Supercritical Fluid

Chromatography:

Past, Present and Future

Pat Sandra

e-seminar November 16, 2011

... I love CO2 as mobile phase

Green

Better than NP-LC

Cheaper than LC

Faster than LC

Unique selectivity

Complementary to RP-LC & HILIC

Compatibility with detectors

MD (Semi)-Prep SFC

but ... Terry Berger in J. Chromatogr. A 1218 (2011) 2320

This new low noise level makes it possible to validate SFC methods for the first time

21st Century Triangle

Throughput

Productivity Resolution

21st Century Triangle

Throughput

Productivity Resolution

Supercritical Fluid

Chromatogtraphy:

Panacea or Pitfall ?

London 2009

Supercritical Fluid

Chromatography:

The Good, the Bad or the Ugly

?

Bruges 2010

Fundamentals

Past (1982 – 2008/2009)

Present (2009 – 2011)

Future

Content

T

SUPER-

CRITICAL

FLUID

P

SOLID LIQUID

GAS

Fundamentals - 1

Courtesy of Dr. Christopher Rayner, University of Leeds

NO phase transition

31.1°C

73.8 bar

Fundamentals - 2 N 0.5 a - 1 k Rs = 4 a 1 + k

N = L/2dp tR = L/u (k + 1) = N.2dp/u (k + 1)

Fundamentals - 3

Modifiers !s

Density

N

k

a

but also on

- Viscosity

- Solubility

Fundamentals - 4

NSFC = NHPLC

uopt SFC = up to 5 times > than uopt HPLC

This is only correct for pure CO2

uopt SFC = up to 2-3 times > than uopt HPLC

For CO2 with 5 to 40% MeOH

DP SFC << DP HPLC

Fundamentals - 5

1

1.5

2

2.5

3

3.5

4

4.5

0 0.5 1 1.5 2 2.5 3 3.5 4

h

SFC

HPLC

mL/min

0 0.08 0.17 0.25 0.34 0.42 0.50 0.58 0.66 cm/s

250 mm x 4.6 mm ID x 5 µm RX-Sil

Fundamentals - 5

Selectivity ?

Multi-mode technique from highly polar to apolar

solutes but in senso stricto NORMAL PHASE

Characterization of Stationary Phases for SFC with the Solvation Parameter Model

C. West & E. Lesellier in Advances in Chromatography 2010 Vol 48, 195-253

All NP chiral and achiral stationary phases

Dedicated SFC stationary phases 2-Et pyridine, triazol, ... DEAP, PPU, DNP (?)

Past 1982 – 2008/2009

1962: Klesper and Turner first used supercritical fluids as mobile phase in 1962 1982: 1st commercial SFC instrument introduced by HP 1986: Capillary SFC developed by Lee Scientific 1992: 2nd commercial SFC instrument introduced by HP HP G1205A SFC - 1995 1992 – 2000: R&D 2000 – 2008/2009: the SFC believers

Chiral separations – Generic method - 1993

h

h

h

3: X= COOCH3

6: X= CH2COOMe

9: X= COOCH3

13: X= CH2COOCH3

O

X

O

X

O

X

O

Y

z

O

X

O

X

1: X= COOCH3

4: X= CH2COOCH3

14: X= CH3

15: X= CH2CH3

7: X= COOCH3

10: X= CH2COOCH3

17: X= CH3

18: X= CH2CH3

2: X= COOCH3

5: X= CH2COOCH3

16: X= CH3

8: Y= CH2COOCH3, Z= H

11: Y= (CH2)2COOCH3, Z= H

12: Y= H, Z= (CH2)2COOCH3

19: Y= H, Z=CH2CH3

O

COOCH3

20

h

O

X

Y

21: X= CH2COOCH3, Y= H

22: X= H, Y= CH2COOCH3

}

C D

E F

A B

0 2 4 6 8 10 12

0

200

400

600

800

1000

1200

1400

1600

1800

2000

Re

sp

on

se

Time (min.)

0 2 4 6 8 10

-200

0

200

400

600

800

1000

1200

1400

1600

1800

Re

sp

on

se

Time (min.)

0 2 4 6 8 10

-200

0

200

400

600

800

1000

1200

Re

sp

on

se

Time (min.)

0 2 4 6 8 10

0

200

400

600

800

1000

Re

sp

on

se

Time (min.)

-2 0 2 4 6 8 10 12 14 16

-100

0

100

200

300

400

500

600

Re

sp

on

se

Time (min.)

0 5 10 15 20 25

0

500

1000

1500

2000

Re

sp

on

se

Time (min.)

