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Agilent Capillary ElectrophoresisSystem — Technical Description

Product Note

and applications literature anda worldwide network of CEspecialists.

The design objective was todevelop a complete easy-to-usesystem that provides high sensitiv-ity diode-array detection, improvedquantitative analysis, fullautomation, the capability ofperforming all modes of capillaryelectroseparation to the highest

standards available (includingCEC*), and at the same timebearing in mind the future require-ments of the user. This has beenachieved by innovative detectordesign, high precision control ofoperational parameters, versatileautomated operational featuresand provision of a fully opera-tional core instrument which isequipped to perform all CEseparation modes and is designedto facilitate low-cost upgrade forCE-MS interfacing.The instrument is controlledthrough the Agilent CE ChemStationusing a self-explanatory, intuitivegraphical user interface. TheChemStation control offers manyadvanced operational features toensure versatility in all capillaryelectrophoretic separations. Everyoperation, from method develop-ment and routine operation todemanding research, has beendesigned to be straightforwardand easily mastered. With thisvision Agilent has created aprecision engineered instrumentallowing the analyst fully featuredoperation from routine analysis tocutting-edge research.

This product note presents thetechnical details of the full CEoffering from Agilent with descrip-tions and examples of relevantfeatures and benefits.

The Agilent CE system is aversatile, fully automated systemfor high performance capillaryelectrophoresis (CE) separations,comprising an Agilent CEinstrument controlled throughan Agilent CE ChemStation.

The Agilent CE system is supportedby a range of specialized capillar-ies, buffers, method developmentand total solutions kits, educational

* for abbreviations see back page

Agilent TechnologiesInnovating the HP Way

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Figure 2The Agilent Capillary Electrophoresis instrument

The Agilent CapillaryElectrophoresis Instrument

The Agilent CE instrument (figure2) contains all functional hardwarefor performing CE separations.The instrument houses a high-voltage power supply, carousel forautosampling and fraction collec-tion, injection system, on-capillaryhigh-sensitivity diode arraydetector, offline buffer replenish-ment station, capillary cartridgeand is capable of accepting an

external gas pressure for perform-ing CEC and CGE. The instrumentincludes features that alloweconomic conversion for CE-MSinterfacing. Each of these isdescribed in detail in the follow-ing sections.

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from valve-induced pressurefluctuations. This results in accu-rate and highly reproducibleinjections (figure 4) as well asexceptional linearity over a broadpressure range, even when injectiontimes are very short (figure 5).When using pressure injection boththe time and the applied pressurecan be selected (time range: 0 to

1000 seconds, pressure range: -50to 50 mbar). Accurate and repro-ducible injection is possible fromvolumes as low as a few µl. ForCEC separations the capillary ispacked with stationary phase andtherefore presents a high back-pressure. In this case the externalpressure source (up to12 bar) can be used to injectsample.

Figure 3Self-correcting pressureinjection profile

Figure 4Reproducibility of corrected peak area for 3sets (N=6) of injections ofp-hydroxyacetophenone separated by CZE

Figure 5Linearity of peak corrected area for MECCseparation of paracetamol with pressureinjected amount (mbar*s)

Sample Injection

Two injection modes are provided:• pressure injection, and• electrokinetic injection.

Although pressure injection is usedmost frequently, electrokineticinjection may be used when thecapillary contains a matrix whichdoes not allow pressure injection(for example with fixed gel CGE),and for selectively injecting onlyone type of ion in non-EOF analy-ses. Injection using either modemay be performed from the longend of the capillary or from thepost-detector (short) end of thecapillary with equal precision.

Pressure InjectionSample introduction into thecapillary is achieved by applyingpressure to the injection vial afterinsertion of the capillary. Thequantity injected is dependent onthe pressure and the length of timethe pressure is applied.

The Agilent CE system uses aunique mechanism which con-stantly controls the pressure andcorrects for the rise-time effects ofvalves and for any leakage duringinjection. Figure 3 shows the profileof pressure against time duringpressure injection. When applied,the pressure to the sample vial isincreased gradually to its maximum(A1). This is then reduced to a valueabout half of the maximum (A2) anda correction time (t2) is inserted.The process is repeated (A3, A4 ) inorder to apply precisely the injec-tion parameters. In this way thetotal area under the graph iscalculated as the product of thepressure and time settings toprovide accurate injection.The pressure system valves onlyoperate at atmospheric pressureavoiding any detrimental effects

Pressure (mbar)

Time [sec]

A1

A2

A3A4

t0 t1 t2 t3 t4

0.62

0.64

0.66

0.68

0.7

0.72

0.74

Set 2 Set 3Set1

0.43% 0.55% 0.39%

Corrected area

0 200 400 600 800 1000

mbar*s

Corrected areaLine of regression

Corrected area35

30

25

20

15

10

5

0

r2 = 0.9999

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Electrokinetic InjectionElectrokinetic injection is per-formed by placing the sample vialat the capillary inlet and applyingan electric field. When the anodeend of the capillary is inserted intothe sample vial the injected aliquotwill not truly represent the samplemake-up. Positively charged ionswill migrate into the capillary dueto their electrophoretic mobilitywhereas neutral species will enterthe capillary through the pumpingaction of the EOF. Anionic specieswill only enter the capillary if theEOF is greater than their ownnegative mobility. Inserting thecathode end into the capillary orreversing the power supplypolarity results in the oppositeprocess. The quantity loaded isdependent on the electroosmoticmobility, the ions electrophoreticmobility and the amount andduration of the applied voltage. Ifthe EOF is suppressed only oneion species will be introduced intothe capillary, depending on thepolarity of the electric field. InCGE with fixed crosslinked gelsapplying sufficient pressure forsample injection may deform thegel or extrude it from the oppositeend. Figure 6 shows the separationof synthetic oligonucleotidesinjected using electrokineticinjection.

