PrefaceModern analytical chemistry frequently requires precise analyticalmeasurements, at verylowconcentrations, withavarietyof instru-ments. A high-resolution separation has to be generally performed withaselectivechromatographicmethodpriortoquantication.Thereforethe knowledge of instrumentation used in chemical analysis today is ofparamount importancetosolvecurrent problems andassurefutureprogressinvariouseldsofscienticendeavor. Theseincludechem-istry, biochemistry, pharmaceutical chemistry, medicinal chemistry,biotechnology, nanotechnology, archaeology, anthropology, environ-mental sciences, andavarietyof otherscienticdisciplines. Thein-struments may be operated by a variety of people in industry,government, oracademicelds, whoseeducational backgrounds canrangefromahighschool diplomatoaPh.D. withpostdoctoral expe-rience. Increasedknowledgerelatingtotheprinciplesof theinstru-mentation and separation methodologies allows optimal usage ofinstrumentationwithmoremeaningful datagenerationthat canbeinterpretedreliably.This book covers the fundamentals of instrumentation as well as theapplications,toleadtobetterutilizationofinstrumentationbyallsci-entistswhoplantoworkindiversescienticlaboratories. It shouldserveasaneducational tool aswell asarstreferencebookforthepracticinginstrumentalanalyst.Thistexthasbeenbroadlyclassiedintosixsections:1. Overview, Sampling, Evaluation of Physical Properties, and Ther-malAnalysis(Chapters14)2. SpectroscopicMethods(Chapters511)3. ChromatographicMethods(Chapters1216)4. ElectrophoreticandElectrochemicalMethods(Chapters1718)5. HyphenatedMethods,UniqueDetectors(Chapters1920)6. Problem Solving and Guidelines for Method Selection (Chapter 21)xxixEachofthesesectionshasoneormorechapterscoveringimportantaspects of thetitledsubject. Everychapter is adistinct entitythatprovides the relevant information deemed necessary by the author. Allchapterscontainimportantinformationaboutapplicationsthatillus-tratetheuseofthemethods. Theyalsoincludeanappropriatesetofreviewquestions.This book is planned to enable the reader to understand the properutilization of instrumentation and various methodologies that arebasedonthem.Itprovidesanunderstandingofhowvariousmethodsmaybecombinedtoobtainacomprehensivepictureofthenatureofsample in question. The reader will also gain a better understanding ofhowtoselectthebestmethodforsolvingagivenproblem.Aimedasthetextforaspecialcourseorgraduatestudents,itpro-vides the necessary background informationfor various laboratorypersonnel. Thetextshouldbeveryuseful formanagersinacademia,industrial,andgovernmentlaboratoriesinterestedinimprovingtheirscienticknowledgeandbackground.September1,2005SatinderAhujaNeilJespersenPrefacexxxSerieseditorsprefaceModerninstrumental analysishasalonghistoryintheeldof ana-lytical chemistry, and that makes it difcult to prepare a book like thisone. Themainreasonisthecontinuousimprovement intheinstru-mentation applied to the analytical eld. It is almost impossible to keeptrackof thelatestdevelopmentsinthisarea. Forexample, atPITT-CON, thelargestworldexhibitioninanalytical chemistry, everyyearseveral newanalytical instruments moresophisticatedandsensitivethanthepreviousversionsarepresentedforthismarket.Modern InstrumentalAnalysisis written to reectthe popularity ofthevariousanalytical instrumentsusedinseveral different eldsofscience.Thechaptersaredesignedtonotonlygivethereadertheun-derstanding of the basics of each technique but also to give ideas on howtoapplyeachtechniqueinthesedifferentelds.Thebookcontains21chaptersandcoverssampling;spectroscopicmethodssuchasnearin-frared,atomicandemissionmethods,nuclearmagneticresonanceandmass spectrometricmethods; separationmethodswithall chromato-graphic techniques; electrochemical methods and hyphenated methods.Thelastchapterofthebook,acomplementarychapter,isveryusefulandis practically orientedto problem-solving, giving guidelines formethodselection.Considering all the chapters indicated above, the book is suitable forawideaudience, fromstudentsat thegraduatelevel toexperiencedresearchersandlaboratorypersonnel inacademia, industryandgov-ernment. It is a good introductory book from which one can then go ontomorespecializedbookssuchastheonesregularlypublishedintheComprehensiveAnalytical Chemistryseries. Suchageneral bookde-scribingmodernanalyticalmethodshasbeenneededsincethestartofthis series andit is obvious that after acertainamount of time, amaximumof10years,anupdateofthebookwillbeneeded.Finally,Iwouldliketospeciallythankthetwoeditorsandall thecontributingxxxiauthorsofthisbookfortheirtimeandeffortsinpreparingthisexcel-lentandusefulbookonmoderninstrumentalanalysis.D.BarceloDepart.EnvironmentalChemistry,IIQAB-CSICBarcelona,SpainSerieseditorsprefacexxxiiChapter1OverviewSatinderAhuja1.1 INTRODUCTIONModern analytical chemistry generally requires precise analyticalmeasurements at verylowconcentrations, withavarietyof instru-ments. Frequently, high-resolutionseparations have to be achievedwithselectivechromatographicmethodspriortoanalyticaldetermina-tions [1,2]. Therefore, the knowledge of instrumentation used in chemi-cal analysis today is of paramount importance to assure future progressin various elds of scientic endeavor. This includes various disciplinesof chemistrysuchasbiochemistry, pharmaceutical chemistry, medic-inal chemistry, biotechnology, and environmental sciences. Instru-mentscanbeoperatedbyavarietyofpeopleinindustry,government,oracademicelds,withawiderangeofeducationalbackgrounds.Theoptimalusageofinstrumentationwithmoremeaningfuldatagenera-tion that can be interpreted reliably is possible only with the improvedknowledgeoftheprinciplesoftheinstrumentationsusedformeasure-ment as well as those utilized to achieve various separations. This bookcovers the fundamentals of instrumentation as well as the applicationsthat should lead to better utilization of instrumentation by all scientistswho plan to work in diverse scientic laboratories. It should serve as aneducational tool as well as arst reference bookfor the practicinginstrumentalanalyst.Thistexthasbeenbroadlyclassiedintothefollowingareas:General information (sampling/sample preparation and basicproperties)SpectroscopicmethodsChromatographicmethodsElectrophoreticandelectrochemicalmethodsComprehensiveAnalyticalChemistry47S.AhujaandN.Jespersen(Eds)Volume47ISSN:0166-526XDOI:10.1016/S0166-526X(06)47001-Xr 2006ElsevierB.V.Allrightsreserved. 1Combinationofchromatographicandspectroscopicmethods,anduniquedetectorsProblemsolvingandguidelinesformethodselection.Toprovideaconciseoverviewofthisbook, thecontentsofvariouschaptersarehighlightedbelow.Alsoincludedhereisanadditionalin-formationthatwill helptoroundoutthebackgroundinformationtofacilitatetheperformanceofmoderninstrumentalanalysis.1.2 SAMPLINGANDSAMPLEPREPARATIONPriortoperformanceofanyanalysis, itisimportanttoobtainarep-resentative sample. Difculties of obtaining such a sample and how it ispossibletodothebest jobinthis areaarediscussedinChapter 2.Another important consideration is to prepare samples for the requiredanalyticaldetermination(s). Samplepreparationisfrequentlyamajortime-consumingstepinmostanalysesbecauseitisrarelypossibletoanalyze a neat sample. The sample preparationgenerally entails asignicantnumberofstepspriortoanalysiswithappropriateinstru-mental techniques, as describedinthis book. This chapter presentsvarious methods used for collecting and preparing samples for analysiswith modern time-saving techniques. It describes briey the theory andpractice of each technique and focuses mainly on the analysis of organiccompoundsofinterest(analytes)inavarietyofmatrices, suchastheenvironment,foods,andpharmaceuticals.1.3 EVALUATIONSOFBASICPHYSICALPROPERTIESSome of the simplest techniques such as determinations of melting andboiling points, viscosity, density, specic gravity, or refractive index canserveasaquickqualitycontroltool(Chapter3).Timeisoftenanim-portant considerationinpractical chemical analysis. Rawmaterialsawaitingdeliveryintanker trucks or tractor-trailers oftenmust beapprovedbeforetheyareofoaded. Observationandmeasurementofbasic physical properties are the oldest knownmeans of assessingchemical purity and establishing identity. Their utility comes from thefactthatthevastmajorityofchemicalcompoundshaveuniquevaluesfortheirmeltingpoints, boilingpoints, density, andrefractiveindex.Most of these properties change dramatically when impurities arepresent.S.Ahuja21.4 THERMALMETHODSThermal analysis measurements are conducted for the purpose of eval-uatingthephysical andchemical changes that maytakeplace inasampleas aresult of thermallyinducedreactions (Chapter 4). Thisrequires interpretationof the events observedina thermogramintermsofplausiblethermalreactionprocesses.Thereactionsnormallymonitoredcanbeendothermic(melting, boiling, sublimation, vapori-zation, desolvation, solidsolid phase transitions, chemical degradation,etc.) or exothermic (crystallization, oxidative decomposition, etc.) innature.Thermalmethodshavefoundextensiveuse inthepastaspartofaprogramofpreformulationstudies, sincecarefullyplannedworkcanbeusedtoindicatetheexistenceofpossibledrugexcipientinter-actions in a prototype formulation. It is important to note that thermalmethodsof analysiscanbeusedtoevaluatecompoundpurity, poly-morphism, solvation, degradation, drugexcipientcompatibility, andawidevarietyofotherthermallyrelatedcharacteristics.1.5 GENERALPRINCIPLESOFSPECTROSCOPYANDSPECTROSCOPICANALYSISThe range of wavelengths that the human eye can detect varies slightlyfromindividual toindividual (Chapter5). Generally, thewavelengthregionfrom350to700 nmisdenedasthevisibleregionofthespec-trum. Ultravioletradiationiscommonlydenedasthosewavelengthsfrom200 to350 nm. Technically, the near-infrared(NIR)region startsimmediately after the visible region at 750 nmand ranges up to2,500 nm.Theclassicalinfraredregionextendsfrom2500 nm(2.5 mm)to50,000 nm(50 mm). Theenergiesof infraredradiationrangefrom48 kJ/mol at 2500 nm to 2.4 kJ/mol at 50,000 nm. These low energies arenotsufcienttocauseelectrontransitions, buttheyaresufcienttocausevibrationalchangeswithinmolecules.Thisiswhyinfraredspec-troscopy is oftencalled vibrational spectroscopy. The principles in-volvedinthesespectroscopictechniquesarediscussedinthischapter.1.6 NEAR-INFRAREDSPECTROSCOPYAs mentioned above, the NIR portion of the electromagnetic spectrum islocatedbetweenthevisible andmid-range infraredsections, roughly7502500nmor13,3334000cm1. ItmainlyconsistsofovertonesandOverview3combinations of the bands found in the mid-range infrared region(4000200cm1). In general, NIR papers did not begin to appear in ear-nest until the 1970s, when commercial instruments became easily avail-ableowingtotheworkoftheUSDepartmentofAgriculture(USDA).Some of these developments are discussedunder instrumentationinChapter 6. After the success of the USDA, food producers, chemical pro-ducers, polymermanufacturers, gasolineproducers, etc. pickeduptheball andran withit.The last to becomeinvolved,mainly forregulatoryreasons,arethepharmaceuticalandbiochemicalindustries.1.7 X-RAYDIFFRACTIONANDFLUORESCENCEAll properlydesignedinvestigationsintothesolid-statepropertiesofpharmaceutical compounds begin with understanding of the structuralaspects