Let’s Get CRITICAL, Supercritical Fluid
Extraction (SFE)
Giorgis Isaac, PhD
Principal Scientist
Giorgis_isaac@waters.com
©2015 Waters Corporation
1
SFE Outline
 Sample Preparation Challenges & Extraction Techniques
 MV-10 ASFE System Introduction & Benefits
 SFE Applications
©2015 Waters Corporation
2
A Fully Supercritical Fluid Process
 No treatment between Extraction, Separation and Isolation
SFE Extraction
Extraction Analysis and
scale-up prediction
Final purity
assessment
©2015 Waters Corporation
Preparative SFC for Purification
3
Sample Preparation Challenges
Sample Preparation
 Sample preparation:
– Often overlooked
– Least developed
– Most time-intensive
– Most error-prone
©2015 Waters Corporation
4
Classical Extraction Methods
Evaporation
Soxhlet Extraction
Distillation
©2015 Waters Corporation
5
Traditional Extraction Methods
Drawbacks
 Limited selectivity
 Thermal degradation of heat-labile compounds
 Oxidative degradation of highly unsaturated compounds
 Organic toxic solvents
– Residual solvents
– Government regulations on the use of organic solvent such as
hexane
©2015 Waters Corporation
6
SFE Outline
 Sample Preparation Challenges & Extraction Techniques
 MV-10 ASFE System Introduction & Benefits
 SFE Applications
©2015 Waters Corporation
7
What is a Supercritical Fluid?
Supercritical fluid has High Diffusivity, Low Viscosity and
Low Surface Tension!
73.8
304.1 K
©2015 Waters Corporation
8
Advantages of SFE
 Low temperature extraction conditions
– Minimal degradation of thermo-labile molecules
 Highly selective
 Solvent power can be varied by control of pressure and temperature
 Low viscosity aids rapid extraction
 Negligible surface tension
 Utilization of non-toxic solvent
– No toxic residue
 Isolation of extracted analytes from extraction medium is readily
accomplished by pressure reduction
©2015 Waters Corporation
9
MV-10 ASFE System Components
Backpressure
Regulator(BPR)
Heat Exchanger
Up to 6 Co-Solvents
Available
Fluid Delivery
Module
Column Oven -up to 10
Extraction Vessels
©2015 Waters Corporation
Fraction Collection Moduleup to 12 collection bottles
(5, 10 or 25 mL)
10
Extraction Modes
 Dynamic – (e.g. coffee maker) continuous supply of fresh fluid
passes over/through the matrix/analyte
– Fluid contamination builds up at the trap
– Volatiles may be blown from the trap
 Static – (e.g. tea cup) fixed amount of fluid is exposed to the
matrix/analyte – mixing by diffusion/re-circulation
– Extraction may not be exhaustive
 Static / Dynamic Combination (Most Popular)
– Pressurize analyte/matrix with fresh fluid for period of time
followed by continuous flow of fresh fluid over analyte/matrix.
©2015 Waters Corporation
11
Extractability Based on Polarity
Increasing Polarity
Nonpolars
Alkanes
Ethers
Esters
Alcohols
Amides
Acids
Amines
Highly polar organics
Inorganic
ions
SFE
Neat CO2
CO2 + modifier
CO2 + modifier + ternary additives
CO2 + modifier + ternary additives + water
Liquid – based extraction methods
Small molecules
Peptides
Large proteins
Increasing Molecular Weight
 One of the largest advantages of SFE: Selectivity
©2015 Waters Corporation
12
Supercritical Fluid Extraction:
Effect of Increasing Density of CO2
100 Bar
200 Bar
3 extract’s CO2 & 1% MEOH
@
100, 200 & 350 bar
Isolated compound
of interest
Isolated compound
of interest
300 Bar
Isolated compound
of interest
©2015 Waters Corporation
13
Effect of extraction T and P on γ–tocopherol yeild
©2015 Waters Corporation
14
Control of Tunable Extraction Parameters
Critical to Optimizing and Reproducibility
CO2 tunable parameters and polarity for selectivity
©2015 Waters Corporation
15
SFE Outline
 Sample Preparation Challenges & Extraction Techniques
 MV-10 ASFE System Introduction & Benefits
 SFE Applications
©2015 Waters Corporation
16
Example 1: Omega-3 From Marine
Origines
 SFE Step
– 90 –150 bar, max 45 °C
– Remove pesticides & heavy metals
– Separate <C18 from >C18 (mainly
saturated and mono-unsaturated FA)
