Waters ACQUITY UPLC Family...2.1 x 50 mm, 1.7 µm 10 min Compatible with HPLC, UHPLC and UPLC...
Transcript of Waters ACQUITY UPLC Family...2.1 x 50 mm, 1.7 µm 10 min Compatible with HPLC, UHPLC and UPLC...
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Waters ACQUITY UPLC Family
UPLC Users Meetings Denmark
April, 26-27th
Frederic Forini
Waters European Headquarters
©2012 Waters Corporation 1
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“Demand for UHPLC systems has continued to outpace demand for other HPLC techniques. UHPLC t f t UHPLC systems are fast becoming the standard tool for research and routine applications ”research and routine applications.
– Strategic Directions International
©2012 Waters Corporation 2
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Industry Trends:Industry Trends:The Market has ChangedThe Market has ChangedThe Market has ChangedThe Market has Changed
Transitioning from HPLC to UPLC Technology
Increasing number of organizations have realized the business and scientific advantages UPLC Technology
Increased availability of UHPLC instruments provides vendor choice
T h l hift h l d i t l t h t b t tili Technology shift has led companies to evaluate how to best utilize their existing HPLC instruments as they continue to invest in, and transition to, newer UPLC systems.
During this transition, a number of challenges have arisen
that need to be addressed
©2012 Waters Corporation 3
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Resolution Power Provides Opportunity Resolution Power Provides Opportunity to Reduce Run Time to Reduce Run Time Without Compromising ResultsWithout Compromising Results
10
m
AU 0.20
0.30
p gp g
min
A
0.00
0.10
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00
1.5
miAU0.20
0.30 HPLC UPLC
Number of samples* 13,500 13,500
Total days 250 38
in
0.00
0.10
0.00 0.50 1.00 1.50 2.50 3.00 3.50Minutes
Total days 250 38
Acetonitrile costs ($100/L) $14,175.00 $715.50
Disposal cost* * $28,350.00 $1,431.00
Total cost $42,530.00 $2,147.00
Cost per sample $3.15 $0.16
Savings per sample $2.99Saving per year $40,383.00
*10 min/analysis, 8 hr/day, 250 working/year
©2012 Waters Corporation 4
** Assume disposal is two times cost of acquiring
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Reducing Environmental Impact Reducing Environmental Impact Without Compromising ResultsWithout Compromising Results
10
m
AU 0.20
0.30 XSelect CSH C18 XP4.6 x 75 mm, 2.5 µm
HPLC System
p gp g
min
A
0.00
0.10
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00
1.5
m
ACQUITY UPLC CSH C182.1 x 50 mm, 1.7 µm
UPLC System
AU
0.20
0.30
minA
0.00
0.10
0.00 0.50 1.00 1.50 2.50 3.00 3.50Minutes
Traditional HPLC System 10 min run time
Uses 10.5 ml of ACN
Required disposal 31.5 ml of solvent
ACQUITY® UPLC® System 1.5 min run time
Uses 0.53 ml of ACN
Required disposal 1.19 ml of solvent
©2012 Waters Corporation 5
(H2O/ACN) (H2O/ACN)
Greener result
Operating costs reduced
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Evolution of ACQUITY UPLC TechnologyEvolution of ACQUITY UPLC TechnologyEvolution of ACQUITY UPLC TechnologyEvolution of ACQUITY UPLC Technology20042004 20052005 20062006 20072007 20082008 20092009 20102010 20112011 20122012
Instrument ACQUITY UPLC‐BinaryL I j
ACQUITY UPLC‐Sample O i
Column Oven‐4 column 90CH /C l
ACQUITY UPLC‐Active CVL k
UPLC PATROL‐Process A l
ACQUITY UPLC‐Local Console C ll
UPLC H‐Class‐Quaternary Di I j
UPLC I‐Class‐BinaryL I j
Stay tuned!
