i HPLC 920 -LC Analytical HPLC 940 -LC

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i. HPLC 920 -LC Analytical HPLC 940 -LC Analytical to Prep HPLC

Varian 900 -LC Series i. HPLC

Agenda • Introduction • • • Introduction to the 9 X 0 -LC series HPLC Total market size by technique and market segment An overview of the 920 -LC Analytical HPLC • • Positioning, competition and target markets • • Features and benefits Applications An overview of the 940 -LC Analytical to Prep and Prep HPLC • • Positioning, competition and target markets • 3 Features and benefits Applications

What is the i. HPLC? • 2 models 920 -LC and 940 -LC • Compact, stylish, automated analytical and semi prep HPLC • Pre-plumbed and factory tested for the highest confidence • Easy to order, install and use – Single Ethernet cable connection • Focused on improved productivity through automation • Updated premium performance hardware • Autosampler, Fraction Collector and Scale up Module for semi-prep • Quaternary pump, precision Column Heater, RI, Fluor, ELSD and autosampler for analytical • Updated Integrated Instrument Software Interface • Eliminate repetition, modernise UI • Consistent • Easy to use with added functionality • Adds to modular range 4

HPLC Demand SDI 2007 • Overall LC is $3. 2 B @ 6% growth • 23000 systems sold in 2006 • New HPLC systems sales (initial) • $1220 M – $764 M Conventional analytical – $180 M Prep – $113 M Fast LC 5

Current Status of Varian HPLC • • • • 6 Installation/setup difficulties No supporting materials Perceptions Ease of ordering Competitive performance Lack of competitive specifications Ease of use Reliability Productivity Validation and Compliance Flexibility/expansibility (especially in semi-prep) Competitive pricing Wide product range – hardware, columns, consumables

Varian 920 -LC Integrated Analytical HPLC 7

Varian 920 -LC Silver Bullets • • Superb Fast LC Performance optimised for sub 3 um columns – Improved throughput without compromising robustness • High sensitivity/Top Line Performance detection systems across UV/PDA/RI/Fluor and ELSD – Unparalleled strength • Unbeatable Injection performance and carryover statistics with 2008 Loopless Technology Autosampler • 8 Premium Analytical Pump Performance and reliability Full system level computer controlled AIQ (validation) • Updated UI for Enhanced User Experience

Varian 920 -LC Analytical HPLC Goals • Aim • Become credible player in this $800 M segment by doubling market share and revenue • Markets • Pharma / Bio. Pharma • Environmental • Industrial • Strategy • Leverage Varian distribution channel to deliver competitive HPLC hardware, consumables, software and services 9

Varian 920 -LC What Is It? • Compact, stylish automated analytical HPLC – Pre-plumbed and factory tested for the highest confidence – Single cable communications • Virtually pulse-free dual piston pump – Does not require a pulse damper • State-of-the-art low pressure quaternary gradient module with built-in degasser • Autosampler with cooling option – Synchronised injection for outstanding retention time reproducibility – Compatible with 200 x 1. 6 ml HPLC vials or 3 microplates 10

Varian 920 -LC What Is It? • Either dual l UV-Vis or PDA detector – Optional second detector - Fluorescence or RI • High precision (<0. 15 o. C) dual column heater available – Low delay solvent pre-heater for improved heat transfer – Optional column switching and electric injection valve • NEW improved instrument interface on Galaxie. TM software • External CVM, ELSD or 356 -LC RI can be plugged directly into the back of the unit. 11

920 -LC for Standard HPLC to Fast HPLC 25 cm x 4. 6 mm Pursuit XRS 5 um 1. 0 ml/min 25 cm x 4. 6 mm Pursuit XRS 5 um 2. 2 ml/min 15 cm x 4. 6 mm Pursuit XRS 5 um 1. 0 ml/min 15 cm x 4. 6 mm Pursuit XRS 5 um 3. 75 ml/min 10 cm x 2. 0 mm Pursuit XRS 2. 8 um Ultra 0. 8 ml/min 5 cm x 2. 0 mm Pursuit XRS 2. 8 um Ultra 0. 4 ml/min 16 x Speed Enhancement 12 5 cm x 2. 0 mm 2. 4 um 1. 3 ml/min

Varian 920 -LC Fast LC PAHs Varian 920 -LC 6 minute Analysis Waters Acquity 7 Minute Analysis 14

