Principles and Practice of Modern Chromatographics Methods

518 Pages·2022·25.085 MB·English

by  Kevin Robards

PRINCIPLES AND PRACTICE OF MODERN
CHROMATOGRAPHIC METHODS
PRINCIPLES
AND PRACTICE
OF MODERN
CHROMATOGRAPHIC
METHODS
SECOND EDITION
K R
EVIN OBARDS
EmeritusProfessor,CharlesSturtUniversity,WaggaWagga,NSW,Australia
D R
ANIELLE YAN
SeniorLecturerinChemistry,CharlesSturtUniversity,WaggaWagga,NSW,Australia
Preface
My(KR)firstencounterwithchromatographywasatthetailendofthe1960swithpaperandthinlayerchroma-
tographyandthenwithagaschromatographmanufacturedintheworkshopsoftheUniversityofNewSouthWales,
Sydney,Australia.Outputfromthegaschromatographwascapturedonachartrecorderwhichconstantlyslippeda
gearorfailedcompletely,abortingachromatographicrun.However,lookingbackIwasfortunateasIdidnothaveto
generatemychromatogramsbyplottingthemfromspotgalvanometerreadingsasdidtheveryearlypioneers.Onthe
otherhand,perhapsIwastheunfortunateoneasIamsurethattheexperienceofthoseearlypioneersdevelopedan
associationwith,andfeelfor,thetechniqueofwhichwelatecomerscanonlydream.Itisappropriatethatthesecond
author(DR)wasinvolvedintheearlydaysoftwo-dimensionalcomprehensiveGCwithPhilMarriottforitwasthe
introduction of this modificationthat heralded theneed fornew demandsoninstrumentation andmethods of data
management.a
Ourbasicgoal in re-writing isunchanged from the first edition and that is to provide:
(cid:129) newusersofchromatographywithanhistoricalcontextandthebasicknowledgeandterminologytogetstartedin
this field, and
(cid:129) experienced users with a revision of the fundamentals of chromatography and its various techniques.
Whytheinterestinterminologyanddefinitions?Thesayingthat“preciselogicaldefinitionsofconceptsareafun-
damentalprerequisitetotrueknowledge”isattributedtoSocrates.Itisdoubtfulthatanindividualcandevelopafull
comprehensionofthescienceofchromatographywithouttheappropriatelanguage.Theterminologyassociatedwith
chromatographyhasoftenbeenusedratherlooselywithmanytextsfailingtodistinguishdifferentterminologiesand
classificationsystems.Clearenunciationanddifferentiationofclassificationsystemswasastrengthofthefirstedition
thathasbeenbuiltuponinthissecondeditionwhichisclearerandmorelogicalparticularlyintheexpositionofchro-
matographic theorywhich has been extensively revised and expanded.
Asinanyarea,thelanguageandassociateddefinitionsmustevolveanddevelopwithprogressfor,indeed,nothing
inthislifeisconstant;changeisuniversal.Theextent,nature,andrateofchangehavedifferedgreatlybothwithinand
betweenthedifferentareasofchromatography.Forexample,gaschromatographyasamaturefieldhasundergone
evolutionarychangeinthelast30yearswhererevolutionarychangehascharacterizedsomeotherareas.Columnsin
gaschromatographyareessentiallythesamenowastheywerein1990althoughnewcolumnchemistrieshavebeen
developed.Onethingthathaschangedistheaccelerationintheabuseofnomenclature.Coupled,hyphenated,multi-
dimensional,etc.havealwayspresentedanomenclatureproblemandtherehavebeennumerousattemptstoresolve
thesituation.Thehistoricaldevelopmentofthefieldofchromatographyandtheindependentdevelopmentandtiming
ofdevelopmentscontributedtotheseproblemswithnomenclature.However,insteadoflearningfrompastmistakes
theproblemhasproliferatedwithtermssuchashighspeed,fast,andmicrogaschromatographybeingusedwithwild
abandonpresumablyinsomeinstancestomakeapapermoreappealingtoreadersandconvinceeditorstoacceptfor
publication. Thisis confusing and obfuscates the ability of anewcomer to integrate with the field.
