6-Supercritical Fluid Chromatography SFC.pptx

Supercritical Fluid Chromatography

Aurelia Leyva and Trang Duong

Chem 230

11/30/2010

1

Chapter 33Supercritical-Fluid Chromatography

Supercritical-fluid chromatography (SFC), in

which the mobile phase is a supercritical

fluid, is a hybrid of gas and liquid

chromatography that combines some of the

best features of each. For certain

applications, it appears to be clearly

superior to both gas-liquid and high-

performance liquid chromatography.

Important Properties of Supercritical Fluids

A supercritical fluid is formed whenever a substance is heated above its critical temperature. At the critical temperature, a substance can no longer be condensed into its liquid state through the application of pressure.

The physical properties of a substance in the supercritical-fluid state can be remarkably different from the same properties in either the liquid or the gaseous state.

Supercritical Fluid• Substance at a T

and P above critical point

• Effuse through solids like a gas and dissolve materials like a liquid

• Close to critical point, small changes in T or P results in large changes in density

www.che.tohoku.ac.jp

T/P Phase Diagram

W.W. Christie and Lipid Technology 4

• Supercritical fluids have solvent power similar to a light hydrocarbon for most solutes. However, fluorinated compounds are often more soluble in scCO2 than in hydrocarbons.

• Solublity increases with increasing density (i.e. with increasing pressure).

• Supercritical fluids can have solvating powers similar to organic solvents, but with higher diffusivities, lower viscosity, and lower surface tension.

• The solvating power can be adjusted by changing the pressure or temperature, or adding modifiers to the supercritical fluid.

• A common modifier is methanol (typically 1-10%) which increases the polarity of supercritical CO2.

SCF Extractions

• The main advantages of using supercritical fluids for extractions is that they are inexpensive, contaminant free, and less costly to dispose of safely than organic solvents.

• Supercritical fluid CO2 is used to extract caffeine from coffee.

• Flavours for brewing are extracted from hops using SCF extraction.

Analytes easily recovered

• Equilibrate at low temp and atmospheric P

• CO2 is most commonly used

• Good for thermally unstable species

SFC Instrumentation

• Solvent delivery system

• Injector

• Column/Column Oven

• Restrictor

• Detector

• Data System

SFC Apparatus

http://www.cee.vt.edu/ewr/environmental/teach/smprimer/sfc/sfc.html

1. Mobile phase (CO2) 5. Column 2. Pump 6. Detector 3. Injection System 7.Chromatogram 4. Oven

13

14

Schematic Diagram of a Packed Column SFC

Restrictor

• Back pressure regulator is very important – or gas P will drop in column

• May let SFC convert to gas before detector

Solvent Delivery System

• Maintains precise mobile phase flow (1 to 10 L/min {OT} or 1 to 10 mL/min {Packed}).

• Aids in the control of the system pressure (up to 60 Mpa).

• Moves mobile phase in the liquid state under pressure through the injector & into the column.

SCF Chromatography• High pressures are used so that the SCF is

much more dense (102-103 x) than a gas• Thus it has better solvating properties• – but still not as dense as a liquid• For chromatography – we want:• low viscosity coefficient (gas)• Low diffusion coefficient (liquid)• The values of these for SCF’s are intermediate• High MW compounds that can’t be separated by

GC can be analyzed by SFC.

• Compound Tc º C Pc atm d*

• CO2 31.3 72.9 0.96

• C2H4 9.9 50.5 ---

• N2O 36.5 72.5 0.94

• NH3 132.5 112.5 0.40

• n-C5 196.6 33.3 0.51

• n-C4 152.0 37.5 0.50

• CCl2F2 111.8 40.7 1.12

• CHF3 25.9 46.9 ----

Supercritical Fluid Chromatography (SFC)

• Hewlett-Packard developed and manufactured the SFC instrument in 1982

• Combinations of desirable characteristics from both GC and HPLC

• Used for compounds:

– Highly polar

– Large MW

– Too thermally labile for GC

– Compounds undetectable for HPLC detector

Hemaxi S. Bhatt et al. / Journal of Pharmacy Research 2009, 2(10),1606-16011

Property Gas (STP) SCF Liquid Density (g/cm 3 ) (0.6-2) x 10 -3 0.2-0.5 0.6-2 Diffusion coefficient (cm 2 /s)

(1-4) x 10 -1 10 -3 x 10 - 4 (0.2-2) x 10 -5

Viscosity (G Cm -1 s -1 ) (1-4) x 10 - 4 (1-3) x 10 - 4 (0.2-3) x 10 -2

24

25

a. Loop injection -Low pressure feed pump needed to fill

the loop -Mostly for preliminary tests of column

performance and elution parameters

b. In-line injection -Flexibility for changing injected volume -High-pressure pump required to inject feed solution -Injected stream dissolved in eluent flow

c. In-column injection -Permits injection of the feed solution

directly onto the column-No dilutions required

Journal of Liquid Chromatography & Related Technologies, 28: 1233–1252, 2005

Types of Injectors

25

Injectors

• Typical HPLC design injectors for packed columns.

• Split/Splitless valve injector (0.01 to 0.05 L injections) for open tubular columns.

• Timed - split injector (0.01 to 0.05 L injections) for open tubular columns.

• Open tubular columns– Injection volumes >96nL– Greater volume affects resolution

• Packed columns– Injection volumes >1uL

Injector Volumes

27

28

ColumnOpen Tubular- Smaller column diameter more efficient- Efficiency decreases at high density (decrease u)

* Decrease column diameter* Increase T (increase diffusion, solubility, volatility)

- Minimal pressure drop effect

J.W. King, H.H. Hill, and M.L. Lee. 1993 28

Packed- Packing reduces permeability and creates flow resistance, L <25cm- Efficiency increases with increasing density (Dm decreases)- Smaller particle size = smaller H, but increase pressure drop, and decrease

permeability- Large P drop effect (density decreases, solubility decreases along column length)

J.W. King, H.H. Hill, and M.L. Lee. 1993

Column

29

-Packed columns are more efficient with higher density analytes.

Retention Time


Packed capillary - Lower volumetric flow rate = more compatible with mass flow detector, greater

sensitivity in conc. detector, easier sample transfer in multi-D system, higher permeability-longer column

Column

30

Stationary Phase• Same as those for GC and LC, with some modification.• Silica/Alumina

• Useful for non-polar compounds• Lead to irreversible adsorption of some polar solutes

• Bonded to provide less adsorptive packing material• Octyl, octadecyl, cyanoalkyl, aminoalkyl, diolakyl• Need organic modifiers to elute analytes

• Widely used polar S Phase• Polysiloxanes-- stable, flexible Si--O bond lead to good diffusion.

• Substituted with chemical groups for selective interaction with analyte• Polymethylsiloxanes--increase efficiency in separating closely related polar

analytes• Cyanopropyl polysiloxanes-useful for compounds with –COOH

31

Stationary phase

• Columns similar to those of HPLC.• Capillary SFC – organic films bonded to

capillaries. • Because SCF’s have low viscosities – can

use very long columns.• Get high resolution in a reasonable time.

