Next Generation Ultra High Pressure Liquid Chromatography (UHPLC) Technologies: Change the Landscape...

55
The world leader in serving science Change the Landscape in LC Next Generation UHPLC Technologies:

Transcript of Next Generation Ultra High Pressure Liquid Chromatography (UHPLC) Technologies: Change the Landscape...

1 Proprietary & Confidential

The world leader in serving science

Change the Landscape in LC

Next Generation UHPLC Technologies:

2 Proprietary & Confidential

Agenda

• Core Enhanced Technology • Delivering better separations • Next generation column technology - Accucore Vanquish • Accucore Vanquish - delivering more results

• Next Generation UHPLC - getting the most out of your column

• How to achieve the best of both worlds for your separation: • Speed versus resolution

• How to manage and successfully process very data rich results

3 Proprietary & Confidential

Core Enhanced Technology Thermo Scientific™ Accucore™ Vanquish™ UHPLC Columns

4 Proprietary & Confidential

• Features • Less bed consolidation • Better packing of particles • Less void volume in column • Reduced pore depth

• Benefits • More efficient chromatography

• Competitive Edge • Bar for bar gives better separations than porous materials

Core Enhanced Technology Delivers: Better Separations

5 Proprietary & Confidential

Liquid Chromatography Particle Design:

2.6 µm 80 Å 2.6 µm 150 Å 4 µm 80 Å 1.5 µm 80 Å

Solid Core Particles

Conventional Fully Porous Non-Porous Solid Core

Reduce Size to improve kinetics at expense of

operating pressure

Low sample capacity

Very high pressure

Small particle kinetics

Reasonable pressure Very High Sample Capacity

Lower Efficiency

Low Sample Capacity

Very High Efficiency

High Sample Capacity

High Efficiency

Core Enhanced Technology Delivers: Better Separations

6 Proprietary & Confidential

What is the Accucore Vanquish?

• Next generation UHPLC column • Founded on state-of-the-art Core Enhanced Technology • 1.5 µm solid core particles • Designed in synergy with the Vanquish UHPLC to deliver:

• Better Separations: high efficiency enables the separation of very complex mixtures • More Results: High efficiency at high flow rates • Easier Interactions: Seamless workflow solution, for simple and easy separations

7 Proprietary & Confidential

• Accucore Vanquish UHPLC columns extend chromatographic efficiency. The solid core particle delivers this giving the flexibility for fast and highly efficient separations.

• Key Components

Core Enhanced Technology Delivers: Better Separations

8 Proprietary & Confidential

• The factors that affect chromatographic efficiency are eddy dispersion, longitudinal diffusion, and resistance to mass transfer - the A, B and C terms respectively from the van Deemter equation.

• Uniform packed bed • Reduced void volume

• Fast mass transfer

Results in Better Separations

Core Enhanced Technology Delivers: Better Separations

9 Proprietary & Confidential

• More efficient at optimum flow than fully porous sub 2 micron

• More efficient at optimum flow than 2.6 micron solid core

• Wider “no-efficiency-loss” range than any other column technology

• Effectively, as solution with Vanquish delivering unmatched resolution at high flows => pure productivity!

Core Enhanced Technology Delivers: Better Separations

10 Proprietary & Confidential

Why 1.5 µm Provides: Better Separations

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00

min

3

2

1

Accucore Vanquish 1.5 µm, 295 000 plates/m

Accucore 2.6 µm, 196 000 plates/m

Fully porous 1.7 µm, 176 000 plates/m

40% efficiency gain

• Efficiency = Better Separations

11 Proprietary & Confidential

Accucore Vanquish Delivers: More Results

40% time saving and Better Separations

Accucore Vanquish C18 1.5 µm 100 x 2.1mm

Solid Core C18 2.6 µm 100 x 2.1mm

Average increase in peak capacity of 220%

Average increase in resolution of 24%

12 Proprietary & Confidential

Accucore Vanquish Delivers: More Results

0.79 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.04-0.7

5.0

10.0

15.0

20.0

25.0

28.4 20140624 Pesticides C18 #108 [modified by ukrun.cell.appslab] 20140714 pesticides C18 Cortecs 1.6µm 20-80B in & minutes 43C 0.65ml/min UV_VIS_1mAU

min

WVL:230 nm

0.50 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.01-2.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

