Supercritical fluid chromatography Sajna.pdf

Supercritical Fluid Chromatography (SFC) is a form of normal phase chromatography that utilizes a supercritical fluid (SCF), typically carbon dioxide ), as the mobile phase. An SCF is defined as a highly compressed fluid that combines the properties of a gas and a liquid. It is created by subjecting a substance to a temperature and pressure beyond its critical point, which is defined by a critical temperature and critical pressure .CO2 is the most common SCF due to its low critical temperature ), low critical pressure , non-toxicity, and low viscosity, which enables faster separations. The use of an SCF mobile phase offers distinct advantages: better solute diffusivity than in liquids, low viscosity, and a dissolving effect dependent on its density.The fundamental principle of SFC lies in adsorption or partition chromatography. Adsorption occurs when a polar stationary phase (like silica or Diol) is used, while partition is the mechanism with a nonpolar stationary phase . Separation is achieved based on the differential interaction of the sample components with the mobile and stationary phases. To enhance the mobile phase's polarity and improve the separation of polar analytes, modifiers, such as methanol or ethanol, are frequently added to the . Stationary phases are broadly classified into Non-polar, Polar, Chiral (used for separating enantiomers), and Specialty phases0.The instrumentation consists of several key components: the Mobile Phase Reservoir a Cooler a Pump (Reciprocating or Syringe type) to force the mobile phase through the system ; an Injection System (loop, in-line, or in-column) for introducing the sample and a Thermostated Oven to heat the fluid to its supercritical state and maintain a precise column temperature. Critically, a Restrictor is installed after the column to maintain the pressure above the critical point, ensuring optimal conditions. Finally, various Detectors are employed, including the Flame Ionization Detector (FID), UV Spectrometric detector, and Mass Spectrometer (MS)17.SFC provides several benefits, including high efficiency, reduced solvent usage, compatibility with thermally sensitive compounds, and environmental friendliness. However, it suffers from a few drawbacks, such as the lack of reproducibility, low UV sensitivity, and a limited ability to handle highly polar or water-soluble compounds19191919. Recent developments include the use of Ultra-High Performance SFC–Mass Spectrometry (UHPSFC-MS) to boost sensitivity and selectivity, and Two-dimensional SFC (2D-SFC) for separating highly complex mixtures20202020. SFC has extensive applications

1. 1
SUPERCRITICAL
FLUID
CHROMATOGRAPHY
Presented by,
Sajna K S
2
nd
Semester M.Pharm (2024 Batch)
Department of Pharmaceutical Chemistry
St. James’ College of Pharmaceutical Sciences , Chalakudy

2. CONTENTS
Introduction
Principle
Instrumentation
Recent advances
Application
Reference
2

3. INTRODUCTION
•Supercritical fluid chromatography is a form of normal phase chromatography that
uses a supercritical fluid such as carbon dioxide as the mobile phase.
•A supercritical fluid (SCF) isa highly compressed fluid that combines the properties of gases and
liquids.
•They are created by increasing temperature and pressure beyond a substance's critical point.
Supercritical fluid
Gas
Liquid
Supercritical
fluid
3

4. Critical point
The critical point is theend point of a
phase equilibrium curve, defined by a
critical pressure T p and critical
temperature P c.
A phase diagram isa graphical representation of the
physical states, or phases, of a substance under
different conditions of temperature, pressure, and
composition.
Critical
temperature (??????
??????)
Critical temperature (of a
substance) can be defined
asthe highest possible
temperature value at which the
substance can exist as a liquid
the temperature at and above
which vapor of the substance
cannot be liquefied, no matter
how much pressure is applied.
Critical
Pressure(??????
??????)
the minimum pressure required
to liquefy a substance at its
critical temperature
The critical pressure of a
substance is thepressure
required to liquefy a gas at its
critical temperature.
4

