Agilent 1290 Infinity LC Next Generation Technology for highest Performance and Flexibility.
HPLC 2009 Dresden, Germany Stefan Schuette Agilent Technologies
Short History of UHPLC 2003 Agilent
1200 Binary
1.8um RRHT Columns
2004 Waters
Acquity
1.7um BEH Columns
2006 Agilent
1200 RRLC
Thermo 2007 Shimadzu
Accela UFLCxr
Jasco
Xtreme-LC
Hitachi
LaChrom Ultra
Scient. Syst. y Ultra HP 2008 Dionex Knauer 2009 PE
RSLC Platin Blue Fl Flexar
10 New UHPLC Systems in 5 Years – All different.
UHPLC Situation 2009 Power Ranges g Power = Pressure x Flow
bar 1200
Pressure Ù Resolution Flow Ù Speed
1000
Waters Acquity 800
Dionex RSLC 600
Agilent RRLC
Thermo Accela 400
Shimadzu UFLCXR 200
Standard LC
0 0
3
1
2
3
4
5
ml/min
UHPLC Situation 2009 10 Systems in 5 Years – All Different! y
Differences
Problems
Debate
• System y Volumes
• Method Transfer • Not possible • Requires re-devel. and -validation • Performance P f Limitations Li it ti • Speed • Resolution • Sensitivity • Precision • Linearity
• Methods: transferable?
• Pressures • Flow Rates • Temperatures T t • Data Rates • Sensitivity • Injection principle • ...
4
• ID: 4.6, 3 or 2.1 mm? • Particle: TP or SP? • Size: STM or larger? • Flow: low or high? • T: low or high? • P: 600 or 1000 bar?
STM = Sub-2um SP = Superficially Porous TP = Totally Porous
Agilent 1290 Infinity LC - Development Objectives System Requirements 1. 2.
Delay Volume: lowest, adjustable
3.
Sensitivity, Precision: better than any (U)HPLC
4.
Injection Principle: variable and fixed loop
5.
5
Power Range: encloses any (U)HPLC
Gradient Behaviour: emulate any (U)HPLC
The Debate is Over • Methods: transferable ! • ID: 4.6, 4 6 3 and 2.1 2 1 mm ! • Size: STM and larger ! a c e TP a and dS SP ! • Particle: • Flow: low and high ! • T: low and high ! • P: 600 and 1000 bar !
STM = Sub-2um SP = Superficially Porous TP = Totally Porous
Agilent 1290 Infinity LC
¾ All analytical columns
Power Range
¾ Method Transfer ¾ Higher Resolution
bar
¾ Faster Analysis
1200
¾ Higher (MS) Sensitivity Agilent 1290 Infinity LC
¾ More viscous solvents ¾ Lower temperatures
1000
Waters Acquity 800
Dionex RSLC 600
Agilent RRLC
Thermo Accela 400
Shimadzu UFLCXR 200
Standard LC
0 0
6
1
2
3
4
5
ml/min
STM = Sub-2um SP = Superficially Porous TP = Totally Porous
Agilent 1290 Infinity LC Power Range g and 4.6mm,, 1.8um TP Columns
50 x 4.6 mm 100 x 4.6 mm
bar
150 x 4.6 mm
1200
250 x 4.6 mm H2O/ACN, 40 °C
1000
800
600
400
200
0 0
7
1
2
3
4
5
ml/min
STM = Sub-2um SP = Superficially Porous TP = Totally Porous
Agilent 1290 Infinity LC
50 x 2.1 mm
Power Range and 2.1mm, 1.8um TP Columns
100 x 2.1 mm 150 x 2.1 mm
0.45 mL/min
250 x 2.1 mm
bar 1200
H2O/ACN, O/ACN 40 °C C
1000
800
600
ZORBAX RRHT SB-C18 2.1 x 50mm H2O/ACN 2 4 l/ i 2.4ml/min T=95 °C 560bar
400
200
0 0
8
1
2
3
4
5
ml/min
STM = Sub-2um SP = Superficially Porous TP = Totally Porous
How to achieve highest Peak Capacity at given Gradient Time and System Pressure? By applying maximum Flow Rate (and Temperature)! • t_g < 5min:
50 mm Column Length
• 5min < t_g < 30min: 100 mm Column Length • t_g > 30min:
P Petersson et al (AZ), P. (AZ) J.Sep.Sci, J Sep Sci 31, 2346-2357, 2008 Page 9
150mm Column Length
D Guillarme et al D. al, Journal of Chromatography A, 1216 (2009) 3232–3243, 2009 June 06, 2005
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
50 x 4.6 mm
Agilent 1290 Infinity LC
100 x 4.6 mm
Power Range g and 2.7um SP Columns
150 x 4 4.6 6 mm 250 x 4.6 mm
bar
150 x 3 mm
1200
250 x 3 mm 150 x 2.1
1000
H2O/ACN, 40 °C 800
2.1x50, T=40C
600
2.1x50, T=80C
400
200
0 0
11
1
2
3
4
5
ml/min
STM = Sub-2um SP = Superficially Porous TP = Totally Porous
Agilent 1290 Infinity LC - Resolution p per Time: Superficially p y Porous Columns Peak Capacity* ~ 170 in 24 sec. „World World Record Record.“
*Pc = based on 2nd peak W(1/2)
Tranquilizer Sample
0.095sec PW
Column: Superficially Porous 0.140secPW
µ 50 x 2.1 mm,, 2.7 µm F = 3.5 ml/min, P = 700 bar Gradient 2-92 % ACN in 0.4 min T= 80 °C
12
1290 Infinity LC and 6460 QQQ Jet Stream - Resolution p per Time: Totally y Porous Columns + MRM (455.29999 -> 165.09961) std-t0-1500ulmin-column3-60C-r003.d x10 5 8 1 6 4
Avg W1/2 = 0.44 sec 11 points across W Rel. Area RSD [%] = 4.0
Verapamil T = 0.54 min
2 + MRM (386.00000 -> 122.09961) std-t0-1500ulmin-column3-60C-r003.d x10 5 1 3
Avg W1/2 = 0.70 sec points across W 20 p Rel. Area RSD [%] = 4.2
2 1
Buspirone p T = 0.22 min
+ MRM (272.20001 -> 215.19922) std-t0-1500ulmin-column3-60C-r003.d x10 4 1 2 Avg W = 0.98 0 98 sec 1/2
28 points across W Rel. Area RSD [%] = 2.2
1.5 1
Dextromethorphan T = 0.30 min
0.5
2 1
Avg W1/2 = 0.37 sec 9 points across W Rel. Area RSD [%] = 7.8
01 0.1
02 0.2
03 0.3
Diclofenac T = 0.73 min
04 0.4 0 5 0.6 0.5 0 6 0.7 0 7 0.8 0 8 0.9 09 Counts vs. Acquisition Time (min)
1
11 1.1
• Column: ZORBAX RRHD SB-C18 • Dimension: 2.1x50, 1.8um • T = 60°C • F = 1.5 mL/min • P = 1070 bar • PW = 0.37 0 37 – 0.98 0 98 sec 114 msec CT, 6 Transitions, 30ms polarity switching
• 9 – 28 Data Points across Peak • Area RSD = 2.2 – 7.8%
- MRM (294.00000 -> 250.00000) std-t0-1500ulmin-column3-60C-r003.d x10 3 1 4 3
Metabolic Stability Assay
12 1.2
• Analysis time < 0.8 min • Run time = 1.5 min
Pharma Impurity Analysis: HPLC to UHPLC - Highest g Speed p with Highest g Sensitivity y & Precision Method Successfully Validated • Metoclopramide Metoclopramide, PN 5990-3981EN, 5990-3981EN G. Vanhoenacker, F. David, P. Sandra • LOD: 0.001 - 0.005 % w/w relative to main compound (0 (0.5-2.5ng 5 2 5ng on on-column) column) 10 - 50x more sensitive than required • RSD 0.05% Impurities: 0.5 - 2.7% RSD 0.005% Impurities: 1.4 - 4.5% 3x / 2x more precise than required
150 x 3.0 mm, 3.5 µm . 14 min
100 x 2 2.1 1 mm mm, 1 1.7 7 µm Pmax = 880 bar 3 min
14
1290 Infinity LC with 1200bar ZORBAX RRHD – Resolution: Totally Porous Columns 20:80 H2O:ACN, F = 0.5 mL/min, T = ambient
Rs3,2: 4.92 N6 : 29600 P 560 b P: bar
mAU 300
150 mm
250
200
α3,2: 1.15 Pw2: 0.015 min Pw6: 0.023 min
150
100
50
0
0
mAU 300
0.5
1. 2 2. 3. 4. 5. 6.