Searching the literature demonstrates a large change in research output since the first edition was written. Then,
research into chromatographic techniques and instrumentation including consumables was relatively prolific; now
itismuchmoresparseexceptinafewisolatedinstances.Theothermajordifferenceacrossallareasofchromatography
hasbeentheembraceofcomputertechnology.Computersystemsnowbothcontroltheinstrumentandprocessand
reporttheresults.Onemightexpectthatthiswouldallowmoretimeforcontemplationofthesystemandhowitfunc-
tions. However, just the opposite has occurred with many chromatographers becoming operators with very little
knowledge of the fundamental processes that control the separation. The result is a generation of ill-prepared
analysts.b,c
aColinF.Poole,J.Chromatogr.A1421(2015)1.
bLauraBush,LCGCNorthAmerica30(8)(2012)656.
cColinF.Poole,andSalwaK.Poole,JournalofChromatographyA,1184(2008)254.ColinPooleJ.Chromatogr.A1250(2012)157.
vii
viii
Preface
It is fortunate that within all this change, some things are constant apart from death and taxes; the fundamental
chemistry and basic principles of chromatography are unaltered from its inception although our understanding
mayhaveimproved.“Thedeepandconcreteknowledgeofbasictheorybehindseparationscienceandmainlychro-
matographictechniquesisfundamentalforanyanalystorlabpractitioner.Theusersofchromatographicinstruments
mustbeawareofthetheoreticalaspectsoftheirtechniquesinordertobeabletodevelopandapplyspecificanalytical
methodsaccordingtotheirneeds”(unknown).Thisbookprovidesthisessentialinformationtoreadersinaclearand
concise style.
Thebookhasbeenextensivelyrevisedandupdatedwiththeadditionofanumberofnewtopicsplusupdatedsche-
maticoverviewsofthecontentofeachchapter.Topiccoverageisbothintegratedandcomprehensivebasedonchro-
matographic bibliometrics and survey reports on the relative usage of chromatographic techniques. However, the
philosophicalapproachhasnotchangedfromthefirstedition.Thebookuseslanguagethatisclearandconcisewith
a style and format that engages the reader and facilitates a deeper understanding – a clear, logical development of
concepts and ideas that avoids the obfuscation of different terminologies and classification systems prevalent in
thearea.Thisapproachmakesiteasierforbothspecialistandnon-specialistreaderstobridgegapsbetweenexisting
knowledgeandtheirneeds(e.g.specialisttomovefromonetechniqueinchromatographytoanother;non-specialistto
obtain acomprehensive overview of chromatography).
Thisbookwillbeofvalueandinteresttobothpostgraduatestudentsandundergraduatestudents,notonlythose
studyingchemistry,butotherscientifictopicsaswell,e.g.biology,pharmacy,forensictoxicology,etc.Itwillbenefitall
practitioners/analystsinvolvedinallfieldsofapplicationsofchromatography;notonlythosewholackthefundamen-
tal background in chromatography but also those who wish to refresh their knowledge with updated information.
Thoseinvolvedincriminalforensiclawwillfinditausefulintroductiontoanareathatfeaturesprominentlyinforensic
cases.dThebookwillbenefitusersofchromatography,eitherthoseexperiencedinoneareaofchromatographythat
need to moveto another, or new users who want to understandthe backgroundof what they do.
Preface to first edition
Chromatographyisanestablishedanalyticalprocedurewithahistoryofusespanningatleastsevendecades.Itis,
nevertheless,acontinuouslyevolvingtechniquewithnewvariantsandmodifiedprocedures.Inmanywaysthishas
led to a plethora of terms that are confusing to the specialist and beginner alike. Most texts currently available are
written for the specialist and concentrate on one particular form of chromatography. This is understandable, given
the volume of information available on each of them but it is not of much help to the user requiring an overview
of developments across all areas of chromatography. Certainly, a unified approach is essential for the novice but
shouldalsobeofassistance tothespecialistsuddenlyfaced with theneedtoswitch from onetechniquetoanother.
