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Varian 940-LC Analytical to Prep HPLC

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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

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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

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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

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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

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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

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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/minAnalytical

21ml/minPrep

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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

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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

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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

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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

Performance

Accuracy and Seamless Simplicity

One Autosampler, all rack formats

No need for multiple injector options

Versatility

Simplicity

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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

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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 AU• Low Drift ~ 1 mAU/hr

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Agilent 1200 Flowcells

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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

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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

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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!!

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Varian 440-LC Fraction Collector

• Fraction Log shows peak location using colour coded rack assignment

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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

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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

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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

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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

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Varian 940-LC Configurations

pump inject detect SUM FC

Modular Isocratic

25mlOpt any

Manual UV/Vis NA NA

Base Grad

25ml

Man inj Dual UV option option

Productive 1 Grad

25mlOpt 50

Auto 5ml +man

Dual UV yes yes

Productive 2 Grad

25mlOpt 50

Auto 5ml +man

PDA yes yes

Prep Grad

100mlOpt 200

pump

manual

Dual UV/Vis NA option

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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

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Varian 940-LC Consumables

• Columns designed for Prep users

• From 3 to 10m 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 HoobinVarian Australia Pty Ltd

679 Springvale Rd Mulgrave VIC Australia

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Varian 940-LC Analytical to Prep Instrument

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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

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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

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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)

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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

• Flow Rate Packing Material

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Instrument ConditionGradient 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.

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Analytical to Preparative

50 μL analytical injection of 5HTP standard

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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

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Instrument Scale up Chromatograms

4 mL preparative injection of legume extract, full scale. Note the extended range absorbance maximum of 8.5 abs

50 μL analytical injection of legume extract

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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

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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

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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

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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

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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

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What About the Modular HPLC?

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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

Price Increases so beware!

Yet, they will come!

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Modules Overview

• Obsoleting

– 220/230/240 ternary pump module

– 340 UV detector

– 355 RI detector

– 363 Fluorescence detector

– 400

– CVO module

– 701 fraction collector

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Varian 356-LC RI Detector

4646

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

Questions??