Chromatographic

Methods
(GC,HPLC, and SFC)
 GROUP 1

Abapo, Julianne V.

Alfeche, Griezel G.

Arnoza, Michael Max Rey C.

Bandolon, Ruffamae C.

Calonia, Denver C.
OBJECTIVES:

• Identify the essential components of a GC, HPLC, and SFC.

• Distinguish GC from HPLC and SFC.

• List different applications of GC, HPLC, and SFC.

Theories Behind Chromatography
Plate Theory
According to the model, the chromatographic column consists of separate layers known
as the theoretical plates. These plates are hypothetical zones or stages that help in
establishing an equilibrium between two phases. Thus, plate theory describes the
properties of chromatographic separation and hence determines the number of
theoretical plates.
Rate Theory Of Chromatography
The theories describes how the analytes move in the stationary phase as the mobile phase flows
through it. Also, the rate theory of chromatography defines the activity in a chromatography column. It
shows that when solute elutes out of the column, it impacts the band shape and is affected by the
elution rate. The rate theory provides information about the shape and breadth of the elution bands
as the mobile phase migrates and flows through a column. It helps understand the process of peak
dispersion and factors impacting band broadening.
Gas Chromatography
Gas Chromatography

Principle

• Gas-solid chromatography uses solid adsorbent as a

stationary phase, while gas-liquid chromatography uses
a thin layer of non-volatile liquid bound to solid support.
Components of a sample are separated according to
their partition co-efficient.
Gas Chromatography

Principle

• Gas-solid chromatography uses solid adsorbent as a

stationary phase, while gas-liquid chromatography uses
a thin layer of non-volatile liquid bound to solid support.
Components of a sample are separated according to
their partition co-efficient.
 Gas Chromatography

Theory

• The separation of compounds is based on the different

strengths of interaction of the compounds with the
stationary phase (“like-dissolves-like”-rule). The
stronger the interaction is, the longer the compound
interacts with the stationary phase, and the more time it
takes to migrate through the column (=longer retention
time)
 Gas Chromatography

Essential Components

1. Sample injection system:

A sample port is used to introduce the sample into the vaporization chamber and a sample splitter is used
to direct excess sample to waste. Commercial gas chromatographs use split and split less injections.
 Gas Chromatography

Essential Components

2. Carrier Gas:
A carrier gas plays a vital role in GC. It should be inert, dry & free of oxygen. Helium, Nitrogen, argon &
hydrogen gases are used as carrier gas depending upon the desired performance & detector being
used .Carrier gas is supplied at high pressure & is passed to instrument at a rapid & reproducible rate.
 Gas Chromatography

Essential Components

3. Separation column:
Open tubular columns or capillary columns & packed columns are used in GC. WCOT columns have a
thin layer of stationary phase coated along the column walls, while SCOT columns have greater sample
capacity and greater column efficiencies. Coated Fused Silica open tubular columns are popular.
 Gas Chromatography

Essential Components

4. Column Oven or Thermostat chambers:

The thermostat oven are there to control the temperature of the column to conduct precise work. The
oven can be operated in two manners: isothermal programming or temperature programming. In
isothermal programming, the temperature of the column is held constant throughout the whole
separation. In the temperature programming method, the column temperature is either increased
continuously or in steps as the separation progresses.
 Gas Chromatography

Essential Components

5. Detectors:
Most common types of detectors used in GC are: Mass Spectrometer , Flame ionization detector (FID),
Electron capture detector (ECD), Thermal conductivity detector (TCD),Atomic emission detector
(AED), Photoionization detector (PID), Chemilumnescence detector. Detector is present at the end of
the column & gives the quantitative measurement of the components of the mixture as they elute in
combination with the carrier gas.
 Gas Chromatography

Essential Components

5. Detectors:
Most common types of detectors used in GC are: Mass Spectrometer , Flame ionization detector (FID),
Electron capture detector (ECD), Thermal conductivity detector (TCD),Atomic emission detector
(AED), Photoionization detector (PID), Chemilumnescence detector. Detector is present at the end of
the column & gives the quantitative measurement of the components of the mixture as they elute in
combination with the carrier gas.
 Gas Chromatography

Essential Components

Mass Spectrometer
 Gas Chromatography

Essential Components

Electron Capture Detector

 Gas Chromatography

Essential Components

Chemilumnescence Detector
 Gas Chromatography

Essential Components

6. Amplification & Recorder system:

These are the last & final components of GC instrumentation. These are meant to record the signals
that come from the detector. These use special electronic circuits the process & amplify the signals so as
to display in an understandable graphical format that represents several peaks of the constituents of the
sample under analysis.
 Gas Chromatography
Applications

GC has wide range of applications in various fields.

