ACQUITY UPLC®: An Analytical Platform for Biopharmaceuticals · 2012-08-13 · ©2011 Waters...
Transcript of ACQUITY UPLC®: An Analytical Platform for Biopharmaceuticals · 2012-08-13 · ©2011 Waters...
©2009 Waters Corporation | COMPANY CONFIDENTIAL ©2011 Waters Corporation
ACQUITY UPLC®: An Analytical Platform for Biopharmaceuticals
John Barackman
Waters Technical Support Specialist
©2011 Waters Corporation 2
Topics
Components of LC Resolution
— Physical
— Chemical
Ultra-Performance Liquid Chromatography
— What is Required; How does it differ from HPLC
Our Waters ACQUITY UPLC Family
Overview ACQUITY UPLC Configurations for
Biopharmaceutical analysis
Tools for Methods Transfer
©2011 Waters Corporation 3
The Components of Resolution
Efficiency Selectivity Retentivity
Physical Van Deemter Equation
— Column length
— Particle size, shape, packing
— Mobile phase flow rate (linear velocity)
System induced Bandspread
Chemical
Dependent upon
— Analyte properties
— Stationary phase properties
— Mobile phase properties
©2011 Waters Corporation 4
The Components of Resolution
Physical
©2011 Waters Corporation 5
The Driving Force for UPLC
The “Van Deemter Equation” published 1956, correlates diffusion,
resistance, particle size and shape with mass transfer leading to
non ideal behaviour in chromatography
H = a(dp) + b(Dm) + c(dp2)(µ) µ(dp) Dm
©2011 Waters Corporation 6
The “Van Deemter Equation” published 1956, correlates diffusion,
resistance, particle size and shape with mass transfer leading to
non ideal behaviour in chromatography
H = a(dp) + b(Dm) + c(dp2)(µ) µ(dp) Dm
Resolu
tion
In
creases
as p
artic
les g
et s
malle
r
Two Important Points Regarding the Van Deemter Plot
©2011 Waters Corporation 7
Two Important Points Regarding the Van Deemter Plot
For particle sizes below approx, 2 µm, the optimum flow rate profiles flattens out. Achieve high throughput by pushing flow rate without significant loss of resolution.
At each particle size range, there is an optimum linear velocity (flow rate) that achieves maximum resolution for that particle size.
©2011 Waters Corporation 8
The Driving Force for UPLC
The Van Deemter Equation is valid for large molecules.
— Diffusion is slower so ideal flow rates are slower
0
0.0005
0.001
0.0015
0.002
0.0025
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2
u (mm/sec)
H (c
m)
3.5 micron particles peptides
1.7 micron particles peptides
Van Deemter Plot For a 1500 Dalton Peptide
3.5 mm
1.7 mm
©2011 Waters Corporation 9
Smaller, Rounder, Narrower Distributions, Better Packing
Particle size, shape, and packing effect diffusivity and the efficiency of mass transfer which, in turn, impacts resolution.
©2011 Waters Corporation 10
10 min
1980’s - 2000
5 – 2.5µ spherical micro-porous
1500-4000 psi
50,000 - 80,000 plates/meter
3.9 x 150mm
Late 1970’s
10µ irregular micro-porous
1000-2500 psi
25,000 plates/meter
3.9 x 300mm
10 min
Evolution of Chromatographic Efficiency
Early 1970’s
40µ pellicular non-porous coated
100-500 psi
1000 plates/meter
1m columns
10 min
Note: Since peaks are narrower for the same mass injected, you gain sensitivity by using smaller particles.
©2011 Waters Corporation 11
Why Not Go Smaller?
©2011 Waters Corporation 12
Chromatographic Resolution: Impacting Efficiency
Efficiency Selectivity Retentivity
Two components of efficiency:
• From the Column Packing
• Everywhere Else (“Extra-Column”)
©2011 Waters Corporation 13
Loss of Resolution Due To Extra-Column Bandspread
In this region, both analytes (blue and red) are not separated [a partial co-elution –
shown as a “purple” band]
System with MORE
Band Spreading
System with LESS
Band Spreading
Better separation More concentrated “Bands”
Higher Sensitivity
Extra-column bandspread is “non-ideal” behavior (loss of resolution) caused by the LC system outside of the column packing and is
“on top of” the column contribution.
