Développement de méthodes en UPC² v1 - Waters Corporation UPC2.… · Chloroform Acetone...

Développement de méthodes en UPC² Développement de méthodes en UPC

Gilles JAOUEN

©2015 Waters Corporation 1

Rappel : principe du CO2 supercritiqueDiagramme de phaseg p

T (⁰C) P (Bar)CO 31 76 CO2 31 76


Rappel : La Chromatographie Convergente UPC²g p g

SFC UltraPerformanceConvergence Chromatographyg g p y

UltraPerformance Convergence Chromatography est le résultatd’avancées technologiques significatives dans l’instrumentationSFC et les chimie de colonnes ce qui conduit à une augmentationSFC et les chimie de colonnes ce qui conduit à une augmentation importante de sélectivités accessibles.

Table. Ordre de grandeur de la diffusion, la viscosité et la

Gas Supercritical Fluid

Liquid

3 1

diffusivité

Density (g/cm3)

10-3 10-1-1Liquid-like

1

Diffusivity (cm2/s)

10-1 10-4-10-3 <10-5

Viscosity 10-4 10-4-10-3 10-2

©2015 Waters Corporation 3Data courtesy of Davy Guillarme, Jean-Luc Veuthey LCAP, University of Geneva, Switzerland

Viscosity (g/cm·s)

10 10 10Gas-like

10

Rappel :Fonctionnement de l’ACQUITY UPC2

PDA detector

Splitter

Column ManagerMake-up

Pump

AuxiliaryInject valve

Back Pressure Regulator(Dynamic and Static)

Pump

Mass Spec

Inject valve

mixerThermo-electric heat exchanger


Waste Modifier CO2Supply CO2

PumpModifier

Pump

Sélectivité: L’apport de la Chromatographie Convergente

SolventPentane, Hexane,

HeptaneSupercritical

COHeptane

Xylene

Toluene

Diethyl ether

Wea

k

CO2

Convergence

Dichloromethane

Chloroform

Acetone

Wg

Chromatography Selectivity Space

Dioxane

THF

MTBE

Ethyl acetate

DMSO

Acetonitrile

I l

Str

ong


Isopropanol

Ethanol

MethanolOrganicModifier

Développement de méthodes UPC²Mode achiralMode achiral


New UPC2 Achiral Phases – Torus™

Highlights– 4 completely new innovative chemistries for UPC2p y

o 1.7 µm particles, 2.1 and 3.0 mm ID– Excellent peak shape – eliminates or reduces need for additives– Added selectivity – wide range of compoundsAdded selectivity wide range of compounds– Improved Robustness


Torus Technologygy

A new particle technology Novel two-stage bonding process yielding high density ligands Novel, two stage bonding process yielding high density ligands The second stage imparts the unique selectivity for each phase

– new interactions with analytes

O SiO

OO

OHNH

N

O OH

ACQUITY UPC2

Torus 2-PIC

ACQUITY UPC2

1 7 µm

High Density Bonding

O SiO

OO

OHN

O OH

ACQUITY UPC2

Torus DEA

ACQUITY UPC2 1.7 µm BEH Particle

O SiO

OOH

NH

O SiO

O OH ACQUITY UPCTorus DIOL

ACQUITY UPC2

Torus 1-AA


US 6,686,035US 7,223,473Others patent pending

OTorus 1-AA

Wide Range of SelectivityGreater S-Value, Greater Orthogonality, g y

1

3 2

568

S-Value Reference

1

2 35

4 72-PIC

S-Value = 584 6

78DEA

3 25

6 8

1 47

DIOL

S-Value = 93

1

3

24

56

7

8

S-Value = 90


Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

1-AA

(1) Uwe Neue, et al. , Journal of Chromatography A, 1127 (2006

Better Peak Shape Without Additives – Torus 2-PIC

1. Ibuprofen2. Theophylline3. Amitriptyline4. Fenoprofen5. Thymine6 P l l

3.0 x 50mm, 1.2mL/min, 5-40% MeOH in four min, 40C, 2175psi

86. Propranolol7. Prednisolone8. Sulfamethoxazole9. Cytosine

Conventional 2-EP 1 7µm3

2 5

61 7

4

9Conventional 2 EP 1.7µm

6

ACQUITY UPC² Torus 2-PIC 1.7µm

12

3 45

7 89

Minutes0.00 0.55 1.10 1.65 2.20 2.75 3.30 3.85 4.40 4.95 5.50


Excellent peak shapes seen on 2-PIC using pure MeOH co-solvent

Torus 2-PICEffect of Basic Additive

1

5

37 3.0 x 100mm

1.5mL/min10% Co-Solvent2500psi

1.Caffiene2.Papaverine3.Amitriptyline4 Fenoprofen4

5

2

6

8

35C4.Fenoprofen5.Thymine6.Prednisone7.Propranolol8.Prednisolone9.Sulfamethoxazole10 Sulfanilamide MeOH6

