Varian 940-LC Analytical to Prep HPLC

Varian 940-LC Silver Bullets The 940-LC one instrument – many uses: High performance automated Analytical HPLC High performance semi-preparative HPLC High performance prep LC

Varian 940-LC Silver Bullets Flexible and capable Wide flow rate from 0.1 ml/min to 200 ml/min Dual path-length flowcells for extended Abs One Injector, analytical or prep volumes (5ul to 4.5ml) Superb fraction collecting Galaxie real-time peak detection Practical collect and inject Much Easier interface, it does what you tell it! Cleaner UI

Goals of the 940-LC Analytical to Prep Aim Become market leader in this $180M segment Double market share Market Continue to focus on Pharma / BioPharma Academic Strategy Deliver innovation in hardware, consumables, software and services

Varian 940-LC Instrument Overview Factory tested, pre-plumbed instrument: Plug and play operation High performance semi-preparative HPLC Gradient Analytical HPLC Autoprep System Choice of UV or PDA detector Dual path flow cell for unmatched range and scalability No compromise performance for prep and analytical – switch with a key stroke – no plumbing or hardware changes needed! The most flexible analytical to prep HPLC available Fast prep Integ fc co;\llection Perform Consumables Ana to skid

Varian 940-LC Instrument Overview Scale-up Module for automated methods transfer from analytical to semi-prep, or switching from Analytical to Prep work Versatile and robust solvent-delivery system New loopless autosampler – wide volume range NEW Fraction collection with Complete range of collection modes such as slope, threshold and time slicing Wide range of vessels, including autosampler rack Comprehensive range of columns and consumables More productivity via GalaxieTM software Valve automation for genuine unattended operation NEW direct control interface

Varian 940-LC Versatility and Performance Easy to change pump heads Versatile solvent delivery systems Powerful pumps allow 4000psi at 25mL/min operation for fast chromatography Ideal for 4.6mm and 1” column Able to use smaller 3 & 5 um particles columns at elevated pressures Improves productivity Save solvents and waste costs Short run times save time High resolution separations = high purity 1ml/min Analytical 21ml/min Prep We have uncovered the market need -

Varian 940-LC Pumps Same range of accessories as PS210/218 5ml Head to 200ml Head Easily interchangeable pressure modules thru removable side panel. Easy access to mixer Self maintenance and versatility features have been completely retained with no compromises – integrated systems are notoriously difficult to maintain so this is Unique

Varian 940-LC Pumps - FAB Feature Advantage Benefit Interchangeable Pump Heads (5 to 200ml/min) Unrivalled flexibility for any application Economical and Convenient with Excellent Performance Open access for ease of maintenance Easy for the user to correct basic issues resulting in more uptime Dependable Simple and system Durability Analytical to Prep on one System One system will do the work of two providing high performance flexibility Performance and Reliability at an Excellent Price/Performance ratio

Varian 940-LC Autosampler Prep version of the 920-LC Sampler 5ul to 4.5ml Injection range No necessity to combine a Manual injector No need to dedicate injector to prep or analytical One Sampler does all! Cooled version Loopless Design Needle in path Design No carryover 2008 Technology – no changing loops

Varian 940-LC Autosampler - FAB Feature Advantage Benefit Needle in Path Design No need for Injection loops No Carryover Versatility, Economical Performance One Injector, large or small inject volumes Simplifies the system, no need to swap components for analytical to prep Accuracy and Seamless Simplicity One Autosampler, all rack formats No need for multiple injector options Versatility Simplicity

Varian 940-LC UV/Vis - PDA Detectors High Sensitivity for Analytical applications Extended absorbance – desensitised, for Prep Applications All in One Cell! NEW 4 x 0.25mm Cell low dead volume Excellent performance at low flow rates for Analytical Excellent Extended Absorbance for Prep

Varian 940-LC Dual Pathlength Flowcell Absorbance Detector Extended absorbance range without re-plumbing (<70 AU) NEW 4mm x 0.25 mm 4mm x 0.15 SuperPrep Low dispersion UV/Vis Detector PDA Detector Low noise ~ +10 mAU Low Drift ~ 1 mAU/hr