©2011 Waters Corporation 14
Sources of Bandspread
Band Spreading will occur:
1. Along the flow path from the injector (“sample band”)
2. Into, through and out of the column (“analyte bands”)
3. Into the detector
3
2
1
©2011 Waters Corporation 15
Sources of Bandspread
Band Spreading will occur:
1. Along the flow path from the injector (“sample band”)
2. Into, through and out of the column (“analyte bands”)
3. Into the detector
3
2
1
• UPLC instruments must be designed specifically to take advantage of the sub-2µm particles
Pumps
Injectors
Tubing
Fittings
Column Heaters
Detectors, flow cells, nebulizers
→ Must minimize extra-column bandspread and increase data
rate
• Using HPLC components of any kind would perform poorly with UPLC columns
• However: HPLC columns work fine on UPLC systems!
©2011 Waters Corporation 16
Putting it All Together UltraPerformance LC®
A Class of Liquid Chromatograph Based On:
— Chromatography columns with very small, pressure-
tolerant particles, narrow size distributions, efficiently
packed
— Instruments designed to take advantage of the small
particles
o Pumps, Injectors, Tubing, Fittings, Column
Heaters, Detectors all designed to withstand the
pressures required, minimize extra-column
bandspread, and utilize the high data rates
required
If the Entire System is Holistically Designed for UPLC
Then:
— Significant improvements in resolution, sensitivity
AND sample throughput are realized
©2011 Waters Corporation 17
402 pesticide residues detected in a 8-minute UPLC run on TQD
Benefits of UPLC
• Resolution • Speed • Sensitivity
©2011 Waters Corporation 18
Benefits of UPLC Maximize Throughput
©2011 Waters Corporation 19
Benefits of UPLC Maximize Resolution
(3.5 µm)
(1.7 µm)
C18 RP Peptide maps at constant L/dp and gradient time.
©2011 Waters Corporation 20
The Components of Resolution
Efficiency Selectivity Retentivity
Chemical • Focus on the
stationary phase
©2011 Waters Corporation 21
ACQUITY UPLC Column Chemistries
Requirements for UPLC Columns:
— Sub–2 µm particles (1.7 µm typical)
— Pressure tolerant (≥ 15,000 psi)
— Efficiently packed (low dispersion)
— Narrow size distribution
Additional Desirable Characteristics:
— Very wide pH stability (pH 1-8 and pH 1-11 typical)
— High lot-to-lot reproducibility
— Wide range of selectivity, retentivity, porosity options
— Scalable from UPLC HPLC PREP
Waters manufactures most of its own particles and columns
©2011 Waters Corporation 22
Ever-Expanding ACQUITY UPLC® Column Offering
BEH C18
BEH C8
BEH Phenyl
BEH Shield RP18
BEH HILIC
BEH Amide
CSH C18
CSH Fluoro-Phenyl
HSS T3
HSS C18
HSS C18 SB
Five particle substrates
• 130Å, 200Å and 300Å BEH [Ethylene Bridged Hybrid], HSS
[High Strength Silica] and CSH [Charged Surface Hybrid]
Wide and growing selection of column chemistries
• 15 stationary phases
• BEH 130Å C18, C8, Shield RP18, Phenyl, HILIC and Amide
• BEH 300Å C18 and C4
• BEH 200Å SEC
• HSS C18, T3, C18 SB
• CSH C18, Fluoro-Phenyl and Phenyl-Hexyl
Proven application-based solutions
• AAA, OST, PST, PrST and Glycan
Transferability between HPLC and UPLC
XBridge HPLC and ACQUITY UPLC BEH columns
HSS HPLC and ACQUITY UPLC HSS columns
XSelect HPLC and ACQUITY CSH columns
VanGuard Pre-columns
eCord Technology
Nano, UPLC, HPLC, Semi-PREP, PREP Scale (most chemistries) CSH Phenyl-Hexyl
©2010 Waters Corporation | COMPANY CONFIDENTIAL 23
Column Chemistries For Biomolecules
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ACQUITY BEH300 C4, 1.7 µm
©2010 Waters Corporation | COMPANY CONFIDENTIAL 24
Column Chemistries For Biomolecules
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Waters Protein-PakTM Hi Res IEX
