60mACQUITY 4 - 19

Overview of the Historical HPLC Technology Advancements
Ultra Performance LCTM

Redefining Separation Science
Advances in science and technology Redefining separation science

Introducing a new concept UPLC
ACQUITY UPLC
The technology developments

Column chemistry and design Hardware developments Software for control and interfacing
The ACQUITY UPLC Systems

Redefining separation science
Application for todays lab work

Advances over current boundaries
2004 Waters Corporation
Pittsburgh Conference 2004

ACQUITY UPLC introduction Editors' Gold Award for Best New Product
Jonathan Hare - Instrumenta, Brian Howard - American Laboratory, Katja Habermueller - GIT Verlag, John Morawski - Waters Corporation, Alex Sands Instrumenta, Bob Stevenson - American Laboratory
Technology Landscape
1960-2003
60 60s
70 70s
80 80s
90 90s
00 00s
Increasing Refinement & Consistency

1959
GPC - Dow Columns & Waters instrumentation
1972 M6000 HPLC Pump 1973

BondapakC18
1967 Waters 1st HPLC
1978 Sep-Paks 1979 WISP
19901990-92 Major ESI & PB advances from 19901990-91 integrators to CE introduced computers 19931993-95 - Turbochrom Introduction of first relational - Maxima database 1981 SFC introduced chromatography software 19831983-85 Millennium API & MS/MS 19931993-94 19851985-87 APcI Advances in 1995 laboratory HP 1100 1996 networking Alliance 1996 Q-Tof 1999 XTerra
19801980-84
2000 ZQ Mass Detector AutoPurification 2002 Atlantis Empower CapLC 2003 Quattro Premier LCT Premier
Particle Size Evolution

Late 1960s 40m pellicular non-porous coated 100-500 psi (7-40 bar) 5,000 plates/meter
10 min.
Early 1970s 10m Irregular micro-porous 1000-2500 psi (70-180 bar) 40,000 plates/meter
10 min.
1980s to present day 3.5 - 5m spherical micro-porous 1500-4000 psi (110-280 bar) 80,000 - 115,000 plates/meter
10 min.
The Promise of van Deemter Plot
Ultra Performance LC
The enabler of productivity

Smaller particles provide:
increased efficiency maintain efficiency over a wider linear velocity ability for both added resolution and increased speed of separation
Smaller Particles
Particles are central to the quality of the separation
Irregular to spherical shaped particles

1974 the enabling technology for modern HPLC. Irregular shape, large diameter, wide particle size distribution
Column Chemistry Evolution
Large Interstitial Spaces or Channels
80s and 90s spherical shape, smaller diameter 5m and 3m, narrow particle size distribution
Much smaller interstitial spaces or channels -- tightly packed
Compressed Chromatography
Compromised resolution
1
5um Reversed Phase Column
* 50 mm column * Higher Flow Rates
2.0 mL/min
1 2
Peak 1 2
Rs -3.3
RT Area %RSD %RSD 0.4 0.1 0.3 0.3
Peak
Rs -2.3
3.0 mL/min.
0.0
1 2
RT Area %RSD %RSD 0.8 0.3 0.6 0.4
Time in Minutes
3.0
Fails Rs Goal of 3 Limitation
Run time is reduced, but required resolution Is lost!

The enabler of productivity

The promise of the van Deemter plot
Smaller Particles
The promise of the van Deemter Plot Simulation
Future 1.7 m hybrid particle 2.1 x 100 mm up to 15,000 psi (1064 bar) 235,000 plates/meter
Combining Speed and Resolution
1 minute
UPLCTM A new category of chromatography We can get a higher quality of information faster
0.75 0.70 0.65 0.60 0.55 0.50 0.45 0.40 AU 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 0.00 0.10 0.20 0.30 0.40 0.50 0.60 Minutes 0.70 0.80 0.90 1.00 1.10
Propiophenone - 0.495
Butyrophenone - 0.603
Hexanophenone - 0.798
Valerophenone - 0.704
Heptanophenone - 0.883
Future: 1.7m hybrid particle 2.1 x 50 mm 1.0 ml/min Gradient ACN/Water UV at 247 nm / 40 pts/sec Backpressure: 14,450 psi
Octanophenone - 0.961
Acetophenone - 0.370
Benzophenone - 0.646
1 minute
Combining Speed, Sensitivity and Resolution
Fundamental Resolution Equation