Theauthorshavetaughtandresearchedextensivelyinbothacademicandindustrialareas.Theintentioninwriting
thistextwastoappealtoaswideanaudienceaspossible.Tothenon-chemistitishopedthatthismaterialwillprovide
aneasy-to-readoverviewinanareathathashadaprofoundeffectinfieldsasdiverseasclinicalchemistry,geology,
andfoodscience.Formanyscientistsengagedintheseareas,theirfirstrealcontactwithchromatographycomeswhen
facedwithananalyticalproblemrequiringtheseparatingpowerthatonlychromatographycanprovide.Totheprac-
tising chromatographer involved in research or routine analyses it will provide an update in those techniques with
which they are less familiar. Students will find the materialsuitableas an undergraduate text.
dhttps://www.paduiblog.com/pa-dui/a-large-problem-in-gas-chromatography-no-uniform-standard-for-gc-run-position-or-composition/.
C H A P T E R
1
Introduction and overview
1.1 Introduction
Thisisaseeminglysimpleandlogicalquestion.Atthemostbasiclevel,chromatogra-
phy is a separation technique or process. Its value depends on the ability to resolve or separate the components of
mixtures with a large number of either similar (molecular size, polarity, etc.) and/or dissimilar analytes. The result
ispresentedinachromatogram,thenatureofwhichisdifferentdependingontheactualprocess.Inpaperchroma-
tography, it is a piece of chromatography paper with a series of visible spots. However, the most common form of
PrinciplesandPracticeofModernChromatographicMethods 1 Copyright©2022ElsevierLtd.Allrightsreserved.
https://doi.org/10.1016/B978-0-12-822096-2.00006-2
2
1. Introductionandoverview
chromatogramisnowacomputer-generatedprint-outcomprisingaseriesofpeaksrisingfromabaselinedrawnona
timeaxis.Eachpeakrepresentsthedetectorresponseforadifferentcompound.Thetimefromthepointofinjectionof
sample into the chromatograph to the apex or peak maximum is referred to as the retention time of that particular
compound.
Anexampleofa2019state-of-the-artseparationbychromatographyisshowninthechromatogramofFig.1.1for
12analytesin6min.Thechromatogram(aplotofdetectorresponseversustime)providesdirectlybothqualitativeand
quantitativeinformation.Eachcompoundinthemixturehasitsowncharacteristicelutionorretentiontime(thetime
pointatwhichthesignalappearsinthechromatogram)underagivensetofconditions(qualitativeanalysis)andthe
areaandheightofeachsignalareproportionaltotheamountofthecorrespondingsubstance(quantitativeanalysis).
This makes chromatography avery powerfulanduseful technique.
Chromatography was originally developed by the Russian botanist M. S. Tswett (1872–1919) (Fig. 1.2) as a tech-
nique for the separation of coloured plant pigments. A clear definition of chromatography is clearly desirable and
should answer our question. Tswett gave a very pragmatic definition [1,2]. However, the first detailed definition
FIG.1.1 Anexampleofthemostcommonformofachromatogram
in which the x-axis represents time and the y-axis is the detector
response.Thechromatogramshowstheseparationof12analytesin
6min.Thepeaksobservednear1minarecomponentsofthesample
solvent and some minor peaks are also seen throughout the
chromatogram.
FIG.1.2 PhotographofMikhailTswett(1872–1919),thesonofaRussianforeignserviceoffi-
cialandanItalianmother.HestudiedattheUniversityofGenevabutthenreturnedtoRussia
beforeworkingatWarsawUniversityandthenbeingevacuatedtoMoscowinWorldWarI.
Credit:Elsevier.
3
1.1 Introduction
appearstobeduetoZechmeister[3]andvarioussubsequentdefinitionshavesincebeenformulated.Ageneralized
definitionwasprovidedin1974[4]andessentiallyconfirmedwithminorrefinementsin1993[5]byaspecialcommit-
teeoftheInternationalUnionofPureandAppliedChemistrywhichregardschromatographyas‘…amethod,used
primarilyforseparationofthecomponentsofasample,inwhichthecomponentsaredistributedbetweentwophases,
oneofwhichisstationarywhiletheothermoves.Thestationaryphasemaybeasolid,liquidsupportedonasolid,ora
gel.Thestationaryphasemaybepackedinacolumn,spreadasalayer,ordistributedasafilm…Themobilephase
maybegaseousorliquid’.Thisdefinitionneglectedthepossibilityofusingasupercriticalfluidasthemobilephase
which highlights the difficulties associated withproviding an adequate definition.
