Agilent capillary electrophoresis systemNew dimensions in capillary electrophoresis

2

The Agilent CE system – new dimensionsin capillary electrophoresis

Capillary electrophoresis (CE), withits high efficiency and resolution,rapid analysis time, plus minimalsample and solvent requirements, is an established technique in manylaboratories. Its flexibility covers a broad range of applications in a wide variety of industries, from drugdiscovery and development throughquality control and ion analysis.

Agilent Technologies is the leadingglobal partner for chromatographyand capillary separations. When youselect Agilent as your partner ofchoice you can be assured of qualityinstrumentation, solutions andservice.

Agilent's CE system is the first choice for capillaryelectrophoresis offeringbenefits such as:

• best in class performance andlegendary Agilent reliability

• integrated Agilent ChemStation 32-bit software

• flexibility in separation techniques,including capillary electrochromato-graphy (CEC) and capillary electro-phoresis mass spectrometry (CE/MS )

• world wide service and support

• laser induced fluorescence (LIF)detection upgrade possible throughAgilent partner, Picometrics, seewww.picometrics.com

Checklist

✓ HPLC-like sensitivity and linearity

✓ HPLC-like reproducibility

✓ Self-correcting injection system

✓ Enhanced integrator

✓ Replenishment system

✓ CEC

✓ Single vendor CE/MS solution

✓ Multitechnique software

✓ Regulatory compliance tools

Multitechnique 32-bit software

• Familiar Agilent ChemStation look-and-feel

• Easy to use graphical user interface

• Increased productivity and reducedtraining costs

HPLC-like sensitivity, linearityand reproducibility

The diode-array detector incorporatedin the Agilent CE system offers

• quantitative and qualitative analysiswith excellent sensitivity

• wide linear detection from 190-600 nm

• full spectral capabilities

In addition, the sensitivity and linearrange can be further enhanced withAgilents's extended light path capillariesand high sensitivity detection cell, orwith the laser induced fluorescence (LIF)detector from Agilent partner Picometrics.

The Agilent CE system uses advancedtechnologies for instrument control suchas automated buffer replenishment.Combined with advanced data processingthis ensures HPLC-like reproducibility.

Fully integrated CE/MSsolutions

Agilent offers a fully integrated CE/MSsolution with all system componentsand support coming from Agilent. TheAgilent CE system combines seam-lessly with all models in the Agilent6000 Series, including quadrupole,time-of-flight, ion trap, triplequadrupole and quadrupole time-of-flight MS systems. A uniquefeature of the CE-MS combinationis their ease of use – the MScontrol is smoothly integrated intothe Agilent ChemStation software.The Agilent spraying system needsno positional adjustments, and theunique design of the MS inletmakes the CE separationconditions independent of the MSoperation conditions.

3

High sensitivitydetection cell

75 µm i.d. capillary

The Agilent high sensitivity detectioncell provides a 10-fold increase insensitivity, unsurpassed spectralfidelity and an extended linear range.All with a unique decoupled celldesign allowing simple capillaryreplacement.

• Agilent 6100 Series quadrupole MS for nominal mass detection

• Agilent 6210 time-of-flight MS for ultra-fast accurate mass

• Agilent 6300 Series ion trap MS for sensitive MSn

• Agilent 6410 triple quadrupole MS for accurate MS/MS quantification

• Agilent 6510 quadrupole time-of-flight MSfor MS/MS structural information withaccurate mass

Time [min]4 6 8 10 12

Absorbance [mAU]

0

25

50

75

100

125

150

175Rf

8.6 kDa16.9 kDa

29 kDa

66 kDa

205 kDa

log MW

0

0.5

1

1.5

2

2.5

0 0.5 1

log MW

4

In today’s demanding pharmaceuticalindustry, fast and robust separationmethods are needed in order toreduce the time to market for newdrugs. Capillary electrophoresis, thepowerful instrumental approach toelectrophoresis, responds to theseneeds in all stages from drug discoverythrough quality control. CE offers fast separations with orthogonalselectivities to chromatographictechniques. In addition, the integrationof CE into the US Pharmacopeia as astandard physical test – initiated in1996, ensures its acceptanceworldwide.

Agilent has made a long-termcommitment to the pharmaceuticalindustry. Changing regulatoryrequirements are monitored all overthe world to ensure you get the rightproduct features and services – alarge number of applications andsolution kits have already beendeveloped as part of this process.

High sensitivity SDS-protein separation by capillary gel electrophoresis.

