Post on 13-Apr-2018
Agilent 1290 Infinity LC
Next Generation Technology for highest Performance and Flexibility.
HPLC 2009HPLC 2009Dresden, GermanyStefan SchuetteAgilent TechnologiesAgilent Technologies
Short History of UHPLC
2003 Agilent 1200 Binary 1.8um RRHT Columns2004 Waters Acquity 1.7um BEH Columns2006 Agilent 1200 RRLC2006 Agilent 1200 RRLC
Thermo Accela 2007 Shimadzu UFLCxr
Jasco Xtreme-LCHitachi LaChrom UltraScient. Syst. Ultra HPy
2008 Dionex RSLCKnauer Platin Blue
2009 PE Fl2009 PE Flexar
10 New UHPLC Systems in 5 Years – All different.
UHPLC Situation 2009Power Rangesg
1200
bar Power = Pressure x Flow
Pressure Resolution
1000WatersAcquity
Pressure ResolutionFlow Speed
600
800Acquity
Dionex RSLC
400
Agilent RRLCThermoAccela
Shimadzu UFLCXR
0
200
Standard LC
3
0 1 2 3 4 5 ml/min
UHPLC Situation 200910 Systems in 5 Years – All Different!y
Problems
• Method Transfer
Differences
• System Volumes
Debate
• Methods: transferable?• Not possible• Requires re-devel.
and -validationP f Li it ti
y
• Pressures
• Flow Rates
T t
• ID: 4.6, 3 or 2.1 mm?
• Particle: TP or SP?• Performance Limitations
• Speed • Resolution • Sensitivity
• Temperatures
• Data Rates
• Sensitivity
• Size: STM or larger?
• Flow: low or high?
• T: low or high?• Sensitivity • Precision• Linearity
• Injection principle
• ...
• T: low or high?
• P: 600 or 1000 bar?
4
STM = Sub-2um SP = Superficially PorousTP = Totally Porous
Agilent 1290 Infinity LC- Development Objectives- Development Objectives
System Requirements The Debate is Over
1. Power Range:encloses any (U)HPLC
• Methods: transferable !
• ID: 4 6 3 and 2 1 mm !2. Delay Volume:
lowest, adjustable
3. Sensitivity, Precision:
ID: 4.6, 3 and 2.1 mm !
• Size: STM and larger !
• Particle: TP and SP !better than any (U)HPLC
4. Injection Principle:variable and fixed loop
a c e a d S
• Flow: low and high !
• T: low and high !
5. Gradient Behaviour:emulate any (U)HPLC
• P: 600 and 1000 bar !
5
STM = Sub-2um SP = Superficially PorousTP = Totally Porous
Agilent 1290 Infinity LCPower Range
All analytical columns
Method Transfer
1200
bar
Higher Resolution
Faster Analysis
Higher (MS) Sensitivity
1000WatersAcquity
Agilent 1290 Infinity LC More viscous solvents
Lower temperatures
600
800Acquity
Dionex RSLC
400
Agilent RRLCThermoAccela
Shimadzu UFLCXR
0
200
Standard LC
6
0 1 2 3 4 5 ml/min
STM = Sub-2um SP = Superficially PorousTP = Totally Porous
Agilent 1290 Infinity LC Power Range and 4.6mm, 1.8um TP Columns 50 x 4.6 mmg ,
1200
bar
100 x 4.6 mm150 x 4.6 mm250 x 4.6 mm
1000H2O/ACN, 40 °C
600
800
400
0
200
7
00 1 2 3 4 5 ml/min
STM = Sub-2um SP = Superficially PorousTP = Totally Porous
Agilent 1290 Infinity LC Power Range and 2.1mm, 1.8um TP Columns
50 x 2.1 mm100 x 2.1 mm
1200
bar
150 x 2.1 mm250 x 2.1 mm
0.45 mL/min
H O/ACN 40 °C
1000
H2O/ACN, 40 C
600
800
400
600
ZORBAX RRHT SB-C182.1 x 50mmH2O/ACN2 4 l/ i
0
2002.4ml/minT=95 °C560bar
8
00 1 2 3 4 5 ml/min
STM = Sub-2um SP = Superficially PorousTP = Totally Porous
How to achieve highest Peak Capacity at given Gradient Time and System Pressure?
• t_g < 5min: 50 mm Column Length
By applying maximum Flow Rate (and Temperature)!
