《复合材料 Composites》课程教学资源（学习资料）第五章 陶瓷基复合材料_fibrous molithic-45

Analytica Chimica Acta 655(2009)8-29 Contents lists available at Science Direct MYTCA Analytica Chimica Acta ELSEVIER journalhomepagewww.elsevier.com/locate/aca Review article Developments and applications of capillary microextraction techniques: A review Hiroyuki Kataoka*, Atsushi Ishizaki, Yuko Nonaka, Keita Saito School of pharmacy, Shujitsu University, 1-6-1, Nishigawara, Okayama 703-8516, Japan ARTICLE INFO A BSTRACT Sample preparation is important for isolating desired components from complex matrices and greatly eceived 14 August 2009 influences their reliable and accurate analysis. Recent trends in sample preparation include miniaturiza- tion, automation, high-throughput performance, and reduction in solvent consumption and operation ccepted 22 September 2009 time. This review focuses on novel microextraction techniques using capillaries for off-line and on-line Available online 26 September 2009 mple preparation Open-tubular trapping (OTT). in-tube solid-phase microextraction(SPME),wire- l-tube SPME, fiber-in-tube solid-phase extraction(SPE). sorbent-packed capillary in-tube SPME and monolithic capillary in-tube SPMe are critically evaluated and applications of these techniques in bio logical, pharmaceutical, environmental and food analyses are summarized. Capillary microextraction o 2009 Elsevier B V. All rights reserved. On-line analysis Automated analysis Contents 1. Introduction 8 2. Capillary microextraction techniques en-tubular d with gas chi 2.1.1. On-line OTr-GC system 2. 1.2. Off-line OrT-GC systems 2. 2. In-tube solid-phase microextraction 2. 2.2. Optimization of parameters.... 2. 2.3. Capillary coatings oo1122345 2.3. Packed capillary in-tube SPME 2.3.1. Wire-in-tube SPMe and fiber-in-tube Spe 5 2.3. 2. Sorbent-packed capillary in-tube SPME 4. Monolithic capillary in-tube SPME 3. Applications of capillary microextraction techniques 3.1. Recent applications to biological and pharmaceutical samples 3.3. Recent applications to food samples 4. Conclusions and future perspectives the complex matrices such as biological, environmental and food samples yet. Among the analytical processes such as sampling, sam- In recent years, sensitivity and specificity of analytical instru- ple preparation, separation, detection and data analysis ments have been achieved, but most of them cannot directly handle preparation is important for isolating desired components from complex matrices and greatly influences their reliable and accurate re, over 80% of analysis time is generally Corresponding author. Tel. +81 86 8342: fax: +81 86 8342. spent on the sampling and sample preparation steps including E-mail address: kataoka@shujitsuac jp(H Kataoka). extraction, concentration, fractionation and isolation of analytes 0003-2670/s-see front matter o 2009 Elsevier B v. All rights reserved. doi:10.1016aca2009.09032

Analytica Chimica Acta 655 (2009) 8–29 Contents lists available at ScienceDirect Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca Review article Developments and applications of capillary microextraction techniques: A review Hiroyuki Kataoka∗, Atsushi Ishizaki, Yuko Nonaka, Keita Saito School of Pharmacy, Shujitsu University, 1-6-1, Nishigawara, Okayama 703-8516, Japan article info Article history: Received 14 August 2009 Received in revised form 19 September 2009 Accepted 22 September 2009 Available online 26 September 2009 Keywords: Sample preparation Capillary microextraction Open-tubular trapping In-tube solid-phase microextraction On-line analysis Automated analysis abstract Sample preparation is important for isolating desired components from complex matrices and greatly influences their reliable and accurate analysis. Recent trends in sample preparation include miniaturiza￾tion, automation, high-throughput performance, and reduction in solvent consumption and operation time. This review focuses on novel microextraction techniques using capillaries for off-line and on-line sample preparation. Open-tubular trapping (OTT), in-tube solid-phase microextraction (SPME), wire￾in-tube SPME, fiber-in-tube solid-phase extraction (SPE), sorbent-packed capillary in-tube SPME and monolithic capillary in-tube SPME are critically evaluated and applications of these techniques in bio￾logical, pharmaceutical, environmental and food analyses are summarized. © 2009 Elsevier B.V. All rights reserved. Contents 1. Introduction ............................................................................................................................... ........... 8 2. Capillary microextraction techniques ............................................................................................................... 10 2.1. Open-tubular trapping coupled with gas chromatography ................................................................................. 10 2.1.1. On-line OTT-GC systems............................................................................................................ 11 2.1.2. Off-line OTT-GC systems ........................................................................................................... 11 2.2. In-tube solid-phase microextraction......................................................................................................... 12 2.2.1. Operation system ................................................................................................................... 12 2.2.2. Optimization of parameters ........................................................................................................ 13 2.2.3. Capillary coatings ................................................................................................................... 14 2.3. Packed capillary in-tube SPME ............................................................................................................... 15 2.3.1. Wire-in-tube SPME and fiber-in-tube SPE ......................................................................................... 15 2.3.2. Sorbent-packed capillary in-tube SPME............................................................................................ 15 2.4. Monolithic capillary in-tube SPME ........................................................................................................... 15 3. Applications of capillary microextraction techniques ............................................................................................... 16 3.1. Recent applications to biological and pharmaceutical samples ............................................................................. 17 3.2. Recent applications to environmental samples .............................................................................................. 17 3.3. Recent applications to food samples ......................................................................................................... 26 4. Conclusions and future perspectives ................................................................................................................ 26 References ............................................................................................................................... ............ 27 1. Introduction In recent years, sensitivity and specificity of analytical instru￾ments have been achieved, but most of them cannot directly handle ∗ Corresponding author. Tel.: +81 86 271 8342; fax: +81 86 271 8342. E-mail address: hkataoka@shujitsu.ac.jp (H. Kataoka). the complex matrices such as biological, environmental and food samples yet. Among the analytical processes such as sampling, sam￾ple preparation, separation, detection and data analysis, sample preparation is important for isolating desired components from complex matrices and greatly influences their reliable and accurate analysis [1]. Furthermore, over 80% of analysis time is generally spent on the sampling and sample preparation steps including extraction, concentration, fractionation and isolation of analytes 0003-2670/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2009.09.032

H Kataoka et aL/ Analytica Chimica Acta 655 (2009 )8-29 [2]. Therefore, sample preparation has been recognized as the have the above advantages over traditional lle and conventional main bottleneck of the analytical process, especially for analysis SPE LPME including single drop microextraction( SDME)is newly of trace components. Efficient sample ation requires that developed sample preparation technique using minimal amounts sample loss be kept to a minimum, so that the analyte can be of solvent Fiber SPMe and SBSe are widely used microextraction covered in good yield; that coexisting components be removed techniques that use a fiber and a stir-bar-coated polymeric station- efficiently; that problems do not occur in chromatography and ary phase, respectively, as extraction devices. These techniques are electrophoresis systems: that the procedure be performed con- usually performed in vessel, and absorption or adsorption of ana- veniently and quickly and that the cost of analysis be kept to a lytes occurs at the outer surface of the extraction device. In contrast, minimum. Previous sample preparation techniques, however, have in-tube in-needle and in-tip SPMe are unique sample preparation been associated with various problems, such as complicated and techniques that use a capillary tube a microsyringe and a pipette time-consuming operations, the requirement for large amounts of tip respectively, as extraction devices. Absorption or adsorption of sample and organic solvents, and difficult automation. Forexample, analytes occurs at the inner, polymer-coated surface and the outer if sample preparation is time-consuming, the number of samples is surface of packed sorbent. limited and multi-step procedures are prone to loss of analytes.Fur Microextraction techniques using a capillary tube have several thermore, use of harmful chemicals and large amounts of solvent advantages over other microextraction techniques, such as minia cause environmental pollution and health hazards for operators, turization, automation, high-throughput performance, on-line d extra-operational costs for waste treatment. coupling with analytical instruments and no solvent consumption. Traditional liquid-liquid extraction(LLE) and conventional A capillary microextraction technique using an open-tubular fused- lid-phase extraction(SPE) have been widely used for the prepa- silica capillary column as extraction device was first developed ration of biological, environmental and food samples 3-8]. Recent in 1986 as"open-tubular trapping(OTT)"[48. In OTT, a gaseous trends in sample preparation have focused on miniaturization, or liquid sample was passed through the open capillary and the automation, high-throughput performance, on-line coupling with volatile organic compounds trapped on the capillary coating were analytical instruments and low-cost operations through extremely analyzed by thermal desorption and gas chromatography (gc) low or no solvent consumption. Minimizing sample preparati Subsequently, OTT was applied to the analysis of polycyclic aro- steps is effective, not only in reducing sources of error but in reduc- matic hydrocarbons in aqueous samples, coupled with GC [49]. steps is also particularly advantageous for measuring trace do n In a similar capillary microextraction technique, in-tube SPME, g time and cost. Using a minimum number of sample preparati Itra-trace analytes in complex matrices. Microextraction tech- the extracted analytes on the capillary were an nalyzed niques, such as liquid-phase microextraction(LPME)(4,5,9-13], by solvent desorption, coupled on-line with high-performance Ind solid-phase microextraction(SPME)3-47, including in-tube liquid chromatography(HPLC)[50]. Although commercial GC cap in-needle, and in-tip SPME and stir-bar sorptive extraction(SBSE), illary columns are usually used for both OTT and in-tube SPMe, Fused silica Polymer coating capillar Fused silica capillary GC capillary segment Fused silica Sorbent packing Fused silica Fig. 1. Devices for capillary microextraction (A)Open-tubular capillary for OTT and in-tube SPMe, (B)fber-packed capillary, (C) sorbent-packed capillary, (D)monolithic

H. Kataoka et al. / Analytica Chimica Acta 655 (2009) 8–29 9 [2]. Therefore, sample preparation has been recognized as the main bottleneck of the analytical process, especially for analysis of trace components. Efficient sample preparation requires that sample loss be kept to a minimum, so that the analyte can be recovered in good yield; that coexisting components be removed efficiently; that problems do not occur in chromatography and electrophoresis systems; that the procedure be performed con￾veniently and quickly; and that the cost of analysis be kept to a minimum. Previous sample preparation techniques, however, have been associated with various problems, such as complicated and time-consuming operations, the requirement for large amounts of sample and organic solvents, and difficult automation. For example, if sample preparation is time-consuming, the number of samples is limited andmulti-step procedures are prone to loss of analytes. Fur￾thermore, use of harmful chemicals and large amounts of solvent cause environmental pollution and health hazards for operators, and extra-operational costs for waste treatment. Traditional liquid–liquid extraction (LLE) and conventional solid-phase extraction (SPE) have been widely used for the prepa￾ration of biological, environmental and food samples [3–8]. Recent trends in sample preparation have focused on miniaturization, automation, high-throughput performance, on-line coupling with analytical instruments and low-cost operations through extremely low or no solvent consumption. Minimizing sample preparation steps is effective, not only in reducing sources of error but in reduc￾ing time and cost. Using a minimum number of sample preparation steps is also particularly advantageous for measuring trace and ultra-trace analytes in complex matrices. Microextraction tech￾niques, such as liquid-phase microextraction (LPME) [4,5,9–13], and solid-phase microextraction (SPME) [3–47], including in-tube, in-needle, and in-tip SPME and stir-bar sorptive extraction (SBSE), have the above advantages over traditional LLE and conventional SPE. LPME including single drop microextraction (SDME) is newly developed sample preparation technique using minimal amounts of solvent. Fiber SPME and SBSE are widely used microextraction techniques that use a fiber and a stir-bar-coated polymeric station￾ary phase, respectively, as extraction devices. These techniques are usually performed in vessel, and absorption or adsorption of ana￾lytes occurs at the outer surface of the extraction device. In contrast, in-tube, in-needle and in-tip SPME are unique sample preparation techniques that use a capillary tube, a microsyringe and a pipette tip, respectively, as extraction devices. Absorption or adsorption of analytes occurs at the inner, polymer-coated surface and the outer surface of packed sorbent. Microextraction techniques using a capillary tube have several advantages over other microextraction techniques, such as minia￾turization, automation, high-throughput performance, on-line coupling with analytical instruments and no solvent consumption. A capillary microextraction technique using an open-tubular fused￾silica capillary column as extraction device was first developed in 1986 as “open-tubular trapping (OTT)” [48]. In OTT, a gaseous or liquid sample was passed through the open capillary and the volatile organic compounds trapped on the capillary coating were analyzed by thermal desorption and gas chromatography (GC). Subsequently, OTT was applied to the analysis of polycyclic aro￾matic hydrocarbons in aqueous samples, coupled with GC [49]. In a similar capillary microextraction technique, in-tube SPME, an aqueous sample was passed through an open capillary and the extracted analytes on the capillary coating were analyzed by solvent desorption, coupled on-line with high-performance liquid chromatography (HPLC) [50]. Although commercial GC cap￾illary columns are usually used for both OTT and in-tube SPME, Fig. 1. Devices for capillary microextraction. (A) Open-tubular capillary for OTT and in-tube SPME, (B) fiber-packed capillary, (C) sorbent-packed capillary, (D) monolithic capillary