Books

1988 Modern SFC Ed. Curt White

1988 SFC Ed. Roger Smith

1990 Analytical SFC & SFE Ed. Milton Lee & Karin Markides

1995 Packed Columns SFC Terry Berger

1998 SFC with Packed Columns Eds. Klaus Anton & Claire Berger

1999 Practical SFC & SFE Eds. Marcel Caude & Didier Thiebaut

…

… the good

Packed column SFC has characteristics which logically

make it superior to LC for most molecules that can be

solvated by SFs.

in Packed Column SFC by T.A. Berger – 1995

Why could SFC not keep its promises ?

… the bad and the ugly (from the cavilers, pettifoggers, etc.)

No fundamental developments

Instability in the SFC instrumentation market

… to difficult ... too many parameters

Features overemphasized

New developments e.g. in column manufacturing, suffered from

reproducibility issues, bleeding in MS, etc.

The detector issue UV .... ELSD, CAD, MS ?

Robustness … IQ/PV ? … from research to QA/QC … validation?

Application notes/publications considered competitive … but not at

all … contraproductive

Present 2009 - 2011

Desire of environmentally conscious techniques

Major instrument companies entered or re-entered

the SFC market

- Agilent Technologies

- Waters

- Jasco

- (Selerity Technologies)

Unique selectivity (NP-LC),

complementary to RP-LC and HILIC

Robustness at the GC and HPLC level

21st Century Triangle

Throughput

Productivity Resolution

Chiral Separations Generic conditions – High Productivity

Cellulose LUX-1 (4.6 x 250 mm, 3 µm),

SF = CO2, Modifier = MeOH with 0.1% TFA + 0.1% DEA isocratic at 30%,

Outlet Pressure = 150 bar, Flow Rate = 2.0 mL/min, Detection = UV 220 nm, Temperature = 50°C

R – 7

S – 7

R = 1.42

R = 1.37

min0 0.5 1 1.5 2 2.5

mAU

0

25

50

min0 0.5 1 1.5 2 2.5

mAU

0

25

50

75

min0 0.5 1 1.5 2 2.5

mAU

0

25

50

75

min0 0.5 1 1.5 2 2.5

mAU

0

25

50

75

LUX C-1

LUX C-2

LUX C-3

LUX C-4

min0 0.5 1 1.5 2 2.5

mAU

0

50

100

min0 0.5 1 1.5 2 2.5

mAU

0

20

40

60

min0 0.5 1 1.5 2 2.5

mAU

0

20

40

min0 0.5 1 1.5 2 2.5

mAU

0

25

50

75

LUX A-2

AD-3

AD-5

AS-5

Chiral – High Throughput

min0 0.5 1 1.5 2 2.5

mAU

0

25

50

min0 0.5 1 1.5 2 2.5

mAU

0

25

50

75

min0 0.5 1 1.5 2 2.5

mAU

0

25

50

75

min0 0.5 1 1.5 2 2.5

mAU

0

25

50

75

LUX C-1

LUX C-2

LUX C-3

LUX C-4

min0 0.5 1 1.5 2 2.5

mAU

0

50

100

min0 0.5 1 1.5 2 2.5

mAU

0

20

40

60

min0 0.5 1 1.5 2 2.5

mAU

0

20

40

min0 0.5 1 1.5 2 2.5

mAU

0

25

50

75

LUX A-2

AD-3

AD-5

AS-5

From 50 to 250 mm

50 to 250 mm

SFC-TOFMS – Sample pooling

2x10

0.250.50.75

+ EIC(250.0) Scan sfcquiral040.d

1

x10

2.55

7.5

+ EIC(247.0) Scan sfcquiral040.d

1

x10

2

4

6+ EIC(455.0) Scan sfcquiral040.d

1

x10

1

2

+ EIC(382.0) Scan sfcquiral040.d Smooth (1)

1

x10

0.51

1.5

- EIC(438.0) Scan sfcquiral043.d

1

2.55

7.5

- EIC(420.0) Scan sfcquiral043.d 1

min1 2 3 4

MS

Ab

und

ance

Alprenolol HCl

Mepivacaine HCl

Verapamil

Warfarin

trans-4-Cotinene carboxyline acid

Bendrofumethiazide

Blood/plasma Extraction LC-MS

Lipid profile

Level (g/mL)

Mean % Recovery % RSD

1.5 (0.01%)

109.6 11.7

7.5 (0.05%)

97.9 5.8

15.0 (0.1%)

