Imran Imran AliAli11
Vinod.K.
Vinod.K. GuptaGupta22
Hassa
Hassann Y.Y. AboAboul-Eul-Eneinnein33
Afza
Afzall HussHussainain11 1
1Department of Chemistry, JamiaDepartment of Chemistry, Jamia Millia Islamia (A Central
Millia Islamia (A Central University) New Delhi, India University) New Delhi, India 2
2Department of Chemistry,Department of Chemistry, Indian Institute of Technology Indian Institute of Technology Roorkee, India
Roorkee, India 3
3Pharmaceutical and MedicinalPharmaceutical and Medicinal Chemistry Department, Chemistry Department, Pharmaceutical and Drug Pharmaceutical and Drug Industries Research Division, Industries Research Division, National Research Centre, National Research Centre, Dokki, Cairo, Egypt Dokki, Cairo, Egypt
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Analysis at trace levels, an ideal area of application for hyphenated techniques, is Analysis at trace levels, an ideal area of application for hyphenated techniques, is steadily gaining importance. Many sample pre-concentration and clean-up methods steadily gaining importance. Many sample pre-concentration and clean-up methods have been hyphenated with core analytical techniques to accomplish the task of have been hyphenated with core analytical techniques to accomplish the task of low level detection
low level detection. The . The presepresent article descrint article describes the bes the state of the state of the art of art of hyphhyphenationenation of
of variovarious us technitechniques such ques such as as solid phase solid phase extraextraction, micro-solction, micro-solid id phasphase e extraextraction,ction, dialysis, and chromatographic modalities
dialysis, and chromatographic modalities etc.etc. with liquid chromatography, gas chro-with liquid chromatography, gas chro-mat
matogrographaphy, y, cacapilpillarlary y eleelectrctrophophoreoresissis, , and and spspectectrosroscopcopic ic metmethodhods. s. BesBesideides,s, attempts have been made to address the hyphenation approach in microfluidic attempts have been made to address the hyphenation approach in microfluidic devi-ces.
ces.
Keywords:
Keywords:Capillary electrophoresis / Gas chromatography / Liquid chromatography / Micro-flu-Capillary electrophoresis / Gas chromatography / Liquid chromatography / Micro-flu-idic
idic devidevicesces // SoliSolidd phasphasee extrextractiactionon //
Re
Receiceivedved:: MarMarchch 4,4, 2002008;8; rerevisvised:March10,ed:March10, 2002008;8; accacceptepted:March17,ed:March17, 20020088 DOI
DOI 10.1002/jssc.200810.1002/jssc.20080012300123
1 Introduction
1 Introduction
Normally, many analytes in biological and Normally, many analytes in biological and environmen-tal samples are present at very low concentrations in the tal samples are present at very low concentrations in the nan
nano o or or levlevel el ranrangesges, , whiwhich ch are beyonare beyond d the reach of the reach of det
detectection ion by by conconvenventiontional al anaanalytlyticaical l insinstrutrumentments.s. Besides, thousands of impurities also present in Besides, thousands of impurities also present in biologi-cal
cal and environmentand environmental al matrimatrices disturb ces disturb analyanalyses and,ses and, hence, sample preparat
hence, sample preparation ion of of biologbiological and ical and envirenviron- on-mental matrices is essential prior to introduction onto mental matrices is essential prior to introduction onto analytical machines. One of the most important trends analytical machines. One of the most important trends
to simplify these complications is the generation of to simplify these complications is the generation of sim-ple, rapid, and reliable procedures for sample ple, rapid, and reliable procedures for sample prepara-tion. Method development and setup requires the use of tion. Method development and setup requires the use of material of known compositions,
material of known compositions, e.g.e.g. certified referencecertified reference materials. Therefore, spiking experiments have to be materials. Therefore, spiking experiments have to be per-for
formedmed forfor metmethodhod quaqualitlityy concontroltrol.. IntIntegregratiationon andand autauto- o-mation of all the steps between sample preparation and mation of all the steps between sample preparation and det
detectection ion sigsignifnificaicantly ntly redreduce uce the the timtime e of of anaanalyslysis,is, increa
increasingsing bothboth reprreproducioducibilitybility andand accuaccuracyracy..
In 1995, the seventh symposium on handling In 1995, the seventh symposium on handling environ-mental and biological samples in chromatography was mental and biological samples in chromatography was held on May 7–10, at Lund, Sweden. This symposium held on May 7–10, at Lund, Sweden. This symposium was in continuation of the series started by the late Dr. was in continuation of the series started by the late Dr. Roland Frei, one of the early visionaries in sample Roland Frei, one of the early visionaries in sample prepa-ration
ration technotechnologies logies inin analyanalytical tical sciensciences.ces. AA survesurveyy ofof thethe papers presented at this symposium indicates that five papers presented at this symposium indicates that five points
points have behave beenen highlhighlighted ighted andand consiconsidered dered asas essenessentialtial during sample preparation. First, the need for a during sample preparation. First, the need for a continu-ous search for new technologies was realized, so that the ous search for new technologies was realized, so that the high cost due to chemicals and experimental labor may high cost due to chemicals and experimental labor may be
be redreducuced. ed. SecSecondondly, ly, the the neeneed d was was recrecognognizeized d forfor increasing sensitivity with better and more selective increasing sensitivity with better and more selective con-centra
centration techniquestion techniques, , which has which has drivedriven n scienscientists totists to examine affinity and immuno-affinity supports that can examine affinity and immuno-affinity supports that can selectively remove compound classes for further selectively remove compound classes for further investi-gation. Thirdly, the development of multidimensional gation. Thirdly, the development of multidimensional chroma
chromatogratographic phic technitechniques ques allowiallowing ng on-linon-line e sampsamplele
clean-clean-up, up, which which proviprovides des severaseveral l advaadvantages includingntages including autom
automation, ation, better better reprreproducioducibilitybility, , and and closeclosed d systesystemm capability was advocated. The fourth point considered capability was advocated. The fourth point considered was the development of better sample preparation was the development of better sample preparation tech-niques enabli
niques enabling more ng more effectieffective ve use of use of biosenbiosensors andsors and
Correspondence:
Correspondence:Professor Hassan Y. Professor Hassan Y. Aboul-EneinAboul-Enein, , PharmaceutPharmaceuti- i-cal
cal and and MediMedicinacinal l ChemChemistristry y DepaDepartmertment, nt, NatiNational onal ReseResearcharch Centre, Dokki, Cairo 12311, Egypt
Centre, Dokki, Cairo 12311, Egypt E-mail:
E-mail:[email protected]@gawab.com Fax:
Fax:+20-2-33370931+20-2-33370931 Abbrevi
Abbreviatioations: ns: AAS,AAS, atomatomic ic absoabsorptirption on specspectromtrometryetry;; AES,AES, atomic emission
atomic emission spectrometrspectrometry;y; DMAE,DMAE, dynamic microwave-as-dynamic microwave-as-sisted extraction;
sisted extraction; DSAE,DSAE,dynamic dynamic sonicationsonication-assisted extraction;-assisted extraction; ECD,
ECD, elecelectron tron captucapture re detedetector;ctor; EF,EF, enrichmeenrichment nt factor;factor; FID,FID, flame ionization detection;
flame ionization detection; GF,GF,gel filtration;gel filtration; HCH,HCH,hexachloro- hexachloro-cyclohexane;
cyclohexane; HGAAS,HGAAS, hydrhydride ide genegeneratiration on atomatomic ic absoabsorptirptionon spectroscopy;
spectroscopy; ICP,ICP,inductivelinductively y coupled plasma coupled plasma spectrometspectrometry;ry; IC,IC, ion
ion chromatograchromatography;phy; IDA,IDA, iminodiacetic acid;iminodiacetic acid; IMAC,IMAC, immobi- immobi-lized metal affinity chromatography;
lized metal affinity chromatography; IPLC,IPLC,ion pair liquid chro-ion pair liquid chro-matography;
matography; ISP-CGC,ISP-CGC, immunoaffinimmunoaffinity ity sample sample pretreatmepretreatment- nt-capillary gas
capillary gas chromatograpchromatography system;hy system; LLE,LLE,liquid–liquid– liquid liquid extrac- extrac-tion;
tion; MMLLE,MMLLE,microporous membranmicroporous membrane liquide liquid –– liquid extractionliquid extraction;; NP,
NP, normal-phase;normal-phase; OTT,OTT, open-tubular trapping;open-tubular trapping; OPPs,OPPs, organo- organo-phosphorus pesticides;
phosphorus pesticides; PAHs,PAHs, polycpolycycliyclic c aromaromatic atic hydrhydrocar- ocar-bons;
bons; PHWE,PHWE, pressurized hot water extraction;pressurized hot water extraction; PCB,PCB,polychlori- polychlori-nated biphenyl;
nated biphenyl; llRPLC,RPLC,micro reverse phase liquid chromatog-micro reverse phase liquid chromatog-raphy;
raphy; SFE,SFE,supercritical fluid extraction;supercritical fluid extraction; SLM,SLM,stratum lacuno-stratum lacuno-sum-moleculare
other sensors because exposure to raw matrices can foul many sensors. The last and the fifth point, which requires considerable attention from the scientist, was the quality movement which has found its way into sam-ple handling. Therefore, extraction, purification, and pre-concentration of the natural samples are very impor-tant and essential operations in separation science, but, of course, involve the use of costly chemicals and time. Moreover, these techniques are not able to prepare sam-ples containing analytes at nano levels. In view of this, a new trend of hyphenationis emerging in which a sample preparation unit is coupled with an analytical instru-ment. This hyphenation technology is the latest develop-ment and future of sample preparation in the present century. In view of these developments, the present article discusses state-of-the-art sample preparation through hyphenation.
2 Sample preparation techniques
Basically, sample preparation is a complex and sensitive step in analysis, which requires considerable expertise, especially when dealing with samples containing ana-lytes at micro or nano level concentrations. Many off-line methods have been used for sample preparation, includ-ing solvent extraction (23%), solid phase extraction (48%), supercritical fluid extraction (11%), immunoaffin-ity extraction (5%), matrix solid phase dispersion (2%), automated solid phase extraction (SPE 2%), dialysis (5%), solid phase micro extraction (3%), and mole mass filtra-tion (1%). To provide a quick impression and to permit comparison, these percentages are shown in Fig. 1.
Solvent extraction (liquid –liquid extraction) is a classi-cal method of sample preparation exploiting unequal distribution of solutes in two immiscible liquid phases. It has been used for the extraction of many compounds of biological and environmental importance [1 – 2]. Extrac-tion from liquid and solid samples is carriedout by using a variety of solvents such as hexane, acetone, acetic acid, benzene, toluene, methanol, acetonitrile, petroleum ether, ethyl ether, iso-octane, pentane, dichloromethane, etc. This method has certain drawbacks such as high con-sumption of costly solvents and time. Besides, the dis-posal of used solvents (environmental hazards) and emul-sion formation are other problems associated with this technique. Of course, solid phase extraction (SPE) is a quite practical method involving the use of reversed phase (C8, C18, etc. silica gel) adsorption phases in the
form of discs and cartridges, but it also has certain draw-backs [3]. SPE requires multiple steps and costly solvents, and is a time-consuming process since the solvent con-centration should be protected from evaporation. Some-times, clotting, channelling, and percolation create problems in sample preparation in this sort of sample
handling modality. Chromatographic techniques are also important and effective methods of sample prepara-tion and include HPLC, GPC, and SFC. However, these are not affordable in all laboratories due to their costly instrumentation and running costs [4, 5]. Besides, mem-brane filtration and dialysis have also been used for sam-ple preparation but they are also limited to certain appli-cations [4, 5]. Due to advances in separation science in the new millennium the demand for analyses is increas-ing at nano or lower level detection limits and scientists in academia and industry as well as government agencies require data at such low limits all over the world [6]. Under such circumstances, the role of sample prepara-tion becomes crucial, and creates a need for greater focus on miniaturization and non-exposure of samples during their preparation. Moreover, rapidity, efficiency, selectiv-ity, reproducibilselectiv-ity, low cost, and low limits of detection are demanded by today's separation science. A literature search and our experience indicate that these demands can be satisfied by hyphenation.
3 Hyphenation technology
Basically, the above-cited methods are used for sample preparation in biological and environmental matrices. However, certain drawbacks make these methods less than ideal since they are not effective in the case of low amounts of samples and consume costly solvents and time. Besides, contamination and poor recoveries may also occur during experiments. The factors underscored the need for hyphenated techniques. Hyphenation is nothing but the coupling of a sample preparation unit with the core analytical instrument. It has been found more effective than conventional methods in terms of efficiency, effectiveness, selectivity, high recoveries, suit-ability for small samples, and for samples containing components that are difficult to analyze and give rise to procedural problems. Therefore, some papers have been published dealing specifically with biological and envi-ronmental samples. The use of hyphenation has been Figure 1. Percentage contribution of different sample prepa-ration techniques (source Toxline and Current Contents ; years: 1997–1998–1999).
classified on the basis of the core analytical technique, as discussed in the following sections.