‐Loop InjectorColumn Oven‐Single 65CDetectors‐TUV and PDA
OrganizerDetectors‐Revised TUV and PDA‐ELSD
‐Heater/Cooler10 – 90C
‐Leak sensors‐Advanced needle designnanoACQUITY‐CapillaryColumn Oven‐Single 90CDetectors
AnalyzerUPLC Open Access‐open bed autosamplerDetectors‐Extended λPDA
Controller ‐Direct Injector‐Met DevH‐Class BIO‐InertnanoACQUITYHDX‐Hydrogen‐Deuterium
‐Loop Injector‐Direct InjectorACQUITY UPSFC‐SFC / NPUPLC PATROL‐Laboratory AnalyzerTRIZAIC
Chemistry
Detectors‐Fluorescence
PDA Deuterium ExchangeUPLC Online SPE
TRIZAIC‐nanofluidicUPLC 2D
Hybrid 1.7 µmBEH C18
Hybrid 1.7 µmBEHShieldRP18BEH C8BEH Phenyl
Hybrid 1.7 µmBEH HILICBEH 300Å C18AccQ‐Tag Ultra
Silica 1.8 µmHSS C18HSS C18 SB
Hybrid 1.7 µmBEH 300Å C4OST C18
Hybrid 1.7 µmBEH AmideBEH Glycan
Hybrid 1.7 µmCSH C18CSH Fluoro‐PhenylCSH Phenyl‐Hexyl
Silica 1.8 µm‐HSS Cyano‐HSS PFP2.5 µm XP
Size Exclusion‐SEC 125Å
y g
Silica 1.8 µmHSS T3
Guard ColumnVanGuard SFC 1.7 µm
2‐ethylpyridineFluoro‐phenylhybrid
µ‐14 chemistriesSize Exclusion‐SEC 200ÅIon Exchange‐High Res Q‐High Res CM‐High Res SP
Stay tuned!
Mass Spec Technology ACQUITY SQD
ACQUITY TQD3rd party MS control
SYNAPT‐Ion Mobility
XEVO TQ MS XEVO QTof
SYNAPT G2
XEVO G2 QTof
XEVO TQ‐S
SYNAPT G2‐SXEVO TQDACQUITY SQD2
Stay tuned!
©2012 Waters Corporation 6
Application Kits
AccQ‐Tag Ultra Amino Acid
Peptide Separations
Oligonucleotide Aflatoxin
Intact Proteins
AccQ‐Tag Ultra AminoAcid Analysis for H‐Class
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UPLC Technology Platform Has Expanded:UPLC Technology Platform Has Expanded:Adopting Across Organization to Achieve Business GoalsAdopting Across Organization to Achieve Business Goalsp g gp g g
I-ClassH-Class H-Class Bio 2D
©2012 Waters Corporation 7
nanoACQUITY HDXPATROLUPC2
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UPLC Technology Platform Has Expanded:UPLC Technology Platform Has Expanded:Adopting Across Organization to Achieve Business GoalsAdopting Across Organization to Achieve Business Goalsp g gp g g
I-ClassH-Class H-Class Bio 2D
©2012 Waters Corporation 8
nanoACQUITY HDXPATROLUPC2
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©2012 Waters Corporation 9
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Transfer Methods With Transfer Methods With MinimalMinimal Disruption to Process…Disruption to Process…MinimalMinimal Disruption to Process…Disruption to Process…
Sub 2 micron Method
HPLC Legacy Method
Sub 2 micron Method
… and Maximum
HPLC Legacy Method
Maximum Impact on
ProductivityHPLC Legacy Method
©2012 Waters Corporation 10
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Ease Ease of Method of Method Transfer Between LC PlatformsTransfer Between LC PlatformsEase Ease of Method of Method Transfer Between LC PlatformsTransfer Between LC Platforms
XSelect HSS T34.6 x 50 mm, 2.5 µm XP
Flow rate = 1.9 mL/min
HPLC
in n /D
aidz
Ura
cil
Gly
citin
Gen
istin
Dai
dze
in
Gly
cite
in
Gen
iste
in
(Daidzin)
Pc = 92
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
1.61
Transfer methods between
XSelect HSS T34.6 x 50 mm, 2.5 µm XP
Flow rate = 1.9 mL/min
ACQUITY UPLC H-Class
P 94
between different LC systems
1.63(Daidzin)
Pc = 94
©2012 Waters Corporation 11
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
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Transferring with Constant L/Transferring with Constant L/dpdp
2.265
4.610
6.916
0.05
XBridge C184.6 x 150 mm, 5 µmAlliance 2695 HPLCL/dp = 30,000
40 min
Transferring with Constant L/Transferring with Constant L/dpdp
12 14 160.00
9 1 4
0.05XBridge BEH C184.6 x 75 mm, 2.5 µm XPAlliance 2695 HPLCL/dp = 30 000
40 min
4X f t th d
3.129
3.731
4.324
0.00
0.05
L/dp = 30,000
XBridge BEH C184.6 x 75 mm, 2.5 µm XPACQUITY UPLC H-Class
10 min4X faster methods
on your HPLC
3.106
3.697
4.275
0.00
0.05
QL/dp = 30,000
ACQUITY UPLC BEH C182.1 x 50 mm, 1.7 µm
10 min Compatible with HPLC, UHPLC and UPLC systems
1.368
1.641
1.902
0.00
