Nuevos Desarrollos en Columnas ACQUITY UPLC & HPLC

Transcription

1 Nuevos Desarrollos en Columnas ACQUITY UPLC & HPLC Bàrbara Bagó Bilbao, 12 Julio Waters Corporation 1

2 Agenda Column Manufacturing and Platform Definitions New HPLC & ACQUITY UPLC Column Chemistries Upcoming Changes to USP Chromatography <621> New 2.5 µm extended Performance (XP) Columns Summary 2011 Waters Corporation 2

3 What makes an LC column reproducible? A) Sorbents B) Packing C) Engineering µm ACQUITY UPLC BEH C µm XBridge BEH C 18 XP Plate Height (H, 4 sigma) 2. 6 µm Core-Shell C Acenaphthene, 2.1 x 50 mm columns 70/30 MeCN/H 2O, 30 C, 254 nm Linear Velocity, u (cm/sec) 2011 Waters Corporation 3

4 Developing a Waters Column: cgmp Registration All Waters manufacturing facilities are registered with the FDA for the manufacture and distribution of class I medical devices, and are ISO 9001:2000 certified The GMP Quality Systems Regulations cover such items as Quality management and organization Production and process controls Packaging and labeling controls Document and record controls As part of the registration process, the FDA may inspect the facilities every two years for compliance Milford MA, USA: Hardware Manufacture Taunton, MA, USA: Particle Synthesis Wexford, Ireland: Device Manufacturing 2011 Waters Corporation 4

5 Developing a Waters Column Hardware Materials Design Optimization Sorbents Chemistries Synthesis Packing Equipment Considerations Optimization Product QC and testing 2011 Waters Corporation 5

6 Developing a Waters Column: Particle Synthesis and Bonding Manufacture of a particle through bonding takes between 4-6 months Each step of the synthesis process quality controlled 2011 Waters Corporation 6

7 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 2011 Waters Corporation 7

8 Developing a Waters Column: Final QC and Inspection Each column Passes: Efficiency testing using a test mixture 2011 Waters Corporation 8

9 Developing a Waters Column: Final QC and Inspection Each column Passes: Efficiency testing using a test mixture COA tracking and QC checks of product conformity All column ecord information is verified against original manufacturing data and testing 2011 Waters Corporation 9

10 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 2011 Waters Corporation 10

11 Agenda Column Manufacturing and Platform Definitions New HPLC & ACQUITY UPLC Column Chemistries Upcoming Changes to USP Chromatography <621> New 2.5 µm extended Performance (XP) Columns Summary 2011 Waters Corporation 11

12 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 HSS C 18 and HSS C 18 SB XBridge ACQUITY UPLC HSS Cyano/PFP columns XSelect TM HSS Cyano/PFP columns 2.5 µm XP columns Nova-Pak ProteinPak TM Pico-Tag TM AccQTag TM Atlantis Atlantis T3 ACQUITY UPLC HSS T3 AccQTag TM Ultra Symmetry 300 Atlantis HILIC Silica Prep OBD Intelligent Speed TM BioSuite NanoEase XBridge HILIC ACQUITY UPLC BEH200 SEC XSelect CSH HPLC columns ACQUITY CSH Columns Viridis SFC Columns ProteinPak High Rs IEX 2011 Waters Corporation 12

13 Begins with Two Fully-Scalable HPLC 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 and 10 µm 1.7 [UPLC], 2.5, 3.5 and 5 µm CSH 1.8 [UPLC], 2.5, 3.5 and 5 µm HSS 2011 Waters Corporation 13

14 Column Particle Platform Definitions BEH Particle Ethylene-Bridged Hybrid 130Å, 200Å and 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 2011 Waters Corporation 14

15 Begin with Two Fully-Scalable HPLC 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 and 10 µm 1.7 [UPLC], 2.5, 3.5 and 5 µm CSH 1.8 [UPLC], 2.5, 3.5 and 5 µm HSS 2011 Waters Corporation 15