©2010 Waters Corporation | COMPANY CONFIDENTIAL 25
Column Chemistries For Biomolecules
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ACQUITY BEH200 SEC, 1.7 µm
For Protein Analysis
©2010 Waters Corporation | COMPANY CONFIDENTIAL 26
Column Chemistries For Biomolecules
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ACQUITY BEH200 SEC, 1.7 µm
For Protein Analysis
ACQUITY BEH300 C18, 1.7 µm
For Peptide Analysis
©2010 Waters Corporation | COMPANY CONFIDENTIAL 27
ACQUITY BEH Amide, 1.7 µm For Oligosaccharide Analysis
Column Chemistries For Biomolecules
©2010 Waters Corporation | COMPANY CONFIDENTIAL 28
ACQUITY BEH Amide, 1.7 µm
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ACQUITY UPLC BEH Glycan, 1.7µm, 2.1 x 150 mm
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ACQUITY UPLC BEH Glycan, 1.7µm, 2.1 x 150 mm
ACQUITY BEH Glycan, 1.7 µm
For N-Linked Glycan Analysis
Column Chemistries For Biomolecules
©2010 Waters Corporation | COMPANY CONFIDENTIAL 29
ACQUITY BEH Amide, 1.7 µm
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ACQUITY UPLC BEH Glycan, 1.7µm, 2.1 x 150 mm
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ACQUITY UPLC BEH Glycan, 1.7µm, 2.1 x 150 mm
ACQUITY BEH Glycan, 1.7 µm
ACQUITY OST C18, 1.7 µm
For Oligonucleotide Analysis
Column Chemistries For Biomolecules
©2011 Waters Corporation 30
Waters Corporation
What Waters Has To Offer
In 2004, Waters Corporation introduced the first true UPLC: the ACQUITY UPLC
©2009 Waters Corporation | COMPANY CONFIDENTIAL ©2011 Waters Corporation
©2009 Waters Corporation | COMPANY CONFIDENTIAL ©2011 Waters Corporation
Classic ACQUITY
High Throughput
Clinical/IVD
MS Inlet
H-Class
H-Class Bio
General Purpose
Methods Transfer
Bio-Analysis Nano ACQUITY
PATrol System
©2009 Waters Corporation | COMPANY CONFIDENTIAL ©2011 Waters Corporation
New Members of the ACQUITY Family
ACQUITY UPLC I-Class
- SFC at UPLC Scale - Complementary to RP, NP, GC
- Flexible Architecture - Lowest System
Dispersion of ANY UPLC - Ultimate MS Inlet
©2011 Waters Corporation 34
An Overview of Selected ACQUITY Systems
ACQUITY System Details
Introduction to the various pump and injector design paradigms, sample organizer, column heaters, and
detector types that make up our family of UPLC systems
©2011 Waters Corporation 35
High Pressure mixing, dual pump
—Two solvents at a time
—Smaller system volume/mixing volume
The BSM: A High-Pressure Mixing System
A high pressure mixer is capable of very efficient gradient formation.
A smaller dwell volume improves system throughput.
Smaller System Volume = Smaller Dwell volume
Multiple Pumps
ACQUITY UPLC
Detector Injector Column
Pump A
Pump B
Mixer
©2009 Waters Corporation | COMPANY CONFIDENTIAL ©2011 Waters Corporation
Standard ACQUITY UPLC System
• Binary Solvent Manager (BSM), High Pressure Mixing
• Sample Manager – Rheodyne Injector (SM)
• Column Heater, Column Manager (CM)
• System Characteristics
o <120 µL dwell volume
o <10 µL bandspread
• Supports all current ACQUITY UPLC detectors
o PDA/PDAeλ, TUV, FLR, ELSD
o SQD, TQD, XEVO MS Platforms
o Empower and MassLynx
• Optimum High-Throughput and MS Inlet BSM
©2011 Waters Corporation 37
Rheodyne Based Injector
SAMPLE
PVDD
SAMPLE /
METERING
SYRINGE
FROM
PUMP
TO
COLUMN
INJECT VALVE
INJECT POSITION
SAMPLE
LOOP
3 2
14
5 6
Needle Overfill IIStep 1: Sample Aspiration
Weak Wash Solvent
Air Gap
Buffer Volume
Sample
Mobile Phase
SOLVENT TYPE
Volumes Represented
are not to Scale
Internal Valve Passage
V = 0.100uLA rheodyne based injector minimized dwell volume and allows for a range of injection modes to meet your particular requirements.
