6-supercritical Fluid Chromatography Sfc 3u4g3

Supercritical Fluid Chromatography Aurelia Leyva and Trang Duong Chem 230 11/30/2010 1

Chapter 33 Supercritical-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 highperformance 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

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

W.W. Christie and Lipid Technology

T/P Phase Diagram

www.che.tohoku.ac.jp

4

• Supercritical fluids have solvent power similar to a light hydrocarbon for most solutes. However, fluorinated compounds are often more soluble in scCO 2 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

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

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

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Schematic Diagram of a Packed Column SFC

14

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 31.3 72.9 • CO2 • C2H4

9.9

Pc atm 0.96

50.5

• N2O

36.5

72.5

• NH3

132.5 112.5

--0.94 0.40

• n-C5

196.6 33.3

0.51

• n-C4

152.0 37.5

0.50

• CCl2F2 • CHF3

111.8 25.9

d*

40.7 46.9

1.12 ----

Supercritical Fluid Chromatography (SFC) Property Density (g/cm 3 ) Diffusion coefficient (cm 2 /s) Viscosity (G Cm -1 s -1 )

Gas (STP) (0.6-2) x 10 -3 (1-4) x 10 -1

SCF 0.2-0.5 10 -3 x 10 - 4

Liquid 0.6-2 (0.2-2) x 10 -5

(1-4) x 10 - 4

(1-3) x 10 - 4

(0.2-3) x 10 -2

•

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

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Types of Injectors 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

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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.

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

• Packed columns – Injection volumes >1uL

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Column Open 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

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28

Column 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

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Column Retention Time

-Packed columns are more efficient with higher density analytes.

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

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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

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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

Mobile Phase Neat Fluid • CO2 low Tc, low toxicity, nonflammability • N2O high degree of nonideality, permits high fluid density when compressed • Isopropyl/diethyl ether separation of polynuclear aromatics and polymers. • Hydrocarbon limited use due to high flammability • n-Pentane separation of oligomers • Flourocarbon low Tc, unique selectivity for certain solutes 3 6

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

36

Mobile Phase 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

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

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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

40

120

0.82 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 &

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 nonpolar 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 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 Ambient Pressure Ionization FID : organics hydrogen-atmosphere FID: organometallics thermionic detector: N, P containing compounds ECD: Halogenated/electronegative compounds photoionization detector: organics with less than 10.2eV ionization potential ion mobility detector: high electron/proton affinity compounds Optical UVD FT-IR Flourescence detector FPD (flame photometric detector): S, P containing compounds CD (chemiluminescence detector): S, and easily oxidizable compounds Element selective plasma emission detector LSD (light scattering detector)

Vacuum MS with (CI, EI, CE, API ionizer)

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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

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

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SFC Comparison

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

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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

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 compounds.

thermally

labile

• Can analyze solutes of much higher molecular weight.

SFC Advantages • 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

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

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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

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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 6 1

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

Chrysene

– 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

GC

SFC

0.70g/mL at 0.005g/mL per min after a 10-min J. W. KING, H. H. HILL, AND M. L. LEE

6 6

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

6 Journal of Chromatographic Science, Vol. 28, January 1990 7

Comparison of HPLC to SFC

Metabolized Camazepam by HPLC (Rat Liver

Microsomes,

37

C, 90 min)

M5, TMZ

Column: Zorbax SB-C18, 150 x 4.6 mm Mobile phase: ACN - 0.02 M phosphate buffer pH 7 ( 40 : 60 V/V ) Flow rate: 1.2 ml/min Detection: 230 nm

CMZ

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

7.571 3.921

1.223 1.325 1.478

13.058

6.160

HPLC Analysis

M 4

M9' NCMZ

0.0

Time (min)

68

15.0

Metabolized Camezapam by SFC (rat

liver

mAU

microsomes,

37

C, 90 min)

CMZ

SFC Analysis Column: Lichrosphere NH 2 , 250 x 4.6 mm Mobile phase: 13% ethanol in CO 2 for 4 min, then 3%/min to 30% Flow rate: 2.5 ml/min Outlet pressure: 150 bar Oven temperature: 30 C Detection: 227 nm

400

350

300

M5 250

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

200

150

100

M4 NCMZ

50

M9'

TMZ

M7

M9

0 1

2

3

4

5

Time (min)

6

7

69 8

9

Identification of Metabolites using UV Spectra

1

2

3

M4

3.703

2.316

CMZ

2.941

M5

mAU 400 350 300 250 200 150 100 50 0

4

5

6

7

8

9

Time

Norm.

