60macquity 4_19
TRANSCRIPT
Ultra Performance LCTM
Redefining Separation Science
Overview of the Historical HPLC
Technology Advancements
©2004 Waters Corporation
ACQUITY UPLC™Advances in science and technology
• Redefining separation science– Introducing a new concept – UPLC™
• The technology developments– Column chemistry and design– Hardware developments– Software for control and interfacing
• The ACQUITY UPLC™ Systems– Redefining separation science
• Application for today’s lab work– Advances over current boundaries
©2004 Waters Corporation
Pittsburgh Conference 2004ACQUITY UPLC™ introduction
Jonathan Hare - Instrumenta, Brian Howard - American Laboratory, Katja Habermueller - GIT Verlag, John Morawski - Waters Corporation, Alex Sands -Instrumenta, Bob Stevenson - American Laboratory
Editors' Gold Award for Best New Product
©2004 Waters Corporation
19591959GPC - Dow Columns & Waters instrumentation
19671967Waters 1st
HPLC
6060’’ss 7070’’ss
19721972M6000 HPLC Pump
19731973µBondapakC18
19781978Sep-Paks
19791979WISP
8080’’ss
19801980--8484Major advances from integrators to computers - Turbochrom- Maxima
19811981SFC introduced19831983--8585API & MS/MS
19851985--8787Advances in laboratory networking
9090’’ss
19901990--9292ESI & PB
19901990--9191CE introduced
19931993--9595Introduction of first relational database chromatography software –
Millennium 19931993--9494
APcI19951995
HP 1100 19961996
Alliance 19961996
Q-Tof 19991999
XTerra
0000’’ss
20002000ZQ Mass DetectorAutoPurification 20022002Atlantis EmpowerCapLC20032003Quattro PremierLCT Premier
Technology Landscape1960-2003
Increasing Refinement & Consistency
©2004 Waters Corporation
1980’s to present day3.5 - 5µm spherical micro-porous1500-4000 psi (110-280 bar)80,000 - 115,000 plates/meter
Early 1970’s10µm Irregular micro-porous1000-2500 psi (70-180 bar)40,000 plates/meter
Particle Size EvolutionLate 1960’s40µm pellicular non-porous coated100-500 psi (7-40 bar)5,000 plates/meter
10 min.
10 min.
10 min.
©2004 Waters Corporation
Smaller ParticlesThe 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
• Particles are central to the quality of the separation
©2004 Waters Corporation
Column Chemistry EvolutionIrregular to spherical shaped particles
1974 the enabling technology for modern HPLC.Irregular shape, large diameter, wide particle size distribution
Large Interstitial Spaces or Channels
80’s and 90’s spherical shape, smaller diameter 5µm and 3µm, narrow particle size distribution
Much smaller interstitial spaces or channels -- tightly packed
©2004 Waters Corporation
Compressed Chromatography Compromised resolution
* 50 mm column * Higher Flow Rates
2.0 mL/min
0.0
1
2
3.0 mL/min.
1
2
Time in Minutes 3.0
1 -- 0.4 0.12 3.3 0.3 0.3
Peak Rs RT %RSD
Area %RSD
1 -- 0.8 0.32 2.3 0.6 0.4
Peak Rs RT %RSD
Area %RSD
Fails Rs Goal of 3Limitation
5um ReversedPhase Column
Run time is reduced, but required resolution Is lost!
