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©2018 Waters Corporation 1 COMPANY CONFIDENTIAL
Tips and Tricks to Perform Efficient
Method Transfer
Jonathan E. Turner
Waters Corp.
©2018 Waters Corporation 2 COMPANY CONFIDENTIAL
When are the Methods Transferred?
R&D
Manufacturer Contract
Labs
CxOs
QC Testing
Lab Method Transfer
©2018 Waters Corporation 3 COMPANY CONFIDENTIAL
Method Validation, Verification and Transfer
<621>
New/Intern
al Methods
USP
Methods
Compendial methods must pass the system
suitability requirement(s), and these need
to be verified with the API and/or Final drug
formulation.
©2018 Waters Corporation 4 COMPANY CONFIDENTIAL
US-FDA Guidance for Industry;
– PAC-ATLS: Post Approval Changes – Analytical Testing Laboratory Sites (1998)
– Comparability protocols (February 2003)
– Changes to an Approved NDA or ANDA (April 2004)
– Changes to an Approved NDA or ANDA; Specifications – Use of Enforcement Discretion for Compendial Changes (November 2004)
– CMC Postapproval Manufacturing Changes Reportable in Annual Reports (March 2014)
– Process validation Guideline (2011)
– Analytical Procedures and Methods Validation for Drugs and Biologics (February 2014)
European Variation guideline
ISPE Technology Transfer Guide
World Health Organization - WHO Annex 7 Technology Transfer
ICH
– Q1A-E Stability Testing of New Drug Substances and Products
– Q2 (R1): Validation of Analytical Procedures: Text and Methodology
– Q8 Pharmaceutical Development
– Q9 Quality Risk Management
– Q10 Pharmaceutical Quality Systems
– Q11 Product Life Cycle Management/applies to API
US Pharmacopeia
– Chapter <1224>, <1225>, <1226>
– Chapter <621> (specific to chromatography)
What are the Regulatory Guidance on Method
Transfer?
©2018 Waters Corporation 5 COMPANY CONFIDENTIAL
How is Modernizing Methods helping with Transfers?
Highly competitive, regulated business environment
• Need to lower costs without compromising product quality while
maintaining regulatory and compliance requirements
• Decrease time to market while maintaining quality of information
Deliver sustainable competitive advantage
• Invest in the correct technologies to achieve business objectives
• Capacity to grow the business and anticipate that need
• Demonstrate fast return on investment
Challenged to increase profitability
• Increasing regulatory pressures, price controls, increased quality
expectations, and competitive pressures
• Pressure to reduce manufacturing costs
• Harmonize approach across sites – simply & manage diverse platform
©2018 Waters Corporation 6 COMPANY CONFIDENTIAL
Method Transfer Approaches
Use existing method and/or monograph
Adjust method within USP Chapter <621> or EP <2.4.46> guidelines
Make changes to an approved method and provide comparison analysis data as defined by the change classification (I, II, III, IV) as outlined in USP <1225>
Re-develop and Re-validate method(s)
©2018 Waters Corporation 9 COMPANY CONFIDENTIAL
Waters
Waters
Waters
Waters
Instrument Characteristics
Detector Flow cell characteristics
Data rate Wavelength range
Injector Configuration: Flow through
needle or fixed loop Injection volume Needle wash(es)
Pump Quaternary vs binary Mixing Pressure Flow rate
Column Compartment Mobile phase pre-heating Heating mode Cooling Number of columns
©2018 Waters Corporation 10 COMPANY CONFIDENTIAL
Instrument Challenges in Methods Transfer
System dwell volume •Understanding of dwell volume and mixing behavior
•Can affect retention time, selectivity and resolution
Temperature Control and Related Effects • Thermal environment of the column both oven temperature and inlet preheating •Thermal mismatch & Transferability
Extra Column Dispersion •Resolution, sensitivity, efficiency and peak capacity
•Strong solvent effects (strong diluent effects)
Chemistry and Columns • base particle
• bonded phase
©2018 Waters Corporation 12 COMPANY CONFIDENTIAL
Extra-column Volume
Dwell Volume
Volume between the point of mixing of solvents and the head of an LC column
Volume between the effective injection point and the effective detection point, excluding the part of the column containing the stationary phase
Where are the different volumes?
