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©2007 Waters Corporation

Waters AAPS 2007 Seminars

November 12-13, 2007

©2007 Waters Corporation 2

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An Introduction to UPLCAn Introduction to UPLC®® Technology: Technology: Improve Productivity and Data Quality Improve Productivity and Data Quality

Doug McCabeDoug McCabe


AgendaAgenda

Introduction: What is UPLC® Technology?

Migrating an HPLC Method to a UPLC® Method

Efficient UPLC® Method Development and Validation

Conclusion


UPLCUPLC®® Technology & The Technology & The Fundamental Resolution EquationFundamental Resolution Equation

))((1k

k14N

Rs+

−=

αα

•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

If N ↑ 3x, Rs ↑ 1.7x

NRs∝

Physical Chemical


Improving Resolution with Smaller ParticlesImproving Resolution with Smaller ParticlesConstant Column LengthConstant Column Length

Efficiency (N), is inversely proportional to Particle Size, dp

Rs ↑ 1.7X N ↑ 3X,dp ↓ 3X,(e.g., 5 μm to 1.7 μm) (i.e., Rs α √N)

∝


0

5

10

15

20

25

30

0 1 2 3 4 5 6 7 8 9 10 11 12

Linear Velocity [mm/s]

HE

TP

(µ

m)

5 µm SunFire™ C18

3.5 µm SunFire™ C18

1.8 µm ACQUITY UPLC® HSS T3

1.7 µm ACQUITY UPLC® BEH C18

UPLCUPLC®® Particles, Particles, van Deemter Curves & van Deemter Curves & Flow RatesFlow Rates

8.407.707.006.305.604.904.203.502.802.101.400.704.6 mm ID

1.801.651.501.351.201.050.900.750.600.450.300.152.1 mm ID

0.420.390.350.320.280.250.210.180.140.110.070.041.0 mm ID

Flow rate (mL/min)


Resolution (and Speed)Resolution (and Speed)Constant Column LengthConstant Column Length

Plates, Flow Rate and Particle Size

10000900080007000600050004000300020001000

1.0 2.0 3.0

Flow Rate{mL/min}

N

1200011000

Smaller Particle

Larger Particle

Smaller Particle Size*Increased N,*Higher, optimal u*Increased pressure

Optimal

Optimal flow rate is inversely proportional to dp

Isocratic analysis time is inversely proportional to F

Rs

Rs ↑ 1.7X, N ↑ 3X, dp ↓ 3X, T ↓ 3X

dp1

Fopt ∝

(e.g., 5 μm to 1.7 μm) (Rs α √N)


Efficiency, N is inversely proportional to the square of Peak Width, W

Peak height is inversely proportional to Peak Width

Peak Width and SensitivityPeak Width and SensitivityConstant Column LengthConstant Column Length

2w1

N∝

w1

Height ∝

Rs ↑ 1.7X, N ↑ 3X, dp ↓ 3X, T ↓ 3X

Sensitivity ↑ 1.7X

(e.g., 5 μm to 1.7 μm) (Rs α √N)


BackpressureBackpressureConstant Column LengthConstant Column Length

Backpressure is proportional to Flow Rate, FR, andinversely proportional to Particle Size squared

Optimal Flow Rate is inversely proportional to Particle Size (further to the right on van Deemter curve)

2dp1

FRΔP ×∝

dp1

FRopt∝

P ↑ 27X (~1/dp3)

dp ↓ 3X,(e.g., 5 μm to 1.7 μm)


Constant Column LengthConstant Column LengthFlow Rate Proportional to Particle SizeFlow Rate Proportional to Particle Size

AU

0.000

0.010

0.020

0.030

0.040

0.050

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 15.00

4.8 µm, 0.2 mL/min, 354 psi

AU

0.000

0.010

0.020

0.030

0.040

0.050

Minutes

0.00 1.00 2.00 3.00 4.00 5.00 6.00

Theory1.7X Resolution

3X Faster1.7X Sensitivity25X Pressure

Reality1.5X Resolution

2.6X Faster1.4X Sensitivity22X Pressure

1.7 µm, 0.6 mL/min, 7656 psi

2.1 x 50 mm columns Too Much Backpressure!


Column Length to Particle Size RatioColumn Length to Particle Size RatioIndicates Maximum Resolution CapabilityIndicates Maximum Resolution Capability

100mm1.7μm

μm

300mm10μm = 30,000

30,000150mm =5μm

100mm =3 33,300

50mm1.7μm

= 29,410

= 58,820

1970’s ~ 30 min.

1990’s ~ 10 min.

2004 ~1 - 2 min.

1980’s ~ 15 min.

L/dp RATIO Typical Run Times

2x Max. Resolution Capability

150mm1.7μm

= 88,230 3x Max. Resolution Capability

30 mm

50 mm

100 mm

150 mm

17,650

29,500

58,820

88,235

L/dp

2.5 μm

3.0 μm

3.5 μm

5.0 μm

8,000

6,667

5,714

4,000

L/dp

IS® Columns(20 mm Length)

dp

Length (L)

The SAME L/dp for 2 columns will produce the SAME Resolution.The difference: shorter columns will produce the separations faster.


