Intelligent RP LC Column Selection & New Technologies Peru October 2016 Ricardo Martínez

Intelligent RP LC Column Selection & New Technologies Peru ctober 2016 Ricardo Martínez 2016 Waters Corporation 1

Agenda LC Fundamentals Columns Quality Particles & Ligands Column Efficiency ph in LC New Tech Columns Summary & Questions 2016 Waters Corporation 2

Escitalopram (ral Solution) Mobile Phase: Buffer 6.1g of monobasic potassium phosphate. To each L of this solution add 1.5 ml of triethylamine. Adjust with phosphoric acid to ph of 2.5. Mobile phase ACN/Buffer (32:68). Column: 4.6 x 250 mm, 5 um, column packed with L1 2016 Waters Corporation 3

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Chromatogram Characteristics Resolution Sensitivity Precision Time Analysis Method mplicity Cost 2016 Waters Corporation 5

Tswett's Experiment (1903) Tall glass open column filled with sand-like particles (alumina, or chalk) Ground-up plant extract Poured into the column and saw colored bands develop as the extract percolated down thru the column Different compounds had separated Different Colored Bands Greek Chroma Graphy -- color -- writing/study of Note: Tswett in Russian means Color 2016 Waters Corporation 6 6

Chromatography Applications pharmaceutical proteomics clinical food safety environmental 2016 Waters Corporation 7

HPLC System for Purification 2016 Waters Corporation 8

Identification and Quantitation Compound Identification Based on Retention Time Acrylamide at 2.85 minutes 2016 Waters Corporation 9

2998 Photodiode Array Detector Features: Spectral Analysis Library Matching Spectral analysis algorithms can distinguish closely related compounds providing reliable library matching. Benefit: Peak Identification Peak 3 - Butalbarbital Library Match - Butalbarbital 2016 Waters Corporation 10

LC/MS/MS Instrument 2016 Waters Corporation 11

AU 286.1 Imp. F 342.0 Imp. A 316.0 Imp. G 300.1 - API 182.1 Imp. 9 196.0 Imp. H 202.0 Imp. C 224.1 Imp. D 258.0 Imp. B PDA-MS Detection 0.04 ACQUITY Arc System 0.02 0.00 0.00 1.50 3.00 4.50 6.00 7.50 9.00 10.50 12.00 13.50 15.00 Minutes Imp. F 213.5 272.8 311.0 API 213.5 272.8 309.1 Imp. A 212.9 263.0 306.0 Imp. G 211.0 271.5 307.9 UV Spectra 286.1 300.1 302.0 342.0 316.0 MS Spectra 288.1 343.9 317.9 Apex Apex Apex Apex 2016 Waters Corporation 12

Identification and Quantitation 10X Area Count Sample A has 10X the concentration of Acrylamide 2016 Waters Corporation 13

Normal vs Reversed-Phase Chromatography Normal Phase Reversed-Phase Stationary Phase Un-bonded lica (Polar) Surface Non-Polar Ligand (C18) Bonded to lica Surface Packing Polarity Polar Non-Polar Mobile Phase Polarity Non-Polar Polar Elution rder Most Non-Polar First Most Polar First Effect of Increasing Mobile Phase Polarity Reduces Retention Time Decreases Retention Time 2016 Waters Corporation 14

Waters Column Product History µbondapak Styragel Spherisorb DeltaPak PrepPak 1958 SymmetryShield XTerra Symmetry ACQUITY UPLC BEH SunFire Columns XTerraPrep XBridge ACQUITY UPLC BEH Amide ACQUITY UPLC BEH Glycan XBridge Amide XSelect HSS HPLC Columns ACQUITY UPLC HSS C 18 and HSS C 18 SB ACQUITY UPLC HSS Cyano & PFP columns XSelect TM HSS Cyano & PFP columns XP 2.5 µm Columns 1984 1992 1998 2002 2006 2008 2010 2012 2013 1964 1973 1976 1979 1986 1994 1999 2003 2004 2005 2007 2009 2011 Nova-Pak ProteinPak TM Pico-Tag TM AccQTag TM Symmetry 300 Atlantis Atlantis HILIC lica Prep BD Intelligent Speed TM BioSuite NanoEase Atlantis T3 ACQUITY UPLC HSS T3 AccQTag TM Ultra BEH130 Columns BEH300 Columns XBridge HILIC ACQUITY UPLC BEH200 SEC XSelect CSH HPLC columns ACQUITY CSH Columns Viridis SFC Columns ProteinPak High Rs IEX ACQUITY UPLC BEH125 SEC ACQUITY UPC 2 Columns CRTECS Columns ACQUITY UPLC BEH450 SEC ACQUITY APC Columns CSH130 Columns 2016 Waters Corporation 15

Quality Systems Minimize Risk with Dependable Column Performance Method Validation kits provide three batches of chromatographic media [derived from different base particles] to judge the quality, reliability and consistency of an analytical method Waters uniquely positioned as an industry partner to minimize risk 2016 Waters Corporation 16

Benchmarking System Performance Principle of benchmarking: Use of a Quality Control Reference Material (QCRM) to evaluate or check key performance criteria by comparison with data generated when the system is known to be in good working order Importance of benchmarking Routine use on the analytical system and control charting the data allows for an understanding of the capability of your system to provide reliable results and is a useful troubleshooting tool. Typical Criteria 1. Retention time range or reproducibility 2. Peak area range or reproducibility 3. Peak tailing range 4. Peak resolution 5. Response 6. System Pressure 2016 Waters Corporation 17

What is a lanol Group? H H H H Comes from the silica gel particle (substrate) used to make reversed-phase packing materials 2016 Waters Corporation 18

lica Gel Pore Structure -H = lanol 2016 Waters Corporation 19

Making a Bonded Phase Material: Monofunctional Synthesis H + H 3 C Cl C CH 3 C8 lane Ligand C C C C C C CH 3 lica Gel Surface H 3 C C CH 3 C C C C C C + HCl CH 3 2016 Waters Corporation 20

C18 Bonded lica Gel Pore H 3 C CH 3 CCCCCCCCCCCCCCCCCC 25 Å Steric Hindrance 2016 Waters Corporation 21

C18 Bonded and Fully Endcapped lica Gel Pore H 3 C CH 3 CCCCCCCCCCCCCCCCCC 25 Å Steric Hindrance H 3 C CH 3 CH 3 Endcap (2 nd Bonding Step) What do you still see? lanols! 2016 Waters Corporation 22

Surface of a lica Gel Bonded-Phase Packing Material H 3 C C 8 alkyl chains H 3 C H 3 C H 3 C H 2 C CH 2 H 2 C CH 2 H 2 C CH 2 H 2 C CH 2 H 2 C CH 2 H 2 C CH 2 H 2 C CH 2 H 2 C CH 2 CH 2 CH 2 H 2 C H 2 C H 2 C residual silanol CH 2 CH 3 CH 2 H 3 C CH H H 3 C H 3 C 3 C 3 CH CH 3 3 H H H H CH 2 CH 2 H 2 C CH 2 endcap CH 2 H 3 C CH 3 CH 3 CH 3 H 3 C CH H 3 H H Note: ~ 30-50% of the surface silanols remain even with high bonding densities 2016 Waters Corporation 23

Why Do You See Poor Peak Shape? Answer: Ionization of lanols Surface silanol charge changes with mobile phase ph H H+ Behaves as a Cation Exchanger (ph 2) (ph 7) Result: Strong interaction (cation exchange) between ionized surface silanols (negative charge) and ionized basic analytes (positive charge) lanols are weak acids 2016 Waters Corporation 24

RP-Ion exchange Hydrophobic Interaction with Bonded Phase Mobile Phase ph < 3 - H - - H - - H - H - - - H - - + HN (CH 3 ) 2 Ion exchange Interaction with Charged tes High lanol Activity - - - - - - - - - - - - -- - + (CH) 2 HN 3 Mobile - Phase ph > 5 N Substrate Protonated - no charge Substrate De-protonated -- Negative Charge Base RP Cation X Base Increased retention and PR Peak Shape SAME CLUMN 2016 Waters Corporation 25

AU Why not just run at ph s less than 3? Selectivity 0.04 0.02 0.00 ph 2.0 0.00 2.00 4.00 6.00 8.00 10.00 Minutes A nortriptyline amitriptyline = A ph 7.0 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 A 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 A 2016 Waters Corporation 26

Different lica Gel Manufacturing Processes Precipitation of sodium silicate: (irregular particles)-µbondapak Sodium silicate polymerized to silicic acid (silica gel) then ground to fine particles SolGel 1 Process: (spherical particles)- Nova-Pak Colloidal silica spheres fused into chromatographic particle lgel 1 Process: (spherical particles)-symmetry Pure silanes polymerized to form chromatographic particle 2016 Waters Corporation 27

Types of Surface lanols Found on lica Gel Vicinal (Bridged) H H Geminal (lanediol) H H H Lone (most active) H 2016 Waters Corporation 28

Acidic Compounds:Two Different C18 s (Different Brands Same Ligand, cont.) Suprofen Ketoprofen and Tolmetin Note: Different selectivity due to different silica particle. Naproxen Col Brand A DS Suprofen Ketoprofen Naproxen Tolmetin Col Brand B DS 4 5 6 7 8 9 10 Minutes 2016 Waters Corporation 29

Aluminum in the lica Gel Lattice Bronsted Acid 3D top view of silica particle surface with silanols pointing upward H H Al H Metal available for chelation 2016 Waters Corporation 30

Correlation Between Base Tailing and Aluminum Content of lica Gel Analyte: Chlorpheniramine Mobile Phase: Acetonitrile/KH 2 P 4 ph 3.0 (20:80) Tailing Factor 4 3 2 "High Purity lica Gel" Region 1 0 100 200 300 400 Aluminum Content, ppm 2016 Waters Corporation 31

