Neural correlates underlying mental calculation in abacus experts: a

12 Pages·2003·0.3 MB·English

1176 II/ELECTROPHORESIS/CapillaryElectrophoresis
Capillary Electrophoresis
S.F.Y. LiandY. S.Wu, NationalUniversityof freesolutionelectrophoresiswithin-linemonitoring,
Singapore,Singapore thefullpotentialwithrespecttocolumnperformance
^ wasnot yetattained.Also,complexityininstrumen-
Copyright 2000AcademicPress
tal design deterred follow-up by ordinary elec-
trophoresispractitioners.
Introduction A milestonefor column-basedelectrophoresiswas
setintheearly1980s,whenJorgensonetal.introduc-
The migration of charged particles under the inSu- ed capillary zone electrophoresis(CZE) with on-col-
enceofanelectricReldwasdiscoveredandcharacter- umnopticaldetection.Theyfoundthatwiththeinner
ized theoretically more than 100 years ago by diameter of the capillaries scaled down to 80(cid:1)m,
Kolrausch et al. Foreseeing the possibility of separ- voltages as high as 30kV could be applied without
ation of charged species through the application of incurringoverheating problems.Thusthe separation
avoltage,theterm‘electrophoresis’wascoinedsoon time for most charged species, from small molecules
after.However,earlyattemptstouseelectrophoresis to macromolecules, was shortened to less than
as an analytical tool were persistently frustrated by 30min, which is comparable to modern chromato-
theexistenceofJouleheating,whichactstodiscount graphic methods. For the Rrst time outstanding
the electrophoreticeffect. Thus a way of combatting column efRciencies of several hundred thousand
the thermal effect during the electrophoretic process plates was routinely obtained. The unprecedented
was needed. By 1950s, Tiselius et al. found that performance,relatively simple instrumentation,con-
avarietyofsubstancessuchasagaroseandpolymeric current with the widespread availability of fused
gels could serve as stabilizing agents in electro- silica capillary columns by the mid-1980s quickly
phoretic analysis owing to their anticonvective aroused the interests of both electrophoresis practi-
properties.Thiseventuallyledtothecreationofslab tionersandchromatographers,thusmakingcapillary
gelelectrophoresis,whichhasbecomeafundamental electrophoresis(CE)oneofthemostexcitingresearch
technique for the study of proteins, DNA fragments areas.Today,ithasbecome anindispensablebranch
and other biomacromolecules in life sciences and of modern separation science. The powerful separ-
biotechnology. Notwithstanding its great success, ation ability of CE was exempliRed in an early
slab gel electrophoresis has its drawbacks with re- electropherogram concerning the resolution of de-
spect to speed and automation when compared rivated peptides originated from egg white lysozyme
with contemporary chromatographic techniques (Figure 1).
such as high performance liquid chromatography This article serves as an introduction to CE. It
(HPLC). coversthe basic principles,variousaspects of instru-
A straightforward way to speed up an electro- mentation,separationmodesandmajorapplications.
phoreticseparationprocessistoapplyhigherelectric Some future trends of CE are discussed in the Rnal
Relds,andthisnecessitatessystemsabletoreleasethe section.
heatgeneratedmore efRciently. Electrophoresiswith
a tube as a separation channel is hence an attractive
Fundamentals
choice since the desired surface-to-volume ratio can
be achieved by simply reducing the tube radius. Per-
ElectrophoreticMigrationofIons
formingelectrophoresisbasedonthetubeformathas
an added advantage in that simultaneous detection The uniform motion of an ion under an electric
may be implemented in a way analogous to HPLC, Reld can be recognized as a result of balancing
thus rendering the entire procedure fast and auto- electromotive and frictional forces of the ion in
matic.RunningelectrophoresiswithatubeconRgura- solution:
tion was initiated by Hjerten as early as the 1960s,
andfurtherattemptedbyVirtanenetal.andMikkers qE"6(cid:1)r(cid:2)u [1]
etal.in1970s.Duringthisperiod,theadoptedinner
diameters of tubes were in the range of 0.2}3mm, where q is the effective charge of the ion concerned,
and thermal effects conRned the applied voltage to EiselectricReld,whileristheion’sStokesradius,(cid:2)is
around 1000}2000V, which was of the same order the dynamic viscosity of the solution, and u is the
as in typical slab gel electrophoresis. As a conse- linear velocity of the ion. The important parameter,
quence, despite these pioneering efforts to perform electrophoretic mobility ((cid:3)), is deRned as the ion’s
II/ELECTROPHORESIS/CapillaryElectrophoresis 1177
Rxed. Hence electrophoretic mobility, the inherent
attributeofanionisdirectlyreSectedbyitsmigration
time. This provides the theoretical basis of using
migration time as a means of identifying an ion
in CE.
