Successfully Scaling and Transferring HPLC and UPLC Methods

Successfully Scaling and Transferring HPLC and UPLC Methods Esa Lehtorinne Esa_Lehtorinne@waters.com Tel: +358-9-5659 6288 Fax: +358-9-5659 6282 Waters Finland Kutomotie 16 00380 Helsinki 2013 Waters Corporation 1

Types of Method Transfer Transfer through different LC instruments Transfer of established method between different instruments with the same or equivalent column Adjustment of method Certain method parameters can be adjusted (within limits) in response to meeting a procedure s system suitability Method equivalency must be verified Change of method Significant change to method in which full validation is required 2013 Waters Corporation 2

Method Transfer Process: Key to Success New method must preserve critical parameters Resolution of all relevant analytes Peak homogeneity/purity Certainty of peak identification Quantitative accuracy and precision Achieve same analytical result, both qualitatively and quantitatively, independent of instrumentation or column type Requirements Column selectivity must be preserved when changing particle size and/or column dimension Column length-to-particle size (L/dp) ratio is maintained Mobile phases, gradient curves and sample concentration is maintained 2013 Waters Corporation 3

Method Transfer Process: Steps for Success Gather information about existing method and results Selected column should keep original method retentivity/selectivity Method Transfer Process: Steps forsuccess Compare LC instruments and differences must be accounted Geometrically scale conditions from original to target method Evaluate results of transferred method Optimize as necessary 2013 Waters Corporation 4

Method Transfer Process: Steps for Success Gather information about existing method and results Column Chemistry (Ligand, Particle Size, Brand), Dimensions Method Transfer Process: Steps For Success Conditions Mobile phase, Flow Rate, Gradient Profile, including reequilibration, Column Temperature Sample Diluent, Concentration, Injection Volume, Molecular Weight Chromatogram Number of Analytes, Retention required, Resolution required Quantitation Limit of Detection, Limit of Quantitation, Linear Dynamic Range, Accuracy, Precision 2013 Waters Corporation 5

Method Transfer Process: Steps for Success Gather information about existing method and results Selected column should keep original method retentivity/selectivity Method Transfer Process: Steps For Success Column Selectivity Chart Provides an assessment of a column s hydrophobicity and base/neutral selectivity Select equivalent column Available at: www.waters.com/selectivitychart 2013 Waters Corporation 6

Scalability Across Technology Platforms: UPLC-to-HPLC Family designed and optimized for ph stability Most MS-compatible HPLC columns on the market Family designed and optimized for selectivity Multiple particle substrates to solve multiple chromatographic problems 1.7 [UPLC], 2.5, 3.5, 5 and 10 µm 1.7 [UPLC], 2.5, 3.5 and 5 µm CSH 1.8 [UPLC], 2.5, 3.5 and 5 µm HSS 2013 Waters Corporation 7

The Widest LC Column Offering Six particle substrates 125Å (SEC), 130Å, 200Å (SEC) 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 16 scalable stationary phases (excl. UPLC SEC & GPC columns UPLC IEX & new CORTECS columns ) BEH 130Å C 18, C 8, Shield RP18, Phenyl, HILIC and Amide BEH 300Å C 18 and C 4 HSS C 18, T3, C 18 SB, PFP and CN CSH C 18, Fluoro-Phenyl and Phenyl-Hexyl Transferability between HPLC and UPLC XBridge HPLC and ACQUITY UPLC BEH columns XSelect HSS HPLC and ACQUITY UPLC HSS columns XSelect CSH HPLC and ACQUITY UPLC CSH columns VanGuard Pre-columns ecord Technology 2013 Waters Corporation 8

UPLC and HPLC Column Equivalency Same Chemistries on Different Particle Sizes UPLC 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 BEH130 C 18 XBridge BEH130 C 18 ACQUITY UPLC BEH300 C 18 XBridge BEH300 C 18 ACQUITY UPLC BEH300 C 4 XBridge BEH300 C 4 ACQUITY UPLC HSS C 18 XSelect HSS C 18 ACQUITY UPLC HSS C 18 SB ACQUITY UPLC HSS T3 ACQUITY UPLC HSS PFP ACQUITY UPLC HSS CN XSelect HSS C 18 SB XSelect HSS T3 XSelect HSS PFP XSelect HSS CN ACQUITY UPLC CSH C 18 XSelect CSH C 18 ACQUITY UPLC CSH Phenyl Hexyl ACQUITY UPLC CSH Fluoro Phenyl XSelect Phenyl Hexyl XSelect Fluoro Phenyl ACQUITY SEC125, SEC200, SEC450 - - - ACQUITY APC AQ & XT - - - Protein-Pak Hi Res CM, SP & Q - - - 2013 Waters Corporation 9