The ACQUITY UPLC SM is a Rheodyne Base
Injector Design
©2009 Waters Corporation | COMPANY CONFIDENTIAL ©2011 Waters Corporation
Standard ACQUITY UPLC System
• Binary Solvent Manager (BSM), High Pressure Mixing
• Sample Manager – Rheodyne Injector (SM)
• Column Heater, Column Manager (CM)
• System Characteristics
o <120 µL dwell volume
o <10 µL bandspread
• Supports all current ACQUITY UPLC detectors
o PDA/PDAeλ, TUV, FLR, ELSD
o SQD, TQD, XEVO MS Platforms
o Empower and MassLynx
• Optimum High-Throughput and MS Inlet
SM
©2009 Waters Corporation | COMPANY CONFIDENTIAL ©2011 Waters Corporation
Rheodyne Loop Injection Loading the Sample into the Loop
Injection Modes: Partial, PLNO, Full Loop
©2009 Waters Corporation | COMPANY CONFIDENTIAL ©2011 Waters Corporation
Rheodyne Loop Injection Pushing the Sample onto the Column
Sample Loop contains Sample, air gaps and weak wash
40
©2011 Waters Corporation 41
ACQUITY Column Heaters
Standard Configuration: • Single Column • Temperature range: 5°C above ambient to 90°C • Up to 4.6 mm diameter, up to 150 mm length columns including filter and guard column Optional Configurations: • Four column capacity Column Manager with column select valves • 30-cm Column Heater/Cooler available for legacy methods
©2011 Waters Corporation 42
High Throughput System
Optional Sample Organizer • Up to 22 microtiter and 10
2mL vial tray capacity
• Up to 8448 Samples
• 15 second plate cycle time
• 4 to 40⁰C temperature
controlled
©2009 Waters Corporation | COMPANY CONFIDENTIAL ©2011 Waters Corporation
ACQUITY UPLC – High Throughput 0.86 min
> 1670 injections per day.
ACQUITY UPLC – High Throughput
©2011 Waters Corporation 44
Intro to the NanoACQUITY UPLC
Flow
Sensor
Modules
Heating/Trapping
Module
Sample
Manager
Binary Solvent
Manager
Auxiliary Solvent
Manager
• Binary high pressure blending without splitting
• 200 nL/min to 100 µL/min
• Low dispersion design, UPLC pressure capable (10,000 psi)
• Tunable UV/Vis, PDA, MS and MS/MS
• Wide range of column selectivity's available
• 75 – 150 µm diameters
• 100 – 250 mm length
• 1.7 – 3.5 µm particle sizes
©2011 Waters Corporation 45
1D Separation – Reverse Phase C18
2000
1500
1000
500
0
Cou
nts
10080604020
Retention Time (min)
7 - 40% ACN Gradient 250 ng Protein Digest Load
250 nL/min 1.7µm BEH C-18, 75µm x 25cm nanoACQUITY with Synapt HDMS
©2011 Waters Corporation 46
NanoACQUITY UPLC 2D System Schematic
Analytical Column Trap
Needle
Trap
Valve 6
5 4
3
2 1
Inject Valve
6
5 4
3
2 1
RP 1
Syringe
nanoBSM
nanoBSM
Waste
XBridge C18
300µm x 5cm
pH 2 Sample
pH 10 Symmetry C18
180µm x 2cm
BEH C18
75µm x 15cm
Plug
Plug
High organic and pH are reduced before trapping
©2011 Waters Corporation 47
2D RP/RP of Rat Brain Digest
11.1% ACN
20.8% ACN
17.4% ACN
14.5% ACN
45% ACN
©2011 Waters Corporation 48
The QSM: A Low-Pressure Mixing System
Low Pressure mixing, single pump
— Solvent is mixed before it is passed
through the pump
Gradient Proportioning Valve
Detector Injector A B
C D
Column Pump
Single Pump Design
ACQUITY UPLC H-Class
Low pressure mixing simplifies the pump design and expands the range of solvents that can be blended. Common GPV/Pump design to many legacy systems.
©2009 Waters Corporation | COMPANY CONFIDENTIAL ©2011 Waters Corporation
ACQUITY UPLC H-Class/H-Class Bio
• Quaternary Solvent Manager (QSM)
• Sample Manager – Flow Through Needle (SM-FTN)
• Column Heater with Active pre-heating
• System Characteristics
— <400 µL dwell volume
— <10 µL bandspread
— 2 ml/min max flow rate
— 15,000 psi max pressure
• Reproduce Common Legacy Methods
• Supports all current ACQUITY UPLC
detectors
• H-Class Bio: All wet-able surfaces made
from biocompatible materials QSM
©2011 Waters Corporation 50
The QSM: A Low-Pressure Mixing System
Low Pressure mixing, single pump
— Solvent is mixed before it is passed
through the pump
Gradient Proportioning Valve
Detector Injector A B
C D
Column Pump
Single Pump Design
ACQUITY UPLC H-Class
Low pressure mixing simplifies the pump design and expands the range of solvents that can be blended. Common GPV/Pump design to many legacy systems.