ACQUITY UPLC H-ClassL/dp = 29,411
ACQUITY UPLC BEH C182 1 x 50 mm 1 7 µm
4.5 min
Transfer to 1.7 µm UPLC for
©2012 Waters Corporation 12
1.319
1.582
1.842
0.00
0.05 2.1 x 50 mm, 1.7 µmACQUITY UPLC I-ClassL/dp = 29,411
4.5 min
UPLC for 9X faster methods
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Fusion to Fusion to ComplyComply withwith QbDQbDPublishedPublished GuidelinesGuidelinesPublishedPublished GuidelinesGuidelines
Quality by Design: A systematic approach to development that begins with predefinedobjectives and emphasizes product and process objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management
ICH Q8(R2) Guidelines
©2012 Waters Corporation 13
*ICH: International Conference on Harmonisation
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ACQUITY UPLC HACQUITY UPLC H--Class Bio AttributesClass Bio AttributesACQUITY UPLC HACQUITY UPLC H Class Bio AttributesClass Bio Attributes
Based on ACQUITY UPLC H-Class True UPLC performance
Based on ACQUITY UPLC H-
Cl
True UPLC performance
Class
Bio-compatible
AutoBlend+simplifiescompatible
fluidics for biological molecules
simplifiesmeth dev
©2012 Waters Corporation 14
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UPLC Technology Platform Has Expanded:UPLC Technology Platform Has Expanded:Adopting Across Organization to Achieve Business GoalsAdopting Across Organization to Achieve Business Goalsp g gp g g
I-ClassH-Class H-Class Bio 2D
©2012 Waters Corporation 15
nanoACQUITY HDXPATROLUPC2
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Extending the ACQUITY UPLC Family…
©2012 Waters Corporation 16
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ACQUITY UPLC IACQUITY UPLC I--Class AttributesClass Attributes
Pinnacle of h t
ACQUITY UPLC IACQUITY UPLC I Class AttributesClass Attributes
True UPLC performance
chromatographic
performanceHT
without compromising
Built on 7 years user-
based design performancedesign innovation
Compatible with existing
Optimizes the with existing
ACQUITY UPLC & H-
Class methods
the performance of any Mass
Spec
©2012 Waters Corporation 17
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ACQUITY UPLC IACQUITY UPLC I--ClassClassThe Pinnacle of Chromatographic The Pinnacle of Chromatographic PerformancePerformance
Highest resolution offered by Sub 2um particles and low
dispersion fluidics
Accelerated complex separations achieved with low system’s dispersion &
faster cycle-time
Best sensitivity reached with lowest carryover
autosamplerp faster cycle-time p
SYNAPT G2 MS
ACQUITY SQD
ACQUITY TQD
XEVO TQ MS
XEVO TQ-S
Xevo TOFSYNAPT G2 MS
SYNAPT G2 HDMS
©2012 Waters Corporation 18
Optical Detectors:
TUV; PDA, FLR, ELSD
Q
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Rugged and Robust Systems Rugged and Robust Systems Designed to Optimize Productivity Designed to Optimize Productivity g p yg p y
ACQUITY UPLC I-Class System ACQUITY UPLC H-Class System
Ultimate Binary Performance Ultimate Quaternary PerformanceUltimate Binary Performance Ultimate Quaternary Performance
Ideal MS Inlet Methods Transfer & Development
Ultra High Throughput Routine Analysis
©2012 Waters Corporation 19
Binary Solvent Management Multiple Solvent Management
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UPLC Technology Platform Has Expanded:UPLC Technology Platform Has Expanded:Adopting Across Organization to Achieve Business GoalsAdopting Across Organization to Achieve Business Goalsp g gp g g
I-ClassH-Class H-Class Bio 2D
©2012 Waters Corporation 20
nanoACQUITY HDXPATROLUPC2
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ACQUITY UPLC with 2D TechnologyACQUITY UPLC with 2D TechnologyACQUITY UPLC with 2D TechnologyACQUITY UPLC with 2D Technology
“The ability to perform two y pdimensional UPLC separations using Waters technology has enabled some of our most challenging assays that require ultra-high sensitivity while maintaining
ll t d d excellent ruggedness and instrument up-time.”