9

10

10.Sulfanilamide

3

MeOH

1

37

45

2 8 MeOHw/ 40mM NH3

©2015 Waters Corporation 12Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

69

10

w/ 40mM NH3

Torus DEA – Good Peak Shapes for Strong Basesg

1 7 T DEA

Column: 3.0 x 100 mmFlow rate: 1.5 mL/min IsocraticMobile Phase: 12% MeOHTemperature: 35°CABPR: 2500 psi

Competitor 1 8µm 2-EP

1.7µm Torus DEA ABPR: 2500 psi

Competitor 1.8µm 2 EPTrimipramineAmitriptylineImipramineNortriptyline

Minutes0.0 0.5 1.0 1.5 2.0 2.5 Minutes0.0 0.5 1.0 1.5 2.0 2.5

No additive needed to give sharp peak shapes for very basic compounds – necessary to take advantage of rapid UPC2


compounds necessary to take advantage of rapid UPCseparations

Torus DIOL– Good Peak Shapes for Acidic Analytesy

4.2e-1

4.4e-1

4.6e-1

4.8e-1

3.0e-1

3.2e-1

3.4e-1

3.6e-1

3.8e-1

4.0e-1

AU

1 6e-1

1.8e-1

2.0e-1

2.2e-1

2.4e-1

2.6e-1

2.8e-1

P iti l I f

4.0e-2

6.0e-2

8.0e-2

1.0e-1

1.2e-1

1.4e-1

1.6e 1 Positional Isomers of Dimethoxybenzoic Acids

Excellent peak shape for a three minute separation of acidic aromatic regioisomers

Time-0.00 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 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30

0.0

2.0e-2


Technology Brief: “Direct separations of the six positional isomers of disubstituted benzoic acids using ACQUITY UPC2 Torus columns”

Torus 1Torus 1--AA for Hydrophobic CompoundsAA for Hydrophobic CompoundsFree Fatty AcidsFree Fatty Acidsyy

Time (min) Flow (mL/min) %B Curve

Initial 1.9 1.8 --

5.0 1.9 11 9

3.0x100mm 1.7µm Torus 1-AACO2/MeOH, 50C, 2100psi BPRUPC2 w/ QDa ESI-

C16:1

C16

C20:4

5.5 1.9 30 1

6.5 1.9 30 11

7.0 1.9 1.8 1

UPC w/ QDa ESI

C17

C17:1 C18:2

C20:2

C20:4

C22:2

Carbon length : number of double bonds

% C14C14:1

C15C15:1

C17 C18

C20 C22C20:1 C22:1 C22:6

C24:1

C10C11 C12

C13


Time (min)0.60 1.00 1.40 1.80 2.20 2.60 3.00 3.40 3.80 4.20 4.60 5.00

0

C6 C8C10

Method Development Strategy for Achiral ACQUITY UPC² Torus ColumnsQ

2) Defined Screening1) Rapid Scouting 3) Optimization

Acidic orMix Analyte

) gGeneric gradient with specified chemistry

and co-solvent

DIOL DIOL w/baseGeneric Gradient:

1.2 mL/min,

2-PIC

Co-solvent

BasicAnalytes DEA DEA

w/base

4-50% MeOH in 3 mins30°C, 2,000 psi

3.0 x 100 mm column

A) If separation criteria is met then proceed to

Temperature

Order of im

p

1-AA /b

/

1-AA / id

NeutralAnalytes

met, then proceed to Optimization if needed

B) If good separation but need better peak shape, then run 2-PIC

Additive

Backpressure

pact

w/base w/acidAnalytesp ,with additive

C) If different separation is needed, proceed to Defined Screening

Backpressure


ACQUITY UPC² Torus Columns Method Development Strategyp gy

2) Defined ScreeningGeneric gradient with specified chemistry

d l t

1) Rapid Scouting 3) Optimization

2 CAcidic or

Mix Analyte

and co-solvent


1.2 mL/min, 4-50% MeOH in 3 mins

30°C 2 000 psi

2-PIC

Co-solvent

Ord


w/base

30 C, 2,000 psi3.0 x 100 mm column

A) If separation criteria is met, then proceed to Optimization if

Temperature

der of impact

1-AA w/base

1-AA w/acid

NeutralAnalytes

pneeded

B) If good separation but need better peak shape, then run 2-PIC with additive

Additive

Backpressure

t



Simplified Column Selectionp

Torus Columns will use a targeted screening approach to method development. The goal is to minimize the effort needed to arrive at a set of conditions that can be optimized


to arrive at a set of conditions that can be optimized.