• 5min < t_g < 30min: 100 mm Column Length
• t_g > 30min: 150mm Column Length
P Petersson et al (AZ) J Sep Sci D Guillarme et al Journal of Chromatography
June 06, 2005Page 9
P. Petersson et al (AZ), J.Sep.Sci, 31, 2346-2357, 2008
D. Guillarme et al, Journal of Chromatography A, 1216 (2009) 3232–3243, 2009
Critical Comparison of TP 1.7um and SP 2.7um Particle Columns under Optimized Ultra-high Pressure Conditions. Xiaoli Wang et al, AstraZeneca, High-Speed LC Symposium, Pittcon 2009
Theory Experiment
Wed, LH5, 11:30am
Agilent 1290 Infinity LC Power Range and 2.7um SP Columns
50 x 4.6 mm100 x 4.6 mm150 x 4 6 mm
1200
g
bar
150 x 4.6 mm250 x 4.6 mm150 x 3 mm
1000
250 x 3 mm150 x 2.1
H2O/ACN, 40 °C
600
800 2.1x50, T=40C
400
6002.1x50, T=80C
0
200
00 1 2 3 4 5
11
ml/min
STM = Sub-2um SP = Superficially PorousTP = Totally Porous
Agilent 1290 Infinity LC- Resolution per Time: Superficially Porous Columnsp p y
Peak Capacity* ~ 170 in 24 sec.World Record “ *Pc = based on 2nd peak W(1/2)
„World Record.
0.095sec PW
0.140secPW
Tranquilizer Sample
Column: Superficially Porous
50 x 2.1 mm, 2.7 µm, µ
F = 3.5 ml/min, P = 700 bar
Gradient 2-92 % ACN in 0.4 min
T= 80 °CT= 80 °C
12
1290 Infinity LC and 6460 QQQ Jet Stream- Resolution per Time: Totally Porous Columns
5x10
4
6
8
+ MRM (455.29999 -> 165.09961) std-t0-1500ulmin-column3-60C-r003.d
1
Metabolic Stability Assay• Column: ZORBAX RRHD SB-C18
VerapamilT = 0.54 min
Avg W1/2 = 0.44 sec 11 points across WRel. Area RSD [%] = 4.0
p y
2
5x10
2
3
+ MRM (386.00000 -> 122.09961) std-t0-1500ulmin-column3-60C-r003.d
1
• Column: ZORBAX RRHD SB-C18 • Dimension: 2.1x50, 1.8um
• T = 60°CBuspironeAvg W1/2 = 0.70 sec 20 points across W
1
2
4x10
2
+ MRM (272.20001 -> 215.19922) std-t0-1500ulmin-column3-60C-r003.d
1
• F = 1.5 mL/min• P = 1070 bar
PW = 0 37 0 98 sec
pT = 0.22 min
pRel. Area RSD [%] = 4.2
Avg W = 0 98 sec
0.5
1
1.5
2
3x10- MRM (294.00000 -> 250.00000) std-t0-1500ulmin-column3-60C-r003.d
1
• PW = 0.37 – 0.98 sec114 msec CT, 6 Transitions, 30ms polarity switching
• 9 – 28 Data Points across Peak• Area RSD = 2.2 – 7.8%
DextromethorphanT = 0.30 min
Avg W1/2 = 0.98 sec 28 points across WRel. Area RSD [%] = 2.2
1
2
3
41
0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 1 1 1 1 2
Area RSD 2.2 7.8%
• Analysis time < 0.8 min• Run time = 1.5 min
DiclofenacT = 0.73 min
Avg W1/2 = 0.37 sec 9 points across WRel. Area RSD [%] = 7.8
Counts vs. Acquisition Time (min)0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2
Pharma Impurity Analysis: HPLC to UHPLC- Highest Speed with Highest Sensitivity & Precisiong p g y
150 x 3.0 mm, 3.5 µmMethod Successfully Validated• Metoclopramide PN 5990-3981EN
14 min
Metoclopramide, PN 5990-3981EN, . G. Vanhoenacker, F. David, P. Sandra
• LOD: 0.001 - 0.005 % w/w relative to main compound (0 5 2 5ng on column)