ElectroosmoticFlow(EOF)
Electroosmosisis a fundamental electrokinetic effect
involving movement of the bulk solution against
a charged solid surface under the inSuence of an
electric Reld. In the case of CE, the capillary inner
wall usually carries negative charges due to the de-
protonation of silanol groups. For the part of the
liquid adjacent to the capillary wall, build-up of
cations takes place to counterbalance the negative
chargesonthecapillarysurface.AccordingtoStern’s
double layer model the solution containing net ca-
tionscanbedividedintotworegions,namelyarigid
layer and a diffuse double layer. The rigid layer is
immediately adjacent to the capillary wall, so the
cationswithinitarelargelyimmobilizedowingtothe
strong electrostatic interaction with the wall. The
diffuselayerisslightlyawayfromthewall,hencethe
Figure1 Capillary zone electrophoresis separation of fluor- cations inside are mobile. Upon the application of
escamine-labelledpeptidesobtainedfromatrypticdigestof re- avoltage,thesecationstogetherwiththeirsurround-
ducedandcarboxymethylatedeggwhitelysozyme.(Adaptedwith
inghydratingwaterwillmigratetowardsthecathode.
permission from Jorgenson JW and Lukacs KD (1981) Zone
The cohesive nature of water causes the whole solu-
electrophoresisinopen-tubularglasscapillaries:preliminarydata
onperformance.JournalofHighResolutionChromatographyand tion inside the capillary to be dragged forward,
ChromatographicCommunications4:230}231.) generating a net Sow across the capillary. This is
named the electroosmotic Sow (EOF). The magni-
tude of the EOF can be described via the Helmultz
linear velocity per unit of electric Reld: equation:
u q (cid:4)(cid:5)
(cid:3)" " [2] (cid:3) " [4]
E 6(cid:1)r(cid:2) eo 4(cid:1)(cid:2)
From eqn [2], it can be seen that the ion’s effec- where (cid:4) is the dielectric constant of the buffer solu-
tive charge, its size and the viscosity of the solution tion, (cid:6) is the zeta-potential across the diffuse layer,
decide ionic mobility. Thus in a given system ionic and (cid:2) is the viscosity of the electrolyte. Unlike con-
mobility is an intrinsic property of an ion. Usually ventional electrophoresis where EOF is regarded as
ionic mobility cannot be directly derived from unfavourable and thus usually suppressed, in CE it
eqn [2], as the parameters are not easily accessible has several importantpositive implications.
quantities. Instead it can be measured based on rel- First, the existence of an EOF offers a simple and
evantexperimentaldata,i.e.howlonganiontakesto highly efRcient way of driving a separation system.
travel through a certain distance under a deRnite Thezeta-potentialisuniformlydistributedwithinan
electric Reld, as follows: extremely narrowcylindricalregion alongthe whole
capillary so the bulk electrolyte solution
u L 1 L 1 L (cid:1)L is pumped out of the capillary with virtually no
(cid:3)" " eff(cid:1) " eff(cid:1) " eff tot [3]
E t E t V/L V(cid:1)t pressure drop (Figure 2). A ‘plug-like’ Sow is ob-
m m tot m
tained, which subsequently contributes to high col-
where L and L are the effective migration length umn performance. This is advantageous over the
eff tot
(frominlettodetectionwindow)andtotalmigration conventional pumping methods such as in HPLC,
length,respectively,Vistheappliedvoltage,andt is where the pressure-based Sow always introduces
m
themigrationtime oftheion. ForaCEsystemoper- aparabolicproRlethusaddingto thelossof column
ated undera constantvoltage, L ,L and V are all efRciency.
eff tot
1178 II/ELECTROPHORESIS/CapillaryElectrophoresis
broadening in CE include longitudinal diffusion, in-
jection-related volume overloading, thermal
effects,electrodispersion,walladsorption,etc.These
bandbroadeningmechanisms are deemed to be ran-
dom and independent events, so that the concept of
summation of variances can be used to evaluate the
contributionsofindividualfactorstotheoverallband
broadeningeffect, that is:
(cid:7)2 "(cid:7)2 #(cid:7)2 #(cid:7)2 #(cid:7)2 #(cid:7)2 #(cid:7)2 [6]
Figure2 The generation of electroosmotic flow (EOF) in a tot diff inj therm wall electr other
silicacapillary.
A brief description of these band broadening factors
is given below.
Second, the presence of EOF affects the apparent
mobilities of ions (Figure 3). In any electrophoretic Longitudinal diffusion In the course of electro-
separationsystemwhereEOFisnotfullysuppressed, phoretictransportationofan analytebandalongthe
theobservedmobilityofachargedspecieswillbethe capillary, the sample molecules will inevitably have
resultantof its effective electrophoreticmobilityand a tendency to enter the surrounding buffer solution
EOF: because of the apparent concentration difference,
leading to a wider and more dilute sample band.