©2004 Waters Corporation
Smaller ParticlesThe enabler of productivityThe promise of the van Deemter plot
©2004 Waters Corporation
Particle Size EvolutionThe promise of the van Deemter Plot
1 minute
Future
1.7 µm hybrid particle2.1 x 100 mmup to 15,000 psi (1064 bar)
235,000 plates/meter
Simulation
CombiningSpeed and Resolution
©2004 Waters Corporation
Particle Size EvolutionUPLCTM A new category of chromatography
Future:1.7µm hybrid particle2.1 x 50 mm1.0 ml/minGradient ACN/WaterUV at 247 nm / 40 pts/secBackpressure: 14,450 psi
Combining Speed,Sensitivity and Resolution
We can get a higher quality of information faster
1 minute
AU
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
Minutes0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10
Ace
toph
enon
e - 0
.370
Pro
piop
heno
ne -
0.49
5
But
yrop
heno
ne -
0.60
3
Ben
zoph
enon
e - 0
.646
Val
erop
heno
ne -
0.70
4
Hex
anop
heno
ne -
0.79
8
Hep
tano
phen
one
- 0.8
83
Oct
anop
heno
ne -
0.9
61
©2004 Waters Corporation
Fundamental Resolution EquationAt constant column length
•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 = N4 (α -1
α ) kk+1( )
System Selectivity RetentivityEfficiency
NRs ∝And, Efficiency (N), is inversely proportional to Particle Size , dp
dpN 1∝So:
Rs ↑ 1.7X N ↑ 3X, dp ↓ 3X,Therefore:
©2004 Waters Corporation
Fixed Column LengthFlow rate proportional to particle size
AU
0.000
0.010
0.020
0.030
0.040
0.050
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 15.00
4.8 µm, 0.2 mL/min, 354 psi
AU
0.000
0.010
0.020
0.030
0.040
0.050
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00
Theory1.7X Resolution3X Faster1.7X Sensitivity25X Pressure
Actual1.5X Resolution2.6X Faster1.4X Sensitivity22X Pressure
1.7 µm, 0.6 mL/min, 7656 psi
2.1 x 50 mm columns
©2004 Waters Corporation
Gradient Peak Capacity EquationPotential of gradient resolution power
-0.005
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50
tg
w ww w
w
wt
1P g+=
Gradient Duration
Peak Width
w ↓, P ↑
Peak capacity is a measure of the separation power of a gradient on a particular column.
©2004 Waters Corporation
GradientDuration
(min)
FlowRate
(mL/min)
Pressure(psi)
Max PeakCapacity
1 0.352 3621 634 0.124 1280 10016 0.088 905 13632 0.062 640 151
Effect of Particle Size on Peak Capacity for UPLC™ Separations*
Gradient Duration
(min)
Flow Rate
(mL/min)
Pressure(psi)
Max Peak Capacity
1 0.249 10852 1084 0.249 10852 17216 0.124 5426 21632 0.088 3837 231
Gradient Duration
(min)
Flow Rate
(mL/min)
Pressure(psi)
Max Peak Capacity
1 0.35 1774 464 0.124 627 7616 0.062 314 10732 0.044 222 121
ACQUITY UPLC™ Columns can provide better Peak Capacity at 1 minute, than a 5µm column at 16 minutes!!
1.0 x 50 mm Columns Pmax = ~11,000 psi
0.00
30.
004
0.00
50.
008
0.01
10.
016
0.02
20.
031
0.04
40.
062
0.08
80.
124
0.17
60.
249
0.35
20.
498
0.70
40.
995
1.40
71.
990
12
48163264
0
50
100
150
200
250
0.00
30.
004
0.00
50.
008
0.01
10.
016
0.02
20.
031
0.04
40.
062
0.08
80.
124
0.17
60.
249
12
48
1632
64
0
50
100
150
200
250
0.00
30.
004
0.00
50.
008
0.01
10.
016
0.02
20.
031
0.04
40.
062
0.08
80.
124
0.17
60.
249
0.35
20.
498
0.70
40.