100 x 2 1 mm 1 7 µm
main compound (0.5-2.5ng on-column) 10 - 50x more sensitive than required
• RSD 0.05% Impurities: 0.5 - 2.7%100 x 2.1 mm, 1.7 µmPmax = 880 bar
RSD 0.005% Impurities: 1.4 - 4.5%
3x / 2x more precise than required
3 min
14
1290 Infinity LC with 1200bar ZORBAX RRHD– Resolution: Totally Porous Columns
Rs3,2: 4.92N6 : 29600P 560 b
20:80 H2O:ACN, F = 0.5 mL/min, T = ambient
150 mmmAU
200
250
300
P: 560 barα3,2: 1.15Pw2: 0.015 minPw6: 0.023 min50
100
150
200
Rs (3,2): 6.75N6 49200
150 +100 mm = 250mm1. Uracil2 A t h
min0 0.5 1 1.5 2 2.5 3 3.5
0
mAU
250
300
N6 : 49200P: 1000 barα3,2: 1.15Pw2: 0.017 min
2. Acetophenone3. Propiophenone4. Butyrophenone5. Valerophenone6. Hexanophenone
2
6
543
100
150
200
250
Pw6: 0.029 min11
min0 0.5 1 1.5 2 2.5 3 3.5
0
50
N = 50 000 in less than 3 minutes
Group/Presentation TitleAgilent Restricted
Month ##, 200XPage 15
N = 50,000 in less than 3 minutes
1290 Infinity LC + 6460 QQQ Jet Stream- Analysis of 300 Pesticides in 15 min.
600 Dynamic MRM Transitions9 replicates analyzed at 200 pg level
Column: 2.1 x 150mm, 1.7umMobile phase: A = H2O w/5 mM ammonium formate + 0.01% formic
B = MeOH w/5 mM ammonium formate + 0.01% formicLC Gradient: 8%B (T=0) to 92%B (T=15 min), hold 3 min
40 Pesticides in 1 minPW ~2 sec FWHM
p y pgMean area %RSD = 3.2 Mean height %RSD= 3.6
( ) ( ),Column Temperature: 70°CLC flow rate: 0.5 ml/min
Agilent MassHunter Workstation
February-2009Page 16
Agilent 1290 Infinity LC”Next Generation Technology”
for highest Flexibility and Performance
• New! 1290 Infinity Binary Pump
• New! 1290 Infinity Autosampler
• New! 1290 Infinity FlexCubeNew! 1290 Infinity FlexCube
• New! 1290 Infinity Column Compartment
N ! 1290 I fi it Di d A D t kt• New! 1290 Infinity Diode-Array Detektor
• New! ZORBAX RRHD Columns
17
1290 Infinity LC Binary Pump1290 Infinity LC Binary Pump1290 Infinity LC Binary Pump1290 Infinity LC Binary Pump
Page 18
1290 Infinity LC Binary Pump-The Next Generation in LC Pump Technology
Highest Speed and Resolution1200 bar @ 2.0 mL/min
1000bar @ 3.5ml/min
800 bar @ 5.0 mL/min
Lowest Delay Volume: 10 µl / 45 µly
Highest Precision and SensitivityLowest „Mixing Noise“
Lowest RT RSDLowest RT RSD
Longest Maintenance Intervalls Automatic Leak-Correction
150 Lit S l t150 Liter Solvent
Convenient OperationAutomatic „Purge“
Page 19
1290 Infinity LC Binary Pump Technology- Maximum Power and Robustness
Page 20
1290 Infinity LC Binary PumpHeat Dissipation and Robustnessp
New Piston Material: SSiC (Sintered Silicon Carbide)SSiC (Sintered Silicon Carbide)
Optimized heat dissipation enables • higher pressure x flow rates (1200bar at 2 ml/min)• longer seal lifetime / maintenance intervalls (>150L)• longer seal lifetime / maintenance intervalls (>150L)• one seal for reversed and normal phase
Multi-layer capillary for optimized heat exchange between pump heads
Page 21
heat exchange between pump heads
1290 Infinity Binary Pump- Lowest Delay Volume and Lowest „Mixing Noise“
1200 Series Channel A
Passive Damper0 - 300 µl
Passive Mixer400 µl
1200 SeriesChannel B Very Low Noise
~ 800 µl
1290 InifinityChanel A
1290 InifinityChanel B
Lowest Noise
+ =Aktive Damping10µl
Jet Weaver Mixer35µl
• min. 50% lower Delay Volume• 10µl / 45µl• 80x / 20x lower
Page 22
• 80x / 20x lower
Agilent Jet Weaver- Next Generation highest-efficient Mixingg g
Highest Mixing Efficiency at lowest Volume
• 35 µl for standard UV Applications
• 100 µl for TFA Applications
Technology:• Proprietary multi-layer, microfluidics Technology• Diffusion bonded stainless steel, edged structures
(100x120um)
23
Active Damping- The Technology and its Advantagesgy g
Active Dampingp g
• Lowest noise at lowest delay volume (10µl/45ul) for highest UV sensitivity
FirmwarePump
Intelligence
Full characterisation of solvent properties
Tuning (realtime optimization basedon static and dynamic parameters) for highest UV sensitivity
in ultra-fast runs.