involved. There is no doubt that the primary tool for the studyofsolid-statecrystallographyisX-raydiffraction.Toproperlycompre-hendthepowerofthistechnique, itisrstnecessarytoexaminetheprocessesassociatedwiththeabilityof acrystallinesolidtoactasadiffractiongrating for the electromagnetic radiationof appropriatewavelength. Chapter 7 separately discusses the practice of X-raydiffractionasappliedtothecharacterizationofsinglecrystalsandtothestudyofpowderedcrystallinesolids.Heavyelements ordinarilyyieldconsiderablymore intense X-rayuorescence (XRF) spectrum, because of their superior uorescentyieldbandsthanthelightelements. Thisfeaturecanbeexploitedtodeterminetheconcentrationof inorganicspeciesinasampleortheconcentrationof acompoundthatcontainsaheavyelementinsomematrix. The background emission detected in an XRF spectrum is usu-allyduetoscatteringofthesourceradiation. Sincethescatteringin-tensity from a sample is inversely proportional to the atomic number ofthescatteringatom, itfollowsthatbackgroundeffectsaremorepro-nouncedfor samples consistinglargelyof second-rowelements (i.e.,organicmolecules ofpharmaceuticalinterest).Hence,background cor-rection routines play a major role in transforming raw XRF spectra intospectrasuitableforquantitativeanalysis.1.8 ATOMICSPECTROSCOPYElemental analysis at the trace or ultratrace level can be performed by anumber ofanalytical techniques;however,atomicspectroscopy remainsS.Ahuja4themostpopular approach.Atomic spectroscopy can be subdivided intothree elds: atomic emission spectroscopy (AES), atomic absorptionspectroscopy(AAS), andatomic uorescence spectroscopy(AFS) thatdifferbythemodeofexcitationandthemethodofmeasurementoftheatom concentrations. The selection of the atomic spectroscopic techniquetobeusedforaparticularapplicationshouldbebasedonthedesiredresult, since each technique involves different measurement approaches.AESexcitesground-stateatoms(atoms) andthenquantiesthecon-centrationsofexcited-stateatomsbymonitoringtheirspecialdeactiva-tion. AAS measures the concentrations of ground-state atoms byquantifyingtheabsorptionofspectralradiationthatcorrespondstoal-lowed transitions from the ground to excited states. AFS determines theconcentrations of ground-state atoms by quantifying the radiative deacti-vationof atoms that havebeenexcitedbytheabsorptionof discretespectral radiation. All of these approaches are discussed in Chapter 8. ItshouldbenotedthatAASwithaameastheatomreservoirandAESwith an inductively coupled plasma have been used successfully to speci-atevariousultratraceelements.1.9 EMISSIONSPECTROSCOPICMEASUREMENTSThetermemissionreferstothereleaseoflightfromasubstanceex-posedtoanenergysourceofsufcientstrength(Chapter9).Themostcommonly observed line emission arises from excitation of atomic elec-trons to higher level by an energy source. The energy emitted by excitedatomsofthiskindoccursatwavelengthscorrespondingtotheenergylevel difference. Sincetheselevelsarecharacteristicfortheelement,theemissionwavelengthcanbecharacteristicforthat element. Forexample, sodiumandpotassiumproducedifferentlinespectra. Emis-sion methods can offer some distinct advantages over absorption meth-ods in the determination of trace amounts of material. Furthermore, itmaybepossibletodetectevensingleatoms.1.10 NUCLEARMAGNETICRESONANCESPECTROSCOPYNuclearmagneticresonance(NMR)spectroscopyisusedtostudythebehaviorof thenuclei inamoleculewhensubjectedtoanexternallyappliedmagnetic eld. Nuclei spinabout the axis of the externallyappliedmagneticeldandconsequentlypossessanangularmomen-tum. Thegroupof nuclei mostcommonlyexploitedinthestructuralOverview5characterizationof small molecules by NMRmethods are the spin1/2nuclei,whichinclude1H,13C,19F,and31P.NMRisamenabletoabroadrangeofapplications. Ithasfoundwideutilityinthepharma-ceutical, medical, andpetrochemical industriesaswell asacrossthepolymer, materials science, cellulose, pigment, and catalysis elds. Thevast diversity of NMR applications may be due to its profound ability toprobebothchemical andphysical properties of molecules, includingchemical structureandmolecular dynamics. Furthermore, it canbeappliedtoliquids,solids,orgases(Chapter10).1.11 MASSSPECTROMETRYMass spectrometry is arguably one of the most versatile analyticalmeasurementtoolsavailabletoscientiststoday, ndingapplicationinvirtually every discipline of chemistry (i.e., organic, inorganic, physical,and analytical) as well as biology, medicine, and materials science(Chapter 11). The technique provides both qualitative and quantitativeinformation about organic and inorganic materials, including elementalcomposition, molecular structure, and the composition of mixtures. Thecombination of the technique with the powerful separation capabilitiesofgaschromatography(GC-MS)andliquidchromatography(LC-MS)ledtothedevelopmentofnewkindsofmassanalyzersandtheintro-ductionofrevolutionarynewionizationtechniques.Thesenewioniza-tiontechniques(introducedlargelyinthepast15years)areprimarilyresponsiblefortheexplosivegrowthandproliferationofmassspectro-metryasanessentialtoolforbiologistsandbiochemists.Theinforma-tion derived from a mass spectrum is often combined with that obtainedfromtechniquessuchasinfraredspectroscopyandNMRspectroscopyto generate structural assignments for organic molecules. The at-tributes of mass spectrometrythat makeit aversatileandvaluableanalytical technique are its sensitivity (e.g., recently, a detectionlimit of approximately 500 molecules, or 800 yoctomoles has beenreported) and its specicity in detecting or identifying unknowncompounds.1.12 THEORYOFSEPARATIONSSeparationmodelsaregenerallybasedonamixtureofempirical andchemical kinetics and thermodynamics. There have beenmany at-tempts to relate known physical and chemical properties to observationS.Ahuja6and to develop a predictive theory. The goal has been to improve ourunderstandingoftheunderlyingphenomenaandprovideanefcientapproachtomethoddevelopment(seeChapter12). Thelatterpartofthegoalisofgreaterinteresttothevastmajorityofpeoplewhomakeuse of separation methods for either obtaining relatively pure materialsor in isolating components of a chemical mixture for quantitativemeasurements.1.13 THIN-LAYERCHROMATOGRAPHYThin-layer chromatography (TLC) is one of the most popular and widelyused separation techniques because it is easy to use and offers adequatesensitivity and speed of separations. Furthermore, multiple samples canberunsimultaneously. Itcanbeusedforseparation, isolation, identi-cation,andquanticationofcomponentsinasample.Theequipmentusedforperformanceof TLC, includingapplicationsof TLCindrugdiscoveryprocess, iscoveredinChapter13. Thistechniquehasbeensuccessfully used in biochemical, environmental, food, pharmacological,andtoxicologicalanalyses.1.14 GASCHROMATOGRAPHYThetermgas chromatography(GC) refers toafamilyof separationtechniques involvingtwo distinct phases: astationaryphase, whichmay be a solid or a liquid; and a moving phase, which must be a gas thatmovesinadenitedirection. GCremainsoneofthemostimportanttools for analysts inavarietyof disciplines. As aroutineanalyticaltechnique,GCprovidesunparalleledseparationpower,combinedwithhighsensitivityandeaseofuse.Withonlyafewhoursoftraining,ananalyst can be effectively operating a gas chromatograph, while, as withother instrumental methods, to fully understand GC may require yearsof work. Chapter 14 provides an introduction and overview of GC, witha focus on helping analysts and laboratory managers to decide when GCisappropriatefortheiranalyticalproblem,andassistsinusingGCtosolvevariousproblems.1.15 HIGH-PRESSURELIQUIDCHROMATOGRAPHYThephenomenal growthinchromatographyislargelyduetotheintro-ductionof the technique called high-pressure liquid chromatography,Overview7which is frequently called high-performance liquid chromatography (bothare abbreviated as HPLC; see discussion in Chapter 15 as to which term ismore appropriate). It allows separations of a large variety of compounds byofferingsomemajorimprovementsovertheclassical columnchromato-graphy, TLC, GC; and it presents some signicant advantages over morerecent techniques such as supercritical uid chromatography (SFC), capil-laryelectrophoresis(CE),andelectrokineticchromatography.1.16 SUPERCRITICALFLUIDCHROMATOGRAPHYSupercriticaluidchromatographyemployssupercriticaluidinsteadof gasorliquidtoachieveseparations. Supercritical uidsgenerallyexist at conditions above atmospheric pressure and at an elevated tem-perature. As a uid, the supercritical state generally exhibits propertiesthat are intermediate to the properties of either a gas or a liqiud.Chapter16discussesvariousadvantagesof SFCoverGCandHPLCandalsoprovidessomeinterestingapplications.1.17 ELECTROMIGRATIONMETHODSThecontributionofelectromigrationmethodstoanalytical chemistryandbiological/pharmaceuticalsciencehasbeenverysignicantinthelastseveral decades. Electrophoresisisaseparationtechniquethatisbasedonthedifferential migrationof chargedcompoundsinasemi-conductive medium under the inuence of an electric eld (Chapter 17).It is the rst method of choice for the analyses of proteins, amino acids,andDNAfragments. Electrophoresis ina capillaryhas evolvedthetechniqueintoahigh-performanceinstrumentalmethod.Theapplica-tionsarewidespreadandincludesmallorganic-, inorganic-, charged-,neutral-compounds,andpharmaceuticals.Currently,CEisconsideredan established tool that analytical chemists use to solve many analyticalproblems. Themajor applicationareas still areintheeldof DNAsequencingandproteinanalysis,aswellaslow-molecularweightcom-pounds (pharmaceuticals). The Human Genome Project was completedmanyyearsearlierthaninitiallyplanned, becauseofcontributionsofCE. TheCEtechnologyhasgrowntoacertainmaturity, whichhasallowed development and application of robust analytical methods. Thistechnique is already describedas general monographs inEuropeanPharmacopoeiaandtheUSP. Eventhoughbothelectromigrationandchromatographicmethodsevolvedseparatelyovermanydecades,theyS.Ahuja8haveconvergedintoasinglemethodcalledcapillaryelectrochromato-graphy.Thistechniquehasalreadyshownalotofpromise.1.18 ELECTROCHEMICALMETHODSThediscussionof electrochemical methodshasbeendividedintotwoareas,potentiometryandvoltammetry.1.18.1 PotentiometryIn potentiometry, the voltage difference between two electrodes ismeasuredwhiletheelectriccurrent betweentheelectrodesismain-tained under a nearly zero-current condition (Chapter 18a). In the mostcommonformsofpotentiometry, thepotentialofaso-calledindicatorelectrodevaries, dependingontheconcentrationoftheanalytewhilethe potential arising from a second reference electrode is ideally a con-stant. Most widely used potentiometric methods utilize an ion-selectiveelectrodemembranewhoseelectrical potential toagivenmeasuringion, eitherinsolutionorinthegasphase, providesananalytical re-sponsethatishighlyspecic. Amultiplicityof ion-selectiveelectrodedesignsrangingfromcentimeter-longprobestominiaturizedmicrom-eter self-containedsolid-state chemical sensor arrays constitute thebasis of modernpotentiometricmeasurements that havebecomein-creasinglyimportantinbiomedical, industrial, andenvironmentalap-plicationelds. Themodernpotentiometricmethodsareuseful foramultitude of diverse applications such as batch determination