introducing agilent’s next generation capillary electrophoresis system · pdf...

Introducing Agilent’s Next Generation Capillary Electrophoresis System

Agilent 7100 Capillary Electrophoresis System

2009 Agilent Innovations Seminar

Agenda

•Basics of Capillary Electrophoresis

• Introduction to the 7100 CE

•CEMS and 3rd Party Detectors

•Applications examples


Capillary Electrophoresis ‐ 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 - limitedby thermal diffusion and convection

In the early 1980s Jorgenson and Lukacs advanced the technique by using 75-μm i.d. fused silica capillaries. Jorgenson also clarified the theory, described the relationships betweenoperational parameters and separation quality, and demonstrated the potential of capillary electrophoresis (CE) as an analytical technique.



Electro-osmotic flow(EOF)



Ways to control EOF


Capillary Electrophoresis ‐Modes of Operation


Capillary Electrophoresis

For charged samples try CE first!

• 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, there’s 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


Agilent TechnologiesThe Leading Provider Of Electrophoresis Platforms

CE – Capillary Electrophoresis• Flexible analytical

system with broadest

range of detectors

Bioanalyzer –Electrophoresis in micro-channels• Analytical system dedicated to

biomolecules and cells

OGE – Offgel Electrophoresis• Semi preparative system for protein separation > 10,000

Installed


Capillary Electrophoresis at Agilent

• Agilent has more than 15 years of CE

experience and CE is one of Agilent’s

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


Agilent 7100 Capillary Electrophoresis System

Smaller, quieter and more energy-efficient

•Unprecedented sensitivity and handling

•100% backward-compatible

with previous generation

CE solutions


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)

Agilent 7100 Capillary Electrophoresis SystemA Next Generation Platform

New!


Detector sensitivity

The new diode array detector offers significantly increased sensitivity. A linear dynamic range of 1x104

combined with a baseline noise of <20 μAU allows detection of impurities as little as 0.05% of a main peak.

When used with Agilent’s bubble cell capillaries, sensitivity is 6 -10x better than competitive instruments!

Peak resolution can be increased due to the 7100 CE’s 40 Hz sampling rate.

0.05% impurity detection with S/N > 6


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


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

New!



Improved robustness

• New vial sensor avoids vial conflicts when loading vials on the fly

• More powerful capillary cooling systemallows higher currents

• Improved replenishment

Vial sensorReduced 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)

New!



• 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!



Agilent 7100 Capillary Electrophoresis SystemCombining the power of Mass Spectrometry

Agilent is the only provider of complete CE-MS portfolio

Agilent‘s CE-MS systems provide: •excellent MS sensitivity•“plug and play" spraying system •most robust spraying technology with thefastest 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


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

Agilent 7100 Capillary Electrophoresis SystemCompatible with External detectors – LIF and CCD

New!


Capillary Electrophoresis Applications

Cation /Anion analysis

Chiral separation

Impurity Analysis

Natural compounds

Small Molecules

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

Antibody purity

Oligonucleotides

Protein separations

Peptide mapping

Bio - Molecules


Biopharma• Antibody analysis• Carbohydrates (heparin)• Oligonucleotides

Pharmaceutical• Chiral and vitamin analysis• Impurity analysis• Ion & counter-ion analysis

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

Capillary Electrophoresis Examples of existing users & applications

• Novartis• Sandoz• Roche• BASF

• US Departm‘t of Justice• Toyota Chemical• Nuclear Power plants• Bundeskriminalamt• FBI

• GE• Amgen• Genentech

Capillary Electrohoresis has become established as the technique of choice for several key applications across many industries


Ion analysis in various matrices using Capillary Electrophoresis

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

Data taken from Page 98 CE-Primer: P/N 5990-3777EN

Agilent Reagent-Kits and ready to use methods are available e.g. for Anions, Cations and organic acids


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

Chiral analysis

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


Chiral analysis example

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

Data taken from Application Note 5989-9810EN

min

mAU

0

200

400

600

800

1000

1200

Cromakalim

Methyl dopa

Isoproterenol

Epinephrine

Doxylamine

3,5-DOMA

Clenbuterol

Chromatographic conditionsCapillary: Effective length 40 cm, total length 48.5 cm, internal diameter 25 μm, BF 5(Agilent part number G1600-60132)

Buffer: 25 mM phosphate/TEA pH 3.3, 5 % (w/v), Heptakis sulpho-ß-cyclodextrin

Detection: 195/10 nmInjection: 500 mbar x sVoltage: 30 kVTemperature: 25 °C


Buffer: 20mM borate pH 9.3Capillary: 64cm (56cm eff)Detection: HSC, 225nm / 20nmInjection: 200mbar*sRun: 20°C, 30kV

impurities reported as % area/area of main peak

min0 1 2 3 4 5 6 7 8 9

mAU

-50510152025303540

0.036 %

0.09 %

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

Impurities Determination by CE

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

Data taken from Application Note 5965-9034E


Biopharma purity check: 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 μm id, BF5(Agilent p/n G1600-60132) cut to a total length of 33 cmBuffer: 600 mM lithium phosphate, pH 2.5. Injection: 1000 mbsTemperature: 20 °CVoltage: –14 kV, current ~53 μADetection: UV, 200 or 195 nm; reference offRun time: 10 minSample concentration: 30 mg/mL heparin.


Oligonucleotide Analysis

min5 10 15 20 25 30 35

0

50

100

150

200High 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 BufferPolymer flush: -7.5 bar, 3 min.

Sample: Antisense oligonucleotide pd(T) 6 - 25Injection: 7 sec, -10 kV.Voltage -25kVTemp 30°CDetection: 260 (8) nm, Ref. off, DNA Filter

Automated high resolution separation of single stranded DNA



CE‐MS Analysis of small molecules

min0 5 10 15 20 250

1000

2000

3000

4000 SIM m/z = 290+304

m/z = 290

m/z = 304

l-hyo

scya

mine

d-hy

oscy

amine

l-sco

polam

ineNatural Alkaloids

CE-MS analysis providing doubtless detection in difficult matrices Data taken from

Application Note 5968-9414E

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

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


Toxicology Drug screening

CE-MS and MSn analysis in heavy matrices providing unambiguous detection of drugs


CE-MS of drugs below 20 ng/µL in whole blood using CE coupled to an Agilent Iontrap MS-system


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


Antibody Analysis by CE and CE‐MS

CE + MS analysis

For detailed info on proteinsHigh performance separationExact MS data on Quad or TrapAccurate Mass by ToF and Q-ToFMS-MS spectra for identification

CE-Separation of FITC labeled IgG (200ng/ml from Serum). Detection by LIF-


Peptide Mapping by CE/MS

min5 10 15 20 25 30

mAU

0

20

40

60

80

100

120

1 mg/ml tryptic digest of human haemoglobin

UV detection at 195nm

min10 20 30 40 50 60

0

2500

5000

7500

10000

12500

15000

17500

20000

22500

m/z500 550

0

1000

3000

5000

541

.3 5

52.3

Abundance

MS detection

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

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


• Most sensitive CE on marketup to 10 x more than other CE instruments

• Widest choice of detectorsDAD, LIF, MS, CCD

• Only vendor with plug & spray ESI-MScomplete CE-MS portfolio

• Only vendor offering buffer replenishment system reproducible throughput and exceptional linearity

• Flexible and fast method developmenteasy method development using CE-Chemstation with graphicaluser interface

Summary Agilent 7100 CE System

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

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


introducing agilent’s next generation capillary electrophoresis system · pdf...

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