Ratio of Column Length-to-Particle Size: Maintaining Separation Power Separation Index Typical HPLC Column 4.6 x 150 mm, 5 µm Column length (L): 150 mm = 150 000 µm Particle Size d p : 5 µm L = 150 000 = 30 000 d p 5 Application Example Efficiency (N) L/dp Ratio 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 2013 Waters Corporation 10

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 Size Resolution Capability 100000 90000 80000 Extremely Difficult 70000 60000 50000 Difficult 40000 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 2013 Waters Corporation 11

Ratio of Column Length-to-Particle Size: Maintaining Separation Power HPLC 5 µm 150 mm L/d p = 30 000 HPLC 3.5 µm 100 mm L/d p = 28 571 UPLC 1.8 µm 50 mm L/dp = 27 778 2013 Waters Corporation 12

Method Transfer Method Transfer Kits are designed to take the guess work out of transferring a method from one LC to another. Sustained selectivity between UPLC and HPLC particle sizes Each kit includes: Access to the ACQUITY UPLC Columns calculator (multi-directional) that enables the customer to transfer from any technology platform to any other technology platform o HPLC-to-UPLC-to-HPLC UPLC and HPLC column with equivalent selectivity and resolving power Method Transfer Assistance Kit Options: 1.7/1.8 µm to 5 µm Transfer kit o 2.1 x 50 mm, 1.7/1.8 µm and 4.6 x 150 mm 5 µm 1.7/1.8 µm to 3.5 µm Transfer kit o 2.1 x 50 mm, 1.7/1.8 µm and 4.6 x 100 mm 3.5 µm 1.7/1.8 µm to 3.5 µm High Rs Transfer kit o 2.1 x 100 mm, 1.7/1.8 µm and 4.6 x 150 mm 3.5 µm 2013 Waters Corporation 13

Method Transfer Process: Steps for Success Gather information about existing method and results Selected column should keep original method retentivity/selectivity Method Transfer Process: Steps For Success To ensure that the retentivity and selectivity of the method on the original LC system is maintained, system differences need to be accounted for Differences in injection mechanism o Injection modes and wash steps Gradient type and differences in system volume o o Maintain the column/system volume ratio to ensure the isocratic hold imparted by the system at the beginning of the gradient is constant Re-equilibration time may also need to be adjusted Differences in column heating o Compare LC instruments and differences must be accounted Column heaters have different heating efficiencies and the differences can impact absolute retention of the components and selectivity 2013 Waters Corporation 14

Injection Mode: Difference In Injection Mechanism ACQUITY UPLC Rheodyne injector ACQUITY UPLC H-Class Flow through needle SAMPLE LOOP SAMPLE NEEDLE Everything that s in the loop will be injected Everything that s in the needle will be injected 2013 Waters Corporation 15

Injection Mode: Difference In Injection Mechanism Sample Injection: Injection volume used on the UPLC ACQUITY H-Class must correspond to the injection volume specified in the UPLC method UPLC method (FL Design) ACQUITY H-Class/Alliance (FTN Design) Full loop with 10 µl loop 10 µl 2.5 µl PLUNO in 10 µl loop 2.5 µl Wash Solvent: Solvent wash rinse the injector after injection to reduce carry-over Wash solvent used on the ACQUITY UPLC H-Class must correspond to (strong) wash specified in the UPLC method UPLC method ACQUITY H-Class/Alliance Weak wash: 85/15 H20/MeOH Strong wash: 5/95 H20/MeOH Wash solvent: 5/95 H20/MeOH 2013 Waters Corporation 16