Scaling HPLC to UPLCScaling HPLC to UPLC®® SeparationsSeparationsConstant L/Constant L/dpdp = Equivalent Resolution= Equivalent Resolution

HP

LC

Sep

ara

tio

ns

UP

LC

®

Sep

ara

tio

n

2.5 µm – 75 mmInjection = 2.5 µL

Flow rate = 0.5 mL/minRs (2,3) = 2.34

5 µm – 150 mmInjection = 5.0 µL







Speed Increases Speed Increases Constant L/dpConstant L/dp

Efficiency, N, is directly proportional to column length, L, and

inversely proportional to particle size, dp:

For same N and, therefore, same Rs

dpL

N∝

N = 1X, L ↓ 3X,dp ↓ 3X, Rs = 1X,

F ↑ 3X, T ↓ 9X Efficiency & Resolution

Remain Unchanged

(e.g., 5 μm to 1.7 μm) (e.g., 150 mm to 50 mm)

(i.e., F increases 3X,L decreases 3X)


Assuming same efficiency, Peak Height is inversely proportional to column length, L:

For same efficiency, column length, L is decreased proportionally to particle size, dp (constant L/dp)

Sensitivity IncreasesSensitivity IncreasesConstant L/dpConstant L/dp

L1

Height =

N = 1X, L ↓ 3X, dp ↓ 3X, Rs = 1X,

Sensitivity ↑ 3X T ↓ 9X, Efficiency & Resolution

Remain Unchanged

(e.g., 5 μm to 1.7 μm) (e.g., 150 mm to 50 mm)

(Increased optimal flow rate & shorter column)

(Peak height increases as peak width and column length decreases)


Backpressure (P) is proportional to Column Length, L:

For constant L/dp, Backpressure is inversely proportional to the square of Particle Size, dp:

Backpressure Increases Backpressure Increases Constant L/dpConstant L/dp

LP∝

2dp1

P∝

P ↑ 9X L ↓ 3X, dp ↓ 3X, (e.g., 5 μm to 1.7 μm) (e.g., 150 mm to 50 mm)


AU

0.00

0.02

0.04

0.06

Minutes

0.00 5.00 10.00 15.00 20.00 25.00 30.00

4.8 µm, 100 mm, 0.2 mL/min

Length Proportional to Particle SizeLength Proportional to Particle SizeSimilar L/dpSimilar L/dp

AU

0.00

0.02

0.04

0.06

Minutes

0.00 1.00 2.00 3.00 4.00

RealitySame Resolution

8X Faster2.5X Sensitivity

11X Back Pressure

1.7 µm, 30 mm, 0.6 mL/min

TheorySame Resolution

9X Faster3X Sensitivity

9X Back Pressure

2.1 mm ID columnsManageable Backpressure

Increase


UPLCUPLC®® Technology & Gradient Technology & Gradient Peak Capacity (Resolution)Peak Capacity (Resolution)

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50

tg

w ww w

w

wt

1Pc g+=

Gradient Duration

Peak Width

w ↓, Pc ↑

Peak capacity is a measure of the separation power of a gradient on a particular column

Pc = # Peaks separated per gradient duration time


UPLCUPLC®® Technology & Gradient Technology & Gradient Peak Capacity (Resolution)Peak Capacity (Resolution)

Peak capacity is affected by:— Gradient duration (tg)

— Flow rates (F)

— Column length (L)

— Particle size (dp)

Remember, the flow rate, column length and particle size all influence the plate count (N in isocratic separations)

By optimizing these parameters, peak capacities can be maximized

Influences peak width


Dependence of Peak Capacity on Dependence of Peak Capacity on Operating ConditionsOperating Conditions

1tt

ΔcB

ΔcB4

tL

Dd

cLt

Dbda

L

1P

g

0

0M

2p0

Mp

+⋅⋅

⋅⋅

⋅⋅+⋅⋅+⋅+=

We can now generate a 3-dimensional plot to examine how the gradient time and flow rate effect the peak capacity of a separation.

wt

1Pc g+=Start with the simple equation for peak capacity

Make a few substitutions

Neue, U. D., Mazzeo, J. R. J. Sep. Sci. 2001, 24, 921-929.Cheng, Y-F., Lu, Z., Neue, U. Rapid Commun. Mass Spectrom. 2001, 15, 141-151.


GradientDuration

(min)

FlowRate

(mL/min)

Pressure(psi)

Max PeakCapacity

1 0.352 3621 634 0.124 1280 10016 0.088 905 13632 0.062 640 151

Effect of Particle Size on Peak Effect of Particle Size on Peak Capacity for UPLCCapacity for UPLC®® SeparationsSeparations

Gradient Duration

(min)

Flow Rate

(mL/min)

Pressure(psi)

Max Peak Capacity

1 0.249 10852 1084 0.249 10852 17216 0.124 5426 21632 0.088 3837 231

Gradient Duration

(min)

Flow Rate

(mL/min)

Pressure(psi)

Max Peak Capacity

1 0.35 1774 464 0.124 627 7616 0.062 314 10732 0.044 222 121

ACQUITY UPLC® Columns can provide better Peak Capacity in 1 minute, than a 5 μm column in 16 minutes!!

1.0 x 50 mm Columns Pmax = ~11,000 psi

0.00

30.

004

0.00

50.

008

0.01

10.

016

0.02

20.

031

0.04

40.

062

0.08

80.

124

0.17

60.

249

0.35

20.

498

0.70

40.

995

1.40

71.

990

12

48163264

0

50

100

150

200

250

0.00

30.

004

0.00

50.

008

0.01

10.

016

0.02

20.

031

0.04

40.

062

0.08

80.

124

0.17

60.

249

12

48

1632

64

0

50

100

150

200

250

0.00

30.

004

0.00

50.

008

0.01

10.

016

0.02

20.

031

0.04

40.

062

0.08

80.

124

0.17

60.

249

0.35

20.

498

0.70

40.

995

12

4816

3264

0

50

100

150

200

250

Peak C

ap

aci

ty

Flow Rate (mL/min)

1.7 μm 3.5 μm 5 μm

108

100 107

Gra

dien

t D

ura

tion

(min

)


High Resolution Peptide Mapping:High Resolution Peptide Mapping:Influence of Particle SizeInfluence of Particle Size

AU

0.00

0.02

0.04

0.06

0.08

AU

0.00

0.02

0.04

0.06

0.08

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 60.00

UPLC® Gradient1.7 µm

Peaks = 168Pc = 360

2.5X Increase

HPLC Gradient5 µm

Peaks = 70Pc = 143

More information in the same amount of time


Summary:Summary:UPLC® Technology: What is it?