Peak Shapes of Chelating Agent (Hinokitiol) Low metals M = metal M n+ High metals 2 4 6 8 Minutes 2016 Waters Corporation 32

Test Compounds for the Measure of Residual lanol Activity N + Amitriptyline pka = 9.4 Basic Compound Acenaphthene Neutral Compound 2016 Waters Corporation 33

Impact of Choosing Two Different C18 Brands on Peak Shape for a Base Note: Different brands (different silica particle) have different silanol behaviors. Waters SunFire C18 Amitriptyline Waters Spherisorb DS2 0 10 20 30 40 50 60 70 80 90 Minutes Note: ph plays a role in peak shape 2016 Waters Corporation 34

Pore ze Most silica gel packings are porous >99% of the surface area is contained within the particle (not on the surface)- Where the chromatography happens. Rules of Thumb The smaller the pore size, the greater the surface area. o (100 Å approx. 300 m 2 /gram) o (300 Å approx. 100 m 2 /gram) The greater the surface area, the greater the retention. A typical 15 cm column holds a surface area of ~100-300 square meters 2016 Waters Corporation 35

lica Gel Pore Structure Analyte MW Pore ze Recommendation < 3,000 60-130 Å (6-13 nm) 3,000 10,000 125-200 Å (12.5-20 nm) >10,000 300 1,000 Å (30-100 nm) Very Large Non-porous Analyte molecule Analyte needs to fit inside a pore to interact with chromatographic surface -H = lanol Note: Pore ze is a distribution 2016 Waters Corporation 36

Selectivity ln [α] amitriptyline/acenaphthene) Reversed-Phase Column Selectivity Chart 3.6 Waters Spherisorb S5 P 3.3 3 2.7 2.4 2.1 1.8 1.5 1.2 0.9 0.6 0.3 0-0.3-0.6 Waters Spherisorb S5CN Nova-Pak CN HP ACQUITY UPLC BEH Phenyl XBridge Phenyl Inertsil Ph-3 Hydrophobicity (ln [k] acenaphthene) ACQUITY UPLC HSS PFP XSelect HSS PFP ACQUITY UPLC HSS C18 SB XSelect HSS C18 SB Waters Spherisorb DS1 Resolve C18 Hypersil Phenyl ACQUITY UPLC CSH Fluoro-Phenyl ACQUITY UPLC BEH C18 XSelect CSH Fluoro-Phenyl XBridge C18 Waters Spherisorb DS2 ACQUITY UPLC HSS CN µbondapak C18 ACQUITY UPLC HSS T3 XSelect HSS CN YMC-Pack YMC J'sphere XSelect HSS T3 Phenyl DS L80 Nucleosil C18 Hypersil CPS Cyano Inertsil CN-3 Nova-Pak Phenyl CRTECS C18+ Hypersil BDS Phenyl YMC J'sphere DS M80 Chromolith Nova-Pak YMC J'sphere DS H80 XTerra YMCbasic RP-18 C18 YMC-Pack DS AQ Phenyl YMC-Pack CN Nova-Pak Luna YMC-Pack Pro C4 Atlantis dc18 C8 Phenyl Hexyl Zorbax XDB C18 YMC-Pack Pro C8 Atlantis T3 ACQUITY UPLC BEH C8 XTerra MS C8 ACT Ace C18 Symmetry C8 YMC-Pack DS-A XBridge C8 Luna Luna C18 (2) C8 (2) YMC-Pack Inertsil DS-3 Pro C18 ACQUITY UPLC CSH Phenyl-Hexyl SunFire C18 SunFire C8 XTerra MS C18 XSelect CSH Phenyl-Hexyl Symmetry C18 SymmetryShield RP8 Zorbax SB C18 XTerra RP18 ACQUITY UPLC BEH Shield RP18 SymmetryShield RP18 XBridge Shield RP18 ACQUITY UPLC CSH ACQUITY UPLC HSS C18 XTerra RP8 XSelect CSH C18 XSelect HSS C18 YMC-Pack PolymerC18-1.5-0.5 0.5 1.5 2.5 3.5 CRTECS C18 2016 Waters Corporation 37

Creating a New Particle Inorganic (licon) Advantages Mechanically strong High efficiency Predictable retention Disadvantages Limited ph range Tailing peaks for bases Chemically unstable Polymer (Carbon) Wide ph range No ionic interactions Chemically stable Mechanically soft Low efficiency Unpredictable retention Hybrid (licon-carbon) Particle Technology 2016 Waters Corporation 38

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1 st Generation Hybrid (Methylloxane/lica) Particles Methyl Groups on Hybrid Surface (Better Peak Shape) and in Hybrid Particle (High ph Life-time) Waters Patented technology US Patent: 6,686,035 B2 Date of Patent: Feb. 3, 2004 MethylPolyethoxysilane (MPES) Tetraethoxysilane (TES) Methyltriethoxysilane (MTES) 2016 Waters Corporation 40

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Ethylene Bridged Hybrid [BEH] Particle U.S. Patent No. 6,686,035 B2 Bridged Ethanes within a silica matrix Anal. Chem. 2003, 75, 6781-6788 2016 Waters Corporation 43

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The Resolution Equation N α 1 k Rs 4 α k 1 Mechanical Contributions Ultra-low dispersion system perate at optimal linear velocity Particle morphology Small particles Well-packed columns Chemical/Physical Contributions Complementary bonded phases Multiple particle substrates Ability to utilize high ph Increase retentivity 2016 Waters Corporation 45

Column Length and Mechanical Separating Power* Columns contain the same packing material, same particle size and same mobile phase, only one is twice as long Additional column length does provide a better separation. However, several costs are incurred: more time (2X) for the analysis, use more solvent, increased back pressure and the longer column costs more to buy. A better approach, would be to try a different particle chemistry/mobile phase combination or a smaller particle size that can create the separation in less time. * This is also called Efficiency 2016 Waters Corporation 47

Particle ze and Mechanical Separating Power* Columns contain the same packing material chemistry, are the same length with the same mobile phase. ne column has particles which are a third the size. Smaller particle sizes provide for a better separation with the same run time. However, back pressure will increase. * This is also called Efficiency 2016 Waters Corporation 48

N = Theoretical Plates A Measure of Efficiency 2 Variances Peak Width/Band Spread = 2 extra column + 2 column Analyte Band Flowing Through Detector Cell The narrower the peak (W), the higher the Plate Count, N Elution Volume Tangents 2016 Waters Corporation 49

Calculation of Column Efficiency 5 Method N 5 18.70 25 1.03 2 V R 16.63 8240 plates V R 18.70 N 5 16. 63 25 1. 44 2 3334 plates 4.4% height W 1.03 Good Column 4.4% height W 1.44 Bad Column 2016 Waters Corporation 50

Waters Particle Technology 60 μm Human Hair (very fine hair) 5 μm Analytical Particles (can fit 12 across hair) Images are on same scale (Bar = 10 μm) 1.7 μm ACQUITY UPLC Particles ptimal Particle ze Distribution For Max Efficiency at a given Pressure 2016 Waters Corporation 51

Plate Count (4 sigma) Efficiency vs. Flow Rate Acenaphthene, 2.1 x 50 mm columns 70/30 MeCN/H 2, 30 C, 254 nm 16000 14000 12000 1.7 µm ACQUITY UPLC BEH C 18 2.5 µm XBridge C 18 2.6 µm Superficially Porous C 18 10000 8000 6000 4000 2000 0 0.0 0.5 1.0 1.5 2.0 2.5 Flow Rate (ml/min) 2016 Waters Corporation 52

Plate Height (H, 4 sigma) van Deemter Curves Plate Height (Non-reduced, H) 25 Acenaphthene, 2.1 x 50 mm columns 70/30 MeCN/H 2, 30 C, 254 nm 1. 7 µm ACQUITY UPLC BEH C 18 20 2. 5 µm XBridge C 18 2. 6 µm Superficially Porous C 18 15 10 5 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Linear Velocity, u (cm/sec) 2016 Waters Corporation 53

UPLC Technology & The Fundamental Resolution Equation Rs N 4 Physical ( α 1)( k ) α k 1 Chemical In UPLC systems, increasing N (efficiency) is the primary focus Selectivity and retentivity are the same as in HPLC Resolution, Rs, is proportional to the square root of N Rs N If N 3x, Rs 1.7x 2016 Waters Corporation 54

AU AU Constant Column Length Flow Rate Proportional to Particle ze 0.050 0.040 0.030 0.020 0.010 0.000 0.050 0.040 0.030 0.020 0.010 0.000 1.7 µm, 0.6 ml/min, 7656 psi 0.00 1.00 2.00 3.00 4.00 5.00 Minutes 4.8 µm, 0.2 ml/min, 354 psi 0.00 2.00 4.00 6.00 8.00 10.00 12.00 Minutes 2016 Waters Corporation 55 6.00 15.00 Reality 1.5X Resolution 2.6X Faster 1.4X Sensitivity 22X Pressure Theory 1.7X Resolution 3X Faster 1.7X Sensitivity 25X Pressure 2.1 x 50 mm columns Very High System Pressure

Resolution (and Speed) Constant Column Length Plates, Flow Rate and Particle ze Rs N 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 Smaller Particle ptimal Larger Particle Smaller Particle ze *Increased N, *Higher, optimal u *Increased pressure 1.0 2.0 3.0 Flow Rate {ml/min} ptimal flow rate is inversely proportional to dp F opt 1 dp Isocratic analysis time is inversely proportional to F dp 3X, N 3X, Rs 1.7X, T 3X (e.g., 5 μm to 1.7 μm) (Rs α N) 2016 Waters Corporation 56