250
200
150
100
1
2
0
0.5
2.5
3
3
4 5
1 1
1.5
2
2.5
3.5
min
Rs (3,2): 6.75 N6 : 49200 P: 1000 bar α3,2: 1.15 Pw2: 0.017 min Pw6: 0.029 min
6 1
0
2
150 +100 mm = 250mm
Uracil A t h Acetophenone Propiophenone Butyrophenone Valerophenone Hexanophenone
50
1.5
3
3.5
min
N = 50 50,000 000 in less than 3 minutes Page 15
Group/Presentation Title Agilent Restricted Month ##, 200X
1290 Infinity LC + 6460 QQQ Jet Stream - Analysis of 300 Pesticides in 15 min. 600 Dynamic MRM Transitions 9 replicates p analyzed y at 200 pg level Mean area %RSD = 3.2 Mean height %RSD= 3.6
40 Pesticides in 1 min Column: 2.1 x 150mm, 1.7um PW ~2 sec FWHM
Mobile phase: A = H2O w/5 mM ammonium formate + 0.01% formic B = MeOH w/5 mM ammonium formate + 0.01% formic LC Gradient: 8%B ((T=0)) to 92%B ((T=15 min), ), hold 3 min Column Temperature: 70°C LC flow rate: 0.5 ml/min
Agilent MassHunter Workstation Page 16
February-2009
Agilent 1290 Infinity LC ”Next Generation Technology” for highest Flexibility and Performance
• New! 1290 Infinity Binary Pump • New! 1290 Infinity Autosampler • New! 1290 Infinity FlexCube • New! 1290 Infinity Column Compartment • New! N ! 1290 IInfinity fi it Di Diode-Array d A D Detektor t kt • New! ZORBAX RRHD Columns
17
1290 Infinity LC Binary Pump
Page 18
1290 Infinity LC Binary Pump -The Next Generation in LC Pump Technology ¾ Highest Speed and Resolution 1200 bar @ 2.0 mL/min 1000bar @ 3.5ml/min 800 bar @ 5.0 mL/min Lowest Delayy Volume: 10 µl / 45 µl
¾ Highest Precision and Sensitivity Lowest „Mixing Noise“ Lowest RT RSD
¾ Longest Maintenance Intervalls Automatic Leak-Correction 150 Lit Liter S Solvent l t
¾ Convenient Operation Automatic „Purge“
Page 19
1290 Infinity LC Binary Pump Technology - Maximum Power and Robustness
Page 20
1290 Infinity LC Binary Pump Heat Dissipation p and Robustness New Piston Material: SSiC (Sintered Silicon Carbide) Optimized heat dissipation enables • higher pressure x flow rates (1200bar at 2 ml/min) • longer seal lifetime / maintenance intervalls (>150L) • one seal for reversed and normal phase
Multi-layer capillary for optimized heat exchange between pump heads
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1290 Infinity Binary Pump - Lowest Delay Volume and Lowest „Mixing Noise“ 1200 Series Channel A 1200 Series
Passive Damper 0 - 300 µl
Channel B
Passive Mixer 400 µl
Very Low Noise ~ 800 µl
1290 Inifinity Chanel A 1290 Inifinity Chanel B
Lowest Noise • min. 50% lower
Aktive Damping 10µl
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+
Jet Weaver Mixer 35µl
Delay Volume =
• 10µl / 45µl • 80x / 20x lower
Agilent Jet Weaver - Next Generation highest-efficient g Mixing 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 gy and its Advantages g