94.0 3.2

Minutes2 4 6 8 10 12 14 16 18 20

mV

-4

-2

0

2

4

6

8

Minutes2 4 6 8 10 12 14 16 18 20

mV

-4

-2

0

2

4

6

8

Th

iou

re

a

PI-

X

Trace Analysis

Claudio Brunelli

Pfizer, Sandwich, UK

Generic method for achiral analysis

Stationary phase

- particle size Bare silica * 1.8, 3 and 5 µm

Column dimensions

- Length

- ID

5, 10, 15 and 25 cm

2, 3 and 4.6 mm

Mobile phase

- CO2

- Modifier

- Additive 1

- Additive 2

° more acidic conditions

° more basic conditions

1 to 5 mL/min

MeOH or EtOH

1 to 5% water

5 to 20 mM ammonium formate or acetate

Formic or acetic acid

Ammonium hydroxide

Restrictor 100 to 200 bar

Detection UV-DAD

MS (Q, QqQ, TOFMS, Q-TOFMS)

ELSD

CAD

* 2-Et pyridine, Diol, Cyano, Chiral columns, etc.

RX-SIL column - 250 mm x 4.6 mm x 5 µm; 40°C; Po 100 bar FLOW = 3 mL/min

Modifier = methanol/water 95/5 (v/v) + 10 mM ammonium formiate Modifier = 10 to 40% in 20 min

Alcohol ethoxylates CH3(CH2)mO(CH2CH2O)nH

0 2 4 6 8 10 12 14 16 18 20

0

20

40

60

80

100

120

140

160

12

20

22

13

6

1621

19

18

15,17

14

4,11

10

7

5

1,3

9

8

2

Alltima Silica

(4.6 x 250, 3 um)

Time (min)

0

20

40

60

80

100

120

140

160

2221

20

19

18

17

16

15

14

12,13

9,11

10

8

7

6

5

4

3

2

1

Princeton Pyridine

(4.6 x 250, 3 um)

AU

254

nm

1) Caffeine

2) Ibuprofen

3) Theophylline

4) Theobromine

5) Thymine

6) Adenine

7) Uracil

8) Fenoprofen

9) Flurbiprofen

10) Cortisone

11) Prednisone

12) Cytosine

13) Hypoxanthine

14) Hydrocortisone

15) Prednisolone

16) Sulfamerazine

17) Sulfamethoxazole

18) Sulfadimethioxime

19) Estriol

20) Sulfaguanidine

21) Sulfaquinoxaline

22) Sulfamethizole

Columns = 4.6 x 250 mm, 3 µm,

Flow Rate 2.5 mL/min,

Modifier = 5 to 30% in 20 min

Modifier = MeOH w. 20 mM NH4OOCH

Pressure = 100 bar,

Temperature = 40 °C

Et-pyridine

Silica

For Research Use Only.Not for use in diagnostic procedures.

RX-SIL column 50 mm x 4.6 mm x 1.8 µm; 40°C; Po 100 bar CO2 FLOW = 2 mL/min

Modifier = methanol/water 95/5 (v/v) + 10 mM ammonium formiate Modifier flow = 0.40 mL/min to 1.30 mL/min in 5 min

Triton X

High Resolution - Pesticides

Column = 4 x Zorbax RX-SIL (4.6 x 250 mm, 5 µm),

Flow rate = 2.0 mL/min, Outlet P = 150 bar

Modifier = MeOH with 20 mM HCOONH4 (2% H2O), 0 – 60 min 5 – 15% modifier.

Detection = 254 nm, Temp = 40°C,

P1

P2 P3 P4

P5

P

6

P

7

P

8

P

9

P10

P11 P12

P13

P14

,

P15

P16

P17

P18

P19

P20 P21

P22 P23

P24

P25

P26

P27

MSD CAD ELSD

BPR

UV Detector

Caloratherm 60 ° C

Heating Element

SS Capillary Tubing

0.12 x 105 mm SS Capillary Tubing

0.12 x 400 mm

LC Pump

Detectors

Column = Zorbax RX-SIL (4.6 x 250 mm, 5 µm), Flow Rate = 2.0 mL/min, Outlet P = 120 bar, SF = CO2, Modifier = MeOH with 20 mM NH4COOH (2% H2O), Modifier gradient = 0-20 min: 5-40%, Column T = 40°C, Make-up = MeOH with 2% H2O at 0.2 mL/min, Caloratherm = 60°C, Detection = UV (254 nm), ELSD (Evap = Neb = 35°C, smoothing = 3.0 s, PMT gain = 1, data rate = 40Hz, Flow Rate = 1.6 SLM).

min0 2 4 6 8 10 12 14 16 18

mAU

0

20

40

60

80

100

120

DAD1 A, Sig=254,4 Ref=360,100 (L:\SFC_AGI...520_ELSD\ORE11138_ELSD 2011-05-20 15-36-33\201105200000015.D)

min0 2 4 6 8 10 12 14 16 18

mV

10

15

20

25

30

ELS1 A, Voltage (L:\SFC_AGI...ORE11138_20110520_ELSD\ORE11138_ELSD 2011-05-20 15-36-33\201105200000015.D)

A) UV 254 nm

B) ELSD

1 2

3

4 5

6

7

8

9

10

1

2

3 4

5 6

7

8

9

10

Theobromine

Theophylline

Cortisone

Prednisone

Hydrocortisone

Prednisolone

Sulfaquinoxaline

Sulfamerazine

For Research Use Only.Not for use in diagnostic procedures.

