2010 Waters Corporation

Transcription

1 UPLC Column Positioning Jurmala September 3-5 Anne Dyrdal CBU 2010 Waters Corporation

2 Waters Column Product History Styragel 1958 µbondapak ACQUITY UPLC BEH SunFire Columns Spherisorb SymmetryShield XTerra DeltaPak PrepPak Symmetry 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 XP 2.5 µm Columns Nova Pak ProteinPak TM Pico Tag TM Atlantis Atlantis T3 ACQUITY UPLC AccQTag TM ACQUITY UPLC HSS T3 BEH125 SEC AccQTag TM Ultra ACQUITY UPC 2 Symmetry 300 Atlantis HILIC Silica Prep OBD XBridge HILIC Intelligent Speed TM BioSuite NanoEase ACQUITY UPLC BEH200 SEC XSelect CSH HPLC columns ACQUITY CSH Columns Viridis SFC Columns ProteinPak High Rs IEX 2010 Waters Corporation 2

3 The Widest UPLC Column Offering Five particle substrates 130Å, 200Å and 300Å BEH [Ethylene Bridged Hybrid], HSS [High Strength Silica] and CSH [Charged Surface Hybrid] All are available in HPLC and UPLC particle sizes Wide and growing selection of column chemistries 15 stationary phases BEH 130Å C 18, C 8, Shield RP 18, Phenyl, HILIC and Amide BEH C 18 BEH C 8 BEH Phenyl BEH Shield RP18 BEH HILIC BEH 300Å C 18 and C 4 BEH 200Å SEC HSS C 18, T3, C 18 SB CSH C 18, Fluoro Phenyl and Phenyl Hexyl Proven application based solutions AAA, OST, PST, PrST and Glycan Transferability between HPLC and UPLC XBridge HPLC and ACQUITY UPLC BEH columns HSS HPLC and ACQUITY UPLC HSS columns XSelect HPLC and ACQUITY CSH columns BEH Amide HSS T3 HSS C 18 HSS C 18 SB CSH C 18 CSH Fluoro Phenyl VanGuard Pre columns ecord Technology CSH Phenyl Hexyl O O Si O O O Si O O O Si O F F F F F 2010 Waters Corporation 3

4 Positioning Attributes to select the right column Retentivity Stability, Low ph Selectivity Stability, high ph Polar Analyte Retention MS Bleed Loadability ph Drift Tolerant Peak Shape Low ph (Base) Re-Equilibration Ease 2010 Waters Corporation 4

5 Waters UPLC Column Platforms Three major column platforms Hybrid Columns ACQUITY UPLC BEH ACQUITY Q UPLC CSH Application Directed Column Chemistries High Added Value Products ACQUITY UPLC HSS T3 ACQUITY UPLC BEH HILIC ACQUITY UPLC BEH Amide High Purity Silica Columns ACQUITY UPLC HSS C 18 ACQUITY UPLC HSS C 18 SB ACQUITY UPLC HSS T3 ACQUITY UPLC PST ACQUITY UPLC PrST ACQUITY UPLC OST ACQUITY UPLC Glycan AccQ Tag Ultra UPLC ACQUITY UPLC BEH200 SEC ACQUITY UPLC BEH300 C 4 ACQUITY UPLC BEH300 C Waters Corporation 5

6 ACQUITY UPLC High Strength Silica (HSS) Particles Why develop a 100% silica UPLC particle? Different application needs require different selectivities Different particles impart selectivity differences Customers asked for: 1. A column that retains and separates small, water-soluble organic molecules HSS T3 Sep A column that offers superior peak shape, increased retentivity and superior acid stability HSS C 18 Jun A column that is truly different in terms of selectivity for basic compounds HSS C 18 SB Jan Waters Corporation 6

7 HSS [High Strength Silica ] Chemistries HSS C 18 High coverage, trifunctionally bonded C 18 [ph 1 8] Universal, high performance C18 Proprietary endcapping technique for superior peak shape and lifetime 1.8 µm UPLC HPLC 3.5 and 5 µm HSS C 18 SB [Selectivity for Bases] HSS T3 Low ligand density, trifunctionally bonded C 18 [ph 2 8] Non endcapped C 18 designed for silanophilic interactions and alternate selectivity with exceptional peak shape for bases Low ligand density, trifunctionally bonded C 18 [ph 2 8] Aqueous compatible C 18 chemistry with long column lifetimes Recommended for maximum polar compound retention by RPLC 2010 Waters Corporation 7

