New Advances in UHPLC -Resolution, Speed & Sensitivity

New Advances in UHPLC -Resolution, Speed & Sensitivity Enhancing Productivity with Agilent s Newest Instrumentation Patrick Cronan LC Applications Scientist Boston, MA September 14, 2012 Page 1

The 1290 Infinity Quaternary LC System How binary pump-like performance can enhance your lab efficiency 1290 Infinity DAD with 60mm cell (& HDR optional) 1290 Infinity TCC with broadest valve selectivity 1290 Infinity Autosampler (with Flexible Cube for lowest carry-over) 1290 Infinity Quaternary Pump Page 2 Page 2 September 24, 2012

Innovation: 1290 Infinity Quaternary Pump Quaternary Pump with Binary Pump-like Performance 1290 Infinity Quaternary Pump Boost Performance Highest accuracy and precision for composition and flow, with exceptionally low delay volume Save Time Accelerates transfer of existing HPLC methods to UHPLC Reduce Costs Outstanding UHPLC performance at HPLC-like costs of ownership Page 3

New Inlet-Mixer - based on JetWeaver technology 4 Page 4 Oct. 2011

1290 Infinity Quaternary Pump Various tools enabling the high performance Switchable Jet Weaver Mixer 4-channel Degasser Multi-Purpose- Valve MCGV (Multi- Channel-Gradient- Valve) Inlet-Weaver Pump Head In-line Filter 5 Page 5

Multipurpose valve functions for highest comfort Standard Application for standard Delay Volume 6 Page 6

Multipurpose valve functions for highest comfort Extra Mixing Volume for lowest baseline ripple (TFA applications) 7 Page 7

Multipurpose valve functions for highest comfort Automatic Purging 8 Page 8

Multipurpose valve functions for highest comfort Automatic filter backflush 9 Page 9

Agilent 1290 Infinity Quaternary Pump Specifications & Benefits Power Range For any kind of analysis Composition Accuracy and Precision < 0.15 % RSD or 0.02 min SD 0.4 % (1-99 % Composition B) High RT precision in gradient runs Flow Accuracy and Precision < 0.07 % RSD or 0.01 min SD 1.0 % or 10 µl High RT precision in isocratic runs Composition Range 1-99 % Wide analytical range Delay Volume < 350 µl For fast quaternary gradients Page 10

Composition Accuracy and Precision Step Gradient Analysis with Tracer Different mixtures (H2O, MeOH, ACN) at different pressures and different flow rates Full application range! 11 January, 26, 2012 Page 11

Flow and Composition Accuracy and Precision Isocratic analysis of Glucocorticoids 1: Hydrocortisone 2: Beclomethasonedi-proprionate 3: Hydrocortisone acetate Column: Agilent ZORBAX Eclipse Plus C18, 4.6 x 100 mm, 5 µm (p/n 959996-902) Mobile phase: Water/ Acetonitrile = 30/70 Flow rate: 1 ml/min Stop time: 5 min Injection vol.: 3 µl (needle wash for 3 sec) Column Temp.: 30 C DAD: 254/10 nm, (ref 400/ 80 nm), 20 Hz, slit 4 nm Page 12

Flow and Composition Accuracy and Precision Isocratic analysis of Glucocorticoids mau 30 25 1 2 RT of compounds very sensitive regarding changes in mobile phase composition! 20 15 10 5 0 1 1.5 2 2.5 3 3.5 3 Water/ACN 29:71 Water/ACN 31:69 Water/ACN 30:70 min Page 13

Accuracy and Precision Isocratic analysis of Glucocorticoids Specification (< 0.07 %) mau 35 30 25 1: Hydrocortisone 2: Beclomethasonedi-proprionate 20 15 10 3: Hydrocortisone acetate 5 0 0 1 2 3 4 min Page 14

Agilent 1290 Infinity Quaternary Pump Specifications & Benefits Power Range For any kind of analysis Composition Accuracy and Precision < 0.15 % RSD or 0.02 min SD 0.4 % (1-99 % Composition B) High RT precision in gradient runs Flow Accuracy and Precision < 0.07 % RSD or 0.01 min SD 1.0 % or 10 µl High RT precision in isocratic runs Composition Range 1-99 % Wide analytical range Page 15