of medi-cally relevant electrolytes (or polyions in undiluted whole blood), in vivoreal time assessment of blood gases and electrolytes with non-thrombogenic implantable catheters, multi-ionmonitoring inindus-trial process control via micrometer solid-state chemical-eldeffecttransistors(CHEMFETs), andthedeterminationof heavymetalsinenvironmentalsamples.1.18.2 VoltammetryThis is adynamic electrochemical technique, whichcanbe usedtostudyelectrontransfer reactions withsolidelectrodes. Avoltammo-gramis the electrical current response that is due to appliedexci-tationpotential. Chapter 18bdescribes the originof the current insteady-state voltammetry, chronoamperometry, cyclic voltammetry, andsquare wave voltammetry andother pulse voltammetric techniques.Overview9Theemployment of thesetechniquesinthestudyof redoxreactionsprovidesveryusefulapplications.1.19 HYPHENATEDMETHODSThecombinationof separationtechniqueswithspectroscopyhaspro-videdpowerfultools.Forexample,UVandvariousotherspectroscopicdetectors have been commonly used in HPLC (or LC), SFC, and CE (seeChapters 1517). To fully utilize the power of combined chromato-graphy-mass spectrometry techniques, it is necessary to understand theinformationandnuancesprovidedbymassspectrometryinitsvariousforms (Chapter 19). In principle, these novel systems have solved manyof the problems associated with structure elucidation of low-levelimpurities. It must be emphasized that the tried-and-true method of off-lineisolationandsubsequent LC-MSisstill themost reasonableap-proach in some cases. In general, some compounds behave poorly, spec-troscopically speaking, when solvated in particular solvent/solventbuffercombinations. Themanifestationof thispoorbehaviorcan be multiple stable conformations and/or equilibration isomers in theintermediate slow-exchange paradigm, resulting in very broad peaks ontheNMRtimescale.Therefore,it canbe envisionedthattheexibilityofhavinganisolatedsampleinhandcanbeadvantageous. Thereareadditional issuesthat canarise, suchassolubilitychanges, theneedfor variabletemperatureexperiments, theneedtoindirectlyobserveheteroatoms other than13C (triple resonance cryogenic probe required),as well as many others. It must be stated that LC-NMR and its variousderivativesareideallysuitedforlookingatsimpleregiochemicalissuesin relative complex systems. In some cases, full structure elucidation ofunknown compounds can be completed. Although this will become moreroutine withsome of the recent advances suchas peaktrappingincombination with cryogenic ow-probes. There are many elegant exam-ples of successful applications of LC-NMRandLC-NMR/MStoverycomplex systems. The principle advantage is that in most cases one cancollectNMRandMSdataonanidenticalsample,thuseliminatingthepossibilityofisolation-induceddecomposition.1.20 UNIQUEDETECTORSSpecial detectors have been developed to deal with various situations. Afewinterestingexamples arediscussedbelow. TheseincludeopticalS.Ahuja10sensors (optrodes), biosensors, bioactivity detectors, and drug detectors.Some of the new developments in the area of lab on chip have also beensummarized.1.20.1 OpticalsensorsChemical sensorsaresimpledevicesbasedonaspecicchemical rec-ognition mechanism that enables the direct determination of either theactivities or concentrations of selectedions and electrically neutralspecies, withoutpretreatmentofthesample. Clearly, eliminatingtheneed for sample pretreatment is the most intriguing advantage ofchemicalsensorsoverotheranalyticalmethods.Opticalchemicalsen-sors(optodesoroptrodes)useachemicalsensingelementandopticaltransduction for the signal processing; thus these sensors offer furtheradvantagessuchasfreedomfromelectrical noiseandeaseof minia-turization,aswellasthepossibilityofremotesensing.Sinceitsintro-duction in 1975, this eld has experienced rapid growth and hasresultedinoptodesforamultitudeofanalytes(seeChapter20a).Opticalbiosensorscanbedesignedwhenaselectiveandfastbiore-actionproduceschemicalspeciesthatcanbedeterminedbyanopticalsensor. Like the electrochemical sensors, enzymatic reactions that pro-duce oxygen, ammonia, hydrogen peroxide, and protons can be utilizedtofabricateopticalsensors.1.20.2 BioactivitydetectorsRoutinemonitoringof foodandwaterforthepresenceof pathogens,toxins, andspoilage-causing microbes is a major concernfor publichealth departments and the food industry. In the case of disease-causingcontamination,theidenticationoftheorganismiscriticaltotrace its source. The Environmental Protection Agency monitors drink-ingwater, ambientwater, andwastewaterforthepresenceoforgan-ismssuchasnonpathogeniccoliformbacteria, whichareindicatorsofpollution. Identifying the specic organismcanaid inlocating thesourceofthepollutionbydeterminingwhethertheorganismisfromhumans, livestock, or wildlife. Pharmaceutical companies monitorwater-for-injectionforbacterial toxinscalledpyrogens, whicharenotremoved by lter-sterilization methods. Chapter 20b discusses the needformethodstoquicklydetect biothreat agents, whichmaybeintro-ducedasbacteria, spores, viruses, rickettsiae, ortoxins, suchasthebotulismtoxinorricin.Overview111.20.3 DrugdetectorsDrugdetectiontechnologiesareusedinvariouslawenforcementsce-narios, whichpresentchallengesintermsofinstrumentationdevelop-ment.Notonlymustbedetecteddrugsvary,buttheamountstobedetectedrangefrommicrogramquantitiesleftasincidental contami-nationfromdrugactivity to kilograms being transportedas hiddencaches.Thelocationsofhiddendrugscreatedifcultiesindetectionaswell. Customs and border agents use drug-detection technology to inter-ceptdrugssmuggledintothecountryinbulkcargoorcarriedbyin-dividuals. Correctional facilitiessearchfordrugsbeingsmuggledintothe prison by mail (e.g., small amounts under stamps) or by visitors, andfurthermoreneedtomonitordrugpossessioninsidetheprison. Lawenforcement representatives may use detectors in schools to nd cachesof dealers. Other users of drug detection technology include aviation andmarinecarriers, postal andcourierservices, privateindustry, andthemilitary. Thetaskistondtherelativelyrarepresenceofdrugsinapopulation of individuals or items, most of which will be negative. Con-sequently,fordrugdetectionpurposes,theabilitytondillicitcontra-bandisofgreaterimportancethanaccuratequantitation.Chapter20cdescribes various instruments that have been developed for drugdetection.MicrochipArrays: Aninterestin developingalabonchiphas ledtosomeremarkableadvancementsininstrumentation[3]. Intheearly1990s, microfabricationtechniques borrowedfromtheelectronic in-dustrytocreateuidpathwaysinmaterialssuchasglassandsilicon[4]. This has led to instrumentation such as NanoLC-MS [5]. The mainfocusofthesemethodsisanalysisofbiologicallysourcedmaterial forwhich existing analytical methods are cumbersome and expensive. Themajor advantages of these methods are that a sample size 100010,000timessmallerthanwiththeconventional systemcanbeused, high-throughput analysis is possible, and a complete standardization of ana-lyticalprotocolcanbeachieved. Alargenumberofsequenceanalyseshave to be performed in orderto build a statistically relevant databaseof sequence variation versus phenotype for a given gene or set of genes[6]. Forexample, patternsofmutationincertaingenesconferresist-ance to HIV for various antiretrovirals. Sequence analyses of many HIVsamplesinconjunctionwithphenotypedatacanenableresearcherstoexploreanddesignbettertherapeuticals.Similarly,therelationshipofmutationincancer-associatedgenes, suchasp53, todiseaseseveritycanbeaddressedbymassivesequenceanalyses.S.Ahuja12DNAprobearraysystemsarelikelytobeveryusefulfortheseana-lyses. Active DNA microchip arrays with several hundred test sites arebeingdevelopedforevolvinggeneticdiseasesandcancerdiagnostics.These approaches can lead to high-throughput screening (HTS) in drugdiscovery.TheHumanGenomeProjecthasbeenamajordrivingforceinthedevelopmentofsuitableinstrumentsforgenomeanalysis. Thecompanies that can identify genes that are useful for drug discovery arelikely to reap the harvest in terms of new therapeutic agents and thera-peuticapproachesandcommercialsuccessinthefuture.1.21 PROBLEMSOLVINGANDGUIDELINESFORMETHODSELECTIONAnalytical chemistryhelpsanswerveryimportant questionssuchasWhat is in this sample, and how much is there? It is an integral partofhowweperceiveand understandthe worldand universearoundus,soithasbothverypractical andalsophilosophical andethical impli-cations. Chapter 21 deals with the analytical approach rather than ori-enting primarily with techniques, even when techniques are discussed.Itisexpectedthatthiswillfurtherdevelopcritical-thinkingandprob-lem-solvingskills and, at thesametime, gainmoreinsight intoanunderstandingandappreciationofthephysicalworldsurroundingus.This chapter will enable the reader to learn the type of thinking neededtosolvereal-worldproblems, includinghowtoselectthebest tech-niquefortheproblem.REFERENCES1 S. Ahuja, Trace and UltratraceAnalysis by HPLC, Wiley, New York, 1992.2 S. Ahuja, ChromatographyandSeparationScience, Academic, SanDiego,2003.3 S.Ahuja,HandbookofBioseparations,Academic,SanDiego,2000.4 Am.Lab.,November.(1998)22.5 PharmaGenomics,July/August,2004.6 T.Kreiner,Am.Lab.,March,(1996)39.Overview13Chapter2SamplingandsamplepreparationSatinderAhujaandDianeDiehl2.1 INTRODUCTIONSamplepreparation(SP)isfrequentlynecessaryformostsamplesandstill remains one of the major time-consuming steps in most analyses. Itcan be as simple as dissolution of a desired weight of sample in a solventprior to analysis, utilizing appropriate instrumental techniques de-scribedinthisbook. However, itcanentail anumberof procedures,which are listed below, to remove the undesirable materials [1] (also seeSection2.3below):1. Removalofinsolublecomponentsbyltrationorcentrifugation2. Precipitationofundesirablecomponentsfollowedbyltrationorcentrifugation3. Liquidsolidextractionfollowedbyltrationorcentrifugation4. Liquidliquidextraction5. Ultraltration6. Ion-pairextraction7. Derivatization8. Complexformation9. Freeze-drying10. Solid-phaseextraction11. Ionexchange12. Useofspecializedcolumns13. Preconcentration14. Precolumnswitching15. MiscellaneouscleanupproceduresTheapplicationof roboticstoenhancelaboratoryproductivityhasmet with some success in laboratories with high-volume repetitive rou-tineassays. However, therequirementsforextensiveinstallation, de-velopment,validation,andcontinuingmaintenanceefforthaveslowedComprehensiveAnalyticalChemistry47S.AhujaandN.Jespersen(Eds)Volume47ISSN:0166-526XDOI:10.1016/S0166-526X(06)47002-1r 2006ElsevierB.V.Allrightsreserved. 