Développement de méthodes UPC²Phases chiralesPhases chirales


Chiral Stationary Phase (CSP)Structures

O

OR

O

HN

R

O

RO

OR

n

OR=

AMY1: Amylose tris-(3,5-dimethylphenylcarbamate)

OO

OR

RO

HN

R=Chiral polysaccharide coating on silica particle

“Chiral Selector” modulatesthe chiral recognition

RO

OR

n

O

CEL1: Cellulose tris-(3,5-dimethylphenylcarbamate)

coat g o s ca pa t c ecreates the chiralenvironment

OO

OR

RO

Cl

HN

R=


RO

OR

n

OR

CEL2: Cellulose tris-(3-chloro-4-methylphenylcarbamate)

Trefoil™ Columns CoverA Wide Range of Selectivities

AMY1 CEL1 CEL2

g

Troger’s Base

B i i iBupivicaine

O

O

Minutes0.00 0.80 1.60 2.40 3.20 4.00

Minutes0.00 0.60 1.20 1.80 2.40 3.00

Minutes0.00 0.80 1.60 2.40 3.20 4.00Trimebutine

O

O

ON


UPC2 Chiral Method Development (MD)Key Success Elementsy

ACQUITY UPC2 Best analytical SFC

f l CS Trefoil™ CSPs: – Trefoil™ AMY1– Trefoil™ CEL1 Highest chiral separation success– Trefoil™ CEL2

2.5 um particles Best in class efficiency vs. 3.0 & 5.0 um 2.1 x 50 mm Max. speed / Min. solvent use

Method– 2 min Screening Method Ballistic general purpose gradient

o PDA/MS detection Peak identification/confirmation

Optimal Combinations of co-solvents & additives favorably modulate chiral recognition


Quicker Method Development Success

SFC Chiral MD Study:Co-Solvent / Additive CombinationImpact on Chiral Resolution SuccessImpact on Chiral Resolution SuccessSystematic Variation of Co-Solvents & Additives 15 Combinations of 4 Co-Solvents:

Condition MeOH EtOH IPA ACN Low pH Mid pH High pH1 X X2 X X3 X X

– Methanol, Ethanol, Isopropanol, Acetonitrile

3 Additives (Low, Mid & High pH)

4 X X5 X X6 X X7 X X8 X X9 X X10 X X11 ‐‐‐ ‐‐‐12 X X13 X X X14 X X X

Single Co-Solvents X 3 Additives

– Trifluroacetic Acid (TFA), Ammonium Acetate (AmAc), Ammonium Hydroxide (AmOH)

14 X X X15 X X X16 X X X17 X X X18 X X X19 X X X20 X X X21 X X X22 X X X23 X X X24 X X X25 X X X

Binary Co-Solvent Blends X 3 Additives

25 X X X26 X X X27 X X X28 X X X29 X X X30 X X X31 X X X X32 X X X X33 X X X X34 X X X X35 X X X X

For 1 CSP:44 combinations x 55 chiral study 36 X X X X

37 X X X X38 X X X X39 X X X X40 X X X X41 X X X X42 X X X X43 X X X X X44 X X X X X45 X X X X X

Ternary Co-Solvent Blends X 3 Additives

Quaternary Co-Solvent Blends X 3 Additives

x 55 chiral study compounds=2,420 runs


Examined 44 Co-Solvent / Additive Combinations(excluded ACN with Ammonium Acetate (AmAc) due to insolubility)

SFC Chiral MD Study:Search for Optimal CombinationsWith Trefoil™ AMY1 ColumnWith Trefoil™ AMY1 Column

UPC2™ chromatography of 55 representative chiral compounds. Each racemate is problem needing a solution (resolution!!!) Each racemate is problem needing a solution (resolution!!!). Examine 2,420 separate analyses (UV and MS ESI +/- per analysis)

using this “Pass” criterion:– Enantiomer Resolution If Rs > 1.2 then compound solved!