8 HSS [High Strength Silica ] Chemistries Positioning HSS C18 and HSS T3 : General purpose columns for low to neutral ph Alternate selectivity to BEH particles HSS C18 SB : Alternate selectivity,especially for basic compounds, to BEH particles HSS T3 : Enhanced RP retention ti for polar molecules l Direct scalability between UPLC Technology and HPLC (no prep) 2010 Waters Corporation 8

9 Waters UPLC Column Platforms Three major column platforms Hybrid Columns ACQUITY UPLC BEH ACQUITY Q UPLC CSH Application Directed Column Chemistries High Added Value Products ACQUITY UPLC HSS T3 ACQUITY UPLC BEH HILIC ACQUITY UPLC BEH Amide High Purity Silica Columns ACQUITY UPLC HSS ACQUITY UPLC PST ACQUITY UPLC PrST ACQUITY UPLC OST ACQUITY UPLC Glycan AccQ Tag Ultra UPLC ACQUITY UPLC BEH200 SEC ACQUITY UPLC BEH300 C 4 ACQUITY UPLC BEH300 C Waters Corporation 9

10 What is HILIC? HILIC Hydrophilic Interaction Chromatography Retention of highly polar analytes not retained by reversed-phase Less interference from matrix components Complementary selectivity to reversed-phase Polar pesticides retain more than hydrophobic interferences Enhanced sensitivity in mass spectrometry High organic mobile phases (> 80% ACN) promotes enhanced ESI-MS response Direct injection of high organic extracts without dilution Facilitates use of lower volume samples Improved sample throughput Direct injection of high organic eluates from PPT, LLE or SPE without the need for dilution or evaporation and reconstitution 2010 Waters Corporation 10

11 BEH HILIC Chemistries ACQUITY UPLC BEH HILIC Unbonded ethylene bridged hybrid particle Particle size: 1.7 µm [UPLC]; 2.5, 3.5 and 5 µm [HPLC] ph range: 1 9; Temp. limit: 45 C Exceptional peak shape and retention for polar basic analytes 1.7 µm UPLC ACQUITY UPLC BEH Amide Alternative selectivity for HILIC separations Particle size: 1.7 µm [UPLC]; 3.5 µm [HPLC] ph range: 2 11; Temp. limit: 90 C Designed for retention of polar acidic, neutral and basic compounds. Ideal column for carbohydrates and glycans HPLC 2.5, 3.5, 5 & 10 µm 2010 Waters Corporation 11

12 Selectivity Comparison BEH Amide vs. BEH HILIC AU ACQUITY UPLC BEH HILIC 2.1 x 100 mm, 1.7 µm 3 Compounds 1. Methacrylic Acid 2. Cytosine 3. Nortriptyline 4. Nicotinic Acid ACQUITY UPLC BEH Amide 2.1 x 100 mm, 1.7 µm AU Minutes Isocratic mobile phase of 90/5/5 ACN/EtOH/10 mm CH 3 COONH 4 in H 2 O with 0.02% CH 3 COOH, flow rate 0.5 ml/min, column temp. 25 C, 1.5 µl injection, 60 µg/ml each compound, sample diluent 75/25 ACN/MeOH, UV 210 nm 2010 Waters Corporation 12

13 Blueberry Jam on 1.7µm BEH Amide Column ACQUITY ELSD using 0.29mL/min at 35 C, Isocratic with 0.2%TEA in 75% ACN 1 P-Toluam mide Food Sugars (1mg/mL) Blueberry Jam Extract (5mg/mL) Minutes 1) Fructose, 2) Glucose, 3) Sucrose, 4) Maltose, 5) Lactose 2010 Waters Corporation 13

14 BEH [Ethylene Bridged Hybrid] HILIC Chemistries - Positioning For enhanced retention of polar compounds when RP columns do not work BEH Amide for alternate t selectivity it to BEH HILIC column and Sugar/Saccharides/Carbohydrates separation Direct scalability between UPLC Technology to HPLC to Prep (only for BEH HILIC) 2010 Waters Corporation 14