Speed Increases Constant L/dp Efficiency, N, is directly proportional to column length, L, and inversely proportional to particle size, dp: N L dp For same N and, therefore, same Rs dp 3X, L 3X, N = 1X, Rs = 1X, (e.g., 5 μm to 1.7 μm) (e.g., 150 mm to 50 mm) F 3X, T 9X (i.e., F increases 3X, L decreases 3X) Efficiency & Resolution Remain Unchanged 2016 Waters Corporation 57

AU AU Length Proportional to Particle ze milar L/dp 0.06 0.04 0.02 0.00 0.06 0.04 0.02 0.00 1.7 µm, 30 mm, 0.6 ml/min 0.00 1.00 2.00 3.00 4.00 Minutes 4.8 µm, 100 mm, 0.2 ml/min 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes 2.1 mm ID columns Reality Same Resolution 8X Faster 2.5X Sensitivity 11X Back Pressure Theory Same Resolution 9X Faster 3X Sensitivity 9X Back Pressure Manageable Backpressure Increase 2016 Waters Corporation 58

Ratio of Column Length-to-Particle ze: Maintaining Separation Power Typical HPLC Column 4.6 x 150 mm, 5 µm Column length (L) = 150 mm = 150,000 µm d p = 5 µm L = 150,000 = 30,000 d p 5 Separation Index Application Example Efficiency (N) L/d p Easy Content Uniformity 5,000 15,000 Moderately Challenging Related compound assay 12,000 30,000 Difficult Impurity profiling 20,000 50,000 Extremely Difficult Metabolite identification 35,000 85,000 2016 Waters Corporation 59

30 mm 50 mm 30 mm 75 mm 50 mm 30 mm 100 mm 75 mm 50 mm 30 mm 150 mm 100 mm 75 mm 50 mm L/dp Ratio 150 mm 100 mm 75 mm 250 mm 150 mm 100 mm 250 mm 150 mm Ratio of Column Length to Particle ze Resolution Capability 100000 90000 80000 50 mm, 1.7 µm 75 mm, 2.5 µm 100 mm, 3.5 µm 150 mm, 5 µm L/d p ~ 30,000 Extremely Difficult 70000 60000 50000 40000 Difficult Impurity 30000 Moderately Challenging 20000 10000 Easy 0 5.0 3.5 0.005 0.0035 0.0025 0.0017 particle size (µm) 2.5 1.7 2016 Waters Corporation 60

Improved Productivity Well packed columns containing smaller particles have more intrinsic efficiency than columns packed with larger particles. N α L dp The nce Typical 4.6 x 100 mm, 3.5 µm Column L = 100,000 µm = 28,571 d p 3.5 µm Replaced with 2.1 x 50 mm, 1.6 µm solid-core Column L = 50,000 µm = 31,250 d p 1.6 µm 2016 Waters Corporation 61

Proper Method Transfer ACQUITY CSH Phenyl-Hexyl 50 mm, 1.7 µm α 3,5 = 1.07 1 2 3 5 4 6 UPLC 0.00 1.00 2.00 3.00 4.00 5.00 XSelect CSH Phenyl-Hexyl 100 mm, 3.5 µm α 3,5 = 1.06 1 2 3 5 4 6 HPLC 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 XSelect CSH Phenyl-Hexyl 150 mm, 5 µm α 3,5 = 1.07 1 2 3 5 4 6 HPLC/Prep 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 2016 Waters Corporation 62

Defining the LC Categories by their System Dispersion How are these categories differentiated? Chromatographic Resolution Increases verall Run Time Decreases Method Sensitivity Increases 2016 Waters Corporation 63

What is at the Root of the Performance Differences across the LC Categories? Dispersion n. Broadening of an analyte band due to both on-column effects (diffusion and mass transfer kinetics which are both dependent on particle size and linear velocity) and system effects (tubing internal diameter (I.D.) and length, connections, detector flow cell volumes, etc.) True separation performance is governed by the system dispersion paired with a flow rate range that yields the highest possible efficiency for a given analytical column 2016 Waters Corporation 64

Maximizing the Separation Performance based on System Dispersion Determine your systems Dispersion Matching the right LC system to the right column will yield the best chromatographic results 2016 Waters Corporation 65

Maximizing the Separation Performance based on System Dispersion Select the appropriate particle size to match the HPLC, UHPLC, or UPLC system 2016 Waters Corporation 66

Maximizing the Separation Performance based on System Dispersion Pair the particle size with the column i.d. that best matches the dispersion of your chromatographic system 2016 Waters Corporation 67

Maximizing the Separation Performance based on System Dispersion Select a flow rate that gives the optimal linear velocity to maximize efficiency for your column characteristics (van Deemter) 2016 Waters Corporation 68

Maximizing the Separation Performance based on System Dispersion The system must be able to operate at the typical back pressures associated with the selected column 2016 Waters Corporation 69

Maximizing the Separation Performance based on System Dispersion The relative cost/per analysis will decrease as you move from HPLC to UHPLC to UPLC due to shorter run times at lower flow rates 2016 Waters Corporation 70

Maximizing the Separation Performance based on System Dispersion HPLC UHPLC UPLC 2016 Waters Corporation 71

4.6 x 75 mm, 2.7 µm AU AU AU 3.0 x 75 mm, 2.7 µm AU AU AU 2.1 x 50 mm, 1.6 µm AU AU AU Dispersion Impact on Performance: Isocratic Separations on HPLC, UHPLC and UPLC 1.00 HPLC 1.00 UHPLC 1.00 UPLC 0.80 0.80 0.80 0.60 0.60 0.60 0.40 0.40 0.40 0.20 0.20 0.20 0.00 0.50 1.00 1.50 2.00 2.50 Minutes 1.00 0.00 1.00 0.50 1.00 1.50 2.00 Minutes 0.00 1.00 0.50 1.00 1.50 2.00 Minutes 0.80 0.80 0.80 0.60 0.60 0.60 0.40 0.40 0.40 0.20 0.20 0.20 0.00 0.50 1.00 1.50 2.00 2.50 1.00 Minutes 0.00 1.00 0.50 1.00 1.50 2.00 Minutes 0.00 1.00 0.50 1.00 1.50 2.00 Minutes 0.80 0.60 0.80 0.60 0.80 0.60 * 0.40 0.20 0.40 0.20 0.40 0.20 * 0.00 0.50 1.00 1.50 2.00 2.50 Minutes 0.00 0.50 1.00 1.50 2.00 Minutes 2016 Waters Corporation 72 0.00 0.50 1.00 1.50 2.00 Minutes * Strong solvent effects

4.6 x 75 mm, 2.7 µm AU AU AU 3.0 x 75 mm, 2.7 µm AU AU AU 2.1 x 50 mm, 1.6 µm AU AU AU Dispersion Impact on Performance: Isocratic Separations on HPLC, UHPLC and UPLC 1.00 HPLC 1.00 UHPLC 1.00 UPLC 0.80 0.80 0.80 0.60 0.60 0.60 0.40 0.40 0.40 0.20 0.20 0.20 0.00 0.50 1.00 1.50 2.00 2.50 Minutes 1.00 0.00 1.00 0.50 1.00 1.50 2.00 Minutes 0.00 1.00 0.50 1.00 1.50 2.00 Minutes 0.80 0.80 0.80 0.60 0.60 0.60 0.40 0.40 0.40 0.20 0.20 0.20 0.00 0.50 1.00 1.50 2.00 2.50 1.00 Minutes 0.00 1.00 0.50 1.00 1.50 2.00 Minutes 0.00 1.00 0.50 1.00 1.50 2.00 Minutes 0.80 0.60 0.80 0.60 0.80 0.60 * 0.40 0.20 0.40 0.20 0.40 0.20 * 0.00 0.50 1.00 1.50 2.00 2.50 Minutes 0.00 0.50 1.00 1.50 2.00 Minutes 2016 Waters Corporation 73 0.00 0.50 1.00 1.50 2.00 Minutes * Strong solvent effects

What Causes Poor Peak Shape with Basic Compounds? lanol Interactions with Basic Compounds 2016 Waters Corporation 74

Impact of poor peak shape in methods development Integration errors Reduced resolution Reduced sensitivity 0 5 10 15 20 25 Minutes 2016 Waters Corporation 75

Influence of Poor Peak Shape on Integration Tailing Factor = 1.00 Recovered Peak Areas 99.9 % 99.8 % 99.6 % Tailing Factor = 1.58 Recovered Peak Areas 97.8 % 95.3 % 92.3 % 2016 Waters Corporation 76

Influence of Poor Peak Shape on Resolution Rs = (t r Peak 2 t r Peak 1)/ 0.5 (w 4.4% Peak 1 + w 4.4% Peak2) Rs = (11.5-10.6)/ 0.5 (2.0 + 0.5) Rs = 0.72 Note: Baseline resolution = 1.5 0 5 10 15 20 25 Minutes 2016 Waters Corporation 77

Tamoxifen: Influence of Poor Peak Shape on Sensitivity AU 0.0004 0.0000 C18 Brand A gnal/noise Ratio A 11.0 B 6.5 AU 0.0004 0.0000 5 10 Minutes 5 10 Minutes C18 Brand B Note: gnal/noise Requirements LQ>10 LD>3 2016 Waters Corporation 78

Factors Affecting the Separation Stationary Phase Factors Type of bonded ligand Ligand Density Concentration of the ligand on the surface of the particle Ligand High Density Low Density Mobile Phase Factors rganic strength adjusted to create appropriate elution times 2016 Waters Corporation 79

