Supercritical Fluid Chromatography Theory, Design and Applications DJ Tognarelli Chromatography Application Scientist JASCO Seminar Series
Supercritical Fluid ChromatographyTheory, Design and Applications
DJ TognarelliChromatography Application Scientist
JASCO Seminar Series
Founding Members
1958
Established at the Optical Research Institute at Tsukuba University, Tokyo
Founding members include: World famous physicist Yoshio Fujioka Nobel Prize winner Shinichiro Tomonaga
(1965 - Physics for QED with Richard Feynman)
1972
JASCO in the USA
HPLC Products
1985
SFC – First dynamic BPR
Dr. Tomonaga
Products
Overview
Section I Introduction to Supercritical Fluids Advantages of SFC
Section II SFC System Preparative Unique Capabilities
Section III Applications
Overview
Section I Introduction to Supercritical Fluids Advantages of SFC
Section II SFC System Preparative Unique Capabilities
Section III Applications
SupercriticalFluid
-56.4
0.51
31.3
7.29
Triple Point
Critical Point
SolidLiquid
Gas
Temperature (oC)
Pres
sure
(MPa
)
Phase Diagram for CO2- Critical temperature (Tc):
the temperature above whicha distinct liquid phase can NOTexist, regardless of pressure
- Critical pressure (Pc): the vapor pressure at the
critical temperature
At T and P above itscritical point (Tc, Pc), a substance exists in
a supercritical fluid state!
Introduction to Supercritical Fluid
Compound Tc (C) Pc (Mpa)
NH3 132 11.28
CO2 31 7.38
N2O 36 7.24
H2O 374 22.06
C3H8 97 4.25
C6H14 234 2.97
CH3OH 239 8.09
C2H5OH 243 6.38
C6H5(CH3) 318 4.11
Critical Temperatures and Pressures
Advantages of SC-CO2
SC-CO2 → Non toxic, non flammable, inexpensive
Low Cp and CtEasy removal of CO2 by decompression
Environmentally friendly (alternative solvent to hazardous organic solvents)Safely used in food and pharmaceutical products.
Gas-liquid 2-phase state
Supercritical State1-phase state
Critical pointRayleigh Scattering
Phase Transition
72.9 atm (bar) / 31.3 C
Diffusivity (cm2/s)
Density (g/cm3)
Viscosity (g/cm · s)
Gas 10-1 10-3 10-4
Supercritical Fluid
10-4 – 10-3Liquid Like
0.2-0.8Liquid Like
10-4Gas Like
Liquid 10-5 - 10-6 1 10-2
Physical Properties
High diffusivity and densitywith low viscosity
Faster flow rates with lower pressure=
1. SFC is a normal phase technique where CO2 replaces hexane
2. Faster separation/analysis time (3 to 5 times faster than HPLC)
SFC Advantages
-1000
100200300400500600700800900
0 1 2 3 4 5 6 7 8 9 10 11 12
Time (min)
mAU
-1000
100200300400500600700800900
0 1 2 3 4 5 6 7 8 9 10 11 12
Time (min)
mAU
-1000
100200300400500600700800900
0 1 2 3 4 5 6 7 8 9 10 11 12
Time (min)
mAU
HPLC
SFC
HT-SFC
Mobile phase: Hexane/Isopropanol (IPA)(60/40)Flow rate: 0.5 mL/minColumn: CHIRALCEL OD (4.6 mm ID x 250 mm L)Column temp.: 40 ºCWavelength: 230 nm
CO2 : 3.0 mL/minModifier: Methanol, 0.1 mL/minColumn: CHIRALCEL OD (4.6 mm ID x 250 mm L)Column temp.: 40 ºCPressure: 20 MPaWavelength: 230 nm
CO2 : 5.0 mL/minModifier: Methanol, 2.5 mL/minColumn: CHIRALPAK OD (4.6 mm ID x 150 mm L)Column temp.: 40 ºCPressure: 20 MPaWavelength: 230 nm
HPLC, SFC, and HT-SFC
System Column ID HPLC Flow Rate SFC Flow Rate
Analytical 3mm, 4.6mm 0.6mL/min, 1mL/min 2mL/min, 5mL/min
Hybrid 4.6mm, 10mm 1mL/min, 5mL/min 5mL/min, 20mL/min
Semi-Preparative 4.6mm, 10mm, 20mm 1mL/min, 5mL/min, 20mL/min 5mL/min, 20mL/min, 90mL/min
Preparative 10mm, 20mm, 30mm 5mL/min, 20mL/min, 40mL/min 20mL/min, 70mL/min, 150mL/min
HPLC vs SFC Systems
SFC is 3 – 5 times faster
