Extending the ACQUITY UPLC Family…
©2011 Waters Corporation
1
©2011 Waters Corporation
2
Introducing ACQUITY UPLC II-Class
The ACQUITY UPLC I-Class
System represents
More recent evolution of UPLC
Based on 7 years of engineering
innovation
Fueled by customer input
Pinnacle of chromatographic
performance
©2011 Waters Corporation
The ACQUITY UPLC I-Class
System accomplishes
Highest throughput without
compromising performance
Maximum Peak Capacity
Enhances the performance of any
Mass Spectrometer
Better data quality
3
ACQUITY UPLC II-Class System
What is it?
Full range of ACQUITY UPLC
Column chemistries
New cells for TUV and PDA
— 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
— New APH with smaller ID
Sample manager
(SM-FTN or SM-FL)
— Less dispersion volume (tubing, valve)
— Improved carry over
Binary solvent manager (BSM)
— 1200 bar
— New flow path with smaller ID
©2011 Waters Corporation
4
ACQUITY UPLC II-Class
The Pinnacle of Chromatographic
Performance
Highest resolution offered
by Sub 2um particles and low
dispersion fluidics
Accelerated complex
separations achieved with
low system’s dispersion &
faster cycle-time
Xevo TOF
ACQUITY SQD
Best sensitivity reached with
lowest carryover
autosampler
SYNAPT G2 MS
SYNAPT G2 HDMS
ACQUITY TQD
XEVO TQ MS
XEVO TQ-S
Optical Detectors:
TUV; PDA, FLR,
ELSD
©2011 Waters Corporation
5
Instrument Contribution to
Bandspread/Extra--Column Effects
Bandspread/Extra
2
2
σ v2,total = σ v2,injector + σ v2, precolumn + σ v2,column + σ v2, postcolumn + σ v2,det ector + τ det
ector ⋅ F
Injection
volume
+
injector
bandspreading
Tubing
between
injector
and
column
Column
volume
+
frits
Tubing
between
column
and
detector
Bandspreading
inside the
detector
cell
+
tubing
Time-based
Bandspreading
in the
Detector
(Sampling
Rate; Time
Constant)
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
6
Extra Colum Volume
Contribution Of Tubing ID
Tubing ID
Volume/foot
Volume/100 cm
0.009’’
12 µl
42 µl
0.005’’
3.5 µl
12.5 µl
0.003’’
1.4 µl
4.6 µl
0.004’’
2.5 µl
8.2 µl
How does it translate to chromatographic performances?
—
—
—
—
Peak capacity is increased (with decreasing ID)
Peak shape is improved
Overal chromatographic performances are strongly impacted
Pressure drop created with tubings is increased
o ∆P α 1/ID4
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
The goal is to maintain the reduced elution volume of resolved UPLC
peaks
©2011 Waters Corporation
7
NEW Binary Solvent Manager
ACQUITY UPLC I-Class BSM
— 1200 bar maximum
— 0.01 - 2 mL/min
— Four solvents (A1 or A2 and B1 or B2)
New pump head, seals, vent valves
New higher pressure mixers
— 50 µL default, 100 µL, and 380 µL
Reliable, robust RT reproducibility
270
260
Average Peak Capacity
High pressure binary mixing
Peak Capacity = 271
Flow Rate = 1.8mL/min
280
250
240
230
220
210
200
400
600
800
1000
1200
Flow Rate (uL/min)
1400
1600
1800
AU
0.20
0.00
©2011 Waters Corporation
0.00
1.00
2.00
Minutes
3.00
4.00
8
Two NEW Sample Managers
Fixed Loop
Flow Through Needle
New EverFlow inject valve design
to enable higher pressures
ACQUITY UPLC H-Class chassis and robust
rotary sample tray/plate mechanism
Compatible with newest Sample Organizer (18
shelves)
New low dispersion fittings, shorter sample
path for the FL (10 µL)
New low dispersion fittings, lower dispersion
needle seal for the FTN (<18 µL)
Low dispersion 1, 2, 5 & 10-µL loop design
Optional extension coils
— Conventional 20, 100, and
250 µL available
System volume <100 µL
Omeprazole 10ng 0403