5. PRINCIPLE
The main principle is adsorption or partition chromatography
It utilizes supercritical fluids like ????????????
?????? as mobile phase for
separation which exhibits both gas and fluid like properties.
5
Mobile phase
(Supercritical
fluid)
Solvent
Sample Stationary
Phase
Separates
based on
their
interaction
Separation occurs
Adsorption
For polar
Stationary
phase
Partition
For Nonpolar
Stationary
phase

6. 6
DENSITY
The dissolving effect of a
supercritical fluid is
dependent on its density
value.
Supercritical fluids are also
better carriers than gases
thanks to their higher
density.
DIFFUSIVITY
solute can show better
diffusivity in a
supercritical fluid than
in a liquid.
The greater diffusivity
gives supercritical
fluids the chance to be
faster carriers
VISCOSITY
Viscosity for a supercritical
fluid is almost the same
as a gas, being
approximately
1
/
10of that
of a liquid.
•These are less resistant
than liquids
The use of supercritical fluid as mobile phase provides:
Mode Stationary phase Mobile Phase Mechanism
Normal Phase SFC Polar (Silica.Diol,Amino)Nonpolar (Supercritical
CO
2+ Small amount of
modifier)
Adsorption
Reverse Phase SFC Nonpolar (C18,CN) Polar (Supercritical
CO
2+ High amount of
modifier)
Partition

7. DISADVANTAGES
•
ADVANTAGES
•High efficiency:
•Environmental friendliness:
•Compatibility with thermally
sensitive compounds
•Wide range of applications:
•Reduced solvent usage:
•Enhanced selectivity:
DISADVANTAGES
•Lack of reproducibility
•Low UV sensitivity
•It does not work for water
solublecompounds
•it is limited for highly polar
compounds and proteins.
•The CO2 tanks required for this
analysis are large, and heavy.
7

8. INSTRUMENTATION
Mobile phase
Modifier
Stationary
phase
Mobile phase
reservoir
Cooler
Pump
Pressure
programming
Injection
systems
Thermostated
oven
Columns
Restrictor
Detector
8

9. MOBILE PHASE
•Modifiersarepolar organic solvents
(e.g., methanol, ethanol, acetonitrile)
added to
•increase its polarity,
•improving the solubility and
•Improve separation of polar analytes
•Carbon dioxide (CO₂) → the most widely
used mobile phase because:
•It has a low critical temperature (31.1
°C) and low critical pressure (73.8 bar).
•It is non-toxic, inexpensive, and inert.
•It provides low viscosity and high
diffusivity, giving faster separations.
MODIFIERS
9
Others:Nitrous oxide ,Alkanes, Hydrocarbons,Xenons

10. STATIONARY PHASE
10
NON-POLAR
PHASE
•Includes alkyl-
bonded
stationary phases
that do not have
hydrophilic
groups.
• These columns
are suitable for
the separation of
hydrophobic
compounds
POLAR PHASE
Include
•silica gel,
•3-aminopropyl
bonded silica
(NH
2),
•3-cyanopropyl
bonded silica
(CN),
•propanediol
bonded silica
(Diol),
CHIRAL PHASE
•These phases are
designed to
separate
enantiomers.
•Forms transient
diastereomeric
complexes with
the analytes by
ohydrogen bonding,
odipole-dipole,
opi-pi interactions.
SPECIALITY PHASE
•Containsnitrogen
heterocycles, like
o2-Ethylpyridine (2-
EP)
oPyridine (PIC),
•Manages the peak
shape of alkaline
compounds
•provides
selectivity for
acidic and neutral
compounds.
The non-moving components within a
column that retain analytes, allowing
for their separation.