Surface of a lica Gel Bonded-Phase Packing Material H 3 C H H 2 C H 3 C H H 3 C H 2 C H 2 C H 2 C H 3 C H 2 C H 2 C CH 2 Polar analytes are not able to energetically fit between ligands can t interact with surface or ligands CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 H 2 C H 2 C H 2 C CH 2 CH 2 CH 2 Residual silanol Endcap H 2 C H 2 C H 2 C CH 2 CH 2 CH 2 CH 3 CH H 3 3 C H 3 C CH 3 CH H 3 C CH 3 H 3 C 3 CH 3 CH H 3 3 C H H H H H H C 8 alkyl chains CH 3 H 3 C H CH 3 H 3 C H 2 C CH 2 Polar analytes easily interact with surface and ligands H 3 C H 2 C CH 2 Residual silanols H 3 C H 2 C H 2 C H 2 C H 3 C H 2 C CH 2 CH 2 CH 2 CH 2 CH 3 CH 2 H 3 C H 3 C CH 3 CH 3 H CH 3 CH H 3 C 3 H H H H H High Coverage High Ligand Density Low Coverage Low Ligand Density Endcap CH 3 CH 3 2016 Waters Corporation 80

Surface of a lica Gel Bonded-Phase Packing Material H 2 C Non-polar portion of analyte interacts with bonded phase H 2 C (hydrophobic) H 3 C H H 2 C H 2 C CH 2 CH 2 CH 2 CH 2 Alkyl chains H CH 3 C 3 H N H NH CH 3 Residual silanols H 3 C CH 3 H Polar portion of analyte interacts with silanols (hydrogen bonding and/or ion exchange) CH 3 CH 3 H Low Coverage Low Ligand Density ~1.65 2016 Waters Corporation 81 N H NH 2 N H H 3 C H H 2 C H 2 C H 2 C H 2 C CH 2 CH 2 CH 2 CH 2 CH 3 H H 3 C Endcap H CH 3 CH 3

What is De-Wetting Hydrophobic Collapse? A: Loss of reversed-phase retention Low organic or pure aqueous mobile phase is expelled from pores (de-wetted) C18 lica-gel Wetted Pore De-Wetted Pore Note: Retentivity is a function of the surface area and ligand density. However, if the surface is non-wetted, then the effective chromatographic surface area is reduced > 95%. Thus reducing the retentivity of the analyte => poor capture = De-wetting (Hydrophobic collapse) Remember! almost all of the surface area is in the pores! 2016 Waters Corporation 82

AU AU AU AU HSS T3 Versus HSS C18: 100% Aqueous Conditions 0.06 0.04 0.02 0.00 0.06 0.04 1 2 3 4 XSelect HSS T3 5 Initial 0.02 0.00 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 0.06 0.04 0.02 0.00 0.15 0.10 0.05 1 2 Pks 1-5 3 0.00 0.00 0.50 1.00 1.50 2.00 Minutes 2.50 3.00 3.50 4.00 Peak i.d.: 1) thiourea 2) 5-Fluorocytosine 3) adenine 4) thymidine-5 -monophosphate 5) thymine Conditions: 10mM Ammonium Formate ph 3; 0.2mL/min; 30ºC; 2.1 x 50 mm 4 2016 Waters Corporation 83 5 After dewetting XSelect HSS C18 Initial After dewetting

Excellent Peak Shape and Retention with mple MS Compatible Mobile Phases The popular void marker Uracil is well retained and is actually peak 3 Conditions Column: Atlantis TM dc 18 4.6 x 150 mm, 5 µm Mobile Phase A: H 2 Mobile Phase B: ACN Mobile Phase C: 100 mm CH 3 CNH 4, ph 5.0 Flow Rate: 1.0 ml/min Gradient: Time Profile (min) %A %B %C 0.0 90 0 10 10.0 84 6 10 Injection Volume: 10 µl Temperature: 30 o C 1 Detection: UV @ 254 nm Instrument:: Alliance V 0 = 1.83 TM 2695, min 2996 PDA 2 3 4 Compounds: 1. Cytosine 2. 5-Fluorocytosine 3. Uracil 4. 5-Fluorouracil 5. Guanine 6. Thymine 7. Adenine 5 6 Note excellent peak shape for strong polar base Adenine at ph 5.0 7 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 Minutes At ph 5.0, the strong polar base Adenine still exhibits excellent peak shape (reason: fully endcapped) Grumbach, Diehl 2016 Waters Corporation 84

Perfect Balance Between Polar and Non-Polar Compound Retention More retention for polar compounds 7 Equal or less retention for non-polar compounds V 0 = 0.98 min 2 8 9 Atlantis dc 18 1 3 4 5 6 10 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 Minutes Conventional C 18 7 Back Marker V 0 = 0.98 min 1 2 3 4 5 6 8 9 10 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 Minutes 2016 Waters Corporation 85

Increased Polar Compound Retention with ACQUITY UPLC HSS T3 Columns Compounds: Conc (mg/ml) 1. Norepinephrine 25 2. Epinephrine 25 3. Dopamine 10 4. 3,4- Dihydroxyphenylacetic acid 25 5. Serotonin (5-HT) 30 6. 5-Hydroxy-3-indoleacetic acid 25 7. 4-Hydroxy-3-methoxyphenylacetic acid (HVA) 25 1 2 3 4 5 6 Conditions: Columns: As Indicated Mobile Phase A: 10mM CH 3 CNH 4, ph 5.0 Mobile Phase B: ACN Flow Rate: 0.438 ml/min Isocratic Mobile Phase Composition: 2% B Injection Volume: 0.7 µl Temperature: 30 o C Detection: UV @ 280 nm Instrument: ACQUITY UPLC TM System with ACQUITY UPLC 2996 PDA 7 ACQUITY UPLC BEH C18 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 Minutes 1 2 3 4 5 NEW ACQUITY UPLC HSS T3 6 7 Increased Retention 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 Minutes 2016 Waters Corporation 86

Polar Retention: Why Does HSS T3 Work? Dominant retention mechanism is reversed-phase (van der Waals forces hydrophobic attraction) Retention maximized using 100% aqueous mobile phases Retention maximized by using reduced C 18 coverage o Polar analytes can fit between C 18 ligands and interact with pores of material o ptimized particle morphology (i.e. pore diameter/volume) Secondary interactions due to residual silanols that are more accessible due to reduced C 18 coverage Cation-exchange interactions Hydrogen bonding interactions 2016 Waters Corporation 87

Different C18 Bonding Levels: Carbon Load Measured as weight of bonded phase per weight of silica (w/w %) Typical range for C18 ligand : 6 to 22% Carbon Load Retention 2016 Waters Corporation 88

Different Bonding Levels Packing Column Volume (ml) lica Density (g/ml) Mass of Packing (g) Carbon Load (%) Carbon in Column (g) Nova-Pak C18 µbondapak C18 1.8 0.8 1.44 7.3 0.11 1.8 0.40 0.72 9.8 0.07 2016 Waters Corporation 89

(ln [a] amitriptyline/acenaphthene) Column Selectivity Comparison Chart (Methanol) 2.8 2.3 Decreasing lanol Activity 1.8 1.3 9.8% with 1.42 µmoles/m 2 µbondapak C18 Waters Spherisorb DS2 11.5% with 2.84 µmoles/m 2 0.8 Nova-Pak C18 7.3% with 2.80 µmoles/m 2 0.3-0.2 Increasing Hydrophobicity Symmetry C18 20% with 3.20 µmoles/m 2 1 1.5 2 2.5 3 3.5 (ln [k] acenaphthene) 2016 Waters Corporation 90

(ln [a] amitriptyline/acenaphthene) Column Selectivity Comparison Chart 2.8 2.3 1.8 1.3 Decreasing lanol Activity Sph1DS-Low 9% Waters Spherisorb DS2 µbondapak C18 14% Sph2DS-Med Sph3DS-High 0.8 Nova-Pak C18 22% 0.3 Increasing Hydrophobicity Symmetry C18-0.2 1 1.5 2 2.5 3 3.5 (ln [k] acenaphthene) 2016 Waters Corporation 91

(ln [a] amitriptyline/acenaphthene) Selectivity Chart 2.7 2.2 NTE: Do you see two columns that look like they are out of place? Hint: upper left quadrant C18 s Resolve C18 Waters Spherisorb DS1 1.7 1.2 0.7 0.2 µbondapak C18 Waters Spherisorb DS2 YMC J'sphere DS L80 Nucleosil C18 YMC J'sphere DS M80 YMCbasic Hypersil DS YMC J'sphere DS H80 YMC-Pack Pro C4 Inertsil C8 Nova-Pak C18 Nova-Pak C8 Kromasil C8 YMC-Pack DS AQ YMC-Pack Pro C8 YMC-Pack DS-A Symmetry C8 Hypersil BDS C8 YMC-Pack Pro C18 Kromasil C18 Inertsil DS-3 Hypersil BDS C18 Inertsil DS-2 Symmetry C18-0.3 1 1.5 2 2.5 3 3.5 4 (ln [k] acenaphthene) 2016 Waters Corporation 92

Base-Deactivated Packings What are they? A class of reversed-phase packing materials that exhibit improved peak shape for basic compounds---especially at ph s 4-7 How are they made? Various techniques in silica gel synthesis and/ or in the bonding process that yields a particle surface with less silanol interaction with basic analytes 2016 Waters Corporation 93

Base-Deactivated Packings a. Metal Impurities (silica gel) a. Lower concentration of Al 3+ and Fe 3+ b. Ligand Density c. Mixed C18 and Amino Ligands d. Embedded polar ligands (advanced ligands) 2016 Waters Corporation 94

Acid, Base, Neutral Chromatographic Evaluation Test at ph 3.0 H N + H 3 C H CH 3 N + Cl + H H N H HC - C H H CH 3 NH 2 Chlorpheniramine pk a = 3.6, 9.2 Propranolol pk a = 9.5 Maleic Acid pk a = 1.9, 6.3 Toluamide Base: Positive Charge Base: Positive Charge Acid: Negative Charge Neutral: No Charge Mobile Phase: 50 mm H3P4-KH2P4 ph 3.0/Acetonitrile (80/20) 2016 Waters Corporation 95