16 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 2011 Waters Corporation 16

17 Ethylene Bridged Hybrid [BEH] Particles U.S. Patent No. 6,686,035 B2 Bridged Ethanes within a silica matrix Anal. Chem. 2003, 75, Waters Corporation 17

18 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 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 2011 Waters Corporation 19

20 Begin with Two Fully-Scalable HPLC 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 and 10 µm 1.7 [UPLC], 2.5, 3.5 and 5 µm CSH 1.8 [UPLC], 2.5, 3.5 and 5 µm HSS 2011 Waters Corporation 20

21 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 2011 Waters Corporation Comparative separations may not be representative of all applications 21

22 Column Particle Platform Definitions BEH Particle Ethylene-Bridged Hybrid 130Å, 200Å and 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 2011 Waters Corporation 22

23 [CSH] and [HSS] Chemistries FOUR general-purpose C 18 columns for low to neutral ph reversed-phase separations CSH C18: Rapid ph switching for method development 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 NEW HSS PFP and HSS 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 2011 Waters Corporation 23

24 Ever-Growing Selectivity Range High Strength Silica [HSS] Particles First silica-based particle (1.8 μm) designed for UPLC applications Fully scalable manufacturing process enabled HPLC particle sizes (2.5, 3.5 and 5 μm) Available with FIVE bonded phases HSS particles benefits: Alternate selectivities (vs. BEH & CSH particle columns) Increased retention Seamless transferability Highest pressure tolerance 5 μm 3.5 μm 2.5 μm 1.8 μm 2011 Waters Corporation 24

25 Column Particle Platform Definitions BEH Particle Ethylene-Bridged Hybrid 130Å, 200Å and 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 2011 Waters Corporation 25

26 Ever-Growing Selectivity Range Charged Surface Hybrid [CSH] Particles Step 1 Step 2 Step 3 CHARGED SURFACE HYBRID PROCESS 2011 Waters Corporation 26

27 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 2011 Waters Corporation 27

28 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) 2011 Waters Corporation 28

29 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 2011 Waters Corporation Comparative separations may not be representative of all applications 29

30 Ever-Growing Selectivity Range Ethylene Bridged Hybrid [BEH]: 2004 ACQUITY BEH C18 XBridge C Waters Corporation 30

31 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 31

32 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 32

33 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 33

34 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 34

35 New XSelect/ACQUITY UPLC Column Chemistries Two Robust and Fully Scalable Chemistries that Increase the Selectivity Range of our XSelect HPLC & ACQUITY UPLC Column Families HSS PFP Exceptional selectivity and retention for positional isomers, halogenated compounds and polar compounds HSS CN Stable, NP-compatible, reproducible CN chemistry available in HPLC and 1.8 µm (UPLC) particle sizes 2011 Waters Corporation 35

36 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 2011 Waters Corporation 36

37 Retention Comparison - Basic Compounds: CSH Fluoro-Phenyl vs. HSS PFP BASIC COMPOUNDS Conditions: Columns: 2.1 x 50 mm Mobile Phase A: 10mM 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 2011 Waters Corporation Comparative separations may not be representative of all applications CSH Fluoro-Phenyl

38 Selectivity at ph 3.0: Ligands that improve chromatographic diversity XSelect HSS PFP HSS PFP: Increased Retentivity vs. CSH Fluoro-Phenyl Selectivity XSelect HSS CN XBridge Phenyl XSelect CSH Phenyl-Hexyl XSelect CSH Fluoro-Phenyl XSelect HSS C 18 SB XBridge C 8 XBridge C 18 XSelect HSS T3 XSelect CSH C 18 XSelect HSS C 18 XBridge Shield RP18 Hydrophobicity 2011 Waters Corporation 38

