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©2007 Waters Corporation
HPLC to UPLCHPLC to UPLC®® Method Migration:Method Migration:An Overview of Key Considerations and An Overview of Key Considerations and
Available ToolsAvailable Tools
Dr. Michael Swartz, Ph. D.Dr. Michael Swartz, Ph. D.
Principal Consulting ScientistPrincipal Consulting Scientist
Worldwide Pharmaceutical Business OperationsWorldwide Pharmaceutical Business Operations
Waters CorporationWaters Corporation
PittCon 2007PittCon 2007
©2007 Waters Corporation 2
Evaluation FormEvaluation FormEvaluation Form
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Please Complete Evaluation FormQualify for drawing to win a $25 AMEX gift card (drawn at end of this session – must be present to win)
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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 Productivity
Optimal velocity range
©2007 Waters Corporation 6
HPLC vs. UPLCHPLC vs. UPLC®®: : Speed, Sensitivity and Speed, Sensitivity and ResolutionResolution
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00
2.1 x 150 mm, 5 µmRs (2,3) = 4.29
12 3
HPLC
20.00
0.26
Abs
orba
nce
at 2
70 n
m
0.00
Minutes0.40 0.80 1.20 1.60 2.00 2.50
2.1 x 50 mm, 1.7 µmRs (2,3) = 4.281
23
8X Speed3.4X SensitivitySame Resolution
0.26
Abs
orba
nce
at 2
70 n
m
0.00
UPLC®
Faster, More Sensitive Methods
Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
3
2.1 x 100 mm, 1.7 µmRs (2,3) = 6.38
1
2
4.5X Speed2X Sensitivity1.5X Resolution
4.50
0.26
Abs
orba
nce
at 2
70 n
m
0.00
UPLC®
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 resolution
oMore information
oMore robust methods
oBetter situational response time (stat samples faster, research decisions with more information, process monitoring, product release)
oMore samples analyzed per system, per scientist
Increased 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?Method Migration Success?
©2007 Waters Corporation 10
Method Migration Success!Method Migration Success!
©2007 Waters Corporation 11
CaffeicCaffeic Acid Derivatives in Echinacea Acid Derivatives in Echinacea PurpureaPurpurea
AU
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0.002
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0.004
0.005
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
au
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Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Original HPLC Method = 35 minXTerra® MS C184.6 x 150 mm, 5 µm
Final UPLC® Method = 5 minACQUITY UPLCTM BEH C182.1 x 50 mm, 1.7 µm
©2007 Waters Corporation 12
ACQUITY ACQUITY ““HPLCHPLC””
10.0
Rs = 1.86
Rs = 2.302.1x100mm 5µ C18
Time in Minutes0.0 10.0
Rs = 3.01
Rs = 6.45
2.1x100mm 5µ C18
ACQUITY (HPLC)
Vendor X HPLC
8 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 conversion
oHPLC 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 ChemistryChemistry
HYDROPHOBICITY (ln [k] acenaphthene)
XTerra® MS C18
Hypersil® Elite C18
Inertsil® ODS-3
Hypersil® HyPurity Elite C18
Luna™ C18YMC-Pack™ Pro C18™ Zorbax® Eclipse® XDB C18Zorbax® Rx C18
Prodigy™ C18
Symmetry® C18
SymmetryShield™ RP18
Supelcosil™ LC-ABZ+Plus
Supelcosil™ LC DB-C18
XTerra® RP18
Luna™ C18(2)
Polaris™ C18-A
-0.2
0
0.2
0.4
0.6
0.8
1 1.5 2 2.5 3 3.5
SELE
CTI
VITY
(ln[α
] am
itrip
tylin
e/ac
enap
hthe
ne) 1.0
1.2
Atlantis® dC18
YMC-Pack™ ODS–AQ™
EXPANDED VIEW
ACQUITY UPLC™ BEHXBridge™
C18
ChromolithTM RP-18
Nucleosil® C181.5
SunFire™ C8
SunFire™ C18
ACQUITY UPLC™ BEHXBridge™
Shield RP18
ACQUITY UPLC™ BEHXBridge™
Phenyl
ACQUITY UPLC™ BEHXBridge™
C8
01/2006 pH 7
©2007 Waters Corporation 17
Determination of Proper Column Determination of Proper Column Dimensions Dimensions
Internal diameter—Generally prefer 2.1 mm
—Only use 1 mm for specific reason
oSeverely sample limited
oDirect flow to mass spectrometer
Length—If primary goal is SPEED
o50 mm length to start
—If primary goal is RESOLUTION
o100 mm length to start
©2007 Waters Corporation 18
Instrument Differences: Instrument Differences: 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 smaller isocratic segment—ADD an initial hold to the gradient table to give the identical
hold.
