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Extending the ACQUITY UPLC Family…
©2011 Waters Corporation 1
©2011 Waters Corporation 2
Introducing ACQUITY UPLC IIntroducing ACQUITY UPLC I--ClassClass
The ACQUITY UPLC I-Class System represents
The ACQUITY UPLC I-Class System accomplishes
g Qg Q
System represents System accomplishes Highest throughput without
compromising performance More recent evolution of UPLC
Based on 7 years of engineering Maximum Peak Capacity
Enhances the performance of any
Based on 7 years of engineering innovation
Fueled by customer inputMass Spectrometer
Better data quality Pinnacle of chromatographic
performance
©2011 Waters Corporation 3
ACQUITY UPLC IACQUITY UPLC I--Class SystemClass SystemWhat is it?What is it?
New cells for TUV and PDA
Full range of ACQUITY UPLC Column chemistries
— 500 nl/10mm
— 250 nl/10 mm (for 1mm ID columns)
Compatible with FLR, ELSD & all MS
Column Manager Flexibility (CH-A or CM-A)
— Single or multiple column
N APH ith ll ID
Sample manager (SM-FTN or SM-FL)
— Less dispersion volume (tubing valve)
— New APH with smaller ID
Binary solvent manager (BSM)
Less dispersion volume (tubing, valve)
— Improved carry over
©2011 Waters Corporation 4
— 1200 bar
— New flow path with smaller ID
Instrument Contribution to Instrument Contribution to Bandspread/ExtraBandspread/Extra--Column EffectsColumn Effectsp /p /
22det
2,det
2,
2,
2,
2,
2, Fectorectorvpostcolumnvcolumnvprecolumnvinjectorvtotalv
Injectionvolume
+injector
Tubingbetweeninjector
and
Columnvolume
+frits
Tubingbetweencolumn
and
Band-spreadinginside thedetector
Time-basedBand-
spreadingin theinjector
band-spreading
and column
frits and detector
detectorcell+
tubing
in theDetector
(SamplingRate; TimeConstant)
Engineering developments have specifically improved dispersion
— Injector design, injector volume, fittings, flow path, sealing surfaces
— Reduced tubing volumes - higher pressure/extended flow rate range
— Improved flow cell dispersion
©2011 Waters Corporation 5
System Band System Band Spreading:Spreading:Effect Effect of of Tubing ID and LengthTubing ID and Length
Tubing with a large ID increases the overall system volume and contributes to band spreading
g gg g
Excessive tubing lengths contribute to band spreading Decreasing tubing ID and length increases system
pressure
Smallest ID is best
Flow Direction
0.020"0.0025" 0.040"
Shortest length is best
©2011 Waters Corporation 6
Extra Colum VolumeExtra Colum VolumeContribution Of Tubing ID Contribution Of Tubing ID
Tubing ID Volume/foot Volume/100 cm
0 009’’ 12 l 42 l
gg
0.009’’ 12 µl 42 µl
0.005’’ 3.5 µl 12.5 µl
0.004’’ 2.5 µl 8.2 µl
0.003’’ 1.4 µl 4.6 µl
How does it translate to chromatographic performances?— Peak capacity is increased (with decreasing ID)Peak capacity is increased (with decreasing ID)
— Peak shape is improved
— Overal chromatographic performances are strongly impacted
— Pressure drop created with tubings is increasedp g
0.003’’ vs 0.004’’: 50% less volume, 3x more pressure
Higher pressure limit is required, just a tool, a technology enabler
Higher pressure is not a goal
©2011 Waters Corporation 7
Higher pressure is not a goal
The goal is to maintain the reduced elution volume of resolved UPLC peaks
Two NEW Sample ManagersTwo NEW Sample Managersp gp g
Fixed Loop Flow Through Needle
