ADVANCED SPECTRAL ANALYSIS

SEMINAR

ON

SUPERCRITICAL FLUID CHROMATOGRAPHY

PRESENTED BY: PRESENTED TO:

GOVIND Dr. SOMASHEKARA P.L

GOVERNMENT COLLEGE OF PHARMACY,

P. KALINGARAO ROAD, SUBBAIAH CIRCLE,
MISSION ROAD
BENGALURU, KARNATAKA-560027
CONTENTS

 Introduction
 Importance of SFC
 Instrumentation
 Application
 Reference
INTRODUCTION

Supercritical fluid chromatography

 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.
 Terminologies
 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.
 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
 Modifiers
 Sample introduction unit
 Stationary phase
 Resictore and back pressure
 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).
Supercritical fluids -Example

FLUID CRITICAL TEMPERATURE CRITICAL PRESSURE(bar)

(°C)
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

 MODIFIERS

 Thesolvent 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.
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.
There are 3 types of injector used in sfc
 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.
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.
 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.
 Split/ splitless valve injector used for open tubular column , allows 0.01-0.05μL sample inj
On-column injectors:
 This type of inlet, the entire sample is injected onto the column
as a liquid, which is later vaporized by temperature
programming of the column or inlet. With on-column inlets, the
analyte is separated from the solvent by thermal and solvent
effects.
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.
 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.

COLUMNS

 Packed columns: contain small deactivated particles so which the

stationary phases adheres the columns.

 Made up of stainless steel

 10-25 cm long.

 More than 100,000 theoretical

plates have been achieved with PC.

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.
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.
Pump
 Flow control is function of pumping systems
and it is necessary for pulseless operation

 The type of pump used is determined by

column type;
 These pumps are normally pressure controlled.
 There are two times of pump used in sfc

1. Syringe pumps
2. reciprocating piston pumps
 SYRINGE PUMP RECIPROCATING PISTON
PUMP
In a syringe pump, a stepper motor drives The reciprocating piston pumps are mostly
the syringe piston by means of a ball of the dual head type with the plungers being
screw to control the outlet pressure of the driven by a noncircular gear.
pump.  

In operation, while one of the pump heads is

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

This filling cannot be done during a run This type of pumps is more suitable in the
as this might lead to serious flow and case of higher flow rates such as those
pressure distortions.   encountered in standard-bore packed
column SFC.
 
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.
 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