Selectivity (50 mm AMY1 screen column)Modulation Example

0 32

p

N

NH

O

SH2N

O

O

AU

0.000.080.160.240.32 H

O

O

Methanol/IsopropanolAmmonium HydroxideRs = 0.74

Sulpiride

AU

0.080.160.240.32

Isopropanol/AcetonitrileAmmonium HydroxideRs = 0.27

0.00

AU

0 150.300.450.60 Ethanol/Acetonitrile

Ammonium AcetateRs = 0.73

0.000.15

AU 0 40

0.600.80 Ethanol/Isopropanol

TFA


A

0.000.200.40

Minutes0.00 0.40 0.80 1.20 1.60 2.00

Rs = 1.47

SFC Chiral MD Study:Search for Optimal CombinationsWith Trefoil™ AMY1 Column (cont )With Trefoil™ AMY1 Column (cont.)

Found:– 34 Chiral compounds were “solved” by at least one combination.34 Chiral compounds were solved by at least one combination.

“Optimal Combination” (def.):– A combination of Co-Solvents / Additives associated with

high chiral resolution successhigh chiral resolution success.

Ask:– What is the fewest number of Optimal Combinations needed to solve

h “ i i l h”these compounds? The “Critical Path”.


AMY1 Critical Path Through “Co-Solvents / Additive Space”pStep Combo % Salvage % Total Success

1 EtOH / ACN / AmAc ---- 56 %

2 EtOH / IPA / TFA 18 % 74 %

3 IPA / ACN / AmOH 14 % 88 %

4 MeOH / IPA / AmOH 6 % 94 %/ /

5 IPA / AmOH 3 % 97 %

6 ACN / TFA 3 % 100 %

90.0%100.0%

ss

% Success by Step

50.0%60.0%70.0%80.0%

Total Succe

All CombosHigh success rate can be achieved High success rate can be achieved with 4 cowith 4 co--solvent / additive combossolvent / additive combos


40.0%1 2 3 4 5 6

Step

What if we had only used single co-solvents?gStep Combo % Salvage % Total Success

1 EtOH / AmOH ---- 44%

2 IPA / TFA 18 % 62%

3 EtOH / TFA 6 % 68%

4 ACN / TFA 6 % 74%/

5 MeOH / AmOH 3 % 77%

6 IPA / AmAc 2 % 79%

90.0%100.0%

ess

% Success by Step

50.0%60.0%70.0%80.0%

Total Succe

All Combos

Single CombosSingle coSingle co--solvents solvents are inferior to coare inferior to co--solvent blendssolvent blends


40.0%1 2 3 4 5 6

Step

are inferior to coare inferior to co solvent blendssolvent blends

Full 3 CSP Method DevelopmentStrategy: AMY1, CEL1, CEL2gy , ,

Expand to 3 CSP’s: AMY1, CEL1, CEL2– Examine 4 Step efficiency for each CSP (2,420 x 3 = 7260 analyses).Examine 4 Step efficiency for each CSP (2,420 x 3 7260 analyses).– Examine 4 Step efficiency for full compound set (55).– Explore most efficient CSP/co-solvent/additive “Critical Path”.

G l 4 t 8 i t Chi l M th d –Goal: 4 step, 8 minute Chiral Method Development. AMY1 CEL1 CEL2

Condition MeOH EtOH IPA ACN Low pH Mid pH High pH1 X X2 X X



For 3 CSP:44 combinations x 55 compounds

X X3 X X4 X X5 X X6 X X7 X X8 X X9 X X10 X X11 ‐‐‐ ‐‐‐12 X X13 X X X14 X X X15 X X X16 X X X17 X X X18 X X X19 X X X20 X X X21 X X X22 X X X

X X3 X X4 X X5 X X6 X X7 X X8 X X9 X X10 X X11 ‐‐‐ ‐‐‐12 X X13 X X X14 X X X15 X X X16 X X X17 X X X18 X X X19 X X X20 X X X21 X X X22 X X X

X X3 X X4 X X5 X X6 X X7 X X8 X X9 X X10 X X11 ‐‐‐ ‐‐‐12 X X13 X X X14 X X X15 X X X16 X X X17 X X X18 X X X19 X X X20 X X X21 X X X22 X X Xx 55 compounds

x 3 CSPs=7,260 runs

23 X X X24 X X X25 X X X26 X X X27 X X X28 X X X29 X X X30 X X X31 X X X X32 X X X X33 X X X X34 X X X X35 X X X X36 X X X X37 X X X X38 X X X X39 X X X X40 X X X X41 X X X X42 X X X X43 X X X X X




