NEW 1200 Infinity Series Raising the Standard in HPLC

NEW 12 Infinity Series Raising the Standard in HPLC Scaling LC performance while keeping method transferability a contradiction? UHPLC for Every Lab! 122 Infinity LC 126 Infinity LC 129 Infinity LC Stefan Schuette June 18, 21 Page 1

Agenda Agilent introduces a new HPLC Standard Scaling Performance while maintaining method transferability 2 Confidential April 21

Agenda Agilent introduces a new HPLC Standard Scaling Performance while maintaining method transferability 3 Confidential April 21

The NEW Agilent 12 Infinity Series Enhanced 129 Infinity LC 129 Infinity LC NEW 12 Series 126 Infinity LC NEW 112 Compact LC 122 Infinity LC Page 4 Page 4

The NEW Agilent 12 Infinity Series Prep- Scale LC new Low- Flow LC new enhanced new new new new new new Page 5

NEW Agilent 12 Infinity Series Raising the Standard in HPLC Highest Value for any Budget 122 Infinity LC Integrated 6 bar 8 Hz Most affordable HPLC and UHPLC 126/129 Technology 126 Infinity LC Modular 6 bar 8 Hz 2x or 1x UV Sensitivity 1% HPLC Compatible HPLC Price UHPLC Capability 129 Infinity LC Modular 12 bar 16 Hz Highest Performance Highest Flexibility HPLC Service Costs Page 6

The new Agilent 12 Infinity Series Pressure [bar] 12 1 8 6 129 Infinity LC All analytical columns Method Transfer Higher Resolution Faster Analysis More viscous solvents Lower temperatures Semi-prep 4 2 126 Infinity Binary LC 126 Infinity Quaternary/Iso LC 122 Infinity LC 2 4 6 8 1 Flow [ml/min] Page 7

Instrument Column Compatibility Column Type Particle UHPLC < 2um (STM) UHPLC Superficial Porous HPLC 3 5um Column Length / mm Short: 3 5 Long: 1 15 3-15 5-3 CoIumn I.D. / mm 2.1 3. 4.6 2.1 3. 4.6 2.1 3. - 4.6 3. - 4.6 Max Pressure / bar 12 6 6 12 6 6 6 6 4 Agilent Column RRHD RRHT RRHT RRHD RRHT RRHT Poroshell Poroshell Various 129B 12bar 126B 6bar 2) 126Q/122G 6bar 1) 2) 1) 11/12 4bar 2) 2) 2) 2) 3) 3) best good accept. compr. Any column of any dimension and particle can be used on 122/126/129 Infinity LC s 12 Infinity Series matches UHPLC columns pressure rating: 129 Infinity LC: 12bar + lowest delay volume (DV) for optimum support of 2.1-4.6mm ID STM (RRHD) 126/122 Infinity LC: 6bar + std DV for optimum support of 3-4.6mm ID STM (RRHT) & SP (Poroshell) STM-short: sub-2µm particles, 3 5mm length STM-long: sub-2µm particles, 1 15mm length HPLC: 3-5 µm particles 1) Because of delay volume. 2) Because of delay volume and pressure. 3) Because of pressure.

NEW: 122 Infinity LC Isocratic and Gradient RRLC at most affordable Price Most affordable access to 126 Infinity Quality & Performance Configurations: 122 Infinity manual isocratic system (upgradeable!) 122 Infinity manual binary gradient system (upgradeable!) 122 Infinity automated binary gradient system Productivity: 1x higher than conventional HPLC systems 6 bar up to 5 ml/min (1 ml/min at 2 bar) 8 Hz detector speed Technology and Quality: 126 and 129 Infinity inside! Degassing: Same Technology/Parts as 129 Bin/126 Quat degasser Pump: Same Technology/Parts as 126 Quat Pump (2-channel) Autosampler: Same Technology/Parts as 126 ALS (no cooling) VWD: Same Technology/Parts as 126/129 VWD Price: markedly below 126 Infinity Isocratic / Quaternary Systems Page 9

NEW: 126 Quaternary LC System Enhanced Quaternary UHPLC at Quaternary HPLC Price * ) > 6, systems sold! Evolution of best-selling 12/11 Series Quat LC* Productivity: 1x higher than 12/11 Quaternary HPLC 6 bar up to 5 ml/min (1 ml/min at 2 bar) 8 Hz Detector Speed Sensitivity: Up to 1x higher than 12/11 Quaternary HPLC 1x by new 126 Infinity DAD with 6 cm flow cell (±.6µAU) 2x by new 126 Infinity VWD and 126 Infinity DAD with 1cm flow cell Technology: Leverage of 129 Infinity LC innovations Pump: New, build-in degassing unit and next generation inlet valves Detectors: Next generation VWD and DAD-technology Price: Similar as 12 Series Quat (4 bar, 2 Hz, ±6 µau) Page 1

NEW: 126 Binary LC System Enhanced Binary RRLC Capability at HPLC Price Successor of 12 Series RRLC (12 SL) Productivity: Same as the highly successful 12 Series RRLC for 2.1 mmid to 4.6 mmid columns, by scalable delay-volume Sensitivity: 1x higher than 12 Series RRLC by new 126 Infinity DAD Autosampler-Performance: <.4 % carry-over and 1 µl injection by new 126 Infinity High Performance Autosampler. 4 µl kit for delay volume reduction. Technology: Leverage of 129 Infinity LC innovations Pump: New next generation inlet valves for better performance Detectors: Next generation 129 Infinity DAD and VWD technology HiP Autosampler: Same technology and parts as 129 Autosampler Price: Markedly lower than 12 Series RRLC Page 11

