Principle of HPLC
Operation
Comparison between GC and HPLC
Typical Industrial Application
Introduction
• HPLC is a form of liquid chromatography used to separate compounds that are
dissolved in solution. HPLC instruments consist of a reservoir of mobile phase, a
pump, an injector, a separation column, and a detector.
• Compounds are separated by injecting a sample mixture onto the column. The
different component in the mixture pass through the column at differentiates due
to differences in their partition behavior between the mobile phase and the
stationary phase. The mobile phase must be degassed to eliminate the formation
of air bubbles.
HPLC system
FOUR TYPES OF LIQUID CHROMATOGRAPHY
• Partition chromatography
• Adsorption, or liquid-solid
• chromatography
• Ion exchange chromatography
• Size exclusion, or gel, chromatography
What Is HPLC?
Basic Principles
5
Invention of Chromatography by M. Tswett
Ether Chromatography
Colors
Chlorophyll
CaCO3
6
Comparing Chromatography to the Flow of a
River...
Light leaf
Heavy stone Water flow
Base
7
Mobile Phase / Stationary Phase
• A site in which a moving phase
(mobile phase) and a non-
moving phase (stationary
Mobile phase) make contact via an
phase interface that is set up.
Strong Weak
• The affinity with the mobile
phase and stationary phase
Stationary
varies with the solute.
Separation occurs due to
phase
differences in the speed of
motion.
8
Three States of Matter and Chromatography
Types
Mobile phase
Gas Liquid Solid
Gas
Stationary
phase Liquid
Gas Liquid
chromatography chromatography
Solid
9
Liquid Chromatography
• Chromatography in which the mobile phase is a liquid.
• The liquid used as the mobile phase is called the “eluent”.
• The stationary phase is usually a solid or a liquid.
• In general, it is possible to analyze any substance that can be
stably dissolved in the mobile phase.
10
Interaction Between Solutes, Stationary Phase, and Mobile
Phase
• Differences in the interactions between the solutes and
stationary and mobile phases enable separation.
Solute
Degree of adsorption,
solubility, ionicity, etc.
Stationary
Mobile phase
phase
11
Column Chromatography and Planar
Chromatography
Separation column
Paper or a
substrate coated
with particles
Packing material
Column Chromatography Paper Chromatography
Thin Layer Chromatography (TLC)
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Separation Process and Chromatogram for
Column Chromatography
concentration
Chromatogram
Output
Time 13
Chromatogram
Intensity of detector signal
tR
Peak tR : Retention time
t0 t0 : Non-retention time
h
A A : Peak area
h : Peak height
Time
14
From Liquid Chromatography to High Performance Liquid
Chromatography
• Higher degree of separation!
Refinement of packing material (3 to 10 µm)
• Reduction of analysis time!
Delivery of eluent by pump
Demand for special equipment that can
withstand high pressures
The arrival of high performance liquid chromatography!
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Flow Channel Diagram for High Performance Liquid
Chromatograph
Detector
Column
Pump Column oven
(thermostatic
column chamber)
Eluent Sample injection unit Drain
(mobile phase) (injector)
Data processor
Degasser
16
Advantages of High Performance Liquid
Chromatography
• High separation capacity, enabling the batch analysis of
multiple components
• Superior quantitative capability and reproducibility
• Moderate analytical conditions
• Unlike GC, the sample does not need to be vaporized.
• Generally high sensitivity
• Low sample consumption
• Easy preparative separation and purification of samples
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Fields in Which High Performance Liquid
Chromatography Is Used
• Biogenic substances • Food products
• Vitamins, food additives, sugars, organic
• Sugars, lipids, nucleic acids, acids, amino acids, etc.
amino acids, proteins,
peptides, steroids, amines, etc. • Environmental samples
• Medical products • Inorganic ions
• Drugs, antibiotics, etc. • Hazardous organic substances, etc.
• Organic industrial products
• Synthetic polymers, additives, surfactants,
etc.
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Picture of HPLC instrument
COMPOSITION OF A LIQUID CHROMATOGRAPH SYSTEM
• Solvent
• Solvent Delivery System (Pump)
• Injector
• Sample
• Column
• Detectors (Diode Array)
• Waste Collector
• Recorder (Data Collection)
HPLC Analysis Parameters
Mobile Phases
Flow Rate
Composition
Injection Volume
Column
Oven Temperature
Wavelength
Time Constant
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Modes of High Performance Liquid
Chromatography
Types of Compounds Mode Stationary Mobile Phase
Phase
Neutrals Reversed C18, C8, C4 Water/Organic
Weak Acids Phase cyano, amino Modifiers
Weak Bases
Ionics, Bases, Acids Ion C-18, C-8 Water/Organic
Pair Ion-Pair Reagent
Compounds not Normal Silica, Amino, Organics
soluble in water Phase Cyano, Diol
Ionics Inorganic Ions Ion Anion or Cation Aqueous/Buffer
Exchange Exchange Counter Ion
Resin
High Molecular Weight Size Polystyrene Gel Filtration-
Compounds Exclusion Silica Aqueous
Polymers Gel Permeation-
Organic
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HPLC Applications
Bioscience
Chemical proteins
peptides
polystyrenes nucleotides
dyes
phthalates
tetracyclines
Pharmaceuticals corticosteroids
antidepressants
barbiturates Consumer Products
lipids
antioxidants
sugars
Environmental
polyaromatic hydrocarbons Clinical
Inorganic ions amino acids
herbicides vitamins
homocysteine
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Got a Problem to Solve, Which separation
technique should use
I need a quantitative
separation of I’ll get
carbohydrates in some on it!
of our products
as soon as possible.
I’ll need a separation
technique.
42
Separation Techniques
I have two separation techniques in my lab,
High Performance Liquid Chromatography
and Gas Chromatography. Which should I use?
43
Comparison of HPLC and GC
Sample Volatility Sample Polarity
HPLC HPLC
• No volatility requirement • Separates both polar and
non polar compounds
• Sample must be soluble
in mobile phase • PAH - inorganic ions
GC GC
• Samples are nonpolar
• Sample must be volatile
and polar
44
Comparison of HPLC and GC
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Comparison of HPLC and GC
Sample Thermal Lability Sample Molecular Weight
HPLC HPLC
• Analysis can take place
• No theoretical upper limit
at or below room
temperature
• In practicality, solubility is
limit.
GC GC
• Sample must be able
to survive high • Typically < 500 amu
temperature injection
port and column
46
Comparison of HPLC and GC
Sample Preparation Sample Size
HPLC HPLC
• Sample must be filtered
• Sample size based upon
column i.d.
• Sample should be in
same solvent as mobile
phase
GC GC
• Solvent must be volatile • Typically 1 - 5 L
and generally lower
boiling than analytes
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Comparison of HPLC and GC
Separation Mechanism Detectors
HPLC HPLC
• Both stationary phase • Most common UV-Vis
and mobile phase take • Wide range of non-
part destructive detectors
• 3-dimensional detectors
• Sensitivity to fg (detector
dependent)
GC GC
• Most common FID,
•Mobile phase is a universal to organic
sample carrier only compounds
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