15thegrowthofroboticsinpharmaceuticallaboratories[2].ManyrobotsinpharmaceuticalR&Dlabshavebeenabandonedbecausetheywerenot sufciently exible or reliable to automate the varying applicationsto warrant the continuing upkeep. The ideal dream of full automation,whereonesimplyinputssamples(tablets, capsules, orotherpharma-ceutical products) and receives viable data with absolute reliability andwithoutfrequenttroubleshooting, isgenerallynotrealizedbylabora-toryrobotics.Thischapterpresentsanoverviewof thevariousmethodsforcol-lecting and preparing samples for analysis, from classical to more mod-ern techniques. We provide below a general overview, outlining some ofthetheoryandpracticeofeachtechnique.Ourfocusismainlyontheanalysis of organic compounds of interest (analytes) inavarietyofmatrices, such as environment, food, and pharmaceuticals. For furtherreading, the analysts are referred to a more detailed discussion of thesetechniquesinvarioustextbooksandkeyreferences[37].2.2 SAMPLINGSuccessful quantitativeanalysisstartswiththesamplecollectionandendswiththeinterpretationofthedatacollectedonthem.Dependingonthenatureofthematrix, varioussamplecollectiontechniquesareemployed. Thesampleshouldberepresentativeofthebulkmaterial.Forexample, withenvironmental samples, whichincludesoil, water,and air, the rst important consideration of representative sampling isrightatthepointofcollectionbecauseyouneedtoconsiderintrinsicheterogeneityofmostmaterials[8].Distributionofmineralsinearth,dispersionofleachatesfromtoxicwaste intoouraquifers,dischargeofdamagingchemicals intostreams andrivers, andthebuildupof airpollutants inurban areasall present complexsamplingchallenges andrequiresample-specicstrategies toobtainclearandunbiasedover-views. Theseconsiderationsbecomeexceedinglyimportant wheneveryou are performing analysis of trace and ultratrace components[1,9,10].Letusconsideradifcultexampleofsamplingofminedmaterials(sand, dolomite, and limestone) used for the manufacture of glass. Thematerials are delivered in railroad cars; each may contain up to 2 tons ofmaterialandaresusceptibletoverticalseparationonthebasisofsizeand density of particles because of vibrations during transportation [8].To obtainarepresentative sample, samplingis done withahollowS.AhujaandD.Diehl16conicalstainlesssteelspear,onaverticalbasis,whichcollectsapprox-imately500geachtime. Thesampleiscollectedatsixdifferentloca-tions per railroad car. This means a total of 3 kg sample is collected perrailroad car, so you can imagine the magnitude of sample size you mayhave at hand if you multiply this by the number of railroad cars. Enor-mous difculty will be encountered to select arepresentative sample ifyou were to analyze 0.5 g portion(s) of the bulk sample. Clearly, it wouldbe necessary to homogenize the sample thoroughly if the analysis of thecompositewastheobjective(seediscussionbelow).2.2.1 SamplesizereductionParticlesizereductionisnecessarytoassurethatvariousmaterialsinthe samplehaveroughlythe same particle size[11]. Evenafter theparticlesizehasbeenmadereasonablyuniform,thesamplesizehastobe reduced to a manageable amount that can be analyzed, e.g., 0.5g froma bulk sample of several kilograms in the example discussed above. Someof theapproachesthathavebeenusedfollow: coningandquartering,proportionaldivider,andstraight-linesampler(fordetailsseeRef.[8]).It is important todivide thesamplewithout discriminationwithrespect tosize, density, or anyother physical characteristics of theparticles.2.2.2 SamplingerrorTherelationshipbetweenthesamplingerrorandparticlesizecanbeseeninFig. 2.1. Variouslinesinthatshowtheerrorrelatingtotheamountsampledineachcase. Itisclearthatalargersamplesizere-duceserrorsignicantly; however, itisnotlogical toworkwithverylarge quantities. The sampling error for the normal sample for analysis,whichis likelyto be anywhere between0.1%and1%of the wholesample, can be seen in Fig. 2.1. The sampling error can be minimized bygrindingtheparticlesdowntonesize, assumingtherearenoissuesrelatingtothestabilityoftheanalytes.Tominimize samplingerrors inthe pharmaceutical industry, aneffective quality assurance approach utilizes sampling at various stagesofthemanufacturingprocesstoprovideabetterindicationofthetruecontent of aproduct. Let us reviewanexampleof samplingtablets(drug product) for potency analysis; i.e., amount of the active ingredientpresent ineachtablet. Abatchof tablets cancontainasmanyasamillion or more tablets made with a high-speed tablet-punchingSamplingandsamplepreparation17machine. Since a relatively small number of tablets are nally tested topassorfail abatch, several testingstepsarebuiltintoreducevari-ationsthatareduetosamplinguncertaintyandthusassurequalityofthebatch. Priortomanufacturingabatchoftablets, theactivephar-maceutical ingredient(API)ismixedcarefullywiththeinertingredi-ents(excipients)toproduceagranulationthatensuresthatthe APIisuniformly dispersed and that it would become more readily available toreachthebloodstreamwhenadministeredtopatients. Duringthede-velopment phase, tests are conducted at various intervals in the mixingprocesstoassureoptimummixingtime. Duringproduction,thegran-ulationistestedtoassurethatreliablemixinghasbeenaccomplishedforeachbatchbeforetabletsaremanufactured. Thetabletsareman-ufactured with a tablet-punching machine, where an individualweighedportionispunchedintoanindividual tablet. Theweight oftheAPIandthe weightofthe tabletcaninuence theaccuracyofthisprocess. Tabletsaresampledatvariousportionsoftheruntoassurethatthetablet-punchingmachineisindeedproducinguniformtabletsinterms of dosage of API. The nal batchneeds tobe sampledtorepresent the whole batch. This sample is drawn from various portionsofthewholebatchtoassurefairrepresentationofthebatch. Anan-alyticalchemistthenselects20tablets(acompositesample),inaran-dom manner, from the bulk sample. The composite sample is powderedtoproduceauniformpowderfromwhichaportionisdrawnfortheanalysis. Duplicateanalysesaregenerallycarriedouttoarriveatthepotencyofthebatch.Fig. 2.1.Plot that shows the relationship between sample error, the number ofparticles in a sample, and different percentages of original material sampled [8].S.AhujaandD.Diehl18Toassuretablet uniformityforexample, theUnitedStatesPhar-macopoeia requires that a sample size of 30 tablets be tested as follows:Test 10 tablets: the batch passes if no tablet is outside of plus or minus15% of specied limits; if one tablet out of 10 is outside 15% of speciedlimits, then 20 more tablets are tested; and no more than 2 tablets canbeoutsidetheplusorminus25%ofthespeciedlimits.Table 2.1 demonstrates howexcellent dosage uniformity canbeachieved[12] forTofranil tablets(10, 25, and50 mg) andPertofranecapsules(10, 25, and50 mg). Of the730individual tabletsanalyzed,over 90% were within 10% of indicated dosage, and all except one tablet(Tofranil 10 mg) were within 15% of the indicated dosage. And all werewithinthe25%limits. Of the590capsulesanalyzed, all werewithin15%oftheindicateddosage.This example demonstrates howvarious testingsteps canassurethat representative results can be obtained, even with a relatively smallsamplesize,especiallywhenstepshavebeentakentoassurethatthewholebatchisreasonablyuniform.Itisimportanttorememberthatthecondenceinournalresultsdependsbothonthesizeofoursample, whichadequatelyrepresentsthe whole batch, and steps that have been taken to assure uniformity ofthe whole batch. In addition, the magnitude of interfering effectsshouldbeconsidered.One should never forget that all sampling investigations are subjecttoexperimentalerror.Carefulconsiderationofsamplingcankeepthiserror toaminimum. Toassurevalidsampling, major considerationshouldbegiventothefollowing[13]:1. Determination of the population or the bulk from which the sampleisdrawn.TABLE2.1VariationofdosageofPertofranecapsulesandTofraniltabletsa%Tabletsorcapsuleswithinindicateddosage72% 75% 710% 715%Dosage(mg) Tablets Capsules Tablets Capsules Tablets Capsules Tablets Capsules10 27.7 32.5 57.4 65.0 90.3 96.7 99.4 100.025 38.1 49.7 79.6 67.8 99.2 94.1 100.0 100.050 45.2 42.0 82.9 88.7 99.0 100.0 100.0 100.0aAdaptedfromRef.[12].Samplingandsamplepreparation192. Procurementofavalidgrosssample.3. Reductionofthegrosssampletoasuitablesampleforanalysis.It is desirable to reduce the analytical uncertainty to one-third or lessofsamplinguncertainty[14]. Interestedreadersshouldconsidersam-plingtheory[15].2.3 SAMPLEPREPARATIONAsurveyontrendsofsamplepreparation(SP)revealedthatpharma-ceuticalandenvironmentalanalystsaremorelikelytoemploysamplepreparationtechniques[16]. Figure2.2showsvarioussampleprepa-rationstepsthatmaybeemployedinsamplepreparation. Mostana-lystsuse1to4stepsforsamplepreparation; however, somecanusemore than 7. Interestingly, a large number of analysts have to performanalysesofcomponentsintheparts-per-millionrangeorbelow.Asdiscussedabove, afterthesamplehasbeencollected, adecisionhastobetakenastowhethertheentiresampleoraportionof thesampleneedstobeanalyzed.Forlow-levelanalytes,theentiresampleis often utilized. When the concentration of the analyteis suspected tobewellaboveadetectionlimit, thenaportionofthecollectedsamplecanbeused. Thesamplemustbehomogenoussothatthedatagen-erated from the subsample are representative of the entire sample. Forexample, asoil sample may need to be sieved, large pieces ground, andthenmixedforthesampletobehomogenous.Thenextcrucial portionof theprocessisthesamplepreparation,which allows the analyte to be detected at a level that is appropriate fordetectionbytheinstrument. Thesamplemustbepreparedinsuchawaythatthematrixdoesnotinterferewiththedetectionandmeas-urement of the analytes. Often, this requires complete separation of thesamplematrixfromtheanalyte(s)ofinterest.Inothercases,theana-lytes can be measured in situ or rst derivatized and then measured insitu. Both the nature of the analyte and the matrix dictate the choice ofsamplepreparation(seeRef.[1]).Table 2.2 shows a comparison of various extraction methods for solidsamples [17]. It appears that one can take anywhere from 0.1 to 24 h forthe extraction process. Microwave-assisted sample preparation requiresminimaltime;however,ifcostisaconsideration,Soxhletextractionisleastcostlybutrequiresthelongestsamplepreparationtime.S.AhujaandD.Diehl20Fig.2.2.Samplepreparationprocedurescommonlyused[16].Samplingandsamplepreparation21TABLE2.2ComparisonofextractionmethodsforsolidsamplepreparationaMethod Samplesize(g)Solventvolume(ml)Temperature(1C)Typicaloperatingpressure(psi)Timeb(h) AutomationlevelNumberofsamplescCostdSonication 2050 100300 Ambient40 Atmospheric 0.51.0 None 1(serial),many(batch)LowTraditionalSoxhlet1020 200500 40100 Atmospheric 1224 None 1(serial) VerylowModernSoxhlet1020 50100 40100 Atmospheric 14 Mostly 6(batch) ModerateSFE 510 1020e50150 20004000 0.51.0 Fully 44(serial) HighPressurizeduid130 1045 50200 15002000 0.20.3 Fully 24(serial),6(batch)HighClosed-vesselmicrowave-assisted25 30 100200 15002000 0.10.2 Mostly 12(batch) ModerateOpen-vesselmicrowave-assisted210 2030 Ambient Atmospheric 0.10.2 Mostly 6(batch) ModerateaAdaptedfromRef.