Chromatography was discovered in 1903 and was used to separated plant pigments.
It is a popular method used to separate mixtures to their individual components.
The history of HPLC began in the 60’s and was known as high pressure liquid chromatography.
The instrumentation and columns improved over the time and it is now known as high
performance liquid chromatography.
HPLC separation technique provides high speed, efficiency and high sensitivity as compared to
A very small volume of sample is injected into a tube packed with a stationary phase (3-5 microns
particles) and active components are separated using high efficiency columns.
From Liquid Chromatography to High Performance
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!
4. Advantages of HPLC
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
5. Principle of HPLC
Separation is based on the analyte’s relative solubility between two
HPLC utilizes different types of stationary phase (typically,
hydrophobic saturated carbon chains), a pump that moves the mobile
phase(s) and analyte through the column, and a detector that provides
a characteristic retention time for the analyte.
Analyte retention time varies depending on the temperature of the
column, the ratio/composition of solvent(s) used, and the flow rate of
the mobile phase.
With HPLC, a pump (rather than gravity) provides the higher
pressure required to propel the mobile phase and analyte through the
densely packed column.
6. COMPONENTS OF HPLC
1.Solvent Reservoir: The contents of mobile phase are present in glass container. In
HPLC the mobile phase or solvent is a mixture of polar and non-polar liquid
components. Depending on the composition of sample, the polar and non-polar
solvents will be varied.
2.Pump: pumps the mobile phase at a specific flow rate in mL/min. The pump
pressure is normally between 400-600 bar.
3.Injector: Introduces the sample into the column (about 5-20 μL).
4.Column: provides separation through high pressure created by the small particles.
5.Detector: it quantifies and identify the sample components and provides
information to the computer.
7. 5. Data Collection Devices or Integrator: Signals from the detector might be gathered
on graph recorders or electronic integrators that fluctuate in many-sided quality and in
their capacity to process, store and reprocess chromatographic information. The PC
coordinates the reaction of the indicator to every part and places it into a chromatograph
that is anything but difficult to interpret.
10. Solvent reservoir and degassing unit
Solvents (mobile phase) – are stored in special reservoirs connected to the
pumping system – must be free of particles that can clog components & free of
bubble forming gases that get trapped in column or detector
Problems caused by dissolved air in the eluent
Unstable delivery by pump
More noise and large baseline drift in detector cell
In 0rder to avoid this problems the solvent must be degassed.
Three basic ways to degas solvents
1) vacuum or suction filter (0.4 or 0.2 μm)
2) ultrasonicate (with vacuum)
3) He purge (spurges units often built in)
Can purchase HPLC solvents & water - still
12. HPLC Pump
The HPLC pump has to have ruggedness and at the same time should be able to
provide reproducible flow characteristics run after run. The operational pressure
limits have a vast range depending upon analysis requirements.
In normal analytical operation the pressure can vary between 2000 – 5000 psi but in
applications covered under UHPLC mode operating pressure can be as high as
15000 – 18000 psi.
An ideal pump should have the following desirable characteristics
Solvent compatibility and resistance to corrosion
Constant flow delivery independent of back pressure
Convenience of replacement of worn out parts
Low dead volume for minimum problems on solvent changeover.
Types of pump in HPLC:
Constant pressure pump
Reciprocating piston pump
14. Constant pressure pump
Provide consistent continuous flow rate through
the column with the use of pressure from a gas
Valving arrangement allows rapid refill of solvent
A low pressure gas source is needed to generate
high liquid pressures.
15. Syringe pump
These are suitable for small bore columns.
Constant flow rate is delivered to column by a motorized
Solvent delivery rate is set by changing voltage on the
These pumps deliver pulseless flow independent of
column backpressure and changes in viscosity but major
disadvantages are limited solvent capacity and limitation
on gradient operation
16. Reciprocating piston pump
Deliver solvent(s) through reciprocating motion of a piston in a
On the back stroke the solvent is sucked in and gets delivered to the
column in the forward stroke.
Flow rates can be set by adjusting piston displacement in each
stroke. Dual and triple head pistons consist of identical piston
chamber units which operate at 1800 or 1200 phase difference.
The solvent delivery of reciprocating pump systems is smooth
because while one pump is in filling cycle the other is in the delivery
High pressure output is possible at constant flow rate and gradient
operation is possible. However, pulse dampening is required for
further elimination of pressure pulses.
18. Advantages and Disadvantages of
High- / Low-Pressure Gradient Systems
High-pressure gradient system
High gradient accuracy
Complex system configuration (multiple pumps required)
Low-pressure gradient system
Simple system configuration
19. HPLC Column
In GC the analyte affinity for the column is influenced by temperature.
