Super Critical Fluid Chromatography M.pharm- 2nd semester P'ceutical Chemistry

1. Supercritical Fluid Chromatography
Presented by-
Pallavi Rushiya
M. Pharm [P'ceutical Chemistry (II nd semester)]
Guru Nanak College Of Pharmacy, Nagpur.

2. C O N T E N T S
Introduction
Instrumentation
Working
Application

3. Introduction
Chromatography is separation technique used for analysing
mixtures of chemicals using stationary phase & mobile
phase.
Supercritical Fluid Chromatography (SFC) is form of normal
phase chromatography that uses supercritical fluid such as
CO2 as mobile phase.
SFC is accepted as column chromatography methods along
with GC & HPLC.

4. Supercritical Fluid
• A supercritical fluid (SF) is phase of material at critical temperature &
critical pressure of material above its critical point.
a) Critical temperature- temperature at which gas cannot become liquid as
long as there is no extra pressure.
b) Critical pressure- minimum amount of pressure to liquefy a gas at its
critical temperature.
c) Critical point- Boundary between 2 phases ( liq. & gas) is completely loss.

5. • It has properties intermediate
between liquid & gas, non
compressible & high density fluid.
• A supercritical fluid provides a
gas like characteristics when it
fills a container & takes a shape
of container.
• On other hand, it behaves like a
liquid because its density
property is near to liquid thus, it
shows similarity to dissolving
effects of liquid.

6. Properties of SF
1. Density-
Se in pressure (at constant temp.) ----- SF density se
Constant pressure ( se temp.) ----- SF density se
2. Viscosity-
Almost same as a gas (approx. 1/10 of that liq.)
Less resistant than liq. towards components flowing through.

7. 3. Diffusivity-
It is parallel with temp. & opposite to pressure.
Increasing pressure affects SF molecules to become closer to each other &
decreases diffusivity.
Greater diffusivity gives SF the chance to be faster carrier for analytical
applications.
➡ Viscosity, density & diffusivity are related to each other.
As se pressure causes rise in viscosity & thus it results in declining
diffusivity.

8. • It is base upon Adsorption & Partition chromatography.
• It is similar to both HPLC & GC, however SFC typically uses CO2 as main mobile
phase.
• SFC is a normal phase chromatographic technique with inherent high speed &
efficiency due to it's mobile phase.
• As, high pressure liquid or supercritical fluid using CO2 is preferred solvent for
its low viscosity & high diffusivity required in separation & determination of
group of compound that are not handled by GC / HPLC.
P r i n c i p l e

9. 1. Lower operating temperature.
2. High diffusion co-efficient & low viscosity.
3. High resolution at low temperature.
4. Improved yield.
5. Analysis of thermolabile or non-volatile
compounds.
6. Providing rapid separation without using
organic solvents.
1. Elevated pressure required.
2. Expensive equipment.
3. Time consuming cleaning.
4. Difficult to separate gas/ liquid during product
collection.
5. Upon depressurization, CO2 rapidly turns into
gas & aerosolizes any dissolved analytes in
process.
Advantages Disadvantages
Supercritical Fluid Chromatography

10.  Mobile phase reservoir
 Stationary phase
 Cooler
 Pump
 Injector
 Thermo stated oven
 Columns
 Restrictor back pressure regulator
 Microprocessor
 Detectors
 Collector
 Data reader
Instrumentation

12. Mobile phase reservoir-
Usually stainless steel cylinder when gases are used.
Pressure regulator cylinder is provided to control flow of gas.
Eg. CO2 stored in cylinder.
Stationary phase-
Silica/ Alumina
• Useful for non polar compounds.
• Leads to irreversible absorption of polar solutes.
 Widely used polar stationary phase are-
1. Polysiloxanes- stable & flexible So bond lead to good diffusion.
2. Polymethylsiloxanes- increase efficiency of polar analytes.
3. Cyanopropylpolysiloxane- useful for compounds with COOH.
Other solvents- NO2, Alkanes, Hydrocarbon, xenons, etc.

13. Cooler-
Used to cool CO2 before pumped into column.
The solvent is pumped as liquid in cooling conditions.
Eg. CO2 is pumped below 5°c at 50 bars of pressure.
Pumps-
Small scale- Reciprocating / Syringe Pumps
Large scale- Diaphragm Pumps
In SFC, pumps are choosen depending upon type of column.
For Packed column- Reciprocating Pump
For Capillary AFC Syringe Pump
Reciprocating Pump- provide easier mixing of MP/ introduction of modifier
liquid.

14. Injectors-
For Packed SFC- Conventional HPLC injector
For Capillary SFC- small volume depends on column diameter therefore,
Pneumatically driven valves are used.
① Loop injector- direct transposition of what is applied.
② Inline injector- requires high pressure.
③ In Column injector- permits injection of feed solution directly onto
column.
Oven-
Maintain MP temperature.
Both column in SFC & Extraction vessel in SFE, are placed inside Oven.
Temp. display with keypads is provided for adjusting temp.

