Techniques and Strategies for Transferring Methods from ... · ©2008 Waters Corporation Techniques and Strategies for Transferring Methods from HPLC to UPLC Dan Root, Ph.D. Systems

©2008 Waters Corporation

Techniques and Strategies for Transferring Techniques and Strategies for Transferring Methods from HPLC to UPLCMethods from HPLC to UPLC

Dan Root, Ph.D.Dan Root, Ph.D.

Systems Marketing LabSystems Marketing Lab

Waters CorporationWaters Corporation

720002520EN

©2008 Waters Corporation 2

IntroductionIntroduction

Implementation

When a lab invests in UPLC® technology their focus moves to the implementation of this new technology in order to reap it’s many benefits.

HPLC methods must be transferred or migrated to the ACQUITY UPLC®

This may seem like a tremendous challenge but doesn’t have to be.

Waters has software tools that can make this transition rapid and seamless.


Method Transfer: a DefinitionMethod Transfer: a Definition

The movement or migration of an HPLC-based method to the ACQUITY UPLC

This is an integrated solution consisting of the instrument AND the sub 2µm particle columns with their wide variety of chemistries.

Only by utilizing the entire package can the user truly reap the maximum benefits of this new technology


In this TalkIn this Talk

-Go through the method transfer sequence for both an Isocratic and Gradient HPLC method.

-Use Waters software tools

-No theory

-No manual calculations

-Process-oriented talk


Transfer SequenceTransfer Sequence

1. Know as much as possible about method to be transferredGoals of method – resolution/speed Method must be adequate to the task

2. Select appropriate column and dimensionsWaters Column Selectivity chart – column chemistryL/dp index – column dimensions

3. Scale the HPLC method to UPLCACQUITY Columns Calculator

4. Input scaled parameters, inject and evaluate

5. Optimize if necessary

6. Get back to work!


Method Sequence Method Sequence

Step 1: Know Your Method


Step 1: Know Your HPLC MethodStep 1: Know Your HPLC Method

USP Fluconazole related compounds, test 1

Flow rate: 0.50 mL/minSample analytes: Fluconazole (10 μg/mL)

Fluconazole Related Substance A (10 μg/mL) Fluconazole Related Substance B (10 μg/mL)Fluconazole Related Substance C (10 μg/mL)

Molecular weight(s): 306.27Sample diluent: water/acetonitrile (80/20)Injection: 20μLDetection: 260 nmColumn Temperature: 40°CMobile phase: isocratic water/acetonitrile (80/20)Solid Phase: Sunfire™ C18Particle Size: 3.5 µmID: 4.6 mmLength: 150 mm


Isocratic ExampleIsocratic ExampleOriginal HPLC methodOriginal HPLC method

AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00

Resolution (A-B) = 16Resolution (B-C) = 2.5Resolution (C-Flu) = 4.6

Critical resolution for B-C must be > 1.5

A

B

C

Fluconazole



Step 2: Select Column Chemistry


Step 2: The ColumnStep 2: The Column

Waters Column selectivity chart

Link:


Icon on DesktopIcon on Desktop


Using the chart: main windowUsing the chart: main window


Using the chart: Vendor name Using the chart: Vendor name SelectionSelection

Vendor


Using the chart: Vendor name Using the chart: Vendor name Selection_WatersSelection_Waters –– columns displayed columns displayed when when mousedmoused overover


Using the chart: Vendor name Using the chart: Vendor name Selection_WatersSelection_Waters –– columns displayed as columns displayed as list list –– sunfiresunfire C18 highlightedC18 highlighted


Using the chart: remember Using the chart: remember SunfireSunfireposition and return to main pageposition and return to main page


Select the Select the SunfireSunfire C18 columnC18 column


Select the Method Development KitsSelect the Method Development Kits


Select Kit 5: ACQUITY UPLCSelect Kit 5: ACQUITY UPLC


ACQUITY HSS T3ACQUITY HSS T3


ACQUITY BEH C18ACQUITY BEH C18


Which one?Which one?

Closest ACQUITY columns to the Sunfire are:

1) ACQUITY HSS T32) ACQUITY BEH C18

Since the HSS and the Sunfire are both Silica columns we’ll select the HSS chemistry. (BEH is a hybrid particle)

HSS T3

BEH C18


Step 2: Selecting the ColumnStep 2: Selecting the Column

So, from the chart:

ACQUITY HSS T3 chemistry

We have the column chemistry selected, now we need the dimensions.

Is there a straightforward way to do this?



