Intro To HPLC system - Lab Training

High Performance Liquid Chromatography (HPLC) is a popular and versatile technique which provides affordable solutions on separation, identification and
quantification of constituents of complex organic samples.

At first sight the assembly of complex modules and bunch of tubings might scare you. However, on gaining an insight into the role of each component the mystery will start unfolding and the HPLC system will turn out to be a user-friendly piece of equipment that if properly looked after will ensure highest accuracy and reliability of results analysis after analysis.

This is a concise introduction to the components of HPLC. It is important to understand the individual component parts and how each contributes to the overall reliability of analysis. Your increased comfort level will make you realize that HPLC is a simple to use system and holds promise of unlimited applications.

Chromatography equipment looks rather intimidating to anyone who has not handled it before, but on a closer look and as you get familiar with the equipment you realize that behind the network of wires, complex plumbing and circuitry is a simple machine with only a few major parts.
Different combinations of these parts namely :
  • Pumps
  • Detectors
  • Injectors yield an infinite number of configurations based on the application.
Just like an understanding of human anatomy makes you conscious of the vital role of each and every body organ towards your well being and vitality. Similarly you need to have a good understanding of the parts of your HPLC system to generate data of highest reliability.
A conceptual understanding of the function of each component will add to your comfort level with your HPLC system. You will ensure long time usage with high reliance on output data.
What is HPLC?
HPLC is a technique for separation, identification and quantification of components in a mixture. It is especially suitable for compounds which are not easily volatalised, thermally unstable and have high molecular weights.
Mobile Phase
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 physico-chemical interactions between the analyte molecules and the stationary phase.
Mobile Phase
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.
Mobile Phase Reservoirs
These are inert containers for mobile phase storage and transport. Generally transparent glass bottles are used so that 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.
see more details in Precautions in the handling of HPLC mobile phase
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.
Injectors
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.
Column
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 and separation efficiency run after run.
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.
See also:Guard columns and their benefits in HPLC analysis - Lab Training
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.

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.
Specialized Versions
Specialized HPLC Systems might also have solvent selection valves, vacuum degasser, auto samplers, column switches, pre or post column derivatization and fraction collectors. These are all covered at length in our online certificate program on HPLC. Certificate Course on HPLC
*lab-training*
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