- It has a medicinal & pharmaceutical applications.

- It is used in food, beverage, flavor & fragrance analysis. It is also helpful in environmental
analysis and monitoring.

- It is used to detect doping of drugs.

- In forensics, it is used in cases of arson, detection of body fluids, for the testing of fiber ,
blood alcohol, detection of poisons , pesticides & also to detect explosives residues.

- It is also useful in Security and chemical warfare agent detection.

 Gas Chromatography
Applications

The application of gas chromatography to environmental analysis:

GC has significant role in the identification & quantification of pollutants of environment. Capillary GC is used
in the analysis of various classes of persistent organic contaminants in air, water, soils, sediments and biota. The
organic pollutant groups like volatile organic compounds (VOCs); polycyclic aromatic hydrocarbons
(PAHs); pesticides; and halogenated compounds such as polychlorinated dibenzo-p-dioxins and dibenzofurans,
polychlorinated biphenyl, terphenyls, naphthalenes and alkanes, organochlorine pesticides, and the
brominated flame retardants, polybrominated biphenyls and polybrominated diphenylethers are analysed by
GC.
 Gas Chromatography
Applications

Application of gas chromatography in food analysis:

Gas chromatography (GC) is widely used in food analysis. Quantitative and qualitative analysis of food
composition, natural products, food additives, flavor and aroma components, a variety of transformation products
and contaminants, such as pesticides, fumigants, environmental pollutants, natural toxins, veterinary drugs, and
packaging materials are done through GC.
 Gas Chromatography
Applications

Application of GC in catalysis:
GC is an analytical tool used to determine the physicochemical properties of solid catalysts and adsorbents,
catalyst evaluation and kinetics, and study of catalytic reactions. It can be used in two ways: in a chromatographic
column, where the properties are estimated by chromatographic parameters such as retention time, retention
volume, band width and shape, and behavior of the chromatographic peak; and in a micro reactor, which provides
a rapid analysis of feed and products of the catalytic process.
 Gas Chromatography
Applications

Application of GC to the qualitative & quantitative Copolyamide analysis:

The gas chromatographic separation of diacids recovered from hydrolyzed copolyamides prepared from
hexamethylenediamine gives both qualitative and quantitative results. The method requires only less than 0.2 gm
samples and requires a caliberation curve for each diamine and diacid. The liberated diacids in the hydrozylate are
esterified with boron triflouride – methanol and recovered in diethyl ether. A second hydrolyzed used is made
caustic and extracted with n-butanol. The residue is gas chromatographed to identify diamines.
 Gas Chromatography

GC analysis of xylene isomers: Xylene isomers are precursors to many chemicals. o-xylene
is a precursor for pthalic anhydride , m- xylene is a precursor for isopthalic acid , p-
xylene is aprecursor for tetrapthalic acid & dimethyl terepthalate. The cresol isomers are
precursors to many chemicals. The chromatogram of a mixture of aromatic & methyl phenol
compounds was generated using an SLB-IL60 ionic liquid column. It’s interation
mechanisms allows the separation of all three xylene isomers & all three cresol isomers.

GC analysis of petroleum products: The petroleum products such as jet fuel petrol, diesel,
and kerosene are also analyzed through GC. Test parameters involves column- supeul –Q
PLOT , oven-35 degree Celsius, 16 degree per min. to 250 degree Celsius, detector – TCD ,
carrier gas – He ,sample-jet fuel. GC analysis of water ib gasoline is also done.
 Gas Chromatography

Identification of hazardous compounds in waste damps

- Quantification of drugs & their metabolites in blood & urine for both
pharmacological & forensic applications
- Identification of reaction products.
- Quantification of pollutants in drinking & waste water.
- Analysis of industrial products for quality control
- Skin sample analysis.
- RNA isolation
- Astro chemistry & geochemical search.
HPLC
 HPLC

Principle

• HPLC is a separation technique used to separate

individual components of a sample based on the
distribution of the analyte between a mobile and
stationary phase.
 HPLC

Theory

• The intermolecular interactions and the packing

material (stationary phase), define their time “on-
column.” Hence, different molecules of the sample
are eluted at varying times, and the separation of
individual components of the sample is achieved.
 HPLC

Essential Components
1. Solvent Reservoir

 Mobile phase contents are contained in a glass reservoir.