H Kataoka et aL/ Analytica Chimica Acta 655 (2009)8-29 new capillaries were recently devised as microextraction devices a vital consideration in the use of an OTT column, whereas equilib- [7, 15,20, 33]. In addition to open-tubular capillaries, fiber-packed, rium extraction necessitates that a portion of the analyte remains sorbent-packed and rod-type monolith capillaries were developed in the sample after passing through the sorbent. OTT and in-tube to improve extraction efficiency and specificity( Fig. 1)[15, 20, 23. SPME are usually used in combination with GC and LC, respe This review focuses on novel capillary microextraction tech- tively. Therefore, for convenience, coupling with GC is called OTT niques for off-line and on-line sample preparation. OTT, in-tube and coupling with LC is called in-tube SPME Fiber-packed capillary PMe, wire-in-tube SPMe, fiber-in-tube SPE, sorbent-packed cap- microextraction, called"fiber-in-tube SPE", is a modified method ry in-tube SPMe and monolithic capillary in-tube SPMe are using capillary tubes(Fig 1B) packed with fibrous rigid-rod hetero ically evaluated, and their applications to biomedical, pharma- cyclic polymers, which increase extraction efficiency by decreasing ceutical, environmental and food analyses are summarized and capillary volume or by increasing the extracting surface. In con- discussed The details of capillary microextraction techniques for trast, pieces of micro-LC capillary columns packed with extracting sample preparation have also been described in books 1, 2, and phase can also be used for sorbent-packed(Fig 1C)and rod-type well-documented reviews 3, 5-7, 13-25]. monolith( Fig. 1D)capillary microextraction. In these techniques, analytes are absorbed or adsorbed at the outer surface of the packed sorbent Open-tubular and packed capillary microextraction tech- 2. Capillary microextraction techniques niques can be applied to the analysis of particulate-free gas and clean water samples, and analytes can be highly enriched by passing Several types of microextraction devices using capillaries have the sample through the capillary. These capillary microextraction been developed. Pieces of open-tubular capillary GC columns techniques are considered advantageous for the pretreatment of (Fig 1A)are used for OTT and in-tube SPME. In these techniques, complex sample matrices prior to chromatographic and capillary analytes are absorbed or adsorbed at the innersurface-coated inter- electrophoretic processes because they enable on-line analysis at nally with a thin film of the extraction phase. Two methods have low operating costs and with no environmental pollution. In this been developed In the first, for complete trapping of analytes, the section, we describe the characteristics and optimization of these volume of sample should not exceed the breakthrough volume In capillary microextraction techniques. the second, sorption is carried out as an equilibrium process. The sample is passed through the capillary column, but the amount of the analyte retained by the stationary phase at equilibrium is 2. 1. Open-tubular trapping coupled with gas chromatography directly related to its concentration in the sample solution. In both the exhaustive and non-exhaustive(equilibrium) methods, ana In OTT, ambient air, solution, or solution headspace is sampled lytes are desorbed with an appropriate solvent and transferred by passing a gas or liquid through the open capillary. The analytes desorbed thermally. Although the design of in-tube SPME appears Retention of the analytes is based on partitioning into the stationary similar to that of oTT for on-line sample preparation, equilibrium phase. The analytes are subsequently desorbed, either with a small versus exhaustive extraction remains a fundamental difference amount of solvent or by thermal desorption. The sample is forced to between the two techniques. The elimination of breakthrough flow through the capillary and analytes reach the trapping medium (A)Extraction mode Helium(carrier gas OTT sample drying Helium(carier gas) N OTT drying Fig. 2. Schematic diagram of on-line open-tubular trapping (A)Extraction mode(trapping). (B)injection mode(desorption).(1)Three-way flow selection valve, (2)six-port witching valve, (3)on/off valve and P is a pressure gauge

10 H. Kataoka et al. / Analytica Chimica Acta 655 (2009) 8–29 new capillaries were recently devised as microextraction devices [7,15,20,33]. In addition to open-tubular capillaries, fiber-packed, sorbent-packed and rod-type monolith capillaries were developed to improve extraction efficiency and specificity (Fig. 1) [15,20,23]. This review focuses on novel capillary microextraction tech￾niques for off-line and on-line sample preparation. OTT, in-tube SPME, wire-in-tube SPME, fiber-in-tube SPE, sorbent-packed cap￾illary in-tube SPME and monolithic capillary in-tube SPME are critically evaluated, and their applications to biomedical, pharma￾ceutical, environmental and food analyses are summarized and discussed. The details of capillary microextraction techniques for sample preparation have also been described in books [1,2,51] and well-documented reviews [3,5–7,13–25]. 2. Capillary microextraction techniques Several types of microextraction devices using capillaries have been developed. Pieces of open-tubular capillary GC columns (Fig. 1A) are used for OTT and in-tube SPME. In these techniques, analytes are absorbed or adsorbed at the inner surface-coated inter￾nally with a thin film of the extraction phase. Two methods have been developed. In the first, for complete trapping of analytes, the volume of sample should not exceed the breakthrough volume. In the second, sorption is carried out as an equilibrium process. The sample is passed through the capillary column, but the amount of the analyte retained by the stationary phase at equilibrium is directly related to its concentration in the sample solution. In both the exhaustive and non-exhaustive (equilibrium) methods, ana￾lytes are desorbed with an appropriate solvent and transferred off-line or on-line to a GC or HPLC column. Analytes can also be desorbed thermally. Although the design of in-tube SPME appears similar to that of OTT for on-line sample preparation, equilibrium versus exhaustive extraction remains a fundamental difference between the two techniques. The elimination of breakthrough is a vital consideration in the use of an OTT column, whereas equilib￾rium extraction necessitates that a portion of the analyte remains in the sample after passing through the sorbent. OTT and in-tube SPME are usually used in combination with GC and LC, respec￾tively. Therefore, for convenience, coupling with GC is called OTT and coupling with LC is called in-tube SPME. Fiber-packed capillary microextraction, called “fiber-in-tube SPE”, is a modified method using capillary tubes (Fig. 1B) packed with fibrous rigid-rod hetero￾cyclic polymers, which increase extraction efficiency by decreasing capillary volume or by increasing the extracting surface. In con￾trast, pieces of micro-LC capillary columns packed with extracting phase can also be used for sorbent-packed (Fig. 1C) and rod-type monolith (Fig. 1D) capillary microextraction. In these techniques, analytes are absorbed or adsorbed at the outer surface of the packed sorbent. Open-tubular and packed capillary microextraction tech￾niques can be applied to the analysis of particulate-free gas and clean water samples, and analytes can be highly enriched by passing the sample through the capillary. These capillary microextraction techniques are considered advantageous for the pretreatment of complex sample matrices prior to chromatographic and capillary electrophoretic processes because they enable on-line analysis at low operating costs and with no environmental pollution. In this section, we describe the characteristics and optimization of these capillary microextraction techniques. 2.1. Open-tubular trapping coupled with gas chromatography In OTT, ambient air, solution, or solution headspace is sampled by passing a gas or liquid through the open capillary. The analytes are trapped on the coating of a short piece of a capillary GC column. Retention of the analytes is based on partitioning into the stationary phase. The analytes are subsequently desorbed, either with a small amount of solvent or by thermal desorption. The sample is forced to flow through the capillary and analytes reach the trapping medium Fig. 2. Schematic diagram of on-line open-tubular trapping. (A) Extraction mode (trapping), (B) injection mode (desorption). (1) Three-way flow selection valve, (2) six-port switching valve, (3) on/off valve and P is a pressure gauge

