©2015 Waters Corporation 1 Let’s Get CRITICAL, Supercritical Fluid Extraction (SFE) Giorgis...

Let’s Get CRITICAL, Supercritical Fluid Extraction (SFE)

Giorgis Isaac, PhD

Principal Scientist

Giorgis_isaac@waters.com

Sample Preparation Challenges & Extraction Techniques

MV-10 ASFE System Introduction & Benefits

SFE Applications

SFE Outline

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

Sample Preparation Challenges

Sample preparation:– Often overlooked

– Least developed

– Most time-intensive

– Most error-prone

Sample Preparation

Classical Extraction Methods

Soxhlet Extraction

Distillation

Evaporation

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

SFE Applications

SFE Outline

What is a Supercritical Fluid?

304.1 K

Supercritical fluid has High Diffusivity, Low Viscosity and Low Surface Tension!

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

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)

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.

Increasing Polarity

Non-polars

Alkanes

EthersEsters

AlcoholsAmides

AcidsAmines Highly polar organics Inorganic

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

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

Effect of extraction T and P on γ–tocopherol yeild

CO2 tunable parameters and polarity for selectivity

Control of Tunable Extraction ParametersCritical to Optimizing and Reproducibility

SFE Applications

SFE Outline

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

Fish Oil

ConcentratedOmega-3

Commercial process - Patented

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

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.

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

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

(A) SFE

(B) Solvent Extraction

Target, m/z= 391, 1.61e7

Targetm/z=391, 1.68e7

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

2.00 4.00 6.00 8.00 10.00 12.00 14.00

Ingenol Standard

Solvent Extract

SFE Extract

Residual Sample after SFE

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

Let’s Get CRITICAL, Supercritical Fluid Chromatography (SFC)

Introduction to ACQUITY UPC2

Giorgis Isaac, PhD

Principal Scientist

Giorgis_isaac@waters.com

SFE Extraction

assessment

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)

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

Evolution of Separation Technology

Gas Chromatography Liquid Chromatography Convergence Chromatography

Capillary GC

Resolution, Sensitivity, Throughput

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

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

DH_Neg_20eV_RP1 80 (0.737) Cm (18:126) 1: TOF MS ES- 2.11e6341.11

290.09

128.04113.00

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

DH_Neg_20eV_RP1 1: TOF MS ES- TIC

1.49e60.74

1.5x107

5.25x107

4.5x106

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

Rehmannia extract(the polar compounds elute near solvent front)

-0.002

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

UHPSFC 60 min

16 min

1.5 min

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

-7.5e-1

-5.0e-1

-2.5e-1

-9.375e-8

2.5e-1

5.0e-1

7.5e-1

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

Goitrin (S-goitrin) Epigoitrin (R-goitrin)

R, S-Goitrin from Isatis Indigotica Fort

(板蓝根 )

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

200.000

400.000

600.000

0.00 5.00 10.00 15.00 20.00 25.00

200.000

400.000

600.000

0.00 5.00 10.00 15.00 20.00 25.00

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.

R C-25

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

UPC2 Analysis of Volatile Compounds from TCM

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

Example 5: UPC2 Analysis of Cannabinoids

CBDVCBDACDGCBDTHCVCBNTHCCBCTHCA

CDBVCBDd8THCd9THCCBCCBNCBGTHCACBDACBGA

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 and UPLC chromatograms of a mixture of 10 cannabinoid standards

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

– 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

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

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

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

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

10:0 14:0

16:018:0

20:0 22:0 24:0

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

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

FFA C8-C36

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

SFE Extraction

assessment

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

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

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

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

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

G4_2_25_2012_006 1: Scan ES+ 391

1.23e8

Extraction, Separation and Purification Simplifies your Sample Complexity

Isolation

Standard

Extraction

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)

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

Thank you!!!

Giorgis_Isaac@waters.com

©2015 Waters Corporation 1 Let’s Get CRITICAL, Supercritical Fluid Extraction (SFE) Giorgis...

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