High Performance Liquid
Chromatography
Theory & Principle
Instrumentation
Applications
Disadvantages
Introduction
High pressure (before 1970) or high
performance (after 1970)
Utilizes a liquid mobile phase to
separate the components of mixture
Components- dissolved in a solvent and
then forced to flow through a
chromatographic column under high
pressure
Types of HPLC Separations (partial list)
Normal Phase: Separation of polar analytes by
partitioning onto a polar, bonded stationary
phase.
Reversed Phase: Separation of non-polar analytes
by partitioning onto a non-polar, bonded
stationary phase.
Adsorption: In Between Normal and Reversed.
Separation of moderately polar analytes using
adsorption onto a pure stationary phase (e.g.
alumina or silica)
Ion Chromatography: Separation of
organic and inorganic ions by their
partitioning onto ionic stationary phases
bonded to a solid support.
Size Exclusion Chromatography:
Separation of large molecules based in
the paths they take through a “maze”
of tunnels in the stationary phase.
HPLC - Instrumentation
Solvent Delivery System (Pump)
Injector (introduce samples)
Column guard
Column (containing stationary phase)
Detectors (“eyes” )
Waste Collector
Recorder (Data Collection)
Schematic Diagram of
HPLC System
Pump System
Ideal pumps:
Ability to generate high pressure
Pulse-free output
Accurate control of flow
Corrosion resistant
Role: Deliver the mobile phase
Two groups of pumps:
Constant pressure
Constant volume
Pump System
Three types of pumps are available:
1. Reciprocating pumps (90% of Commercial
HPLC; produce pulse flow)
2. Displacement pumps (produce flow that are
independent of viscosity and back pressure)
3. Pneumatic pumps (cannot do gradient and
pressure less than 2000psi)
Injectors
Most common
injector is
sample loop
(5-500uL; 0.5-5uL)
Injection - usually 5 to 1000 L volumes, all
directly onto the column
not much worry about capacity since the columns
have a large volume (packed).
Injector is the last component before the
column(s)
6-PORT Rotary Valve is the standard manual
injector
Automatic injectors are available
Two positions, load and inject in the typical
injector
Injection loop internal volume determines
injection volume.
Inject (move the sample
loop into the mobile
phase flow)
Columns
Analytical column variables
Length (10-30 cm)
ID (4-10mm)
Packing (many kinds)
Particles sizes (3-10 µm); pore sizes
Most common columns 25cm x 4.6 id with
5µm particles
Preparative columns
Analytical vs Preparative HPLC
Analytical HPLC Preparative HPLC
• Sample goes from • Sample goes from
detector into waste. detector into
fraction collector
• Goal: Quantification
and/or identification • Goal: Isolation
of compounds and/or purification
of compounds
Source: Huber and Majors. 2007.
Stationary Phase
Stationary phases are particles which are
usually about 1 to 20 m in average diameter
(often irregularly shaped)
In Adsorption chromatography, there is no
additional phase on the stationary phase particles
(silica, alumina, Fluorosil).
In Partition chromatography, the stationary phase
is coated on to (often bonded) a solid support
(silica, alumina, divinylbenzene resin)
Polar (stationary phase for normal phase
HPLC):
Silica, alumina
Cyano, amino or diol terminations on the bonded
phase
Non-Polar (stationary phase for Reversed
Phase HPLC)
C18 to about C8 terminations on the bonded phase
Phenyl and cyano terminations on the bonded
phase
Mixtures of functional groups can be used!!
Packed particles in a column require:
Frits at the ends of the column to keep the
particles in
Filtering of samples to prevent clogging with
debris
High pressure pumps and check-valves
Often a “Guard Column” to protect the analytical
column
Guard column:
• A miniature column, ideally
packed with the same packing
material as the analytical
column.
• Installed in front of an
analytical column to protect it
from strongly retained
impurities and prolong the its
shelf life.
Detectors
Visualize separated compounds and translate
the concentration changes into signals
Using every conceivable physical and
chemical properties
Characteristics of an ideal detector
Adequate sensitivity
Good stability and reproducibility
Gives linear response to analysts that have several
ranges magnitudes
Short response time
High reliability and ease of use
Similarity in response toward all analyst
Non-destructive
Two basic types of detectors
Bulk property detectors response to a
mobile-phase bulk property
Refractive index
Dielectric constant
Density
Solute property detectors response to
solute property
Spectroscopy (IR, UV, MS, Fluorescence)
Popular Type Detectors
UV/Visible absorption detectors
Fluorescence
Refractive Index (RI)
Electrochemical (ED)
UV/Vis Absorption Detector
Usually utilize typical UV-VIS lamps and 254 nm
default wavelength
Can be set to other wavelengths.