(cid:3) "(cid:3) #(cid:3) [5] AccordingtoEinstein’sdiffusionequation,banddis-
obs ep eo
persiondue to longitudinaldiffusionis a functionof
diffusion coefRcient and time:
Under normal conditions,with EOF directs towards
the cathode, obviously cations will be accelerated,
(cid:7)2 "2D t [7]
whileanionswill be decelerated.If the magnitudeof diff m
the EOF exceeds the mobilities of the anions, the
Under an ideal situation, longitudinal diffusion be-
anionswill beswepttowardsthedetectionside,thus
comes the only unavoidable band broadening pro-
allowing the simultaneous analysis of cationic and
cess. Therefore it deRnes the maximum attainable
anionic species. As the magnitude and direction of
column efRciency in CE. Based on chromatographic
EOFwillaffect howlongtheanalytesstayinsidethe
theory, the maximum obtainable theoretical plates
separation capillary, manipulation of EOF often be-
(N) can be derived as follows:
comesacoreissueforeffecting asatisfactoryresolu-
tion.SincetheformationofEOFinvolvestwophases
(capillarywallandrunningbuffer),anymodiRcation N"L2" L2 " L2
totheirchemistrieswillbringaboutachangeinEOF. (cid:7)2 2Dmt 2Dm(cid:1)(L/v)
L2 (cid:3)V
Causesof BandBroadening " " [8]
2D (cid:1)[(L (cid:1)L )/(cid:3)V] 2D
m eff tot m
Asinachromatographicprocess,inelectrophoresisit
is necessary to contain the ionic species within nar- Thus the maximum column efRciency in CE is pro-
row bands while creating sufRcient mobility differ- portional to the mobility and voltage, while
ences.Hownarrowabandisdependsnotonlyonthe inversely proportional to the diffusion coefRcient.
various dispersive factors inherent to the elec- Considering that mobilities of ions range
trophoreticprocess, but also on how well the whole between10(cid:1)4and10(cid:1)3cm2V(cid:1)1s(cid:1)1,diffusioncoefR-
process is performed. The common causes of band cients from 10(cid:1)7 to 10(cid:1)5cm2s(cid:1)1, and an applied
voltage up to 104V, the attainable theoretical
platenumberwouldbeintheorderof105}106,which
is much higher than any conventional HPLC ap-
proach. Eqn [8] also suggests that in principle CE
shouldbewellsuitedfortheseparationofhigh-mass
charged particles such as biopolymers, since their
diffusion coefRcients are extremely low. This has
been demonstrated in the most successful resolution
Figure3 EffectofEOFontheapparentmobilitiesofanionsand ofDNAfragmentsandproteinswhereplatenumbers
cations. of 106 have been reached. It should be emphasized
II/ELECTROPHORESIS/CapillaryElectrophoresis 1179
thateqn [8]isonlyvalidunderthepreconditionthat equivalent to the superimposition of a parabolic
longitudinal diffusion plays a predominant role proRle to the otherwise plug-like ion boundaries
among the various band broadeningmechanisms. In and bulk Sow, as any temperature gradient will be
other words, to achieve the maximum column efR- translatedintoviscosityandmobilitygradientsinthe
ciency, the electrophoretic separation should be car- solution.TominimizetheinSuenceofthermaleffects
ried out in such a way that all the other potential on the overall column efRciency, it is imperative to
band dispersions are curbed well below the magni- limit the heat generation while maximizing the heat
tude of the longitudinaldiffusion effect. dissipation. In this regard, the use of narrow bore
capillaries is particularly recommended because it
Injection related volume overload During sample favours the above two aspects simultaneously. Ac-
injection,aRnitevolumeofsampleisplacedontothe cording to eqn [10], for a certain CE system, heat
capillary.Thelengthofthisstartingplugwillcontrib- generationmayalsobecontrolledthroughbalancing
ute directly to the Rnal band width. Treating the the buffer compositionand separation potential.
originalbandasrectangularinshape,thevarianceof
this plug can be expressedby: Wall adsorption effect The capillary surface, like
most solid surfaces, never behaves in a completely
(cid:1) (cid:2)
(cid:7)2 " linj 2 [9] inertmannertoforeigncompounds.InHPLC,it has
inj 12 been well known that peak anomalies are often the
result of some speciRc interaction (e.g. hydrogen
wherelinjistheinitialpluglength.Asaruleofthumb, bonding) between the residual silanol groups and
loss of efRciency due to any extraneous dispersion analytes. While similar adverse effects cannot be
factor should be kept within 10% of the maximum ruled out, in CE the problem is exacerbated by the
theoretical column efRciency. Assuming a fact that under a typical operation condition, the
moderateplatenumberintheorderof105asdeRned silanolgroupsalongthewallaremostlydeprotonated
by longitudinal diffusion, it can be easily estimated to give a negatively charged capillary surface. When
that the acceptable injection length should be a few an analyte with a positive charge travels along the
millimetres. For the commonly employed capillaries capillary, the electrostatic force will tend to attract
withinnerdiameterbetween50and75(cid:1)m,theabove the analytes onto the wall, causing additional band
length is equivalent to only a few nanolitres.So it is broadening. This is a feature of the analysis of pro-
obvious that CE’s high column efRciency will pose teins owing to their low diffusion coefRcients and
very stringent restriction on the sample size. Any multiple charge sites. SigniRcant efforts have been
attempttoincreasetheinjectionvolumeinanaimto made to tackle this problem, mostly through the
enhance detection sensitivity may result in a signiR- suppression of EOF or complete reversal of the
cant loss of column efRciency. charge status of the capillary wall.