Injection ProgramThe Agilent CE Chemstation allowsthe flexibility of defining a numberof functions during the injectionpart of a method. Availableparameters for definition are:• inlet and outlet vials,

injection pressure (-50 to50 mbar for 0 to 1000 s),

• injection voltage (-30 to 30 kVfor 0 to1000 s), and

• wait time before or afterinjection.

This allows a variety of injectiontechniques, for example:• injection of a buffer plug after

the sample plug to minimizesample loss through thermalexpansion,

• injections from multiple vialsreadily allowing the use oftransient ITP or stacking toimprove sensitivity,

• injection from multiple vialsfor online spiking for peakidentification, and

• washing the capillary end bydipping into water and waitingthus reducing the risk ofsample carryover.

Short-end InjectionFull control over sample-vialaccess allows the use of the post-detector capillary length as theeffective separation length. Usingthis functionality, the sample isplaced at the outlet end of thecapillary and a negative pressure isapplied for a set time. By reversingthe polarity of the electric field,the short, 8.5-cm length ofcapillary may be used forseparation with the same degree ofprecision as for normal injection(figure 7). This may be used for:• quick purity check,• rapid screen of analytical

parameters, and• very short analysis time.For more information seeAgilent publication number5963-3403E.

Capillary PVA-coatedleff 24.5 cmid100 µm

Buffer Agilent oligonucleotide bufferInject 7s @ -10kVVoltage -25 kVTemperature 30 °CDetection 260 / 8 nm

DNA filter

Figure 6Oligonucleotide separation using the AgilentOligonucleotide Analysis Kit (part number5063-6530)

Absorbance [mAU]

5 10 15 20 25 30 35

0

10

20

30

40

50

60

Time [min]

5

Electrode

Buffer replenishment

Prepuncher

Capillary

Sample or buffer evaporation isavoided by the combination ofcarousel thermostating and theuse of fully-sealed vials. The vialcaps are pierced in the vialstations immediately beforeinsertion of the capillary andelectrodes or the replenishmentneedle.

Sample vials can be exchanged atany time during an analysis orduring a sequence so that theactual sample capacity can beincreased. For routine analysisonly two vial locations are neededfor the running buffer. Formethod development more than 20different buffers can be tested in a

sequence. In addition, a separatewaste vial can be selected so thatall flushings from preconditioningcan be collected separately toprevent fouling of the runningbuffer. Random access to the vialsenables a sequence of multipleanalyses to be made from a singlesample with different buffersystems which is mandatory forautomated method development.

Random access to all vials alsoallows the carousel to operate asboth buffer and sample tray and tofunction as a fraction collector(see page 10).

Figure 7Short-end injection for fast analysisof paracetamol (1), caffeine (2) and4-hydroxyacetophenone (3) by MECC

Figure 8Sample carousel andlift stations

Sample Carousel

The Agilent CE system is equippedwith a 48-position carousel(figure 8). The carousel can bethermostated simply by connect-ing an external water bath to theinstrument (10 to 40 °C). Two viallift stations are situated around thecarousel to place vials at thecapillary inlet and outlet. Further,a third vial lift station can auto-matically replenish, fill or emptyany vial offline before or duringthe analysis.

The carousel design allowsrandom access to any vial at alltimes. The carousel can be ac-cessed during analyses becausethe vials are lifted above thecarousel when electrophoresis isperformed. The anode and cathodevials can be emptied and refilledwith buffer offline so that novaluable sample locations in thecarousel are occupied by addi-tional buffer vials, thus maximiz-ing the sample vial capacity.

Time [min]0.5 1 1.5

2

2.5

Absorbance [mAU]

0

2

4

6

8

10

Repetitive analyses1

3

6

is of particular importance whenthe buffer is chosen for propertiesother than its buffering capacity(for example in indirect detectiontechniques) or when the chosenbuffer is not a true buffer but isacting merely as an electrolyte. Inaddition when running a series ofsamples at high pH (> 7) thepumping action of the EOF canreduce the buffer volume of theinlet vial — this can result inlaminar flow in the capillary due tounequal vial levels. On averagebuffer replenishment should beperformed every three to fiveanalyses.

High Sample ThroughputThe buffer reservoir for replenish-ment has a capacity of 500 mlwhich ensures sufficient capacityfor > 300 analyses when replenish-ing both inlet and outlet vial aftereach run (figure 9).

Figure 9Extended operation using buffer replenisment Capillary id 50 µm

Length 56 cmBuffer 89 mM Tris-Borate, pH 8.2Injection 100 mbarsVoltage 25 kVCurrent 13 µA

Capillary Cassette andAlignment Interface

The Agilent CE system is equippedwith a cassette that is easilyopened without using tools andallows quick exchange of capillar-ies (figure 10). This allows rapidexchange of capillaries whenchanging methods. Further, theability to rapidly remove andinstall a new capillary ensuresminimum downtime, with theentire process taking less than1 minute.