[17].bTotalprocessingtimepersamplefromweighingtocollection.cMaximumnumberofsamplesthatcommercialinstrumentscanhandle;serialmeansonesampleprocessedatatime,andbatchmeansmultiplesamplesatatime.dVerylow lessthan$1000;low lessthan$10,000;moderate $10,00020,000;high morethan20,000.eSolventvolumewhenorganicmodierisusedtoaffectpolarity.S.AhujaandD.Diehl222.3.1 TypesofsamplesOur focus in this chapter is on the analysis of organic analytes in sam-ple matrices that are organic and/or inorganic in nature. These organicanalytescanbesubclassiedintovolatile,semivolatile,ornonvolatile.Thematrixcanbegas(orvolatilesamples), solid, orliquid. Boththeanticipated concentration of the analyte and the type of sample dictatethe instrumentation that can be used, as well as the sample preparationtechniquerequired.2.3.1.1 VolatilesamplesVolatilesamplesareusuallyanalyzedbygaschromatography. Therearemanydifferentsamplepretreatmentmethodsforgases.Letusre-viewthesetechniques:GrabsamplingAgaseoussampleispulledintoanemptycontainersuchasametalcanister. Inthelaboratory, thesampleisoftenchilledtoisolatethevolatilecompounds. Thecontainermaythensimplyberinsedwithasolventtocapturethesecompounds. Thesolventcanthenbedirectlyinjectedintoasuitableinstrument foranalysis, suchas agas chro-matograph(GC).Solid-phasetrappingA gaseous sample is passed through a solid material, such as silica gelor polyurethane foam (PUF), in a tube. A glass ber lter is often put infrontofthesolidsupporttocaptureparticle-phaseconstituents,whilethevapor-phasecompoundsarecapturedonthesolidsupport.Thisisusedforsemivolatileanalytes, suchaspolycyclicaromatichydrocar-bonsandpesticides.Thesolidsupportisthenusuallyextractedinthelabwithasolvent(seetechniquesdescribedlaterinthischapter),andthenthetechniquesusedforliquidsamplesarefollowed.LiquidtrappingInthiscase,thegasisbubbledthroughaliquidthathasanafnityfortheanalyteofinterest.Afteracertainperiodoftime,theliquidisthenanalyzed,oftendirectlybyinjectionintoaGC.Head-spacesamplingThe solid or liquid sample is placed into a glass vial, which is heated,withenoughemptyspaceinthevial abovethesampletoallowtheanalyte to reachequilibriumbetweenthe gas and solid (or liquid)phase. The gas phase is sampled and analyzed, usually by GC. This typeof samplingisgenerallyusedforanalyzingtraceamountsof volatilesamples.Samplingandsamplepreparation23PurgeandtrapThistechniqueissimilartohead-spacesamplinginwhichthesam-pleisplacedinaheatedvial.However,thehead-spacevaporsarecon-tinually removed by a ow of inert gas. This gas is then trapped eitheron a solid support or is cooled and then thermally desorbed into the GC.This procedure is used preferably over head-space sampling if theanalytesareinlowconcentrationorhaveunfavorablepartitioncoef-cients. An interesting application of this technique is in the area of foodqualitycontrolspecicallyinthewineindustry[18]. 2,4,6-Trichlo-roanisole(TCA)istheanalyteresponsibleforcorktaintinbottlesofwine. Inthisresearch, samplesofwineandcorkwereextractedwithpentane, rotary evaporated, and reconstituted in an aqueous solution ofsodiumchlorideandsulfuricacid, andthensubmittedtopurgeandtrap,withgaschromatographicanalysis.Anothermethodthatutilizespurge and trap is the analysis of volatile organic compounds in drinkingwater[19].Compoundsthatcontainmultiplehalogenssuchasdichlo-robromomethanearepotentiallyharmfultohumans.Inthisresearch,heliumwaspassedthroughwatersamplestostripout theanalytes,whichweretrappedinacapillarytrapandthermallydesorbedinapurgeandtrapsystem.2.3.1.2 SolidsamplesSolidsamples oftenrequiremorecreativityinpreparation. Theap-proachof dissolve, dilute, andshoot(inject intoachromatograph) isquitecommonfortheanalysisofdrugsubstances. Inadrugproduct(tabletsorcapsules) whereAPIcanbeeasilysolubilized, theprocesscanbereducedtodissolve,lter,andshootaswell.However,fordrugproducts, a more elaborate process of grind, extract, dilute, and lter isgenerallyemployedtoextracttheAPIfromthetablets(asolidmatrixcomposedofinactiveingredients). Ashighaccuracyandprecisionaremandatoryinassaysfordrugproducts, volumetricasks(25500ml)areusedtoattainverypreciseandsufcientvolumesforsolubilizingthe API in the case of the tablets. This approach is straightforward forimmediate-releasedosageformsbutcanbechallengingforcontrolled-releaseproductsandformulationsoflow-solubilityAPIs[2].2.3.1.2.1 PretreatmentstepsBefore many of the techniques forprocessing solid samples can be em-ployed,thesolidsamplemustbehomogenized. Therearemanyphys-icaltechniquestoaccomplishthisgoal.First,grinding,suchaswithamortarand pestleor withamechanicalgrinderputs the sampleintoaS.AhujaandD.Diehl24neparticulatestate,makingtheextractiontechniquesmoreeffectiveandmoreefcient. However, ifthermallylabileanalytesarepresent,caremust betakentoavoidheatinginducedbyfriction. For softersamples, ball milling is the preferred grinding method. In this case, thesampleisplacedinacontainer; porcelain, steel, orsomeotherinertmaterialisaddedtothecontainer, andtheentiredeviceisshakenorrotated.Forverystickysamples,liquidnitrogenmaybeusedtoforcethesampleintoasolidbrittlestate.An additional step must be taken for samples that are wet, such as asoilsampleorplanttissue. Theamountofwaterpresentinasamplecanaffectthetotalreportedconcentration.Therefore,samplesareof-ten analyzed for water content before analysis. In this case, samples areweighed, dried (usually in an oven), and then reweighed. Care must betaken if the analytes are volatile or if the sample may decompose underheating.More elaborate sample preparation is often needed for complexsample matrices, e.g., lotions and creams. Many newer SP technologiessuch as solid-phase extraction (SPE), supercritical uid extraction(SFE), pressurized uid extraction, accelerated solvent extraction(ASE), and robotics are frequently utilized (see Ref. [2]). Dosage formssuch as suppositories, lotions, or creams containing a preponderance ofhydrophobic matrices might require more elaborate SPandsamplecleanup,suchasSPEorliquidliquidextraction.Forsamplesthataremorecomplex, sometypeofliquid-extractionprocesstoremovetheanalytesof interestmayberequired. Manyofthesetechniques have beenaroundfor over 100years. These tech-niquesarewidelyacceptedbyvariousglobalregulatoryagencies,suchastheEPAintheUSA, andthereforehavenot changedmuchovertime. Someimprovements toreducetheamount of organicsolventsusedhavebeenmadeandaredetailedbelow:Solid liquidextractionThis is one of the simplest extraction methods. The sample is placedin a vessel, and the solvent is added to dissolve the sample components.Thecontainerisshakenforagivenperiodof time, andthenthein-solublecomponents areremovedbylteringor centrifugation. Thisprocess works well if the analytes are readily soluble in the solvent andnot tightlyboundtothematrix. Thesampleisoftenheatedtoboil/reuxtospeeduptheprocessifthermallabilityisnotaproblem.Solution solventextractionTheanalytesof interest aresolubilizedinaqueoussolvent at thedesiredpHandextractedwithanorganicsolvent(seeSection2.3.1.3Samplingandsamplepreparation25formoredetailsonliquidliquidextraction). Letusreviewaparticu-larlychallengingsamplepreparationtechniqueforwater-solublevita-minsfrommultivitamintablets, whichentailsdiversityofanalytesofvariedhydrophobicities andpKa. Water-solublevitamins (WSVs) in-cludeascorbicacid(vitaminC), niacin, niacinamide, pyridoxine(vita-minB-6), thiamine(vitaminB-1), folicacid, riboavin(vitaminB-2),and others [20]. While most WSVs are highly water soluble, riboavin isquitehydrophobic andinsolubleinwater. Folic acidis acidic, whilepyridoxineandthiaminearebasic. Inaddition, ascorbicacidislightsensitive and easily oxidized. The SP for fat-soluble vitamins (FSVs) A,E, and D presents a different set of challenges. The extraction strategyemploys a two-step approach, using mixed solvents of different polarityandacidityasfollows. Atwo-phaseliquidliquidextractionsystemisused to isolate the FSV into hexane, while retaining other analytes andexcipientsintheaqueouslayer. Acappedcentrifugetesttuberatherthanaseparatoryfunnelisusedtominimizeextractiontimeandsol-vent volumes. Thenal hexanesolutionisinjecteddirectlyintotheHPLCsystemwithamobilephaseconsistingof85%methanol/water.Chromatographic anomalies frominjecting a stronger solvent areavoidedbyreducingtheinjectionvolumeto5 ml.SonicationSonicationhelpsimprovesolidliquidextractions. Usuallyanelyground sample is covered with solvent and placed in an ultrasonic bath.Theultrasonicactionfacilitatesdissolution, andtheheatingaidstheextraction. TherearemanyEPAmethodsforsolidssuchassoilsandsludgesthatusesonicationforextraction. Thetypeofsolventusedisdeterminedbythenatureof theanalytes. This techniqueis still inwidespread use because of its simplicity and good extraction efciency.Forexample, inresearchtodeterminetheamountofpesticideinairafterapplicationtoricepaddysystems, airsamplescollectedonPUFwere extracted by sonication, using acetone as the solvent. The extrac-tionrecoverieswerebetween92%and103%[21].SoxhletextractionThis extractionmethodremains themost widelyusedmethodofextractionforsolidsamples.Thesampleisplacedinathimble,whichsitsinsidetheSoxhletapparatus. Aconstantreuxingof thesolventcausestheliquidtodripontopofthesampleandleachthroughthethimble(takinganalytewithit).Thedesignofthesystemissuchthatonce the liquid reaches a certain level, a siphoning mechanism returnsthe liquid to the boiling vessel. The liquid continues boiling andreuxingfor1824 h. Whilethisisaslowprocess, oncethesetupisS.AhujaandD.Diehl26complete, the operator can walk away and return to it at the end of theoperation.Oneoftherequirementsofthisapproachisthattheanalytesmustbe stable at the boiling point of the solvent, since the analytes collect intheask. Thesolvent mustshowhighsolubilityfortheanalyteandnoneforthesamplematrix.Sincethisisoneoftheoldestmethodsofsamplepreparation, therearehundredsof publishedmethodsforallkindsofanalytesinasmanymatrices.Forexample,XAD-2resin(sty-rene-divinylbenzene) thatwasusedtocollectairsamplestomonitorcurrent usage of pesticides in Iowa was Soxhlet-extracted for 24 h withhexane/acetone [22]. This is common in environmental sample analysis,andone will see rows androws of these systems inenvironmentallaboratories. OnedisadvantageofSoxhletextractionistheamountofsolventconsumed, thoughmodernapproachestosolidsampleextrac-tionhavetriedtoreducetheamountofsolventused.Inrecent years, several modications toSoxhlet extractions havebeendeveloped.Twosuchmethodsareautomatedfocusedmicrowave-assistedSoxhlet extraction(FMASE) [23,24] andultrasound-assistedSoxhlet extraction [25]. In one experiment with the FMASE setup [23],thesamplecartridgeisirradiatedbymicrowaves, allowingforfasterextraction. Soil samplescontaminatedwithpolychlorinatedbiphenylswereextractedbyFMASEandconventional Soxhlet extraction. TheFMASEmethodproduceddatathatareasaccurateandpreciseastheconventionalmethod.Thebenetsofthisnewermethodarethetime-savings(70 minversus24 h) andtherecyclingof 75%of theorganicextractionsolvent. Similarly, utilizingultrasound-assistedSoxhletex-traction[25] for determiningthetotal fat content inseeds, suchassunowerandsoybeans, resultedindatacomparabletoconventionalSoxhlet extraction, with RSDs less than 1.5%, and with extraction timesof90 minversus12 h.Acceleratedsolventextraction(ASE)Inthistechnique,organicsolventsathightemperatureunderpres-sureareusedtoextractanalytes. Theprocessconsistsofplacingthesample in a stainless-steel vessel, introducing the solvent, pressurizingthe vessels, heating of the sample cell under constant pressure, extrac-tion,transferringtheextracttoasealedvialwithafreshwashofthesolidsample, nitrogenpurge of the cell, andthenloadingthe nextsample. Withcommercial systems, thisprocessisall automated. An-other advantage of this system is the reduction in both extraction times(1020 min versus 1824 h) and solvent usage (20 ml versus 150500 ml)from a traditional Soxhlet extraction. The US EPA has certied an ASESamplingandsamplepreparation27extraction method for the extraction of polychlorinated biphenyls,PAHs, organochloride and organophosphorus pesticides