— Rand Jenkins, Director of Research
©2012 Waters Corporation 21
and Development Pharmaceutical Product Development
(PPD)
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ACQUITY Systems with 2D TechnologyACQUITY Systems with 2D Technology
Characterize Increase
Q y gyQ y gy
True UPLC performance
Characterize the most complex samples
Increase selectivity
and sensitivity
Eliminate unwanted
interferences
Mobile phase
flexibility
The first ever ACQUITY UPLC System with 2D Technology that
©2012 Waters Corporation 22
provides the proven UPLC benefits of Resolution, Sensitivity and Productivity, now for multidimensional applications.
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2D Chromatography With 2D Chromatography With UPLCUPLCWhat Is This?What Is This?What Is This?What Is This?
2D chromatography is a generic concept (from a hardware point of view): Ypoint of view):– UPLC systems configured with 2 or more solvent managers
– One or several HP valves might be required
S l i h b i d
AC
QU
ITY
– Several temperature zones might be required
Three types of applications:
3 types of applications
R l 2D LC Online Sample Parallel C l
©2012 Waters Corporation 23
Real 2D LC Online Sample Preparation Column Regeneration
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Technology EnablersTechnology EnablersWhat Is Required?What Is Required?qq
New ACQUITY 2D Software for Flexibility– Multi-pump multi-valveMulti pump, multi valve
– Basic User Interface
MassLynx softwaref f– Specific version for 2D
CM-A Required– Two HP valves
– Different temperature zones
Configurations with:– ACQUITY UPLC– ACQUITY UPLC
– ACQUITY H-Class
– ACQUITY H-Class Bio
CQ C Cl
©2012 Waters Corporation 24
– ACQUITY UPLC I-Class
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2D Chromatography With 2D Chromatography With UPLCUPLCWhat Is This?What Is This?What Is This?What Is This?
3 types of applicationsyp pp
Real 2D LC Online Sample PreparationParallel Column Regenerationp g
Column 2 is being regenerating while column 1 is used for the analysis
Increase sample throughput
More flexibility, Faster ROI,
Faster Decision Making, Less Waiting Time…
©2012 Waters Corporation 25
Hardware
Requirements
(2 SM, 2x6PHPV)
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2D Chromatography With 2D Chromatography With UPLCUPLCWhat Is This?What Is This?What Is This?What Is This?
