60mACQUITY 4_19

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


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


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


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.

Ultra Performance LC™


The Promise ofvan Deemter Plot


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


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


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!


Smaller ParticlesThe enabler of productivityThe promise of the van Deemter plot


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


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


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:


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


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.


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)


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


HPLC and UPLCTM


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


HPLC and UPLCTM


10.0

0.30

AU

FS Rs = 4.71

Rs = 9.15

2.1x100mm 1.7µm ACQUITY UPLC

0.33

AU

FS


Rs = 3.52

Rs = 1.82

2.1x30mm 1.7µm ACQUITY UPLCScaled Gradient

Same Resolution as HPLC, Less Time

ACQUITY UPLCTM

ACQUITY UPLCTM


HPLC and UPLCTM


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


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


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?]


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


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



The Challenges of Column Chemistry


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


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


Tetraethoxysilane(TEOS)

Polyethoxysilane(PEOS)

Classical High Purity Silica gel particle synthesis


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


1st Generation Hybrid Particle Where we were in 1999

Methyl Groups on Hybrid Surface(Better Peak Shape)

andin Hybrid Particle(High pH Life-time)


MethylPolyethoxysilane(MPEOS)

Methyltriethoxysilane(MTEOS)

Waters Patented technologyUS Patent: 6,686,035 B2Date of Patent: Feb. 3, 2004


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


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


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


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


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


0.30

AU

FS


ACQUITY UPLCTM ChemistryThe same reproducibility as HPLC

N = 25



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


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


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™


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


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



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


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


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


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


7.00e4


7.00e4

ACQUITY UPLC™Carry over studied with rat urine LC/MS

Peak Area = 97963

Peak Area = 747309

Peak Area = 96015


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


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


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


ACQUITY UPLCTM

eCord™ technology

eCord™


eCord™ Technology Waters quality control results

Paperless Certificate of Analysis & Performance Chromatogram Data


eCord™ Technology User history file


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


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


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


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


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


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


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



Integrated software for the future, today

Speed

Sensitivity

Resolution

Innovation



Interactive System Monitor and Control Interface


ACQUITY UPLCTM ConsoleAccess 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


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



The Benefits of:

Speed

Resolution

Sensitivity



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


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™



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



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



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


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


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

Speed

Sensitivity

Resolution

Innovation

Questions?

60mACQUITY 4_19

Documents

Transcript of 60mACQUITY 4_19