Differences in System Volume: Low vs. High Pressure Mixing Multiple/Dual Pumps (Binary) High Pressure Mixing Smaller System Volume = Smaller Dwell volume Solvent mixing: Pump A Pump B Mixer Injector Column Detector After going through the pump Or pre-mixed solvents only Smaller system volume Minimal dispersion Single Pump (Quaternary) - Low Pressure Mixing Larger System Volume = Larger Dwell volume Solvent mixing: Before going through the pump A B C D Gradient Proportioning Valve Pump Injector Column Detector Larger system volume Auto Blend : For mobile phase generation 2013 Waters Corporation 17

System Volume Timing Offset Solvent Composition at Mixer Solvent Composition at Column Head { } Actual mobile phase profile on original system measured at the column inlet 0 Injection t g x Time System volume creates an offset before the solvent composition change reaches the inlet of column (i.e. an isocratic hold at the beginning of every gradient) 2013 Waters Corporation 18

Different System Volumes Effect on Separation Original Instrument System Volume 0.9 ml Smaller Volume System Volume 0.35 ml Larger Volume System Volume 1.4 ml Target System with smaller volume (less isocratic hold time) Target System with larger volume (longer isocratic hold time) 2013 Waters Corporation 19

% System Volume Recommended Method for Measurement 1. Remove column 2. Use Acetonitrile as A, and Acetonitrile with 0.05 mg/ml Uracil as B 3. Monitor 254 nm 4. Use the flow rate in the original method and the intended flow rate on the target instrument 5. Collect 100 % A baseline for 5 min 6. At 5.00 min, program a step to 100 % B, and collect data for an additional 5 min 100 % Asymptotic 100.00 90.00 80.00 70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00 50 % Liftoff Black programmed Blue observed 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 2013 Waters Corporation 20

AU System Volume Recommended Method for Measurement 7. Measure absorbance difference between 100 % A and 100 % B 8. Measure time at 50 % of that absorbance difference 50 % Absorbance = 0.3582 AU 0.70 0.65 0.60 0.55 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 Total absorbance = 0.7164 AU 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 Minutes 2013 Waters Corporation 21

AU System Volume Recommended Method for Measurement 9. Calculate time difference between start of step and 50 % point 10. Multiply time difference by flow rate Programmed time = 5.00 minutes 0.70 0.65 0.60 0.55 0.50 5.69 min - 5.00 min 0.69 min 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 50 % Time = 5.69 minutes System Volume: 0.69 min. x 1.5 ml/min. = 1.04 ml 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 Minutes 2013 Waters Corporation 22

Gradient Type: Compensating for System Volumes Compare system volumes This volume should be converted to Column Volumes (CVs) for the best comparison If target system gives smaller isocratic segment ADD an initial hold to the gradient table to give the identical hold If target system gives larger isocratic segment Use the pre-injector volume feature (if available) 2013 Waters Corporation 23

ACQUITY UPLC Columns Calculator ACQUITY UPLC Columns Calculator handles these calculations 2013 Waters Corporation 24

Differences In Column Heating: Active vs Passive Pre-Heater Module Turn off Active Pre-Heater (APH) if thermolabile compounds are present ACQUITY UPLC H-Class: APH 2013 Waters Corporation 25

ACQUITY UPLC H-CLASS: HPLC Simplicity UPLC Performance Delivers the proven business impact of UPLC into routine testing laboratories Will reproduce established HPLC methods while enabling seamless transfer to UPLC The system of choice for method development Replaces conventional HPLC as the chromatographic system of choice Future-proof your LC laboratory with the widest range of chemistry, detection and services HPLC simplicity UPLC performance 2013 Waters Corporation 26

Method Transfer Process: Steps for Success Gather information about existing method and results Selected column should keep original method retentivity/selectivity Method Transfer Process: Steps For Success Compare LC instruments and differences must be accounted Geometrically scale conditions from original to target method To ensure good chromatographic performances, geometrically scaled conditions need to be accounted for Mobile phase and sample diluents Injection volume Temperature Flow rate 2013 Waters Corporation 27