New EverFlow inject valve designNew EverFlow inject valve designto enable higher pressures
ACQUITY UPLC H-Class chassis and robust rotary sample tray/plate mechanism
New low dispersion fittings, shorter sample path for the FL (10 µL)
Compatible with newest Sample Organizer (18 shelves)
New low dispersion fittings, lower dispersion needle seal for the FTN (<18 µL)
Low dispersion 1, 2, 5 & 10-µL loop design
— Conventional 20, 100, and250 µL available
Optional extension coils
System volume <100 µL100
Omeprazole 10ng 0403 MRM of 1 Channel ES+ 346.083 > 198.068 (Omeprazole)
7.06e7
System volume <95 µL
100
0.56 0.58 0.60 0.62 0.64 0.66 0.68 0.70 0.72 0.74 0.76 0.78 0.80 0.82 0.84 0.86 0.88
%
0
Blank1 10ng 0403 MRM of 1 Channel ES+ 346.083 > 198.068 (Omeprazole)
4.42e3
103
0.20
5
0.24
9
0.27
6
0.29
4
08
psi
16200.00
17100.00
18000.00
©2011 Waters Corporation 8
Time0.56 0.58 0.60 0.62 0.64 0.66 0.68 0.70 0.72 0.74 0.76 0.78 0.80 0.82 0.84 0.86 0.88
%
0
0.1
0.13
6 0.30
8
13500.00
14400.00
15300.00
Minutes0.00 0.15 0.30 0.45
II--Class FTN vs. IClass FTN vs. I--Class FL:Class FL:Comparison SummaryComparison Summaryp yp y
ACQUITY I-Class FTN I-Class FL
Dwell Volume* 120 µL 100 µL 95 µL
Bandspread* 12 µL <9 µL <7 µL
Carryover 0.005 % 0.001 % 0.002 %
P ecision <0.3% Full loop <1% 0.2 to 1.9 μL <0.3% Full loop Precision p<1.0% PLUNO
μ<0.5% 2 to 10 μL
p<1.0% PLUNO
Cycle Time < 15 sec(with load ahead)
< 15 sec(with load ahead)
< 15 sec(with load ahead)
User Simplicity 3 injection modesloop changes
Single injection mode
3 injection modesloop changes
©2011 Waters Corporation 9
*Measured with a complete system – Measurement on module only doesn’t make sense
A1
Slide 9
A1 Note we have chnaged the spec on teh cycle time both SM's are the same and edited seaker notes to reflect this.Author; 05-10-2011
Comparison SummaryComparison Summaryp yp y
©2011 Waters Corporation 10
*Measured with a complete system – Measurement on module only doesn’t make sense
A2
Slide 10
A2 Note we have chnaged the spec on teh cycle time both SM's are the same and edited seaker notes to reflect this.Author; 05-10-2011
Column OvensColumn Ovens
NEW Column Heater NEW Column Manager
Active solvent pre-heating
Two (2) Columns plumbed right or left only
Active solvent pre-heating
New 0.003”/0.075 mm I.D. tubing
Robust 1200 bar fitting New 0.003”/0.075 mm I.D. tubing
Robust 1200 Bar fitting
For method development or multi-
Robust 1200 bar fitting
Excellent method transfer between ACQUITY UPLC I-Class Systems
CH-30A is compatible where HPLC method supportcolumns must be supported
©2011 Waters Corporation 11
ACQUITY UPLC IACQUITY UPLC I--Class SystemClass SystemOptical Detection (TUV & PDA)Optical Detection (TUV & PDA)p ( )p ( )
ACQUITY UPLC I-Class, TUV & PDA
500nL flow cell has been optimized for reduced dispersion
Flow cell body is the samey
Inlet and outlet tubing i.d. has been reduced
0025” ID inlet PEEK tubing .0025 ID inlet PEEK tubing
.0035” ID outlet PEEK tubing
250nL / 183 µm version for 1mm chemistry (10 mm pathlength)
©2011 Waters Corporation 12
ELSD & FLR are compatible with I-Class
Isocratic Separation ComparisonIsocratic Separation Comparison-- 2.1 x 50 mm column 2.1 x 50 mm column
Instrument Method Name: SampleName: 1290 TCA 50 inj 6
0.44
8
0.55
1 0.68
4
93448.00
60.00
UHPLC low volume configuration; 21 L d b d d0.