3 types of applicationsyp pp
Real 2D LC Online Sample Preparation Parallel Column Regeneration
Trap & Elute At Column Dilution
Column 2 is being regenerating while
column 1 is used for the analysis
Sample of interest istrapped and
concentrated on the cartridge
Impurities can betrapped on the
cartridge
Increase sample throughput
Increased samplethroughput
Increased robustness
Increased samplethroughput
Increased robustness
More flexibility
Fast ROI…
©2012 Waters Corporation 26
Hardware
Requirements
(2 SM, 1x6PHPV)
Hardware
Requirements
(2 SM, 1x6PHPV)
Hardware
Requirements
(2 SM, 2x6PHPV)
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Increase Sample Loading and Increase Sample Loading and Sensitivity with Trap and BackSensitivity with Trap and Back--Transfer Configuration Transfer Configuration
100MRM of 6 Channels ES+
327 > 269.91.80e5
2.14
20 uL Injection Clozapine 1 ppb on 1D System
gg
Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50
%
0
250ul Injection on 1 dimension
%
100MRM of 6 Channels ES+
327 > 269.94.28e5
2.73
250 uL Injection on 1D System Peak DistortionVolume Overload
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50
%
0
%
100MRM of 6 Channels ES+
327 > 269.91.97e6
250 uL Injection on 2D System
©2012 Waters Corporation 270.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50
0
Loading 1th dimension Gradient Elution 2nd dimension
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2D Chromatography With 2D Chromatography With UPLCUPLCWhat Is This?What Is This?What Is This?What Is This?
3 types of applicationsyp pp
Real 2D LC Online Sample PreparationParallel Column
Regeneration
Heart Cutting Comprehensive 2D Trap & Elute At Column Dilution
g
Column 2 is being regenerating while column 1 is used for the analysis
Sample of interest istrapped and
concentrated on the cartridge
Impurities can betrapped on the cartridge
An additional pump is configured for
“reconditioning” the sample plug in aqueous solvent before injection
onto the column
Increase sample throughput
Increased samplethroughput
Increased robustness
Increased samplethroughput
Increased robustness
Might be used to simplifysample preparation More flexibility
©2012 Waters Corporation 28
Hardware
Requirements
(2 SM, 1x6PHPV)
Hardware
Requirements
(2 SM, 1x6PHPV)
Might be required in combination with heart
cutting
Hardware
Requirements
(2 SM, 2x6PHPV)
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Compound X in 100% ACN at 1 ppb Compound X in 100% ACN at 1 ppb with ACQUITY System with 2D with ACQUITY System with 2D TechnologyTechnologygygy
100MRM of 6 Channels ES+
278.1 > 105 (Amytriptyline)4.01e4
2.69
2 66
20 uL Injected onto 1D System
%
0
2.66
0.35 2.58
2.912.94
3.01
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.000
MRM of 6 Channels ES+ 278.1 > 105 (Amytriptyline)
6.44e50.83
250 uL Injected onto 1D System
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
00.31
3.49
%
100MRM of 6 Channels ES+
278.1 > 105 (Amytriptyline)1.04e6
7.00250 uL Injected onto 2D System with At-Column Dilution
5 % Water line A
©2012 Waters Corporation 29
Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
0
5 % Water line A95 % Water line B50 uL mixer post injector
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2D Chromatography With 2D Chromatography With UPLCUPLCWhat Is This?What Is This?What Is This?What Is This?
3 types of applicationsyp pp
Real 2D LC Online Sample PreparationParallel Column
Regeneration
Heart Cutting Comprehensive Trap & Elute At Column Dilution
g
Column 2 is being regenerating while column 1 is used for the analysis
1/Sample passes throughthe first column
2/Portions of the effluent are sent to the second column
1/Sample passes throughthe first column
2/All or Portions of the effluent are sent to the
second column
Sample of interest is trappedand concentrated on the
cartridge
Impurities can be trapped on the cartridge
An additional pump is configured for
