“The only source of knowledge is experience”
— Albert Einstein
Chromatography equipment look rather intimidating to anyone who has not handled them 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 and 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. The present module is intended to serve this very purpose and in simple terms you will appreciate the role of each part and its contribution to overall system efficiency.
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.
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. 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.
We now introduce you to the significance and role of each component part of the HPLC system.
HPLC 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.
HPLC Mobile Phase Reservoirs
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.
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 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.
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.
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
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.
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. These are all covered at length in our upcoming online certificate program on HPLC.
The first three modules have covered general course introduction, evolution of chromatography and a brief introduction to HPLC component parts. After going through these modules you would have felt an urge to know more about this popular technique.
You’ll be pleased to know that an elaborate certificate programme is also available online. The certificate program is designed to provide you exposure to the technique through 18 sequential steps.To find more click on the link http://lab-training.com/product/join-our-certificate-course-on-hplc/
The next module will introduce you to the types of stationary phases used in columns for separation of compounds.
Did you get here from a link from a friend, or Twitter? This lesson is part 3 of 10 parts High Performance Liquid Chromatography Free e-course. To get more information about it and sign up Click here.