Agilent 7100 Capillary Electrophoresis System

Introducing Agilents Next Generation Capillary Electrophoresis System

2009 Agilent Innovations Seminar

Agenda

BasicsofCapillaryElectrophoresis Introductiontothe7100CE CEMSand3rd PartyDetectors Applicationsexamples

2009 Agilent Innovations Seminar

CapillaryElectrophoresis basics
Historical background, current status and applications of CE Electrophoresis as a separation technique was introduced by Tiselius in 1937. For this work he was awarded a Nobel Prize. Separation efficiency in free solution - limited by thermal diffusion and convection In the early 1980s Jorgenson and Lukacs advanced the technique by using 75-m i.d. fused silica capillaries. Jorgenson also clarified the theory, described the relationships between operational parameters and separation quality, and demonstrated the potential of capillary electrophoresis (CE) as an analytical technique.
2009 Agilent Innovations Seminar Page 3

CapillaryElectrophoresis basics
Electro-osmotic flow (EOF)

2009 Agilent Innovations Seminar

CapillaryElectrophoresis basics
Ways to control EOF

2009 Agilent Innovations Seminar

CapillaryElectrophoresis ModesofOperation

2009 Agilent Innovations Seminar

CapillaryElectrophoresis ForchargedsamplestryCEfirst!
CE offers fast separations with exceptional efficiency & resolution for analytical challenges that often can only be met with difficulty by LC For charged substances like biomolecules, small basic or acidic drugs and ions, theres nothing faster or easier In addition, CE uses low amounts of sample and much less buffer than LC CE complements LC for analysis of small charged, chiral or polar molecules & ions
Orthogonal technique to LC Low consumption of sample and buffer

2009 Agilent Innovations Seminar

AgilentTechnologies
TheLeadingProviderOfElectrophoresisPlatforms CE Capillary Electrophoresis
Flexible analytical system with broadest range of detectors

Bioanalyzer Electrophoresis in micro-channels

Analytical system dedicated to bio- molecules and cells

OGE Offgel Electrophoresis

Semi preparative system for protein separation
0 0,00 > 1 lled Insta
2009 Agilent Innovations Seminar

CapillaryElectrophoresisatAgilent

Agilent has more than 15 years of CE experience and CE is one of Agilents core technologies (4000 systems installed) Agilent pioneered the proprietary bubble cell capillaries & high sensitivity cell which enable access to higher sensitivity levels CEC Introduced in 1996 CEMS Introduced in 1997, compatible with all Agilent LCMS platforms
2009 Agilent Innovations Seminar

Agilent7100CapillaryElectrophoresisSystem

Smaller, quieter and more energy-efficient Unprecedented sensitivity and handling 100% backward-compatible with previous generation CE solutions

2009 Agilent Innovations Seminar

Agilent7100CapillaryElectrophoresisSystem
ANextGenerationPlatform
Improved detection New detector (1200 Series LC-based) with increased sensitivity 1024 diode array elements, 1 nm resolution Up to 40 Hz measurement rate for better peak resolution (previously 10 Hz)
New!

2009 Agilent Innovations Seminar

Agilent7100CapillaryElectrophoresisSystem
ANextGenerationPlatform
Detector sensitivity
The new diode array detector offers significantly increased sensitivity. A linear dynamic range of 1x104 combined with a baseline noise of <20 AU allows detection of impurities as little as 0.05% of a main peak. When used with Agilents bubble cell capillaries, sensitivity is 6 -10x better than competitive instruments! Peak resolution can be increased due to the 7100 CEs 40 Hz sampling rate.
0. impur 05% it y d e with S tection /N > 6

Conditions:

1 M 4-hydroxy-aceto-phenon with impurity injected at 50 mbar x 5 seconds; 3 x 50 m bubble cell capillary; baseline noise: at 2 seconds response time

2009 Agilent Innovations Seminar

Agilent7100CapillaryElectrophoresisSystem
ANextGenerationPlatform
Easier handling, reliability & service New instrument is 30% lighter with a 25% smaller footprint which saves bench space Modular architecture enables easy servicing & maintenance Calibration tools for electrodes help to avoid electrode damage Easy lamp exchange 2x lamp lifetime (>2000h), available as standard & high brightness lamp
2009 Agilent Innovations Seminar

New!