Varian 920 -LC Pump • Dual piston quaternary pump produces fast gradients – even at 200 ml/min • Virtually pulse-free pumping without pulse damper • Outstanding gradient compositional accuracy give retention time precision at the <1% level • Built-in vacuum degas maintains pump prime and enhanced injection reliability • Overlay of 10 chromatograms • 10% steps at 1 ml/min 15 • Accurate flow from 0. 1 to 10 m. L/min for 2 to 4. 6 mm columns

Varian 920 -LC Pump - FAB Feature Advantage Benefit Dual Piston Quaternary Capability Market leading precision and accuracy Performance benchmark Built in Degasser and Enhanced run to run Pulse Free Flow reproducibilty Optional Semi-Micro Kit 16 Dependable Confidence in results Performance with Reliability Fast LC capability

Varian 920 -LC Autosampler • Needle-in-flow path geometry for low carry-over (<0. 01%) • Typical inj precision (< 0. 3% at 10 ml) • Optimized flow paths for fast injection turn-around • Synchronised injection for best RT reproducibility • Programmable wash setting for low carry-over • Injection volumes from 1 ul to 50 m. L • Cooled option available 17

Varian 920 -LC Washout Performance 125 ug/ml Caffeine Blank injection <0. 01% carryover 18

920 -LC Autosampler How Does it Work? 19

Varian 920 -LC Injection Synchronization 20

Varian 920 -LC Autosampler - FAB Feature Advantage Needle in Path Design No need for Injection Versatility loops No Carryover Performance Synchronised injections with Pump Stroke cycle Enhanced retention time reproducibilty One Autosampler, all No need for multiple rack formats injector options 21 Benefit Performance Accuracy Versatility Simplicity

Sensitive UV-Vis Detectors Typical Agilent VWD S/N ~ 25 Typical Varian 920 -LC UV Vis S/N ~ 38 22

Varian 920 -LC UV/Vis - PDA Detectors - FAB Feature Benefit Best signal to Noise Enhanced sensitivity and detection limits Performance Tool Free maintenance Easy to change lamps and flow cells Simplicity Range of cells for ul/min to Litres/min 23 Advantage Fits all applications from LCMS to Benchtop Prep Versatility and Performance

Varian 920 -LC Fluorescence Detector • Sensitive Fluorescence detector (S/N > 600 Raman H 2 O peak baseline method) • Time programmable l for increase sensitivity • Automatic l validation with built-in Hg lamp • Low 12 ml dead volume flowcell for improved chromatography 24

Varian 920 -LC Fluorescence Detector - FAB Feature Benefit Improved Sensitivity (S/N > 600 Raman) Lower limits of detection Performance Automatic l calibration ( Hg lamp) Greater accuracy of analysis Confidence in results; Dependable Low dead volume cell ( 12 ul volume) 25 Advantage Sharper peaks, enhanced resolution Performance

$Varian 920 -LC Refractive Index Detector • Same as 356 -LC Refractive Index Standalone$ Varian 920 -LC Refractive Index Detector • Same as 356 -LC Refractive Index Standalone • Low Volume, flow efficient cell for Superior Peak Shape • Stable, Long Life and High Intensity LED • Temperature Controlled Optics for minimised baseline drift and outstanding detection limits • Complete automation thru Galaxie Software 26

Varian 920 -LC RI Detector - FAB Feature Benefit Low Volume Cell Minimal Peak Dispersion giving enhanced resolution Performance and Accuracy High Intensity, long life LED Maintenance Free for Dependability, consistant long term Durability and performance Confidence in results Temp Controlled optics 27 Advantage Minimises baseline drift for lower settling times, better sensitivity Performance and Ecconomy – saves the analyst precious time

Varian 380 -LC and 385 -LC ELSD • Varian Product numbers • 2 to 3 Times More Sensitive • NEW Glass Nebuliser for Improved Reproducibility • 40 Hz Data Rate for Fast LC • Onboard timetable program to enable parameter change thru a run 28

Varian 380 -LC and 385 -LC ELSD - FAB Feature Benefit Improved Sensitivity Lower limits of detection Performance NEW Glass Nebuliser Greater run to run reproducibility Confidence in results; Performance 40 Hz Data Rate 29 Advantage Suitable for Fast LC Performance coupled with 920 -LC in Fast LC operation

