2012 Waters Corporation 1

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1 An Introduction to Waters HPLC and ACQUITY UPLC Column Technologies for small molecule analysis Esa Lehtorinne Tel: Fax: Waters Finland Kutomotie Helsinki 2012 Waters Corporation 1

2 Agenda Column Manufacturing and Platform Definitions New HPLC & ACQUITY UPLC Column Chemistries New 2.5 µm extended Performance (XP) Columns Summary 2012 Waters Corporation 2

3 What makes an LC column reproducible? A) Sorbents B) Packing C) Engineering 2012 Waters Corporation 3

4 Developing a Waters Column: Quality By Design Behave like our customers in regulated industries US Food and Drug Administration registered o cgmp s o Class 1 medical devices ISO 9000:2001 ISO 13485:2003 Own and control every step of the process Raw materials to final product o Very few suppliers do this or are capable of doing this Understand the parameters that make a difference.. and then control them So what does this mean 2012 Waters Corporation 4

5 Developing a Waters Column Hardware Materials Design Optimization Sorbents Chemistries Synthesis Packing Equipment Considerations Optimization Product QC and testing 2012 Waters Corporation 5

6 Developing a Waters Column: Hardware Manufacture Waters produces most of our hardware from raw materials Each step of the machining process is quality controlled Product hardware must pass: Material specifications Engineering tolerances Cosmetic Standards 2012 Waters Corporation 6

7 Developing a Waters Column: Particle and Sorbent Manufacture All Waters modern chemistry products are synthesized from raw materials allowing complete process control and traceability Waters manufacturing philosophy: Small batches ensures process understanding Provide synthesis batches that reflect product need requirements <2 kg scale (nano, capillary, analytical) 10+ kg process scale (preparative, bulk) Completely scalable and predictable through state-of-the-art process control 2012 Waters Corporation 7

8 Developing a Waters Column: Column Packing Computer controlled real time packing optimization Each column is packed on a dedicated custom designed packing station Optimized for slurry flow Optimized for packing pressure columns packed annually Equals over 800 columns per working day 2012 Waters Corporation 8

9 Developing a Waters Column: Final QC and Inspection Each column Passes: Rigorous cosmetic standards Efficiency testing using a test mixture COA tracking and QC checks of product conformity Final packaging and documentation 2012 Waters Corporation 9

10 Agenda Column Manufacturing and Platform Definitions New HPLC & ACQUITY UPLC Column Chemistries New 2.5 µm extended Performance (XP) Columns Summary 2012 Waters Corporation 10

11 Waters Column Product History µbondapak Styragel Spherisorb PrepPak 1958 DeltaPak SymmetryShield Symmetry ACQUITY UPLC BEH SunFire Columns XTerra XTerraPrep ACQUITY UPLC BEH Amide ACQUITY UPLC BEH Glycan XBridge Amide XSelect HSS HPLC Columns ACQUITY UPLC HSSC 18 and HSS C 18 SB XBridge ACQUITY UPLC HSS Cyano & PFP columns XSelect HSS Cyano & PFP columns XP 2.5 µm Columns Nova-Pak ProteinPak Pico-Tag AccQTag Symmetry 300 Atlantis Atlantis HILIC Silica Prep OBD Intelligent Speed BioSuite NanoEase Atlantis T3 ACQUITY UPLC HSS T3 AccQTag Ultra XBridge HILIC ACQUITY UPLC BEH125 SEC BEH450 SEC ACQUITY UPC 2 ACQUITY UPLC BEH200 SEC XSelect CSH HPLC columns ACQUITY CSH Columns Viridis SFC Columns ProteinPak High Rs IEX 2012 Waters Corporation 11

12 Begin with Two Fully Scalable Column Platforms Family designed and optimized for ph stability Most MS-compatible HPLC columns on the market Family designed and optimized for selectivity Multiple particle substrates to solve multiple chromatographic problems 1.7 [UPLC], 2.5, 3.5, 5 & 10 µm BEH 1.7 [UPLC], 2.5, 3.5 & 5 µm CSH 1.8 [UPLC], 2.5, 3.5 & 5 µm HSS 2012 Waters Corporation 12