26.4 20140624 Pesticides C18 #78 [modified by ukrun.cell.appslab] 20140630 Pesticides C18 43GW 1.5 20-80 in 7 min 43C 0.65ml/min UV_VIS_1mAU

min

WVL:230 nm

Accucore Vanquish C18 1.5 µm 100 x 2.1 mm

Competitor C18 1.6 µm 100 x 2.1mm

• Accucore Vanquish vs. Competitor 1.6µm Solid Core Column

Average increase in peak capacity of 11%

Average increase in resolution of 6%

13 Proprietary & Confidential

Accucore Vanquish Delivers: More Results

0.55 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.01-2.0

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

24.0 20140624 Pesticides C18 #93 [modified by ukrun.cell.appslab] 20140711 Pesticides C18 kinetex 1.7 20-80B in 7min 43C 0.65ml/min UV_VIS_1mAU

min

WVL:230 nm

0.50 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.01-2.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

26.4 20140624 Pesticides C18 #78 [modified by ukrun.cell.appslab] 20140630 Pesticides C18 43GW 1.5 20-80 in 7 min 43C 0.65ml/min UV_VIS_1mAU

min

WVL:230 nm

Accucore Vanquish C18 1.5 µm 100 x 2.1 mm

Competitor C18 1.7 µm 100 x 2.1 mm

• Accucore Vanquish vs. Competitor 1.7µm Solid Core Column

Average increase in peak capacity of 22%

Average increase in resolution of 17%

14 Proprietary & Confidential

Accucore Vanquish Delivers: More Results

• Rapid Screening of 47 Drugs of Abuse in <4min

Overlaid selected-reaction monitoring chromatograms showing detection of 47 compounds

15 Proprietary & Confidential

• Separation of isobaric compounds

Accucore Vanquish Delivers: More Results

Chromatographic resolution of naltrexone and 6-acetylcodine with common precursor (342 u) and product ions

Chromatographic resolution of codeine and hydrocodiene with common precursor (300 u) and product ions

Chromatographic resolution of N-desmethyltapentadol and MDEA with out common precursor and product ions

16 Proprietary & Confidential

Accucore Vanquish Delivers: More Results

• Rapid Screening of 36 Veterinary Antibiotics in <5min

Overlaid selected-reaction monitoring chromatograms showing detection of 36 compounds

17 Proprietary & Confidential

Core Enhanced technology Delivers: More Results

• Reproducibility • The advanced bonding and automated packing technology used in the

manufacture of Accucore Vanquish UHPLC columns results in exceptionally reproducible chromatography.

4000 injections @ 500 bar

18 Proprietary & Confidential

Next Generation UHPLC Thermo Scientific™ Vanquish UHPLC System

19 Proprietary & Confidential

Vanquish – System Overview

• Integrated modularity • Viper-based (MP35N), tool-free fluidics • Biocompatible, iron-free flow path • Support of state-of-the-art column technology • New column thermostatting technology • Enhanced LC/MS support • Portable system control with tablet • Reduced system height • Revolutionary drawer design • Removable doors for easy access

20 Proprietary & Confidential

VPump – All New Pump

• Industry-leading performance: up to 1500 bar / 22 000 psi • Designed for lowest pressure pulsation • Intelligent piston position control and

ultra-low pump GDV (< 40 µL) • Individual linear piston drives (parallel) • Automatic purge unit • 2 ×3 solvent channels • Fingertight check valves and capillaries • New sealing system for higher uptime at

high pressures

21 Proprietary & Confidential

VPump – Flow-Pressure Footprints

• The Vanquish system expands our future‐proof flow‐pressure footprint even further

Pres

sure

[bar

]

1250

1034

800

620

10 100 1 10 100 1 2 4 6 8 10

Logarithmic Scale Linear Scale

20 nL/min

50 µL/min

Flow Rate

3 5 7 9 mL/min µL/min nL/min

1500

0

22 Proprietary & Confidential

VPump – Adaptive Thermal Effect Compensation

• Adaptive Thermal Effect Compensation: ATEC

• Fast pre-compression with a very long hold phase thanks to the Vanquish pumping concept • ~3 seconds at 1 mL/min

• Real-time monitoring of the volume contraction effect

• Solvent cool-down in the piston chamber

• Extrapolate the monitored volume contraction effect during the solvent delivery phase