995
12
4816
3264
0
50
100
150
200
250
Peak
Cap
acity
Flow Rate (mL/min)
1.7 µm 3.5 µm 5 µm
108
100 107
Grad
ient D
urat
ion(m
in)
©2004 Waters Corporation
HPLC and UPLCTM
Leveraging the theory
2.1x100mm 4.8µm
HPLC
0.30
AU
FS
Time in Minutes0.0 10.0
Rs = 4.71
Rs = 9.15
2.1x100mm 1.7µm ACQUITY UPLC™More Resolution
ACQUITY UPLCTM
0.30
AU
FS
10.0
Rs = 1.86
Rs = 2.30
©2004 Waters Corporation
HPLC and UPLCTM
Leveraging the theory
10.0
0.30
AU
FS Rs = 4.71
Rs = 9.15
2.1x100mm 1.7µm ACQUITY UPLC
ACQUITY UPLCTM
0.33
AU
FS
Time (min)0.0 3.5
2.1x30mm 1.7µm ACQUITY UPLCScaled gradient
Same resolution as HPLC, less time
ACQUITY UPLCTM
©2004 Waters Corporation
HPLC and UPLCTM
Leveraging the theory
10.0
0.30
AU
FS Rs = 4.71
Rs = 9.15
2.1x100mm 1.7µm ACQUITY UPLC
0.33
AU
FS
Time in Minutes0.0 3.5
Rs = 3.52
Rs = 1.82
2.1x30mm 1.7µm ACQUITY UPLCScaled Gradient
Same Resolution as HPLC, Less Time
ACQUITY UPLCTM
ACQUITY UPLCTM
©2004 Waters Corporation
HPLC and UPLCTM
Leveraging the theory
0.33
AU
FS
Time (min)0.0 3.5
2.1x30mm 1.7µm ACQUITY UPLC Scaled gradient, same resolution as HPLC, less time
1.6
0.25
AU
FS
Time0.0
ACQUITY UPLCTM
1.6
0.25
AU
FS
Time (min)0.0
Rs = 1.84
2.1x30mm 1.7µm ACQUITY UPLCScaled gradient, double flow, half the gradient time
similar resolution, much less time
©2004 Waters Corporation
High Resolution Peptide MappingInfluence of particle size on resolution
AU
0.00
0.02
0.04
0.06
0.08
AU
0.00
0.02
0.04
0.06
0.08
Minutes
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
UPLC™1.7 µm
Peaks = 168Pc = 360
2.5X increase
HPLC4.8 µm
Peaks = 70Pc = 143
©2004 Waters Corporation
High Resolution Peptide MappingInfluence of particle size on sensitivity
AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
Column: 2.1 X 50 mm 1.7 µm, ACQUITY UPLCTM C182.1 x 50 mm 5.0 µm, Prototype Bridged Hybrid C18
Flow: 0.8 mL/min (1.7 µm), 0.3 mL/min (5.0 µm)Temperature: 38 oCGradient (5min & 30 min): 5-50% ACN with 0.018% Trifluoroacetic AcidDetector: UV at 214 nm, 40 points/sec, Tc = 0.1Injection size: 10 uLSample: MassPREPTM Peptide Mixture [who am i?]
©2004 Waters Corporation
Ultra Performance LC™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
©2004 Waters Corporation
Incredible ProductivityHigher throughput
80,00010,000# of Samples Run per Year
324Cycle time (min)
UPLC™HPLC
Assume that an HPLC is running about 67% of the year, or 4,000 hours
8x as many samples run in a year on one UPLC system
©2004 Waters Corporation
ACQUITY UPLC™Innovation in column technology
• Sub 2µm particlesPorous for optimum mass transferBridged hybrid particle* required because of high strength and outstanding chromatographic performance Innovative sizing technology for narrow particle size distribution
• Column hardwareNew frit technology* to retain particles Fittings optimized for high pressure operation
• Packing technologyNew column packing processes to optimize stability
• eCord™New information chip to store column history *patent pending
©2004 Waters Corporation