The cassette contains two feet thatmake leveling of the capillary endseasy and straightforward. Theprecision engineered capillaryalignment interface ensures auto-alignment of the capillary detec-tion window when inserting thecassette into the detector opening.Because capillary alignment canaffect sensitivity it is importantthat the capillary is properlyaligned in the incident light path.The dimensions of the slit are ofmajor importance in reducingstray light and therefore increasingsensitivity and detector lineardynamic range (for more informa-tion see Agilent publicationnumber 5963-1891E). Alignmentinterfaces are available for allstandard capillary dimensions andin addition special interfaces areavailable for use with the AgilentExtended Light Path capillaries(see table 1).

Buffer Replenishment

The Agilent CE system is equippedwith an automated buffer replen-ishment station which can accessany vial before or during ananalysis, empty it, and refill it withfresh buffer from a large volumereservoir (about 500 ml).Replenishment can be pro-grammed to refill vials to a presetlevel to automatically correctunequal vial levels. For moreinformation see Agilent publica-tion number 5963-3296E.

Increased ReproducibilityDuring electrophoresis, electroly-sis of the buffer solutions resultsin changes in buffer compositionand pH. This can result in unstableelectroosmotic flow and can alsocause changes in selectivity. This

Absorbance [mAU]

0

10

20

30

40

Run # 35

Run # 105

Run # 200

Run # 265

1 4

2

3

1 Acetaminophen2 Nicotinic acid3 Acetyl salicyclic acid4 Dihydroxybenzoic acid

Time [min] 100

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The alignment interfaces are color-coded similarily to Agilent capillar-ies to ensure correct selection.Any capillary from a third-partyvendor may be used with thesystem, provided that it has aminimum total length of 33 cmwith a minimum post-detectionwindow length of 8.5 cm and astandard outer diameter of365 µm. The design of the align-ment interface and capillarycassette also provides the neces-sary protection when using packedcapillaries for capillaryelectrochromatography.

Figure 10Capillary cassetteMinimum total length = 33 cm

Minimum effective length = 8.5 cm

Capillary id 75 µmLength 80.5 cmBuffer 105 mM phosphate, pH 2.5Injection 150 mbarsVoltage 30 kVTemperature 35 °CCurrent 130 µAPower 4.8 W/m

Capillary Thermostatting

The Agilent CE system providesstable and efficient capillarythermostating through the use ofhigh-velocity recycling air flow(10 m/s) which is thermostaticallyregulated by a Peltier element.Capillary temperature is one of themany operational parameterswhich can be optimized in capil-lary electrophoresis. Temperatureaffects an analyte’s electrophoreticmobility in CZE separations,partition coefficients in MECC andCEC and even the injected samplevolume through its affects onliquid viscosity. Therefore, stabletemperature control is extremely

Figure 11Reproducibility ofpeptide map of humanhormone throughefficient thermostatingunder high powerconditions

important to ensure precision,reproducibility and selectivity. Byactively monitoring and correctingfor variations in ambienttemperature, the system isindependent of externaltemperature fluctuations. Thecapillary temperature can beregulated between 10 degreesbelow ambient (minimum 4 °C) upto 60 °C. This efficient high-velocity air flow thermostating iscapable of ensuring stable tem-peratures and effective capillarycooling even when operating withhigh concentration, high powerbuffers (figure 11), for moreinformation see Agilentpublication number 5963-3975E.The actual temperature of thecapillary compartment is recordedthroughout the analysis and can bestored with analysis data. Theability to achieve rapidtemperature equilibrium allowsthe use of temperature gradients.

Time [min]10 15 20 25

250

0

AbsorbancemAU [200 nm]

1

2

3

Reproducibility (%RSD)

Peak 1 Peak 2 Peak 3Migrationtime 0.36 % 0.60 % 0.33 %

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adapted from LC detectors, theoptical design of the Agilent CEdetector has been specificallydeveloped for UV-visible absor-bance detection in capillary-shaped flow cells. In conjunctionwith the capillary alignment inter-faces the optical design of thedetector is optimized for bothstandard capillaries and forAgilent Extended Light Pathcapillaries. Wavelength calibrationis performed automatically on re-placing the capillary cassette andcan be performed manually at anytime. The response time of thedetector is sufficient to detect andacquire spectra of high efficiencypeaks with peak widths less than0.02 min.

High Sensitivity and LinearDynamic RangeThe design of the capillary align-ment interfaces reduces theamount of stray light reaching thedetector allowing a linear dynamicrange up to 500 mAU with a 75-µmid capillary, with less than 1 %deviation from linearity. Thistypically covers a concentrationrange of three orders of magni-

tude. The linear dynamic range ofthe detector allows detection of awide range of analyte concentra-tions without resorting to sampledilution. This provides great utilityin the analysis of trace impurities inthe presence of a major componentand alsofor determination of chiral excess.With the use of the AgilentExtended Light Path capillaries thelinear dynamic range is extendedsignificantly beyond 500 mAU(figure 12).

Spectral Acquisition for PeakIdentification and Peak PurityAnalysisThe Agilent CE ChemStation built-indiode array detector allows arange of tasks. Simple spectra,isoabsorbance plots, 3-D plots,and other evaluation can take placeonline while analysis is in progress.For more information see Agilentpublication number 5963-6977E.