39 PFP Column Performance Comparison Basic Compound Mixture ACQUITY UPLC HSS PFP, 1.8 µm, 2.1 X 50mm 3 RPH Ave = Compounds 1. Trimethoprim 2. Nordoxepin 3. Doxepin 4. Nortriptyline 5. Imipramine 6. Amitriptyline 7. Trimipramine Phenomenex Kinetex PFP, 1.7 µm, 2.1X50 mm RPH Ave = System: ACQUITY UPLC H-Class with ACQUITY UPLC PDA Detector Mobile Phase A: 10mM NH4COOH, ph3.0 Mobile Phase B: ACN Isocratic Mobile Phase: 60:40; A:B Flow Rate: 0.5 ml/min Injection Volume: 1 µl Column Temperature: 30 C UV Detection: 254 nm Waters Corporation Comparative separations may not be representative of all applications 39

40 XSelect HSS/ACQUITY UPLC HSS CN: A Phase for Non-Polar Compounds Provides the chromatographer with a phase for: Low hydrophobicity for rapid elution of hydrophobic analytes Unique selectivity Normal-phase compatible Chromatographic benefits: Significantly less retention than C18 (typically requires lower % organic mobile phase) Compatible with highly aqueous phases Stable, low-bleed in LC-MS separations 2011 Waters Corporation 40

41 Reversed-Phase Retention: Steroids Separation XSelect HSS CN 5 µm 2.1 x 150 mm Hydrocortisone 2. Corticosterone 3. β - Estradiol 4. Progesterone Isocratic: 40% ACN 0.20 XSelect HSS CN 3.5 µm 2.1 x 100 mm ACQUITY UPLC HSS CN 1.8 µm 2.1 x 50 mm Waters Corporation 41

42 Normal-Phase Retention: Steroids Separation XSelect HSS CN 5 µm 2.1 x 150 mm Hydrocortisone 2. Corticosterone 3. β - Estradiol 4. Progesterone 0.15 XSelect HSS CN 3.5 µm 2.1 x 100 mm Minutes ACQUITY UPLC HSS CN 1.8 µm 2.1 x 50 mm Isocratic: 83% Hexane / 5% DCM / 12% IPA 2011 Waters Corporation 42

43 3 Particles and 14 Chemistries ACQUITY UPLC, XBridge, XSelect Ligand Type Available Particle Sizes Ligand Density Carbon Load Endcap Style ph range Trifunctional C ,2.5,3.5,5, µmol/m 2 18% Proprietary 1-12 Trifunctional C 8 1.7,2.5,3.5,5, µmol/m 2 13% Proprietary 1-12 Monofunctional Embedded Polar Group 1.7, 2.5, 3.5, µmol/m 2 17% TMS 2-11 Trifunctional C 6 Phenyl 1.7,2.5,3.5,5, µmol/m 2 15% Proprietary 1-12 Unbonded 1.7, 2.5, 3.5, Trifunctional Amide 1.7, 2.5, 3.5, µmol/m 2 12% None 2-11 Trifunctional C , 2.5, 3.5, µmol/m 2 11% Proprietary 2-8 Trifunctional C , 2.5, 3.5, µmol/m 2 15% Proprietary 1-8 Trifunctional C , 2.5, 3.5, µmol/m 2 8% None 2-8 Trifunctional Propylfluorophenyl 1.8, 2.5, 3.5, µmol/m 2 7.5% None 2-8 Launch Date Monofunctional Diisopropyl Cyano 1.8, 2.5, 3.5, µmol/m 2 5.5% None 2-8 Trifunctional C , 2.5, 3.5, µmol/m 2 15% Proprietary 1-11 Trifunctional Propylfluorophenyl 1.7, 2.5, 3.5, µmol/m 2 10% None 1-8 Jan 2008 Trifunctional C 6 Phenyl 1.7, 2.5, 3.5, µmol/m 2 14% Proprietary Waters Corporation 43