Particle TechnologyInorganic vs polymer based material
Advantages Disadvantages
Inorganic (Silicon)
• Mechanically strong• High efficiency• Predictable retention
• Limited pH range• Tailing peaks for bases• Chemically unstable
Polymer (Carbon)
• Wide pH range• No ionic interactions• Chemically stable
• Mechanically ‘soft’• Low efficiency• Unpredictable retention
©2004 Waters Corporation
Tetraethoxysilane(TEOS)
Polyethoxysilane(PEOS)
Classical High Purity Silica gel particle synthesis
©2004 Waters Corporation
Particle TechnologyCreating a hybrid material
Advantages Disadvantages
Inorganic (Silicon)
• Mechanically strong• High efficiency• Predictable retention
• Limited pH range• Tailing peaks for bases• Chemically unstable
Polymer (Carbon)
• Wide pH range• No ionic interactions• Chemically stable
• Mechanically ‘soft’• Low efficiency• Unpredictable retention
Hybrid (EthylSiloxane/Silica) Particle Technology
©2004 Waters Corporation
1st Generation Hybrid Particle Where we were in 1999
Methyl Groups on Hybrid Surface(Better Peak Shape)
andin Hybrid Particle(High pH Life-time)
Tetraethoxysilane(TEOS)
MethylPolyethoxysilane(MPEOS)
Methyltriethoxysilane(MTEOS)
Waters Patented technologyUS Patent: 6,686,035 B2Date of Patent: Feb. 3, 2004
©2004 Waters Corporation
Introducing 2nd Generation Hybrid:Bridged EthylSiloxane/Silica Hybrid Particles
Bridged EthanesIn Silica Matrix
Anal. Chem. 2003, 75, 6781-6788
Waters Patented TechnologyNo. 6,686,035 B2
Tetraethoxysilane(TEOS)
Bis(triethoxysilyl)ethane(BTEE)
+4
Polyethoxysilane(BPEOS)
Si
EtO
EtO OEtEtO
Si
EtOEtO
CH2EtO
CH2Si
OEt
OEtOEtSi
EtO
O
CH2 CH2
Si O
Si
EtO
OEt
Si O
O
OEtO
Si
O
Si
OEt
O
OOEt
Et
Et
n
©2004 Waters Corporation
New 2nd Generation HybridBridged EthylSiloxane/Silica Hybrid Particles
• Bridged Ethanes in Hybrid Matrix • Improved Strength• Improved Efficiencies• Improved Peak Shape • Wider pH Range• Used in ACQUITY UPLC™ Columns• (1.7µm particle size)
Anal. Chem. 2003, 75, 6781-6788
©2004 Waters Corporation
2nd Generation Hybrid Particle 2004…where we are now
• Where we were (XTerra®)– Good pressure tolerance– Good pH range– Good efficiency– 2.5 µm particles
• Where we are now (Bridged Hybrid)– Higher pressure tolerance– Wider pH range– Enhanced efficiency– 1.7 µm particles
Wall
Surface
Wall
Surface
OSi
OSi
OSi
OSi
OSi
OSi
OSi
OSi
OSi
O
OH OH OH OH OH H3C CH3OH OH
O O O O O O O
OSi O Si
OSi
OSi
OSi
OSi
OSi
OSi
OSi
OH3C CH3 O O O O O O O
Si
SiO
O
O
OH
O
CH3H3CH3CCH3
The 1.7 µm ACQUITY UPLCTM particle is the most technologically advanced particle ever produced and was designed specifically for the ACQUITY UPLCTM System.
OSi
OSi
OSi
OSi
OSi
OSi
OSi
OSi
OSi
O
OH OH OH OH OH H2C CH2OH OH
O O O O O O O
OSi O Si
OSi
OSi
OSi
OSi
OSi
OSi
OSi
OH2C CH2 O O O O O O O
Si
SiO
OO
OH
O
CH2H2CH2CCH2
©2004 Waters Corporation
ACQUITY UPLCTM ChemistryThe same ruggedness as HPLC
Stability Testing Conditions:1000 one minute gradients from 10 to 90% methanolTemperature: 55 °CMax. pressure: 8500 psiFlow rate: 1.3 ml/min.
