation

1.2.3 Automation of Sample Preparation

1.3 Sample Extraction Techniques

1.3.1 Liquid-Phase Microextraction

1.3.2 Solid-Phase Extraction

1.3.3 Solid-Phase Microextraction

1.3.4 Fiber SPME

1.3.5 In-tube SPME

1.3.6 Other Sorbent Microextraction Techniques for HPLC

1.4 Conclusions

References

1.1 Introduction

Although highly efficient analytical instruments for the endpoint determination of analytes have been developed of late, most of these instruments cannot handle complex matrices directly, such as biological, environmental, and food samples. Among various analytical steps such as sampling, sample preparation, separation, detection, and data analysis, sample preparation is essential for the extraction, isolation, and concentration of target analytes from samples. Sample preparation is one of the most labor-intensive and time-consuming operations in sample analysis. In addition, it requires large amounts of sample and organic solvents, is difficult to automate, and is the most error-prone part of the process, greatly influencing the precision and accuracy of the overall analysis. Therefore, sample preparation has been regarded as the main bottleneck in the development of sensitive, selective, and precise analytical methods, especially for the analysis of trace components in complex sample matrices. Sample preparation strategies include the exhaustive or nonexhaustive extraction of analytes from matrices. Moreover, on-line coupling with the separation system is regarded as an important goal. This chapter focuses on efficient sample preparation techniques for high-performance liquid chromatography (HPLC), especially emphasizing automation using column switching techniques. The details of sample preparation techniques have been described in recent books [1–14] and reviews [15–55].

1.2 Overview

1.2.1 Objectives of Sample Preparation

The objectives of the analytical method will indicate how much time and effort will be necessary for sample preparation. Therefore, efficient sample preparation should minimize sample loss, resulting in high yields of target compounds, removal of unwanted substances, and ensurance of continuous operation of chromatographic systems. Moreover, these procedures should be rapid and convenient, while reducing the cost of analysis and solvent consumption. In many cases, the capability of the system to handle smaller initial sample sizes is essential, particularly when the amounts of sample are limited. The main objectives of sample preparation can be classified into three groups (Table 1.1) [1,9,11,12]: (1) modification of the sample matrix to enable injection into a chromatographic system, (2) clean-up and purification to remove impurities, and (3) analyte enrichment. An ideal sample preparation method should be simple and rapid; result in efficient quantitative and reproducible analyte recovery; be specific for analytes through the efficient removal of coexisting compounds; provide high sample throughput; utilize fewer operation steps to minimize analyte losses, and be solvent-free, inexpensive, easy to automate, and compatible with chromatographic systems. However, no universal sample preparation technique is suitable for all sample types, because sample preparation is dependent on both the analytes and sample matrices, as well as on the subsequent analytical technique. In developing an analytical method, the analyst must choose the one(s) most suitable. This choice, in turn, will depend on several parameters, including the sample matrix; the nature, physical and chemical properties, and concentrations of the analytes; and the analytical instruments available.

Table 1.1

Objectives of Preparing Samples for High-Performance Liquid Chromatography

1.2.2 Classification of Sample Preparation

Typical analytical processes for sample analysis are shown in Fig. 1.1 [11,12]. Various stages of these analytical processes prepare samples for HPLC. Processes can be generally classified into four operations: (1) release of analytes from a matrix, (2) liquid handling, (3) removal of endogenous compounds and extraction of analytes, and (4) enhancement of the sensitivity and selectivity of detection (Table 1.2) [11,12]. A sample matrix may be solid, particulate, or a mixture of organic compounds in an aqueous solution, and may consist of many components such as macromolecules and small molecules. In the case of a biological sample, the analytes bound to protein, glucuronide, and/or sulfate must be initially released from the matrix by hydrolysis, using an enzyme, acid, or base. Liquid handling procedures mainly provide the links among operation groups, and may be required especially prior to the extraction and isolation of analytes. These procedures can often be the rate-limiting steps in sample preparation processes, because they are both labor-intensive and tedious. Injection of samples containing large amounts of protein constituents into a chromatographic column results in a rapid deterioration of the ability of the column to separate components, increase in background interferences, and dramatic increase in column backpressure. Therefore, protein precipitation and ultrafiltration are required to remove proteins from biological samples prior to extraction. Precipitation by denaturing with organic solvents, inorganic acids, or salts is effective for protein removal. The membrane filters used for ultrafiltration are efficient, although care must be taken to avoid binding of the analyte to the membrane. Dialysis can also separate an analyte from its matrix by diffusion through a semipermeable membrane, an alternative to separation by centrifugal force during ultrafiltration.

Fig. 1.1 Stages of analytical process for chromatography.

Table 1.2

Stages of Sample Preparation for High-Performance Liquid Chromatography

Methods used to extract and isolate analytes from sample matrices prior to chromatography include liquid-liquid extraction (LLE), ultrasonic extraction, microwave-assisted extraction, pressurized-liquid extraction, supercritical fluid extraction, matrix solid-phase dispersion, liquid-phase microextraction (LPME), solid-phase extraction (SPE), solid-phase microextraction (SPME), and membrane-based extraction. Of these, SPE and SPME are the most useful sample preparation techniques for chromatography, as they can efficiently produce clean extracts for analysis. The details of the main extraction techniques for HPLC are described in Section 1.3. Other sample preparation techniques include the derivatization of analytes, which can enhance sensitivity and/or selectivity using precolumn or postcolumn reactions (see Chapter 2). Selective detectors for HPLC are also effective.

1.2.3 Automation of Sample Preparation

The final aims of sample preparation are the isolation and purification of the analyte and its introduction to a chromatographic column in a manner compatible with the instrument. Sample preparation, combined with the techniques mentioned above, is usually performed with an off-line or on-line system. Sample preparation methods tend toward the development of miniaturized systems and automated methods, making the development of new efficient extraction devices and automated equipment a key to the final success of the analysis. The advantages of automating sample preparation include freeing personnel from routine tasks, reducing the error resulting from sample preparation by different individuals, reducing hands-on and total analysis time, allowing operations to be run overnight, improving the quality and reproducibility, reducing exposure to hazardous material (e.g., radioactivity, toxic chemicals), increasing sample throughput and productivity per instrument, increasing accuracy and precision, and being applicable for on-line analysis.