44 What is HILIC? HILIC - Hydrophilic Interaction Chromatography Term coined in 1990 to distinguish from normal-phase* HILIC is a variation of normal-phase chromatography without the disadvantages of using solvents that are not miscible in water Reverse reversed-phase or aqueous normal-phase chromatography Stationary phase is a POLAR material Silica, hybrid, cyano, amino, diol, amide The mobile phase is highly organic (> 80% ACN) with a smaller amount of aqueous mobile phase Water (or the polar solvent(s)) is the strong, eluting solvent *Alpert, A. J. J.Chromatogr. 499 (1990) Waters Corporation 44

45 Benefits of HILIC Retention of highly polar analytes not retained by reversed-phase Less interference from non-polar matrix components Complementary selectivity to reversed-phase Polar metabolites/impurities/degradants retain more than parent compound Enhanced sensitivity in mass spectrometry High organic mobile phases (> 80% ACN) promotes enhanced ESI-MS response Direct injection of PPT supernatant without dilution Facilitates use of lower volume samples Improved sample throughput Direct injection of high organic extracts from PPT, LLE or SPE without the need for dilution or evaporation and reconstitution 2011 Waters Corporation 45

46 When To Use HILIC ESI-MS Response excellent poor polar HILIC Reversed-phase Normal-phase non-polar Compound Index When to Use HILIC: Need improved retention of hydrophilic or ionizable compounds Need improved MS response for polar or ionizable compounds Need improved sample throughput for assays using organic extraction 2011 Waters Corporation 46

47 Stationary Phases for HILIC separations Hybrid HILIC Columns (ph range 1 11) ACQUITY UPLC BEH Amide XBridge TM Amide ACQUITY UPLC BEH HILIC XBridge TM HILIC Silica HILIC Columns (ph range 1 5) Atlantis HILIC Silica 2011 Waters Corporation 47

48 Waters HILIC screening strategy ACQUITY UPLC BEH HILIC 2.1 x 50 mm, 1.7 µm ph 3 ph 9 ACQUITY UPLC BEH Amide 2.1 x 50 mm, 1.7 µm Atlantis HILIC Silica 2.1 x 50 mm, 3 µm Optimization Where do I start? Initial scouting gradient from 95 to 50% acetonitrile over 5 minutes At least 5% should be a polar solvent (i.e., water or methanol) 2011 Waters Corporation 48

49 Implementing the Approach: Example 1, Water Soluble Vitamins N NH 2 HO N CH 3 OH H 3 C HO N OH OH OH N O N O O O H 3 C N O NH OH Nicotinamide Pyridoxal Riboflavin Nicotinic acid O H 3 C N CH 3 N + N NH S 2 Thiamine Cl - OH HO HO HO Ascorbic acid O OH O H 3 C H 2 N H 2 N H 2 N O P O - O O H 3 C NH O H 3 C O H 3 C O OH H OH O N N CH 3 Co + CH 3 N N H 3 C N N N CH 3 CH 3 O NH 2 O NH 2 CH 3 CH 3 O NH 2 H 2 N N N H O N N Folic Acid NH O HO HO O NH O B Waters Corporation 49

50 Stationary Phase Selectivity at Low ph: Water-soluble Vitamins 1 BEH Amide ph All 3 columns yield different selectivity 1,6 4 5 BEH HILIC BEH Amide has greatest resolution of all peaks 3, , Minutes 7 Atlantis HILIC Silica Compounds 1. Nicotinamide 50 µg/ml 2. Pyridoxine 50 µg/ml 3. Riboflavin 30 µg/ml 4. Nicotinic acid 50 µg/ml 5. Thiamine 50 µg/ml 6. Ascorbic Acid 50 µg/ml 7. B12 50 µg/ml 8. Folic Acid 25 µg/ml 2011 Waters Corporation 50

51 Commercial Food Samples Using XBridge BEH Amide Column XBridge BEH Amide, 3.5µm, 4.6 x 250mm Isocratic: 75% ACN with 0.2% TEA 1.0mL/min, 15 µl injection volume Strawberry Smoothie Hot Cross Buns White Bread Lamb Curry Meal Food Sugar Standard Minutes 1) p-toluamide 2) Fructose 3) Glucose 4) Sucrose 5) Maltose 6) Lactose 2011 Waters Corporation 51