Agilent7100CapillaryElectrophoresisSystem
ANextGenerationPlatform
Improved robustness New vial sensor avoids vial conflicts when loading vials on the fly More powerful capillary cooling system allows higher currents Improved replenishment Reduced cost of ownership through better diagnosis Self test of instrument with Agilent LabAdvisor software EMF (early maintenance feedback) indicates when parts need to be replaced (e.g. up-, down-movements of lifters)
2009 Agilent Innovations Seminar

New!

Vial sensor

Agilent 7100 Capillary Electrophoresis System

ANextGenerationPlatform
Software redesign. Updated interface Easier method setup Maintained graphical User Interface from previous system New driver architechture enables integration of MS & CE software (planned!)
New!

2009 Agilent Innovations Seminar

Agilent7100CapillaryElectrophoresisSystem
CombiningthepowerofMassSpectrometry
Agilent is the only provider of complete CE-MS portfolio Agilents CE-MS systems provide: excellent MS sensitivity plug and play" spraying system most robust spraying technology with the fastest switching between LC- and CE-MS Key applications: Pharmaceutical impurity analysis CE-QQQ, CE-Trap Metabolomic studies CE-TOF Glycan & protein analysis CE-TOF

Agilent 6000 Series mass spectrometers

2009 Agilent Innovations Seminar

Agilent7100CapillaryElectrophoresisSystem
CompatiblewithExternaldetectors LIFandCCD
Partner solutions: Picometrics: LIF detector ISTech: CCD Direct installation of detection cell into cassette Signal in Chemstation Plug & play setup: analog in, A/D converter in new CE Applications: LIF: Biomolecules www.picometrics.com CCD: Ion analysis. www.istech.at
New!

2009 Agilent Innovations Seminar

CapillaryElectrophoresisApplications
CE, CEC & CE-MS capabilities with a single instrument offers a wide range of applications as well as an orthogonal technique to LC or LC-MS

Cation /Anion analysis Chiral separation Impurity Analysis Natural compounds Small Molecules

Antibody purity Oligonucleotides Protein separations Peptide mapping Bio - Molecules

2009 Agilent Innovations Seminar

CapillaryElectrophoresis
Examplesofexistingusers&applications
Pharmaceutical
Chiral and vitamin analysis Impurity analysis Ion & counter-ion analysis

Novartis Sandoz Roche BASF USDepartmtofJustice ToyotaChemical NuclearPowerplants Bundeskriminalamt FBI

CapillaryElectrohoresishasbecome establishedasthetechniqueofchoice forseveralkeyapplicationsacross manyindustries

Chemical

Food quality control (beer) illegal drugs analysis Water analysis Forensic

Biopharma

Antibody analysis Carbohydrates (heparin) Oligonucleotides

GE Amgen Genentech

2009 Agilent Innovations Seminar

Ionanalysisinvariousmatricesusing CapillaryElectrophoresis

Fast analysis in complex matrices with minimum sample preparation (often dilution only)

Agilent Reagent-Kits and ready to use methods are available e.g. for Anions, Cations and organic acids
Data taken from Page 98 CE-Primer: P/N 5990-3777EN

2009 Agilent Innovations Seminar

Chiralanalysis
CE can provide Organic Chemists with rapid screening of chiral compounds and chiral excess determinations with minimal use of expensive chiral selectors.

In CE the chiral selector is simply added to the buffer. This allows fast method development and rapid analyses at lowered cost. Chiral CE compares very favorably with HPLC
Data taken from Page 149 CE-Primer: P/N 5990-3777EN

2009 Agilent Innovations Seminar

Chiralanalysisexample
A single method can be applied to a wide range of analytes.

Chromatographic conditions Capillary: Effective length 40 cm, total length 48.5 cm, internal diameter 25 m, BF 5 (Agilent part number G1600-60132) Buffer: 25 mM phosphate/TEA pH 3.3, 5 % (w/v), Heptakis sulpho--cyclodextrin Detection: 195/10 nm Injection: 500 mbar x s Voltage: 30 kV Temperature: 25 C

Doxylamine mAU 1200 1000 800 600 400 200 0 min

Data taken from Application Note

Epinephrine 3,5-DOMA Isoproterenol

Methyl dopa

Clenbuterol

Cromakalim

5989-9810EN

2009 Agilent Innovations Seminar

ImpuritiesDeterminationbyCE
Rapid high resolution separation with a different selectivity than LC methods.
mAU 40

Use of Agilent High sensitivity Cell provides accurate main component and low level impurity determination in a Regulated Environment.