 The mobile phase, or solvent, in HPLC, is usually a mixture of polar
and non-polar liquid components whose respective concentrations
are varied depending on the composition of the sample.
 HPLC

Essential Components
2. Pump

 A pump aspirates the mobile phase from the solvent reservoir and
forces it through the system’s column and detector.
 Depending on several factors, including column dimensions, the
particle size of the stationary phase, the flow rate and composition
of the mobile phase, operating pressures of up to 42000 kPa (about
6000 psi) can be generated.
 HPLC

Essential Components
3. Sample Injector

 The injector can be a single injection or an automated injection

system.
 An injector for an HPLC system should provide an injection of the
liquid sample within the range of 0.1-100 mL of volume with high
reproducibility and under high pressure (up to 4000 psi).
 HPLC

Essential Components
4. Columns

 Columns are usually made of polished stainless steel, are between

50 and 300 mm long, and have an internal diameter between 2 and
5 mm.
 They are commonly filled with a stationary phase with a particle size
of 3–10 µm.
 Columns with internal diameters of less than 2 mm are often called
micro bore columns.
 Ideally, the temperature of the mobile phase and the column should
be kept constant during an analysis.
 HPLC

Essential Components
5. Detector

 The HPLC detector, located at the end of the column, detects the
analytes as they elute from the chromatographic column.
 Commonly used detectors are UV-spectroscopy, fluorescence,
mass-spectrometric and electrochemical detectors.
 HPLC

Essential Components
6. Data Collection Devices

 Signals from the detector may be collected on chart recorders or

electronic integrators that vary in complexity and their ability to
process, store and reprocess chromatographic data.
 The computer integrates the detector’s response to each
component and places it into a chromatograph that is easy to read
and interpret.
 HPLC
Applications

The HPLC has developed into a universally applicable method so that it finds its use in almost all areas of
chemistry, biochemistry, and pharmacy.

 Analysis of drugs
 Analysis of synthetic polymers
 Analysis of pollutants in environmental analytics
 Determination of drugs in biological matrices
 Isolation of valuable products
 Product purity and quality control of industrial products and fine chemicals
 Separation and purification of biopolymers such as enzymes or nucleic acids
 Water purification
 Pre-concentration of trace components
 Ligand-exchange chromatography
 Ion-exchange chromatography of proteins
 High-pH anion-exchange chromatography of carbohydrates and oligosaccharides
SFC
SFC

Principle

• SCF`s principles are similar to those of high

performance liquid chromatography (HPLC),
however SFC typically employs fluids that are
raised above their critical pressure (Pc) and critical
temperature (Tc) as mobile phases. Carbon
dioxide, CO2, is the supercritical fluid most
commonly used as a mobile phase for SFC. It is
capable of solvating mostly nonpolar analytes.
Because the supercritical phase represents a state
in which liquid and gas properties converge,
supercritical fluid chromatography is sometimes
called "convergence chromatography."
SFC

Theory
• Supercritical fluid Chromatography is based on
supercritical fluids (SF) having densities and dissolving
capacities similar to those of certain liquids, but lower
viscosities and better diffusion properties. Accordingly,
SF used as mobile phases in chromatography should
act both as substance carriers like the mobile phases in
gas chromatography (GC) and also dissolve these
substances like the solvents in liquid chromatography
(HPLC).
 SFC

Essential Components
1. Solvent Delivery

Carbon dioxide is delivered in high pressure cylinders for analytical and lab-
scale instruments, or in high capacity tanks for larger units. If the bottle is
equipped with a dip tube, liquid CO2 can be withdrawn without any
significant pressure drop due to liquid phase evaporation, but at high CO2
flow rates, there is a risk of freezing the bottle. Liquid modifiers used as
polar additives in supercritical fluid chromatography are typically HPLC or
analytical grade solvents.
 SFC

Essential Components
2. Pumping System

Piston or membrane pumps are used for SFC systems, with a sub-
cooler between the CO2 source and the pump's suction valve to
avoid cavitation.
 SFC

Essential Components
3. Feed Injection

Three different modes of injection can be applied

a. Loop injection is a direct transposition of what is applied in

analytical SFC. A low pressure feed pump is used to fill the loop.
This is mostly recommended for preliminary tests of column
performance and elution parameters.
 SFC

Essential Components
3. Feed Injection

Three different modes of injection can be applied

b. An “in-line” injection mode is more versatile: the system offers

a better flexibility for changing the injected volume. A high-pressure
pump is required to inject the feed solution, but the injected stream
is dissolved in the eluent flow.
 SFC

