HPLC is a technique for separation, identification and quantification of components in a mixture. It is especially suitable for compounds which are not easily volatilized, thermally unstable and have high molecular weights.
The instrumentation of the HPLC system is quite complex there are so many critical parts in HPLC system.
Following are the different parts of the HPLC system:
Solvent Reservoir
Tubing
Pumps
Injector system
Guard column
Analytical column
Detectors
Recorders & integrator
Waste collector
The liquid phase is pumped at a constant rate to the column packed with the stationary phase. Before entering the column the analysis sample is injected into the carrier stream. On reaching the column the sample components are selectively retained on the basis of physio-chemical interactions between the analyte molecules and the stationary phase. The mobile phase moving at a steady rate elutes the components based on the operating conditions. Detection techniques are employed for detection and quantification of the eluted components.
Details about the part of the HPLC system:
These are inert containers for mobile phase storage and transport. Generally transparent glass bottles are used so as to facilitate visual inspection of mobile phase level inside the container. Stainless steel particulate filters are provided inside for removal of particulate impurities in the mobile phase if any.
Mobile phase serves to transport the sample to the system. Essential criteria of mobile phase are inertness to the sample components. Pure solvents or buffer combinations are commonly used. The mobile phase should be free of particulate impurities and degassed before use.
Following are the characteristic of the mobile phase:
-It should be inert to mobile phase.
-Made up of stainless steel or glass .
-Capacity:500ml or more than that.
-Degasing: to prevent formation of gas bubble in the detector .it is done by various methods: By distillation system, by vacuum pumping system, by heat solvent with stirring,
Tubing:
The nature of the tubing used to connect all parts of the system deserves some attention.
The inside diameter of the tubing prior to the injection device is not critical but the tubing should be inert have the ability to withstand pressure & be able to carry sufficient volume.
Pumps:
Variations in flow rates of the mobile phase effect elution time of sample components and result in errors. Pumps provide constant flow of mobile phase to the column under constant pressure
Ideal requirement:
Produce very high pressure 5000-10000 psi.
Produce pulse free output.
Flow control and flow reproducible.
Flow rate of m.p. should be in the range of 0.1-10 ml/min.
All material of construction should be corrosion free like s.s., Teflon.
2 types of pumps available
1. Constant flow rate.
2. Constant pressure.
Injector System:
Injectors are used to provide constant volume injection of sample into the mobile phase stream. Inertness and reproducibility of injection are necessary to maintain high level of accuracy.
Ideal requirement:
Sample should be injected in a narrow plug.
Size of sample should be variable.
Should be reproducible.
System must
2 types of the injector system are there
Septum Injector and loop Injector
Guard Column:
Guard column has very small quantity of adsorbent and improves the life
of the analytical Column. They do not have any contribution in the separation.
It also acts as a pre filter to
remove particulate matter, if any, and other material.
Guard column has the same
material as that of the analytical columns.
Analytical Columns:
A column is a stainless steel tube packed with stationary phase. It is a vital component and should be maintained properly as per supplier instructions for getting reproducibility separation efficiency run after run.
Analytical column is that important part of HPLC technique which decides the efficiency of separation.
There are several stationary phases available depending upon the technique or mode of separation used.
Column material:
The columns are made up of either Stainless steel, glass, polyethylene and PEEK( poly ether ,ether ketone).
Column length: Varies from 5cm to 30cm
Column diameter: Ranges from 2mm to 50mm
Particle size : From 1μ to 20μ
Particle nature: Spherical, uniform sized, porous materials are used.
Column Oven
Variation of temperature during the analytical run can result in changes of retention time of the separated eluting components. A column oven maintains constant column temperature using air circulation. This ensures a constant flow rate of the mobile phase through the column.
Detector
A detector gives specific response for the components separated by the column and also provides the required sensitivity. It has to be independent of any changes in mobile phase composition. Majority of the applications require UV-VIS detection though detectors based on other detection technique are also popular these days.
Ideal requirement:
High sensitivity.
Reproducible response &give response to all analyte.
Response should be linear over wide range of concentration.
Should not be sensitize to flow rate fluctuation or temp. Change or change in composition.
Capable of withstand at high pressure.
Data Acquisition & Control
Modern HPLC systems are computer based and software controls operational parameters such as mobile phase composition, temperature, flow rate, injection volume and sequence and also acquisition and treatment of output.
These are the main parts of a basic HPLC system more specialized equipment might also have solvent selection valves, vacuum degasser, auto samplers, column switchers, pre or post column derivatization and fraction collectors.
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