13 Column Particle Platform Definitions BEH Particle Ethylene-Bridged Hybrid 130Å, 200Å & 300Å HSS Particle High Strength Silica 100Å CSH Particle Charged Surface Hybrid 130Å Designed for Industry-Leading Chemical (ph) Stability Designed for Increased Retentivity and Selectivity Designed for Maximum Selectivity and Sample Loading All three substrates are fully scalable between UPLC and HPLC platforms 2012 Waters Corporation 13

14 XBridge HPLC Columns General-purpose HPLC and UPLC columns for small molecule separations Broadest range of compound classes When buffers are exclusively used Where high ph stability is most important Where ultra-low MS bleed is desired Direct scalability between UPLC Technology, analytical HPLC and preparative HPLC 2012 Waters Corporation 14

15 Ethylene Bridged Hybrid [BEH] Particle U.S. Patent No. 6,686,035 B2 Bridged Ethanes within a silica matrix Anal. Chem. 2003, 75, Waters Corporation 15

16 Ethylene Bridged Hybrid [BEH] Chemistries Rugged, reproducible, fully-scalable column chemistries for reversed-phase and HILIC separations BEH C18: First column choice, widest ph range, LC/MS BEH C8: For hydrophobic compounds, widest ph range, LC/MS BEH Shield RP18: Embedded carbamate group, alternate selectivity BEH Phenyl: Most stable phenyl column, wide ph range BEH HILIC: Unbonded, rugged BEH particle for HILIC separation of very polar bases BEH Amide: General-purpose HILIC column for very polar compounds such as sugars, saccharides, carbohydrates, etc Waters Corporation 16

17 Industry Leading ph stability Column lifetimes in acidic mobile phases Extremely acidic mobile phases hydrolyse the bonded ligand of conventional LC sorbents Reduces analyte retention Test conditions: 1 % TFA in water (ph 1.0) at 80 o C. Comparative separations may not be representative of all applications Waters Corporation 17

18 Industry Leading ph stability Column lifetimes in alkaline mobile phases High ph mobile phases (>ph 10) rapidly dissolve silica-based stationary phases Only modern hybrid-based sorbents, [BEH] and [CSH], extend the usable mobile phase ph range from 1-12 Comparative separations may not be representative of all applications Waters Corporation 18

19 The importance of mobile phase ph: Rapid Method Development Using a wide mobile phase ph range is an effective approach to change compound selectivity Increase selectivity for: Acids (Green/Blue) Bases (Red/Yellow) Neutrals (Peak 2) are largely unaffected by mobile phase ph 2012 Waters Corporation 19

20 High Strength Silica [HSS] Chemistries First silica-based particle (1.8 µm) designed for UPLC applications Fully scalable manufacturing process enabled HPLC particle sizes (2.5, 3.5 & 5 µm) Three general-purpose C 18 columns for low to neutral ph reversedphase separations HSS C18: Superior peak shape & acid stability HSS C18 SB: Alternate Selectivity for Basic (SB) compounds HSS T3: Enhanced reversed-phase retention of polar compounds PFP and CN Chemistries Designed for alternate selectivities vs. C 18 chemistries HSS PFP: Exceptional selectivity and retention for positional isomers, halogenated compounds and polar basic compounds HSS CN: Stable, normal phase-compatible, reproducible CN chemistry 2012 Waters Corporation 20

21 Charged Surface Hybrid [CSH] Particles Step 1 Step 2 Step 3 CHARGED SURFACE HYBRID PROCESS 2012 Waters Corporation 21

22 Charged Surface Hybrid [CSH] Chemistries Three innovative CSH chemistries that provide alternate selectivity and improved performance low ionic strength mobile phases CSH C18: Superior peak shape for bases, increased loadability, alternate selectivity vs. conventional C18 columns CSH Phenyl-Hexyl: High performance phenyl column, best in additive-based mobile phases, different selectivity vs. BEH Phenyl CSH Fluoro-Phenyl: Truly different selectivity, optimized for low ph separations, enhanced retention of acidic compounds 2012 Waters Corporation 22

23 Charged Surface Hybrid [CSH] Particles Designed for LC/MS Use mobile phase additives at low ionic strength Formic acid versus TFA exposure Ideal for isolation/purification Superior peak shape for basic compounds at high mass load Use volatile additives versus ion-pairing reagents and buffers Rapid method development Prefer to work at low ph with occasional high ph work Switch back/forth between low & high ph (additives) 2012 Waters Corporation 23

