Short course SFC Fundamentals - Waters Corporation Users...©2015 Waters Corporation 3 Evolution of Separation Technology Gas Chromatography Liquid Chromatography GC Capillary GC New

©2015 Waters Corporation 1

Short course

-

SFC Fundamentals

Waters European User Meeting on SFC, Purification & Related Technologies

December 3-4, 2015

Isabelle François, UPC²/SFC & Strategic Separations Business Development Europe & India


Fundamentals on supercritical fluid chromatography

(SFC) and convergence chromatography

Practical aspects – Instrument and methodologies

Application examples

Agenda


Evolution of Separation Technology

Gas Chromatography Liquid Chromatography

GC

Capillary GC

HPLC

UPLC, UHPLC

SFC

New generations (UPC²)

Supercritical Fluid Chromatography

SFC


Advancements in Gas Chromatography

Gas Chromatography

Since the advent of the use of fused-silica capillaries in the late 1970’s, only small incre-mental advancements in GC have occurred.

GC Capillary GC

Minimal development in gas chromatography over the past 30 years


Advancements in Liquid Chromatography

18,000 psi

30,000 psi

130,000 psi

0

50,000

100,000

150,000

200,000

250,000

300,000

350,000

400,000

450,000

500,000

5 µm 3.5 µm 1.7 µm 1.4 µm 1.0 µm

Pla

tes/

me

ter

Particle Size

Liquid Chromatography

The advent of UPLC in 2004 has revolutionized liquid chromato-graphy, providing significant advancement in sensitivity, resolution and throughput

LC UltraPerformance LC [UPLC] or UHPLC

The practical and functional limits of LC efficiency (N) have been improved through reduced system dispersion and smaller particles.

UPLC, UHPLC

70% more efficiency than 1.7 µm

100% more efficiency than 3.5 µm


Advancements in Supercritical Fluid Chromatography


SFC Newer generations? New era? UltraPerformance Convergence Chromatography (UPC²)

Data courtesy of Davy Guillarme, Jean-Luc Veuthey LCAP, University of Geneva, Switzerland

YES!


Separation Technology Overview

Gas Chromatography

Liquid Chromatography


Separation achieved by a temperature gradient

•High efficiency [N] • Virtually no limitation to column length

•Limited selectivity [α]

• Limited stationary phase options

Separation achieved by a solvent gradient

•Limited efficiency [N] • Limited due to pressure drop across column

• High selectivity [α] • Different modes: reversed-phase, normal-phase, SEC, IEX, affinity, ion pair, HILIC, GPC…etc.

Separation achieved by density/solvent gradient

•High efficiency [N] • Low viscosity allows longer columns and/or smaller particles

•High selectivity [α]

• Wide variety of stationary phase and mobile phase (modifier / additive) • Pressure and temperature as additional parameters to alter selectivity

GC

LC

SFC


History of SFC

• Introduced by Klesper et al. in J. Org. Chem.

‘High Pressure Gas Chromatography Above Critical

Temperatures’

• Initially performed on capillary columns

• First capillary SFC available on the market (Hewlett-Packard)

based on work of Berger and Gere

• CO2 only – density gradients

• GC-like with applications limited to non polar compounds

• SFC is involved in the ‘green chemistry’ global effort

• Acceleration of instrumental developments

• 2012 – Waters introduces UPC²

• Extended to packed column SFC with improved and

optimized instrumentation

• Introduction of polar modifier to CO2

• LC-like with development of applications for more polar

compounds


Advantages of a Supercritical Fluid

Data courtesy of Davy Guillarme, Jean-Luc Veuthey LCAP, University of Geneva, Switzerland


Why CO2 ?

