Deep dive into the flow

Develop and transfer robust

HPLC methods

Application Scientist
Agilent Technologies

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Agenda

Method development

• Overview
• Hands-on method development
• Automated method scouting
• Automated method development

Method transfer
• Overview
• Delay volume
• Extra column volume/Dispersion

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HPLC method development overview

1. Define the method goals

2. Choose detectors

3. Choose LC mode

4. Prepare sample

5. Develop the method

6. Optimize conditions

7. Validate the method

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Define the method goals

• What is known about the sample?

 Number of compounds present
 Chemical/physical properties (structure; molecular weight; pKa values; solubility; stability…)
 Concentration
 Matrix
• What information do you want from the sample?
 Detect if the compound is present
 Quantify the compound
 Identify the compound
 Characterize the compound
 Purify the compound
• Literature search (don’t believe everything you read)

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Choose detectors
Chemical properties of the compound; detection limits needed

• Ultraviolet/Visible light detector (UV)

 Compound must have chromophore or can be derivatized
• Fluorescence detector (FLD)
 UV absorbing compounds that can fluoresce
• Refractive Index (RI)
 Universal (only isocratic methods)
• Evaporative light scattering detector (ELSD)
 Universal (semi-volatile or non-volatile)
• Mass spectrometer (MS)
 Compound can be ionized

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Choose LC modes
Compound type, solubility and molecular weight; sample matrix

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Prepare sample
Sample prep options

• Filtration (particulate level)

• Ultrafiltration (molecular level)
• Centrifugation
• Drying or freeze-drying (lyophilized)
• Precipitation
• Liquid-liquid extraction
• Solid phase extraction (primitive prep LC)
• Derivatization
• .....and chopping, crushing, dissecting, etc.

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Develop the method
Where to start?

Critical method parameters (CMPs) Critical method attributes (CMAs)

• Column chemistry • Peak resolution
• Solvent types • Specificity
• Gradients • Sensitivity
• Column temperature • Run time
• pH • …
• …
 Consider compound chemistry
 Optimize selectivity
 Use a scouting gradient

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Hands-on method development

Step 1. Start with EC-C18

Step 2. Generic gradient (5-95% ACN) Common additives
Step 3. Change the gradient 1. 0.1% Formic Acid (pH ~2.7)
2. 10 mmol Ammonium Acetate (adj. pH 5)
Step 4. Change modifiers, temperatures 3. 0.1% Ammonium Hydroxide (pH ~10)
4. 0.1% Trifluoroacetic acid (pH ~1.5, MS)
Step 5. Change from ACN to MeOH 5. 150 mmol Sodium Phosphate (adj. pH 3, MS)

Step 6. Choose a different column

…. more steps
Starting Recommendation
Poroshell 120 EC-C18
Change Selectivity Change Selectivity For Many Early No retention at 98+% Acidic Solvents Basic Solvents
Slightly Significantly Eluters Aqueous in RP (pH < 2) (pH >6)
1. Poroshell 1. Poroshell 1. Poroshell SB-Aq 1. Poroshell HILIC-Z 1. Poroshell SB- 1. Poroshell
Phenyl-Hexyl Bonus-RP 2. Poroshell PFP 2. Poroshell PFP C18 HPH-C18
2. Polaris C18-A 2. Poroshell PFP 3. Poroshell HILIC-Z 3. Poroshell HILIC- 2. Poroshell SB-Aq 2. PLRP-S
3. Poroshell EC-C8 3. Pursuit XRs OH5 3. PLRP-S 3. Poroshell
Diphenyl 4. Poroshell SB-C8 HPH-C8

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Automated LC method scouting

CMPs
• Column chemistry
• Solvent types
• Gradients
• Column temperature
• pH
• …

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Scalable LC method development solutions
1260 Infinity II Method Development 1290 Infinity II Method Development

Column Capacity

Four columns in one MCT Eight columns in one MCT

External Solvent
Selection Valves

A B C D A B C D
One solvent selection valve (15 mobile phases) Two solvent selection valves (26 mobile phases)