Thermal gradient effect An electrophoretic pro- Electrodispersion Electrodispersion is the result of
cess is always accompanied by certain amount of Ohm’s law during the electrophoretic separation. It
thermal effects due to the passage of a current mayappearintwoinstances.First,iftheconductivity
through the resistive medium (Joule heating). For of the injection plug is larger than that of the buffer
a CE system,the electrical power (P) responsiblefor solution, an isotachophoresiseffect will occur to di-
the generation of heat can be estimated through the lutetheoriginalbandtill theconductivityisequalto
following equation: the surrounding buffer. Second, during the separ-
ation,an analyte zone is ‘submerged’ into the buffer
P"V(cid:1)I"V2"V2(cid:1)r2(cid:8)c [10] solution. Any mismatch of its mobility with its co-
R L ions in the buffer will render the local electric Reld
where V is the applied voltage, r and L are capillary different from that in the normal running buffer. If
inner radius and length, respectively, while (cid:8) and themobilityofthesampleionislargerthanthatofits
c are respectively the molar conductivity and co-ions,thenthere will be a lower electrical Reld for
the concentration of the electrolyte solution. While theanalytezone.Thusanysampleionsdiffusingout
heat generation is uniform for the whole electrolyte ofthezonewillexperienceahigherelectricReld,and
solution,heatdissipationisapparentlynot:thenearer theseionswillspeedupalongthemigrationdirection.
the electrolyte is to the capillary wall, the faster is This causes the ions at the rear to re-enter the zone,
the heat transferred out to the surroundings. Con- whereastheionsatthefrontwilldriftawayfromthe
sequently,atemperaturegradientisgenerallypresent zone.Theaccumulativeeffectofsuchphenomenonis
in the radial direction of the capillary, which is the formation of a tailing band showing a sharp
1180 II/ELECTROPHORESIS/CapillaryElectrophoresis
supplyis connectedvia the platinumelectrodes.Fol-
lowing the introduction of sample at the capillary
inlet, a high voltage is applied, thus driving the
analytes to travel inside the capillary with different
velocities.Somewhereclosetothecapillaryoutlet,an
on-linedetectorisinstalledtomonitortheseparation
process. The resulting signals are fed to the data
acquisition device, and Rnally the result is presented
in the form of an electropherogram.
Figure4 Mobilitymismatch-inducedbandbroadening. Apart from these fundamental components, com-
mercialCEinstrumentsarecommonlyequippedwith
some dedicated facilities, such as an autosampler,
trailingboundarybutabroadenedleadingboundary. pressure regulating unit, capillary thermostatting,
By a similar argument, if the analyte ion is of lower and comprehensive supporting software. These
mobilitythantheco-ion,afrontingbandisexpected addedfunctionsallowasequentialanalysisofdiffer-
(Figure 4).Thehigherthesampleconcentrationcom- entsamplesunderprespeciRedconditions,thusensur-
pared to the buffer concentration, the more pro- ing better reproducibility, accuracy and higher
nouncedarethenonuniformitieswithrespecttocon- throughput. Two modern commercial systems are
ductivity and Reld strength, and eventually more se- shown in Figure 6.
vere is the band asymmetry.
From the above discussion it is obvious that, to
HighVoltagePower Supply
preventpossiblelossofcolumnefRciencyduetoelec-
trodispersion, the conductivity of the injection plug A high voltage power source delivering stable DC
and the actual sample concentrationshouldbe sufR- potential of $30kV will satisfy the requirement of
ciently low. Theoretical study had shown that, to most CE applications. Many power supplies offer
conRne the electrodispersion-related band broaden- additional features such as polarity switching, con-
ing at a level comparable to longitudinal diffusion, stant potential/current setting, and an interlocked
thesampleconcentrationshouldbetwoordersbelow
the buffer concentration. To some extent, it is the
electrodispersion that limits the mass loadability of
a CE system.
Instrumentation
CE can be performed with relatively simple instru-
mentation as depicted in Figure 5. A capillary con-
taininganappropriateseparationmediumspanstwo
buffer reservoirs, to which the high voltage power
Figure6 CommercialCEsystems.(A)BenchtopCEsystem.
Figure5 Instrumental setup of a capillary electrophoresis Photographcourtesy of Bio-Rad Laboratories.(B) Portable CE
system. system.PhotographcourtesyofCEResourcesPte.Ltd.