©2018 Waters Corporation 13 COMPANY CONFIDENTIAL
Solvent Composition at Mixer
Solvent Composition at Column Head
Actual mobile phase profile on original system measured at the column inlet
0
Injection
x
Dwell volume creates an offset before the solvent composition change reaches the inlet of column
(i.e., an “isocratic hold” at the beginning of every gradient). This volume can be thought of in terms of column volumes.
tg
{ }
Time
What is System Dwell Volume?
©2018 Waters Corporation 14 COMPANY CONFIDENTIAL
𝑡𝐷 = 𝑡50% 𝐴𝑢 − 0.5 𝑡𝐺
𝑉𝐷 = 𝑡𝐷 𝐹
T50% Au
50%
100%
tG
Time (min)
Flow (ml/min)
%A
%B
-- 1.00 100 0
5 1.00 100 0
25 1.00 0 100
30 1.00 0 100
35 1.00 100 0
A: Water B: Water with 10 mg/mL caffeine : 273 nm
%
0.00
20.00
40.00
60.00
80.00
100.00
Minutes
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00
tG: Gradient Time tD: Dwell Time VD: Dwell Volume
tD= (16.0 min – 5.0 min (isocratic hold) – (0.5 x 20.0 min) = 1.0 min
VD= (1.0 min x 1.0 mL/min) = 1.0 mL
Isocratic
hold
Determining Dwell Volume
©2018 Waters Corporation 15 COMPANY CONFIDENTIAL
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0.30
0.60
0.90
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0.00
0.30
0.60
0.90
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0.00
0.30
0.60
0.90
Minutes
3.75 4.50 5.25 6.00 6.75 7.50
Method Transfer from HPLC to HPLC Minimal Dwell Volume Difference
Agilent 1100 (Quaternary) LC System VD -1.3mL
Alliance HPLC System VD -1.16mL
Alliance HPLC System -adjusted for gradient delay differences (0.14 mL)
USP Res = 2.2
USP Res = 2.5
Analyte
1 Rel impurity A
2 Rel Impurity G
3 Ondansetron
4 florifenicol
5 busprione
6 coumarin
7 Rel Compound C
8 protriptyline
9 Rel Compound D
10 flavone
1 2
3
4 5
6
7
8
9
10
USP Res = 2.4
Gradient Separation, 4.6 x 150 mm, 3.5 μm Column
Comparable retention times and resolution for critical pair
©2018 Waters Corporation 16 COMPANY CONFIDENTIAL
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0.00
0.06
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0.05
Minutes
1.50 3.00 4.50 6.00 7.50
Minimal difference in retention time observed for systems with similar dwell volumes
Agilent 1260 Quaternary System Vd= 1.13 mL
ACQUITY Arc System Vd= 1.19 mL
1 2
3
4
5
6
7
1 2
3
4
5
6 7
Peak No
Agilent 1260 ACQUITY
Arc Retention
Time Δ
1 2.03 2.06 0.03
2 2.47 2.47 0.00
3 2.79 2.78 -0.01
4 3.26 3.25 -0.01
5 3.85 3.85 0.00
6 5.5 5.48 -0.02
7 6.31 6.29 -0.02
Method Transfer: UHPLC to ARC Systems with Similar Dwell Volumes
©2018 Waters Corporation 17 COMPANY CONFIDENTIAL
*** Dwell volumes vary with pressure
Pump A
Pump B
Gradient Proportioning Valve
Detector Injector A B
C D
Column Pump
Binary pump Quaternary pump
Detector Injector Column Mixer
Mixer
Dwell Volume
System Dwell Volume (mL)
Agilent 1100 Quat Series LC 1.290***
Alliance HPLC w/2998 1.145
ACQUITY UPLC H-Class with 30 cm Column Heater (30CH-A)
n/a
Agilent 1260Q Infinity
DAD VL+
1.170***
ACQUITY Arc 1.100 or 760
Agilent 1290Q Infinity
1µL v/10mm MaxLight FC
0.263
ACQUITY UPLC H-Class with Column Heater, analytical FC 0.375
ACQUITY UPLC I-Class SM-FTN with Column Heater 0.0725
©2018 Waters Corporation 18 COMPANY CONFIDENTIAL
Measured Vd = 375 L
Measured
Vd = 1150 L
After injection- Amount of solvent to deliver after making injection before starting
gradient (ie. increases gradient hold)
Amount of solvent to deliver before
making injection (i.e.. shortens gradient hold)
HPLC UPLC UPLC HPLC
Tools for Gradient Adjustment between
HPLC to UHPLC to UPLC Systems
©2018 Waters Corporation 19 COMPANY CONFIDENTIAL
0.25
ACQUITY UPLC H-Class Separation
Alliance HPLC no gradient adjustments for dwell volume
Alliance HPLC Using Gradient SmartStart pre-inject function (add – adjustment factor)
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0.15
0.20
0.25
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0.04
0.06
0.08
0.10
0.12
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0.00
0.02
0.04
0.06
0.08
0.10
0.12
Minutes
2.00 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 5.20 5.40 5.60 5.80 6.00
0.12
0.12
Retention time shifted
Effect of Gradient Adjustment on Method
Transfer
©2018 Waters Corporation 20 COMPANY CONFIDENTIAL
Chromatographic data of the metoclopramide API at 0.5 mg/mL with 1.0% of related substances for the method transfer from an Agilent LC System to an ACQUITY Arc System.