3.1 Hyphenation in liquid chromatography
Basically, liquid chromatography represents a landmark in the history of separation science and sample prepara-tion is a key issue in this area. Many workers have attempted to hyphenate sample preparation units with liquid chromatographs and some important research work is discussed herein. Johansen et al. [7] described the hyphenation of Automated Sequential Trace Enrichment of Dialysates (ASTED) system with HPLC for analysis of antidepressant drugs in plasma. In this system the pro-tein and particles were purified through a semi-perme-able membrane and collection of the drug molecules on a trace enrichment column (TEC) was followed by HPLC analyses on a Supelcosil column (15064.6 mm). The ASTED system consisted of a cellulose acetate dialysis membrane and interactions of analytes with the cellu-lose acetate membrane have been reported for basic drugs such as the opiate derivative pholcodine, benzodia-zepines, and the neuroleptic drug clozapine [8 – 10]. Most of the antidepressant drugs showed ionic and hydropho-bic interactions with the membrane and were selected as model substances to investigate more closely the ability of cationic surfactants to inhibit analyte-membrane interactions. The author optimized ASTED– HPLC condi-tions to achieve maximum recoveries by adding cationic surfactants to the donor solution in the dialyser, by the effect of chain length and concentration of cationic sur-factants, pH of the donor solution, and the volume of the acceptor solution. Furthermore, the authors used a che-mometric approach via factorial design and response surface modeling for optimization strategies. The devel-oped unit was applied successfully for monitoring mian-serine, imipramine, desimipramine, amitriptyline, and nortriptyline drugs in human plasma. Dialysis was per-formed for 12.8 minutes after which the six port valve was switched to the injection position and the enriched analytes were loaded onto TEC with HPLC mobile phase (acetonitrile – methanol– 0.005 M ammonium phosphate buffer (pH 7.0) (70:15:15 v/v/v)) at a flow rate of 1.5 mL/ min The limits of detection of the reported drugs in human plasma were in the range of 17 – 39 nM/L with UV detection.
Cheng et al. [11] studied the biotransformation of D -aspartic acid into L-aspartic acid with the help of SPE– HPLC hyphenation. The column used in HPLC was of ligand exchange type allowing the chiral separation of D -and L-aspartic acid. The mobile phase used for SPE was
5 mM sodium-1-octanesufonate (pH 2.2) at 0.1 mL/min flow rate while HPLC eluent was 0.25 mM CuSO4(pH 3.6)
at a flow rate of 0.5 mL/min. Kema et al. [12] developed an automated on-line SPE– HPLC method for profiling of
plasma indoles tryptophan, 5-hydoxytryptophan (5-HTP), serotonin, and 5-hydroxyindoleacetic acid (5-HIAA) com-pounds for diagnosing of carcinoid tumors in patients. The SPE cartridge consisted of hydrophobic polystyrene resin and the analytes were enriched on SPE due to vari-ous interactions such as hydrogen bonding, van der Waals forces, steric effects etc. The fluorometric detector permitted detection of several metabolically related indole derivatives. HPLC conditions were Inertsil column (25063 mm) with mobile phase of different ratio of 50 mM potassium dihydrogen phosphate adjusted to pH 3.3 with phosphoric acid with acetonitrile as eluent. This set-up is shown in Fig. 2, indicating a coupling of SPE and HPLC along with its working mechanism. The SPE cartridge is pre-conditioned with acetonitrile, dipo-tassium EDTA in water (5g/L), and water at 3 mL/min flow rate; followed by autosampling of the enriched ingredients onto the HPLC column. By the time chroma-tographic separation on the analytical column is com-plete, the SPE unit has been made ready for the next sam-ple preparation and injection. The authors advocated this hyphenation as an emerging technique due to its direct injection procedure in combination with column switching that offered the possibility of combining sam-ple pre-purification, concentration, and analysis simulta-neously.
Hasselstrom et al. [13] described a fully automated on-line SPE–HPLC–UV system for quantification of quetia-pine, an antipsychotic drug, in human serum. SPE was conducted on a C2packing and the mobile phase used for
HPLC was MeOH–20 mM NH4CH3COO (pH 5.0) (99:1, v/v)
at a flow rate of 1.0 mL/min with 257 nm. Similarly, Man-drioli et al. [14] also studied quetiapine by SPE– HPLC Figure 2. Schematic representation of on-line SPE system coupled to HPLC with fluorescence detection, on top SPE with conditioning of the cartridge and sample injection, the middle panel represents the system during backward-flush elution of the cartridge and the bottom panel shows the sys-tem during regeneration of the cartridge [12].
hyphenated separation. Kato et al. [15] also described an on-line solid-phase extraction method, coupled with HPLC –MS-MS for the determination of 16 phthalate metabolites in human urine. The method employed a conventional analytical column for the chromato-graphic separations of these analytes and the mobile phase used was A (0.1% acetic acid in water) and B (0.1% acetic acid in acetonitrile) at a flow rate of 0.35 mL/min. The limits of detection ranged from 0.11 to 0.90 ng/mL. A similar set-up was described by Kuklenyik et al. [16] for the extraction and measurement of perfluorinated organic acids and amine in human serum and milk. A 12-lL volume of the reconstituted serum or milk extract was auto-injected on to HPLC at a 300-lL/min flow rate with 20 mM ammonium acetate (pH 4) in water and methanol as mobile phase. The HPLC gradient program (14 min) was started at 60% methanol in the mobile phase followed by an increase in organic content to 80% in 0.5 min, which was kept for 9 min. Later on, the mobile phase organic content was decreased in 0.5 min to 60% methanol, where it was kept for 3 min to equili-brate the column. Furthermore, the same group [17] described an automated on-line hyphenation of SPE with HPLC– MS for the extraction and measurement of isofla-vones and lignans in urine. The mobile phases used were 10 mM ammonium acetate (pH 6.5) and methanol–ace-tonitrile (50:50 v/v) at a flow rate of 0.8 mL/min, respec-tively. The detection limits were in the range of 0.2– 0.7 ng/mL. These authors described these hyphenations as an innovation in separation science.
