Date post: | 17-Jan-2016 |
Category: |
Documents |
Upload: | cory-mccarthy |
View: | 225 times |
Download: | 1 times |
©2015 Waters Corporation 1
Let’s Get CRITICAL, Supercritical Fluid Extraction (SFE)
Giorgis Isaac, PhD
Principal Scientist
©2015 Waters Corporation 2
Sample Preparation Challenges & Extraction Techniques
MV-10 ASFE System Introduction & Benefits
SFE Applications
SFE Outline
©2015 Waters Corporation 3
A Fully Supercritical Fluid Process
SFE Extraction
Extraction Analysis and scale-up predictionFinal purity
assessment
Preparative SFC for Purification
No treatment between Extraction, Separation and Isolation
©2015 Waters Corporation 4
Sample Preparation Challenges
Sample preparation:– Often overlooked
– Least developed
– Most time-intensive
– Most error-prone
Sample Preparation
©2015 Waters Corporation 5
Classical Extraction Methods
Soxhlet Extraction
Distillation
Evaporation
©2015 Waters Corporation 6
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
Traditional Extraction Methods Drawbacks
©2015 Waters Corporation 7
Sample Preparation Challenges & Extraction Techniques
MV-10 ASFE System Introduction & Benefits
SFE Applications
SFE Outline
©2015 Waters Corporation 8
What is a Supercritical Fluid?
304.1 K
73.8
Supercritical fluid has High Diffusivity, Low Viscosity and Low Surface Tension!
©2015 Waters Corporation 9
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 10
MV-10 ASFE System Components
Fluid DeliveryModule
Up to 6 Co-SolventsAvailable
Column Oven -up to 10 Extraction Vessels
BackpressureRegulator(BPR)
Heat Exchanger
Fraction Collection Module-up to 12 collection bottles(5, 10 or 25 mL)
©2015 Waters Corporation 11
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 12
Increasing Polarity
Non-polars
Alkanes
EthersEsters
AlcoholsAmides
AcidsAmines Highly polar organics Inorganic
ions
Neat CO2 SFECO2 + modifier
CO2 + modifier + ternary additives
CO2 + modifier + ternary additives + water
Liquid – based extraction methods
Small molecules Peptides Large proteins
Increasing Molecular Weight
Extractability Based on Polarity
One of the largest advantages of SFE: Selectivity
©2015 Waters Corporation 13
3 extract’s CO2 & 1% MEOH @ 100, 200 & 350 bar
Isolated compound of interest
100 Bar200 Bar
300 Bar
Supercritical Fluid Extraction:Effect of Increasing Density of CO2
Isolated compound of interest
Isolated compound of interest
©2015 Waters Corporation 14
Effect of extraction T and P on γ–tocopherol yeild
©2015 Waters Corporation 15
CO2 tunable parameters and polarity for selectivity
Control of Tunable Extraction ParametersCritical to Optimizing and Reproducibility
©2015 Waters Corporation 16
Sample Preparation Challenges & Extraction Techniques
MV-10 ASFE System Introduction & Benefits
SFE Applications
SFE Outline
©2015 Waters Corporation 17
SFE Step– 90 –150 bar, max 45 °C
– Remove pesticides & heavy metals
– Separate <C18 from >C18 (mainly saturated and mono-unsaturated FA)
– Remove cholesterol
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. – No thermal stress
– No oxidation
Example 1: Omega-3 From Marine Origines
SaturatedMonounsaturatedOmega-6
EPA
DHA
EPA
DHA
SaturatedMonounsaturatedOmega-6
Fish Oil
ConcentratedOmega-3
Commercial process - Patented
©2015 Waters Corporation 18
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%)
Example 2: Stevia Extraction
©2015 Waters Corporation 19
Conventional extraction methods for stevioside involve – aqueous or alcohol extraction
– precipitation and coagulation with filtration
– clean-up step
– crystallization and drying
Stevioside Extraction
Choi, Y. H. et. al, Chromatographia, 55, 716-620, 2002.
