igh
P erformance
L iquid
C hromatography

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.
This unit is equipped
with two pump units
and a UV/Vis
detector.
The gradient is
controlled via the
pump controllers.
LC
methods are not
as sensitive to
temperature.
Columns are
commonly
mounted outside
the instrument.
Solvents
• All solvents should be ‘HPLC’ grade.
– This is a type of reagent grade material.
– It has been filtered using a 0.2 μm filter.
• Filtered solvent helps extend pump life by
preventing scoring. It also reduces the chances
of a column plugging.
Solvent degassing
All solvents should be degassed prior to use.
This reduces the chances of bubbles being formed in
the column or detector. Oxygen present at high
pressure can also cause a problem.
Methods that can be used
! Displacement with a less soluble gas
! Applying a vacuum
! Heating the solvent.
HPLC Injectors
In order to introduce a sample
onto the column for analysis,
a special valve called the
injector must be used to
transfer the sample into the
pressurized system
Injectors may look different from the outside, but internally, most
are 6-port rotary valves. These valves consist of a fixed body (the
"stator") plus an internal seal that rotates (the "rotor").
Three internal passages connect alternate pairs of external ports.
The valves can switch between two positions, referred to as the
"inject" and "load" positions, respectively. In the load position, the
pump is connected to the column, and the sample inlet is
connected to one end of a piece of tubing, called the sample loop.
The other end of the sample loop is connected to the waste port.
Rotation results in reconnecting the various lines that enter the
valve, so that a sample volume can be inserted into the mobile
phase that flows from the pump to the column inlet.
The column
HPLC has seen significant improvement over
the last 20 years primarily due to improved
column technology.
Packings are more uniform and smaller.
Phases are commonly chemically bound to
the packing.
Packing methods have improved.
Pump types:
•Isocratic pump - delivers constant mobile phase composition;
•solvent must be pre-mixed;
•lowest cost pump
•Gradient pump -delivers variable mobile phase composition;
•can be used to mix and deliver an isocratic mobile phase or a
gradient mobile phase
􀂙 Reciprocation pumps
--- advantages: small internal volume, high output pressures,
ready adaptability to gradient elution and constant flow rates
--- disadvantages: produce a pulsed flow, cause baseline noise.
􀂙 Displacement pumps
--- advantages: output is pulse free
--- disadvantages: limited solvent capacity (<250 mL)
inconvenience when change solvents
􀂙 Pneumatic pumps:
--- advantages: inexpensive, output is pulse free
--- disadvantages: limited solvent capacity, not amenable to
gradient elution and limit pressures <2000 psi
Packings
Originally, these were irregular silica and
alumina. A range of synthetic, regularly
shaped packings are now available.
! Porous - channels through packing
!!
Superficially porous - rough surface
!!
Smooth - bead like.
Packing size
As packing size is decreased, efficiency and
pressure requirements are increased.
! Common diameters for analytical work
diameter ! plates
"" 10 μm "" 5000
"" 5 μm "" 9000
"" 3 μm" " 15,000
All are for a 15 cm x 4.6 mm column
Column body
Typically consist of stainless steel with a high
precision internal bore.
Some manufacturers offer column inserts
- " don’t need to repurchase the column
" fittings.
Others offer columns where the external
body can be compressed to improve packing
efficiency.
HPLC column examples
Column stationary phases
Today, most packing fall
into four classes.
Silica or alumina
Bound phases on either
alumina or silica.
Gels
Controlled-pore glass or
silica
Absorption phases - alumina
! common mobile phases
! hexane, chloroform, 2-propanol.
! example application - amines.
silica
! common mobile phases
! hexane, chloroform, 2-propanol.
! example applications - ethers, esters,
! porphyrins, fat-soluble vitamins.
Partition phases
Can be broken down into
! Normal phase - polar materials bound to the support.
! Reverse phase - non-polar materials bound to the
support.
! Mixed phase - ! may have some of each.
Partition phases
Normal
! Amino (-NH2)
! Cyano (-CN)
! Diol (glycidoxy-ethylmethoxysilane)
Reverse
! C-2 or RP-2 (-Si-CH2CH3)
! C-8 or RP-8 (-Si-(CH2)7CH3)
! C-18 or RP-18 (-Si-(CH2)17CH3)
Increasing the C number results in a thicker, more retentive
phase
Ion exchange phases
Strong cation ! - sulfonic
acid group
Strong anionic !quarternary amine
Weak anion ! - primary
amine
Weak cation ! - COOH
Size exclusion phases
Gels - organic or aqueous based
Controlled-pore - silica or glass
Must be selected based on pressure
requirements and size range
required for your application.
Capillary and Microbore columns.
Several companies have begun offering
columns with smaller ID.
Microbore column - 1 mm ID, packed
column.
Capillary column - < 1 mm ID, internal
bound phase.
These columns require smaller solvent flows,
reduced sample size and improved detector
design.
Capillary and Microbore columns. Capillary
and Microbore columns.
Aromatic Compounds
mobile phase!2% ethylacetate in
hexane
flow rate " 4 μl/min
column ""Fusica II, 300μm I.D. x
! ! ! 25 cm silica
sample
! 1. toluene
! 2. nitrobenzene
! 3. acetophenone
! 4. 2,6-dinitrobenzene
injection! ! 60 nl
detection! ! UV 254 nm
Silica based columns
Silica is the ideal support for HPLC columns. It
offers a large mechanical stability, excellent
physicochemical surface properties, a wide range
of bonding chemistries and is compatible
with a broad range of organic solvents.
pH stability
In general, HPLC columns are stable within a pH range of
2 to 8. If you are measuring a pH value, the measurement
must be done in the aqueous media before mixing the
eluent with organic solvents.
Modern HPLC columns can be used outside that pH
range. The new bonding chemistries allow use down to pH
1 for some stationary phases. However, please check
vendor’s product information before using silica based
column outside the pH range of 2 to 8. However, best
lifetimes are obtained between pH 2.0 and pH 6.8.
Mechanical stability
Stationary phases based on silica are
mechanically very stable. The packed columns
show no pressure limit and can be used at more
than 40 MPa (6000 psi) without any problem.
However, please avoid pressure shocks on the
column. Pressure shocks lead to channelling in
the column, which results in peak splitting in the
corresponding chromatogram.
Mobile phases (Eluents)
Silica based stationary phases are compatible with
all organic solvents in the above mentioned pH
range. .
• Filter all prepared buffer through a 0.5 μm filter
before using them in your HPLC system
The use of non pure solvents in HPLC causes
irreversible adsorption of impurities on the column
head. These impurities block adsorption sites, change
the selectivity of the column and lead to peak
splitting in the chromatogram. In gradient elution,
impurities cause so called “Ghost Peaks”.
Ghost peaks are peaks that always appear in the same
position on the chromatogram. Their origin is not the
sample, but the impurities from the solvents or
solvent additives. Therefore, it is highly
recommended to run a gradientwithout injection in
the beginning of each method to determine the ghost
peaks
To avoid irreversible adsorption at the column head,
you should always use a pre-column.
The use of a pre-column increases the life time of a
column dramatically. In addition, a pre-column can
filter solid parts stemming from pump seals or
injection rotors. An alternative to a pre-column is an
in-line filter. These filters are attached directly to the
column. These filters get rid of solid parts in the eluent
but will not avoid irreversible adsorption of organic
impurities
Detector Systems
Virtually every chemical and physical
property that can be measured in
solution has been look at.
Detectors fall roughly into two classes
Bulk property - measures an overall change in the
mobile phase.
Solute property - measures a solute specific property.
Properties of a good detector
A detector must provide
• high sensitivity, low detection limits,
• linearity,
•Reproducibility.
This is true for any detector.
Each detector will have specific advantages and will
vary as to peak shape and spread, noise and
flow/temperature dependence they have.
UV/Vis detector
A solute property detector.
Sample must exhibit absorption in UV/Vis range.
Solvent must not absorb significantly at the
measured wavelength.
Types " Filter photometer - single
• Variable wavelength
• Multiwavelength.
Refractive index detector
Bulk property detector - general purpose.
Based on refraction of light as it passes from
one media to another. Presence of a solute
changes the refractive index of the solvent.
Heat of absorption detector
A small amount of heat is released
when a sample absorbs on a suitable
surface.
This detector can measure this.
Electrochemical detectors
A number of properties have been evaluated
Detector types
! ! Dielectic constant
! ! Amperometric
! ! Conductometric
! ! Polarographic
! ! Potentiometric
Dielectric constant detector
Bulk property detector.
Measures changes in polarity of the liquid phase
passing through the cell.
Conductometric detector
Measures conductivity of the solvent. Useful for
solutions of ions.
Amperometric detectors
Most frequently applied type of electrochemical
detector.
A known potential is applied across a set of
electrodes - typically a glassy carbon type.
Ability to oxidize or reduce a species can be
measured.
Typically limited to working with a specific class
of materials per analysis