The Agilent CE Chemstation, whichcontrols the system, enables fullspectral information to be retrievedas well as single wavelength andmultiple wavelength detection.

Figure 12Analysis of trace impurities in polylysine

High-Voltage Power Supply

The Agilent CE system has apower supply capable of applyingvoltages over the range from-30 kV to +30 kV. The high-voltagepower supply is the heart of anyCE instrument and as such it mustsupply a stable voltage during thecourse of an analytical run. This isachieved by constantly monitoringthe applied voltage throughout theanalysis and actively correcting forvoltage fluctuations. The actualvoltage applied during the run canbe recorded and viewed duringanalysis and stored with otheranalytical data. The power supplyprovides for rapid rise and decaytimes of around 1 second, which isof particular importance wherefraction collection of peaks isperformed (see page 10). Thesystem has safety features built inwhich limit maximum current andpower levels of 300 µA and 6 Wrespectively. The polarity of theapplied voltage is fully controlledby software providing a truepositive or negative polarity withthe outlet electrode held at groundpotential. The software controlenables the construction ofvoltage, current or power gradi-ents, and the application of voltageramps which is of great impor-tance when analysing heat-sensitive analytes in low conduc-tivity solutions.

High Sensitivity Diode ArrayDetector

The Agilent CE system is equippedwith a high-sensitivity diode arraydetector (table 3). In CE the capil-lary itself acts as the detector flowcell. Unlike many detectors in CEinstrumentation, which have been

Absorbance [mAU]

1200

1000

800

600

400

200

0

5 10 15 20 25 30 35 40

Time [min] Time [min]

9.5 10 10.5 11 11.5 12 12.5

14

12

8

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6

4

2

0

Absorbance [mAU]

9

Spectral library searches and peakpurity algorithms can greatlyfacilitate method development inCE. The peak purity algorithm isespecially useful for differentiatingpeak shapes distorted by mobilitydifferences between analyte andrun buffer from those that repre-sent actual impurities (figure 13).

Capillary id Bubble Optical Alignment slit Color(µm) factor pathlength (µm) dimensions (µm) code

25 5 125 120 x 80 Black50 1 50 40 x 620 Green

3 150 145 x 145 Red75 1 75 55 x 620 Blue

2.7 202 200 x 150 Yellow100 1 100 55 x 620 Blue

Figure 14Bubble cell with dye front pushed through tohighlight the extended path

Table 1Capillary internal diameters, bubble factorsand alignment interface dimensions

Figure 13Peak-purity analysis

zone expands radially (across thecapillary) and contracts longitudi-nally (along the capillary), keepingthe total volume constant. This

results in an unchanged zoneconcentration but an increase inoptical path length correspondingto the extension factor (figure 16).

Agilent Extended Light PathCapillaries

To further enhance sensitivity,Agilent Extended Light Pathcapillaries (bubble cell) can beused with the Agilent CE system.These capillaries contain anexpanded diameter section(bubble) at the point of detection(figure 14). The bubble manufac-turing process is computercontrolled to provide tolerances ofless than 3 %. Agilent providescapillaries with a range of normaland extended light paths (table 1).This design results in correspond-ing increases in sensitivity overconventional straight capillaries ofthe same id, for more informationsee Agilent publication number5963-1889E. In the region of thebubble the electrical resistance isreduced and thus the field de-creases. Concomitant to this is aproportional decrease in flowvelocity due to the expandedvolume of the bubble. When thezone front enters the bubble itsvelocity decreases and the zonecontracts or stacks. The sample

min8.12 8.22 8.32 8.42

--> Spectral Data contains impurity <--

min2 4 6 8 10 12 14 16 18

Absorbance [mAU]

50100150200250

nm250 350 450 550

Electropherogram

Spectra overlay Wavelength overlay

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25 µm id capillaries with extendedlight paths of 125 µm can be usedfor applications which use highpower buffers e.g. high salt or SDSconcentrations (see figure 15).The dimensions of the alignmentinterfaces used with the bubblecells are designed specifically tomaintain light throughput by beingextended radially, and to maintainresolution by being reducedaxially. This maintains sensitivitywithout sacrificing resolution.A detailed description is givenin Agilent publication number5963-1889E.

Figure 17Spectral filter foroligonucleotideanalysis

Figure 15Separation of protein standards in a25 µm id capillary with bubble using 150 mMphosphate, ph 7.0 with 200 mM ammoniumsulfate

Figure 16Mechanism of zoneconcentration usingAgilent Extended LightPath capillaries

Capillary Gel ElectrophoresisOperation

High Pressure for ViscousReplaceable GelsThe Agilent CE system can acceptan external gas supply up to15 bar which is internally regulatedfrom 2 to 12 bar. Provision ofhigher pressure is of benefit in theCGE analysis of proteins andoligonucleotides where highlyviscous replaceable gels are used.When using a viscous polymermatrix, necessary for high resolu-tion SDS-PAGE, dsDNA andoligonucleotide separations,capillaries of smaller internaldiameter (� 50 µm) require rela-tively high pressure (about8 bar) in order to replace thecapillary volume within a reason-able inter-analysis time frame. Withthe high-pressure option availableas a core composite of the Agilent

CE system, such separations in thebioscience field are made moreaccessible. High pressure is alsoinvaluable for flushing 25 µm idcapillaries.

