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The use of HPLC for separation & detection of amino acids

in plasma
The separation and detection of amino acids in biological fluids is an important
practice in clinical laboratory medicine. It is necessary in monitoring and assessing
the nutritional requirements and status of health. The benefits of this process are, it
helps to evaluate the nutritional requirements of patients, helping to study about the
amino acid metabolism in different situation. In earlier years amino acids present has
been analysed by ion exchange chromatography with post column ninhydrin
detection. These experiments performed on instruments which are costly and time
consuming. Alternatively amino acids can be analysed by reversed phase High
Performance Liquid Chromatography (HPLC).
Introduction of HPLC
HPLC is a separation technique used to separate the components present in a
mixture to identify and analyse each components. It was developed from open
column chromatography in 1970.The various techniques used for HPLC are:
Reverse phase chromatography, size exclusion chromatography, Ion exchange
chromatography. This method uses an extremely high pressure (up to
3000psi).HPLC is highly efficient and has very fast speed of resolution. The greater
advantage is that may employ the principles of adsorption, partition, ion exchange
and affinity chromatography.
Instrumentation of HPLC
The instrumentation required to perform HPLC are;

Solvent reservoir
High pressure pump
Sample injector
Column
Detector

The solvent reservoir is to store the mobile phase and it is generally made up of
glass. It must have a provision for degassing the solvents. All solvents to be used in
HPLC must be pure since degasser is used to avoid the formation of bubbles in
detector.
Solvent pumping system is used to pump the mobile phase through the column. It
provides a reproducible high pressure which needs to obtain high resolution, high
speed analyses. They made up of chemically resistant materials to mobile phase.
Reciprocating pumps are normally used as solvent pumping system.
Sample injection is an automated process which is an important factor in
achieving a satisfactory resolution. The columns for HPLC are usually made up of

stainless steel. They are commonly filled with a stationary phase. Mobile phase
temperature and column should be kept constant during an experiment.
Detector is used to detect the individual molecules that elute from the column and
converting into an electrical signal. Refractive index detector, Fluorescence detector
and UV absorption detector are some detectors used in HPLC.
The type of separation is based on the choice of the mobile phase. There are two
types. Such as Isocratic HPLC and Gradient HPLC.In isocratic HPLC, the
composition of the mobile phase is kept fixed throughout the process. In Gradient
HPLC, mobile phase composition changed during separation.
Factors affecting HPLC
The column can be affected by particle size, flow rate, thickness of stationary phase,
mobile phase viscosity; sample size. These factors are affecting certain parameters
of HPLC.such as;
Retention time: Time taken for a compound to travel through the column. It depends
on the pressure used, nature of stationary phase and column temperature.
Band brodening: Reduces efficiency, resolution
Stationary phase: Porous silica is used as one of the base material for stationary
phase.
Resolution: Measure of extent of separation of two components and the base line
separation achieved.
Separation of Amino acids in plasma
The amino acid sample inject to HPLC sample injector system fitted with a guard
column at a constant temperature. Individual amino acids can be separated by
reverse phase HPLC and detected based on the retention time establish for each
amino acid present in plasma sample under defined experimental condition. Different
amino acids will produce different level of peaks and by using that separation of
amino acids can be done efficiently.