1.31
0
1.62
8
2.46
0mA
U
0.00
12.00
24.00
36.00 21 μL measured band spreadAverage USP Plates = 6016
Instrument Method Name: TCA isocratic 50_50 0p8 SampleName: tca 50
0.44
4
0.55
0
0.68
5
0.93
3
1.32
0
646
5
AU
0 024
0.036
0.048
0.060ACQUITY UPLC; 12 μL measured band spreadAverage USP Plates = 7163
1.6
2.48
5
0.000
0.012
0.024
Instrument Method Name: TCA isocratic 50_50 0p8 SampleName: tca 50
01 6
0.10
ACQUITY UPLC I Cl
0.41
5 0.5 0
0.63
6
0.89
1
1.26
2
1.56
4
2.37
3
AU
0.02
0.04
0.06
0.08 ACQUITY UPLC I-Class;5 μL band spreadAverage USP Plates = 11,356
©2011 Waters Corporation 13
0.00
Minutes0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80
Lower Lower DDispersion Further Enhances ispersion Further Enhances ACQUITY UPLC I Class PerformanceACQUITY UPLC I Class PerformanceQQ
ACQUITY UPLC
ACQUITY UPLC I-Class
©2011 Waters Corporation 14
Impact of Low Dispersion:Impact of Low Dispersion:Increase in SensitivityIncrease in Sensitivityyy
1.87x105
1.04x105
©2011 Waters Corporation 15
ACQUITY UPLC I Class Performance ACQUITY UPLC I Class Performance vsvs Competitive UHPLCCompetitive UHPLCpp
1400.00
UHPLC
0.29
4
0.33
9
mA
U
800.00
1000.00
1200.00
0.19
2
0.22
6
0.25
8
0.32
1
0.39
1
m
200.00
400.00
600.00
0.00
Minutes0.140 0.168 0.196 0.224 0.252 0.280 0.308 0.336 0.364 0.392 0.420 0.448 0.476 0.504 0.532
0.29
2
0.33
71.00
1.20
1.40
ACQUITY UPLCI-Class
0.19
5
9
0.25
7
9
0.38
7
AU
0.40
0.60
0.80
©2011 Waters Corporation 16
0.22
0.31
0.00
0.20
Minutes0.140 0.168 0.196 0.224 0.252 0.280 0.308 0.336 0.364 0.392 0.420 0.448 0.476 0.504 0.532
II--Class/FTN 2,1mm ID Column Class/FTN 2,1mm ID Column vsvs1mm1mm
1mm column w/1 µl Inj. volume
0.0275AU
0.007AU
2,1mm column w/2 µl Inj. volume
0.013AU
0.0035AU
h h ff h i i
©2011 Waters Corporation 17
1 mm ID chromatography offers the same retention time, same peak capacity but much more sensitivity with even less injection volume!
SMSM--FTN: UV Carryover PerformanceFTN: UV Carryover Performanceyy
Chlorhexidine Dioctyl Phthalate CaffeineWash Solvent1% Formic Acid in MeOH
Wash SolventAcetonitrile
Wash Solvent80/20 Water/MeOH
AU
0.70
1.40
AU
0.70
1.40
AU
0.60
1.20
Concentration0.25mg/mL
Concentration0.5mg/mL
Concentration0.25mg/mL
0.00
Minutes0.00 0.50 1.00 1.50 2.00
0.00
Minutes0.00 1.00 2.00 3.00 4.00 5.00
0.00
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00
AU
0.006
0.012
AU 0.0012
0.0024
AU
0.008
Standard TraceBlank Trace
Standard TraceBlank Trace
0.000 0.0000
0 60 1 20 1 80 2 40 3 00
0.000
0.004
0 50 1 00 1 50 2 00
Standard TraceBlank Trace
©2011 Waters Corporation 18
Minutes1.00 2.00 3.00 4.00 5.00
For concentrations within linear range of detector(<2AU): No Quantifiable Carryover
Minutes0.60 1.20 1.80 2.40 3.00
Minutes0.50 1.00 1.50 2.00
ACQUITY UPLC IACQUITY UPLC I--ClassClassSMSM--FTN: UV Carryover PerformanceFTN: UV Carryover Performanceyy
Chlorhexidine Dioctyl Phthalate CaffeineWash Solvent1% Formic Acid in MeOH
Wash SolventAcetonitrile
Wash Solvent80/20 Water/MeOH
AU
1.00
2.00
AU
1.00
2.00Concentration10mg/mL(40x Higher)
Carryover0.00093%
Concentration10mg/mL(20x Higher)
Carryover0.00020%
AU
1.00
2.00Concentration10mg/mL(40x Higher)
Carryover0.00034%
0.00
Minutes0.00 1.00 2.00 3.00 4.00 5.00
0.00
Minutes0.00 0.60 1.20 1.80 2.40 3.00
0.00
Minutes0.00 0.50 1.00 1.50 2.00
AU
0.002
AU
0.005
0.010
AU
0.008
Standard TraceBlank Trace
Standard TraceBlank Trace
0.000
0.001
0.000
0.000
0.004
Standard TraceBlank Trace
©2011 Waters Corporation 19
To quantify carryover: High concentrations, above detection linear range, are injected.