UV

Single Quad TIC +

Single Quad TIC -

... a real drug discovery sample

Sensitive determination of impurities in achiral pharmaceuticals by

supercritical fluid chromatography using the Agilent 1260 Infinity Analytical

SFC System Publication Number 5990-6413EN

High resolution separations by supercritical fluid chromatography

using a coupled column approach with the Agilent 1260 Infinity

Analytical SFC System Publication Number 5990-6934EN

Comparison of UV detection limits between the Agilent 1260 Infinity

Analytical SFC System and an Agilent 1200 Series LC System

Publication Number 5990-9195EN

Chiral impurity analysis and enantiomeric excess determination

with the Agilent 1260 Infinity Analytical SFC System

Publication Number 5990-5969EN

Agilent 1260 Infinity SFC/MS Solution Publication Number 5990-7972EN

The future ...

CO2 is a super fluid as mobile phase

Super fluid chromatography?

SFC instrumentation

New analytical possiblities

New challenges in understanding

the mechanisms

...

A B

Column Zorbax RSiL (250 mm x 4.6 mm x 5 µm dp) - Flow 1 mL/min

Green HILIC

HILIC Ethanol/

Ammonium formate

20 mM (95/5)

HILIC Acetonitrile/

Ammonium formate

20 mM (95/5)

1. uracil, 2. thymine, 3. guanine, 4. cytosine, 5. adenine

A. Pereira, A.J. Giron, E. Admasu and P. Sandra, J. Sep. Sci. 33, 834-837 (2010)

For Research Use Only.Not for use in diagnostic procedures.

1.5 ml/min of CO2 2.5 ml/min of CO2

Column Zorbax RX SiL (250 mm x 4.6 mm x 5 µm dp) - Flow 1 mL/min

Green HILIC

HILIC Ethanol/

Ammonium formate

20 mM (95/5)

HILIC Acetonitrile/

Ammonium formate

20 mM (95/5)

1. uracil, 2. thymine, 3. guanine, 4. cytosine, 5. adenineFor Research Use Only.Not for use in diagnostic procedures.

Liquid CO2Check valve

Modifier

Column

Fixed restrictor

OVEN

Optional Heater

P. Sandra, K. Sandra, A. Pereira, G. Vanhoenacker and F. David, LC.GC Europe 23 (5), 242-259 (2010).

A. Pereira, F. David, G. Vanhoenacker, C. Brunelli and P. Sandra, LC.GC North America, November Issue (2011).

Modular simplified system

min0 2 4 6 8

mAU

0

100

200

300

min0 2 4 6 8

mV

0

100

200

300

min0 2 4 6 8

mAU

500

540

580

min0 2 4 6 8

mV

0

100

200

300

A

B

C

D

1

2

2

2

3

3

3 4

4

4 5

5

5 6

6

67

7

7 8

8

89

9

9UV 254 nm

UV 254 nm

ELSD

ELSD

120 bar FR

NO restriction

6x10

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

+APCI TIC Scan Frag=225.0V SFC501.8-006.d

-2

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

DAD1 - A:Sig=254,16 Ref=360,16 SFC501.8-006.d

Acquisition Time (min)

0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95

mA

UC

ou

nts

1

2

2

3

3

4

45

5

6

6

7

7 8

89

9

363.1

303.1

195.0

A

B

A) APCI - positive mode, B) UV at 254 nM

Column RX Sil 50 mm x 4.6 mm x 1.8 µm at 40°C.

CO2 Flow 3.7 mL/min. Modifier methanol at 0.7 mL/min

NO restrictor but outlet pressure at ~ 80 Bar

1) Acetone

2) Theophylline

3) Caffeine

4) Cortisone

5) Prednisone

6) Hydrocortisone,

7) Prednisolone

8) Sulfaquinoxaline

9) Sulfamerazine

For Research Use Only.Not for use in diagnostic procedures.

Alberto Pereira

Frank David

Melissa Dunkle

Gerd Vanhoenacker

Claudio Brunelli

Roman Szucs

William Farrell

Selerity Technologies (Jody Clark)

The SFC believers

You for your attention