• Lowest RT RSD for most precise peak ID
on static and dynamic parameters)
+
precise peak ID
• Automatic compensation of small leaks for longer maintenance intervalls
Dedicated µ-processor for realtime piston controlHighest resolution pump drives:65000 steps, 300 pL/step, 50x better
HardwareSmooth motion
control maintenance intervalls and lower RT RSDIndependent piston drives
control
Page 24
1290 Infinity LC – UFLC Retention Time Precision
RT RSD = 0.005% !„World Record.“
P i i P k 1 P k 2 P k 3 P k 3 P k 5 P k 6 P k 7 P k 8 P k 9Precision Peak 1 Peak 2 Peak 3 Peak 3 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9
% RSD RT 0.016 0.0077 0.0052 0.0058 0.0058 0.0054 0.0059 0.0057 0.0080
% RSD Area 0 40 0 39 0 43 0 47 0 40 0 46 0 44 0 52 0 56Phenone Standard, 10 Consecutive Runs0.5 µl 0.40 0.39 0.43 0.47 0.40 0.46 0.44 0.52 0.56
2.1 x 50mm RRHD SB C18, 1.8µm1.2ml/min, 0.5ul, 40C
80Hz, 245/10nm, REF 360/80nm5 - 95% ACN in 0.5min
Page 25
1290 I fi it Di d A D t kt1290 I fi it Di d A D t kt1290 Infinity Dioden-Array Detektor1290 Infinity Dioden-Array Detektor
June 06, 2005Page 26
1290 Infinity Diode-Array Detector- The Next Generation in UV-Vis Detection
GratingMirror
Programmableslit
Deuterium lamp
slit
Deuterium lamp
1024 elementdiode-array
Max-Light Cartridge CellNext Generation Flow Cell Design
Non-Coated Fused Silica Optofluidic Waveguides
Page 27
1290 Infinity Diode Array Detector- highest sensitivity under any condition
1200 RRLC (no mixer, damper)5-95% ACN, 0.5 ml/min 80C, 40 Hz, 230nm/360nm
1290 Infinity DADNext Level Sensitivity and Productivity
More automated integration at any condition
1200 DAD SL
More automated integration at any condition2 – 20x higher sens with 1 cm Cell8 – 80x higher sens with 6 cm UHS Cell
Page 28
1290 Infinity Diode Array Detector- Ultra Sensitivity by 6cm Flow Cell
mAU
DAD1 B, DAD1A, DAD: Signal A, 251 nm/Bw:10 nm Ref 360 nm/Bw:100 nm (F:\AE4-ANTHRACENE\ANTHRA-STD-CEL7.D)DAD1 B, DAD1A, DAD: Signal A, 251 nm/Bw:10 nm Ref 360 nm/Bw:100 nm (F:\AE4-ANTHRACENE\ANTHRA-HS-CEL07.D)
Anthracene, 16.4pg/µlW t /ACN 30/70
35
40
Water/ACN = 30/700.5ml/min, 1µl injection36C, 251nm, PW>0.05min
4 x Higher Sensitivity
20
25
30
High-Sensitivity Standard Cell (1cm)
Ultra High-Sensitivity Cell (6cm)S/N: 2339
10
15
High Sensitivity Standard Cell (1cm)S/N: 612
min0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8
0
5
Page 29
1290 Infinity Autosampler1290 I fi it Fl C b
1290 Infinity Autosampler1290 I fi it Fl C b1290 Infinity FlexCube1290 Infinity FlexCube
Page 30
1290 Infinity Autosampler- Next Generation Variable Loop Injector
AdvantagesLower volume (80ul)
Highest precision w/o loop change from 0.1 – 40ul
No loss of sample
Lowest Carry Over
<0.004% Chlorhexidine
New Cartridge Loop
Page 31
g p
1290 Infinity FlexCube- Extending the capabilities g p
Syringe PumpRail for
Valve option 1 Valve option 2Low pressureValve
external Valve
Applications:a) Automated needle seat back-flushing for lower carry overb) Fixed loop injection mode for fastest cycle timesc) and more ...........
