fundamentals and advancements in analytical sfc ·

©2012 Waters Corporation 1

Acquity UPSFC

Granada, 23 Febrero 2012

Alberto Méndez Applications Specialist

[email protected]


Remember Smaller Particles - Enabler of Productivity


SFC Column Efficiency Curves

2.5 µm XBridge™ HILIC, 3.0x50 mm



1.7 µm ACQUITY BEH HILIC, 3.0x50 mm

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


Resolution Comparison

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

AU

0.00

0.05

0.10

0.15

0.20

0.25

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

1.7 µm

5 µm 3.0x100 mm Column 2 mL/minute CO2/Methanol Gradient


Effect of System Volume

0.46

4

0.51

3

0.62

0

0.64

6

0.70

0

0.82

6

0.85

50.

870

0.90

9

0.94

3

1.03

9

1.07

3

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.47

1

0.53

9

0.65

20.

670

0.89

8

0.92

60.

946

0.98

5

1.01

9

AU

0.00

0.10

0.20

0.30

0.40

Minutes0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10

3.0x100 mm, 1.7 µm, at 3 mL/minute, CO2/MeOH gradient

non-optimized

optimized


Effect of System Volume

0.62

0

0.64

6

AU

0.00

0.02

0.04

0.06

0.65

2

0.67

0

AU

-0.010

0.000

0.010

0.020

0.030

0.040

0.050

Minutes0.590 0.600 0.610 0.620 0.630 0.640 0.650 0.660 0.670 0.680 0.690

non-optimized

optimized


ACQUITY UPSFC™ Design Objectives

Develop an Analytical SFC System based upon proven UPLC® technology

Purposeful Innovation: – Peltier cooling of BSM pump heads – Accurate, linear and precise injections – CO2 management and pressure control – Reduced system volume – Detection sensitivity

Meaningful Impact: – Total solution – Reduced Chiral/Achiral separation times – Reduced cost per injection – System validation


ACQUITY UPSFC™ Design Objectives

Column Manager CM-A

SFC PDA Detector

SFC Manager

Sample Manager - FL

SFC BSM Pump


18 Compounds Mixture 6 Overlaid Injections

3-B

enzo

ylpy

ridin

e - 1

.187

Cho

leca

lcife

rol -

1.4

77

6-H

ydro

xyfla

vano

ne -

1.76

6

4-N

itroa

nilin

e - 2

.179

Caf

fein

e - 2

.308

Theo

phyl

line

- 2.7

70

Car

bam

azep

ine

- 3.3

89

Est

radi

ol -

3.59

4 4,4'

-Bip

heno

l - 3

.695

Cor

tison

e - 4

.285

Pre

dnis

one

- 4.5

92

Ace

tam

inop

hen

- 4.8

56

Sul

fisox

azol

e - 5

.014

Hyd

roco

rtiso

ne -

5.29

2

Sul

fam

ethi

zole

- 5.

487

Pre

dnis

olon

e - 5

.647

Sul

faph

enaz

ole

- 5.8

15

4-A

min

ophe

nyl S

ulfo

ne -

7.18

5

AU

0.00

0.02

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

0.32

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Consistent peak widths High resolution <0.4% RSD in RT

2.1 x 150 mm Viridis Hybrid 1.7 µm @ 1.20 mL/min @ 45 °C A=Carbon Dioxide B=Methanol UV 230 nm (compensated 400-500 nm) 2 µL injection Gradient 2% B to 16% B over 7 minutes


Pea

k1 -

2.53

9

AU

0.00

0.02

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

0.32

0.34

0.36

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00

1.5% MeOH

Pea

k1 -

4.44

1

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00

Pea

k1 -

1.4

46

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00

2.5% MeOH

Pea

k1 -

1.80

6

AU

0.00

0.02

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

0.32

0.34

0.36

0.38

0.40

0.42

0.44

0.46

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00

2.0% MeOH

Mobile phase strength is a non-linear function of modifier concentration

The first small additions of modifier have a large effect on retention

1.0% MeOH

Precision at Low % Co-Solvent

Viridis Hybrid 2.1x150 mm @ 1 mL/min


1-Couma

rin - 0.8

40

2-Flavon

e - 1.116

3-Caffein

e - 1.247

4-Thymin

e - 1.584

5-Papave

rine - 1.6

64

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

Minutes0.00 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

1-Coumar

in - 0.788

2-Flavone

- 1.072

3-Caffeine

- 1.209

4-Thymin

e - 1.559

5-Papave

rine - 1.64

1

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

Minutes0.00 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

Gradient Performance

RT %RSD: <0.15 Area %RSD: <0.67

1-20% B back to 1% B

RT %RSD: <0.34 Area %RSD: <0.69

1.5-20% B back to 1.5% B

Viridis® BEH 2-Ethylpyridine, 3 x 100 mm, 1.7 µm 2.00 mL/min, 55 °C, 1958 psi (135 Bar) Injection Volume = 3 µL, 10 repeated injections PDA 280 nm compensated (500-600 nm) 20 Hz, TC = 0.2 sec