0
.27
6
18000.00
0
.2
0
5
0
0.56
0.58
Blank1 10ng 0403
0
.24
9
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
0.60
0.62
0.64
0.66
0.68
0.70
0.72
0.74
0.76
0.78
0.80
0.82
0.84
100
0
.1
3
6
0
.3
0
8
0.1
0
3
p
si
16200.00
15300.00
0.2
9
4
%
System volume <95 µL
17100.00
MRM of 1 Channel ES+
346.083 > 198.068 (Omeprazole)
7.06e7
100
0.86
0.88
MRM of 1 Channel ES+
346.083 > 198.068 (Omeprazole)
4.42e3
%
14400.00
13500.00
0.00
0.15
0.30
0.45
Minutes
0
©2011 Waters Corporation
Time
0.56
0.58
0.86
0.88
9
A1
I-Class FTN vs. II-Class FL:
Comparison Summary
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 %
Precision
<0.3% Full loop <1% 0.2 to 1.9 µL <0.3% Full loop
<1.0% PLUNO <0.5% 2 to 10 µL <1.0% PLUNO
Cycle Time
< 15 sec
< 15 sec
< 15 sec
(with load ahead) (with load ahead) (with load ahead)
User Simplicity
3 injection modes
loop changes
Single injection
mode
3 injection modes
loop changes
*Measured with a complete system – Measurement on module only doesn’t make sense
©2011 Waters Corporation
10
Slide 10
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
Column Ovens
NEW Column Heater
Active solvent pre-heating
New 0.003”/0.075 mm I.D. tubing
Robust 1200 bar fitting
Excellent method transfer between
ACQUITY UPLC I-Class Systems
CH-30A is compatible where HPLC
columns must be supported
©2011 Waters Corporation
NEW Column Manager
Active solvent pre-heating
Two (2) Columns plumbed right or
left only
New 0.003”/0.075 mm I.D. tubing
Robust 1200 Bar fitting
For method development or multimethod support
11
ACQUITY UPLC II-Class System
Optical Detection (TUV & PDA)
ACQUITY UPLC I-Class, TUV & PDA
500nL flow cell has been optimized for
reduced dispersion
Flow cell body is the same
Inlet and outlet tubing i.d. has been
reduced
.0025” ID inlet PEEK tubing
.0035” ID outlet PEEK tubing
250nL / 183 µm version for 1mm
chemistry (10 mm pathlength)
ELSD & FLR are compatible with I-Class
©2011 Waters Corporation
12
ACQUITY UPLC II-Class
The Pinnacle of Chromatographic
Performance
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
autosampler
Reduced dispersion
volume
Higher peak capacity
Less matrix effect
Less ion suppression
More information from
complex samples
Higher sensitivity with
sharper and more
concentrated peaks
©2011 Waters Corporation
13
BEH UPLC Technology
Using pH to Increase Resolution
Ranitidine Plasma
17MAR2010_PR_119
0.91
%
100
1: MRM of 1 Channel ES+
TIC (Ranitidine)
2.24e5
Greater peak retention
Basic Separation
0
0.50
17MAR2010_PR_119
1.00
1.50
2.00
2.50
3.00
1.95
100
%
2.20
3.50
4.00
3: Parents of 184ES+
TIC
1.17e9
2.93
0
0.50
17MAR2010_PR_119
1.00
1.50
1.94
100
%
2.00
2.50
3.50
2.21
0
0.50
1.00
1.50
2.00
4.00
2: MS2 ES+
TIC
1.84e9
2.88
1.84
0.24
3.00
2.50
3.00
3.50
Time
4.00
Ranitidine Plasma
17MAR2010_PR_103
%
100
1: MRM of 1 Channel ES+
TIC (Ranitidine)
3.25e4
0.25
Acidic Separation
0.56
0
0.50
17MAR2010_PR_103
1.00
1.50
100
2.00
3.00
3: Parents of 184ES+
2.91
TIC
1.35e9
2.50
3.00
2: MS2 ES+
2.91
TIC
1.31e9
2.27
%
1.98
2.50
0
0.50
17MAR2010_PR_103
1.00
1.50
%
100
2.00
1.98
2.27
0.21
2.43 2.62
©2011 Waters Corporation
0
Time
0.50
1.00
1.50
2.00
2.50
3.00
14
Isocratic Separation Comparison
- 2.1 x 50 mm column
1.628
24.00
UHPLC low volume configuration;
21 µL measured band spread
Average USP Plates = 6016
2.460
0.934
0.551
0.448
36.00
1.310
mAU
48.00
0.684
Instrument Method Name: SampleName: 1290 TCA 50 inj 6
60.00
12.00
0.00
Instrument Method Name: TCA isocratic 50_50 0p8 SampleName: tca 50