At constant column length
Rs
N 4
System Efficiency
-1
)(
k k+1
Selectivity
Retentivity
In UPLC systems, N (efficiency) is the primary driver Selectivity and retentivity are the same as in HPLC Resolution, Rs, is proportional to the square root of N
Rs N
And, Efficiency (N), is inversely proportional to Particle Size , dp
So:
1 N dp
dp 3X, N 3X, Rs 1.7X
Therefore:
Flow rate proportional to particle size

0.050 0.040 0.030 AU 0.020 0.010 0.000 0.00 2.00 4.00 6.00 8.00 Minutes 10.00 12.00
Fixed Column Length
4.8 m, 0.2 mL/min, 354 psi

Theory 1.7X Resolution 3X Faster 1.7X Sensitivity 25X Pressure Actual 1.5X Resolution 2.6X Faster 1.4X Sensitivity 22X Pressure
15.00
0.050 0.040 0.030 AU 0.020 0.010 0.000 0.00 1.00 2.00 3.00 Minutes 4.00 5.00
1.7 m, 0.6 mL/min, 7656 psi
6.00
2.1 x 50 mm columns
Potential of gradient resolution power Peak capacity is a measure of the separation power of a gradient on a particular column.
0.035 0.030 0.025 0.020 0.015 0.010 0.005 0.000 -0.005 0.00
Gradient Peak Capacity Equation
tg P =1+ w
Gradient Duration
w , P
tg
w
0.10 0.20 0.30 0.40 0.50 0.60
w
0.70
w
0.80 0.90
Peak Width
1.00 1.10 1.20 1.30 1.40 1.50
Effect of Particle Size on Peak Capacity for UPLC Separations*

1.0 x 50 mm Columns 1.7 m
250 200 150 100 50 0
0.003 0.004 0.005 0.008 0.011 0.016 0.022 0.031 0.044 0.062 2 0.088 0.124 0.176 1 0.249
Pmax = ~11,000 psi
3.5 m
250 200 150 100 50 0
0.003 0.004 0.005 0.008 0.011 0.016 0.022 0.031 0.044 0.062 0.088 0.124 0.176 0.249 0.352 0.498 0.704 0.995 64 32 16 8
5 m
250 200 150 100 50
108
4 8
64 32 16
Peak Capacity
100
107
Gr ad ien t (m Dura in) tio n
0
0.003 0.004 0.005 0.008 0.011 0.016 0.022 0.031 0.044 0.062 0.088 0.124 0.176 0.249 0.352 0.498 0.704 0.995 1.407 1.990 1 2
64 32 16 8 4
Flow Rate (m
L/min)
Gradient Duration (min) 1 4 16 32
Flow Rate (mL/min) 0.249 0.249 0.124 0.088
Pressure Max Peak (psi) Capacity 10852 10852 5426 3837 108 172 216 231
Flow Rate (mL/min) 0.352 0.124 0.088 0.062
Flow Rate (mL/min) 0.35 0.124 0.062 0.044
ACQUITY UPLC Columns can provide better Peak Capacity at 1 minute, than a 5m column at 16 minutes!!
Leveraging the theory
HPLC and UPLCTM
0.30 AUFS
Rs = 1.86
Rs = 2.30
2.1x100mm 4.8m
HPLC
0.30
Rs = 9.15
10.0
AUFS
Rs = 4.71
2.1x100mm 1.7m ACQUITY UPLC More Resolution
ACQUITY UPLCTM
0.0
Time in Minutes
10.0