Composition Range Shallow gradient at low organic concentration CH 3 CH 3 O N N O N N N H N 3 C O H 1: Theophylline H 3 C N N O CH 3 2: Caffeine Column: Agilent Poroshell 120 EC-C18, 4.6 x 50 mm, 2.7 µm (p/n 699975-902) Mobile phase: A = Water / B = Acetonitrile Gradient: Flow rate: Stop time: 2 12 % B in 5min 2 ml/min 5 min Injection vol.: 5 µl Column Temp.: 40 C DAD: 254/10 nm, (ref. 360/ 100 nm), 20 Hz Page 16

Composition Range Shallow gradient at low organic concentration mau CH 3 CH 3 100 80 1 O N N N H N 3 C O H 1: Theophylline O H 3 C N N N N O CH 3 2: Caffeine 60 40 2 20 0 2 2.5 3 3.5 4 4.5 min Page 17

Composition Range Shallow gradient at low organic concentration 0.5 Retention time precision 0.4 RSD RT (%) 0.3 0.2 Specification (< 0.15 %) Theophylline Caffeine 0.1 0 1290 Infinity Quat. System Page 18

Composition Range Shallow gradient at low organic concentration Retention time precision 0.5 2.4 0.4 RSD RT (%) 0.3 0.2 Specification (< 0.15 %) Theophylline Caffeine 0.1 0 1290 Infinity Quat. System 1260 Infinity Quat. System Quat. UHPLC Other vendor Page 19

Composition Range Shallow gradient at low organic concentration mau 250 Step gradient 1 % steps from 0 to 10 % 200 150 100 50 0 1290 Infinity Quaternary system Quaternary UHPLC from other vendor 0 10 20 30 40 50 Page 20

Ternary/Quaternary Gradients Blend Assist You need different concentrations of modifiers in your analysis, would like to have just one stocksolution and do online dilution to profite from the quaternary mixing capability of your pump? Here is a simple tool BlendAssist! Water Water 1% TFA Desired method conditions - example: 1. 5 to 95% gradient of ACN with 0.1% TFA in Water and 0.08% TFA in ACN 2. 20 80% gradient of ACN with 0.5% TFA in Water and 0.4% TFA in ACN Without BlendAssist you need to either pre-mix the required solvents or by using stock-solutions of TFA in Water and ACN to program complex gradients (%A, B, C, D). ACN ACN 1% TFA With BlendAssist: just program your binary organic/aqueous gradient and define the dilution factor! Page 21

Ternary/Quaternary Gradients Blend Assist - simple Tool for Online-Dilution of Modifiers and Gradient Set-Up BlendAssist - Solvent/modifier channel definition Page 22

Buffer Advisor Software Quaternary mixing 23 Page 23 September 24, 2012

1260 Infinity Bio-inert HPLC What is similar compared to standard 1260 Infinity Quaternary LC? Pumping performance, gradient performance Pumping Specifications Detector, Autosampler Performance and Specs Overall System Robustness TCC specs 600 bar also for fast or high resolution Bioseparations Modular flexibility maintained 24 Page 24 September 24, 2012

1260 Infinity Bio-inert HPLC What is different compared to standard 1260 Infinity Quaternary LC Pump titanium based, no SST Autosampler metal-free Capillaries and connections metal-free Detector Flow Cell metal-free ph-range 1-13, short term ph 14 Active Seal wash included in pump Low pressure is possible Wide ph range High salt tolerance Lowest surface activity Bio-Inertness 25 Page 25 September 24, 2012

New Capillary Design Ensures Bio-inertness Capillaries: Needle: Metal cladded PEEK capillary New capillary technolgy enables 600 bar AND is completely metal free!! SST housing for robustness PEEK and ceramic for metal free sample path 26 Page 26 September 24, 2012

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Buffer Advisor Software Workflow 1: Select Buffer and Gradient Solution 2: Enter Gradient Timetable 3: Review Composition of Stock Solutions and Adjust 4: Gradient Display Section: Process and Review Gradient 29 Page 29 September 24, 2012

Experimental Situation Page 30

Buffer Advisor Software Workflow Page 31

Buffer Advisor Software Timetable import into Chemstation 32 Page 32

Buffer Advisor Software ph scouting 33 Page 33

Buffer Advisor Software stable ph 34 Page 34

30x wider linear UV range - Quantification of widely different concentration levels in one single run Agilent 1200 Infinity High Dynamic Range (HDR) Solution Page 35