Chromatographic technique similar to HPLC

– Was introduced to replace NP -> unpolar solvent had to be mimicked by

supercritical fluid -> CO2 has very low dipole

Mobile phase is supercritical fluid + one or more co-solvents

– CO2 is miscible with all organic solvents

– CO2 is the most common supercritical fluid

– MeOH is the most common co-solvent

Substance Critical Temp

oC Critical Pressure (bar)

Comments

Carbon Dioxide 31 74 Physical state easily changed

Water 374 221 Extreme conditions needed

Methanol 240 80 Extreme temperature needed

Ammonia 132 111 Highly corrosive

Freon 96 49 Environmentally unfriendly

Nitrous Oxide 37 73 Oxidizing agent


Why CO2 ?

CO2 reaches supercritical state at 31.1°C and 73.8 bar

– Its physical state can be easily manipulated

CO2 is non toxic, non flammable

CO2 is chemically pure, stable and non-polar solvent

CO2 is compatible with LC detectors

CO2 is as a Green Solvent

– Environmentally neutral:

• Recovered e.g. from industrial and fermentation plants

– Avoids the production of CO2 that would have been generated from disposal of the solvents it replaces

– Less time and energy are used to evaporate fractions to get to pure analytes as CO2 is a gas at room temperature


Green chromatography using SFC?

Courtesy of A. Grand-Guillaume Perrenoud, D. Guillarme, Pr J-L. Veuthey, University of Geneva


CO2 Phase Diagram

Critical point of CO2

Tc = 31.1°C

Pc = 73.8 bar


Initial SFC work: playing with density (often on

capillary columns)

Working isothermically: T = ct; Increase P

Working isobarically: P = ct; Decrease T

Density increase resulting in solvent strength increase

Reprinted from ‘Scheidingstechnieken – Een inleiding’ by prof. Dr. Pat Sandra

Pressure as a function of density at different isotherms. For CO2 : Pc = 72.8 atm, Tc = 31.1°C, c = 0.466 g/mL.

SFC IN THE PAST AND TODAY


Advantage:

Coupling with typical GC detectors e.g. FID possible

(ASTM in petroleum / petro-chemical industry)

No solvents necessary, no solvent waste

Disadvantage:

Limited to relatively apolar analytes



In general: separation / elution is achieved by solvent / modifier gradient (comparable to LC)

Pressure and temperature are kept constant, but different values provide different

selectivities

Elution by increasing the polarity of the mobile phase with polar solvent (up to typically 40%)


Typical modifiers:

Acetonitrile, methanol, isopropanol, ethanol, tert methylbutylether, hexane, heptane, ...

Additives:

Isopropylamine, diethylamine, ammoniumformate, formic acid, acetic acid, ammonium

hydroxide ...

Critical conditions can no longer be obtained!

But ... No problem: if separation is ok, it is ok to work under subcritical/superfluid

conditions!

When adding even small percentages of solvent:

Supercritical fluid chromatography becomes subcritical fluid chromatography!


On identical column, retention decreases by:

Reducing pressure

PARAMETERS INFLUENCING RETENTION

P (outlet) = 180 bar

P (outlet) = 130 bar


AU

0.110

0.120

0.130

0.140

0.150

0.160

0.170

0.180

Minutes

9.40 9.60 9.80 10.00 10.20 10.40 10.60 10.80

AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

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

AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00

110 Bar

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Minutes

7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00

AU

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

Minutes

6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50

Original: 130 Bar

AU

0.090

0.100

0.110

0.120

0.130

0.140

0.150

0.160

0.170

0.180

0.190

Minutes

9.00 9.20 9.40 9.60 9.80 10.00 10.20 10.40 10.60 10.80

150 Bar

AU

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

Minutes

6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50

AU

0.110

0.120

0.130

0.140

0.150

0.160

0.170

0.180

Minutes

9.10 9.20 9.30 9.40 9.50 9.60 9.70 9.80 9.90 10.00 10.10 10.20 10.30 10.40 10.50

Courtesy of David Clicq, UCB, Belgium

AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

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



Increasing solvent% or using different modifier


15

14 5 9

13

12 3 6

11

10

16

4 2

8

7

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

Minutes

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

14

5 9

13

12

63 11

410

16

2

8

7

AU

0.00

0.10

0.20

0.30

Minutes

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

Modifier MeOH (0.1% HCOOH)

