Chromatographic Methods and

Hyphenated Techniques
JOSHUA RICHARD T. PAYOPANIN, RMT
INTENDED LEARNING OUTCOMES

At the end of the session, students will be able to:

1. Understand different principles in chromatography
2. Identify apparatus and instruments in chromatography
3. Recognize different applications in chromatography
4. Distinguish disadvantages of different types of
chromatography
CHAPTERS OVERVIEW

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
reactive (H2), and High Separation
Time (N2)
Figure 1 Block diagram of a gas-chromatographic apparatus.
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).
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 inside diameters of 2 to 4 mm.
⮚Prepared from naturally occurring diatomaceous Figure. 5 Capillary GC Column
earth.
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
Chromatogra
m
Liquid Phases for Gas-
Liquid Chromatography
• Desirable properties for the immobilized liquid phase include
⮚ low volatility
⮚ thermal stability
⮚ chemical inertness
⮚ solvent character-characteristics such thatkand
αvaluesfor 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 SP 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.
 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

Effect of
flow rate
and
packing
size in
plate
height
 • 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.

High- ⮚Advantages of reciprocating pumps include small

internal volume, high output pressure (up to 10,000
Performance psi), ready adaptability to gradient elution, and constant

Liquid
flow rates.
• Column
Chromatograp ⮚Most columns range in length from 10 to 30 cm and have

hy
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
measurements.

Figure 8. UV Detector for HPLC

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
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.
Additional
materials
• HPLC Virtual Lab Simulation
- https://www.labster.com/simulations/hplc/

• Caffeine extraction
- https://www.labster.com/simulations/liquid-liquid-extraction-extract-caffeine-from-your-everyday-
drinks/

• Isolation of Plant Pigments by Column Chromatography

- https://vlab.amrita.edu/?sub=3&brch=64&sim=160&cnt=211

• Separation of Plant Pigments through Paper Chromatography

• https://amrita.olabs.edu.in/?sub=79&brch=17&sim=124&cnt=4
 1. Harris, D. C., &; Lucy, C. A.
(2016). Quantitative Chemical
Analysis, 9th ed. New York:
W.H. Freeman.

REFERENCES 2. Skoog, D. A., West, D. M.,

Holler, F. J., &; Crouch, S. R.
(2014). Fundamentals of
Analytical Chemistry. Australia:
Brooks/Cole Cengage Learning.
THE
END