If target system gives larger isocratic 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 Method
Original Column: 4.6 x 150 mm, 5 μm@ 1.5 mL/min
Abs
orba
nce
254
nm
Minutes
1
23
0 10 20 30
Resolution (1,2) = 12Resolution (2,3) = 28
Ambient temperature: 21- 22°CFlow rate: 1.50 mL/minSample analytes: 1. Caffeine (100mg/mL),
2. Hydroquinidine (33mg/mL), 3. 3-Aminobenzophenone (39mg/mL)
Molecular weight(s): Less than 500Sample diluent: DMSOInjection: 10μLDetection: 254nmMobile phase: A: 0.05% TFA in water
B: 0.05% TFA in acetonitrile
Objective: Maintain resolution while increasing speed
©2007 Waters Corporation 20
Method MigrationMethod MigrationColumn Comparison: Injection VolumesColumn Comparison: Injection Volumes
2.1 x 50mm
Sample volume too largefor smaller column volume
20μL injection/0.19mL = 11%UPLC
4.6 x 150mm 20μL injection/2.49mL = 0.8%HPLC
©2007 Waters Corporation 21
AU
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1.00
1.20
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
Method Migration Method Migration Column Comparison: Injection VolumesColumn Comparison: Injection Volumes
Column transfer from 4.6mm to 2.1mm i.d.No 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 0.5 – 1 µL—If calculated volume too small for injection, dilute 5 - 10x with
initial strength mobile phase
—Typically 5 µL maximum injection on 2.1 x 50 mm
©2007 Waters Corporation 23
Method MigrationMethod MigrationCalculate Injection VolumeCalculate Injection Volume
Scaling a 10µL injection on 4.6 x 150mm to 2.1 x 50mm
Target injection volume =
Original injection volume X Target Column VolumeOriginal Column Volume
0.0760.192.49
=
10μL x 3.14 x 1.12 x 503.14 x 2.32 x 150
=
10μL x10μL x
= 0.8μL
©2007 Waters Corporation 24
Method Migration ExampleMethod Migration ExampleFlow RateFlow Rate
For Migration:—First, adjust flow rate proportional to column diameter
squared for constant linear velocity (geometrically scaled)
—Second, adjust Gradient Table to maintain the same number of column volumes of solvent through the target column
—Finally, adjust flow 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 Geometry
d2Target
d2 Original
Target Flow Rate = Original Flow Rate x π x r2 of Targetπ x r2 of Original
This reduces to:
Target Flow Rate = Original Flow Rate x
1.5mL/min. X 2.12
4.62 = 0.31mL/min.
Scaling a 1.5mL/min flow rate on 4.6x150mm to 2.1x50mm*
*Note: this assumes same particle size
So:
©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 Profile
Gradient Step
Time Since Injection
Flow Rate
%A %B Curve
Initial 0 1.5 95 5 *
6
1
1
2 15 1.5 5 95
3 20 1.5 5 95
4 30 1.5 95 5
©2007 Waters Corporation 28
Gradient SegmentsGradient SegmentsExpress as Column VolumesExpress as Column Volumes
For 15 min at 1.5mL/min on a 4.6 x 150mm column
Gradient Volume = Flow Rate x Time = 1.5mL/min x 15min = 22.5mL
Column Volume = π x r2 x L = 3.14 x 0.232 x 15.0 = 2.49mL
Gradient Duration (cv) = Gradient VolumeColumn Volume
Gradient Duration = 22.5mL2.49mL
= 9.03 cv
©2007 Waters Corporation 29
Original Gradient Profile for ScalingOriginal Gradient Profile for Scaling
StepTime Since Injection
Flow Rate
%A%B
CurveSegment Duration
(min)
Segment Duration
(cv)
Initial 0 1.5 95 5 *
6
1
1
0
2 15 1.5 5 95
0
15
5
9.03
10
3 20 1.5 5 95 3.01
4 30 1.5 95 5 6.02
©2007 Waters Corporation 30
Scaling Gradient Step TimeScaling Gradient Step TimeMaintain Duration (Maintain Duration (cvcv))
Original Step 2: 15 min @ 1.5 mL/min with duration of 9.03cv
Calculate Target Step 2: (keeping duration @ 9.03cv)
Column Volume = π x r2 x L = 3.14 x 0.1052 x 5.0 = 0.17mL
Gradient Step Volume = Duration (cv) x Target Column Volume
= 9.03cv x 0.17mL = 1.54mL
Gradient Step Time = Gradient Step Volume/Flow Rate
= 1.54mL / 0.31 mL/min =
5 min
©2007 Waters Corporation 31
Scaled Gradient ProfileScaled Gradient Profile2.1x50mm Column2.1x50mm Column
Gradient
Step
Time Since
Injection
Flow Rate
%A
%B
CurveSegment Duration
(min)
Segment Duration
(cv)
Initial 0 0.31 95 5 *
6
1
1
0
2 5 0.31 5 95
0
5.0
1.67
9.03
3.33
3 6.67 0.31 5 95 3.01
4 10 0.31 95 5 6.02
Adjust time for same number of column volumesper gradient segment