15 Waters UPLC Column Platforms Three major column platforms Hybrid Columns ACQUITY UPLC BEH C 18 ACQUITY UPLC BEH C 8 ACQUITY Q UPLC BEH Phenyl ACQUITY UPLC BEH Shield RP18 ACQUITY Q UPLC CSH Application Directed Column Chemistries High Added Value Products ACQUITY UPLC HSS T3 ACQUITY UPLC BEH HILIC ACQUITY UPLC BEH Amide ACQUITY UPLC PST ACQUITY UPLC PrST ACQUITY UPLC OST ACQUITY UPLC Glycan AccQ Tag Ultra UPLC ACQUITY UPLC BEH200 SEC ACQUITY UPLC BEH300 C 4 ACQUITY UPLC BEH300 C 18 High Purity Silica Columns ACQUITY UPLC HSS 2010 Waters Corporation 15

16 BEH [Ethylene Bridged Hybrid] Reversed Phase Chemistries BEH C18 Trifunctionally bonded C18 Widest ph range for maximum selectivity [ph 1 12] Superior peak shape and efficiency in buffered mobile phases BEH C8 Trifunctionally bonded C8 Wide ph range [ph 1 12] Lower hydrophobicity than C18 BEH Shield RP18 Monofunctionally bonded, embedded carbamate phase [ph 2 11] Alternate selectivity compared to alkyl chain columns 1.7 µm UPLC HPLC 2.5, 3.5, 5 & 10 µm BEH Phenyl Trifunctionally bonded C6 Phenyl Wide ph range [1 12] Complementary selectivity it for aromatic species 2010 Waters Corporation 16

17 BEH RP Chemistries - Positioning General purpose columns applicable to a broadest range of compound classes when buffers are exclusively l used where high ph stability is most important Direct scalability between UPLC Technology to HPLC to Prep Designed for ph Stability 2010 Waters Corporation 17

18 Industry Trends: Current State of Reversed-phase Separations Advances in stationary phase design Hybrid particle technology o Extended usable ph range [1-12] o Exceptional peak shape and efficiency o Rugged and reliable column life Sub 2 µm particle technology o Improvements in resolution, sensitivity and speed of analysis Pellicular [core-shell] particles Instrument platform of choice UltraPerformance LC with UV and mass spectrometry [UPLC/MS/[MS]] /[ o Requires volatile mobile phases Excludes typical UV-based buffers [i.e., phosphate buffers] o Preference towards low ionic strength additives[i.e., formic acid, acetic acid, ammonium hydroxide] Avoid preparation of buffers if possible 2010 Waters Corporation 18

19 Defining the Problem: Modern High Purity Stationary Phases Lack of selectivity choices for method development Poor mass loading of charged cationic [basic] solutes in low ionic strength additives under low ph mobile phases due to limited sample capacity High tailing factors Poor signal intensity Elution [retention] time shift after exposure to a higher ph mobile phase* 1 Irreproducible assay performance when performing method screening o Low/high ph switching with un-buffered mobile phases *1 Marchand, D.H., et al., J. Chromatogr. A 2003, 1015, Waters Corporation 19

20 Waters UPLC Column Platforms Three major column platforms Hybrid Columns ACQUITY UPLC BEH ACQUITY Q UPLC CSH C 18 ACQUITY UPLC CSH Phenyl Hexyl ACQUITY UPLC CSH Fluoro Phenyl Application Directed Column Chemistries High Added Value Products ACQUITY UPLC HSS T3 ACQUITY UPLC BEH HILIC ACQUITY UPLC BEH Amide ACQUITY UPLC PST ACQUITY UPLC PrST ACQUITY UPLC OST ACQUITY UPLC Glycan AccQ Tag Ultra UPLC ACQUITY UPLC BEH200 SEC ACQUITY UPLC BEH300 C 4 ACQUITY UPLC BEH300 C 18 High Purity Silica Columns ACQUITY UPLC HSS 2010 Waters Corporation 20

21 Charged Surface Hybrid (CSH ) Technology CSH Technology Charged Surface Hybrid bid 2010 Waters Corporation 21

22 CSH [Charged Surface Hybrid] Chemistries ACQUITY UPLC CSH C18 Mid/high coverage trifunctional C18 End capped High ph stable Superior peak shape for bases Lower retentivity for bases (vs. BEH C18) ph range: 1 11 ACQUITY UPLC CSH Fluoro Phenyl Most different selectivity (vs. CSH C18 & Phenyl Hexyl) Excellent reproducibility Not end capped Superior peak shape and lower retentivity for bases ph range: 1 8 ACQUITY UPLC CSH Phenyl Hexyl Trifunctional phenylhexyl End capped High ph stable Superior peak shape for bases Different selectivity (vs. BEH Phenyl) ph range: µm UPLC HPLC 3.5, 5 µm 2010 Waters Corporation 22