Separation is based on the analyte’s
relative solubility between two liquid
phases
Mobile Phase
Stationary Phase
Solvent
Bonded Phase

Normal Phase.
- Polar stationary phase and non-polar
solvent.
• Reverse Phase.
- Non-polar stationary phase and a polar
solvent.
Methan0l
CH3OH
• Acetonitrile
CH3CN

• Tetrahydrofuran
• Water
H 2O
Solid Support - Backbone for bonded phases.
 Usually 10µ, 5µ or 3µ silica or polymeric particles.
 Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support.
 Extremely stable
 Reproducible
 Guard - Protects the analytical column:
 Particles
 Interferences
• Analytical - Performs the separation.
 Prolongs the life of the analytical column

Ethyl Silyl
-Si-CH2-CH3
• C-8
Octyl Silyl
-Si-(CH2)7-CH3
• C-18
Octadecyl Silyl
-Si-(CH2)17-CH3

C-2
• CN
Cyanopropyl Silyl
-Si-(CH2)3-CN
Gradient
Controller
•
Column
Pump
Mobile Phases
Detector
Injector

UV
 Single wavelength (filter) [610, 8330]
 Variable wavelength (monochromator) [8316, 8325]
 Multiple wavelengths (PDA) [555]



Fluorescence [610]
Electrochemical [605]
Mass Spectrometric [8325]
Restek® ULTRA C-18 and CN Columns (250mm x 4.6mm, 5µ),
Mobile Phase: (1:1 Methanol:Water), 1.5 mL/min.
A
Supelcosil LC-PAH Columns.
B
Conditions: A: 150mm x 4.6mm, 5µ.
Conditions: B: 50mm x 4.6mm, 3µ.
Flow Rate: 1.5 mL/min
Flow Rate: 3.0 mL/min