Waters Spherisorb DS2 Standard C18 Mobile Phase: 50 mm H3P4-KH2P4 ph 3.0/Acetonitrile (80/20) Maleic Acid Toluamide Chlorpheniramine Propranolol 0 4 8 12 16 20 24 28 32 Minutes 2016 Waters Corporation 96

Repulsion of Cations with Technique C - + NH 3 (CH 2 ) 11 CH 3 Reduced silanol effect on basic analyte resulting in better peak shape H 3 C H N CH 3 + Repulsion of cationic base resulting in shorter retention times H N + Cl Waters 97 2016 Waters Corporation 97

Propranolol Waters Spherisorb DS2 versus Waters Spherisorb DSB (same silica gel) Acenaphthene Base-Deactivated DSB Technique C Acenaphthene Standard C18 Ligand DS2 Amitriptyline 0 60 50 Minutes 100 150 98 2016 Waters Corporation 98

Maleic Acid Toluamide Technique C: Pros and Cons (Better for Bases Poor for Acids) Mobile Phase: 50 mm H3P4-KH2P4 ph 3.0/Acetonitrile (80/20)? Waters Spherisorb DSB Base Deactivated Waters Spherisorb DS2 Chlorpheniramine Propranolol 0 4 8 12 16 20 24 28 32 99 Minutes 2016 Waters Corporation 99

Maleic Acid Toluamide Technique C: Pros and Cons Mobile Phase: 50 mm H3P4-KH2P4 ph 3.0/Acetonitrile (80/20) Maleic Acid Waters Spherisorb DSB Base Deactivated Waters Spherisorb DS2 Propranolol 0 4 8 12 16 20 24 28 32 100 Minutes 2016 Waters Corporation 100

Making an Embedded Polar Bonded Phase Material: Two-Step Synthesis H H H H H H H H H + Cl Cl N H NH 2 NH 2 C NH 2 Cl + Amide (CH 2 ) n Cl CH 3 C Amination Step with trifunctional silane (CH 2 ) n CH 3 Acylation Step Note: Residual amine on particle surface due to steric hindrance (incomplete acylation). 2016 Waters Corporation 101

Characteristics of Two-Step Synthesis Process Inexpensive process: aminopropyl silane and acyl chlorides readily available Incomplete secondary reaction due to steric hinderance resulting in the presence of an aminopropyl group Packing materials show anion exchange properties that are undesirable (unusual retention of acidic compounds) Batch to batch reproducibility issues 2016 Waters Corporation 102

Toluamide Chlorpheniramine Propranolol Two-Step Synthesis Embedded Polar Bonded Phase Column Mobile Phase: 50 mm H3P4-KH2P4 ph 3.0/Acetonitrile (80/20) Note: Great Peak Shape for the Bases Maleic Acid? 0 2 4 6 8 10 12 14 Minutes Note: Technique D using Two-Step Synthesis Process with residual amines. 2016 Waters Corporation 103

Chlorpheniramine Propranolol Two-Step Synthesis Embedded Polar Bonded Phase Column Mobile Phase: 50 mm H3P4-KH2P4 ph 3.0/Acetonitrile (80/20) Maleic Acid 0 2 4 6 8 10 12 14 Minutes 2016 Waters Corporation 104

Toluamide Embedded Polar Ligand: Two Step Synthesis Note: milar results to Technique C: Mixed C18 and Amino Ligands Mobile Phase: 50 mm H3P4-KH2P4 ph 3.0/Acetonitrile (80/20) Chlorpheniramine Propranolol Supelcosil LC-ABZ+Plus Technique D: Two-Step Maleic Acid Waters Spherisorb DSB Technique C 0 2 4 6 8 10 12 14 16 18 20 Minutes 2016 Waters Corporation 105

Making an Embedded Polar Bonded Phase Material: ne-step Synthesis H + Monofunctional silane H 3 C Cl CH 3 C N H (CH 2 ) 7 CH 3 lica Gel Surface H 3 C CH 3 C N H (CH 2 ) 7 + HCl Carbamate group built into starting silane CH 3 Note: No residual amines on silica gel surface 2016 Waters Corporation 106

Possible Mechanisms of Embedded Polar Ligand Me Me C H N C 8 H 17 Analyte interacting with polar group H X R Me H Me HN C 8 H 17 Analyte competing with polar group for silanol H X R Me Me H H C H N C 8 H 17 H X R Polar group increases H 2 concentration in surface layer 2016 Waters Corporation 107

Embedded Polar Ligand: Possible Mechanism Polar group increases water concentration in surface layer - H 3 C CH 3 H H C N H (CH 2 ) 7 CH 3 2016 Waters Corporation 108

Water Layer Embedded Polar Ligand: Possible Mechanism Reason 1 Water layer increases dielectric constant, reducing ionic interactions Particle Reduced retention of bases Reduced peak tailing Both reasons given describe why the Shield RP18 boning provides excellent peak shape observed for bases Reason 2 The carbamate group may hydrogen bond with silanol groups, shielding analytes from interacting with them 2016 Waters Corporation 109

Maleic Acid Toluamide Chlorpheniramine Propranolol SymmetryShield RP18 Mobile Phase: 50 mm H3P4-KH2P4 ph 3.0/Acetonitrile (80/20) 0 2 4 6 8 10 12 14 Minutes Technique D: ne-step process (great peak shape for acids and bases) 2016 Waters Corporation 110

Embedded Polar Ligand versus Linear Alkyl Ligand on lica Gel Amitriptyline SymmetryShield RP18 TF USP = 1.1 Symmetry C18 TF USP = 1.9 15 25 0 10 20 30 Minutes Note: Reduced retention, 25 > 15 min. and improved peak shape for the base. 2016 Waters Corporation 111

Acidic Compounds:Embedded Polar versus Alkyl Linear Ligands S S K T K N T N F F D In D In Ib Symmetry C18 Suprofen = S Ketoprofen = K Naproxen = N Tolmetin = T Fenoprofen = F Diclofenac = D Indomethacin = In Ibuprofen = Ib SymmetryShield RP18 Ib 0 4 8 12 16 20 Minutes Note: No change in mobile phase. 2016 Waters Corporation 112

Chromatographic Impact of 1990 s Embedded Polar Ligands Reduced retention and decreased tailing of basic compounds Unique selectivity Improved water wettability of the chromatographic bed (hydrogen bonding of water to the particle surface) and reduced risk of hydrophobic collapse 2016 Waters Corporation 113

Percent Un-Ionized Retention Factor (k) Reversed-Phase Retention Map: The Impact of ph on Ionizable Compounds 100 40 Maximum acidic compound retention range Maximum basic compound retention range 90 80 70 35 30 Acid Neutral 60 25 50 40 30 20 20 15 10 10 0 5 0 Base 0 2 4 6 8 10 12 ph ph Range for lica Particles ph Range for lica Particles ph Range for Hybrid Particles 2016 Waters Corporation 114

Capacity Factor (k) Relating Retention Maps to Chromatography Reversed-Phase Retention Map 14 13 Neutral B A N ph 2 12 11 10 9 Acid 0 1 2 3 8 7 6 5 4 3 2 1 Base 0 0 1 2 3 4 5 6 7 8 9 10 11 12 ph 2016 Waters Corporation 115

Capacity Factor (k) Relating Retention Maps to Chromatography Reversed-Phase Retention Map 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 Acid Base Neutral 0 1 2 3 4 5 6 7 8 9 10 11 12 ph B A N ph 2 0 1 2 3 A,B N ph 5 0 1 2 3 2016 Waters Corporation 116

Capacity Factor (k) Relating Retention Maps to Chromatography Reversed-Phase Retention Map 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 Acid Base Neutral 0 1 2 3 4 5 6 7 8 9 10 11 12 ph 0 1 2 3 0 1 2 3 A B A,B A B N N N ph 2 ph 5 ph 10 0 1 2 3 Minutes Note: ph is a powerful selectivity tool 2016 Waters Corporation 117

Capacity Factor (k) Impact of ph on the Retention of a Zwitterionic Compound 120 100 Aqueous pk a = 4.3 Aqueous pk a = 9.5 80 Negative Charge 60 40 Positive Charge B + A Dual Charge B A - 20 0 0 1 2 3 4 5 6 7 8 9 10 11 12 H N H + B + A - ph Fexofenadine (Antihistamine) CH 3 H CH 3 C - 2016 Waters Corporation 118

Tailing Factor Peak Shape ver Wide ph Range (Strong Base -- Amitriptyline pka 9.4) 4 Low ligand density and high metal content silica gel: Conventional C18 3 2 High ligand density and high purity silica gel: Modern C18 1 2 3 4 5 6 7 8 Buffer ph Ideal Behavior Pure Polymer No lanols 2016 Waters Corporation 119

AU AU AU The Importance of Mobile Phase ph: Rapid Method Development 3 4 5 ph 2 1.70 1.60 1.50 2 1.40 6 1.30 1.20 1.10 1.00 0.90 0.80 1 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 Minutes 1.70 1.60 3 4 5 1.50 2 6 ph 7 1.40 1.30 1.20 1 1.10 1.00 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 1.80 3 2 Minutes 1.60 5 ph 12 6 1.40 4 1.20 1.00 0.80 0.60 1 0.40 0.20 0.00 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 Minutes Using a wide mobile phase ph range is an effective approach to change compound selectivity Increase selectivity for: Acids (Green/Brown) Bases (Red/Yellow) Neutrals (Peaks 2 & 5) are largely unaffected by mobile phase ph 2016 Waters Corporation 120

General Retention and Mobile Phase ph Rules To achieve stable chromatographic retention times Stay outside the ± 2 ph units from the analytes pk a value Always measure ph before adding the organic solvent 2016 Waters Corporation 121