©2015 Waters Corporation 20
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 compoundso Less compound interference, more column loading capacity
SFE Extract
Solvent Extract
Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
%
0
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
%
0
(A) SFE
(B) Solvent Extraction
Target, m/z= 391, 1.61e7
Targetm/z=391, 1.68e7
©2015 Waters Corporation 21
Example 4: Selective Extraction of Ingenol from Euphorbia Plant
Selectivity is key to efficient sample preparation
Time2.00 4.00 6.00 8.00 10.00 12.00 14.00
%
0
2.00 4.00 6.00 8.00 10.00 12.00 14.00
%
1
2.00 4.00 6.00 8.00 10.00 12.00 14.00
%
0
2.00 4.00 6.00 8.00 10.00 12.00 14.00
%
0
Ingenol Standard
Solvent Extract
SFE Extract
Residual Sample after SFE
©2015 Waters Corporation 22
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 23
Let’s Get CRITICAL, Supercritical Fluid Chromatography (SFC)
Introduction to ACQUITY UPC2
Giorgis Isaac, PhD
Principal Scientist
©2015 Waters Corporation 24
A Fully Supercritical Fluid Process
SFE Extraction
Extraction Analysis and scale-up predictionFinal purity
assessment
Preparative SFC for Purification
No treatment between Extraction, Separation and Isolation
©2015 Waters Corporation 25
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 26
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 27
Evolution of Separation Technology
Gas Chromatography Liquid Chromatography Convergence Chromatography
GC
Capillary GC
HPLC
UPLC
SFC
UPC2
Resolution, Sensitivity, Throughput
©2015 Waters Corporation 28
How the ACQUITY UPC2 System Works
Inject valve
AuxiliaryInject valve
Column Manager
PDA detector
Back Pressure Regulator(Dynamic and Static)
Waste Modifier CO2 Supply
CO2 Pump
Modifier Pump
mixerThermo-electric heat exchanger
Make-upPump
Mass Spec
Splitter
©2015 Waters Corporation 29
UPC2RPLC
Example 1: Separating Polar Compounds (Catalpol/stachyose/sucrose/D-mannitol/yellow glucoside)
m/z50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950
%
0
100
DH_Neg_20eV_RP1 80 (0.737) Cm (18:126) 1: TOF MS ES- 2.11e6341.11
290.09
128.04113.00
89.03
181.07
133.02
191.02
195.02
245.10308.10
665.21
503.16
387.11
342.11
343.12
361.11
407.12 470.15
455.10425.10
471.12
549.17
504.16
505.16
632.20
550.17
617.15551.16633.18
711.22
666.22
683.22
712.22
827.26
794.25713.22
779.20
845.27 873.27
874.27 956.31 974.34
Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
DH_Neg_20eV_RP1 1: TOF MS ES- TIC
1.49e60.74
0.68
1.5x107
3x106
5.25x107
4.5x106
3x107
3.0x107
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
EIC, 181
EIC, 361
EIC, 341
EIC, 685
EIC, 665
ES-, TIC
HO
OH
OH
OH
OH
OH
O
HO
HO
OH
O
OH
O
H
H
OH
H
O
HO
OOH
OH
OH
CH2OH
OO
CH2OH
CH2OH
OH
OH
O
OH
OH
OH
HO
O
OOH
HO
HO
O
O
OH
OH
OO
HO
HO
OH
OH
O
OH
HO
OOH
OHO
OH
HO
OOH
O
HOHO
O
OH
OHO
OH
OH
OH
-3.26
-4.61
-4.49
-6.45
-6.64
Rehmannia extract(the polar compounds elute near solvent front)
©2015 Waters Corporation 30
AU
-0.002
0.000
0.002
0.004
0.006
0.008
Minutes
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40
Example 2: Fat Soluble Compounds Lycopene and -Carotene
LycopeneLogP=11.11 L. Zhang et al. / Food Chemistry 132 (2012) 2112–2117
H. Li et al. / Food Chemistry 132 (2012) 508–517
-caroteneLogP=10.68
UPLC
HPLC
UHPSFC 60 min
16 min
1.5 min
©2015 Waters Corporation 31
Example 3: Chiral Separations
Time0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
AU
-1.25
-1.0
-7.5e-1
-5.0e-1
-2.5e-1
-9.375e-8
2.5e-1
5.0e-1
7.5e-1
1.0
1.25
1.5
1.75
2.0
2.25
2.5
2.75
3.0
3.25
3.5
3.75
4.0
4.25
2.47
0.85
2.90
60 min 6 min
“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
(S)
NH
O
S
(R)
NH
O
S
Goitrin (S-goitrin) Epigoitrin (R-goitrin)
R, S-Goitrin from Isatis Indigotica Fort
(板蓝根 )
©2015 Waters Corporation 32
Taking Advantage of Low Viscosity
Coupling columns in series is one of the benefits of working with low viscosity solvents
Time0.00 5.00 10.00 15.00 20.00 25.00
LS
U
0.000
200.000
400.000
600.000
0.00 5.00 10.00 15.00 20.00 25.00
LS
U
0.000
200.000
400.000
600.000
0.00 5.00 10.00 15.00 20.00 25.00
LS
U
0.000
250.000
500.000
750.000(A) 150 mm
(B) 250 mm
(B) 400 mm
Column Length
(mm)t1 (min) t2 (min) w0.5,1 w0.5,2
Isomeric ratio
(peak1/peak2)*Rs Increase
Theor.
Increase
150 7.14 7.95 0.34 0.39 0.62 1.31 0 0
250 11.21 12.54 0.44 0.48 0.64 1.71 31% 29%
400 18.20 20.33 0.55 0.58 0.64 2.23 70% 63%*, where t is the retention time and w0.5 is the peak width at half height
Table 1. Comparison of three SFC runs with different column lengths.