UPLC® Technology is based on chromatographic theory (not marketing) and utilizes:— Small, pressure-tolerant particles that are efficiently

packed into short (fast) or long (high resolution) columns

— An LC system that can operate at the optimal linear velocity (and resulting pressure) for these particles and possesses minimal system volume and fast, responsive detectors that do not negatively affect efficiency

UPLC® Technology provides information in less time and, hence, at a lower cost


AgendaAgenda




Conclusion


Example: Migrating an HPLC Method to a Example: Migrating an HPLC Method to a UPLCUPLC®® MethodMethod

AU

0.00

0.10

0.20

0.30

0.40

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

UPLC® MethodPc = 85

HPLC MethodPc = 94

AU

0.00

0.10

0.20

0.30

0.40

Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00


Method Migration/Conversion: Method Migration/Conversion: HPLC to UPLCHPLC to UPLC®® TechnologyTechnology

Why Convert HPLC Methods to UPLC® Technology?— Acquire results in less time and/or with more resolution

o More information - faster

o More robust methods – greater confidence

o Better situational response time (stat samples faster, research decisions with more information, process monitoring, product release)

o More samples analyzed per system, per scientist


Channel: W2996 238.0nm-1.2; Processed Channel: W2996 PDA 238.0 nm at 1.2; Injection: 3; Date Acquired: 10/5/2006 10:12:29 AM EDT; Result Id: 1318; Processing Method: Simvastatin BEH 4_6 x 250

9.28

1

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

USP USP HPLCHPLC Separation of Separation of SimvastatinSimvastatin

O

OO

OOH

CH3 CH3 CH3

CH3

CH3H

Plates = 12,112USP Method Requirements

k’ > 3.0N > 4,500Tf < 2.0

HPLC:RT = 9.28 min

k’ = 4.1N = 12,112

Tf = 1.1


UPLCUPLC®® Method Conversion Method Conversion ChoicesChoices

Sim

vast

atin

- 1.

412

AU

0.00

0.07

0.14

0.21

Minutes0.00 0.50 1.00 1.50

Sim

vast

atin

- 1.

921

AU

0.00

0.07

0.14

0.21

Minutes0.00 0.50 1.00 1.50 2.00 2.50

Sim

vast

atin

- 0.

234

AU

0.00

0.03

0.06

0.09

Minutes0.00 0.50 1.00 1.50

Maximum EfficiencyMaximum Efficiency Equal EfficiencyEqual Efficiency Fastest AnalysisFastest Analysis

Efficiency = 12874Efficiency = 17685 Efficiency = 977

UPLC®:RT = 1.41 min

k’ = 4.9N = 12,874

Tf = 1.1

HPLC:RT = 9.28 min

k’ = 4.1N = 12,112

Tf = 1.1

USP Req.k’ > 3.0

N > 4,500Tf < 2.0


Critical Caveat of Method Critical Caveat of Method ConversionConversion

The new method will be different from the original method—Operating conditions, e.g., flow rate

—Run time

—Appearance

—Of course, the objective was an improved method

The new method must preserve critical parameters—Complete resolution of all relevant analytes

—Peak homogeneity/purity

—Certainty of peak identification

—Quantitative accuracy and precision


Method Conversion ProcessMethod Conversion ProcessSteps for SuccessSteps for Success

1. Gather information about existing method and results

2. Select new or target column— Chemistry

— Dimensions

3. Compare instruments

4. Calculate method conversion conditions

5. Evaluate results of transfer

6. Optimize as required


1. Gather Required Information 1. Gather Required Information Original Method & ResultsOriginal Method & Results

Column — Chemistry (ligand, brand,

particle size)— Dimensions

Conditions— Mobile phase— Flow rate— Gradient profile, including

regeneration and reequilibration

— TemperatureSample— Diluent— Concentration— Molecular weight(s)— Injection volume

Chromatogram— Number of peaks

— Retention

— Resolution (critical pairs)

Quantitation— Limit of detection

— Limit of quantitation

— Linear dynamic range

— Accuracy

— Precision


2. Select Target Column 2. Select Target Column ACQUITY UPLCACQUITY UPLC®® Column ChemistriesColumn Chemistries

UPLC® Column Chemistries— ACQUITY UPLC® BEH C18— ACQUITY UPLC® BEH Shield RP18— ACQUITY UPLC® BEH C8 — ACQUITY UPLC® BEH Phenyl— ACQUITY UPLC® BEH HILIC— ACQUITY UPLC® HSS T3— ACQUITY UPLC® HSS C18— ACQUITY UPLC® HSS C18 SB

Check Column Selection Chart for closest match to original— Chromatographic test at pH 7— Provides an assessment of a column’s hydrophobicity and

base/neutral selectivity— Can be used to select “equivalent” columns for methods transfer


Waters ReversedWaters Reversed--Phase Column Phase Column Selectivity ChartSelectivity Chart