Modifier MeOH/ACN 1/1 (0.1% HCOOH)




Decreasing temperature Inverse correlation between

retention and temperature when compared to HPLC


AU

0.00

0.10

0.20

0.30

0.40

0.50

AU

0.00

0.10

0.20

0.30

0.40

0.50

Minutes

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

40°C

50°C



Courtesy of C. West, E. Lesellier, ICOA University of Orléans


H2O---Methanol---Acetonitrile---isopropanol---CH2Cl2---Hexane

Silica C18, C12, C8 Phenyl hexyl

Cyano propyl Propyl phenyl PGC Fluorinated Phases

Silica Amino, Cyano Diol, PVA, Ethylpyridine, Polyamide

Reversed phase LC Normal phase LC

CO2


Benzodiazepines

I. Midazolam II. Flunitrazepam III. Lormetazepam IV. Clorazepate V. Nitrazepam VI. Oxazepam

AU

0.00

0.05

0.10

0.15

0.20

Minutes

0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60 4.00

Steroids

1. Androstenedione 2. Mestanolone 3. Testosterone 4. Stanozolol

Van Deemter curve H = f(u)

0.0

20.0

40.0

60.0

80.0

100.0

0.0 2.0 4.0 6.0 8.0 10.0

H (

µm

)

u (mm/s)

uopt

1

2

4

3

0.60 min

1

2

4

3

1.90 min

AU

0.00

0.05

0.10

0.15

0.20

Minutes

0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60 4.00

2.75 min

I

II

IV III

V VI 0.85 min

I II

IV

III

V VI

Generic conditions

2-EP, 150 x 4.6mm, 5m. CO2-MeOH, 3.5mL/min

Oven temp @ 40 C BPR @ 200bar

UV detection @ 220nm

Generic conditions

2-EP, 150 x 4.6mm, 5m. CO2-MeOH, 10mL/min

Oven temp @ 40 C BPR @ 200bar

UV detection @ 220nm

3 x uopt

Only 25% efficiency loss

SOME THEORY … DIFFUSIVITY, VISCOSITY, SPEED AND EFFICIENCY



Low viscosity Low pressure drop

Efficiency can be increased by: Decreasing particle size

High flow rates can be applied with little to no efficiency loss

2.5 µm XBridge™ HILIC, 3.0x50 mm



1.7 µm ACQUITY BEH , 3.0x50 mm


Isocratic Conditions CO2/Methanol 85:15 130 bar outlet pressure


Low viscosity Low pressure drop

Efficiency can be increased by: Increasing column length by

coupling columns

BZD 1. Diazepam 2. Midazolam 3. Flunitrazepam 4. Lormetazepam 5. Flurazepam 6. Alprazolam 7. Triazolam 8. Clorazepate 9. Bromazepam 10. Nitrazepam 11. Clonazepam 12. Oxazepam 13. Lorazepam 14. Clozapine 15. Olanzapine

N

N R2

R3 R4

R5

R1

150 x 4.6mm, 5m

30bar

2 1

3

4

5 + 6

7 8

9

10 + 11

12

13 + 14

15 Pc = 63

450 x 4.6mm, 5m

2 1 3

4

6

7 8 9 11 12

13 14

80bar

5 10

15

Pc = 108

Analytical conditions : CO2-MeOH gradient mode, 4mL/min ; PrincetonSFC 2EP 150 x 4.6mm, 5m; Oven temp @ 40 C ; BPR @ 150bar ; UV @ 220nm




Best injection solvent = weak mobile phase constituent

In real life – Compromise between

– Sample solubility

– Peak shape

– Sample stability

– Compound retention

Even more important in prep SFC

Dissolution solvent

THF/heptane (70/30)