400

Spectrum of M4 Spectrum of M5 Spectrum of CMZ

350 300 250 200 150 100 50 0 200 225 250 275 300 325 350 375 Time (min)

70

CMZ

1

2

4.461

mAU 400 350 300 250 200 150 150 100 0

2.316

Identification of Metabolites Using UV Spectra

3

4

5

6

7

8

9

Time (min)

Norm.

Spectrum Spectrum

of the peak at 4.461 min of CMZ

400 350 300

CMZ

250 200 150 100 50 0 200 225 250 275 300 325 350 375

71

Effect of Organic modifier

Tricyclic Antidepressants

6.Buclizine

7. Benactyzine OH

CH

N

N

CH 2

C(CH ) 3 3 CCOCH2CH2N(C2H 5) 2

8. Hydroxyzine

Cl

CH

9.Perphenazine Cl

N

N

O

CH2CH2OCH 2CH2OH

10.Thioridazine CH 3

CH2CH2CH2 N

N Cl

N

CH2CH2OH

N CH2CH 2 SCH 3

N S S

72

Effect of Organic modifier

Packed Column Analysis of Tricyclic Antidepressants (9.5 ppm each; 5 3

mAU 15

l injected) 1. 2. 3. 4. 5.

Conditions: Column : 4.6 x 250 mm Lichrosphere Cyanopropyl (5 m) Mobile phase: CO 2 + 10% MeOH + 0.5% Isopropylamine Flow rate: 3 ml/min Column temperature: 50 C Outlet pressure: 200 bar 1 Detector: UV - 254 nm

Amitriptyline Imipramine Nortriptyline Desipramine Protriptyline

4 5

10 2

5

0

0.50

1.00

1.50

2.00

2.50

3.00

3.50 Time

4.00

4.50

5.00

5.50

6.00

(min)

10% Methanol

73

6.50

7.00

20% Methanol

Packed Column Analysis of Tricyclic Antidepressants (9.5 ppm each; 5 ul injected) mAU

25

20

3

Conditions: Column : 4.6 x 250 mm Lichrosphere Cyanopropyl (5 u m) Mobile phase: CO 2 + 20% MeOH + 0.5% Isopropylamine Flow rate: 3 ml/min 1 Column temperature: 50 C Outlet pressure: 200 bar 2 Detector: UV - 254 nm

4

15

1. 2. 3. 4. 5.

Amitriptyline Imipramine Nortriptyline Desipramine Protriptyline

5

10

5

0 0.50

1.00

1.50 2.00 Time (min)

2.50

3.00

74

3.50

Same columns used in HPLC can be used in SFC

Steroids

H 3C

OH H3C

CH 3

OH

CH 3 H3C

C=O

O

HO

CH 3

Testosterone

Estradiol CH 2 O H

H3C O

O

Progesterone

O

H3 C

C=O OH

CH 3 HO

CH 2 O H

Estrone HO H3C

H3 C

O

Cortisone

C=O OH

OH CH 3

CH 3

OH OH

CH 3

O

Hydrocortisone

HO

Estriol

CH 3

O

17 Methyltestosterone 75

Packed Column Analysis of Steroids on Different Silica Columns 2.1 x 200 mm Hypersil 60 C, 3 mL/min, 250 bar, 6% methanol

R es p o n se

215 nm Progesterone, Methyltestosterone, Testosterone Estrone, Estradiol, Cortisone, Hydrocortisone, Estriol

2.1 x 250 mm Lichrosphere Si-60 70 C, 2.5 mL/min, 200 bar, 20% methanol 210 nm 0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80 Time

2.00

2.20

2.40

2.60

2.80

3.00

(min)

76

3.20

3.40

Packed Column SFC for Pharmaceutical Applications Needs: € Product quality assurance € Fast method development

Chiral Purity Analysis

€ Complementary techniques

1.06% -l-

€ High throughput

220 nm

Key Applications: € € € €

Chiral compounds Excipients Drugs Drug/metabolites

carbobenzyloxy-d,l-alanine

1.76% -d4.0

4.50

5.0

5.5 Time (min)

220 nm

6.0

6.5

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

77

Effect of Additive on Chiral Separation of Propranolol by Packed Column SFC mAU 60 50 40

without additive

100 ppm R,S-propranolol Chiralcel OD 4.6 x 250 mm, 10 30% methanol in CO 2 2 ml/min 200 bar outlet pressure 30 C oven temperature

m particles

30 20 10 0

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 Time (min)

78

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

100 ppm R,S-propranolol

150

Chiralcel OD 4.6 x 250 mm, 10 m particles 30% methanol with 0.5% isopropylamine in CO 2 2 ml/min 200 bar outlet pressure 30 C oven temperature

with additive

100

50

0

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

Time (min)

79

7.00

7.50

Fast Chiral Separation of Propranalol by Packed Column SFC mAU 300

250

200

250x 4.6mm,10

R

m Chiracel OD

40 C, 250 bar, 4 mL/min 30% (Methanol + 0.5% Tripropylamine)

S

150

100

Rs = 3.5 50

0

Time(min)

80

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

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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.