Koster et al. [18] reported the analysis of lidocaine in urine by an on-line SPME – LC method. A polydimethylsi-loxane (PDMS) coated fiber was directly immersed into buffered urine with optimized contact time, pH, ionic strength, and temperature. The extraction yields were 22% in about 45 min with a reproducibility of a5%
expressed as relative standard deviation. The detection limits were 25 – 1000 ng/mL. Volmer et al. [19] studied the eleven corticosteroid and two steroid conjugations in a urine sample by SPME–LC–MS. Several SPME optimiza-tion factors such as polarity of fibres, extracoptimiza-tion time and effect of ionic strength, were investigated, and their impact on the SPME/LC/MS technique was studied. The method was sensitive with detection limits between 4 and 300 ng/mL and precision between 4.9 and 11.1% RSD. Kim et al. [20]. developed sol-gel titania-based coat-ing capillary micro extraction (CME) coupled with HPLC for the extraction and analyses of polycyclic aromatic hydrocarbons, ketones, and alkylbenzenes at high pH. To perform CME–HPLC, a so-gel TiO2–PDMS capillary was
installed in an HPLC injection port as an external sam-pling loop. HPLC conditions were ODS column (25064.6 mm) with acetonitrile– water (80:20v/v) as mobile phase. The target analytes were extracted on-line by passing the aqueous sample through this sampling
loop. The sol-gel titania–PDMS coated capillaries were used for on-line extraction and HPLC analysis of poly-cyclic aromatic hydrocarbons, ketones, alkylbenzenes, and a wide range of other less volatile or thermally labile compounds [21] that are not amenable to GC separation. Hashi et al. [22] described the determination of polycyclic aromatic hydrocarbons (PAH) in the atmospheric partic-ulates by using on-line enrichment coupled with fast high-performance liquid chromatography with fluores-cence detection. The limits of detection of PAH were in the range of 0.02–0.23 ng/mL with recoveries between 87 and 12% for spiked atmospheric particulate sample. The mobile phase used was acetonitrile–water (72:28, v/v) at a flow rate of 1.0 mL/min. Altun et al. [23] developed and validated a method for local anesthetics in human plasma through on-line MEPS by using a cation-exchanger with a flow rate of 0.20 mL/min.
Abdel-Rehim [24] developed and validated a new sensi-tive, selecsensi-tive, and accurate on-line micro-extraction in packed syringe (MEPS) technique hyphenated with HPLC for the determination of lidocaine, prilocaine, ropiva-caine, and mepivacaine in human plasma. The extrac-tion recoveries were in the range of 60–90%. Veuthey et al. [25] described on-line solid phase extraction to achieve nano analysis of drugs in biological samples. In this hyphenation technique, the single column performs two functions, i.e. extraction and separation. The column was connected to a detection system via a switching valve. The sample was directly injected on to the extraction support with and after the extraction, the valve was switched, and analytes were transferred to the detector with the eluting mobile phase followed by extraction support re-equilibration. According to the authors, the method was simple and several applications have been published for the direct analysis of biofluids. Quintana et al. [26] described an automated on-line hyphenation of SPE– HPLC incorporating multi syringe flow injection analysis (MSFIA), bead injection, and lab-on-valve (BI– LOV) prior to HPLC. The potential of the novel MSFI–BI– LOV hyphenation for on-line handling of complex envi-ronmental and biological samples prior to reversed-phase chromatographic separations was assessed for the expeditious determination of five acidic pharmaceutical residues viz. ketoprofen, naproxen, bezafibrate, diclofe-nac, and ibuprofen along with one metabolite, i.e. sali-cylic acid, in surface water, urban wastewater, and urine. The column used was an Xterra RP-18 (3.96150 mm) with the mobile phases A: MeOH–water (20:80, v/v) and B: MeOH– water (95/5, v/v), both containing 0.1% (v/v) for-mic acid at flow rates of 1.0 mL/min. The detection limit was 0.02– 0.67 ng/mL.
Clarkson et al. [27] described hyphenation of solid-phase extraction with liquid chromatography and nuclear magnetic resonance: application for identifica-tion of flavonol glycosides (kaempferol 3-O-(6-O-a-L
-rham-nopyranosyl)-b-D-glucopyranoside; kaempferol 3-O-(2,6-di-O-a-L-rhamnopyranosyl)-b-D-glucopyranoside; quercetin
3-O-(2,6-di-O-a-l-rhamnopyranosyl)-b-D-glucopyranoside (rutin); and isorhamnetin, 3-O-(6-O-a-L
-rhamnopyranosyl)-b-D-glucopyranoside) and three 5-a-cardenolides (coro-glaucigenin 3-O-6-deoxy-b-D-allopyranoside;
coroglaucige-nin 3-O-(4-O-b-D-glucopyranosyl)-6-deoxy-b-D -glucopyrano-side; 39-O-acetyl-39-epiafroside) were identified. Zhang et
al. [28] described an automated on-line hyphenation of SFC–2-D–HPLC–MS for sample preparation, separation, detection, and identification of the fruiting bodies of Ganoderma lucidum, and at least 73 components in the extract were resolved with a calculated peak capacity of up to 1643. The SFE and 2-D HPLC systems were fitted with a Hypersil-CN (5 lm, 1200A, 15064.6 mm id) and Chromolith Flash columns, respectively. In the first dimensional separation, the binary mobile phase was composed of A (water) and B (methanol) with a flow rate of 0.1 mL/min In the second dimensional separation, the mobile phase was composed of C (water) and D (acetoni-trile) with a flow rate of 4 mL/min. The same group reported a simple SFE–HPLC system for comprehensive analyses of traditional Chinese medicines [29]. However, for complex samples, it is impossible to separate all com-ponents by one-dimensional chromatography. There-fore, two-dimensional HPLC has been developed and was regarded as a powerful technique for the separation of proteins, peptides, polymers, natural products, and other complex mixtures by different workers [30–43]. Taylor et al. [44] established an on-line SFE–HPLC–UV/ ESI-MS technique for the quantitative analysis of Hyper-forin pertoratum.
Ritter et al. [45] described the hyphenation of an elec-trolytic on-line eluent generation device with high per-formance anion-exchange chromatography coupled with UV detection for the determination of a wide range of intracellular metabolites from mammalian cells. The detection wavelength of the UV detector was switched from 220 to 260 nm and the detection limits were in the range of mM. Two Dionex AS11 analytical columns (25062 mm id) were used with 0.35 mL/min as mobile phase flow rate. Tuytten et al. [46] described an on-line automated SPE– HPLC– ESI-MS method for targeted metabolomic analysis of urinary modified nucleoside levels. The unit comprised a boronate affinity column as a trapping device, a hydrophilic interaction chromatog-raphy (HILIC) separation, and information-dependent MS detection modes. The system was applied to biological samples, detecting a number of modified nucleosides. Clarkson et al. [47] described HPLC–SPE–NMR hyphena-tion for structural elucidahyphena-tion of some natural products. Lambert et al. [48] described the identification of natural products by using SPE–HPLC coupling with Cap-NMR. This coupling was used for identification of sesquiter-pene lactones and esterified phenylpropanoids present
in an essentially crude plant extract (toluene fraction of an ethanolic extract of Thapsia garganica fruits).