AU
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
Minutes
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75
ACQUITY UPLCTM C18 Column2.1 x 30mm 1.7 µm
Black: BeforeRed: After
©2004 Waters Corporation
0.30
AU
FS
Time in Minutes0.0 10.0
ACQUITY UPLCTM ChemistryThe same reproducibility as HPLC
N = 25
Ultra Performance LCTM
Redefining Separation Science
Overview of the Historical HPLC
Technology Advancements
©2004 Waters Corporation
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
©2004 Waters Corporation
ACQUITY UPLCTM
Binary Solvent Manager – Flow Path
Filter/ Mixer/ Tee Assembly
“A” Pump Accumulator
B2
“A” Pump Solvent Select Valve
“B” Pump Solvent Select Valve
B1A2
Solvent A1
“A” Pump Primary
“B” Pump Primary
“B” Pump Accumulator
Six ChannelDegasser
Vent Valve To Sample Manager
©2004 Waters Corporation
ACQUITY UPLCTM
Isocratic compositional reproducibilityA
U
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
0.020
0.022
0.024
0.026
0.028
0.030
0.032
0.034
0.036
0.038
0.040
Minutes1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10
Pro
pyl P
arab
en -
1.38
9
But
yl P
arab
en -
1.85
1
Column: 2.1x100, 1.7µm ACQUITY Chemistry
Mobile Phase: Isocratic 70/30 MeOH/Water, Auto Blend
Flow Rate: 0.3 mL/min
Temperature: 30˚C
Sample: 10ug/mL of propyl- and butyl-paraben in 20/80 MeOH/H2O
Injection Volume: 1.2 µL
Detection: UV at 254nm
Acquisition Rate: 20 points/second
Filtering Constant: 0.1
N = 15 injections with Auto·Blend™
©2004 Waters Corporation
ACQUITY UPLCTM
Binary Solvent Manager gradient stepsAU
0.16
0.18
0.20
0.22
0.24
0.26
0.28
0.30
0.32
0.34
0.36
0.38
Minutes8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00
10% Gradient Steps Expanded for 40/50/60 Steps
©2004 Waters Corporation
AU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
0.26
0.28
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00
0% B
3.0% B
1.0% B
2.0% B
ACQUITY UPLCTM
Binary Solvent Manager gradient steps
1% Step Gradient ~ 7100 psi Backpressure
Bob putting on cts
©2004 Waters Corporation
Ultra Performance LCTM
The same reproducibility as HPLCA
U
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
Minutes0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40
Acet
anilid
e -0
.677
Acet
ophe
none
-0.8
37
Prop
ioph
enon
e-0
.951
Buty
roph
enon
e-1
.035
Benz
ophe
none
-1.0
64
Vale
roph
enon
e-1
.111
Hex
anop
heno
ne-1
.182
Hep
tano
phen
one
-1.2
47
Oct
anop
heno
ne-1
.307
Gradient Reproducibility N = 34
©2004 Waters Corporation
ACQUITY UPLCTM
Sample Manager
• 0.1 – 50µL 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 40ºC, two plate sample compartment• Optional Sample Organizer
New Technologies - all-new injection process
©2004 Waters Corporation
ACQUITY UPLCTM
Sample Manager precisionA
U
-0.0005
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
0.0030
0.0035
0.0040
0.0045
Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40
Pro
pyl P
arab
en -
1.37
But
yl P
arab
en -
1.81
9
Overlay of 15 x 0.2µL Injections
©2004 Waters Corporation
1000 ng/mL, Plasma, 5 ms
0.20 0.60 1.00 1.40 Time0
100
%
0
100
%
0
100
%
CalP5ms0328058 MRM of 3 Channels ES+ 314.1 > 210.20.63
CalP5ms0328058 MRM of 3 Channels ES+
309.1 > 205.20.63
CalP5ms0328058 MRM of 3 Channels ES+ 260.2 > 116.00.48
6.91e6
4.97e7
6.27e6
Plasma Blank
0.20 0.40 0.60 0.80 1.00 1.20 1.40Time0
100
%
0
100
%
0
100
%