Checklist

Method:

✓ Fast separation

✓ Robust

✓ Low cost per analysis

✓ Regulatory compliance tools

✓ Simultaneous quantification of main component and tracecontaminants

Hardware:

✓ Agilent high sensitivity detectioncell

✓ Replenishment for unattendedoperation

✓ CEC capability

✓ Single vendor CE/MS solution

Software:

✓ Multitechnique software

✓ Self-explanatory user interface

✓ Direct access to instrument control

✓ Enhanced integrator for CE peaks

✓ Mobility calculation for improvedreproducibility

✓ Integrated CE/MS control

Vitamins

CE solutions – for discovery,development and quality control of drugs

Proteins

5

Chiral drug compounds

Regulatory authorities are increasinglyrequesting that the pharmaceuticalindustry assess the enantiomericpurity of their chiral building blocks,intermediates and final drugsubstances. This, coupled with theever increasing demands imposed on the analyst to reduce methoddevelopment times, makes chiral CE a very attractive technique. It offersunprecedented chiral discriminationthrough its high resolving power, chiral selector versatility and rapidautomated method development.When used with the high sensitivitydetection cell the simultaneousquantification of the main componentand trace impurities below 0.1 % ispossible without sample overloading.

Peptides

Chirals5 10 15 20

0

20

40

60

80

100

120

140

160

1

23

4

5

67

8

Time [min]

Absorbance[mAU] at 200 nm

1: VHLTPVEK2: DRVYIHPF [Angiotensin II]3: SIGSLAK [Somatostatin]4: AGCKNFFWKTFTSC5: ELYENKPRRPWIL [(Trp)-Neurotensin]6: LIEGKRPW [Xenopsin]7: WMDF8: YGGFL [(Leu)-Enkephalin]

Electropherogram of a peptide sample analyzed by capillary zone electrophoresis.

Electropherogram of trace impurities in a chiral drug compound.

GlycoproteinsAbsorbance [mAU]

500

400

300

200

100

0

0.05 % area/area

S-form

R-form

0 10 20 30 Time [min]

6

2.5

2

1.5

1

0.5

0

-0.5

Absorbance [mAU]

Time [min]

Chlo

ride

Sulfa

te

Nitr

ate

Nitr

ite

Oxa

late

Citr

ate Fl

uorid

e

Form

ate

2.52 3 3.5 4 4.5

Capillary electrophoresis is a truealternative to ion chromatography. Its main features include:

• unmatched analysis time –enhancing your sample throughput

• high resolving power – allowinganalysis of complex samples in onerun

• typically less than 1 ml of solventrequired with only a small amountof waste – saving money andhelping to protect the environment

• no need for specialized columns,precolumns and membranes – easyinstrument setup and maintenance

• often no laborious samplepreparation needed – just sampledilution

• only nanoliter amounts of samplesinjected – allowing differentmethods or analytical techniques tobe applied to one sample

Power plants

Ionic contamination in primary andsecondary circuit water leading tometal corrosion, is a major problem forthe nuclear power generating industry.For this reason low levels of small

anions and cations must be monitored.CE provides a rapid and facile methodfor their determination in the low ppbrange.

Semiconductor industry

Capillary electrophoresis, as a micro-analytical technique, features merits in the determination of contaminantson wafer surfaces. As the diameterincreases, the financial value of the wafers increases exponentially,therefore it is mandatory to apply a great variety of different analysistechniques when working on onesingle wafer. This is possible with CE due to the low sample volume.Moreover, by changing only thecomposition of the electrolyte, a broadvariety of analytes in different matricescan be determined. CE covers a rangefrom ionic to non-ionic analytes thatare important in the semiconductorindustry.

Electropherogram of contaminants on semiconductor wafer surfaces.

Electropherogram of ionic contaminants in circuit water.

Absorbance [mAU]

3 4 5 6 7 8 9Time [min]

Bro

mid

eCh

lorid

eSu

lfate

Nitr

ate

Nitr

iteO

xala

te

Chlo

rate

Fluo

ride

Form

ate

Phos

phat

e

Ace

tate

Checklist

✓ Ease of sample preparation

✓ Low sample consumption

✓ Fast separation

✓ Minimal maintenance

✓ Low cost of ownership

✓ Low ppb sensitivity

CE solutions – the alternative for ions

7

Pulp and paper industry

Recycling for cost reduction andenvironmental concerns necessitatesthe monitoring of Kraft liquors foranionic content. The caustic nature ofthe samples results in labor intensivemaintenance procedures. CE analysesare three times faster and requireminimal sample preparation comparedto ion chromatography.

Plating bath industry

In the plating bath industry, themonitoring of additives in bathsolutions or waste is essential forquality control and cost saving. Theanalysis by capillary electrophoresis isadvantageous over ion chromatographywith respect to sample preparation,resolution and simplicity.