Lin et al. [49] described hyphenation of in-tube solid-phase micro-extraction (SPME) and pressure-assisted CEC (p-CEC) by installing a poly(methacrylic acid-co-ethylene glycol dimethacrylate) monolithic capillary at a six-port valve in a CEC system. Theobromine, theophylline, and caffeine were chosen as model drugs to facilitate compar-ison with the results obtained by in-tube SPME–HPLC. The detection limits of these three analytes were improved more than 100 times when compared with direct analysis by l-HPLC. Besides the above-cited meth-ods of sample preparation, some modalities of liquid chromatography have also been used as sample prepara-tion methods and hyphenated with HPLC. Only one article on size exclusion chromatography coupled with HPLC is cited. Pomazal et al. [50] described analyses of cop-per, iron, manganese, and zinc in blood samples by exploiting the hyphenation of SEC with an HPLC–ICP-AES unit. Besides, this device was also used to monitor metalloproteins in erythrocytes and blood plasma sam-ples. Optimization was achieved via parameters like pH, flow rate, and salt concentration. For optimizing experi-ments, blood samples from one female subject were used and the direct determination of the elements was per-formed by ICP-AES on blood fractions of ten different sub-jects to obtain the average concentration ranges.
3.2 Gas chromatography
Gas chromatography is considered the best choice for analysis of volatile compounds, including several agri-cultural, industrial, and other chemical compounds. Of course, many xenobiotics are present at trace concentra-tions and cannot be analyzed directly and this circum-stance compels scientists to perform sample preparation, i.e. to adopt pre-concentration and hyphenation approaches. Many sample preparation methods have been coupled with GC for analyses of various species and these include LLE, SPE, membrane, etc.
Membrane extraction is considered to be one of the best extraction techniques because it has the important advantage that the sample and the extractant can contin-uously be kept in contact without physical mixing, thus providing the basis for a continuous, real-time process permitting automation and on-line connection to instru-ments [51]. Consequently, membrane techniques have advanced during few decades to a stage permitting the solution of numerous analytical problems. These tech-niques allow the simultaneous extraction and enrich-ment of analytes and typically facilitate selective extrac-tion at trace levels while consuming small amounts of solvents. Automated on-line liquid – liquid membrane extraction (LLME) has also been reported for determina-tion of PCB [52] and of anesthetics in blood [53]. For PCB
determinations, Barri et al. [52] designed a miniaturized membrane extraction card (referred to as the ESy card) connected to a GC injector via an electromechanical installation which controls pre-treatment and triggers the GC instrument. The EF (enrichment factor) exhibited by this hyphenation was between 33 and 40 for PCBs in river water. Shen et al. [53] used a sample processor sys-tem consisting of an auto-sampling injector, dilutors, and a six-port valve connected to a GC injector loop for achieving EF up to 50 for some local anesthetics in blood plasma.
Abdel-Rehim [24] developed and validated a sensitive, selective, and accurate on-line sample preparation tech-nique for the determination of lidocaine, prilocaine, ropivacaine, and mepivacaine in human plasma. The on-line micro-extraction unit was a packed syringe (MEPS; silica gel C2) coupled with GC–MS. The plasma samples
(50–1000 lL) were drawn through the syringe by an auto-sampler and passed through the solid support, resulting in their adsorption onto the solid phase. The solid phase was then washed once with water (50 lL) to remove proteins and other interfering material. The MEPS technique differed from commercial solid-phase extraction (SPE) in the way in which the packing was inserted directly into the syringe, and not into a separate column. MEPS was capable of handling sample amounts from10 to 1000 lL of plasma, urine, or water in GC appli-cations. MEPS took only about one minute for each sam-ple with greater robustness than the SPME technique, and gave recoveries between 60 and 90%. GC experimen-tation conditions were 908C column temperature for 3 min followed by an increase up to 2808C at a rate of 508C per min; with helium as carrier gas at 2.0 mL/min flow rate.
Li et al. [54] described an hyphenation of SPE with pro-grammable temperature vaporizers– large volume injec-tion/gas chromatography/mass spectrometry (PTV– LVI/ GC/MS) for on-line sample preparation and separation of semi-volatile organic compounds (pesticides and herbi-cides) in a variety of water samples. The authors utilized this unit for real life samples of chlorinated tap water, well water, and river water. Furthermore, optimization achieved minimum limit of detection (0.1 lg/L) with rela-tive recoveries in the range of 70–120% and a relarela-tive standard deviation of less than 15%. The schematic repre-sentation of an SPE Twin–PAL PTV–LVI/GC/MS system is shown in Fig. 3. Pawliszyn et al. [55–59] developed solid phase micro extraction (SPME) methods for non-volatile compounds. A fused silica rod with a polymeric coating on the surface was employed as the extraction medium for the adsorption of volatile analytes from aqueous sam-ple solutions. The SPME fiber was inserted into the GC injector port for desorption and analyses of the analytes. Brossa et al. [60] described an automated on-line SPE– GC–MS set-up for the determination of a group of
endo-crine disruptors in water samples. The chromatographic column used was HP-5 MS (28 m6250 lm id) and the limits of detection of the method were between 0.001 and 0.036 lg/L.
3.3 Hyphenation in capillary electrophoresis
Nowadays, capillary electrophoresis (CE) is valued as a versatile technique exhibiting high speed, high sensitiv-ity, lower limits of detection, and low running costs, and represents a major trend in analytical science; the num-ber of publications on this technique has increased expo-nentially [61 – 64]. CE is suitable for samples that may be difficult to separate by liquid chromatography and gas chromatography, or at least complements these tech-niques since the principles of separation are different. The lower detection limits of CE lead to the possibility of separating and characterizing very small quantities of materials, which normally require pre-concentration and sample preparation strategies, especially in unknown matrices. Therefore, some on-line methods have been reported from time to time to achieve the goal of micro level separation and detection in CE.