Page 32
1290 Infinity Autosampler plus FlexCube- Fixed loop injection modep j
AdvantagesLow Delay volume (10 µl)y ( µ )
Overlapped injection with lowest carry over
Page 33
1290 Infinity LC System Delay Volume- Configure the sytem to your needs
Configuration Delay Vol.* / µl
1290 P 101290 Pump 10
1290 Pump + Fixed Loop1 (for MS) 20
1290 Pump + Jet Weaver + Fixed1 Loop (for UV) 551290 Pump Jet Weaver Fixed Loop (for UV) 55
1290 Pump + Variable Loop (for MS) 90
Vendor A1 90
1290 Pump + Jet Weaver + Variable Loop (for UV) 120
RRLC Pump + Variable Loop (for MS) 260
RRLC Pump + Damper/Mixer + Variable Loop (for UV) 740-940
* Delay Volume: 10 % step gradient Definition preliminary data Delay Volume: 10 % step gradient Definition, preliminary data1 5 µl Loop
Page 34
1290 Infinity1290 Infinity1290 InfinityThermostatted Column Compartment1290 InfinityThermostatted Column Compartment
Page 35
1290 Infinity Therm. Column Compartment- Next level of Versatility, Automation and PerformanceNext level of Versatility, Automation and Performance
• New generic valve drive • New quick change valve heads (2/6 2/10 9/8 )• New quick-change valve heads (2/6, 2/10, 9/8, ...)
with RFID tags (type, pressure, switches)• Two independent temperatures zones with
improved thermal insulationimproved thermal insulation• New Door Open Sensor• New Capillary Guides and Leak Funnel• Improved Low Dispersion Heat Exchangers• Improved Low Dispersion Heat Exchangers
1290 Infinity LC Solutions- Flexible Automation
Method Development and Multi-Method• Up to 3 clustered Compartments for up
to 8 Columns 26 Solvents 6 T zonesto 8 Columns, 26 Solvents, 6 T-zones• MD: ChromSword and AutoChrom• Method-Screening: Scouting Wizard • Multi-Method: ChemStation, EZAccess
Alternating Column Regeneration• 2000 Samples/day with overlapped
injections and offline data analysis
Online Sample Preparation• Enrichment• Stripping
37
Stripping
Gradient Behaviour and Method Transfer – Emulating any (U)HPLC System
Scatter Plot
100
UV Signal
80
90
100
Method Transfer
50
60
70
Same RT of all Peaks
Same Resolution all Peak Pairs
20
30
40
1290 Infinity LC1200 RRLC (damper + mixer)
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2
0
10 1290 Infinity LC (+ damper + mixer + capillary)
Delay Volume (ml)
Page 38
Agilent 1290 Infinity LC- Meeting the Development Objectives- Meeting the Development Objectives
Requirements The Debate is Overq
1. Power Range:encloses any (U)HPLC
• Methods: transferable !
• ID: 4 6 3 and 2 1 mm !2. Delay Volume:
lowest, adjustable
3. Sensitivity, Precision:
• ID: 4.6, 3 and 2.1 mm !
• Size: STM and larger !
• Particle: TP and SP !y,better than any (U)HPLC
4. Injection Principle:variable and fixed loop
Particle: TP and SP !
• Flow: low and high !
• T: low and high !p
5. Gradient Behaviour:emulate any (U)HPLC
g
• P: 600, 1000 bar and higher!
39
STM = Sub-2um SP = Superficially PorousTP = Totally Porous
Agilent 1290 Infinity LC
More Powerful
Resolution and Speed: 1200 bar (2 ml/min)800 bar (5 ml/min)20 µl Delay Volume
1200bar RRHD Columns N 50 000 in 3min
Sensitivity UV : 2 – 20x with 10 mm Cell
1200bar RRHD Columns N = 50,000 in 3minRT Precision: 0.005% RSD (1min run)
8 – 80x with 60 mm CellCondition independent
Sensitivity MS/MS: < 1 fg with Jet StreamCarry Over: < 0 004% Chlorhexidine
More Sensitive
Carry Over: < 0.004% Chlorhexidine
Supports all analytical columns Enables Method transfer from/to any (U)HPLC
MoreFlexible
Flexible Injection: flow-through and fixed loopHighest-Throughput Solution (> 2000 samples/day)Method Development and Multi-method Solutions: up to 8 columns 26 solvents 6 temperature zones
40
up to 8 columns, 26 solvents, 6 temperature zones
Agilent 1290 Infinity LCg y
June 06, 2005Page 41