Step 2: Column Dimensions


Step 3: Selecting the Column DimensionsStep 3: Selecting the Column Dimensions

L/dp RATIO

Column Length/Particle Diameter = Dimensionless #

30,000150mm =5 μm =150,000μm5 μm

We use this ratio as a means of comparing the ‘resolving power’ of columns.

Example:

If you keep the L/dp ratio the SAME for 2 columns, you will obtain the SAME Resolution.


Isocratic Example Isocratic Example Original HPLC columnOriginal HPLC column

Solid Phase: Sunfire™ C18

Particle Size: 3.5 µm

ID: 4.6 mm

Length: 150 mm

Calculate: dp150,000 μm

3.5 μm= = 43,000L


Isocratic ExampleIsocratic ExampleL/L/dpdp Comparison for LC ColumnsComparison for LC Columns

5.0

3.5

2.5

1.8μmUPLC®

HSS

1.7μmUPLC®

BEH

Ldp

4,000

5,700

8,000

11,110

20mm

6,000

8,600

12,000

16,670

17,650

30mm

10,000

14,300

20,000

27,770

29,410

50mm

20,000

28,600

55,556

58,820

100mm

50,00030,000

71,40042,900

83,333

88,230

250mm150mm


Isocratic Example Isocratic Example Original HPLC columnOriginal HPLC column

So we now have a column chemistry and the column dimensions:

HSS T3 1.8 µm 2.1 x 100 mm

This was the hardest part!

Now we scale the method parameters…



Step 3: Scale Method Parameters to the ACQUITY UPLC


Isocratic SeparationIsocratic Separation


Isocratic SeparationsIsocratic Separations


Fill In Isocratic HPLC ConditionsFill In Isocratic HPLC Conditions


Change in Maximum PressureChange in Maximum Pressure


Maximum Pressure ChangedMaximum Pressure Changed


Calculate UPLCCalculate UPLC®® ConditionsConditions


UPLCUPLC®® ResultsResults

2.1 x 100 mm column at a flow rate of 0.738 ml/min with an injection volume of 2.8 µl



Step 4: Inject and Evaluate


Step 4: Inject and Evaluate: Step 4: Inject and Evaluate: The Original HPLC Method ProfileThe Original HPLC Method Profile

AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00



A

B

C

Fluconazole


The Transferred MethodThe Transferred Method

AU

0.000

0.010

0.020

0.030

0.040

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00


Resolution (A-B) = 12.2Resolution (B-C) = 6Resolution (C-Flu) = 3.6A

B

C

Fluconazole


Parameter Comparison: Parameter Comparison: Isocratic ExampleIsocratic Example

Original Transferred

Flow rate: 0.50 mL/min 0.738 mL/minInjection: 20μL 2.8 µLDetection: 260 nm 260 nmSolid Phase: Sunfire™ C18 HSS T3Particle Size: 3.5 µm 1.8 µmID: 4.6 mm 2.1 mmLength: 150 mm 100 mmRun Time: 15 minutes 3 minutes (80% faster!)


Resolution (A-B) = 12.2Resolution (B-C) = 6.0Resolution (C-Flu) = 3.6



Step 5: Optimize if Necessary


Transfer Sequence Recap: IsocraticTransfer Sequence Recap: Isocratic

1. Know as much as possible about method to be transferred

Goals of method – resolution/speed

Method must be adequate

2. Selected appropriate column

Waters Column Selectivity chart

L/dp index – column dimensions

3. Scaled the HPLC method to the ACQUITY UPLC

ACQUITY Columns Calculator

4. Injected and evaluated

5. Optimization was not necessary

Transfer Successful! …now for a gradient method



Step 1: Know Your Method


Gradient Example Gradient Example Step 1 Step 1 -- Original HPLC methodOriginal HPLC method

Flow rate: 1.00 mL/minSample analytes: peak #1 - 2-Acetylfuran (4 μg/mL)

peak #2 - Acetanilide (4 μg/mL) peak #3 - Acetophenone (4 μg/mL) peak #4 - Propiophenone (1 μg/mL)peak #5 - Butylparaben (1 μg/mL)peak #6 - Benzophenone (1 μg/mL)

Molecular weight(s): roughly from ~200 - 300Column temperature: 40°CSample Diluent: 10/90 acetonitrile/waterInjection: 15 μLDetection: 254 nmMobile phase: A: 0.1% TFA in water

B: 0.1% TFA in acetonitrile

Column: Atlantis T3 5µm 4.6 x 150 mm


Gradient Example Gradient Example Original HPLC gradient profileOriginal HPLC gradient profile