1.5 bar = 0.25%

6.0 bar = 1.00%

ATEC on

ATEC off

Water/ACN, 5/95, 600 bar, 2.0 mL/min

Outstanding low pulsation even at maximum pressure

23 Proprietary & Confidential

Vanquish – A Completely New Developed Autosampler

• Metering device replaces syringe • Fine aspiration drive for ultra precise dosage

• Sample pre-compression for outstanding retention time precision

• Newly developed ceramic valve • Life-time DLC (diamond like carbon) coating

• 3-position 5-port

• Advanced air-to-air cooling • Extended sample capacity

• 4 sample racks and/or well plates

• Optimized fluidic design

24 Proprietary & Confidential

VAS – Sample Cooling

• Air-to-Air cooling concept • No liquid coolant

• Sample heat removed by circulating compartment air • Fan and Peltier element

• Closed sample compartment • Efficient cooling due to insulation from the environment • Deposited humidity will be actively removed • Magnetic door lock opens when power is off

• Cooling performance • 4 – 40 °C (down to ≥ 23 K below ambient)

25 Proprietary & Confidential

Column: Hypersil Gold, 1.9 µm, 2.1 × 100 mm System: Vanquish System Mobile Phase: A–water B–acetonitrile Gradient: Flow rate: 260 µL/min Pressure: 300 bar (max.) Comp. Vol: 0 µL and 3 µL Temperature: 25 ºC Injection: 1 µL Detection: pump pressure sensor

120

150

200

250

320 1 - 02_ID02A_R02_VAS_SETTINGS_wo_leak_comp Pump_Pressurebar

1

0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 10,0120

150

200

250

320 2 - 02_ID02A_R02_VAS_SETTINGS_wo_leak_comp Pump_Pressurebar

min

2

Time [min]

% A %B

0 60 40

0.4 60 40

4.0 0 100

4.8 0 100

4.9 60 40

10.0 60 40

w/o pre-compression

w/ pre-compression

Inject command at 0.1 min to track the pressure drop

-> isocratic step from 0 to 0.4 min (cycle completed)

Experimental Results – Pre-Compression

26 Proprietary & Confidential

Experimental Results – Pre-Compression Column: Hypersil Gold, 1.9 µm, 2.1 × 100 mm System: Vanquish System Mobile Phase: A–water B–acetonitrile Gradient: Flow rate: 260 µL/min Pressure: 300 bar (max.) Comp. Vol: 0 µL and 3 µL Temperature: 25 ºC Injection: 1 µL (N = 6) Detection: pump pressure sensor

Time [min]

% A %B

0 60 40

0.4 60 40

4.0 0 100

4.8 0 100

4.9 60 40

10.0 60 40

0,00 1,00 2,00 3,00 4,00 5,00 6,00-100

200

400

700 mAU

min

654321

1

2

3

45

6

7

8 910

WVL:254 nmw/o pre-compression

0,00 1,00 2,00 3,00 4,00 5,00 6,00-100

200

400

700 mAU

min

654321

1

2

3

45

6

7

8 910

WVL:254 nmw/ pre-compression

Ret. Time Precision [%] 1 2 3 4 5 6 7 8 9 10

w/o pre-comp. 0.040 0.026 0.050 0.096 0.035 0.033 0.033 0.039 0.044 0.040

w/ pre-comp. 0.008 0.009 0.010 0.007 0.008 0.007 0.006 0.005 0.006 0.009

27 Proprietary & Confidential

VAS – Injection Valve

• Innovative new valve design by Thermo Fisher Scientific • High pressure capability of 1517 bar • Compact valve design for optimized flow path

• Bio-compatible valve • Stator and rotor made of ceramics

• Long-life DLC coating • Valve is available as an entire assembly

• Valve is compatible with Viper fittings only • Needle seat is directly installed on the injection valve

• Integrated needle seat connection to the valve • Reduces extra column volume

Port Connected Component 1 Inlet capillary from the pump 2 Outlet capillary to the column 3 Needle seat (pre-installed) 4 Wash port waste (pre-installed) 6 Metering device (pre-installed)

