SUPERCRITICAL FLUID

EXTRACTION
CHROMATOGRAPHY
P R E S E N T E D B Y:
K AV YA S H R E E
I M. PHARM
D E P T. O F P H A R M A C E U T I C A L C H E M I S T RY
CONTENTS
Introduction
Importance of SFC
Instrumentation
Application
INTRODUCTION
Triple point
A temperature and pressure at which solid, liquid and
gaseous phase of a pure substance can coexist.

Critical point
A point where all the respective intensive properties of
liquid and vapor phases merge.

Supercritical fluid
Is any substance at a temperature and pressure above
its critical point.
INTRODUCTION
It is a combination of High Performance Liquid Chromatography
and Gas Chromatography.
Supercritical fluid chromatography (SFC) is a method of
chromatographic separation in which the mobile phase is a fluid
in a supercritical or a subcritical state.
 Supercritical fluid -Properties

Above critical temperature a substances can only exist as a fluid.

This fluid shares both liquid and vapour properties; it is capable of dissolving other materials like
liquid also it is compressible and will expand uniformly in container like vapour.

There is no phase change separating the supercritical fluid from the liquid and vapor phases. Thus,
it is possible to change a fluid from liquid to vapor (or vice versa) without a phase transition.

SCFs has high density due to which they have ability to dissolve higher molecular weight and non-
volatile molecules.
PRINCIPLE
Supercritical chromatography uses supercritical fluids as mobile phase.
Hence, the principle is based on Triple point, Critical point and of Supercritical
Fluid.

Example:

CO2 is a gas at normal temperature and pressure and like all gases, below a
certain critical temperature, further increasing the pressure results in the
formation of a liquid.(31.3°C, 72.9 bar)
PRINCIPLE
If a gas is compressed above, it’s critical pressure and critical
temperature, increases the density of the fluid.
At the critical temperature and pressure, the density of the gas
phase and the liquid phase are the same.
This state is neither a gas nor a liquid but is a supercritical fluid.
Use of a compound in this fluid state as a chromatographic mobile
phase provides different properties than when it is used as either a
gas or a liquid in GC or HPLC.
WHY SFC?
 SFC is inherently faster than LC because of the lower viscosity and higher diffusion
rates in supercritical fluids.

 High diffusivity leads to band spreading.

 When a molecule dissolves in a supercritical medium, the process resembles

volatilization but at a much lower temperature than under normal circumstances.
As a result, compounds such as high molecular weight compounds, thermally
unstable species, polymers, and biological molecules can be brought into a much
more fluid state than that of a normal liquid solution of these same molecules.
WHY SFC?
The operating costs are small in SFC.
CO2 is inexpensive to acquire and may be safely disposed by
venting.
The total liquid waste generated is small because only the modifier
is collected for disposal.
Because water is not used in the mobile phases, columns are
virtually free of hydrolysis degradation and have very long lifetimes.
WHY SFC?
This chromatogram compares
the performance characteristics
of a packed column when
elution is performed with
supercritical CO2 vs. a
conventional HPLC mobile
phase.
The rate of elution is
approximately 4 times faster
using SFC.
INSTRUMENTATION
INSTRUMENTATION
MOBILE PHASE
The most commonly used mobile phase for SFC is Carbon dioxide (CO2).
It is odourless, nontoxic, readily available, and inexpensive when compared with
other chromatographic solvents.
Carbon dioxide’s critical temperature of 31.3°C and its pressure of 72.9 bar at the
critical point permits a wide selection of temperatures and pressure.
Disadvantage
CO2 has inability to elute very polar or ionic compounds.
This can be overcome by adding a small portion of a second fluid called a Modifier
fluid. (Alcohols, cyclic ethers, acetonitrile and chloroform).
INSTRUMENTATION

MODIFIERS
The solvent characteristics of a fluid can be modified by adding a
modifier or entrainer.
Modifiers can also be added to develop other characteristics;

 Toluene is used to enhance the aromatic character.

 Tributyl phosphate is added to improve interaction with metals.
 Alcohols are added to modify the polarity of the molecules.
Supercritical fluids -Example
FLUID CRITICAL TEMPERATURE (°C) CRITICAL PRESSURE(bar)