Firmware
Pump Intelligence
Full characterisation of solvent properties Tuning (realtime optimization based on static and dynamic parameters)
+
Hardware
Smooth motion control
Page 24
Dedicated µ-processor for realtime piston control Highest resolution pump drives: 65000 steps, 300 pL/step, 50x better Independent piston drives
Active Damping p g • Lowest noise at lowest delay volume (10µl/45ul) for highest UV sensitivity in ultra-fast runs. • Lowest RT RSD for most precise peak ID • Automatic compensation of small leaks for longer maintenance intervalls and lower RT RSD
1290 Infinity LC – UFLC Retention Time Precision RT RSD = 0.005% ! „World Record.“
P Precision i i P k 1 Peak Peak P k2
P k3 Peak
P k3 Peak
P k5 Peak
P k6 Peak
P k7 Peak
P k8 Peak
P k9 Peak
% RSD RT
0.0077
0.0052
0.0058
0.0058
0.0054
0.0059
0.0057
0.0080
0 39 0.39
0 43 0.43
0 47 0.47
0.016
% RSD Area 0 40 0.40 0.5 µl
Page 25
Runs 0 40 Phenone 0.40 0 46 Standard, 0.46 0 44 10 Consecutive 0.44 0 52 0.52 0 0.56 56
2.1 x 50mm RRHD SB C18, 1.8µm 1.2ml/min, 0.5ul, 40C 80Hz, 245/10nm, REF 360/80nm 5 - 95% ACN in 0.5min
1290 Infinity I fi it Dioden-Array Di d A D Detektor t kt
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June 06, 2005
1290 Infinity Diode-Array Detector - The Next Generation in UV-Vis Detection Grating
Mirror
Programmable slit
Deuterium lamp 1024 element diode-array
Next Generation Flow Cell Design Max-Light Cartridge Cell
Non-Coated Fused Silica Optofluidic Waveguides
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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 DAD
Next Level Sensitivity and Productivity ¾ More automated integration at any condition 1200 DAD SL
Page 28
¾ 2 – 20x higher sens with 1 cm Cell ¾ 8 – 80x higher sens with 6 cm UHS Cell
1290 Infinity Diode Array Detector - Ultra Sensitivity by 6cm Flow Cell 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) mAU
40
Anthracene, 16.4pg/µl W t /ACN = 30/70 Water/ACN 0.5ml/min, 1µl injection 36C, 251nm, PW>0.05min
4 x Higher Sensitivity
35
Ultra High-Sensitivity Cell (6cm) S/N: 2339
30
25
20
High-Sensitivity High Sensitivity Standard Cell (1cm) S/N: 612
15
10
5
0 0
Page 29
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
min
1290 Infinity Autosampler 1290 Infinity I fi it FlexCube Fl C b
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1290 Infinity Autosampler - Next Generation Variable Loop Injector
Advantages ¾ Lower volume (80ul) ¾ Highest precision w/o loop change from 0.1 – 40ul ¾ No loss of sample ¾ Lowest Carry Over 2000 samples/day) ¾ Method Development and Multi-method Solutions: up to 8 columns columns, 26 solvents solvents, 6 temperature zones
40
Agilent g 1290 Infinity y LC
Page 41
June 06, 2005
