Supercritical Fluid Chromatography

(SFC)

Introduction

SFC is a hybrid of gas and liquid chromatography

that combines some of the best features of both.

In SFC, the sample is carried through a separating column by

a supercritical fluid where the mixture is divided based on the

interaction between analytes and a stationary phase in the

column.

Mobile phase in SFC :

Fluid in supercritical state supercritical fluid

What is a supercritical fluid (SF) ?

SF is a material above its critical point (Pc and Tc).

It is not a gas, or a liquid, although it is sometimes referred to as a

dense gas.

SF generally exist at conditions above its critical temperature (Tc)

and pressure (Pc).

SF has densities, viscosities and other properties that are

intermediates between those of the substance in its gaseous and

liquid state

Tc: temperature above which a distinct liquid phase does not exist regardless of pressure

Pc: - minimum pressure required to liquefy a gas at its critical temperature

- vapor pressure at critical temperature

SF:

- diffusivity much higher than a liquid readily penetrates

porous and fibrous solids

- Low viscosity (equal to gas)

Phase diagram temperature/pressure of CO2

Important Properties of Supercritical fluids (SF)

– Remarkable ability to dissolve large non-volatile molecules

e.g., supercritical CO2 can dissolve n-alkanes containing over 30 carbon atoms

related to their high densities

– Dissolved analytes are easily recovered

equilibrate with atmosphere at relatively low temperatures

e.g., analyte in supercritical CO2 can be recovered by reducing the pressure and

allowing the CO2 to evaporate

– No need for organic solvents

environmentally friendly

– Inexpensive, innocuous and non-toxic

– Higher diffusion coefficients and lower viscosities (compared to liquid)

faster and higher resolution separations

Components of SFC

Fig. Schematic of a SFC installation using a HPLC packed column.

A commercial SFC installation

Novasep preparative SFC systems - Cold liquid CO2 is pumped

(1 2).

- Prior to entering the column, it is

heated and becomes supercritical

(23).

- Because of its low viscosity, the

pressure at the column outlet is

almost identical to the pressure at

the column inlet (3 4).

- At the column outlet, the mobile

phase is decompressed and

heated and becomes gaseous

(45)

- Products are recovered in

cyclones of appropriate design.

- The gaseous CO2 is then

cleaned and cooled down and

returned to the tank.

SFC Columns Two types of analytical columns used in SFC:

(1) Capillary columns of fused silica coated with cross-linked chemically bonded

stationary phases, that are used in GC are equally applicable in SFC.

(2) Packed columns developed for high performance liquid chromatography (HPLC)

are being used with SFC.

(a) Silica gel, the major material for current phases

Rigid porous (or nonporous) particles

Spherical particles

Irregularly-shaped particles

(2) Packed columns developed for high performance liquid chromatography (HPLC) are

being used with SFC.

(b) Bonded silica

Organochlorosilane

- If R is a polar functional group, the stationary phase will be polar.

i.e. cyano (–C2H4CN), diol (–C3H6OCH2CHOHCH2OH), or amino

(–C3H6NH2)

- If R is a non polar functional group, the stationary phase will be non polar.

i.e. n-octyl (C8) or n-octyldecyl (C18) hydrocarbon chain

(c) Other stationary phases of varying polarity

Aluminium oxide Al2O3

Zirconium oxide ZrO2

Styrene-divinylbenzene

Hydroxymethylstyrene

Porous graphite

SFC Mobile Phase CO2 is the primary mobile phase used in SFC.

The advantage ofCO2 as the mobile phase is

- low cost,

- low interference with chromatographic detectors,

- nontoxic,

- low critical temperature (31.1 oC),

- inflammability and that it can permit a flame ionisation detector to be used, with all

the benefits in terms of ease of use, linearity and sensitivity

Disadvantage of carbon dioxide is

- inability to elute polar or ionic compounds.