23 New Way to think about selectivity ph 3 ammonium formate/acetonitrile gradient g on BEH C 8 or CSH Fluoro-Phe enyl k R² = R² = k g on BEH C 18 (kg: gradient retention factor) Selectivity (S) Similar il selectivity i S-value = 6 Different selectivity S-value = 65 Higher S-values = larger selectivity differences 100* 1 R 2 BAA JET PCI without berberine and PSA 2010 Waters Corporation 23

24 Wide Selectivity Range for ACQUITY CSH Columns XBridge C18 3.5µm XSelect CSH C18 3.5µm 19 S XBr C18 11 XSelect / CSH Columns Were 0 XSelect CSH Fluoro-Phenyl l 3.5µm Designed for Selectivity S XBr C18 36 S XS CSH C XSelect CSH Phenyl-Hexyl 3.5µm S XBr C18 20 S XS CSH C S XS CSH Fluoro-Phenyl Minutes 2010 Waters Corporation 24

25 Improving LC Column performance : Unexpectedly High Tf For Basic Compounds Current State of Reversed-Phase Separations Unexpectedly high tailing factors for analytical mass loads of basic analytes in low ionic strength, low ph mobile phases due to mass overload Slow equilibration at low ph Retention time shifts in low ph mobile phases after exposure to high ph (e.g., > ph 7) mobile phases Introducing Charged Surface Hybrid (CSH ) Technology Achieving D.V. Performance McCalley / J. Chromatogr. in Acidic, A Low-Ionic-Strength 1075 (2005) Mobile Phases Tailing Factors and Loading Capacity for Bases µg on a 4.6 x 150 mm Retention Time Changes after High ph Exposure. Stability Acid stability Base Stability Overlaid chromatograms for 1.25, 0.25 and 0.05 µg nortriptyline in A= 0.02M formic acid in water ph 2.75, B= acetonitrile 0.04M formic acid in water 50:50 (v/v) on 4.6 mm x 150 mm column Waters Corporation 25

26 Implementing Mobile Phase ph Switching: Monitoring Column Performance Most systematic screening protocol in method development evaluates high and low ph mobile phases. Screen multiple columns and organic modifiers at ph 3 and ph 10 Stationary phase must be re-equilibrated equilibrated when exposed to a new set of conditions With low ionic strength mobile phases [i.e., formic acid, ammonium hydroxide], column performance [retention and selectivity] can change * 1 Slow surface equilibration at low ph Inconsistent selectivity can impact open access systems and method transfer *1 Marchand, D.H., et al., J. Chromatogr. A 2003, 1015, Waters Corporation 26

27 Low ph to High ph to Low ph Method Development N a m e P u rp le P u r p l e L C / M S T r a c e S e p : 0 4 : 5 8 L N B R e f Q C w e ll 2 :1 In le t m e th o d Q C : D io d e A r r a y R a n g e : e e % Formic Acid 1. 6 e + 2 New Column e AU 1. 2 e e e Amitript tyline 1. Low ph 6. 0 e e e Name Tony W Cook LNB Ref TM hph4 In le t m e th o d JSB JSB AU AU 1.3e+2 1.5e+2 1.2e+2 1.1e e+2 1.0e+2 1.0e+2 9.0e+1 8.0e+1 7.5e+1 7.0e+1 6.0e+1 5.0e+1 5.0e+1 2.5e+1 4.0e+1 3.0e+1 2.0e+1 1.0e T im e % 10 mmformic Ammonium Acid 0.61 Column Bicarbonate Previously Exposed New to Column High ph Purple LC/M S Trace 0.93 Amitrip ptyline Am mitriptyline Note: Increased RT & Peak Shape Loss for Amitriptyline Oct :36:37 w e ll 2 : : Diode Array Range: 1.891e+2 3: Diode Array Range: 1.333e+2 Metho od Develop ment 3. Low ph 2. High ph Time Tim e Waters Corporation 27

