This document discusses supercritical fluid chromatography (SFC). SFC uses supercritical fluids like carbon dioxide as the mobile phase. Carbon dioxide is most widely used as it is non-toxic, inexpensive, and has a critical temperature and pressure that are easily reached. SFC works on the principles of adsorption and partition chromatography. It can be used to analyze and purify low to moderate weight compounds, including chiral separations. SFC instrumentation includes pumps to deliver the mobile phase, an oven for temperature control, various injectors, columns, a backpressure regulator, and detectors. SFC finds applications in fields like pharmaceuticals and has advantages over HPLC like using less toxic solvents.

1. PRINCIPLE &
INSTRUMENTATION &
APPLICATION OF SFC
Presented by,
Anbu Dinesh Jayakumar ,
M.Pharmacy (Semester-II)

2. CONTENTS
Definition
Principle of SFC
Stationary & Mobile phases used in SFC
Instrumentation
Working
Drawbacks of SFC
Application
Conclusion
Reference

3. Chromatography is an separation method used for analysing
mixtures of chemicals using stationary phase and mobile phase.
Chromatography – mobile phase
Gas chromatography - GAS
Liquid chromatography - LIQUID
Supercritical fluid chromatography – SUPERCRITICAL FLUID

5. Supercritical fluid chromatography (SFC) is a form of normal phase chromatography that
uses a supercritical fluid such as CARBON DIOXIDE AS THE MOBILE PHASE.
It is used for the analysis and purification of low to moderate molecular weight, thermally
labile molecules and can also be used for the separation of chiral compounds.
 Triple point is the temperature and pressure at which
solid, liquid & vapour phases of a particular
substance coexist in equilibrium.
 The critical point is the point at which two phases of a
substance initially become indistinguishable from one
another.
 It is the end point of a phase equilibrium curve, defined by
a critical pressure Tp and critical temperature Pc.
 At this point, there is no phase boundary.

6. PRINCIPLE INVOLVED IN SFC
 The Principle is based on ADSORPTION AND PARTITION CHROMATOGRAPHY.

7. LIQUIFIED GAS
CARBON DIOXIDE
(COMPRESSED& HEATED)
DECOMPRESSION(BELOW
CRITICALPRESSURE)
SUPERCRITICAL FLUID+
MODIFIER/ CO-SOLVENT
CHROMATOGRAPHIC
COLUMN
TEMPERATURE
NEAR CRITICAL
TEMPERATURE
ELUTION
PRESSURE &
TEMPERATURE
INCREASES
SELECTIVITY
GAS
PHASE
separation with pure CO2 eluent, the
solvent power of the fluid is decreased ,
leading to solute precipitation and eluent-
solute separation.
PRODUCT
+CO2

8.  Mostly used are Silica/Alumina
 Useful for Non-polar
compounds
WIDELY USED POLAR STATIONARY PHASE ARE
 Polysiloxanes - stable, flexible Si--O bond lead to good
diffusion.
 Polymethylsiloxanes - increase efficiency in separating
closely related polar analytes
 Cyanopropyl polysiloxanes - useful for compounds with –
COOH
Mostly Carbon-dioxide is used.
Disadvantage of carbon dioxide is its inability to elute very polar or ionic compounds.
It is overcomed by adding of Modifier fluid. (Alcohols, cyclic ethers, acetonitrile and chloroform)
Modifier fluid improves the solvating ability and enhances selectivity of the separation.

9. OTHER SFC SOLVENTS
– NITROUS OXIDE - Similar in solvating and separation properties to CO2(expensive)
– 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.

10. INSTRUMENTATION
a) PUMPS
b) OVEN
c) INJECTORS
i) LOOP INJECTOR
ii) INLINE INJECTOR
iii) INCOLUMN INJECTOR
d) COLUMN
e) BACK PRESSURE RESTRICTOR
f) DETECTOR

11. a) PUMPS
 Flow controlling is an function of pumping systems flow control is necessary and pulseless operation .
 Type of pump used in super critical fluid is choosed by column type.
 For packed columns - reciprocating pumps are used.
 For capillary columns- super critical fluid syringe pumps are used.
 Reciprocating pumps allows easier mixing of mobile phase or introduction of modifier fluids.

12. b) OVEN
 A Thermostated column oven is required for precise temperature control of the mobile
phase.
 Conventional GC or LC ovens are generally used.

13. c) INJECTORS
Injector - For packed SFC A Conventional HPLC Injection System is adequate,
For capillary column sfc, the small volume depends on column diameters.
Small volume must be quickly injected into column.
Therefore Pneumatically Driven Values Are Used.
A) LOOP INJECTORS
B) INLINE INJECTOR
C) INCOLOUMN INJECTOR

14. i) LOOP INJECTORS
– It is a direct transposition of what is applied in analytical SFC
– Used mostly for preliminary tests of column performance and elution
parameters.

15. ii) INLINE INJECTOR
– More Versatile
– System Offers Better Flexibility For Changing Injected Volume.
– High Pressure Pump Required To Inject Feed Solution.
– Injected Stream Dissolves in Diluent Flow

16. iii) INCOLOUMN INJECTOR
 An Alternative System
Permits Injection Of Feed Solution Directly Onto Column
No Dilutions Required

17. d) COLUMN
There are two types of analytical columns used in SFC,
– Packed columns contain small deactivated particles so which the stationary phases
adheres the columns are conventionally stainless steel.
– Capillary columns are open tubular columns of narrow internal diameter made off
used silica with the stationary phase bonded to the wall of the column.