129 Infinity LC More Affordable Access to the World s Best LC NEW: UHPLC Productivity with HPLC Service Costs New services pricing similar to 12 Series, attractive contracts Based on proven robustness 2x higher than 11 at introduction Increased parts lifetime longer maintenance intervalls new design-for-supportability technology for faster maintenance, diagnostics and repair NEW: More Flexibility, Performance and Affordability New Multi-method/Walk-up and Method Development Solution New high-capacity solution for highest-throughput applications New lowest-carry over solution (<.1%, <1ppm with 129 FlexCube) More affordable entry-level configurations using same technology 12 Page 12

NEW: 126 Infinity Diode Array Detector 2x or 1x higher Sensitivity at similar Price NEW: Successor of 12 DAD and DAD SL Sensitivity: Same as 129 Infinity DAD 1-cm flow cell: 2x higher than 12 DAD/12 DAD SL 6-cm flow cell: 1x higher than 12 DAD/12 DAD SL Technology: Same as 129 Infinity DAD 8Hz (instead of 16 Hz on 129 DAD) 4nm fixed slit (instead of variable slit on 129 DAD) Price: Markedly lower than 129 DAD and 12 DAD SL Similar to 12 DAD 13 Page 13

History of DAD Sensitivity Gain - The last 3 years Sensitivity Gain 126 / 129 Infinity Diode Array Detector (1986) (1988) (1995) (26) (21) Detector Model (Intro Year) Page 14

Optofluidic Waveguides: Max-Light Flow Cells Total-internal reflection in a non-coated fused silica fiber 126 / 129 Infinity DAD Mirror Grating Programmable or fixed slit Deuterium Lamp Max-Light Cartridge Cell 124 element diode-array Page 15

11.4 x Higher Sensitivity 129 Infinity DAD compare to 12 Series DAD SL 3 25 2 mau 126/129 Infinity DAD Columns: 15 x 4.6mm Zorbax SB C18, 5µm Sample: Anthracene: 835 pg/µl Mobile phase: A: Water, B: Acetonitrile Elution: isocratic 8 % B Injection volume: 5 µl Flow: 1.5 ml/min DAD: 251/4nm, Ref= 45/8nm, 2.5Hz, slit width 4nm 15 126/129 DAD 6 mm 12 DAD SL 1mm 1 5 12 Series DAD / DAD SL Height [mau] 28.876 4.938 Noise [mau].98.19 Signal/ noise 2944 259 Increase 11.4 x 1 2 3 4 5 min 16 Confidentiality Label July 8, 21

11.4 x Higher Sensitivity 129 Infinity DAD compared to 12 Series VWD A/B 3 25 2 mau 126/129 Infinity DAD Columns: 15 x 4.6mm Zorbax SB C18, 5µm Sample: Anthracene: 835 pg/µl Mobile phase: A: Water, B: Acetonitrile Elution: isocratic 8 % B Injection volume: 5 µl Flow: 1.5 ml/min DAD: 251/4nm, Ref= 45/8nm, 2.5Hz, slit width 4nm VWD: 251 nm, 2.5 Hz 15 126/9 DAD 6 mm 12 VWD 1mm 1 5 12 Series VWD A/B Height [mau] 28.876 4.8 Noise [mau].98.191 Signal/Noise 2944 253 Increase 11.6 x 1 2 3 4 min 17

NEW 12 Infinity Series More workflow-automation and application-based solutions New New Analytical SFC Bio-inert HPLC Nano LC & Cap LC Preparative LC & LC/MS GPC SEC Isocratic LC New New Multi-method and Method Development High-throughput LC & LC/MS Nanoflow HPLC-Chip MS 18 Page 18

NEW 126 Infinity Bio-inert HPLC Solution Infinitely Better for Bio-molecule Analysis New 1% Bio-inert Metal-free sample flow path (instrument and column) Stainless steel free solvent flow path Widest ph range (1-13, short-term 14) 1x sensitivity and 6bar power range Lowest LOD and highest resolution per time From lowest pressure up to highest pressure STM columns Unique resolution for protein applications Bio-analysis and Bio-purification Analytical flow and semi-prep fraction coll. up to 1 ml/min Most complete offering for NBE Characterization Therapeutic protein analysis using SEC, IEX, peptide mapping Page 19

NEW 126 Infinity Analytical SFC System Infinitely Better for Supercritical Fluid Chromatography New Highest Analytical SFC Performance HPLC-like Sensitivity (1x over existing instruments) Precision and Dynamic Range >1, for accurate quantitation of.1 % level impurities UHPLC-like speed on 1.8µm RRHT/RRHD columns Lowest Operating Cost, Green Chemistry 1 15x lower operating costs by compatibility with standard grade CO 2 instead of liquid SFC grade CO 2 Lowest solvent consumption and waste generation Makes routine analytical SFC a reality! Ease of use and Reliability Highest investment protection by modular, reversible design ChemStation control, data analysis and reporting Agilent warranty and service quality Single Vendor Solution Single Vendor Support Page 2