Su et al. [65] studied the analysis of riboflavin in beer by CE – LED coupled with stacking micellar electrokinetic chromatography (MEKC) as pre-concentration tech-niques. The detection limit reported was 1.0 ng/mL with 38000 theoretical plates per meter. Hsieh et al. [66] hyphenated sweeping-micellar electrokinetic chroma-tography with CE to analyze trans-resveratrol in red wine. The CE buffer was methanol–water solution (25:75, v/v) and the system was operated at 77 kV with detection at a wavelength of 369 nm; the detection limit was 5 ppb. Fang et al. [67] described an on-line centrifuge micro-Figure 3. Schematic representation of an SPE Twin–PAL-PTV– LVI/GC/MS system. S1: upper PAL head and syringe, S2: lower PAL head and syringe, C1: upper PAL control unit, C2: lower PAL control unit, FC: sample flow cell, W1: wash station for upper PAL syringe, W2: wash station for lower PAL syringe, SR: solvent reservoir, SS: standard station, SV: on-line sample spiking vial, ET: sample extraction tray, CT: eluate collection tray, ST: standard tray, WS: water sam-ple source and WW: water waste container [54].
extraction back-extraction field-amplified sample injec-tion capillary electrophoresis system (CME–OLBE–FASI-CE) for determining trace ephedrine derivatives in urine and serum. CME and OLBE–FASI were two separate con-centration units. The detection limits of this set-up were between 0.15 to 0.25 ng/mL on using photodiode array UV detection at 192 nm. The separations were achieved on an uncoated fused-silica capillary (50.2 cm650 lm id). Zhang et al. [68] developed hyphenation of immobi-lized metal affinity chromatography with capillary elec-trophoresis (IMAC– CE) for on-line concentration and analysis of peptides and proteins. The polymer mono-lithic immobilized metal affinity chromatography (IMAC) materials were prepared by an iminodiacetic acid (IDA) type adsorbent covalently bonded with monolithic rods of macroporous poly(glycidyl methacrylate-co-ethyl-ene dimethacrylate). Cu(II) was subsequently introduced into the support via interaction with IDA. Liu et al. [69] described a microdialysis hollow fiber as a macromole-cule trap for on-line coupling of solid phase micro-extrac-tion and capillary electrophoresis for analysis of protein samples. The detection limit was 3.0610– 7M with UV
absorbance detection. Kuban and Karlberg [70] have
reported an on-line dialysis/FIA– CE sample clean-up pro-cedure for metal ion analysis; with coupling via a spe-cially designed interface (Fig. 4). Samples were continu-ously pumped into a dialysis unit and the outgoing acceptor stream containing the analytes was allowed to fill a rotary injector in the FIA part of the system. Multi-ple samMulti-ple injections were possible in one electropho-retic run, and the entire analytical procedure could easily be mechanized. The repeatability of the unit was in the range of 1.6–3.3% (n = 7). This unit was applied in a wide range of real samples with complicated matrices like milk, juice, slurry, and liquors from the pulp and paper industry.
3.4 Hyphenation in spectroscopy
As in case of chromatography and capillary electrophore-sis, pre-concentration and sample preparation tech-niques were also hyphenated with spectroscopic instru-ments leading their capabilities to analyze samples of low volume or having poor ingredients. Many modalities of spectroscopy have been reported in the literature of identification of various inorganic and organic species. The most important spectroscopic techniques are atomic absorption spectrometry (AAS), inductively coupled plasma spectrometry (ICP), nuclear magnetic resonance spectrometry (NMR), atomic emission spectroscopy (AES), mass spectrometry (MS), infrared (IR), atomic fluores-cence spectrometry (AFS) etc. Normally, the detection lim-its of these techniques ranged from mg to lg and if applied in biological and environmental samples having low concentrations of ingredients, the analytical results become inadequate. Sample pre-concentration and prep-aration are the tools used by analytical scientist to over-come such challenging problems. In view of these facts, some papers have addressed on-line hyphenation of sam-ple pre-concentration and preparation techniques hyphenated with spectroscopic techniques. Sometimes, chelation of metal ions with suitable reagents enhanced the detection [71– 73].
Danesi [74] described a simplified model for the car-rier-facilitated transport of metal ions through hollow fiber supported liquid membranes. Yang et al. [75] reported the clean-up, extraction, and enrichment of numerous metals including Pb, Cu, Cr, La, Ce, Zn, and Co by HF-SLME and coupled it with AAS and ICP-MS as shown in Fig. 5. Katarina et al. [76] described an on-line hyphen-ation of sample preparhyphen-ation method by using a computer controlled pre-treatment system (Auto-Pret AES) coupled with ICP-AES for the sample pretreatment and determi-nation of trace metals in water samples. This system enabled determination of trace metals at the ppt level. Fan et al. [77] synthesized diphenylcarbazone-functional-ized silica gel for SPE able to withstand 1– 6 mol/L HCL or H2SO4as well as common organic solvents. This SPE was Figure 4. Schematic representation of FIC– CE coupling,
(X): cross-sectional view of the FIA–CE interface and (Y): schematic diagram of the FIA–CE system used for on-line sample dialysis, (S): sample, (A): acceptor stream, (E): elec-trolyte, (M): dialysis membrane, (D): UV detector, (V1): injec-tion valve in filling posiinjec-tion, (V2): injecinjec-tion valve in inject posi-tion, (W): waste, (C): capillary, (Pt): platinum electrodes, and (HV): high-voltage supply [70].
used for the extraction of Hg(II) selectively from eight metal ions with similar characteristics such as Cd(II), Ni(II), Co(II), Mn(II), Pb(II), Zn(II), Cu(II), and Fe(III). A micro-column packed with diphenylcarbazone-function-alized silica gel was coupled with flow-injection (FI) spec-trophotometry for the selective separation, pre-concen-tration, and determination of Hg(II) in six different ciga-rette samples with detection limit of 0.90 ng/mL.
Motomizu et al. [78] described an on-line flow injection inductively coupled plasma atomic emission spectrome-ter (FI– ICP-AES) system using anion- and cation-exchange resin disks for the speciation of chromium species in fresh water. Two kinds of ion exchange resin disks packed in line-filters were fixed and serially connected on the loop of each six-way valve. Five milliliters of a sam-ple solution (pH 4.5) was introduced into the system. Anionic chromate ion, Cr(VI), was collected on the anion-exchange resin disk while cationic chromium ion, Cr(III), was collected on the cation-exchange resin disk. The col-lected species were then sequentially eluted by 2 M nitric acid and nebulized to the plasma of ICP-AES. The detec-tion limit of Cr(VI) and Cr(III) were 0.04 and 0.02 lg/L respectively. This method was applied to the speciation of Cr(III) and Cr(VI) in fresh water samples. Similarly, Jit-manee et al. [79} reported arsenic speciation in fresh water by using inductively coupled plasma-atomic emis-sion spectrometry coupled with pre-concentration sys-tem containing solid phase anion exchange resin. Two miniaturized columns with a solid phase anion exchange resin, placed on two 6-way valves were used for
the solid-phase collection/concentration of arsenic(III) and arsenic(V), respectively. The limit of detection for both As(III) and As(V) were 0.1 lg/L. In the same year, Sumida et al. [80] described on-line pre-concentration spe-ciation of Cr(III) and Cr(VI) by using dual mini-columns coupled with plasma-atomic emission spectrometry in water samples. Cr(III) was collected on the first column packed with iminodiacetate resin. Cr(VI) in the effluent from the first column was reduced to Cr(III), which was collected on the second column packed with iminodiace-tate resin.
