Techniques and Strategies for Transferring
Methods from HPLC to UPLC
Dan Root, Ph.D.
Systems Marketing Lab
Waters Corporation
720002520EN
©2008 Waters Corporation
Introduction
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.
©2008 Waters Corporation
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Method 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
©2008 Waters Corporation
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In 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
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Transfer Sequence
1. Know as much as possible about method to be transferred
Goals of method – resolution/speed
Method must be adequate to the task
2. Select appropriate column and dimensions
Waters Column Selectivity chart – column chemistry
L/dp index – column dimensions
3. Scale the HPLC method to UPLC
ACQUITY Columns Calculator
4. Input scaled parameters, inject and evaluate
5. Optimize if necessary
6. Get back to work!
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Method Sequence
Step 1: Know Your Method
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Step 1: Know Your HPLC Method
USP Fluconazole related compounds, test 1
Flow rate:
Sample analytes:
Molecular weight(s):
Sample diluent:
Injection:
Detection:
Column Temperature:
Mobile phase:
Solid Phase:
Particle Size:
ID:
Length:
0.50 mL/min
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)
306.27
water/acetonitrile (80/20)
20μL
260 nm
40°C
isocratic water/acetonitrile (80/20)
Sunfire™ C18
3.5 µm
4.6 mm
150 mm
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Isocratic Example
Original HPLC method
0.090
0.080
0.070
Resolution (A-B) = 16
Resolution (B-C) = 2.5
Resolution (C-Flu) = 4.6
Critical resolution for
B-C must be > 1.5
A
C
0.060
AU
0.050
0.040
0.030
0.020
Fluconazole
B
0.010
0.000
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
Minutes
8.00
9.00
10.00
11.00
12.00
13.00
14.00
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Method Sequence
Step 2: Select Column Chemistry
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Step 2: The Column
Waters Column selectivity chart
Link:
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Icon on Desktop
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Using the chart: main window
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Using the chart: Vendor name
Selection
Vendor
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Using the chart: Vendor name
Selection_Waters – columns displayed
when moused over
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Using the chart: Vendor name
Selection_Waters – columns displayed as
list – sunfire C18 highlighted
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Using the chart: remember Sunfire
position and return to main page
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Select the Sunfire C18 column
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Select the Method Development Kits
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Select Kit 5: ACQUITY UPLC
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ACQUITY HSS T3
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ACQUITY BEH C18
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Which one?
Closest ACQUITY columns to the Sunfire are:
1) ACQUITY HSS T3
2) 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
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Step 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?
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Method Sequence
Step 2: Column Dimensions
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Step 3: Selecting the Column Dimensions
L/dp RATIO
Column Length/Particle Diameter = Dimensionless #
Example:
150mm = 150,000μm
5 μm
5 μm
=
30,000
We use this ratio as a means of comparing the
‘resolving power’ of columns.
If you keep the L/dp ratio the SAME for 2
columns, you will obtain the SAME Resolution.
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Isocratic Example
Original HPLC column
Solid Phase:
Sunfire™ C18
Particle Size:
3.5 µm
ID:
4.6 mm
Length:
150 mm
Calculate:
L
dp
=
150,000 μm
=
3.5 μm
43,000
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Isocratic Example
L/dp Comparison for LC Columns
L
30mm
50mm
100mm
150mm
17,650
29,410
58,820
88,230
11,110
16,670
27,770
55,556
83,333
2.5
8,000
12,000
20,000
3.5
5,700
8,600
14,300
28,600
42,900
71,400
5.0
4,000
6,000
10,000
20,000
30,000
50,000
dp
20mm
250mm
1.7μm
UPLC®
BEH
1.8μm
UPLC®
HSS
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Isocratic Example
Original 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…
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Method Sequence
Step 3: Scale Method Parameters to the
ACQUITY UPLC
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Isocratic Separation
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Isocratic Separations
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Fill In Isocratic HPLC Conditions
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Change in Maximum Pressure
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Maximum Pressure Changed
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Calculate UPLC® Conditions
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UPLC® Results
2.1 x 100 mm column at a
flow rate of 0.738 ml/min with
an injection volume of 2.8 µl
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Method Sequence
Step 4: Inject and Evaluate
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Step 4: Inject and Evaluate:
The Original HPLC Method Profile
0.090
0.080
0.070
Resolution (A-B) = 16
Resolution (B-C) = 2.5
Resolution (C-Flu) = 4.6
Critical resolution for
B-C must be > 1.5
A
C
0.060
AU
0.050
0.040
0.030
0.020
Fluconazole
B
0.010
0.000
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
Minutes
8.00
9.00
10.00
11.00
12.00
13.00
14.00
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The Transferred Method
0.040
Resolution (A-B) = 12.2
Resolution (B-C) = 6
Resolution (C-Flu) = 3.6
Critical resolution for
B-C must be > 1.5
A
AU
0.030
C
0.020
0.010
Fluconazole
B
0.000
0.00
0.50
1.00
1.50
Minutes
2.00
2.50
3.00
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Parameter Comparison:
Isocratic Example
Flow rate:
Injection:
Detection:
Solid Phase:
Particle Size:
ID:
Length:
Run Time:
Original
Transferred
0.50 mL/min
20μL
260 nm
Sunfire™ C18
3.5 µm
4.6 mm
150 mm
15 minutes
0.738 mL/min
2.8 µL
260 nm
HSS T3
1.8 µm
2.1 mm
100 mm
3 minutes (80% faster!)