Linearity - 1-10µL injections AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

0.100

0.110

0.120

0.130

0.140

0.150

0.160

Minutes1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60

Peak 1 - Coumarin Peak 2 - Flavone Peak 3 - Caffeine Peak 4 - Thymine Peak 5 - Papaverine

Column 4.6 x 150 mm Viridis Silica 2-EP, 5 um; Flow Rate : 3.00 mL/min; Column Temp = 55°C,A=CO2 B=Methanol 5-20% B Grad over 5 min followed by a return to 5% B over 1 min; ABPR = 1958 psi (135 Bar) PDA 280 nm compensated (500-600 nm) 20 Hz, TC = 0.2 sec


Linearity - 1-10µL injections

Coumarin: y = 43495x - 4326.9 R² = 0.9999 Flavone: y = 28758x - 2908 R² = 0.9999

Caffeine: y = 52213x - 5212.3 R² = 0.9999

Papaverine: y = 14608x - 2097.1 R² = 0.9994 Thymine: y = 26624x - 2774.5 R² = 0.9999

0

100000

200000

300000

400000

500000

600000

0 2 4 6 8 10

Are

a C

ou

nts

uL Injected

Coumarin

Flavone

Caffeine

Thymine

Papaverine

Linear (Coumarin)

Linear (Flavone)

Linear (Caffeine)

Linear (Thymine)

Linear (Papaverine)


ABPR Performance: “FINE” Control

0.54

8

1.24

3

2.85

9

4.65

6

0.55

3

1.25

9

2.90

6

4.73

4

0.55

1

1.25

2

2.88

4

4.70

0

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 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

ABPR @ 1980 psi ABPR @ 2000 psi ABPR @ 2020 psi


(Cathode)

(Anode)

(Glass board)

Organic Light Emitting Diode (OLED))

OLED Separation and Impurity Profiling


OLED Degradation Investigation with LC/UV/MS

TCTA

Flrpic

α-NHP

BAlq

Flrpic Structural Isomers

Normal Phase LC Separation


Standards Flrpic

1.01

9

1.36

0

1.46

3

1.61

9

1.93

5

2.74

6

3.62

2

4.12

4

AU

0.00

0.03

0.06

0.09

0.12

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Flrpic

N

FF

N

F

F

Ir

N

F

F

N

F

F

Ir

N

F

F

N

F

F

Flrpic Isomer

N

F

Ir

N

F

F

N

F

F

Flrpic -F

N

F

Ir

N

F

N

F

F

Flrpic -2F

Flrpic -3F

N

F

Ir

N

F

N

F


Standards and Spectra Flrpic and MS ES+ m/z Values

1.00

3

1.34

51.

448

1.60

01.