No. Analyte
1 Impurity F
2 Metoclopramide
3 Impurity A
4 Impurity G
5 Impurity 9
6 Impurity H
7 Impurity C
8 Impurity D
9 Impurity B
AU
0.000
0.015
0.030
AU
0.000
0.015
0.030
Minutes0.00 1.50 3.00 4.50 6.00 7.50 9.00 10.50 12.00 13.50 15.00
Agilent 1260 Infinity LC System
ACQUITY Arc System
13
4
2
56
7
8
9
13
4
2
5 6
7
8
9
Agilent LC System
ACQUITY Arc LC System
Replicate Established Methods: Arc Multi-flow PathTM Technology
©2018 Waters Corporation 21 COMPANY CONFIDENTIAL
Effects of Instrument Dispersion with Method
Transfer
©2018 Waters Corporation 22 COMPANY CONFIDENTIAL
Instrument dispersion is the broadening of the
analytical band due to the instruments flow
path volume
– It’s part of all LC systems, and varies significantly
depending on the configuration
Any place where the analytical band “moves”
adds to the instruments dispersion
– Injector
– Tubing
o Pre-column
o Post-column
– Oven design
– Flow cell volume
Instrument (System) Dispersion
What is it & Where is it
©2018 Waters Corporation 23 COMPANY CONFIDENTIAL
Simple Approach
– Replace column with low volume union
– Run following method conditions:
o 7:3 Water:Acetonitrile at 0.3mL/min
o Sampling rate: 40Hz, λ = 273 nm
o Sample: 0.16 mg/mL Caffeine
9:1 Acetonitrile:water, 1 µL injection
– Measure peak width at 13.4 % (4σ) or 4.4% (5σ) of the peak
height
Calculate the Dispersion
Extra column dispersion (µL) = peak width (min) * flow rate
(µL/min)
How to Measure Instrument Dispersion
A more complex approach of measuring dispersion: Gritti_Guiochon_Accurate measurements of true column efficiency Journal of Chromatography A, 1327 (2014) 49– 56.
Low volume union
©2018 Waters Corporation 24 COMPANY CONFIDENTIAL
Instrument Dispersion Differences
Figure 2. Bandspread results for three systems. Results are based on 4σ peak width.
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0.06
0.09
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0.08
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0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60
ACQUITY UPLC = 3.9 µL
ACQUITY Arc UHPLC = 12.8 µL
Alliance HPLC = 37.3 µL High
Medium
Low
©2018 Waters Corporation 25 COMPANY CONFIDENTIAL
Extra Column Dispersion Measurements
Measurements may vary from system to system.