0.30
Rs = 9.15
HPLC and UPLCTM
2.1x100mm 1.7m ACQUITY UPLC

AUFS
Rs = 4.71
ACQUITY UPLCTM
10.0
0.33 AUFS
2.1x30mm 1.7m ACQUITY UPLC Scaled gradient Same resolution as HPLC, less time
ACQUITY UPLCTM
0.0 Time (min) 3.5

0.30
Rs = 9.15
HPLC and UPLCTM
2.1x100mm 1.7m ACQUITY UPLC

AUFS
Rs = 4.71
ACQUITY UPLCTM
10.0
0.33
Rs = 3.52
AUFS
Rs = 1.82
2.1x30mm 1.7m ACQUITY UPLC Scaled Gradient Same Resolution as HPLC, Less Time
ACQUITY UPLCTM
0.0 Time in Minutes 3.5

0.33 AUFS
HPLC and UPLCTM
2.1x30mm 1.7m ACQUITY UPLC Scaled gradient, same resolution as HPLC, less time
ACQUITY UPLCTM
0.0 0.25 AUFS Time (min) 3.5
2.1x30mm 1.7m ACQUITY UPLC Scaled gradient, double flow, half the gradient time similar resolution, much less time
0.25 AUFS
Rs = 1.84
0.0 Time
1.6
0.0
Time (min)
1.6
Influence of particle size on resolution

0.08
High Resolution Peptide Mapping
0.06
AU
0.04
0.02
HPLC 4.8 m Peaks = 70 Pc = 143
0.00 0.08
0.06
AU
0.04
0.02
UPLC 1.7 m Peaks = 168 Pc = 360

5.00 10.00 15.00 20.00 25.00 30.00 Minutes 35.00 40.00 45.00 50.00 55.00 60.00
0.00 0.00
2.5X increase
Influence of particle size on sensitivity

Column: 2.1 X 50 mm 1.7 m, ACQUITY UPLCTM C18 2.1 x 50 mm 5.0 m, Prototype Bridged Hybrid C18 Flow: 0.8 mL/min (1.7 m), 0.3 mL/min (5.0 m) Temperature: 38 oC Gradient (5min & 30 min): 5-50% ACN with 0.018% Trifluoroacetic Acid Detector: UV at 214 nm, 40 points/sec, Tc = 0.1 Injection size: 10 uL Sample: MassPREPTM Peptide Mixture [who am i?]
0.030
High Resolution Peptide Mapping
0.025
0.020
0.015 AU 0.010 0.005 0.000

Speed, Resolution, Sensitivity
Increased speed and sensitivity with the same resolution Increased resolution and sensitivity at the same speed Maximum speed with enhanced sensitivity with sufficient resolution
Incredible Productivity
Higher throughput
Assume that an HPLC is running about 67% of the year, or 4,000 hours
HPLC Cycle time (min) # of Samples Run per Year 24 10,000
UPLC 3 80,000
8x as many samples run in a year on one UPLC system

The Challenges of Column Chemistry
Innovation in column technology Sub 2m particles

Porous for optimum mass transfer Bridged hybrid particle* required because of high strength and outstanding chromatographic performance Innovative sizing technology for narrow particle size distribution
ACQUITY UPLC
Column hardware
New frit technology* to retain particles Fittings optimized for high pressure operation
Packing technology
New column packing processes to optimize stability
eCord
New information chip to store column history
*patent pending
Particle Technology
Inorganic vs polymer based material
Advantages Inorganic (Silicon) Polymer (Carbon)

Mechanically strong High efficiency Predictable retention Wide pH range No ionic interactions Chemically stable
Disadvantages
Limited pH range Tailing peaks for bases Chemically unstable Mechanically soft Low efficiency Unpredictable retention
Silica gel particle synthesis
Classical High Purity
Polyethoxysilane (PEOS)
Tetraethoxysilane (TEOS)
Creating a hybrid material
Particle Technology
Advantages Inorganic (Silicon) Polymer (Carbon)