Minutes0.60 1.20 1.80 2.40 3.00
Minutes1.00 2.00 3.00 4.00 5.00
Minutes0.50 1.00 1.50 2.00
Carryover Performance: Carryover Performance: Vendor A, ACQUITY Vendor A, ACQUITY UPLC, ACQUITY UPLC, ACQUITY UPLC IUPLC I--Class with Class with XevoXevo TQTQ--SSUPLC, ACQUITY UPLC, ACQUITY UPLC IUPLC I Class with Class with XevoXevo TQTQ SS
High Conc. Standard at Top of Linear Range of DetectorMethod Requirements Carryover to be <20% of LLOQ
0.0136%of LLOQ
LLOQ > s/n = 10LLOD > s/n = 3
n/d n/d n/dTherefore, method Requires <0.001% Carryover
0.0020%
High Conc. Standard Above Linear Range of Detector
Carryover
0.00470%
0.00052% 0.00041%0.00120% 0.00087%20s wash on vendor
A and with multiple
©2011 Waters Corporation 20
pvalve switches!
Transferring Methods ACQUITY UPLC Transferring Methods ACQUITY UPLC to ACQUITY UPLC Ito ACQUITY UPLC I--ClassClass
Important things to remember— Passive versus active heating
Passive versus active heating
— System volume differences
— System dispersion differences
Binary versus Quaternary gradients— Binary versus Quaternary gradients
©2011 Waters Corporation 21
Original Barbiturates Separation Original Barbiturates Separation ACQUITY UPLC HACQUITY UPLC H--Class SystemClass SystemACQUITY UPLC HACQUITY UPLC H Class SystemClass System
1 – Allobarbital2 – Phenobarbital3 – Butalbarbital4 – Mephobarbital5 – Amobarbital6 - Secobarbital
©2011 Waters Corporation 22
UPLC Method on ACQUITY UPLC HUPLC Method on ACQUITY UPLC H--Class SystemClass SystemClass SystemClass System
Sample: Barbiturates Mix @ 20μM
Column: 2.1 x 50mm ACQUITY UPLC BEH C18, 1.7μmColumn: 2.1 x 50mm ACQUITY UPLC BEH C18, 1.7μm
Injection Volume: 1.5μL
Temperature: 45°C
Fl R t 0 64 L/ iFlow Rate: 0.64mL/min
Mobile Phase A: 10mM Ammonium Bicarbonate Buffer pH 10.0
Mobile Phase B: Acetonitrile
Gradient: Please refer to the table below
Detection Wavelength: 245nm
Time (min) %A %BTime (min) %A %B
Initial 90 10
1.70 68 32
©2011 Waters Corporation 23
1.71 90 10
Gradient Hold Insert for Gradient Hold Insert for ACQUITY UPLC IACQUITY UPLC I--ClassClassACQUITY UPLC IACQUITY UPLC I--ClassClass
©2011 Waters Corporation 24
System System Volume Difference Volume Difference Not AdjustedNot Adjusted
1 2
0.28
System System Volume Difference Volume Difference Not AdjustedNot Adjusted
ACQUITY UPLC H-Class
3
4
5
6
AU
007
0.14
0.21
0.00
0.07
ACQUITY UPLC I Cl
1
2
3
4
5
6
U
0.21
0.28ACQUITY UPLC I-Class
AU
000
0.07
0.14
©2011 Waters Corporation 25
0.00
Minutes0.00 0.30 0.60 0.90 1.20 1.50
Adjusted Gradient Table forAdjusted Gradient Table forACQUITY UPLC IACQUITY UPLC I--Class SystemClass System
ACQUITY UPLC H-Class Gradient Table
ACQUITY UPLC IACQUITY UPLC I--Class SystemClass System
BEFORE NOW
Time (min) %A %B
Initial 90 10
1.70 68 32
Q
1.71 90 10
Gradient Hold insert of 262uL at 640uL/min Flow Rate translates to
ACQUITY UPLC I-Class Gradient Table
Flow Rate translates to 0.40 minutes
Time (min) %A %B
Initial 90 10
0.40 90 10
2 10 68 32
©2011 Waters Corporation 26
2.10 68 32
2.11 90 10
System System Volume Difference Volume Difference AdjustedAdjusted