YMC-Pack™ ODS-A™

YMC J'sphere™ ODS–H80

ACQUITY UPLC® BEHXBridge™

C18

(ln [k] acenaphthene)102007

SunFire ™ C18

YMC-Pack™ PolymerC18™

Hypersil® CPS Cyano

YMC-Pack™ CN

Waters Spherisorb® S5 P

Hypersil® BDS PhenylNova-Pak® Phenyl

YMC-Pack™Phenyl

Hypersil® PhenylInertsil® Ph-3

YMC-Pack™ Pro C4™

YMCbasic™

Symmetry® C8YMC-Pack™ Pro C8™

Nova-Pak®C8

XTerra® MS C18 Symmetry® C18

YMC-Pack™Pro C18™

Inertsil® ODS-3

Nova-Pak®C18

YMC J'sphere™ODS–L80 Nucleosil® C18

Waters Spherisorb® ODS2

Waters Spherisorb® ODS1Resolve® C18

µBondapak™ C18

YMC-Pack™ ODS–AQ™

YMC J'sphere™ ODS–M80

Inertsil® CN-3

Waters Spherisorb® S5CN

Nova-Pak® CN HP

SymmetryShield™ RP8

SymmetryShield™ RP18

XTerra® RP8

XTerra® RP18

-0.6

-0.3

0

0.3

0.6

0.9

1.2

1.5

1.8

2.1

2.4

2.7

3

3.3

3.6

-1.5 -0.5 0.5 1.5 2.5 3.5

(ln [α

] am

itrip

tylin

e/ac

enap

hthe

ne)

XTerra® MS C8 Luna ® C18 (2)

XTerra ®Phenyl Luna ™

Phenyl Hexyl

ChromolithTM

RP-18Atlantis® dC18 Zorbax® XDB C18

ACT Ace® C18

Zorbax® SB C18

SunFire ™ C8

Luna®C8 (2)

ACQUITY UPLC® BEHXBridge™Shield RP18

ACQUITY UPLC® BEH XBridge™

C8

ACQUITY UPLC® BEH XBridge™

Phenyl

Atlantis® T3

ACQUITY UPLC® HSS T3

ACQUITY UPLC® HSS C18

ACQUITY UPLC® HSS C18 SB


3. Required Information 3. Required Information Original InstrumentOriginal Instrument

Mode of gradient generation—Single pump with gradient proportioning valve (GPV)

—Dual pump

—Brand and model number

System volume (dwell volume or delay volume)—Value and method used to measure

Injection mechanism

Mode of detection


3. System Volumes of Pumping 3. System Volumes of Pumping SystemsSystems

DetectorInjector Column

Pump 1

Smaller System Volume = Smaller Dwell volume

Pump 2

Mixer

Multi-Pump (High Pressure)

Gradient Proportioning Valve

DetectorInjectorAB

CD

Column Solventdelivery

Larger System Volume = Larger Dwell volume

Single Pump (Low Pressure)

Volume: From where mobile phase is mixed, to

where it enters the column.


Compensating for System Volume Compensating for System Volume DifferencesDifferences

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

ACQUITY UPLC® Columns Calculator handles this compensation


3. Instrument Comparison3. Instrument ComparisonInjectionInjection

We assume—The specified volume of sample is delivered to the

column

—The sample composition is not altered

—There is no carryover

System differences affect—Volume of sample required

—Absolute amount injected

—Sample carryover


ACQUITY UPLCACQUITY UPLC®® SystemSystemSample ManagerSample Manager

Minimize sample dispersion during injection

Reduce cycle time

Preserve—Accuracy

—Precision

—Low carryover

—Sample format flexibility

Dual wash system with a strong wash followed by a weak wash


3. Instrument Comparison 3. Instrument Comparison DetectionDetection

We assume—Response is specific

—Response is linear with concentration

—Detector does not alter peak shape

System differences affect—Specificity

—Limit of detection (LOD)

—Limit of quantitation (LOQ)

—Linear dynamic range

—Band-broadening


Detection with UPLCDetection with UPLC®® SeparationsSeparationsUV, ELSD & FLRUV, ELSD & FLR

Only Waters ACQUITY UPLC® TUV or PDA—Minimize band-broadening

—Provide adequate sampling rate (S/N)

—Accurate wavelength

UPLC® ELSD and FLR detectors also available

Sensitivity and dynamic range are affected by reduced peak volume and by improved resolution


Data Acquisition RatesData Acquisition RatesImpact on UV DataImpact on UV Data

Minutes0.50 0.52 0.54 0.56 0.58 0.60 0.62 0.64 0.66 0.68 0.70 0.72 0.74

1 pt/s

2 pt/s

5 pt/s

40 pt/s

20 pt/s

10 pt/s

How many Data Points is

enough?

Simple Rule:15-20 Points per

Peak

The RATE the points are collected is

determined by how wide the peak is in

TIME at the baseline.

If a peak is only 1 second wide, then you need to collect

20 points in 1 second (20 Hz)

Peaks are ~ 1-2 seconds wide


Detection with UPLCDetection with UPLC®® Separations Separations MS and MS/MSMS and MS/MS

Waters MS systems designed for UPLC®

separations—Z-spray source minimizes band-broadening

—Quattro Premier XE, LCT Premier XE and Q-Tof Premier

—SQD & TQD designed specifically for ACQUITY UPLC®

system and separations

—ZQ single quadrupole data acquisition is compatible with UPLC® separations

Other Waters MS detectors can be operated with compatible sampling rates


Ready to Migrate: Target Conditions Ready to Migrate: Target Conditions Mobile Phase and Injection ParametersMobile Phase and Injection Parameters

Use exactly the same mobile phase—Alter only after evaluating transfer if optimization is

required

Use exactly the same sample—Same concentration

—Same diluents

ACQUITY UPLC® System needle wash— Use final gradient conditions as strong needle wash

(200 µL)

— Use initial gradient conditions as weak needle wash (600 µL)


AU

0.00

0.10

0.20

0.30

0.40

Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00

4. Example: Original HPLC Method:4. Example: Original HPLC Method:Caffeic Acid Derivatives of Echinacea Purpurea

Pc = 94

1 23

4

5

Name Retention Time USP Resolution1. Caftaric acid 5.712. Chlorogenic acid 7.07 4.203. Cynarin 13.96 21.194. Echinacoside 16.54 10.165. Cichoic acid 20.32 17.14


