920-LC Analytical HPLC 940-LC Analytical to Prep HPLC iHPLC 920-LC Analytical HPLC 940-LC Analytical to Prep HPLC

Varian 900-LC Series iHPLC

Agenda Introduction An overview of the 920-LC Analytical HPLC Introduction to the 9X0-LC series HPLC Total market size by technique and market segment An overview of the 920-LC Analytical HPLC Features and benefits Positioning, competition and target markets Applications An overview of the 940-LC Analytical to Prep and Prep HPLC

What is the iHPLC? 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

HPLC Demand SDI 2007 Overall LC is $3.2B @ 6% growth 23000 systems sold in 2006 New HPLC systems sales (initial) $1220M $764M Conventional analytical $180M Prep $113M Fast LC

Current Status of Varian HPLC 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

Varian 920-LC Silver Bullets Premium Analytical Pump Performance and reliability 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 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 $800M segment by doubling market share and revenue Markets Pharma / BioPharma Environmental Industrial Strategy Leverage Varian distribution channel to deliver competitive HPLC hardware, consumables, software and services

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

Varian 920-LC What Is It? Either dual l UV-Vis or PDA detector Optional second detector - Fluorescence or RI High precision (<0.15oC) 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 GalaxieTM software External CVM, ELSD or 356-LC RI can be plugged directly into the back of the unit.

920-LC for Standard HPLC to Fast HPLC 25cm x 4.6mm Pursuit XRS 5um 1.0ml/min 25cm x 4.6mm Pursuit XRS 5um 2.2ml/min 15cm x 4.6mm Pursuit XRS 5um 1.0ml/min 15cm x 4.6mm Pursuit XRS 5um 3.75ml/min 10cm x 2.0mm Pursuit XRS 2.8um Ultra 0.8ml/min 5cm x 2.0mm Pursuit XRS 2.8um Ultra 0.4ml/min 16 x Speed Enhancement 5cm x 2.0mm 2.4um 1.3ml/min

920-LC for Standard HPLC to Fast HPLC 25cm x 4.6mm Pursuit XRS 5um 1.0ml/min 25cm x 4.6mm Pursuit XRS 5um 2.2ml/min 15cm x 4.6mm Pursuit XRS 5um 1.0ml/min 15cm x 4.6mm Pursuit XRS 5um 3.75ml/min 10cm x 2.0mm Pursuit XRS 2.8um Ultra 0.8ml/min 5cm x 2.0mm Pursuit XRS 2.8um Ultra 0.4ml/min 16 x Speed Enhancement 5cm x 2.0mm 2.4um 1.3ml/min

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

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 Accurate flow from 0.1 to 10 mL/min for 2 to 4.6 mm columns Overlay of 10 chromatograms 10% steps at 1ml/min

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

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 1ul to 50mL Cooled option available

Varian 920-LC Washout Performance 125 ug/ml Caffeine Blank injection <0.01% carryover

920-LC Autosampler How Does it Work?

Varian 920-LC Injection Synchronization

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

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

Varian 920-LC UV/Vis - PDA Detectors - FAB Feature Advantage 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 Fits all applications from LCMS to Benchtop Prep Versatility and Performance

Varian 920-LC Fluorescence Detector Sensitive Fluorescence detector (S/N > 600 Raman H2O 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

Varian 920-LC Fluorescence Detector - FAB Feature Advantage 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 ( 12ul volume) Sharper peaks, enhanced resolution

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

Varian 920-LC RI Detector - FAB Feature Advantage Benefit Low Volume Cell Minimal Peak Dispersion giving enhanced resolution Performance and Accuracy High Intensity, long life LED Maintenance Free for consistant long term performance Dependability, Durability and Confidence in results Temp Controlled optics 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 40Hz Data Rate for Fast LC Onboard timetable program to enable parameter change thru a run

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

Heated Column Compartment Temperature range from ambient + 10oC up to 65oC Temperature reproducibility +0.15oC 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 250mm columns Option with electrically actuated column switching valve and injection valve