24 Benefits of CSH Technology: Loading Comparison ACQUITY UPLC CSH C x 50 mm, 1.7 µm Quetiapine (base) Propiophenone (neutral) AU AU Fully porous silica C x 50 mm, 1.8 µm Quetiapine (base) Propiophenone (neutral) 0.0 AU Kinetex C x 50 mm, 1.7 µm Quetiapine (base) Propiophenone (neutral) Minutes 2012 Waters Corporation Comparative separations may not be representative of all applications 24

25 Ever-Growing Selectivity Range Ethylene Bridged Hybrid [BEH]: 2004 ACQUITY BEH C18 XBridge C Waters Corporation 25

26 Ever-Growing Selectivity Range Ethylene Bridged Hybrid [BEH]: 2005 ACQUITY BEH Phenyl XBridge Phenyl ACQUITY BEH C8 XBridge C8 ACQUITY BEH C18 XBridge C18 ACQUITY BEH Shield RP18 XBridge Shield RP Waters Corporation 26

27 Ever-Growing Selectivity Range [BEH + HSS]: 2008 ACQUITY HSS C18 SB XSelect HSS C18 SB ACQUITY BEH Phenyl XBridge Phenyl ACQUITY BEH C8 XBridge C8 ACQUITY BEH Shield RP18 XBridge Shield RP18 ACQUITY HSS T3 XSelect HSS T3 ACQUITY BEH C18 XBridge C18 ACQUITY HSS C18 XSelect HSS C Waters Corporation 27

28 Ever-Growing Selectivity Range [BEH + HSS + CSH]: 2010 ACQUITY HSS C18 SB XSelect HSS C18 SB ACQUITY CSH Fluoro-Phenyl ACQUITY BEH Phenyl XSelect CSH Fluoro-Phenyl XBridge Phenyl ACQUITY BEH C8 XBridge C8 ACQUITY CSH Phenyl-Hexyl XSelect CSH Phenyl-Hexyl ACQUITY BEH Shield RP18 XBridge Shield RP18 ACQUITY HSS T3 XSelect HSS T3 ACQUITY BEH C18 XBridge C18 ACQUITY HSS C18 XSelect HSS C18 ACQUITY CSH C18 XSelect CSH C Waters Corporation 28

29 Ever-Growing Selectivity Range [BEH + CSH + HSS]: 2011 ACQUITY HSS C18 SB XSelect HSS C18 SB ACQUITY HSS PFP XSelect HSS PFP ACQUITY HSS CN XSelect HSS CN ACQUITY CSH Fluoro-Phenyl ACQUITY BEH Phenyl Xselect CSH Fluoro-Phenyl XBridge Phenyl ACQUITY BEH C8 XBridge C8 ACQUITY CSH Phenyl-Hexyl XSelect CSH Phenyl-Hexyl ACQUITY BEH Shield RP18 XBridge Shield RP18 ACQUITY HSS T3 XSelect HSS T3 ACQUITY BEH C18 XBridge C18 ACQUITY HSS C18 XSelect HSS C18 ACQUITY CSH C18 XSelect CSH C Waters Corporation 29

30 Column Selectivity Choices Caffeic Acid Derivatives , BEH C 18 BEH C 8 BEH Shield RP18 BEH Phenyl 5 CSH Phenyl- Hexyl CSH Fluoro-Phenyl 5 CSH C 18 HSS CN Conditions : Columns: 2.1 x 50 mm Mobile Phase A: 0.1 % CF 3 COOH in H 2 O Mobile Phase B: 0.08 % CF 3 COOH in ACN Flow Rate: 0.5 ml/min Gradient: Time Profile Curve (min) %A %B Injection Volume: 1.0 µl Sample Diluent: 50:50 H 2 O: MeOH with 0.05 % CF 3 COOH Sample Conc.: 100 µg/ml Temperature: 40 o C Detection: 330 nm Sampling rate: 40 pts/sec Time Constant: 0.1 Instrument: Waters ACQUITY UPLC, with ACQUITY UPLC TUV HSS PFP HSS T3 HSS C 18 HSS C 18 SB Compounds 1. Caftaric acid 2. Chlorogenic acid 3. Cynarin 4. Echinacoside 5. Cichoric acid Minutes 2012 Waters Corporation Comparative separations may not be representative of all applications 30