History of DAD Linearity Gain The last 30 years 45.0 40.0 > 30 35.0 1200 Infinity HDR-DAD 30.0 >30X Linearity With HDR Gain Linearity 25.0 20.0 15.0 10.0 5.0 0.0 1.4 1.7 2.0 1.4 1040 to 1090 1090 to 1050 1050 to 1100 1110 to 1200 1200 to 1290/60 1200 to HDR 2.9 (1986) (1988) (1995) (2006) (2010) (2011) Detector Model (Intro Year) 36 Page 36 September 24, 2012

Optofluidic Waveguides: Max-Light Flow Cells Total-internal reflection in a non-coated fused silica fiber Mirror 1260 / 1290 Infinity DAD Grating Programmable or fixed slit Deuterium Lamp Max-Light Cartridge Cell 10 mm or 60 mm path length 1024 element diode-array 37 Page 37 September 24, 2012

Effects of path length increase Conventional flow cells: 10 mm pathlength small geom. cell volume (13 µl) high light transmission Peak Dispersion 60 mm pathlength large geom. cell volume (78 µl) lower light transmission - Loss of resolution - Loss of signal height Max-Light High Sensitivity cell: Optofluidic waveguides (total internal reflection) 60 mm pathlength 4 µl V dispersion volume 38 Page 38 September 24, 2012

10x Higher Sensitivity 1290/1260 Infinity DAD compare to 1200 Series DAD SL 30 mau Columns: 150 x 4.6mm Zorbax SB C18, 5µm Sample: Anthracene: 835 pg/µl Mobile phase: A: Water, B: Acetonitrile Elution: isocratic 80 % B 25 20 1260/1290 Infinity DAD Injection volume: 5 µl Flow: 1.5 ml/min DAD: 251/4nm, Ref= 450/80nm, 2.5Hz, slit width 4nm 15 1260/1290 DAD 60 mm 1200 DAD SL 10mm 10 5 0 1200 Series DAD / DAD SL 0 1 2 3 4 5 min Height [mau] 28.876 4.938 Noise [mau] 0.0098 0.0190 Signal/ noise 2944 259 Increase 11.4 x Page 39 39 September 24, 2012

30x Wider Linear Range with HDR-DAD 3.7 mm and 60 mm and Max-Light flow cell Detector Signal [AU/cm] 3.7 mm path-length 60 mm path-length Computing the signals from 60 mm path-length for the low concentration 3.7 mm path-length for the high concentration Conc [µg/ml] 40 Page 40 September 24, 2012

1290/1260 High Dynamic Range HDR Increasing Linear UV Range by >30x DAD 1: DAD 2: 60mm Clustered DAD 3.7mm Technology Cluster of two DADs DAD 1: 60 mm Cell for low concentrations DAD 2: 3.7 mm Cell for high concentrations Output: One, combined signal, normalized to 10 mm path length (HDR range: 0.6 x 10-6 to 6.7 AU/cm) vs for 1200 Series: 7 x 10-6 to 2 AU/cm) Control / Usability Like standard 1290/1260 DAD: Control, data analysis, reporting Investment Protection Existing 1290/1260 DAD can be upgraded (by 2nd DAD) Existing 1100/1200 Systems can be upgraded (by two 1290 or 1260 DAD) 41 Page 41 September 24, 2012

Fixed Dose Combinations From Roche Switzerland Fixed dose combinations: A combination of two or more APIs in a fixed ratio of doses Agilent 1200 (black): API 2: 10 µl injected Agilent 1200 (red): API 1: 100 µl injected Infinity HDR-DAD (blue): API 1 and 2 simultaneously: 70 µl injected API 2 API 1 42 Page 42 September 24, 2012

Fixed Dose Combinations From Roche Switzerland With the Agilent 1200 Infinity HDR- DAD we significantly decreased our turnaround time. We also get lower LODs and better area precision Dr. Rose-Marie Meier F. Hoffmann - La Roche AG Infinity HDR-DAD Agilent 1200 DAD 43 Page 43 September 24, 2012

2 Injections needed if a conventional DAD is used mau Paracetamol 1000 Vitamin C 800 600 1µl injection to determine the first 3 peaks 400 200 Caffeine Chlorphenamine? 0 0 1 2 3 4 5 min mau 3800 3000 Vitamin C Paracetamol Detector saturation Outside the linear range 5µl injection to determine the last peak and further impurities The first 2 peaks are out of linear range 2000 Chlorphenamine 1000 Caffeine 0 0 1 2 3 4 5 min 44 Page 44 September 24, 2012