Heated Column Compartment • Temperature range from ambient + 10 o. C up to 65 o. C – Temperature reproducibility +0. 15 o. C – Ensures excellent analytical reproducibility – Uses a low delay volume pre-heater for efficient heat transfer to mobile phase – Spacious column compartment easily accommodates 2 x 250 mm columns – Option with electrically actuated column switching valve and injection valve 30

Outstanding Stability 31

Reproducibility of 50 Injections Reproducibility <0. 8% over 50 injections and 12 hours 32

Varian 920 -LC Column Heater - FAB Feature Advantage Benefit Low Volume Solvent Pre-Heater Eliminates thermal column effects and enhances resolution/method transfer Performance Outstanding Stability Provides greater and Accuracy method robustness Spacious compartment with easy access 33 Dependable, Confidence in results Accurate Simple and Convenient Fast column changes, room to work

Varian 920 -LC Software Usability • User friendly Galaxie. TM software • Simplified, modern, consistent Instrument interface • Modular 11 pages • 920 -LC 4 pages!! • One page real-time status and chromatogram • Advanced integration and calibration • New IMS capabilities • Automated instrument validation • Reduces downtime and operating costs 34 • Fusion AE

Varian 920 -LC AIQ (Validation) AIQ (Analytical Instrument Qualification) • Fully PC controlled IQ and OQ (not PQ) • Puts the Varian 920 -LC at the forefront of regulated capabilities • Consistent, reproducible methodology • Includes pumps, autosampler, detectors, column heater • Chargeable option • Fully documented 35

Varian 920 -LC User interface - FAB Feature Advantage Benefit Modern, User friendly UI ( 11 pages to 4!) Simple to use and an Simplicity coupled enjoyable experience with Stylish Appearance Shockwave Graphics Visually pleasing with Stylish appearance for 2008 Software Flashing instrument Technology status Instrument maintenance Software 36 Automated instrument validation/testing Ensures instrument Accuracy and Performance

Varian 920 -LC Concept - FAB Feature Advantage Benefit 1 st Fully Integrated i. HPLC System Unique and Interesting concept Prestige resulting in elevated HPLC interest/prospects Easy to Use, Install and Maintain Creates a new level of customer satisfaction Product Confidence and enhanced repeat business Compact and Stylish High level of instrument capability in Unique footprint 37 Desirable to own, Convenient to use

Varian 920 -LC Concept - FAB Feature Advantage Benefit Single Part Numbers Easy to quote One Power, Communications cable and “On” Button Easy to configure, clean and tidy look Simplicity with enhanced Appearance Pre-Plumbed and “Ready” out of the Box 38 Much simpler to sell, and easier experience Fast installation so Convenient and you are quickly able Simple to use the instrument

Varian 920 -LC Concept - FAB Feature Benefit Sample tray access without opening the main doors System looks good at all times without exposing internals Clean Appearance Door trays and holders for columns and accessories Provides space to tidily store the many parts used daily Clean Appearance Tray Storage 39 Advantage Provides additional storage space for Software CD, tools, columns etc… Clean Appearance and Versatility

Varian 920 -LC Concept - FAB Feature Benefit Solvent Tray capacity Shape of instrument provides ample space for solvents Convenience and Safety Ski Slopes for easy tray insertion/removal Eliminates tray location errors Provides Autosampler Confidence and Durability Single Centralised Waste port 40 Advantage No need for multiple waste vessels positioned around the instrument Clean Appearance and Simplicity. Only 1 waste to empty

Varian 920 -LC Concept - FAB Feature Leak Detector Ensures the system is Performance and “leak free” and Confidence performing to the max Modern Manual Injectors Push button valve positioning with automatic return, eliminate operator error Bottle Management 41 Advantage Benefit Ability to fill reservoirs Provides a Safety and empty waste mechanism for Confidence in overnight runs Even Simpler to use, eliminates user error improving Confidence in results

Varian 920 -LC Concept - FAB Feature Benefit Tool Tips, Help and Videos Extensive library of Mouse tool tips and Online help Makes the instrument Simple to use and Convenient to access New Brochures Physical material to Convenient and present to customers Powerful messages to explain to customer Updated Data Sheets, Price List and App Notes 42 Advantage Supporting materials Provides Confidence to leave with the in product range and customer Simple to Quote