Variables that can affect bandspread or extra column dispersion-
– Tubing ( ID >length)
– Flow cells
– Preheating
* Multiple Pre-heater configurations
** Multiple Flow cells
System Band Spread (µL) 5
Agilent 1100 Quat Series LC 27-31*
Alliance HPLC w/2998 43-45**
ACQUITY UPLC H-Class with 30 cm Column Heater (30CH-A) 14-26*
Agilent 1260Q Infinity
DAD VL+
31-41*
ACQUITY Arc 25-30
Agilent 1290Q Infinity
1µL v/10mm MaxLight FC
20-21*
ACQUITY UPLC H-Class with Column Heater, analytical FC 8
ACQUITY UPLC I-Class SM-FTN with Column Heater 7.5
©2018 Waters Corporation 26 COMPANY CONFIDENTIAL
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1.00
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0.50 1.00 1.50 2.00
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0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00
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1.00
Minutes
0.50 1.00 1.50 2.00
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1.00
Minutes
0.50 1.00 1.50 2.00
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1.00
Minutes
0.50 1.00 1.50 2.00
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0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00
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0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00 2.50
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0.20
0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00 2.50
AU
0.00
0.20
0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00 2.50
UHPLC Extra column dispersion ~25 µL
UPLC Extra column dispersion< 10 µL
HPLC Extra column dispersion >30 µL
2.1
x 5
0 m
m,
1
.6 µ
m
3.0
x 7
5 m
m,
2
.7 µ
m
4.6
x 7
5 m
m,
2
.7 µ
m
*
k’ =1
*
Strong solvent effects
Dispersion Impact on Performance Isocratic Separation on HPLC, UHPLC and UPLC
©2018 Waters Corporation 27 COMPANY CONFIDENTIAL
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0.40
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1.00
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0.50 1.00 1.50 2.00
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0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00
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1.00
Minutes
0.50 1.00 1.50 2.00
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0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00
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0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00
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0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00
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0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00 2.50
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0.00
0.20
0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00 2.50
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0.00
0.20
0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00 2.50
Strong solvent effects
UHPLC > 3.0 mm ID Extra column dispersion ~25 µL
UPLC > 2.1 mm ID Extra column dispersion< 12 µL
HPLC > 4.6 mm ID Extra column dispersion >30 µL
2.1
x 5
0 m
m,
1
.6 µ
m
3.0
x 7
5 m
m,
2
.7 µ
m
4.6
x 7
5 m
m,
2
.7 µ
m
*
k’ =1
*
Dispersion Impact on Performance Isocratic Separation on HPLC, UHPLC and UPLC
©2018 Waters Corporation 28 COMPANY CONFIDENTIAL
Column: C18 2.1x 50 mm
USP Assay for Diclazuril
Dispersion Impact on Performance Gradient Separation on UHPLC and UPLC
AU
0.000
0.012
0.024
0.036
0.048
AU
0.000
0.012
0.024
0.036
0.048
Minutes
1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00
ARC Extra column dispersion 25 µL
H-Class Extra column dispersion< 10 µL
1
2 3
4 5 6
USP Res= 1.5
USP Res= 2.0 USP Res= 2.7
USP Res= 1.8
No Compound
1 6 carboxylic acid
2 6-carboxamide
3 Diclazuril
4 Ketone
5 4-amino Derivative
6 Des-cyano derivative
©2018 Waters Corporation 29 COMPANY CONFIDENTIAL
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-0.02
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0.02
0.04
0.06
0.08
0.10
0.12
0.14
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0.02
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0.08
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0.14
Minutes
2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40
System: Alliance HPLC with 2998 (Blue) Detector; Gradient: 5-90% B in 4min
Column: CORTECS C18+ 2.1 x 75 mm 2.7 µm, Column
Gradient adjusted for dwell volume differences using integrated software
10 mm Flow Cell Analytical Flow Cell
8 mm Flow Cell, μbore flow cell
USP Res = 1.6 – 2.5
USP Res = 1.1- 1.8
Post Column Dispersion Effect