“reconditioning” the sample plug in aqueous solvent before injection onto the
column
Increase sample throughput
Increased selectivity Increased peak capacityIncreased sample
throughput
Increased robustness
Increased samplethroughput
Increased robustness
Might be used to simplifysample preparation More flexibility
©2012 Waters Corporation 30
Hardware
Requirements
(2 SM, 1x6PHPV)
Hardware
Requirements
(2 SM, 1x6PHPV)
Hardware
Requirements
(2 SM, 1x6PHPV)
Hardware
Requirements
(2 SM, 1x6PHPV)
Might be required in combination with heart
cutting
Hardware
Requirements
(2 SM, 2x6PHPV)
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Typical LC Separation versus Typical LC Separation versus Separation Using ACQUITY UPLC with Separation Using ACQUITY UPLC with 2D Technology2D Technologygygy
T i l LC S ti ACQUITY UPLC ith 2D T h l
desired peptides(co-eluting)
phospholipids interference
Typical LC Separation ACQUITY UPLC with 2D Technology
desired peptidesp p
no phospholipids
©2012 Waters Corporation 31
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UPLC Technology Platform Has Expanded:UPLC Technology Platform Has Expanded:Adopting Across Organization to Achieve Business GoalsAdopting Across Organization to Achieve Business Goalsp g gp g g
I-ClassH-Class H-Class Bio 2D
©2012 Waters Corporation 32
nanoACQUITY HDXPATROLUPC2
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ACQUITY UPCACQUITY UPC22 SystemSystemACQUITY UPCACQUITY UPC SystemSystem
©2012 Waters Corporation 33
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©2012 Waters Corporation 34
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ACQUITY UPCACQUITY UPC22
The technique Fi t SFC
ACQUITY UPCACQUITY UPC
True UPLC performance
The technique of choice for normal phase
analysis
First SFC system
designed to fully utilize
sub 2u sub 2u chemistries
UPSFC capability with LC
Familiarity
The technique of choice for
chiraltiseparations
Minimi e cost pe anal sis b ed cing sol ent cons mption b
©2012 Waters Corporation 35
Minimize cost per analysis by reducing solvent consumption by up to 80%
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What is a Supercritical Fluid ?What is a Supercritical Fluid ?pp
Gas - like low viscosity confers high diffusivity and limits pressure drop
Liquid - like high density ensures good solvent properties
High diffusion coefficient enhances kinetic performance of SFC
©2012 Waters Corporation 36
High diffusion coefficient enhances kinetic performance of SFC
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Supercritical Fluid ApplicationsSupercritical Fluid Applicationsp ppp pp
Chromatography (SFC) Analysis & Purification Chiral Separation Chiral Separation Normal Phase
Extraction (SFE) Bioactive compounds Nutraceuticals
SupercriticalCO2
Nutraceuticals Spices and aromatic Decaffeination Decontamination or cleaning
Val able molec les f om
SF Particle Design(RESS & SAS) Polymerization Cristallization Impregnation Valuable molecules from
waste
Reaction
Impregnation
©2012 Waters Corporation 37
Reaction Hydrogenation Hydroformylation Carboxylation
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Why COWhy CO22 ??yy 22
CO2 reaches supercritical state at 31.1°C and 73.8 bar – Its physical state can be easily manipulatedp y y p
CO2 is non toxic, non flammable
CO2 is chemically pure, stable and non-polar solvent, also compatible with LC detectors
Carbon Dioxide as a Green Solvent– Recovered from industrial and fermentation plants
d h d f h ld h b d f– Avoids the production of CO2 that would have been generated from disposal of the solvents it replaces.
– Less time and energy are used to evaporate fractions to get to pure analytes
©2012 Waters Corporation 38
analytes.
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What is Supercritical Fluid What is Supercritical Fluid Chromatography (SFC) ?Chromatography (SFC) ?g p y ( )g p y ( )
CO2 is used as the mobile phase in combination with one or more organic solvent– Wide range of solvent strength