11. MOBILE PHASE
RESERVOIR
•Mainly stainless steel
cylinder.
•Provided with pressure
regulator to control flow
of gas.
•Used to cool CO₂ before pumping
into column
•Usually CO₂ is pumped below
5℃
11
COOLER

12. PUMP
12
RECIPROCATING PUMP
A reciprocating pump works byconverting
mechanical energy into liquid flow using a
piston or plunger moving back and forth inside a
cylinder,
During the suction stroke, the piston moves back,
creating a vacuum and drawing liquid into the
cylinder through a suction valve.
During the discharge stroke, the piston moves
forward, closing the suction valve and opening a
discharge valve to force the liquid out.
SYRINGE PUMP
•It works byusing a motor-driven mechanism to
precisely push a syringe plunger, delivering
fluids or medications at a controlled and
accurate rate
•A syringe is loaded into the pump, the user sets
the desired flow rate and volume using the
pump’ssoftware,and amotor moves the
plunger at a constant speed to infuse the fluid
over a specific duration.

13. INJECTION SYSTEMS
13
INJECTION
SYSTEM
Loop injection
A low pressure feed
pump is used to fill
a loop which is
passed to column
In line injection
A high pressure
pump is used
along with eluent
flow
In column
injection
Directly into
column

14. THERMOSTATED OVEN
14
A thermostat column oven is employed for maintenance of precise
temperature of the mobile phase
A Column is place inside the oven
The temperature display pads is provided for adjustment of temperature
The cooled fluid from pump should be heated to achieve supercritical state
before entering into the column

15. COLUMNS
•Mainly there are 2 types
15
These are most used and are coated
with cross linked silica material for a
stationary phase
The less than 100 micrometre inner
diameter capillaries were used
especially with supercritical
hydrocarbons
The typical packed capillary columns in
SFC can be defined by the I.D. in the range
of 0.05 – 0.5 mm, length from 10 cm to
several meters, andparticle size of 5 μm
The capillaries suffer from several
problems arising from capillary fragility
the cracks resulting from high pressure
improper handling or cutting were
reported
OPEN TUBULAR COLUMN PACKED COLUMN

16. Restrictors in
SFC Systems
Purpose of Restrictors
Restrictors maintain SFC pressure above the critical
point, ensuring optimal conditions for chromatography
and detection.
TWO TYPES:
Capillary Restrictors
Capillary restrictors create resistance using tube length
and diameter, ideal for controlling low flow rates in
SFC systems.
Mechanical Restrictors
Mechanical restrictors use adjustable valves or
pneumatic systems to handle higher flow rates and
precise pressure control.
16

17. DETECTORS
Varies depending upon:
1.Mobile phase
composition
2.Column type
3.Flow Rate
4.Ability to withstand high
pressure
17
Flame ionization
detector
Flame photometric
detector
UV Spectrometric
detector.
Mass spectrometer
Refractive index
detector
Detectors used

18. 18
Flame ionization detector
operates by introducing a sample into a hydrogen-air
flame,
ionizes organic compounds.
These ions are then collected by an electrode, creating a
current
The magnitude of this current is directly proportional to the
number of carbon atoms in the organic sample that passes
through the detector

19. UV Visible spectrophotometer
19
Pass sample
through a
transparent
flow cell
UV light is
directed on
to flow cell
Sample interacts with
this light absorbing a
portion of it
Measure the absorption
of light at specific
wavelengths
Calculate the
variation of light
intensity
between mobile
phase and
phase with
sample

20. MASS SPECTROMETER
20
It operates by
creating an ionized
spray of sample
Separate the ions
based on
mass/charge ratio
The mass/charge
ratio is plotted
against abundance
Creates a fingerprint
for chemical
identified
The chemical
fingerprint is matched
against a database to
know which
compound it is.

21. 21
WORKING
Pump generating
liquid CO2
Modifier
Pump
Injector
DetectorWaste
Sample
CO2
Cylinder
Reservoir for
modifier
Dampener Mixing
column
Oven
Backpressure
regulator
Chromatographic
column