Target Conditions Use exactly the same mobile phase Alter only after evaluating transfer if optimization is required Use exactly the same sample Same concentration, same diluents Geometrically scale injection volume to volume of column Capacity proportional to surface area and internal solvent volume Suggested minimum injection volume on the instrument is 0.5 1 µl If the calculated volume is too small for injection, dilute 5 10x with initial strength mobile phase Typically 5 µl maximum injection on 2.1 x 50 mm 2013 Waters Corporation 28

Scaling Injection Volume Guideline: Injection volume should be less than 5 % of column volume. Aim for < 1 % and experimentally determine if you can go higher based on chromatographic conditions. 4.6 x 150 mm 2.49 ml 20 µl injection/2.49 ml = 0.8 % 2.1 x 50 mm 0.17 ml 20 µl injection/0.17 ml = 12 % If you inject too much, the result will be poor peak shape due to overload 2013 Waters Corporation 29

Target Conditions Temperature directly affects every chromatographic mechanism Indeed, in method transfer, temperature must be kept constant Pre-heating is essential o Solvent is in original (HPLC) column for approximately 1.5 minutes o Solvent is in optimized target (UPLC) column for 15 seconds o Less time for heat transfer Adapt the flow rate 1/ Adjust flow rate proportional to column diameter squared for constant linear velocity (geometrically scaled) 2/ Adjust gradient table to maintain the same number of column volumes of solvent through the target column 3/ Adjust flow rate (linear velocity) for smaller particle o Analyte molecular weight must be considered 2013 Waters Corporation 30

ACQUITY UPLC Columns Calculator ACQUITY UPLC Columns Calculator handles these calculations 2013 Waters Corporation 31

Method Transfer Process: Steps for Success Gather information about existing method and results Selected column should keep original method retentivity/selectivity Method Transfer Process: Steps For Success Compare LC instruments and differences must be accounted Geometrically scale conditions from original to target method Evaluate results of transferred method Optimize as necessary 2013 Waters Corporation 32

3 Ways To Transfer Method Scenario 1 : Maximizing Asset Utilization Applying existing HPLC methods onto HPLC and H-Class systems Adapting HPLC methods to different systems design Scenario 2 : Transferring a HPLC method to UPLC systems Turning a legacy, HPLC method into a UPLC one Scenario 3: Transferring a UPLC method to HPLC systems Taking advantage of UPLC for fast and efficiently developping a method Transfer this method to labs still equipped with HPLC systems 2013 Waters Corporation 33

Scenario 1, Maximizing Asset Utilization: Adapting HPLC Methods to Different Systems Design Future-proofing your lab Invest in new instrumentation that can run both legacy HPLC methods and UPLC methods for new projects Goal Transfer existing HPLC method to a different LC system Must compensate for system dwell volume differences 2013 Waters Corporation 34

Scenario 1: LC Instrument Transfer Original Instrument System Volume 0.9 ml Column Volume 4.6 x100 mm : 1.66 ml Conversion in column volumes : 0.9/1.66 =0.54 cv Target System with smaller volume (less isocratic hold time) Must compensate with an isocratic hold to preserve separation Smaller Volume System Volume 0.35 ml Column Volume 4.6 x100 mm : 1.66 ml Conversion in column volumes : 0.35/1.66 =0.21 cv 2013 Waters Corporation 35

Scenario 1: HPLC to HPLC Method Transfer Dwell Volume To preserve the gradient profile when transferring from one instrument to another, the system dwell volume must be considered. 2013 Waters Corporation 36

Scenario 1: HPLC to HPLC Method Transfer Gradient Column Volumes To preserve the gradient profile, the number of gradient column volumes for each step, should be maintained. As a result, the gradient time table has been adjusted. 2013 Waters Corporation 37

Why Adding an Isocratic Hold? Injection Alliance System with larger dwell volume (longer isocratic hold time) Injection ACQUITY UPLC H-Class 1/Isocratic Hold 2/Tubing System with smaller dwell volume (less isocratic hold time) 2013 Waters Corporation 38

Scenario 1, The Result: HPLC to HPLC Method Transfer Future-proof your lab Run HPLC methods on ACQUITY UPLC H-Class Flexibility to run both HPLC and UPLC methods Relative RT to Clozapine Peak H-Class HPLC Impurity D 0.867 0.865 Impurity C 0.898 0.895 Impurity A 0.951 0.950 Clozapine 1.000 1.000 Impurity B 1.507 1.513 2013 Waters Corporation 39