52 Agenda Column Manufacturing and Platform Definitions New HPLC & ACQUITY UPLC Column Chemistries Upcoming Changes to USP Chromatography <621> New 2.5 µm extended Performance (XP) Columns Summary 2011 Waters Corporation 52

53 What is the USP? The United States Pharmacopeia (USP) is an independent, official public standards-setting authority Prescription and OTC medicines Healthcare products Food ingredients Dietary supplements The USP-NF is the official authority FDA-enforceable standards Enforcement of USP standards is the responsibility of FDA and other government authorities in the U.S. and elsewhere USP has no role in enforcement The U.S. Federal Food, Drug, and Cosmetics Act designates the USP NF as the official compendia for drugs marketed in the United States 2011 Waters Corporation 53

54 The Beginning of A Significant Change to Chromatography <621> Chromatography <621> describes the adjustments allowed in the chromatography system when system suitability test fails In early 2009, Waters was approached to write a Stimuli Article to modernize/add more flexibility to selecting LC columns in Chromatography <621> In the summer of 2009 Dr. Uwe Neue et. al. wrote a paper (article) that basically describes UPLC technology and method transfer Dr. Neue s calculations are also the basis of the ACQUITY UPLC Columns Calculator 2011 Waters Corporation 54

55 The Beginning of A Significant Change to Chromatography <621> Stimuli Article PF 35(6) [Nov-Dec 2009] Are you aware of this Article? What does the Stimuli Article propose? 2011 Waters Corporation 55

56 What is the PF 35(6) [Nov-Dec 2009] Stimuli Article? Stimuli Article PF 35(6) [Nov-Dec 2009] allows the flexibility to change the column dimensions and/or particle size as long as an equivalent (or better) separation is achieved The ability to change LC columns in a method was restrictive and outdated What if the prescribed column is no longer available? What if a faster (more modern) separation can be obtained with another column (e.g., UPLC)? When particle size and/or diameter of the column is changed in the method, the flow rate may need adjustment Waters Corporation 56

57 Allowable Adjustments in Chromatography <621> Variable Allowable Changes Before Stimuli Article PF 35(6) After Stimuli Article PF 35(6) Particle Size -50% Any allowed as long as column length to Column Length ±70% particle size (l/dp) is maintained (±25%) Flow Rate ±50% Adjust for length, particle size and column ID Column ID Any allowed Any allowed Injection Volume Any reduction Any allowed Column Temperature ±10% ±10% Mobile Phase ph ±0.2 unit ±0.2 unit When Stimuli Article PF 35(6) [Nov-Dec 2009] Officially Becomes Part of Chromatography <621>, Analysts Will Have More Flexibility to Utilize Modern Chromatographic Techniques (e.g., UPLC technology) 2011 Waters Corporation 57

58 Effect of Stimuli Article PF 35(6) [Nov- Dec 2009] Change OVERVIEW: Adjustments in column length, internal diameter, particle size, and flow rate can be used in combination to give equivalent conditions (same N), but with differences in pressure and run time. The table below lists some of the more popular column configurations to give equivalent efficiency (N), by adjusting these variables. 1 Column Length (mm) Column ID (mm) Particle Size (dp, µm) Relative Values l/dp F N Pressure Run Time , , , , , , , , , EXAMPLE: If a monograph specifies a 4.6 x 150 mm, 5-µm column operated at 1.5 ml/min, the same separation may be expected with a 2.1 x 75 mm, 2.5-µm column operated at 1.5 ml/min x 0.4 = 0.6 ml/min, along with a pressure increase of about 4 times and a reduction in run time to about 30% of the original Waters Corporation 58

