hplc hyphenated

Imran Ali1

Vinod.K. Gupta2

Hassan Y. Aboul-Enein3

Afzal Hussain1

1Department of Chemistry, JamiaMillia Islamia (A CentralUniversity) New Delhi, India

2Department of Chemistry,Indian Institute of TechnologyRoorkee, India

3Pharmaceutical and MedicinalChemistry Department,Pharmaceutical and DrugIndustries Research Division,National Research Centre,Dokki, Cairo, Egypt

Review

Hyphenation in sample preparation: Advancementfrom the micro to the nano world

Analysis at trace levels, an ideal area of application for hyphenated techniques, issteadily gaining importance. Many sample pre-concentration and clean-up methodshave been hyphenated with core analytical techniques to accomplish the task oflow level detection. The present article describes the state of the art of hyphenationof various techniques such as solid phase extraction, micro-solid phase extraction,dialysis, and chromatographic modalities etc. with liquid chromatography, gas chro-matography, capillary electrophoresis, and spectroscopic methods. Besides,attempts have been made to address the hyphenation approach in microfluidic devi-ces.

Keywords: Capillary electrophoresis / Gas chromatography / Liquid chromatography / Micro-flu-idic devices / Solid phase extraction /

Received: March 4, 2008; revised: March 10, 2008; accepted: March 17, 2008

DOI 10.1002/jssc.200800123

1 Introduction

Normally, many analytes in biological and environmen-tal samples are present at very low concentrations in thenano or level ranges, which are beyond the reach ofdetection by conventional analytical instruments.Besides, thousands of impurities also present in biologi-cal and environmental matrices disturb analyses and,hence, sample preparation of biological and environ-mental matrices is essential prior to introduction ontoanalytical machines. One of the most important trends

to simplify these complications is the generation of sim-ple, rapid, and reliable procedures for sample prepara-tion. Method development and setup requires the use ofmaterial of known compositions, e. g. certified referencematerials. Therefore, spiking experiments have to be per-formed for method quality control. Integration and auto-mation of all the steps between sample preparation anddetection significantly reduce the time of analysis,increasing both reproducibility and accuracy.

In 1995, the seventh symposium on handling environ-mental and biological samples in chromatography washeld on May 7–10, at Lund, Sweden. This symposiumwas in continuation of the series started by the late Dr.Roland Frei, one of the early visionaries in sample prepa-ration technologies in analytical sciences. A survey of thepapers presented at this symposium indicates that fivepoints have been highlighted and considered as essentialduring sample preparation. First, the need for a continu-ous search for new technologies was realized, so that thehigh cost due to chemicals and experimental labor maybe reduced. Secondly, the need was recognized forincreasing sensitivity with better and more selective con-centration techniques, which has driven scientists toexamine affinity and immuno-affinity supports that canselectively remove compound classes for further investi-gation. Thirdly, the development of multidimensionalchromatographic techniques allowing on-line sampleclean-up, which provides several advantages includingautomation, better reproducibility, and closed systemcapability was advocated. The fourth point consideredwas the development of better sample preparation tech-niques enabling more effective use of biosensors and

Correspondence: Professor Hassan Y. Aboul-Enein, Pharmaceuti-cal and Medicinal Chemistry Department, National ResearchCentre, Dokki, Cairo 12311, EgyptE-mail: [email protected]: +20-2-33370931

Abbreviations: AAS, atomic absorption spectrometry; AES,atomic emission spectrometry; DMAE, dynamic microwave-as-sisted extraction; DSAE, dynamic sonication-assisted extraction;ECD, electron capture detector; EF, enrichment factor; FID,flame ionization detection; GF, gel filtration; HCH, hexachloro-cyclohexane; HGAAS, hydride generation atomic absorptionspectroscopy; ICP, inductively coupled plasma spectrometry; IC,ion chromatography; IDA, iminodiacetic acid; IMAC, immobi-lized metal affinity chromatography; IPLC, ion pair liquid chro-matography; ISP-CGC, immunoaffinity sample pretreatment-capillary gas chromatography system; LLE, liquid – liquid extrac-tion; MMLLE, microporous membrane liquid – liquid extraction;NP, normal-phase; OTT, open-tubular trapping; OPPs, organo-phosphorus pesticides; PAHs, polycyclic aromatic hydrocar-bons; PHWE, pressurized hot water extraction; PCB, polychlori-nated biphenyl; lRPLC, micro reverse phase liquid chromatog-raphy; SFE, supercritical fluid extraction; SLM, stratum lacuno-sum-moleculare