NEW: 12 Infinity Series Agilent Value Promise Infinitely Better Investment Protection Full module compatibility Mix 122, 126, 129 Infinity LC with 11 and 12 Series and future systems! Stepwise upgrade now and in future: Exchange Detector only for higher sensitivity and detection speed. Exchange Pump and Autosampler only for ultra-high resolution LC on latest 1.8 µm rapid resolution column technology Exchange Column Compartment only to upgrade to a fully automated multi-method or method development solution supporting up to 8 columns and 24 solvents. Page 21

NEW: Instrument Control Framework (ICF) The Infinitely Better 3 rd Party Connectivity Solution NEW Adapter NEW ICF RC.NET LC Driver RC.NET LC Driver RC.NET LC Driver 3rp Party Supplier Agilent: 11/ 112/ 12/ 122/ 126/ 129 Advantages for Customers Immediate Control Plug and Play (like windows printer driver) Full and Robust Control all Features Supported by Agilent Drivers Future Proof Control Backwards compatibility of Funtional Extension Advantages for Application Software Suppliers Cost Savings Develop one Adapter instead of 5 Drivers, No more instrument purchase Customer Satisfaction Immediate control, No Feature Missing Risk Reduction Use tested Agilent Driver Page 22

Agenda Agilent introduces a new HPLC Standard Scaling performance while maintaining method transferability 23 Confidential April 21

Scaling Performance while maintaining Method Transferability Why Important? Performance Sample Throughput, Productivity Sample Knowledge, Data Quality, Security Method Transfer Run established, validated methods unchanged and get same results Match System of Client or Customer From R+D to QA From QA to R+D Confidential 24 April 21

How to achieve highest Peak Capacity at given Gradient Time and System Pressure? By applying maximum Flow Rate (and Temperature)! t_g < 5min: 5 mm Column Length 5min < t_g < 3min: 1 mm Column Length t_g > 3min: 15mm Column Length P. Petersson et al (AZ), J.Sep.Sci, 31, 2346-2357, 28 D. Guillarme et al, Journal of Chromatography A, 1216 (29) 3232 3243, 29 Page 25 June 6, 25

12 Infinity Series Power Range and 3-4.6mm, 1.8um TP columns bar 5 x 4.6 mm 5 x 3. mm 1 x 4.6 mm 15 x 4.6 mm 25 x 4.6 mm H 2 O/ACN, 4 C ml/min 26 STM = Sub-2um SP = Superficially Porous TP = Totally Porous

Scaling Performance: 126 Quat / 122 Grad UHPLC Performance for EVERY LAB! 1x productivity increase and improved data quality! Conventional 4.6x15, 5µm Resolution peak 5 = 4.2 Run time 11 min 2 4 6 8 1 min Resolution optimized 3.x1mm, 1.8µm Resolution peak 5 = 7.16 Run time 7 min.9ml/min, 46bar 2 4 6 8 1 min Speed and resolution optimized 3.x5mm, 1.8µm Resolution (peak 5) = 4.79 Run time 1.1min 2.4ml/min, 54bar 2 4 6 8 1 min Page 27

Scaling Performance: 126 / 122 Infinity UHPLC Performance for EVERY LAB! Customer Example Isocr. Impurity Method 4 Impurities 7 Impurities 7 Impurities Zoom of critical time range @ 7min 2 Not Baseline Separated! 6 Not Baseline Separated! All 7 Baseline Separated! 4.6 x 15, 5um 93 bar N = 7259 R_S = 1.15 S/N = 42 4.6 x 15, 3.5um 165 bar N = 14862 R_S = 1.37 S/N = 5 4.6 x 15, 1.8um 49 bar N = 28669 R_S = 1.8 (+ 57%) S/N = 44

Different Aspects of Method Transfer From Instrument to Instrument -> with method/instrument change: Isocratic Hold / Pre-injection, Vol. Modification -> available tools w/o Method or Instrument change : none so far From column dimension to column dimension (e.g. 4.6 -> 2.1, 5 mm -> 1 mm) -> recalculate flow rates, recalculate gradient times, adjust connection capillaries and flow cells, due to delay volume results may vary, method might need revalidation -> available tools: Agilent Method translator From particle size to particle size (HPLC -> UHPLC) -> often associated with a change of instruments due to constraints in max. pressure, extra-column band broadening etc.. Some fine-tuning of method usually required due to frictional heating effects -> available tools : Agilent Method translator, Third Party Method development SW (ACD Labs, ChromSword etc.) From one eluent type or phase chemistry to an other -> see Method development -> available tools : Method development SW (Chromsword, ACD Labs, etc.)