1. SFC is a normal phase technique where CO2 replaces hexane
2. Faster separation/analysis time (3 to 5 times faster than HPLC)
3. Easy removal of mobile phase (typically alcohol)
4. Using up to 95% less solvent – Cost Savings
SFC Advantages
Tocopherol (Vitamin E)
HPLCColumn : Finepak SIL-5 (4.6mm I.D. x 250mmL)Eluent : n-Hexane / IPA / CH3COOH (99 / 1 / 0.1)Flowrate : 1.0 mL/min Temperature : 40 deg.CINJ.VOL. : 10 µLWavelength range (PDA) : 200 - 650 nm
SFCColumn : SFCpak SIL (4.6mm I.D. x 250mmL)CO2 : 3.0 mL/minSolvent (EtOH) : 0.2 mL/min Pressure : 20.0 MPaTemperature : 80 deg.CINJ.VOL. : 10 µLWavelength : 280 nm
0.0
1.5E+05
3.0E+05uV
5.0 10.0 15.0 20.0 [min]
0.0
2.0E+04
4.0E+04
6.0E+04 1: Tocopherol α 2: Tocopherol β
3: Tocopherol γ 4: Tocopherol δ
12
3
4
12
3
4
HPLC (14min) SFC (8min)Amount Price Amount Price
Solvent 26.600L $2356 9.680L $1633Column 2cm 2cmFlow rate 19mL/min 60.5mL/minTime 23 hours 13 hoursSolvent disposal cost is not included
Column : CrestPak C18S (4.6mm I.D. x 150mmL)Eluent : A; MeOH / H2O (75 / 25)
: B; /.EtOAc / CH3CN (50 / 50)Flowrate : 1.5 mL/min Temperature : 30 deg.CINJ.VOL. : 15 µLWavelength : 270 nm
Time(min) A(%) B(%)0.0 90 10
27.0 0 10032.0 0 100
Polymer Additives
Column : SFCpak SIL (4.6mm I.D. x 250mmL)CO2 : 3.0 mL/minSolvent (EtOH) : 0.3 mL/min Pressure : 20.0 MPaTemperature : 60 deg.CINJ.VOL. : 10 µLWavelength : 280 nm
0.0
4.0E+04
8.0E+04
uV
10.0 20.0 30.0 [min]0.0
6.0E+04
1.2E+05
1
2
3
1
23
1: Irganox 1010 2: Irganox 1076 3: Irgafos 168
HPLC (42min) SFC (9min)Amount Price Amount Price
Solvent 267.750L $26,902 21.760L $3,944Column 3cm 3cmFlow rate 42.5mL/min 136mL/minTime 70 hours 15 hoursSolvent disposal cost is not included
30.0
[min]
20.0
10.0
0.0
6.0E+04
1.2E+05
0.0
4.0E+04
8.0E+04
uV
1. SFC is a normal phase technique where CO2 replaces hexane
2. Faster separation/analysis time (3 to 5 times faster than HPLC)
3. Easy removal of mobile phase (typically alcohol)
4. Using up to 95% less solvent – Cost Savings
5. Higher selectivity especially in chiral separation with wider array of columns and co-solvents
6. Longer column lifetime
SFC Advantages
Solvents and Columns
Stationary Phase Choices
Silica
2-ethylpyridine
Cyano
Aminopropyl
Diol
Amide
PFP
Phenyl
C18 < C8 Reversed-phase range
Normal phase range
SFC range
Solvent
Solvent Polarity [P’]
Pentane, Hexane, Heptane 0.1
Xylene 2.5
Toluene 2.4
Diethyl ether 2.8
Dichloromethane 3.1
Chloroform 4.1
Acetone 5.1
Dioxane 4.8
THF 4.0
MTBE 2.5
Ethyl acetate 4.4
DMSO 7.2
Acetonitrile 5.8
Isopropanol 3.9
Ethanol 4.3
Methanol 5.1
Water 10.2
1. SFC is a normal phase technique where CO2 replaces hexane
2. Faster separation/analysis time (3 to 5 times faster than HPLC)
3. Easy removal of mobile phase (typically alcohol)
4. Using up to 95% less solvent – Cost Savings
5. Higher selectivity especially in chiral separation with wider array of columns and co-solvents
6. Longer column lifetime
7. Similar methodology to HPLC
SFC Advantages
Method Principles
Sample : CaffeineColumn : SCFpak SIL (4.6 mm I.D. x 250 mmL)Pressure : 20.0 MPaTemperature : 60 deg.C
0.0
6.0E+04
1.2E+05uV
0.0
1.0E+05
2.0E+05
4.0 8.0 12.0 16.0 [min]0.0
1.5E+05
3.0E+05
10%
15%
20%
Same methodology used in HPLC applies to SFC Gradient Isocratic Column Solvent (with additives)
Structurally similar compounds
Chiral compounds
Degradants
Metabolites
Increased efficiency makes SFC a superior technique for separating these types of compounds.