Spectral FilterLinear polyacrylamide (LPA) isused in a variety of capillary gelelectrophoresis applications and isalso used in some coated capillar-ies. However, LPA is susceptible todegradation when exposed to highintensity light sources at low UVwavelengths. In order to facilitatethe use of LPA and LPA-coatedcapillaries without resorting tochanging the detector, Agilent hasdeveloped spectral filters designedspecifically for use in nucleic acidanalysis. For more information,

see Agilent publication 5963-9807E. This filter allows higherlight trans-mission levels than areavailable with conventionalinterference filters which give lighttransmission of about 15 % found infixed wavelength detectors. Foranalysis of nucleic acids thespectral filter provides high light

Absorbance [mAU]

50

40

30

20

10

0

0 2.5 5 7.5 10 12.5 15 17.5 20

Time [min]

50 µm 150 µm

22

33

11

10.00%

30.00%

50.00%

70.00%

190 290 390 490 590Wavelength [nm]

Transmission

60.00%

40.00%

20.00%

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transmission at the detectionwavelength range of 254 – 260 nm(bandwidth 4 nm) with lowertransmission levels at 360 nmwhich is necessary for cassetterecognition (figure 17). The lightthroughput at 260 nm is about 68 %transmission. This is much largerthan is found with fixed wave-length detectors, which useintereference filters. In this casethe light throughput is around 15 %resulting in significantly higherbaseline noise. The spectral filteris also recommended for use withthe Agilent ssDNA kit whre itsignificatntly extends its lifetime(figure 18). For more informationsee Agilent publication number5963-9870E.

Figure 18Long term stability testing of CGE foroligonucleotide analysis using spectral filter

Fraction Collection usingPressure Elution

Pressure elution of fractions fromthe capillary can be used in most CEmodes. The user must inputinformation on the time taken formigration of the buffer from theinlet to the detection window usingthe elution pressure of 50 mbar(figure 18). This low pressureensures minimum distortion ofother analyte zones still within thecapillary and allows collection ofmultiple fractions from a single run.The software calculates when thepeak will reach the end of thecapillary and places the collectionvial at the outlet end at the appropri-ate time. Fractions may be collectedin as little as 5 µl of solvent. Figure18 shows the reinjection of apeptide fraction collected bypressure elution from separation ofa protein tryptic digest.

Fraction Collection

Although in capillary electrophore-sis only very small quantities ofanalytes are injected, fractioncollection can be desirable forfurther analysis using a differentCE mode, protein sequencing,mass spectrometry or other offlineanalytical techniques. TheAgilent CE system offers fullyautomated, software-controlledfraction collection in threedifferent modes. Fractions maybe collected by pressure elution,by electrokinetic elution or fromCIEF analyses. The fractioncollection mode is selected fromthe Instrument menu of thegraphical user interface, for moreinformation see Agilentpublication numbers 5963-1251Eand 5963-3506E .

In each case the DAD signal isused as the peak sensor, allowngtime-independent fraction collec-tion. In all three modes up to fourpeaks may be collected individu-ally by defining their migrationtime, or multiple peaks over a settime range can be collected. Thepeak detector may also be used tospecify collection of those peakswhich have an absorbance above auser-defined threshold. Becausethe sample carousel also acts asthe fraction collection vial holder,the location of the vial for fractioncollection can be specified. Wheremultiple fractions are beingcollected the Agilent CE instru-ment will collect the first fractionin the specified vial and subse-quent fractions in correspondinglyincremented vials. For fractioncollection it is essential that thepower supply decays in < 1 s.

mAU

5 10 15 20

0

10

20

30

40

50

60

70

80Run # 73

Run # 1

Run # 49

Exemplary RSD (n=6)for migration time: 0.35%

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Figure 21Window for enteringelectrokinetic elutionfraction collectionparameters

Figure 19Window for enteringpressure elution fractioncollection parameters

Figure 22MALDI-TOF analysis of single fraction collected from CGE oligonucleotideseparation

Figure 20Reinjection of pressure collected fraction from peptide map single run usingCZE

Fraction Collection usingElectrokinetic Elution

In this case the user is promptedfor the capillary dimensions andtime of the peaks of interest

(figure 21). Electrokinetic elutionof fractions is particularly appro-priate when the capillary containsa pressure resistant matrix, forexample, fixed gels, viscouspolymers or packed solid phaseparticles (figure 22).

20 25 30 35 40

0

80

Absorbance [mAU 200 nm] Peak 4

reinjected

Time [min]20 30 40

1

23

4

5

6CE

X Y2

4

Time [min]

Time [min]20 30 40

0

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2600 3000 3400m/z

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1100

1300

1500

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Abundance

3230.1Da

Mass peak fromsingle fractioncollection

Fraction collected

13

Time [min]14 16 18 20 22 24 26

0

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50

Absorbance [mAU]

75

100

125

150 7.2 6.6 5.9 5.4

WQQQSYLDSGIK

15

10

5

0

2 2.5 3 3.5 4Time [min]

Absorbance [mAU]

Figure 23Window for inputting CIEFfraction collection parameters

Fraction Collection from CIEF

In CIEF pressure and voltage areapplied simultaneously to mobilizethe separated components whilemaintaining the integrity of the

Figure 24Refocussing of proteins isolated frommicropreparative by CIEF

Figure 25Graphical representation of EOF generation inCEC

sharply focused zones. Using thefraction collection option for CIEFthe user provides information onthe capillary dimensions andmigration time using pressure(figure 23).