31 Expanding Retention Capability: XSelect HSS/ACQUITY UPLC HSS PFP Provides chromatographer with multiple selectivity mechanisms including: hydrogen bonding dipole-dipole aromatic (pi-pi) hydrophobic (reversed-phase) Target Analyses: positional isomers, halogenated compounds and polar compounds Compared to Similar Fluorinated ligands: HSS PFP: LONGER retention of basic compounds CSH Fluoro-Phenyl: SHORTER retention of basic compounds HSS PFP exhibits TRUE PFP behavior 2012 Waters Corporation 31

32 Retention Comparison - Basic Compounds: CSH Fluoro-Phenyl vs. HSS PFP BASIC COMPOUNDS Conditions: Columns: 2.1 x 50 mm Mobile Phase A: 10 mm NH 4 COOH, ph 3.0 Mobile Phase B: MeOH Flow Rate: 0.4 ml/min Gradient: Time Profile (min) %A %B Inj. Volume: 1 µl Sample Diluent: H 2 O Temperature: 30 C Detection: 260 nm Sampling Rate: 40 points/sec Filter Response: Normal Instrument: ACQUITY UPLC Compounds 1. Aminopyrazine 2. Pindolol 3. Quinine 4. Labetalol 5. Verapamil 6. Diltiazem 7. Amitriptyline ACQUITY UPLC PDA 2 1 HSS PFP HSS PFP Retains Basic Compounds Longer than CSH Fluoro-Phenyl 2012 Waters Corporation Comparative separations may not be representative of all applications CSH Fluoro-Phenyl

33 Summary Waters has an ongoing commitment to developing highperforming column chemistries The XBridge columns family was developed to be the standard column for reliability and reproducibility The XSelect column family was developed for the method developer to maximize selectivity choice Continue to expect new products that are designed for purpose to solve the next generation LC challenges 2012 Waters Corporation 33

34 Agenda Column Manufacturing and Platform Definitions New HPLC & ACQUITY UPLC Column Chemistries New 2.5 µm extended Performance (XP) Columns Summary 2012 Waters Corporation 34

35 Maximize HPLC Productivity How to Best Use Existing HPLC Systems While Transitioning to UPLC Technology Increasingly, more organizations realize the business and scientific advantages UPLC Technology However, this technology shift has led companies to evaluate how to best utilize their existing HPLC instruments as they continue to invest in and transition to, newer UPLC systems. Smaller particles ( µm) Core-shell columns 2012 Waters Corporation 35

36 extended Performance 2.5 µm Columns Packed in ultra-low dispersion hardware to minimize band spreading Designed to withstand high pressure 4.6 mm ID capable of 9,000 PSI 2.1 & 3.0 mm IDs compatible with UPLC pressures Flexibility in configurations 2.1, 3.0 & 4.6 mm ID o 2.1 & 3.0 mm incorporating ecord technology 30, 50, 75, 100 & 150 mm lengths Packed with XBridge [BEH] and XSelect [CSH & HSS] 2.5 µm particles and chemistries 14 scalable stationary phases 2012 Waters Corporation 36

37 Challenges Still Arise: HPLC, UHPLC and UPLC What are the challenges? Need to utilize existing LC assets while improving throughput Method transfer between different LC platforms Selectivity choice for challenging separations Solvent cost reduction is minimal due to the requirement of mm ID columns to overcome system dispersion. Transition strategy between different LC platforms? Cannot transfer regulated methods without full revalidation Would like to reduce backpressure when possible 2012 Waters Corporation 37

38 Flexible Options to Improve LC Productivity 2012 Waters Corporation 38

39 Understanding Column Performance: Improving Separations in the Column Eddy dispersion (A-Term) Increase bed homogeneity o Improve column packing o Narrow particle size distribution Decrease particle size Axial Dispersion (B-Term) Increase speed of mobile phase B H = A+ + u Cu Mass transfer kinetics (C-Term) Increase diffusion speed (elevated temperature) Decrease diffusion distance (smaller particles) Limit diffusion distance (core-shell particles) 2012 Waters Corporation 39

40 Understanding Mass Transfer [Diffusion]: Influence of Particle Size Diffusion distance is shorter with decreasing particle size resulting in a narrower, more efficient, chromatographic band 2012 Waters Corporation 40