Mechanically strong High efficiency Predictable retention Wide pH range No ionic interactions Chemically stable
Disadvantages
Limited pH range Tailing peaks for bases Chemically unstable Mechanically soft Low efficiency Unpredictable retention
Hybrid (EthylSiloxane/Silica) Particle Technology

1st Generation Hybrid Particle
Where we were in 1999
Methyl Groups on Hybrid Surface (Better Peak Shape) and in Hybrid Particle (High pH Life-time)
Waters Patented technology
US Patent: 6,686,035 B2 Date of Patent: Feb. 3, 2004
MethylPolyethoxysilane (MPEOS)
Methyltriethoxysilane (MTEOS)
Introducing 2nd Generation Hybrid: Bridged EthylSiloxane/Silica Hybrid Particles

Waters Patented Technology No. 6,686,035 B2
Bridged Ethanes In Silica Matrix
EtO CH2 CH2 OEt O Si Si O Si EtO

O
OEt Si O Et O
O Si OEt O
4
O Et
EtO
Si
Si OEt EtO EtO
EtO EtO Si EtO
OEt CH2 Si OEt CH2 OEt
OEt
Polyethoxysilane (BPEOS)
Anal. Chem. 2003, 75, 6781-6788
Bis(triethoxysilyl)ethane (BTEE)
New 2nd Generation Hybrid Bridged EthylSiloxane/Silica Hybrid Particles

Bridged Ethanes in Hybrid Matrix Improved Strength Improved Efficiencies Improved Peak Shape Wider pH Range Used in ACQUITY UPLC Columns (1.7m particle size)
Anal. Chem. 2003, 75, 6781-6788
2nd Generation Hybrid Particle

Surface
O O H Si O O Si O O H Si O Si O O H Si O H O H C H 3 C H 3 O H O H O H
2004where we are now

O H O H O O Si O Si O O H Si O H O H H C 2 C H 2 Si O Si O O O O H Si O H Si O H O
Surface
O O
Si Si Si Si Si Si Si O O O O O O O C H HC O O 3 C O O O HC 3 O H 3 3 Si O O O Si O O Si O O Si O O Si O O Si O O Si O
Si O
Si O
Wall
Si
H H C C 3 3
Wall
H H C C 2 2
Si Si Si O O O O H C O 2 C O O H 2 Si Si Si Si O O O O O O O O O
Si O C H O HC 2 O 2 O O Si O O Si O
Si O
Where we were (XTerra)

Good pressure tolerance Good pH range Good efficiency 2.5 m particles
Where we are now (Bridged Hybrid)

Higher pressure tolerance Wider pH range Enhanced efficiency 1.7 m particles
The 1.7 m ACQUITY UPLCTM particle is the most technologically advanced particle ever produced and was designed specifically for the ACQUITY UPLCTM System.
ACQUITY UPLCTM Chemistry

The same ruggedness as HPLC
Stability Testing Conditions: 1000 one minute gradients from 10 to 90% methanol Temperature: 55 C Max. pressure: 8500 psi Flow rate: 1.3 ml/min.
0.35 0.30 0.25 AU 0.20 0.15 0.10 0.05 0.00 0.00 0.25 0.50 0.75 Minutes
ACQUITY UPLCTM C18 Column 2.1 x 30mm 1.7 m Black: Before Red: After
1.00
1.25
1.50
1.75
The same reproducibility as HPLC
ACQUITY UPLCTM Chemistry
0.30
N = 25
AUFS 0.0
Time in Minutes
10.0
Overview of the Historical HPLC Technology Advancements

ACQUITY UPLCTM
A holistic approach
ACQUITY UPLCTM
Binary Solvent Manager
Low volume flow path Serial/parallel Flow Path Four solvent choice A1, A2, B1, B2 UPLC pressure capabilities
Materials S/W algorithms
User diagnostics
Binary Solvent Manager Flow Path