Outstanding Stability

Reproducibility of 50 Injections Reproducibility <0.8% over 50 injections and 12 hours

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 and Accuracy Provides greater method robustness Dependable, Confidence in results Accurate Spacious compartment with easy access Fast column changes, room to work Simple and Convenient

Varian 920-LC Software Usability User friendly GalaxieTM 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 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

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

Varian 920-LC Concept - FAB Feature Advantage Benefit 1st Fully Integrated iHPLC 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 Desirable to own, Convenient to use

Varian 920-LC Concept - FAB Feature Advantage Benefit Single Part Numbers Easy to quote Much simpler to sell, and easier experience 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 Fast installation so you are quickly able to use the instrument Convenient and Simple

Varian 920-LC Concept - FAB Feature Advantage 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 Tray Storage Provides additional storage space for Software CD, tools, columns etc… Clean Appearance and Versatility

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

Varian 920-LC Concept - FAB Feature Advantage 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 present to customers Convenient and Powerful messages to explain to customer Updated Data Sheets, Price List and App Notes Supporting materials to leave with the customer Provides Confidence in product range and Simple to Quote

Varian 920-LC Ordering Matrix pump inject detector Oven Detector 2 option Options Modular Isocratic 210-LC 10ml Manual 325 PDA, RI, ELSD 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 ELSD opt High Performance Gradient 00-100892-00 Auto cooled Development 00-100893-00 PDA Std Research 00-100894-00

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

Varian 920-LC Consumables Excellent range of HPLC Columns providing industry leading resolution and robustness Pursuit Pursuit XRS Polymeric Phases - PL From 2mm to 2” ID Prepacked Other Speciality Phases NEW 2.8 Ultra Column AND 2.4um Columns for Fast or High Res LC 2.8um Ultra is optimised for Speed 2.4um gives further enhancements in Resolution

2.8um Vs 2.4 um – High Res or Extra Speed 2.4um 3mm x 5cm for RESOLUTION 2.8um 2mm x 5cm for speed

Varian 920-LC Positioning

UPLC, Fast LC and the Varian 920-LC

But smaller particles give greater back pressure!! 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!!

Van Deemter Plot 3.5 mm 5 mm 1.8 mm HETP (cm/plate) Linear velocity u cm/sec 0.000 0.005 0.010 0.015 0.020 0.025 0.035 0.030 0.040 0.045 0.0 0.2 0.4 1.6 1.0 1.2 1.4 0.6 0.8

Fast LC Productivity 25cm x 4.6mm Pursuit XRS 5um 1.0ml/min 10cm x 2.0mm Pursuit XRS 2.8um Ultra 0.8ml/min 5cm x 2.0mm Pursuit XRS 2.8um Ultra 0.4ml/min 16 x Speed Enhancement 5cm x 2.0mm 2.4um 1.3ml/min

Market Size for Fast LC - 2007 Total Systems market Size 2007 = US$1.3b (SDI 2006)

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

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

Small particle porous column packing 1.7 – 3.0 mm in diameter 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

Theory UPLC or Fast LC Small particle porous column packing 1.7 – 3.0u 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

Advantages of Fast LC Price Flexibility Reliability 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

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

Typical Speed Performance of a UPLC Separation

Typical Speed Performance of Fast LC 2.4 minutes Fanta Orange Drink Sugar Standard Conditions Mobile Phase: 10mm Sodium Phosphate ph6.0/ACN gradient Detection: UV Detection Temperature: 40°C Flow-Rate: 1,2mL/min Column: Varian Ultra 50x2,0mm

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

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).