II/ELECTROPHORESIS/CapillaryElectrophoresis 1181
antielectrical shock loop. In the commercial CE in- injection volumes of individual components can
struments, the power supply is designed with digital be calculated throughthe following equation:
communicationcapability,sothat moreinformation
suchasacurrentcurvecanbetrackedandretrieved, ((cid:3)#(cid:3) )(cid:1)r2Vt
V " i eo [12]
and the applied voltage can be programmed. inj L
SeparationCapillary
where (cid:3),(cid:3) are the mobilities of the analyte and
i eo
Although capillaries made of glass or polymer EOF,respectively,andVistheinjectionvoltage.For
(e.g.TeSonorNylon)havefoundoccasionalapplica- electrokineticinjectiontheinjectedamountsofdiffer-
tion,fusedsilicacapillariesareusedpredominantlyin ent analytes are dictated by the mobilities of the
CE,largelyduetotheirstrength,Sexibilityandmost respective analytes. Thus it is different from hy-
importantlytheirexcellentUVtransparency.Usually drodynamic injection, where the sample plug is of
the fused silica capillary is coated with a layer of entirelythe samecompositionas the original sample
polyimide to enhance its durability. For on-column solution. To avoid sample injection bias, hydro-
optical detection a small segment of this coating dynamic injection is preferred. However, in some
needs to be removed to provide the detection win- circumstances hydrodynamic injection may be im-
dow. The most commonly used CE capillaries have practicable (e.g. owing to the high viscosity or low
inner diameter between 20 and 75(cid:1)m, outer dia- permeability of the separation medium). Elec-
meter 100}400(cid:1)m and are about 30}100cm in trokinetic injection is then the only viable option,
length. suchas incapillarygel electrophoresis.Onthe other
hand, sometimes electrokinetic injection may be ex-
SampleInjection
ploitedinfavourofCEoperation.Forexample,itcan
ToachievehighcolumnefRciencyandgoodquantit- be employed to diminish the interference of the
ative results, sample injection must be performed in sample matrix if the components concerned are of
areproduciblemanner.Sincetheinjectionvolumein low mobilities. Moreover, if the sample is of low
CEisinthe nanolitrerange,whichprecludestheuse conductivity, then sample enrichment during elec-
of conventional injection methods, alternative ap- trokineticinjectionispossiblebytakingadvantageof
proaches have to be pursued. Hydrodynamic injec- the sample-stackingeffect.
tionandelectrokineticinjectionhaveturnedouttobe
the most widely employed sampling techniques. Detection
Hydrodynamicinjectionintroducesasamplebased
InCE, sampleseparationis accomplishedinan elec-
onapressuredifferenceinthetwosidesofthecapil-
trolyte solution, so detection strategies analogous to
lary.Foralaboratory-builtinstrument,thisisrealized
HPLC are adopted. Compared with HPLC, CE col-
bysimplyliftingupthesamplevialtogetherwiththe umn efRciencies are at least one order of magnitude
capillary inlet for a certain period of time (typically
higher,whichsuggeststhat solutebandswill be nar-
a few seconds).The hydrodynamicforce will siphon
row and the average concentration of the analyte
a band of sample solution into the capillary. For
zoneisseveraltimeshigherthanforHPLC.Asfaras
commercialinstruments,thepressuredropiscreated
aconcentration-sensitivedetectorconcerned,thisim-
byeitherapplyingpressureattheinletside,orimpos-
pliesalargerdetectionsignaloutput.However,sofar
ingavacuumattheoutletvial.Theinjectionvolume
for CE the concentration sensitivity is usually lower
can be calculated based on Poiseuille’s law:
thanitsHPLCcounterparts.Thisapparentcontradic-
tion stems mainly from the small sample size,
(cid:9)P(cid:1)r4t (cid:10)g(cid:1)r4(cid:9)ht which poses great difRculties for detection. The fun-
V " " [11]
inj 8(cid:2)L 8(cid:2)L damental detection schemes in CE fall into three
categories: optical (UV absorptive and Suorescent)
where (cid:9)P and (cid:9)h are the pressure and height differ- detection,electrochemicaldetection,andvarioushy-
ences,respectively,whiler,t,(cid:2)andLrepresentcapil- phenating techniques (typically mass spectrometric
lary inner radius, injection time, solution viscosity detection).Thesensitivitiesofthe different detection
and capillary total length, respectively. methods are compared in Figure 7.
Electrokineticinjectionisbasedonthetransporta-
tionofsampleionsbyelectrophoreticmovementand UV adsorbance This is the most commonly used
EOF. Normally a lower voltage than that for separ- detection method for CE, mainly because of its sim-
ationpurposeisappliedforacertainamountoftime plicity. It is frequently implemented by modifying
to allow analytes to migrate into the capillary. The a HPLC-type UV detector. The normal Sow cell is
1182 II/ELECTROPHORESIS/CapillaryElectrophoresis
Figure7 ComparisonofsensitivitiesofvariousCEdetectionmethods.(AdaptedwithpermissionfromLandersJP(ed.)Handbookof
CapillaryElectrophoresis,2ndedn,chap.10,pp.425}448.BocaRaton,FL:CRCPress.)
removed and the capillary is mounted directly be- focusingofexcitationlightandmoreeffectivecollec-
tweentheincidentlensandphotocell,inconjunction tion of emission light. An important development in
with a corresponding aperture. Such on-column de- CESuorescencedetectionistheintroductionoflaser-
tection conRguration, though easy to execute, pro- based excitation sources. Owing to its outstanding
vides only a moderate detection sensitivity with the coherence, a powerful laser source can be focused
lowerdetectionlimitsaround10(cid:1)5to10(cid:1)6molL(cid:1)1, into an extremely sharp beam to illuminate the core
as it is associated with two major problems. First, partofthecapillary,thusproducingemissionlightof
since the capillary is illuminated radially, the max- high intensity. As a result, detection limits of
imumopticalpathisequivalenttotheinnerdiameter 10(cid:1)9}10(cid:1)11molL(cid:1)1 can be obtained routinely,
of the capillary. This severely reduces the achievable whichisatleasttwoordersofmagnitudemoresensi-
absorbance according to Lambert}Beer’s law. Sec- tivethantheconventionalapproach.Singlemolecule
ond,duetoitsuniquecylindricalgeometry,thecapil- detection has been demonstrated by Rne-tuning the
lary tends to act like a lens to defocus the incoming CE system and utilizing laser-induced Suorescence
light, thus posing difRculties in light orientation and (LIF) detection. A major drawback of Suorescence
collection.Asaconsequence,signalnoiseandnonlin- detection is that the number of analytes with native
earity are further aggravated. By bending the capil- SuorescenceisfarlessthanthatwithUVabsorbance.
