tips and tricks to perform efficient method transfer€¦ · b: water with 10 mg/ml caffeine : 273...

©2018 Waters Corporation 1 COMPANY CONFIDENTIAL

Tips and Tricks to Perform Efficient

Method Transfer

Jonathan E. Turner

Waters Corp.

[email protected]


When are the Methods Transferred?

R&D

Manufacturer Contract

Labs

CxOs

QC Testing

Lab Method Transfer


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.


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?


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


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)


System Factors that Impact Method Transfer


Many Different Systems for the Laboratory


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


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


Effects of Dwell Volume in Method Transfer


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?


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?


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


AU

0.00

0.30

0.60

0.90

AU

0.00

0.30

0.60

0.90

AU

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


AU

0.00

0.06

AU

0.00

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


*** 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


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


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


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)

AU

0.00

0.05

0.10

0.15

0.20

0.25

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

AU

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


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


Effects of Instrument Dispersion with Method

Transfer


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


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


Instrument Dispersion Differences

Figure 2. Bandspread results for three systems. Results are based on 4σ peak width.

AU

0.00

0.03

0.06

0.09

AU

0.00

0.03

0.06

0.09

AU

0.00

0.04

0.08

0.12

Minutes

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


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*


DAD VL+

31-41*

ACQUITY Arc 25-30


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


AU

0.00

0.20

0.40

0.60

0.80

1.00

Minutes

0.50 1.00 1.50 2.00

AU

0.00

0.20

0.40

0.60

0.80

1.00

Minutes

0.50 1.00 1.50 2.00

AU

0.00

0.20

0.40

0.60

0.80

1.00

Minutes

0.50 1.00 1.50 2.00

AU

0.00

0.20

0.40

0.60

0.80

1.00

Minutes

0.50 1.00 1.50 2.00

AU

0.00

0.20

0.40

0.60

0.80

1.00

Minutes

0.50 1.00 1.50 2.00

AU

0.00

0.20

0.40

0.60

0.80

1.00

Minutes

0.50 1.00 1.50 2.00

AU

0.00

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

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


AU

0.00

0.20

0.40

0.60

0.80

1.00

Minutes

0.50 1.00 1.50 2.00

AU

0.00

0.20

0.40

0.60

0.80

1.00

Minutes

0.50 1.00 1.50 2.00

AU

0.00

0.20

0.40

0.60

0.80

1.00

Minutes

0.50 1.00 1.50 2.00

AU

0.00

0.20

0.40

0.60

0.80

1.00

Minutes

0.50 1.00 1.50 2.00

AU

0.00

0.20

0.40

0.60

0.80

1.00

Minutes

0.50 1.00 1.50 2.00

AU

0.00

0.20

0.40

0.60

0.80

1.00

Minutes

0.50 1.00 1.50 2.00

AU

0.00

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

AU

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


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


AU

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

AU

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

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


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


AU

0.00

0.05

0.10

0.15

0.20

0.25

Minutes

0.50 1.00 1.50 2.00 2.50 3.00

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

AU

0.00

0.10

0.20

0.30

0.40

Minutes

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

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

Minutes

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


Challenges of Thermal Distortion with Method

Transfer


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

AU

0.000

0.012

0.024

0.036

0.048

AU

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

AU

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


USP Res= 1.5 36° C

38° C

40° C

48° C

USP Res= 1.3

Agilent 1100

AU

0.00

0.15

0.30

0.45

AU

0.00

0.15

0.30

0.45

AU

0.00

0.15

0.30

0.45

AU

0.00

0.15

0.30

0.45

Minutes

8.25 8.80 9.35 9.90 10.45 11.00

6

7

6

7

6 +7

7

6

AU

0.00

0.15

0.30

0.45

AU

0.00

0.15

0.30

0.45

AU

0.00

0.15

0.30

0.45

AU

0.00

0.15

0.30

0.45

Minutes

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


AU

0.00

0.15

0.30

0.45

Minutes

4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

AU

0.00

0.15

0.30

0.45

Minutes

4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

AU

0.00

0.15

0.30

0.45

Minutes

4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

AU

0.00

0.15

0.30

0.45

Minutes

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


Column Factors Influencing Method Transfer


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


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


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


AU

0.00

0.02

0.04

0.06

0.08

AU

0.00

0.05

0.10

AU

0.00

0.02

0.04

0.06

Minutes

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%


tR3 %RSD NMT 2.0%: 0.5%


tR3 %RSD NMT 2.0%: 0.5%

Meets all regulatory accepted guidelines

Meets all regulatory accepted guidelines

USP Method Transfer

Across Different Instrument Platforms


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


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


What’s Important for Chromatography

Pore Diameter (PD)

Pore Volume (PV)

Surface Area (SA)

Particle composition

Particle Size

Particle Surface charge


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


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

http://cms.bluerainmedia.com:8086/ColumnCoach/existingcolumn/column


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.


Acknowledgements

Paula Hong

Neil J Lander

Eric Grumbach

Patricia McConville

Bill Nyquist

Hillary Hewitson

Dick Andrews

Mike Jones

tips and tricks to perform efficient method transfer€¦ · b: water with 10 mg/ml caffeine : 273...

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