All compounds typically used on Normal Phase HPLC
Polar Compounds
Those that are difficult to retain in HPLC
Orthogonality
When other separation techniques don’t work.
When To Try SFC?
Overview
Section I Introduction to Supercritical Fluids Advantages of SFC
Section II SFC System Preparative Unique Capabilities
Section III Applications
SFC1
23
MS
1. Cooled CO2 Pump2. High Pressure Flow Cell3. Back Pressure Regulator
SFC System
HPLCMS
SFC Hardware Requirement 1
Pump
Liquid CO2 is required for SFC Typically from a cylinder for analytical SFC Bulk CO2 for Prep SFC
To maintain the liquid CO2, the pump head must be cooled Peltier cooled for analytical pumps Circulated chiller for Prep pumps
Otherwise it is a HPLC pump
1. CO2 Pump
Pump heads (-10 °C)
Peltier cooling
Recirculating Chiller Head (-10 °C)
Insulating Cooling Block
Automatic
Shut-Off Valves
CO2 Physical Properties
SFC Hardware Requirement 2
Detector Flow Cell
HPLC pressure is between the pumps and column Flow cell is low pressure
SFC pressure is between the pumps and back pressure regulator Flow cell must be high pressure
Detector is identical, just a different flow cell
2. High Pressure Flow Cell
HPLC Detector + High Pressure Flow Cell
25.0
15.4
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Retention Time [min]
-1000
0
1000
2000
Inte
nsity
[µV]
Performance and Sensitivity
# Peak Name tR Area Height S/N NTP Resolution Symmetry Factor1 Peak-001 1.662 2688 858 25.0 7250 7.341 1.3672 Peak-002 2.472 2751 530 15.4 4767 N/A 1.076
10uL Injection
0.05% flurbiprofen test
SFC Detectors
Circular DichroismVariable Wavelength UV Photodiode Array Fluorescence
Flame Ionization(FID)
Evaporative Light Scattering(ELSD)
Mass Spectrometry
SFC Hardware Requirement 3
Back Pressure Regulator
SFC requires a back pressure regulator This maintains the critical pressure for CO2 to stay in a supercritical fluid state The performance of this is critical to the separation efficiency and reproducibility
If this restrictor is open or bypassed, then you would have HPLC
• Pressure control without changing flow rate of SCF.
• Pressure programming vs. time
• Self cleaning of precipitated substances
• Low dead-volume
• Patented
Valve seatValve needle
Needle-drive solenoid
Needle seal Return spring
Heater
FLOW
Gap adjustment screw
Electrical-Feedback Regulator
3. Back-Pressure Regulator
Back-Pressure Regulator Performance
Caffeine ConditionsFlowrate 3mLs/minCO2/MeOH 85/15Back pressure 150 barColumn Silica 4.6x150, 5um5uL Injection
0.169% Retention Time RSD
Preparative SFC Fraction Collection
(Prep Column ID)2
(Analytical Column ID)2Prep Flow Rate
Analytical Flow Rate=Scale-Up
CO2
Sample(and solvent)
Sample solutiondissolved in CO2+Modifier
Open-Bed Fraction Collection
Recovery 95%+
Unique Capabilities
Back Pressure Regulation
Parallel SFC
Stacked Injections
Back Pressure Regulation
0.0
1.5E+04
3.0E+04uV
0.0
5.0E+04
1.0E+05
1.5E+05
5.0 10.0 15.0 [min]0.0
1.0E+05
2.0E+05
10.0 MPa
15.0 MPa
20.0 MPa
Sample : CaffeineColumn : SCFpak SIL (4.6mm I.D. x 250mmL)CO2 : 3.0 mL/minSolvent (EtOH) : 0.5 mL/min Temperature : 60 deg.C
Back Pressure Regulation is unique to SFC and can provide additional separation optimization as the pressure changes the density.