– Remove cholesterol
Fish Oil
EPA
Saturated
Monounsaturated
Omega-6
DHA
 SFC Step
– 90 –150 bar, max 45 °C
– Selectivity according to C-chain length
AND to number of double bonds
– Highly purified concentrates up to
over 99% per individual FA
 Omega-3 FA are handled under CO2
atmosphere at temperatures below
45 C.
Concentrated
Omega-3
Saturated
Monounsaturated
Omega-6
EPA
DHA
– No thermal stress
– No oxidation
©2015 Waters Corporation
Commercial process - Patented
17
Example 2: Stevia Extraction
 Stevioside isolated from Stevia rebaudiana has been proposed
as a promising sweetener because of its low calorie content and
relatively low toxicity
– Stevioside and rebaudioside A have about 300 times the relative
sweetness intensity of 0.4% (w/v) sucrose
 2 major diterpene glycosides
– stevioside (5–18%)
– rebaudioside A (2–4%)
©2015 Waters Corporation
18
Stevioside Extraction
 Conventional extraction methods for stevioside involve
– aqueous or alcohol extraction
– precipitation and coagulation with filtration
– clean-up step
– crystallization and drying
©2015 Waters Corporation
Choi, Y. H. et. al, Chromatographia, 55, 716-620, 2002.
19
Example 3: SFE Increases Specificity
 Using Super Critical Fluid as an extraction solvent provides a
mechanism to increase specificity
– By varying the extraction conditions, we can reduce the amount of
unwanted interference compounds
o
Less compound interference, more column loading capacity
(A) SFE
%
Target, m/z= 391, 1.61e7
SFE Extract
0
0.50
1.00
1.50
2.00
2.50
3.00
3.50
(B) Solvent Extraction
Target
m/z=391, 1.68e7
%
Solvent Extract
0
0.50
©2015 Waters Corporation
4.00
1.00
1.50
2.00
2.50
3.00
3.50
Time
4.00
20
Example 4: Selective Extraction of Ingenol
from Euphorbia Plant
Selectivity is key to efficient sample preparation
%
Residual Sample after SFE
0
2.00
4.00
6.00
8.00
10.00
12.00
14.00
%
SFE Extract
0
2.00
4.00
6.00
8.00
10.00
12.00
14.00
%
Solvent Extract
1
2.00
4.00
6.00
8.00
10.00
12.00
14.00
%
Ingenol Standard
0
2.00
©2015 Waters Corporation
4.00
6.00
8.00
10.00
12.00
Time
14.00
21
Summary
 SFE provides an appealing sample preparation technique which:
– Improves extraction efficiency and reduces extraction time vs. other
sample preparation techniques
– Reduces costly and hazardous solvent consumption
– Is environmentally compatible
– Does not require pre-concentration prior to analysis
– Automated
– Can selectively extract specific fractions of a complex sample
– Operates at lower temperatures than PFE, MAE and soxhlet
– Wider selectivity range with use of co-solvents
 SFE simplified for the end user: MV10-ASFE
©2015 Waters Corporation
22
Let’s Get CRITICAL, Supercritical Fluid
Chromatography (SFC)
Introduction to ACQUITY UPC2
Giorgis Isaac, PhD
Principal Scientist
Giorgis_isaac@waters.com
©2015 Waters Corporation
23
A Fully Supercritical Fluid Process
 No treatment between Extraction, Separation and Isolation
SFE Extraction
Extraction Analysis and
scale-up prediction
Final purity
assessment
©2015 Waters Corporation
Preparative SFC for Purification
24
UltraPerformance Convergence Chromatography
(UPC2)TM
Convergence Chromatography is a category of separation science that
provides orthogonal and increased separation power, compared to liquid or
gas chromatography, to solve separation challenges.
UltraPerformance Convergence Chromatography [UPC2]TM is a
holistically designed chromatographic system that utilizes liquid CO2 as a
mobile phase with one or more co-solvents to leverage the chromatographic
principles and selectivity of normal phase chromatography.