Agilent 1200 Flowcells

Varian 940-LC UV/Vis - PDA Detectors - FAB Feature Advantage Benefit NEW 4x0.25 Low Volume Cell Optimised for Analytical to Prep, no need to change Cells Performance and Versatility and Dependable Tool Free maintenance Easy to change lamps and flow cells Simplicity Range of cells for ul/min to Litres/min Fits all applications from Analytical to Benchtop Prep Versatility and Performance

Varian 440-LC Fraction Collector Fully controlled by software for foolproof collection by slope, level or timeslice Known fluid path for accurate fraction collection Software display of collected fractions and source injections Reliable operation Compatible with a wide range of containers Belart and other optional racks Microvial to 50ml tube capacity Autosampler rack compatibility provides practical collect and re-inject capabilities

Varian 440-LC Fraction Collector We chose not to collect Peak 3 Peaks 1,2,4,5,6,7 collect with exceptional accuracy Excellent algorithm, and simple delay volume calculation Slope/Threshold based on real numbers!!

Varian 440-LC Fraction Collector Fraction Log shows peak location using colour coded rack assignment

Varian 440-LC Fraction Collector - FAB Feature Advantage Benefit Modern Design, improved movement and hardware Significant improvement ergonomically on 701 Pleasing Appearance with Stylish fast Collector movement Simplified user interface Easy to use, does what you ask it to do both manually and automated Simplicity and precious Dependability for customer fractions Inject/Collect and Re-inject Collect into A/sampler rack then re-insert into the Sampler for Fraction Purity Determination Provides Confidence in Fraction Puritys without the need to do any liquid transfers

Varian 445-LC Scale-up Module Optimized flow paths for automated scale-up Separate optimised flow paths for Prep and Analytical columns Solvent selection valves for 4 solvents ( 2 per pump) Integrated priming valves for easy solvent changes Two Integrated Push Button Manual valves for Analytical and prep Injections

Varian 445-LC Scale Up Module( SUM) - FAB Feature Advantage Benefit 2 Solvent Selection valves for solvent switching and flexibility Can prime up 4 most popular solvents thus saving time on re-priming Simplicity and Dependability Analytical and Prep Columns Positions for Automated Scale Up or Switching Seamless and simple transfer from an Analytical analysis to Prep Simplicity and Versatility Integrated Manual Valves which are dedicated for Analytical and Prep Push button valve positioning with automatic return, eliminate operator error Even Simpler to use, eliminates user error improving Confidence in results

Varian 940-LC Software Usability Software designed for Prep users Enhanced manual control One page status and chromatogram Real time fraction collection display Advanced peak sensing for better fraction collection Purity displays for absorbance detection – standard! Documentation and audit trails for traceability and regulatory needs

Varian 940-LC Configurations pump inject detect SUM FC Modular Isocratic 25ml Opt any Manual UV/Vis NA Base Grad Man inj Dual UV option Productive 1 Opt 50 Auto 5ml +man yes Productive 2 PDA Prep 100ml Opt 200 manual Dual UV/Vis

940-LC Pricing Market driven pricing Similar pricing to existing systems at the same level Opportunity to move system price up with valuable options General move to higher ASP systems

Varian 940-LC Consumables Columns designed for Prep users From 3 to 10mm media Wide range of C4 to C18 Silica and Polymer media From 2mm to 2” ID Unique solutions for dedicated prep with load and lock systems Matched Analytical to Prep columns and packing's to assure smooth scale up Solutions for hardware, software and consumables

Applications of the 940-LC

Using Linear Scale-up Principles and the Varian 940-LC with Scale-Up Module for the Isolation of 5-Hydroxytryptophan from Griffonia Simplicifolia Dennis Hoobin Varian Australia Pty Ltd 679 Springvale Rd Mulgrave VIC Australia

Varian 940-LC Analytical to Prep Instrument

Introduction Griffonia simplicifolia is a tall bushy vine found in Ghana, Nigeria, Liberia, Ivory Coast, Cameroon and Congo The vine produces pods containing 3 large legumes The legumes are a rich source of 5-hydroxytryptophan (5HTP) 5-hydroxytryptophan produced by the human body from the essential amino acid L-hydroxytryptophan (LT) 5-HTP has been clinically used to increase serotonin production to combat many mental illnesses like depression and schizophrenia among others