IPA

MeOH

J. Fairchild, J. Hill, P.C. Iraneta, LCGC North America 31 (2013) 326.


UPC² Similar Set-up as in UPLC

Inject valve

Auxiliary Inject valve

Column Manager

PDA detector

Back Pressure Regulator (Dynamic and Static)

Waste Modifier CO2 Supply CO2

Pump Modifier

Pump

mixer Thermo-electric heat exchanger


Inject valve

Auxiliary Inject valve

Column Manager

PDA detector

Back Pressure Regulator (Dynamic and Static)

Waste Modifier CO2 Supply CO2

Pump Modifier

Pump

mixer Thermo-electric heat exchanger

Make-up Pump

Mass Spec

Splitter

UPC² - MS Similar Set-up as in UPLC-MS


UPC2: Compatibility with all Waters MS Technologies

For ultimate CC-MS performance, ACQUITY UPC2 System coupled with: ACQUITY QDa - Single quadrupole detector for robust and routine

performance Xevo TQ-S - Ultimate sensitivity

Xevo G2-S Qtof and Synapt G2-S - Qualitative and quantitative results from a single

platform

Bringing MS to NP separations!


Binary Solvent Manager:

– Mobile phase:

o A: Supercritical CO2

o B: Methanol (MeOH)

– Flow rate: 1.8 mL/min

– End time: 7 min

– Gradient: see table

Column:

– ACQUITY UPC² BEH 100 mm L x 3 mm i.d. X 1.7 µm dp

– T = 40°C

Sample manager:

– Strong wash: IPA

– Weak wash: MeOH

– Loop volume: 10 µL

– Injection volume: 1 µL

Convergence manager:

– P (outlet, APBR): 130 bar

PDA:

– Acquisition rate: 10 or 20 Hz

Typical Example of an SFC / UPC² Method


Addressing Selectivity: RP and NP Chromatography

Solvent

Pentane, Hexane, Heptane

Xylene

Toluene

Diethyl ether

Dichloromethane

Chloroform

Acetone

Dioxane

THF

MTBE

Ethyl acetate

DMSO

Acetonitrile

Isopropanol

Ethanol

Methanol

Water

Stationary Phase

Silica / BEH

2-ethylpyridine

Cyano

Aminopropyl

Diol

Amide

PFP

Phenyl

C18 < C8

Reversed-phase and

HILIC Selectivity

Space

Normal Phase Selectivity

Space


SFC or UPC² Selectivity Space

Addressing Selectivity: SFC or UPC²

Solvent

Pentane, Hexane, Heptane

Xylene

Toluene

Diethyl ether

Dichloromethane

Chloroform

Acetone

Dioxane

THF

MTBE

Ethyl acetate

DMSO

Acetonitrile

Isopropanol

Ethanol

Methanol

Water

Stationary Phase

Silica / BEH

2-ethylpyridine

Cyano

Aminopropyl

Diol

Amide

PFP

Phenyl

C18 < C8

Weak

Str

ong

Supercritical CO2

Organic Modifier

Additional parameters for selectivity finetuning (density): - Pressure - Temperature


ACQUITY UPC² Torus Columns Method Development Strategy available

1-AA w/base

Acidic or Mix Analyte

Basic Analytes

2) Defined Screening Generic gradient with specified chemistry

and co-solvent

DIOL

DEA DEA

w/base

1-AA w/acid

Neutral Analytes

DIOL w/base

1) Rapid Scouting

Generic Gradient: 1.2 mL/min,

4-50% MeOH in 3 mins 30°C, 2,000 psi

3.0 x 100 mm column A) If separation criteria is

met, then proceed to Optimization if needed

B) If good separation but

need better peak shape, then run 2-PIC with additive

C) If different separation is

needed, proceed to Defined Screening

3) Optimization

2-PIC

Co-solvent

Temperature

Additive

Backpressure

Decre

asin

g im

pact


ACQUITY UPC2 Trefoil™ Chiral Column Technology

ACQUITY UPC2 Trefoil AMY1

– Amylose tris-(3,5-dimethylphenylcarbamate)

ACQUITY UPC2 Trefoil CEL1

– Cellulose tris-(3,5-dimethylphenylcarbamate)


– Cellulose tris-(3-chloro-4-methylphenylcarbamate)

Screening strategy available!