SFC Columns

• Open tubular (derived from GC)

- Large # theoretical plates (~X500) - Easier to control pressure (low P drop)

• Packed (derived from HPLC)

- Faster analysis

- Higher flow rates

- Higher sample capacity

Open Tubular Columns

• Smaller than GC capillary columns, typically 50 m i.d., 10 to 20 m in length

• MP must be more stable due to extreme conditions of supercritical fluids

Packed Columns

• Similar to HPLC columns (10, 5, or 3 m porous particles)

• Silica based chemically bonded phases

• Typically 10 cm long X 4.6 mm i.d

36

Mobile PhaseNeat Fluid

• CO2 low Tc, low toxicity, non-

flammability• N2O high degree of nonideality,

permits high fluid density when compressed

• Flourocarbon low Tc, unique

selectivity for certain solutes

• Hydrocarbon limited use due to high flammability

• n-Pentane separation of oligomers

• Isopropyl/diethyl ether separation of polynuclear aromatics and polymers.


36

Mobile Phase

Caution when using mixed fluids to assure that the components are miscible over the range of T and P used.

Mix Fluid: Addition of polar organic modifier-Enhance solubility of polar analyte-Reduce retention volume-Eliminate strong interaction between absorptive site and polar solute

-Will modify polarity of eluent (asso. with ε and polarity index)-k and α affected by modifier identity and concentration

37

SFC and Retention

• Retention dependent on temperature, pressure, mobile phase density, and composition of the stationary and mobile phase.

• Complex interactions and not easily predictable.

• For supercritical fluids

- solvating properties similar to liquids - viscosity closer to gases

• Solvating power density

Temperature/Pressure Effects

• At lower P, > T, < solubility

• At higher P, > T, > solubility

-> T, Pv of solute > solute solubility

-< fluid density < solubilizing power

• > T, < solvent • >P, > solvent

Supercritical CO2 Density

• P (MPa) T (oC) (g/cm3)

7.3 40 0.22

7.3 80 0.14

7.3 120 0.12

40 40 0.96

40 80 0.82

40 120 0.70

Solvent Programming

• Programming is very useful in controlling solvent strength.

• Variations in P (density), T, and mobile phase composition.

• Density programming is most widely used (not simple relationship, T & P).

-> density, > solubility, < retention

- Combined T & P programming to control and thereby solubility and diffusion

SFC Mobile Phases

• Generally non-polar compounds with low

to moderate critical properties

- CO2, N2O, ethane, pentane

• Normal phase type separations

- non-polar mp and low polarity sp

(substrate + amino, diol, or cyano

groups)

• Elution = function of molecular mass &

polarity

Carbon Dioxide: SFC Solvent

• Low Tc

- operating T as low as 40oC

• Moderate Pc and c of 0.448g/cm3

- reach high with P < 40 MPa

• Safe to use

- nontoxic, nonflammable, noncorrosive, inert

• Detector compatible

• Wide range

CO2 as mobile phase

• Commonly used• Transmits UV• Colourless• Odourless• Non-toxic• Relatively cheap• Suitable Tc – T is generally kept ~10o

above the Tc.

Other SFC Solvents

• Nitrous Oxide - Similar in solvating and separations properties to CO2

• Alkanes - less safe and not as detector compatible than CO2

- better solvent characteristics for non-polar solutes

• Halocarbons, xenon, etc. - specialty applications only

• More polar solvents for highly polar & high molecular weight compounds

Solvent Modifiers

• Add organic modifiers to > solvent strength

- methanol

- isopropanol

- dichloromethane

- THF

- acetonitrile

• Mobile phase has solvating power which can be adjusted -

• Can vary composition (organic additives), T and P.

• Rapid equilibration times.• Lower T in SFC than GC – good if

thermally unstable.• Solvates polymers• More environmentally friendly – no organic

solvents to dispose of or recycle

Detectors

Ambient Pressure IonizationFID : organicshydrogen-atmosphere FID: organometallicsthermionic detector: N, P containing compoundsECD: Halogenated/electronegative compoundsphotoionization detector: organics with less than 10.2eV ionization potentialion mobility detector: high electron/proton affinity compounds

OpticalUVD FT-IR Flourescence detector FPD (flame photometric detector): S, P containing compoundsCD (chemiluminescence detector): S, and easily oxidizable compoundsElement selective plasma emission detectorLSD (light scattering detector)

VacuumMS with (CI, EI, CE, API ionizer)

Can use either GC or LC detectors with adaptations-LC detector: in close cell, fluid maintain under pressure, but cooled liquid-GC detector: in open cell, fluid decompressed to gas

49

Detectors

• Most any detector used in GC or HPLC can be used.

• FID and UV detectors commonly used.

• Coupled Detectors

- MS

- FTIR

Detectors

• UV absorption and FID are most common• UV absorption with packed columns• FID with capillary SFC


SFC Comparison

52

SFC Comparison

J.W. King, H.H. Hill, and M.L. Lee. 1993 54

SFC Separations• SFC is a hybrid of gas and liquid

chromatography that combines some of the best features of each

• As in HPLC, variation of the mobile phase composition affects separation

• In SFC, mobile phase affinity for the analyte is a function of mobile phase density

• Density is controlled by controlling system pressure

• Highly polar samples are not easy to handle (high critical parameters & high reactivity)

SFC Advantages vs HPLC• Supercritical fluids have low viscosities

- faster analysis (5 to 10 X faster)

- less pressure drop across the column - the use of open tubular columns is feasible

• Column lengths from 10 to 20 m are used

• Can be used with a wide range of sensitive detectors

• Resolving power is ~5X that of HPLC

SFC Advantages vs GC

• Can analyze non-volatile, polar, or

adsorptive solutes without

derivatization.

• Can analyze thermally labile

compounds.

• Can analyze solutes of much higher

molecular weight.

• Provides rapid separations without the use of organic solvents

• Uses environmentally conscious technology

• Faster separation process– Decrease in resistance to mass transfer in column

• High resolution at lower temperature

• Low viscosity

• High diffusion coefficient– Greater diffusibility

SFC Advantages


59

SFC Disadvantages• SCF is a recent chromatography technique that requires

development in the following areas: Method development Hardware development Pump design Injection methodology

• Expensive technology Pressure & Temperature

• Cleaning is time consuming

Ind. Eng. Chem. Res. 2000, 39, 4531-4535

60

Applications• Fossil Fuels and Hydrocarbons• Agrichemicals• Drugs and Pharmaceuticals• Polymers• Explosives and Propellants• Lipids• Carbohydrates• Industrial Chemicals• Foods and Flavors• Natural Products• Metal Chelates and Organometallic Compounds• Enantiomers

61

Separation of polyaromatic hydrocarbons in coal tars

• Gradient of 50 – 100% acrylonitrile (CH2CHCN) over 40 minutes

GC: T gradient, 80C to 250C over the 50-minute retention time

SFC

• A pressure ramp is used in the solvent, such that solvent density rose from 0.225g/L to 0.7 g/ml over the retention period of 120 minutes.