The ACQUITY UPC2 System is built utilizing proven UPLC Technology to
enable scientists to address routine and complex separation challenges while
delivering reliability, robustness, sensitivity and throughput.
Accepted in the scientific community as:
UHPSFC: UltraHigh Performance Supercritical Fluid Chromatography
SFC: Supercritical Fluid Chromatography (NOT Science Fiction
Chromatography)
©2015 Waters Corporation
25
What Does Supercritical Fluid Mean To The
Chromatographer?
 Lower viscosity means higher optimal flow rates
– About 4 times higher than LC
 Higher flow rates means
– Faster equilibration times
– Faster transit time on column
– Lower operating pressures allows for the ability to
work with multiple columns in series
 Adjusting pressure adjusts solvating strength
– In LC, we adjust solvent composition and temperature
– In SFC pressure adjustment provides an additional
variable to work with
©2015 Waters Corporation
26
Evolution of Separation Technology
Gas Chromatography
Liquid Chromatography
Convergence Chromatography
GC
HPLC
SFC
Capillary GC
UPLC
UPC2
Resolution, Sensitivity, Throughput
©2015 Waters Corporation
27
How the ACQUITY UPC2 System Works
Splitter
PDA detector
Column Manager
Make-up
Pump
Mass Spec
Auxiliary
Inject valve
Back Pressure Regulator
(Dynamic and Static)
Inject
valve
mixer
Thermo-electric
heat exchanger
Waste
Modifier
©2015 Waters Corporation
CO2
Supply
CO2
Pump
Modifier
Pump
28
Example 1: Separating Polar Compounds
(Catalpol/stachyose/sucrose/D-mannitol/yellow glucoside)
RPLC
UPC2
DH_Neg_20eV_RP1
1: TOF MS ESTIC
1.49e6
0.74
1.5x107
%
Rehmannia extract
(the polar compounds
elute near solvent front)
0.68
3x106
5.25x107
0
-3.26
EIC, 181
-4.61
EIC, 361
-4.49
EIC, 341
Time
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
DH_Neg_20eV_RP1 80 (0.737) Cm (18:126)
1: TOF MS ES2.11e6
341.11
100
4.5x106
-6.45
EIC, 685
665.21
3x107
-6.64
EIC, 665
%
711.22
503.16
ES-, TIC
549.17
387.11
666.22
290.09
128.04
113.00
133.02
89.03
181.07 191.02
343.12
195.02
245.10
0
50
308.10
361.11
3.0x107
712.22
342.11
407.12
470.15
455.10
425.10
504.16
471.12 505.16
632.20
683.22
827.26
845.27
550.17
713.22
633.18
551.16 617.15
873.27
794.25
779.20
874.27
956.31 974.34
m/z
100
150
200
250
©2015 Waters Corporation
300
350
400
450
500
550
600
650
700
750
800
850
900
950
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
Minutes
29
Example 2: Fat Soluble Compounds
Lycopene and -Carotene
HPLC
0.008
UHPSFC
0.006
-carotene
LogP=10.68
Lycopene
LogP=11.11
60 min
L. Zhang et al. / Food Chemistry 132 (2012) 2112–2117
AU
0.004
UPLC
0.002
0.000
-0.002
0.00
0.20
0.40
0.60
0.80
Minutes
1.00
1.20
1.40
1.5 min
H. Li et al. / Food Chemistry 132 (2012) 508–517
©2015 Waters Corporation
16 min
30
Example 3: Chiral Separations
2.47
4.25
4.0
2.90
3.75
3.5
3.25
3.0
2.75
2.5
2.25
2.0
AU
1.75
1.5
1.25
1.0
7.5e-1
5.0e-1
2.5e-1
0.85
-9.375e-8
-2.5e-1
-5.0e-1
-7.5e-1
-1.0
-1.25
0.00
0.50
1.00
1.50
60 min
R, S-Goitrin from
Isatis Indigotica Fort
(板蓝根)
(S)
O
(R)
Goitrin (S-goitrin)
2.50
3.00
3.50
4.00
4.50
5.00
5.50
Time
6.00
6 min
O
S
S
N
H
2.00
N
H
Epigoitrin (R-goitrin)
“手性高效液相色谱法测定板蓝根中表告依春和告依春含量”, 林瑞超,
2010, Chinese Journal of Chromatography, 28 (10), 1001-04
©2015 Waters Corporation
31
Taking Advantage of Low Viscosity
(A) 150 mm
750.000
LSU
 Coupling columns in series is one of
the benefits of working with low
viscosity solvents
500.000
250.000
0.000
0.00
5.00
10.00
15.00
20.00
25.00
(B) 250 mm
LSU
600.000
400.000
200.000
0.000
0.00
5.00
10.00
15.00
20.00
LSU
600.000
25.00
(B) 400 mm
400.000
200.000
0.000
0.00
5.00
10.00
15.00
20.00
Time
25.00
Table 1. Comparison of three SFC runs with different column lengths.