59 How Might These Changes Benefit Chromatographers and Organizations? Chromatographic Benefits More flexibility to change the column dimensions and/or particle size as long as an equivalent (or better) separation is achieved A more restrictive, science-based approach to change a column or method that requires an equivalent separation (performance) be obtained Organizational Benefits Analyses can continue even if the prescribed column is no longer available A faster, greener and more sustainable separation can be obtained using a more modern separation technique 2011 Waters Corporation 59

60 What Does This Revision Status Change Mean? It means: Sufficient changes to revised General Chapter <621> Chromatography were made that require public comment (again) Comment period will begin 01-Mar-2012 in PF 38(2) No changes to <621> will occur in 2012 extended Performance (XP) Columns are compliant with current <621> guidelines NOW 2011 Waters Corporation 60

61 Agenda Column Manufacturing and Platform Definitions New HPLC & ACQUITY UPLC Column Chemistries Upcoming Changes to USP Chromatography <621> New 2.5 µm extended Performance (XP) Columns Summary 2011 Waters Corporation 61

62 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 and 3.0 mm IDs compatible with UPLC pressures Flexibility in configurations 2.1, 3.0 and 4.6 mm ID (2.1 and 3.0mm incorporating ecord technology) 30, 50, 75 and 100 mm lengths 14 scalable stationary phases Packed with XBridge [BEH] and XSelect [CSH and HSS] 2.5 µm particles and chemistries BEH C 18, Shield RP18, C 8, Phenyl, HILIC and Amide CSH C 18, Phenyl-Hexyl and Fluoro-Phenyl HSS C 18, T3, C 18 SB, Cyano and PFP 2011 Waters Corporation 62

63 Improved Productivity AU Tolmetin Naproxen Fenoprofen Indomethacin Diclofenac XSelect CSH C x 100 mm, 3.5 µm HPLC System 3.5 µm Minutes AU XSelect CSH C 18 XP 4.6 x 75 mm, 2.5 µm HPLC System 2.5 µm XP X Faster than 3.5 µm HPLC Minutes AU ACQUITY UPLC CSH C x 50 mm, 1.7 µm UPLC System 1.7 µm X Faster than 3.5 µm HPLC 5X Faster than 2.5 µm HPLC Minutes Waters Corporation 63

64 Resolution, Throughput and Sensitivity: Comparing 2.5 µm XP and 1.7 µm UPLC Columns AU Tolmetin Naproxen Fenoprofen Indomethacin Diclofenac XSelect CSH C 18 XP 2.1 x 75 mm, 2.5 µm Flow rate: 0.54 ml/min 1.7 µm ACQUITY UPLC columns provide 2X increased speed and 20% higher sensitivity compared to 2.5 µm XP columns while maintaining selectivity AU Minutes ACQUITY UPLC CSH C x 50 mm, 1.7 µm Flow rate: 0.8 ml/min Minutes A: water with 0.1% formic acid B: acetonitrile with 0.1% formic acid NSAID mixture ( ug/ml) UV detection at 270 nm Gradient (2.5µm): 35%B to 65%B over 4.85 min, hold for 0.66 min, reequilibrate at 35%B. Gradient (1.7µm): 35%B to 65%B over 2.20 min, hold for 0.3 min, reequilibrate at 35%B. Both columns were run on an ACQUITY UPLC H-Class system 2011 Waters Corporation 64

65 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 = 40 15% increase in Rs and Pc 20% higher sensitivity Minutes Both columns were run on an ACQUITY UPLC H-Class system 2011 Waters Corporation 65

66 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 2011 Waters Corporation 66

67 Efficiency vs. Flow Rate Observations: Plate Count (4 sigma) µm ACQUITY UPLC BEH C µm XBridge BEH C 18 XP 2.6 µm Core-Shell C 18 When plotting efficiency vs. flow rate, it is clear that a smaller particle size will always perform better than a larger particle size 2.5 µm fully-porous and 2.6 µm core-shell particles provide the same efficiency 2000 Acenaphthene, 2.1 x 50 mm columns Flow Rate (ml/min) What about back pressure? 70/30 MeCN/H 2 O, 30 C, 254 nm 2011 Waters Corporation 67