Instrumentation Varian 940-LC Semi-Prep HPLC (part no 02102201-00) Binary gradient pumps 128 position autosampler with 5mL syringe Scale Up Module to allow seamless scalability from Analytical to Semi-preparative applications Optimised Dual path flowcell Diode Array detector Accurate Fraction collection Columns - Varian XRs C18 Analytical column -150 mm, 4.6 mm ID, 5u Preparative column - 150 mm, 21.2 mm ID, 5u

Instrument Features Scale Up Module Fully Automated unit allowing switching between analytical and preparative columns software-controlled module switches from analytical to preparative operation to ensure that chromatograms are correlated Flow cell optimized dual path flowcell that spans analytical to prep flow rates (1–25 mL/min) the dual path flowcell design provides the ability to work over an extended absorbance range (mAu to tens of Au)

Instrument Conditions Scaling Up Scaleable System Elements Absolute System Elements Flow Rate Packing Material Tubing Area Column Length Sample Injection Volume Sample concentration Solvent Consumption Gradient Slope Run Time Back Pressure

Instrument Condition Gradient conditions Mobile phase 85% A (H2O) 15% B (MeOH) Flow rates: 1 mL/min (analytical) 21.2 mL/min (preparative) Injection volume 50 μL analytical–4 mL preparative Dual Array PDA detector: monitoring 2 channels at 230 and 280 nm.

Analytical to Preparative 50 μL analytical injection of 5HTP standard

Diode Array Chromatogram Diode array display. Quadrant 1: Chromatogram. Quadrant 2: 3D diode array data. Quadrant 3: Isogram. Quadrant 4: UV spectrum derived from chromatogram at cursor position

Instrument Scale up Chromatograms 50 μL analytical injection of legume extract 4 mL preparative injection of legume extract, full scale. Note the extended range absorbance maximum of 8.5 abs

Purity Check Fraction between 3 and 3.5 min in the preparative chromatogram was collected resulting in a fraction volume of 10.1 mL Reinjection of the collected fraction onto the Analytical column results in a single peak chromatogram. Overlayed with the PDA peak purity trace, the flat top of the purity trace indicates a pure compound

Discussion A good estimate of the amount of the % of HTP in dry Griffonia seeds can be made The entire 10 mL fraction contains 17.2 mg 1gm of seeds contains 215mg of 5HTP Approx. 20% dry weight G. Simplicifolia seeds are a very rich source of 5HTP

Conclusion Varian 940-LC is a powerful automated system to allow easy development of HPLC preparative separations Developing an analytical method – seamless scale up to an optimised preparative columns Excellent correlations between analytical and preparative chromatograms Confidence in the purity of fractionated material

Varian 940-LC Summary The 940-LC one instrument – many uses High performance automated Analytical HPLC High performance semi-preparative HPLC Versatility Pumping system able to operate at analytical flow rates and scalable up to prep flows Dual path-length flow cell technology for unmatched range Scale-up Module for automated methods transfer Advanced fraction collection with full software control Complete range of columns and consumables Prep enhanced Galaxie TM software and valve automation for genuine unattended operation

Status of Varian HPLC at Release Installation/setup  Perceptions  Ease of ordering  Operational flexibility  Competitive performance  Competitive specifications  Ease of use  Reliability  Productivity  Validation and Compliance  Flexibility/expansibility (especially in semi-prep)  Competitive pricing  Wide product range – hardware, columns, consumables 

What About the Modular HPLC?

Modules Overview What about modules? Low price manual, isocratic systems After market module business Odd configurations/applications not served by iHPLC Retaining 210/218, SD-1 and 212 pumps 325/335/356 detectors CVM 500 Still available but no need to actively sell due to iHPLC 420 for Online SPE 410/430 Yet, they will come! Price Increases so beware!

Modules Overview Obsoleting 220/230/240 ternary pump module 340 UV detector 355 RI detector 363 Fluorescence detector 400 CVO module 701 fraction collector

Varian 356-LC RI Detector

Conclusion for 900 Series Compact and Stylish Integrated Systems Retain flexibility and maintenance features Top Tier hardware performance Top Tier User Interface – pleasant experience Simplicity in a box – Install , Use and Maintain 1st Truly Integrated HPLC’s on the Market 46 46

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