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

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Change the Landscape in LC
Next Generation UHPLC Technologies:

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

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

• 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

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

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

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

• 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

• 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!

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

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%

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_1
mAU
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_1
mAU
min
WVL:230 nm
100 x 2.1 mm
Competitor C18 1.6 µm
100 x 2.1mm
• Accucore Vanquish vs. Competitor 1.6µm Solid Core Column

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_1
mAU
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_1
mAU
min
WVL:230 nm
100 x 2.1 mm
Competitor C18 1.7 µm
100 x 2.1 mm
• Accucore Vanquish vs. Competitor 1.7µm Solid Core Column

• Rapid Screening of 47 Drugs of Abuse in <4min
Overlaid selected-reaction monitoring chromatograms showing detection of 47 compounds

• Separation of isobaric compounds
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

• Rapid Screening of 36 Veterinary Antibiotics in <5min
Overlaid selected-reaction monitoring chromatograms showing detection of 36 compounds

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

Next Generation UHPLC
Thermo Scientific™ Vanquish UHPLC System

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

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

VPump – Flow-Pressure Footprints
• The Vanquish system expands our future‐proof flow‐pressure
footprint even further
Pressure[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/minµL/minnL/min
1500
0

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

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

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)

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_Pressure
bar
1
0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 10,0
120
150
200
250
320
2 - 02_ID02A_R02_VAS_SETTINGS_wo_leak_comp Pump_Pressure
bar
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

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
4
5
6
7
8 9
10
WVL:254 nm
w/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
4
5
6
7
8 9
10
WVL:254 nm
w/ 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

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)

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

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

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

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

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
70 °C 80 °C73 °C 76 °C
70 °C
70 °C 90°C
70 °C

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

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

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 ilic a L ig h t P ip e F lo w C e ll
A ir
F u s e d S ilic a P ip e F ib e r O p t ic sF ib e r O p t ic s
Fiber Optics Fiber OpticsFused Silica Pipe
Fused Silica LightPipe Flow Cell
Air

VDAD – Flow Cell Operating Principle
Inlet
Outlet
Light
Flow path
Flow cell locks
Glass body Flow cell FluidicsFiber optics

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

How to achieve the best of both worlds for your separation

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_2
mAU
min
WVL:254 nm
Flow: 0.400 ml/min
ACN 0.1%FA: 5.0 %
35.0
85.0
5.0
WVL:254 nm

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_2
mAU
min
WVL:254 nm
Flow: 0.400 ml/min
ACN 0.1%FA: 5.0 %
35.0
85.0
5.0
Routine Targeted Methods

How to manage and successfully process very data-rich results

Chromeleon 7 Streamlines Chromatography
Thermo Scientific™
Dionex™ Chromeleon™ 7
Software

Chromeleon 7 Streamlines Chromatography
Now also for Mass Spectrometry

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

Modern and Familiar User Interface in Console
Navigation Pane
Category Bars

Modern and Familiar User Interface in Console
Integrated
Sequence Editor
MiniPlots™

And Chromatography Studio - All Experiment Details
Navigation Pane
Ribbon
Category Bars

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

Cobra Peak Detection Algorithm
And Your Integration Is Done!

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?

SmartPeaks Integration Assistant
1. Activate SmartPeaks™ Wizard
2. Select Area of Interest
3. Select the Integration
You Want

SmartPeaks Integration Assistant
And Your Integration Is Done!

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

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

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

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

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