Agrichemical Application• Pesticide detection• GC conditions:

– 15m x 200pm– Open tubular column– 0.15 q film thickness– Temp. program from 40 to

200°C at l0°C/min, then 4°C/min to 240°C

• SFC conditions:– 10m x 50pm– Open tubular column– 0.15 pm film thickness– CO2 100°C– Density program from 0.55 to 0.70g/mL at 0.005g/mL per min after a 10-min

GC SFC

Chrysene

J. W. KING, H. H. HILL, AND M. L. LEE

66

Natural Products Application• Cholesterol content of a fish oil

• Content of capsule was emptied into injection solvent n-hexane

• The oil-laden solution was directly injected into SFC & separated using density program

• Two peaks were eluted with in program density interval 0.40-0.42 g/mL

– Found by spiking the above solution with known amounts of alpha-tocopherol (vitamin E) and cholesterol

Journal of Chromatographic Science, Vol. 28, January 1990

67

68

Metabolized Camazepam by HPLC(Rat Liver Microsomes, 37 C, 90 min)

Time (min)0.0 15.0

1.32

51.

478

3.92

1

7.57

1

13.0

586.16

0

1.22

3

M

M9'

4

NCMZ

CMZ

M5, TMZ

Flow rate: 1.2 ml/minDetection: 230 nm

Column: Zorbax SB-C18, 150 x 4.6 mmMobile phase:ACN - 0.02 M phosphate

HPLC Analysis

buffer pH 7 ( 40 : 60 V/V )Camazepam(CMZ)Norcamazepam(NCMZ)Temazepam(TMZ)

M5,M4, M9' metabolites

Comparison of HPLC to SFC

69

Metabolized Camezapam by SFC(rat liver microsomes, 37 C, 90 min)

CMZ

M5

M4

TMZM7

M9'M9

mAUmAU

50

100

150

200

250

300

350

400

0

1 2 3 4 5 6 7 8 9

Time (min)

Camazepam (CMZ)

Norcamazepam (NCMZ)Temazepam (TMZ)M4, M5, M7, M9, M9' metabolites

Column: LichrosphereMobile phase: 13% ethanol in CO

Flow rate: 2.5 ml/minOutlet pressure: 150 barOven temperature: 30Detection: 227 nm

then 3%/min to2 for 4 min,

SFC Analysis

NH 2 , 250 x 4.6 mm

30%

C

NCMZ

70

Identification of Metabolites using UV Spectra

Time

Norm.Norm.Spectrum of M5

50

100

150

200

250

300

350

400

0200 225 250 275 300 325 350 375

2.3

16

M5

CMZ

2.9

41

M4

3.7

03

mAUmAU

050

100150200250300350400

Time (min)

6 82 4 5 9731

Spectrum of M4

Spectrum of CMZ

71

Identification of Metabolites Using UV Spectra

Time (min)

0

50

100

150

200

250

300

350

400

CMZ

Norm.Norm.Spectrum of CMZ

200 225 250 275 300 325 350 375

2.31

6

mAU

150200250300350400

4.46

1

CMZ

41 5 63 7 8 92

150100

0

Spectrum of the peak at 4.461 min

72

Tricyclic Antidepressants

N N

S

N

Cl

CCOCH CH N(C2 2 2H5)2

OH

O

CH CH2 2

SCH 3

N

CH 3

CH CH OCH CH OH2 2 2 2

CH CH OH2 2CH CH CH2 2 2

C(CH )3 3CH2

Cl

S

N Cl

N N

CH N N

CH

Benactyzine6.Buclizine 7.

8.Hydroxyzine

10.Thioridazine9.Perphenazine

Effect of Organic modifier

73

Packed Column Analysis of Tricyclic Antidepressants

2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00

Time (min)

0

5

10

15

mAU

2.001.501.000.50

45

3

2

1. Amitriptyline2. Imipramine3.4. Desipramine5. Protriptyline

1

NortriptylineColumn : 4.6 x 250 mm LichrosphereMobile phase: COFlow rate:Column temperature: 50Outlet pressure: 200 barDetector: UV - 254 nm

2 + 10% MeOH + 0.5% Isopropylamine

C3 ml/min

Conditions:Cyanopropyl (5 m)

5(9.5 ppm each; l injected)

10% Methanol

Effect of Organic modifier

74

Packed Column Analysis of Tricyclic Antidepressants

1.00 1.50 2.00 2.50 3.00 3.50Time (min)

0

5

10

15

20

25

mAU

0.50

1. Amitriptyline2. Imipramine3.4. Desipramine5. Protriptyline

Nortriptyline1

2

3

4

5

Column : 4.6 xMobile phase: CO + 20% MeOH + 0.5% IsopropylamineFlow rate:Column temperature: 50 COutlet pressure: 200 barDetector: UV - 254 nm

2250 mm Lichrosphere

3 ml/min

Conditions:

Cyanopropyl (5 um)

ppm each; 5 injected)(9.5 ul

20% Methanol

75

Steroids

17 Methyltestosterone

Cortisone

Estradiol

3

Testosterone

3

Progesterone

Hydrocortisone

Estriol

3

3

CH3OH

CH3

O

CH3

O

O H

C=O

CH2 O H

3CHO

CH3

OH

CHOH

O

CH3

H C3 OH

O

CH3

H CC=O

CH3

O

OO HH

CH3

C=O

CH2 O H

3CH

O

OH

H

H

OHC

O

O

H

H C

Estrone

Same columns used in HPLC can be used in SFC

76

Packed Column Analysis of Steroids on Different Silica Columns

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

Res

po

ns

e

60 C, 3 mL/min, 250 bar, 6% methanol2.1 x 200 mm Hypersil

215 nm

210 nm

70 C, 2.5 mL/min, 200 bar, 20% methanol2.1 x 250 mm Lichrosphere Si-60

Progesterone,Estrone,Estriol

Estradiol, Cortisone, Hydrocortisone,Methyltestosterone, Testosterone

Time (min)

77

Packed Column SFC for Pharmaceutical Applications

carbobenzyloxy-d,l-alanine

20% ETOH(+0.2% TPA)/CO2 1ml/min250 x 4.6 mm Chiralcel-OD

4.0 4.50 5.0 5.5 6.0 6.5Time (min)

Chiral Purity Analysis

1.06% -l-

1.76% -d-

220 nm

220 nm

Needs:

€ Chiral compounds

€ Excipients

€ Drugs

€ Drug/metabolites

Key Applications:

€ Productquality assurance

€ Fast method development

€ High throughput

€ Complementarytechniques

78

Effect of Additive on Chiral Separation of Propranolol by Packed Column SFC

Time (min)0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

0

10

20

30

40

50

mAU

60

without additive100 ppm R,S-propranolol

30% in CO2 ml/min200 bar outlet pressure30 C oven

2methanol

temperature

10 m particlesChiralcel OD 4.6 x 250 mm,

79

Effect of Additive on Chiral Separation of Propranolol by Packed Column SFC

Time (min)

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

0

50

100

150

with additive100 ppm R,S-propranolol

30% in CO2 ml/min200 bar outlet pressure30

2

C oven

methanol with 0.5% isopropylamine

temperature

10 m particlesmm,Chiralcel OD 4.6 x 250

80

Fast Chiral Separation of Propranalol by Packed Column SFC

4030% (Methanol + 0.5%Tripropylamine)

Time(min)

C, 250 bar, 4 mL/min

Rs= 3.5

S

R

mAU

300

250

200

150

100

50

0

250x 4.6mm,10 mChiracel OD

References• M. Perrut. Supercritical Fluid Applications: Industrial Developments

and Economic Issues. Ind. Eng. Chem. Res., 39, 12, 2000

• J. W. King. Applications of Capillary Supercritical Fluid Chromatography-Supercritical Fluid Extractions to Natural Products. Journal of Chromatographic Science, 28, 1990

• W. Majewski, E. Valery, O. Ludemann-Hombourger. Principle and Applications of Supercritical Fluid Chromatography. Journal of Liquid Chromatography & Related Technologies, 28, 1233–1252, 2005

• H. S. Bhatt, G. F. Patel, N. V. Vekariya1, S. K. Jadav. Super critical fluid chromatography-an overview. Journal of Pharmacy Research, 2(10),1606-16011, 2009

• J. W. King, H. H. Hill, M. L. Lee. Analytical Supercritical Fluid Chromatography and Extraction. Ind. Eng. Chem. Res., Vol. 39, No. 12, 1993

81

Capillary SFC• capillary SFC can be used to separate the analytes of interest from a

relatively nonvolatile sample matrix without resorting to sample preparation prior to chromatography.

• capillary SFC include the separation of reaction products from higher molecular weight starting materials and the deformulation of commercial products.

• Capillary SFC, when coupled with micro-scale SFE also permits the characterization of small samples, such as portions of single seeds and extractables from single, live insects.

82Journal of Chromatographic Science, Vol. 28, January, 1990 82

PROBLEMS

• What controls the pressure in SFC?• The restrictor

• Why is a modifier fluid needed?• To help elute polar analytes

• List one advantage of SFC compared to HPLC and GC? • can analyze high MW, non-volatile compound, higher resolution compare to GC (see slide

17)

• Compare the plate number (N) between an open tubular (Ds = 1x10-6cm2/s, dc = 50um, df = 0.25um, L = 10m, u = 5.8cm/s) and packed column (ko = 0.5, dp = 5um, L = 0.1m, u = 2.1cm/s). k=2, and Dm = 2x10-4cm2/s.

• OT (N) = 19000, Packed (N)= 9100

83

Important Info Web pages• http://www.google.com/imgres?imgurl=http://www.chem.leeds.ac.uk/People/CMR/images/scco24.jpg&imgrefurl=http://

www.chem.leeds.ac.uk/People/CMR/criticalpics.html&usg=__y5nA5mu0N3cnYx3-cqlUrb7dKik=&h=294&w=308&sz=54&hl=en&start=3&zoom=1&um=1&itbs=1&tbnid=chFV9_BtWriMMM:&tbnh=112&tbnw=117&prev=/images%3Fq%3Dsupercritical%2Bfluid%26um%3D1%26hl%3Den%26sa%3DN%26tbs%3Disch:1

• http://en.wikipedia.org/wiki/Supercritical_fluid

84


Combines some advantages of liquid chromatography with the high efficiency

of gas chromatography.

Instrumentation and Operating VariablesInstruments for supercritical-fluid chromatography are similar in design to high-performance liquid chromatography.

ColumnsBoth packed columns and open tubular columns are used in supercritical fluid chromatography. Packed columns have the advantages of greater efficiency per unit time and the capability of handling larger sample volumes. Packed columns for SFC can be much longer than those for HPLC, providing well over 100,000 plates. Because of the low viscosity of supercritical media, columns can be much longer than those used in liquid chromatography, and column lengths of 10 to 20 m and inside diameters of 50 or 100 m are common.

Mobile PhasesThe most widely used mobile phase for supercritical-fluid chromatography is carbon dioxide. It is an excellent solvent for a variety of nonpolar organic molecules. In addition, it transmits in the ultraviolet and is odorless, nontoxic, readily available, and remarkably inexpensive relative to other chromatographic solvents. Its critical temperature of 31oC and its pressure of 73 atm at the critical temperature permit a wide selection of temperatures and pressures without exceeding the operating limits of modern high-performance liquid chromatographic equipment.

Detectors

A major advantage of supercritical-fluid chromatography is that the sensitive and universal detectors of gas-liquid chromatography are applicable to this technique as well. For example, the convenient flame ionization detector of gas-liquid chromatography can be applied by simply allowing the supercritical carrier to expand through a restrictor and into a hydrogen flame, where ions formed from the analytes are collected at biased electrodes, giving rise to an electrical current.

Supercritical-Fluid Chromatography versus Other Column Methods

Several physical properties of supercritical fluids are intermediate between the properties of gases and liquids. As a consequence, this new type of chromatography combines some of the characteristics of both gas and liquid chromatography. Thus, like gas chromatography, supercritical-fluid chromatography is inherently faster than liquid chromatography because of the lower viscosity and higher diffusion rates in the mobile phase.

Applications

It is applicable to a class of compounds that is not readily amenable to either gas-liquid or liquid chromatography. These compounds include species that are nonvolatile or thermally unstable and, in addition, contain no chromophoric groups that can be used for photometric detection. Separation of these compounds is possible with supercritical-fluid chromatography at temperatures below 100 oC; furthermore, detection is readily carried out by means of the highly sensitive flame ionization detector.

CHAPTER 29Supercritical Fluid Chromatography

• The mobile phase is a supercritical fluid (a fluid above its critical T and critical pressure)

• Supercritical fluid properties (density, viscosity, and refractive index) vary with T & P

Supercritical Fluids• At temperatures and pressures above its critical

temperature and pressure (critical point), a substance is called a supercritical fluid. The critical temperature is the temperature above which a distinct liquid phase cannot exist. The vapor pressure at its critical temperature is its critical pressure.

• Where supercritical fluids exist: The forces from the kinetic energy of the molecules exceeds the forces from condensing influence of the intermolecular forces, so no distinct liquid phase exists

SFC Mobile Phases

• Mobile phases should have critical parameters that are easily reached using chromatographic pumps and ovens common to currently used instrumentation.

• Advantages of supercritical fluids over carrier gasses and liquid mobile phases are in its solubility properties, physical properties, and detector compatibility.

Injectors

• Typical HPLC design injectors for packed columns.