©2018 Waters Corporation 30 COMPANY CONFIDENTIAL
System HPLC UHPLC UPLC
Dispersion > 40 μL 22 – 29 μL <20 μL
Particle Size 3.5 μm, 5 μm, 10 μm (Prep) 2.x μm < 2 μm
Routine Pressure < 4000 psi < 10000 psi < 18000 psi (I-Class)
Column ID 4.6 mm (3.0 mm) 3.0 mm (2.1 mm) 2.1mm (1.0 mm)
Column Length 75 - 250 mm 50 mm - 100mm ≤ 150 mm
Matching Column Configuration with LC
Systems
Increased flexibility and sample characterization
©2018 Waters Corporation 32 COMPANY CONFIDENTIAL
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Strong solvent effect related to k’ or peak volume
Injection volume <15% of peak volume if diluent = starting mobile phase, lower if strong solvent diluent used
Additional strategies: add post injector volume, change to weaker sample diluent
Sample diluent: MeOH Injection volume: 7.2 µL Column: 4.6 x 75 mm column Isocratic separation
Measured extra column dispersion – 9 µL
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0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
Measured extra column dispersion – 11 µL
peak distortion due to strong solvent effects
Scale to 3.0 x 75 mm column, 3.1 µL injection
Lower injection volume From 7.2 to 3 µL
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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 1.60 1.70 1.80 1.90 2.00 2.10 2.20
USP Allowable Changes
Dispersion Effects with
Low k` and High Organic Solvent
©2018 Waters Corporation 34 COMPANY CONFIDENTIAL
Challenges of Thermal Distortion with Method
Transfer
©2018 Waters Corporation 35 COMPANY CONFIDENTIAL
Transfer to HPLC No mobile phase pre-heating
Transfer to HPLC Added inlet tubing for passive pre-heating
Fronting peaks, broadening
Improved peak shape and efficiencies
Original UPLC Method Active pre-heater
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0.000
0.012
0.024
0.036
0.048
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0.000
0.012
0.024
0.036
0.048
Minutes
3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
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0.000
0.015
0.030
0.045
0.060
Minutes
4.00 5.00 6.00 7.00 8.00 9.00 10.00
0.60
0.48
0.48
Thermal Distortion Impact on Methods Transfer
©2018 Waters Corporation 36 COMPANY CONFIDENTIAL
USP Res= 1.5 36° C
38° C
40° C
48° C
USP Res= 1.3
Agilent 1100
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0.30
0.45
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0.30
0.45
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8.25 8.80 9.35 9.90 10.45 11.00
6
7
6
7
6 +7
7
6
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0.30
0.45
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0.45
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8.25 8.80 9.35 9.90 10.45 11.00
ACQUITY UPLC H- Class
6
7
6
7
6 +7
7
6
Effect of Mobile Phase Pre-Heating Thermal Effects in Different Instruments
©2018 Waters Corporation 38 COMPANY CONFIDENTIAL
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4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
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4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
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4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
Effect of Mobile Phase Pre-Heating on Methods Transfer
Column temperature: 46 C
Pre-heating Agilent 1100
No Pre-heating Agilent 1100
Pre-heating ACQUITY UPLC H-Class
No Pre-heating ACQUITY UPLC H-Class
√ Transferred
√ Transferred
X X
Can not be transferred
©2018 Waters Corporation 40 COMPANY CONFIDENTIAL
1k
k
α
1α
4
NRs
Mechanical Contributions
System Dispersion Fittings/Connections
Dwell volume Particle size
Well-packed columns
Chemical/Physical Contributions
Complementary bonded phases
Multiple particle substrates Ability to utilize high pH
Increase retentivity
Resolution Equation
Efficiency
©2018 Waters Corporation 41 COMPANY CONFIDENTIAL
L/dp is directly related to the resolving power
– Higher the L/dp, higher the efficiency (N), higher resolving power
What the USP had in mind was the resolving power (N) of the various column dimensions
maintain the same result using geometric scaling of column dimension and flowrate
Length
(L, mm)
Column Diameter
(dc, mm)
Particle Size (dp, m)
Relative Values
L/dp F N Pressure Run Time
250 4.6 10 25,000 0.5 0.8 0.2 3.3
150 4.6 5 30,000 1.0 1.0 1.0 1.0
150 2.1 5 30,000 0.2 1.0 1.0 1.0
100 4.6 3.5 28,600 1.4 1.0 1.9 0.5
100 2.1 3.5 28,600 0.3 1.0 1.9 0.5
75 4.6 2.5 30,000 2.0 1.0 4.0 0.3
75 2.1 2.5 30,000 0.4 1.0 4.0 0.3
50 4.6 1.7 29,400 2.9 1.0 8.5 0.1
50 2.1 1.7 29,400 0.6 1.0 8.5 0.1
Maintaining Efficiency