H Kataoka et aL/ Analytica Chimica Acta 655 (2009 )8-29 coated onto the walls by diffusion. The thermal stability of GC sta- smoothly evaporate during analyte transfer For example, the on- tionary phases allows collected analytes to be thermally desorbed line OTt/GC technique, using a commercial GC capillary coated with from a trap after sampling. These analytes can be desorbed directly polydimethylsiloxane(PDMs), has been used for the isolation and onto a GC column for analysis, avoiding dilution of the sample with enrichment of organic pollutants in air matrices [58]. solvent. The likelihood of sample cross-contamination and possible degradation are minimized because intermediate sample handling 2.1.2. Of-line OTT-GC eps are eliminated. Another advantage is that even very volatile The off-line oTt/GC method is used for the sampling and enrich- compounds can be enriched at ambient temperature, omitting the tof volatile compounds in air and water samples. These include need for a cryogenic refocusing step. Although the OTT approach methods for the microextraction of volatile organic compounds overcomes some mechanical stability problems inherent to con-(VOCs)using an inside needle capillary adsorption trap device 54 ventional SPME fibers, it involves complex instrumental setup and and a new high-performance cryofocusing system for the capil- unfavorable sampling conditions, for example high pressure drops lary microextraction of VOCs in aqueous matrices and headspace from long traps and limited flow-rates. An additional disadvan- GC applications [61. In the latter device, the compounds tage is the need for dry purging with nitrogen after extraction, to centrated in a fused-silica transfer capillary with the aid of liqui completely remove the water from the capillary walls. Several OTT nitrogen. A glass tube liner(ca 25 cm x 1. 5 mm i.. ) is inserted into approaches, involving off-line or on-line coupling with GC, have the heated (200'C)injector of the gas chromatograph in place been described 49, 52-71 of the standard glass liner, and also extends further through a liq- uid nitrogen container made with styroform-like material Inside 1. 1. On-line OTT-GC syste his glass tube, the fused-silica transfer line passing through the schematic diagram of an on-line OTT GC system 56] is shown oven door is connected like a pre-column to the analytical high in Fig. 2. In this system, water samples are pumped through an resolution GC column. It can move fast between the heated and OTT capillary(2 m x 0.32 mm i.d. )coated with a 5-um thick sta- cooled zones; when this movement starts, cryofocused analytes tionary phase by an HPLC pump at a flow-rate of 1.5 mLmin-. The are injected"at once"resulting in symmetrical and sharp injection sampling time required to reach equilibrium is determined exper- bands with"zero"carryover. This cryofocuser resulted in repeat imentally. After sampling, the OTT capillary is dried by purging for able and accurate injections, which helped GC separations of VOcs 5 min with 1 mL min nitrogen to completely remove the water. coming from solid, gaseous or aqueous matrices. The high repro- he retained analytes are thermally desorbed by heating the cap- ducibility of the system depends on the high temperature stability illary under stop flow conditions. The analytes released from the of the heated cooled zones and on the negligible thermal mass per stationary phase of the capillary are transferred into the void vol- unit length of fused-silica. ume of the column, the six-port valve is switched to the injection Sol-gel capillary microextraction methods have been developed position, and the analytes are transferred to the analytical column for the solventless preconcentration of trace analytes 57, 64-70 by the flow of a carrier gas. The amount injected can be varied by The capillary with the extracted analytes can then be connected varying the time during which the six-port valve is left in the injec- to the inlet end of the gc column using a two-way press-fit tion position. Finally, the analytes are separated on the gC column. fused-silica connector housed inside the gC injection port. the The on-line OTT/GC system is also applied to the concentration and analytes can be desorbed from the extraction capillary by rapid analysis of gaseous samples [55] A second on-line OTT/GC system uses phase-switching port. The desorbed analytes are transported down the system by 49, 52,53], in which the analytes are adsorbed or absorbed from the flow of helium and further focused at the inlet end of the a methanol-water mobile phase to the stationary phase of an Ott GC column maintained at 30C. Several sol-gel coatings, includ capillary. Subsequently, the aqueous phase is removed by purg ing poly(dimethylsiloxane)(PDMS), poly(ethylene glycol)(PEG). ing the trap with nitrogen and the analytes are desorbed with polytetrahydrofuran, cyano-PDMS, and organic-inorganic hybrid organic solvent and transferred to the GC system using a pro- materials, have been developed for the extraction of non-polar, grammed temperature vaporizing(PTv)injector as interface. The moderately polar and polar compounds. Sol-gel technology can main advantage of analyte enrichment based on capillary column is fine tune the selectivity of a sol-gel coating simply by chang the ability to completely and reliably remove water after sampling ing the relative proportions of organic and inorganic components by purging a short plug of gas through the capillary In principle of the sol solution For extraction, aqueous samples are prepared the otr capillary has two disadvantages compared with packed by further diluting to ng/mL concentrations. a thermally condi columns: (1)its retention power is generally weaker and (2)sam- tioned capillary(10-40 cm x 0.25 mm i.d. )can then be vertically ling flow-rate is limited due to the slow diffusion of analytes in connected to the lower end of the gravity-fed sample dispenser. a water. The retention power of an OTT capillary (i. e a 2 m piece of 50 mL aliquot of aqueous sample is then poured into the dispenser the gC column)can be greatly enhanced by swelling the station- from its top end, and allowed to flow through the microextrac ary phase with an organic solvent prior to sampling 52] allowing tion capillary under gravity. While passing the sample through the practical use of OTT for on-line extraction-GC of aqueous sam- the capillary, the analytes can be sorbed by the sol-gel coating time becon about 2.5 mL For large sample volumes, the sampling on the inner wall of the capillary. Extraction requires 30-40 min time becomes unacceptably long due to the requirement for low for establishment of equilibrium. The capillary is then detached sampling flow-rate(ca 0.1 mLmin ) Obviously, flow-rate should from the dispenser and the residual sample droplets are removed not exceed a certain threshold, ca 0.2 mLmin-l, or breakthrough by touching one end of each microextraction capillary tube with willoccur immediately Alternatively, a thick-film stationary phas a tissue. The capillary can then be installed in the gC injection will increase the breakthrough volume. For example, much higher port, with 3 cm of its lower end protruding into the gC oven. sample flow-rates, i. e. up to 4 mLmin, are allowed for coiled or This end is then interfaced with the inlet of a GC capillary col- stitched columns, because deformed capillaries induce a secondary umn using a deactivated two-way press- fit quart connector. The flow, enhancing radical dispersion[53]. water should not dissolve analytes are thermally desorbed from the capillary by rapidly rais- in the organic solvent, or should the organic solvent dissolve in ing the temperature of the injector. Although low detection limits water. In the former case, water would be injected into the GC sys- were achieved for the analysis of phenols, alcohols, and amines tem, while in the latter case the swollen stationary phase would water [ 57] the procedure could not be automated and, therefore, lose(part of)the swelling agent. Finally, the organic solvent should was highly time-consuming. Recently, a high pH-resistant surface-

H. Kataoka et al. / Analytica Chimica Acta 655 (2009) 8–29 11 coated onto the walls by diffusion. The thermal stability of GC sta￾tionary phases allows collected analytes to be thermally desorbed from a trap after sampling. These analytes can be desorbed directly onto a GC column for analysis, avoiding dilution of the sample with solvent. The likelihood of sample cross-contamination and possible degradation are minimized because intermediate sample handling steps are eliminated. Another advantage is that even very volatile compounds can be enriched at ambient temperature, omitting the need for a cryogenic refocusing step. Although the OTT approach overcomes some mechanical stability problems inherent to con￾ventional SPME fibers, it involves complex instrumental setup and unfavorable sampling conditions, for example high pressure drops from long traps and limited flow-rates. An additional disadvan￾tage is the need for dry purging with nitrogen after extraction, to completely remove the water from the capillary walls. Several OTT approaches, involving off-line or on-line coupling with GC, have been described [49,52–71]. 2.1.1. On-line OTT-GC systems A schematic diagram of an on-line OTT/GC system [56] is shown in Fig. 2. In this system, water samples are pumped through an OTT capillary (2 m × 0.32 mm i.d.) coated with a 5-m thick sta￾tionary phase by an HPLC pump at a flow-rate of 1.5 mL min−1. The sampling time required to reach equilibrium is determined exper￾imentally. After sampling, the OTT capillary is dried by purging for 5 min with 1 mL min−1 nitrogen to completely remove the water. The retained analytes are thermally desorbed by heating the cap￾illary under stop flow conditions. The analytes released from the stationary phase of the capillary are transferred into the void vol￾ume of the column, the six-port valve is switched to the injection position, and the analytes are transferred to the analytical column by the flow of a carrier gas. The amount injected can be varied by varying the time during which the six-port valve is left in the injec￾tion position. Finally, the analytes are separated on the GC column. The on-line OTT/GC system is also applied to the concentration and analysis of gaseous samples [55]. A second on-line OTT/GC system uses phase-switching [49,52,53], in which the analytes are adsorbed or absorbed from a methanol–water mobile phase to the stationary phase of an OTT capillary. Subsequently, the aqueous phase is removed by purg￾ing the trap with nitrogen and the analytes are desorbed with organic solvent and transferred to the GC system using a pro￾grammed temperature vaporizing (PTV) injector as interface. The main advantage of analyte enrichment based on capillary column is the ability to completely and reliably remove water after sampling by purging a short plug of gas through the capillary. In principle, the OTT capillary has two disadvantages compared with packed columns: (1) its retention power is generally weaker and (2) sam￾pling flow-rate is limited due to the slow diffusion of analytes in water. The retention power of an OTT capillary (i.e. a 2 m piece of the GC column) can be greatly enhanced by swelling the station￾ary phase with an organic solvent prior to sampling [52], allowing the practical use of OTT for on-line extraction-GC of aqueous sam￾ples, up to about 2.5 mL. For large sample volumes, the sampling time becomes unacceptably long due to the requirement for low sampling flow-rate (ca. 0.1 mL min−1). Obviously, flow-rate should not exceed a certain threshold, ca. 0.2 mL min−1, or breakthrough will occur immediately. Alternatively, a thick-film stationary phase will increase the breakthrough volume. For example, much higher sample flow-rates, i.e. up to 4 mL min−1, are allowed for coiled or stitched columns, because deformed capillaries induce a secondary flow, enhancing radical dispersion [53]. Water should not dissolve in the organic solvent, or should the organic solvent dissolve in water. In the former case, water would be injected into the GC sys￾tem, while in the latter case the swollen stationary phase would lose (part of) the swelling agent. Finally, the organic solvent should smoothly evaporate during analyte transfer. For example, the on￾line OTT/GC technique, using a commercial GC capillary coated with polydimethylsiloxane (PDMS), has been used for the isolation and enrichment of organic pollutants in air matrices [58]. 2.1.2. Off-line OTT-GC systems The off-line OTT/GC method is used for the sampling and enrich￾ment of volatile compounds in air and water samples. These include methods for the microextraction of volatile organic compounds (VOCs) using an inside needle capillary adsorption trap device [54] and a new high-performance cryofocusing system for the capil￾lary microextraction of VOCs in aqueous matrices and headspace GC applications [61]. In the latter device, the compounds are con￾centrated in a fused-silica transfer capillary with the aid of liquid nitrogen. A glass tube liner (ca. 25 cm × 1.5 mm i.d.) is inserted into the heated (∼200 ◦C) injector of the gas chromatograph in place of the standard glass liner, and also extends further through a liq￾uid nitrogen container made with styroform-like material. Inside this glass tube, the fused-silica transfer line passing through the oven door is connected like a pre-column to the analytical high￾resolution GC column. It can move fast between the heated and cooled zones; when this movement starts, cryofocused analytes are injected “at once” resulting in symmetrical and sharp injection bands with “zero” carryover. This cryofocuser resulted in repeat￾able and accurate injections, which helped GC separations of VOCs coming from solid, gaseous or aqueous matrices. The high repro￾ducibility of the system depends on the high temperature stability of the heated/cooled zones and on the negligible thermal mass per unit length of fused-silica. Sol–gel capillary microextraction methods have been developed for the solventless preconcentration of trace analytes [57,64–70]. The capillary with the extracted analytes can then be connected to the inlet end of the GC column using a two-way press-fit fused-silica connector housed inside the GC injection port. The analytes can be desorbed from the extraction capillary by rapid temperature programming of 100 ◦C min−1 of the GC injection port. The desorbed analytes are transported down the system by the flow of helium and further focused at the inlet end of the GC column maintained at 30 ◦C. Several sol–gel coatings, includ￾ing poly(dimethylsiloxane) (PDMS), poly(ethylene glycol) (PEG), polytetrahydrofuran, cyano-PDMS, and organic–inorganic hybrid materials, have been developed for the extraction of non-polar, moderately polar and polar compounds. Sol–gel technology can fine tune the selectivity of a sol–gel coating simply by chang￾ing the relative proportions of organic and inorganic components of the sol solution. For extraction, aqueous samples are prepared by further diluting to ng/mL concentrations. A thermally condi￾tioned capillary (10–40 cm × 0.25 mm i.d.) can then be vertically connected to the lower end of the gravity-fed sample dispenser. A 50 mL aliquot of aqueous sample is then poured into the dispenser from its top end, and allowed to flow through the microextrac￾tion capillary under gravity. While passing the sample through the capillary, the analytes can be sorbed by the sol–gel coating on the inner wall of the capillary. Extraction requires 30–40 min for establishment of equilibrium. The capillary is then detached from the dispenser and the residual sample droplets are removed by touching one end of each microextraction capillary tube with a tissue. The capillary can then be installed in the GC injection port, with ∼3 cm of its lower end protruding into the GC oven. This end is then interfaced with the inlet of a GC capillary col￾umn using a deactivated two-way press-fit quart connector. The analytes are thermally desorbed from the capillary by rapidly rais￾ing the temperature of the injector. Although low detection limits were achieved for the analysis of phenols, alcohols, and amines in water [57], the procedure could not be automated and, therefore, was highly time-consuming. Recently, a high pH-resistant surface-

