HPLC- Instrumentation, types,Advantages and Disadvantages

1. High Performance Liquid
Chromatography
Dr Sumitha J
Associate Professor
JBAS College for Women,Chennai-18.

2. HPLC is an analytical technique used to separate, identify and
quantify each component in a mixture
HPLC is an advanced form of Column Liquid Chromatography
which uses high-pressure withstanding metal columns instead
of low-pressure glass columns.
Solvent seeps through a column under gravity in Column liquid
Chromatography but is forced under high pressure(+/- 400
atmospheres)

3. image Source: Sartorius AG.

4. image Source: Liquid Chromatography Laboratory Services (HPLC) - S&N Labs.

5. There is a separation column between a stationary and a mobile phase
Stationery Phase-Granular material with small porous particles in the
separation column
Mobile Phase is a solvent or solvent mixture which is forced at high
pressure through the separation column. The most common solvents used
are methanol and acetonitrile
Retention time is variable and it depends on the interactions between the
stationary phase, the molecules being analyzed, and the solvent(s) used
Solvent seeps through a column under gravity in Column liquid
Chromatography but is forced under high pressure(+/- 400 atmospheres)

6. The sample is injected into the mobile phase
fl
ow from the pump to
the separation column using a syringe through a sample loop(made
of stainless steel)
the individual components of the sample migrate through the
column at different rates because they are retained to a varying
degree (due to their interactions with the stationary phase)
After leaving the column, the individual substances are detected by
a suitable detector and passed on as a signal to the HPLC software
on the computerMobile Phase-.is a solvent or solvent mixture
which is forced at high pressure through the separation column
At the end of this operation/run, a chromatogram in the HPLC
software on the computer is obtained.

7. The development of the pump system plays a crucial role in the sensitivity of
HPLC
The pump is positioned in the upper stream of the system and generates a
fl
ow
from the solvent reservoir into the system.
High-pressure generation is a “standard” requirement of pumps
The pumps should provide a consistent pressure at any condition at a controllable
and reproducible
fl
ow rate.
Most pumps used in current systems generate the
fl
ow by the back-and-forth
motion of a motor-driven piston.
Because of this piston motion, it produces “pulses”.
The Pump

8. An injector is placed next to the pump.
The simplest method is to use a syringe to introduce the
sample into the
fl
ow.
Auto-injector system is also widely used that allows repeated
injections in scheduled timing.
Injector

9. The separation is performed inside the column.
The recent columns are often prepared in a stainless steel
housing, instead of glass columns since it is tolerant towards
a large variety of solvents.
The packing material generally used is silica or polymer gels
compared to calcium carbonate.
The eluent used for LC varies from acidic to basic solvents.
Column

10. A detector is used to observe the obtained separation.
The composition of the eluent depends on whether the
analyte is present or not. The presence of an analyte changes
the composition of the eluent.
The main job of the detector is to measure these differences.
This difference is monitored as a form of an electronic signal
Commonly used detectors are UV-spectroscopy,
fl
uorescence,
mass-spectrometric and electrochemical detectors.
Detector

11. Signals from the detector may be collected on chart recorders
or electronic integrators that vary in complexity and their
ability to process, store and reprocess chromatographic data.
The computer integrates the detector’s response to each
component and places it into a chromatograph that is easy to
read and interpret
Recorder

12. The eluent used for LC analysis may contain gases such as
oxygen causing an unstable baseline.
Degasser uses special polymer membrane tubing with
numerous small pores on the surface aids in removing gases.
Degasser

13. The LC separation is often largely in
fl
uenced by the column
temperature.
In order to obtain repeatable results, it is important to keep
consistent temperature conditions.
Also for some analysis, such as sugar and organic acid, better
resolutions can be obtained at elevated temperatures (50 to 80°C).
Thus columns are generally kept inside the column oven (column
heater)
Column Heater

15. Normal phase:
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 compounds.
Reverse phase
The column packing is non-polar (e.g C18), and the mobile phase is
water and miscible solvent (e.g methanol).
It can be used for polar, non-polar, ionizable, and ionic samples.
Types of HPLC

16. Ion exchange:
Column packing contains ionic groups and the mobile phase is a
buffer. It is used to separate anions and cations.
Size exclusion:
Molecules diffuse into pores of a porous medium and are
separated according to their size relative to the pore size.
Large molecules elute
fi
rst and smaller molecules elute later.
Types of HPLC

17. Analysis of drugs, synthetic polymers and pollutants
(environmental analytics)
Determination of drugs in biological matrices & Product purity
quality control of industrial products and
fi
ne chemicals
Separation and puri
fi
cation of biopolymers such as enzymes or
nucleic acids
Water puri
fi
cation
Pre-concentration of trace components
Applications of HPLC

18. Speed
Ef
fi
ciency
Accuracy
Versatility
Precise
Advantages of HPLC

19. Cost
Complexity
HPLC does have low sensitivity for certain compounds, and some
cannot be detected as they are irreversibly adsorbed.
Volatile substances are better separated by gas chromatography
Dis-Advantages of HPLC