Parallel SFC
4X or 5X Throughput
-1000
100200300400500600700800900
0 1 2 3 4 5
mAU
-1000
100200300400500600700800900
0 1 2 3 4 5
mAU
1 2 3 4 5
1 2 3 4 5
Stacked Injections
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
-100
1 2 3 4 5
1 2 3 4 5
Stacked injections eliminate the time between injection and start of the first peak for all injections except the first significantly reducing the total purification time.
Overview
Section I Introduction to Supercritical Fluids Advantages of SFC
Section II SFC System Preparative Unique Capabilities
Section III Applications
Applications
Pharmaceuticals Natural products Foods Pesticides and herbicides Surfactants Lipids Cosmetics Polymers Petroleum Explosives Propellants Gasoline, diesel and bio-diesel fuels
Pharmaceuticals
Achiral and Chiral Molecules
LC/MS vs SFC/MS Pinkston et. al. screened 2153 compounds. Eluted and Detected. 87% by SFC/MS 90% by LC/MS Ammonium Acetate aid in retention of very
polar and ionic analytes
SFC method time of 4 minutes
LC method time of 6 minutes
*LC screen required nearly 3 additional days.
0.0 2.0 4.0 6.0 8.0 10.0 Retention Time [min]
-5000
0
5000
Inte
nsity
[µV]
Fraction1Fraction2 Fraction3
Pharmaceutical – Chiral Purification
0.0 2.0 4.0 6.0 8.0 10.0 12.0 Retention Time [min]
0
100000
200000
300000
Inte
nsity
[µV]
Fraction1 Fraction2
0.0 2.0 4.0 6.0 8.0 10.0 Retention Time [min]
-10000
0
10000
20000
Intensit
y [µV]
UV
CD
∆CD/ ∆UV
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 Retention Time [min]
0
50000
100000
Intensi
ty [µV
]
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 Retention Time [min]
0
50000
100000
Intensi
ty [µV
]
Enantiopurity 99.8 ± 0.5% Enantiopurity 99.6 ± 0.5%
Fraction 1 Fraction 3
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 Retention Time [min]
0
50000
100000
150000
Inte
nsity
[µV
]
Fraction 1 Fraction 2
Purity 98 ± 0.5% Purity 93 ± 0.5%
Pharmaceutical – Chiral
Enantiomer 1Isomer B
Enantiomer 2 isomer B
16391
14342
Time (min)15 20 25 30 35 40
mAU
Time (min)15 20 25 30 4035
0
45
Isomer A (25 min)
Impurity (37 min)
Enantiomer 2 of isomer B(45.4 min)
Enantiomer 1 of isomer B(41.1 min)
10
20
15
5
100 200 300 400 500 600 700 800 m/z
%
%
%
0
100
%
100
100
100
0
0
0
393452
785
393
785
393
785
389
778448
N
F
X
N
F
* X
Isomer BMz = 392
Isomer AMz = 392
MS
UV
CD
Pharmaceutical – In Vitro Metabolism
VerapamilVerapamil is an L-type calcium channel blocker ofthe phenylalkylamine class. It has been used as avasodilator during cryopreservation of blood vessels,and is a class 4 antiarrhythmic, more effective thandigoxin in controlling ventricular rate. Verapamil hasalso been used in the treatment of hypertension,angina pectoris, cardiac arrhythmia, and mostrecently, cluster headaches.
C27H38N2O4 MW = 454.2832
Sample PrepA sample of in vitro verapamil 25uM and 10uM wasincubated for 30 minutes at 37°C with 2mg/mL protein and aNADPH regenerating system. The reaction was quenchedwith an equal volume of acetonitrile and the sample wascentrifuged. The sample supernatant was transferred to a200uL sample vial.
Metabolism
TIC and Enhanced Product Ion Spectra TIC and Enhanced Product Ion Spectra
Verapamil Norverapamil 1st and 2nd pair (active metabolite)
SFC-MSMS
Pharmaceutical – Potency
WarfarinWarfarin is a coumarin anti-coagulant and is marketed as the racemate. The entantiomers are differentially metabolized by human cytochromes P450(CYP). R-warfarin is metabolized primarily by CYP1A2 to 6- to 8-hydroxywarfarin and by CYP3A4 to 10-hydroxywarfarin. S-warfarin is metabolized primarily by CYP2C9 to 7-hydroxywarfarin. The S(-)-form is the more potent isomer.