35 30 25 20 15 10 5 0 -5 0 1 2 3 4 5 6 7 0.036 %

Buffer: 20mM borate pH 9.3 Capillary: 64cm (56cm eff) Detection: HSC, 225nm / 20nm Injection: 200mbar*s Run: 20C, 30kV impurities reported as % area/area of main peak

0.09 %

9 min
Data taken from Application Note

5965-9034E

2009 Agilent Innovations Seminar

Biopharmapuritycheck:Heparin
High speed high resolution impurity check for pharmaceuticals
CE is the ideal tool for separation of biomolecules (proteins, peptides, nucleic acids and carbohydrates) and encouraged by regulatory authorities as an accepted orthogonal technique
Capillary: Extended light path capillary 25 m id, BF5 (Agilent p/n G1600-60132) cut to a total length of 33 cm Buffer: 600 mM lithium phosphate, pH 2.5. Injection: 1000 mbs Temperature: 20 C Voltage: 14 kV, current ~53 A Detection: UV, 200 or 195 nm; reference off Run time: 10 min Sample concentration: 30 mg/mL heparin .
Data taken from Application Note

5990-3517E
2009 Agilent Innovations Seminar

OligonucleotideAnalysis
Automated high resolution separation of single stranded DNA
200

150

High pressure capability allows the use of very viscous gels

Capillary: PVA coated, id=100um, leff=24.5 cm; Polymer Solution A/B (w/wo organic additive), Oligonucleotide Buffer Polymer flush: -7.5 bar, 3 min. Sample: Antisense oligonucleotide pd(T) 6 - 25 Injection: 7 sec, -10 kV. Voltage -25kV Temp 30C Detection: 260 (8) nm, Ref. off, DNA Filter

100

50

0 5 10 15 20 25 30 35 min

Data taken from Application Note

5988-4303E

2009 Agilent Innovations Seminar

CEMSAnalysisofsmallmolecules
ol a l-hmin dyo e hy s os c y cya a m mi ne in e
25 min
Data taken from Application Note 5968-9414E
2009 Agilent Innovations Seminar

Other MS types (QQQ, ToF, QToF) can be controlled via Chemstation plus Masshunter software packages

4000 3000 2000 1000 0

0

SIM m/z = 290+304 m/z = 290 m/z = 304

5 10 15 20

CE-MS analysis providing doubtless detection in difficult matrices

l-s

co p

CE-MS with SQMS detection offering complete access via ChemStation software including Sheath liquid handling from separate LC-pump

Natural Alkaloids

ToxicologyDrugscreening
CE-MS and MSn analysis in heavy matrices providing unambiguous detection of drugs
CE-MS of drugs below 20 ng/L in whole blood using CE coupled to an Agilent Iontrap MS-system

Data taken from Application Note

5989-2911E

2009 Agilent Innovations Seminar

Preckel, T. et al, The Peak, LCGC, March 2009, Pub# 5990-3986EN

2009 Agilent Innovations Seminar

AntibodyAnalysisbyCEandCEMS
CE + MS analysis For detailed info on proteins High performance separation Exact MS data on Quad or Trap Accurate Mass by ToF and Q-ToF MS-MS spectra for identification
CE-Separation of FITC labeled IgG (200ng/ml from Serum). Detection by LIF-

2009 Agilent Innovations Seminar

PeptideMappingbyCE/MS

CE-MS analysis + DAD data providing maximum information per run

UV detection at 195nm
mAU

MS detection
22500 20000
3000 Abundance 5000 541.3 552.3 550

1 mg/ml tryptic digest of human haemoglobin

120

17500

100
15000

1000 0 500

80

m/z

12500 10000 7500

60

40
5000

20

2500 0 10 20 30 40 50 60 min

0 5 10 15 20 25 30 min

Agilent CE/MS solution is fully integrated at software and hardware level. The most robust Electrospray-MS solution on the market
2009 Agilent Innovations Seminar

SummaryAgilent7100CESystem
Agilent's next generation 7100 CE system builds on a successful HP/Agilent history in Capillary Electrophoresis. Offering evolutionary improvement compared with the older CE system for sensitivity, flexibility of methods, ease of use and with complete backwards compatibility
Most sensitive CE on market up to 10 x more than other CE instruments Widest choice of detectors DAD, LIF, MS, CCD Only vendor with plug & spray ESI-MS complete CE-MS portfolio Only vendor offering buffer replenishment system reproducible throughput and exceptional linearity Flexible and fast method development easy method development using CE-Chemstation with graphical user interface

More information at www.agilent.com/chem/ce

2009 Agilent Innovations Seminar