Journal of Chromatographic Science, Vol.

28, January, 1990

82 8 2

PROBLEMS •

What controls the pressure in SFC? •

•

Why is a modifier fluid needed? •

•

To help elute polar analytes

List one advantage of SFC compared to HPLC and GC? •

•

The restrictor

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

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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.ht ml&usg=__y5nA5mu0N3cnYx3cqlUrb7dKik=&h=294&w=308&sz=54&hl=en&start=3&zoom=1&um=1&itbs=1&tbnid=chFV9_BtWriMMM:&tbnh=112&tbnw=117&prev=/im ages%3Fq%3Dsupercritical%2Bfluid%26um%3D1%26hl%3Den%26sa%3DN%26tbs%3Disch:1

•

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

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Supercritical Fluid Chromatography Combines some advantages of liquid chromatography with the high efficiency of gas chromatography.

Instrumentation and Operating Variables Instruments for supercritical-fluid chromatography are similar in design to high-performance liquid chromatography. Columns Both 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 Phases The 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

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 gasliquid 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 o C; furthermore, detection is readily carried out by means of the highly sensitive flame ionization detector.

CHAPTER 29 Supercritical 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

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.

Chapter 8 Chromatography with Supercritical Fluids

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

Mode of Operation: Elution chromatography

.

Elution Chromatography: A Chromatogram

Different Mobile Phases Mass transport high

Solvent power high

Schoenmakers, Uunk 1987

SFC: Stationary Phases Composition

Trade name

Polysiloxane R, R': 100 % methyl 95 % methyl, 5 % phenyl 94 % methyl, 1 % vinyl, 5 % phenyl 25 % cyanopropyl, 50 % methyl, 25 % phenyl polyethylene glycol ( CH2  CH2  O  )n

Application separation according molecular weight

to

OV-1, SE-30 OV-3, SE-52 SE-54 OV-225 Carbowax 20 M

separation according to polarity

SFC: Different Gases as Mobile Phase

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; Temperature 296.15 K; Flow rate 670 cm 3/min at STP (after Pickel /23/).

SFC: Different Modifiers 1 = caffeine; 2 = theophylline; 3 = theobromine; 4 = xanthine (Randall 1984).

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

SFC: Influence of Pressure and Temperature

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

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

SFC: Pressure And Density Programming

Chromatograms For Different Amounts of Injection

Concentration

Analytical injection

Overloading by volume

Overloading by concentration

Time

Adsorption Isotherms And Corresponding Chromatograms

SFC: Flow Scheme of Apparatus

Elution Chromatography: A Chromatogram

Capacity Ratio

k    tr  tm  / tm  nis / nim . tm = residence time in the mobile phase tr = retention time of the solute k' = capacity ratio  = volumetric phase ratio Vs / Vm Vs = the volume of the stationary phase, to,. Vm = the volume of the mobile phase

Vs K e k  K  , Vm 

n s K e k   K e  , nm  e

Vm  nm vm

and Vs  ns v s and     e vm  / v s .

e = molar phase ratio v = molar volume of a phase V = total volume of a phase

k   K e

vm Vs K e vm  . v s Vm  vs

Capacity Factors Capacity factors of paraffines as a function of density (after Mollerup et al. /18/).

Chromatographic Separation

with n = number of stages for p:

Fi cim  Vm  Vs / K i

  vi  n  1 exp   2n 2 n 



2



.

Chromatographic Separation Maximum of the peak:

Number of theoretical plates:

vi  n ; V n  vi ; Vm  Vs / K i

Points of inflection:

vi,left  n  n and vi,right  n  n Points of intersection with the base line:

vi,left  n  1  2 n and vi,right  n  1  2 n ;

Chromatographic Separation Width of peak:

b  4 n.

Time at which the peak maximum appears

tr i  1 / ki  ni or tr i  ki ni . Number of equilibrium stages

2

 4 tr i   . ni    bi 

Chromatographic Separation

Chromatographic Separation Selectivity

si j  ki / k j .

Resolution

Ri j 

2 trj  tri  bi  b j

.