3.5 Hyphenation in microfluidic devices
Microfluidic devices are an innovation in separation sci-ence as they can be used to analyze samples of low vol-ume and having low-concentration ingredients. Among various methods using microfluidic devices, nano-liquid chromatography (NLC) and nano-capillary electrophore-sis (NCE) are the two most important techniques, and are used to achieve separations at nano levels. During a liter-ature survey we found few papers dealing with on-line chip-based sample preparation methods in NLC and NCE. Attempts have been made to discuss these in the follow-ing paragraphs.
Huynh et al. [81] described the first hyphenation of micro-dialysis NCE system for monitoring the hydrolysis of fluorescein mono-b-D-galactopyranoside (FMG) by b-D
-galactosidase. The layout of the microdialysis/microchip CE device shown in Fig. 6 indicates channel lengths, volt-age scheme, perfusate (20 mM sodium phosphate buffer, pH 7.4). Furthermore, the same authors [82] presented an on-line microdialysis sampling unit coupled with NCE. The authors used this set-up for amino acid and peptide analyses. Wilson et al. [83] reported an on-line desalting of macromolecule (betaine-type amphoteric or zwitter-ionic surfactant solutions) using a two-layered laminar flow system due to differential diffusion of analytes. Wheeler et al. [84] developed an on-line sample prepara-tion method for MALDI-MS, which depended on an elec-tro-wetting-on-dielectric-based technique. Ramsey et al. Figure 5. Schematic of hollow fiber column
pre-concentra-tion unit with ICP-AES [75].
Figure 6. Schematic representation of on-line hyphenation of micro-dialysis sample prepara-tion unit with NCE [81].
[85] coupled SPE to micellar electrokinetic chromatogra-phy to give a system permitting completely automated extraction, elution, injection, separation, and detection steps, respectively and separately. The authors reported fast analysis of rhodamine B yielding pre-concentration factors of more than 200 in less than 5 min with a 60 femtomolar detection limit. A schematic representa-tion of this hyphenarepresenta-tion is shown in Fig. 7, clearly indi-cating sample preparation and separation components. Legendre et al. [86] described a chip-based on-line solid-phase extraction (SPE) for DNA and polymerase chain reaction in NLC. The amount injected was 600 nL of blood sample. Xiao et al. [87] presented a sample prepara-tion method on a PDMS/glass chip coupled to gas chro-matography. The authors tested this assembly for anal-ysis of ephedrine from aqueous solution and reported good reproducibilities of extraction and analysis.
Sample stacking has recently come to be regarded as the best pre-concentration technique in capillary electro-phoresis, and has been tested in the NCE format [88]. Many modifications have been made to sample stacking, which include field-amplified sample stacking (FASS) [89– 91], large-volume sample stacking (LVSS) [92, 93], pH-mediated stacking [94, 95], and micellar electrokinetic chromatography (MEKC) stacking [96 – 98]. Some reviews have been published on sample stacking techniques for a wide variety of compounds [88, 99–108]. Terabe et al.
[109– 111] developed cation- and anion-selective exhaus-tive injection-sweeping-micellar electrokinetic chroma-tography (CSEI-, ASEI-sweeping-MEKC) methods for increased sensitivity and detection. Britz-McKibbin et al. [112, 113] designed an on-line focusing method based on different mobilities of cationic analytes between back-ground electrolyte (BGE) and sample matrix, which is called velocity difference-induced focusing (V-DIF). Cong et al. [114] reported on-line sample pre-concentration using field amplified stacking injection in NCE. Accord-ing to the authors, pressure-driven flows into or from the branch channels, due to bulk velocity, can be used for liquid transportation in the channels. The detection sen-sitivity was improved 94-, 108-, and 160-fold for fluores-cein-5-isothiocyanate, fluorescein disodium, and 5-car-boxyfluorescein, respectively, relative to a traditional method. Similarly, Zhang and Yin [115] developed multi-T microchip integrated field amplified sample stacking (FASS) coupled with NCE. According to the authors, a volumetrically defined large sample plug was formed in one step within 5 s by negative pressure in the headspace of two sealed sample waste reservoirs. The authors reported precisions in migration time and RSD as 3.3% and 1.3% for rhodamine123 (Rh123) and fluorescein sodium salt, respectively. Schematic representation of the channel design of a multi-T microfluidic chip and NCE system with negative pressure large volume sample injection is shown in Fig. 8.
Jung et al. [116] designed, fabricated, and characterized a novel field-amplified sample stacking (FASS)-NCE chip having photo-initiated porous polymer for the analyses of fluorescein and bodipy. Furthermore, the authors Figure 7. Schematic representation of chip-based solid
phase extraction-MEKC device. (a): Layout of the entire device and (b): expanded view of the extraction region of the device. The dotted lines represent the direction of fluid flow during extraction; the solid line signifies flow during elution/ injection. (Narrow channels areca . 55lm wide, the column chamber is ca . 210 lm wide with all channels ca . 15lm deep.) [85].
Figure 8. Schematic representation of (a): channel design of the multi-T microfluidic chip and (b): NCE with negative pres-sure large volume sample injection. SP: Syringe pump, V: 3-way valve, HV: high-voltage power supply, T: T-shape con-nector [115].
Table 1.Applications of hyphenation in sample preparation of different compounds in various matrices.
Compounds Matrices Hyphenation modalities LOD Refs.