An Optional 6-Way Solvent Select Valve can be fitted into the QSM to
aid methods development. (A, B, C, D1->D6)
©2009 Waters Corporation | COMPANY CONFIDENTIAL ©2011 Waters Corporation
Another Benefit of Auto•Blend Rapid Method Scouting
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0.05% TFA – 5% D
0.1% TFA – 10% D
0.025% TFA – 2.5% D
©2011 Waters Corporation 52 *Available THF/Hexane Kit Installed
Using Auto•Blend for Complex Gradient Delivery
Time
(min)
%
Water
%
Acetonitr
ile
%
THF*
%
Methano
l
0.0 64 29 4 3
5.0 56 34 7 3
9.0 41 38 7 14
Aldehydes and Ketones as DNPH Derivatives
©2010 Waters Corporation | COMPANY CONFIDENTIAL 53
Auto•Blend Plus™ Technology AQUITY H-Class/H-Class Bio
Program methods directly in units of pH and Molarity
Calculation of required proportions from physical constants
— pH is calculated using Henderson-Hasselbalch equation with
pKa provided
— Typically use pKa corrected for salt concentration
OR:
— Empirical calibration table covering operating range of buffer
and salts selected
o Can produce a near true linear pH gradients as required
Independent gradients for pH and salt concentration
©2010 Waters Corporation | COMPANY CONFIDENTIAL 54
Instrument Control Method Auto•Blend™ to Auto•Blend Plus™
Click to convert the IM to Auto•Blend PlusTM
©2010 Waters Corporation | COMPANY CONFIDENTIAL 55
Typical Gradient Table Auto•Blend™ Plus
QSM percent table is converted to units of pH and Salt concentration.
The allowed pH and [salt] ranges are based on the respective buffer
system selected
©2010 Waters Corporation | COMPANY CONFIDENTIAL 56
Benefit of Auto●Blend Plus™ Rapid Methods Development
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pH 6.1
pH 7.6
pH 7.1
pH 7.0
pH 6.9
A mixture of proteins was separated using cation exchange chromatography- alpha-Chymotrypsinogen A (peak A), Ribonuclease A (peak B), and Cytochrome C (peak C) –
A
A
A+B
A
A
B
B
B
B
C
C
C
C
C
©2011 Waters Corporation 57
Flow-Through Needle Based Injectors (FTN)
Wash Solvent
Purge Solvent
©2011 Waters Corporation 58
ACQUITY UPLC H-Class/H-Class Bio Direct Inject Sampling with the FTN
Sample Manager – Flow Through Needle (SM-FTN)
No re-configuration necessary across the entire UPLC injection
volume range
— Up to 10uL injection standard
— Up to 1000uL with extensions
No wasted sample and low carryover
— Needle and loop are part of flow path
o Low carryover performance
Good precision, linearity, and accuracy
Full range of ANSI plates and vials
H-Class Bio: Wet-able surfaces are
made of biocompatible materials
A flow-through injector design, while adding some dwell volume provides a single, simple injection model and provides a wide linear range, with good accuracy and high precision. Common injection model to many legacy systems.
SM-FTN
©2011 Waters Corporation 59
Sample Manager –FTN Sample Loading
Inject Valve Load Position
©2011 Waters Corporation 60
Sample Manager –FTN Sample Injection
©2010 Waters Corporation | COMPANY CONFIDENTIAL 61
SM-FTN Wide Linearity Range
Benzocaine Tetracaine Procaine R2=0.999991 R2=0.999997 R2=0.999989
©2010 Waters Corporation | COMPANY CONFIDENTIAL 62
SM-FTN Repeatability
Method: ACQUITY UPLC BEH200 1.7 µm 4.6x150mm 50mM KPO4, 100mM KCl, pH 6.8 40⁰C, 0.5 mL/min flow, 220 nm detection
©2010 Waters Corporation | COMPANY CONFIDENTIAL 63
3.5 AU Full Scale
Stress Injection @ 2mg/mL 1st Blank Injection
Carryover = 0.0002%
3.5 AU Full Scale
SM–FTN Low Carryover
©2011 Waters Corporation 64
ACQUITY UPLC H-Class Bio What’s different from the H-Class?