O O
OH
CH3
O
C19H16O4 MW = 308.1049
Continued
The blood sample (approximately 4 mL) was collected from a patient that had taken 1.75 mg Q.D. Taro Pharmaceutical’s warfarin sodium USP. The whole blood was mixed with 10 mL of acetonitrile-isopropanol mix (1:1), vortexed for 20 minutes, and centrifuged for 20 min at 5,000 rpm at ambient temperature. The supernatant was filtered with 0.2-mm syringe filter and transferred to a 1.8-mL auto-sampler vial for SFC/MS/MS analysis.
SFC MS TIC and EPI. TIC of Racemic Warafin 10ng/ul, 10uL injection (top) and enhanced product ion spectra of each enantiomer (bottom 2) . Conditions: AD-H 4.6 x 250nm. Gradient 10% to 50% Methanol with 0.3% TFA over 18minutes at 3mL/min at 30°C.
warfarin MRM result.rdb (warfarin 1): "Linear" Regression ("1 / (x * x)" weighting): y = 33.2 x + 245 (r = 0.9894)
1000.0 2000.0 3000.0 4000.0 5000.0 6000.0 7000.0 8000.0 9000.0 1.0e4Concentration, ng/mL
0.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
3.0e5
3.2e5
3.4e53.5e5
Area, c
ounts
MRM Calibration Curve using Peak 1
SFC MS MRM. 2 MRMs of Taro warfarin sodium USP 1 mg pill dissolved in10 mL methanol, 0.45mm-filtered, 10-uL injection (top). 2 MRMs of the patient’s blood that had taken 1.75mg of O.D. Taro Pharaceutical’swarfarin sodium USP pill (bottom)
1.63mg warfarin in blood
Polymers – SFC & SFEQualitative and Quantitative
Chimassorb 81
Tinuvin 327
Irgafos 168 Phosphate
Irgafos 168
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
Retention Time [min]
0
500000
1000000
1500000
Inte
nsity
[µV]
SFCColumn : Luna C18 (4.6mm x 150mmL)CO2 : 2.7 mL/minSolvent (MeOH): 0.3 mL/min Pressure : 250 barTemperature : 75 deg.CINJ.VOL. : 20 µLWavelength : 220 nm
Irgafos 168 Tinuvin 326
Chimassorb 81
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00
Concentration[mg/mL]
0
10000000
20000000
Inte
nsity
Chimassorb 81 - CH9
Calibration for Chimassorb 81
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
Retention Time [min]
0
500000
1000000
1500000
Inten
sity [
µV]
Contour Plot
Standard 2
Standard 3
Chimassorb 81
Standard 2
Standard 3
Tinuvin 327
Standard 2Standard 3
Irgafos 168
Extraction Results
Chimassorb 81
Tinuvin 326
Unknown
Irganox 1010
Irgafos 168
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
Retention Time [min]
0
500000
1000000
Inte
nsity
[µV
]
0.11% Irgafos 1680.45% Tinuvin 3260.30% Chimassorb 81
Fraction collected from extraction
SFE extraction. 500mgs of polymer film was cut into squares and packed in a 10mL extraction vessel.ConditionsPressure: 250 barStatic Extraction Time: 5 minutesDynamic Extraction Time: 5 minutes
Petrochemicals – Olefin Analysis
8.0 8.5 9.0 9.5 10.0 Retention Time [min]
10000
20000
Inte
nsity
1.0% Olefins-3 - CH13.5% Olefins-3 - CH16.0% Olefins-3 - CH18.5% Olefins-3 - CH112% Olefins-3 - CH117% Olefins-3 - CH125% Olefins-3 - CH1
0.0 5.0 10.0 15.0 20.0 25.0 Concentration [%]
0
200000
400000
600000
800000
Inte
nsity
Olefins - CH1
ASTM D6550
Column : Silica 4.6 x 250mm, 5um: Silver 4.6 x 50mm, 5um
CO2 : 3.0 mL/min Pressure : 100 barTemperature : 35 CINJ.VOL. : 0.5 µLDetection : Flame Ionization (FID) 1
0.0 2.0 4.0 6.0 8.0 10.0 Retention Time [min]
20000
40000
60000
Commercial gasoline contained 1.0% olefins
Petrochemicals – Diesel
ASTM D5186Column : Silica 4.6 x 250mm, 5umCO2 : 3.0 mL/min Pressure : 100 barTemperature : 35 CINJ.VOL. : 0.5 µLDetection : Flame Ionization (FID)
1
2
0.0 1.0 2.0 3.0 4.0 5.0 6.0 Retention Time [min]
20000
40000
60000
80000
Commercial gasoline contained 59.3% non-aromatics and 40.7% aromatics.