> 100 chromatographic conditions > 1300 chromatographic conditions

∆T Temperature ∆T
Zones
Two independent temperature zones Two independent temperature zones

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Agilent Method Scouting Wizard

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Agilent Method Scouting Wizard

• Define project
Choose scouting combinations and
base method.
• Select columns
All installed columns are shown
automatically.
• Select solvents
Pump types and valves are
automatically detected.
• Define gradients
Select between different gradients and
temperatures.
• Review and select screening
methods
Check for incompatible combinations.
• Set up samples
Define injection volumes and number of
repetitions.

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MSW – Select the Column

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Submit the scouting sequence

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Example: Separation of 16 pesticides on reversed phase columns

Name ID L (mm) Particle Flow Time

(mm) Size (um) Factor Factor
Bonus RP 2.1 100 1.8 0.208 0.667

SB-C18 2.1 100 1.8 0.208 0.667

SB-C8 2.1 100 1.8 0.208 0.667

Eclipse Plus C18 2.1 150 1.8 0.208 1.000

SB-C8 4.6 50 5 1.000 0.333

SB-C18 4.6 50 3.5 1.000 0.333

Bonus RP 4.6 50 3.5 1.000 0.333

2 mobile phase combinations: water-methanol; water-acetonitrile

3 column temperatures: 40; 50; 60 °C

• 88 methods (42 analytical methods, 46 automatically created system flushing and column equilibration methods)
• Duplicate sample injections => 84 sample data files
• Total run time:1 day and 15 hours

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Evaluate the screening results

No. of Minimum Minimum

peaks resolution symmetry

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Example: Separation of 16 pesticides on reversed phase columns
Evaluate screening results-bubble plot

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Example: Separation of 16 pesticides on reversed phase columns
Evaluate screening results-table

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Example: Separation of 16 pesticides on reversed phase columns
Evaluate screening results-chromatograms

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Example: Separation of 16 pesticides on reversed phase columns
Evaluate screening results-chromatograms

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Method development
Traditional approach can lead to method transfer issues

Traditional Approach
One-factor-at-a-time Variability in critical method
± 50% ± 0.2
attributes (resolution, tailing, etc)

Method specifications:
Buffer solutions prepared by
Slope 14.5% MeOH
20mM buffer
different operators deliver different
pH 7 results
45oC
1.0 mL/min
± 10%
Small changes in pH, temperature
± 10% or flow rate show a large effect
Method fails robustness
± 10oC

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Method development
Traditional approach vs Quality by Design (QbD) approach

Traditional Approach QbD Approach

One-factor-at-a-time Systematic statistical

± 50% ± 0.2

Method specifications:
Slope 14.5% MeOH
20mM buffer
pH 7
45oC
1.0 mL/min
± 10%

± 10%
Method fails robustness
± 10oC Design space
Working within the design space will ensure the method‘s robustness

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Method development
Integrating QbD principle

• Statistical software: R, SAS, JMP…

• Dedicated software packages for chromatographic method development
• ChromSwordAuto
• Fusion QbD
• ACD/AutoChrom

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Automated method development
QbD approach (3rd party software: ChromSwordAuto)

ChromSwordAuto 5.1 package :

• ChromSwordAuto Scout: Method
screening
• ChromSwordAuto Developer:
Rapid and fine method optimization
for small and large molecules
• ChromSword AutoRobust:
Robustness studies and method
improvement
• ReportViewer: Data browsing,
processing, and projects
management

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Automated method development – 16 pesticides
QbD approach (3rd party software: ChromSwordAuto)

Initial gradient Optimized gradient Final gradient

26 April 20, 2022 DE44337.984212963 Tech Overview: 5994-0373EN

Automated method development – 16 pesticides
QbD approach (3rd party software: ChromSwordAuto)

For robustness studies, full factorial

design was applied.
All possible combinations of flow
rate, temperature, and concentration
of solvent A (%). The applied
gradients were exactly parallel, with
1 % distance between them.