O
O
HO
OO
OHO
HO
O
HO OH
HOH3C
HO H
S
C-25
O
O
HO
OO
OHO
HO
O
HO OH
HOH3C
HO H
R C-25
©2015 Waters Corporation 33
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 34
UPC2 Analysis of Volatile Compounds from TCM
254nm
2.5min
RSD<1%
组分 1
组分 2
Overlay of 6 replicates
UPC2 analysis for the isomers– Analysis time
2.5 minutes
– Direct analysis, easy sample prep
– Easy scale up to Prep
©2015 Waters Corporation 35
Example 5: UPC2 Analysis of Cannabinoids
UPLC
UPC2
CBDVCBDACDGCBDTHCVCBNTHCCBCTHCA
CDBVCBDd8THCd9THCCBCCBNCBGTHCACBDACBGA
CB
GV
- 3
.088
CB
D -
3.1
58
d8T
HC
- 3
.235
d9T
HC
- 3
.294
CB
C -
3.6
06
CB
N -
3.8
08
CB
G -
3.9
05
TH
CA
- 4
.420
CB
DA
- 4
.520
CB
GA
- 4
.811
AU
-0.02
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
Minutes
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
UPC2
UPC2 and UPLC chromatograms of a mixture of 10 cannabinoid standards
©2015 Waters Corporation 36
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
Example 6: UPC2 Analysis of Lipid
Aqueous layerSalts, polar metabolites
Organic layerDissolved Lipids
Extract
Suspended and dissolved material
©2015 Waters Corporation 37
Lipid Analysis Work Flow GC, LC and UPC2
Sample• Extraction • FAMEs
Derivitization• Ready for
GC/MS analysis
Sample• Extraction• Direct UPC2
analysis
Sample• Extraction• Evaporate to
dryness• Reconstitution• Ready for UPLC
analysis
GC UPLCUPC2
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
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
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
©2015 Waters Corporation 38
Fast and Simple Free Fatty Acid Analysis Using UPC2
Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
%
0
100
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
%
0
100
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
%
0
100
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
%
0
100 311.30283.26
227.20
199.17
197.81
367.36
311.29283.26
227.20
199.17197.81
367.36
367.36311.30283.26
255.23
227.20
199.17197.81
339.33
311.29
283.26
255.23
227.20199.17197.81 171.14
367.36339.33
1 to 25 % B
5 to 25 % B
15 to 25 % B
20 to 25 % BUPC2 Conditions:A= CO2
B=MeOH in 0.1% FAColumn= ACQUITY UPC2 HSS C18 SB 1.8µm (2.1 x 150 mm)Flow rate= 0.6 mL/minColumn temp= 50 ºCP=1600 psi
Peak m/z FA
1 143.10 C8:02 171.14 C10:03 199.17 C12:04 227.20 C14:05 255.53 C16:06 283.26 C18:07 311.30 C20:08 339.33 C22:09 367.36 C24:0
12:0
10:0 14:0
16:018:0
20:0 22:0 24:0
©2015 Waters Corporation 39
Separation of FFA C8-C36 from Algae Extract
Time-0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
%
0
100 x16255.23
227.20
465.47
283.27
423.42395.39
367.36
299.20
311.30 451.45
479.48
479.45
507.48455.24
16:0
34:0
23:0
FFA C8-C36
32:0
2 min
©2015 Waters Corporation 40
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 41
A Fully Supercritical Fluid Process
SFE Extraction
Extraction Analysis and scale-up predictionFinal purity
assessment
Preparative SFC for Purification
No treatment between Extraction, Separation and Isolation
©2015 Waters Corporation 42
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
Example 7: Extraction, Separation and Isolation of Schisandra Berry Extracts Using SFE and SFC
©2015 Waters Corporation 43
UPC2 separation of crude SFE extractSFE
UPC2/PDA 220 separation of collected fraction
Extraction, Separation and Isolation of Schisandra Berry Extracts using SFE and SFC
UPC2/QDa UPC2/PDA 220
Prep SFC separation of crude SFE extract
©2015 Waters Corporation 44
Time0.20 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
%
0
G4_SolE_002 1: Scan AP+ TIC
4.10e7
Time0.20 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
%
2
G4_MPLC_023 1: Scan AP+ TIC
6.65e7
Time0.20 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
%
0
100
G4_2_25_2012_006 1: Scan ES+ 391
1.23e8
Extraction, Separation and Purification Simplifies your Sample Complexity
Isolation
Standard
Sim
ple
r
Extraction
©2015 Waters Corporation 45
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 workflowo Direct injection of organic solvents/extractso Reduces solvent usageo 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
Summary UHPSFC (UPC2)
©2015 Waters Corporation 46
Acknowledgements
Waters Natural Products teamDr. Dhaval Patel Waters SingaporeDr. Lirui (Kevin) Qiao Waters, China
Dr. Jimmy Yuk Waters Corporation Dr. Kate Yu Waters Corporation Dr. Kerri Smith Waters CorporationMr. James Traub Waters Corporation
Mr. Ronan Cleary Waters Corporation Mr. Darcy Shave Waters Corporation Mr. Andrew Aubin Waters Corporation
©2015 Waters Corporation 47
Thank you!!!