1.646
0.024
ACQUITY UPLC;
12 µL measured band spread
Average USP Plates = 7163
2.485
0.933
0.685
0.036
1.320
AU
0.048
0.550
0.444
0.060
0.012
0.000
ACQUITY UPLC I-Class;
5 µL band spread
Average USP Plates = 11,356
2.373
0.04
1.564
0.06
1.262
0.636
0.891
AU
0.08
0.501
Instrument Method Name: TCA isocratic 50_50 0p8 SampleName: tca 50
0.415
0.10
0.02
0.00
0.00
©2011 Waters Corporation
0.40
0.80
1.20
1.60
Minutes
2.00
2.40
2.80
15
Lower Dispersion Further Enhances
ACQUITY UPLC I Class Performance
ACQUITY UPLC
ACQUITY UPLC I-Class
©2011 Waters Corporation
16
Impact of Low Dispersion:
Increase in Sensitivity
1.87x105
1.04x105
©2011 Waters Corporation
17
ACQUITY UPLC I Class Performance
vs Competitive UHPLC
UHPLC
1400.00
1200.00
0.294
1000.00
mAU
0.339
800.00
0.391
0.258
0.192
600.00
0.226
0.321
400.00
200.00
0.00
0.140
0.168
0.196
0.140
0.168
0.196
0.224
0.252
0.280
0.308
0.336
Minutes
0.364
0.392
0.420
0.448
0.476
0.504
0.532
0.420
0.448
0.476
0.504
0.532
0.292
1.20
0.337
ACQUITY UPLC
I-Class
1.40
0.195
1.00
AU
0.257
0.80
0.387
0.60
0.229
0.319
0.40
0.20
0.00
©2011 Waters Corporation
0.224
0.252
0.280
0.308
0.336
Minutes
0.364
0.392
18
ACQUITY UPLC II-Class
The Pinnacle of Chromatographic
Performance
Highest resolution offered
by Sub 2um particles and low
dispersion fluidics
Reduced dispersion
volume
Higher peak capacity
Less matrix effect
Less ion suppression
More information from
complex samples
Higher sensitivity with
sharper and more
concentrated peaks
©2011 Waters Corporation
Accelerated complex
separations achieved with
low system’s dispersion &
faster cycle-time
Best sensitivity reached with
lowest carryover
autosampler
Multi-pump design,
reduced dwell volume
Increased sample
troughput
Fast ballistic gradients
Real 1mm column ID
separation
No compromises with
chromatographic fidelity
19
Accelerated analyses
Ballistic Gradients
10 Second Gradient
Peak Capacity = 46
RETENTION TIME SD = 12ms (for 6 replicates)
0.50
AU
½ Height Width = 88ms
0.00
0.00
©2011 Waters Corporation
0.05
0.10
Minutes
0.15
0.20
2 mL/min @ 90°C
20
Accelerate Ballistic Separations
ACQUITY UPLC and ACQUITY UPLC II-Class
– 1 mm I.D. column
0.110
ACQUITY UPLC
0.483
0.457
Instrument Method Name: 30sec_10_95p600mL_90C ACQUITY
Average peak
capacity at 5σ = 57
0.419
0.205
0.044
0.503
0.351
0.066
0.158
AU
0.088
0.022
0.000
Instrument Method Name: 20sec_NO hold 10_95_p904mL_90C
0.261
ACQUITY UPLC I-Class
Average peak capacity
at 5σ = 70
0.276
0.213
0.117
AU
0.066
0.098
0.088
0.241
0.170
0.110
0.044
0.022
0.000
0.00
©2011 Waters Corporation
0.10
0.20
0.30
0.40
Minutes
0.50
0.60
0.70
Enhanced backpressure limits allows the use of optimum flow
rates for 2.1 and 1.0 mm I.D. columns packed with 1.7 µm
particles resulting in best speed and resolution.
21
I-Class/FTN 2,1mm ID Column vs
1mm
0.0275AU
1mm column w/
1 µl Inj. volume
0.007AU
0.013AU
2,1mm column w/
2 µl Inj. volume
0.0035AU
1 mm ID chromatography offers the same retention time,
same peak capacity but much more sensitivity with even
less injection volume!
©2011 Waters Corporation
22
Metabolites of Ibuprofen Performance Flexibility
1.0 mm ID
RT= 2.63
RT= 2.20
2.1 mm ID
1 mm ID chromatography
offers additional
FLEXIBILITY, when use of
costly solvents, higher
viscosity solvents or for some
sample limited analyses
2.1 mm ID
1.32 x 106
1.0 mm ID
2.52 x 106
©2011 Waters Corporation
23
The ACQUITY UPLC II-Class System Optimizes
Peak Capacity by Minimizing Dispersion and
Providing the Necessary Pressure Range.