1.2.3.1 Robotic sample preparation systems

Automation is playing an important role in allowing scientists to meet the high throughput demands of today's research environment [3]. Various combinations of robotics, liquid handling workstations, and/or improved formats such as microplate sample preparation were introduced to allow high-speed analyses [3,56]. Several procedures involved in liquid handling can be automated with robotic systems (e.g., Zymate, RapidTrace), including pipetting, weighing, diluting, aspirating, filtering, heating, evaporating, mixing, washing, and capping. Well-based microplate-type workstations for LLE and SPE, such as Quadra96 and Biomek, are also available for off-line automated sample preparation. Headspace HS- and direct immersion DI-fiber SPME techniques can be performed manually or automatically and used routinely in combination with chromatographic systems. Commercially available autosamplers, such as Combi-PAL (CTC, Switzerland), MPS 2 (Gestel Inc., Germany), TriPlus (Thermo Fisher Scientific, Italy), and Concept 96 (PAS Technology, Germany), can be easily programed to perform various sample preparation steps such as dilution, agitation, and extraction. These autosamplers provide a number of advantages, including shorter times for routine analysis and method development, faster sample throughput, and greater reproducibility. In these automated systems, samples are agitated by fiber vibration or sample tray rotation, whereas the most common technique for manual fiber SPME is magnetic stirring. The Concept 96 robotic system is especially suitable for automated high-throughput analysis with a multifiber SPME configuration.

1.2.3.2 Column switching sample preparation

Column switching techniques [3,13,57,58] using two or more separation columns, multiport valves, and one or more pumps, are useful for trace enrichment, automated sample cleanup, and rapid on-line analysis by dynamic extraction or transfer of the extract to the HPLC system. The advantages and disadvantages of column switching systems for sample preparation are summarized in Table 1.3. On-line column switching sample preparation techniques, such as SPE [59,25,60–64] and turbulent flow chromatography (TFC) [65,66] using a cartridge column and in-tube SPME [67,68] using a capillary column, are usually combined with HPLC. In SPE and TFC, target fractions from the first column are transferred selectively on-line to one or more second columns with different properties, allowing further separation by zone cutting and flushing techniques. In in-tube SPME, column-switching techniques can be used to determine the analytes in complex matrices by direct sample injection into the HPLC or after simple sample treatment such as filtration. Thus, on-line procedures can limit contact with dirty and hazardous samples, and reduce sample contamination and loss. Furthermore, column-switching techniques can improve the sensitivity, resolution of complex sample portions, and sample throughput. The success of column switching depends on the analyte properties and the stationary-phase materials. Biocompatible or functional polymer coating materials used in micro/nanosample preparation devices include conventional hydrophobic supports, restricted access media (RAM) [69–72], monolithic materials [73–77], immunoaffinity adsorbents (IAAs) [78–81], molecularly imprinted polymers (MIPs) [82–88], sol-gel porous silica [89,90], ionic liquids [91,92], and multiwalled carbon nanotubes [93–97], with each developed for specific and efficient extraction (Fig. 1.2) [58]. The details of these extraction sorbents are described, along with specific sample preparation techniques in Section 1.3.

Fig. 1.2 Novel extraction sorbent materials for sample preparation. (A) Restricted access media, (B) monolithic polymers, (C) immunosorbent materials, (D) molecularly imprinted polymers, and (E) multiwalled carbon nanotubes.

Table 1.3

Advantages and Disadvantages of Column Switching Systems for Sample Preparation

1.3 Sample Extraction Techniques

Traditional LLE, on the basis of partition constant of the analytes between liquid phases of low mutual solubility, has been used to extract various compounds from aqueous samples. The main disadvantages of this technique are the large volume of organic solvents normally required, limited preconcentration factors, and tedious procedures. Because the scientific community has shown increased interest in developing environmentally friendly laboratory methods, efforts are underway to replace toxic with nontoxic reagents. Sample extraction techniques that may be more useful and convenient as efficient pretreatments, when combined with analysis by HPLC, include LPME [20,33,36,37,47,98–123], SPE [15,21,32,33,37,41,47,61–64,124–128], SPME [20,21,32,33,41,47,67,68,129–148], and related microextraction techniques such as stir-bar sorptive extraction (SBSE) [36,47,149–154], thin-film microextraction (TFME) [140,155], solid-phase dynamic extraction (SPDE) [32,136,143], microextraction in a packed syringe (MEPS) [33,156–161], and in-tip SPME [32,143,144] (Table 1.2). The main advantages and disadvantages of these sample extraction techniques are summarized in Table 1.4. This section will address the principles, procedures, and characteristics of these techniques.

Table 1.4

Advantages and Disadvantages of Main Sample Extraction Techniques

1.3.1 Liquid-Phase Microextraction

LPME using minimal amounts of solvent is a quick and inexpensive sample extraction technique. LPME normally involves a small volume of water-immiscible solvent and an aqueous phase containing analytes; the acceptor phase can be immersed directly in, or suspended above, the sample for headspace extraction. The volume of the acceptor phase is in the microliter or submicroliter range. The high sample volume-to-acceptor phase volume ratio can result in high enrichment factors. LPME can be subclassified as dispersive liquid-liquid microextraction (DLLME) [98–103], single-drop microextraction (SDME) [36,104–109], and hollow-fiber (HF) LPME [110–123]. The advantages and disadvantages of LPME are summarized in Table 1.4. Solvent consumption in LPME was 99% lower than that with LLE, and there was no need to evaporate solvent or reconstitute analytes prior to injection into the instrument.

1.3.1.1 DLLME

DLLME [99−104] is a miniaturized LLE technique based on a ternary component solvent system composed of a certain amount of sample, a disperser solvent, and an extraction solvent. As shown in Fig. 1.3, DLLME involves several steps: (a) A volume of sample is placed in a tube with a conical bottom. (b) The appropriate mixture of microliter volumes of water-immiscible extraction (hydrophobic) solvent and a few submilliliter volumes of water-miscible disperser (hydrophilic) solvent are rapidly injected, with a syringe, into an aqueous sample containing analytes. (c) Following through mixing, a cloudy solution forms in the test tube. During this step, analytes are extracted rapidly and with high efficiency into the finely dispersed droplets of the organic phase, owing to the large surface area between the extracting solvent and aqueous sample. The final step is the centrifugation of the cloudy solution; depending on the density of the extracting solvent, the analyte (d) sediments on the bottom of the test tube or (e) floats at the top of the solution. Finally, the enriched analytes in very fine tiny droplets are injected into the chromatographic system. DLLME is a popular, environmentally benign sample preparation technique, because it is fast, inexpensive, easy to perform with a high enrichment factor, and consumes small volumes of organic solvents. The amount of analyte in the extracting solvent depends on the solvent type, volume, ionic strength, pH, and extraction time. Optimization of these parameters is required for high enrichment and recovery of the analytes. The distribution or partition constant of a compound is defined as the ratio of its concentrations in the extracting solvent to its concentration in the aqueous sample solution at equilibrium. Extracting solvents should have a high distribution constant, good analyte selectivity, and low miscibility with aqueous solutions. Other properties of interest include their boiling point, density, interfacial tension, viscosity, corrosiveness, flammability, toxicity, stability, availability, and cost. In general, high-density solvents such as carbon tetrachloride, chloroform, and tetrachloroethylene; low-density solvents such as 1-dodecanol, 1-undecanol, 1-octanol, and cyclohexane; and ionic liquids such as 1-butyl-3-methylimidazolium hexafluorophosphate are used as extracting solvents. However, halogenated hydrocarbons are incompatible with mobile phases for reversed-phase HPLC. Increasing the extraction solvent volume also enhances the recovery of the analyte. In contrast, the dispersing solvent should be miscible with the extraction solvent and the aqueous sample. Common dispersing solvents include methanol, acetonitrile, acetone, and tetrahydrofuran. If the volume of the dispersing solvent is too low, the cloudy state is not formed properly; whereas, if the volume is too high, the polarity of the extracting phase would be reduced, decreasing the analyte partition constant, and markedly reducing the extraction efficiency. In most cases, the dispersing solvent to sample ratio is about 1:5–1:10 (v/v).