Varian 920 -LC Ordering Matrix pump inject detector Modular Isocratic 210 -LC 10 ml Manual 325 Isocratic 00 -100890 -00 Dual piston pump 10 ml head Auto Dual l UV/Vis Opt FL, RI ELSD Gradient 00 -100891 -00 Quat, Grad, Degas Auto Dual l UV/Vis Opt FL, RI ELSD opt High Performance Gradient 00 -100892 -00 Quat, Grad, Degas Auto cooled Dual l UV/Vis Opt FL, RI ELSD opt Development 00 -100893 -00 Quat, Grad, Degas Auto PDA Std FL, RI ELSD opt Research 00 -100894 -00 Quat, Grad, Degas Auto cooled PDA Std FL, RI ELSD opt 43 Oven Detector 2 option Options PDA, RI, ELSD

Varian 920 -LC Target Markets • Strong historical position for Varian in analytical • Varian has the leading range of analytical technologies: spectroscopy, chromatography including consumables, data and services • Target Users • Environmental • Food/Beverage • Industrial • Generic Pharmaceutical * • Academia and Biotech • Same (but wider) target groups with a stronger product offering *40% of Regulated Pharma has significant competitive barriers due to installed software base 44

Varian 920 -LC Consumables • Excellent range of HPLC Columns providing industry leading resolution and robustness • Pursuit XRS • Polymeric Phases - PL • From 2 mm to 2” ID Prepacked • Other Speciality Phases • NEW 2. 8 Ultra Column AND 2. 4 um Columns for Fast or High Res LC • 2. 8 um Ultra is optimised for Speed • 2. 4 um gives further enhancements in Resolution 45

2. 8 um Vs 2. 4 um – High Res or Extra Speed 2. 4 um 3 mm x 5 cm for RESOLUTION 2. 8 um 2 mm x 5 cm for 46 speed

Varian 920 -LC Positioning

UPLC, Fast LC and the Varian 920 -LC

What is Fast LC? • Involves using LC columns packed with very small (sub 3 mm diameter) particles • Smaller particles column are more efficient giving greater resolution per unit length • For the same separation (at the same flow rate) can use a shorter column which reduces run time for improve productivity • As Van Deemter plot is flatter can operate at higher flow rates efficiently But smaller particles give greater back pressure!! 49

Van Deemter Plot 0. 045 0. 040 0. 035 0. 030 HETP (cm/plate) 0. 025 5 mm 0. 020 0. 015 3. 5 mm 0. 010 1. 8 mm 0. 005 0. 000 0. 2 0. 4 0. 6 0. 8 Linear velocity u cm/sec 50 1. 2 1. 4 1. 6

Fast LC Productivity 25 cm x 4. 6 mm Pursuit XRS 5 um 1. 0 ml/min 25 cm x 4. 6 mm Pursuit XRS 5 um 2. 2 ml/min 15 cm x 4. 6 mm Pursuit XRS 5 um 1. 0 ml/min 15 cm x 4. 6 mm Pursuit XRS 5 um 3. 75 ml/min 10 cm x 2. 0 mm Pursuit XRS 2. 8 um Ultra 0. 8 ml/min 5 cm x 2. 0 mm Pursuit XRS 2. 8 um Ultra 0. 4 ml/min 16 x Speed Enhancement 51 5 cm x 2. 0 mm 2. 4 um 1. 3 ml/min

Market Size for Fast LC - 2007 52 Total Systems market Size 2007 = US$1. 3 b (SDI 2006)

Fast LC • Fast LC market estimated at $160 mill growing rapidly • Many names • • 53 Ultrahigh Pressure LC Rapid Resolution LC High Resolution LC Fast LC

Target Markets for UPLC and Fast LC UPLC • Research Pharma – Drug discovery • QA/QC Pharma – Not as successful as first thought • No. 1 use - separation front end for MS 54

Theory UPLC or Fast LC • Small particle porous column packing 1. 7 – 3. 0 mm in diameter • Increased flow rates • Increased pressure • Increased column temperatures Advantage: • Improved resolution of peaks in complex samples • Improved sensitivity for low concentration or limited samples • Reduced analysis times 55

Theory UPLC or Fast LC • Small particle porous column packing 1. 7 – 3. 0 u in dia. • Increased flow rates • Increased pressure • Increased column temperatures Disadvantage: • Increased back pressure – special hardware requirements needed, micro leaks become a problem = Poor reproducibility • Band spreading in the column decreases – the HPLC will need to account for this • High acquisition rates for detector collection • Expensive • Proprietary columns in some instances 56