Ease of Method Transfer Between LC Platforms XSelect HSS T3 4.6 x 50 mm, 2.5 µm XP Flow rate = 1.9 ml/min P c = 92 ACQUITY UPLC H-Class XSelect HSS T3 4.6 x 50 mm, 2.5 µm XP Flow rate = 1.9 ml/min P c = 94 Transfer methods between different LC systems 2013 Waters Corporation 40

3 Ways To Transfer Method Scenario 1 : Maximizing Asset Utilization Applying existing HPLC methods onto HPLC and H-Class systems Adapting HPLC methods to different systems design Scenario 2 : Transferring a HPLC method to UPLC systems Turning a legacy, HPLC method into an UPLC one Scenario 3: Transferring an UPLC method to HPLC systems Taking advantage of UPLC for fast and efficiently developping a method Transfer this method to labs still equipped with HPLC systems 2013 Waters Corporation 41

Galantamine Scenario 2: Turning a Legacy, HPLC Method into a UPLC One HPLC Separation on Alliance HPLC System XBridge TM C18 4.6 x 100 mm, 3.5 µm Tailing = 1.60 Rs = 7.61 1 2 4 5 Method Transfer - Reduce Analysis Time Take advantage of sub 2 µm particle technology and transfer to ACQUITY UPLC H-Class Criteria USP Tailing < 2.0, Rs (galantamine/impurity 4) > 4.5 2013 Waters Corporation 42

Scenario 2: LC Instrument Transfer Original Instrument System Volume 0.9 ml Column Volume 4.6 x100 mm : 1.66 ml Conversion in column volumes : 0.9/1.66 =0.54 cv Larger Volume System Volume 0.35 ml Target System with larger volume in cv (longer isocratic hold time) Column Volume 2.1 x50 mm : 0.17 ml Conversion in column volumes : 0.35/0.17 =2.06 cv 2013 Waters Corporation 43

Scenario 2: Turning a Legacy, HPLC Method into a UPLC One L/dp To preserve the separation power of the gradient, L/dp must be matched 2013 Waters Corporation 44

Scenario 2: Turning a Legacy, HPLC Method into a UPLC One Injection Volume To preserve the mass and volume load on column, the injection volume must be scaled appropriately Pre-Injector Volume To preserve the gradient profile, the pre-injector volume must be used. This allows the gradient to start before the injection is triggered. 2013 Waters Corporation 45

Why Using a Pre-injection Volume? Injection Alliance System with larger dwell volume (longer isocratic hold time) Injection ACQUITY UPLC H-Class System with smaller dwell volume (less isocratic hold time) 2013 Waters Corporation 46

Galantamine AU Galantamine Scenario 2, The Result: Turning a Legacy, HPLC Method into a UPLC One 0.090 HPLC Separation on Alliance HPLC System 0.080 0.070 0.060 0.050 XBridge TM C18 4.6 x 100 mm, 3.5 µm 0.040 0.030 Tailing = 1.60 Rs = 7.6 Adjustment of method Reduce analysis time 4.3X while preserving separation integrity Tailing and Resolution Criteria were met 0.020 0.010 0.000 4 5 2 1 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 Minutes 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 UPLC Separation on ACQUITY UPLC H-Class Criteria USP Tailing < 2.0, Rs (galantamine/impurity 4) > 4.5 1 2 4 5 ACQUITY UPLC BEH C18 2.1 x 50 mm, 1.7 µm Tailing = 1.43 Rs = 7.2 2013 Waters Corporation 47

Transferring with Constant L/dp XBridge C 18 4.6 x 150 mm, 5 µm Alliance 2695 HPLC L/dp = 30 000 40 min XBridge BEH C 18 4.6 x 75 mm, 2.5 µm XP Alliance 2695 HPLC L/dp = 30 000 10 min 4X faster methods on your HPLC XBridge BEH C 18 4.6 x 75 mm, 2.5 µm XP ACQUITY UPLC H-Class L/dp = 30 000 10 min Compatible with HPLC, UHPLC and UPLC systems ACQUITY UPLC BEH C 18 2.1 x 50 mm, 1.7 µm ACQUITY UPLC H-Class L/dp = 29 411 4.5 min ACQUITY UPLC BEH C 18 2.1 x 50 mm, 1.7 µm ACQUITY UPLC I-Class L/dp = 29 411 4.5 min Transfer to 1.7 µm UPLC for 9X faster methods 2013 Waters Corporation 48