5951.00Initial

69551.0201

11

11

6

Curve

5951.025.13

5951.0304

9551.0252

%B%AFlow Rate

Time since injection

Gradient Step


Gradient Example: Original HPLC Gradient Example: Original HPLC MethodMethod

AU

0.00

0.20

0.40

0.60

0.80

1.00

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

1 2 3

4

5 6

10.06

5.205

15.04

23.33

7.802

1

ResolutionPeak



Step 2: Select Column


Step 2: Select the Column Step 2: Select the Column ChemistryChemistry


Gradient example: Atlantis T3 Gradient example: Atlantis T3 selectedselected


Gradient example: ACQUITY HSS T3 Gradient example: ACQUITY HSS T3 closest matchclosest match


Step 2: Selecting the ColumnStep 2: Selecting the Column

So, from the chart:

ACQUITY HSS T3 chemistry

What about the dimensions?

What is goal of method – In this case, speed



Step 2: Column Dimensions


Step 3: Column DimensionsStep 3: Column Dimensions

Solid Phase: Atlantis T3

Particle Size: 5.0 µm

ID: 4.6 mm

Length: 150 mm

Calculate: dp150,000 μm

5 μm= = 30,000L


Gradient ExampleGradient ExampleL/L/dpdp Comparison for LC ColumnsComparison for LC Columns

5.0

3.5

2.5

1.8μmUPLC®

HSS

1.7μmUPLC®

Packed

Ldp

4,000

5,700

8,000

11,110

20mm

6,000

8,600

12,000

16,670

17,650

30mm

10,000

14,300

20,000

27,770

29,410

50mm

20,000

28,600

55,556

58,820

100mm

50,00030,000

71,40042,900

83,333

88,230

250mm150mm


Step 3: The ColumnStep 3: The Column

So the column chemistry and the column dimensions are:

HSS T3 1.8 µm 2.1 x 50 mm

Now we scale the gradient parameters to the ACQUITY UPLC



Step 3: Scale Method Parameters to the ACQUITY UPLC


Gradient ExampleGradient Example
















Gradient Example: PrintGradient Example: Print--out out



Step 4: Inject and Evaluate


Gradient Example: Original HPLCGradient Example: Original HPLC

AU

0.00

0.20

0.40

0.60

0.80

1.00

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

1 2 3

4

5 6

10.06

5.205

15.04

23.33

7.802

1

ResolutionPeak


Gradient Example: Transferred MethodGradient Example: Transferred Method

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

10.0

5.20

15.0

23.3

7.80

Resolution

UPLC

6

5

4

3

2

1

Peak

1 2 3

4

56


Parameter Comparison: Parameter Comparison: Gradient ExampleGradient Example

Original Transferred

Flow rate: 1.0 mL/min 1.287 mL/minInjection: 15μL 1.0 µLDetection: 254 nm 254 nmSolid Phase: Atlantis T3 HSS T3Particle Size: 5.0 µm 1.8 µmID: 4.6 mm 2.1 mmLength: 150 mm 50 mmRun Time: 30 minutes 3.86 minutes (87% faster!)

10.0

5.20

15.0

23.3

7.80

Resolution

UPLC

8.006

9.205

15.24

18.23

8.902

1

Resolution

HPLC

Peak



Step 5: Optimize if Necessary


Transfer Sequence Recap: GradientTransfer Sequence Recap: Gradient

1. Know as much as possible about method to be transferred

Goals of method – resolution/speed

Method must be adequate

2. Selected appropriate column

Waters Column Selectivity chart

L/dp index – column dimensions

3. Scaled the HPLC method to the ACQUITY UPLC

ACQUITY Columns Calculator

4. Injected and evaluated

5. Optimization

Another Successful Transfer!


SummarySummary

In both examples we followed a series of straight-forward steps to rapidly and effectively transfer HPLC methods to the ACQUITY UPLC.

The resultant transferred methods were significantly faster and maintained or improved the original HPLC method resolutions.

The Column Selectivity Chart and ACQUITY Columns Calculator are simple, useful tools that will enable rapid, seamless implemenation of the ACQUITY UPLC.


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It provides easy data tracking and complete organization of validation data and results monitored by the built-in oversight of automated error checking.

MVM reduces the time and costs required to perform chromatographic method validation by as much as 80%.

Because MVM allows the entire chromatographic method validation process to be efficiently performed within Empower 2, fewer software applications need be deployed, validated, and maintained

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Documents

Techniques and Strategies for Transferring Methods from ... · ©2008 Waters Corporation Techniques and Strategies for Transferring Methods from HPLC to UPLC Dan Root, Ph.D. Systems