Advantages of Fast LC • Price – Binary gradient LC with autosampler and detector ~ 50 -60% of UPLC cost • Flexibility – Operates as standard HPLC and in Fast LC mode • Allows all LC applications to be run • Reliability – Reproducibility of results 57

Advantages of UPLC • Speed – Reduced analysis times = cost savings • Flexibility – Optimum performance as front end for MS • Performance – Improved sensitivity 58

Typical Speed Performance of a UPLC Separation 59

Typical Speed Performance of Fast LC Fanta Orange Drink Conditions Mobile Phase: 10 mm Sodium Phosphate ph 6. 0/ACN gradient Detection: UV Detection Temperature: 40°C Flow-Rate: 1, 2 m. L/min Column: Varian Ultra 50 x 2, 0 mm Sugar Standard 60 2. 4 minutes

Current Systems on the market Waters Acquity • High pressure – quoted to 15, 000 psi but generally run at around 10, 000 psi • Low dispersion • Small particle size columns • Small columns – generally <2 mm in dia. • Binary gradient system – 4 solvent selection per pump • Typical column temp of 30°C to 50°C 61

Current Systems on the market Agilent 1200 RRLC • High pressure – quoted to 9, 000 psi but generally run at lower pressures • Low dispersion • Small particle size columns • Flow rate maximum of 5 ml/min • Binary gradient system – 4 solvent selection per pump • Typical column temp of 30°C to 100°C • Can switch between Fast LC and normal LC (variable dead volume control). 62

Current Systems on the market Thermo Accela • High pressure – quoted to 15, 000 psi only with flow rates of 1 -1000 m. L/min • Low dispersion • Compatible with small particle size columns • Promoted as conventional and Ultrahigh press LC – max flow rate of 1 ml/min limits conventional HPLC • Quaternary pumping system 63

Current Systems on the market Others Shimadzu • Conventional LC that shows 2. 2 mm particle column applications – Fast LC approach Jasco • UPLC system utilising 15000 psi pressure capability and column temperatures to 60°C Hitachi • 9000 psi pressure capability with 90°C temperature performance. Variation on their L 2000 series HPLC 64

Van Deemter Plot 0. 045 0. 040 0. 035 0. 030 HETP (cm/plate) 0. 025 5 mm 0. 020 0. 015 3. 5 mm 0. 010 2. 4 mm 0. 005 1. 8 mm 0. 000 0. 2 0. 4 0. 6 0. 8 Linear velocity u cm/sec 65 1. 0 1. 2 1. 4 1. 6

Trends in Fast LC • • • 66 Movement away from sub 2 mm particles owing to – Ultrahigh back pressures & reproducibility – High temperatures & column lifetime – Data acquisition rates 2. 4 mm and 2. 8 mm particles allows – Improved resolution without ultrahigh back pressures – Use of modified conventional HPLC (reduced delayed volumes) – Use of more moderate conditions e. g. temp & pressure Many column and instrument companies developing products – Shimadzu – Phenomenex – Varian

16 PAHs by UPLC and Fast LC Waters Acquity UPLC Mobile Phase: Acetonitrile Water Gradient Detection: UV@254 nm Temperature: 40°C Flow-Rate: 0, 8 m. L/min Column: Pursuit XRs C 18 3 um, 100 x 2 mm Varian 920 -LC Fast LC 67

Soft Drinks Using 920 -LC Fanta Orange Drink Conditions Mobile Phase: 10 mm Sodium Phosphate ph 6. 0/ACN gradient Detection: UV Detection Temperature: 40°C Flow-Rate: 1, 2 m. L/min Column: Varian Ultra 50 x 2, 0 mm Sugar Standard 2. 4 minutes 68

Alkylated Phenones Using 920 -LC Conditions Mobile Phase: Acetonitrile Water Gradient Detection: UV Detection Temperature: 60°C Flow-Rate: 1, 6 m. L/min Column: Varian Ultra 3 um 50 x 2, 0 mm 69 1. 4 minutes

Anthocyanadins Using 920 -LC Conditions Mobile Phase: -solvent A Water phosphoric acid 0, 3% -Solvent B Acetonitrile -Gradient separation from 88% A to 78% B in 2, 5 min Detection: DAD 525 nm Temperature: 55°C Flow-Rate: 0. 9 ml/min Column: HP 3 C 18 CP Injection volume : 1µl sample Delphinidin, Cyanidin, Petunidin, Pelargonidin, Peonidin, Mavidin 70