laryintoZ-shapeorcreatingabubblefeatureonthe Therefore,tomakeuseofthis highlysensitivedetec-
capillary body, the optical pathlength can be con- tion scheme, derivatization of analytes by tagging
siderably increased. These efforts, coupled with with a Suorescent group is often needed.
improved detection optics, enhance the detection
limits to 10(cid:1)7molL(cid:1)1. The capillary can also be Indirect photometric detection For compounds
connected to an HPLC-type Sow cell (pathlength without any chrophormore, indirect detection offers
&1mm) as in the HP system. asimple,effectivewaytotakeadvantageofthe con-
venienceofon-columnopticaldetection.HereaUV-
Fluorescence detection Fluorescence detection is so absorbing or Suorescent compound with the same
farthemostsensitivedetectionmodeavailabletoCE sign as the sample ions is added to the running
due to the fact that the measurement is performed buffer to provide a stable background. During the
against a ‘dark’ background,and Suorescence inten- electrophoretic separation, the sample ions will dis-
sityislesspathlengthdependentbutdirectly propor- place a certain amount of background ions due to
tional to excitation power. On-column Suorescent electroneutrality requirements. Thus the passage of
detection can also be realized throughadaptation of a sample zone through the detection window will
anHPLC-typedetector.Onceagain,duetothepres- appearasanegativepeak.Inprinciple,thequantiRca-
ence of the lens effect of the capillary, improvement tionofasampleinsuchawayinvolvesdifferentiating
of the detection optics is necessary to ensure better asignalfromtwolargeresponses,hencethedetection
II/ELECTROPHORESIS/CapillaryElectrophoresis 1183
sensitivity is lower than that for direct photometric fabricate microelectrodes and mount them into an
detection. To achieve an acceptable detection limit, extremely small space, usually deRned by the separ-
the background concentration should be kept low. ation capillary. While the current state-of-the-art
Undersuchcircumstance,anymismatchofmobilities allows the preparation of microelectrodes with di-
betweenthesampleionsandbackgroundco-ionswill mensionsdownto several (cid:1)m, the installationof the
give rise to a considerable electrodispersion, hence microsensing elements to the detection region is
leading to severely distorted peaks. Therefore, a task requiring complicated microfabrication tech-
selectingasuitableCEbackgroundisofspecialsignif- niques and great patience. All three types of electro-
icance for the implementation of indirect optical chemicaldetectionhaveshowntheirfeasibilityinCE
detection. with typical detection limits in the range of
10(cid:1)7}10(cid:1)9molL(cid:1)1.
Electrochemicaldetection Inelectrochemicaldetec-
tion, sample bands are monitored in terms of an Hyphenation with mass spectrometry Following
electrical signal. Depending on the application, the thegreatsuccessininterfacingHPLCwithmassspec-
measured quantity can be conductivity, voltage or trometry,itisalogicalmovetoexplorethepotential
current. Accordingly it is referred to as conduc- of CE-MS. For a successful implementation of
timetry,potentiometryoramperometry,respectively. CE-MS,keepingaproperelectricalcontactattheMS
Unlike optical detection in which the measurement side is essential. This has been achieved through
output depends strongly upon the available volume, a variety of means, such as contacting via coaxial
inelectrochemicaldetectionthesignalrelatesonlyto sheathSow, or througha liquid junction. Sheathless
the part of solution that is directly contacting the contact has been realized by attaching the metal-
electrode surfaces. Thus, wherever volume insufR- coated separation capillary tip directly to the ion
ciency precludes the employmentof photometricde- sourceemitter.Forsampleionizationandtransporta-
tection,e.g.incasewhereultra-narrowborecapillary tion,theelectrosprayionizationinterface(ESI)ispre-
is used to facilitate fast separation, electrochemical dominantlyadopted, mainly because ESI is operated
detection may still be a viable choice. Moreover, as underalmostatmosphericpressure,thusnotconSict-
long as the analyte is electrochemically active, the ing with CE separation. Moreover, ESI ionization
detection can be performed directly without involv- workswith electrostatically induced nebulization,in
ing derivatization, as it may be for photometric which compounds are ionized with different charge
detection. Due to these advantages, electrochemical status depending on their relative molecular masses
detection gradually gains its popularity in CE andshapes.Inprinciple,thisallowsdeterminationof
practice. relative molecular mass for a large range of com-
There are two major difRculties involvedwith im- pounds, from small molecules to large biopolymers
plementing electrochemical detection for CE. The (such as proteins and polysaccharides). Thus it is
Rrstcomesfromthefactthat,itisnoteasytomakean applicabletoalltypesofCEanalytes.Asforthemass
electrochemicaldetectorfunctionwellinthepresence analyser, the high efRciency of CE separation de-
ofahighvoltage,becausetheseparationvoltagewill mands a mass analyser with a fast scan rate. The
producenoisethatswampstheminuteresponseofthe time-of-Sight (TOF) mass spectrometer is probably
analytes. Thus, decoupling of the separation electric the most promising candidate, but the quadrupole