28 Proprietary & Confidential

Charger – Extended Sample Capacity

• Optional rack loader to further increase the sample capacity

• Sample thermostatting 4 – 40 °C • Sample rack format recognition

via barcode • Inventory scan each time the door is

closed

• Sample capacity • 9 additional sample racks or deep

well plates • 20 additional shallow well plates

29 Proprietary & Confidential

VAS and Charger – Rack Type Recognition

• Automated barcode recognition • Encoded information

• Presence and orientation of the sample containers

• The rack and well plate type • Status of sample containers in the sampler

and charger will be available in CM panel

• Inventory scan any time the doors

are closed

• Implementation for Thermo Scientific sample racks

30 Proprietary & Confidential

VTCC – All New Column Compartment

• High temperature accuracy and precision to host 2 columns (max. ID 10mm, max. L 300mm, 5 - 120˚C)

• Support of forced air and still air operating modes (method transfer)

• Actively controlled pre-heater • Slim, vertical compartment design • Extendable to multiple columns and

temperature zones (up to 3 modules) • Upgradeable with up to 2 switching valves • “Contact free” column holder clip design • Sturdy “plug-in” column-ID tags

31 Proprietary & Confidential

Still Air • Backpressure (≤ 400 bar) • Low heat transfer to column • Cold incoming solvent

• Cold incoming solvent cools column near to solvent temperature over time

• Peak broadening, higher retention

Forced Air • Backpressure (≤ 400 bar) • Controlled thermal conditions at

column surface • Cold incoming solvent

• Axial and radial temperature gradient

• Peak broadening, higher retention

Operating Principle: Forced Air vs. Still Air Heating

22 °C 45 °C 65 °C 70 °C 70 °C

70 °C

22 °C 22 °C

70 °C

32 Proprietary & Confidential

Still Air • Backpressure (≥ 400 bar) • Solvent pre-heated

• Reduced axial temperature gradient

• Self-heating by friction • Almost no radial temp. gradient • Axial temperature gradient

• Sharp peak, less retention

Forced Air • Backpressure (≥ 400 bar) • Solvent pre-heated

• Reduced axial temperature gradient

• Self-heating by friction • Radial temperature gradient • Low axial temperature gradient • Peak broadening, less retention

Operating Principle: Forced Air vs. Still Air Heating

70 °C 80 °C 73 °C 76 °C

70 °C

70 °C 90°C

70 °C

33 Proprietary & Confidential

Still Air • At a backpressure ≥ 400 bar

frictional heating leads to: • Increasing effluent temperature

due to friction • Radial homogeneous flow • That leads to narrower peaks • and reduced retention/changed

selectivity Increased separation performance

Forced Air • At a backpressure ≥ 400 bar

frictional heating leads to: • Increasing effluent temperature due

to friction • Concentrated flow in center of

column • Peak broadening due to radial

temperature and flow gradient Worse separation performance

Operating Principle: Forced Air vs. Still Air Heating

34 Proprietary & Confidential

VDAD – All New Diode Array Detector

• Entirely new optical design with Thermo Scientific™ Vanquish™ LightPipe™ flow cell • 10 and 60 mm detection path length

• Significantly improved detector

linearity range and signal-to-noise ratio

• Active control of the spectrograph temperature

• Variable slit for highest spectral resolution

35 Proprietary & Confidential

VDAD – LightPipe Flow Cell Setup

• Fused Silica • Very narrow silica walls (0.05 mm) surrounded by air

• Total reflection at the outside of the fused silica at the interface silica – air (n = 1)

• Virtually no influence of the mobile phase on the total reflection

F u s e d S i l i c a L i g h t P i p e F l o w C e l l

A i r

F u s e d S i l i c a P i p e F i b e r O p t i c s F i b e r O p t i c s Fiber Optics Fiber Optics Fused Silica Pipe

Fused Silica LightPipe Flow Cell

Air

36 Proprietary & Confidential

VDAD – Flow Cell Operating Principle

Inlet Outlet

Light

Flow path

Flow cell locks

Glass body Flow cell Fluidics Fiber optics

37 Proprietary & Confidential

Outstanding Sensitivity And Linearity

Nevirapine impurities on Thermo Scientific Syncronis™ C18, 1.7µm, 2.1mm ×100mm

• Impurities as low as 0.008% of active pharmaceutical ingredient (API) area

• Exceptional linearity for API and impurity quantitation

• Very fast flow and high pressure (800µL/min at max. 890bar)

• 10 mm flow cell

• Very easy method transfer from UltiMate 3000 series

0 60 72 -500

0

3,500

R² = 0.9998

0

1000

2000

3000

0 0.4 0.8 Nevirapine [mg/mL]

mAU

30

Nevirapine

18 72

-2

0

5 mAU

seconds

1

2

3 4

5 6 7 8

mAU

seconds

38 Proprietary & Confidential

How to achieve the best of both worlds for your separation

39 Proprietary & Confidential

Exploratory Untargeted Methods

Generic Method for Herbal Extracts: Ginkgo Extract on Coupled Dionex Acclaim RSLC C18 2.2µm, 2.1mm × 250mm (500mm effective column length)

• Boosted sample

loadability on column

• Perfect exploratory

method (generic)

• Do I miss peaks?