REPRODUCIBILITY INCREASED RESOLUTION


When to Use SFC or Convergence Chromatography

Normal phase

Compounds with no

retention in RPLC

Compounds degrading

in H2O

Lipidomics

Lipids in food

stuffs

Orthogonal in

comparison

to C18

Cosmetics

Future scale-up

Vitamins

Simplified

sample prep

Chiral = no-brainer

Structural Isomers



Normal phase

Compounds with no

retention in RPLC

Compounds degrading

in H2O

Lipidomics

Lipids in food

stuffs

Orthogonal in

comparison

to C18

Cosmetics

Future scale-up

Vitamins

Simplified

sample prep

Chiral = no-brainer

Structural Isomers


Elution of CLA under NPLC and UPC2 conditions.

Peaks: 1) C18:2 t11 t13; 2) C18:2 t9 t11; 3) C18:2 t8 t10; 4) C18:2 t10 c12; 5) C18:2 c9 t11;

6) C18:2 c9 c11.

Column: Chrompack ChromSpher 5 Lipids 4.6x250 mm, 5µm, three in series

Column temp.: 50°C

NPLC UPC2

1 2 5 4

3

6

1

2

3

4 5 6

Normal phase

SEPARATION OF METHYL ESTERS OF CONJUGATED LINOLEIC ACID (CLA) ISOMERS STANDARDS

46 min 27 min

Structural Isomers

With permission of Eric Mignolet, UCL, Belgium

Gradient has eliminated wrap arounds ! (occuring in NPLC mode due to isocratic mode)


Normal phase

Chiral = no-brainer

ORGANIC SYNTHESIS UPC² - FASTER TIME TO RESULT AND INCREASED PROFIT

NPLC on AD-H

Heptane/IPA (9/1), isocratic

230 nm

9

UPC² on AD-3

Gradient: 5-40% Isopropanol


Chiral separations with faster time to result and increased profit

HPLC

UPC²


UPC² Return on Investment Calculation Estimated Saving Analysis UPC² vs HPLC

Parameters UPC² HPLC

Number of Injections 1000 1000 Per System

Sample Preparation Time (min) Same in both the systems

Run Time (min) 4.5 20 Injection to Injection

Total Run Time Required (Hr) 75 333.33

No. of Days 9.38 41.67 8 Hrs Per Day Per Instrument

No. of Days Saved 32.29 days saved using UPC²

Method Flow Rate (ml/min)

MTBE consumption/1000 Run (lit)

Methanol consumption/1000 Run (lit)

CO2 Consumption/1000 Run (lit)

FA Consumption/ 1000 Run (lit)

TFA Consumption/ 1000 Run (lit)

Total Cost (Rs:)/1000 runs

Total Cost (Rs:)/ run

Customer’s LC Method

1.5 9.0 21.0 - 0.15 - 27300.02 27.3

Waters UPC2

Method

3.0 - 6.07 7.43 - 0.01 2847.83 2.85

% Productivity Upon Investment increased 89.56%

*All values rounded off to two decimal places

*% Improvement in productivity upon investment = (Previous investment-Current investment) x 100/Previous Investment


Sample pooling and QDa for increased throughput and profitability in chiral analyses

Analyse 6 components in one screening Use the power of QDa to distinguish compounds Screening is shortened 6 times!

