Comparison of SFC, HPLC, and Chiral techniques for the ... · PDF fileComparison of SFC, HPLC,...

Comparison of SFC, HPLC, and Chiral techniques for the separation of diastereomers of a diverse

set of small molecules

Katalin Ebinger and Harold N. Weller

Bristol-Myers Squibb CompanyResearch and Development

Synthesis and Analysis Technology

Purpose of the study

Diastereomer separation has a key importance in the Pharmaceutical Industry.

• Isomer separation generally favors unmodified silica columns1, but modified stationary phases, including Chiral columns, using NPLC, RPLC or SFC conditions, frequently in isocratic mode, have also been reported for diastereomer separation.

New developments in SFC instrumentation (SFC-MS, open bed fraction collector for batch purification) has led to renewed interest in SFC for complex separations.

2

1 Introduction to Modern Liquid Chromatography, L..R. Snyder, J.J. Kirkland, J.W. Dolan, 3rd edition, (2010).

Design and Goals Success rates for separation of 258 synthetic

diastereomer pairs were compared using three techniques, and two stationary phases per technique:

1. Reverse Phase Non-Chiral HPLC

2. Reverse Phase Chiral HPLC Separation

3. Non-Chiral SFC

3

Selection of stationary phases

Column dimension: 4.6 x 150 mm, 5 µmSelection : Literature and Experimental data

Selection of stationary phasesLiterature data - Columns selection for HPLC

1. Reverse Phase Non-Chiral HPLC SeparationRP columns under HPLC conditions are tested, and documented by vendors, with consideration of their orthogonality1 two commonly used columns were selected:

XBridge™ C18 (Waters Corporation, Milford MA, USA)

Synergi 4 µm Polar RP (Phenomenex, Torrance CA, USA)

2. Reverse Phase Chiral HPLC Separation2

Ultron ES-OVM (Shinwa Kyoto, Japan), protein based

Chiralcel OJ-RH (Daicel Chemical Industries Tokyo,Japan), cellulose based

1 L. R. Snyder, J. W. Dolan, and P. W. Carr, The hydrophobic subtraction model of reversed phase column selectivity, J. Chromat. A, 1060, 77-116, (2004).2 K. Valko (Ed.) Separation Methods in Drug Synthesis and Purification, Handbook of Analytical Separation Volume 1, Recent development in liquid chromatographic enantioseparation. M. Lammerhofer and W.Lindner 337-437, (2000).

5

Selection of stationary phasesExperimentation required for column selection - SFC

Literature data1 and our own observations suggested that retention factors can shift significantly on certain stationary phases when ammonium acetate additive is used.

This effect may have resulted from interaction between the additive and the stationary phases and/or ion pair formation between the additive and the analyte.

1 J. Zheng, T. Glass , L.T. Taylor, J. David Pinkston, Study of the elution mechanism of sodium aryl sulfonates on bare silica and cyano bonded phase with methanol-modified carbon dioxide containing an ionic additive J. Chromat. A, 1090, 155-164, (2005).

6

Examples of Retention Factor Shift

0

2

4

6

8

10

12

14

0 50 100

Rete

ntio

n Fa

ctor

(k')

Injection number

Zorbax-SIL

0

2

4

6

8

10

12

14

16

18

0 50 100

Ret

entio

n Fa

ctor

(k')

Injection number

DEAP

7

SD=0.62

SD=0.32

SD=0.41

SD=0.1

SD=0.11

SD=0.09SD=0.08

SD=0.46

SD=0.09

SD=0.03

Retention Factors for a 5 component text mixture injected 100 times consecutively and eluted using the same linear gradient.