18. e) BACK – PRESSURE DEVICE OR RESTRICTOR
 It is used to maintain desired pressure in column by pressure adjustable
diaphragm or controlled nozzle, so that, same column outlet pressure is
maintained irrespective of mobile phase pump flow rate.
 It keeps mobile phase super critical throughout separation and often must be
heated to prevent clogging
 Pressure restriction is placed either at the end of the column or after detector.

19. f) DETECTOR
Most any detector used in GC or HPLC can be used.
a) Flame photometric detectors
b) Flame ionization detectors
c) Refractive index detectors
d) Ultraviolet-visible spectrophotometric detectors
e) Light scattering detectors

20.  Mobile phase (liquid) is pumped into the supercritical
region by heating it above its supercritical temperature
before entering into the analytical column.
 It passes through an injection valve where the sample is
introduced into the supercritical stream and then into the
analytical column.
 It is maintained supercritical as it passes through the
column and into the detector by a pressure restrictor
placed either after the detector or at the end of the column.
 The restrictor keeps the mobile phase supercritical
throughout the separation and heated to prevent clogging.
 Both variable and fixed restrictors are available.
WORKING

21.  It requires HIGH PRESSURE operating conditions (High-pressure vessels are expensive and
bulky) , It is difficult to maintain pressure (backpressure regulation).
 Reason :Supercritical fluids are highly compressible and their physical properties change
with pressure - such as the pressure drop across a packed-bed column.
 Automated backpressure regulators can maintain a constant pressure in the column even if
flow rate varies.
 It is difficult to separate gas/liquid separation during collection of product.
 Upon depressurization, the CO2 rapidly turns into gas and aerosolizes any dissolved analytes
in the process.
DRAWBACKS

24. APPLICATIONS

26. SEPARATION OF TOFISOPAM

27.  Tofisopam is a member of the 2,3-benzodiazepine compound family used in the treatment
of ANXIETY AND ALCOHOL WITHDRAWAL.
 Due to its Stereogenic center at C(5)-atom it exists as TWO ENANTIOMERS (R(+) AND
S(–)).
 Upon dissolution, its diazepine ring system will exist in two boat conformations, leading to
two conformers for each enantiomer .
 The Driving force for conformer transition is attributed to the steric repulsion effect
between C(4)-methyl and C(5)-ethyl groups.
As a result of the pharmacological interest in Tofisopam, It is essential to separate the four
isomers in order to evaluate their different biological activities of the drug.

28.  Agilent 1260 Infinity Analytical SFC System is used.
 It consists of A5 fusion module for CO2 pre- and post
conditioning and a modified Agilent 1260 Infinity Binary
LC System for accurate and constant metering of the
mobile phase.
 The system (G4309A) consisted of the following modules:
 SFC Fusion A5 module
 Agilent 1260 Infinity SFC Binary Pump
 Agilent 1260 Infinity Standard Degasser
 Agilent 1260 Infinity Standard Autosampler
 Agilent 1260 Infinity Diode Array Detector with high
pressure SFC flow cell
INSTRUMENTATION

29. Chromatographic conditions
 Flow rate - 3.0 mL/min,
 Temperature of the column compartments at 35 °C and
 Back pressure of CO2 supercritical fluid at 150 bar.

30. MeOH appears to be the most versatile co-solvent in resolution of
Tofisopam isomers
 The best resolution of the four peaks was achieved with
CHIRALPAK IA and CHIRALPAK ID.
Analysis times can be as short as 3 minutes (Figure 2 (a)) or 5
minutes (Figure 2 (b)), respectively.
Elution order is the same on CHIRALPAK IA,CHIRALPAK
IC, and CHIRALPAK ID.
Co-elutionof two P(Ax.) and one P(Eq.) is observed on
CHIRALPAK IB

31.  SFC was developed in the 1960s, and some commercially available instruments were
introduced to the market in the beginning of the 1980s.
 When users found that instruments were difficult to operate, and poorer performance than
HPLC were obtained, the interest in SFC considerably dropped.
 There is, nowadays, increasing demand for new, environmentally friendly processes, trends to
banish some classes of solvents (ozone depletion), to prevent or reduce the direct and indirect
effects of emissions of volatile organic compounds (VOCs) in the environment and on human
health.
 All these reasons favour the use of SFC.
 Considering the numerous advantages of Prep-SFC, especially for rapid chiral separations of
pharmaceutical compounds, its revival in the near future is very likely.
CONCLUSION

32. REFERENCE
 Super critical fluid chromatography “Skoog” instrumental analysis Pg: 935 to 940.
 Supplement and Cumulative Index, Edited by Bryant W. Rossiter and Roger C. Baetzold
 Supercritical Fluids and Nanotechnology Opportunities for Multidisciplinary Collaborative Research
“A Presentation to Air Force Research Laboratory” Edwards, CA by B. Cherhoudi, PhD
 Principle and Applications of Supercritical Fluid Chromatography by Wieslaw Majewski a ,
Eric Valery a & Olivier Ludemann‐ Hombourger a NovaSep SAS, Pompey, France