Filter/ Mixer/ Tee Assembly Vent Valve
ACQUITY UPLCTM
To Sample Manager
A Pump Primary
A Pump Accumulator
B Pump Primary
B Pump Accumulator
B Pump Solvent Select Valve A Pump Solvent Select Valve Solvent A1 A2 B1 B2 Six Channel Degasser
ACQUITY UPLCTM
Isocratic compositional reproducibility N = 15 injections with AutoBlend
0.040 0.036 0.034 0.032 0.030 0.028 0.026 0.024 0.022
Propyl Paraben - 1.389
0.038
Column:
2.1x100, 1.7m ACQUITY Chemistry Isocratic 70/30 MeOH/Water, Auto 0.3 mL/min 30C 10ug/mL of propyl- and butylparaben in 20/80 MeOH/H2O 1.2 L UV at 254nm 20 points/second
Butyl Paraben - 1.851
Mobile Phase: Blend Flow Rate: Temperature: Sample: Injection Volume: Detection: Acquisition Rate:
AU
0.020 0.018 0.016 0.014 0.012 0.010 0.008 0.006 0.004 0.002 0.000 1.20 1.30 1.40 1.50 1.60 1.70 1.80
Filtering Constant: 0.1
1.90
2.00
2.10
Minutes
Binary Solvent Manager gradient steps
ACQUITY UPLCTM
0.38
0.36
0.34
10% Gradient Steps Expanded for 40/50/60 Steps
0.32
0.30
0.28 AU 0.26 0.24 0.22 0.20 0.18 0.16 8.00 8.50 9.00 9.50 10.00 10.50 11.00 Minutes 11.50 12.00 12.50 13.00 13.50 14.00 2004 Waters Corporation
ACQUITY UPLCTM
Binary Solvent Manager gradient steps
0.28 0.26 0.24 0.22 0.20 0.18 0.16 AU 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 Minutes 14.00 16.00 18.00 20.00 22.00 24.00
Bob putting on cts
3.0% B
1% Step Gradient ~ 7100 psi Backpressure

1.0% B 0% B
2.0% B
The same reproducibility as HPLC

Gradient Reproducibility N = 34
0.80
Propiophenone - 0.951
0.70
Acetophenone - 0.837
Butyrophenone - 1.035
Benzophenone - 1.064
Acetanilide - 0.677
Hexanophenone - 1.182
0.60
Valerophenone - 1.111
Heptanophenone - 1.247
0.50
0.40
0.30
0.20
0.10
0.00 0.60
0.70
0.80
0.90
1.00
1.10
1.20
1.30
Octanophenone - 1.307
AU
1.40
Minutes
ACQUITY UPLCTM
Sample Manager New Technologies - all-new injection process 0.1 50L injection range Fast cycle time
25 sec no wash, <60 sec dual wash
Pressure-assist sample injection Needle-in-Needle Sampling Needle calibration sensor Low sample carry over Positive feedback transducer 4 to 40C, two plate sample compartment Optional Sample Organizer
Sample Manager precision Overlay of 15 x 0.2L Injections

0.0045 0.0040 0.0035 0.0030 0.0025
ACQUITY UPLCTM
Propyl Paraben - 1.37
0.0020 0.0015 0.0010 0.0005 0.0000 -0.0005 0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
Butyl Paraben - 1.819

2.00
AU
2.20
2.40
Minutes
Carry over studied with rat urine LC/MS

1000 ng/mL, Plasma, 5 ms
CalP5ms0328058
100
ACQUITY UPLC
Plasma Blank
CalNt5ms0328059
100
0.63
MRM of 3 Channels ES+ 314.1 > 210.2 6.91e6
Peak Area = 97963