68 Back Pressure Comparison: Fully Porous vs. Core-Shell µm XBridge BEH C 18 XP Observations: Pressure (psi) µm Core-Shell C µm fully-porous and 2.6 µm superficially porous produce the same back pressure Flow Rate (ml/min) 2.1 x 50 mm columns 70/30 MeCN/H 2 O, 30 C, 254 nm 2011 Waters Corporation 68

69 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 2011 Waters Corporation 69

70 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 2011 Waters Corporation 70

71 Resources that Enable Success: Online Tools Column selection tools and advisors 2011 Waters Corporation 71

72 Resources that Enable Success: Columns Selectivity Chart Quickly compare columns based on: Retention Selectivity Similarity USP Designation Map method development kits based on: Waters MD kits Non-Waters columns Also available as a web-based tool Waters Corporation 72

73 Resources that Enable Success: Waters Column Advisor Make informed decisions based on: Method requirements Column attributes Choose recommended columns based on your most important criteria Visit: Waters Corporation 73

74 Resources that Enable Success: Technology Primers Four easy to read and understand books covering the basic concepts of: Waters Corporation 74

75 Resources that Enable Success: LC Columns Wall Chart Easily compare and relevant column data based on Ligand type Usable ph range Temperature limits Carbon Load Contains data for both HPLC and all UPLC column configurations EN 2011 Waters Corporation 75

76 Resources that Enable Success: LC Column Family Brochures XP Columns XBridge Columns XSelect Columns ACQUITY UPLC Columns EN EN EN EN 2011 Waters Corporation 76

77 Adding a New Business Competency 2011 Waters Corporation 77

78 WATERS ANALTYICAL STANDARDS & REAGENTS An Introduction Waters Corporation Waters Corporation Confidential COMPANY CONFIDENTIAL

79 Benefits To Laboratory Increased Productivity Save time on preparation by using pre-made, ready-to-use products Achieve consistent, repeatable results and reduce assay re-runs Add reassurance to your analyses through consistent practice and trending Maintain optimum performance for equipment and improve system up-time Eliminate raw material sourcing, stocking, storage, and inventory management Minimize variations across instruments, users, labs, site locations, geographies Enhanced Analytical Confidence Reassurance in your analytical results by using Waters products manufactured, traceable, and certified to exacting specifications. Confidence in reproducibility as Waters products offer consistency from vial to vial and batch to batch. Built-In Compliance Our products are developed, manufactured and documented to the highest accreditation levels in a state-of-the-art custom built facility. o ISO 9001, ISO/IEC 17025, ILAC Guide 13, ISO 43-1, ISO Guide 34 Standardizing your processes with Waters fully compliant products will simplify your compliance burdens 2011 Waters Corporation 79

80 The Products 2011 Waters Corporation 80

81 Thank You For Your Time and Attention 2011 Waters Corporation 81

82 APPENDIX: ADDITIONAL SLIDE RESOURCES AND INFORMATION 2011 Waters Corporation 82

83 Columns First range of sub-2-µm column chemistries Designed and tested for the ACQUITY UPC 2 system High efficiency Speed Resolution ACQUITY UPC 2 Columns offered in 3.5 µm column chemistries Maximum flexibility to tackle challenging routine analyses Maximize plate counts and selectivity 2011 Waters Corporation 83

84 Why SFC? Reduce Solvent Costs Better, Faster & Easier Chiral Separations Need for Orthogonal Separations Increase Speed & Throughput Issues with Range of Polarity Limit Exposure to Toxic Solvents Green Chemistry 2011 Waters Corporation 84