©2007 Waters Corporation 32
Smaller ParticlesSmaller ParticlesThe Enabler of ProductivityThe Enabler of Productivity
Optimal velocity range
©2007 Waters Corporation 33
Estimate Optimum Flow RateEstimate Optimum Flow RateUPLCUPLC®®
Consider 1.7µm target particle (2.1mm ID column)
Assume temperature and viscosity transferred
Adjust flow rate based on van Deemter curve and approximate molecular weight
— ~0.6 mL/min for smaller molecules
oaverage 500 dalton (molecular weight) molecules
— ~0.1 mL/min for larger molecules because diffusion is slower
oe.g., ~2,000 dalton peptides
©2007 Waters Corporation 34
Scaling for UPLCScaling for UPLC®® Flow RateFlow RateStep Time to Maintain Duration (Step Time to Maintain Duration (cvcv))
Original Step 2: 15 min.@ 1.5 mL/min with Duration of 9.03cv
Calculate Target Step 2: (keeping duration @ 9.03cv)
Target Column Volume (2.1 x 50) = 0.17mL
Gradient Step Volume = Duration (cv) x Target Column Volume
= 9.03cv x 0.17mL = 1.54mL
Gradient Step Time = Gradient Step Volume / UPLC™ Flow Rate
= 1.54mL / 0.60 mL/min. =
2.6min
©2007 Waters Corporation 35
Optimized Gradient ProfileOptimized Gradient Profile
Gradient
Step
Time Since
Injection
Flow Rate
%A
%B
CurveSegment Duration
(min)
Segment Duration
(cv)
Initial 0 0.6 95 5 *
6
1
1
0
2 2.61 0.6 5 95
0
2.61
0.87
9.03
1.74
3 3.48 0.6 5 95 3.01
4 5.22 0.6 95 5 6.02
Select 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: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 Method
Original Column: 4.6 x 150 mm, 5 μm@ 1.5 mL/min, 30 minute cycle time
Abs
orba
nce
254
nm
Minutes
1
23
0 10 20 30
Resolution (1,2) = 12Resolution (2,3) = 28
Ambient temperature: 21- 22°CFlow rate: 1.50 mL/minSample analytes: 1. Caffeine (100mg/mL),
2. Hydroquinidine (33mg/mL), 3. 3-Aminobenzophenone (39mg/mL)
Molecular weight(s): Less than 500Sample diluent: DMSOInjection: 10μLDetection: 254nmMobile phase: A: Water
B: acetonitrileC: 1.0% TFA in water
Gradient: (A/B/C) 90/5/5 to 0/95/5 in 15 minutes (Curve 6) hold for 5 minutes then to initial conditions (curve 11) hold for 10 minutes
©2007 Waters Corporation 38
Method ConversionMethod ConversionScaled UPLCScaled UPLC®®
Minutes
Abs
orba
nce
254
nm
0 10 20 30
1
23
Resolution 1,2 = 11Resolution 2,3 = 26
Column Temp: 30ºCSample analytes: 1. Caffeine (100mg/mL),
2. Hydroquinidine (33mg/mL), 3. 3-Aminobenzophenone (39mg/mL)
Molecular weight(s): Less than 500Sample diluent: DMSOInjection: 0.8μLDetection: 254nmMobile phase: A: 0.05% TFA in water
B: 0.05% TFA in acetonitrileGradient: (A/B) 95/5 to 5/95 in 2.61 minutes (Curve 6)
hold for 0.87 minutes then to initial conditions (curve 11)hold for 1.74 minutes
ACQUITY UPLC BEH C18, 1.7 µm,2.1 x 50 mm @ 0.6mL/min
©2007 Waters Corporation 39
Method ConversionMethod ConversionScaled UPLCScaled UPLC®® MagnifiedMagnified
Abs
orba
nce
254
nm
Minutes
0 2.6 5.2
1
23
UPLC™: 2.1 x 50 mm column, 1.7μm@ 0.6mL/min, 5.2 minute cycle time
Resolution 1,2 = 11Resolution 2,3 = 26
©2007 Waters Corporation 40
Original HPLC MethodOriginal HPLC MethodCritical resolutionCritical resolution
Minutes
Resolution 1,2=12Resolution 2,3=28
Abs
orba
nce
254
nm
1
23
0 10 20 30
Baseline and presence of unknown “peaks”
©2007 Waters Corporation 41
UPLCUPLC®® Magnified ViewMagnified ViewCritical resolutionCritical resolution
Resolution 1,2=11Resolution 2,3=26
Abs
orba
nce
254
nm
Minutes
0 2.6 5.2
1
23
Same resolving power and sensitivity
©2007 Waters Corporation 42
ACQUITY UPLCACQUITY UPLC®® CalculatorCalculator
©2007 Waters Corporation 43
Method ConversionMethod ConversionSignificantlySignificantly Different Column ChemistryDifferent Column Chemistry
When column chemistries are different, try a modified method development approach
Proposed Strategy 1:—Understand general column selectivity differences
—Follow chemistry and system scaling protocols
—Generic gradients : 10 to 90 % organic solvent
—Run a short gradient (3 min) and a long gradient (6 min)
—Optimization tools (simulation software) are useful
Proposed Strategy 2:—Follow method development protocols
At what point does method conversion become method development?