In HPLC the solvent strength affects an analytes retention on column. Therefore,
analogous to temp programming in GC, do solvent programming in HPLC This
is also referred to as gradient elution.
Gradient elution dramatically improves the efficiency of separation.
- usually stainless steel
- can be PEEK (poly ether ether ketone)
- may cost $200-$1000 packed
- Length 10-30 cm, ID 4-10 mm
- Packings are 3, 5, or 10 μm particle size
- Most common 25 cm, 5 μ, 4.6 mm ID
- N = 40,000 to 60,000
-Normally packed under 6000 psi pressure at factory as a slurry
Guard columns are normally used before the analytical column to protect & increase lifetime of
column – operator usually slurry or dry packs short guard column regularly with same or similar
packing used in analytical column (old column material) – can purchase guard systems, cartridges,
21. Types of HPLC based on column type
Normal phase HPLC
Reverse phase HPLC
Size exclusion HPLC
Ion exchange HPLC
Column packing is polar (e.g. silica) and the mobile phase is non polar. It is used for
water-sensitive compounds, geometric isomers, cis-trans isomers and chiral
The column packing is non-polar (e.g. C18) , mobile phase is water+ miscible solvent (e.g
methanol). It can be used for polar, non polar, ionizable and ionic samples.
Column packing contains ionic groups and the mobile phase is buffer. It is used to
separate anions and cations.
Molecules diffuse into pores of a porous medium and are separated according to
their relative size to the pore size. Large molecules elute first and smaller molecules
24. Sample injectors
Injectors serve to introduce required sample volume accurately into the HPLC system.
Sample injection into the moving mobile phase stream in HPLC is quite different from
injection into a gas stream in Gas Chromatography as precise injection is required against
high back pressure. In such a situation it is not possible to simply inject using a syringe
Precautions to take while injecting sample in HPLC
Prime injector with solvents to be used but it should be ensured that solvent is
compatible with solvent used earlier.
Needle wash between samples will prevent carry over between injections.
Before start and at end of analysis ensure tubing is completely washed of buffers
or previously used solvents.
Do not forget to feed the vial number correctly on auto sampler rack and list out
the sequence correctly in the computer.
26. Manual injector: Injection method
Syringe measurement method
It is desirable that no more than half the loop volume is
Loop measurement method
It is desirable that at least 3 times the loop volume is
1) Filter based UV-vis detector – Typically set at 254 nm using the most prominent band in
Hg spectrum – can also use 313, 365, 334 nm and other lines as well
2) Variable wavelength detectors – use continuous source like (D2 or H2) & a
monochromator, select any λ, less sensitive
3) PDA - D2 or H2 source, disperse & focus on diode array, get complete spectrum every 1 sec,
powerful, expensive, less sensitive, lots of data generated
4)Fluorescence detector – normally fixed wavelength filter fluorometer excitation filter &
emission filter can be changed for particular λ of interest gives selectivity based on:
- ability to exhibit fluorescence
- excitation wavelength
- emission wavelength
Variable λ monochromator based fluorescence detectors also available
Filter based detectors usually more sensitive
5) Refractive index detector (RI) -
responds to nearly all solutes but has poor sensitivity – detects changes in refractive index as
sample passes through as long as solute has different RI than solvent analogous to TCD in GC.
• Amperometric – fix potential & measure current (i)
• Conductometric – measure conductivity
• Coulometric – fix potential & integrate i
• Voltammetric – vary potential & measure i
• Potentiometric – measure potential
Can use 2 or 3 electrode design with Pt or carbon electrodes (glassy C or C paste)
Electrochemical detector is nearly universal.
Other HPLC detectors
• LC-MS using thermospray – new popularity (pharmaceuticals)
• Evaporative light scattering - polymers
• LC-plasma emission or ICP-MS
35. Applications of HPLC
The HPLC has several applications in the fields of pharmacy, forensic, environment
and clinical. It also helps in the separation and purification of compound [57-83].
Pharmaceutical Applications: The pharmaceutical applications include
controlling of drug stability, dissolution studies and quality control.
Environmental Applications: Monitoring of pollutants and detecting
components of drinking water.
Forensic Applications: Analysis of textile dyes, quantification of drugs and
steroids in biological samples.
Food and Flavour Applications: Sugar analysis in fruit juices, detecting
polycyclic compounds in vegetables, analysis of preservatives.
Clinical Applications: Detecting endogenous neuropeptides, analysis of
biological samples like blood and
Book of Instrumental Methods of Chemical Analysis of Gurdeep R. Chatwal & Sham
K. Anand, Pg. no. 2.264.