Capillary Electro-chromatography (CEC)

CEC is a true fusion of CE and LCand is, at present, an emergingtechnique in the first excitingstages of investigation. Thetechnique may be used to separateneutral molecules, or mixtures ofcharged and/or neutral species.Similarly to LC, separations areachieved through partition be-tween a mobile phase and astationary phase. The motive forcewhich propels neutral moleculesthrough the capillary bed is theelectroosmotic flow (EOF). Thebenefits of this are that the EOFhas a uniform flow-velocity profileunlike LC which provides a morevariable flow profile with anincreased contribution to band

broadening from eddy diffusion.The more uniform velocity flowprofile greatly reduces this majorcontribution to band broadening.

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Figure 26Part of the graphical userinterface for CEC operationwindow

Column Spherisorb ODS1, 3 µm250 x 0.1 mm

Mobile phase 80/20 ACNTris-HCl50 mM, pH 8

Voltage 25 kVInjection 5 k, 3 sPressure 10 bar both sidesTemperature 20 °CPlate number 50,000 – 60,000Symmetry 0.95 – 0.98

Figure 27CEC separation of polyaromatic hydrocarbons

to either or both of the bufferreservoirs. The external gaspressure source should be oil-freeair or nitrogen.

The CEC operational mode isselected from the graphical userinterface which changes toindicate that an external highpressure source is connected andto indicate the external pressuresupplied (figure 26). The AgilentCE system controls both the rate

Since the flow is generated withinthe capillary, longer columnscontaining very small particles (<3 µm) may be used which areprohibited by pressure constraintsin LC. For a comprehensivedescription of the technique seeAgilent publication number 5964-5930E. Typically, in CEC separa-tions, microbubble formation canoccur as a consequence of apply-ing high voltage across a packedcapillary bed. This phenomenoncan be avoided by ubiquitousapplication of pressure to bothinlet and outlet vials whicheliminates bubble formation andseparation breakdown, whileintroducing no laminar flowbecause of the equally appliedpressure.

The Agilent CE system is designedto internally regulate an externalpressurized gas supply (� 15 bar)to provide single or dual-vialpressurization at 2 to 12 bar.Regulated pressure can be applied

of application of pressure and thepressure applied. All CEC function-ality is included in the Agilent CEinstrument hardware and softwarecontrol is provided as standard inthe Agilent CE ChemStation.Although an external gas supply isneeded for high-pressure and CECoperation (see figure 27), theAgilent CE instrument is fullyfunctional for all other CE modeswithout this external gas pressure.

Absorbance [mAU]

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0

1 2 3 4 5 6 7 8Time [min]

Thio

urea

Nitr

oben

zene

Nap

htha

lene

Biph

enyl

Anth

race

ne

Fluo

rant

hene

Fluo

rene

15

Figure 28Single point instrument controlfor CE-MS

Capillary Electrophoresis/Mass Spectrometry

The interfacing of capillary electro-phoresis with mass spectrometricanalysis is widely viewed as anoptimum conjunction of separationpower with a universal detectionprinciple which also provideshighly selective identification data.The Agilent CE instrument is fullyfitted as standard with the featuresnecessary for conversion toCE-MS operation. This providesa versatile and fully automatedfront end for CE-MS separations.Designing the Agilent CE instrumentsuch that it has built-in featureswhich enable its conversion toCE-MS capability greatly reducesthe cost of opting for CE-MSanalysis.

The Agilent CE-MS adapter kit(part number G1603A) includes allparts to allow interfacing of theAgilent CE to most ESI-MS instru-ments and includes:• capillary cassette designed to

allow thermostating of thecapillary throughout its positionwithin the instrument (figure 29),

• ground cable,• capillary safety sleeve,• warning labels, and

• plastic UV capillary interface toallow tandem UV/MS detection(figure 30).

The capillary can be passedthrough the DAD for methoddevelopment or directly into theMS for routine operation. TheCE-MS probe for non-Agilent MSshould be purchased from the MSvendor. The Agilent CE sprayer kit(part number G1607A) is designed

to interface perfectly with theAgilent 1100 Series MSD and theEsquire-LC Ion Trap LC-MS (n). TheAgilent CE ChemStation suppliedas standard already contains thenecessary software for CE-MSoperation. CE-MS option may beselected from the GUI toolbar.When using CE-MS software (partnumber G2201AA) the Agilent1100 Series MSD and the Agilent1100 Series isocratic pump used tosupply the sheath liquid can becontrolled directly from the GUI asshown in figure 28 (For moredetailed information refer topublication number 5968-1328E).

Figure 29CE-MS cassette design andits position within theinstrument

Air cooling

Detectorinterface

Cassette

Agilent

Optional outletfor regular CE mode

Inlet

Capillary

to MS

Sheath flowsettings, ... ... data acquisition

settings

spray chambersettings, and...

16

Figure 30CE-MS of peptide mixture

Figure 31Graphical userinterface for CE

Agilent CE ChemStation

The Agilent CE ChemStationcomprises instrument control anddata handling software, personalcomputer and printer. The soft-ware runs within Microsoft®

Windows and MS-DOS® operatingenvironments. Data analysisprocedures range from standardintegration protocols and report-ing to advanced spectral librarysearches and peak-purity analysis.The Agilent CE ChemStation ispart of a standardized platform forrunning CE, GC and LC instru-ments from Agilent Technologies.