Fat Soluble Vitamins Using 920 -LC Chromatogram 260 nm Conditions Mobile Phase: A Water 0; 1% formic acid B CH 3 CN 0, 1% Formic acid gradient t 0 min B 80% ; t 1. 5 min B 80%; t 2 min B 100% T 12 min B 100% Detection: UV 260 nm & Max. Plot Chromatogram Max. Plot Temperature: 50°C Flow-Rate: 0, 7 m. L/min Column: Pursuit C 18 3 µm 100 x 2, 1 mm Injection volume 25 µL, sample Retinol, vitamin D 2, D 3, E, E Ac, K 1 71

Fast Sugars Using 920 -LC Fanta Orange Drink Sprite Conditions Sugar Standard 72 Mobile Phase: Acetonitrile Water 75: 25 Detection: RI Detection Temperature: 40°C Flow-Rate: 2, 5 m. L/min Column: Polaris NH 2 5 um 150 x 4, 6 mm

Varian 920 -LC Conclusion • UPLC does not currently suit all applications • Restricted column choices • Demanding H/W are chemistry requirements • Fast LC with 2. 4 mm to 3 mm particle columns provides flexibility • Compatible with 920 -LC – Low delay volume kit e. g mixer, flowcell, tubing • Wide choice of chemistries • Price competitive (can purchase 2 Fast LC for UPLC) 73

Varian 920 -LC Target Markets

SDI 2006 Market by Industry Split 75

Varian LC Sales by Industry Type 76

Varian 920 -LC Target Markets Environmental • Build-on Spectroscopy, GC-MS & LC-MS products – Superb gradient pumping system – Sensitive Fluoro detector for PAH’s 77

Varian 920 -LC Target Markets Industrial • Traditionally strong Varian market • Leverage profile of GC, Spectroscopy and Polymer Lab products • Large range of systems • Isocratic system with UV, RI, PL columns, standards & Galaxie add-in ideal for low temp GC applications • 920 -LC has a wide range of detection incl: – UV, PDA, FL, RI, ELSD 78

Varian 920 -LC Competitors

Varian 920 -LC Market Competitors • Shimadzu • Waters • Agilent • Dionex • The 920 -LC presents a significant step forward for the Varian 80

Varian 920 -LC Compared to the Waters Alliance HPLC

Waters Alliance • Consists of 2695 Separations Module • Pump, autosampler and controller • Options of – 2996 Diode Array Detector – 2487 Dual wavelength Detector – 2475 multi-wavelength Detector – 2414 RI Detector – 432 Conductivity Detector – 2420 ELS Detector – 2465 Electrochemical Detector 82

Varian 920 -LC Compared to Waters Alliance Maximum flowrate Varian 920 -LC 5 ml/min 10 ml/min 120 HPLC vials 200 HPLC vials Maximum pressure Autosampler Capacity Microplate Capability NA 3 plates Maximum Column Heater Temp 65 o. C 60 o. C Number of Detectors None Sample Carryover 83 Up to 2 built-in <0. 01% <0. 1%

Varian 920 -LC compared to the Agilent 1200 Series LC

Agilent 1200 Quaternary Pump G 1354 A • Dual piston, low pressure quaternary gradient pump • Electromechanical check valve • Variable piston stroke • Pulse damper between primary and secondary piston • Upgrade? ? of 1100 series • Requires degasser module 85

Agilent 1200 Autosamplers • Option of • G 1329 A standard Autosampler capable of 100 HPLC vials • G 1367 B Enhanced Autosampler compatible with either 2 microplates or 108 HPLC vials • Standard carryover <0. 1% but can be improved to <0. 01% using needle wash • Both have peltier cooled option 86

Agilent 1200 Detectors • Wide range of detectors • • • 87 Variable Wavelength Detector Multi-Wavelength Detectors Diode Array Detector Fluorescence Detector RI Detector

Varian 920 -LC Compared to Agilent 1200 Pulse Damper Yes Not Required Microplate compatibility Not Compatible Up to 3 96 well plates Autosampler Capacity 100 HPLC vials 200 HPLC vials Dual l UV-Vis Capability NA Yes Carryover <0. 1% <0. 01% UV-Vis l range 190 – 600 D 2 only 190 – 900 D 2 & QI UV detector noise 88 Varian 920 -LC + 0. 75 x 10 -5 Au + 0. 5 x 10 -5 Au