3 Ways To Transfer Method Scenario 1 : Maximizing Asset Utilization Applying existing HPLC methods onto HPLC and H-Class systems Adapting HPLC methods to different systems design Scenario 2 : Transferring a HPLC method to UPLC systems Turning a legacy, HPLC method into a UPLC one Scenario 3: Transferring a UPLC method to HPLC systems Taking advantage of UPLC for fast and efficiently developping a method Transfer this method to labs still equipped with HPLC systems 2013 Waters Corporation 49

Scenario 3 : Transferring a UPLC Method to HPLC Systems Maximize Asset Utilization Transfer from UPLC to another department/contract partner that has a bank of HPLC instruments Goal Transfer UPLC method to HPLC while maintaining selectivity 2013 Waters Corporation 50

Scenario 3: LC Instrument Transfer Original Instrument System Volume 0.35 ml Column Volume 2.1 x50 mm : 0.17 ml Conversion in column volumes : 0.35/0.17 =2.06 cv Target System with smaller volume (less isocratic hold time) Must compensate with an isocratic hold to preserve separation Smaller Volume System Volume 0.9 ml Column Volume 4.6 x100 mm : 1.66 ml Conversion in column volumes : 0.9/1.66 =0.54 cv 2013 Waters Corporation 51

Transferring a UPLC Method to HPLC Systems Considerations To preserve the separation power of the gradient, L/dp must be matched System dwell volume must be measured 2013 Waters Corporation 52

Transferring a UPLC Method to HPLC Systems Injection Volume To preserve the mass and volume load on column, the injection volume must be scaled appropriately Gradient Column Volumes To preserve the gradient profile, the number of gradient column volumes for each step, should be maintained. 2013 Waters Corporation 53

Why Having an Initial Column Volume? Injection Alliance System with larger dwell volume (longer isocratic hold time) Injection ACQUITY UPLC H-Class System with smaller dwell volume (less isocratic hold time) 2013 Waters Corporation 54

Scenario 3, The Result: Transferring a UPLC Method to HPLC Systems Maximize Asset Utilization Transfer between HPLC and UPLC Sustained selectivity between particle sizes Relative RT to Clozapine Peak H-Class HPLC Impurity D 0.867 0.865 Impurity C 0.890 0.895 Impurity A 0.939 0.950 Clozapine 1.000 1.000 Impurity B 1.500 1.513 2013 Waters Corporation 55

Method Transfer Summary Define instrument characteristics Injection Mechanism (FL vs FTN; Injection Volume, Wash Solvents, APH...) Define the best equivalent column and efficiency Column selectivity chart ( www.waters.com/selectivitychart ) USP L Designation Keep L/dp Ratio constant Define the scenario used HPLC/HPLC HPLC/UPLC UPLC/HPLC Convert the method accordingly Use the Acquity Calculator to define appropriate CVs and compensate differences in instrument volumes Optional: Confirm the chemistry (separation) with the HPLC column on the Acquity system Optional: Run a scaled gradient (without linear velocity change) on the Acquity column Optional: Set the flow rate (and scale the gradient) to use the best part of the Van Deemter curve Adjust the flow rate to obtain the best resolution 2013 Waters Corporation 56

Summary Legacy HPLC methods can be easily transferred between HPLC instrumentation and the ACQUITY UPLC H-Class Facilitated by quaternary solvent, low-pressure mixing and flow through needle design of ACQUITY UPLC H-Class HPLC Methods can be transferred to take advantage of sub 2 µm UPLC technology Compensate for differences in system dwell volume Utilize method transfer kits for sustained selectivity between particle sizes Transfer UPLC methods to HPLC to maximize existing asset utilization Facilitated by quaternary solvent, low-pressure mixing and flow through needle design Utilize method transfer kits for sustained selectivity between particle sizes 2013 Waters Corporation 57

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