H Kataoka et aL/ Analytica Chimica Acta 655 (2009)8-29 bonded organic-inorganic hybrid zirconia coating was developed the capillary can be easily blocked. Therefore, to prevent plugging to analyze polycyclic aromatic hydrocarbons(PAHs), ketones, and of the capillary column and flow lines, it is necessary to filter or Idehydes in aqueous samples 66 centrifuge sample solutions before extraction. Although yields are generally low, these compounds may be extracted reproducibly using an autosampler, and all extracts may be introduced into an 2. 2. In-tube solid-phase microextraction LC column after in-tube spme In-tube SPME [ 3, 15, 24, 50 is an effective sar technique that uses an open-tubular capillary column as an SPME 2.2.1. Operation system device and can be coupled on-line with HPLC or LC-MS. It was In in-tube SPMe, organic compounds can be directly extracted developed to overcome some problems related to the use of con- and concentrated in the stationary phase of the column by introduc- entional fiber SPMe, such as fragility low sorption capacity, and ing the sample, using a programmed autosampler, untilequilibrium bleeding from thick-film coatings of fiber. Unlike fiber SPMe, where is achieved or until extraction is sufficient. After desorption, the a sorbent coating on the outer surface of a small-diameter solid analytes can be directly transferred to an LC column. For static rod serves as the extraction medium, in-tube SPme typically uses desorption, a solvent is drawn into the capillary and the desorbed a piece of fused-silica capillary with a stationary phase coating analytes are sent into the injection loop of the valve. If desorp- its inner surface(e. g, a short piece of GC column) for extraction. tion is efficient in the initial mobile phase, the mobile phase is tube SPMe has also been termed "coated capillary microextrac- directly passed through the capillary to the column for dynamic tion". This method can directly extract target analytes in aqueous desorption. The procedures of in-tube SPMe, including extraction, matrices and concentrate the analytes into the internally coated concentration, desorption, and injection, can be easily automated tationary phase of a capillary. the analytes can then be desorbed using a conventional autosampler. In-tube SPme operation sys by introducing a stream of mobile phase, or by using a static des- tems can be categorized as flow through extraction systems(coated orption solvent when the analytes are more strongly adsorbed to capillary microextraction)[76. in which solutions are passed con- the capillary coating. The desorbed compounds can subsequently tinuously in one direction through an extraction capillary column; be injected into the LC column for analysis. One alternative to or as draw/eject extraction systems (in-tube SPME)[0]. in which coated fiber is an internally coated capillary, through which the sample solution is repeatedly aspirated into and dispensed from this technique is that it enables automation of the SPME-HPlc diagrams of on-line in-tube SPME systems. Although their designs process, allowing extraction, desorption and injection to be per- appear similar to those of on-line OTT systems using a switching ormed continuously using a standard autosampler. In addition, it technique, equilibrium versus exhaustive extraction remains a fun- has lower detection limits compared with fiber SPME-HPLC sys- damental difference between the two methods. The elimination of tems. Automated sample handling procedures not only shorten breakthrough is a vital consideration in the use of an OTT column. be used with all GC commercial columns, thus increasing the A flow through capillary microextraction system [76, 77 is number of stationary phases, allowing a wide field of applica- shown in Fig. 3. The complete analytical system consists of an tion. Although these stationary phases are unsuitable for extraction automatic six-port valve, two pumps (sample pump and wash of polar compounds, the problem can be improved by deriva- pump) and an LC system. The capillary column is installed in tization of compounds [72-75. The automated on-line in-tube the switching six-port valve. The enrichment procedure is divided SPME-assisted derivatization technique has been developed for into 4 steps: conditioning, extraction, washing and desorption the analysis of dimethylamine by extraction/derivatization with The extraction capillary column is rinsed and conditioned with derivatizing agent previously coated on capillary [72]. In-tube Mili-Q water(wash pump, 1 min, 1.5mLmin-l) During extraction privatization techniques improve detectability through increasing( Fig 3A), the six-port valve is switched to the""LORD"position, selectivity and sensitivity, and enhance the separation of analytes and the aqueous sample is pumped through the column(sample with poor chromatographic behavior. The main disadvantage of pump, 5 min, 4.0 mLmin-1) This is followed by a washing step the tech is the requirement for very clean samples, because (wash pump), in which the capillary column is rinsed for 1 min (B) Inject position(desorption) sⅸ port valve Capillary column Sample Wash LC column pump pump LC column pump pump Detector Fig. 3. Schematic diagram of automated on-line coated capillary microextraction(flow through extraction system).(A)Load position(extraction),(B)inject positio

12 H. Kataoka et al. / Analytica Chimica Acta 655 (2009) 8–29 bonded organic–inorganic hybrid zirconia coating was developed to analyze polycyclic aromatic hydrocarbons (PAHs), ketones, and aldehydes in aqueous samples [66]. 2.2. In-tube solid-phase microextraction In-tube SPME [3,15,24,50] is an effective sample preparation technique that uses an open-tubular capillary column as an SPME device and can be coupled on-line with HPLC or LC–MS. It was developed to overcome some problems related to the use of con￾ventional fiber SPME, such as fragility, low sorption capacity, and bleeding from thick-film coatings of fiber. Unlike fiber SPME, where a sorbent coating on the outer surface of a small-diameter solid rod serves as the extraction medium, in-tube SPME typically uses a piece of fused-silica capillary with a stationary phase coating on its inner surface (e.g., a short piece of GC column) for extraction. In-tube SPME has also been termed “coated capillary microextrac￾tion”. This method can directly extract target analytes in aqueous matrices and concentrate the analytes into the internally coated stationary phase of a capillary. The analytes can then be desorbed by introducing a stream of mobile phase, or by using a static des￾orption solvent when the analytes are more strongly adsorbed to the capillary coating. The desorbed compounds can subsequently be injected into the LC column for analysis. One alternative to a coated fiber is an internally coated capillary, through which the sample flows or is drawn repeatedly. The main advantage of this technique is that it enables automation of the SPME-HPLC process, allowing extraction, desorption and injection to be per￾formed continuously using a standard autosampler. In addition, it has lower detection limits compared with fiber SPME-HPLC sys￾tems. Automated sample handling procedures not only shorten the total analysis time, but are more accurate and precise than manual techniques. Another important advantage is that it can be used with all GC commercial columns, thus increasing the number of stationary phases, allowing a wide field of applica￾tion. Although these stationary phases are unsuitable for extraction of polar compounds, the problem can be improved by deriva￾tization of compounds [72–75]. The automated on-line in-tube SPME-assisted derivatization technique has been developed for the analysis of dimethylamine by extraction/derivatization with derivatizing agent previously coated on capillary [72]. In-tube derivatization techniques improve detectability through increasing selectivity and sensitivity, and enhance the separation of analytes with poor chromatographic behavior. The main disadvantage of the technique is the requirement for very clean samples, because the capillary can be easily blocked. Therefore, to prevent plugging of the capillary column and flow lines, it is necessary to filter or centrifuge sample solutions before extraction. Although yields are generally low, these compounds may be extracted reproducibly using an autosampler, and all extracts may be introduced into an LC column after in-tube SPME. 2.2.1. Operation system In in-tube SPME, organic compounds can be directly extracted and concentrated in the stationary phase of the column by introduc￾ing the sample, using a programmed autosampler, until equilibrium is achieved or until extraction is sufficient. After desorption, the analytes can be directly transferred to an LC column. For static desorption, a solvent is drawn into the capillary and the desorbed analytes are sent into the injection loop of the valve. If desorp￾tion is efficient in the initial mobile phase, the mobile phase is directly passed through the capillary to the column for dynamic desorption. The procedures of in-tube SPME, including extraction, concentration, desorption, and injection, can be easily automated using a conventional autosampler. In-tube SPME operation sys￾tems can be categorized as flow through extraction systems (coated capillary microextraction) [76], in which solutions are passed con￾tinuously in one direction through an extraction capillary column; or as draw/eject extraction systems (in-tube SPME) [50], in which the sample solution is repeatedly aspirated into and dispensed from an extraction capillary column. Figs. 3 and 4 illustrate schematic diagrams of on-line in-tube SPME systems. Although their designs appear similar to those of on-line OTT systems using a switching technique, equilibrium versus exhaustive extraction remains a fun￾damental difference between the two methods. The elimination of breakthrough is a vital consideration in the use of an OTT column, whereas equilibrium extraction requires a portion of the analyte to remain in the sample after passing through the sorbent. A flow through capillary microextraction system [76,77] is shown in Fig. 3. The complete analytical system consists of an automatic six-port valve, two pumps (sample pump and wash pump) and an LC system. The capillary column is installed in the switching six-port valve. The enrichment procedure is divided into 4 steps: conditioning, extraction, washing and desorption. The extraction capillary column is rinsed and conditioned with Mili-Q water (wash pump, 1 min, 1.5 mL min−1). During extraction (Fig. 3A), the six-port valve is switched to the “LORD” position, and the aqueous sample is pumped through the column (sample pump, 5 min, 4.0 mL min−1). This is followed by a washing step (wash pump), in which the capillary column is rinsed for 1 min Fig. 3. Schematic diagram of automated on-line coated capillary microextraction (flow through extraction system). (A) Load position (extraction), (B) inject position (desorption)

H Kataoka et aL Analytica Chimica Acta 655(2009)8-29 (A)Load position(extraction) (B)Inject position(desorption) PEEK tube Column connector Injection loop Capillary njection loop (0) Capillary capillary njection Metering column needle n Au oler Waste d Waste° Mobile phase DA LC column Detector Workstation Detector Workstation Fig. 4. Schematic diagram of automated on-line in-tube solid-phase microextraction(draw/eject extraction system)(A)Load position(extraction).(B)inject position (desorption). DAD is photodiode array detector and MSD is mass detector. with Mili-Q water(1.5 mLmin-1)to remove remaining matrix and The computer controls the drawing and ejection of sample solu norganic residues from the capillary. For desorption( Fig. 3B), the tion, switching of the valves, control of peripheral equipment such x-port valve is switched to the" INJECT"position, and the lc elu- as the HPLC and mSD, and analytical data processing, thus redue acetonitrile-Mili-Q water containing 1% acetic acid(85+15), ing labor and enhancing precision. In addition, a large number of is passed through the column for 4 min. For this step, the flow-rate samples can be autor lly processed by the autosampler with- of the lC pump was reduced to 100 HLmin-I to reduce the back out carryover, because the injection needle and capillary column pressure of the analytical column on the capillary. The desorbed are washed in methanol and the mobile phase before the sample is analytes are transferred to the analytical column for separation and extracted. detected using a UV or tandem mass selective detector(MS-MS). In a similar system, using double column switching valves [78]. the 2.2.2. Optimization of parameters extraction and analysis segments were independent, enabling In-tube SPme depends on the distribution coefficient of each pid, simultaneous performance of several runs, thus shortening analyte as well as its affinity for the fiber SPME, making it important and the time for the whole analysis can be shortened. These sys- the rapidity and efficiency of extraction s y phase to optimize analysis time, with the separation process of the previous sample to raise the distribution factor in the stationa tems, however, may cause some systematic problems, including ciency of the extraction depend on the extraction rate the sample contamination of the switching valve with sample solution because volume, the ph of the sample the type of stationary phase, and the capillary column is directly fixed on the six-port valve. This may the internal diameter, length, and film thickness of the capillary result in inaccurate quantitative information and overestimation of column. Several commercially available capillary columns, which differ In the draw/eject extraction system(Fig. 4), an extraction capil- according to the selectivity of the stationary phase, internal diam ry column is placed between the injection loop and the injection eter, length and film thickness, have been developed. For example, needle of the HPLC autosampler. The capillary connections are facil- a low polarity column with a methyl silicon liquid-phase selec itated by using a 2.5-cm long sleeve of 1/16in polyether ether tively retains hydrophobic compounds, whereas a high polarity ketone(PEEK)tubing at each end of the capillary fixed with 1/16- column with a polyethylene glycol liquid-phase selectively retains in SS unions(0. 25 mm bore stainless steel nuts)and ferrules. An hydrophilic compounds. Since the internal diameter, length and injection loop is installed to prevent sample contamination of the film thickness of the column and other dimensions affect the metering pump and switching valve Building in UV, diode arrays amount of sample that can be loaded and the amount of compound or fluorescence detectors between the HPLC and the MsD, can that can be extracted these parameters should be chosen carefully. enhance the multi-dimensional and simultaneous multidetections, The thin(usually <1 um)coating in such capillaries often results in improving analyte identification. As shown in Fig. 4A, a computer low stationary phase loading reducing sample capacity and extrac controls the injection syringe, which repeatedly draws and ejects tion sensitivity, although extraction equilibrium is quickly attaine sample from the vial, with the analytes partitioning from the sam in the relatively thin layers. Although increasing the film thickness ple matrix into the stationary phase until equilibrium is almost of the stationary phase may solve this problem, it is extremely dif- reached. Subsequently, the extracted analytes can be directly des- ficult to reliably immobilize thicker coatings using conventional orbed from the capillary coating by mobile phase flow or by an approaches, and conventionally prepared GC coatings do not bind A Pirated desorption solvent after switching the six-port valve chemically to the fused-silica capillary inner surface. This lack ig. 4B). It is therefore necessary to prevent plugging of the cap- of chemical bonds is mainly responsible for low solvent stabil- illary columns and flow lines during the extraction, as well as to ity, preventing effective hyphenation of in-tube SPME techniques remove particles from samples by filtration before extraction. The that employ nic or organo-aqueous mobile phases. In add desorbed analytes are then transported to the HPLC column for sep- tion, although large amounts of compound can be extracted their aration and detected using a UV or mass selective detector(MSD). quantitative desorption from capillary columns may be difficult by