fromsoil,Method3545[26].Supercriticaluidextraction(SFE)The popularity of this extraction method ebbs and ows as the yearsgoby. SFEistypicallyusedtoextract nonpolartomoderatelypolaranalytes fromsolid samples, especially in the environmental, foodsafety, andpolymersciences. Thesampleisplacedinaspecial vesselandasupercriticalgassuchasCO2ispassedthroughthesample.Theextractedanalyteisthencollectedinsolventoronasorbent. Thead-vantages of this technique include better diffusivity and low viscosity ofsupercritical uids, which allow more selective extractions.One recentapplicationof SFEistheextractionof pesticideresiduesfromhoney[27]. In this research, liquidliquid extraction with hexane/acetone wastermed the conventional method. Honey was lyophilized and thenmixed with acetone and acetonitrile in the SFE cell. Parameters such astemperature, pressure, andextractiontimewereoptimized. There-searchersfoundthat SFEresultedinbetterprecision(lessthan6%RSD),lesssolventconsumption,lesssamplehandling,andafasterex-tractionthantheliquidliquidmethod[27].SFE may also be coupled to GC and HPLC systems [28] for a simpleon-line extraction and analysis system. Lycopene is determined in foodproductssuchastomatoesandtomatoproducts,usingSFEcoupledtoHPLCwithanHPLCcolumnusedfor trapping and analysis. Themethod is short, requires small sample amounts, and has good linearityand sensitivity. Because the entire systemis closed, there is littlechanceforthelycopenetodegrade.Microwave-assistedextraction(MAE)Most averagehomescontainamicrowaveoven. Researchershavetakenthatsametechnologyandappliedittosampleextraction.Usingmicrowave technology with acidic solutions has become a commonplacereplacement for traditional aciddigests. The sample is placedinaclosed,chemical-resistantvesselandheatedinamicrowave.Thispro-cessismuchfasterthanhot-platetechniquesandhasbecomewidelyacceptedbysuchagenciesastheUSEPA.Combiningmicrowaveheatingwithsolvent extraction(MAE) hasgainedstatusinthescienticcommunity. Compoundsabsorbmicro-waveenergyapproximatelyinproportiontotheirdielectricconstants:the higher the constant, the higher the level of absorptionof themicrowaveenergy. TherearetwowaysofrunningMAE: usingami-crowave-absorbingsolventwithahighdielectricconstantoruseof aS.AhujaandD.Diehl28non-microwaveabsorbingsolventoflowdielectricconstant. Withthesolvent that absorbs microwave energy, the solvent and sample are in aclosedvessel.Thiscausesthesolventtoheattoaboveitsboilingpointandforcesaquickextractionundermoderatepressure. Inthenon-microwaveabsorbingapproach,thesampleandsolventareinanopenvessel.Inthiscase,thesampleitselfabsorbstheenergyandheatsup.Theanalytesarereleasedfromthehotsampleintothecoolsurround-ingliquid.TheadvantagesofMAEareshortextractiontimes(10 min),extrac-tion ofmany samples atone time(up to 14,depending onthe system),and less organicsolvent consumed. Inone recentstudy [29],MAE wasusedtoextractpaclitaxel fromIranianyewtrees. Theneedlesof thetreewereair-driedandground. Theneedleswerecoveredwithmeth-anolwater and placed in the MAE apparatus. Extractions took916 min. TheextractswerelteredandanalyzedbyHPLC. Furtheroptimizationofthemethodresultedinlessthan10%RSDsforpreci-sionandgreaterthan87%recovery. TheoverallbenetsoftheMAEmethodarereducedextractiontimes(1520 minversus17 h),minimalsamplehandling,and7580%reductioninsolventconsumption[29].2.3.1.3 LiquidsamplesLiquidsamples, otherthanthosethatareinherentlyliquid, canarisefromthesolidsampleextractiontechniquesdescribedabove.Asmen-tioned previously, sometimes a simple dilute-and-shoot approach can beutilized, i.e., add solvent to the sample and then inject directly into theinstrument. Other times, evaporationof residual liquidcanbe uti-lizedthenthesampleiseitherdirectlyinjected, orif thesampleisevaporated to dryness, a new solvent can be added. Often, however, theresidual matrix causes interference and the following techniques can beemployedforfurthersamplecleanup.MicrodialysisAsemipermeablemembraneisplacedbetweentwoliquids,andtheanalytestransferfromoneliquidtotheother. ThistechniqueisusedforinvestigatingextracellularchemicaleventsaswellasforremovinglargeproteinsfrombiologicalsamplespriortoHPLCanalysis.LyophilizationAqueoussamplesarefrozen, andwaterisremovedbysublimationunder vacuum; this procedure is epecially good for nonvolatile analytes.FiltrationLiquidis passedthrough alterpaper or membraneto removesus-pendedsolids.Samplingandsamplepreparation29CentrifugationA liquid sample with solids is spun at high speed to force the solids tothe bottomof the vessel. The liquidis decantedandoftenfurtherprocessed.Liquid liquidextraction(LLE)Liquidliquid extraction is a form of solvent extraction in which thesolventsproducetwoimmiscibleliquidphases.Theseparationofana-lytesfromtheliquidmatrixoccurswhentheanalytepartitionsfromthe matrixliquid phase to the other. The partition of analytes betweenthe two phases is based on their solubilities when equilibriumisreached. Usually, oneof thephases is aqueous andtheother is animmiscible organic solvent. Large, bulky hydrophobic molecules like topartitionintoanorganicsolvent, whilepolarand/orioniccompoundsprefertheaqueousphase.Areviewof several classicequilibriumequationsisinorder. TheNernst distributionlawstates that aneutral species will distributebetweentwoimmisciblesolventswithaconstant ratioof concentra-tions.KD Co=CaqwhereKDisthedistributionconstant, Coistheconcentrationof theanalyte in the organic phase, and Caq is the concentration of the analytein the aqueous phase. Another equation that is more appropriate is thefractionofanalyteextracted,E,E CoVo=CoVo CaqVaq KDV=1 KDVwhere Vo is the volume of the organic layer, Vaq the volume of aqueousphase,andVthephaseratioVo=Vaq:Extractionisanequilibriumprocess,andthereforeaniteamountofsolutemightbeinbothphases,necessitatingotherprocessingstepsormanipulationofthechemicalequilibria.Theorganicsolventshouldpreferablybeinsolubleinwater,shouldbeofhighvolatilityforeasyremoval,andshouldhaveagreatafnityfor the analytes. There are several mechanisms for increasing KD suchaschangingtheorganicsolventsotheanalytemorereadilypartitionsintothatlayer, manipulationofthechargestatetomaketheanalyteunchargedandmoresolubleintheorganiclayer,orsaltingouttheanalytefromtheaqueouslayerbyaddinganinert,neutralsalttotheaqueouslayer.Theactualextractionisusuallycarriedoutinaseparatoryfunnel,with tens of milliliters of solvents. Most extractions require two to threeS.AhujaandD.Diehl30extractions, resulting in large volumes of solvent that must be removed,oftenbyrotaryevaporation. Evenwiththelargeamountsofsolventsgenerated, LLEisperceivedaseasytodoandhasbeenusedfortheextraction of acyclovir in human serum [30] and amphetamine in urine[31].Oneadvanceintheareaof LLEistheuseof solidsupportsthatfacilitatethepartitioningoftheanalyte(s)ofinterest. LLEextractionmethods involving nonpolar matrices often suffer from the formation ofemulsions, andusingthesolidsupport isapossiblesolution. Inonestudy, polychlorinatedbiphenyls, dioxins, andfurans wereextractedfrom the lipid fraction of human blood plasma [32], using diatomaceousearthasthesolidsupport. Longglasscolumns(30cm) werepackedwithseverallayersofChem-Elut(aVarianproduct)andsodiumchlo-ride. Theplasmasamples weredilutedwithwater andethanol andpassed over the columns. A mixture of isopropanol and hexane (2:3) waspassedoverthecolumnandtheLLEwasperformed. It canbecon-cluded that the LLE with the solid support is easier to perform and canbeappliedtootherlipidheavymatricessuchasmilk[32].2.4 SOLID-PHASEEXTRACTIONOneofthemostwidelyusedtechniquesacrossalldisciplinesissolid-phase extraction (SPE). A simple approach to SPE is shown in Fig. 2.3.Thesecolumnsproviderapidextraction,minimizeemulsionproblems,andeliminatemostsamplehandling.Fig.2.3.Rapidextractionwithsolid-phasecolumns.Samplingandsamplepreparation31Thebasictechniqueofremovingundesiredcomponentsfromaso-lutionwiththehelpofsolidshasbeenusedforsamplecleanupforalong time. The recoveries from a quick cleanup for waste solvents basedon a sample ltration through a Florisil and sodium sulfate column aregiven in Table 2.3. It can be seen from the data in this table that a largenumberofpesticidesareretainedbyFlorisil. Thesecanberecoveredforappropriateanalysis.Moresophisticatedmodicationshavebeendevelopedrelativelyre-cently to remove various matrix effects from the sample (see BiologicalMatricesinSection2.4). Forexample, asolidsorbent issandwichedbetween two frits in a barrel-type device. The particles are often irreg-ularly shaped particles of 2040 mm in diameter, which allows for betterow through the device. The same mechanisms that apply to HPLC alsoapplytoSPE. However, becauseof thelargeparticlediameters, theefciencyismuchlowerthaninHPLC.TABLE2.3FlorisilltrationefciencyaCompound Sample concentration(ppm)%RecoverybStandarddeviationAldrin 2 93 9.5BHC 2 86 6.1Lindane 2 92 13.5Chlordane 2 79 4.7DDD 2 69 16.0DDE 2 92 11.0DDT 2 89 7.2Dieldrin 2 88 3.5Endosulfan 2 91 7.6Endrin 2 98 7.6Heptachlor 2 97 2.9Toxaphene 3 90 16.5Arochlor1016 20 93 2.0Arochlor1221 20 95 6.4Arochlor1232 20 100 8.2Arochlor1242 20 93 8.3Arochlor1248 10 95 9.1Arochlor1254 10 86 9.7Arochlor1260 10 87 12.2aAdaptedfromPedersen,B.A.andHiggins,G.M.,LCGC,6(1988)1016.bAveragerecoveryfromtriplicateanalysis.S.AhujaandD.Diehl32SPEisaneleganttechniquebecauseofthevarietyofwaysthede-vices canbeused. Over half of theuses of SPEaretoremoveanyunwanted matrix fromthe analytes of interestin other words,separatetheanalytesfromeverythingelseinthesample. Theinter-ferences either pass throughthe sorbent unretainedor become ad-sorbed to the solid support. This technique has been used in just abouteveryapplicationareafrombioanalytical analysesinthepharmaceu-tical industrytoremoval of dyes fromfoodsamples totheremovalofhumicacidsfromsoilsamples. ThesecondmajorbenetofSPEistheconcentrationoftheanalytesintraceanalysis. Forexample, sev-eral liters of river water can be passed over a few grams of sorbenttheanalytes adsorb to the sorbent, to be later eluted with a small volume ofsolution. Other uses include solvent switching or phase exchange,inwhichthesampleisswitchedfromonetypeofsolventtoanother,often fromaqueous to organic; and solid-phase derivatization inwhich the SPEsorbent is coated with a special reagent, such asdinitrophenylhydrazine (DNPH). DNPH devices contain acidiedreagent DNPHcoatedonasilicasorbent. Airsamplesarecollected,and these SPEdevices are used for quantitationof aldehydes andketones by reactionto formthe hydrazone derivative. DNPH-silicais speciedinseveral EPAprocedures for the analysis of carbonylcompoundsinair.Besidesthebarrelcongurations,anexplosionofotherformatshasbecomeavailableovertheyears.Onetypeofdeviceincludesadiskormembranethatcontainsasmallamountofparticulatematerial. Thisformathastheadvantagethatlargeamountsofsamplecanbepassedoverthediskinmuchlesstimethaninlargerbedmasses. However,largeholdupvolumescanbeobserved. Tofacilitateautomated, high-throughput SPE, 96-well and384-well plates havebecomecommon-place in the pharmaceutical industry. Typically, 260 mg of sorbent arecontainedineachwellmakingfastSPEof biological uidssuchasurine andplasmapossible. Other advances inthe format include amicroelution plate design in which small volumes (25 ml) of solvent