• Split/Splitless valve injector (0.01 to 0.05 L injections) for open tubular columns.

• Timed - split injector (0.01 to 0.05 L injections) for open tubular columns.

Supercritical Fluid Chromatography SFC

Chromatographic Fundamentals

Practical Verification of SFC

Theoretical Description of SFC / Scale-up

SFC on a Preparative Scale: Examples Prostaglandins, Tocopherols DHA / DPA, Phytol

On-line Analysis with SFC

Continuous Chromatography: SMB

Chapter 8

Chromatography with Supercritical Fluids

.

Mode of Operation: Elution chromatography

Elution Chromatography: A Chromatogram

Mass transport high

Solvent power high

Schoenmakers, Uunk 1987

Different Mobile Phases

Composition Trade name Application

Polysiloxane

R, R':

separation according tomolecular weight

100 % methyl OV-1, SE-3095 % methyl, 5 % phenyl OV-3, SE-5294 % methyl, 1 % vinyl, 5 % phenyl SE-5425 % cyanopropyl, 50 % methyl,25 % phenyl

OV-225

polyethylene glycol( CH2 CH2 O )n

Carbowax 20 M separation according to po-larity

SFC: Stationary Phases

Separation of aromatic hydrocarbons with different gases as mobile phase. Aromatic hydrocarbons: 1= benzene; 2 = naphthalene; 3 = fluorene; 4 = anthracene;5 = pyrene. Gases: a = carbon dioxide (CO2); b = nitrous oxide (N2O); c = propane (C3H8); d =propylene (C3H6); Column: 30 x 4.6 mm, unmodified silica gel. Initial pressure 12 MPa; Temperature296.15 K; Flow rate 670 cm3/min at STP (after Pickel /23/).

SFC: Different Gases as Mobile Phase

1 = caffeine;2 = theophylline;3 = theobromine;4 = xanthine(Randall 1984).

Variation of capacity ratios ofpolycyclic aromatic compounds due to modifier concentration (1.4-dioxane) in the mobilephase (n-pentane).P at column outlet 3.6 MPa;T = 513.15 K(Leyendecker et al. 1986).

SFC: Different Modifiers

Variation of retention times with temperatureof polycyclic aromatic components in n-butane at 4.5 MPa.1 = naphthalene; 2 = anthracene; 3 = pyrene; 4 = chrysene (Klesper and Leyendecker 1986).

Variation of retention timesof chrysene with pressure.Mobile phase n-butane(Klesper, Leyendecker 1986).

SFC: Influence of Pressure and Temperature

SFC: Pressure And Density Programming

Overloading by volume

Analytical injection

Overloading by concentration

Con

cent

ratio

n

Time

Chromatograms For Different Amounts of Injection

Adsorption Isotherms And Corresponding Chromatograms

SFC: Flow Scheme of Apparatus

Elution Chromatography: A Chromatogram

.// imismmr nntttk

,

e

m

s K

V

VKk

tm = residence time in the mobile phasetr = retention time of the solutek' = capacity ratio = volumetric phase ratio Vs / Vm

Vs = the volume of the stationary phase, to,.Vm = the volume of the mobile phase

,e

e

m

se

K

n

nKk

./andand smesssmmm vvvnVvnV

.s

me

ms

sme v

vK

Vv

VvKk

e = molar phase ratiov = molar volume of a phaseV = total volume of a phase

Capacity Ratio

Capacity factors of paraffinesas a function of density(after Mollerup et al. /18/).

Capacity Factors

.

2exp

2

1

/

2

n

nv

nKVV

Fc i

ism

iim

with n = number of stages for p:

Chromatographic Separation

Maximum of the peak: ;nvi

Number of theoretical plates:

;/ ism

i KVV

Vvn

Points of inflection:

nnvnnv rightilefti ,, and

Points of intersection with the base line:

;21and21 ,, nnvnnv rightilefti


Width of peak:

.4 nb

Time at which the peak maximum appears

.or/1 iiiriiir nktnkt

Number of equilibrium stages

.4

2

i

iri b

tn


./ jiji kks Selectivity

.

2

ji

rirjji bb

ttR

Resolution

Resolution of two peaks of similar compounds

.1

1

4

2/1

k

k

s

snR

ij

ijij


.1

116

2

2

k

k

s

sRn

ij

ijij


,1

822 2

2

uDk

dk

u

DdH

isi

Fiimps

Van Deemter


Height of theoretical stage Hs

for SFC and HPLCfor packed columnswith different particle diameters(after Gere et al.)


SFC Analytical Scale, hp

Influence of temperature

20 MPa; mobile phase:CO2/methanol (5.3 wt.%);column: 125 x 4 mm; 5 m LiChrosorb Si 60.

Preparative separation

Chromatograms of fractions

Upnmoor

1992

Separation of Prostaglandins

Shapes of peaks under overloading conditionsChromatograms of -tocopherol mixture under overloading conditionsUpnmoor, Brunner, 1992

Separation of Tocopherols

Influence of modifier concentration

Solutions of -tocopherol in chloroform.Injected volume: 10 ml;mobile phase: CO2/methanol;15 MPa; 293 K; column: 125 x 4 mm;5 m LiChrosorb Si 60.Upnmoor 1992

Separation of Tocopherols

82

84

86

88

90

92

94

96

98

0 1 2 3 4 5 6 7

250 x 4.6 pS 250 x 8.0 pS

specific productivity DHA [mg/cm3 h]

Are

a D

HA

G

C [

%]

1mg DHA/(h,cm3) * 500 ml = 0,5 g DHA/h

Some kg DHA: Fully automatized plant !

RF=0,842

Productivity: DHA / DPA Separation by SFC

Dynamic axial compressed SFC column;

Dimensions:ID = 30 mm, length of packing: 0 to 190 (type I), 0 to 450 mm (type II) Pmax 400 bar, Tmax 200 °C.

SMB- Plant: Separation Columns

SFC, Preparative Scale

Rotating column Rotating ports

Continuous Chromatography

ExtractA + D

RaffinateB + D

FeedA + B + D

Desorbent D

Zone 1Purification of Adsorbent

Zone 3Enrichment of B

Zone 4 Purification of Desorbent

Zone 2Enrichment of A

True Moving Bed (TMB) Process

Principle of Simulated Countercurrent Separation

Mazzotti, ETH-Z

ExtractA+D

RaffinateB+D

FeedA+B

DesorbensD

Concentration A, B

Simulated Moving Bed-Process

Gottschall: PREP 95

Performance SMB vs Elution (99.5 % Purity)

Preparative SMB-Plant

Depta, 2000

0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,60

10

20

30

40

50

concentration [mg/ml]

q [

mg

iso

mer/m

l stat

ion

ary

ph

ase]

Measurements

20

22

24

26

28

30

32

34

dq/dc

dq

/dc

0,0 0,5 1,0 1,5 2,0 2,5 3,00

20

40

60

80

100

120

dq/dc

concentration [mg/ml]

q [

mg

iso

mer/m

l stat

ion

ary

ph

ase]

Measurements

30

32

34

36

38

40

42

44

dq

/dc

Adsorption isotherms for Phytol cis- and trans- isomer (black lines) and derivatives (red lines). 225 bar, 40 °C, 1.8 mass% isopropanol as modifier.