85 ACQUITY UPC 2 Columns BEH 2-EP (2-Ethylpyridine) Good retention, peak shape and selectivity BEH Heightened interaction with polar groups such as phospholipids CSH Fluoro-Phenyl Good retention of weak bases Alternate elution orders for acidic and neutral compounds HSS C 18 SB Analysis of glycerides across vertical markets (Pharmaceutical, Food, Chemical Materials) 2011 Waters Corporation 85

86 Different Selectivity Coumarin 2. Flavone 3. Caffeine 4. Thymine 5. Papaverine 6. Sulfamethoxazole 7. Cytosine 8. Sulfamethizole BEH 2-EP BEH CSH Fluoro-Phenyl Column: 4.6 x 150mm, 5 µm 5-40% MeOH without additive 3.0mL/min at 40 C, 150 bar Injection Volume: 3µL Minutes 2011 Waters Corporation 86

87 Batch Testing All ACQUITY UPC 2 Columns have SFC batch test results reported on the Certificate of Analysis (CoA) All ACQUITY UPC 2 Columns have an attached e-cord 2011 Waters Corporation 87

88 HILIC COLUMN CHEMISTRIES 2011 Waters Corporation 88

89 Stationary Phases for HILIC separations Hybrid HILIC Columns (ph range 1 11) ACQUITY UPLC BEH Amide XBridge TM Amide ACQUITY UPLC BEH HILIC XBridge TM HILIC Silica HILIC Columns (ph range 1 5) Atlantis HILIC Silica 2011 Waters Corporation 89

90 Waters HILIC screening strategy ACQUITY UPLC BEH HILIC 2.1 x 50 mm, 1.7 µm ph 3 ph 9 ACQUITY UPLC BEH Amide 2.1 x 50 mm, 1.7 µm Atlantis HILIC Silica 2.1 x 50 mm, 3 µm Optimization Where do I start? Initial scouting gradient from 95 to 50% acetonitrile over 5 minutes At least 5% should be a polar solvent (i.e., water or methanol) 2011 Waters Corporation 90

91 Implementing the Approach: Example 1, Water Soluble Vitamins N NH 2 HO N CH 3 OH H 3 C HO N OH OH OH N O N O O O H 3 C N O NH OH Nicotinamide Pyridoxal Riboflavin Nicotinic acid O H 3 C N CH 3 N + N NH S 2 Thiamine Cl - OH HO HO HO Ascorbic acid O OH O H 3 C H 2 N H 2 N H 2 N O P O - O O H 3 C NH O H 3 C O H 3 C O OH H OH O N N CH 3 Co + CH 3 N N H 3 C N N N CH 3 CH 3 O NH 2 O NH 2 CH 3 CH 3 O NH 2 H 2 N N N H O N N Folic Acid NH O HO HO O NH O B Waters Corporation 91

92 Stationary Phase Selectivity at Low ph: Water-soluble Vitamins 1 BEH Amide ph All 3 columns yield different selectivity 1,6 4 5 BEH HILIC BEH Amide has greatest resolution of all peaks 3, , Minutes 7 Atlantis HILIC Silica Compounds 1. Nicotinamide 50 µg/ml 2. Pyridoxine 50 µg/ml 3. Riboflavin 30 µg/ml 4. Nicotinic acid 50 µg/ml 5. Thiamine 50 µg/ml 6. Ascorbic Acid 50 µg/ml 7. B12 50 µg/ml 8. Folic Acid 25 µg/ml 2011 Waters Corporation 92

93 Carbohydrate Analysis Using the XBridge Amide Column 2011 Waters Corporation 93

94 Commercial Food Samples Using XBridge BEH Amide Column XBridge BEH Amide, 3.5µm, 4.6 x 250mm Isocratic: 75% ACN with 0.2% TEA 1.0mL/min, 15 µl injection volume Strawberry Smoothie Hot Cross Buns White Bread Lamb Curry Meal Food Sugar Standard Minutes 1) p-toluamide 2) Fructose 3) Glucose 4) Sucrose 5) Maltose 6) Lactose 2011 Waters Corporation 94