Fig. 1.3 Schematic diagram of dispersive liquid-liquid microextraction.

1.3.1.2 SDME

SDME [36,105−110] is based on the suspension of a single drop of organic solvent from the end of a microsyringe needle in an aqueous solution (Fig. 1.4A–C). Different modes of SDME have been developed, including direct immersion (DI)-SDME and headspace (HS)-SDME. In DI-SDME, a microliter of organic solvent is drawn into a microsyringe, and the needle of the microsyringe is inserted through the sample vial septum and immersed in the liquid sample. A droplet of organic solvent is therefore suspended at the tip of the needle in a stirred aqueous sample (Fig. 1.4A). After extraction, the droplet containing the analytes extracted by passive diffusion can be directly injected into a chromatographic system. Solvents for SDME usually include n-octyl acetate, isoamyl alcohol, undecane, octane, nonane, and ethylene glycol. SDME usually involves a two-phase extraction system, in which analytes are extracted from an aqueous sample into an organic phase, resulting in >100-fold enrichment and significant sample clean-up. Another variation is three-phase DI-SDME (single drop liquid-liquid-liquid microextraction), in which analytes are first extracted from an aqueous sample matrix (donor phase) into an organic solvent (organic membrane) layered over the donor phase and then back-extracted into an aqueous drop (acceptor phase) suspended from the tip of a microsyringe into the organic membrane (Fig. 1.4B). This permits water-soluble analytes to be extracted by manipulating the pH of the donor and acceptor phases, and allows the final analysis to be performed by reversed-phase HPLC. HS-SDME, in which the organic droplet is held above the aqueous sample solution, is suitable for the extraction of volatile and semivolatile analytes (Fig. 1.4C). In this mode, the analytes are distributed among three phases, that is, the water sample, the headspace, and organic solvent. Mass transfer in the aqueous phase is the rate-determining step in the extraction process, and a high stirring speed of the sample solution facilitates mass transfer among the three phases. The HS-SDME method results in a high degree of enrichment, >200-fold. The main disadvantage of the SDME technique is the instability of the drop, which may limit the reproducibility of the extraction (Table 1.3).

Fig. 1.4 Configurations of single-drop microextraction and hollow-fiber liquid-phase microextraction. (A) DI-SDME (two-phase), (B) DI-SDME (three-phase), (C) HS-SDME, (D) HF-LPME (three-phase), (E) HF-LPME (two-phase), and (F) rod-shaped HF-LPME (two-phase).

1.3.1.3 HF-LPME

HF-LPME (Fig. 1.4D–F) [111−125] using a polymeric membrane was developed to improve the stability of the drop in SDME. The membrane is usually made of polypropylene or another porous hydrophobic material compatible with a broad range of organic solvents. The pore size of cellulose acetate 0.2 μm causes the membrane to strongly immobilize the organic solvent in the pores. Common steps in HF-LPME are: (1) cleaning of the HF, (2) conditioning of the HF by impregnating it with the extraction solvent, (3) adding a specific volume of the solvent into the HF, (4) immersing the HF in the sample for a definite time, and (5) aspirating the preconcentrated sample for analysis. In HF-LPME, an organic solvent is immobilized within the wall pores of the U-shaped HF, resulting in a supported liquid membrane, and an aqueous acceptor solution is held within its lumen. Analytes are extracted into an intermediary organic phase and subsequently into the aqueous phase (Fig. 1.4D). Another mode of HF-LPME is based on a two-phase system, in which both the wall pores and the HF lumen are filled with an organic solution (Fig. 1.4E). HF-LPME has recently attracted attention for in-vial extraction in a rod-like configuration (Fig. 1.4F). The extraction process depends on the partition constant of the analytes. A static or dynamic extraction mode can be combined easily with chromatography, depending on the membrane module. Static membrane-assisted extraction typically uses hollow fibers or membrane bags, both of which are usually disposable. The HF-LPME method is capable of high enrichment (>100-fold), as well as on-line coupling to chromatography and other instrumental systems. In on-line systems, the entire extract is transferred to the chromatographic system, providing favorable sample detectability.

1.3.2 Solid-Phase Extraction

SPE [15,21,32,33,37,41,47,61–64,124–128] is a simple and commonly used sample preparation technique for the extraction and concentration of analytes from liquid samples. The fundamental concept of SPE is similar to that of liquid chromatography. SPE is based on the partitioning of compounds between a solid (extraction) phase and a liquid (sample) phase, with the intermolecular forces between the phases influencing retention and elution. The advantages and disadvantages of SPE are summarized in Table 1.4.

1.3.2.1 SPE devices and processing steps

Various SPE devices are commercially available, including syringe barrel columns, cartridges, disk membranes, and multiwell plates (Fig. 1.5). The syringe barrel and cartridge columns are made of polypropylene (PP) or glass, with the sorbents held in place by porous frits made of PP or polytetrafluoroethylene (PTFE). These formats are usually used in combination with vacuum manifolds. The volume of the syringe barrel depends on the sample volume, and the amount of sorbent determines the sample capacity. Disk and membrane type SPEs were developed to avoid the drawbacks of syringe barrel columns. Because small-diameter disks are also easy to prepare, these disks are also favored for handling small-volume samples. Particle-loaded membranes are also available in a conventional cartridge format. These techniques minimize the volume of the sorbent bed, resulting in smaller elution volumes and highly concentrated extracts. This trend is also apparent from the microscale formats of SPE pipette tips. The SPE process consists of four consecutive steps: (1) conditioning of the sorbent (i.e., solvation; activation of functional groups), (2) sample loading, (3) rinsing to remove the undesired constituents of the sample matrix, and (4) analyte elution by selective desorption with a suitable solvent (Fig. 1.6). During method development, a suitable sorbent and rinsing and elution solvents must be selected on the basis of characteristics of the analytes and the matrix. The final extraction solvent should be compatible with the HPLC system; if not, the solvent should be evaporated and the residue redissolved in a suitable solvent. SPE automation can be performed off- or on-line. In off-line automation, SPE cartridges are not directly connected to the chromatographic system. The use of 96- or 384-well format multiwall plates for SPE automated with a robotic liquid handling system facilitates high throughput analysis.