H. Kataoka et al. / Analytica Chimica Acta 655 (2009) 8–29 13 Fig. 4. Schematic diagram of automated on-line in-tube solid-phase microextraction (draw/eject extraction system). (A) Load position (extraction), (B) inject position (desorption). DAD is photodiode array detector and MSD is mass detector. with Mili-Q water (1.5 mL min−1) to remove remaining matrix and inorganic residues from the capillary. For desorption (Fig. 3B), the six-port valve is switched to the “INJECT” position, and the LC elu￾ant, acetonitrile–Mili-Q water containing 1% acetic acid (85 + 15), is passed through the column for 4 min. For this step, the flow-rate of the LC pump was reduced to 100 L min−1 to reduce the back pressure of the analytical column on the capillary. The desorbed analytes are transferred to the analytical column for separation and detected using a UV or tandem mass selective detector (MS–MS). In a similar system, using double column switching valves [78], the extraction and analysis segments were independent, enabling the rapid, simultaneous performance of several runs, thus shortening analysis time, with the separation process of the previous sample and the time for the whole analysis can be shortened. These sys￾tems, however, may cause some systematic problems, including contamination of the switching valve with sample solution because the capillary column is directly fixed on the six-port valve. This may result in inaccurate quantitative information and overestimation of analyte. In the draw/eject extraction system (Fig. 4), an extraction capil￾lary column is placed between the injection loop and the injection needle of the HPLC autosampler. The capillary connections are facil￾itated by using a 2.5-cm long sleeve of 1/16 in. polyether ether ketone (PEEK) tubing at each end of the capillary, 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. Building in UV, diode arrays or fluorescence detectors between the HPLC and the MSD, can enhance the multi-dimensional and simultaneous multidetections, improving analyte identification. As shown in Fig. 4A, a computer controls the injection syringe, which repeatedly draws and ejects sample from the vial, with the analytes partitioning from the sam￾ple matrix into the stationary phase until equilibrium is almost reached. Subsequently, the extracted analytes can be directly des￾orbed from the capillary coating by mobile phase flow or by an aspirated desorption solvent after switching the six-port valve (Fig. 4B). It is therefore necessary to prevent plugging of the cap￾illary columns and flow lines during the extraction, as well as to remove particles from samples by filtration before extraction. The desorbed analytes are then transported to the HPLC column for sep￾aration and detected using a UV or mass selective detector (MSD). The computer controls the drawing and ejection of sample solu￾tion, switching of the valves, control of peripheral equipment such as the HPLC and MSD, and analytical data processing, thus reduc￾ing labor and enhancing precision. In addition, a large number of samples can be automatically processed by the autosampler with￾out carryover, because the injection needle and capillary column are washed in methanol and the mobile phase before the sample is extracted. 2.2.2. Optimization of parameters In-tube SPME depends on the distribution coefficient of each analyte as well as its affinity for the fiber SPME, making it important to raise the distribution factor in the stationary phase to optimize the rapidity and efficiency of extraction. The selectivity and effi- ciency of the extraction depend on the extraction rate, the sample volume, the pH of the sample, the type of stationary phase, and the internal diameter, length, and film thickness of the capillary column. Several commercially available capillary columns, which differ according to the selectivity of the stationary phase, internal diam￾eter, length and film thickness, have been developed. For example, a low polarity column with a methyl silicon liquid-phase selec￾tively retains hydrophobic compounds, whereas a high polarity column with a polyethylene glycol liquid-phase selectively retains hydrophilic compounds. Since the internal diameter, length and film thickness of the column and other dimensions affect the amount of sample that can be loaded and the amount of compound that can be extracted, these parameters should be chosen carefully. The thin (usually <1 m) coating in such capillaries often results in low stationary phase loading reducing sample capacity and extrac￾tion sensitivity, although extraction equilibrium is quickly attained in the relatively thin layers. Although increasing the film thickness of the stationary phase may solve this problem, it is extremely dif- ficult to reliably immobilize thicker coatings using conventional approaches, and conventionally prepared GC coatings do not bind chemically to the fused-silica capillary inner surface. This lack of chemical bonds is mainly responsible for low solvent stabil￾ity, preventing effective hyphenation of in-tube SPME techniques that employ organic or organo-aqueous mobile phases. In addi￾tion, although large amounts of compound can be extracted, their quantitative desorption from capillary columns may be difficult by

H Kataoka et aL/ Analytica Chimica Acta 655 (2009)8-29 石西园店可 extension of the sample bandwidth(peak broadening and tailing) The optimal length of a capillary column is 20-100 cm and the opti mal length of a flow through in draw/eject extraction system is 50-60 cm. The internal diameter of a capillary used in combina tion with HPLC is 0. 25 or 0. 32 mm with a short widebore capillary (0.53 mm)used in combination with capillary electrophoresis(CE), because widebore capillaries are resistant to pressure. Although capillary columns with chemically bonded or cross-linked liquid phases are very stable in water and organic solvents, they readily deteriorate in the presence of strong inorganic acids or alkalis. How- ever, capillary columns are generally stable for the mobile phase usually used in HPLC. In in-tube SPME, complete equilibrium extraction is generally 落二三莴手R的导 not obtained fo y analyte, because these analytes are partially desorbed into the mobile phase during the ejection step For flow through extraction systems, the volume of sample passed through a capillary is usually 0. 2-2 mL, and the optimum extraction flow-rate is 0. 25-4 mLmin-, depending on the capacity of the column. In contrast, an increase in the number and volume of draw/eject cycle can enhance the extraction efficiency of in-tube SPMe, but band- width may widen, and peak broadening has often been observed. A column 20-80 cm in length is usually used for effective extraction. For a capillary column (inner diameter 0. 25 mm, length 60 cm) with a volume of 30uL and an injection needle of 10 HL, optimal condi- tions include a draw/ejection volume of 30-40-FL, a draw/ejection flow-rate of50-100-HLmin-I and 10-15 draw/ejection cycles [15] Generally, it is possible to increase the extraction efficiency of ana- lyte into a stationary phase in Spme by changing the ph and salt level of the sample solution. Acidic and basic compounds can be effectively extracted from acidic and alkaline sample solutions respectively. However, the stability of each compound at the ph 目创|导N,,=,,,=,, of the sample solution must be determined beforehand. Although salting out increases extraction efficiency in fiber SPMe, the salt deposits can clog the column during in-tube SPME. Furthermore, the presence of a hydrophilic solvent such as methanol in the sam- ple decreases the extraction efficiency by increasing the solubility of the compound in the sample, but methanol concentrations ,, <5% have little effect on extraction efficiency. The amount of com- pound extracted into the stationary phase is dependent on the 2. 2.3. Capillary coatings GC capillary column coatings were initially compared with 至三R,,,, uncoated fused-silica(a retention gap capillary) as extraction devices for in-tube SPMe. These comparisons included silica mod ified with PDMs (e.g, SPB-1, PTE-5 and SPB-5) or polyethylene glycols(PEG), such as Omegawax 250 and Supelcowax, porous DVB and Supel-Q-Plot 3, 79-81. The relatively polar poly(ethylene glycol)(PEG) coating(Omegawax 250)is sufficiently bonded and cross-linked to prevent loss of phase when solvent is passe through the capillary, showing the highest yield of analyte. Si ica modified columns have been found more suitable for the analysis of non-polar compounds. Use of the adsorptive coated cap- illary Supel-Q-PLOT(DVB polymeric material) has recently been found to be more efficient for the analysis of estrogens, because 9=,, of its large surface area, enhancing mass-transfer kinetics, and because estrogens are of intermediate polarity [82]. Table 1 shows a comparison of some commercially available GC capillary columns used in the extraction of environmental pollutants, food contami nants and biological compounds For most organic comp porous polymer-type capillary column(Supel-Q PLOT) showed bet- ter extraction efficiency than the other liquid-phase type capillary columns(CP-Sil 5CB, CP-Sil 19CB, and CP-Wax 52CB) As the PLot column has a large adsorption surface area and a thick-film layer, the amount extracted was greater than that with liquid-phase type columns. However, patulin was effectively extracted with another

