Introducing Agilent s Next Generation Capillary Electrophoresis System

Transcription

1 Agilent 7100 Capillary Electrophoresis System Introducing Agilent s Next Generation Capillary Electrophoresis System

2 Agenda Basics of Capillary Electrophoresis Introduction to the 7100 CE CEMS and 3 rd Party Detectors Applications examples

3 Capillary Electrophoresis basics Historical background, current status and applications of CE Electrophoresis as a separation technique was introduced by Tiselius in 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-μm 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. Page 3

4 Capillary Electrophoresis basics Electro-osmotic flow (EOF)

5 Capillary Electrophoresis basics Ways to control EOF

6 Capillary Electrophoresis Modes of Operation

7 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

8 Agilent Technologies The 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 bio- molecules and cells OGE Offgel Electrophoresis Semi preparative system for protein separation > 10,000 Installed

9 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

10 Agilent 7100 Capillary Electrophoresis System Smaller, quieter and more energy-efficient Unprecedented sensitivity and handling 100% backward-compatible with previous generation CE solutions

11 Agilent 7100 Capillary Electrophoresis System A Next Generation Platform Improved detection New detector (1200 Series LC-based) New! with increased sensitivity 1024 diode array elements, 1 nm resolution Up to 40 Hz measurement rate for better peak resolution (previously 10 Hz)

12 Agilent 7100 Capillary Electrophoresis System A Next Generation Platform Detector sensitivity 0.05% impurity detection with S/N > 6 The new diode array detector offers significantly increased sensitivity. A linear dynamic range of 1x10 4 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. 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

13 Agilent 7100 Capillary Electrophoresis System A Next Generation Platform Easier handling, reliability & service New instrument is 30% lighter with a 25% smaller footprint which saves bench space New! 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

14 Agilent 7100 Capillary Electrophoresis System A Next Generation Platform Improved robustness New! 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 Vial sensor 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)

15 Agilent 7100 Capillary Electrophoresis System A Next Generation Platform Software redesign. Updated interface New! Easier method setup Maintained graphical User Interface from previous system New driver architechture enables integration of MS & CE software (planned!)

16 Agilent 7100 Capillary Electrophoresis System Combining 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 the fastest switching between LC- and CE-MS Key applications: Pharmaceutical impurity analysis Metabolomic studies Glycan & protein analysis CE-QQQ, CE-Trap CE-TOF CE-TOF Agilent 6000 Series mass spectrometers

17 Agilent 7100 Capillary Electrophoresis System Compatible with External detectors LIF and CCD 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 New! Applications: LIF: Biomolecules CCD: Ion analysis.

18 Capillary Electrophoresis Applications 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

19 Capillary Electrophoresis Examples of existing users & applications Pharmaceutical Chiral and vitamin analysis Impurity analysis Ion & counter-ion analysis Chemical Food quality control (beer) illegal drugs analysis Water analysis Forensic Novartis Sandoz Roche BASF US Departm t of Justice Toyota Chemical Nuclear Power plants Bundeskriminalamt FBI Capillary Electrohoresis has become established as the technique of choice for several key applications across many industries Biopharma Antibody analysis Carbohydrates (heparin) Oligonucleotides GE Amgen Genentech

20 Ion analysis in various matrices using Capillary Electrophoresis Fast analysis in complex matrices with minimum sample preparation (often Agilent Reagent-Kits and ready to use methods are available e.g. for Anions, Cations and organic acids dilution only) Data taken from Page 98 CE-Primer: P/N EN

21 Chiral analysis 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 EN

22 Chiral analysis example 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 μm, BF 5 (Agilent part number G ) Buffer: 25 mm phosphate/tea ph 3.3, 5 % (w/v), Heptakis sulpho-ß-cyclodextrin mau Doxylamine 3,5-DOMA Epinephrine Methyl dopa Isoproterenol Detection: 195/10 nm Injection: 500 mbar x s Voltage: 30 kv Temperature: 25 C Clenbuterol Cromakalim 0 min Data taken from Application Note EN

23 Impurities Determination by CE Rapid high resolution separation with a different selectivity than LC methods. Use of Agilent High sensitivity Cell provides accurate main component and low level impurity determination in a Regulated Environment. mau % Buffer: 20mM borate ph 9.3 Capillary: 64cm (56cm eff) Detection: HSC, 225nm / 20nm Injection: 200mbar*s Run: 20 C, 30kV impurities reported as % area/area of main peak 0.09 % min Data taken from Application Note E

24 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 G ) 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 μa Detection: UV, 200 or 195 nm; reference off Run time: 10 min Sample concentration: 30 mg/ml heparin. Data taken from Application Note E

25 Oligonucleotide Analysis Automated high resolution separation of single stranded DNA 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 30 C Detection: 260 (8) nm, Ref. off, DNA Filter min Data taken from Application Note E

26 CE MS Analysis of small molecules 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 Natural Alkaloids 4000 SIM m/z = m/z = 290 l-scopolamine l-hyoscyamine d-hyoscyamine m/z = min CE-MS analysis providing doubtless detection in difficult matrices Data taken from Application Note E

27 Toxicology Drug screening CE-MS and MS n 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 Data taken from Application Note E

28 Preckel, T. et al, The Peak, LCGC, March 2009, Pub# EN

29 Antibody Analysis by CE and CE MS 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-

30 Peptide Mapping by CE/MS CE-MS analysis + DAD data providing maximum information per run UV detection at 195nm MS detection mau mg/ml tryptic digest of human haemoglobin Abundance m/z min min Agilent CE/MS solution is fully integrated at software and hardware level. The most robust Electrospray-MS solution on the market

31 Summary Agilent 7100 CE System Most sensitive CE on market up to 10 x more than other CE instruments Widest choice of detectors DAD, LIF, MS, CCD 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 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