Resolution of two peaks of similar compounds

n1 / 2 sij  1 k  Rij  . 4 sij 1  k 

Chromatographic Separation

sij   1  k     n  16 R  .   s 1  k   ij  2 ij



2

Chromatographic Separation 2  8 k d 2  Dim i F Hs  2 d p   2 u, u  1  ki Dis

Van Deemter

Chromatographic Separation Height of theoretical stage Hs for SFC and HPLC for packed columns with different particle diameters (after Gere et al.)

SFC Analytical Scale, hp

Separation of Prostaglandins Preparative separation

Influence of temperature

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

1992

Chromatograms of fractions

Separation of Tocopherols

Shapes of peaks under overloading conditions

Chromatograms of -tocopherol mixture under overloading conditions Upnmoor, Brunner, 1992

Separation of Tocopherols 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

Influence of modifier concentration

Productivity: DHA / DPA Separation by SFC 250 x 4.6 pS 250 x 8.0 pS

98

Area DHA GC [%]

96 94 92 90

RF=0,842

88

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

86 84 82

0

1

2

3

4

5

6

7

3

specific productivity DHA [mg/cm h]

Some kg DHA: Fully automatized plant !

SMB- Plant: Separation Columns

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.

SFC, Preparative Scale

Continuous Chromatography Rotating column

Rotating ports

True Moving Bed (TMB) Process

Desorbent D Extract A+ D Feed A+B+D Raffinate B+D

Zone 1 Purification of Adsorbent Zone 2 Enrichment of A Zone 3 Enrichment of B Zone 4 Purification of Desorbent

Principle of Simulated Countercurrent Separation

Mazzotti, ETH-Z

Simulated Moving Bed-Process Concentration A, B Desorbens D Extract A+D Feed A+B Raffinate B+D

Performance SMB vs Elution (99.5 % Purity)

Gottschall: PREP 95

Preparative SMB-Plant

Depta, 2000

Adsorption Isotherms Isotherms exhibit a point of inflection for each isomer.

c(b1  2b 2c) q  qs 1  b1c  b 2c 2

50

30 30 28 20

26 24

10

Measurements

0 0,0

0,2

0,4

0,6

0,8

1,0

1,2

concentration [mg/ml]

22 1,4

20 1,6

42

100 80

dq/dc

40 38

60

36

40

34 Measurements

20 0 0,0

0,5

1,0

1,5

2,0

2,5

concentration [mg/ml]

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

32 3,0

30

dq/dc

32

dq/dc

q [mgisomer/mlstationary phase]

40

44

120

dq/dc

q [mgisomer/mlstationary phase]

34

Batch-Simulations feed concentration: 2 mg/ml 5 mg/ml 10 mg/ml 20 mg/ml 50 mg/ml

concentration [mg/ml]

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

3

4

5

6

7

8

9

retention time [min] Experimental and simulated phytol chromatogramssymbols: experimental data; lines: simulations.

SMB-Simulation 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.

ci ci  ci 1   qi u 0 u  ci  Dap 2  t z z z  t 2

SMB process modeled with four key parameters: the net flow ratios mj: Ruthven, Storti.

mzone

TMB SMB shift Qzone  Qsolid  Qzone t  Vcolumn total   Qsolid (1   ) Vcolumn (1   total )

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

m*zone 

SMB Qzone  mobile _ phaset shift  Vcolumn total

Vcolumn (1   total )

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 : •

•

any type of isotherm equation • axial dispersion • number of used columns change in mobile phase density

SMB-Simulation: Phytol Separation black triangles:infinite dilution situation and infinite number of theoretical plates

36 34

36 34

operating point

32 30 28 26

same parameter set as operating point in figure 5

raffinate (cisisomer) pure extract (transisomer) pure raffinate + extract pure

24 24 25 26 27 28 29 30 31 32 33 34

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

32 30 28 26 24 24 25 26 27 28 29 30 31 32 33 34

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

SMB-Simulation: Phytol Separation black triangles:infinite dilution situation and infinite number of theoretical plates

36 34

36 34

operating point

32 30 28 26

same parameter set as operating point in figure 5

raffinate (cisisomer) pure extract (transisomer) pure raffinate + extract pure

24 24 25 26 27 28 29 30 31 32 33 34

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

32 30 28 26 24 24 25 26 27 28 29 30 31 32 33 34

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

SMB-Simulation: Phytol Separation Influence of pressure drop: 35

35

30

30 raffinate (cisisomer) pure

25

extract (transisomer) pure raffinate + extract pure

20 20

25

30

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

25 20 20

25

30

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*

Experimental Results of Ibuprofen Separation 11 10

m3

low concentration in Feed linear Adsorption isotherm Ideal model

8 7

7

8

Run O

2,5

1 2

3

9

9 m2

10

11

Run M

Run N

2

2,0

1

1,5

1

1,0

0,5

0,0

0 1

2

3

4

5

6

7

Extract Feed Raffinate

8

0

1

2

3

Extract

4

5

6

7

Feed Raffinate

8

1

2

3

4

5

6

7

8

Extract Feed Raffinate

Separation of Ibuprofen

12 11 10 9 8 7 6 5 4 3 2 1 0 -10

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

6

4

2

0 0

10

20

extract

30

40

50

length [cm]