i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

2040 I. Ali et al. J. Sep. Sci. 2008, 31, 2040 – 2053

J. Sep. Sci. 2008, 31, 2040 –2053 Sample Preparations 2041

other sensors because exposure to raw matrices can foulmany sensors. The last and the fifth point, whichrequires considerable attention from the scientist, wasthe quality movement which has found its way into sam-ple handling. Therefore, extraction, purification, andpre-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, anew trend of hyphenation is emerging in which a samplepreparation unit is coupled with an analytical instru-ment. This hyphenation technology is the latest develop-ment and future of sample preparation in the presentcentury. In view of these developments, the presentarticle discusses state-of-the-art sample preparationthrough hyphenation.

2 Sample preparation techniques

Basically, sample preparation is a complex and sensitivestep in analysis, which requires considerable expertise,especially when dealing with samples containing ana-lytes at micro or nano level concentrations. Many off-linemethods 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 permitcomparison, these percentages are shown in Fig. 1.

Solvent extraction (liquid–liquid extraction) is a classi-cal method of sample preparation exploiting unequaldistribution of solutes in two immiscible liquid phases. Ithas been used for the extraction of many compounds ofbiological and environmental importance [1–2]. Extrac-tion from liquid and solid samples is carried out by usinga variety of solvents such as hexane, acetone, acetic acid,benzene, toluene, methanol, acetonitrile, petroleumether, 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 thistechnique. Of course, solid phase extraction (SPE) is aquite practical method involving the use of reversedphase (C8, C18, etc. silica gel) adsorption phases in theform 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 createproblems in sample preparation in this sort of sample

handling modality. Chromatographic techniques arealso important and effective methods of sample prepara-tion and include HPLC, GPC, and SFC. However, these arenot affordable in all laboratories due to their costlyinstrumentation 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 inthe new millennium the demand for analyses is increas-ing at nano or lower level detection limits and scientistsin academia and industry as well as government agenciesrequire 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 focuson miniaturization and non-exposure of samples duringtheir preparation. Moreover, rapidity, efficiency, selectiv-ity, reproducibility, low cost, and low limits of detectionare demanded by today's separation science. A literaturesearch and our experience indicate that these demandscan be satisfied by hyphenation.

3 Hyphenation technology

Basically, the above-cited methods are used for samplepreparation in biological and environmental matrices.However, certain drawbacks make these methods lessthan ideal since they are not effective in the case of lowamounts of samples and consume costly solvents andtime. Besides, contamination and poor recoveries mayalso occur during experiments. The factors underscoredthe need for hyphenated techniques. Hyphenation isnothing but the coupling of a sample preparation unitwith the core analytical instrument. It has been foundmore effective than conventional methods in terms ofefficiency, effectiveness, selectivity, high recoveries, suit-ability for small samples, and for samples containingcomponents that are difficult to analyze and give rise toprocedural problems. Therefore, some papers have beenpublished 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, asdiscussed in the following sections.