Enter Existing HPLC Enter Existing HPLC Gradient Conditions Gradient Conditions

1. Enter column, system & analyte information

2. Enter HPLC gradient Time & %B

3. Select Pmax for UPLC®

separation

4. Press Calculate

HPLC results given here

Calculated for you


Five UPLCFive UPLC®® Gradient Choices GivenGradient Choices Given

Five Gradient Choices:

GeometricallyScaled

1. HPLC Linear Velocity2. UPLC® Linear Velocity

3. User Definedor

Optimally Scaled4. Maximum Peak

Capacity5. Shortest Analysis

Time

Select View for Detailed Gradient Profiles


UPLCUPLC®® Method Gradient Profiles Method Gradient Profiles ChoicesChoices

Original HPLC Gradient Method

Geometrically ScaledHPLC Linear Velocity

Geometrically ScaledUser Defined Flow Rate

Geometrically ScaledUPLC® Linear Velocity

Maximum Peak Capacity Equivalent

Analysis Time

Equivalent Peak Capacity Shortest

Analysis Time


Calculator Choice 1 (default):Calculator Choice 1 (default):Geometrically Scaled Gradient at HPLC Linear Velocity

Geometrically Scaled UPLC® Gradient at HPLC Linear Velocity:Identical Column Volumes per Time Segment

Flow rate scaled for column ONLY

Original HPLC GradientUPLC® Gradient:

HPLC Linear Velocity


AU

0.00

0.10

0.20

0.30

0.40

Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00

AU

0.00

0.10

0.20

0.30

0.40

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00

Calculator Choice 1 (default):Calculator Choice 1 (default):Geometrically Scaled Gradient at HPLC Linear Velocity

HPLC Method

UPLC® MethodChoice 1

Pc = 94

Pc = 99

Similar Resolution35.00

NameRetention Time

USP Resolution

caftaric acid 5.71chlorogenic acid 7.07 4.20cynarin 13.96 21.19echinacoside 16.54 10.16cichoic acid 20.32 17.14

NameRetention Time

USP Resolution

caftaric acid 1.99chlorogenic acid 2.38 3.97cynarin 4.85 25.00echinacoside 5.93 15.09cichoric acid 7.11 19.62


Calculator Choice 2:Calculator Choice 2:Geometrically Scaled Gradient at UPLC® Linear Velocity

Geometrically Scaled UPLC® Gradient at UPLC® Linear Velocity:

Identical column volumes per Time SegmentFlow rate scaled for column AND particle size

Original HPLC GradientUPLC® Gradient:

UPLC® Linear Velocity


AU

0.00

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0.20

0.30

0.40

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

Calculator Choice 2:Calculator Choice 2:Geometrically Scaled Gradient at UPLC® Linear Velocity

NameRetention Time

USP Resolution

caftaric acid 5.71chlorogenic acid 7.07 4.20cynarin 13.96 21.19echinacoside 16.54 10.16cichoic acid 20.32 17.14

HPLC Method

NameRetention Time

USP Resolution

caftaric acid 0.71chlorogenic acid 0.87 3.96cynarin 1.72 22.12echinacoside 2.06 11.40cichoric acid 2.44 14.28

UPLC® MethodChoice 2

Pc = 94

Pc = 85

Similar Resolution

AU

0.00

0.10

0.20

0.30

0.40

Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00


UPLCUPLC®® Method Gradient Profiles Method Gradient Profiles ChoicesChoices

AU

0.00

0.10

0.20

0.30

0.40

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00

AU

0.00

0.10

0.20

0.30

0.40

Minutes

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00

AU

0.00

0.10

0.20

0.30

0.40

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

Original HPLC Gradient Method

Geometrically ScaledHPLC Linear Velocity

Geometrically ScaledUPLC® Linear Velocity

Equivalent Peak Capacity Shortest

Analysis Time

Maximum Peak Capacity Equivalent

Analysis Time

Similar Selectivities Different Selectivities


SummarySummaryHPLC to UPLC® Method Conversion

Methods can be moved directly from HPLC to ACQUITY UPLC® technology for— Improved resolution

— Improved speed

— Improved detectability

Attention to detail leads to success

ACQUITY UPLC® Columns Calculator facilitates process

Converting to UPLC® technology/methodology increases profitability by lowering cost of sample analysis and various connected operating costs


AgendaAgenda




Summary


Pharmaceutical Application AreasPharmaceutical Application Areas

Forced Degradation Studies

Stability Indicating Studies

Impurity Profiling and Identification

Quantitative Bioanalysis

Batch Scale Up Studies

Batch Comparison

Raw Material Acceptance

Finished Product Release Testing

Cleaning Validation

Patent Protection


UPLC Methods Development Protocol2.1 x 50 mm, 1.7 µm, 0.5 mL/minpH 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 minSCREENING TIME 2 Hours/ Hybrid column

x 3 columns

1 Hour/ Silica columnx 1 column

TOTAL SCREENING TIME 7 HOURS

UPLC® Screening: 6.1X faster than 5.0 µm HPLC Column

Why Develop Methods with UPLCWhy Develop Methods with UPLC®® Technology?Technology?Time Savings Versus 5 µm HPLC Column

EQUIVALENT HPLC Methods Development Protocol, 5 µm4.6 x 150 mm, 5 µm, 1.0 mL/minpH 3/ acetonitrile TimeFlow ramp 5 minColumn conditioning (2 blank gradients) 79.2 minSample injection (2 replicates) 79.2 minpH 3/ methanolFlow ramp 5 minColumn conditioning (2 blank gradients) 79.2 minSample injection (2 replicates) 79.2 minColumn purge 43.2 minpH 10/ acetonitrileFlow ramp 5 minColumn conditioning (2 blank gradients) 79.2 minSample injection (2 replicates) 79.2 minpH 10/ methanolFlow ramp 5 minColumn conditioning (2 blank gradients) 79.2 minSample injection (2 replicates) 79.2 minColumn purge 43.2 min