Fig. 1.5 Various formats in solid-phase extraction. (A) Syringe barrel, (B) column cartridge, (C) pipette tip, (D) disk membrane, (E) 96-well microtiter plate, and (F) on-line cartridge.

Fig. 1.6 Schematic diagram of solid-phase extraction.

1.3.2.2 On-line column switching SPE

On-line techniques, with direct connection to the chromatographic system, do not require further sample handling between the trace-enrichment and separation steps, and are suitable for fully automated techniques. On-line fully automated SPE equipment uses a column-switching system, with the precolumn fixed in the sample loop within the injection device. The sample is preconcentrated by loading a comparatively large sample volume with retention of the analytes by the solid phase. Fig. 1.7 shows a single column-switching configuration with a 6-port valve setup. The on-line column switching system can be operated in both straight-flush and back-flush modes. In the straight-flush mode (Fig. 1.7A), the sample is injected onto the extraction column and undesirable components are directly discharged to waste (position 1). By rotating the 6-port valve to position 2, the analyte-containing fraction is transferred onto the analytical column, followed by separation and detection of the analytes. In this mode, the flow of the analytes from the extraction column to the analytical column is in the same direction as the solvent flow and utilizes a single pumping system. However, the analytical column cannot be cleaned or conditioned during the extraction. Therefore, a long time is required to elute the analytes and other components of the matrix from the extraction and separation columns. In contrast, the back-flush mode utilizes a flow direction opposite to that of the mobile phase for the separation through the extraction column (Fig. 1.7B). During extraction, the switching-valve is in position 1. The sample is injected into the extraction column along with the loading mobile phase for extraction. Simultaneously, the analytical column is conditioned with mobile phase. After the 6-port valve is switched to position 2, analytes are eluted in the back-flush mode from the extraction column by mobile phase and then transferred to the separation column. During analyte separation, the extraction column is reequilibrated with the loading mobile phase, making the system ready for injection of the next sample. As the analytes retained on the extraction column front are directly diverted to the separation column, dispersed analyte bands due to the large injection volume are refocused. This minimizes peak tailing on the separation column.

Fig. 1.7 Schematic diagrams of on-line column switching solid-phase extraction coupled with HPLC. (A) Straight-flush column switching mode and (B) back-flush column switching mode.

1.3.2.3 Sorbent selection and coating materials for SPE

A wide range of chemically modified sorbents (e.g., silica gel, synthetic polymer resins) enable precise group separation based on different types of nonpolar, polar, and ionic interactions, for example, reversed-phase (C2, C8, C18, CH, PH, BP), normal-phase (CN, SI, diol, almina, florisil), anion-exchange (SAX, DEA, NH2, PSA), and cation-exchange (SCX, PRS, CBA). The interactions between the analytes and the sorbent phase include hydrophobic interactions such as van der Waals forces, and hydrophilic interactions such as dipole-dipole, hydrogen bonding, and π-π interactions. Appropriate sorbents are selected to optimize the extraction. The sample solvent (aqueous or organic), the analyte type (nonpolar, polar, or ionized), and the ionization conditions (strong or weak acid or base) provide a logical guide for method selection (Fig. 1.8). Various SPE devices are now commercially available, including diatomaceous earth Extrelut, Chem Elut, Bond Elut Certify, and Chromabond mixed-mode columns. Mixed-phase extraction columns (e.g., Bond-Elut Certify and Chromabond) show good recoveries and allow retention of all functional groups and different polarities. Bond-Elut Certify, a mixture of C8 and SCX, has both hydrophobic and ion exchange properties, and is suitable for the extraction of basic compounds. Hydrophilic-lipophilic balanced (HLB) copolymers show good retention of both polar and nonpolar compounds, in that the hydrophilic N-vinylpyrrolidone increases the water wettability of the polymer, and the lipophilic divinylbenzene (DVB) provides the reversed-phase retention necessary to retain analytes. SPE disks (Empore disk cartridge) can be used as well to rapidly extract analytes from liquid samples, with elution by the mobile phase following direct injection into the HPLC system.

Fig. 1.8 Sorbent selection for solid-phase extraction. SAX , strong anion exchanger (trimethylaminopropyl); DEA , diethylaminopropyl; NH 2 , aminopropyl; PSA , N-propylethylenediamine; SCX , strong cation exchanger (benzenesulfonylpropyl); PRS , sulfonylpropyl; CBA , carboxymethyl; C18 , octadecyl; C8 , octyl; C2 , ethyl; CH , cyclohexyl; PH , phely: BP , biphenyl; CN , cyanopropyl; SI , silica gel; FL , florisil; RP , reversed phase; NP , normal phase; IE , ion exchanger.

A biocompatible SPE device packed with alkyl-diol-silica (ADS) particles was developed as a RAM column for size exclusion (Fig. 1.2A). In this type of column, the internal surface is covered with a bonded reversed-phase material and the external surface is covered with a nonadsorptive, but hydrophilic material. This dual-phase column can effectively separate the analytes from macromolecules in the sample matrix; small molecules enter the pores of the hydrophobic reversed-phase region and become retained, whereas proteins and larger matrix components are excluded by the outer, hydrophilic phase and pass through the column as waste. IAAs consist of biological antibodies covalently linked to silica, controlled-size glass particles, or agarose or other soft gels, and result in high affinity and selective antigen-antibody interactions (Fig. 1.2C). The antibodies can be compound-specific, but usually have cross-reactivity with structural analogs, and are denatured by contact with organic solvents. Therefore, these adsorbents should be used under mild SPE conditions with regard to the selection of pH and organic solvents. However, they are expensive, and only a limited number are commercially available. MIPs, obtained from functional monomers and template molecules by polymerization, are tailor-made adsorbents with high selectivity for target molecules or their structural analogs due to their molecular-recognition mechanisms (Fig. 1.2D), making MIPs useful for the sensitive and specific analysis of analytes. MIPs are sufficiently stable in organic solvents, at high temperatures, and over a wide pH range, as well as being easily prepared and inexpensive. However, the complete recovery of analytes may be difficult and residual template material may leak into sample extracts, leading to incorrect quantification, especially at trace levels. Different modes of MIP-based SPE have been demonstrated, including various modes of off-line and on-line SPE for preconcentration or pretreatment of analytes, as well as for conventional SPE where the MIP is packed into columns or cartridges.