3.1 Hyphenation in liquid chromatography

Basically, liquid chromatography represents a landmarkin the history of separation science and sample prepara-tion is a key issue in this area. Many workers haveattempted to hyphenate sample preparation units withliquid chromatographs and some important researchwork is discussed herein. Johansen et al. [7] described thehyphenation of Automated Sequential Trace Enrichmentof Dialysates (ASTED) system with HPLC for analysis ofantidepressant 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 ona trace enrichment column (TEC) was followed by HPLCanalyses on a Supelcosil column (15064.6 mm). TheASTED system consisted of a cellulose acetate dialysismembrane and interactions of analytes with the cellu-lose acetate membrane have been reported for basicdrugs such as the opiate derivative pholcodine, benzodia-zepines, and the neuroleptic drug clozapine [8–10]. Mostof the antidepressant drugs showed ionic and hydropho-bic interactions with the membrane and were selected asmodel substances to investigate more closely the abilityof cationic surfactants to inhibit analyte-membraneinteractions. The author optimized ASTED –HPLC condi-tions to achieve maximum recoveries by adding cationicsurfactants to the donor solution in the dialyser, by theeffect of chain length and concentration of cationic sur-factants, pH of the donor solution, and the volume of theacceptor solution. Furthermore, the authors used a che-mometric approach via factorial design and responsesurface modeling for optimization strategies. The devel-oped unit was applied successfully for monitoring mian-serine, imipramine, desimipramine, amitriptyline, andnortriptyline drugs in human plasma. Dialysis was per-formed for 12.8 minutes after which the six port valvewas switched to the injection position and the enrichedanalytes were loaded onto TEC with HPLC mobile phase(acetonitrile–methanol–0.005 M ammonium phosphatebuffer (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 inhuman plasma were in the range of 17–39 nM/L with UVdetection.

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 ofligand exchange type allowing the chiral separation of D-and L-aspartic acid. The mobile phase used for SPE was5 mM sodium-1-octanesufonate (pH 2.2) at 0.1 mL/minflow 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 anautomated 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 polystyreneresin and the analytes were enriched on SPE due to vari-ous interactions such as hydrogen bonding, van derWaals forces, steric effects etc. The fluorometric detectorpermitted detection of several metabolically relatedindole derivatives. HPLC conditions were Inertsil column(25063 mm) with mobile phase of different ratio of50 mM potassium dihydrogen phosphate adjusted topH 3.3 with phosphoric acid with acetonitrile as eluent.This set-up is shown in Fig. 2, indicating a coupling ofSPE and HPLC along with its working mechanism. TheSPE cartridge is pre-conditioned with acetonitrile, dipo-tassium EDTA in water (5g/L), and water at 3 mL/minflow rate; followed by autosampling of the enrichedingredients 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 advocatedthis hyphenation as an emerging technique due to itsdirect injection procedure in combination with columnswitching 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 wasconducted on a C2 packing and the mobile phase used forHPLC 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 systemcoupled to HPLC with fluorescence detection, on top SPEwith conditioning of the cartridge and sample injection, themiddle panel represents the system during backward-flushelution 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 anon-line solid-phase extraction method, coupled withHPLC–MS-MS for the determination of 16 phthalatemetabolites in human urine. The method employed aconventional analytical column for the chromato-graphic separations of these analytes and the mobilephase 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. Asimilar set-up was described by Kuklenyik et al. [16] forthe extraction and measurement of perfluorinatedorganic acids and amine in human serum and milk. A 12-lL volume of the reconstituted serum or milk extractwas auto-injected on to HPLC at a 300-lL/min flow ratewith 20 mM ammonium acetate (pH 4) in water andmethanol as mobile phase. The HPLC gradient program(14 min) was started at 60% methanol in the mobilephase followed by an increase in organic content to 80%in 0.5 min, which was kept for 9 min. Later on, themobile phase organic content was decreased in 0.5 minto 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 withHPLC–MS for the extraction and measurement of isofla-vones and lignans in urine. The mobile phases used were10 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 hyphenationsas an innovation in separation science.

Koster et al. [18] reported the analysis of lidocaine inurine by an on-line SPME–LC method. A polydimethylsi-loxane (PDMS) coated fiber was directly immersed intobuffered urine with optimized contact time, pH, ionicstrength, and temperature. The extraction yields were22% in about 45 min with a reproducibility of a5%expressed as relative standard deviation. The detectionlimits were 25–1000 ng/mL. Volmer et al. [19] studied theeleven corticosteroid and two steroid conjugations in aurine sample by SPME–LC–MS. Several SPME optimiza-tion factors such as polarity of fibres, extraction timeand effect of ionic strength, were investigated, and theirimpact on the SPME/LC/MS technique was studied. Themethod was sensitive with detection limits between 4and 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 HPLCfor the extraction and analyses of polycyclic aromatichydrocarbons, ketones, and alkylbenzenes at high pH. Toperform CME –HPLC, a so-gel TiO2 –PDMS capillary wasinstalled 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) asmobile phase. The target analytes were extracted on-lineby passing the aqueous sample through this sampling