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

Current Systems on the market Others Shimadzu Conventional LC that shows 2.2 mm particle column applications – Fast LC approach Jasco UPLC system utilising 15000psi pressure capability and column temperatures to 60°C Hitachi 9000psi pressure capability with 90°C temperature performance. Variation on their L2000 series HPLC

Van Deemter Plot 3.5 mm 5 mm 2.4 mm 1.8 mm HETP (cm/plate) Linear velocity u cm/sec 0.000 0.005 0.010 0.015 0.020 0.025 0.035 0.030 0.040 0.045 0.0 0.2 0.4 1.6 1.0 1.2 1.4 0.6 0.8

Trends in Fast LC 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 Varian 920-LC Fast LC Mobile Phase: Acetonitrile Water Gradient Detection: UV@254nm Temperature: 40°C Flow-Rate: 0,8mL/min Column: Pursuit XRs C18 3um, 100x2mm Varian 920-LC Fast LC

Soft Drinks Using 920-LC Conditions Fanta Orange Drink 2.4 minutes Fanta Orange Drink Sugar Standard Conditions Mobile Phase: 10mm Sodium Phosphate ph6.0/ACN gradient Detection: UV Detection Temperature: 40°C Flow-Rate: 1,2mL/min Column: Varian Ultra 50x2,0mm

Alkylated Phenones Using 920-LC 1.4 minutes Conditions Mobile Phase: Acetonitrile Water Gradient Detection: UV Detection Temperature: 60°C Flow-Rate: 1,6mL/min Column: Varian Ultra 3um 50x2,0mm

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:0.9ml/min Column:HP 3 C18 CP Injection volume : 1µl sample Delphinidin, Cyanidin, Petunidin, Pelargonidin, Peonidin, Mavidin

Fat Soluble Vitamins Using 920-LC Chromatogram 260 nm Chromatogram MaxPlot Conditions Mobile Phase: A Water 0;1% formic acid B CH3CN 0,1% Formic acid gradient t 0min B 80% ; t 1.5 min B80%; t 2min B 100% T 12 min B100% Detection: UV 260 nm & MaxPlot Temperature: 50°C Flow-Rate: 0,7mL/min Column: Pursuit C18 3 µm 100 x 2,1mm Injection volume 25 µL, sample Retinol, vitamin D2,D3,E, E Ac, K1

Fast Sugars Using 920-LC Conditions Fanta Fanta Orange Drink Sprite Sugar Standard Conditions Mobile Phase: Acetonitrile Water 75:25 Detection: RI Detection Temperature: 40°C Flow-Rate: 2,5mL/min Column: Polaris NH2 5um 150x4,6mm

UPLC does not currently suit all applications 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)

Varian 920-LC Target Markets

SDI 2006 Market by Industry Split

Varian LC Sales by Industry Type

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

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

Varian 920-LC Competitors

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

Varian 920-LC Compared to the Waters Alliance HPLC

Consists of 2695 Separations Module Options of 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

Varian 920-LC Compared to Waters Alliance Maximum flowrate 5 ml/min 10 ml/min Maximum pressure Autosampler Capacity 120 HPLC vials 200 HPLC vials Microplate Capability NA 3 plates Maximum Column Heater Temp 60oC 65oC Number of Detectors None Up to 2 built-in Sample Carryover <0.1% <0.01%

Varian 920-LC compared to the Agilent 1200 Series LC

Agilent 1200 Quaternary Pump G1354A 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

Agilent 1200 Autosamplers Option of G1329A standard Autosampler capable of 100 HPLC vials G1367B 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

Wide range of detectors Agilent 1200 Detectors Wide range of detectors 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 Carryover <0.1% <0.01% UV-Vis l range 190 – 600 D2 only 190 – 900 D2 & QI UV detector noise + 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

Can be analysed by GC, GC-MS Compared to GC, HPLC offers 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

Varian 920-LC Analytical HPLC (0010089300) Instrumentation Varian 920-LC Analytical HPLC (0010089300) Quaternary gradient pump with 4 channel degasser 200 position autosampler with 100 mL syringe Thermostated column compartment Diode Array detector Optional Fluorescence detector (99100xxx00) Varian Pursuit® 3 PAH 100 x 4.6 mm column with 3mm packing material (A7001100X046)

Instrument Conditions - Gradient Conditions Time (min) Flow rate (ml/min) % Water % Acetonitrile 0.0 1.3 40 60 3.0 7.0 10 90 13.3 100 16.0 16.1 21.0