43 X X X X X44 X X X X X45 X X X X X



AMY1 + CEL1 + CEL2Optimal Path ScreenUsing All Combos

Step Combo % Salvage % Total Success

1 AMY1 EtOH/IPA/ACN AmAc 45 5 %1 AMY1-EtOH/IPA/ACN-AmAc ---- 45.5 %

2 CEL1-MeOH/IPA-TFA 27.3 % 72.7 %

3 CEL2-EtOH/ACN-TFA 13.6 % 86.4 %

4 AMY1-EtOH/IPA-TFA 9.1 % 95.5 %

5 CEL1-IPA-TFA 4.5 % 100.0%

80.0%90.0%100.0%

ess

% Success by Step

40.0%50.0%60.0%70.0%80.0%

Total Succe

All Combos


30.0%1 2 3 4 5 6

Step

AMY1 + CEL1 + CEL2 Optimal Path Screen Using Single Co-Solvent Combos

Step Combo % Salvage % Total Success1 CEL1 EtOH TFA 43 2 %1 CEL1-EtOH-TFA ---- 43.2 %2 AMY1-EtOH-AmOH 22.7 % 65.9 %3 CEL2-EtOH-TFA 11.4 % 77.3 %4 AMY1 IPA A A 4 5 % 81 8 %4 AMY1-IPA-AmAc 4.5 % 81.8 %5 AMY1-EtOH-TFA 4.5 % 86.4 %6 CEL1-IPA-TFA 4.5 % 90.9 %

90.0%100.0%

s

% Success by Step AMY1+CEL1+CEL2: Recommend Co-Solvent

Mixtures

40.0%50.0%60.0%70.0%80.0%

Total Success

All Combos

Single Combos

For 3 CSPs, CoFor 3 CSPs, Co--Solvent Solvent mixturesmixtures are are better than single Cobetter than single Co--Solvents (ca. 14%)Solvents (ca. 14%)


30.0%1 2 3 4 5 6

Step

Trefoil™ Chiral Method Development:Strategy DetailsS a egy e a s

Use all three 2.1x50mm Trefoil columns and the ballistic screening method on ACQUITY UPC² to implement the following screening method on ACQUITY UPC to implement the following Trefoil Optimal Path Screen (4 steps):

S l 2 Minute Screening MethodStep Columns & Blends Solv

Line1 AMY1-EtOH/IPA/ACN-AmAc B1

2 Minute Screening MethodTrefoil 2.1x50 mm columns @ 40°C 1.2 mL/min flow rate ABPR = 3200 psi

Use indicated blend with:

2 CEL1-MeOH/IPA-TFA B23 CEL2-EtOH/ACN-TFA B34 AMY1 EtOH/IPA TFA B4

Use d cated b e d t• 20 mM AmAc OR • 0.2% TFA

Equilibrate: 3% B for 0.5 minGradient: 3 to 60% B over 1.5 min 4 AMY1-EtOH/IPA-TFA B4 Hold: 60% B for 0.5 min

2.5 min Cycle Time per Step


ACQUITY UPCACQUITY UPC22 Trefoil Columns Trefoil Columns for for ChiralChiral SeparationsSeparationspp

Highlights– Designed for the ACQUITY UPC2 Systemg Q y

o Transition away from Normal Phase LC– Broad selectivity for separation of a wide range of chiral

compounds– High speed and efficiency

o Fast gradients - 2.5 µm particles, 2.1 and 3.0 mm ID– Excellent mechanical stability– Mass spectrometer compatible

o Single Quad for Method developmento Triple Quad for DMPK studiesp Qo Tof for accurate mass, impurity ID

Chiral & Achiral analysis using same mobile phases and same t


system

Conclusion

L’UPC² a démontré ses bénéfices en tant que:– Technique complémentaire à la LC (sélectivité très différente)– Alternative à la phase normale– Technique simple d’utilisation et avec une bonne performance– Système à coût réduit d’utilisation

L’UPC² s’applique à un nombre important de domaines:– Séparations chirales– Chimie de synthèse– Produits finis dans le pharmaceutique par exemple– Industrie chimique (tensio-actifs, composés apolaires…)– Les lipides

b d’– Et bien d’autres… Flexibilité de la détection

– Optique : UV, PDA, Fluo, DEDLMS l W


– MS : toute la gamme Waters

Développement de méthodes en UPC² v1 - Waters Corporation UPC2.… · Chloroform Acetone...

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Transcript of Développement de méthodes en UPC² v1 - Waters Corporation UPC2.… · Chloroform Acetone...