1 2
0.28
yy jj
ACQUITY UPLC H-Class
3
4
5
6
AU
007
0.14
0.21
0.00
0.07
ACQUITY UPLC I-Class
1 2
3 4
5
6
U
0.21
0.28ACQUITY UPLC I-Class
AU
000
0.07
0.14
©2011 Waters Corporation 27
0.00
Minutes0.00 0.30 0.60 0.90 1.20 1.50
Average of 6 Replicate InjectionsAverage of 6 Replicate InjectionsAverage of 6 Replicate InjectionsAverage of 6 Replicate Injections
USP Resolution
Barbiturates Mix ACQUITY UPLC H-Class
ACQUITY UPLC I-Class
Peak 1 and 2 5.70 5.77
Peak 2 and 3 10.74 10.53
Peak 3 and 4 13.30 13.33
Peak 4 and 5 5.32 5.32
Peak 5 and 6 11.28 11.34
©2011 Waters Corporation 28
Transferring Methods ACQUITY UPLC Transferring Methods ACQUITY UPLC to ACQUITY UPLC Ito ACQUITY UPLC I--ClassClass
1.00
Peaks Resolution
2-3 3.54ACQUITY UPLC
AU
0.00
0.50 3-4 4.56
7-8 2.02
AU
0.50
1.00 Peaks Resolution
2-3 3.27
3-4 5.20
7 8 2 37
ACQUITY UPLC I-Class – Same Conditions
0.00
1.00
7-8 2.37
Peaks Resolution
2 3 3 61ACQUITY UPLC I-Class – Optimized Transfer*
AU
0.00
0.50
2-3 3.61
3-4 4.80
7-8 2.13
©2011 Waters Corporation 29
Minutes0.00 0.50 1.00 1.50 2.00
*Compensated for System Volume (20µL) and Temperature Differences (8°C)
Transferring Methods ACQUITY UPLC Transferring Methods ACQUITY UPLC to ACQUITY UPLC Ito ACQUITY UPLC I--ClassClass
Important things to remember— Passive versus active heating
Passive versus active heating
— Small system volume differences
— System dispersion differences
©2011 Waters Corporation 30
I‐Class
©2011 Waters Corporation 31
Third party instrument Third party instrument control – MS
March 2012
©2011 Waters Corporation 32
ABSciexABSciex /Danaher/Danaher
ACQUITY BSMNanoAcquity UPLC H‐Class UPLC I‐Class
ABI
Analyst V1.42
XX
No 2D
Supports ACQUITY UPLC – Driver Pack v1.40 & June 2011 Driver PackSupports nanoACQUITY UPLC/1D – Driver Pack v1.41
Analyst V1.51
XX
No 2D X X
Enabled with June 2011 Driver PackHot fix v1.52 – supports all previously tested drivers
Analyst V1.60
Not Tested Not Tested Not Tested Not TestedWindows 7 version 32 bit – not currently released – pending date
©2011 Waters Corporation 33
HystarHystar / / BrukerBrukeryy
ACQUITY BSMNanoAcquityUPLC H‐ClassUPLC I‐Class
Bruker
Hystar V3.2 X
XSupports ACQUITY UPLC – Driver Pack v1.40Supports nanoACQUITY UPLC/1D – Driver Pack v1 41
SR2X
No 2DSupports nanoACQUITY UPLC/1D – Driver Pack v1.41
Hystar V3 2 44 XWith plug in Version 1.5 (Planned on Dec 2011)
Hystar V3.2.44 SR2
XX
No 2DX X
©2011 Waters Corporation 34
ThermoFisherThermoFisher//DionexDionex
ACQUITY BSM NanoAcquity UPLC H‐Class UPLC I‐Class
Thermo
XcaliburV 2.1
XX
No 2D
Supports ACQUITY UPLC – Driver Pack v1.30Supports nanoACQUITY UPLC/1D – Driver Pack v1.31June 2011 SR2 / March 2011 Driver Packs ‐ Not currently tested ‐ in process [Waters ‐Milford]‐ Issues may delay introduction
XcaliburX
XNo
X
ACQUITY UPLC, ACQUITY UPL I‐Class – June 2011 Driver Pack nanoACQUITY UPLC– March 2011 Driver Pack [v1 42] –
V 2.2X
No 2DNo
No FTNMarch 2011 Driver Pack [v1.42]
©2011 Waters Corporation 35
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