Chromatography
Chapter 8 | BIO 1103
Far Eastern University, BIO1103/4 Cluster AY 2021 – 2022
Discussion Outline
• Gas Chromatography
• GC Column
• Liquid Phases for Gas Liquid Chromatography
• Applications of Gas-Liquid Chromatography
• High-Performance Liquid Chromatography
• High-Performance Partition Chromatography
• High-Performance Adsorption Chromatography
• High-Performance Ion-Exchange Chromatography
• High-Performance Size-Exclusion Chromatography
• HPLC vs GLC
• Solid Phase Extraction
• Solid Phase Microextraction
Gas Chromatography
Types of GC
• Gas-liquid chromatography (GLC)
• Gas-solid chromatography (GSC).
Carrier Gas System
• Helium is the most common
mobile phase, although argon,
nitrogen, and hydrogen are also
used.
• Disadvantage of other gases:
expensive (Ar), Chemically
Figure 1 Block diagram of a gas-chromatographic apparatus. reactive (H2), and High
Separation Time (N2)
Gas Chromatography
Sample Injection System
• The sample port (Figure 2) is ordinarily
about 50°C above the boiling point of the
least volatile component of sample sizes
range from a few tenths of a microliter to
20 mL. Capillary columns require
samples that are smaller by a factor of
100 or more.
• Commercial gas chromatographs with
capillary columns incorporate such
splitters and also allow for sample
injection without splitting when packed
columns are used (split /splitless
injection systems).
Gas Chromatography
DETECTORS
Flame-Ionization Detectors
• FID exhibits a high sensitivity, a large
linear response, and low noise
Thermal Conductivity Detectors
• The limitation of the thermal
conductivity detector is its relatively
low sensitivity.
Electron-Capture Detectors
• Most widely used detectors for
environmental samples because this
detector selectively responds to
halogen-containing organic compounds. Figure 3. A typical flame ionization
detector.
Gas Chromatography
DETECTORS
Electron-Capture Detectors
• Electron-capture detectors are highly
sensitive and have the linear response
of the detector, however, is limited to
about two orders of magnitude.
Hyphenated Methods of Detection
Thermionic Detectors
• Nitrogen and phosphorous-containing
compounds produce increased currents
in a flame in which an alkali metal salt
is vaporized.
Figure 4. A typical thermal conductivity
detector.
Gas Chromatography
DETECTORS
Electrolytic Conductivity Detector
• Compounds containing halogens,sulfur,
or nitrogen are mixed with a reaction
gas in a small reactor tube. The
products are then dissolved in a liquid,
which produces a conductive solution.
Hyphenated Methods of Detection
Photoionization Detectors
• Molecules are photoionized by UV
radiation. The ions and electrons
produced are then collected, and the
resulting current is measured.
Figure 4. A typical thermal conductivity
detector.
GC Column
Capillary, or Open Tubular,
Columns
• Currently, the most widely used
capillary columns are fused-silica
open tubular columns (FSOT
columns).
• Their inside diameters are typically
from 0.1 to 0.5 mm.
Packed Columns
• Packed columns are fabricated from
glass or metal tubing; they are
typically 2 to 3 m long and have Figure. 5 Capillary GC Column
inside diameters of 2 to 4 mm.
• Prepared from naturally occurring
GC Column
Column Thermostating
• Reproducible retention times require
control of the column temperature to
within a few tenths of a degree
Figure. 5 Capillary GC Column
Effect of Temperature on Gas Chromatogram
Liquid Phases for Gas-Liquid Chromatography
• Desirable properties for the immobilized liquid phase include
• low volatility
• thermal stability
• chemical inertness
• Solvent character-characteristics such that k and α values for the
solutes to be resolved fall within a suitable range.
• Polar stationary phases contain functional groups such as － CO, －
OH, unsaturation (double- or triple-bond), － NO₂, and － CN.
Hydrocarbon-type stationary phases and dialkyl siloxanes are
nonpolar.
• Generally, the polarity of the stationary phase should match that
of the sample components.