Column Length
t1 (min)
t2 (min)
w0.5,1
w0.5,2
150
7.14
7.95
0.34
0.39
250
11.21
12.54
0.44
400
18.20
20.33
0.55
(mm)
Isomeric ratio
Theor.
*Rs
Increase
0.62
1.31
0
0
0.48
0.64
1.71
31%
29%
0.58
0.64
2.23
70%
63%
(peak1/peak2)
Increase
*, where t is the retention time and w0.5 is the peak width at half height
©2015 Waters Corporation
32
Example 4: Analysis of Volatile Compounds from TCM
 Target analytes are lipophilic α/β cis/trans isomers
 Currently separate by GC, 29 minutes
– Difficult sample prep: need derivitization
– can not be scaled up for purification
©2015 Waters Corporation
33
UPC2 Analysis of Volatile Compounds from TCM
Overlay of 6
replicates
 UPC2 analysis for
the isomers
组分1
– Analysis time 2.5
minutes
254nm
RSD<1%
– Direct analysis,
easy sample prep
组分2
– Easy scale up to
Prep
2.5min
©2015 Waters Corporation
34
Example 5: UPC2 Analysis of Cannabinoids
 UPC2 and UPLC chromatograms of a mixture of 10 cannabinoid
standards
UPLC
UPC2
UPC2
©2015 Waters Corporation
CBDV
CBDA
CDG
CBD
THCV
CBN
THC
CBC
THCA
CDBV
CBD
d8THC
d9THC
CBC
CBN
CBG
THCA
CBDA
CBGA
35
Example 6: UPC2 Analysis of Lipid
 Liquid-liquid extraction using chloroform/MeOH
– Folch method / Blight and Dyer method
 RPLC & HILIC
– Phase transfer required to be able to inject onto RP and HILIC system
 UPC2
– Phase transfer process can be eliminated by injecting the organic
extract directly onto the UPC2 system
©2015 Waters Corporation
36
Lipid Analysis Work Flow GC, LC and UPC2
Sample




GC
Sample
UPC2
• Extraction
• FAMEs Derivitization
• Extraction
• Ready for GC/MS analysis
• Direct UPC2 analysis
Free fatty acids are typically
derivatized to form Fatty Acid
Methyl Esters (FAMEs)~ 1hr
Risk of rearrangement of the FA
and contamination
Low volatile very long chain fatty
acids (>24 carbon atoms) are
difficult to analyze
Analysis time ~30 min
©2015 Waters Corporation



Organic extract can be injected
directly to the system
Single methodology to separate
complex inter and intra lipid class
Faster baseline separation of
lipids based on chain length and
number of double bonds ~ 5min
Sample
UPLC
• Extraction
• Evaporate to dryness
• Reconstitution
• Ready for UPLC analysis

Analyzed by both HILIC & RP

HILIC separates lipid classes by
polar head group

Phase transfer required before
injection

RP separates based on acyl chain
length and number of double
bonds ~ 20min
37
Fast and Simple Free Fatty Acid Analysis
Using UPC2
283.26 311.30
100
367.36
20 to 25 % B
%
227.20
199.17
197.81
0
0.50
1.00
1.50
283.26
100
311.29
2.00
2.50
3.00
3.50
4.00
367.36
15 to 25 % B
%
227.20
197.81
199.17
0
0.50
1.00
1.50
16:0
100
18:0
283.26
2.00
20:0
311.30
2.50
22:0
339.33
%
10:0
3.00
3.50
24:0
5 to 25 % B
14:0
227.20
12:0
197.81
199.17
0
0.50
1.00
1.50
2.00
2.50
3.00
3.50
311.29
100
283.26
227.20
0
0.50
©2015 Waters Corporation
1.00
1.50
2.00
2.50
3.00
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
367.36
255.23
197.81 171.14 199.17
m/z
4.00
339.33