Method Transferability how is this possible? Run Method and Get Same Result? 122 Infinity 12 Series? 126 Infinity 11 Series 129 Infinity Page 3

Method Transferability: 126 Quat / 122 Grad 1% HPLC Methods Compatibility Riskless Replacement Run existing conventional HPLC methods and get same result! Column: 4.6 x 15 mm, 5 µm Gradient: min 2 % B 1min 95 % Flow rate: 1 ml/min 126 / 122 Infinity LC Quat / Gradient system 11 / 12 Series Quaternary system 2 4 6 8 1 min Page 31

Seamless Method Transfer 126/122 Infinity By unchanged critical specification 11/12 Series HPLC 126 Infinity Quaternary LC 12 Series RRLC Std. 126 Infinity Binary LC Max Flow Rate 5 ml/min 5 ml/min 5mL/min 5 ml/min Delay Volume 6 9 µl 6 8 µl 6 8 µl* 6 8 µl* Capillary ID.17mm.17mm.17mm.17mm Disp. Vol. w/o cell 15µl 15µl 15µl 15µl Injection Principle Variable Loop Variable Loop Variable Loop Variable Loop Inj.Volume Std/Ext. 1 / 15 µl 1 / 15 µl 1 / 15 µl 1 / 15 µl Area RSD <.25 % <.25 % <.25 % <.25 % Oven Design A A A A Column Length 3 mm 3 mm 3 mm 3 mm Optimized for 3 4.6mm ID Columns * Smaller delay volumes possible Page 32

Agilent 129 Infinity LC Power Range and 2.1mm, 1.8um TP Columns bar.45 ml/min 5 x 2.1 mm 1 x 2.1 mm 15 x 2.1 mm 25 x 2.1 mm H 2 O/ACN, 4 C ZORBAX RRHT SB-C18 2.1 x 5mm H 2 O/ACN 2.4ml/min T=95 C 56bar ml/min 33 STM = Sub-2um SP = Superficially Porous TP = Totally Porous

Seamless Method Transfer What if critical specification change...? * Optimized for 2.1-4.6mm ID ** Optimized for 2.1mm ID 11/12 Series HPLC 126 Infinity Quaternary LC 12 Series RRLC Std. 126 Infinity Binary LC 126 Infinity Binary LC 129 Infinity Binary LC Max Flow Rate 5 ml/min 5 ml/min 5mL/min 5 ml/min 5 ml/min 5 ml/min Delay Volume 6 9 µl 6 8 µl 6 8 µl* 6 8 µl* 12 µl** 1 11 µl* Capillary ID.17mm.17mm.17mm.17mm.12mm**.12mm* Disp. Vol. w/o cell 15µl 15µl 15µl 15µl 7.5µL** 7.5µL* Injection Principle Variable Loop Variable Loop Variable Loop Variable Loop Variable Loop Variable Loop Inj.Vol. Std/Ext. 1 / 15 µl 1 / 15 µl 1 / 15 µl 1 / 15 µl 1 / 15 µl 1 µl Area RSD <.25 % <.25 % <.25 % <.25 % <.25 % <.25 % Oven Design A A A A A A Column Length 3 mm 3 mm 3 mm 3 mm 3 mm 3 mm * Smaller delay volumes possible Page 34

Definition of Delay / Dwell / Transition Volume Scatter Plot 9 8 Transition Volume 9% of step height 7 6 5 Dwell volume @ 5 % of step 4 3 2 1 Delay Volume @ 1% of step 1% of step height.2.4.6.8 1 1.2 1.4 1.6 1.8 Delay volume (ml)

Consequencs of different Delay Volumes 12 RRLC versus 129 Infinity System Line Chart 97 7 86 61 7 62 % organic modifier 74 53 62 44 %organ ic modifie r 5 36 38 27 27 19 Programmed gradient 129 Infinity System 12 RRLC / SL System 53 45 36 28 19 15 1 3 2.5 1 1.5 2 2.5 3 3.5 4 4.5 Volume [ml] 1 2 36

Method Transferability: 129 Infinity LC Consequences of a lowest delay volume system %B Lowest delay volume, accuray and precision of 129 Infinity pump allows to match any other gradient behavior! time Page 37

Method Transferability: 129 Infinity LC Today s Bridging Approaches 1. Add physical Volume (plumbing solution) 129 + 1ul dwell vol. 11/12 Quat Pump 2. Programmed Isocratic Hold * 12 RRLC 129 + 9ul hold Page 38 Page 38

Programmed Isocratic Hold Limitations Original gradient response traces Gradient shifted by isocratic hold 1 9 8 7 6 5 4 3 2 1 2 4 6 8 1 12 14 129 Infinity 12 SL 1 9 8 7 6 5 4 3 2 1 2 4 6 8 1 12 14 129 Infinity 12 SL 2 18 16 14 12 1 8 6 4 2 3 4 5 129 Infinity 12 SL 1 98 96 94 92 9 88 86 84 82 8 11 11.5 12 12.5 13 129 Infinity 12 SL

Method Transfer Low to Large DV System Elements to consider Gradients are delayed AND bent by transition volume characterization 2. transition offset 3. delay Composition accuracy: - e.g. quaternary vs. binary pump concept - systematic offset 1.

How to overcome Limitations? TT-a Delay/Transition a F a How to turn Delay/Transition b into Delay/Transitition a? TT-a Delay/Transition b F b

Execution Concept TT-a input Method temporary TT-b H a-b F a = = F b F 12 F129 output F b No Change on Method / TT or Instrument!