Simple filter detectors no longer widely used
adjustable wavelength units are cost-
effective.
Compounds with strong UV/Vis chromophores
Compounds with conjugated or nonconjugated
double bonds; aromatic molecules.
Note: Chromophore is a group of atoms and electrons forming part of an organic
molecule that causes it to be coloured.
UV/UV-VIS detectors are most frequently used to measure
components showing an absorption spectrum in the
ultraviolet or visible region.
A UV detector employs a deuterium discharge lamp (D2
lamp) as a light source, with the wavelength of its light
ranging from 190 to 380 nm.
If components are to be detected at wavelength longer than
this, a UV-VIS detector is used, which employs an
additional tungsten lamp (W lamp).
Advantage: Simple, reliable, inexpensive,
compatible with gradient elution and non-
destructive
Disadvantage: Not as sensitive as
fluorescence detection, ED, not-universal
(only for molecules with chromophores)
Samples of use: vitamins, carotenoids,
phytonutrients
Fluorescence Detectors
Compounds with fluorophors
By nature (carbamate pesticides, aflatoxins, vitamins,
amino acids)
By post-column derivatization
Advantage: Highly sensitive (femtomole = 10-15),
low background, highly selective
Disadvantage: Perceived difficulty of its use,
more instrumental variables to account for
during optimization, changes in fluorescence
can occur with pH and viscosity
Samples of use: drugs and aflatoxins
Refractive Index Detectors
Compounds that do not have strong UV/Vis
chromophores, fluorophours, electrochemical
activity or ionic conductivity
Advantage: Universal in nature
Disadvantage: Lack of sensitivity; impractical for
gradient elution; instability of base line
Samples of use: organic acids, sugars, fungal
metabolites, oligosaccharides
Responds to analytes changing the RI of the
mobile phase
requires a separate reference flow of mobile phase
Extremely temperature sensitive, usually
heated
sensitive to temp changes of +/- 0.001 °C
No longer really widely used
Absorbance detectors are relatively cheap.
Useful for process work, on-line monitoring,
etc.
Recorder/Data Collections
Many recoding devices are available:
Strip-chart recorder (retention time/Peak
areas or peak height)
Integrator
Computer controlled data collections
The Mobile Phase in HPLC...
Must do the following:
solvate the analyte molecules and the solvent they are
in
be suitable for the analyte to transfer “back and forth”
between during the separation process
Not too compressible (causes pump/flow
problems)
Free of gases (which cause compressability problems)
Must be:
compatible with the instrument (pumps, seals,
fittings, detector, etc)
compatible with the stationary phase
readily available (often use liters/day)
of adequate purity
spectroscopic and trace-composition usually!
Elution Condition
Elution can be performed isocratically or with
a gradient
Isocratic elution is performed with a constant
solvent (pure or constant mixture)
In gradient elution the solvent mixture is
altered during the run if one solvent is not
satisfactory
In a reverse-phase separation, the solvent is
made gradually more polar (higher ε°)
Isocratic versus Gradient Elution
Isocratic elution has a constant mobile phase
composition
Can often use one pump!
Mix solvents together ahead of time!
Simpler, no mixing chamber required
Limited flexibility, not used much in research
mostly process chemistry or routine
analysis.
Gradient elution has a varying mobile phase
composition
Uses multiple pumps whose output is mixed
together
often 2-4 pumps (binary to quarternary
systems)
Changing mobile phase components changes the
polarity index
can be used to subsequently elute
Increasing ratio of acetonitrile during the
analysis behaves like temperature
programming in GC
Segmented gradient diagram
HPLC Separation
Separation in based upon differential
migration between the stationary and
mobile phases.
Mobile Phase - carries the sample through the
stationary phase as it moves through the
column.
How does separation takes place ?
The Chromatogram
to - elution time of unretained peak
tR- retention time - determines sample identity
tR
tR
mAU Area is proportional
to the quantity of analyte.
to
Injection time
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
Applications
Carbohydrates
1. fructose
2. Glucose
3. Saccharose 5
4. Palatinose
5. Trehalulose mAU
2
3
6. isomaltose 1 4
6
Zorbax NH2 (4.6 x 250 mm)
time
70/30 Acetonitrile/Water
1 mL/min Detect = Refractive Index
Disadvantages
Only one sample can be analyzed at one time
Difficult to optimize separations
Column performance is very sensitive, which
depends on the method of packing.
No universal and sensitive detection system is
available
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
•Sample must be volatile •Samples are nonpolar
and polar
Comparison of HPLC and GC
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
•Typically < 500 amu
to survive high
temperature injection
port and column
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
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
GC GC
•Most common FID,
•Mobile phase is a universal to organic
sample carrier only compounds