740 minSCREENING TIME 12.3 Hours/ Hybrid column

x 3 columns

6.15 Hours/ Silica columnx 1 column

TOTAL SCREENING TIME 43 HOURS


Automated Method Development and Automated Method Development and ValidationValidation

Automated Method Development— ACQUITY UPLC® Column

Manager, 4 column selection device

— ACQUITY UPLC® Binary Solvent Manager, solvent select valves

Automated Method Validation— Empower® 2 Method Validation

Manager (MVM) streamlines method validation process


Column Chemistry

Solvent pHα

Selectivity

Selectivity ToolsSelectivity Tools

αSelectivity


Effect of Mobile Phase pHEffect of Mobile Phase pH

Affects only analytes with ionizable functional groups— Amines

— Carboxylic acids

— Phenols

Some compounds contain one or more ionizable function

Strongest selectivity effects can be caused by pH changes


pH

0

5

10

15

20

25

30

35

40

0 2 4 6 8 10 12

Ret

entio

n Fa

ctor

(k)

Acid

Base

Neutral

Note: Retention of neutral analytes not affected by pH

Increasedbase retention

Increased acid retention

Silica pH Range

Hybrid Particle pH Range

Neue et. al. American Laboratory 1999 (22) 36-39.

ReversedReversed--Phase Retention Map:Phase Retention Map:The Importance of Mobile Phase pHThe Importance of Mobile Phase pH

BEH

HSS


Column ChemistryLigand & Base particle

Solvent pH

αSelectivity


αSelectivity


Waters UPLCWaters UPLC®® Particles OverviewParticles Overview

Ethylene Bridged Hybrid (BEH) Particles—Wide pH range (1-12)—Five chemistries—Seamless HPLC → UPLC® method migration – with same

selectivity as XBridgeTM HPLC columns—130Å and 300Å pore diameters

High Strength Silica (HSS) Particles—ONLY UPLC®-certified 100% silica particle—Three C18 chemistries —Developed specifically for UPLC® applications—Packed, tested and guaranteed compatibility with at

pressures up to 15,000 psi (1000 bar)


The The ChemistriesChemistries of UPLCof UPLC®®

Technology Technology

Sep2006

Jun 2007

Dec2007

Mar2004

Mar2005

Mar2005

Mar2005

Dec2005

LaunchDate


BEH C18

BEH C8

BEH Shield RP18

BEH Phenyl

1

43

5 8,9,137 10116

1412

1 43

52

7 810

9 116

141312

14352

87 109116 141312

2

1

4

35 8 710,11

9 61413122

HSS T31 435

7,810

9 116 1413122

Conditions : Columns: ACQUITY UPLC® BEH 2.1 x 100 mm, 1.7 µm

ACQUITY UPLC® HSS 2.1 x 100 mm, 1.8 µmMobile Phase A: H2OMobile Phase B: MeOHFlow Rate: 0.5 mL/min Isocratic: 28% MeOHInjection Volume: 5.0 µLSample Concentration: 10 µg/mLTemperature: 50 oCDetection: UV @ 254 nmSampling rate: 20 pts/secTime Constant: 0.1Instrument: ACQUITY UPLC®with ACQUITY UPLC® PDA

HSS C18102

9431

8765

14131211

Selectivity ChoicesSeparations of Separations of NitroaromaticsNitroaromatics

Compounds1. HMX2. RDX3. 1,3,5-TNB4. 1,3-DNB5. NB6. Tetryl7. TNT8. 2-Am-4,6-DNT9. 4-Am-2,6 DNT10. 2,4-DNT11. 2,6-DNT12. 2-NT13. 4-NT14. 3-NT

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00

1122

33 44 5,65,677

88 99 1010 1111

1212 1313 1414 HSS C18 SB


Different LigandsDifferent Ligands::Different SelectivitiesDifferent Selectivities

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


Low pH Selectivity DifferencesLow pH Selectivity Differences35%ACN/65% 15.4 mM HCOONH35%ACN/65% 15.4 mM HCOONH44, pH 3.0, pH 3.0

Y-AXIS X-AXISMaterial k (Tol) α (Ami/Tol) ln k (Tol) ln α (Ami/Tol)

BEH C18 13.01 0.365 2.57 -1.01

BEH C8 8.27 0.469 2.11 -0.76

BEH Shield RP18 11.19 0.256 2.42 -1.36

BEH Phenyl 6.60 0.613 1.89 -0.49

BEH 300 C18 5.97 0.409 1.79 -0.89

HSS C18 20.45 0.299 3.02 -1.21

HSS T3 17.91 0.393 2.89 -0.93HSS C18 SB 9.23 0.842 2.22 -0.17

BEH C8

BEH Phenyl

Shield RP18

BEH C18

BEH 300 C18

HSS T3HSS C18

HSS C18 SB

1.0

1.5

2.0

2.5

3.0

3.5

-1.75 -1.25 -0.75 -0.25 0.25

ln α (ami/tol) pH 3.0

lnK

(to

luen

e)


ACQUITY UPLCACQUITY UPLC®® USP USP ““LL””Designation Designation –– 2007/20082007/2008

August 2007: ACQUITY UPLC® Columns meet USP requirements are now officially considered “L” columns


Column Chemistry

Solvent pH

αSelectivity


αSelectivity


Solvent PropertiesSolvent Properties

Methanol—Weaker eluent

—H-bonding solvent

Acetonitrile—Aprotic solvent

—Stronger eluent

—Lower viscosity


Automated Method Development and Validation Automated Method Development and Validation Example: Example: ParoxetineParoxetine and Related Compoundsand Related Compounds