Method Transferability: 129 Infinity LC System Emulation Technology (planned for 211)* Concept H a-b Emulated System A Select System to be emulated by a simple mouse click: Agilent 11 Series Agilent 12 Series RRLC Agilent 126 Infinity LC Agilent 122 Infinity LC Non-Agilent HPLC / UHPLC * Existing 129 Infinity will be upgradeable Page 43

NEW Agilent 12 Infinity Series Summary http://www.agilent.com/chem/infinity NEW 122 and 126 Infinity LC New HPLC Standard 1x Productivity and 1x Sensitivity 1% HPLC Compatible Riskless Replacement HPLC Price UHPLC Performance Enhanced 129 Infinity LC Highest Performance and Flexibility by any measure New: Highest UHPLC productivity HPLC Service Costs New: highest-productivity solutions and more affordable entry-level configurations NEW 12 Infinity Series Continuum of UHPLC Solutions starting at < 2k$ Scalable and Compatible across entire 12 Infinity Series and 11/12 Series Common technology and quality for highest robustness and reliability Method transferability and compatibilty within entire series and 11/12 Series New workflow automation and application-based solutions incl. Bio-inert HPLC and SFC New ICF for seamless integration into 3rd party software 44 Page 44

Agilent s Analytical LC Portfolio Compatible and Scaleable across entire Series Full module and methods compatibility Run 11/12 Methods on 126/129 Infinity LC Highest investment protection Upgrade 11/12 with 126/129 Modules Page 45 Page 45

Introduction Modern UHPLC instruments are optimized for fast separations in small diameter columns. Delay volumes have been reduced by a factor of 5-1 compared to conventional HPLC instrumentation. Due to differences in pump and mixer design differences in gradient formation exist between different types of HPLC instruments Approaches have been evaluated to overcome these problems and a novel solution is proposed to provide method compatibility between HPLC and UHPLC is proposed. 46

Comparison of Gradient Runs on Different Instruments mau 8 6 4 2.256 129 Infinity 1.629 1.98 2.46 2.84 2.887 3.16 3.352 4.71 1 2 3 4 5 min mau 8 6 4 2.34 12 SL Binary Pump 2.524 3.18 3.616 4.19 4.14 4.381 4.549 5.327 1 2 3 4 5 min % 8 6 4 2 Gradient: 5%B to 95%B, TG: 5 min 1 2 3 4 5 min Column: Zorbax SB-C18, 2,1x5 mm, 1,8µ Mobile phase: A = water / B = acetonotrile Sig: 254,1nm; Ref: 36,1nm Temperatue: 4 C Inj.vol.: 1µl Flow rate:,5 ml/min Sample: Sigma

Current Common Solutions add physical Volume (plumbing solution) Programmed isocratic hold Page 48

Plumbing solution : Comparison 11/12 Quaternary Pump vs 129 Infinity mau 12 8 4 129 Infinity 3.928 5.516 6.62 7.472 7.783 8.234 8.929 9.556 1.116 2 4 6 8 1 mau 12 8 4 129 + 1µl dwell volume * 4.495 6.12 8.377 7.214 8.66 8.827 9.521 1.146 1.75 min 2 4 6 8 1 mau 12 8 4 11/12 Quaternary Pump 1.194 4.454 % 2 4 6 8 1 1 8 6 4 2 2 4 6 8 1 6.56 7.177 8.42 8.358 8.816 9.521 1.157 1.728 min min Gradient: 5%B to 95%B, TG: 1 min min Column: Zorbax SB-C18, 4,6x15 mm, 3,5µ Mobile phase: A = water / B = acetonotrile Sig: 254,1nm; Ref: 36,1nm *experimentally obtained dwell volume value Temperatue: 4 C Inj.vol.: 5µl Flow rate: 1,5 ml/min Sample: RRLC check out

Plumbing solution : Comparison 11/12 Binary Pump vs 129 Infinity mau 12 8 4 129 Infinity 3.928 5.518 6.621 7.474 7.785 8.236 8.932 9.56 1.12 2 4 6 8 1 mau 12 8 4 129 + 1µl dwell volume * 2 4 6 8 1 mau 12 8 4 11/12 Binary Pump 4.494 4.536 6.12 6.14 8.377 7.215 8.66 8.828 9.522 1.146 8.39 7.245 8.86 8.837 9.521 1.136 1.73 1.689 min min % 1 8 6 4 2 2 4 6 8 1 min Gradient: 5%B to 95%B, TG: 1 min 2 4 6 8 1 min Column: Zorbax SB-C18, 4,6x15 mm, 3,5µ Mobile phase: A = water / B = acetonotrile Sig: 254,1nm; Ref: 36,1nm *experimentally obtained dwell volume value Temperatue: 4 C Inj.vol.: 5µl Flow rate: 1,5 ml/min Sample: RRLC check out

Plumbing solution : Comparison 11/12 Binary Pump vs 129 Infinity mau 8 6 4 2 1.55 1.143 129 Infinity mau 1 2 3 4 5 6 7 8 9 8 6 4 2 1.54 129 + 1µl dwell volume* 11/12 Binary Pump 4.194 4.69 5.435 6.23 6.475 6.957 7.615 8.856 4.732 5.199 6.34 6.83 7.77 7.553 8.2 9.448 mau 1 2 3 4 5 6 7 8 9 8 6 4 2 1.17 1.331 4.768 5.226 6.52 6.843 7.88 7.561 8.29 % 1 2 3 4 5 6 7 8 9 8 6 4 2 9.432 min min min Gradient: 5%B to 95%B, TG: 1 min 2 3 4 5 6 7 8 9 min Column: Zorbax SB-C18, 4,6x15 mm, 3,5µ Mobile phase: A = water / B = acetonotrile Sig: 254,1nm; Ref: 36,1nm *experimentally obtained dwell volume value Temperatue: 4 C Inj.vol.: 5µl Flow rate: 1,5 ml/min Sample: Sigma