Isotherms exhibit a point of inflection for each isomer.

221

21s cbcb1

)cb2b(cqq

Adsorption Isotherms

3 4 5 6 7 8 90,0

0,2

0,4

0,6

0,8

1,0

1,2feed concentration:

2 mg/ml 5 mg/ml 10 mg/ml 20 mg/ml 50 mg/ml

conc

entr

atio

n [m

g/m

l]

retention time [min]

Experimental and simulated phytol chromatogramssymbols: experimental data; lines: simulations.

Batch-Simulations

Model: equilibrium, axially dispersed plug flow with variable velocity of mobile phase,Pressure drop: Ergun equation,Properties of mobile phase (CO2) calculated with equation of state.

t

q

z

cD

z

uc

z

cu

t

c iiapi

ii

1

02

2

SMB process modeled with four key parameters: the net flow ratios mj:

Ruthven, Storti.

)1()1( totalcolumn

totalcolumnshiftSMB

zone

solid

solidTMBzone

zone V

VtQ

Q

QQm

SMB-Simulation

SMB- SFC: Volume-flow is a function of column length.Therefore, net flow ratios are not constant in each zone.

)1(_*

totalcolumn

totalcolumnshift

phasemobileSMBzone

zone V

VtQm

New parameter:

Representation of SMB-SFC process in a (m2*-m3

*)-plane,

solution of mass balance equations with finite difference method [Kniep et al.], adapted to variable velocity of mobile phase.

The algorithm is fast enough to calculate the region of complete separation in the (m2

*-m3*)-plane numerically, taking into account:

• any type of isotherm equation• axial dispersion

• number of used columns• change in mobile phase density

SMB-Simulation

242526272829303132333424

26

28

30

32

34

36

242526272829303132333424

26

28

30

32

34

36

operating point

raffinate (cis-isomer) pure

extract (trans-isomer) pureraffinate +extract pure

black triangles:infinite dilution situation and infinite number of theoretical plates same parameter set

as operating point in figure 5

Region of complete separation for phytol Cfeed=5.0 mg/ml 230 bar, no pressure drop, columns: 2/2/2/2; 300 plates per column

Columns: 1/1/1/1; 1000 plates per column

SMB-Simulation: Phytol Separation

20 25 30

20

25

30

35

20 25 30

20

25

30

35

Influence of pressure drop:

raffinate (cis-isomer) pure

extract (trans-isomer) pureraffinate +extract pure

Region of complete separation for phytol, infinite dilution, columns: 2/2/2/2; 300 plates per column, 230 bar, no pressure drop

Same as in left figure but calculations with pressure drop

Pressure drop leads to a shift of the complete separation region to lower values of m2

* and m3*

SMB-Simulation: Phytol Separation

1 2 3 4 5 6 7 80

1

2

Run N

Extract FeedRaffinate

1 2 3 4 5 6 7 80

1

Run M

1 2 3 4 5 6 7 80,0

0,5

1,0

1,5

2,0

2,5

Run O

ExtractFeedRaffinate Extract FeedRaffinate

7 8 9 10 117

8

9

10

11

m3

m2

low concentration in Feedlinear Adsorption isothermIdeal model

1 23

Experimental Results of Ibuprofen Separation

-10 0 10 20 30 40 50 60 70 80

0123456789

101112

peak

are

a [m

V*m

in]

conc

entr

atio

n [g

/l]

length [cm]

Sim S(+) Sim R(-) Exp S(+) Exp R(-)

0

2

4

6

raffinateextract

140 mgRacemate/min; 2/2/3/1 configuration

Separation of Ibuprofen

Verunreinigungen PhytolisomereConditions of separation:

240 bar, 50°C,column 4 x 250 mm packed withLiChrospher 100 (Silica),flow 2,56 g carbon dioxide / min,modifier 3 wt.- % EtOH,productivity 45 mg/(ml, h).

17mg pur

0,85 mg in Hexan

OH

CH3

CH3

CH3

CH3H H CH3

Phytol

• Diterpene-alcohol,• Intermediate for vitamin E, K1• esterified lipophilic compound

of chlorophyll

144


145

Breaking the PurificationBottleneck

BERGER SFC

HPLC

A Mettler-Toledo Company

146

Supercritical fluid chromatography

• Supercritical fluid is used as the mobile phase.

• SFC combines advantages of gas and liquid chromatography.

• Critical temperature exists in which a substance can no longer be converted into the liquid phase even if higher pressures are applied.

• The vapour pressure belonging to the critical point is the critical pressure.

• The fluid is in a supercritical state close to the critical point and has characteristics which lie somewhere between those of gases and liquids.

• Characteristics particularly important for chromatography on the critical point are the density, the viscosity, and the diffusion coefficient.

147

• Typically supercritical fluids are used at densities ranging from 0.1 to 0.8 of their liquid density and 100 - 1000 times greater than that of a gas at ambient temperature.

• Pressures range from 50 atm to 500 atm.• Diffusion coefficients of supercritical CO2 varies between 10-4 and 10-3 cm2s-

1.• Liquids typically have diffusivities of less than 10-5 cm2s-1.• The viscosities of supercritical fluids mirror their diffusivities and are typically

10 - 100 times lower than for liquids.

• Supercritical fluids are not only used for chromatography but also for supercritical fluid extraction.1) caffeine-free coffee can be produced by the extraction from coffee 2)cigarettes with a low nicotine content can be produced in

the extractive separation of nicotine from tobacco

148

Instrumentation

• Precise temperature setting must take place in the column.• This is achieved with a thermostatted column oven as in GC.• To achieve subambient temperatures cooling with an auxiliary CO2 tank is

employed- Usually to ~ -10 to -50C.

• Pressure in system must be precisely monitored since the density of the supercritical fluid alters with the pressure and pressure differences lead directly to a change in the capacity factors.

• Higher pressure provides greater density.• This causes elution power of the mobile phase to increase and shorter retention

times are observed. • Pressure programming in SFC as a gradient in the same way we are familiar

with it for temperature in GC or the composition of the mobile phase in HPLC.

149

The range of solvating power of practical supercritical fluids for SFC is of primary importance, and ultimately defines the limits of the application.

The solubility of the analytes typically increases with density, and a maximum rate of increase in solubility with pressure is generally observed near the critical pressure where the rate of the increase of density with pressure is the greatest.

Increase pressure--> Increase density---> should decrease kIncrease temperature--> Decrease density --> should increase k to a certain temperature until other variables start playing predominant role.

150

Stationary Phases

• Stationary phase can be either a packed column or be set up in a capillary.