loop. The sol-gel titania–PDMS coated capillaries wereused for on-line extraction and HPLC analysis of poly-cyclic aromatic hydrocarbons, ketones, alkylbenzenes,and a wide range of other less volatile or thermally labilecompounds [21] that are not amenable to GC separation.Hashi et al. [22] described the determination of polycyclicaromatic hydrocarbons (PAH) in the atmospheric partic-ulates by using on-line enrichment coupled with fasthigh-performance liquid chromatography with fluores-cence detection. The limits of detection of PAH were inthe range of 0.02–0.23 ng/mL with recoveries between87 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] developedand validated a method for local anesthetics in humanplasma 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, selective, and accurate on-line micro-extraction inpacked syringe (MEPS) technique hyphenated with HPLCfor the determination of lidocaine, prilocaine, ropiva-caine, and mepivacaine in human plasma. The extrac-tion recoveries were in the range of 60–90%. Veuthey etal. [25] described on-line solid phase extraction to achievenano analysis of drugs in biological samples. In thishyphenation technique, the single column performs twofunctions, i. e. extraction and separation. The column wasconnected to a detection system via a switching valve.The sample was directly injected on to the extractionsupport with and after the extraction, the valve wasswitched, and analytes were transferred to the detectorwith the eluting mobile phase followed by extractionsupport re-equilibration. According to the authors, themethod was simple and several applications have beenpublished for the direct analysis of biofluids. Quintana etal. [26] described an automated on-line hyphenation ofSPE–HPLC incorporating multi syringe flow injectionanalysis (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 theexpeditious determination of five acidic pharmaceuticalresidues 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) andB: 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 limitwas 0.02–0.67 ng/mL.

Clarkson et al. [27] described hyphenation of solid-phase extraction with liquid chromatography andnuclear 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; quercetin3-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 etal. [28] described an automated on-line hyphenation ofSFC –2-D–HPLC–MS for sample preparation, separation,detection, and identification of the fruiting bodies ofGanoderma lucidum, and at least 73 components in theextract were resolved with a calculated peak capacity ofup to 1643. The SFE and 2-D HPLC systems were fittedwith a Hypersil-CN (5 lm, 1200A, 15064.6 mm id) andChromolith Flash columns, respectively. In the firstdimensional separation, the binary mobile phase wascomposed of A (water) and B (methanol) with a flow rateof 0.1 mL/min In the second dimensional separation, themobile phase was composed of C (water) and D (acetoni-trile) with a flow rate of 4 mL/min. The same groupreported a simple SFE–HPLC system for comprehensiveanalyses 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 wasregarded as a powerful technique for the separation ofproteins, peptides, polymers, natural products, andother 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 coupledwith UV detection for the determination of a wide rangeof intracellular metabolites from mammalian cells. Thedetection wavelength of the UV detector was switchedfrom 220 to 260 nm and the detection limits were in therange of mM. Two Dionex AS11 analytical columns(25062 mm id) were used with 0.35 mL/min as mobilephase flow rate. Tuytten et al. [46] described an on-lineautomated SPE–HPLC–ESI-MS method for targetedmetabolomic analysis of urinary modified nucleosidelevels. The unit comprised a boronate affinity column asa trapping device, a hydrophilic interaction chromatog-raphy (HILIC) separation, and information-dependent MSdetection modes. The system was applied to biologicalsamples, detecting a number of modified nucleosides.Clarkson et al. [47] described HPLC–SPE–NMR hyphena-tion for structural elucidation of some natural products.Lambert et al. [48] described the identification of naturalproducts 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 ofan 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-ethyleneglycol dimethacrylate) monolithic capillary at a six-portvalve in a CEC system. Theobromine, theophylline, andcaffeine 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 wereimproved more than 100 times when compared withdirect analysis by l-HPLC. Besides the above-cited meth-ods of sample preparation, some modalities of liquidchromatography have also been used as sample prepara-tion methods and hyphenated with HPLC. Only onearticle on size exclusion chromatography coupled withHPLC is cited. Pomazal et al. [50] described analyses of cop-per, iron, manganese, and zinc in blood samples byexploiting the hyphenation of SEC with an HPLC–ICP-AES unit. Besides, this device was also used to monitormetalloproteins 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 usedand 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 foranalysis of volatile compounds, including several agri-cultural, industrial, and other chemical compounds. Ofcourse, 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 hyphenationapproaches. Many sample preparation methods havebeen coupled with GC for analyses of various species andthese include LLE, SPE, membrane, etc.