Reld from the detection system has to be done to massspectrometeriscurrentlytheworkhorseforCE-
facilitatethemeasurement.Theinsulationofthesep- MSowingtoitscommercialavailabilityandrelative-
arationvoltagecanberealizedbyintroducingafrac- ly low cost. Its insufRcient scan speed may be com-
tureoragapstructureinthecapillarythroughwhich pensated partially through the manipulation of the
the electrical grounding is made, such that the seg- electric Reld, i.e. when a certain analyte is migrating
ment behind the fracture or the gap can be used for out of the capillary, the applied voltage can be re-
theaccommodationoftheelectrochemicalelectrodes. duced temporarily, such that the analyte of interest
Alternatively, to shield off the high voltage, the de- will remain in the ionization chamber for a longer
tecting elements may be mounted immediately out- time. With careful optimization of parameters, CE-
side the outlet of the separation capillary. A more MShasshowndetectionlimitsintheattomolerange
elegantwayistoetchthecapillaryendtogeneratean (10(cid:1)18molmass).
enlarged conical cuvette from where the detecting
electrodesareinstalled.Inthiswaytheinterferenceof
Separation Formats of CE
high voltage can be eliminated because the electric
Reld decays very rapidly before the enlarged conical One of the advantages of CE is that many types of
part. The second difRculty stems from the need to separation can be performed with the instrument
1184 II/ELECTROPHORESIS/CapillaryElectrophoresis
describedinFigure 2.Thisismadepossiblebysimply CapillaryGelElectrophoresis(CGE)
altering the separation medium inside the capillary
For this format, polymeric networks are present
andutilizinganappropriatebuffersystem.Thecom-
alongtheelectrophoreticpathwayofanalytes,which
monly employed separation formats in CE can be
causes chargedspecies to be resolved onthe basis of
divided into the following.
theirphysicalsizesratherthancharge-to-massratios.
Accordinglythisseparationprocessisalsocommonly
known as ‘molecular sieving’. It is particularly suit-
CapillaryZoneElectrophoresis(CZE) ablefortheseparationofbiomacromoleculesconsist-
ing of numerous repeat charge units, such as DNA
Among the various CE formats CZE is the simplest
fragments,SDS-denaturedproteins and polysacchar-
andmost popular.Here a homogenousfree solution
ides(Figure 8).CGEmaybeconsideredasanadapta-
isemployedtomaintainaconstantelectricReldalong
tionoftraditionalgelelectrophoresisintoitscapillary
the capillary. Ionic species are separated inside this
format. However, traditional gel electrophoresis use
supporting solution according to their different
onlycross-linkedpolyacrylamide(socalled‘chemical
charge-mass ratios, thus forming segregate zones.
gel’), while for CGE both cross-linked polymer gels
The desired separation selectivity can be achieved
and various polymer solutions can be employed to
by simply optimizing the parameters of the carrier
createthesievingpores.Theuseofpolymersolutions
electrolyte particularly pH, as well as ionic
instead of chemical gels facilitates renewing of the
strength and the type and concentration of various
sieving medium by simply Sushing out the original
EOF modiRers. A number of secondary equilibria
solution,and reRlling with another.It also alleviates
are available to strengthen the separation selectivity
sometechnicalproblemsassociatedwithchemicalgel
further.
such as gel shrinkage, bubble formation and matrix
Although CZE is commonly performed with an
collapse, hence improving the separation repro-
aqueousbuffer,itcanalsobeimplementedinanon-
ducibility.
aqueous medium by using organic solvents and
compatible conductive salts. The effect of replacing
water with an organic solvent in CE can be under-
stood from the fact that organic solvents possess
signiRcantly different viscosities and dielectric con-
stants compared with water. Thus, changes in the
magnitude of EOF and migration behaviours of
charged analytes are expected in non-aqueous CE,
which can be seen from eqns [2] and [4]. Further-
more, organic solvents differ in their capacity to
stabilize equilibria. Thus, in organic media, the
chargestatusoforganicanalytescanbedramatically
different from that in an aqueous medium, hence
leadingtoquitedifferentseparationselectivity.Addi-
tionally, it is evidenced that organic media are ca-
pableofpromotingcertainmechanismssuchasinclu-
sion interaction and ion-pair formation, which en-
hance possibilities of achieving the desired separ-
ation.Moreover,withawaterdominatedbuffer,itis
verydifRculttoconductCEseparationsofhydropho-
bic analytes.Undersuchcircumstances,switchingto
a non-aqueous buffer system would provide an efR-
cient solution. Finally, it is noted that, when an or-
ganic medium is utilized for CZE separations, the
electrophoretic current is reduced considerably. As
a result, even though capillaries of relatively wide
inner diameter are employed, Joule heating is at Figure8 CGE separation of poly(uridine 5(cid:11)-phosphate).
amanageablelevel,thusenablingtheenhancementof (AdaptedwithpermissionfromYinHF,LuxJAandSchomburgG
(1990) Production of polyacrylamide gel filled capillaries for
detection sensitivity through the usage of wide bore
capillarygelelectrophoresis(CGE):influenceofcapillarysurface
capillaries. In short,non-aqueousCZE is an attract-
pretreatmentonperformaceandstability.JournalofHighResolu-
ive means for extending the applicability of CE. tionChromatography15:624}627.)