Retention factor shift overtimeZorbax SIL Princeton DEAP

Caffeine

Caffeine

Sulconazole

Perphenazine

Propranolol

Bendroflum

Sulconazole

Bendroflum

Propranolol

Perphenazine

Inj#1

Inj# 100

Process for selecting only two columns for SFC

1. Start with 24 columnsa) Retention factor reproducibility on 24 columns

b) Orthogonality consideration on 24 columns

c) Column selectivity parameters from the literature

2. Reduce column number to 10 columnsa) Separation of a subset of 33 diastereomeric

mixtures on 10 selected columns

3. Selection of the final two columns

Columns Used in the Study

10

Short Name

Full Name Vendor Short Name Full Name Vendor

Kromasil Sil Kromasil 60-5SIL Eka-AkzoNobel, Eka Chemicals AB,Sweden

SiliCycle XDB1-CN

SiliCycle®SiliaChrom ™ XDB1CN

SiliCycle Inc., Quebec City, Canada

Zorbax Sil Zorbax SIL Agilent Technologies Wilmington, DE, USA

SiliCycle HILIC SiliCycle®SiliaChrom ™ HILIC

SiliCycle Inc., Quebec City, Canada

Zorbax Rx Sil Zorbax Rx- SIL Agilent Technologies Wilmington, DE, USA

Viridis EP Viridis™ SFC 2-Ethylpyridine

Waters Corporation, Milford MA, USA

Luna Sil Luna Silica (2) Phenomenex, Torrance CA, USA

Princeton EP* PrincetonSFC 2-Ethylpyridine

Princeton Chromatography, Princeton NJ, USA

Viridis Sil Viridis™ SFC Silica


Zorbax NH2 Zorbax NH2 Agilent Technologies Wilmington, DE, USA

Princeton Sil PrincetonSFC SILICA


Princeton DEAP PrincetonSFC DEAP Princeton Chromatography, Princeton NJ, USA

YMC PVA-Sil YMC-Pack PVA-SIL-NP

YMC Co., Ltd. Japan PrincetonHA-Dipyridyl

PrincetonSFCHA-Dipyridyl


Atlantis HILIC Atlantis® HILIC Silica


Princeton DNP PrincetonSFC DNP Princeton Chromatography, Princeton NJ, USA

Xbridge HILIC XBridge™ HILIC Waters Corporation, Milford MA, USA

Zorbax TMS Zorbax TMS Agilent Technologies Wilmington, DE, USA

Luna HILIC Luna 5um HILIC Phenomenex, Torrance CA, USA

Synergi Polar RP Synergi 4 um Polar RP Phenomenex, Torrance CA, USA

Princeton CN-Diol

PrincetonSFC 2CN:Diol


Xbridge Amide XBridge™ Amide Waters Corporation, Milford MA, USA

Zorbax SB-CN

Zorbax SB-CN Agilent Technologies Wilmington, DE, USA

Cosmosil PYE Cosmosil 5U PYE Nacalai USA. Inc.,San Diego, CA, USA

Retention factor reproducibility on 24 columns24 columns, selected from 4 column-type categories, were evaluated for retention reproducibility. (4.6 x 150 mm, 5 µm) A test mix of 5 components was used

• Sulconazole (weak base, not ionized in ammonium acetate) • Caffeine (weak base, not ionized in ammonium acetate)• Bendroflumethiazide (weak acid, not ionized in ammonium acetate)• Propanolol (strong base, protonated in ammonium acetate) • Perphenazine (strong base, protonated in ammonium acetate)

The test mix was injected on each column 100 times consecutively and eluted using the same linear gradient method.

• CO2 / Methanol + 10 mM NH4OAc• 5% to 60% Methanol from 1-7 minutes + 1.5 min @ 60%• Flow rate 5 g/min, Inlet pressure 100 bar

Retention factors of 100 injections of all 5 components on each columns were recorded and the standard deviations (SD) were calculated and compared.

11

Retention factor reproducibility on 24 columnsComparison of standard deviations of k’

SD of k’ = Standard Deviation of the retention factor based on 100 injection

* = Selected for further study

Significant retention factor drift is observed on some columns over time.

12

Si-Diol CN Amine Others

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Krom

asil S

il

Zorb

ax S

il

Zorb

ax R

x Si

l*

Luna

Sil

Virid

is S

il

Prin

ceto

n Si

l

YMC

PVA

Sil *

Atla

ntis

HIL

IC

Xbrid

ge H

ILIC

*

Luna

HIL

IC*

Prin

ceto

n C

N-D

iol

Zorb

ax S

B-C

N*

Silic

ycle

XD

B1-C

N

Zorb

ax N

H2*

Viri

dis

EP

Prin

ceto

n EP

*

Prin

ceto

n D

EAP

Prin

ceto

n H

A-D

ipyr

idyl

*

Prin

ceto

n D

NP

Zorb

ax T

MS

Syne

rgi P

olar

RP*

Xbrid

ge A

mid

e

Cos

mos

il PYE

*

Silic

ycle

HIL

IC

SD o

f k'

Caffeine Propranolol Perphenazine Sulconazole Bendroflumethazide

* ** ** * *** *

Retention Orthogonality for 24 ColumnsRetention Factors for 5 components on each of 24 columns

Other ColumnTypes

Amine Columns Cyano Columns

Silica & DiolColumns Retention factors

(distance from center), vary significantly from column to column, even for columns with nominally similar chemistry.