28 Implementing Mobile Phase ph Switching: Monitoring Column Performance Performance in 0.1% formic acid/acetonitrile gradient MAINTAINS Retention Time and Peak Shape After High ph Exposure. AU 0.18 XSelect CSH C µm Before high ph exposure After high h ph exposure AU Metoprolol Amitriptyline neutral phthalates h min 20% RT change 25% RT change 64% peak height loss 81% peak height loss Gemini-NX C 18 3 µm Before high ph exposure After high ph exposure Metoprolol Amitriptyline neutral phthalates min 2010 Waters Corporation 28

29 Why Haven t I Seen This Before? Remember: when using low ph buffers as part of your method development protocols (e.g., ammonium formate, sodium phosphate, etc.) these ph switching effects do not occur ph switching effects only occur when using acidic, low ionic strength additives ALL high ph-capable columns EXCEPT ACQUITY CSH and XSelect Columns exhibit this behavior 2010 Waters Corporation 29

30 CSH Technology: Controlled Surface Charge Yields High Performance AU AU Minutes ACQUITY CSH C µm 0.05 % formic acid Peak capacity = Fused Core C µm 0.05 % formic acid Peak capacity = 26 Column: 2.1 x 50 mm MP A: water MP B: acetonitrile MP C: 2% formic acid Gradient: 25-35%B in 2 min, 35 95%B from 2-3 min; [2.5% C held constant] Inj. Vol.: 5 µl Sample Conc.: 10 µg/ml Detection 254 nm Sampling rate: 20 Hz Filter: 0.1 sec System: ACQUITY UPLC H-Class with ACQUITY UPLC PDA and SQD Tricyclic antidepressants: 1. Doxepin 2. Desipramine 3. Imipramine 4. Nortriptyline 5. Amitriptyline 6. Trimipramine Minutes 2010 Waters Corporation 30

31 CSH [Charged Surface Hybrid] Chemistries - Positioning Use additives AND buffers Isolation/purification Prefer to work at low ph with occasional high ph work Switch back/forth between low & high ph (additives) LC/MS laboratory Seeking additional ACQUITY UPLC column selectivities Direct scalability between UPLC Technology to HPLC to Prep Designed for Selectivity 2010 Waters Corporation 31

32 Waters UPLC Column Platforms Three major column platforms Hybrid Columns ACQUITY UPLC BEH ACQUITY UPLC CSH Application Directed Column Chemistries High Added Value Products ACQUITY UPLC HSS T3 ACQUITY UPLC BEH HILIC ACQUITY UPLC BEH Amide ACQUITY UPLC PST ACQUITY UPLC PrST ACQUITY UPLC OST ACQUITY UPLC Glycan AccQ Tag Ultra UPLC ACQUITY UPLC BEH200 SEC ACQUITY UPLC BEH300 C 4 ACQUITY UPLC BEH300 C 18 High Purity Silica Columns ACQUITY UPLC HSS 2010 Waters Corporation 32

33 Bioseparation Columns Sub 2 µm particle specifically designed For UPLC QC Tested with relevant biomolecules to achieve unmatched: Sensitivity i i Resolution Method Reproducibility Characterization of: Amino Acids Peptides Proteins Glycans Synthetic oligonucleotides 2010 Waters Corporation 33

34 Bioseparation Columns ACQUITY UPLC BEH200 SEC Column Determines aggregation levels for monoclonal antibodies up to 10X faster than HPLC SEC methods Fully optimized i column chemistry Eliminates high salt concentration mobile phases QC tested with relevant proteins, unmatched batch-to-batch b h consistency increased confidence in validated QC release methods Analyte pi MW 1. Thyroglobulin, 3 mg/ml , IgG, 3 mg/ml (Vicam) , BSA, 5 mg/ml , Myoglobin, 2 mg/ml 6.8, , Uracil, 0.1 mg/ml N/A Minutes 2010 Waters Corporation 34

35 Bioseparation Columns Protein Separation Technology: BEH 300Å C 4 and C Å pores C 4 and C 18 Ligands available Minimal undesirable secondary interactions Minimal detectable carryover at elevated temperature separations E D BEH 300Å C 18 A B C F BEH 300Å C 4 A B C D E F A. Ribonuclease B. Cytochrome c C. BSA D. β-lactoglobulin E. Enolase F. Phosphorylase b Minutes Shorter chain gives narrower peaks, particularly for the later eluting components Waters Corporation 35