14 H. Kataoka et al. / Analytica Chimica Acta 655 (2009) 8–29 Table 1 Ability of commercially available capillary coatings to extract various compounds by in-tube SPME. Compound DB-1 (0.25 m) CP-Sil 5CB (5 m) DB-17 (0.25 m) CP-Sil 19CB (1.2 m) Omega wax (0.25 m) CP-Wax 52CB (1.2 m) Pora PLOT amine (10 m) Supel-Q PLOT (17 m) Carboxen 1006 (15 m) Ref. Nonyl phenol 9.7 – 10.4 – 3.5 – – 21.6 – [161] Bisphenol A 1.6 – 1.8 – 1.2 – – 22.8 – [161] Dibutyl phthalate 8.6 – 11.0 – 2.7 – – 24.1 – [161] Di-2-ethylhexyl phthalate 3.6 – 5.1 – 4.0 – – 5.8 – [161] Genistein 1.1 – 2.3 – 7.2 – – 11.9 – [166] Patuline – 0.4 – 0.4 – 0.2 11.8 0.8 30.3 [163] Aflatoxin B1 – 3.1 – 2.5 – 2.8 23.7 56.2 2.8 [164] -Estradiol 0.8 – 2.4 – 1.8 – – 30.0 – [82] Testosterone – 13.3 – 9.3 – 4.2 20.5 44.9 5.8 Cortisol – 8.8 – 8.4 – 9.5 – 15.9 5.9 [122] Nicotine – 3.6 – 3.0 – 2.3 50.5 21.4 26.7 [123] Atrazine 1.5 – 2.3 – 1.1 – – 58.8 12.0 [149] Perfluorooctane sulfonate – 0.4 – 1.9 – 1.2 57.5 12.8 5.9 Phenanthrene – 50.8 – 63.7 – 17.7 – 54.8 1.1 Benzo(a)pyrene – 28.7 – 61.9 – 44.7 – 49.2 2.6 Each data represents relative ratio of peak height of compound by in-tube SPME against that by direct injection. extension of the sample bandwidth (peak broadening and tailing). The optimal length of a capillary column is 20–100 cm and the opti￾mal length of a flow through in draw/eject extraction system is 50–60 cm. The internal diameter of a capillary used in combina￾tion with HPLC is 0.25 or 0.32 mm with a short widebore capillary (0.53 mm) used in combination with capillary electrophoresis (CE), because widebore capillaries are resistant to pressure. Although capillary columns with chemically bonded or cross-linked liquid phases are very stable in water and organic solvents, they readily deteriorate in the presence of strong inorganic acids or alkalis. How￾ever, capillary columns are generally stable for the mobile phase usually used in HPLC. In in-tube SPME, complete equilibrium extraction is generally not obtained for any analyte, because these analytes are partially desorbed into the mobile phase during the ejection step. For flow through extraction systems, the volume of sample passed through a capillary is usually 0.2–2 mL, and the optimum extraction flow-rate is 0.25–4 mL min − 1, depending on the capacity of the column. In contrast, an increase in the number and volume of draw/eject cycles can enhance the extraction efficiency of in-tube SPME, but band￾width may widen, and peak broadening has often been observed. A column 20–80 cm in length is usually used for effective extraction. For a capillary column (inner diameter 0.25 mm, length 60 cm) with a volume of 30 L and an injection needle of 10 L, optimal condi￾tions include a draw/ejection volume of 30–40-L, a draw/ejection flow-rate of 50–100- L min − 1 and 10–15 draw/ejection cycles [15] . Generally, it is possible to increase the extraction efficiency of ana￾lyte into a stationary phase in SPME by changing the pH and salt level of the sample solution. Acidic and basic compounds can be effectively extracted from acidic and alkaline sample solutions, respectively. However, the stability of each compound at the pH of the sample solution must be determined beforehand. Although salting out increases extraction efficiency in fiber SPME, the salt deposits can clog the column during in-tube SPME. Furthermore, the presence of a hydrophilic solvent such as methanol in the sam￾ple decreases the extraction efficiency by increasing the solubility of the compound in the sample, but methanol concentrations of ≤5% have little effect on extraction efficiency. The amount of com￾pound extracted into the stationary phase is dependent on the concentration of the compound in the sample. 2.2.3. Capillary coatings GC capillary column coatings were initially compared with uncoated fused-silica (a retention gap capillary) as extraction devices for in-tube SPME. These comparisons included silica mod￾ified with PDMS (e.g., SPB-1, PTE-5 and SPB-5) or polyethylene glycols (PEG), such as Omegawax 250 and Supelcowax, porous DVB and Supel-Q-Plot[3,79–81]. The relatively polar poly(ethylene glycol) (PEG) coating (Omegawax 250) is sufficiently bonded and cross-linked to prevent loss of phase when solvent is passed through the capillary, showing the highest yield of analyte. Sil￾ica modified columns have been found more suitable for the analysis of non-polar compounds. Use of the adsorptive coated cap￾illary Supel-Q-PLOT (DVB polymeric material) has recently been found to be more efficient for the analysis of estrogens, because of its large surface area, enhancing mass-transfer kinetics, and because estrogens are of intermediate polarity [82] . Table 1 shows a comparison of some commercially available GC capillary columns used in the extraction of environmental pollutants, food contami￾nants and biological compounds. For most organic compounds, the porous polymer-type capillary column (Supel-Q PLOT) showed bet￾ter extraction efficiency than the other liquid-phase type capillary columns (CP-Sil 5CB, CP-Sil 19CB, and CP-Wax 52CB). As the PLOT column has a large adsorption surface area and a thick-film layer, the amount extracted was greater than that with liquid-phase type columns. However, patulin was effectively extracted with another

H Kataoka et aL/ Analytica Chimica Acta 655 (2009 )8-29 porous polymer-type capillary column(Carboxen-1006 PLOT)but is convenient for coupling of miniaturized samples to micro-or not Supel-Q PLOT For the extraction of nicotine and perfluorooc scale separation technologies such as micro-LC and-CE The ne sulfonate, a CP-Pora PLOT amine gave superior extraction configuration of the on-line preconcentration is the same as that efficiency because of its affinity to relatively polar compounds In of the coated capillary microextraction system described in Section contrast, CP-Sil 19CB (liquid-phase type capillary) was superior for 2.2.1, with construction of these systems involving two Microfeeder aromatic hydrocarbons, although the film was thin. MF-2 pumps equipped with MS-GAN microsyringes. Several research groups have attempted to synthesize new A comparison of PDMs columns packed with Zylon(a materials to improve extraction efficiency and selectivity. These fibrous rigid-rod heterocyclic polymer; poly (p-phenylene -2, 6 nclude the preparation of a series of electrochemical coating benzobisoxazole) fibers[89 and stainless steel wire 88 for the based on polypyrrol(PPY) by an oxidative polymerization method determination of antidepressant drugs showed that up to 246 fila- [183]. The extraction efficiencies of PPY coatings were better than ments could be packed into a capillary, with an optimum packing those of commercial GC columns, due to the numerous types of density of 52% of the lumen volume. Using this system, analytes interactions between these multifunctional (i.e. T-T, polar, hydro- could be optimally desorbed in 2 FL of acetonitrile, with about gen bonding and ionic interactions)coatings and the analytes. 4 nL being injected for CE. Compared with wire-in-tube techniques Another advantage of electrochemical polymer-coated over cor in combination with micro-LC, The fiber-in-tube SPE technique in mercial capillaries for in-tube SPME is the ability to manipulate combination with CE resulted in a 3-76-fold increase in precon- extraction efficiency and selectivity by regulating the thickness of centration, depending on whether analytes interacted significantly the coating (ie the number of electrochemical polymer cycles). with the sorbent fiber in the lumen of the capillary. Zylon has Chemically or electrochemically deposited PPY coatings have been also used in the same way for determination of phthalates in oupled to either HPLC [84 for determination of aromatics and wastewater[91-93 Exhaustive extraction was achieved through a anions, respectively. The sensitivity and selectivity of these coatings combination of the increased sorbent capacity and reduced sample for in-tube extraction can be adjusted by altering film thickness In volume. Desorption into the mobile phase was very efficient for the contrast, a simple Spme device has been fabricated for use in on- fiber-in-tube method, eliminating the need for a separate desorp- line immunoaffinity capillaries [85 Immunoaffinity-SPME, which tion solvent, although in this case the mobile phase itself contained combines the inherent selectivity of antibodies and the advantages a high proportion of organic solvent(90% methanol). of SPME, is prepared by immobilization of an antibody in in-tube SPME, using a sensitive, selective, and reproducible method Impor- 2.3.2. Sorbent-packed capillary in-tube SPMe tant aspects of the optimization of in-tube SPMe conditions and An alternative approach using a small section of capillary packed evaluation of capacity of immunoaffinity capillaries have been with microsphere beads is similar to SPE. Although this technique described [85]. Furthermore, sol-gel titania-PDMS-coated capillar- is easy to implement in existing autosampler systems, sorbent s have been used for on-line in-tube SPme and the analysis of packed capillaries can easily break under high pressure. When PAH, ketones, and alkylbenzenes [ 86]. and this method can be easily liquid samples are analyzed by direct immersion, the main dis- automated by using standard HPLC equipment. More recently, new advantage of this technique is that even very tiny particles are ic liquid-mediated sol-gel coatings were developed for capillary able to block the capillaries, making it necessary to use very microextraction of PAHs[87] clean samples. Phases better suited to the extract relatively polar compounds from aqueous samples have been developed to ed capillary in-tube SPME enhance the sensitivity and overall utility of capillary microex traction methods. These include a molecularly imprinted polymer 2.3.1. Wire-in-tube SPME and fiber-in-tube SPE (MIP), consisting of cross-linked synthetic polymers produced by Several methods have been developed to increase extraction copolymerizing a monomer with a cross-linker in the presence of a efficiencies and extend this method to microscale applications, template molecule as an in-tube SPMe adsorbent [ 90. A capillary including"wire-in-tube SPME, using modified capillary columns packed with MiP particles in an 8-cm PEEK tube (inner diameter with inserted stainless steel wires [88]; and"fiber-in-tube SPE", 0.76 mm) has been used for the selective analysis of B-blockers using capillary tubes packed with fibrous rigid-rod heterocyclic biological fluids. In addition, a highly biocompatible SPME-capillary polymers(Fig. 1B)89, 90. These techniques, which require fixed packed with alkyl-diol-silica particles(ADS) particles was deve sample volumes, have also been called miniaturized SPE, rather oped as a restricted access material(RAM)(94]. The bifunctionality than SPME. These distinctions are important, as SPME is an equi- of the aDs extraction phase prevented fouling of the capillary by librium extraction technique where sample volume is significantly adsorbed protein while simultaneously trapping the analytes in the arger than sorbent capacity, with calibration based on the par- hydrophobic porous interior. this oach required a simplified titioning or affinity of each analyte for sorbent For wire-in-tube apparatus compared with existing RAM column switching proce- SPME, internal capacity can be significantly reduced by insertion of dures, as well as overcoming the need for ultrafiltration a narrow stainless steel wire into the extraction capillary while the deproteinization step prior to handling biological samples, thus fur- For fiber-in-tube SPE, several hundred fine filaments of polymeric pre-concentrate resulted in low-ng/mL detection limit ability to surface area of the polymeric coating material remains the same. ther minimizing sample preparation requirements. the ability to materials packed longitudinally into a short polyether ether ketone (PEEK) capillary tube serve as the extraction medium. This tech- 2.4. Monolithic capillary in-tube SPME ique not only can reduce the internal void volume of the extraction capillary but the fine polymer filaments can be employed as the An alternative approach consists of in-tube SPme using mono- extraction medium. Because the filaments are arranged parallel lithic capillary columns comprised of one piece of organic polymer to the outer tubing, narrow coaxial channels can form inside the or silica with a unique flow through double-pore structure. Capil capillary. therefore, fiber-in-tube spe device involves a reduced laries with monolithic sorbents can be easily synthesized in situ pressure drop during the extraction and desorption compared with initiated thermally or by radiation, using a mixture of monomer. a conventional particle-packed SPE cartridge. Furthermore, the cross-linker and proper porogenic solvent. It generally results effective interaction of the sample solution with a number of fine in monolithic structures with different functional groups that fibrous extraction capillaries suggests further miniaturization as are biocompatible and pH-stable. A C18-bonded monolithic sil- a microscale sample preconcentration device, as this technology ica column, prepared by in situ hydrolysis and polyc