Many Components Re-Engineered for Bio-Compatibility
— Primary & accumulator pump heads
— Check-valves
— UPLC Valves (Vent, Injector, CM, etc..) & SSV
— Mixer housing, mixer manifold, sinkers
— SM Needle Assembly and Injector Port
— CM-A Active Pre-Heater (APH) Assembly
— UPLC tubing assemblies
— PDA & TUV Flow Cells
Bio-Compatible Materials:
— Minimize corrosion (particularly to halides)
— Improve sample recovery (particularly for bio-molecules)
— Minimize iron adducts (IEX and LCMS)
— Limit oxidation
— High strength, durable
Wet-Able Surfaces are made principally from Titanium and Nickel-
Cobalt Alloys
©2011 Waters Corporation 65
ACQUITY UPLC I-Class System Optimized for Low Dispersion
Binary Solvent Manager (BSM)
— 18,000 psi capable
Sample Manager (Rheodyne or FTN)
— Fixed-Loop (FL) Sample Manager
— Flow-Through-Needle (FTN) Sample Manager
Column Management (2 options)
— Single Column Heater
— Dual Column Manager
o Max 2 columns
o Optional 2D Technology feature
Detection
— MS
— TUV or PDA only – new lower dispersion flow cells
All Existing ACQUITY UPLC Chemistries
— Plus optimum platform for 1 mm ID columns
©2011 Waters Corporation 66
Instrument Contribution to Extra-Column Effects
Engineering developments have specifically improved dispersion in every part of the system flow path to allow the I-Class to minimize system induced band spread:
— Injector, fittings, flow path, sealing surfaces, reduced tubing volumes, improved flow cell dispersion
— ~ 95 uL dwell volume, < 7 uL band-spread
22
det
2
,det
2
,
2
,
2
,
2
,
2
, Fectorectorvpostcolumnvcolumnvprecolumnvinjectorvtotalv
Injection volume
+ injector band-
spreading
Tubing between injector
and column
Column volume
+ frits
Tubing between column
and detector
Band- spreading inside the detector
cell +
tubing
Time-based Band-
spreading in the
Detector (Sampling Rate; Time Constant)
©2011 Waters Corporation 67
ACQUITY UPLC Systems with 2D Technology Solves Key Challenges
Increase Selectivity & Sensitivity
Eliminate Unwanted
Interferences
Mobile Phase Flexibility
Characterize the Most Complex Samples
©2011 Waters Corporation 68
2D Capability Available on All ACQUITY UPLC System (New Systems and Upgrades)
ACQUITY UPLC H-Class
System with 2D
Technology
ACQUITY UPLC I-
Class System with
2D Technology
ACQUITY UPLC
System with 2D
Technology
©2011 Waters Corporation 69
Flexibility of ACQUITY UPLC Systems with 2D Technology
Typical Experiments that can be Performed with these Systems
— Trapping
— Heart Cutting
— Parallel Column Regeneration
— At-Column Dilution (ACD)
©2011 Waters Corporation 70
ACQUITY UPLC Systems with 2D Technology Example Flow Path (Trapping Config Shown)
©2011 Waters Corporation 71 Time
3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40
%
0
100
MRM of 6 Channels ES+
327 > 269.9
1.46e6
4.31
250 uL Clozapine 1 ppb 2D – 3 min gradient
250 uL Clozapine 1 ppb 1D – 3 min gradient
Peak distortion Volume Overload
20 uL Clozapine 1 ppb 1D – 3 min gradient
Increase Sample Loading and Sensitivity with Trap and Back-Transfer Configuration
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HPLC to UPLC Methods Transfer
Tools to Help Make Methods Transfer Easier
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Column Selector and Calculator Calculator Tools
Transfer Existing HPLC Methods to the H-Class/H-Class Bio Redevelop Existing HPLC Method to UPLC Methods
Using our Column Calculator and Column Selector Tools (Free! www.waters.com)
Quickly scale/translate between column selectivity, column size, particle diameters, injection volumes, gradient times and system dwell volumes
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Transfer Methods Between UPLC and
HPLC with Ease
Utilize existing Assets
Run HPLC methods on ACQUITY UPLC H-Class
Future-proof your lab
Transfer from HPLC-to-UPLC
©2009 Waters Corporation | COMPANY CONFIDENTIAL ©2011 Waters Corporation
Transferring HPLC Methods Across
Multiple HPLC and UPLC Systems
©2009 Waters Corporation | COMPANY CONFIDENTIAL ©2011 Waters Corporation
Questions?