Common Liquid Stationary Phase for GLC
Applications of Gas-Liquid
Chromatography
QUALITATIVE ANALYSIS
• a chromatogram provides but a single piece
of information about each species in a
mixture (the retention time)
• qualitative analysis of complex samples of
unknown composition is limited.
• limitation has been largely overcome by
linking chromatographic columns directly with
ultraviolet, infrared, and mass spectrometers.
QUANTITATIVE ANALYSIS
• Quantitative GC is based on comparison of
either the height or the area of an analyte
peak with that of one or more standards.
• The Internal-Standard Method Figure 6. Gas Chromatography
System
High Performance Liquid
Chromatography
The most versatile and widely used type of
elution chromatography.
Types of HPLC:
• Partition, or liquid-liquid,
chromatography
• Adsorption, or liquid-solid,
chromatography
• Ion-exchange, orion, chromatography
• Size-exclusion chromatography
• Affinity chromatography.
Figure 7. HPLC
system
High Performance Liquid
Chromatography
Effect of flow rate and packing size in plate height
High Performance Liquid
Chromatography
PUMPING SYSTEM
• The requirements include (1) the generation of pressures of up to 6000 psi (lb/in.),
(2) pulse-free output, (3) flow rates ranging from 0.1 to 10 mL/min, (4) flow
reproducibilities of 0.5% relative or better,and (5) resistance to corrosion by a
variety of solvents.
• Advantages of reciprocating pumps include small internal volume, high output
pressure (up to 10,000 psi), ready adaptability to gradient elution, and constant
flow rates.
COLUMN
• Most columns range in length from 10 to 30 cm and have inside diameters of 4 to
10 mm. Column packings typically have particle sizes of 5 or 10 μm. Columns of
this type often contain 40,000 to 60,000 plates/m.
• high-performance microcolumns with inside diameters of 1 to 4.6 mm and lengths
of 3 to 7.5 cm have become available. These columns, which are packed with 3- or
5-μm particles, contain as many as 100,000 plates/m
High Performance Liquid
Chromatography
DETECTOR
• The most widely used detectors
for liquid chromatography are
based on absorption of ultraviolet
or visible radiation.
• HPLC/MS systems can identify
the analytes exiting from the
HPLC column.
• Several electrochemical
detectors have also been
introduced that are based on
potentiometric, conductometric,
and voltammetric Figure 8. UV Detector for HPLC
measurements.
High Performance Partition Chromatography
Types of High-Performance Partition Chromatography
• Normal Phase Chromatography (Non-Polar MP, Polar SP)
• Reverse Phase Chromatography (Polar MP, Non-Polar SP)
In the reversed-phase method, the most polar component elutes
first, and increasing the mobile phase polarity, hence weakening
its solvent strength, increases the elution time.
High Performance Adsorption Chromatography
• HPAC was based on adsorption of analyte species on a solid
surface
• Finely divided silica and alumina are the only stationary phases
that find extensive use for adsorption chromatography.
• A particular strength of adsorption chromatography not shared by
other methods is its ability to resolve isomeric mixtures such as
meta- and parasubstituted benzene derivatives.
High Performance Ion-Exchange Chromatography
Types of HPIEC
• Suppressor based
- Conductivity detectors have many of the properties of the
ideal detector.
• Single column
- This approach depends on the small differences in
conductivity between sample ions and the prevailing eluent
ions.
High Performance Size-Exclusion
Chromatography
• Size-exclusion, or gel, chromatography is a powerful technique
that is particularly applicable to high-molecular-weight species
• Chromatography based on the hydrophilic packings is sometimes
called gel filtration, whereas techniques based on hydrophobic
packings are called gel permeation.
HPLC vs GLC
Solid Phase Extraction
• The SPE consists of four
steps—column conditioning,
sample sorption, rinsing, and
elution to desorb solutes.
Solid Phase Microextraction
• SPME uses a coated fused-silica fiber to
draw organic compounds from the
ambience by diffusion.
• Alternatively, use of convection or
agitation helps to speed up a deep
sampling process. Headspace is another
popular method, in which the SPME fiber
laying in a confined space draws analytes
from a sample of soil or water by diffusion.