280
270
Average Peak Capacity
260
0.20
250
240
230
220
AU
210
200
400
600
800
1000
1200
Flow Rate (uL/min)
1400
1600
1800
Peak Capacity = 271
Flow Rate = 1.8mL/min
0.00
0.00
©2011 Waters Corporation
1.00
2.00
Minutes
3.00
4.00
24
ACQUITY UPLC II-Class
The Pinnacle of Chromatographic
Performance
Highest resolution offered
by Sub 2um particles and low
dispersion fluidics
Reduced dispersion
volume
Higher peak capacity
Accelerated complex
separations achieved with
low system’s dispersion &
faster cycle-time
Multi-pump design,
reduced dwell volume
Less matrix effect
Increased sample
troughput
More information from
complex samples
Real 1mm column ID
separation
Less ion suppression
Higher sensitivity with
sharper and more
concentrated peaks
©2011 Waters Corporation
Fast ballistic gradients
Best sensitivity reached with
lowest carryover
autosampler
Minimised carry-over
Real benefit of higher
sensitivity
Better quantitation
Extended linear range
of calibration curves
No compromises with
chromatographic fidelity
25
SMSM-FTN: UV Carryover Performance
Chlorhexidine
Dioctyl Phthalate
Wash Solvent
1% Formic Acid in MeOH
Wash Solvent
Acetonitrile
Concentration
0.25mg/mL
Wash Solvent
80/20 Water/MeOH
Concentration
0.5mg/mL
AU
0.70
0.00
0.70
0.60
0.00
0.00
0.00
1.00
2.00
Minutes
3.00
4.00
0.00
5.00
Concentration
0.25mg/mL
1.40
AU
1.20
AU
1.40
Caffeine
0.50
1.00
1.50
2.00
Mi nutes
2.50
3.00
0.00
0.50
1.00
Minutes
Standard Trace
Blank Trace
0.012
0.0024
1.50
2.00
Standard Trace
Blank Trace
AU
AU
0.006
AU
0.008
0.0012
0.004
Standard Trace
Blank Trace
0.000
1.00
2.00
Minutes
3.00
4.00
0.0000
0.000
5.00
0.60
1.20
Mi nutes
1.80
2.40
3.00
0.50
1.00
Minutes
1.50
2.00
For concentrations within linear range of detector(<2AU): No Quantifiable Carryover
©2011 Waters Corporation
26
ACQUITY UPLC II-Class
SMSM-FTN: UV Carryover Performance
Chlorhexidine
Dioctyl Phthalate
Wash Solvent
1% Formic Acid in MeOH
Wash Solvent
Acetonitrile
AU
AU
0.00
2.00
Mi nutes
3.00
4.00
5.00
0.00
Carryover
0.00034%
1.00
0.00
0.00
1.00
Concentration
10mg/mL
(40x Higher)
2.00
Carryover
0.00020%
1.00
1.00
0.00
Concentration
10mg/mL
(20x Higher)
2.00
Carryover
0.00093%
Wash Solvent
80/20 Water/MeOH
AU
Concentration
10mg/mL
(40x Higher)
2.00
Caffeine
0.60
1.20
Minutes
1.80
2.40
0.00
3.00
Standard Trace
Blank Trace
0.010
0.002
0.50
1.00
Mi nutes
1.50
2.00
Standard Trace
Blank Trace
0.008
Standard Trace
Blank Trace
0.000
1.00
2.00
Minutes
3.00
4.00
5.00
AU
AU
AU
0.005
0.001
0.004
0.000
0.000
0.60
1.20
Minutes
1.80
2.40
3.00
0.50
1.00
Minutes
1.50
2.00
To quantify carryover: High concentrations, above detection linear range, are injected.
©2011 Waters Corporation
27
Carryover Performance:
ACQUITY UPLC II-Class with Xevo TQTQ-S
Omeprazole 10 ng/mL
Blank Injection
Carryover was reduced to <0.0005% of the highest standard (10 ng/mL)
Assurance that the quantitation represents only the presence of analyte, not
carryover
— Regulation is <20% of LOWEST CALIBRANT
— Calibration was achieved from 500 fg/mL to 10 ng/mL
— Linear quantification >4 orders of magnitude
©2011 Waters Corporation
28
Carryover Performance: 1290, ACQUITY
UPLC, ACQUITY UPLC II-Class with Xevo TQ
TQ-S
Method Requirements
Carryover to be <20%
of LLOQ
High Conc. Standard at Top of Linear Range of Detector
0.0136%
LLOQ > s/n = 10
LLOD > s/n = 3
Therefore, method
Requires <0.001%
Carryover
n/d
0.0020%
n/d
n/d
High Conc. Standard Above Linear Range of Detector
0.00470%
20s wash on the
1290 with multiple
valve switches!
©2011 Waters Corporation
0.00052% 0.00041%
0.00120%
0.00087%
29
I-Class
©2011 Waters Corporation
30