The yellow area is the optimum resolution range,

where measured resolution is above 2.0 for all
peak pairs.

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 Automated method development
QbD approach (3rd party software: Fusion QbD)

Chromatography-centric QbD Software for

LC, LC-MS, and SFC Method Development

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QbD workflow with Fusion QbD software

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Automated method development – peptide mapping
QbD approach (3rd party software: Fusion QbD)

Screening

30 April 20, 2022 DE44337.984212963 App Note: 5991-6338EN

Automated method development – peptide mapping
QbD approach (3rd party software: Fusion QbD)

Optimization

31 April 20, 2022 DE44337.984212963 App Note: 5991-6338EN

Automated method development – peptide mapping
QbD approach (3rd party software: Fusion QbD)

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What is method transfer?
From particle size to particle size (HPLC to UHPLC)
 often associated with a change of instruments due to constraints in max. pressure, extra-
column band broadening etc.. Some fine-tuning of method might be required due to frictional
heating effects
 available tools: Method translator, Method development SW (S-Matrix, ACD Labs,
ChromSword, etc.)

From one eluent type or phase chemistry to another

 Method development
 available tools: Method development SW (ACD Labs, ChromSword, S-Matrix etc.)

From column dimension to column dimension (e.g. 4.6 to 2.1 mm i.d., 50 mm to 100 mm)
 recalculate flow rates, recalculate gradient times, adjust connection capillaries and flow
cells, due to delay volume results may vary, method might need revalidation
 available tools: Method translator

From Instrument to Instrument

 Run established, validated methods unchanged and get same results
 available tools w/o method or instrument change: ISET

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Method transfer considerations

140

120

Legacy method
Response (mAU)

100

80

60

40
Method transfer — What went wrong?
20

0 1 2 3 4 5 6 7

Retention time (min)

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Potential instrument related method transfer factors

Delay volume Extra column volume

• Flow cell design
Detector • Data rate
Volume between the
effective injection point
and the effective detection
Column • Solvent pre-heating
point, excluding the part of Thermostat • Control range
the column containing the
stationary phase.
• Design: flow-through
or fixed loop
Autosampler • Injection volume
Volume between the point • Needle wash
of solvent mixing and the
column head • Quaternary or binary pump
Pump • Pressure x flow rate
• Mixing behavior

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Method transfer between different HPLC systems
Impact of delay volume and mixing behavior

Programmed 1290 Infinity II 1260 Infinity

gradient step High Speed Pump Binary Pump
90 %

Gradient slope/
response

Mixing behavior
What happens with a
programmed gradient?
Delay volume

10 %
time
1200 Series Total delay volume of the system (sum
of capillaries, mixer, cells, valves..)
1290 Infinity II

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Method transfer between different HPLC systems
Impact of delay volume and mixing behavior

mAU Gradient differences due to different dwell

600 volumes and different mixing behavior
1290 Infinity LC
500

400 1100 Series Quaternary LC

300

200

100

Results from a tracer experiment

0

0 1 2 3 4 5 6 7 8 9 min

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Method transfer between different HPLC systems
Approach 1: adding an isocratic hold

1290 Infinity LC
mAU
Still gradient differences due to 1 min isocratic hold
different mixing behavior

400
1100 Series Quaternary LC

300

200

100

Results from a tracer experiment

0

0 1 2 3 4 5 6 7 8 9 min

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Method transfer between different HPLC systems
Approach 1: adding an isocratic hold

1260 Infinity LC

1290 Infinity LC
+ 900 µl hold

• Low consistency
• Requires manual determination of the dwell volume/ isocratic hold
(systems equipped with dampeners the dwell volume is pressure dependent and variable)
• Requires modification of the methods

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Method transfer between different HPLC systems
Approach 2: adding a physical capillary

1290 Infinity LC

mAU
Almost consistency of both 1 mL loop installed

gradient curves

400
1100 Series Quaternary LC

300

200

100

Results from a tracer experiment

0

0 1 2 3 4 5 6 7 8 9 min

40
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Method transfer between different HPLC systems
Approach 2: adding a physical capillary

1290 + 1000 μL dwell vol.