By adding a small portion of a second fluid, modifier, this can be overcome. Modifiers

are generally an organic fluid (such as alcohols ,cyclic , etc) which are completely

miscible with carbon dioxide. Modifiers improve the solvating ability of the SCF and

sometimes enhance separation selectivity

Typical Supercritical mobile phase

SFC Injection

-For packed SFC, a typical LC injection valve is commonly used

load

Inject

- In capillary SFC, small sample volumes must be quickly injected into the column and

therefore pneumatically driven valves are used.

Carrier

Waste/Drain

Sample

in

Column

Six-way injection valve Injection of identical volume of sample

Loading Injection

SFC Pump The type of high pressure pump used in SFC is determined by the column type.

- For packed columns, reciprocating pumps are generally used

Reciprocating pumps allow easier mixing of the mobile phase or introduction of modifier

fluids.

- For capillary columns, syringe pumps are used.

Syringe pumps provide consistent

pressure for a neat mobile phase.

Double-plunger micro pump (Reciprocating pump)

Stroke length 1.0 mm

Plunger diameter 2.5 mm

Stroke volume 5 ml/stroke

Pumping mechanism

dual head

reciprocation

SFC Detector

FID (flame ionization detector) and MS detector.

The restrictor is installed before the detector,

UV-absorption, fluorescence or light diffusion.

The restrictor is installed after the detector.

Restrictor: a device installed in the SFC system to control pressure of

the fluid during the chromatographic process

The major difference in SFC and conventional LC equipment is

the pumping systems as well as the safety features installed to maintain higher

pressure.

Unique SFC equipment differences are:

1. Carbon dioxide tank for mobile phase supply

- Equipped with a pressure relief value and rupture disk

2. High-pressure pump

- Chiller to maintain mobile phase in liquid state

3. High-speed injector

4. Pressure restrictor

- High-pressure tubing

5. High-pressure flow cell for UV detection

6. Solvent collection device with ability to vent to a laboratory hood or elephant trunk.

SFC Vs LC Instrumentations

Advantages of SFC compared to LC and GC - SFC can separate compounds that are not conveniently handled by GC or LC.

non-volatile or thermally labile

and

contain no functional group that makes possible detection in LC using

spectroscopic or electrochemical techniques

< up to 25% of all separation problems fall into this category

< examples include: polymers, fossil fuels, pesticides, foods, drugs, etc

- Separations are faster then LC

- Run at lower temperature than GC

- Beneficial in industrial scale purification

Faster elution Reduction in

peak width

Reduction in

elution time

Reduction in

peak width

Reduction in

elution time

Effects of Pressure: Pressure increases results in reduced elution time

- increase in density of mobile phase

- effects retention or capacity factor (k’)

- pressure changes analogous to gradient elution in LC and GC

SFC is more and more used in research and development laboratories

and pilot plants of the pharmaceutical and fine chemical industries.

SFC is particulary interesting for the purification of :

chiral compounds actives or intermediates from complex mixtures lipophilic

compounds

Application of SFC

The ability to vary selectivity by programming the parameters P

(pressure) and T (temperature) rather than by modifying the chemical

composition of the eluent.

The range of compounds analysed by SFC includes lipids and oils,

emulsifiers, oligomers and polymers (compounds of molecular mass

greater than 1000 which cannot be studied in GC)

(1) Chiral separation

The use of supercritical fluids to separate enantiomers is one of the most

important tasks in several areas of research, especially pharmaceuticals and

agrochemicals.

It is well known that the two enantiomeric forms of a molecule can display

dramatically different biological activity.

Example : Chiral separation of four triazole

pesticides by supercritical fluid chromatography

- Showed the effects of

different organic modifiers on

the resolution

and retention via k value

k : 1 - 5

R greater than 1.5

Good if

(2) Polymer separation

Column temperature: 800C,

Pressure: 19.6 MPa.

Coulmn: Inertsil Ph-3

Consistent flow rate of CO2 : 3.0 ml/min

Polyprenol or 1,4-polyprenyl alcohols has 30 monomers

(3) High throughput screening of pharmaceuticals