– Composition from 0 to 100% of organic modifier (usually methanol)
A chromatographic technique similar to HPLC A chromatographic technique similar to HPLC
Unique Features Compressible mobile phase Compressible mobile phase Cooling device for CO2 pump Pressure Resistant UV Flow Cell Back Pressure Regulator
©2012 Waters Corporation 39
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Fast Separation AnalysisFast Separation Analysis
Van Van DeemterDeemter curvecurve H = f(u)H = f(u)80.0
100.0
p yp y
20 0
40.0
60.0
H (µ
m)
uopt
Generic conditionsGeneric conditions2‐EP, 150 x 4.6mm, 5m. CO2‐MeOH, 3.5mL/minOven temp @ 40 C BPR @ 200bar
Generic conditionsGeneric conditions2‐EP, 150 x 4.6mm, 5m. CO2‐MeOH, 10mL/minOven temp @ 40 C BPR @ 200bar
3 x uopt
Only 25% efficiency loss
0.0
20.0
0.0 2.0 4.0 6.0 8.0 10.0u (mm/s)
UV detection @ 220nmUV detection @ 220nm
BenzodiazepinesBenzodiazepinesI. MidazolamII. FlunitrazepamIII. LormetazepamIV Cl
0.15
0.20SteroidsSteroids
1. Androstenedione2. Mestanolone3. Testosterone4 S l l
1
23 1
0.15
0.20I II
III
IV. ClorazepateV. NitrazepamVI. Oxazepam
AU
0.00
0.05
0.104. Stanozolol
4
0.60 min
24
3
1.90 min
AU
0.00
0.05
0.10
2.75 min
III
IVIII
V VI0.85 min
IVV
VI
©2012 Waters Corporation 40
Minutes0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60 4.00
Minutes0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60 4.00
Courtesy of A. Grand-Guillaume Perrenoud, D. Guillarme, Pr J-L. Veuthey, University of Geneva
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High Resolution AnalysisHigh Resolution Analysis
BZDBZD 150150 x 4 6mm 5x 4 6mm 5 mmAnalytical conditions : Analytical conditions : CO2‐MeOH gradient mode, 4mL/min ; PrincetonSFC 2EP 150 x 4.6mm, 5m; Oven temp @ 40 C ; BPR @ 150bar ; UV @ 220nm
g yg y
BZDBZD1. Diazepam2. Midazolam3. Flunitrazepam
150150 x 4.6mm, 5x 4.6mm, 5mm30bar30bar
213 5 + 6
710 + 11
P 634. Lormetazepam5. Flurazepam6. Alprazolam7. Triazolam8. Clorazepate9 Bromazepam
21
48
912 13 + 14
15
Pc = 63
9. Bromazepam10. Nitrazepam11. Clonazepam12. Oxazepam13. Lorazepam14. Clozapine
450450 x 4.6mm, 5x 4.6mm, 5mm80b80b
p15. Olanzapine
N
NR2
R3R4
R1
213
4
6
78 9 1112
1314
80bar80bar
510
15
Pc = 108
©2012 Waters Corporation 41
R5
Courtesy of A. Grand-Guillaume Perrenoud, D. Guillarme, Pr J-L. Veuthey, University of Geneva
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RememberRemember::Smaller Particles the Enabler of ProductivitySmaller Particles the Enabler of Productivityyy
©2012 Waters Corporation 42
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SFC Column Efficiency CurvesSFC Column Efficiency Curvesyy
5.0 µm XBridge™ HILIC, 3.0x50 mm
2.5 µm XBridge™ HILIC, 3.0x50 mm
3.5 µm XBridge™ HILIC, 3.0x50 mm
µ g ,
1.7 µm ACQUITY BEH , 3.0x50 mm
Isocratic Conditions
©2012 Waters Corporation 43
CO2/Methanol 85:15 130 bar outlet pressure
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Resolution ComparisonResolution Comparisonpp
0 14
0.16
5 µm 3.0x100 mm Column
AU
0.08
0.10
0.12
0.14µ
2 mL/minuteCO2/Methanol Gradient
0.00
0.02
0.04
0.06
0.20
0.25 1.7 µm
AU
0 05
0.10
0.15
©2012 Waters Corporation 44
0.00
0.05
Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
-
©2012 Waters Corporation 45
-
UPLC Technology Platform Has Expanded:UPLC Technology Platform Has Expanded:Adopting Across Organization to Achieve Business GoalsAdopting Across Organization to Achieve Business Goalsp g gp g g
I-ClassH-Class H-Class Bio 2D
©2012 Waters Corporation 46
nanoACQUITY HDXPATROLUPC2
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nanoACQUITYnanoACQUITY
Inlet of choice N UPLC
nanoACQUITYnanoACQUITY
True UPLC performance
Inlet of choice for High
Resolution MS systems
Nano UPLC for 3rd party MS systems
System of choice for sample limited l
Direct flow, non-split system
applications
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UPLC Technology Platform Has Expanded:UPLC Technology Platform Has Expanded:Adopting Across Organization to Achieve Business GoalsAdopting Across Organization to Achieve Business Goalsp g gp g g