Applications of the 920 -LC

Improved Analysis of 18 Polycyclic Aromatic Hydrocarbons using a Varian 920 -LC Analytical HPLC and Pursuit® 3 PAH Column By Phuong Truong Varian Australia Pty Ltd 679 Springvale Rd Mulgrave VIC Australia

Varian 920 -LC Analytical HPLC 91

Introduction • PAH’s are carcinogenic contaminants produced as a by-product of combustion – Found in drinking water, soils and sludges • Can be analysed by GC, GC-MS • Compared to GC, HPLC offers • Easier sample preparation • Improved sensitivity with FLD • Better selectivity • PAH’s are difficult to analyse because of their structural similarity • Results in long gradient runs of 25 to 30 minutes 92

Instrumentation • Varian 920 -LC Analytical HPLC (0010089300) • • Quaternary gradient pump with 4 channel degasser 200 position autosampler with 100 m. L syringe Thermostated column compartment Diode Array detector • Optional Fluorescence detector (99100 xxx 00) • Varian Pursuit® 3 PAH 100 x 4. 6 mm column with 3 mm packing material (A 7001100 X 046) 93

Instrument Conditions - Gradient Conditions Time (min) Flow rate % Water % Acetonitrile (ml/min) 0. 0 40 60 3. 0 1. 3 40 60 7. 0 1. 3 10 90 13. 3 1. 3 0 100 16. 0 1. 3 0 100 16. 1 1. 3 40 60 21. 0 94 1. 3 40 60

Instrument Conditions - Fluorescence Detector Conditions Time (min) Excitation Emission (nm) 0. 0 323 5. 6 250 375 7. 1 270 410 8. 5 265 380 10. 5 280 420 14. 7 95 270 293 498

Results Component UV LOD (mg/L) Fluoro LOD (mg/L) USEPA RDL (mg/L) Naphthalene 2 0. 04 1. 8 Acenaphthylene 5 NA 2. 3 1 -Methylnaphthalene 5 0. 05 NR 2 -Methylnaphthalene 5 0. 06 NR Acenaphthene 8 0. 03 1. 8 Fluorene 2 0. 003 0. 21 Phenanthrene 0. 5 0. 04 0. 64 Anthracene 0. 5 0. 06 0. 66 1 0. 14 0. 21 Fluoranthene 96

Results Component UV LOD (mg/L) Fluoro LOD (mg/L) USEPA LOD (mg/L) Pyrene 1 0. 02 0. 27 Benzo(a)anthracene 1 0. 013 0. 5 0. 04 0. 15 Benzo(b)fluoranthene 1 0. 018 Benzo(k)fluoranthene 1 0. 017 Benzo(a)pyrene 1 0. 023 dibenzo(ah)anthracene 2 0. 01 0. 03 Benzo(ghi)perylene 2 0. 01 0. 076 0. 5 0. 02 0. 043 Chrysene Indenol(123 -cd)pyrene 97

18 PAH by Varian 920 -LC & Pursuit 3 PAH 98

16 PAH by Dionex HPLC • Gradient separation at 1. 5 ml/min with Fl Detection 99

17 PAH by Grace Vydac 201 TP 5 Column • Flow rate 1. 5 ml/min 100

Conclusion • The Varian 920 -LC and Pursuit 3 PAH column are capable of separating all 18 PAH’s in 15 minutes • The reproducible gradient delivery and thermostated column compartment ensure excellent retention time reproducibility • With the optional built-in fluorescence detector, detection limits exceed the requirements of USEPA method 8310 • Offers improvements in throughput compared to alternative HPLC separations (Grace Vydac 201 TP 5 column) 101

Analysis of Ibuprofen in Tablets According to the US Pharmacopoiea using a Varian 920 -LC Analytical HPLC and Pursuit®XRs column By Phuong Truong Varian Australia Pty Ltd 679 Springvale Rd Mulgrave VIC Australia