1 to 25 % B
Peak
4.00
367.36
255.23
%
UPC2 Conditions:
A= CO2
B=MeOH in 0.1% FA
Column= ACQUITY UPC2 HSS C18
SB 1.8µm (2.1 x 150 mm)
Flow rate= 0.6 mL/min
Column temp= 50 ºC
P=1600 psi
3.50
Time
4.00
38
Separation of FFA C8-C36 from Algae Extract
16:0
255.23
100
FFA C8-C36
x16
465.47
32:0
479.48
%
283.27
23:0
395.39
227.20
423.42
367.36
299.20
479.45
451.45
311.30
0
-0.00
0.20
0.40
0.60
0.80
1.00
1.20
34:0
507.48
1.40
1.60
1.80
455.24
Time
2.00
2 min
©2015 Waters Corporation
39
UHPSFC/MS of Both Polar and Non-Polar Lipid
Classes in 6 min
Miroslav Lísa and Michal Holčapek ; Anal. Chem., 2015, 87 (14), pp 7187–7195
©2015 Waters Corporation
40
A Fully Supercritical Fluid Process
 No treatment between Extraction, Separation and Isolation
SFE Extraction
Extraction Analysis and
scale-up prediction
Final purity
assessment
©2015 Waters Corporation
Preparative SFC for Purification
41
Example 7: Extraction, Separation and
Isolation of Schisandra Berry Extracts Using
SFE and SFC
 Berries of schisandra (Schisandra chinensis) have been used for
medicinal purposes in TCM
– Extraction: MV 10- ASFE
– Separation: UPC2
– Scale up: Prep 100q SFC System
©2015 Waters Corporation
42
Extraction, Separation and Isolation of
Schisandra Berry Extracts using SFE and SFC
SFE
UPC2 separation of crude SFE extract
UPC2/PDA 220
UPC2/PDA 220 separation of collected fraction
©2015 Waters Corporation
UPC2/QDa
Prep SFC separation of crude SFE extract
43
Extraction, Separation and Purification
Simplifies your Sample Complexity
1: Scan AP+
TIC
4.10e7
%
G4_SolE_002
Extraction
0
Time
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
1: Scan AP+
TIC
6.65e7
%
G4_MPLC_023
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
Time
4.00
1: Scan ES+
391
1.23e8
%
100
Simpler
2
G4_2_25_2012_006
Isolation
0
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
Time
4.00
Standard
©2015 Waters Corporation
44
Summary UHPSFC (UPC2)
 UPC² SIMPLIFIES the workflow
– Combines multiple techniques (GC/NP/RP) into ONE analytical plat form
– Reduces sample prep and analysis times to streamline the analytical
workflow
o Direct injection of organic solvents/extracts
o Reduces solvent usage
o No derivitization required for free fatty acid analysis
 UPC² separates compounds with STRUCTURAL SIMILARITY
– Optical isomers, positional isomers, structural analogs, conjugates
 UPC² provides ORTHOGONALITY
– Complementary separation provides confidence in identifying compound
of interest
©2015 Waters Corporation
45
Acknowledgements
Waters Natural Products team
 Dr. Dhaval Patel
Waters Singapore
 Dr. Lirui (Kevin) Qiao
Waters, China
 Dr. Jimmy Yuk
Waters Corporation
 Dr. Kate Yu
Waters Corporation
 Dr. Kerri Smith
Waters Corporation
 Mr. James Traub
Waters Corporation
 Mr. Ronan Cleary
Waters Corporation
 Mr. Darcy Shave
Waters Corporation
 Mr. Andrew Aubin
Waters Corporation
©2015 Waters Corporation
46
Thank you!!!
Giorgis_Isaac@waters.com
©2015 Waters Corporation
47