1.3.3 Solid-Phase Microextraction

SPME [20,21,32,33,41,47,67,68,129–148] is an effective sample preparation technique for integrating several operations such as sample collection, extraction, analyte enrichment, and isolation from sample matrices, and has been used to extract analytes from gaseous, liquid, and solid samples. SPME technology is a nonexhaustive technique based on the partition equilibrium of analytes between the sample matrix and the extraction phase. The SPME process consists of two basic steps: (1) partitioning of analytes between the extraction phase and the sample matrix, and (2) subsequent desorption of concentrated extracts into an analytical instrument. Because sampling, extraction, preconcentration, and sample introduction into an analytical instrument can be performed in a single step, SPME methods provide quantitative or semiquantitative data results.

SPME can be roughly classified as static in-vessel and dynamic in-flow microextraction. Available configurations for SPME devices are illustrated in Fig. 1.9. SPME usually involves fibers and capillary tubes coated with an appropriate stationary phase. Fiber SPME (Fig. 1.9A) is the most widely used technique, in which the analyte in the sample is directly extracted onto an outer fiber coatings by absorption or adsorption. Alternative static in-vessel microextraction techniques include stir bar sorptive extraction (SBSE) (Fig. 1.9B), and TFME (Fig. 1.9C). In-tube SPME is an efficient dynamic in-flow microextraction technique that uses a capillary column as an extraction device (Fig. 1.9D), with sample analytes extracted directly onto an inner capillary coatings. Other methods include syringe SPME such as solid-phase dynamic extraction (SPDE) (Fig. 1.9E-a) and microextraction in a packed syringe (MEPS) (Fig. 1.9E-b), and in-tip SPME (Fig. 1.9F). These SPME techniques can be performed manually or automatically and are easily coupled to chromatographic systems.

Fig. 1.9 Configurations of various devices for solid-phase microextraction and related microextraction techniques. (A) Fiber SPME, (B) SBSE, (C) TFME, (D) in-tube SPME, (E) syringe SPME, and (F) in-tip SPME.

1.3.4 Fiber SPME

Fiber SPME [20,21,32,33,41,47,129–148], using a fused-silica fiber externally coated with an appropriate stationary phase, has not only been used routinely in combination with GC, but also coupled directly with HPLC to analyze low volatility or thermally labile compounds not amenable to GC analysis. A fiber SPME device consists of a fiber assembly with a built-in extraction fiber inside a needle and an assembly holder (Fig. 1.9A). Retractable fused-silica rod fibers (1 or 2 cm long) coated on the outer surface with a thin film of extraction phase, consisting of a liquid polymer and/or a solid sorbent are commercially available. StableFlex-type fibers consist of a flexible fused-silica core and are less breakable. In addition, new superelastic metal fiber assemblies have been developed to enhance durability, shape memory, and provide more robust performance. Although SPME has the maximum sensitivity at the partition equilibrium, there is a proportional relationship between the amount of analyte adsorbed by the SPME fiber and its initial concentration in the sample before partition equilibrium. Therefore, full equilibration is not necessary for quantitative analysis by SPME. Two modes of fiber SPME are used to extract analytes: HS-SPME, which is used to extract volatile and semivolatile analytes, and DI-SPME, which is used to extract nonvolatile analytes (Fig. 1.10). Both of these techniques can be coupled with on-line and off-line solvent desorption methods to remove the analytes from the fiber. Because HS-SPME does not include direct contact with the sample, fiber damage caused by aggressive or irreversibly adsorbed matrix components is lower with HS- than with DI-SPME. The advantages and disadvantages of fiber SPME are summarized in Table 1.4. In the absence of contamination and damage, fibers can be generally used to analyze more than 100 samples, with metal fibers used to analyze up to 500 samples. A hollow-fiber membrane may be used to protect the SPME fiber from insoluble components in the sample.

Fig. 1.10 Schematic diagrams of solid-phase microextraction coupled with HPLC. (A) Extraction step for HS-SPME, (B) extraction step for DI-SPME, (C) on-line solvent desorption using SPME interface, and (D) off-line solvent desorption and direct syringe injection into HPLC.

1.3.4.1 Fiber SPME processing steps for HPLC

Before analyzing the samples, fibers can be cleaned by insertion into a heated auxiliary injection port or a syringe cleaner and immersed in a flow of clean gas. A sample is placed into a vial, which is sealed with a septum-type cap. The sample is often stirred with a small stirring bar to increase the rate of extraction and shorten the equilibration time. In HS-SPME, the fiber in the headspace is exposed to gaseous, liquid, or solid samples (Fig. 1.10A), whereas, in DI-SPME, the fiber is directly immersed in liquid samples (Fig. 1.10B). The SPME needle is inserted through the septum and the fiber is extended through the needle into the sample. This results in the partition of analytes directly from the sample matrix into the stationary-phase coated onto the surface of the fiber. After a suitable extraction time, the fiber is withdrawn into the needle, and the needle is removed from the septum and inserted directly into an SPME-HPLC interface consisting of a six-port injection valve and a special desorption chamber at the load position under ambient pressure (Fig. 1.10C). Changing the injector to the inject position brings the mobile phase into contact with the fiber, resulting in desorption of the analytes, and their delivery on-line to the HPLC column for separation. Two desorption techniques, dynamic and static desorption, can be used to remove the analytes from the fiber. In dynamic desorption, the analytes are removed by a moving stream of mobile phase. When the analytes are more strongly adsorbed to the fiber, the fiber can be soaked in the mobile phase, or another strong solvent, for a specified time allowing static desorption before injection onto the HPLC column. In these desorption methods, all the extracted analyte is transferred directly to the separation column for quantification, with the relationship between the amount of analyte extracted onto the SPME fibers and its initial concentration in the sample being proportional. Alternatively, the analyte can be desorbed from the fiber coating by off-line solvent desorption (Fig. 1.10D). The solvent solution, including the desorbed analytes is then injected into the HPLC system.