Ten columns (*) were chosen for further study.

Test Compounds:Caffeine

SulconazolePropanolol

PerphenazineBendroflumethiazide

14


Other ColumnTypes








15


Other ColumnTypes








16

Column selectivity parameters from the literature1

Reduced column number - 10 selected columns.(Retention factor reproducibility, orthogonality consideration and literature data on 24 columns)

1 C. West, E. Leslellier, Orthogonal screening system of columns for supercritical fluid chromatography J. Chromatogr. A., 1203, 105-113, (2008).

0.00

0.05

0.10

0.15

0.20

0.25

Zorb

ax R

x Si

l*

YMC

PVA

Sil *

Xbrid

ge H

ILIC

*

Luna

HIL

IC*

Zorb

ax S

B-C

N*

Zorb

ax N

H2*

Prin

ceto

n EP

*

Prin

ceto

n H

A-D

ipyr

idyl

*

Syne

rgi P

olar

RP*

Cos

mos

il PYE

*

SD o

f k'

Caffeine Propranolol Perphenazine Sulconazole Bendroflumethiazide

Separation of a subset of 33 diastereomeric mixtures on 10 selected columns

Separation success rates with consideration of orthogonality were used to select the final two columns.

* Cosmosil PYE

* Xbridge HILIC0

5

10

15

20

25

30

YMC

PVA

Sil

Zorb

ax R

X Si

l

XBri

dge

HIL

IC

Cos

mos

il PY

E

Zorb

ax N

H2

Pola

r RP

Zorb

ax S

B-C

N

Prin

ceto

n H

A

Dip

yrid

yl

Luna

HIL

IC

Prin

ceto

n EP

Num

ber o

f mix

ture

s se

para

ted

Resolution > 1 Resolution > 2

20

* *

Final Columns Used for the Separation Study

21

Reverse Phase Non-Chiral HPLCXBridge™ C18 (Waters Corporation, Milford MA, USA)

Synergi 4 µm Polar RP (Phenomenex, Torrance CA, USA)

Reverse Phase Chiral HPLC2

Ultron ES-OVM (Shinwa Kyoto, Japan), protein based

Chiralcel OJ-RH (Daicel Chemical Industries Tokyo,Japan), cellulose based

Non-Chiral SFCXBridge™ HILIC (Waters Corporation, Milford MA, USA)

Cosmosil PYE (Nacalai USA. Inc.,San Diego, CA, USA)

Diastereomer Separation Study

258 synthetic diastereomer mixtures were analyzed.

All mixtures were analyzed on six columns • 2 Reverse Phase Non-Chiral HPLC• 2 Reverse Phase Chiral HPLC• 2 Non-Chiral SFC

0

10

20

30

40

50

60

-1 0 1 2 3 4 5 6 7 8 9 10

Freq

uenc

y

cLogP

cLogP Distribution of Test Compounds

1 T.I. Oprea, Property Distribution of drug-related chemical databases. J. Computer-Aided Molecular Design, 14, 251-264, (2000).

22

ACD cLogP data for these mixtures was consistent with typical “drug like” compoundscommonly encountered in pharmaceutical research1

Methods for diastereomer separation studyType Column Organic

ModifierBuffer Flow

Rate(ml/min)

Gradient Time (min)

Gradient retention

factor1

(k*)

Gradient Steepness

1 (Gs)(%/min)

Temp(0C)

Gradient Range

(% Organic)

MS Ionizatio

n

HPLCNon-Chiral

XbridgeC-18

4.6 x 150

CH3OH NH4OAc 1.2 10 2.2 9.2 25 20-95 ESI+

HPLCNon-Chiral

Polar RP4.6 x 150

CH3OH NH4OAc 1.2 10 2.2 9.2 25 20-95 ESI+

HPLCChiral

Ultron ES-OVM

4.6 x 150

CH3CN NH4OAc 1.0 17 5.8 3.5 35 10-50 ESI+

HPLCChiral

Chiralcel OJ-RH

4.6 x 150

CH3OH TFA 1.0 11 1.8 11.4 35 10-95 ESI+

SFCNon-Chiral

Cosmosil PYE

4.6 x 150

CH3OH NH4OAc 5.0 6 4.7 4.2 35 5-60 APCI+

SFCNon-Chiral

Xbridge HILIC

4.6 x 150

CH3OH NH4OAc 5.0 6 4.7 4.2 35 5-60 APCI+

23

1 L.R. Snyder, J.J. Kirkland, J.L.Glajch Practical HPLC Method Development , Second edition (1997).

Diastereomer Separation Results

24

Diastereomer pairs were separated for all 258 compounds on one or more of the 6 columns used.