Retention Time Variability Influence of ph Non-Column Influences: ph Neutrals: No Influence Acids: Reduced Retention with Increasing ph Bases: Increased Retention with Increasing ph Up to 10% Change in Retention per 0.1 ph Unit (largest shift within +/- 1 ph unit of pka) 2016 Waters Corporation 122

log (k') Impact n Retention Factor (k) By Changing Mobile Phase ph -- HPLC In REVERSED-PHASE Mode 100 Acid (Un-Ionized) 10 Note: Column Particle,Temperature and % rganic Held Constant ph Limit of lica gel Base (Un-Ionized) Neutral 1 0.1 Base (Ionized) 0 2 4 6 8 10 12 14 ph Acid (Ionized) Range of poor reproducibility due to steep slopes - small ph change -- large retention change 2016 Waters Corporation 123

ph Test for Robustness Poor Results Conditions Column: XTerra RP 18 4.6 x 100 mm, 5 µm Mobile Phase A: Mobile Phase B: Flow Rate: 20mM Ammonium Acetate (ph 5.0 to 5.8) or 20mM Ammonium Bicarbonate (ph 6.8 to 7.0) ACN 0.5 ml/min Isocratic Mobile Phase Composition: 40% A; 60% B Injection volume: 10µL Temperature: Detection: Instrument: 30 o C UV @ 230 nm Alliance TM 2690, 996 PDA 0.40 0.30 0.20 0.10 0.00 0.40 0.30 0.20 0.10 0.00 0.40 0.30 0.20 0.10 0.00 1 3 2 ph 5.0 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 1 3 2 1 3 2 ph 5.2 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 ph 5.4 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 Method developed at ph = 5.0 Zone of reproducibility risk for ph control What is peak 1? 2? 3? 0.40 0.30 0.20 0.10 0.00 1 3 2 ph 5.6 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 Grumbach, Diehl 2016 Waters Corporation 124

ph Test for Robustness Poor Results Conditions Column: XTerra RP 18 4.6 x 100 mm, 5 µm Mobile Phase A: Mobile Phase B: Flow Rate: 20mM Ammonium Acetate (ph 5.0 to 5.8) or 20mM Ammonium Bicarbonate (ph 6.8 to 7.0) ACN 0.5 ml/min Isocratic Mobile Phase Composition: 40% A; 60% B Injection volume: 10µL Temperature: Detection: Instrument: 30 o C UV @ 230 nm Compounds 1. p-toluamide -neutral 2. Lidocaine - base 3. Ibuprofen- acid Alliance TM 2690, 996 PDA Method developed at ph = 5.0 Zone of reproducibility risk for ph control What is peak 1? 2? 3? 0.40 0.30 0.20 0.10 0.00 0.40 0.30 0.20 0.10 0.00 0.40 0.30 0.20 0.10 0.00 0.40 0.30 0.20 0.10 0.00 1 3 2 ph 5.0 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 1 3 2 1 3 2 ph 5.2 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 ph 5.4 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 1 3 2 ph 5.6 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 Grumbach, Diehl 2016 Waters Corporation 125

log (k') Impact on Retention Factor (k) on Robustness By Changing Mobile Phase ph Range - HPLC In Pure REVERSED-PHASE Mode 100 ph Limit of lica gel 10 Acid (Un-Ionized) Base (Un-Ionized) Neutral 1 Base (Ionized) Acid (Ionized) 0.1 0 2 4 6 8 10 12 14 ph Greatly improved reproducibility due to flattening of curves Robustness Note: Column Particle, Temperature and % rganic Held Constant 2016 Waters Corporation 126

ph Test for Robustness Good Results 2016 Waters Corporation 127

log (k) Impact of Retention Factor (k) on Prep LC In Pure REVERSED-PHASE Mode ph Limit of lica gel 100 10 Acid (Un-Ionized) Base (Un-Ionized) Neutral 1 Base + (Ionized) Acid - (Ionized) 0.1 0 2 4 6 8 10 12 14 Maximum Loading for Prep is when analyte is in the UN-INIZED Form ACID at Low ph BASE at High ph ph Note: Column Particle, Temperature and % rganic Held Constant 2016 Waters Corporation 128

Superior Mass Load Capacity without Sacrificing Purity or Speed! ph 10 Loadability Study a Mixture of Three Basic Compounds XBridge 82 x In Un-ionized form! 2016 Waters Corporation 129

(ln [a] amitriptyline/acenaphthene) Basic Compounds 2.8 2.3 W aters Spherisorb DS1 Resolve C18 1.8 µbondapak C18 W aters Spherisorb DS2 1.3 YMC J' sphere DS L80 Amine additives required with buffers N ucleosil C18 YMC J' sphere DS M80 Hypersil DS YMC J' sphere DS H80 0.8 N ova-pak C18 YMC-Pack DS AQ 0.3 Hypersil BDS C18 YMC-Pack Pro C18 Buffers required Kromasil C18 Inertsil DS-3 Inertsil DS-2 Symmetry C18-0.2 1 1.5 2 2.5 3 3.5 (ln [k] acenaphthene) 2016 Waters Corporation 130

Impact of rganic Solvent Composition on Retention Factor (k), ph, and pk a k k HA,0 e B HA c k 1 10 A,0 pk e B a c HA c ph 10 c pk a c ph c rganic Solvent Composition c Retention Factor k Note: k, pk a and ph are a function of the organic solvent composition Slope of relationship between Log k and % rganic B 2016 Waters Corporation 131

Impact of rganic Concentration on the pk a of the Analyte In general: Basic Compounds: pk a will decrease with the addition of an organic solvent Acidic Compounds: pk a will increase with the addition of an organic solvent The specific change in pk a will be compound dependent 2016 Waters Corporation 132

Amitriptyline + NH CH 3 N CH 3 CH 3 CH 3 Ionized Un-ionized pka (H 2 0) 9.4 apparentpk a (20% ACN) 8.5 apparentpk a (30% ACN) 8.3 apparentpk a (40% ACN) 8.0 Note: you cannot determine the degree or the direction of the pk a shift with out experimentally running each organic concentration 2016 Waters Corporation 133

Naproxen CH 3 CH CH 3 C - H 3 C H 3 C Un-ionized Ionized pka (H 2 0) 4.2 apparent pk a (20% ACN) 4.6 apparent pk a (30% ACN) 4.8 Note: you cannot determine the degree or the direction of the pk a shift with out experimentally running each organic concentration 2016 Waters Corporation 134

What is the practical application of this information? For new methods development: aqueous buffers at ph 2 (for acidic analytes) and ph 10 (for basic analytes) when mixed with organic solvents may provide stable chromatographic regions where most analytes will be unionized Caution: Must consider Un-ionized analyte hydrophobicity rganic solvent strength (Methanol versus THF) 2016 Waters Corporation 135

ph Limitations of lica Based Packing Materials Hydrolysis of Bonded Ligand Dissolution of silica particle 10 1 0 2 4 6 8 10 12 14 ph 2016 Waters Corporation 136

Hydrolysis of a Bonded Phase Material: Monofunctional Ligand H + H 3 C Cl C CH 3 C C C C C C CH 3 H 3 C C CH 3 C C C C C C CH 3 Low ph Mobile Phase (hydrolysis of ligand) + HCl H + H 3 C H C CH 3 C C C C C C CH 3 lane Bleed 2016 Waters Corporation 137

Making a Bonded Phase Material: Multifunctional Synthesis H H H + Cl Cl C8 Trichlorosilane Ligand CH 3 Cl H H CH 3 Multi-Point Attachment - usually not 3 + HCl 2016 Waters Corporation 138

Making a Bonded-Phase Material: Trifunctional Synthesis Notice: need to break 3 bonds before H H H lane bleeds CH 3 CH 3 CH 3 1 and 2-point attachments to silica gel surface More hydrolytically stable Risks: Notice creation of additional silanols from the trifunctional synthesis Attachment of silanes may not be attached to silica gel surface Potential for poor batch-to-batch reproducibility 2016 Waters Corporation 139

Peak Shape: Monofunctional lane versus Trifunctional lane Risk: trifunctional silane can increase silanols lica gel particle Monofunctional C18 amitriptyline TF = 1.8 0 1 2 3 lica gel particle Trifunctional C18 amitriptyline TF = 2.6 0 1 2 3 Minutes 2016 Waters Corporation 140

Characteristics of Multifunctional Synthesis Multiple bonds to silica gel surface More resistant to hydrolysis at ph s <2 More difficult for reproducible synthesis Higher silanol activity Capable of higher ligand density 2016 Waters Corporation 141

Changes in Ligand Density Due to Bonding Technique Changes in ligand density due to ligand length and multifunctional silanes Ligand Monofunctional Synthesis (Ligand Density) Trifunctional Synthesis (Ligand Density) C18 3.2 µm/m 2 4.2 µm/m 2 C8 3.5 µm/m 2 5.5 µm/m 2 C4 3.8 µm/m 2 4.9 µm/m 2 Note: Values from different column manufacturers data sheets 2016 Waters Corporation 142

Trifunctional Synthesis: Higher Ligand Densities, Greater Hydrolytic Stability H CH 3 H H H Bonded but not actually CH 3 reacted to substrate CH 3 H H H CH 3 CH 3 CH 3 Risks: Notice creation of additional silanols from the trifunctional synthesis Attachment of silanes may not be attached to silica gel surface Potential for poor batch-to-batch reproducibility 2016 Waters Corporation 143

Solubility of lica in Water (ppm) Mobile Phase ph and Column Life-time Even when bonded with ligands it will still dissolve 240-220- 200-180- 160-140- 120-100- 80-60- 40-20- 0- lica Solubility Curve lica ph 2-8 Polymer ph 2-12 At ph >8 silica dissolves 1 2 3 4 5 6 7 8 9 10 ph Elevated Temperature causes more rapid failure ph > 8 causes VIDING for traditional silica/ bonded particles 2016 Waters Corporation 144