Peaks: 1. n-hexadecane, 2. toluene, 3. tetralin, 4. naphthalene.
Environmental – Pesticides
CO2 : 5.0 mL/minCo-Solvent : MeOH w/ Amm. Ac. gradient to 50% over 7minPressure : 100 barTemperature : 40 CWavelength : 220nm
CarbarylMW 201.22
CarbofuranMW 221.25
DiuronMW 233.09
ChlorpyriphosMW 350.59
MalathionMW 330.36
CoumaphosMW 362.77
1ppm
10ppm
100ppm
Chlopyriphos
Malathion
Coumaphos
Carbofuran
Carbaryl
Diuron
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5
Retention Time [min]
2000
3000
4000
5000
Inten
sity
6 MIX 0.5PPM - CH1
6 MIX 0.1PPM - CH1
6 MIX 0.05PPM - CH1
6 MIX 0.02PPM - CH1
6 MIX 2PPB - CH1
Carbaryl
MS - SIM
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0
Retention Time [min]
0
2000
4000
6000
8000
229 - CH1241 - CH1257 - CH1281 - CH1284 - CH1288 - CH1297 - CH1302 - CH1303 - CH1304 - CH1313 - CH1317 - CH1328 - CH1330 - CH1331 - CH1335 - CH1345 - CH1372 - CH1394 - CH1407 - CH1503 - CH1
26 pesticides
US and EU Require
Environmental – PAHs
Detection limit(S/N=3)
UV 7040 fg
FL 26 fg
-100
0
100
Inte
nsity
[µV]
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
Retention Time [min]
-10000
0
10000
Inte
nsity
[µV]
AnthraceneApprox. 300 times
more sensitive
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0Retention Time [min]
0
10000
20000
30000
Inte
nsity
[µV]
1
23+4
5
67 8 9
1011 12 13 14 15 16
2500 pg each
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0Retention Time [min]
0
10000
20000
30000
Inte
nsity
[µV]
1
2
3+4
5
6
7
8
9
10
11
12
13
14
15
16
25 pg each
POLYAROMATIC HYDROCARBONS ANTHRACENE
Cannabis – Potency and Purification
CO2 +EtOH : 6.0 mL/min (Gradient)Pressure : 100 barTemperature : 25 CINJ.VOL. : 10 µLDetection : 220nm
Terpenes_C6 C18 x2 + IB N-5 40C 80bar - CH1
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0Retention Time [min]
0
1. CBDV2. CBN3. Delta-8-THC4. CBC enantiomer 15. CBD6. Delta-9-THC7. CBC enantiomer 28. THCV9. CBG10. CBDA11. CBDVA12. THCA13. CBGA
MS < 3min
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Slide Number 1Founding Members�Products�Overview�Overview�Introduction to Supercritical Fluid�Critical Temperatures and Pressures�Advantages of SC-CO2�Phase Transition�Physical Properties�SFC Advantages �HPLC, SFC, and HT-SFC �HPLC vs SFC Systems�SFC Advantages �Tocopherol (Vitamin E)�Polymer Additives�SFC Advantages �Solvents and Columns�SFC Advantages �Method Principles�When To Try SFC?�Overview�SFC System�SFC Hardware Requirement 11. CO2 Pump�CO2 Physical Properties�SFC Hardware Requirement 22. High Pressure Flow Cell��Performance and Sensitivity��SFC Detectors�SFC Hardware Requirement 33. Back-Pressure Regulator�Back-Pressure Regulator PerformancePreparative SFC Fraction Collection��Unique Capabilities�Back Pressure Regulation�Parallel SFC�Stacked Injections�Overview��Applications�Pharmaceuticals�Pharmaceutical – Chiral Purification�Pharmaceutical – Chiral�Pharmaceutical – In Vitro Metabolism�Metabolism��Pharmaceutical – Potency �Continued�Polymers – SFC & SFE�Qualitative and Quantitative�Extraction Results�Petrochemicals – Olefin Analysis�Petrochemicals – Diesel�Environmental – Pesticides�Environmental – PAHs�Cannabis – Potency and Purification�JASCO Educational Resources�Thanks for attending!��Questions?