HPLC to UPLC Method Migration:Method Migration: An ...

1 2007 Waters CorporationHPLC to UPLCHPLC to uplc Method migration : Method Migration: An overview of Key Considerations and An overview of Key Considerations and Available ToolsAvailable ToolsDr. Michael Swartz, Ph. Michael Swartz, Ph. Consulting ScientistPrincipal Consulting ScientistWorldwide Pharmaceutical Business OperationsWorldwide Pharmaceutical Business OperationsWaters CorporationWaters CorporationPittCon 2007 PittCon 2007 2007 Waters Corporation 2 Evaluation FormEvaluation FormEvaluation FormWe Appreciate Your Opinion Please Complete Evaluation Form Qualify for drawing to win a $25 AMEX gift card (drawn at end of this session must be present to win)AND Qualify to win a Gold Package registration to Inform 2007 ($1595 value; one winner per day; winner to be notified post-Pittcon)Thank You! 2007 Waters Corporation 3 OutlineOutline uplc overview Principles of Method migration Examples Software Tools Available Summary 2007 Waters Corporation 4 Ultra Performance LCUltra Performance LC A New Class of Separation Science Based on chromatography columns with very small particles Based on instruments designed to take advantage of the small particles Provides Improved Resolution, Speed, and Sensitivity with no Compromises Suitable for Chromatographic Applications in General Appropriate for developing new methods Appropriate for improving existing methods 2007 Waters Corporation 5 Smaller ParticlesSmaller ParticlesThe Enabler of ProductivityThe Enabler of ProductivityOptimal velocity range 2007 Waters Corporation 6 hplc vs.

2 UPLCHPLC vs. uplc : : Speed, Sensitivity and Speed, Sensitivity and x 150 mm, 5 mRs (2,3) = at 270 x 50 mm, mRs (2,3) = SensitivitySame at 270 Faster, More Sensitive x 100 mm, mRs (2,3) = Speed2X at 270 Faster, More Sensitive, Higher Resolution methods 2007 Waters Corporation 7 Method Conversion From Method Conversion From hplc to hplc to ACQUITY ACQUITY UPLCUPLC Why Convert hplc methods to uplc ? Get faster results with more resolutionoMore informationoMore robust methodsoBetter situational response time (stat samples faster, research decisions with more information, process monitoring, product release)oMore samples analyzed per system, per scientistIncreased Productivity 2007 Waters Corporation 8 Migrating MethodsMigrating methods The New Method Must Preserve Complete resolution of all relevant analytes Peak homogeneity/purity Certainty of peak identification Quantitative accuracy and precision 2007 Waters Corporation 9 Method migration Success?

3 Method migration Success? 2007 Waters Corporation 10 Method migration Success! Method migration Success! 2007 Waters Corporation 11 CaffeicCaffeicAcid Derivatives in Echinacea Acid Derivatives in Echinacea hplc Method = 35 minXTerra MS x 150 mm, 5 mFinal uplc Method = 5 minACQUITY UPLCTMBEH x 50 mm, m 2007 Waters Corporation 12 ACQUITY ACQUITY HPLCHPLC = = 5 C18 Time in = = 5 C18 ACQUITY ( hplc )Vendor X HPLC8 Diuretics + impurity 2007 Waters Corporation 13 ACQUITY UPLCACQUITY uplc Transfer of hplc methodTransfer of hplc Method It is possible to transfer most hplc methods to ACQUITY systems with attention to details Instrument design and performance differences could result in result differences Some adjustments may be required But:This is not taking advantage of uplc !! 2007 Waters Corporation 14 Migrating methods FromMigrating methods FromHPLC to UPLCHPLC to uplc Method Transfer Between hplc SystemsoFirst step towards Method conversionoHPLC to ACQUITY as hplc Method migration or Conversion hplc separation to an ACQUITY uplc separation Method Optimization Separation goals Method verification Method Development Systematic approach 2007 Waters Corporation 15 Keys for hplc to UPLCKeys for hplc to uplc Method Method migration SuccessMigration Success Proper Column Choice Chemistry Dimensions Keep in Mind Instrument Differences Gradient delay volume Detector data rates Proper Geometric Scaling Optimize for uplc Don t Forget LC Theory 2007 Waters Corporation 16 Determination of Proper Column Determination of Proper Column ChemistryChemistryHYDROPHOBICITY (ln [k] acenaphthene)