• Adjust method to MS

scanning capabilities

• 30 ºC/1370 bar max.

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0-100

0

100

200

300

400

500

600

700 20140418_Acclaim_500mm #1 Ginko XT (Dave) UV_VIS_2mAU

min

WVL:254 nm

Flow: 0.400 ml/min

ACN 0.1% FA: 5.0 %

35.0

85.0

5.0

WVL:254 nm

40 Proprietary & Confidential

Generic Method for Herbal Extracts => Gingko Extract on Accucore Vanquish C18 1.5µm x 2.1mm x 100mm

• 5 x faster separation

• Sharper peaks

• 27 % more peaks per unit time

• Improved peak height

• Same peak pattern now

with shorter column!

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0-200

500

1,000

1,500

2,000

2,500 20140422_WYCHEPROOF_ 100MM #18 Ginko XT (Dave) UV_VIS_2mAU

min

WVL:254 nm

Flow: 0.400 ml/min

ACN 0.1% FA: 5.0 %

35.0

85.0

5.0

Routine Targeted Methods

41 Proprietary & Confidential

How to manage and successfully process very data-rich results

42 Proprietary & Confidential

Chromeleon 7 Streamlines Chromatography

Thermo Scientific™ Dionex™ Chromeleon™ 7 Software

43 Proprietary & Confidential

Chromeleon 7 Streamlines Chromatography

Now also for Mass Spectrometry

44 Proprietary & Confidential

Chromeleon 7 - Operational Simplicity

• Operational Simplicity™ is the core design principle

• Major design rules: • Minimize number of steps needed to perform any task

• Make all steps easy to understand and easy to use

• Minimize time needed to perform any task

• Three major features using Operational Simplicity™: • Modern user interface

• Peak detection and integration tools

• eWorkflows

45 Proprietary & Confidential

Modern and Familiar User Interface in Console

Navigation Pane

Category Bars

46 Proprietary & Confidential

Modern and Familiar User Interface in Console

Integrated Sequence Editor

MiniPlots™

47 Proprietary & Confidential

And Chromatography Studio - All Experiment Details

Navigation Pane

Ribbon

Category Bars

48 Proprietary & Confidential

Cobra Peak Detection Algorithm

1. Start Cobra™ Wizard

3. Select the Narrowest Peak to be Integrated 4. Select the Smallest Peak

to be Integrated

2. Define the Integration Range

49 Proprietary & Confidential

Cobra Peak Detection Algorithm

And Your Integration Is Done!

50 Proprietary & Confidential

SmartPeaks – A Revolution in Ease of Use

• There are many ways to integrate unresolved peaks

Is this what we want?

Is this what we want?

51 Proprietary & Confidential

SmartPeaks Integration Assistant

1. Activate SmartPeaks™ Wizard

2. Select Area of Interest 3. Select the Integration You Want

52 Proprietary & Confidential

SmartPeaks Integration Assistant

And Your Integration Is Done!

53 Proprietary & Confidential

eWorkflows

• eWorkflows automate all chromatography processes

• Takes you from samples to results in only a few steps • Defines:

• Instruments on which the analysis can be run • All associated files:

• Instrument methods • Processing methods • Reports • Spectral libraries • Documents with description of method

• Template for sequence name and storage location • Custom variables • Rules for sequence layout

54 Proprietary & Confidential

eWorkflow for a Regulated USP/EP Method

1. Choose eWorkflow, Select Instrument, and Click “Launch”

2. Enter Number of Samples and Start Position

3. Sequence is created and can be immediately run

55 Proprietary & Confidential

eWorkflow for a Regulated USP/EP Method

This eWorkflow Also:

• Processes all data

• Calculates all results

From Sample to Result in the Shortest Time Possible