Plumbing solution : Comparison 12 SL Binary Pump vs 129 Infinity mau 8 6 4 2.256 129 Infinity 1.629 1.98 2.46 2.84 2.887 3.16 3.352 4.71 mau 1 2 3 4 5 8 6 129 + 8µl dwell volume * 4 2.262 mau 1 2 3 4 5 8 6 12 SL Binary Pump 4 2.34 2.538 2.524 3.29 3.18 3.616 3.676 4.92 4.177 4.19 4.14 4.381 4.453 4.549 4.622 5.362 5.327 min min % 1 2 3 4 5 8 6 4 2 Column: Zorbax SB-C18, 2,1x5 mm, 1,8µ Mobile phase: A = water / B = acetonotrile Sig: 254,1nm; Ref: 36,1nm Temperatue: 4 C Inj.vol.: 1µl Flow rate:,5 ml/min Gradient: 5%B to 95%B, TG: 5 min 1 2 3 4 5 *experimentally obtained dwell volume value Sample: Sigma min min

Column: Zorbax EC-C18, 4.6x5 mm, 1,8µ Mobile phase: A = water / B = acetonotrile Gradient 5 95 % B in 5 min Sample: RRLC checkout sample Isocratic Hold: Comparison 12 SL / 129 Infinity 5 min Gradient @ 1 ml/min 12 SL + Mixer + damper 129 + 86 µl Delay volume 129 + 9 µl Delay volume 129 + 94 µl Delay volume

Isocratic Hold: Comparison 12 SL / 129 Infinity 1 min Gradient @ 1 ml/min (Alkylphenones) 12 SL + Mixer + damper 129 + 86 µl Delay volume 129 + 9 µl Delay volume 129 + 94 µl Delay volume Column: Zorbax EC-C18, 4.6x5 mm, 1,8µ Mobile phase: A = water / B = acetonotrile Gradient 5 95 % B in 5 min Sample: RRLC checkout sample

Isocratic Hold: Comparison 12 SL / 129 Infinity 1 min Gradient @ 2 ml/min (Alkylphenones) 12 SL + Mixer + damper 129 + 86 µl Delay volume 129 + 9 µl Delay volume 129 + 94 µl Delay volume Column: Zorbax EC-C18, 4.6x5 mm, 1,8µ Mobile phase: A = water / B = acetonotrile Gradient 5 95 % B in 1 min

Isocratic Hold: Comparison 12 SL / 129 Infinity 2 min Gradient @ 1 ml/min (Alkylphenones) 12 SL + Mixer + damper 129 + 86 µl Delay volume 129 + 9 µl Delay volume 129 + 94 µl Delay volume Column: Zorbax EC-C18, 4.6x5 mm, 1,8µ Mobile phase: A = water / B = acetonotrile Gradient 5 95 % B in 5 min Sample: RRLC checkout sample

Isocratic Hold: Comparison 12 SL / 129 Infinity 2 min Gradient @ 2 ml/min (Alkylphenones) 12 SL + Mixer + damper 129 + 86 µl Delay volume Column: Zorbax EC-C18, 4.6x5 mm, 1,8µ Mobile phase: A = water / B = acetonotrile Gradient 5 95 % B in 5 min Sample: RRLC checkout sample

Relative Difference in Retention Time between 12 SL and 129 Infinity with different programmed delays Scatter Plot StopFlow - 1 tg - 5 StopFlow - 1 tg - 1 StopFlow - 1 tg - 2 2 1-1 -2 StopFlow - 2 tg - 5 StopFlow - 2 tg - 1 StopFlow - 2 tg - 2 2 1-1 -2 2 4 6 8 1 2 4 6 8 1 2 4 6 8 1 Column: Zorbax EC-C18, 4.6x5 mm, 1,8µ Mobile phase: A = water / B = acetonotrile Gradient 5 95 % B in 5 min Sample: RRLC checkout sample Peak Number

Difference in Resolution of Adjacent Peaks Scatter Plot StopFlow - 1 tg - 5 StopFlow - 1 tg - 1 StopFlow - 1 tg - 2 3 2 1-1 -2-3 -4-5 StopFlow - 2 tg - 5 StopFlow - 2 tg - 1 StopFlow - 2 tg - 2 3 2 1-1 -2-3 -4-5 2 4 6 8 1 2 4 6 8 1 2 4 6 8 1 Column: Zorbax EC-C18, 4.6x5 mm, 1,8µ Mobile phase: A = water / B = acetonotrile Gradient 5 95 % B in 5 min Sample: RRLC checkout sample Peak Number

129 Infinity Compatibility / Emulation Mode Hypothesis: Any instrument with a low delay volume (e.g. 129 Infinity) can emulate an instrument with a larger delay volume. e.g. Agilent 11/12 / 12 SL <-> 129 Infinity w small/large Jet Weaver Use Cases: Backward compatibility : Methods from larger delay volume systems can be run unchanged on the 129 Infinity in emulation mode with same results Method development: 129 Infinity in emulation mode can be used to develop methods for other systems Possible implementation Determine the transfer functions between 129 and the system to be emulated Implement an Algorithm that calculates gradient shape for emulation mode (FW/SW) Internally execute a modified timetable to emulate stating system

Execution Concept I TT-a Delay/Transition a F a TT-a Delay/Transition b F b

Execution Concept TT-a input Method temporary TT-b H a-b F a = = F b F 12 F129 output F b No Change on Method or Instrument!