1.3.4.2 Optimization of fiber SPME methods

The amount of analyte extracted onto the fiber depends on the polarity and thickness of the stationary-phase coating on the fiber, the extraction time and temperature, the agitation and pH of the sample solution, the addition of salt to the sample, and the concentration of analyte in a sample. These SPME parameters should be optimized for each analyte and matrix. Various fiber coatings are available in thicknesses from 7 to 150 μm. Although fibers coated with thicker films require a longer time to achieve extraction equilibrium, they may be more sensitive as they can extract greater amounts of analyte. Thick and thin coat fibers are suitable for the extraction of volatile and semivolatile compounds, respectively. Salting out by the addition of salts such as sodium chloride increases the extraction efficiency. The partitioning of analytes between the sample and the fiber is also strongly affected by pH for analytes with different pKa values. The effects of extraction temperature depend on the volatility of the analytes and differ for HS- and DI-SPME. A sample may be agitated by stirring to enhance extraction efficiency in nonequilibrium situations. Although full-equilibration is not necessary for accurate and precise analysis, extractions time and other SPME parameters should be consistent. Six-port injection valves or special desorption chambers are used for HPLC, requiring additional solvent or mobile phase flow to transfer the analytes to the analytical instrument. Rapid and complete desorption of analytes using minimal solvent is important for optimizing SPME/HPLC methods. It is unnecessary to remove particles before extraction because they are removed by washing the fiber with water before insertion into the desorption chamber. Although the fiber-SPME/HPLC method also has the advantage of eliminating the solvent peak from the chromatogram, peak broadening is sometimes observed because analytes can be slow to desorb from the fiber.

1.3.4.3 Fiber coating materials

Commercially available SPME fibers are coated with a liquid polymer and/or a porous solid sorbent by immobilization on fused silica fibers as nonbonded, bonded, partially cross-linked, or highly cross-linked films (Fig. 1.11). Nonbonded phases are stable when used with some water-miscible organic solvents, but slight swelling may occur when used with nonpolar solvents. Bonded phases are stable with all organic solvents except for some nonpolar solvents. Partially cross-linked phases are stable in most water-miscible organic solvents and some nonpolar solvents. Highly cross-linked phases are similar to partially cross-linked phases, except that some bonding to the core may occur with the former. Fibers of suitable polarity and thickness are selected based on the affinity of the fiber coating for an analyte. Examples include apolar polydimethylsiloxane (PDMS) for nonpolar analytes, more polar polyacrylate (PA) for polar analytes (especially phenols), PDMS/divinylbenzene (PDMS/DVB) for polar analytes (especially amines), Carboxen (CAR; carbon molecular sieve)/PDMS (CAR/PDMS) for volatile/low molar mass analytes, Carbowax (CW; polyethylene glycol)/DVB (CW/DVB) for polar analytes (especially alcohols), CW/templated resin (CW/TPR) for polar analytes, and DVB/CAR/PDMS for a broad range of analytes. Among commercial fibers, PDMS and PA phases have linear structures and low specific surface areas. In contrast, mixed coatings, such as PDMS/DVB, CW/DVB, PDMS/CAR, CW/TPR, and DVB/CAR/PDMS, have large specific surface areas and can be used to extract low molecular mass and polar analytes. The retention capacity of these coatings is increased due to the mutually potentiating effects of adsorption and distribution to the stationary phase. In mixed coatings, PDMS and CW are used as glues to hold the particles in place.

Fig. 1.11 Polarities and retention capacities of various commercially available SPME fibers.

Similar to SPE sorbents, unique fiber coatings (Fig. 1.2), developed for biomedical analyses include polypyrrole (PPY), RAMs, immunosorbents, MIPs, sol-gel porous silica and ionic liquid, and various new biocompatible coating phases. PPY coatings are intrinsic conducting polymers, which are positively charged and can efficiently extract polar compounds, aromatic compounds, and anionic species. Their enhanced extraction efficiencies are likely due to the numerous types of interactions between these multifunctional (i.e., acid-base, π-π, and dipole interactions; ion-exchange; hydrogen bonding) coatings and the analytes. Commercial 35-μm LiChrospher RP-18 ADS particles glued to cleaned stainless steel wires have been used for direct extraction from biological samples by RAM SPME. Antibodies covalently immobilized on the surface of fused-silica fibers are used for selective and sensitive immunoaffinity SPME. MIPs are cross-linked synthetic polymers synthesized by copolymerization of a monomer with a cross-linker in the presence of a template molecule, and are used to coat SPME fibers. In contrast, sol-gel coatings have been introduced to overcome some of the problems of commercial fibers, such as solvent instability and swelling, low operating temperatures, and stripping of coating. In the sol-gel coating technique, hydroxyl-terminated siloxane polymers or mixed polymers with polyethylene or polypropylene glycols are bonded to Si-OH groups on the fused-silica surface. Although fiber SPME is not suitable for the extraction of polar analytes from samples with a polar matrix, this problem may be mitigated by using new sorbent coatings such as ionic liquids, polyethylene glycol, polypyrrole composite, and polycaprolactone.

1.3.5 In-tube SPME

In-tube SPME [32,67,68,129,133,137,143,144,147,148] is an efficient sample preparation technique that uses a length of open tubular capillary column with a stationary-phase coating as an extraction device (Fig. 1.9D). The extraction phase consists of either an inner surface coating or a sorbent bed, thereby overcoming fiber-related limitations of conventional fiber SPME, including fragility, low sorption capacity, and bleeding from thick-film coatings. In-tube SPME devices exhibit higher mechanical stability than fiber SPME devices, and are easily attached to an autosampler injection system. In-tube SPME typically uses a short inner wall-coated fused-silica open tubular capillary (Fig. 1.9D-a). In addition, fiber-packed (Fig. 1.9D-b), sorbent-packed (Fig. 1.9D-c), and rod-type porous monolith (Fig. 1.9D-d) capillaries with unique phases were developed to improve extraction efficiency and specificity. The capillaries are generally reusable without plugging or breaking and without exfoliation of coating materials. In-tube SPME processing systems can be categorized as flow-through systems (Fig. 1.12A and B), in which sample solutions are continuously passed in one direction through a capillary column; or as repeated draw/ejection system (Fig. 1.12C and D), in which sample solutions are repeatedly aspirated and dispensed from a capillary column under computer control. The advantages and disadvantages of in-tube SPME are summarized in Table 1.4. The main advantage is the ability to automate a series of processes, allowing continuous extraction, desorption, and injection by column switching using a standard autosampler and on-line coupling to an HPLC system. The main disadvantage is the tendency of the capillary to clog. This can be avoided by removing interfering phases such as particles or macromolecules, by filtration or centrifugation before extraction. Moreover, the enrichment factor of in-tube SPME is lower than that for fiber SPME, but in-tube SPME can be made reproducible by using an autosampler.

Fig. 1.12 Schematic diagrams of automated in-tube solid-phase microextraction coupled with HPLC. (A, C) Extraction and (B, D) desorption.