60

70

raffinate

80

peak area [mV*min]

concentration [g/l]

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

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

H

CH3 H

CH3

CH3 OH

CH3 CH3

17mg pur 0,85 mg in Hexan

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

Verunreinigungen

Phytolisomere

Supercritical Fluid Chromatography

144

A Mettler-Toledo Company

Breaking the Purification Bottleneck

BERGER SFC

HPLC

145

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.

146

• 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 cm2s1 . • 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

147

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.

148

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

149

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 m.

150

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.

151

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 presence of target - NCD to quantify on nitrogen content -FTIR

152

Effect of temperature on retention factor

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 ing Tc, k values increase significantly.

153

154

Effect of temperature on retention factor 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. 155

156

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, 2179-2185 However 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.

157

158

Effect of temperature on resolution

N    1   k2    R s     4     k2  1  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.

159

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.

160

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.

161

162

163

A Mettler-Toledo Company

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

164

Schematic Diagram of a Packed Column SFC A Mettler-Toledo Company

Breaking the Purification Bottleneck

165

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 166

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

167

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

A Mettler-Toledo Company

Conditions: 2.5 ml/min [MeOH +0.4% ibam] in CO2, 35°C, 120 bar, 4.6 x 150 mm, 5µm Zymor CN Limits of SFC Peptides with Very Limited Solubility in Methanol Yield Square Topped Peaks 80% modifier Which Indicates Limited Solubility in the Mobile Phase Not to Scale

40%

60%

0.5

1

1.5

2

Retention Time, minutes

2.5

3

3.5

4

4.5

4 168

min

Some Peptides Elute Just Fine

Limits of SFC A Mettler-Toledo Company

Separation of a Small Pepetide: Actinomycin D mAU

800 mAU

1400

Spectrum 1200

UV Absorbance At 240 nm

205-500 nm

1000

800

25% Methanol + 0.1% TFA in CO2 2.5 ml/min, 35°C, 120 bar 4.6 x 150 mm, 5µm Zymor CN

600

400

200

250

300

350

400

0

0.5

1

1.5

2

2

2.5

Retention Time, minutes

3

3.5

4

4 169

450

nm

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 170

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 171 • extracts from natural products

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 172

Polarity Range of Solute Families hydrocarbons PAH's ethers esters aldehydes and ketones alcohols phenols acids monofunctional fatty acids hydroxy acids polyacids

tertiary

anilines benzylamines sulfonamides phenylureas sulfonylureas triazines carbamates organochlorine organophosphorus primary amines amines secondary amines

Polarity Rangesof Stationary Phases C 18 < C 8

phenyl CN silica NH 2 diol SA

Appropriate Mobile PhaseComposition pure

carbon

dioxide CO 2 +

methanol CO 2 + methanol

+

additives

173

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 174

THE CHROMATOGRAPHY DILEMMA A Mettler-Toledo Company



Breaking the Purification Bottleneck

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

175

A Mettler-Toledo Company



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

176

A Mettler-Toledo Company



THE CHROMATOGRAPHY DILEMMA (cont.)

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

177

Berger SFC Separation of Antidepressants A Mettler-Toledo Company

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)

Antidepressants, 2 µL 20 ppm each

Amitriptyline Imipramine Nortriptyline Desipramine Protryptyline

UV Absorbance At 220 nm

0

0.25

0.5

0.75

1

1.0

1.25

1.5

1.75

178 minutes Retention Time,

min

2.0

The Analytical SFC Opportunity A Mettler-Toledo Company

 Breaking the Purification Bottleneck

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

179

SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

Supercritical Fluid Chromatography

• 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 Compound CO2 NH3 H2O CH3OH C2H5OC2H5

Critical Temp. (oC) 31.3 132.3 374.4 240.5 193.6

Critical Press. (atm) 72.9 111.3 226.8 78.9 36.3

Critical Dens. (g/mL) 0.448 0.24 0.344 0.272 0.276

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

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

2

2

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, HPLC3gives 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 d e t e c t o r H ig h V o lt a g e S u p p ly 2 0 -3 0 k V

F u s e d S ilic a C a p illa r y 5 0 -10 0 cm 15 -10 0 m I D B oth b eakers f ille d w it h a buffer