Effects of High ph Mobile Phases Surface Modified lica Particles Hybrid Particles Complete dissolution of lica Catastrophic column failure Short lifetimes Slow rate of surface dissolution Incorporated methyl groups uncovered slows rate of dissolution Longer column lifetimes 2016 Waters Corporation 145

Mechanical -- Column Packing Material Flush Well packed column 2016 Waters Corporation 146

Good Plate Count Results (Instrument K, Column K, Connections K) Well packed column 2016 Waters Corporation 147

Triethylamine.temperature.pH 2016 Waters Corporation 148

Column Bed Collapse Mechanical Poor Plate Count Instrument K, Connections K Voided column - Channeling Packing Material Settled or dissolved 2016 Waters Corporation 149

Column Collapse (voiding) (due to shock / high ph {dissolution of particle}) All Peaks Distorted Voids - high back pressure, distorted and/or double peaks 2016 Waters Corporation 150

Extended ph Range of XBridge/BEH Columns mplifies Methods Development (ne Column Instead of Three) Low ph silica gel column High ph polymeric column Intermediate ph silica gel column Broad ph range Xbridge/BEH columns 1 2 3 4 5 6 7 8 9 10 11 12 ph 2016 Waters Corporation 151

Different Ligands: Different Selectivity Changes in hydrophobicity Longer alkyl chain will provide greater retention Changes in silanol activity Affect peak asymmetry and influences secondary interactions Changes in hydrolytic stability Longer column lifetimes with greater number of ligand attachment points to the particle surface Changes in ligand density Influences sample loadability 2016 Waters Corporation 152

Initial Phases Chosen for Selectivity 2016 Waters Corporation 153

Two Fully-Scalable LC Column Platforms Family designed and optimized for ph stability Family designed and optimized for selectivity 1.7 [UPLC], 2.5 XP, 3.5, 5 and 10 µm 1.7 [UPLC], 2.5 XP, 3.5, 5 and 10 µm CSH 1.8 [UPLC], 2.5, 3.5 and 5 µm HSS 2016 Waters Corporation 154

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XBridge/BEH Family Family designed and optimized for ph stability Excellent general-purpose HPLC and UPLC columns for small molecule separations Broadest range of compound classes Widest ph range (1 12) Widest temperature range Ultra-low MS bleed Direct scalability between UPLC Technology, analytical HPLC and preparative HPLC 1.7 [UPLC], 2.5 XP, 3.5, 5 and 10 µm 2016 Waters Corporation 156

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. 2016 Waters Corporation 157

Industry Leading ph Stability Column lifetimes in acidic mobile phases Shorter bar equals longer lifetime under low ph conditions Test conditions: 1% TFA in water (ph 1.0) at 80 o C. Comparative separations may not be representative of all applications. 2016 Waters Corporation 158

Industry Leading ph stability Column lifetimes in alkaline mobile phases High ph mobile phases (>ph 10) rapidly dissolve silica-based stationary phases nly 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. 2016 Waters Corporation 159

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XSelect HSS Family Highest pressure tolerance; first silica-based particle (1.8 μm) designed for UPLC applications Alternate selectivities (vs. BEH & CSH particle columns) Increased retention 1.8 [UPLC], 2.5 XP, 3.5 and 5 µm HSS 2016 Waters Corporation 162

HSS Chemistries For 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 HSS CN: Stable, normal phase-compatible, reproducible CN chemistry HSS PFP: Exceptional selectivity and retention for positional isomers, halogenated compounds and polar basic compounds 2016 Waters Corporation 163

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Charged Surface Hybrid [CSH] Particles Step 1 Step 2 Step 3 CHARGED SURFACE HYBRID PRCESS 2016 Waters Corporation 165

Column Selectivity Choices Caffeic Acid Derivatives 3 1 2 4 1 3 2 4 1 2 4 1 2 4 3 3 1 2 4 1 2 4 1 2 3 5 5 5 3 3,4 5 5 BEH C 18 BEH C 8 BEH Shield RP18 BEH Phenyl 5 CSH Phenyl- Hexyl CSH Fluoro-Phenyl 5 CSH C 18 Conditions : Columns: 2.1 x 50 mm Mobile Phase A: 0.1% CF 3 CH in H 2 Mobile Phase B: 0.08% CF 3 CH in ACN Flow Rate: 0.5 ml/min Gradient: Time Profile Curve (min) %A %B 0.0 92 8 6 0.1 92 8 6 4.45 50 50 7 4.86 10 90 6 5.0 92 8 6 6.0 92 8 6 Injection Volume: 1.0 µl Sample Diluent: 50:50 H 2 : MeH with 0.05% CF 3 CH Sample Conc.: 100 µg/ml Temperature: 40 o C Detection: UV @ 330 nm Sampling rate: 40 pts/sec Time Constant: 0.1 Instrument: Waters ACQUITY UPLC, with ACQUITY UPLC TUV 1 2 4 3 5 HSS CN 1 2 1 2 4 1 2 3 4 5 1 2 4 3 3 3 4 5 5 5 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 0.0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Minutes 2016 Waters Corporation Comparative separations may not be representative of all applications 166

Selectivity Comparison ACQUITY UPLC BEH C 18 1 2 6 4 3 5 7 1. 1-pyrenesulfonic acid 2. Flavone 3. Imipramine 4. Fenoprofen 5. Amitriptyline 6. Diclofenac 7. ctanophenone ACQUITY UPLC CSH C 18 3 5 1 2 4 6 7 ACQUITY UPLC CSH Phenyl-Hexyl 3 5 2 4 6 1 7 ACQUITY UPLC CSH Fluoro-Phenyl 3 5 4 2 6 7 1 0.00 1.00 2.00 3.00 4.00 5.00 All columns 2.1 x 50 mm, 1.7 µm Minutes 2016 Waters Corporation 167

NT All Peaks are Created Equally! Most common peak shapes Gaussian peak shape due to single interaction with the surface Linear isotherm Exponential tail due to secondary interactions Acidic silanols Shark fin peak shape due to mass overload Convex isotherm 2016 Waters Corporation 168

Conventional and CSH Technology High Purity C 18 columns Prednisone Caffeine C 18 Columns 1.7 µm BEH 1.7 µm CSH milar peak shape for neutrals Prednisone Caffeine Amitriptyline Metoprolol Expected P c With CSH Technology & Small Particles! Amitriptyline Metoprolol 0 100 200 300 400 500 600 700 800 900 Peak Capacity Dramatically Improved Peak Shape for Bases! 2016 Waters Corporation 169

AU AU AU Benefits of CSH Technology: Loading Comparison 3.0 2.0 ACQUITY UPLC CSH C 18 2.1 x 50 mm, 1.7 µm Quetiapine (base) Propiophenone (neutral) 1.0 0.0 3.0 2.0 1.0 Fully porous silica C 18 2.1 x 50 mm, 1.8 µm Quetiapine (base) Propiophenone (neutral) 0.0 3.0 2.0 1.0 Competitive Core-Shell C 18 2.1 x 50 mm, 1.7 µm Quetiapine (base) Propiophenone (neutral) 0.0 0.6 0.8 1.0 1.2 1.4 1.6 Minutes 2016 Waters Corporation Comparative separations may not be representative of all applications 170

AU Imipramine AU Imipramine CSH Technology: Influence of Sample Loading on Trace Impurity Detection 0.10 0.08 0.06 0.04 0.02 0.00 0.10 0.08 ACQUITY CSH C 18 1.7 µm 0.1 % formic acid 2.0 % 1.0 % 0.5 % 0.1 % Amitriptyline 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 Minutes Superficially Porous C 18 1.7 µm 0.1 % formic acid bservations CSH Technology enables superior peak shape and efficiency in low ionic strength mobile phases Improved sensitivity for trace level impurity analysis 0.06 0.04 0.02 0.00 2.0 % 1.0 % 0.5 % 0.1 % 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 Minutes Imipramine concentration held constant at 0.5 mg/ml; 0.1% formic acid mobile phase 2016 Waters Corporation 171

AU Imipramine AU Imipramine AU Imipramine CSH Technology: Influence of Sample Loading on Trace Impurity Detection 0.015 A 0.1% impurity ACQUITY CSH C 18 0.1% formic acid 0.000 0.015 B 0.1% impurity Superficially porous C 18 0.1% formic acid 0.000 0.015 C 0.1% impurity Superficially porous C 18 0.05% TFA 0.000 0.00 0.60 1.20 1.80 2.40 3.00 Minutes 2016 Waters Corporation 172

Intensity Imipramine Intensity Imipramine CSH Technology: Influence of Sample Loading on Trace Impurity Detection 8x10 8 Total Ion Chromatogram 0.1% impurity (m/z 278.3) ACQUITY UPLC CSH C 18 0.1% formic acid 4x10 8 0 0.60 1.20 1.80 2.40 3.00 Minutes 8x10 8 Total Ion Chromatogram UV Trace Superficially porous C 18 0.05% TFA 4x10 8 0.1% impurity 0 0.60 1.20 1.80 2.40 3.00 Minutes 2016 Waters Corporation 173

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 0.12 XSelect CSH C 18 3.5 µm Before high ph exposure After high ph exposure 0.06 0.00 AU 0.18 0.12 Metoprolol 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 min 20% RT change 64% peak height loss Amitriptyline 25% RT change 81% peak height loss neutral phthalates Hybrid Coated C 18 3 µm Before high ph exposure After high ph exposure 0.06 0.00 Metoprolol Amitriptyline neutral phthalates 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 min 2016 Waters Corporation 174

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 2016 Waters Corporation 175