Courtesy of Alex Brien, Victoria Coulthard, Reach Separations, UK


0.00 0.50 1.00 1.50 2.00

AMY1 CEL1 CEL2

Sulindac

Praziquantel

Metoprolol

0.00 0.50 1.00 1.50 2.00 0.00 0.50 1.00 1.50 2.00 Minutes Minutes Minutes

Normal phase

Chiral = no-brainer

Waters Chiral Phases 2.5 µm particles


AU

0.00

0.08

0.16

0.24

0.32

AU

0.00

0.08

0.16

0.24

0.32

AU

0.00

0.15

0.30

0.45

0.60

AU

0.00

0.20

0.40

0.60

0.80

Minutes

0.00 0.40 0.80 1.20 1.60 2.00

Selectivity Modulation

Methanol/Isopropanol Ammonium Hydroxide Rs = 0.74

Isopropanol/Acetonitrile Ammonium Hydroxide Rs = 0.27

Ethanol/Acetonitrile Ammonium Acetate Rs = 0.73

Ethanol/Isopropanol TFA Rs = 1.47

Sulpiride

ACQUITY UPC2 Trefoil AMY1 Column

Normal phase

Chiral = no-brainer


ACQUITY UPC2 Trefoil™ Chiral Column Technology

ACQUITY UPC2 Trefoil AMY1

– Amylose tris-(3,5-dimethylphenylcarbamate)


– Cellulose tris-(3,5-dimethylphenylcarbamate)


– Cellulose tris-(3-chloro-4-methylphenylcarbamate)

Screening strategy available!



Normal phase

Compounds with no

retention in RPLC

Compounds degrading

in H2O

Lipidomics

Lipids in food

stuffs

Orthogonal in

comparison

to C18

Cosmetics

Future scale-up

Vitamins

Simplified

sample prep

Chiral = no-brainer

Structural Isomers


DHEA

T

A E

OA OE T

THE

Cortol

OTHF ATHF

P

Prog

P3

P2

Simplified

sample prep

Structural Isomers

Direct injection of samples needing derivatization or handling in GC

Eg urinary steroids (alternative is derivatization and GC)

Easy MS coupling!


Peak m/z FA

1 143.10 C8:0

2 171.14 C10:0

3 199.17 C12:0

4 227.20 C14:0

5 255.53 C16:0

6 283.26 C18:0

7 311.30 C20:0

8 339.33 C22:0

9 367.36 C24:0

Simplified

sample prep

Structural Isomers

UPC2 Conditions: A= CO2

B=MeOH in 0.1% HCOOH Column= ACQUITY UPC2 HSS C18 SB 1.8µm (2.1 x 150 mm) Flow rate= 0.6 mL/min Column temp= 50 ºC

Direct injection of samples needing derivatization or handling in GC

Eg fatty acids (alternative is methylderivatization and GC)

Easy MS coupling!



Normal phase

Compounds with

no retention in

RPLC

Compounds degrading

in H2O

Lipidomics

Lipids in food

stuffs

Orthogonal in

comparison

to C18

Cosmetics

Future scale-up

Vitamins

Simplified

sample prep

Chiral = no-brainer


Separation of isomers

UPLC – 1 peak

UPC² – 2 peaks

UPLC – low retention + tailing

UPC² - higher retention + sharp peaks

Compounds with no

retention in RPLC

Courtesy of Arjen Gerssen, Rikilt, Netherlands

Structural Isomers

Problematic compounds

in UPLC

Easy MS coupling!



Normal phase

Compounds with no

retention in RPLC

Compounds degrading

in H2O

Lipidomics

Lipids in food

stuffs

Orthogonal in

comparison

to C18

Cosmetics

Future scale-up

Vitamins

Simplified

sample prep

Chiral = no-brainer


Within SFC

1

32

3

4

56

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

3233

34

35

36

37

38

39

40

41

42

4344

45

46

47

48

49

50

51 52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

6768

69

70

71

72

73

74

75

76

77

78

79

e

s

a -b

v

Non-polar

columns Aromatic

Mixed-mode Polar columns

Orthogonality



ACQUITY UPC2 Column Management: Selectivity Choices for AChiral Methods

: CSH PFP

AU

0.000

0.012

0.024

0.036

0.048

: HSS C18 SB

AU

0.000

0.012

0.024

0.036

0.048

: BEH HILIC

AU

0.000

0.012

0.024

0.036

0.048

: 2-EP

AU

0.000

0.012

0.024

0.036

0.048

Minutes

0.00 0.60 1.20 1.80 2.40 3.00 3.60 4.20 4.80 5.40 6.00

ACQUITY UPC2 BEH 2-EP 1.7 µm

ACQUITY UPC2 BEH 1.7 µm

A B C(1,2)