• Packed columns are typical HPLC columns

• In fused silica capillaries the stationary phase is applied in the form of siloxanes as liquids or chemically bonded to the inner wall.

• The usual measurement of the capillaries are lengths of between 10 and 20 m with inner diameter of between 0.05 and 10 mm and film densities of 0.05 to 1 mm.

151

Mobile Phases• Carbon dioxide is most frequently used as the mobile phase.

• Cheap, nonpoisonous, odorless and does not absorb in the UV range up to 190 nm.

• The critical CO2 data are such that the temperature and the pressure can be varied over relatively wide ranges.

• Organic modifiers are often employed such as methanol, ethanol, IPA, n-propanol, dioxane.

• The addition of these modifiers however change the critical point of the system.

• Typically at even low levels of organic modifier the supercritical region is traversed. That is because the addition of the modifier shifts the critical point to higher temperatures and pressures.

152

Detectors

• FID detection

• Coupling of mass spectrometry is considerably easier to achieve by comparisons to LC and has been used.

• UV, IR, Fluorescence, Flame photometric, TCD and ECD can also be employed.

- DAD for purity assessment - MS to verify presence of target - NCD to quantify on nitrogen content -FTIR

153

At constant pressure solute retention in SFC depends on temperature in a very characteristic manner.

Example: Ref N.Wu, J.Medina, J.Bradshaw, M.Lee, J.Microcolumn Separations, 12(8) 454-461 (2000)

• At moderate pressures (150 atm) increasing the temperature from ambient to critical (Tc,31.3oC), k values decrease and reach a minimum which resulted from an increase in solute solubility with temperature in liquid carbon dioxide.

• After passing Tc, k values increase significantly.

Effect of temperature on retention factor

155

1- The density of CO2 and solvent power decreases as the temperature increases, which results in an increase in k.

2- The vapor pressure of the analyte also increases at higher temperature, leading to an increase in solubility and thus a decrease in k.

• The second effect over compensates the first around 100oC(1/T=0.0028), resulting in a maximum in retention factor.

• At temperatures higher than 100oC , the second effect becomes more pronounced and k values decrease with increasing temperature.

• At high pressure the decrease in density with increasing temperature is not as important as the effect of temperature on solvating power. Therefore plots of k vs. 1/T are usually more flat. The two variables effects may compensate each other.

• Results indicate that low temperature is favored for fast chiral separation in SFC, at which both low retention factor and high selectivity can be obtained.

Effect of temperature on retention factor

157

Effect of temperature on efficiency

Efficiency generally increases with an increase in temperature

1) Mobile phase diffusivity increase with temperature at a constant pressure and increases N.2) Decreased density at higher temperatures lead to a drop in mobile phase viscosity which increases N.3) Retention factor increases which will increase N.

R.Stringham, J.Blackwell, Anal.Chem., 1996, 68, 2179-2185However it has been seen that efficiency has increased with temperature to a point and then levels off. Then as the critical temperature is transversed column efficiency declines. This is attributed to the adsorption of solvent to the stationary phase. The presence of this adsorbed layer would slow the kinetics of binding and hence a decline in column efficiency.

159

sR

1

1

4 2

2

k

kN

Resolution depends on column efficiency, solute retention and selectivity.

In SFC we can vary the variables to obtain the desired resolution.Ex. Increase the temperature will

* Decrease the CO2 density* May increase or decrease the retention factor* Vapor pressure of the analyte also increases* Increases rate of diffusion (increased efficiency)

Depending on what variable/variables predominate the Rs could improve or not.

Effect of temperature on resolution

160

Low temperature is beneficial because chiral selectivity usually increases with decreasing temperature

SFC often provides higher resolution per unit time than LC because mobile phases have low viscosities and small mass transfer resistance which lead to higher optimum linear velocities.

161

Effect of pressure

Pressure has a little effect on the selectivity

Pressure affects linear velocity, efficiency, and retention factor.

Therefore resolution depends on the effective pressure.

Retention factor decreases with increasing pressure in SFC since the density of CO2 and solvent power increases.

At increasing pressures mobile phases have greater viscosities and greater mass transfer resistance which may lead to decreased efficiency.

164

Breaking the Purification Bottleneck

What is Packed Column SFC?

A separation technique similar to HPLC Mostly same components as HPLC Almost always uses carbon dioxide as the main component in the mobile phase Almost always uses binary or ternary mixtures as mobile phase Usually perform gradient elution changing the composition of the mobile phase


165


Schematic Diagram of a Packed Column SFC


166

Practical Advantages of SFC

• SFC is Normal Phase– complimentary to reversed phase HPLC

• Diffusion is much Faster– higher speed/throughput-more samples/day– more rapid re-equilibration-shorter cycle time

• Non-Linear Solvent Strength-first small addition of polar additives dramatically increases solvent strength

• Viscosity is Much Lower– lower pressure drop, higher flows, or longer

columns possible

167

What Compounds Can SFC Separate?

• Any solute soluble in methanol or a less polar organic solvent will elute in SFC.

• Strong organic acids and bases require a modifier and an additive in the mobile phase.

• Most salts of organic acids and bases elute.• Small lipophylic peptides elute• With carbon dioxide based fluids, molecular

weights up to 15,000

168

min0.5 1 1.5 2 2.5 3 3.5 4 4.5

80% modifier

60% 40%

Vanomycin is Not Very Soluble in MeOH or in MeOH/CO2 Mixtures

Not to

Scale

Retention Time, minutes4

Conditions: 2.5 ml/min [MeOH +0.4% ibam] in CO2, 35°C, 120 bar, 4.6 x 150 mm, 5µm Zymor CN

Peptides with Very Limited Solubility in Methanol Yield Square Topped Peaks Which Indicates LimitedSolubility in the Mobile Phase

Limits of SFC


169

0.5 1 1.5 2 2.5 3 3.5 4

0

25% Methanol + 0.1% TFA in CO2

2.5 ml/min, 35°C, 120 bar4.6 x 150 mm, 5µm Zymor CN

4

Retention Time, minutes

UVAbsorbanceAt 240 nm

800 mAU

Actinomycin DSeparation of a Small Pepetide:

2

nm250 300 350 400 450

mAU

200

400

600

800

1000

1200

1400

Spectrum205-500 nm

Limits of SFC Some Peptides Elute Just FineA Mettler-Toledo Company

170

What Compounds Will NOT Elute in SFC?