2 Injections needed if a conventional DAD is used mau 1 Chlorphenamine DAD1 A, Sig=254,20 Ref=380,80 (SOP 98 GRI...0FEB\HDR_GRIPPO_OLD_20FEB 2012-02-20 10-40-57\GRIPPO_1UL09.D) DAD1 A, Sig=254,20 Ref=380,80 (SOP 98 GRI...0FEB\HDR_GRIPPO_OLD_20FEB 2012-02-20 10-40-57\GRIPPO_5UL10.D) 0.8 mau 20 Chlorphenamine 0.6 0.4 Imp 4 17.5 0.2 Imp 3 15 0 12.5-0.2 10 4.6 4.7 4.8 4.9 5 5.1 5.2 5.3min 7.5 5 2.5 0 Imp 3 Red: 5µl injection with 21.8mAU peak height for Chlorphenamine Blue: 1µl injection with 3.7mAU peak height for Chlorphenamine Imp 4 4 4.5 5 5.5 6 min 45 Page 45 Confidentiality Label September 24, 2012

Comparison of conventional DAD vs. HDR DAD, 5µl injection mau 4000 3500 3000 Vitamin C Detector saturation Outside the linear range Paracetamol Red conventional DAD: 2 peaks out of linear range with 5µl injection 2500 Green HDR-DAD: 2000 All peaks within linear range (up to 6AU) 1500 1000 500 0 Caffeine Chlorphenamine 0 1 2 3 4 5 min 46 Page 46 September 24, 2012

Comparison of LOD mau 1.3 1.2 More reliable automated peak integration HDR DAD 1.1 1 0.9 Conventional DAD 0.8 0.7 0.6 0.5 3.9 4 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 min Compound LOD with S/N=3 for Conventional DAD LOD with S/N=3 for HDR- DAD solution Chlorphenamine ~1ng ~0.1ng 47 Page 47 September 24, 2012

mau 20 15 Comparison of conventional DAD and HDR DAD, 5µl injection DAD1 K, Sig=254,20 Ref=380,80 (SOP 98 GRI...B\HDR_GRIPPO_HDR_21FEB 2012-02-21 07-51-02\GRIPPO_HDR5UL09.D) DAD1 A, Sig=254,20 Ref=380,80 (SOP 98 GRI...0FEB\HDR_GRIPPO_OLD_20FEB 2012-02-20 10-40-57\GRIPPO_5UL09.D) Vitamin C Paracetamol Caffeine Red conventional DAD: determination of impurities and chlorphenamine in a second run Chlorphenamine Blue HDR DAD: dermination of all peaks in one run 10 Imp 1 Imp 2 Imp 3 Imp 4 5 0 1 2 3 4 5 min 48 Page 48

Comparison of area precision 3 Comparison of Area Precision 14.908 13.013 5.37 5.723 2.5 RSD Area (%) 2 1.5 1 HDR Signal Conv.DAD signal (1µl) Conv.DAD (5µl) 0.5 0 HDR DAD 5µl injection volume for detemination of all compounds (blue) Conventional DAD: 1µl injection volume for determination of Vit C, paracetamol and Caffeine (orange) 5µl injection volume for determination of Chlorphenamine and further impurities (green) 49 Page 49 September 24, 2012

Intelligent System Emulation Technology (ISET) Page 50 Page 50 September 24, 2012

What is ISET? - Two components A. Best in class performance of the 1290 Infinity LC Broad power range, ultra-low delay volume, superior sensitivity, unmatched flow and composition accuracy Peak 3 Overlay of 10 runs RSD = 0.005 % B. The revolutionary ISET emulation algorithm Full characterization of target LC systems Creation of emulation function F A Page 51 Page 51

Method Transferability: 1290 Infinity LC - System Emulation Technology Concept F A Emulated System A Select System to be emulated by a simple mouse click: Agilent 1100 Series Agilent 1200 Series LC Agilent 1260 Infinity LC Agilent 1220 Infinity LC Alliance (2012) Page 52 Page 52

Intelligent System emulation technology - How it works (1290 to 1200) mau 400 350 300 250 200 Injection Same gradient conditions by moving the gradient and by emulating the gradient mixing behavior. 150 100 50 Programmed gradient 1290 gradient 1200 gradient 0 0 0.5 1 1.5 2 2.5 3 3.5 min Page 53 Page 53