0
0 100
CalP5ms0328058
0.63
CalNt5ms0328059
100
Peak Area = 747309

0
CalP5ms0328058
100
0.48
CalNt5ms0328059
100
Peak Area = 96015

0
0.20
0.60
1.00
1.40
Time
0.20
0.40
0.60
0.80
1.00
1.20
1.40
Time
ACQUITY UPLCTM
Sample Organizer Supports high throughput/high capacity Temperature controlled (4 to 40C) Expands capacity to either:
22 microtiter plates 8,448 sample wells 15 mid height plates 8 deep well plates 388 2-ml vials 196 4-ml vials
Access to any sample location Small footprint 10.5 inches (27cm)

ACQUITY UPLCTM Sample Manager - Column Heater

Integrated with Sample Manager
65C upper limit
Pivot positioning
Stacked mode w/ optical detector Swung out mode w/ MS detector
Flow path distance optimized

Minimizes dispersion Column may be accessed from either side
Swung out mode to interface with MS

HPLC Column Compartment

Heater and eCord technology
Paperless tracking of column history Holder, tether and chip permanently attached to column Microchip encased by 16mm stainless steel can Nonvolatile read/write memory
Tether eCord reader
Holder Fixed column manufacturing data - Unique column identification - Certificate of Analysis - QC test data Variable column usage data - Column use data - Gathered through life of column
Encased 16mm Microchip
ACQUITY UPLCTM
eCord technology
eCord
Waters quality control results
eCord Technology
Paperless Certificate of Analysis & Performance Chromatogram Data
eCord Technology
User history file
Technology challenges Faster eluting peaks require:

Higher data rates Lower cell volume
UPLC Detection
Smaller peaks require maximum S/N

High light throughput/transmission Faster digital filter time constants
UPLC separations require low dispersion flow cells

Maintain peak shape Without generating high backpressures
UPLC Flow Cell Design

Low-index Teflon AF tube
Light Guided UPLC flow cells

10 mm pathlength, 0.010, 500 nL volume The flow cell channel is the inside of a low-index Teflon AF tube Total internal reflection at walls, like optical fiber cladding
Teflon AF water Teflon AF
d
ACQUITY Ultra Performance LC

2.1x100mm 400 L/min
26.01 21.10 20.31 4.20 5.61 6.49 7.33 7.93 5.15 12.71 17.13 14.51 16.32 18.26 19.43 23.15 21.66 22.23 24.40 25.17 26.51 27.34 28.77 28.12
Low dispersion UPLC optics
100
0.67 22.92
0.78 2.09 8.62 2.57 1.30 3.55
11.20
No UV cell
0
0.69 0.81 23.05
100
2.58 11.62
UV cell in line
9.11 5.86 6.13 7.06 7.67 8.38 14.78 9.99 13.65 12.96 15.39 21.73 22.30 21.19 18.38 19.50 17.32 20.40 16.60
23.30 26.01
%
1.37
23.51
4.88 3.73 4.36
24.45 25.84 25.09
26.61 27.32 27.51 29.18
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
28.00
Time 30.00
ACQUITY Ultra Performance LC
Expanded region of MS dispersion
100
11.20
No UV cell
0.43mins
0
11.62
100
UV cell in line
%
10.93 11.28
0.43mins
10.80
11.00
11.20
11.40
11.60
11.80
12.00
Time 12.20
Data Acquisition Rates

Impact on UV chromatography data
0.080 0.070 0.060 0.050
1 pt/s 2 pts/s 5 pts/s 10 pts/s 20 pts/s 40 pts/s
AU
0.040 0.030 0.020 0.010 0.000 0.50 0.52 0.54 0.56 0.58 0.60 0.62 0.64 0.66 0.68 0.70 0.72 0.74
Minutes
Data Acquisition Rates

Impact on LC/MS chromatography Alprazolam in Rat Plasma, 10 ng/mL, 5 ul Injection
100 ms Dwell Time, 10 ms Delay 0.63 Convert the x axis to scan number % % 0.63 Peas Area = 16791 Peak Width = 1.8 s Points Across Peak = 7
0.25
0.75
1.25
1.75
Time 0
100
105
110
115
120
125
130
Scan
5 ms Dwell Time, 5 ms Delay Peak Area = 16262 0.63 Convert the x axis to scan number % % 0.63 Peak Width 1.8 s Points Across Peak = 60
0.25
0.75
1.25
1.75
Time 0
1100
1200
1300
1400
Scan
UPLC Systems Technology