L/dp, Column Length to Particle Size Ratio
©2018 Waters Corporation 42 COMPANY CONFIDENTIAL
1. Related Compound C 2. Zidovudine 3. Related Compound B
2
2
1
1
3
3
XBridge C18 4.6 x 250 mm, 5µm Column efficiency (N Peak 3 ): 2957 Flow rate: 1.0 mL/min Resolution Peak 2,3= 3.9 Tailing Peak 2= 1.12
CORTECS C18 4.6 x 100 mm, 2.7µm Column efficiency (N Peak 3 ): 2758 Flow rate: 1.5 mL/min Resolution Peak 2,3= 3.5 Tailing Peak 2= 1.16
• Changes made are within USP <621> allowable adjustments (USP37-NF32 S1, August 2014)
• 4X faster run with 94% solvent savings • Re-validation not required
HPLC System 5 µm 2.7 µm Column
Legacy Column
Modern Column
USP <621> Allowable Changes Maintaining L/dp/Efficiency
©2018 Waters Corporation 43 COMPANY CONFIDENTIAL
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0.04
0.06
0.08
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0.00 2.00 4.00 6.00 8.00 10.00
1. Loratidine Related Compound A 2. Loratidine Related Compound B 3. Loratidine
3.0 x 100 mm CORTECS C8 2.7 µm 0.80 mL/min
UHPLC
4.6 x 150 mm XBridge BEH C8 5 µm 1.00 mL/min
HPLC
2.1 x 50 mm CORTECS UPLC C8 1.6 µm 0.60 mL/min
UPLC
10x Faster 10x Solvent Savings
1 2
3
1 2
3
1 2
3
USP Method Conditions
System Suitability Rs 2,1 NLT 1.5: 2.7
tR3 %RSD NMT 2.0%: 0.7%
System Suitability Rs 2,1 NLT 1.5: 3.5
tR3 %RSD NMT 2.0%: 0.5%
System Suitability Rs 2,1 NLT 1.5: 2.5
tR3 %RSD NMT 2.0%: 0.5%
Meets all regulatory accepted guidelines
Meets all regulatory accepted guidelines
USP Method Transfer
Across Different Instrument Platforms
©2018 Waters Corporation 44 COMPANY CONFIDENTIAL
USP “L” classification is a general term for columns that
meet a certain chemical/bonding criteria
– There are over 600 columns that meet the L1 class criteria
USP “L” Column Classifications
L Classification
for a C18 column
©2018 Waters Corporation 45 COMPANY CONFIDENTIAL
Selectivity Differences Within the Same “L” Class
“L1” Classified Columns
Minutes 0.00 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 15.00 16.00 17.00 18.00 19.00 20.00
Conditions: ACN/ 15.4 mM Ammonium Formate pH 3 (35/65); 0.25 mL/min; 30 °C; 2.1x50 mm columns
5 6
4 1 3 2
4 1 3 2
4 1 3
2
5 6
4 1 3
2
5 6 4 1 3
2
5 6 4 1 3 2
5 6
5 6
1. Uracil
2. Pyrenesulfonic Acid
3. Promethazine
4. Amitriptyline
5. Butylparaben
6. Naphthalene
Do not expect that
two L1 columns
from different
vendors will
perform the same!
Vendor A
Vendor B
Vendor C
Vendor D
Vendor E
Vendor F
©2018 Waters Corporation 46 COMPANY CONFIDENTIAL
What’s Important for Chromatography
Pore Diameter (PD)
Pore Volume (PV)
Surface Area (SA)
Particle composition
Particle Size
Particle Surface charge
©2018 Waters Corporation 47 COMPANY CONFIDENTIAL
Understanding Bonded Phases
Full Coverage C18 – General purpose, balance non-polar retention for acids, bases, and
neutrals
Mid Coverage C18 – Balanced retention for polar and non-polar compounds
C8 – For the retention of strongly hydrophobic compounds
Embedded Polar C18 – Different selectivity, with improved peak shape fore basic compounds
Phenyl Hexyl – Different selectivity especially for aromatic compounds
Petafluorophenyl Propyl – Different selectivity especially for basic compounds
Cyano – Different selectivity especially for polar molecules
Amide (HILIC Phase) – Different selectivity for polar basic/polar acid molecules
©2018 Waters Corporation 48 COMPANY CONFIDENTIAL
4-in-1 new tool:
– Find an alternative Reversed-
phase column
– Compare Reversed-phase
column retention and selectivity
– Search column by USP
designation
– Column recommendation by
compound class
Matching Column Selectivity Tool
The Column Coach
©2018 Waters Corporation 49 COMPANY CONFIDENTIAL
Simplifying Methods Transfer:
Summary
Dwell volume can impact gradient method transfer
– Tools such as Waters Columns Calculator and Gradient SmartStart can
assist in methods transfer across different instruments
Extra-column dispersion can impact separations
– Greater dispersion can affect resolution, particularly for sub 2-μm columns
and low k’ compounds
– Lower dispersion and larger injection volumes can lead to strong solvent
effects
Temperature effects can impact transferability across different instruments
– Inlet pre-heating provides improved temperature control
The column chemistry; understanding the attributes of both the base particle
and bonded phase
– Column selection tools can help with selecting columns.
©2018 Waters Corporation 50 COMPANY CONFIDENTIAL
Acknowledgements
Paula Hong
Neil J Lander
Eric Grumbach
Patricia McConville
Bill Nyquist
Hillary Hewitson
Dick Andrews
Mike Jones