Redefining HPLC purificationAgilent 1200 Series Purification Platform

Christian Gotenfels

2

Contents

Chapter Page

The role of preparative HPLC in the pharmaceutical industry3

Agilent’s portfolio4

Hardware5 - 12

Software13 - 17

Techniques

Agilent’s patented delay sensor _ _____________________________18

Delay calibration – how does it work? ____________19

Mass-based fraction collection20

Recovery strategies ___________________________________21

Advantages

Agilent’s CAN network22

Agilent’s integrated safety concept23

Summary 24

3

Preparative HPLC…for purification is fast enough to keep pace in drug discovery process

Preparative HPLC in the pharmaceutical industry Challe

nges

Pharmaceutical companies are striving to develop faster and more efficient ways to generate new drug candidates against the increasing number of biological targets.In order to achieve this goal improvements are required in the following areas:

• High throughput synthesis

• High throughput purification

• High throughput screening

Challenges for the pharmaceutical industry:

4

Agilent’s approach - dedicated systems

Agilent 1200 Series purification system (PS)

Analytical scale (AS)

Preparative scale (PS)

Syste

ms

Agilent 1200 Series purification system (AS)

For flow rates < 5 (10) mL/min

For flow rates < 100 mL/min

Designed for various sample quantities, optimized for the high recovery and purity

5

Agilent 1200 Series purification systemsSyste

ms

General system hardware features: • Modular approach for

maximum flexibility

• Compact, stackable design (typical bench space for MS-based fraction collection only 2 m)

• Early Maintenance Feedback (EMF), a time-saving maintenance tool alerts when maintenance is due

• Analytical and preparative columns with different selectivities for specific purification tasks

• Integrated safety concept for walk away confidence

6

Agilent 1200 Series purification system (AS)

• Purification of sample amounts up to the low mg range

• for flow rates below 5 (10) mL/min

• Minimum delay volumes for high separation performance resulting in high recovery and purity

- Isocratic - Binary- Quaternary

- Standard- Well-plate- Preparative- Thermostatting- Manual

- VWD- MWD- DAD- MS- UIB for third party detectors

Pumps

Injectors

- Thermostatted compartment- Column organizer

- Valves, columns and supplies

Column holder

Detectors

Fractioncollectors

Hardw

are

Purification systems (AS)designed for:

- Fraction collector (AS)

7

• Purification for sample amounts < 1 g

• Flow rates up to 100 mL/min

Agilent 1200 Series purification system (PS)

- Preparative- Gradient - Extension

- Preparative- Thermostatting- Manual

- Fraction collector (PS)

Pumps

Injectors

Column holder

Detectors

Fractioncollectors

- Column organizer

- Valves- Columns and supplies

- MWD- DAD- MS- UIB for third party detectors

Hardw

are

Purification systems (PS)designed for:

8

• Flow rates up to 100 mL/min at 400 barwithout changing pump heads

• Precise high pressure binary gradient formationwith low delay volume (700 µl) for preparativeand analytical applications down to 0.5 mL/min

• Automated seal wash using a peristaltic pump forextended seal lifetime

• Pressure under and over sensing turns off the pump if the column gets blocked or leakages occurs

• Automated electro-magnetic purge valve

Agilent 1200 Series preparative pumpHardw

are

Preparative pumps deliver:

9

• Injection from a variety of sample containers

• Dual fixed loop concept

• High draw speed for fast injection of high sample volumes

• Needle wash mechanism for lowest carry over

Agilent 1200 Series dual loop autosampler PSHardw

are

Dual loop autosampler PS delivers:

10

Preparative scale (PS) < 100 mL/min• 1200 Series fraction collector

Preparative Scale (PS)

• 1200 Series fraction collector Analytical Scale (AS)

• 1200 Series micro fraction collector

Agilent 1200 Series fraction collectors

Analytical scale (AS) < 10 mL/min

Micro scale < 100 µL/min

Hardw

are

Fraction collectors:

11

Dispersion (peak broadening) between detector and fraction collector results in poor recovery and purity.

The selection of the appropriate flow inner diameter in the collectors delivers the best recovery and purity.

mD

LFr

24

42

Volumetric peak varianceF: Flow rateDm: Molecular diffusion coefficientL: Capillary length

The reason for 3 dedicated fraction collectors- Influence of peak dispersion

Techn

iques

Derived from Aris, Taylor and Golay equation

Click here to see animation

Dispersion:

Capillary inner diameter

12

• Well plates (96 and 384)

• Vials and Eppendorf tubes

• Small or large tubes (up to 45 ml)

• Customized racks

• Funnel trays for collection of large fraction volumes

• Fraction collection can be increased by using 3 fraction collectors in parallel

Agilent 1200 Series fraction collectors

- Accessories

Well plate tray

Multi-funnel trays

Tubes trays

Small vials tray

Customized rack

Hardw

are

Fraction collector accessories:

13

Dedicated software to match your purification needs

Agilent’s purification software solutions

Advanced solution

Easy management of high samples numbers.