Detectors:
Optical and/or Mass Spec Tunable UV or Photodiode Array Optimized flow cell for UPLCTM High speed detection Low dispersion design UPLC pressure capabilities
Ultra performance by design
Column Manager:
Innovative pivot design for MS Interface Column temperature control eCord technology
Sample Manager:
Low dispersion XYZZ Format Fast cycle times Low carryover Plates and/or vials Optional Sample Organizer UPLC pressure capabilities
Binary Solvent Manager:

High pressure blending Binary gradients Four solvent choices On-line degassing Low dispersion design UPLC pressure capabilities
Integrated software for the future, today
Speed Sensitivity Resolution Innovation
Interactive System Monitor and Control Interface
ACQUITY UPLCTM Console
Access to status and control
ACQUITY UPLCTM Console
Access to status and control
ACQUITY UPLCTM
Optional FlexCart
Portability Self contained power strip Computer/monitor/keyboard extension Solvent waste storage Height adjustable to optimize mass spec interfacing
Waters Connections INSIGHT

Connections INSIGHT creates a new standard for instrument service and support through Integrated Device Management (IDM) IDM provides:
Uni-directional communication from intelligent system Secure communication System usage tracking Proactive service Effective distribution of data across enterprise
ACQUITY UPLCTM
Internet
Provides users:
Increased equipment uptime Confidence in results Increased productivity through proactive/predictive reporting
Waters Connections INSIGHT
The Benefits of: Speed Resolution Sensitivity
Speed increases sample throughput UPLCTM increases speed by up to 9X

0.24
1. Thiourea2. - 0.046 toluene - 0.088 3. propylbenzene - 0.137 4. butylbenzene - 0.182 5. hexylbenzene - 0.360
0.22 0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 0.00
UPLCTM
3. propylbenzene - 1.742 4. butylbenzene - 2.413 2. toluene - 1.034
HPLC
No. of components : 5 Complete Separation :
6.00 min
5. hexylbenzene - 5.058
0.50
1. Thiourea - 0.430
1.00
1.50
2.00
2.50
3.00 Minutes
3.50
4.00
4.50
5.00
5.50
6.00
UPLC
0.20
No. Of components:5 Complete Separation:
0.60 min
AU
0.10
0.00 0.05 0.10 0.15 0.20 0.25
0.30 Minutes
0.35
0.40
0.45
0.50
0.55
0.60
Optimized quantitation with MS/MS
UPLCTM with MS/MS
2.61
100
0.67
100
1 ppb, and 10 ppb 6 Injection each

%
1 ppb, and 10 ppb 6 Injection each
HPLC
Time
UPLC
2.40 2.80 3.20
2 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 Time 1.20
1.60
2.00
Average S/N of six 1 ppb injection = 32.9

Average S/N of six 1 ppb injection = 131.5
High resolution peptide mapping Phosphorylase b Tryptic Digest

0.024 0.022 0.020 0.018 0.016 0.014 0.012 AU 0.010 0.008 0.006 0.004 0.002 0.000
ACQUITY UPLCTM C18, 2.1 x 100 mm, 1.7 m Pc = 589
0.00
10.00
20.00
30.00
40.00
50.00
60.00 Minutes
70.00
80.00
90.00
100.00
110.00
120.00
Combined Speed, Sensitivity, Resolution 60% more speed 30% more sensitivity 70% more resolution
5.0 m
2.00 4.00 6.00 8.00 Minutes 10.00 12.00
0.050 0.040 0.030 AU 0.020 0.010 0.000 0.00 0.050 0.040 0.030 AU 0.020 0.010 0.000 0.00 1.00 2.00 3.00 Minutes 4.00
15.00
1.7 m
5.00 6.00
7.00
Increased throughput with improved quality of information