©2007 Waters Corporation 44
Automated Method DevelopmentAutomated Method Development
Automated Method Development—ACQUITY UPLC Column Manager, 4
column selection device
—ACQUITY UPLC Binary Solvent Manager, solvent select valves
©2007 Waters Corporation 45
UPLCUPLC®® Systematic ScreeningSystematic Screening
Four ACQUITY UPLC Chemistries 2.1 x 50 mm, 1.7 µm: —ACQUITY UPLCTM BEH C18
—ACQUITY UPLCTM BEH Shield RP18
—ACQUITY UPLCTM BEH C8
—ACQUITY UPLCTM BEH Phenyl
Solvents: —Acetonitrile
—Methanol
Buffers: —pH 3 ammonium formate
—pH 10 ammonium bicarbonate
©2007 Waters Corporation 46
Experimental MatrixExperimental MatrixpH 3, Acetonitrile pH 10, Acetonitrile
C18
Shield RP18
C8
Phenyl
pH 3, Methanol pH 10, Methanol
©2007 Waters Corporation 47
Automated Column andAutomated Column andSolvent Scouting Solvent Scouting
©2007 Waters Corporation 48
Total Time and Solvents CalculatedTotal Time and Solvents Calculated
©2007 Waters Corporation 49
Can I transfer the method to other Can I transfer the method to other systems that have a standard column systems that have a standard column heater?heater?
AU
0.00
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0.10
0.15
0.20
0.25
0.30
AU
0.00
0.10
0.20
0.30
Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40
ACQUITY UPLC® with TUV & standard Column
Heater
ACQUITY UPLC® with PDA & Column
Manager
©2007 Waters Corporation 50
Develop Methods Faster with UPLCDevelop Methods Faster with UPLC®®::Time SavingsTime Savings
EQUIVALENT HPLC Methods Development Protocol4.6 x 150 mm, 5 µmpH 3/ acetonitrile TimeFlow ramp 5 minColumn conditioning (2 blank gradients) 80 minSample injection (2 replicates) 80 minpH 3/ methanolFlow ramp 5 minColumn conditioning (2 blank gradients) 80 minSample injection (2 replicates) 80 minColumn purge 35 minpH 10/ acetonitrileFlow ramp 5 minColumn conditioning (2 blank gradients) 80 minSample injection (2 replicates) 80 minpH 10/ methanolFlow ramp 5 minColumn conditioning (2 blank gradients) 80 minSample injection (2 replicates) 80 minColumn purge 35 min
730 min
SCREENING TIME 12.2 Hours/columnx 4 columns
TOTAL SCREENING TIME 48.8 HOURS
UPLC Methods Development Protocol2.1 x 50 mm, 1.7 µmpH 3/ acetonitrile TimeFlow ramp 5 minColumn conditioning (2 blank gradients) 11 minSample injection (2 replicates) 11 minpH 3/ methanolFlow ramp 5 minColumn conditioning (2 blank gradients) 11 minSample injection (2 replicates) 11 minColumn purge 6 minpH 10/ acetonitrileFlow ramp 5 minColumn conditioning (2 blank gradients) 11 minSample injection (2 replicates) 12 minpH 10/ methanolFlow ramp 5 minColumn conditioning (2 blank gradients) 11 minSample injection (2 replicates) 11 minColumn purge 6 min
120 min
SCREENING TIME 2 Hours/columnx 4 columns
TOTAL SCREENING TIME 8 HOURS
©2007 Waters Corporation 51
ConclusionConclusion
Strategies and Tools For HPLC to UPLC® Method Migration—Column chemistry and dimensions
—Instrument considerations
—Proper scaling
—Optimization
—Redevelopment
—Method migration/conversion calculator
©2007 Waters Corporation 52
AcknowledgementsAcknowledgements
Diane Diehl
Eric Grumbach
Jeff Mazzeo
Uwe Neue
Michael Jones
Andy Aubin
Craig Dobbs
And:
©2007 Waters Corporation 54
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