4 XTerra MS C18 Hypersil Elite C18 Inertsil ODS-3 Hypersil HyPurity Elite C18 Luna C18 YMC-Pack Pro C18 Zorbax Eclipse XDB C18 Zorbax Rx C18 Prodigy C18 Symmetry C18 SymmetryShield RP18 Supelcosil LC-ABZ+PlusSupelcosil LC DB-C18 XTerra RP18 Luna C18(2)Polaris (ln [ ] amitriptyline/acenaphthene) dC18 YMC-Pack ODS AQ EXPANDED VIEWACQUITY uplc BEHXB ridge C18 ChromolithTMRP-18 Nucleosil C8 SunFire C18 ACQUITY uplc BEHXB ridge Shield RP18 ACQUITY uplc BEHXB ridge PhenylACQUITY uplc BEHXB ridge C801/2006pH 7 2007 Waters Corporation 17 Determination of Proper Column Determination of Proper Column Dimensions Dimensions Internal diameter Generally prefer mm Only use 1 mm for specific reasonoSeverely sample limitedoDirect flow to mass spectrometer Length If primary goal is SPEEDo50 mm length to start If primary goal is RESOLUTIONo100 mm length to start 2007 Waters Corporation 18 Instrument Differences: Instrument Differences.

5 Compensating for System VolumesCompensating for System Volumes Compare system volumes This volume should be converted to column volumes for the best comparison If target system gives smallerisocratic segment ADD an initial hold to the gradient table to give the identical hold. If target system gives largerisocratic segment No exact compensation is possible Chromatographic effect of extra isocratic hold usually small 2007 Waters Corporation 19 Method migration ExampleMethod migration ExampleOriginal hplc MethodOriginal hplc MethodOriginal Column: x 150 mm, 5 m@ mL/minAbsorbance 254 nmMinutes1230102030 Resolution (1,2) = 12 Resolution (2,3) = 28 Ambient temperature: 21- 22 CFlow rate: mL/minSample analytes: 1. Caffeine (100mg/mL),2. Hydroquinidine (33mg/mL), 3. 3-Aminobenzophenone (39mg/mL) Molecular weight(s): Less than 500 Sample diluent: DMSOI njection: 10 LDetection: 254nmMobile phase:A: TFA in waterB: TFA in acetonitrileObjective: Maintain resolution while increasing speed 2007 Waters Corporation 20 Method MigrationMethod MigrationColumn Comparison: Injection VolumesColumn Comparison: Injection x 50mmSample volume too largefor smaller column volume20 L = 11% x 150mm20 L = 2007 Waters Corporation u t e migration Method migration Column Comparison: Injection VolumesColumn Comparison.

6 Injection VolumesColumn transfer from to injection volume scaling 2007 Waters Corporation 22 Method migration Example Method migration Example Injection Volume ConsiderationsInjection Volume Considerations 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 1 L If calculated volume too small for injection, dilute 5 - 10x with initial strength mobile phase Typically 5 L maximum injection on x 50 mm 2007 Waters Corporation 23 Method MigrationMethod MigrationCalculate Injection VolumeCalculate Injection VolumeScaling a 10 Linjection on x 150mm to x 50mmTarget injection volume=Original injection volumeXTarget Column VolumeOriginal Column x x x x 150=10 Lx10 Lx= L 2007 Waters Corporation 24 Method migration ExampleMethod migration ExampleFlow RateFlow Rate For migration .