Instrumental ControlThe Agilent CE system is operatedfrom the Agilent CE Chemstationusing a graphical user interface(figure 31). A schematic diagramof the Agilent CE instrument

guides the user through theprogrammable parameters. Thisinterface also allows direct accessto all method parameters, includ-ing control of the vial tray, capil-lary flushing, and so on. The useris informed about the actual statusof the analysis by real time indica-tors.

CE Mode SelectionThe CE operational mode may beselected from the GUI. This allowsoperation in all normal CE modesand additionally includes CEC,CGE and CE-MS. The GUIchanges to indicate the opera-tional mode selected.

200

Time [min]0 2 4 6 8 10

0

50

100

150

250

300

5 10 15 20 25

10000

20000

30000

40000

50000

Abundance

Time [min]

Angiotensin IIDRVYIHPFMW 1046.2

UVAbsorbance [mAU]

Angiotensin IDRVYIHPFHLMW 1296.5

MS-TIC

Angiotensin IAngio-tensin II

17

Method and sequenceprogrammingMethod programming takes placefrom a single screen using easy torecognize icons (figure 32). Theintuitive design of this interfacereduces the time necessary forfamiliarization and facilitates rapidtraining. Table 2 lists all themethod parameters that can beprogrammed. Individual methodsand sample series can be com-bined in a sequence to fullyautomate any series of analysismethods and samples.

Figure 32Method set-up window

Sequences for MethodDevelopment and High SampleThroughputSequences may be constructed tocontain any number of methodsand to analyze numerous samples.This is of major benefit in methoddevelopment where numerousmethods with varying operationalparameters may be used tooptimize a separation. Additionallydifferent buffer vials may beprogrammed within methods. Thisallows automated method develop-ment which may be performedunattended.

Full access is available to thesample tray during operationallowing samples to be removedand replaced during a sequencethereby increasing the samplethroughput available. Additionallysequences can be updated ormodified while the sequence isrunning.

Data Analysis and Reports

The ChemStation provides forautointegration, integration andmanual integration.• Autointegration can be used to

set up initial integration para-meters based on characteristicsignals.

• Integration integrates theelectropherogram according tothe values in the integrationevents table. This may be usedto adjust integration results bychanging integration events andreintegrating the signal.

• Manual integration may beperformed.

The user has a choice of 15different report styles or maychoose to customize the report tosuit their own requirements(table 3). There are a number ofCE specific parameters which areincluded in the report and CEspecific functions for calibration.

CE Specific data reports andanalysis:

• corrected area (peak area /time),

• apparent mobility and effectivemobility, (see publicationnumber 5968-2232E)

• MW determination from SDS-protein or DNA analyses,

• pI determination for CIEFanalyses.

Quantitative reporting of peakareas as corrected peak area (area/time) is required for accuratereporting of peaks separated byCE, and is mandatory for chiralanalyses. Data analysis optionsalso include the ability to performsequence summary reports ofcalibrated compounds. This is amajor benefit in performingprecision calculations for systemsuitability.

18

Table 2Programmableinstrumentalparameters

Function Parameters/ Settings Values

Power supply Polarity Positive or NegativeVoltage 0 – 30 kVCurrent 0 – 300 µAPower 0 – 6 W

Capillary conditioning Inlet /outlet vial Vial number 1– 48Wait 0 – 1000 minVoltage 0 – 30 kVCurrent 0 – 300 µAPower 0 – 6 WFlushing 1 barPressure 0 – 50 mbar (programmable)High pressure 2 – 12 bar (includes HV)

Both: Inlet/outlet 2 – 12 bar with/outsimultaneous voltage

Buffer replenishment Inlet/outlet Vial number 1– 48Time 0 – 1000 minBuffer level 0 – 1.8 cm

Injection Pressure 0 – 50 mbar0 –1000 s

Electrokinetic 0 – 30 kV0 – 300 µA0 – 6 W0 – 1000 s

Program Vial number 1 – 48Voltage (0 – 30 kV)Current (0 – 300 µA)Power 0 – 6 WPressure 0 –50 mbarFlushing 1 barHigh pressure 2 –12 barWait 0 –1000 min

Detector Wavelength 190 – 600 nmReference wavelength 190 – 600 nmBandwidth 2 – 400 nmPeak width 0.01 – 1 minResponse time 0.1 – 20 sSpectral data rate 0.16 – 5 spectra/sSignals 5 simultaneouslySpectra All spectra

All in peakPeak baseline and apexThreshold

Capillary cassette Temperature 10 degrees below ambient to 60°C(not below 4°C)Full capillary details entered in a table.

Sample carousel Temperature 10 – 40 °CVials 1– 48

Timetable Voltage -30 – 30 kVPower 0 – 6 WCurrent 0 – 300 µAInlet vial 1 – 48Outlet vial 1 – 48Capillary temperature 10 degrees below ambient to 60 °CLower current alarm limitPolarity Positive or negativePressure 0 – 50 barHigh pressure 2 – 12 bars with/out simultaneous voltage

Fraction collection ElectrokineticPressureCIEF

Figure 33Typical electropherogram for OQ/PV testsample (p-hydroxyacetophenone) illustratingshort run time and spectra

Time [min]0.5 1 1.5 2 2.5 3 3.5 4 4.5

mAU [192/4nm]

0

10

20

30

40

Wavelength [nm]200 300 400

mAU

010203040506070

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Report Style Features

Short Quantitative text results of all integrated signals

Electropherogram + short Electropherogram plus quantitative text results

Electropherogram + detail Electropherogram, quantitative text and calibration curves

Header + short File header, quantitative text results.