Instrument Conditions - Fluorescence Detector Conditions Time (min) Excitation (nm) Emission 0.0 270 323 5.6 250 375 7.1 410 8.5 265 380 10.5 280 420 14.7 293 498

Results Component UV LOD (mg/L) Fluoro LOD USEPA RDL Naphthalene 2 0.04 1.8 Acenaphthylene 5 NA 2.3 1-Methylnaphthalene 0.05 NR 2-Methylnaphthalene 0.06 Acenaphthene 8 0.03 Fluorene 0.003 0.21 Phenanthrene 0.5 0.64 Anthracene 0.66 Fluoranthene 1 0.14

Results Component UV LOD (mg/L) Fluoro LOD USEPA LOD Pyrene 1 0.02 0.27 Benzo(a)anthracene 0.01 0.013 Chrysene 0.5 0.04 0.15 Benzo(b)fluoranthene 0.018 Benzo(k)fluoranthene 0.017 Benzo(a)pyrene 0.023 dibenzo(ah)anthracene 2 0.03 Benzo(ghi)perylene 0.076 Indenol(123-cd)pyrene 0.043

18 PAH by Varian 920-LC & Pursuit 3 PAH

16 PAH by Dionex HPLC Gradient separation at 1.5 ml/min with Fl Detection

17 PAH by Grace Vydac 201 TP 5 Column Flow rate 1.5 ml/min

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)

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

Introduction 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 L1 type (C18) column Valerophenone internal standard In this work the recommended column was compared to a 100 x 4.6 x 3 mm column

Instrumentation Varian 920-LC Analytical HPLC (0010089300) Quaternary gradient pump with 4 channel degasser 200 position autosampler with 100 mL syringe Thermostated column compartment Diode Array detector Columns Varian Pursuit®XRs columns feature 100Å silica, high sample loadability, extended pH range, outstanding resolution and excellent column lifetime Varian Pursuit® XRs 100 x 4.6 mm x 3mm column (A6011100X046) Varian Pursuit® XRs 250 x 4.6 mm x 5mm column (A6010250X046)

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

Definitions – Resolution and Selectivity (a) mAU

Chromatographic Resolution and Selectivity 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 5mm Column Ibuprofen and Internal Std with 250 mm x 5 mm column

Standards – Pursuit®XRs 250 x 4.6 x 5mm Column Internal Std and Ibuprofen related compound C with 250 mm x 5 mm column

Results – Pursuit®XRs 250 x 4.6 x 5mm Column Resolution (USP > 2.5) Assymetry (USP < 2.5) Selectivity Ibuprofen 1.29 I/S in Ibuprofen solution 7.3 1.00 I/S 1.06 Ibuprofen related C solution 5.1 1.01 1.18

Standards – Pursuit®XRs 100 x 4.6 x 3mm Column Ibuprofen and Internal Std with Pursuit® XRs 100 mm x 3 mm column

Standards – Pursuit®XRs 100 x 4.6 x 3mm Column Internal Std and Ibuprofen related compound C with Pursuit® XRs 100 mm x 3 mm column

Results – Pursuit® XRs 100 x 4.6 x 3mm Column Resolution (USP > 2.5) Assymetry (USP < 2.5) Selectivity Ibuprofen 1.27 I/S in Ibuprofen solution 5.3 1.01 1.29 I/S 1.13 Ibuprofen related C solution 4.0 1.10 1.18

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

Spectral Confirmation using PDA Detector

Data for Ibuprofen Tablets % of labelled Concentration of Ibuprofen Assymetry of Ibuprofen Assymetry of I/S Resolution between Ibuprofen and Valerophenone I/S 101.9% 1.27 1.08 5.4

Conclusion The Varian 920-LC and Pursuit® XRs 3mm 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)

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 GalaxieTM software with a simpler, consistent instrument interface for real productivity Has automated AIQ and maintenance tools

Questions??