Membrane extraction is considered to be one of thebest extraction techniques because it has the importantadvantage that the sample and the extractant can contin-uously be kept in contact without physical mixing, thusproviding the basis for a continuous, real-time processpermitting automation and on-line connection to instru-ments [51]. Consequently, membrane techniques haveadvanced during few decades to a stage permitting thesolution 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 ofsolvents. Automated on-line liquid –liquid membraneextraction (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 miniaturizedmembrane extraction card (referred to as the ESy card)connected to a GC injector via an electromechanicalinstallation which controls pre-treatment and triggersthe GC instrument. The EF (enrichment factor) exhibitedby this hyphenation was between 33 and 40 for PCBs inriver 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 forachieving EF up to 50 for some local anesthetics in bloodplasma.

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 anauto-sampler and passed through the solid support,resulting in their adsorption onto the solid phase. Thesolid phase was then washed once with water (50 lL) toremove proteins and other interfering material. TheMEPS technique differed from commercial solid-phaseextraction (SPE) in the way in which the packing wasinserted directly into the syringe, and not into a separatecolumn. MEPS was capable of handling sample amountsfrom 10 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 for3 min followed by an increase up to 2808C at a rate of508C per min; with helium as carrier gas at 2.0 mL/minflow 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 ofsemi-volatile organic compounds (pesticides and herbi-cides) in a variety of water samples. The authors utilizedthis unit for real life samples of chlorinated tap water,well water, and river water. Furthermore, optimizationachieved minimum limit of detection (0.1 lg/L) with rela-tive recoveries in the range of 70–120% and a relativestandard deviation of less than 15%. The schematic repre-sentation of an SPE Twin–PAL PTV–LVI/GC/MS system isshown in Fig. 3. Pawliszyn et al. [55–59] developed solidphase micro extraction (SPME) methods for non-volatilecompounds. A fused silica rod with a polymeric coatingon the surface was employed as the extraction mediumfor the adsorption of volatile analytes from aqueous sam-ple solutions. The SPME fiber was inserted into the GCinjector 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 chromatographiccolumn used was HP-5 MS (28 m6250 lm id) and thelimits of detection of the method were between 0.001and 0.036 lg/L.

3.3 Hyphenation in capillary electrophoresis

Nowadays, capillary electrophoresis (CE) is valued as aversatile technique exhibiting high speed, high sensitiv-ity, lower limits of detection, and low running costs, andrepresents 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 bedifficult to separate by liquid chromatography and gaschromatography, or at least complements these tech-niques since the principles of separation are different.The lower detection limits of CE lead to the possibility ofseparating and characterizing very small quantities ofmaterials, which normally require pre-concentrationand sample preparation strategies, especially inunknown matrices. Therefore, some on-line methodshave been reported from time to time to achieve the goalof micro level separation and detection in CE.