canbeusedtoeluteasample, aidinginsampleconcentrationandelimi-nationoftheneedtoevaporateandreconstitute.Therstreversed-phaseSPEsorbentswerebasedonsilicagelpar-ticles, similartotheparticlesusedinHPLC. AnumberofphasesareavailablerangingfromC8toC18toanion-andcation-exchangefunc-tionalities. Recentadvancesinparticletechnologyhaveincludedpol-ymeric materials that combine the benets of a water-wettable particletoretainpolaranalyteswithareversed-phase, hydrophobicmoietytoSamplingandsamplepreparation33retainlargebulkymolecules.Thesepolymersareavailablewithmanytypes of ion-exchange moieties, allowing for selective retention of acidsandbases.Thepractical applicationofSPEisrelativelystraightforward. Thesorbentisrstwashedwithorganicsolventtobothremoveanyresid-ual componentsremainingfrommanufacture(althoughtodaysSPEphasesareextensivelycleaned)andtoallowforproperwettingofthechromatographicsurface. Thisisthenfollowedbyequilibrationwithan aqueous solvent. The sample is then loaded onto the sorbent.Usually, vacuumisappliedtofacilitateowthroughthecartridgedependingonthenatureof thesamplepreparationstep, theanalyteeitherpassesthroughthecartridgewhilethematrixcomponentsareretained, ortheanalyteisretainedonthesorbent. Subsequentwashstepsarethenusedtolockon theanalytetothematerial. Forex-ample,ifabasicanalyteispassedthroughacation-exchangesorbent,an acid wash is used to ensure that the base is in its charged state so itcanstick tothecartridge. Further washsteps, oftenorganic sol-vents, arethenusedtoremovetheunwantedanalytesfromthesor-bent. Anal elutionstepof anorganic solvent, sometimes withanacidic or basic modier, is used to elute the analyte of interest from thesorbent.MethoddevelopmentinSPEbeginswithananalysisofthesamplematrixaswellasthesampleanalytes.Oneofthemostefcientmech-anisms of SPEis to use mixed-mode ionexchange sorbents. If theanalytesarebases,cationexchangeisthemostselectiveSPEmode.Ifthe analytes are acids, anion exhange is the most efcient. However, itissometimeseasiertocapturethematrixcomponentsandallowtheanalytestopassthroughthesorbentunretained.Solid-phasemicroextraction(SPME)Inthe1990s, Pawliszyn[3]developedarapid, simple, andsolvent-freeextractiontechniquetermedsolid-phasemicroextraction. Inthistechnique,afused-silicaberiscoatedwithapolymerthatallowsforfastmasstransferbothintheadsorptionanddesorptionofanalytes.SPMEcoupledwithGC/MShasbeenusedtodetectexplosiveresiduesin seawater and sediments from Hawaii [33]. Various bers coated withcarbowax/divinylbenzene, polydimethylsiloxane/divinylbenzene, andpolyacrylateareused. TheSPMEdevices aresimplyimmersedintothewatersamples.Thesedimentsamplesarerstsonicatedwithace-tonitrile,evaporated,andreconstitutedinwater,andthensampledbySPME. The device is then inserted into the injection port of the GC/MSsystemandtheanalytes thermallydesorbedfromtheber. VariousS.AhujaandD.Diehl34parameterssuchasdesorptiontemperature, sampleadsorptiontime,andpolymercoatinghavebeenevaluated.SPMEhas alsobeenusedintheareaof head-space samplingofvolatile compounds suchas avors andfragrances [34,35]. Intheseexperiments, theanalytesareadsorbedontothepolymer-coatedberthat isincontact withtheheadspaceinthevial. TheberistheninsertedintotheinjectionportofaGCandthermallydesorbed.BiologicalmatricesProteinprecipitationisusedroutinelyinbioanalyticallaboratoriesin the pharmaceutical industry. Plasma is mixed with an excess (3 to 5times) of organic solvent (typically acetonitrile or methanol) or an acidsuch as formic acid. The proteins precipitate out of solution, the sampleiscentrifuged, andthesupernatantisanalyzed. Whilethismethodisrelatively fast and simple, the extract contains salts and lipids that caninterferewithsubsequentanalyses.Often,asecondtechniquesuchasSPE is used for further cleanup. Table 2.4 exhibits various samples thatTABLE2.4Samplesanalyzedbysolid-phaseextractionaSample Matrix Solid-phasecolumnDetectability(ng/ml)3-Methoxy-4-hydroxyphenylglycol Plasma Alumina 110Oxytetracycline Fishtissues C-8 510bDoxefazepam Plasma C-18 0.1cCortisol Urine C-18Basicdrugs Plasma CN TherapeuticlevelsD9-Tetrahydrocannabinol Plasma C-18 2,100pgIbuprofen Plasma C-2 1.3cRanitidine Plasma, otheruidsCN 2Chlorpromazineandmetabolites Plasma C-8Cyclotrimethylenetrinitramine BiologicaluidsC-18Sotalol Plasma,urineC-8 10CyclosporinA Serum,urine CyanopropylCyclosporine Blood C-18 10Growthfactors Urine C-1 2001400-foldenrichmentCarbamazineandmetabolites Plasma C-18 50aAdaptedfromRef.[10].bValueisinng/g.cValueisin mm/ml.Samplingandsamplepreparation35canbeanalyzedbySPEfromdifferentmatrices. Thetypeofsamplesincludesurine,plasma,blood,variousbiologicaluids,andshtissue.Thedetectabilitylevelsareinng/mlrange.Threeimportantconcernsdrivesamplepreparationfrombiologicalmatrices[36]:

removinginterferences,

concentratinganalyte(s)ofinterest,and

improvinganalyticalsystemperformance.InnovativeSPEtechnologyemployingmicroelutionplatedesignisshown in Fig. 2.4. Tip design of the well plate provides a smaller surfaceFig. 2.4.Schematicofthemicroelutionplatedesign.Tipdesignofthe96-wellplateaffords asmaller surfaceareaandlarger depth, morelikeanHPLCcolumn, andpermitsgoodowthroughtheplateandlowholdupvolumeontheorderofnanoliters[36].Fig.2.5.Typesofmicropipettetips[37].S.AhujaandD.Diehl36areaandalargerdepthsimilartoanHPLCcolumnandpermitsgoodowthroughtheplate withlowholdupvolume. This designallowsutilizationof volumes as lowas 25 ml. Higher sensitivity has beenachievedforanalysisofamitriptyline-spikedplasmasamplesat0.1 ng/mlthanwithprotein-precipitatedsamples.TABLE2.5CommonstrategiestoacceleratesamplepreparationaStrategy AimSpecicdetection Avoidresponseofunwantedorincreasedresponseoftargetanalyte(s)Largevolumeinjection MiniaturizationofsamplepreparationAutomation BulkprocessingCombinedanalysis EfciencyimprovementaAdaptedfromRef.[38].Fig. 2.6. Chromatogramshowingthe simultaneous analysis of 16PAHs (A), 7chlorobenzenes (B), 10 organochlorine pesticides (C), and 7 PCBs (D). Column:RapidMS (10 ml 530 mm i.d., 0.12 mm df, Varian, The Netherlands. GC oven:501C(1 min) to1901C(3 min) at301C/minto2801Cat401C/min. MS: time-selectedSIMmodewithdual ionconrmation. Analyteconcentrationlevelswerebetween50and175ppb[38].Samplingandsamplepreparation37The micropipette tip containing solid phases is a relatively newsamplepreparationtechniquethat permitshandlingof microlitertosubmicroliteramountsofliquidsamples,usingthetechniquesofSPE,dialysis, andenzymedigestion. Variousphases(reversed-phase, afn-ity, size-exclusion, etc.) are packed, embedded, or coated on the walls ofpipette, permitting liquidsamples to be transferredwithout unduepressuredroporplugging(Fig.2.5).2.5 ENHANCEDSAMPLETHROUGHPUTSomecommonthroughputenhancementstrategiesforenvironmentalsamplesaregiveninTable2.5(fordetails,seeRef.[38]).Theproblemsrelatingtoincreasedcontaminationlevelsand/orin-sufcient sensitivity may be overcome by using matrixsolid phase dis-persion, MSdetectioninselectedionmonitoring(SIM) mode, and/orlargevolumeinjection. AnexampleofcombinedanalysisthatutilizesspecicdetectionisshowninFig.2.6.Itentailssimultaneous analysesofPAHs,PCBs,chlorobenzene,andorganochlorinepesticidesinsoil.2.6 SUMMARYANDCONCLUSIONSA variety of methods for the sample collection and preparation of gases,solids, andliquidshavebeencoveredinthischapter. Eachtechniquediscussedinvolvesseparatingtheanalytesofinterestfromthecompo-nents of the matrix. Selection of the best sample preparation techniqueinvolvesunderstandingthephysical andchemical propertiesof boththeanalytesandthematrix. Whilemanyofthetechniquesdiscussedhere have been utilized over the years, advances are being continuouslymade. A careful reading of the available methodologies is recommendedpriortoselectingasuitablemethodology.REFERENCES1 S. Ahuja, Trace and Ultratrace Analysis by HPLC, Wiley, New York, 1992.2 S. Ahuja and M.W. Dong, Handbook of Pharmaceutical Analysis by HPLC,Elsevier,Amsterdam,TheNetherlands,2005.3 J. Pawliszyn, Sampling and Sample Preparation for Field and LaboratorySamples,Elsevier,Amsterdam,TheNetherlands,2002.4 S. Mitra, Sample Preparation Techniquesin Analytical Chemistry, Wiley,NewYork,2003.S.AhujaandD.Diehl385 T.C. Lo, M.H.I. BairdandC. Hanson, Handbookof Solvent Extraction,KriegerPublishing,Melbourne,FL,1991.6 D.A.Wells,HighThroughputBioanalyticalSamplePreparation:MethodandAutomationStrategies,Elsevier,TheNetherlands,2003.7 R.E.Majors,LCGC,13(1995)742.8 R.F.Cross,LCGC,18(2000)468.9 S. Ahuja, UltratraceAnalysisofPharmaceuticalsandOtherCompoundsofInterest,Wiley,NewYork,1986.10 S. Ahuja, Trace andResidue Analysis inKirk-Othmer EncyclopediaofChemicalTechnology,Wiley,NewYork,1997.11 R.E.Majors,LCGC,16(1998)438.12 S. Ahuja, Study on Dosage Variations on Individual Capsules and Tabletsof Desipramine and Imipramine Hydrochloride. Presented at A.Ph.A.Meeting,Miami,FL,May,1968;J.Pharm.Sci.57(11) (1968)19791982.13 B.KratochvilandJ.K.Taylor,Anal.Chem.,53(1981)924A.14 W.J.Youden,J.Assoc.Off.Anal.Chem.,50(1967)1007.15 P.M.Gy,LCGC,12(1994)808.16 R.E.Majors,LCGC,20(2002)1098.17 R.E.Majors,LCGC,17(1999)S8.18 N.Campillo,N.Aguinaga,P.Vinas,I.Lopez-GarciaandM.Hernandez-Cordoba,J.Chromatogr.A,1061(2004)8591.19 N.Campillo,P.Vinas,I.Lopez-Garcia,N.AguinagaandM.Hernandez-Cordoba,J.Chromatogr.A,1035(2004)18.20 M.W.Dong,G.D.MillerandR.K.Paul,J.Chromatogr.,14(1996)794.21 F. Ferrari, D.G. Karpouzas, M. Trevisanand ECapri, Environ. Sci.Technol.,39(2005)29682975.22 A. Peck and K. Hornbuckle, Environ. Sci. Technol., 39 (2005) 29522959.23 J. Luque-GarciaandM. LuquedeCastro, J. Chromatogr. A, 998(2003)2129.24 S. Morales-Munoz, J. Luque-Garcia and M. Luque de Castro, J.Chromatogr.A,1026(2004)4146.25 J.Luque-GarciaandM.LuquedeCastro,J.Chromatogr.A,1034(2004)237242.26 K. Wenzel, B. Vrana, A. Hubert and G. Schuurmann, Anal. Chem., 76(18)(2004)55035509.27 S. Rissato, M. Galhiane, F. Knoll andB. Apon, J. Chromatogr. A, 1048(2004)153159.28 J. Pol, T. Hyotylainen, O. Ranta-Aho and M. Riekkola, J. Chromatogr. A,1052(2004)2531.29 M. Talebi, A. Ghassempour, Z. Talebpour, A. Rassouli and L. Dolatyari, J.Sep.Sci.,27(2004)11301136.30 G. Bahrami, S. Mirzaeei andA. Kiani, J. Chromatogr. B, 816 (2005)327331.31 S.Wang,T.WangandY.Giang,J.Chromatogr.B,816(2005)131143.Samplingandsamplepreparation3932 H.Wingfors, M. Hansson,O.Papke,S. Bergek,C.A.NilssonandP. Hag-lund,Chemosphere,58(2005)311320.33 F. Monteil-Rivera, C. BeaulieuandJ. Jawari, J. Chromatogr. A, 1066(2005)177187.34 C. Bicchi, C. Cordero andP. Rubiolo, J. Chromatogr. Sci., 42 (2004)402409.35 M. Rosario-Ramirez, M. Estevez, D. Morcuende andR.J. Cava, Agric.FoodChem.,52(2004)76377643.36 Z. Lu, C. Mallet, D.M. Diehl and Y. Cheng, LCGC, 22(Supplement) (2004).37 A.ShuklaandR.E.Majors,LCGC,23(2005)646.38 J. Vercammen, K. Francois, M. Wolf, Vermeulen, and K. Welvaert, LCGC,23(2005)594.FurtherReadingP.Gy,SamplingforAnalyticalPurposes,Wiley,England,1998.C.J.KoesterandA.Moulik,Anal.Chem.,77(2005)37373754.S. Mitra (Ed.), Sample Preparation Techniques in Analytical Chemistry, Wiley,NewJersey,2003.S.C.Moldoveanu,JChromatogr.Sci.,42(2004)114.F.Settle(Ed.),HandbookofInstrumentalTechniquesforAnalyticalChemis-try,Prentice-Hall,NewJersey,1997.REVIEWQUESTIONS1. Developamethodforextractingpesticidesfromsoilsamples,andexplainthereasoningforselectingthatmethod.2. Developamethodformeasuringtheamountsof