GLP + short Header, sample information, instrument conditions, logbook, electropherogram, quantitative results

GLP + detail Header, sample information, instrument conditions, logbook, electropherogram, quantitative results and calibrationcurves

Short + spectrum Instrument conditions, electropherogram, quantitative results and peak purity plots

Detail + spectrum Header, instrument conditions, electropherogram, quantitative results and peak purity plots

Full Header, sample information, instrument conditions, electropherogram, quantitative results and peak purity plots

Library search Produces a calibrated report including library search results, peak numbers, ,migration times, librarysearch matchfactors, amounts, compound names

Performance Migration time, peak are, peak height, signal description, true half-height peak-width, symmetry, efficiency andresolution

Performance + library search Combines the performance and library search styles

Performance + noise Combines the performance report style with noise calculations

Performance + extended All parameters from the peak performance calculation and individual plots of each peak. Additionally - peak startand end, skew, excess, peak width, USP tailing factor, tie interval between data points, number of data points,statistical moments, plates, plates per metre, selectivity and resolution; all method relevant information (instrument,capillaries, sample and acquisition parameters and electropherogram)

HPCE mobility Quantitative text results plus apparent mobility

Provision for RegulatoryCompliance

Operation Qualification /Performance Verfication(OQ/PV)In the use of capillary electro-phoresis in regulatory compliantenvironments there is an increas-ing demand for operationalqualification/performance verifica-tion (OQ/PV). Using experience inregulatory issues and in capillaryelectrophoresis, Agilent Technolo-gies has developed a kit whichprovides instructions, methodsand materials for performing OQ/PV for the Agilent CE system. The

Table 3Report styles available fromAgilent CE ChemStation

procedure tests the followinginstrumental parameters:• temperature accuracy and

stability,• voltage accuracy and stability,• detector noise and drift,• detector wavelength accuracy

internally (holmium oxidefilter),

• detector wavelength accuracyexternally (using test analyte),

• detector linearity,• injector reproducibility,• injector linearity, and• replenishment system

functionality.

The tests has been developed toidentify and test functions of anAgilent CE system which shouldbe validated for regulatory compli-ance.

Installation Qualification (IQ)Agilent can also supply theappropriate test procedures andcertification necessary forinstallation qualification as an aidto the validation of methods andoperation within regulatorycompliant environments.

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Capillary Electrophoresis Kitsand Accessories

Agilent Technologies offers a rangeof capillaries, buffers, kits formethod development and totalsolutions for CE analysis (table 4).Full details and ordering informa-tion may be found in the AgilentCE supplies catalog. Additionallynumerous applications andeducational literature are availablein the form of application notesand booklets, CE and GLP prim-ers, and scientific publications(figure 35).

Agilent TechnologiesApplication Support

Agilent Technpologies applicationsupport includes educational andapplications materials:• primers and CD-ROMs covering

introduction, theory, applications and GLP requirements,

• a wide range of applicationnotes in bioscience, food andpharmaceutical analysis, and

• an international network of CEspecialists.

Capillaries • Bare fused silica capillaries of varying dimensions with precision cut endsfor accurate and precise quantitation

• Agilent Extended Light Path capillaries for high sensitivity applications• PVA coated capillaries for protein analysis and suppressed EOF analyses• CEP coated capillaries for suppressed EOF analysis and replaceable gel CGE• ODS packed capillaries for CEC

Buffers • CZE buffers covering a wide pH range• MECC buffers for CE of neutral molecules• Gel buffers for DNA analysis

Kits • Chiral method development kit• dsDNA analysis kit• ssDNA analysis kit• Inorganic anion analysis kits• Organic acids analysis kit• OQ/PV and IQ kits for regulatory compliance• Cation solutions kit• Forensic anion solutions kit• Plating bath analysis kit

Table 4Kits and accessories for CE available fromAgilent Technologies

List of abbreviations

CE Capillary electrophoresisCZE Capillary zone electrophoresisMECC Micellar capillary electrochromato-

graphyCGE Capillary gel electrophoresisCIEF Capillary isoelectric focussingCE-MS Capillary electrophoresis/mass

spectrometryOQ/PV Operational qualification/

performance verificationIQ Installation qualificationCEC Capillary electrochromatographyLC Liquid chromatographyGUI Graphical user interfaceSDS-PAGE Sodium dodecylsulfate-

polyacrylamide gel electrophoresisMW Molecular weightLPA Linear polyacrylamidedsDNA Double-stranded deoxyribonucleic

acid

Many of the scientific articles andapplication notes may be accessedthrough the Agilent home page onthe worldwide web at:http://www.agilent.com/chem

Agilent TechnologiesInnovating the HP Way

The information contained in this note issubject to change without notice.

Microsoft® and MS-DOS® are U.S. registeredtrademarks of Microsoft Corporation.

Copyright © 1996-2000, Agilent TechnologiesAll Rights Reserved. Reproduction, adaptationor translation without prior written permissionis prohibited, except as allowed under thecopyright laws.

Publication Number 5965-6512E