Su et al. [65] studied the analysis of riboflavin in beer byCE–LED coupled with stacking micellar electrokineticchromatography (MEKC) as pre-concentration tech-niques. The detection limit reported was 1.0 ng/mL with38000 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 awavelength 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: washstation for upper PAL syringe, W2: wash station for lowerPAL 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 urineand serum. CME and OLBE–FASI were two separate con-centration units. The detection limits of this set-up werebetween 0.15 to 0.25 ng/mL on using photodiode arrayUV detection at 192 nm. The separations were achievedon an uncoated fused-silica capillary (50.2 cm650 lmid). Zhang et al. [68] developed hyphenation of immobi-lized metal affinity chromatography with capillary elec-trophoresis (IMAC–CE) for on-line concentration andanalysis 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 monolithicrods of macroporous poly(glycidyl methacrylate-co-ethyl-ene dimethacrylate). Cu(II) was subsequently introducedinto 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 proteinsamples. The detection limit was 3.0610 – 7 M with UVabsorbance 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 outgoingacceptor stream containing the analytes was allowed tofill a rotary injector in the FIA part of the system. Multi-ple sample injections were possible in one electropho-retic run, and the entire analytical procedure couldeasily be mechanized. The repeatability of the unit wasin the range of 1.6–3.3% (n = 7). This unit was applied ina wide range of real samples with complicated matriceslike milk, juice, slurry, and liquors from the pulp andpaper 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 oflow volume or having poor ingredients. Many modalitiesof spectroscopy have been reported in the literature ofidentification of various inorganic and organic species.The most important spectroscopic techniques are atomicabsorption spectrometry (AAS), inductively coupledplasma spectrometry (ICP), nuclear magnetic resonancespectrometry (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 ifapplied in biological and environmental samples havinglow concentrations of ingredients, the analytical resultsbecome 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 techniqueshyphenated with spectroscopic techniques. Sometimes,chelation of metal ions with suitable reagents enhancedthe detection [71–73].

Danesi [74] described a simplified model for the car-rier-facilitated transport of metal ions through hollowfiber supported liquid membranes. Yang et al. [75]reported the clean-up, extraction, and enrichment ofnumerous metals including Pb, Cu, Cr, La, Ce, Zn, and Coby HF-SLME and coupled it with AAS and ICP-MS as shownin Fig. 5. Katarina et al. [76] described an on-line hyphen-ation of sample preparation method by using a computercontrolled pre-treatment system (Auto-Pret AES) coupledwith ICP-AES for the sample pretreatment and determi-nation of trace metals in water samples. This systemenabled 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 orH2SO4 as 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-linesample dialysis, (S): sample, (A): acceptor stream, (E): elec-trolyte, (M): dialysis membrane, (D): UV detector, (V1): injec-tion valve in filling position, (V2): injection 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 eightmetal ions with similar characteristics such as Cd(II),Ni(II), Co(II), Mn(II), Pb(II), Zn(II), Cu(II), and Fe(III). Amicro-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 injectioninductively coupled plasma atomic emission spectrome-ter (FI– ICP-AES) system using anion- and cation-exchangeresin disks for the speciation of chromium species infresh water. Two kinds of ion exchange resin diskspacked in line-filters were fixed and serially connectedon 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 nitricacid 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/Lrespectively. This method was applied to the speciationof Cr(III) and Cr(VI) in fresh water samples. Similarly, Jit-manee et al. [79} reported arsenic speciation in freshwater by using inductively coupled plasma-atomic emis-sion spectrometry coupled with pre-concentration sys-tem containing solid phase anion exchange resin. Twominiaturized columns with a solid phase anionexchange 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 forboth 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-columnscoupled with plasma-atomic emission spectrometry inwater samples. Cr(III) was collected on the first columnpacked with iminodiacetate resin. Cr(VI) in the effluentfrom the first column was reduced to Cr(III), which wascollected 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. Amongvarious methods using microfluidic devices, nano-liquidchromatography (NLC) and nano-capillary electrophore-sis (NCE) are the two most important techniques, and areused to achieve separations at nano levels. During a liter-ature survey we found few papers dealing with on-linechip-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 ofmicro-dialysis NCE system for monitoring the hydrolysisof fluorescein mono-b-D-galactopyranoside (FMG) by b-D-galactosidase. The layout of the microdialysis/microchipCE 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 anon-line microdialysis sampling unit coupled with NCE.The authors used this set-up for amino acid and peptideanalyses. Wilson et al. [83] reported an on-line desaltingof macromolecule (betaine-type amphoteric or zwitter-ionic surfactant solutions) using a two-layered laminarflow 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-linehyphenation of micro-dialysis sample prepara-tion unit with NCE [81].