While the IUPAC definition regards chromatography as a ‘method’, the Scientific Council on Chromatography,
Russian Academy of Sciences [6] definedchromatography as follows:
(cid:129) Science of intermolecular interactions and transport of molecules or particles in a system of mutually immiscible
phases movingrelative to each other;
(cid:129) Processof multiple differentiated repeated distribution of chemicalcompounds (or particles), as a result of
molecular interactions, between mutually immiscible phases (one of which is stationary)moving relative to each
otherleadingtoformationofconcentrationzonesofindividualcomponentsoforiginalmixturesofsuchsubstances
or particles; and
(cid:129) Methodofseparationofmixturesofsubstancesorparticlesbasedondifferencesinvelocitiesoftheirmovementina
systemof mutually immisciblephases moving relative to each other.
Thisdefinitionrecognizesthatchromatographyissimultaneouslyaprocess,amethod,andabranchofscience.Itis
identifiedas‘anewbranchofscience’[7]andas‘abodyofknowledgethatisnowtoolargeformanyscientiststofully
grasp?’Thisreferenceisanexcellenttributetothepioneersandbuildersofchromatographyandtotheirachievements.
Whilethedefinitionmightappearirrelevant,inactualfact,Socratesheldtheviewthatthe‘preciselogicaldefinitionsof
concepts are a fundamental prerequisite to true knowledge’. Indeed, there has been considerable debate on this
topic [8].
Novákapproachesthisproblemfromadifferentperspectiveandprovidesaphenomenologicaldefinition,amolec-
ularkineticdefinitionandvariousworkingdefinitions[9].Whatisofinterestisthatwitheachsuccessivedefinitionthe
criteriaforaprocesstobecalledchromatographyhavegenerallybeenliberalized.Thisisnotsurprisingaschroma-
tography,likemostscientificdisciplines,iscontinuouslyevolving.Thusweshouldnotallowourselvestobedistracted
by the need for a clear and concise definition, but rather regard chromatography as a group of separation methods
whichareundergoingcontinuousdevelopmentandrefinement.Alternatively,itisalsoappropriatetoseechromatog-
raphy[10]asaunifiedscientificdiscipline:‘the“bridge”—asacentralscience—akeyfoundationbuiltonthetwentieth
century formajor advancesand discoveries yet to comeacross manysciences of the twenty-firstcentury’.
Theoriginsoftheword‘chromatography’arenolessobscure[11,12].A1952paper[11]commentedontheuseofthe
wordchromatographyforoveracenturyandahalfpriortoTswett’suseofthetermalthoughwithadifferent con-
notation. In Tswett’s papers, it was coined by combining two Greek words, chroma, ‘colour’ and graphein, ‘to write’
selectedtoindicatetheindividualcolouredbandsobservedbyTswettinhisseparations(Fig.1.3).Atthesametime
Tswettemphasizedthatcolourlesssubstancescanbeseparatedinthesameway.However,itmaywellbethatTswett,
whowasinvolvedinabittercontroversywithhispeers,gavereferencetotheGreekwordsonlyasanexcuseforas
Purnell[13]states‘…itwouldbenicetothinkthatTswett,whosename,inRussian,meanscolour,tookadvantageof
the opportunity to indulge his sense of humour’. The Germanic transcription of his Cyrillic name, Tswett is mostly
used but it occasionallyappears as the Englishtranscription, Tsvet.
Irrespectiveofsuchconsiderationschromatographyisauniversalandversatiletechnique.Whilethemorelimited
IUPACdefinitionjustifiesthoseinvolvedinapplicationsofchromatographyinfieldsasdiverseasmedicineandengi-
neering,thebroaderconceptofascientificdisciplinelegitimizesbasicresearchinthefieldwhichisanessentialsupport
for the applied aspects. It is equally applicable in all areas of chemistry and biochemistry, biology, quality control,
research, analysis, preparative scale separations, and physicochemical measurements. It can be applied with equal
successonthemacroandmicroscale.Chromatographyisusedindustriallyinthepurificationofsuchdiversemate-
rials as cane sugar, pharmaceuticals, and rare earths. On the other hand, it is widely used in the laboratory for the
separationofminutequantitiesofsubstance, asintheinitialchromatographicexperimentsleadingtothediscovery
of element number 100 which involved only about 200 atoms. This surely represents one of the most remarkable
achievements of modern science [14].