XSelect CSH Family Family designed and optimized for selectivity Designed for LC/MS Improved peak shape for basic analytes under low-ionic strength acidic mobile phases Reduces overload effect Rapid equilibration; prevents retention time drift with changes in ph Multiple particle substrates to solve multiple chromatographic problems 1.7 [UPLC], 2.5 XP, 3.5, 5 and 10 µm CSH 2016 Waters Corporation 176

What Column(s) Should I Choose? CSH Columns Use additives AND buffers Impurity Profile work 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 Designed for Selectivity 2016 Waters Corporation 177

CSH Chemistries For general-purpose C 18 columns for low to neutral ph reversedphase separations CSH C18: Rapid ph switching for method development 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 2016 Waters Corporation 178

Widest Selection of Unique Particle ffering BEH Technology HSS Technology CSH Technology Unparalleled ph stability Mobile phase and temperature versatility Seamless scalability UPLC to HPLC Maximum retention Particle and ligand selectivity Seamless scalability UPLC to HPLC Exceptional loading capacity Superior peak shape for basic analytes Seamless scalability UPLC to HPLC Waters is the NLY company that has fully porous and solid-core sub-2-µm particle columns 2016 Waters Corporation 179

extended Performance Columns 2.5 mm 2016 Waters Corporation 180

Addressing the Challenges: 2.5 µm extended Performance Columns Addressing the challenges Utilize XP 2.5 µm columns to improve productivity or achieve higher resolution Compatible with bandspread volumes of UHPLC systems Implement XP 2.5 µm columns to decrease backpressure or increase flow rate 2016 Waters Corporation 181

Flexible ptions to Improve LC Productivity 2016 Waters Corporation 182

Allowable Adjustments in Chromatography <621> Variable Allowable Changes Particle ze -50% Column Length ±70% Flow Rate ±50% Column ID Any allowed Injection Volume Any reduction Column Temperature ±10% Mobile Phase ph ±0.2 unit System Suitability. USP 37. Pag 306-308 2016 Waters Corporation 183

USP Assay: Levonorgestrel and Ethinyl Estradiol Isocratic HPLC Method System Suitability Requirements Column: Injection Volume: Flow Rate: Mobile Phase: Detection Wavelength: XBridge C 8 4.6 x 150 mm 5 μm (L7) 50 μl 1.00 ml/min 35:15:45 ACN:MeH:Water UV 230 nm USP Resolution: NLT 2.5 Peak Area Precision: NMT 2.0% RSD Questions Can we transfer this simple method? What combination of column and system? What compromises (if any) must be made? What conditions provide the greatest benefits? 2016 Waters Corporation 184

AU ethinyl estradiol levonorgestrel riginal Isocratic USP Assay: 4.6 x 150 mm, 5 μm (Alliance HPLC) 0.070 Flow Rate: 1.00 ml/min Pressure: 1400 psi Run time: 8.00 min System Suitability Requirements USP Resolution: NLT 2.5 Peak Area Precision: NMT 2.0% RSD 0.060 0.050 USP Res: 6.9 0.040 0.030 0.020 0.010 0.000 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 Minutes L/dp = 30000 N = 1X Rs = 1X Run time = 1X Pressure = 1X N Remember: Rs 2016 Waters Corporation 185 L dp N F opt 1 Run Time L F 1 dp

AU ethinyl estradiol levonorgestrel AU ethinyl estradiol levonorgestrel Isocratic Method Transferred to 4.6 mm XP Columns (Alliance HPLC) 0.070 0.060 0.050 0.040 0.030 4.6 x 75 mm XP 2.5 μm Flow Rate: 1.00 ml/min Pressure: 2600 psi Run time: 4.00 min USP Res: 6.5 Could not run at SCALED flow rate due to system pressure limitations Results: Rs = 0.9X Run time = 0.5X Pressure = 2X 0.020 0.010 0.000 0.060 0.050 0.040 0.030 0.020 0.010 0.000 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 Minutes 4.6 x 50 mm XP 2.5 μm Flow Rate: 2.00 ml/min Pressure: 3600 psi Run time: 1.30 min USP Res: 5.0 4.00 1.30 > 50% flow rate change not <621> compliant LWER L/dp column (20000) to reduce pressure: Results: Rs = 0.7X Run time = 0.16X Pressure = 2.9X 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2016 Waters Corporation Minutes 186

ethinyl estradiol AU levonorgestrel AU ethinyl estradiol levonorgestrel Isocratic Method Transferred to ACQUITY UPLC H-Class System 0.08 0.06 0.04 0.02 2.1 x 75 mm XP, 2.5 μm Flow Rate: 0.42 ml/min Pressure: 4400 psi Run Time: 2.00 min USP Res: 7.9 Results: Rs = 1.1X Run time = 0.25X Pressure = 3.1X 0.00 0.06 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 Minutes 2.1 x 50 mm UPLC, 1.7 μm Flow Rate: 0.61 ml/min Pressure: 7700 psi Run Time: 0.90 min. Maximum benefits obtained on ACQUITY UPLC System 0.04 0.02 USP Res: 7.0 Results: Rs = 1X Run time = 0.1X Pressure = 5.5X 0.00 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 Minutes 2016 Waters Corporation 187

Column Parameters Column length More length increases separation power Increases runtime Column ID Smaller ID increases sensitivity Reduces injection volume Particle size Smaller particle size equals more efficiency Increases backpressure L/dp ratio A measure of separation power Equal values of L/dp are equivalent efficiency 2016 Waters Corporation 188

Summary Waters is committed to manufacturing high-quality consumables that ensure reproducible results year after year XBridge BEH Columns designed for ph stability and method flexibility XSelect HSS and CSH Columns provide maximum retention, selectivity and excellent peak shape for basic analytes 2.5 µm XP columns bridge the gap between HPLC and UPLC Compatible with HPLC, UHPLC and UPLC system platforms Maximize HPLC productivity 2016 Waters Corporation 189

Column ptions 2016 Waters Corporation 190

System Comparisons Breeze 2 HPLC Alliance HPLC ACQUITY Arc HPLC/UHPLC ACQUITY H-Class UPLC ACQUITY I-Class UPLC ACQUITY M-Class UPLC 1.6 µm for ultimate efficiency at UPLC backpressure 1.7/1.8 µm for high efficiency and selectivity 2.7 µm for maximum HPLC performance XP 2.5 µm for HPLC selectivity 5 µm for low backpressure and legacy methods 1.8 µm for high efficiency, retention and selectivity 2016 Waters Corporation 191

Resources that Enable Success: Technology Primers 715001531 715002531 715002099 715002098 715003405 2016 Waters Corporation 192

Questions 2016 Waters Corporation 193

Escitalopram (ral Solution) Escitalopram pk a =9.78 Mobile Phase: Buffer 6.1g of monobasic potassium phosphate. To each L of this solution add 1.5 ml of triethylamine. Adjust with phosphoric acid to ph of 2.5. Mobile phase ACN/Buffer (32:68). Column: 4.6 x 250 mm, 5 um, column packed with L1 2016 Waters Corporation 194

Column ptions 2016 Waters Corporation 198

Dibucaine Reported pk a = 8.9 Eluent: 1.2 g sodium lauryl sulfate (sodium dodecyl sulfate), 0.2g sodium acetate, 2.0 ml triethylamine in 300 ml water, adjust ph to 5.6 and add 700 ml methanol Column: 3.9 x 300 mm L1. Tailing Factor NMT 3.0 2016 Waters Corporation 199

Column ptions 2016 Waters Corporation 202

Quinidine pka 8.56 (at 25 C) Solution A: Add 35 ml of methanesulfonic acid to 20 ml of glacial acetic acid, and dilute with water to 500 ml Solution B: Dissolve 10 ml of Diethylamine in water to obtain 100 ml of solution Mobile Phase Acetonitrile, Solution A, Solution B, and Water (10:1:1:40) Column 4.6 x 250 mm, 5 um, L1. 2016 Waters Corporation 203

Column ptions 2016 Waters Corporation 204

Tioconazole (Imidazole Antifungal Basic Compound) and Related Compounds A, B, and C Predicted pka = 6.19 Structures for Related Compounds A-C Unknown Mobile Phase: (Note- Prepare the Mobile phase fresh daily.) Mix 400 ml of acetonitrile, 400 ml of methanol, and 280 ml of water. Add 2 ml of ammonium hydroxide and mix. Column: A 4 mm x 10-cm pre-column that contains packing L4 (silica gel), installed between the pump and the injector (replaced daily) and a 5-mm x 25-cm analytical column that contains packing L1. 2016 Waters Corporation 205

Column ptions 2016 Waters Corporation 207

Succinylcholine Nicotinic acetilcholine receptor agonist Mobile Phase: Prepare a solution in water containing 3.85 g per L of sodium 1-pentanesulfonate anhydrous, 2.9 g per L of sodium chloride, and 1% (v/v) of 1 N sulfuric acid. Prepare a filtered and degassed mixture of Buffer solution and acetonitrile (95:5) Column: A 4.6-mm x 25-cm, 5 um, analytical column that contains packing L1. 2016 Waters Corporation 208

Surface of a lica Gel Bonded-Phase Packing Material H 2 C Non-polar portion of analyte interacts with bonded phase H 2 C (hydrophobic) H 3 C H H 2 C H 2 C CH 2 CH 2 CH 2 CH 2 Alkyl chains H CH 3 C 3 H N H NH CH 3 Residual silanols H 3 C CH 3 H Polar portion of analyte interacts with silanols (hydrogen bonding and/or ion exchange) CH 3 CH 3 H Low Coverage Low Ligand Density ~1.65 2016 Waters Corporation 210 N H NH 2 N H H 3 C H H 2 C H 2 C H 2 C H 2 C CH 2 CH 2 CH 2 CH 2 CH 3 H H 3 C Endcap H CH 3 CH 3

Column ptions 2016 Waters Corporation 211