• Any solutes requiring an aqueous environment

• any solutes requiring a buffered or ionic aqueous environment– I.E., Biomolecules such as Proteins

will NOT elute• inorganic salts will not elute• With carbon dioxide, few solutes over 15,000

MW elute

171

Sample Matrix?• Usually solution in organic solvent

– methanol or less polar allows larger injections

– can use DMSO – small (i.e., 0.5-1 µL) aqueous samples

direct• often reduced sample prep because SFC is

compatible with both lipids and polar molecules

• pills dissolved with excipients, etc. present• extracts from natural products• extracts from fermentation

172

Why Carbon Dioxide?• Miscible with much more polar solvents-wide range

of solvent strength available.– program composition from 0 to 100% organic

modifier

• Low operating temperatures (i.e., 30-50°C) Compatible with most HPLC and GC Detectors

• Relatively safe– product of human respiration– can use with the FID– easy disposal– low purchase price

• Lack of a superior alternative

173

phenylureas

triazinescarbamates

alcohols

phenols

benzylaminessulfonamides

hydrocarbons

PAH's

esters

acidsmonofunctional fatty acids

hydroxy acids

organophosphorusorganochlorine

sulfonylureas

aldehydes and ketones

ethers

anilines

polyacids

tertiary amines aminesamines

primary

secondary

< C 8C 18

CNsilica

diolSA

NH 2

phenyl

carbon dioxidepure

CO 2 ++CO 2

methanolmethanol + additives

Appropriate Mobile PhaseComposition

Polarity Rangesof Stationary Phases

Polarity Range of Solute Families

174

Instrumentation• Berger Instruments dual pump SFC with extended

flow range to 10 mls/min.• Autosampler: 1,2, 4 ml or microtiter plate accessory,

partial to full loop, multiple solvent, or sample washes, up to 2.5 ml syringe, cooling option

• Diode array detector, or variable wavelength UV• Solvent switching valve, column switching valve

available• ECD, NPD, FID GC like detectors available• Full function Chemstation for instrument control, data

acquisition, data analysis, report generation

175


THE CHROMATOGRAPHY DILEMMA

Gas Chromatography (GC) Old, well established (30 years)

>250,000 units in place; >500,000 sold; ~20,000 units/yr.

currently sold Fast, high resolution, wide range of sensitive

detectors Inexpensive @ $10K-$30K/unit Inexpensive to operate Non-polluting APPLICABLE TO ~10% OF ALL MOLECULES


176

THE CHROMATOGRAPHY DILEMMA (cont.)

High Pressure Liquid Chromatography (HPLC) Well established (20 years)

>100,000 units in place; >250,000 sold; ~20,000 units/yr.

currently sold Slow, moderate resolution, limited range of

insensitive detectors Relatively expensive @ $25K-$60K/unit Expensive to operate Polluting APPLICABLE TO ~90% OF ALL MOLECULES


177

Supercritical Fluid Chromatography (SFC) Relatively new (5-10 years)

>1,000 units in place; >2,000 sold; ~200 units/yr. currently sold 3-5 times faster than LC 2-3 times better resolution than LC Uses both GC and LC detectors Hardware is priced 1.3 times LC pricing Inexpensive to operate Non-polluting APPLICABLE TO ~40% OF ALL MOLECULES


THE CHROMATOGRAPHY DILEMMA (cont.)

178

Berger SFC Separation of Antidepressants


min0.25 0.5 0.75 1 1.25 1.5 1.75

Retention Time, minutes 2.00

UV AbsorbanceAt 220 nm

Antidepressants, 2 µL 20 ppm each

5 ml/min 2.5-55% {MeOH + DMEA] in CO2 at 30%/min

35°C, 100 bar, 2 µl injected

Column: 4.6 x 150 mm, 5µm (ES Industries CN-BD Column)

AmitriptylineImipramineNortriptylineDesipramineProtryptyline

1.0

179


The Analytical SFC Opportunity

Pharmaceutical Chiral Drugs CombiChem/High Throughput Screening

Linked closely to the success of PrepSFC, SFC/MS

and quantitative detection Petrochemical

Aromatics in Diesel Fuel Other ASTM methods awaiting approval

Agricultural and Other Chemical Products


SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

CompoundCritical Temp.

(oC)Critical Press.

(atm)Critical Dens.

(g/mL)CO2 31.3 72.9 0.448NH3 132.3 111.3 0.24H2O 374.4 226.8 0.344CH3OH 240.5 78.9 0.272C2H5OC2H5 193.6 36.3 0.276


• SFC is a hybrid of HPLC and GC– Uses HPLC like equipment – Detector is usually a flame ionization detector, although

mass selective, and spectrophotmetric detectors can be used

• Useful for compounds that – Are non volatile or thermally unstable and– Cannot be used with spectophotometric or

electrochemical detectors

• Properties of supercritical fluids

Supercritical Fluid Chromatography-I• Instrumentation

– Similar to HPLC as pressures are like that of HPLC– Need an oven like GC to keep temperature above critical

temperature of mobile phase– A restrictor is required at the end of the column to maintain

the column pressure above that of the critical pressure of the mobile phase

– Instead of temperature programming used in GC, pressure programming, which increases super critical fluid density, is used to modify the solvent power of the mobile phase to control retention times

– Columns• Wall coated open tubular columns made from fused silica• Usually chemically bonded liquid stationary phase• 0.05-0.10 mm ID• Film thickness: 0.05-1.0 mm• 10-60 m long• Packed HPLC columns can also be used

3

Supercritical Fluid Chromatography -II• Mobile phase used most is CO2 or CO2 with some dissolved methanol

which increases the solubility of polar compounds– Nontoxic– Low TC and PC– Easily volatilized at the end of the separation, leaving solute analytes in the

gas phase for flame ionization detection• The FID does not respond to CO2, whereas the mass spec does

• Comparison to HPLC and GC: based on the fact that supercritical fluids have properties intermediate between gases and liquids– Faster elutions than HPLC because the viscosity of mobile phase less than

liquids allowing for higher flow rates– Lower band broadening than HPLC but greater than GC because the

diffusion coefficient of solutes in supercritical fluids is intermediate between that found for gases and liquids

• The overall result is that resolution is greater than HPLC but less than GC• Speed of analyses is greater than HPLC but less than GC

• Some figures of merit by example– See Fig. 27-3 in Skoog, et al.

• At m = 0.6 cm/s, SFC gives HETP of 0.013 mm, HPLC gives 0.039 mm– This means zone broadening is reduced by

• HETPminimum is 0.15 cm/s for HPLC, 0.6 cm/s for SFC– This means the same resolution can be obtained in one-forth the time

– See Fig. 27-4 in Skoog, et al.

SCF-III• One disadvantage of SFC is that selectivity cannot be modified

in the same way as with HPLC by using gradient elution. This is important for large, nonvolatile molecules

• Applications: see Figs. 27-6, 27-7, 27-8 in Skoog, et al.• Capillary Zone Electrophoresis• Electrophoresis is a separation that is produced by differential

migration of species in an electric field– In pure electrophoresis, this is not a chromatographic separation

because no partitioning of dissolved analytes occurs between a mobile and stationary phase

– It is generally applied to ionic substances, although some new techniques allow separation of neutral molecules by combining electrophoresis with chromatography

H igh Volt ageS upply

2 0 - 30 kV

det ect or

F used S ilicaCapillar y

5 0 - 10 0 cm15- 10 0 m I D

Bot h beaker sfi lled wit h a

buff er

6-Supercritical Fluid Chromatography SFC.pptx

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