The achievement of such separations demonstrates the role of chromatography not only in chemistry but also in
science and medicine where the importance of chromatography is indisputable. Two Nobel Prizes in Chemistry (to
A. Tiselius of Sweden in 1948 and to A.J.P. Martin and R.L.M. Synge of Great Britain in 1952) have been awarded
4
1. Introductionandoverview
FIG. 1.3 Illustration (idealized) of classical
column chromatography showing the column
priortoadditionofsampleandatfourdifferent
stages of development illustrating the separa-
tion of three analytes from a mixture. Sand is
addedtothetopofthecolumntominimizedis-
turbance to the sorbent as sample and mobile
phaseareadded.Thisisessentiallythesystem
asusedbyTswettinhisoriginalexperiments.
Priorto1935thecolumnpackingwasremoved
from the column after use and the separated
zones were extracted in order to recover the
‘pure’components.
forworkdirectlyinthefieldofchromatography.Inaddition,chromatographyplayedavitalroleinworkleadingto
the awardof afurther 25Nobel Prizes in the 62yearsbetween 1937 and 1999, and 19 between 2000and 2007[10].
Theuseandimportanceofchromatographytosocietycanbeillustratedinmanyways.Thevalueofsalesofchro-
matographic equipment demonstrates a direct economic importance to the community. However, there are many
other economic impacts that are difficult to assess. For example, the use of chromatography in ensuring the health
oftheenvironment,qualityofourfood,waterandairsupply,andclinicalmonitoring,amongothers,arelesstangible
andmoredifficulttoassignadollarvalue.Equipmentsalesalsoprovideanindirectmeasureofhowwidelychroma-
tography is used. Another measure is the use of scientific publications but this reflects research output rather than
routine dailyuse occurring in governmentorganizationsand departments, food and pharmaceutical industrylabo-
ratories,hospitals,racingclubs,sportingorganizations,themobilesciencelabinFerrari’sF1garage(theovenwitha
30-mcoilinsideisagaschromatograph)[15],thePhilaelanderonComet67Plocatedover500millionkilometresfrom
Earth [16],or any one of themanylaboratories worldwideusing chromatography.
Theauthorsofapaperontheself-imageofchemistsbetweentheyears1950and2000[17]identifiedthe1960sasthe
period of ‘chromatographic takeover.’ They noted the existence at that time of two worlds, ‘that of traditional
chemistry, basically unchanged for two or three centuries, and that of modern chemistry, with a plethora of new
and powerfulphysical methods’with chemists seeing themselves with a foot ineach of thetwo worlds.Hopefully,
there are atleastsome chemists still straddling both of these worlds.
1.2 Coverage
Inthisbookweexaminealltechniquesthatfitthedefinitionofchromatographyprovidedearlierinthischapter.The
orderofpresentationoftopicshaschangedfromthefirsteditiontobetterreflectthedistinctionbetweenplanarand
columntechniques.Newchaptershavebeenaddedonhyphenatedtechniquesandpreparativechromatographywhile
the chapteron theory has been extended and re-written.
References have been updated and extended and partly for this reason but also reflecting the greater ease of
literature searching, the bibliography with each chapter has been deleted.
Electrophoretic techniques bear some similarity to chromatography in that a mobile and stationary phase are
involved and, on this basis, it is easy to argue for the inclusion of modern electrophoretic techniques in the current
book.Sixtypesofcapillaryelectroseparation(Fig.1.4)canbeidentifiedas:capillaryzoneelectrophoresis(CZE),cap-
illarygelelectrophoresis(CGE),micellarelectrokineticcapillarychromatography(MEKC),capillaryelectrochromato-
graphy (CEC), capillary isoelectric focusing (CIEF),andcapillary isotachophoresis (CITP) [18].
5
1.3 Chromatographicseparationsimplyexplained
FIG.1.4 Flowchartshowingthecategoriza-
tionofelectrophoretictechniques.