Resolution (A-B) = 16
Resolution (B-C) = 2.5
Resolution (C-Flu) = 4.6
Resolution (A-B) = 12.2
Resolution (B-C) = 6.0
Resolution (C-Flu) = 3.6
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Method Sequence
Step 5: Optimize if Necessary
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Transfer 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
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Method Sequence
Step 1: Know Your Method
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Gradient Example
Step 1 - Original HPLC method
Flow rate:
Sample analytes:
1.00 mL/min
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):
Column temperature:
Sample Diluent:
Injection:
Detection:
Mobile phase:
roughly from ~200 - 300
40°C
10/90 acetonitrile/water
15 μL
254 nm
A: 0.1% TFA in water
B: 0.1% TFA in acetonitrile
Column:
Atlantis T3 5µm 4.6 x 150 mm
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Gradient Example
Original HPLC gradient profile
Curve
Gradient
Step
Time since
injection
Flow
Rate
%A
%B
Initial
0
1.0
95
5
1
20
1.0
5
95
6
2
25
1.0
5
95
6
3
25.1
1.0
95
5
11
4
30
1.0
95
5
11
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Gradient Example: Original HPLC
Method
1.00
5
0.80
0.60
AU
4
0.40
1
2
0.20
6
Peak
Resolution
1
2
7.80
3
23.3
4
15.0
5
5.20
6
10.0
3
0.00
0.00
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
Minutes
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Method Sequence
Step 2: Select Column
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Step 2: Select the Column
Chemistry
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Gradient example: Atlantis T3
selected
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Gradient example: ACQUITY HSS T3
closest match
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Step 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
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Method Sequence
Step 2: Column Dimensions
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Step 3: Column Dimensions
Solid Phase:
Atlantis T3
Particle Size:
5.0 µm
ID:
4.6 mm
Length:
150 mm
Calculate:
L
dp
=
150,000 μm
=
5 μm
30,000
©2008 Waters Corporation
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Gradient Example
L/dp Comparison for LC Columns
L
30mm
50mm
100mm
150mm
17,650
29,410
58,820
88,230
11,110
16,670
27,770
55,556
83,333
2.5
8,000
12,000
20,000
3.5
5,700
8,600
14,300
28,600
42,900
71,400
5.0
4,000
6,000
10,000
20,000
30,000
50,000
dp
20mm
250mm
1.7μm
UPLC®
Packed
1.8μm
UPLC®
HSS
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Step 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
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Method Sequence
Step 3: Scale Method Parameters to the
ACQUITY UPLC
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Gradient Example
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Gradient Example
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Gradient Example
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Gradient Example
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Gradient Example
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Gradient Example
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Gradient Example
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Gradient Example
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Gradient Example: Print-out
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Method Sequence
Step 4: Inject and Evaluate
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Gradient Example: Original HPLC
1.00
5
0.80
0.60
AU
4
0.40
1
2
0.20
6
Peak
Resolution
1
2
7.80
3
23.3
4
15.0
5
5.20
6
10.0
3
0.00
0.00
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
Minutes
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Gradient Example: Transferred Method
0.40
5
Peak
6
UPLC
1
AU
0.30
4
0.20
0.10
1
2
Resolution
2
7.80
3
23.3
4
15.0
5
5.20
6
10.0
3
0.00
0.00
0.50
1.00
1.50
2.00
Minutes
2.50
3.00
3.50
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Parameter Comparison:
Gradient Example
Flow rate:
Injection:
Detection:
Solid Phase:
Particle Size:
ID:
Length:
Run Time:
Peak
Original
Transferred
1.0 mL/min
15μL
254 nm
Atlantis T3
5.0 µm
4.6 mm
150 mm
30 minutes
1.287 mL/min
1.0 µL
254 nm
HSS T3
1.8 µm
2.1 mm
50 mm
3.86 minutes (87% faster!)
Resolution
Resolution
UPLC
HPLC
2
7.80
8.90
3
23.3
18.2
4
15.0
15.2
5
5.20
9.20
6
10.0
8.00
1
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Method Sequence
Step 5: Optimize if Necessary
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Transfer 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!
©2008 Waters Corporation
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Summary
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.
©2008 Waters Corporation
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Empower 2 Method Validation
Manager
ƒ Waters Method Validation Manager Software is designed to
streamline the set-up, execution, calculation and reporting
of a method validation.
ƒ It provides easy data tracking and complete organization of
validation data and results monitored by the built-in
oversight of automated error checking.
ƒ MVM reduces the time and costs required to perform
chromatographic method validation by as much as 80%.
ƒ 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
ƒ Many other powerful features.
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