High Performance Liquid
Chromatography
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
COMPOSITION OF A LIQUID
CHROMATOGRAPH SYSTEM
Solvent
Solvent Delivery System (Pump)
Injector
Sample
Pre column and Column
Detectors (Diode Array)
Waste Collector
Recorder (Data Collection)
Picture of HPLC instrument
Uses of HPLC
To determine vitamin content and nutritional quality of food (I.e.
Vitamin C)
Spoilage detection and process control of foods (i.e. pyruvic
acid and lactose in milk)
To detect food additive (i.e. benzoic acid, adulteration of
fumaric acid as synthetic malic acid in juices)
HPLC Chromatograph injectors
The function of the injector is to place the sample into
the high-pressure flow in as narrow volume as possible
so that the sample enters the column as a
homogeneous, low-volume plug.
To minimize spreading of the injected volume during
transport to the column, the shortest possible length of
tubing should be used from the injector to the column.
When an injection is started, an air actuator rotates the
valve: solvent goes directly to the column; and the
injector needle is connected to the syringe. The air
pressure lifts the needle and the vial is moved into
position beneath the needle. Then, the needle is lowered
to the vial.
HPLC columns
The column is one of the most important components of the
HPLC chromatograph because the separation of the sample
components is achieved when those components pass through
the column.
The High performance liquid chromatography apparatus is made
out of stainless steel tubes with a diameter of 3 to 5mm and a
length ranging from 10 to 30cm.
Normally, columns are filled with silica gel because its particle
shape, surface properties, and pore structure help to get a good
separation.
Silica is wetted by nearly every potential mobile phase, is inert to
most compounds and has a high surface activity which can be
modified easily with water and other agents.
Silica can be used to separate a wide variety of chemical
compounds, and its chromatographic behavior is generally
predictable and reproducible.
Picture of an HPLC column
The analytes interacting most
strongly with the stationary
phase will take longer to pass
through the system than those
with weaker interactions.
These interactions are usually
chemical in nature, but in some
cases physical interactions can
also be used.
WHAT AFFECTS SYSTEM
Column Parameters Instrument Parameters
Column Material Temperature
Stationary Phase Flow
Coating Material Signal
Sample Sensitivity
Detector
WHAT AFFECTS SYSTEM
Sample Parameters
Concentration
Matrix
Solvent Effect
Sample Effect
Several column types
(can be classified as )
Normal phase
Reverse phase
Size exclusion
Ion exchange
Normal phase
In this column type, the retention is
governed by the interaction of the polar
parts of the stationary phase and solute.
For retention to occur in normal phase, the
packing must be more polar than the
mobile phase with respect to the sample
Example application: Detection of fat-
soluble vitamins and polyphenol.
Reverse phase
In this column the packing material is relatively
nonpolar and the solvent is polar with respect to
the sample.
Retention is the result of the interaction of the
nonpolar components of the solutes and the
nonpolar stationary phase.
Typical stationary phases are nonpolar
hydrocarbons, waxy liquids, or bonded
hydrocarbons (such as C18, C8, etc.) and the
solvents are polar aqueous-organic mixtures
such as methanol-water or acetonitrile-water.
Reverse phase
Example applications: Protein and amino
acids detection, water- or fat-soluble
vitamins.
Size exclusion
In size exclusion the HPLC column is
consisted of substances which have
controlled pore sizes and is able to be
filtered in an ordinarily phase according to
its molecular size.
The mobile phase in this mode is chosen
for sample solubility, column compatibility,
and minimal solute–stationary phase
interaction. Otherwise, it has little effect on
the separation.
Ion exchange
In this column type the sample
components are separated based upon
attractive ionic forces between molecules
carrying charged groups of opposite
charge to those charges on the stationary
phase.
The mobile phase in ion-exchange HPLC
is usually an aqueous buffer , and solute
retention is controlled by changingmobile
phase ionic strength and/or pH.
Detectors
Instrument in the chromatographic
system which senses the presence of a
compound passing through, and
provides an electronic signal to a
recorder or computer data station.
Output is usually peaks, that is, the
chromatogram.
Types of Detectors
Absorbance (UV Evaporative Light
with Filters, UV with Scattering Detector
Monochromators) (ELSD)
IR Absorbance Electrochemical
Fluorescence Mass-
Spectrometric
Refractive-Index
Photo-Diode Array
Types of Detectors:
UV – Ultraviolet light
Lamp
Grating/Lens - Wave length
FlowCell
PhotoDiode - Differential Light Output
RI – Refractive Index
Universal analyte detector
Solvent must remain the same throughout separation
VERY temperature sensitive
Sometimes difficult to stabilize baseline
FD – Fluorescence
Excitation wavelength generates
fluorescence emission at a higher
wavelength
Analytes must have fluorophore group
Can react analyte with fluorophore reagent
Very sensitive and selective
More difficult methods transfer
Results very dependent upon separation
conditions
MS – Mass Spec
Mass to charge ratio (m/z)
Allows specific compound ID
Several types of ionization techniques
electrospray, atmospheric pressure
chemical ionization, electron impact