CalNt5ms0328059 MRM of 3 Channels ES+ 314.1 > 210.2
7.00e4
CalNt5ms0328059 MRM of 3 Channels ES+ 309.0 > 205.2
7.00e4
CalNt5ms0328059 MRM of 3 Channels ES+ 260.2 > 116.0
7.00e4
ACQUITY UPLC™Carry over studied with rat urine LC/MS
Peak Area = 97963
Peak Area = 747309
Peak Area = 96015
©2004 Waters Corporation
ACQUITY UPLCTM
Sample Organizer
• Temperature controlled (4 to 40ºC)
• Expands capacity to either:– 22 microtiter plates
8,448 sample wells– 15 mid height plates– 8 deep well plates
388 2-ml vials196 4-ml vials
• Access to any sample location
• Small footprint – 10.5 inches (27cm)
Supports high throughput/high capacity
©2004 Waters Corporation
ACQUITY UPLCTM
Sample Manager - Column Heater
• Integrated with Sample Manager– 65°C 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
©2004 Waters Corporation
HPLC Column CompartmentHeater 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
– 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
eCord™ reader
Encased 16mm
Microchip
Holder
Tether
©2004 Waters Corporation
eCord™ Technology Waters quality control results
Paperless Certificate of Analysis & Performance Chromatogram Data
©2004 Waters Corporation
UPLC™ DetectionTechnology challenges
• Faster eluting peaks require:– Higher data rates– Lower cell volume
• 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
©2004 Waters Corporation
UPLC™ Flow Cell DesignLow-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
αTeflon® AF
water
dTeflon® AF
©2004 Waters Corporation
ACQUITY Ultra Performance LC™Low dispersion UPLC optics
Time2.00 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 30.00
%
0
100
%
0
100 0.67
22.92
0.78
2.09
1.30
11.208.62
2.57
3.55 4.205.615.15 7.336.49 7.93
21.6621.10
20.3117.13
14.5112.71 16.32
19.4318.26
22.23
26.01
23.15
24.40
25.17
26.51
27.34
28.7728.12
0.69 23.050.81
2.58
1.37
11.62
9.11
6.135.864.883.734.36
7.06 7.678.38 9.99
21.7321.1918.3817.3214.78
13.6512.96 16.6015.39
19.5020.40
22.30
23.30
26.01
23.5124.45
25.8425.09
26.61
27.32
27.5129.18
UV cell in line
No UV cell
2.1x100mm400 µL/min
©2004 Waters Corporation
ACQUITY Ultra Performance LC™Expanded region of MS dispersion
Time10.80 11.00 11.20 11.40 11.60 11.80 12.00 12.20
%
0
100
%
0
100
11.20
11.62
11.2810.93
No UV cell
UV cell in line
0.43mins
0.43mins
©2004 Waters Corporation
AU
0.000
0.010
0.020
0.030
0.040
0.050
0.060
0.070
0.080
Minutes0.50 0.52 0.54 0.56 0.58 0.60 0.62 0.64 0.66 0.68 0.70 0.72 0.74
Data Acquisition RatesImpact on UV chromatography data
1 pt/s2 pts/s5 pts/s10 pts/s20 pts/s40 pts/s
©2004 Waters Corporation
Data Acquisition RatesImpact on LC/MS chromatography
Peak Area = 16262
Peak Width 1.8 s
100 ms Dwell Time, 10 ms Delay
0.25 0.75 1.25 1.75Time0
%
0
%
0.63
0.63
100 105 110 115 120 125 130Scan0
%
0.63
1100 1200 1300 1400Scan0
%
0.63
Peas Area = 16791
Peak Width = 1.8 s
Points Across Peak = 7
5 ms Dwell Time, 5 ms Delay
Points Across Peak = 60
0.25 0.75 1.25 1.75Time
Convert the x axis to scan number
Convert the x axis to scan number
Alprazolam in Rat Plasma, 10 ng/mL, 5 ul Injection
©2004 Waters Corporation
UPLC™ Systems TechnologyUltra performance by design
Detectors:Optical and/or Mass SpecTunable UV or Photodiode ArrayOptimized flow cell for UPLCTM
High speed detectionLow dispersion designUPLC pressure capabilities
Column Manager:Innovative pivot design for MS InterfaceColumn temperature controleCord technologySample Manager:
Low dispersion XYZZ’ FormatFast cycle timesLow carryoverPlates and/or vialsOptional Sample OrganizerUPLC pressure capabilities
Binary Solvent Manager:High pressure blendingBinary gradientsFour solvent choicesOn-line degassingLow dispersion designUPLC pressure capabilities
©2004 Waters Corporation
Ultra Performance LCTM
Integrated software for the future, today
Speed
Sensitivity
Resolution
Innovation
Ultra Performance LC™
Redefining Separation Science
Interactive System Monitor and Control Interface
©2004 Waters Corporation
ACQUITY UPLCTM
Optional FlexCart
• Portability
• Self contained power strip
• Computer/monitor/keyboard extension
• Solvent waste storage
• Height adjustable to optimize mass spec interfacing
©2004 Waters Corporation
ACQUITY UPLCTM
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
• Provides users:– Increased equipment uptime– Confidence in results– Increased productivity through
proactive/predictive reporting Waters Connections® INSIGHT™
Internet
©2004 Waters Corporation
Ultra Performance LCTM
Speed increases sample throughput
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
1. T
hiou
rea
-0.4
30
2. to
luen
e -1
.034
3. p
ropy
lben
zene
-1.
742
4. b
utyl
benz
ene
-2.4
13
5. h
exyl
benz
ene
-5.0
58
No. of components : 5Complete Separation : 6.00 min
0.18
0.20
0.22
0.24
1. T
hiou
rea
-0.0
462.
tolu
ene
-0.0
883.
pro
pylb
enze
ne -
0.13
74.
but
ylbe
nzen
e -0
.182
5. h
exyl
benz
ene
-0.3
60
UPLCTM HPLC
AU
0.00
0.10
0.20
Minutes0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60
UPLC™No. Of components:5Complete Separation: 0.60 min
UPLCTM increases speed by up to 9X
©2004 Waters Corporation
UPLCTM with MS/MS Optimized quantitation with MS/MS
Average S/N of six 1 ppb injection = 32.9
1.60 2.00 2.40 2.80 3.20Time
100
%
2.61
1 ppb, and 10 ppb
6 Injection each
HPLC
Average S/N of six 1 ppb injection = 131.5
0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20Time2
100
%
0.67
1 ppb, and 10 ppb
6 Injection each
UPLC™
©2004 Waters Corporation
Ultra Performance LCTM
High resolution peptide mapping
ACQUITY UPLCTM C18, 2.1 x 100 mm, 1.7 µmPc = 589
AU
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
0.020
0.022
0.024
Minutes
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00 120.00
Phosphorylase b Tryptic Digest
©2004 Waters Corporation
Ultra Performance LCTM
Combined Speed, Sensitivity, Resolution
AU
0.000
0.010
0.020
0.030
0.040
0.050
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00
AU
0.000
0.010
0.020
0.030
0.040
0.050
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 15.00
5.0 µm
1.7 µm
60% more speed30% more sensitivity70% more resolution
Increased throughput with improved quality of information
©2004 Waters Corporation
Ultra Performance LCTM
Speed, Sensitivity and Resolution
0.25
AU
Minutes0.0 1.6
Resolution: 1.7X increase
Sensitivity: 3X increase
Speed: 9X faster separationsA
U
0.00
0.02
0.04
0.06
0.08
0.10
Minutes0.0 5 10 15 20 25 30 35 40 45 50 55 60
PC = 360
AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
©2004 Waters Corporation
Making UPLC™ a Reality TodayEssential technology achievements
• 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
©2004 Waters Corporation
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
©2004 Waters Corporation
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