15. Columns-
Once sample is injected into supercritical stream, it is carried into analytical
column that contains highly viscous liquid (SP) into which analytes can be
temporarily adsorbed & then released based on their chemical nature.
Properties-
SP used in column should not be soluble with supercritical CO2.
SP like Octadecylsilyl (C18) Aminopropyl bonded silica, Decylsilyl (C8) are used
with SFC.
Microprocessor-
Controls variables like pumping pressure, oven temp. & detector
performance.

16. Restrictor-
Used to maintain required pressure in column by pressure adjustable
diaphragm/ controlled nozzle.
Placed either at end of column or after detector.
Detector-
Similar to HPLC & GC.
1. Flame ionization detector (FID)
2. UV Spectrophotometric detector (UVD)
3. Flame Photometric detector (FPD)
4. Refractive index detector (RID)
5. Light scattering detector ( LSD)
Also FTIR detector, fluorescence emission spectrometer & Thermionic
detector are compatible in SFC.

17. Collector-
Extract is obtained as fine particles through aerosol like device & collected.
Data recorder-
Computing system with suitable software that can display chromatogram &
other parameters.
Calibrations & system suitability parameters can conventionally determined
using software.

18. ① In SFC, CO2 is cooled by the help of cooler & introduced into column
which is placed in oven.
② Temp. is maintain by temp. programming to achieve critical point.
③ The restrictor maintain the pressure thus MP is converted to supercritical
fluid.
④ Sample is carried through a separating column by supercritical fluid
where the mixture is divided into unique bands based on amount of
interaction between individual analytes & SP in column.
⑤ Separated analytes reach the detector, the signals are detected & their
identities & quantities are determined.
Working

19. Applications
I. For separation of chiral molecules & uses the same column as a standard
HPLC system.
II. For achiral separation & purification in p'ceutical industry.
III. For identification & quantification of pesticides in food products like
canned foods, vegetables & fruits.
IV. Applied for separation of large no. of enamtiomers, diastereoisomers &
geometrical isomers.
V. SFC finds use in impurity profiling while it is coupled with mass detector.
VI. Explosive & propellants, industrial Chemicals, Metals & chelates &
Organic Compounds.

20. THANK
YOU

21. Multiple Choice Question
1) A supercritical fluid (SCF) is any substance at a ____
a. Temperature & pressure below its critical point.
b. Temperature & pressure above its critical point.
c. Temperature & pressure below its triple point.
d. Temperature & pressure same its critical point.
2) A supercritical fluid chromatography is hybrid of ____
a. GC - SC
b. GC - LC
c. HPTLC - GC
d. LLC - SLC

22. 3) Chromatography is the process of identification, purification &
separation of components of mixture on basis of ____
a. Difference in their solubility.
b. Difference in their affinity for MP & SP.
c. Difference in their concentration of solution.
d. Difference in their viscosity.
4) Density of supercritical fluid are in the range of ____ g/cm^3.
a. 0.01 - 0.05
b. 0.2 - 0.5
c. 4 - 6
d. 0.6 - 2

23. 5) Modifiers like alcohols are added in CO2 ____
a. To make it non-toxic
b. To elute non-polar solute
c. To achieve critical point early
d. To elute polar solvent
6) SCF having ____ properties.
a. Density
b. Viscosity
c. Density, Viscosity & Others
d. Both a & b

24. 7) When pressure is increased in SFC, the elution time ____
a. Increased
b. Decreases
c. Remain same
d. Depends upon mixture used being separated.
8) In SFC, mobile analyte affinity for analyte is a function of ____
a. Mobile phase density
b. Mobile phase viscosity
c. Mobile phase solubility
d. All of the above

25. 9) Retention factor depends upon ____
a. Temperatures & pressure
b. Mobile phase density
c. Composition of SP & MP
d. All of the above
10) For Capillary SFC, injectors used is ____
a. HPLC design injector
b. Time split injector
c. Pneumatic injector
d. All of the above

26. 11) In SFC what kind of pump is not used ?
a. Syringe pump
b. Reciprocating pump
c. Pneumatic pump
d. None of the above
11) Caffeine from Coffee can be separated best using ____
a. Chloroform
b. Carbon Tetrachloride
c. Water
d. Supercritical CO2

27. 12) select correct statement -
a. Supercritical temperature is the above which substance can no longer exists
as a gas, no matter how much temperature is applied.
b. Supercritical temperature is the above which substance can no longer exists
as a liquid, no matter how much pressure is applied.
c. Supercritical temperature is the above which substance can no longer exists
as a solid, no matter how much pressure is applied.
d. Supercritical temperature is the above which substance can no longer exists
as a liquid, no matter how much temperature is applied.
13) Why CO2 is preferred SFC mobile phase ?
14) How the lower viscosity of SF is advantageous over liquid in
Chromatography ?