Flexible for workflow integration

Agilent Purification Software

Standard solution

Standard functionality for easy system usage

21 CFR Part 11 compliancewith Security Pack

Agilent ChemStation

Software

Walk-up solution

System management for secure access,

Ideal for non-expert users

Agilent Easy Access Software

Remote data browsing and purity reports at your desk

Agilent Data Browser

Softw

are

Software solutions:

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• Full system control via graphical user interface

• Standard purification functionality for easy system usage

• Sophisticated trigger options

• Fraction preview helps to identify the right trigger parameter

• Fraction task provides straightforward purification data review

• ChemStation Security Pack supports 21 CFR Part 11 compliance

Standard solution

– Agilent ChemStation

Softw

are

Agilent ChemStation delivers:

15

Advanced solution

– Agilent purification software

Softw

are

Additional to the standard software solution: • Color coded graphical user interface

for easy sample setup and sample/fraction tracking

• Sophisticated export/import functionality integrates the system into the workflow

• Extended reporting capabilities

• Navigation panel for fast data review

16

• Flexible administrator tools to set up user access, queue tracking and project management

• Simple sample submission and status checking

• E-mail notification of results

• Rapid confirmation of molecular weight and target ion presence

Walk-up solution

– Agilent Easy Access software

Softw

are

Agilent Easy Access software delivers:

17

• Allows fast and efficient remote data review

• Data file tree structure for easy navigation

• Display of sample purity

Data browsing and purity reports

– Agilent data browser

Softw

are

Agilent data browser delivers:

18

Agilent’s patented delay sensor

– automated calculation of the delay time

Techn

iques

Fraction delay sensor:

• Comparable to a simple detector in the fraction collector

19

Delay calibration

– how does it work?

Click here to see animation

Techn

iques

Delay calibration:

The time between peak detection and fraction collection is called ‘delay time’. This parameter must be known for correct fraction collection.

The Agilent fraction delay sensor provides an automated and precise way to measure this time.

• Whenever the main flow, or the make-up flow, is changed the system must be re-calibrated

• The fraction delay sensor turns delay calibration into a simple, automated task

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Active splitting

– mass based fraction collection

Techn

iques

• A broad range of variable split ratios for optimizing applications, from 25:1 to 100,000:1

• The constant, accurate split ratios are unaffected by viscosity, temperature and tubing length

• Minimal post-column delay volumes minimizes dispersion and delay times

• Split ratios does not influence fraction delay times. This eliminates the need to recalibrate the system

Active splitting provides:

Click here to see animation

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Agilent offers three strategies to ensure recovery of valuable compounds:

1. Recovery valve for up to 12 recovery locations

2. Third party fraction collectors can be controlled by an external contact board

3. Agilent fraction collectors with software-controlled funnel trays, for 40, 80 or 120 funnels

Recovery strategies

Recovery device

Techn

iques

Fraction collector 1 Fraction collector 2

Peak Peak

Waste Waste

Fraction collector 1 Fraction collector 2

Detector

Valve

Column

Click here to see animation

22

Controlled Area Network

- System Intelligence

The design of the 1200 Series modules delivers:

• Operation independent of PC

• Real-time data processing • Fractions are triggered by inter-module communication without computer delay due to CPU usage or network traffic

• Automatic tray-recognition prevents overflow of fractions

Advan

tage

s

Click here to see animation

23

Standard in all 1200 Series modules:

• Leak detection

• Safe leak handling

• Pressure under sensing in pumps Providing walk-away confidence

• Forced fume extraction for the fraction collector

Allows system use outside a fume cupboard

Integrated safety conceptAdvan

tage

s

Health and safety:

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• Dedicated systems with low delay volumesOptimized for high recovery and purity and desired throughput

• Software solutions for different user needsAllows you to configure your own purification system

• Automated delay volume calibrationFor easy and fast system set-up

• Solutions for recovery collectionEnsures that your valuable sample can never be lost

• Integrated safety conceptSafe leak handling/detection and forced fume extraction gives you walk-away confidence

• System Intelligence with CAN networkData processing in real time for instantaneous and precise fraction collection

• Single-vendor solutionsFor all your equipment, columns, supplies and support

Agilent 1200 Series purification platform

- key features

Summ

ary

25

For the latest information visit:

www.agilent.com/chem/purification