H. Kataoka et al. / Analytica Chimica Acta 655 (2009) 8–29 15 porous polymer-type capillary column (Carboxen-1006 PLOT) but not Supel-Q PLOT. For the extraction of nicotine and perfluorooc￾tane sulfonate, a CP-Pora PLOT amine gave superior extraction efficiency because of its affinity to relatively polar compounds. In contrast, CP-Sil 19CB (liquid-phase type capillary) was superior for aromatic hydrocarbons, although the film was thin. Several research groups have attempted to synthesize new materials to improve extraction efficiency and selectivity. These include the preparation of a series of electrochemical coatings based on polypyrrol (PPY) by an oxidative polymerization method [83]. The extraction efficiencies of PPY coatings were better than those of commercial GC columns, due to the numerous types of interactions between these multifunctional (i.e. –, polar, hydro￾gen bonding and ionic interactions) coatings and the analytes. Another advantage of electrochemical polymer-coated over com￾mercial capillaries for in-tube SPME is the ability to manipulate extraction efficiency and selectivity by regulating the thickness of the coating (i.e. the number of electrochemical polymer cycles). Chemically or electrochemically deposited PPY coatings have been coupled to either HPLC [84] for determination of aromatics and anions, respectively. The sensitivity and selectivity of these coatings for in-tube extraction can be adjusted by altering film thickness. In contrast, a simple SPME device has been fabricated for use in on￾line immunoaffinity capillaries [85]. Immunoaffinity-SPME, which combines the inherent selectivity of antibodies and the advantages of SPME, is prepared by immobilization of an antibody in in-tube SPME, using a sensitive, selective, and reproducible method. Impor￾tant aspects of the optimization of in-tube SPME conditions and evaluation of capacity of immunoaffinity capillaries have been described [85]. Furthermore, sol–gel titania-PDMS-coated capillar￾ies have been used for on-line in-tube SPME and the analysis of PAH, ketones, and alkylbenzenes [86], and this method can be easily automated by using standard HPLC equipment. More recently, new ionic liquid-mediated sol–gel coatings were developed for capillary microextraction of PAHs [87]. 2.3. Packed capillary in-tube SPME 2.3.1. Wire-in-tube SPME and fiber-in-tube SPE Several methods have been developed to increase extraction efficiencies and extend this method to microscale applications, including “wire-in-tube SPME”, using modified capillary columns with inserted stainless steel wires [88]; and “fiber-in-tube SPE”, using capillary tubes packed with fibrous rigid-rod heterocyclic polymers (Fig. 1B) [89,90]. These techniques, which require fixed sample volumes, have also been called miniaturized SPE, rather than SPME. These distinctions are important, as SPME is an equi￾librium extraction technique where sample volume is significantly larger than sorbent capacity, with calibration based on the par￾titioning or affinity of each analyte for sorbent. For wire-in-tube SPME, internal capacity can be significantly reduced by insertion of a narrow stainless steel wire into the extraction capillary while the surface area of the polymeric coating material remains the same. For fiber-in-tube SPE, several hundred fine filaments of polymeric materials packed longitudinally into a short polyether ether ketone (PEEK) capillary tube serve as the extraction medium. This tech￾nique not only can reduce the internal void volume of the extraction capillary, but the fine polymer filaments can be employed as the extraction medium. Because the filaments are arranged parallel to the outer tubing, narrow coaxial channels can form inside the capillary. Therefore, fiber-in-tube SPE device involves a reduced pressure drop during the extraction and desorption compared with a conventional particle-packed SPE cartridge. Furthermore, the effective interaction of the sample solution with a number of fine fibrous extraction capillaries suggests further miniaturization as a microscale sample preconcentration device, as this technology is convenient for coupling of miniaturized samples to micro- or nano-scale separation technologies such as micro-LC and -CE. The configuration of the on-line preconcentration is the same as that of the coated capillary microextraction system described in Section 2.2.1, with construction of these systems involving twoMicrofeeder MF-2 pumps equipped with MS-GAN microsyringes. A comparison of PDMS columns packed with Zylon® (a fibrous rigid-rod heterocyclic polymer; poly(p-phenylene-2,6- benzobisoxazole) fibers [89] and stainless steel wire [88] for the determination of antidepressant drugs showed that up to 246 fila￾ments could be packed into a capillary, with an optimum packing density of 52% of the lumen volume. Using this system, analytes could be optimally desorbed in 2 L of acetonitrile, with about 4 nL being injected for CE. Compared with wire-in-tube techniques in combination with micro-LC, The fiber-in-tube SPE technique in combination with CE resulted in a 3–76-fold increase in precon￾centration, depending on whether analytes interacted significantly with the sorbent fiber in the lumen of the capillary. Zylon® has also used in the same way for determination of phthalates in wastewater [91–93]. Exhaustive extraction was achieved through a combination of the increased sorbent capacity and reduced sample volume. Desorption into the mobile phase was very efficient for the fiber-in-tube method, eliminating the need for a separate desorp￾tion solvent, although in this case the mobile phase itself contained a high proportion of organic solvent (90% methanol). 2.3.2. Sorbent-packed capillary in-tube SPME An alternative approach using a small section of capillary packed with microsphere beads is similar to SPE. Although this technique is easy to implement in existing autosampler systems, sorbent￾packed capillaries can easily break under high pressure. When liquid samples are analyzed by direct immersion, the main dis￾advantage of this technique is that even very tiny particles are able to block the capillaries, making it necessary to use very clean samples. Phases better suited to the extraction of relatively polar compounds from aqueous samples have been developed to enhance the sensitivity and overall utility of capillary microex￾traction methods. These include a molecularly imprinted polymer (MIP), consisting of cross-linked synthetic polymers produced by copolymerizing a monomer with a cross-linker in the presence of a template molecule, as an in-tube SPME adsorbent [90]. A capillary packed with MIP particles in an 8-cm PEEK tube (inner diameter 0.76 mm) has been used for the selective analysis of -blockers in biological fluids. In addition, a highly biocompatible SPME-capillary packed with alkyl-diol-silica particles (ADS) particles was devel￾oped as a restricted access material (RAM) [94]. The bifunctionality of the ADS extraction phase prevented fouling of the capillary by adsorbed protein while simultaneously trapping the analytes in the hydrophobic porous interior. This approach required a simplified apparatus compared with existing RAM column switching proce￾dures, as well as overcoming the need for ultrafiltration or another deproteinization step prior to handling biological samples, thus fur￾ther minimizing sample preparation requirements. The ability to pre-concentrate resulted in low-ng/mL detection limits. 2.4. Monolithic capillary in-tube SPME An alternative approach consists of in-tube SPME using mono￾lithic capillary columns comprised of one piece of organic polymer or silica with a unique flow through double-pore structure. Capil￾laries with monolithic sorbents can be easily synthesized in situ, initiated thermally or by radiation, using a mixture of monomer, cross-linker and proper porogenic solvent. It generally results in monolithic structures with different functional groups that are biocompatible and pH-stable. A C18-bonded monolithic sil￾ica column, prepared by in situ hydrolysis and polycondensation

H Kataoka et aL/ Analytica Chimica Acta 655 (2009)8-29 High- voltage po power Detector Packed capillary >Waste Pressure regulator Monolithic capillary ix-port!(A) LC column Detector ord position position Pump Mobile phase Fig. 5. Schematic diagram of monolithic capillary in-tube SPME coupled with(A)uHPLC or (B)pcEC. of alkoxysilane [95, showed preconcentration efficiencies higher a syringe. Valve A is switched from the lOAd to the INJECT position than those obtained by conventional in-tube SPMe. various mono- for a given time interval in the extraction step and then returned to lithic capillaries have been developed based on poly(methacrylic the Load position immediately. The carrier solution continues to acid-ethylene glycol dimethacrylate)(MMA-EGDMA)[96-110]. flow through the capillary to eliminate the residual sample solution ly(glycidyl methacrylate-co-ethylene dimethacrylate)(GMA- and remove un-retained matrix The analytical mobile phase then o-EDMA)[111, 112] and poly(acrylamide-vinylpyridine-N, N- desorbs the extracted analytes from the monolithic capillary to the methylene bisacrylamide)(AA-VP-Bis)[113-115, and these have analytical column by switching valve B to the INJECT position. After been applied to in-tube SPME in combination with LC or CE. switching valve B back to the LOAd position, the flow of the mobile Hydrophobic main chains and acidic pendant groups (from phase is increased to initiate chromatographic separation. At the MAA monomers)make these monolithic polymers superior for same time the capillary is eluted by the mobile phase, and subse- xtracting basic analytes from aqueous matrices. Moreover, the quently conditioned by carrier solution until the next extraction. biocompatibility of these monolithic structures allows the direct For CEC analysis, a CEC capillary is inserted between the splitter analysis of biological samples with no other manipulation except and the outlet electrolyte chamber. for dilution and or centrifugation, simplifying the entire dete An alternative approach consists of an in-line coupled SPME nination procedure 96, 100, 109. Through its pyridyl group pillary zone electrophoresis ( CZe) using a continuous bed poly (AA-VP-Bis)is expected to show strong hydrophobic and monolithic RAM capillary insert [117. Hyperlink robust bio- n-exchange interactions with acidic compounds. The newly compatible spme devices were interfaced with a capillary zone developed monolithic capillary showed excellent reusability and electrophoresis system and fully automated analysis for sam- high stability under extreme pH conditions during extraction. In ple preconcentration, desorption, separation and quantification of addition, a hybrid organic-inorganic octyl monolithic silica was analytes. The RAM based SPME approach was able to simultane- developed for in-tube SPME for on-line preconcentration coupled ously separate proteins from a biological sample, while directly to capillary HPLC [116. When hybrid silica monolithic capillary was extracting the active components from a natural drug. Recently, an used as a pre-column, the sample volume that could be injected in-capillary microextraction method was described, which used a into the system was as high as 1 mL, with a simultaneous increase monolithic polymer based on butyl methacrylate and dVB forming in column efficiency. in situ inside a capillary column [ 118. This ted ue was applied A monolithic column is very suitable for in-tube SPME medium to the preconcentration of carbamate pesticides prior to their sep- because of its unique features, including a low pressure drop allow- aration by micellar electrokinetic chromatography(MEKC). ing a high flow-rate to achieve high-throughput, and total porosity higher than that of a particle column. Fig. 5 shows the schematic diagram of an in-tube SPME-pHPLC or-pressure assisted capillary 3. Applications of capillary microextraction techniques electrochromatography (pCEC)system[105]. The system consists of two six-port valves, a stainless steel sample loop(1 mL total Capillary microextraction techniques have been successfully volume), two microflow pumps and uHPLC or PCEC(containing applied to the determination of a variety of compounds in a high-voltage power supply and a splitter), with pHPLC and pcEC biological, environmental and food samples. Since their initial each including a UV/Vis detector Pump A is combined with valve development, applications of these techniques have increased, with A and the stainless steel sample loop to act as the pre-extraction about 116 papers in the literature 39 in the analysis of biological segment, and two segments are connected by a PEEK tube between and pharmaceutical samples, 66 in the analysis of environmen alves A and B Before the extraction, the carrier solution is driven tal samples, and 16 in food analysis(Fig. 6). In this section, we by pump A to flow through the capillary for conditioning. At the summarize these applications and discuss the characteristics of the same time, the sample loop is filled with the sample solution using approaches employed

16 H. Kataoka et al. / Analytica Chimica Acta 655 (2009) 8–29 Fig. 5. Schematic diagram of monolithic capillary in-tube SPME coupled with (A) HPLC or (B) pCEC. of alkoxysilane [95], showed preconcentration efficiencies higher than those obtained by conventional in-tube SPME. Various mono￾lithic capillaries have been developed based on poly(methacrylic acid-ethylene glycol dimethacrylate) (MMA-EGDMA) [96–110], poly(glycidyl methacrylate-co-ethylene dimethacrylate) (GMA￾co-EDMA) [111,112] and poly(acrylamide-vinylpyridine-N,N - methylene bisacrylamide) (AA–VP-Bis) [113–115], and these have been applied to in-tube SPME in combination with LC or CE. Hydrophobic main chains and acidic pendant groups (from MAA monomers) make these monolithic polymers superior for extracting basic analytes from aqueous matrices. Moreover, the biocompatibility of these monolithic structures allows the direct analysis of biological samples with no other manipulation except for dilution and/or centrifugation, simplifying the entire deter￾mination procedure [96,100,109]. Through its pyridyl groups, poly(AA-VP-Bis) is expected to show strong hydrophobic and ion-exchange interactions with acidic compounds. The newly developed monolithic capillary showed excellent reusability and high stability under extreme pH conditions during extraction. In addition, a hybrid organic–inorganic octyl monolithic silica was developed for in-tube SPME for on-line preconcentration coupled to capillary HPLC [116].When hybrid silicamonolithic capillary was used as a pre-column, the sample volume that could be injected into the system was as high as 1 mL, with a simultaneous increase in column efficiency. A monolithic column is very suitable for in-tube SPME medium because of its unique features, including a low pressure drop allow￾ing a high flow-rate to achieve high-throughput, and total porosity higher than that of a particle column. Fig. 5 shows the schematic diagram of an in-tube SPME-HPLC or -pressure assisted capillary electrochromatography (pCEC) system [105]. The system consists of two six-port valves, a stainless steel sample loop (1 mL total volume), two microflow pumps and HPLC or pCEC (containing a high-voltage power supply and a splitter), with HPLC and pCEC each including a UV/Vis detector. Pump A is combined with valve A and the stainless steel sample loop to act as the pre-extraction segment, and two segments are connected by a PEEK tube between valves A and B. Before the extraction, the carrier solution is driven by pump A to flow through the capillary for conditioning. At the same time, the sample loop is filled with the sample solution using a syringe. Valve A is switched from the LOAD to the INJECT position for a given time interval in the extraction step and then returned to the LOAD position immediately. The carrier solution continues to flow through the capillary to eliminate the residual sample solution and remove un-retained matrix. The analytical mobile phase then desorbs the extracted analytes from the monolithic capillary to the analytical column by switching valve B to the INJECT position. After switching valve B back to the LOAD position, the flow of the mobile phase is increased to initiate chromatographic separation. At the same time the capillary is eluted by the mobile phase, and subse￾quently conditioned by carrier solution until the next extraction. For CEC analysis, a CEC capillary is inserted between the splitter and the outlet electrolyte chamber. An alternative approach consists of an in-line coupled SPME￾capillary zone electrophoresis (CZE) using a continuous bed monolithic RAM capillary insert [117]. Hyperlink robust bio￾compatible SPME devices were interfaced with a capillary zone electrophoresis system and fully automated analysis for sam￾ple preconcentration, desorption, separation and quantification of analytes. The RAM based SPME approach was able to simultane￾ously separate proteins from a biological sample, while directly extracting the active components from a natural drug. Recently, an in-capillary microextraction method was described, which used a monolithic polymer based on butyl methacrylate and DVB forming in situ inside a capillary column [118]. This technique was applied to the preconcentration of carbamate pesticides prior to their sep￾aration by micellar electrokinetic chromatography (MEKC). 3. Applications of capillary microextraction techniques Capillary microextraction techniques have been successfully applied to the determination of a variety of compounds in biological, environmental and food samples. Since their initial development, applications of these techniques have increased, with about 116 papers in the literature, 39 in the analysis of biological and pharmaceutical samples, 66 in the analysis of environmen￾tal samples, and 16 in food analysis (Fig. 6). In this section, we summarize these applications and discuss the characteristics of the approaches employed.