36 Tranferability from UPLC Technology to HPLC 2010 Waters Corporation 36

37 UPLC and HPLC Column Equivalency UPLC 1.7/ 1.8 µm HPLC ACQUITY UPLC BEH C 18 XBridge C 18 ACQUITY UPLC BEH C 8 XBridge C 8 ACQUITY UPLC BEH Shield RP18 ACQUITY UPLC BEH Phenyl ACQUITY UPLC BEH HILIC ACQUITY UPLC BEH Amide = = = = = = = = = = = = = = XBridge Shield RP18 XBridge Phenyl XBridge HILIC XBridge Amide ACQUITY UPLC BEH300 C 18 XBridge BEH300 C 18 ACQUITY UPLC BEH300 C 4 XBridge BEH300 C 4 ACQUITY UPLC HSS C 18 HSS C 18 ACQUITY UPLC HSS C 18 SB ACQUITY UPLC HSS T3 HSS C 18 SB HSS T3 ACQUITY UPLC CSH C 18 XSelect C 18 ACQUITY UPLC CSH Phenyl Hexyl ACQUITY UPLC CSH Fluoro Phenyl XSelect Phenyl Hexyl XSelect Fluoro Phenyl 2010 Waters Corporation 37

38 Introducing CORTECS Columns Featuring 1.6 µm Solid-Core Particles 2010 Waters Corporation 38

39 Introducing CORTECS Columns herald an unprecedented level of efficiency and sensitivity for UPLC columns. Featuring 1.6 μm silica-based solid-core particles and ultra-low dispersion hardware, CORTECS UPLC Columns enable higher peak capacities and thereby greater resolution and improved throughput to meet even the most challenging demands Waters Corporation 39

40 CORTECS Columns UPLC Columns featuring 1.6 µm solid-core silica particles Key benefits: Highest efficiency UPLC Column (>35% vs fully porous sub-2-µm columns) Improved performance at similar backpressure Increased throughput 3 Chemistries: C18+ C18 HILIC 2010 Waters Corporation 40

41 10 Year Evolution of UPLC Particle Technology BEH Technology First ACQUITY UPLC particle Wide ph and temperature range HSS Technology Enhanced retention Particle and ligand selectivity CSH Technology Designed for selectivity Improved basic peak shape at low ph Waters Corporation 41

42 10 Year Evolution of UPLC Particle Offering BEH Technology HSS Technology CSH Technology CORTECS Solid-Core Technology First ACQUITY UPLC particle Enhanced retention Designed for selectivity Increased efficiency and resolution Wide ph and temperature range Particle and ligand selectivity Improved basic peak shape at low ph Higher throughput What allowed us to take solid-core particle technology to the next level of performance? 2010 Waters Corporation 42

43 The CORTECS Solid-Core Particle Compared to Fully Porous Particles CORTECS Solid-core d core = 1.12 µm d p = 1.6 µm ρ = core diameter / particle diameter Only thin outer layer contains the pores with the chromatographic surface The center core is nonporous The outer shell is typically bumpy not pretty The particles size distribution is narrower Rho, = 1.12/1.60 = % Porous Volume ρ = 0 fully yporous particle ρ = 1 nonporous particle 2010 Waters Corporation 43

44 Enabling Expertise Differentiating Advancements in UPLC Columns Bulk Synthesis Rugged sub-2-µm solid-core particles Highest Efficiencies Engineering UPLC column hardware Low band broadening Column Packing Ultra-stable packed column beds Proprietary packing processes Software Paperless tracking of column history with ecord TM technology 2010 Waters Corporation 44

45 Where we were Since 2004, fully porous ACQUITY UPLC 1.7 µm BEH C 18 Has been our most efficient particle Started us down the path of sub-2-µm particles and the development of UPLC Technology, which was needed to demonstrate its efficiency Plates (4 sigma) 16,000 12,000 8,000 14,150 ACQUITY UPLC 1.7 µm BEH C 18 4, Flow Rate (ml/min) 2.1 x 50 mm column. A standard ACQUITY UPLC I-Class using 70% Acetonitrile in H 2 O at 30 C with 0.5 µl injections from a 1 µl FL injector 2010 Waters Corporation 45

46 However, as of today 20, ,700 39% higher efficiency 4 sigma) 16,000 12,000 14,150 or up to 3x faster! Plates ( 8,000 ACQUITY UPLC 1.7 µm BEH C 18 CORTECS UPLC 1.6 µm C , Flow Rate (ml/min) 2.1 x 50 mm column. A standard ACQUITY UPLC I-Class using 70% Acetonitrile in H2O at 30 C with 0.5 µl injections from a 1 µl FL injector 2010 Waters Corporation 46