1.3.5.1 In-tube SPME processing systems

In flow-through systems, the complete analytical system consists of an automatic six-port valve, two pumps (a sample pump and a wash pump), and an HPLC system. The capillary column is installed in the switching six-port valve. The enrichment procedure involves four steps: conditioning, extracting, washing, and desorbing. The capillary column for extraction is washed and conditioned with water. During extraction (Fig. 1.12A), the six-port valve is switched to the load position, and the aqueous sample is pumped through the column. The capillary column is then washed with water to remove any remaining matrix and residues from the capillary. For desorption (Fig. 1.12B), the six-port valve is switched to the inject position, and the mobile phase is passed through the capillary column (dynamic desorption). Systematic problems can include contamination of the switching valve with the sample solution due to the direct connection of the capillary column to the six-port valve.

In the repeated draw/ejection system, a capillary column for extraction is placed between the injection loop and the injection needle of the HPLC autosampler. A 2.5-cm long sleeve of 1/16 in. polyether ether ketone (PEEK) tubing is connected to each end of the capillary and fixed with 1/16-in. SS unions (0.25-mm bore stainless steel nuts) and ferrules. An injection loop is installed to prevent sample contamination of the metering pump and switching valve. During the extraction (Fig. 1.12C), the injection syringe is computer programmed to repeatedly draw and eject sample from the vial until the analyte from the sample matrix almost reaches partition equilibrium with the stationary phase. After switching the six-port valve, the extracted analytes are directly desorbed from the capillary coating by the mobile phase (dynamic desorption) or by an aspirated desorption solvent (static desorption) (Fig. 1.12D), and transferred to the separation column. In this system, a large number of samples can be processed automatically by the autosampler without carryover, because the injection needle and the capillary column are washed in methanol and the mobile phase before the sample is extracted.

1.3.5.2 Optimization of in-tube SPME methods

In-tube SPME depends on the distribution constant of each analyte, and low and high polarity columns selectively retain hydrophobic and hydrophilic compounds, respectively. Although the use of thick-film capillaries often increases sample capacity and extraction efficiency, it is difficult to reliably immobilize thick-film coatings on the inner surface of fused-silica capillary columns using conventional approaches. The lack of chemical bonding is mainly responsible for low solvent stability, preventing reliable coupling of in-tube SPME techniques that use organic or organo-aqueous mobile phases. Capillary columns with chemically bonded or cross-linked liquid phases are stable in water and organic solvents, and more stable in a mobile phase typically used in HPLC. The internal diameter, length, and coating film thickness of the capillary column can affect the amount of analyte extracted. A capillary column 50–100-cm long is optimal for extraction. Less than this length, the extraction efficiency is reduced, whereas more than this length, peak broadening is observed. Although a stationary phase consisting of a thicker film and a longer column can extract a larger amount of analytes, it may be more difficult to desorb these analytes from the capillary column. PLOT-type columns have a larger surface area and a thicker layer than liquid-coated columns, and can extract a larger amount of analytes. In flow-through extraction systems, the sample volume passed through a capillary is usually 0.2–2 mL, and the optimum extraction flow rate is 0.25–4 mL/min, depending on the capacity of the column. In draw/ejection systems, an increase in the number of draw/eject cycles can enhance extraction efficiency, but peak broadening is often observed. Optimal conditions for a 60-cm capillary column with a 0.25-mm inner diameter include a draw/ejection volume of 30–40 μL, a draw/ejection flow rate of 50–100 μL/min, and 10–15 draw/ejection cycles. With lower flow rates, extractions require a long time, and at higher rates, bubbles can form on the inside of the capillary and the extraction efficiency is reduced. Furthermore, changing the pH of the sample solution may increase the extraction efficiency of analytes by the stationary phase. The presence of a hydrophilic solvent, such as methanol, in the sample solution reduces the extraction efficiency, unless concentrations ≤5% are used. Since the analytes extracted onto the capillary coating are easily statically or dynamically desorbed without a special SPME-HPLC interface, carryover is lower for in-tube SPME compared with fiber SPME, and may be eliminated in the former.

1.3.5.3 Capillary coating materials

Fused silica modified columns are more suitable for the analysis of nonpolar compounds. For most organic compounds, porous polymer-type capillary columns such as Supel-Q PLOT (DVB polymeric material) provide higher extraction efficiencies than other liquid-phase-type capillary columns such as CP-Sil 5CB, CP-Sil 19CB, and CP-Wax 52CB on account of their relatively large surface area. Some compounds were more effectively extracted by other PLOT-type columns such as Carboxen-1006 PLOT and CP-Pora PLOT amine.

Several unique phases, including PPY-coated capillaries, capillaries packed with MIP particles in PEEK tubes, and biocompatible SPME capillaries packed with ADS particles as a RAM, have been developed to improve the extraction efficiency and selectivity. PPY-coated capillaries have higher extraction efficiencies than GC capillary columns for some samples. ADS particles prevented fouling of the capillary by protein adsorption while trapping analytes in the hydrophobic porous interior. In addition, a simple SPME device was fabricated for use in on-line immunoaffinity capillaries. In-tube SPME, with monolithic capillary columns consisting of one piece of organic polymer or silica with a unique flow-through double-pore structure, has also been explored. Monolithic capillary columns with hydrophobic main chains and acidic pendant groups (e.g., poly(methacrylic acid-ethylene glycol dimethacrylate)) are superior for the extraction of basic analytes from an aqueous matrix. Other techniques include wire-in-tube SPME, using modified capillary columns with inserted stainless steel wires, and fiber-in-tube SPME, using PEEK tubes packed with fibrous rigid-rod heterocyclic polymers. These methods have shown improved extraction efficiency when extended to microscale applications.

1.3.6 Other Sorbent Microextraction Techniques for HPLC

1.3.6.1 Static in-vessel microextraction

SBSE [36,47,149–154], using a coated magnetic stir bar (Fig. 1.9B), is an efficient sample preparation technique that overcomes the limited capacity of SPME fibers. PDMS-coated stir bars are now commercially available (Gerstel, Germany). These stir-bars are 10- and 40-mm long, are coated with 55 and 219 μL of PDMS liquid phase, respectively, and possess a thicker extraction layer (0.3–1.0 mm), resulting in 50–250-fold increase in the volume of the extraction phase compared with fiber SPME. The 10-mm stir bars are suitable for stirring samples of 10–50 mL, whereas the 40-mm stir bars are better for sample volumes of up to 250 mL. For DI-SBSE, the stir bar is placed in a suitable volume of liquid sample in a vial, which is then stirred until the partition equilibrium is reached. The extraction time is determined by the sample volume, stirring rate, temperature, and stir bar dimensions, and must be optimized for each application. After extraction, the stir bar is easily removed with tweezers, rinsed with purified water to remove adsorbed sugars, proteins, and other sample components, and dried with clean paper tissue to remove residual water droplets. For HS-SBSE, the stir bar is suspended above the liquid or solid sample in a closed vial; special devices to hold the stir bar are available. The solute may be desorbed from the bar by back extraction with a small volume of solvent for HPLC, followed by the introduction of the extracted analytes into the HPLC system.