Food and beverages industry

Validation of CE for organic acidanalysis in grape juice and wine hasenabled replacement of other methodslike ion chromatography (IC), or HPLC.Major advantages are, speed ofanalysis – six major organic acids inless than 13 minutes; instrumentalsimplicity; its adaptability in a routineQC lab – currently running 24 hours; no sample cleanup; low cost of suppliesand maintenance, and minimal wastedisposal.

2 2.5 3 Time [min]

Green liquor1:200 dilution in water

S2O

32-

Cl- SO4

2-

S2-

SO32-

(Data kindly provided by Dwayne Van, Longview Fibre, Longview WA, USA)

Electropherogram of anions in Kraft liquor.

Electropherogram of additives in plating bath solution.

Absorbance [mAU]

-15

-20

-25

-30

-35

-40

Sulfa

te

Mal

ate

Phos

phat

eA

ceta

te

Hyp

opho

sphi

te

Lact

ate

Nic

kel

2 3 4 5 Time [min]

Electropherogram of organic acids in grape juice.

Absorbance [mAU]

-20

-25

-30

-35

-40

-45

Chlo

ride Su

lfate

Tart

rate

Mal

ate

Citr

ate

Pyru

vate

Succ

inat

e

Phos

phat

eLa

ctat

eA

ceta

te

3 4 5 6 7 8 Time [min]2

8

Capillary electrochromatography(CEC) is a fusion of the techniques ofcapillary electrophoresis and liquidchromatography, resulting in anincrease in efficiency by as much as an order of magnitude overconventional HPLC.

This has three specific benefits:

• separation of closely relatedcompounds

• shorter run times and increasedsample throughput, and

• substitution of gradient HPLCmethods by isocratic CEC.

CEC uses capillary columns packedwith LC stationary phases. As in LC,CEC separations are achieved by thepartition of a solute between themobile and stationary phases. Theadditional separation by mobilitiesresolves charged and neutralcomponents.

In CEC, the pressure driven flow of LCis removed and replaced by anelectrically driven flow – the electroosmotic flow (EOF). This means:

• no pump with moving mechanicalparts or seals,

• elimination of Eddy diffusion,resulting in increased efficiency, and

• column length and particle size thatare not restricted by a pressure limit.

Robust, longterm operation of CECcolumns requires pressurization ofboth capillary ends. This is available asa core function of the Agilent CEsystem.

CEC’s vastly increased resolving powercoupled to the high speed analysiswhich this technique also offers, isessential in a modern pharmaceuticalindustry which demands the separationof increasingly complex mixtures.

Checklist

✓ Microanalytical technique

✓ Separation of closely relatedcompounds without gradient

✓ High sample throughput

✓ Chromatographic selectivity plusseparation mechanism by charge

CEC – for chromatographic selectivityand highest efficiencies

Separation of an EPA PAH standard by CEC.

8

6

4

2

0

1 2 3 4 5 6 7 8 9 Time [min]

Absorbance [mAU]

1

2

3, 45

6

7

8

9 10

11

1213

1415

16

Radius

Flow velocity

Velocity profileChannel Particle

LC

CEC

Pressure drive

Electro osmotic drive

Absorbance[mAU]

Time [min]

9

Agilent's spraying system

Fully integrated and

automated, Agilent's

CE/MS software

guarantees ease of use

from method development

to data processing.

CE/MS combines the short analysistime and high separation efficiency of CE with the molecular weight andstructural information from the MS.The technique has been successfullyused for the analysis of biopolymers,drugs and drug metabolites, andagrochemicals, among others.

Unlike other systems, Agilent offers a fully integrated solution – all thesystem components come from onevendor and are controlled by onesoftware package. In addition,Agilent's unique design featuressurpass previous limitations ofCE/MS.

• The spraying system from Agilent is arranged at right angles and needs no positional adjustments,enabling very simple and stableoperation and allowing the use ofmoderate amounts of conventionalelectrophoretic buffers or additives,for example phosphate orcyclodextrins.

• The nebulizer is operated at groundpotential increasing the effectivefield strength over the separationcapillary and making the CEseparation conditions independentfrom the MS operation conditions.

• The high-efficiency solvent dryingsystem ensures excellent sensitivity.

• The Agilent CE system integratesseamlessly with all models in theAgilent 6000 Series, includingquadrupole, time-of-flight, ion trap,triple quadrupole and quadrupoletime-of-flight MS systems.

• An adapter kit facilitates connectionof the Agilent CE system to any MSinstrument.

CE/MS – one vendor, one software, one solution

Peptide separation with simultaneous UV and MS detection.