II/ELECTROPHORESIS/CapillaryElectrophoresis 1185
CapillaryIsotachophoresis(CITP) ampholyteswith isoelectric pointsin a certain range
andincloseproximitytoeachother.Suchamixture
InCITP,aleadingelectrolyteofhighermobilitythan
is commonly formed by a series of synthetic poly-
anyofthesamplecomponentsisRlledintothesepar-
amino polycarboxylic acids. The capillary is Rlled
ationcapillary and outlet reservoir,while a termina-
with the carrier ampholyte solution and a small
ting electrolyte of lower mobility than any of the
amount of sample, and dipped into the buffer reser-
sample components is placed in the inlet reservoir.
voirs that contain acid and base, respectively. Under
The sample is applied from the capillary inlet. This
the electric Reld, different ampholytes will migrate
arrangement ensures that there will be no mixing of
along the capillary until they reach positions corre-
sample with the two electrolytes during the whole
sponding to their pI values, where they stand still.
electrophoretic process. As a consequence, upon
CollectivelyastablepHgradientisformedacrossthe
applicationofanelectric Reld,themainchangehap-
capillary. The sample components, too, will migrate
pens inside the sample band itself, i.e. the sample
untiltheyRndpositionsequivalenttotheirpIvalues.
components tend to ‘queue up’ according to their
When such a steady state is obtained, an immobiliz-
effectivemobilityorders.Thesecomponentsarerelo-
ation step (by utilizing electrophoretic movement or
catedwithcorrespondingchangesintheirconcentra-
pressure) is performed, and the separated analyte
tions and band lengths. Ultimately a series of con-
bands will be forced out and passed through the
secutive ‘pure zones’ containing only the individual
detector. If the carrier ampholyte mixture is well
substancesareformed.Thisisthesteadystateasthere
prepared and a sufRcient time is allowed for the
willbenomorechangesforthesampleexceptthatall
electricfocusingprocess,averyhighcolumnefRcien-
thezoneswillcontinuetomigrateoutofthecapillary
cy can be realized.
with an identical velocity (hence the term ‘iso-
tachophoresis’). Unlike the other types of CE separ-
ation,in CITPthe concentrationof aspeciRczoneis MicellarElectrokineticChromatography(MEKC)
predetermined by the concentration of the leading
Neutralspeciescanneverberesolvedwithtraditional
electrolyte and the relative mobility of the ion of
electrophoretic techniques since they have no elec-
interest with respectto the leading electrolyte, while
trophoreticmobility.AfterthebirthofCE,thisprob-
the sample amount is reSected by the zone length.
lemwassolvedbyTerabeetal.throughtheaddition
Due to the unique stepwise increase in electric Reld
of a charged surfactant to the running buffer. Typi-
starting from the leading electrolyte side, which
cally a negatively charged surfactant is added at
prevents a component from drifting off its own
a concentration above its critical micelle concentra-
band, sharp boundaries can be maintained more
tion (CMC). Under such circumstances, micelles are
readily. Therefore CITP can be implemented using
formed, which allow neutral compounds to be re-
tubes of relatively large i.d. (e.g. 0.2}0.8mm).
tained based on their hydrophobicities. Under an
Nevertheless, CITP as an analytical tool has largely
electrical Reld, the negatively charged micelles move
lostitspopularitysincetheadventofCZE,probably
toward the anode side, while a strong EOF moves
due to the fact that a thorough knowledge of the
toward the cathode side (Figure 9). As EOF usually
sample is required before the separation. Further-
exceeds the electrophoretic mobility of micelles, the
more, the isotachopherogramis usuallystep-shaped,
micelles will eventually be swept out in the same
and the steps are not time related, thus rendering
automatic identiRcation and quantiRcation difRcult.
CITP is often applied as a sample pre-concentration
step before CZE separations to enhance detection
sensitivity.
CapillaryIsoelectricFocusing(CIEF)
Thisisanotherexampleofanadaptationofaconven-
tionalelectrophoresisprincipletoacapillaryformat.
CIEF exploits differences in isoelectric points (pIs),
auniquecharacteristicofamphotericcompoundsun-
derwhichitsacidicandbasicgroupsdissociatetoan
equal extent, so that the whole molecule exhibitsno
net charge. It is only suitable for the separation of
compounds like amino acids, peptides and proteins.
The operation of CIEF relies on a mixture of carrier Figure9 SeparationmechanismofMEKC.