Method Development— Use systematic screening protocol

— Paroxetine (API) concentration: 0.2 mg/mL in 50:50 MeOH:H2O

— Related compounds at 10% concentration of API for easy identification during scouting

Method Optimization — Related compounds at 0.1% concentration of API

Paroxetinem.w. 374.8


ParoxetineParoxetine Related CompoundsRelated Compounds

Paroxetine HCl

— (-)-trans-4R-(4'-fluorophenyl)-3S-((3',4‘methylenedioxyphenoxy)methyl)piperidine

Paroxetine related compound B

— (trans-4-phenyl-3-[(3,4-methylenedioxy)phenoxymethyl]-piperidine HCl

Paroxetine related compound D

— (cis)-paroxetine HCl

Paroxetine related compound F

— Trans (-)-1-methyl-3-[(1,3-benzodioxol-5-yloxy)methyl]-4-(4-fluorophenyl)piperidine

Paroxetine related compound G

— [(+/-)Trans-3-[(1,3-benzodioxol-5-yloxyl)methyl]-4-(4”-fluorophenyl-4’phenyl)piperidine HCl


2. Column Chemistry

3. Solvent 1. pHα

Selectivity

Systematic Screening:Systematic Screening:Combining Chemical FactorsCombining Chemical Factors

αSelectivity


Stationary Phase Selectivity:Stationary Phase Selectivity:ParoxetineParoxetine

CM, ESG

Methanol pH 3.0

Poor resolution of paroxetine and Related Compounds (RC)

Observation:

Investigate high pHAction:

AU

0.00

0.10

0.20

0.30

0.40

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

ACQUITY UPLC® BEH C18

AU

0.00

0.10

0.20

0.30

0.40

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

AU

0.00

0.10

0.20

0.30

0.40

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

AU

0.00

0.10

0.20

0.30

0.40

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

ACQUITY UPLC®

BEH Shield RP18

ACQUITY UPLC® BEH Phenyl

ACQUITY UPLC® HSS T3

B

GD FPa

roxe

tine

B GD

FParo

xetin

e

BGD

FParo

xetin

e

B

GD

FParo

xetin

e


pH Selectivity:pH Selectivity:ParoxetineParoxetine

CM, ESGCM, ESG

Better retention and resolution of API from RC due to neutral charge state of analytes at alkaline pH

Observation:

Select pH 10 due to better separation

Compare stationary phase selectivity with pH 10 buffer

Actions:

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

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

pH 3.0Methanol

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

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

pH 10Methanol

BGD

F

Paro

xetin

e

B GD F

Paro

xetin

e


Stationary Phase Selectivity:Stationary Phase Selectivity:ParoxetineParoxetine

CM, ESG

Methanol pH 10.0

Any column may provide successful separation

Observation:

Select ACQUITY UPLC® BEH C18

Compare selectivity between organic modifiers

Actions:

AU

0.00

0.10

0.20

0.30

0.40

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


AU

0.00

0.10

0.20

0.30

0.40

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

AU

0.00

0.10

0.20

0.30

0.40

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

ACQUITY UPLC® BEH Shield RP18

ACQUITY UPLC® BEH Phenyl

B GD F

Paro

xetin

e

B GD F

Paro

xetin

e

B GD F

Paro

xetin

e


Solvent Selectivity:Solvent Selectivity:ParoxetineParoxetine


CM, ESG

Methanol is weaker elution solvent resulting in greater retention

Better resolution exhibited with acetonitrile as organic modifier

Observations:

Select acetonitrile as organic modifier

Optimize separation using appropriate concentration of RC

Actions:

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

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

AcetonitrilepH 10.0

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

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

MethanolpH 10.0

BG

D FPa

roxe

tine

BGD F

Paro

xetin

e


Related Compounds at Related Compounds at 0.1% Concentration of 0.1% Concentration of ParoxetineParoxetine

Inadequate resolution among paroxetine and related compounds B and D due to disparate levels of concentration

Observation:

Change gradient slopeAction:

AU

0.00

0.02

0.04

0.06

0.08

0.10

Minutes2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00

AU

-0.003

-0.002

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

Minutes2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00

Related Compounds at 10%

Related Compounds at 0.1%

D

G

F

B

Paro

xetin

e

DG

F

B

Paro

xetin

e


AU

-0.003

-0.002

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

Minutes2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00

AU

0.002

0.003

0.004

0.005

0.006

0.007

0.008

Minutes2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00

Method Optimization:Method Optimization:Gradient SlopeGradient Slope


CM, ESG

5 Minute Gradient5%-90%


Acetonitrile pH 10.030oC

Marginal improvement in separation of impurities from parent compound with shallow gradient slope

Observations:

Alter gradient endpoint to produce shallower slope

Action:

DG

FB

Paro

xetin

e

DG

FB

Paro

xetin

e


AU

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

Minutes2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00

AU

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

Minutes2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00

Method Optimization:Method Optimization:Gradient SlopeGradient Slope

CM, ESG



Acetonitrile pH 10.030oC

Resolution remains inadequate with shallow gradient slope

Observations:

Investigate column temperature

Action:

D

GFB

Paro

xetin

e

DG F

B

Paro

xetin

e


Method Optimization:Method Optimization:Column TemperatureColumn Temperature

CM, ESG

Acetonitrile pH 10.0

Higher temperature improves separation of RC from paroxetine

Peak shape improves as temperature increases

Observations:

Select 60 oC for best resolution and peak shape

Action:

AU

0.002

0.004

0.006

0.008

Minutes2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00

AU

0.002

0.004

0.006

0.008

Minutes2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00

AU

0.002

0.004

0.006

0.008

Minutes2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00

30 oC

45 oC

60 oCD G FB Pa

roxe

tine

D G FB Paro

xetin

e

D G FB

Paro

xetin

e


Final Method Final Method ParoxetineParoxetine and Related Compounds at 0.1%and Related Compounds at 0.1%

AU

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.010

Minutes2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00

D G FBPa

roxe

tine

Compound USP RsRelated compound BParoxetine 1.95Related compound D 3.07Related compound G 13.00Related compound F 6.74

ACQUITY UPLC® BEH C18,, 2.1 x 50 mm, 1.7 µmMobile phase A: 20 mM NH4HCOO3, pH 10Temperature: 60 oC5 Min Gradient: 20%-65% ACNFlow rate: 0.5 mL/minInjection Volume: 4 µLDetection: UV @ 295 nm

AU

0.00

0.10

0.20

0.30

0.40

0.50

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00


Why Validate?Why Validate?