Speed, Sensitivity and Resolution

0.25
Speed: 9X faster separations
AU
0.0
Minutes
1.6
0.030 0.025 0.020 AU 0.015 0.010 0.005 0.000
Sensitivity: 3X increase
0.10 0.08 0.06 AU 0.04 0.02 0.00 0.0

PC = 360
Resolution: 1.7X increase
10
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60
Minutes
Essential technology achievements
Making UPLC a Reality Today
Small, pressure-tolerant particles High pressure fluidic modules (up to15,000 psi) Minimized system volumes and optimized flow paths Reduced cycle times Negligible carryover sample management High speed detectors (optical and mass) Software designed for system integration
Novel communication protocols Advanced diagnostics
Ultra Performance LC Redefining Separation Science

UPLC benefits:
Familiarity of separation mechanism Same chromatographic principles Speed, sensitivity, resolution Method transfer, revalidation is fast
More information about your samples More productivity
ACQUITY UPLCTM
System attributes
Familiarity of separation mechanism

Same chromatographic principles
Expanded application
More information, minimized system volume, connectivity to MS
Confident results
Reliability built in, advanced diagnostics support Compliant ready solutions Connections INSIGHTTM
Increased productivity
Faster chromatography More information per separation Higher laboratory throughput
Redefining separation science
Questions?
Speed Sensitivity Resolution Innovation

60mACQUITY 4 - 19

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Overview of the Historical HPLC Technology Advancements

Ultra Performance LCTM

Advances in science and technology Redefining separation science

The technology developments

The ACQUITY UPLC Systems

Application for todays lab work

Pittsburgh Conference 2004

Increasing Refinement & Consistency

1972 M6000 HPLC Pump 1973

1967 Waters 1st HPLC

1978 Sep-Paks 1979 WISP

2004 Waters Corporation

Particle Size Evolution

2004 Waters Corporation

The Promise of van Deemter Plot

The enabler of productivity

Particles are central to the quality of the separation

2004 Waters Corporation

Irregular to spherical shaped particles

Column Chemistry Evolution

Large Interstitial Spaces or Channels

Much smaller interstitial spaces or channels -- tightly packed

2004 Waters Corporation

* 50 mm column * Higher Flow Rates

RT Area %RSD %RSD 0.4 0.1 0.3 0.3

RT Area %RSD %RSD 0.8 0.3 0.6 0.4

Fails Rs Goal of 3 Limitation

Run time is reduced, but required resolution Is lost!

The enabler of productivity

2004 Waters Corporation

The promise of the van Deemter Plot Simulation

Particle Size Evolution

Combining Speed and Resolution

Particle Size Evolution

Combining Speed, Sensitivity and Resolution

Fundamental Resolution Equation

Flow rate proportional to particle size

Fixed Column Length

4.8 m, 0.2 mL/min, 354 psi

1.7 m, 0.6 mL/min, 7656 psi

2004 Waters Corporation

Gradient Peak Capacity Equation

Effect of Particle Size on Peak Capacity for UPLC Separations*

Pmax = ~11,000 psi

Gr ad ien t (m Dura in) tio n

Gradient Duration (min) 1 4 16 32

Flow Rate (mL/min) 0.249 0.249 0.124 0.088

Gradient Duration (min) 1 4 16 32

Flow Rate (mL/min) 0.352 0.124 0.088 0.062

Gradient Duration (min) 1 4 16 32

Flow Rate (mL/min) 0.35 0.124 0.062 0.044

2004 Waters Corporation

Leveraging the theory

HPLC and UPLCTM

2.1x100mm 1.7m ACQUITY UPLC More Resolution

Leveraging the theory

HPLC and UPLCTM

2.1x100mm 1.7m ACQUITY UPLC

2004 Waters Corporation

Leveraging the theory

HPLC and UPLCTM

2.1x100mm 1.7m ACQUITY UPLC

2004 Waters Corporation