Execution Concept I TTBL-a step response F a input x Transfer Function (complex) = output output input = Transfer Function (complex) = F a F b

Possible Workflow Schematics CFR-21part11 IVD Pharma GMP FDA Company (custom) guidelines - Custom settings SOP, Method critical pair performance specification e.g. or equivalent equipment Application needs in GMP-environment System Suitability ---- - Execute as e.g. 15 - Execute as e.g. 12-SL Details = niceto-know deviations Rare case no passed? documented Audit trail yes Run-sheet Data files comments allowed to use

Conclusions Due to minimized delay volumes in modern UHPLC instrumention, methods developed on standard HPLC instruments will not give the same results in retention time and resolution when run on an UHPLC system Adding physical volumes plumbing solution can lead to more comparable results but is not user friendly and requires exact knowledge of the difference in system delay volumes. Adding an isocratic hold tho the method also does not always yield the desired result as it can correct for delay in gradient start but not for gradient shape. A concept is proposed that allows to perform a SW/FW driven emulation of a variety of instruments on the Agilent 129 Infinity system. Systems can be pre-characterized but also tools for characterization of unknown systems an be provided. 65

12 Series TCC SL Dispersion: New 1.6µl- versus standard 3µl-Heater mau 25 2 Sample: Flow rate: Isocratic Standard Sample.5ml/min 1.6 µl Heater Eluent: Water/ACN = 3/7 Column: 2.1x1mm, RRHT SB-C18 3 µl Heater 3.181 3.227 15 1 5 1.235 1.216 1.339 1.358 1 1.5 2 2.5 3 min Page 66 12 Rapid Resolution System Agilent Restricted December 2, 25

12 Series TCC SL Dispersion: New 1.6µl- vs. standard 3µl-HeatEx. Flow rate Evaluated Peak 1.6µl HeatEx. 5 C 3µl HeatEx. 5 C 1.5ml/min Rs of peak 2 3.19 2.73 1.5ml/min PW 5sigma of peak 4 in min.31.33 1ml/min Rs of peak 2 3.96 (+26%) 3.15 1ml/min PW 5sigma of peak 4 in min.67 (-13 %).76.5ml/min Rs of peak 2 3.29 2.86.5ml/min PW 5sigma of peak 4 in min.42.44.2ml/min Rs of peak 2 3.51 3.14.2ml/min PW 5sigma of peak 4 in min.192.197 Page 67 12 Rapid Resolution System Agilent Restricted December 2, 25

12 Series TCC SL ACR 2Ps/1Pt Valve and Heaters/Cooler for Alternating Column Regeneration 2-position/1-port micro-valve SL U-shaped post-column cooler 1.5 µl Heater for column 1 Heater for column 2 Two columns in alternate usage Page 68 12 Rapid Resolution System Agilent Restricted December 2, 25

Benefit 1 Fastest LC Pharma Impurity Method Pat Sandra & Frank David, RIC 4.6 x 5mm RRHT SB-C18 35% ACN at.6 min to 95% ACN at 2.5 min 3 ml/min, 4 C (45 bar) DAD SL: 13 µl cell 4 Hz Page 69 12 Rapid Resolution System Agilent Restricted December 2, 25

Scaling Performance: 126 Binary UHPLC Performance for EVERY LAB! DAD1 A, Sig=254,4 Ref =of f (51119A\SIG13.D) mau 2.5 2 1.5 1.5 -.5-1 5 1 15 2 min DAD1 A, Sig=254,4 Ref=off (514D\LC_J2.D) Norm. 2.5 2 1.5 1.5 -.5 4.6 x 15, 5um 93 bar N = 7259 R_S = 1.15 S/N = 42-1 5 1 15 2min Column:15x4.6 mm 5µm Pressure: 93 bar N: 8213 Height: 1.25 mau S/N: 42.3 R s = 1.15 Height = 1.25 Noise = 24uAU t r = 14.9 min (1 st epimer) N ptp : 2.4 1-2 mau 4.6 x 15, 3.5um 165 bar Column:15x4.6 mm 3.5µm N = 14862 Pressure: 165 bar N: 14862 R_S = 1.37 S/N = 5 Height:1,34 mau S/N: 5.7 R s = 1.37 Height = 1.34 t r = 15.3 min (1 st epimer) Noise = 2uAU N ptp : 2 1-2 mau N 3 25 2 15 1 5 Norm. 3 2.5 2 1.5 1.5 -.5-1 N 1 d p.2.3.4.5.6 1/dp DAD1 A, Sig=254,4 Ref=off (517D\LC_X1.D) 1 min 4.6 x 15, 1.8um 49 bar Column:15 x 4.6 mm 1.8µm Pressure: 49 bar N: 28669 N = 28669 Height:1.78 R_S = 1.8 (+57%) S/N: 43.6 S/N = 44 R s = 1.8 t r = 17.2 (1 st epimer) Height = 1.8 N ptp : 3 1-2 Noise = 3uAU