[85] coupled SPE to micellar electrokinetic chromatogra-phy to give a system permitting completely automatedextraction, elution, injection, separation, and detectionsteps, respectively and separately. The authors reportedfast analysis of rhodamine B yielding pre-concentrationfactors of more than 200 in less than 5 min with a60 femtomolar detection limit. A schematic representa-tion of this hyphenation 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 chainreaction in NLC. The amount injected was 600 nL ofblood 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 reportedgood reproducibilities of extraction and analysis.

Sample stacking has recently come to be regarded asthe 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 electrokineticchromatography (MEKC) stacking [96–98]. Some reviewshave been published on sample stacking techniques for awide 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 forincreased sensitivity and detection. Britz-McKibbin et al.[112, 113] designed an on-line focusing method based ondifferent mobilities of cationic analytes between back-ground electrolyte (BGE) and sample matrix, which iscalled velocity difference-induced focusing (V-DIF). Conget al. [114] reported on-line sample pre-concentrationusing field amplified stacking injection in NCE. Accord-ing to the authors, pressure-driven flows into or from thebranch channels, due to bulk velocity, can be used forliquid 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 traditionalmethod. Similarly, Zhang and Yin [115] developed multi-T microchip integrated field amplified sample stacking(FASS) coupled with NCE. According to the authors, avolumetrically defined large sample plug was formed inone step within 5 s by negative pressure in the headspaceof two sealed sample waste reservoirs. The authorsreported precisions in migration time and RSD as 3.3%and 1.3% for rhodamine123 (Rh123) and fluoresceinsodium salt, respectively. Schematic representation ofthe channel design of a multi-T microfluidic chip andNCE system with negative pressure large volume sampleinjection is shown in Fig. 8.

Jung et al. [116] designed, fabricated, and characterizeda novel field-amplified sample stacking (FASS)-NCE chiphaving photo-initiated porous polymer for the analysesof fluorescein and bodipy. Furthermore, the authors


Figure 7. Schematic representation of chip-based solidphase extraction-MEKC device. (a): Layout of the entiredevice and (b): expanded view of the extraction region of thedevice. The dotted lines represent the direction of fluid flowduring extraction; the solid line signifies flow during elution/injection. (Narrow channels are ca. 55 lm wide, the columnchamber is ca. 210 lm wide with all channels ca. 15 lmdeep.) [85].

Figure 8. Schematic representation of (a): channel design ofthe 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 matricesTrazodone 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 –

ED10 fmol/lL [121]

Ciprofloxacin Biological samples Dialysis –RPLC–fluorescence 0.1 nM [122]Amino acids Beverages and feed stuff Dialysis –RPLC–fluorescence 1–278 lg/L [123]Fluoroquinolones Fortified tissue 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 plasma Dialysis –RPLC–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, tissueSFE –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-

dialysatesDialysis –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 matricesEndocrine 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]Organic acids Aerosol particles SFE –NPLC–GC–MS 0.4 ng/m3 [167]


described a 1000-fold signal increase during detection.This polymer material provided a region of high flowresistance, which allowed electromigration of sampleions resulting in pre-concentration. Lichtenberg et al.[117] developed a microchip device for field amplifica-tion stacking (FAS), which allowed the formation of com-paratively long, volumetrically defined sample plugswith a minimal NCE bias. The authors studied fluidiceffects; arising from solutions with mismatched ionicstrengths, in chip-based electrokinetically. Furthermore,these authors developed a new chip layout for full col-umn stacking with subsequent sample matrix removalby polarity switching. Some important hyphenationexamples are given in Table 1.

4 Concluding remarks

In the present scenario of separation science advance-ment, hyphenation is continuously gaining importancefor 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 thedevelopment of hyphenation techniques is not yet com-plete and is still progressing. Briefly, the art of hyphena-tion in separation science will be in great demand duringthe 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]RetardantsOrganophosphorus esters Air particulates DMAE–SPE –GC–NPD 90.9 –186.2 pg/m3 [175]Organophosphorus Air particulates DSAE–SPE –GC–NPD 0.1 ng/m3 [176]EstersSulfonamide 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 – 4 ng/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]

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