anewpharma-ceuticalactiveingredientinplasma.3. ExplainnewtechniquesforimprovingSoxhletextraction.4. Explainmicrowave-assistedextraction.S.AhujaandD.Diehl40Chapter3Evaluation of basic physical propertiesNeilJespersen3.1 INTRODUCTIONSome of the simplest techniques and instruments are valuable tools forchemical analysis. This chapter is designedtoremindstudents thatsimple, rapidmethods are advantageous inmany situations. Thesemethodsareoftenusedforqualitycontrolpurposes.Themethodsdis-cussed here are melting and boiling points, viscosity, density or specicgravityandrefractiveindex.Timeisoftenanimportantconsiderationinpracticalchemicalana-lysis. Raw materials awaiting delivery in tanker trucks or tractor-trailersoftenmustbeapprovedbeforetheyareofoaded. Batchesof productpreparedbycompoundersonthefactoryoormust beanalyzedandapprovedbeforethepackagingprocesscanbegin.Analysisisoftenper-formedagainonpackagedproductsbeforetheyaresenttotheware-house. To maintaina smoothly running manufacturing system, theanalytical chemist must have rapid, informative methods to assay a widevariety of samples. Also important in quality control is the establishmentof standardsthat areagreed upon by allinvolved in themanufacturingprocess. Lax enforcement of standards brings into question the utility oftheanalysisandthreatensproductquality.Observationandmeasurementofphysicalpropertiesaretheoldestknownmeansof assessingchemical purityandestablishingidentity.Theirutilitycomesfromthefact that thevast majorityof chemicalcompounds have unique values for their melting points, boiling points,density and refractive index. In addition, viscosity and conductance arerapidmethodsforoverall propertiesof substances. TheHandbookofChemistry andPhysics has approximately 100 tables listing the refrac-tiveindex,density,specicgravity,viscosityandconductivityofaque-ous solutions of common inorganic and organic solutes at varyingconcentrations. Most of theseproperties alsochangedramaticallyifComprehensiveAnalyticalChemistry47S.AhujaandN.Jespersen(Eds)Volume47ISSN:0166-526XDOI:10.1016/S0166-526X(06)47003-3r 2006ElsevierB.V.Allrightsreserved. 41impuritiesarepresent,thusmakingthemidealforqualitycontrolcri-teria. Additional, rapidmethods suchas Fourier transforminfraredspectrometer(FT-IR)andrapidgasandliquidchromatographictech-niquesarealsousedinqualitycontrol,andarediscussedelsewhere.3.2 THEORY3.2.1 MeltingandboilingpointsMelting occurs when a crystal structure is heated to the point when thekinetic energyof themolecules is sufcient toovercomethelatticeenergy of a crystal. The lower the lattice energy, the lower is the melt-ingpoint of asubstance. Boilingoccurs whenthetemperatureof aliquid is increased to the point when the vapor pressure of the liquid isthe same as the ambient atmospheric pressure. Both of these propertiesaresummarizedintheheatingcurveasshowninFig. 3.1. Meltingisthetransitionfromthesolidphasetotheliquidphase. Inparticular,themeltingprocessoccursataxedtemperaturewhentheliquidandsolid phases are both present in the sample. Similarly, boiling occurs atABCDETemperatureHeat AddedTmTbFig.3.1.Heatingcurveforapuresubstanceatconstantpressure.SegmentArepresents heatingthe solid; segmentB the liquidandsolid inequilibrium asmelting occurs; segment C the heating of the liquid; segment D vaporizing theliquid where liquid and vapor are in equilibrium and segment E heating of thevapor.TmandTbrepresentthemeltingandboilingpoints,respectively.N.Jespersen42a xed temperature as long as the liquid and vapor phases are in equi-librium.Figure 3.1 illustrates that the melting temperature varies little withpressure. Boilingpointsontheotherhandcanchangemoresigni-cantly with pressure changes. As a result, we dene the normal boilingpoint (and also the normal melting point) as those measured when theatmosphericpressureis1.00 atm.Themeltingpointandboilingpointswill varywithexternal atmo-spheric pressure as shown in the phase diagram in Fig. 3.2. The effect ofimpurities on melting and boiling points is understandable when we seethat an added impurity has the effect of decreasing the triple point of thesubstancealongthesolidgasequilibriumlineinFig.3.2.Theresultisalwaysadecreaseinthemeltingpointandanincreaseintheboilingpointofasubstance.Forasmallgroupofsubstances,thequantitativerelationship of the freezing point depression and the boiling pointelevationisknownandhasbeenusedtodeterminemolecularmassesusingDT km kgsolute=MMsolutekgsolvent where MMsoluterepresents the molecular mass of a non-dissociatingsolute.liquidsolidvaporPressureTemperatureFig. 3.2. A stylized phase diagram for a simple pure substance. The dashed linerepresents1atmpressureandtheintersectionwiththesolidliquidequili-briumlinerepresentsthenormalboilingpointandtheintersectionwiththeliquidvaporequilibriumlinerepresentsthenormalboilingpoint.Evaluationofbasicphysicalproperties433.2.2 ViscosityViscositymeasurementorrheology.Viscosityisanimportantpropertyof manyuids, particularlyconsumer products. Pancakesyrupthatdoes not appear thick orarunny shampoohas littleconsumerappeal. Viscosity is also an important measurement for liquid polymers.Itisameasureofpolymerchainlengthandbranching. Ontheotherhand, viscosity does not have the sensitivity to detect small amounts ofimpuritiesinpuresubstance.Therearetwovariablesusedinthedescriptionofuidow:shearstressandshearstrain.StressismeasuredinunitsofPascalsandthestrainisdimensionless.A Newtonian uid is one in which the ratio of shear stress to the rateofshearstrainisconstant.Thisparameteristheviscosity Z.Thatis,Z shear stressshear strainThe unit of viscosity is the poise (g cm1s1). Kinematic viscosity v isoftenusedandisdenedasn Z=rwhere r is the density of the uid. The unit of kinematic viscosity is theStokes (cm2s1), manyconsumer products arespeciedinterms oftheir kinematic viscosityincentistokes (cst). Theviscosityof waterranges from 1.79 centipoise (cP) at 01C to 0.28 cP at 1001C. Olive oil hasa viscosity of 84 cP and glycerin has a viscosity of 1490 cP, both at 201C.3.2.3 DensityDensityisthemassperunitvolume.r mass=volumeDensityisalsodependent ontemperatureandtabulatedvaluesofdensity are valid only at the specied temperature. A related but moreversatileis thespecicgravity. This is theratioof thedensityof asubstancetothedensityofwateratthesametemperature.Specific gravity rsample.rwaterThedensityof asubstanceinanydesiredunits(e.g. g/cm3orKg/quart) can be obtained by multiplying the specic gravity by the densityofwaterinthoseunits(i.e.e.g.g/cm3orKg/quartrespectively).N.Jespersen443.2.4 RefractiveindexRefractiveindexisameasureofthevelocityoflightinairdividedbythe velocity of light in the compound of interest. Accurate measurementofrefractiveindicesrequirescarefultemperaturecontrol. Therefrac-tiveindexissensitivetothepurityofthesubstance.3.2.5 ConductivityConductanceofasolutionisameasureofitsioniccomposition.Whenpotentialsareappliedtoapairofelectrodes, electrical chargecanbecarriedthroughsolutionsbytheionsandredoxprocessesattheelec-trode surfaces. Direct currents will result in concentration polarizationat theelectrodesandmayresult inasignicant changeinthecom-positionofthesolutionifallowedtoexistforasignicantamountoftime. Conductance measurements are therefore made using alternatingcurrents to avoid the polarization effects and reduce the effect of redoxprocessesiftheyarereversible.Chargeconductancedependsupontheionsinvolved. Theabilityofan ion to carry charge through a solution is dependent upon its transportnumber, alsoknownasthetransferencenumber. Thisindicatesthatsomeionswillbemoreeffectiveinconductingchargethanothers.Hy-drogenions andhydroxide ions are almost 10 times as effective attransporting charge as other ions. Table 3.1 tabulates these transferencenumbers. As a result, conductance is not useful for specic analyses, butis useful for assessing the overall nature of a solution. Conductance hasits major use in assessing the ionic composition of water and monitoringofdeionizationordistillationprocesses. Consumerproductsmayhavecharacteristicsthatcanbeevaluatedbyconductivityalso.3.3 INSTRUMENTATION3.3.1 MeltingandboilingpointsMeltingpoint instruments consist of aresistance heater toincreasetemperature, asampleholderandatemperature-measuringdevice. Inits simplest form, an oil bath can be heated with a Bunsen burner, whilea capillary tube with a few milligrams of sample is attached to a thermo-meterwiththesamplenexttothemercurybulb.Heatingis most oftenachievedusingelectrical resistivedevices toheat a relatively massive metal block. This ensures that the temperatureEvaluationofbasicphysicalproperties45measuredatonepointontheblockbythetemperaturesensoristhesameasthatexperiencedbythesample.Lowerheatingratesalsohelpassurethatthesensorandthesampleareatthesametemperature.Visual observationis thehistorical methodfor observingmeltingpoints.Someinstrumentsusephotoelectricmethodstodetectchangesthat are interpreted as the onset of melting. Instruments that digitallyrecordimagesof themeltingprocessalongwithautomaticallydeter-miningthemeltingpointareavailable.Thelatterprovidesacheckontheautomatedprocessandalsoapermanentrecord.Temperaturemeasurement was historicallydone withamercurythermometer. Moderninstruments haveelectronictemperaturesen-sors that canbe coupledwithdigital temperature readouts. Digitaltemperature monitoring also allows the operator to record the observedmeltingpointwiththepressof akeypadbutton. Datacanbestoredwithinthe instrument or transmitted to a computer or laboratoryinformationmanagementsystem(LIMS).3.3.2 AccuracyandprecisionThe accuracy and precision of melting point determinations depend onseveral factors. CalibrationofthetemperaturesensoristherstandTABLE3.1Examplesofsomeionicconductivitydata.SumofanionicandcationicconductivitiesmaybeusedasestimatesofsaltconductivityCation Ionicconductivity Anion Ionicconductivity104m2Smol1104m2Smol1Ag+61.9 Br78.11/3Al3+61 Cl69.31/2Cu2+53.6 1/2CO3269.31/2Fe2+54 ClO464.61/3Fe3+68 F55.4H+349 1/2HPO4233K+73.48 H2PO4331/3La3+69.7 1/3PO4369.0NH4+73.5 MnO461.31/2Pb2+71 NO371.421/2UO22+32 OH1981/2Zn2+52.8 SO4280.0N.Jespersen46foremost requirement foraccurateresults. Checksof thecalibrationcanbemadeusingstandardreferencematerials.Thesecondfactorthataffectstheaccuracyandprecisionofthere-sultsistheuniformityofthetemperaturethroughoutthesystem. Inparticular, the temperature sensor and the sample must be at the sametemperature. In systems where the temperature is changing, it is dif-culttoassurethatallcomponentsareatthesametemperature.Heatmustowfromtheheatertothesensorandsampleandthenintothesensorandsample.Inmanyinstrumentstheelectricalresist-anceheater heats arelativelylargemetal blockthat acts as aheatreservoir, andthenheat fromthemetal blockis transferredtothetemperature sensor and sample. Using a thermometer and a sample inacapillarytubeinthisexample, thereareconsiderationsofheatowthrough the glass bulb of the thermometer and the capillary tube. Onlyafter traveling through the glass of the thermometer and capillary tubewill themercuryandsamplereceiveanyof theheat that startedattheheater. Eachofthesesegmentshasathermalgradientwherethehighesttemperaturewillalwaysbetheheaterandthelowesttemper-aturewillbethethermometerorthesample.Unfortunately,thether-malgradie