47 CORTECS Chemistry Overview Chemistry C 18 + C 18 HILIC Intended Use General purpose, high-efficiency, reversed-phase column. A positively charged surface delivers excellent peak shape for basic compounds at low ph. General purpose, high-efficiency, reversed-phase column. Balanced retention of acids, bases and neutrals at low and mid-range ph. High-efficiency column designed for retention of extremely polar analytes. Offers orthogonal selectivity vs. C 18 columns. Ligand Type Trifunctional C 18 Trifunctional C 18 None Surface Charge Modification + None None Endcap Style Proprietary Proprietary None Carbon Load 5.7% 6.6% None Ligand Density 2.4 µmol/m µmol/m 2 None ph Range Temperature Limits Low ph = 45 C High ph = 45 C Low ph = 45 C High ph = 45 C Low ph = 45 C High ph = 45 C 2010 Waters Corporation 47

48 Increased Efficiency of CORTECS Columns 2010 Waters Corporation 48

49 Practical Benefits of Increased Efficiency Benefits of higher efficiency separations include: Improved peak shape and resolution Higher sample throughput: o Comparable separations using faster flow rates or o Comparable separations using a shorter column 2010 Waters Corporation 49

50 Higher Efficiency leads to Sharper Peaks, Better Resolution of Local Anesthetics Lidocaine 2. Butacaine 3. Tetracaine 4. Procaine 5. Procainamide ACQUITY BEH HILIC 2.1 x 50mm 1.7 µm AU 0.10 USP Resolution 2,3 : CORTECS UPLC HILIC 2.1 x 50 mm 1.6 µm AU 0.10 USP Resolution 2,3 : Minutes 2010 Waters Corporation 50

51 Higher Throughput Sulfa Drugs: Double the Flow Rate AU Sulfathiazole Competitor Fully-porous C 18 at 0.5 ml/min 2. Sulfamerazine 2.1 x 50 mm 1.8 µm Sulfamethazine Gradient time: 4.2 min 4. Sulfamethoxypyridazine Runtime: 6 min 5. Sulfachloropyridazine Sulfamethoxazole 7. Sulfasoxazole 5 P c = peaks per minute (gradient) Comparable peak capacities (Pc) in half the time CORTECS UPLC C 18 + at 1.0 ml/min 2.1 x 50 mm 1.6 µm Gradient time: 2.1 min Runtime: 3 min P c = peaks per minute (gradient) Note: the gradient is scaled to account for the change in flow rate Comparative separations may not be representative in all applications Waters Corporation 51

52 Higher Throughput Sulfa Drugs: Using a Shorter Column 2.1 x 100 mm 1.7 µm Competitor Solid-Core C 18 Gradient time: 8.4 min Runtime: 12 min P c = peaks per minute (gradient) Comparable peak capacities (Pc) in 2.1 x 50 mm 1.6 µm CORTECS UPLC C 18 + half the time Gradient time: 4.2min Runtime: 6 min P c = peaks per minute (gradient) Note: the gradient is scaled to account for the change in column length Comparative separations may not be representative in all applications Waters Corporation 52

53 Impact of System Dispersion on CORTECS Column Efficiency ACQUITY UPLC I-Class 5.5 µl USP N: 18,000 ACQUITY UPLC H-Class 12 µl USP N: 11,700 53% Increase in CORTECS Column Efficiency on the ACQUITY UPLC I Class System Acetonitrile/ Water (70/30 v/v), 0.4 ml/ min, 30 o C, 0.5 µl injection. Peak i.d.: Acetone, Naphthalene, Acenaphthene 2.1 x 50 mm CORTECS C 18 Column 2010 Waters Corporation 53

54 Summary CORTECS Column family features 1.6 µm solid-core silica particles Enabled by over 10 years of designing, synthesizing and packing sub-2-µm particles Set the bar as the new efficiency standard for UPLC columns Three unique chemistries for selectivity and exceptional peak shape 2010 Waters Corporation 54

55 Summary Higher efficiency leads to improved resolution and faster throughput Lowest dispersion systems result in ultimate efficiency and performance CORTECS 1.6 µm particle size chosen to maximize efficiency while pairing seamlessly with UPLC systems Most recent addition to an ever-evolving evolving sub-2-µm UPLC particle family 2010 Waters Corporation 55

56 2010 Waters Corporation