Carbon dioxide 31.3 72.9

Nitric oxide 36.5 71.7

Ammonia 132.5 112.5

n- butane 152.0 37.5

Water 647K 221

 INSTRUMENTATION
SAMPLE INTRODUCTION UNIT
The column performance is correlated directly with proper sample introduction from a
narrow plug.
For packed SFC a conventional HPLC injection system is adequate, but for a capillary
column SFC, the small volume depends on column diameters and small volume must be
quickly injected into column.
Loop injectors are used which allows 2 μL -100 μL sample injection.
Split/ splitless valve injector used for open tubular column , allows 0.01-0.05μL sample
inj.
INSTRUMENTATION
Loop Injectors
Sophisticated modern method with good precision.
Sample is introduced in the column without causing interruption to mobile phase
flow.
Volume of sample ranges between 2 μl to over 100 μl.
INSTRUMENTATION
Split/ split less injection
In this method, the gas flow passes through
the septum purge and the split vent. This
configuration is called the split mode because
some of the gas in the injector exits though
the split vent.
If the concentration of an analyte is high,
when the sample contains very small
amounts of analyte, spitless injection is used.
It is sensitive method.
INSTRUMENTATION
STATIONARY PHASE
The columns can be much longer than those used in LC, and column lengths of 10 to
20 m and inside diameters of 50 or 100 μm.
For difficult separations, columns 60 m or longer have been used
Widely used polar stationary phase are Polysiloxanes - stable, flexible Si--O bond
leads to good diffusion.
Polymethyl siloxanes - increase efficiency in separating closely related polar analytes
Halocarbons, xenon etc. - Specialty applications only. More polar solvents for highly
polar &high molecular weight compounds.
INSTRUMENTATION
COLUMNS- WORKING
The analytical column which contains a highly viscous liquid (stationary
phase) into which the analytes can be temporarily adsorbed and then
released based on their chemical nature.
This temporary retention causes some analytes to remain longer in the
column and is what allows the separation of the mixture.
INSTRUMENTATION- COLUMNS
Packed columns: contain small deactivated particles so which the
stationary phases adheres the columns are conventionally stainless
steel.
 10-25 cm long.

 More than 100,000 plates

have been achieved with PC.

INSTRUMENTATION- COLUMNS
Capillary columns: are open tubular columns of narrow internal
diameter made of fused silica with the stationary phase bonded to
the wall of the column.
INSTRUMENTATION
RESTRICTORS/ BACK PRESSURE DEVICE
The restrictor in SFC is needed to maintain the pressure in the column above the
critical point.
The restrictors used in SFC can be classified into two general categories:

1. Fixed restrictors
2. Variable restrictors
Systems with variable restrictors can control the pressure and mass flow rate
independently,
These are recommended for the SFC separation of complex samples with pressure
programming.
 INSTRUMENTATION
PUMP
Pumping systems pressure control is necessary
also pulse less operation is important.
The type of pump used is determined by column
type;
Syringe pumps and reciprocating piston pumps
are nowadays the most widely used fluid delivery
systems in SFC.
These pumps are normally pressure controlled.
 SYRINGE PUMP RECIPROCATING PISTON PUMP
In a syringe pump, a stepper motor drives the The reciprocating piston pumps are mostly of
syringe piston by means of a ball screw to the dual head type with the plungers being
control the outlet pressure of the pump. driven by a noncircular gear.
 

When the pump chamber becomes In operation, while one of the pump heads is
empty, the piston is rapidly withdrawn to delivering the fluid, the other is filling the fluid
refill with fluid. into the pump chamber.
In this way, the resultant fluid flow becomes
continuous and (almost) pulseless.
 

This filling cannot be done during a run as this This type of pumps is more suitable in the case
might lead to serious flow and pressure of higher flow rates such as those encountered
distortions.   in standard-bore packed column SFC.
 
INSTRUMENTATION
OVEN
A thermostated column oven is required for precise temperature
control of the mobile phase.
A constant temperature (variation ± O.1 °C) must prevail in the entire
oven at any time of a positive or negative temperature gradient.
The columns are very sensitive to even slight variations in
temperature, which can result in peak shape deformation, peak
splitting, or irreproducible retention times.
 INSTRUMENTATION
DETECTORS
The most popular detectors in packed column SFC are the UV detector and the flame
ionization detector (FID).
There are three basic categories of detector for SFC based on the operating pressure
and the interface required:

Low-pressure detectors; Ex:Flame-ionization detector (FID), Thermionic detector (TID).

Column pressure; Ex: Ultraviolet absorption detector (UV), Fluorescence detector

In-between-pressure detectors; Ex: Refractive index detector (RI)

 
APPLICATION
The SFC technique has been applied to a wide variety of materials, including natural
products, drugs, foods, pesticides and herbicides, surfactants, polymers and polymer
additives, fossil fuels, and explosives and propellants.
SFC is now commonly used for achiral separations and purifications in the
pharmaceutical industry
SFC is used in the petroleum industry for the determination of total aromatic content
analysis as well as other hydrocarbon separations.
The increased diffusion rates of SFC over LC eluents leads to higher resolution and
sharper peaks to enable accurate measurements of enantiomeric purity.
SFC also used in industry for separation of chiral molecules and uses the same columns
as a standard HPLC systems.
REFERENCE
1. Ewing’s Analytical instrumentation handbook, Marcel dekker, third edition
2005.
2. D.A Skoog, F.J. Holler and S.R Crouch, Principle of instrumental analysis,2007.

3. Practical supercritical fluid chromatography and extraction, edited by Marcel C

and Didier T, Volume- 2, 1999.
4. J.W. Robinson, E.M.S Frame, G.M Frame-II, Instrumental analysis, Sixth edition,
Marcel Dekker,2005