One of the biggest revolutions in analytical chemistry and one of the most widely used chromatography techniques, the High Performance Liquid Chromatography (HPLC) is machine that allows one to isolate, identify and quantify chemical components in mixtures. It was once known as ‘high pressure liquid chromatography’ as it relies on a pump to pass liquids at a high and constant pressure. As seen in the above image, the HPLC does not take up much space and may be stacked with the exception of the monitor. Here, it is opted to be split into two.
Before we get into further details, let’s take a look at a brief history of its origins.
The origins of HPLC dates back to the early 1900s when a Russian botanist decided to fill an open glass column with particles such as calcium carbonate (powdered chalk) and alumina, then pouring a sample (solvent extract of homogenized plant leaves) into the glass column followed by the addition of a pure solvent. With the help of gravity, the solvent was pulled down the column where different coloured bands started forming, as some components were moving faster than others. Each band represented one compound within the mixture. This technique was called “chromatography” by its founder, Mikhail Tsvet, deriving the name from the Greek word “chroma” and “graph”, which literally translates to “colour writing”. Ironically, the Russian name “Tsvet” means colour in Russian. Coincidence? I think not.
Since then, various scientists have contributed on the expansion of the technique such as the theory of partition in chromatography, paper-chromatography and gel permeation which has shaped the HPLC into what we know of it today. The improvements made to the technique allowed for easier interpretation and data collection, accuracy and variety in application. Shortly thereafter, there was a demand for better resolution and high speed analyses of non-volatile samples which led to the development of HPLC. The first commercial HPLC was manufactured by Waters Corporation, known as the ALC 1000 in the late 1960s. Companies joined the HLPC bandwagon and improved the technology by adding more features which would allow for more functions or easier access such as ion-exchange systems to separate ions and polar molecules based on their charge.
So how does the separation of compound even work? Each compound has different strengths of chemical attraction to particles. The stronger the attraction to the particles, the slower the speed of its movement. Likewise, compounds that have a stronger attraction to solvents moved faster.
To use the HPLC, one would have to start by choosing one or more solvents. The pump then mixes the solvent if more than one is chosen and disperses it into the system at a constant rate. This phase is known as the mobile phase, simply because the solvents move. As the solvent flows through the system, it passes through the auto-sampler, which injects a sample of a mixture into the stream. The next component involved is a column, also known as the stationary phase. This is where the components within a substance get separated from one another. In reference to Tsvet’s experiment, the column here is just a metallic version of the glass column and also contains particles.
The nature of the components of the compounds then travel through this tube, and the amount of time it is retained in the column will be recorded. After that, the mixture further passes more detectors such as UV and fluorescence. The natures of the compounds vary in sensitivity towards these wavelengths and will be stained in accordance to how much they can absorb the UV light and fluorescence.
The reactions that take place in the stationary phase is recorded and displayed on a monitor using a software called CDS. The CDS acts as the main controller and data collector. Instructions are given to the HPLC here. The results of the chromatograph are then generated in graphs, with each peak representing one compound. Each peak is generated at a different time, depending of their rate of retention within the column. Active ingredients in the sample would generate a higher peak, signifying its high concentration in comparison to the other compounds in the sample. The graphs also have baselines whereby a purity of a sample can be tested. If the graph does not return to the baseline, it signifies impurity.
HPLC is used in a variety of fields such as biomedical research, cosmetics, energy, food and environmental industries. It mainly serves a purpose in quality control, as compounds are easily identified if the peak of the pure sample and the peak of a certain compound in a mixture is formed at exactly the same retention time.
The machine can be found in CB21 in the red building, under the Faculty of Engineering. Permission is needed prior to access. You may just ask one of the technicians in CB01 if you need assistance or further information about the equipment.
by Marini Shariff