Non-Chiral SFC separated a higher percentage of diastereomers with resolution greater than one relative to the other two techniques studied.

05

10152025303540

XbridgeC18

SynergiPolar RP

UltronES-OVM

ChiralcelOJ-RH

XbridgeHILIC

CosmosilPYE

Pct.o

f Com

poun

ds S

epar

ated

Resolution >1.0 Resolution >2.0

Reverse Phase Non-Chiral HPLC

Reverse PhaseChiral HPLC

Non-Chiral SFC

Summary

SFC methods using relatively inexpensive non-chiral columns provided equivalent or superior diastereomer separation compared with the methods using more expensive chiral columns.

25

Acknowledgements

The authors thank the SATT Synthesis Analysis and Technology Team for their support.

26

Additional slides for possible questions

Following slides only needed in the event of specific questions.

Diastereomer Separation Results

01020304050607080

XbridgeC18

SynergiPolar RP

Ultron ES-OVM

ChiralcelOJ-RH

XbridgeHILIC

CosmosilPYE

Pct.o

f Com

poun

ds S

epar

ated

Resolution >0.0 Resolution >1.0 Resolution >2.0

Reverse Phase Non-Chiral HPLC

Reverse PhaseChiral HPLC

Non-Chiral SFC

Change of retention factors on YMC PVA Sil Column

0

1

2

3

4

5

6

7

8

1 21 41 61 81 101 121 141 161 181Injection Number

SD o

f k'

Caffeine Propranolol Sulconazole Perphenazine Bendroflumethazide

Based on 200 injection

Caffeine Propranolol Sulconazole Perphenazine Bendroflumethazstdev inj1-100 0.11 0.11 0.06 0.19 0.04stdev inj100-200 0.01 0.04 0.02 0.04 0.04stdev inj1-200 0.09 0.10 0.05 0.15 0.04

Modifier effect – Silica column and Modified silica surface

Modifier effect on Viridis Silica column

0.000.100.200.300.400.500.600.70

Newcol +0.1 %TFA

Newcol+0.1%TFA+1% water

Newcol+0.1%TFA (after water

was on thecolumns)

0.1%TFA(after10 mM

NH4OAc was oncolumn)

10mM NH4OAc

Sd o

f k'

Modifier effect on Viridis EP column

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

Newcol +0.1 % TFA Newcol+0.1%TFA+1% water

Newcol+0.1% TFA(after water was on

the columns)

0.1%TFA (after10mM NH4OAc was

on column)

10mM NH4OAc

SD o

f k'

Caffeine Propranolol Perphenazine Sulconazole Bendroflumethazide

Silica Surface

Modified silica Surface

TFA NH4OAc

Modifier effect – Silica surface Viridis Sil0.1 % TFA 0.1% TFA+1 %Water 0.1% TFA+on column run

NH4OAc additive beforeNH4OAc additive

CaffeineCaffeine

Caffeine

BendroflumBendroflum

Propranolol

Propranolol

Sulconazole

Perphenazine

SulconazolePerphenazine

Sulconazole

Perphenazine

Propranolol

Caffeine

Sulconazole

Bendroflum

Perphenazine

Memory effect of the Silica column after using Ammonium salt additive

Bendroflum

Propranolol

Modifier effect – Bonded Silica surface Viridis EP0.1 % TFA 0.1% TFA+1 %Water 0.1% TFA+on column run

NH4OAc additive beforeNH4OAc additive

Caffeine

BendroflumPropranolol

Sulconazole

PerphenazinePerphenazinePerphenazine

CaffeineCaffeine Caffeine

Sulconazole

Propranolol

Sulconazole Sulconazole

Perphenazine

PropranololBendroflum Bendroflum

Propranolol

Bendroflum

No memory effect on bonded silica surface after using Ammonium salt additive

Comparison of SFC, HPLC, and Chiral techniques for the ... · PDF fileComparison of SFC, HPLC,...

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