Biological matrices
Trazodone Plasma SPE – GC – FID 3 lg/L [118]
Benzodiazepines Plasma SPE – GC – NPD 5 – 25 lg/L [119]
Benzodiazepines Plasma ISP – CGC 0.5 – 2 lg/L [120]
Biogenic amines Cells and culture medium Dialysis–RPLC–fluorescence– ED
10 fmol/lL [121]
Ciprofloxacin Biological samples Dialysis –RPLC –fluorescence 0.1 nM [122] Amino a cids Beverages a nd f eed s tuff Dialysis – RPLC – fluorescence 1 – 278 lg/L [123] Fluoroquinolones Fortified t issue Dialysis – RPLC – fluorescence 2.5 – 5 ng/g [124]
Sterols Oils and fats LLE – GC – FID 0.04 – 0.08 ng/100 g [125]
Methylenedioxylated amphet-amines
Plasma and serum samples Dialysis– RPLC– fluorescence 10 lL [126]
Clozapine & N -desmethylcloza-pine
Human plasma Dialysis –RPLC –UV 0.050 –0.055 lmol/L [10]
Verapamil & Norverapamil Human p lasma Dialysis – R PLC – fluorescence 5 lg/L [127]
Local anesthetics Plasma SLM – GC 1 lg/L [128]
Ciprofloxacin Biological samples Dialysis –RPLC –fluorescence 0.1 nM [122] Phenytoin, Carbamazepine &
Phenobarbitone
Plasma Dialysis – RPLC – UV 0.1 – 0.8 lg/L [129]
Tramadol Human plasma RAM – MIP –RPLC –fluoresence a10 ng/mL [130]
Arsenic species Urine Dialysis – IC – HGAAS 1.0 – 2.18 lg/L [131]
Ropivacaine & metabolite Plasma Dialysis – lRPLC – MS 0.1 nM [132]
Ropivacaine Urine SLM – IPLC – UV 2 – 18 nM [133]
Phenols Plasma SLM – LC – biosensor 50 lg/L [134]
Meropenem Rat bile Dialysis – lRPLC – UV 0.1 mg/L [135]
Bambuterol Plasma SLM – lRPLC – UV 80 nM [136]
Atrazine mercapturate Urine SPE – RPLC 0.0 5ng/mL [137]
& planar PCBs Biological samples Blood, milk, tissue
SFE–NPLC–UV a0.3 – 7.4 ng/g [138]
N -Methylcarbamates Food PHWE – PRLC – fluorescence 1 lg/mL [139]
Piritramide Human plasma and urine SPE – LC/MS/MS 0.05 ng/mL [140]
Cyproterone acetate Human plasma RAM – RPLC – MS/MS 300 pg/mL [141]
Sugars and organic acids Foods and beverages Dialysis – RPLC – RI 0.6 – 0.41 lg/g [142] Fluconazole Blood and dermal rat
micro-dialysates
Dialysis –RPLC –UV 0.10 mg/L [143]
Bismuth, cadmium & lead Urine SPE – GF – AAS 0.002 – 0.013 ng/mL [144]
Cadmium Biological RM SPE – ICP-AES 0.05 ng/mL [145]
Environmental matrices
Endocrine disruptors Water SPE – GC – MS 0.1 – 20 ng/L [146]
HCH & ethers Water LLE – GC – ECD or FID 20 – 480 pg/L [147]
Organic pollutants Water SPE – GC – ECD 4.1 – 6.3 ng/L [148]
Phenols Water SPE – GC – FID 1 – 27 ng/L [149]
Phenols Water SPE – GC – FID 0.3 – 2 lg/L [150]
OPPs Water SPE – GC – AED 2 – 5 ng/L [151, 152]
Micro contaminants Water SPE – GC – FTIR 100 – 1000 ng/L [153]
Micro contaminants Water SPE – GC – MS 0.2 – 20 ng/L [154, 155]
Pesticides Water LLE – GC – AED 1 – 5 lg/L [156]
Pesticides Water SPE – GC – MS 2 – 20 ng/L [157]
Pesticides Water SPE – GC – MS a1 lg/L [158]
Pesticides Water SPE – RPLC – MS – MS 0.4 – 13 ng/L [159]
Endocrine disruptors Water SPE – GC – MS 0.1 – 20 ng/L [142]
Triazines & OPPs Wastewater SPE – GC – NPD or MS 15 – 25 ng/L (NPD) 1.5 ng/L (MS)
[160]
Alkylthio-s-triazineherbicides River water SLM –RPLC –UV 0.03 lg/L [161]
Vinclozolin Water MMLLE – NPLC – UV 1 lg/L [162]
Cationic surfactants Aqueous samples MMLLE –NPLC –UV 0.7 –5 lg/L [163]
Drugs Water SFE – RPLC – UV – MS 200 ppb [164]
Wine aroma compounds Wine SFE – GC – FID 0.8 – 3.4 lg [165]
Organic compounds Aerosol particles SFE – NPLC – GC – MS 0.02 – 0.04 ng/m3 [166]
described a 1000-fold signal increase during detection. This polymer material provided a region of high flow resistance, which allowed electromigration of sample ions resulting in pre-concentration. Lichtenberg et al. [117] developed a microchip device for field amplifica-tion stacking (FAS), which allowed the formaamplifica-tion of com-paratively long, volumetrically defined sample plugs with a minimal NCE bias. The authors studied fluidic effects; arising from solutions with mismatched ionic strengths, in chip-based electrokinetically. Furthermore, these authors developed a new chip layout for full col-umn stacking with subsequent sample matrix removal by polarity switching. Some important hyphenation examples are given in Table 1.
4 Concluding remarks
In the present scenario of separation science advance-ment, hyphenation is continuously gaining importance for the determination of analytes at micro or lower con-centration levels. This technique is more useful in biolog-ical samples where the volumes of matrices are low, e.g. blood of infants, cerebrospinal fluid, DNA, and other hor-mone and enzyme samples. It has been observed that the development of hyphenation techniques is not yet com-plete and is still progressing. Briefly, the art of hyphena-tion in separahyphena-tion science will be in great demand during the present century.
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Compounds Matrices Hyphenation modalities LOD Refs.
PAHs Aerosol particles SFE – NPLC – GC – MS 0.02 – 0.04 ng/m3 [168]
PAHs Soil and sediment PHWE – MMLLE – GC – FID 0.65 – 1.66 lg/g [169]
PAHs Soil and sediment PHWE – MMLLE – GC – FID 0.11 – 1.22 lg/g [170]
PAHs Sediment PHWE – NPLC – GC – FID 0.01 lg/g [171]
PAHs Sediment SFE – GC – MS a0.2 lg/g [172]
PAHs Soil SFE – RPLC – UV 0.2 – 4 ng [173]
Brominated flame Sediment PHWE – NPLC – GC – FID 0.70 – 1.41 ng/g [174]
Retardants
Organophosphorus e sters Air p articulates DMAE – SPE – GC – NPD 90.9 – 186.2 pg/m3 [175]
Organophosphorus Air particulates DSAE –SPE –GC –NPD 0.1 ng/m3 [176]
Esters
Sulfonamide antibiotics & pesti-cides
Natural water SPE – RPLC – MS/MS 0.5 – 5 ng/L [177]
Hexavalent chromium Colourants Dialysis – IC – UV 5 lg/L [178]
Explosives Filters SFE – RPLC – UV 9.5 – 56.8 ng/filter [179]
Selenium Cu alloys, Ni sponge SPE – AAS 0.2 ng/mL [180]
Cadmium SRM river water SPE – GF – AAS 2610– 4ng/mL [181]
Cr(III) & Cr(VI) Fresh water SPE – ICP-AES 0.02 – 0.04 ng/mL [78]
As(III) & As(V) Fresh water SPE – ICP-AES 0.1 ng/mL [79]
Cr(III) & Cr(VI) River water SPE – ICP-AES 0.08 – 0.15 ng/mL [80]
Tap water Wastewater
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