In SBSE, typical stirring times to attain equilibration are between 30 and 60 min. Compared with other microextraction techniques, including conventional fiber SPME and LPME, SBSE is more efficient and shows better reproducibility in extracting nonpolar and medium-polarity compounds from liquid samples. The main disadvantage of SBSE is that the procedure must usually be performed manually. Each stir bar can be used for 20 to more than 50 extractions, depending on the matrix and care in handling. In addition to PDMS, several types of polymeric phases such as PPY, RAM, and carbon adsorbents have been examined to cover a wide range of polarities. Compared with conventional PDMS stir bars, dual-phase twisters using different carbon-based adsorbents as an additional concentrating phase enhanced the recovery of volatile and/or polar compounds. PDMS/PPY-coated stir bars have high extraction efficiency (sensitivity and selectivity) based on both adsorption (PPY) and sorption (PDMS) mechanisms. Furthermore, new stir bars, consisting of PDMS and other polymer phases with a range of polarities, such as poly(methacrylic acid stearyl ester-ethylene dimethacrylate) and polypyrrole, have been used for the isolation of six steroid sex hormones from urine for analysis by HPLC-DAD.

TFME (Fig. 1.9C) [140,155] is an improved version of fiber SPME with increased extraction rates and lower detection limits. A flat thin film of PDMS membrane, cut into the shape of a house and mounted onto a stainless steel wire for support (Fig. 1.9C-a), and with a high surface area-to-volume ratio, has a greater mass uptake than conventional SPME configurations with a similar sampling time. High-throughput SPME using 96-blade TFME (Fig. 1.9C-b) was developed for the simultaneous extraction of analytes. A 96-well plate is placed on a shaker and agitated, and subsequently washed with an appropriate solution to remove unwanted substances. The blades are subsequently transferred to a second 96-well plate containing the desorption solvent. These TFME steps are performed by automated robotic workstations, and the extracted analytes can be introduced easily into chromatographic systems. Polymer and silica-based coatings for the 96-blade system were developed for the simultaneous extraction of hydrophobic and hydrophilic compounds from complex sample matrices.

1.3.6.2 Dynamic in-flow microextraction

SPDE [32,136,143], using an internally coated microsyringe needle (Fig. 1.9E-a), is an inside-needle technique for vapor and liquid sampling. This technique involves the use of stainless steel needles (8 cm) coated with a 50-μm film of PDMS and 10% activated carbon. Mixtures are sampled dynamically by passing the headspace through the tube using a syringe. This technique is mainly used in combination with GC analysis.

MEPS (Fig. 1.9E-b) [33,156–161] is a newly developed technique for on-line sample preparation for HPLC. This form of miniaturized SPE uses a procedure similar to in-tube SPME and SPDE. In MEPS, approximately 1 mg of solid packing material is inserted into a syringe (100–250 μL) as a plug with a filter on both sides, and analytes are tapped on the packed bed. After conditioning with methanol and water, sample solutions are drawn through the syringe three times (50 μL each) by the autosampler, which pumps the sample in and out. After washing with water to remove any interfering materials such as proteins, the extracted analytes are eluted with the mobile phase directly into an HPLC injector. Silica-based (C2, C8, C18) RAM and MIPs can be used for adsorption. In addition, MEPS can be used to analyze small samples, requiring only 1 min per sample. MEPS is easier to automate than SPE and is more rugged than SPME. Moreover, MEPS is more stable and has a higher recovery rate (60%–90%) than SPME (1%–10%). MEPS can be used without difficulty for complex matrices.

In-tip SPME [32,143,144], using pipette tips packed with sorbent (Fig. 1.9F), is a miniaturized SPE technique, with sample preparation occurring on the packed bed. Although extraction is usually performed off-line, packed pipette tips allow several samples to be handled in parallel. In-tip SPME is easily automated using 96-well extraction plates and a robot. Usually, however, only part of the sample is injected into the chromatograph. The demands on sorbents in polypropylene pipette tips are largely the same as those for SPE. Silica and monolith particles with relatively large through-pores are usually used as sorbents, as backpressure must not be too high. Moreover, the adsorbent plug in the pipette should be physically stable because the end of the tip has a dead volume. Silica particles of high quality are quite expensive, whereas methacrylate sorbents are inexpensive to prepare and are stable over a wide pH range. High-throughput in-tip SPME methods have been developed using 96-well pipette tips with a chemically bonded monolithic methacrylate sorbent, and a Personal Pipettor robot (Apricot Designs, Monrovia, California).

1.4 Conclusions

Despite developments in high-performance analytical instruments, sample preparation is usually necessary to extract, isolate, and concentrate analytes from complex matrices prior to chromatographic analysis. Sample preparation is also one of the most tedious and time-consuming steps, as well as the most error-prone part of the process. Therefore, selecting and optimizing suitable sample preparation methods are key to the final success of these analyses, and the choice of an appropriate procedure markedly influences the reliability and accuracy of the analysis. Developments of efficient extraction procedures have shown clear trends toward the simplification of sampling and sample preparation methods, increases in their reliability and precision, and the elimination of clean-up steps.

Among the sample preparation techniques discussed in this chapter, SPE using cartridges, disks, or multiwell plates is the most popular for pretreatment prior to chromatographic analysis and has reduced many of the limitations of traditional LLE methods. Current trends in sample preparation techniques include miniaturization, automation, high-throughput, and on-line coupling with analytical instruments. Minimizing the number of sample preparation steps can significantly reduce sources of error, effectively save time and operating costs, and enhance the ability to measure trace and ultra-trace analytes in complex matrices. Efficient microextraction techniques as pretreatment for liquid chromatography include DLLME, SDME, HF-LPME, fiber SPME, in-tube SPME, SBSE, TFME, SPDE, MEPS, and in-tip SPME. LPMEs (DLLME, SDME, and HF-LPME) using minimal amounts of solvent to provide extracts highly enriched for analytes with excellent clean-up of endogenous compounds. Static in-vessel microextractions (fiber SPME, SBSE, and TFME) result in the absorption or adsorption of analytes on the outer surface of the extraction device. In contrast, dynamic in-flow microextraction methods (in-tube