Varian 920 -LC Analytical HPLC 103

Introduction • • • 104 Assays of active pharmaceutical ingredients (API) are common in the pharmaceutical industry • Often used to quantitate the API and contaminants Ibuprofen is a NSAI in widespread use around the world 4 -isobutylacetophenone (Ibuprofen related compound C) is a breakdown product of Ibuprofen • It can adversely affect the central nervous system • The USP requires Ibuprofen related compound C to be determined at < 0. 1% per tablet USP prescribe • a 250 mm x 4. 6 mm ID x 5 mm L 1 type (C 18) column • Valerophenone internal standard In this work the recommended column was compared to a 100 x 4. 6 x 3 mm column

Instrumentation • • 105 Varian 920 -LC Analytical HPLC (0010089300) • Quaternary gradient pump with 4 channel degasser • 200 position autosampler with 100 m. L syringe • Thermostated column compartment • Diode Array detector Columns • Varian Pursuit®XRs columns feature 100Å silica, high sample loadability, extended p. H range, outstanding resolution and excellent column lifetime – Varian Pursuit® XRs 100 x 4. 6 mm x 3 mm column (A 6011100 X 046) – Varian Pursuit® XRs 250 x 4. 6 mm x 5 mm column (A 6010250 X 046)

Instrumental Conditions Mobile Phase Flow rate 2 ml/min Wavelength 254 nm Temperature 106 1% chloroacetic acid in water p. H 3/Acetonitrile 40: 60 v/v Ambient

Definitions – Resolution and Selectivity (a) m. AU 107

Chromatographic Resolution and Selectivity • • 108 Resolution R is similar to selectivity but takes into account peak width Baseline resolution is when R=1. 5 Capacity factor Selectivity a

Standards - Pursuit®XRs 250 x 4. 6 x 5 mm Column 109 • Ibuprofen and Internal Std with 250 mm x 5 mm column

Standards – Pursuit®XRs 250 x 4. 6 x 5 mm Column 110 • Internal Std and Ibuprofen related compound C with 250 mm x 5 mm column

Results – Pursuit®XRs 250 x 4. 6 x 5 mm Column Resolution (USP > 2. 5) Ibuprofen I/S in Ibuprofen solution 111 Selectivity 1. 29 7. 3 I/S Ibuprofen related C solution Assymetry (USP < 2. 5) 1. 00 1. 29 1. 06 5. 1 1. 01 1. 18

Standards – Pursuit®XRs 100 x 4. 6 x 3 mm Column 112 • Ibuprofen and Internal Std with Pursuit® XRs 100 mm x 3 mm column

Standards – Pursuit®XRs 100 x 4. 6 x 3 mm Column 113 • Internal Std and Ibuprofen related compound C with Pursuit® XRs 100 mm x 3 mm column

Results – Pursuit® XRs 100 x 4. 6 x 3 mm Column Resolution (USP > 2. 5) Ibuprofen I/S in Ibuprofen solution 114 Selectivity 1. 27 5. 3 I/S Ibuprofen related C solution Assymetry (USP < 2. 5) 1. 01 1. 29 1. 13 4. 0 1. 18

Ibuprofen Tablet Chromatogram of Ibuprofen tablet sample spiked with I/S using a Pursuit® XRs 100 x 3 mm column 115

Spectral Confirmation using PDA Detector 116

Data for Ibuprofen Tablets % of labelled Concentration of Ibuprofen 101. 9% 117 Assymetry of Ibuprofen 1. 27 Assymetry of I/S 1. 08 Resolution between Ibuprofen and Valerophenone I/S 5. 4

Conclusion • The Varian 920 -LC and Pursuit® XRs 3 mm columns significantly improve throughput of an Ibuprofen assay compared to the USP recommended columns • The calculated concentration of Ibuprofen in the tablet complies with USP requirements of nominal + 10% • The sensitivity of 920 -LC PDA detector ensures the LOD for the Ibuprofen related C compound is 10 fold lower than USP • The PDA detector allowed positive confirmation the unknown peak in the tablet was Ibuprofen related compound C (4 -isobutylacetophenone) 118

Varian 920 -LC All Your HPLC needs • The 920 -LC – Is a robust high performance automated Analytical HPLC – Fast LC compatible using 2. 4/2. 8 mm columns – Has a state-of-art LP quaternary gradient solvent delivery system – A high capacity, low carry-over autosampler with leading performance specifications – Features sensitive UV-Vis and PDA detection technology for unmatched performance – An option of second detector fitted – Fluoro or RI – Uses highly capable Galaxie. TM software with a simpler, consistent instrument interface for real productivity 119 – Has automated AIQ and maintenance tools

Questions? ?