41 Understanding Mass Transfer [Diffusion]: Superficially Porous Particles What SPP producers claim : Diffusion distance is short because the analyte band can only diffuse into the porous layer of material Let s do the test! 2012 Waters Corporation 41

42 Understanding Performance: Fully-Porous vs. Core-Shell Particles Similar Efficiencies Linear Velocity, u [cm/sec] 2012 Waters Corporation 42

43 Understanding Performance: Fully-Porous vs. Core-Shell Particles Similar Backpressures 2012 Waters Corporation 43

44 Performance Comparison: Fully-Porous vs. Core-Shell Particles XSelect CSH C 18 XP 2.5 µm, 4.6 x 75 mm Imipramine concentration held constant at 0.5 mg/ml; 0.1 % formic acid moible phase Kinetex C µm, 4.6 x 75 mm Imipramine Imipramine 2012 Waters Corporation 44

45 Impact of Particle Size on Column Performance AU Tolmetin R s = 5.5 Naproxen Fenoprofen XSelect CSH C 18 XP 2.1 x 75 mm, 2.5 µm Flow rate = 0.54 ml/min Pressure = 5,300 PSI Peak Capacity = Minutes Indomethacin Diclofenac 40 % lower backpressure 1.7 µm ACQUITY UPLC columns provide 15 % improvement in resolution and peak capacity and 20 % higher sensitivity compared to XP 2.5 µm columns R s = 6.3 ACQUITY UPLC CSH C x 75 mm, 1.7 µm Flow rate = 0.54 ml/min AU Pressure = 9,200 PSI Peak Capacity = % increase in Rs and Pc 20 % higher sensitivity Minutes Both columns were run on an ACQUITY UPLC H-Class system 2012 Waters Corporation 45

46 Performance Comparison: Goldenseal Root Extract Particle size alone does not dictate separation performance. Core-Shell C µm 2.1 x 50 mm Interaction between stationary phase and analytes is critical. ACQUITY UPLC BEH C µm 2.1 x 50 mm Mobile Phase A: 0.1 % FA in H 2 O Mobile Phase B: 0.1 % FA in ACN Flow Rate: 0.6 ml/min Gradient: 0 to 40 % B in 6 min, hold Injection Volume: 0.5 µl Column Temperature: 30 C Detection: 210/254 nm Instrument: Waters ACQUITY UPLC ACQUITY UPLC TUV detector Sample: Goldenseal extract Column: 2.1 x 50 mm, 1.7 µm 1. Hydrastine 2. Canadine 3. Canadaline 4. Berberine 2012 Waters Corporation 46

47 Impact of Instrument Dispersion on Column Performance HPLC System XSelect CSH C 18 XP 4.6 x 75 mm, 2.5 µm AU Minutes Tolmetin Naproxen Fenoprofen Indomethacin Diclofenac ACQUITY UPLC H-Class XSelect CSH C 18 XP 4.6 x 75 mm, 2.5 µm Ultra-low UPLC instrument dispersion enhances column performance, even with larger 4.6 mm ID columns AU % decrease in peak width % increase in peak height Minutes 2012 Waters Corporation 47

48 Summary: Matching Column Configurations with LC Systems System HPLC UPLC Particle Size 3.5 µm, 5 µm XP 2.5 µm 1.7/1.8 µm UPLC, XP 2.5 µm, 3.5 µm, 5 µm 1.7/1.8 µm UPLC, XP 2.5 µm, 3.5 µm, 5 µm Routine Pressure < 4000 psi < 4000 psi < psi (H-Class) < psi (I-Class) Column ID 4.6 mm 4.6 mm 2.1 & 3.0 mm* 1.0 & 2.1 mm* Column Length 250 mm 75 mm 150 mm* 150 mm* (*) additional UPLC-based bioseparation column configurations also available 2012 Waters Corporation 48

49 Summary Waters ongoing commitment to high-quality consumables manufacturing ensures reproducible results year after year XP 2.5 µm columns bridge the gap between HPLC and UPLC and are compatible with HPLC, UHPLC and UPLC system platforms Wide range of scalable chemistries, column lengths and IDs Use 4.6 mm ID XP columns with Alliance HPLC systems and 2.1 mm ID XP and UPLC columns with ACQUITY UPLC systems Maximize the efficiency, performance and throughput of ANY LC system 2012 Waters Corporation 49

50 2012 Waters Corporation 50

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