Benefit 3 The Most Flexible LC in the World! Support of RRLC and HPLC on Std and Narrow bore columns 1. The 6-8µl standard delay volume configuration allows to run both standard bore RRLC and conventional HPLC methods without need of re-validation. 2. The 12µl low delay volume configuration provides uncompromised compatibility with narrow bore LC and LC/MS applications in both RRLC and HPLC. mau 8 7 6 5 4-25% RT! -2% RT! 12 RRLC Low delay volume configuration (12µl) RT-Shifts up to 25%, difficult Peak tracking Limited compatibility with HPLC methods Typically requires method re-validation 3 2 1 11 Binary 1 2 3 4 5 6 7 min Page 71 12 Rapid Resolution System Agilent Restricted December 2, 25

Competition Waters Acquity + Alliance Application Range Universal vs. two Expert Systems 12 RRLC Acquity Alliance RRLC Narrow YES YES NO (6) RRLC Std YES NO (1) Limited (5) HPLC Narrow YES NO (2) Limited (4) HPLC Std YES NO (3) YES (1) Max flow rate of 2ml/min (2) Max Column length of 2mm (3) Max Column length of 2mm and no std delay volume configuration => RT shifts of up to 3% => re-validation required (4) Delay volume too large (~1ml) (5) No 1.7um column with 3 4.6mm ID, 4bar/6C limits (6) Del. vol. too large, 4bar/6C limits Page 72 12 Rapid Resolution System Agilent Restricted December 2, 25

Competition Waters Acquity 2.1mm Speed-Benchmark Method 12 RRLC: Better Performance at significant lower pressure!! mau 7 6.62 12 RRLC System Waters Acquity Anal. Time.949 min.914 min Resolution 3.69 3.5 Precision.25.94 %RSD.18.3 %RSD Pressure 44 bar 7 bar (+6%) 5 4 3 2.29.37.55.664.725.821.98.986 1.2.4.6.8 1 min Sample: Phenones Test Mix Columns: 12 RRLC: SB-C18, 2.1x5 RRHT Waters: BEH, 2.1x5 Flow Rate: 1ml/min Gradient: 35-95% ACN in.9min Temperature: 5 Injection volume: 1µl Injection Technique: ADVR, OI, MCO WL: 245nm Data Rate: 8Hz Page 73 12 Rapid Resolution System Agilent Restricted December 2, 25

Product Overview 12 RRLC System Configurations Be Prepared!! Column dimension Flow Rate Gradient Eluent / Modifier Temperature Highest Speed, Res, or best Compromise? System Configurator* Pump delay volume Connecting Capillaries Heat Exchangers Flow Cell *Available by February Page 74 12 Rapid Resolution System Agilent Restricted December 2, 25

Gradient Behaviour and Method Transfer Emulating any (U)HPLC System 1 UV Signal Scatter Plot 9 8 7 6 5 Method Transfer Same RT of all Peaks Same Resolution all Peak Pairs 4 3 2 1 129 Infinity LC 12 RRLC (damper + mixer) 129 Infinity LC (+ damper + mixer + capillary).2.4.6.8 1 1.2 1.4 1.6 1.8 2 Delay Volume (ml) Page 75

Effects of path length increase Conventional flow cells: 1 mm pathlength 13 µl geom. volume Peak Dispersion - Loss of resolution - Loss of signal height 6 mm pathlength 78 µl geom. volume Max-Light High Sensitivity cell: Optofluidic waveguides (total internal reflection) 6 mm pathlength 4 µl σ V dispersion volume

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 29 Theory Experiment Wed, LH5, 11:3am

Agilent 129 Infinity LC Power Range and 2.7um SP Columns bar 5 x 4.6 mm 1 x 4.6 mm 15 x 4.6 mm 25 x 4.6 mm 15 x 3 mm 25 x 3 mm 15 x 2.1 H 2 O/ACN, 4 C 2.1x5, T=4C 2.1x5, T=8C ml/min 78 STM = Sub-2um SP = Superficially Porous TP = Totally Porous

Impurity Analysis Tramadol 12 Quat-DAD versus 126 Quat-DAD mau 1 DAD1 A, Sig=27,8 Ref=4,1 (126 DATA...COLU\12QUAT_TRAMADOL 21-4-26 1-34-21\12QUAT_TRAM8.D) DAD1 A, Sig=27,8 Ref=4,1 (126 DATA...6 NOISE 12APRIL 21-4-26 13-52-43\126 TRAMADOLE2_1MM5.D) DAD1 A, Sig=27,8 Ref=4,1 (126 DATA...6 NOISE 12APRIL 21-4-26 12-4-3\126 TRAMADOLE_6MM9.D) Imp A Tramadol 126 with 6mm cell Imp C Imp B.5 Imp D 126 with 1mm cell -.5 12 with 1mm cell 1 1.5 2 2.5 3 3.5 min Impurities:.2 -.3% Column: 5x4.6mm Zorbax RRHT SB-C18, 1.8µm, 6bar F = 1.2mL/min