7 First, adjust flow rateproportional to column diameter squared for constant linear velocity (geometrically scaled) Second, adjust Gradient Tableto maintain the same number of column volumes of solvent through the target column Finally, adjustflow rate (linear velocity) for smaller particle oAnalyte molecular weight must be considered 2007 Waters Corporation 25 Method MigrationMethod MigrationScale the Flow Rate to Column GeometryScale the Flow Rate to Column Geometryd2 Targetd2 OriginalTarget Flow Rate = Original Flow Rate x x r2of Target x r2of OriginalThis reduces to:Target Flow Rate = Original Flow Rate X a rate on to **Note: this assumes same particle sizeSo: 2007 Waters Corporation 26 Method migration ExampleMethod migration ExampleGradient ProfileGradient Profile Express gradient duration in percent change per column volume (cv) units Calculate each segment as a number of column volumes Calculate time required to deliver the same number of column volumes to the target column at the chosen flow rate 2007 Waters Corporation 27 Original Gradient ProfileOriginal Gradient ProfileGradient StepTime Since InjectionFlow Rate%A% * 2007 Waters Corporation 28 Gradient SegmentsGradient SegmentsExpress as Column VolumesExpress as Column VolumesFor 15 min at on a x 150mm columnGradient Volume = Flow Rate x Time = x 15min = Volume = x r2x L = x x = Duration (cv)

8 = Gradient VolumeColumn VolumeGradient Duration = cv 2007 Waters Corporation 29 Original Gradient Profile for ScalingOriginal Gradient Profile for ScalingStepTime Since InjectionFlow Rate%A%BCurveSegment Duration (min)Segment Duration (cv) * 2007 Waters Corporation 30 Scaling Gradient Step TimeScaling Gradient Step TimeMaintain Duration (Maintain Duration (cvcv))Original Step 2: 15 min @ mL/min with duration of Target Step 2: (keeping duration @ ) Column Volume = x r2 x L = x = Step Volume = Duration (cv) x Target Column Volume= = Step Time = Gradient Step Volume/Flow Rate = / mL/min = 5 min 2007 Waters Corporation 31 Scaled Gradient ProfileScaled Gradient ColumnGradient StepTime Since InjectionFlow Rate%A%BCurveSegment Duration (min)Segment Duration (cv) * 5 95 time for same number of column volumesper gradient segment 2007 Waters Corporation 32 Smaller ParticlesSmaller ParticlesThe Enabler of ProductivityThe Enabler of ProductivityOptimal velocity range 2007 Waters Corporation 33 Estimate Optimum Flow RateEstimate Optimum Flow RateUPLCUPLC Consider m target particle ( ID column)

9 Assume temperature and viscosity transferred Adjust flow rate based on van Deemter curve and approximate molecular weight ~ mL/min for smaller moleculesoaverage 500 dalton (molecular weight) molecules ~ mL/min for larger molecules because diffusion is , ~2,000 dalton peptides 2007 Waters Corporation 34 Scaling for UPLCS caling for uplc Flow RateFlow RateStep Time to Maintain Duration (Step Time to Maintain Duration (cvcv))Original Step 2: 15 min.@ mL/min with Duration of Target Step 2: (keeping duration @ ) Target Column Volume ( x 50) = Step Volume = Duration (cv)x Target Column Volume= = Step Time = Gradient Step Volume / uplc Flow Rate = / mL/min. = 2007 Waters Corporation 35 Optimized Gradient ProfileOptimized Gradient ProfileGradientStepTime Since InjectionFlow Rate%A%BCurveSegment Duration (min)Segment Duration (cv) 5* 95 uplc flow rate and adjust time to maintain same number of column volumes per segment 2007 Waters Corporation 36 Method Conversion Process: Method Conversion Process.

10 Steps for SuccessSteps for Success Refer to current chromatography Observe system volumes, solvents and detection technique Define objectives/room for improvement Select column dimensions scale flow for linear velocity 50 mm length for speed 100mm length for complex samples/resolution Scale injection volume to column dimensions Use a gradient and flow scaled from current Method Adjust gradient to accommodate system differences, column differences and particle size differences Use a gradient and flow rate scaled for uplc Review steps 2007 Waters Corporation 37 Method Conversion: Method Conversion:Original hplc MethodOriginal hplc MethodOriginal Column: x 150 mm, 5 m@ mL/min, 30 minute cycle timeAbsorbance 254 nmMinutes1230102030 Resolution (1,2) = 12 Resolution (2,3) = 28 Ambient temperature: 21- 22 CFlow rate: mL/minSample analytes: 1. Caffeine (100mg/mL),2.