Themeansofdrivingthemobilephaseinliquidchromatographicseparationshasevolvedfromgravityinclassical
columnchromatographythroughcapillaryactioninpaperandthinlayerchromatographytohydrodynamicpressure
inhigh-performanceliquidchromatography.However,inthecaseofelectrophoresis,solutemigrationisalsounder
the influence of anexternal force in the form of an electric field.
Ofthesixtechniques,CECisatruehybridofelectrophoresisandchromatographyandastrongerargumentcould
bemadeforitsinclusion.TheCECprocesstakesplaceinacapillarycolumn,containingaselectedstationaryphase,
wherethemobilephaseisdeliveredbyanelectro-osmoticflowcontrolledbytheapplicationofarelativelyhighelectric
field.CECpresentsanumberofadvantagesrelativetohigh-performanceliquidchromatography(HPLC)[19]butthe
most significant consideration is the improved chromatographic efficiency. The flow rate in CEC is independent of
particlediameterandcolumnlengthandthereisnopressuredependencyunlikeHPLC,wherethecolumnpressure
isinverselyproportionaltotheparticlediametersquaredanddirectlyproportionaltocolumnlength,So,CEChasthe
potentialtogeneratehigherplatecountsthanHPLC.Theplug-likeflowvelocityprofileintheelectro-drivensystem
further reduces band dispersion and thus achieves a higher efficiency [20] to the parabolic or Gaussian profile
associated with hydraulic-driven flow in HPLC.
TherearedivergentviewsontheutilityofCEC.Accordingtoonesourcepublishedin2018[21],interestinopen-
tubularCEC(OT-CEC)continuestothrivewhereasa2009paperposedthequestion‘whateverhappenedtocapillary
electrochromatography?’[22].Cantheseviewsbereconciledorhasthetechniqueexperiencedaresurgencebetween
2009and2017?Between1998and2009therewasanaverageofaround150publishedpapersperyear.Thetechnique
doesappeartohavehadarenaissancesince2009basedonpublicationnumbersbutthesearestillrelativelyfewcom-
paredwithHPLCandCEChasfailedtobecomeamainstreamseparationtechnique[20].Lookingatthetitlesofpub-
lished papers gives a feeling that it is a technique seeking application areas. The development of novel stationary
phases[23]hasalwaysbeentheresearchfocus.ThetheoreticalbasisofCEChasbeenfirmlyestablishedsothataspect
isnotanimpedance.Ratherthemajorissuesinthelackofwidespreadacceptancearetheabsenceofdedicatedcom-
mercialinstruments[24]withthefeaturesneededtocompetewithHPLCandCE,andthelackofapplicationswhere
establishedmethodsfail.However,scientistsarestillinvestigatingtheparametersforCECanddevelopingapplica-
tions despite the lack of commercial support. Indeed, the development of rapid, effective, and selective chiral sepa-
ration methods is getting increasingly important for drug quality control, pharmacodynamic and pharmacokinetic
studies, and toxicologicalinvestigations [25] and this is one area in which CEC may compete.
WehaveexcludedtreatmentofCECinthisbookfortworeasons.Firstly,themeansofdrivingthemobilephasein
electrophoresisisnotencapsulatedbythedefinitionwehavegivenofchromatography.Secondly,CEChasnotdevel-
oped into a routine technique or established particular niche markets. The authors acknowledge the potential CEC
offersandrecognizethatitsinclusioninthenexteditionofthisbookmaybewarrantedtotheexclusionor,morelikely,
reductionin treatment of another technique(s).
1.3 Chromatographic separation simply explained
Thedefinitionofchromatographyimpliesthattheseparationofthecomponentsofasampleoccursbydistribution
ofthecomponentsbetweentwophases,oneofwhichisstationary(asolidorliquid)andtheothermovingormobile(a
liquid,gasorsupercriticalfluid).Consideratwo-component mixturewhich isintroducedattime,t ,intoamoving
o
phasewhichisincontactwithasecondphase,thestationaryphase.Acontinuoussupplyoffreshmobilephaseisthen
providedtotransportthesamplecomponentsthroughthestationaryphase.Astheanalytescomeintocontactwiththe
stationary phase, they distribute or partition between the two phases depending on their relative affinities for the
phases as determined by molecular structures and intermolecular forces. This process is depicted in Fig. 1.5 where