1100/1200 Quat Pump

• Good consistency
• Manual determination of dwell volumes required (issues of a variable dwell volume
with systems containing damperners)
• All mechanical changes are laboriously and not flexible

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Method transfer between different HPLC systems
Agilent Solution: Intelligent System Emulation Technology (ISET)
mAU
400

350

Injection
300
Software controlled compensation of dwell
250 volume differences and synchronization of
mixing behaviors
200

150

100 Programmed gradient

1290 gradient
50
1200 gradient
0
0 0.5 1 1.5 2 2.5 3 3.5 min

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Method transfer between different HPLC systems
Agilent Solution: Intelligent System Emulation Technology (ISET)

1200 Quat
110

100

90

80

70

60

50

40

30

20
rel. Response [%]

10

0
0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25 4.5 4.75 5 5.25 5.5 5.75 6 6.25 6.5 6.75 7 7.25 7.5 7.75 8 8.25 8.5 8.75 9 9.25 9.5 9.75 10 10.25 10.5 10.75 11 11.25 11.5 11.75 12
Retention time [min]

1290 Binary no ISET

110

100

90

80

70

60

50

40

30

20
rel. Response [%]

10

0
0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25 4.5 4.75 5 5.25 5.5 5.75 6 6.25 6.5 6.75 7 7.25 7.5 7.75 8 8.25 8.5 8.75 9 9.25 9.5 9.75 10 10.25 10.5 10.75 11 11.25 11.5 11.75 12
Retention time [min]

110

100

90 1290 Binary with ISET

80

70

60

50

40

30

20
rel. Response [%]

10

0
0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25 4.5 4.75 5 5.25 5.5 5.75 6 6.25 6.5 6.75 7 7.25 7.5 7.75 8 8.25 8.5 8.75 9 9.25 9.5 9.75 10 10.25 10.5 10.75 11 11.25 11.5 11.75 12
Retention time [min]

43
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Method transfer between different HPLC systems
Extra column volume (dispersion)

 General:
• Column
• Tubing (i.d., length, internal surface)
• Connectors (unions, tees, bulkhead fittings)
• Guard columns and/or inline particle filters
• Switching valves for autoSPE, column selection, column regeneration
 Sampler:
• Diluent strength and injection volume
• Sample aspirating needle and loading/transfer port
• Sampler switching valve(s) contacting sample
 Detection:
• Inlet heat exchangers, flow cell volume and geometry

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Resources — Primers

5990-7595EN
The LC Handbook
Guide to LC Columns and Method Development
5991-2359EN
Two Dimensional Liquid Chromatography

5990-3777EN
High Performance Capillary Electrophoresis

5991-5509EN
Supercritical Fluid Chromatography
5989-6639EN
Principles in Preparative HPLC

5991-3326EN
Sample Preparation Fundamentals for Chromatography

5980-1397EN
Fundamentals of UV-visible Spectroscopy

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Resources for Support
• Collection of LC resources:
https://community.agilent.com/technical/lc/w/wiki/4916/collecti
on-of-lc-hplc-resources
• LC Troubleshooting Poster:
https://www.agilent.com/cs/library/posters/public/5994-
0709EN-poster-LC-troubleshooting-agilent.pdf
• Agilent support resources:
https://community.agilent.com/resources/w/wiki/4920/collectio
n-of-support-resources
• Agilent University: http://www.agilent.com/crosslab/university
• Agilent resource center:
http://www.agilent.com/chem/agilentresources
• InfinityLab Supplies Catalog (5991-8031EN)
gc-column-support@agilent.com
• Your local FSE and Specialists lc-column-support@agilent.com
• Youtube – Agilent Channel spp-support@agilent.com
spectro-supplies-support@agilent.com
• Sales and support phone assistance (US and Canada):
1-800-227-9770 Phone Tree Navigation Assistance

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 Thank you!

Questions?
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