Intelligent System Emulation Technology ISET Implementation Select pump Select sampler Page 54 Page 54

Pesticides Column: 3 x 50 mm, 2.7 µm Poroshell Run time: 3.5 min mau 800 700 600 500 Metamitron Chloridazone R s = 1.87 Cyanizine Simazine Chlortoluron Diuron Propazine Terbutylazine Prometryn 1290 System with iset 400 300 200 R s = 1.15 1290 System 100 0 R s = 1.90 1 1.5 2 2.5 3 3.5 1100 Binary System min 55 Page 55

Pesticides Column: 3 x 50 mm, 2.7 µm Poroshell Run time: 3.5 min 56 Page 56

Pesticides Column: 3 x 50 mm, 2.7 µm Poroshell Run time: 3.5 min 57 Page 57

Generic emulation - Limitations Example: 1290 to Vendor X mau 350 300 Injection 250 Generic emulation: Move the gradient 200 150 100 50 Programmed gradient 1290 gradient 0 0 0.5 1 1.5 2 2.5 3 3.5 min Vendor X Page 58 Page 58

Generic emulation - Limitations Example: 1290 to Vendor X mau 350 300 Injection 250 200 150 100 50 0 0 0.5 1 1.5 2 2.5 3 3.5 Generic emulation: Move Move the the gradient gradient Does not consider the gradient mixing behavior (different curves) or delay volume changes because of pressure changes for pumps with damper. min Programmed gradient 1290 gradient Vendor x Page 59 Page 59

Method Transfer Possibilities Today - A) Adding physical volume (plumbing solution) Example: 1290 Infinity LC 1290 + 1000 L dwell vol. 1100/1200 Quat Pump Advantages Works well for generic, slower analysis No Method change Disadvantages Requires hardware change, no flexibility Hardware change difficult in validated environment Requires manual determination of the dwell volume (Pumps with damper the dwell volume is not constant and depends on pressure) Page 60 Page 60

Method Transfer Possibilities Today - B) Isocratic Hold Example: 1290 to 1200 mau 350 300 Injection 250 200 150 Isocratic hold: move the gradient Does not consider the gradient mixing behavior (different curves). 100 50 Programmed gradient 1290 gradient 0 0 0.5 1 1.5 2 2.5 3 3.5 min 1200 gradient Page 61 Page 61

Method Transfer Possibilities Today - B) Isocratic Hold Example: 1290 to 1200 1200 Series LC Binary Pump 1290 Infinity LC + 860 µl Delay Volume 1290 Infinity LC + 900 µl Delay Volume 1290 Infinity LC + 940 µl Delay Volume Page 62 Page 62

Method Transfer Possibilities Today - B) Isocratic Hold Example: 1290 to 1200 1260 Infinity LC 1290 Infinity LC + 900 µl hold Advantages Flexibile, Isocratic Hold is part of the method Disadvantages Works not in all cases Requires manual determination of the dwell volume/ isocratic hold (Pumps with damper the dwell volume is not constant and depends on pressure) Requires modification of the methods (should be avoided in validated environment, but doesn t require revalidation USP Chapter <621>) Page 63 Page 63

Method transfer, pesticide example 2.1x100 mm Zorbax Eclipse Plus, 1.8 µm column, flow: 0,8 ml/min (A) 1100 Quaternary 400 bar 1 2,3 4 5 6,7 8 9 10,11,12 13 14 15 16 (B) 1290 Infinity 1200 bar (C) 1290 Infinity 1200 bar with ISET Page 64 Page 64 September 24, 2012

1290 Quaternary Pump: Full 1290 Infinity LC Power range Summary Highest composition precision and accuracy across pressure and flow rate range Full application range with binary-like pump performance! 1290 Systems using ISET: Achieves higher productivity for method development Simplify your instrument to instrument method transfer while minimizing costs Run your legacy methods with ISET and use UHPLC speed for faster separations HDR DAD Solution: Determination of low and high dose drugs is possible in one run Improved Precision and Lower Detection Limits as compared to convention DAD Using Buffer Advisor with BioInert Quat System: Shortens and simplifies the workflow for ph scouting by using pre-validated buffer systems and dynamically mixed gradients Excellent tool using for automated ph scouting and accurate ion exchange chromatography 65 Page 65

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