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Testing The Purity of Sample - Docx 2

This document discusses using high performance liquid chromatography (HPLC) to test the purity of samples. It explains that HPLC uses smaller stationary phase particles and higher pressures than other forms of chromatography. It also describes how normal phase HPLC uses a polar column and mobile phase, while reverse phase uses a non-polar column and more polar mobile phase. The document outlines the components of an HPLC system, including pumps that maintain high pressure, injection valves, and various detector types like UV-visible and refractive index.

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Sri Kiran Chakravarthy
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63 views3 pages

Testing The Purity of Sample - Docx 2

This document discusses using high performance liquid chromatography (HPLC) to test the purity of samples. It explains that HPLC uses smaller stationary phase particles and higher pressures than other forms of chromatography. It also describes how normal phase HPLC uses a polar column and mobile phase, while reverse phase uses a non-polar column and more polar mobile phase. The document outlines the components of an HPLC system, including pumps that maintain high pressure, injection valves, and various detector types like UV-visible and refractive index.

Uploaded by

Sri Kiran Chakravarthy
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
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TESTING THE PURITY OF SAMPLE

BY USING HPLC
High performance liquid chromatography (HPLC) is a relatively new
technique when compared with gas chromatography, paper
chromatography or thin layer chromatography. It is a form of liquid
chromatography with the main difference being that much smaller and
more uniform stationary phase particles are used. The spaces between
the individual particles are very small and therefore higher pressures
are required to maintain a suitable flow. Another name for this
technique is high-pressure liquid chromatography. In recent years
most HPLC analysis is carried out using bonded phase columns. The
solid stationary phase support (usually silica) has the liquid stationary
phase chemically attached to it via covalent bonds. This means the
liquid part of the stationary phase cannot be easily washed off by
the mobile phase. This has made it possible to use a large variety of
mobile phases and thus has made many difficult separations possible.
Background :
HPLC is based on partition chromatography. The components of a
mixture partition between the liquid film, that serves as the stationary
phase and the mobile phase passing by the stationary phase.
Components that are more like the stationary phase stay on the
column longer than components that are more like the mobile phase.
Size, polarity, and similarity of functional groups affect the degree of
attraction between the individual components in the mixture and the
stationary phase.
In normal phase chromatography the column is polar
and the mobile phase in non-polar . Alumina and silica gel are the
most common normal phase materials. The column is non-polar and
the mobile phase is more polar. In the early days of HPLC liquid
films similar to those used in GC were coated on an inert support.
They worked fine as long as the mobile phase was very polar (water),
but even solvents as polar as methanol tended to move the stationary
phase liquid. Now essentially all reverse phase columns have bonded
phases. A silane material is reacted with small (5 μm), uniform silica
beads. A covalent bond is formed between the silica surface and the
organic silane . The two most common phases are C-8 and C-18,
which have 8 carbon or 18 carbon long straight chain alkanes attached
to the silica. If the phases were not bonded solvents like acetonitrile or
hexane would destroy the column, but with bonded phases these
solvents are commonly used for separations or for cleaning
The heart of an HPLC system is the pump. The pump
forces the mobile phase through the column at a constant flow rate.
Within the pump system are devices to remove pressure pulses caused
by the individual strokes of the pump piston. The pump must be
capable of producing high pressure since the backpressure of the
columns can be substantial.
With the GC the sample is injected through a rubber septum and is
immediately vaporized into the gas stream. Injection in HPLC is not
as straightforward. The mobile phase is forced through the column by
very high pressures (>1000 PSI) and any injection through a septum
would result in a leak .A special injector valve is used to divert the
flow from the pump through a sample loop and then to the column.
The sample in the sample loop is washed onto the column. Since the
sample loop is a section of stainless steel or plastic tubing, with a
fixed internal volume, reproducible injections are possible.
Detectors :
fixed wavelength UV, variable wavelength UV-visible, diode array
UV-visible, refractive index, fluorescence, IR and several types based
on electrochemical techniques. The output from the detector goes to
the computer, which serves as a peak integrator. The computer
processes the data and gives retention times, peak areas, and
concentrations if the method contains calibration information.

The objective behind taking up this project is


 To test the purity of sample by using HPLC.
 Explain the differences between normal phase and
reverse phase HPLC.
 Describe the types of detector systems used in
HPLC.

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