MW 1060.2 BradykininMW 1296.5 Angiotensin IMW 1672.9 NeurotensinMW 1046.2 Angiotensin IIMW 379.4 VYMMW 555.6 Leu-EnkephalinMW 573.7 Met-Enkephalin

UV:

Abs

orba

nce

[m

AU

]

Time [min]

MS:

Abu

ndan

ce

(M+

2H)2+

530

.9

(M+

3H)3+

433

.1

(M+

3H)3+

558

.5

(M+

2H)2+

523

.8 (M+

H)+ 3

80.3

(M+

H)+ 5

56.2

(M+

H)+ 5

74.2

10

Typically, there are five steps requiredfor full instrument, method and datavalidation. Agilent offers softwaretools and kits to facilitate andautomate these steps for the AgilentCE system.

Step 1: Qualify your vendor and yourvendor’s design

Agilent provides documented evidencethat the CE system has been validatedduring development according tostandardized procedures.

Step 2: Qualify the instrument in yourlaboratory before beginning operation

Follow the appropriate test proceduresand certification for InstallationQualification (IQ) and OperationQualification/Performance Verification(OQ/PV). Agilent offers a kit whichprovides instructions, methods andmaterials for performing OQ/PV for the Agilent CE system.

Step 3: Validate your analyticalmethod

The Agilent ChemStation includessystem suitability software forautomated method validation.

Step 4: Qualify system performanceduring routine operation

Agilent’s software features automateddaily system suitability testing, with an optional database featuring onlinequality control charts for documentingperformance parameters.

Step 5: Ensure data security, integrityand traceability

The software has a password-protected user access. It savesinstrument conditions and logbookstogether with raw data in checksum-protected binary files.

CE – for highest confidence in your results

DAD function• wavelength accuracy• linearity• noise and drift

Injector reproducibility and linearity

Temperature stability

Replenishment functionality

Voltage stability

11

Type Specification

Dimensions Width 42.5 cm, (16.8 in) Height 57.5 cm, (22.7 in) Depth 52 cm, (20.5 in)Weight 52 kg, (115 lb)

Environment Temperature: 5–40 °CHumidity: up to 80 %, at 30 °C (non-condensing)

Power requirements Line voltage:100/120/220/240 VAC ; +5 %, -10 %, 650 VALine frequency: 50 (48–55) Hz; 60 (57–66 ) Hz

Pressure system Programmable with 0–50 mbar bidirectionalFlushing with 1 bar or with high pressure 2–12 bar bidirectionalVial pressurization with high pressure 2–12 bar

Safety features Current leak detection; low current limitSafety sensors at door and cover disabling high voltageDiagnostic functions

Electrophoresis power Voltage range: setable 0 to ± 30 kV supply Current: setable 0–300 µAPower: setable 0–6 W Operation under constant voltage, current or power Programmable polarity switch

Injection modes Self correcting injection system with injection from both endsProgrammable range: up to 10,000 secondsPressure: 0–50 mbarElectrokinetic: 0–30 kV

Autosampler/ 48-position carousel. All vials are randomly accessible from fraction collector cathode and anode end of capillary. Temperature control with

external waterbath with vial temperature from 10–40 °C (non-condensing conditions, minimum waterbath temperature + 1 °C)

Replenishment Satellite station for refilling anode and cathode buffer vials with fresh buffer for automatic continuous operation and buffer selectable buffer levelling

Vials 100 µL sample vials, 1 mL or 2 mL buffer vials (polypropylene or glass) with resealing snap caps

Capillary cassette Forced-air temperature-controlled with Peltier element Temperature range: 10 °C below ambient to 60 °C

(± 0.1 °C) with a min. of 4 °CMinimum total capillary length: 33 cmCapillary compatibility 365 µm o.d.

Detector Real time UV-Visible diode-array detector (190–600 nm)Wavelength accuracy: 1 nm Response time: 0.1 to 10 s (8 choices) Light source: prealigned deuterium lampSignals: up to five signals simultaneously, full spectral acquisition

with Agilent ChemStation

Raw data channels Detector signals, voltage, current, power, capillary temperature and pressure.

System control Operating with graphical user interface under Microsoft® Windows® 2000 or XPTime programmable parameters: voltage, current, power, polarity pressure, inlet and

outlet vial capillary temperature, pre and post-run conditioning with pressure and/or voltage,replenishment, fraction collection

CE specific software Mobility report, time corrected areas, pI calibration and bio polymer size calibration

Instrument specifications

Microsoft and Windows are U.S. registered trademarks of Microsoft Corporation.

© Copyright 2007 Agilent Technologies Published November 1, 2007Publication Number 5989-7137EN

Learn more:www.agilent.com/chem/ce

www.agilent.com/chem/ce-ms

Buy online:www.agilent.com/chem/store

Find an Agilent customer center in your country:www.agilent.com/chem/contactus

U.S. and Canada 1-800-227-9770

agilent_inquiries@agilent.com

Europeinfo_agilent@agilent.com

Asia Pacificadinquiry_aplsca@agilent.com

Agilent capillary electrophoresis system