supercritical fluid chromatography: past, present and future · supercritical fluid chromatography:...
TRANSCRIPT
... 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
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 - 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
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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
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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
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
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50
min0 0.5 1 1.5 2 2.5
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75
min0 0.5 1 1.5 2 2.5
mAU
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50
75
min0 0.5 1 1.5 2 2.5
mAU
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25
50
75
LUX C-1
LUX C-2
LUX C-3
LUX C-4
min0 0.5 1 1.5 2 2.5
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100
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75
LUX A-2
AD-3
AD-5
AS-5
Chiral – High Throughput
min0 0.5 1 1.5 2 2.5
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50
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LUX C-1
LUX C-2
LUX C-3
LUX C-4
min0 0.5 1 1.5 2 2.5
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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
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-2
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8
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mV
-4
-2
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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
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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
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2221
20
19
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9,11
10
8
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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
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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
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10
1
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5 6
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8
9
10
Theobromine
Theophylline
Cortisone
Prednisone
Hydrocortisone
Prednisolone
Sulfaquinoxaline
Sulfamerazine
For Research Use Only.Not for use in diagnostic procedures.
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
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min0 2 4 6 8
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A
B
C
D
1
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2
2
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3 4
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67
7
7 8
8
89
9
9UV 254 nm
UV 254 nm
ELSD
ELSD
120 bar FR
NO restriction
6x10
0.1
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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
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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
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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.