D

G H

9

F

A B C

D

G H

9

F

A B

C D

G H

9 F

A B

C D

G

H*

9

F

Unknown [M+H] = 266

Unknown [M+H] = 272

*Imp “H” is a broad peak under the unknown

Unknown [M+H] = 226

Unknown [M+H] = 226

ACQUITY UPC2 CSH Fluoro-Phenyl 1.7 µm

ACQUITY UPC2 HSS C18 SB 1.7 µm

A number of achiral stationary phase choices are available to tune selectivity for method development

ORTHOGONALITY WITHIN SFC Orthogonality


With RPLC

Within SFC

1

32

3

4

56

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

3233

34

35

36

37

38

39

40

41

42

4344

45

46

47

48

49

50

51 52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

6768

69

70

71

72

73

74

75

76

77

78

79

e

s

a -b

v

Non-polar

columns Aromatic

Mixed-mode Polar columns

Orthogonality



15

14 5 9

13

12 3 6

11

10

16

4 2

8

7

AU

0.00

0.20

0.40

0.60

Minutes

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


Pharma sample UCB on ACQUITY UPLC BEH C18 – 210 nm

Pharma sample UCB on ACQUITY UPC² BEH – 210 nm

Orthogonal in

comparison to C18

Application at UCB – Comparison of current UPLC method with UPC²


Courtesy of Alex Brien and Victoria Coulthard Reach Separations, UK

Orthogonal in

comparison to C18

Application at Reach Separations, UK

UPLC

ACQUITY UPLC BEH C18

UPC²

ACQUITY TORUS 2-PIC

ADDITIONAL PEAK IN UPC²!



Normal phase

Compounds with no

retention in RPLC

Compounds degrading

in H2O

Lipidomics

Lipids in food

stuffs

Orthogonal in

comparison

to C18

Cosmetics

Future scale-

up

Vitamins

Simplified

sample prep

Chiral = no-brainer

Structural Isomers


Screening on analytical SFC, purification on preparative SFC (eg Medicinal Chemistry, organic synthesis laboratories)

Best separation / conditions

During / after organic synthesis route

Screening on analytical SFC system

Chiral Achiral

Chiral

Columns

Modifiers

Achiral

Columns

Modifiers

UPC² - QDa UPC² - QDa

8-15 columns

Purification

Scale up to prep SFC for purification

Assess purity of obtained fractions

Prep SFC-MS 100 with QDa

Chiral Achiral



Screening on analytical SFC, purification on preparative SFC (eg Medicinal Chemistry, organic synthesis laboratories)

Best separation / conditions

During / after organic synthesis route

Screening on analytical SFC system

Chiral Achiral

Columns

Modifiers

Columns

Modifiers


8-15 columns

Purification

Waters ACQUITY Oven CM30-S

Up to 8 or 15 columns


When scaled up to preparative mode, SFC represents even higher advantages when

compared to the liquid phase variant.

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 0.14 0.16 0.18

0.00 Minutes

0.5 1.0

1.5 2.0 2.5 3.0

1

2

3

4 5

6

a)

Minutes 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0

b) 19 x 150 mm, 5 µm ~ 83 mL/min

3.0 x 50 mm, 1.7 µm 3.0 mL/min

10 stacked injections of Bucetin (2 enantiomers)

Increased productivity via stacked injection

Purification


Screening on analytical SFC, purification on preparative SFC (eg Medicinal Chemistry)

Advantages:

Less evaporation

Less man power

Lower solvent consumption

Greener

Less waste

Less costs

...