H Kataoka et aL/ Analytica Chimica Acta 655(2009)8-29 related alkaloids in urine and saliva [123]. These compounds were effectively extracted on a CP-pora PLOT capillary(concentrated 20-46-fold in comparison with direct injection), and the detection limits were 15-40 pg mL-I. This method was useful for monitoring tobacco smoking, for estimating the uptake of nicotine and tobacco- related toxicants, for understanding the pharmacologic effects of nicotine and nicotine addiction, and for optimizing nicotine depen- A fiber-in-tube SPME technique has been developed using short capillaries packed longitudinally with several hundred polymer filaments as extraction media [88.89. This method, in combination with HPLC and CE, was used to analyze tricyclic antidepressants in urine samples. PEEK capillaries packed with propranolol MiP particles were used for the selective analysis of lockers from biological fluids 9o Using this method, precon centration of sample increased its sensitivity, yielding a limit of detection of 0.32 ug mL-l by UV detection, excellent reproducibil- ity(rsd< 5%)and column reusability(500 injections)over a fairly wide linear dynamic range(0.5-100 ug mL- )in serum samples. A Fig.6.Publications in capillary microextraction techniques searched from Med- biocompatible in-tube SPME method using PEEK capillaries packed (keywords: capillary microextraction, in-tube solid-phase microextraction and pen-tubular trapping: the end of june, 2009 present). with ADs particles has also been used for direct extraction of several benzodiazepines in serum samples [124 In addition, an 3.1. Recent applications to biological and pharmaceutical samples used for in-tube spme of the selective serotonin inhibitor fluox- An overview of applications of capillary microextraction tech etine in serum samples [85). Several on-line monolithic capillary niques in biological and pharmaceutical analysis is shown in I-tube SPME methods have been developed for the determi- nation of forensic and therapeutic drugs such as amphetamines Table 2. Most of these methods have been used to analyze forensic [99, 100]. angiotensin ll receptor agonists [98, 103, 104] and opiates nd therapeutic drugs in biofluids such as urine and serum. These 102, 106]in biological fluid samples. Most of these poly mono- methods show high chromatographic selectivity, linearity. precI- lith microextraction(PMME)methods use a poly(MMA-EGDMA) sion, and sensitivity, in line with international criteria for validation procedures of applications such as therapeutic drug monitoring. for all analytes. Fig. 7 shows the electropherograms obtained by clinical toxicology, forensic toxicology, bioavailability, and phar- PMME-CE of blank and analytes(ephedrine and pseudoephedrine) The on-line in-tube SPME method can be applied to polar and spiked human plasma and urine samples [106 Comparing the ele non-polar drugs in liquid samples and can be coupled with var- of the direct injection of analytes spiked plasma and urine sam ious analytical methods, such as HPLC and LC-MS. For example, ples(Fig. 7A(a)and B(a), a dramatic peak height enhancement was an in-tube SPME coupled with HPLC-tandem Ms was develope for the determination of six butyrophenone derivatives in plasm ound, and no interfering peak was observed. Recently, monolithic samples using an open-tubular DB-17 capillary as extraction device from biological samples, while directly extracting the active com- [119). This system was on-line and fully automated, with a total nents of caffeine, paracetamol and acetylsalicylic acid from the run time of approximately 25 min per sample following minimal drug NeoCitramonum (1171 lo evaporation or reconstitution was required. A Supel-Q PLOT capillary coated with porous dVB polymer gave superior extrac- 3. 2. Recent applications to environmental samples tion efficiency (Table 1)as an in-tube spme device for the analysis of phthalates, alkylphenols, bisphenol A and cortisol [120-122]. As shown in Fig. 6, capillary microextraction techniques have Recently, an automated on-line in-tube SPMe coupled with LC-Ms been applied to many environmental samples such as air and water. was developed for the determination of nicotine, cotinine and because these samples are comparatively clean and easily treated. SmaU (B) PEE 56789min Fig. 7. Ele grams of ephedrine and pseud ne obtained by PMME-CE of spiked (c))and urine(b(c))at 0.5 ug mL-: PMME-C 5μgmL-1 by CEw rnal standard), berberine. The is was added after PMME with the concentrati ection of 0.2 mL of samne: PE, pseudoephedrine rile-acetic acid (100: 0.2, v/v) is injected via the monolithic capillary tube at 0. 1 mLmin-l and the eluate is injected into the CECE -silica capillary(50 um id x 60 cm): separation buffer, 0. 1 M phosphate electrolyte(pH 2.5)and 10% acetonitrile(v/v); Uv absorbance detection at 214 nm From Ref [106] with permissi

H. Kataoka et al. / Analytica Chimica Acta 655 (2009) 8–29 17 Fig. 6. Publications in capillary microextraction techniques searched from Med￾line (keywords: capillary microextraction, in-tube solid-phase microextraction and open-tubular trapping; the end of June, 2009 present). 3.1. Recent applications to biological and pharmaceutical samples An overview of applications of capillary microextraction tech￾niques in biological and pharmaceutical analysis is shown in Table 2. Most of these methods have been used to analyze forensic and therapeutic drugs in biofluids such as urine and serum. These methods show high chromatographic selectivity, linearity, preci￾sion, and sensitivity, in line with international criteria for validation procedures of applications such as therapeutic drug monitoring, clinical toxicology, forensic toxicology, bioavailability, and phar￾macokinetics. The on-line in-tube SPME method can be applied to polar and non-polar drugs in liquid samples and can be coupled with var￾ious analytical methods, such as HPLC and LC–MS. For example, an in-tube SPME coupled with HPLC–tandem MS was developed for the determination of six butyrophenone derivatives in plasma samples using an open-tubular DB-17 capillary as extraction device [119]. This system was on-line and fully automated, with a total run time of approximately 25 min per sample following minimal sample preparation. In contrast to off-line SPE and LLE methods, no evaporation or reconstitution was required. A Supel-Q PLOT capillary coated with porous DVB polymer gave superior extrac￾tion efficiency (Table 1) as an in-tube SPME device for the analysis of phthalates, alkylphenols, bisphenol A and cortisol [120–122]. Recently, an automated on-line in-tube SPME coupled with LC–MS was developed for the determination of nicotine, cotinine and related alkaloids in urine and saliva [123]. These compounds were effectively extracted on a CP-pora PLOT capillary (concentrated 20–46-fold in comparison with direct injection), and the detection limits were 15–40 pg mL−1. This method was useful for monitoring tobacco smoking, for estimating the uptake of nicotine and tobacco￾related toxicants, for understanding the pharmacologic effects of nicotine and nicotine addiction, and for optimizing nicotine depen￾dency treatment. A fiber-in-tube SPME technique has been developed using short capillaries packed longitudinally with several hundred polymer filaments or wires as extraction media [88,89]. This method, in combination with HPLC and CE, was used to analyze tricyclic antidepressants in urine samples. PEEK capillaries packed with propranolol MIP particles were used for the selective analysis of -blockers from biological fluids [90]. Using this method, precon￾centration of sample increased its sensitivity, yielding a limit of detection of 0.32 g mL−1 by UV detection, excellent reproducibil￾ity (RSD 500 injections) over a fairly wide linear dynamic range (0.5–100 g mL−1) in serum samples. A biocompatible in-tube SPME method using PEEK capillaries packed with ADS particles has also been used for direct extraction of several benzodiazepines in serum samples [124]. In addition, an immunoaffinity capillary containing immobilized antibody was used for in-tube SPME of the selective serotonin inhibitor fluox￾etine in serum samples [85]. Several on-line monolithic capillary in-tube SPME methods have been developed for the determi￾nation of forensic and therapeutic drugs such as amphetamines [99,100], angiotensin II receptor agonists [98,103,104] and opiates [102,106] in biological fluid samples. Most of these polymer mono￾lith microextraction (PMME) methods use a poly(MMA-EGDMA) monolithic capillary, which showed high extraction efficiencies for all analytes. Fig. 7 shows the electropherograms obtained by PMME-CE of blank and analytes (ephedrine and pseudoephedrine) spiked human plasma and urine samples [106]. Comparing the elec￾tropherogram obtained by PMME-CE (Fig. 7A(c) and B(c)) to that of the direct injection of analytes spiked plasma and urine sam￾ples (Fig. 7A(a) and B(a)), a dramatic peak height enhancement was found, and no interfering peak was observed. Recently, monolithic RAM capillaries were used to simultaneously separate proteins from biological samples, while directly extracting the active com￾ponents of caffeine, paracetamol and acetylsalicylic acid from the drug NeoCitramonum [117]. 3.2. Recent applications to environmental samples As shown in Fig. 6, capillary microextraction techniques have been applied to many environmental samples such as air and water, because these samples are comparatively clean and easily treated. Fig. 7. Electropherograms of ephedrine and pseudoephedrine obtained by PMME-CE of spiked plasma (A (c)) and urine (B (c)) at 0.5 g mL−1; PMME-CE of blank plasma (A (b)) and urine (B (b)) sample; and directly analysis of spiked plasma (A (a)) and urine (B (a)) at 5 g mL−1 by CE without treatment. Peaks: E, ephedrine; PE, pseudoephedrine; IS (internal standard), berberine. The IS was added after PMME with the concentration of 2 g mL−1. PMME is carried out by ejection of 0.2 mL of sample solution at a flow-rate of 0.2 mL min−1 using a poly(MAA-EGDMA) monolithic capillary tube (0.53 mm i.d. × 2 cm). For desorption, 0.1 mL acetonitrile–acetic acid (100:0.2, v/v) is injected via the monolithic capillary tube at 0.1 mL min−1 and the eluate is injected into the CE. CE conditions are as follows: uncoated fused-silica capillary (50 m i.d. × 60 cm); separation buffer, 0.1 M phosphate electrolyte (pH 2.5) and 10% acetonitrile (v/v); UV absorbance detection at 214 nm. From Ref. [106] with permission.