Ensures that analytical methodology is accurate, reproducible and robust over the specific range that an analyte will be analyzed

Provides assurance of reliability

FDA Compliance

Good Science!!

"The process of providing documented evidence demonstrating that something (the method or procedure) does what it is intended to do; is suitable for its intended purpose."


Method ValidationMethod Validation

Waters cannot define that for you

You must define or follow your own corporate policies

However, there are some strategies colleagues are using today — Full Blown Validations (conservative approach)

— Cross validations

o SLAP technique– perform selectivity, linearity, area, and precision tests

Relate specifications to critical quality attributes— Propose acceptance criteria based on scientific rationale

— Relationships established from DOE and prior knowledge

Which Validation Steps need to be performed after a method has been converted to UPLC® technology?

Automation tools can be used to facilitate the process


New Method Validation Manager New Method Validation Manager Option For Empower 2Option For Empower 2

"The industry is very much in need of a workflow-based, configurable system that allows users to implement an organization’s method validation practices. An approach that seamlessly implements method validation requirements but is inherently flexible and manages the data, effectively standardizes an often tedious and time-consuming process“— James Morgado, Pfizer Global R&D

Method Validation Manager option automates the laborious method validation process within Empower 2 Software

Realize time savings of 50% – 80% in this formerly manual and error-prone iterative process


Data Acquisition& Processing

Time consuming, repetitive tasks consisting of Time consuming, repetitive tasks consisting of several sequential stepsseveral sequential stepsFaster and Easier Method ValidationFaster and Easier Method Validation

Corporate Method ValidationSOP

MethodValidationManager

Analytical Method Validation Process with Analytical Method Validation Process with Method Validation ManagerMethod Validation Manager

PrepareStandards & Samples

Data Management

Create SampleSequence

Calculation Statistical Results

Reports Compiled


Automated Method Validation Manager:Automated Method Validation Manager:ParoxetineParoxetine Validation ResultsValidation Results

pass0.00 – 2.57% RSDVariance Component < 5 %RSD Retention Time

(Buffer strength, Additive Conc., Column Temperature, Flow Rate, Injection Volume)

Method Robustness

Retention Time

pass0.06 – 1.64% RSDVariance Component < 2 %RSD Peak Area

(Buffer strength, Additive Conc., Column Temperature)

Method Robustness

Peak Area

pass0.05% of active at 0.2 mg/mL (s/n 2.2 – 6.23)

Impurities 0.1% of active at 0.2 mg/mLLOD of impurities

pass

pass

pass

Analyst 1.33% RSD

Instrument 7.72% RSD

Column 0.00% RSD

Variance Component < 10 %RSD Peak Area

(Analyst, Instrument, Column)

Intermediate Precision

pass97 – 102 %80 – 120 %Accuracy

pass

pass

0.9999

1.74% RSD

R2 > 0.995

Residuals < 2.0% RSD

Linearity

Pass/FailReported ValueAcceptance CriteriaParameter


Methods Development and Validation Timeline:Methods Development and Validation Timeline:Instrument Time

UPLC® Methods Development and Validation Timeline2.1 x 50 mm, 1.7 µm, 0.5 mL/min

Screening Time4 Columns, 2 Organics, 2 pH’s 7.0 hours

OptimizationGradient Slope and Temperature 1.7 hours

ValidationAccuracy, linearity, repeatability,Reproducibility, LOD/LOQ, Intermediate precision, robustness 21.1 hours

TOTAL TIME 29.8 HOURS


Method Validation ManagerMethod Validation ManagerBenefitsBenefits

Save time over entire process and less error prone— Data management is handled by Empower 2, not by user

— Automatic data checks performed at each step of the workflow

— Data approvals can be configured at each step of the workflow

— Calculations done in Empower 2

o No transfer to spreadsheets or other software

o No transcription error / No need to check data transfer

o No need to validate spreadsheet functions

o Multi-component analysis and batch processing of validation results

— Report templates can be used to standardize the report format

o Automatic report generation

o Ease of Review


Summary:Summary:Efficient UPLCEfficient UPLC®® Method Development and ValidationMethod Development and Validation

Achieve more resolution, faster by utilizing sub-2 µm UPLC®

columns at optimal linear velocities with full pressure capabilities up to 15,000 PSI

Principles of methods development remain the same

UPLC® column chemistries provide a broad range of selectivity to successfully develop methods efficiently

UPLC® Technology allows for faster methods development and validation

UPLC® Technology, Empower® 2 and Method Validation Manager software can significantly improve laboratory productivity and compliance


AgendaAgenda




Conclusion


ConclusionConclusion

UPLC® Technology provides more reliable information FASTER and at a LOWER cost per analysis

UPLC® Technology is NOT:— Just fast LC (speed WITHOUT resolution)

— 2.X µm HPLC particles packed into short (≤100 mm) HPLC column hardware that are run on an HPLC system under HPLC operating pressures (beware of these claims)

UPLC® Technology and Empower 2 software can improve productivity and compliance by streamlining method development and validation protocols


THANK YOU


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