Purification



Normal phase

Compounds with no

retention in RPLC

Compounds degrading

in H2O

Lipidomics

Lipids in

food stuffs

Orthogonal in

comparison

to C18

Cosmetics

Future scale-up

Vitamins

Simplified

sample prep

Chiral = no-brainer Structural Isomers


TG LPE

LPC

PG PE

PC

IS1

IS2

CE

Chol

FA

1,3-DG

1,2-DG

MG LacCER GlcCER

CER

SM

16 lipid classes + IS1 + IS2

separated in 6 min Nonpolar lipids

Polar lipids

Lipidomics

UPC² - Synapt

Courtesy of Michal Holcapek and Miroslav Lisa, University of Pardubice, Czech Republic

SFC/MS OF BOTH POLAR AND NONPOLAR LIPID CLASSES IN BRAIN EXTRACTS

Easy MS coupling!



Normal phase

Compounds with no

retention in RPLC

Compounds degrading

in H2O

Lipidomics

Lipids in food

stuffs

Orthogonal in

comparison

to C18

Cosmetics

Future scale-up

Vitamins

Simplified

sample prep

Chiral = no-brainer

Structural Isomers

BUT NOT ONLY THESE ...


GC

LC

polarity

Log

MW

‘Bridging the gap between GC and LC’ ???

SFC

Which gap ?

Application gap No

Speed gap Yes

Detection gap Yes

‘Mildness’ gap Yes

Sustainability gap Yes

Hans-Gerd Janssen Unilever and professor @ University of Amsterdam, NL

Testimonial Hans-Gerd Janssen, Unilever


Compound coverage in SFC SFC does not have many unique applications.

Chiral separation is (the only?) one.

But SFC has many applications it can do ‘better’.

SFC is faster, SFC is greener, Fine tuning is easier, etc.

SFC can provide a frame of reference for GC:

Do these labile compounds degrade?

Are these adsorptive compounds lost?

SFC has a very broad compound coverage.

Hans-Gerd Janssen, Unilever


SFC: broad distribution; LC: three series; GC: three series

Food emulsifier by NPLC, NP-SFC and GC

E3

E7

E11

Time (min) 2.00 4.00 6.00 8.00 10.00 12.00 14.00

Time (min) 10.00 20.00 30.00

B

E3

E7

E11

SFC-MS

LC-MS

GC-MS

SFC-MS dot plots help in identifying what you see.

SFC-MS

m/z

Retention time

Hans-Gerd Janssen, Unilever


UPC2-MS

– complementary technique to RP-UHPLC-MS

– presently requires initial screening step

UPC2-MS a crucial tool in Open Access – increases chemistries amenable

to Open Access approach

As easy to use as RP-UHPLC-MS

SFC-MS Niche Application, Complementary Technique or a Tool Every Lab Needs?

JOHN LANGLEY, PROFESSOR AT UNIVERSITY OF SOUTHAMPTON


Reduce Solvent Costs

Green Chemistry

Orthogonal Separations

Increase Speed &

Throughput

Easier & Faster Chiral

Separations

Issues with Range of Polarity

Isomer separations

Simplify Workflow

CONCLUSIONS

SFC / UPC²


Thank You! Acknowledgements

• Davy Guillarme, Alexandre Grand-Guillaume Perrenoud, Jean-Luc Veutthey, University of Geneva, Switzerland

• Caroline West, University of Orléans, France

• David Clicq, UCB, Belgium

• Alex Brien, Victoria Coulthard, Reach Separations, UK

• John Langley, University of Southampton, UK

• Hans-Gerd Janssen, Unilever and University of Amsterdam, Netherlands

• Eric Mignolet, UCL, Belgium

• Michal Holcapek and Miroslav Lisa, University of Pardubice, Czech Republic • You for your attention and belief in the technology!

Documents

Short course SFC Fundamentals - Waters Corporation Users...©2015 Waters Corporation 3 Evolution of Separation Technology Gas Chromatography Liquid Chromatography GC Capillary GC New