22. RECENT
ADVANCES
22
1.Ultra-High Performance Supercritical Fluid Chromatography–
Mass Spectrometry
• combines the separation power of supercritical fluid
chromatography with the sensitivity and selectivity of mass
spectrometry.
• MS detection then identifies analytes based on their mass-
to-charge ratio (m/z), providing both qualitative and
quantitative information.
2.Two dimensional SFC
•Multidimensional SFC was employed for the separation of
complex mixtures of high boiling-point hydrocarbons that could
not be separated by gaschromatography.
• 2D-SFC using CO
2or SF
6as amobile phasewith flame-
ionization detection (FID) became the method of choice for
such applications.
• Early practitioners implemented heart-cutting 2D-SFC by
using a single pump and flow-switching valves to direct mobile
phase flow.
•The flow can go through a single dimension while pausing the
separation in the second dimension

23. APPLICATION
1.Aromatic content of fuels by SFC
•Aniche applicationofSFCis the rapid determination of the content of aromatics in fuels, with high
precision and reproducibility.
•Employs a packed silica column, of length 25 cm, with CO
2asmobile phaseand an FID as detector
2.Chiral separations
•SFC rapidly became an interesting tool to substituteNPLCforchiral separations, due to its
• higher efficiency,
•faster analysis times
• reduced environmental impact, and
•lower costs.
•In comparison to GC,supercritical fluid chromatographyyields better chiral selectivity
•Separations are performed at lower temperatures than GC (selectivity mostly decreases at higher
temperatures).
• These conditions also reduce the probability of racemization during separation.
23

24. APPLICATION
24
Comparison of chiral separation in GC and SCF using the same capillary column
cis-heptachlorepoxide cis-chlordane
α-hexachlorocyclohexane
A number of chiral
compounds of pharmaceutical
andagrochemicalimportance
have been analyzed

25. APPLICATION
3. Application of SFC for bioanalysis: Drugs of abuse, doping control, and
toxicological analysis
•SFC-MS/MS is being used for the analysis of drugs of abuse in forensic
environments and prohibited substances according to antidoping regulations.
•Recent forensicapplications of SFCinclude the analysis of
(synthetic)cannabinoids,chiral separationofamphetamineenantiomers, and
analysis of different new psychoactive substances
•A SFC-UV method using rather less conventionalpureACNasmobile
phasemodifier for the analysis of natural andsynthetic cannabinoidsand their
metabolites in human urine afterdesalting
25

26. APPLICATION
4. Analysis of citrus coumarins:
•Comparison of the results obtained by SFC to those obtained by HPLC
❖Both the analyses allowed a complete separation of the six polymethoxylated flavones (PMFs) known to be present
in sweet orange oil. The SFC analysis was completed in less than 6min, while the HPLC analysis took more than
25min. This represents a reduction in the analysis time by a factor of four.
26
HPLC chromatogram of the polymethoxylated flavones of sweet orange oil SFC chromatogram of the polymethoxylated flavones of
sweet orange oil

27. APPLICATION
5. Group type analysis of petroleum products by SFC:
❖Group-type analysis refers to the separation and quantification of the hydrocarbon groups, i.e.,
saturates,olefins,aromatic hydrocarbons.
❖SFCis very efficient for group-type separations because, as inGC, flame-ionization detection can be used and
does not require correction of response versus the hydrocarbon type. InLCthe use of refractometric detection
requires a calibration that can be inaccurate[10,36].
❖SFC allows group-type separations of middle distillates and heavy fractions.
.
27

28. REFERENCES
1.Pavan M V, Raja, Andrew R, Supercritical fluid
chromatography. In physical methods in chemistry and
Nano science. ChemLibre Texts.2022.3(1);22-28.
2.Marty M, Supercritical fluid chromatography and its
advantages, Pharmaceutical Analytical Chemistry
2023,8(4);34-38.
3.Plachka K. Pilarova V. Horacek O, Columns in analytical
scale supercritical fluid chromatography; From
traditional to conventional Chemistries, Journal of
Separation Science, 2023,46 (18);1-22.
4.Samy M, Fathy M. Fathalla A, Supercritical fluid
Chromatography :A Powerful tool for simultaneous
determination of antihypertensive drug combination in
tablet dosage forms, Journal chromatography
Science.2022,1(2);1-17.
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29. THANK YOU
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