643

1.92

0

2.73

0

3.60

4

4.10

2

Inte

nsity

1.8x107

3.6x107

5.4x107

7.2x107

9.0x107

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

1.345 Peak 2 - SQ 1: MS Scan 1: 300.00-1000.00 ES+, Centroid, CV=Tune

743.93

745.90

Inte

nsity

0.0

9.0x105

1.8x106

2.7x106

3.6x106

m/z734.40 737.10 739.80 742.50 745.20 747.90 750.60 753.30 756.00 758.70


743.97

745.91

Inte

nsity

0.0

1.2x106

2.4x106

3.6x106

4.8x106

m/z738.00 740.00 742.00 744.00 746.00 748.00 750.00 752.00 754.00


743.90

745.90

746.97

Inte

nsity

0

1x106

2x106

3x106

4x106

m/z739.20 741.30 743.40 745.50 747.60 749.70 751.80 753.90 756.00


761.97

763.93

765.08

Inte

nsity

0.0

7.0x106

1.4x107

2.1x107

2.8x107

m/z757.80 759.60 761.40 763.20 765.00 766.80 768.60 770.40 772.20


743.96

745.95

747.05

Inte

nsity

0.0

6.0x106

1.2x107

1.8x107

2.4x107

m/z738.00 740.00 742.00 744.00 746.00 748.00 750.00 752.00 754.00 756.00


725.97

727.94

729.01

Inte

nsity

0.0

1.6x106

3.2x106

4.8x106

6.4x106

m/z722.40 723.80 725.20 726.60 728.00 729.40 730.80 732.20 733.60 735.00


804.01

805.99

806.96

Inte

nsity

0.0

1.2x106

2.4x106

3.6x106

4.8x106

m/z802.80 803.70 804.60 805.50 806.40 807.30 808.20 809.10 810.00


761.95

763.92

765.01

Inte

nsity

0.0

7.0x106

1.4x107

2.1x107

2.8x107

m/z759.60 760.80 762.00 763.20 764.40 765.60 766.80 768.00 769.20

m/z=763.9 m/z=763.9 m/z=746.0 m/z=727.9 m/z=706.0

m/z=745.9 m/z=745.9 m/z=763.9

ES+


Comparison MS/UV Flrpic Mixture

1.01

9

1.36

0

1.46

3

1.61

9

1.93

5

2.74

6

3.62

2

4.12

5

AU

0.00

0.06

0.12

0.18

0.24

1.00

3

1.34

5

1.44

8

1.59

8

1.92

0

2.73

0

3.60

4

4.10

2Inte

nsity

1.8x107

3.6x107

5.4x107

7.2x107

9.0x107

1.91

9

2.73

1

4.10

4Inte

nsity

0.0

6.0x106

1.2x107

1.8x107

2.4x107

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

Conditions: 3.0 x 100mm Viridis BEH 2-EP, 1.7µm; OLED Mixture prepared at 0.1mg/mL in THF; Inj. Volume:2 µL; Temperature:60°C; ABPR: 130 bars; Flow: 2mL/min, CO2, MeOH (ammonium formate) 10 to 25% gradient


Major Constituents in OLED Mixture

1.00

9

1.42

1

1.92

0

2.02

8

2.47

1

AU

0.000

0.025

0.050

0.075

0.100

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

Flrpic Area = 0.4% Rt = 0.1%

TCTA Area = 1.1% Rt = 0.1%

Balq (a) Area = 1.1% Rt = 0.1%

α-NHP Area = 0.4% Rt = 0.1%

*Minor impurities of 1% or lower yielded values typical of impurity analysis: Area %RSD < 5% Rt %RSD < 0.2%


There are hundreds of FSV formulations on the market

Vitamin A Only

Vitamins A + D

Vitamin E

Vitamin K

Vitamins K + D

Vitamin D Only

Fat Soluble Vitamins


1.20

71.

304

Retin

yl A

ceta

te -

1.79

71.

938

2.44

0

3.21

8Re

tinol

Pal

mita

te -

3.35

4

Retin

ol -

3.82

6

4.64

4

5.80

7

6.37

6

AU

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

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Vitamin A Forms


0.29

7 0.41

1

0.79

6

0.94

3

1.15

51.

207

1.30

4

1.51

61.

586 1.

797

1.93

8

2.15

3 2.44

0

2.88

6

3.02

6

3.21

83.

268

4.05

54.

144

4.39

14.

493

4.64

44.

701

4.93

75.

064

5.45

8

5.67

9

5.80

7

6.01

0

6.17

7

6.37

6

6.69

3

AU

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

0.055

0.060

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50

Vitamin A Forms (Zoom)


AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Fat Soluble Vitamins

Red - MS TIC (ESi+) Blue - UV at 317 nm Black - UV at 262 nm


Chiral Method Development with MS

Speeding up chiral method development can be achieved using Mass Spectrometry

Thanks to the selectivity of MS detection, several chiral mixtures can be injected together and the chiral resolution is confirmed using the MS data


IC, 4.6 x 150mm, 5 µm 3 ml/min CO2 / Methanol 5-45% - 4 min 45% - 1 min 45-5% - 0.5 min 5% - 0.5 min 40 °C, 120 bar Oxfendazole & Pantoprazole sodium hydrate (0.2 mg/mL)

Chiral Method Development with MS


Conclusions

SFC is a highly orthogonal analytical tool, compatible with many detection techniques, and applicable in any field

Technique of choice for normal phase methods replacement – uses up to 80% less solvent, low solvent cost and disposal

UPSFC™ , designed with proven UPLC® technology, provides measurable increases in productivity for UV, MS and ELSD based work flows

UPSFC™ capability with LC familiarity

Complete solution for SFC analysis including chemistry, software, service, and support


Thank You for Your Attention and…

Many thanks to: -Michael D Jones -Andy Aubin -Magdalena Abad -Rui Chen -Stéphane Dubant -Isabelle Durieux

fundamentals and advancements in analytical sfc ·

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