Essential Components
3. Feed Injection

Three different modes of injection can be applied

c. The “in-column” injection mode is an alternative which permits

injection of the feed solution directly onto the column, without any
dilution. The selected injection mode depends on the application,
but the right selection is crucial to avoid injection profile distortion
that may occur due to partial solute precipitation or to the injected
solvent used to dissolve the feed.
 SFC

Detection
SFC is compatible with both HPLC and GC detectors:
a. On line pressure withstanding detectors

UV variable wavelength detectors

 SFC

Detection
SFC is compatible with both HPLC and GC detectors:
a. On line pressure withstanding detectors

UV diode array detectors

 SFC

Detection
SFC is compatible with both HPLC and GC detectors:
a. On line pressure withstanding detectors

Special chiral detectors

 SFC

Detection
SFC is compatible with both HPLC and GC detectors:
b. Off line low pressure detectors

Mass spectrometric detector

 SFC

Detection
SFC is compatible with both HPLC and GC detectors:
b. Off line low pressure detectors

Light scattering detector

 SFC

Detection
SFC is compatible with both HPLC and GC detectors:
b. Off line low pressure detectors

Flame ionization detector

 SFC
Applications

• Supercritical fluid chromatography (SFC) is commonly used in industry for achiral separations
and purifications in the pharmaceutical industry.
• It can be used to separate complex hydrocarbon mixtures, oligomeric mixtures of polypropylene
glycols, and drug compounds such as antibiotics, prostaglandins, steroids, taxol, vitamins,
barbiturates, non-steroidal anti-inflammatory agents.
  
Characteristics Of Each Chromatographic Methods

Gas Chromatography

 Gas chromatography (GC) is one of the popular chromatography techniques to separate volatile
compounds or substances. The mobile phase is a gas such as helium, and the stationary phase is
a high-boiling liquid that is adsorbed on a solid.

• Carrier gases are used to move the solutes through the column.

• The mobile phase in GC is usually a carrier gas, which may be helium (an inert gas) or nitrogen
(an unreactive gas).
  
Characteristics Of Each Chromatographic Methods

HPLC

 HPLC method has a column that’s: high sensitivity, small sample size, fast analysis speed, high
separation efficiency and that Columns can be used repeatedly.

 HPLC uses grade water is ultra-pure water with low UV-absorbance wherein specific Conductivity (16-18
mega ohms) is maintained by water purification systems.

 Liquid as the mobile phase, and a high-pressure infusion system is used to pump a single solvent
with different polarities.
  
Characteristics Of Each Chromatographic Methods

SFC

 Similar to gas and liquid chromatography, but using a supercritical fluid as mobile phase.

 Faster stationary phase and mobile phase mass transfer.

 SFC has characteristics between those of LC and GC.

 SFC is a powerful tool for lipid analysis when using alkyl bonded or polar stationary phases.
 
 
 
 EVALUATION  

IDENTIFICATION

1. Chromatography is a physical method that is used to separate and analyze _________.

2. In which type of chromatography is the stationary phase held in a narrow tube and the
mobile phase forced through it under pressure?
3. Gas chromatography can be performed in which of the following ways?
4. In chromatography, the stationary phase can be _________ supported on a solid.
5. Back diffusion occurs mostly in what type of chromatographic method?
6. Absorption in chromatography is a process of?
7. The GC trace obtained after an experiment is called?
8-10. Give one example of each chromatographic method (GC, HPLC, and SFC).

Differentiate between GC, HPLC, and SFC. (5 points).

References
HPLC
HPLC- Definition, Principle, Parts, Types, Uses, Diagram (microbenotes.com)
High Performance Liquid Chromatography (HPLC) : Principle, Types, Instrumentation and Applications - Laboratoryinfo.com
HPLC Working Principle: Types and Applications – StudiousGuy
GC
(PDF) Gas Chromatography -A Brief Review (researchgate.net)
Gas Chromatography Theory (ucla.edu)
SCF
Supercritical Fluid Chromatography - Yuan - Major Reference Works - Wiley Online Library
Majewski, W., Valery, E., & Ludemann‐ Hombourger, O. (2005). Principle and Applications of Supercritical Fluid Chromatography. Journal of Liquid Chromatography & Related Technologies, 28(7-8), 1233–1252. doi:10.1081/jlc-200053039 10.1081/jlc-200053039

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