I-ClassH-Class H-Class Bio 2D
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nanoACQUITY HDXPATROLUPC2
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PATROL Laboratory Analysis SystemPATROL Laboratory Analysis System
Seamless Mi i i
PATROL Laboratory Analysis SystemPATROL Laboratory Analysis System
True UPLC performance
Seamless transfer of optimized
methods from the Process L b t Mf
Minimize error,
optimize methods,
and improve Lab to Mfg floor
and improve workflow
Expand laboratory capabilities from QC to
Bridges pilot plant to mfg
Process Development
Inc ease th o ghp t fo the Reaction Monito ing/Optimi ation
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Increase throughput for the Reaction Monitoring/Optimization Labs
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Sample Work Flow ComparisonSample Work Flow ComparisonACQUITY & PATROL AtlineACQUITY & PATROL AtlineQQ
LOADVIAL
ASPIRATESAMPLE
INJECTSAMPLE
ACQUITY SAMPLE
MANAGER
LOADVIAL
VERIFYVIAL
READBARCODE
VALIDATESAMPLE
ASPIRATESAMPLE
DILUTESAMPLE
INJECTSAMPLE
PATROLPROCESSSAMPLE
MANAGER
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Product Life CycleProduct Life CycleAnalytical SupportAnalytical SupportAnalytical SupportAnalytical Support
DATA and INFORMATICSSpecific to ManufacturingSpecific to Manufacturing
VibrationalSpectroscopy
©2012 Waters Corporation 51
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Product Life CycleProduct Life CycleAnalytical SupportAnalytical SupportAnalytical SupportAnalytical Support
DATA and INFORMATICS
©2012 Waters Corporation 52
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UPLC Technology Platform Has Expanded:UPLC Technology Platform Has Expanded:Adopting Across Organization to Achieve Business GoalsAdopting Across Organization to Achieve Business Goalsp g gp g g
I-ClassH-Class H-Class Bio 2D
©2012 Waters Corporation 53
nanoACQUITY HDXPATROLUPC2
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Hydrogen Deuterium Exchange (HDX)Hydrogen Deuterium Exchange (HDX)What is it?What is it?
Measures changes in protein conformation
Application Space– Drug Binding
– Protein Product Conformation and Stability
– Protein-Protein Interactions
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Typical HDX WorkflowTypical HDX Workflowypyp
D OAt various times,
li t D O
Add D2O, pH 7
D2O
D2O
D2O
D2O
D2O
D2OD2O
LabelingEquilibrate
move an aliquotto quench buffer
Quenched
D2O
D2O
D2O
D2O
D2O
D2OD2O
Labeling25 C, pH 7
Equilibrate25 C, pH 7
QuenchedpH 2.5
++ +
Mass
UPLCElectrosprayMass Spectrometry
+ +
Analyzer
Peptides
Online Digestion
0° C
©2012 Waters Corporation 55
p y
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nanoACQUITY UPLC System nanoACQUITY UPLC System with HDX Technologywith HDX Technology
nanoACQUITY UPLC System with HDX Technology
gygy
consisting of:
nanoACQUITY Binary Solvent Manager
Auxiliary Solvent Manager
HDX Manager (replaces sample manager)
nanoACQUITY UPLC System with HDX Automation
consisting of:
ACQUITY Bi S l t MnanoACQUITY Binary Solvent Manager
Auxiliary Solvent ManagerHDX Manager (replaces sample manager)
Leap HDX Automation Manager (not shown)
©2012 Waters Corporation 56
Leap HDX Automation Manager (not shown)
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nanoACQUITY UPLC HDX ManagernanoACQUITY UPLC HDX ManagernanoACQUITY UPLC HDX ManagernanoACQUITY UPLC HDX Manager
Injection PortInjection PortInjection PortInjection Port Injection ValveInjection ValveInjection ValveInjection Valve Trap ValveTrap ValveTrap ValveTrap Valve
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Column HeaterColumn Heater(Not Visible)(Not Visible)
Column HeaterColumn Heater(Not Visible)(Not Visible) Analytical ColumnAnalytical ColumnAnalytical ColumnAnalytical Column
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©2012 Waters Corporation 59