Separating Complex Mixtures: How They Work and Why It Is Important


Do you know how High Performance Liquid Chromatography works and its applications?

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What do you think is the mobile phase in High Performance Liquid Chromatography?

a gas
a solid

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Do you know how Gas Chromatography works and its applications?

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What do you think is the mobile phase in Gas Chromatography?

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Throughout this post, I will be explaining three commonly used techniques of separation that play an important role in the world we live today. The goal is to help you gain a better understanding of how these methods work and why they are important.


Note: In HPLC, the stationary phase is a solid, or a liquid supported on a solid. The mobile phase is a liquid or gas which flows through the stationary phase and carries the components of the mixture(the sample being tested on) with it.

HPLC is a highly improved version of column photography. Opposed to column chromatography, the solvent is forced through a column under high pressures of up to 400 atmospheres. This allows chemists to use much smaller particle sizes for the column packing material which allows for a greater surface area for interactions between the stationary phase and the molecules flowing past it. Before I move on to explain how the process works, it’s important to recognise that there are two types of HPLC. There is the normal phase and the reversed phase, which is used much more often than the former.

In normal phase HPLC, the column is filled with tiny silica (SiO2) particles and the solvent is non-polar. A typical column has an internal diameter of 4.6 mm and a length of 150 to 250 mm. Polar compounds in the mixture being passed through the column will stick longer to the polar silica than non-polar compounds. Although named “normal” it is not the commonly performed type of HPLC. Reversed phase HPLC is actually the most commonly used form of HPLC.

In reversed phase HPLC, the column size is the same but the silica (stationary phase) is modified to make it non-polar by attaching long chains of hydrocarbons to its surface. Instead of non-polar, a polar solvent such as a mixture of water and methanol is used. As a result, there is a strong attraction between the polar solvent and polar molecules in the mixture being passed through the column. Polar molecules will therefore most of their time moving with the solvent and non-polar molecules with tend to form attractions with the hydrocarbon groups because of van der Waals dispersion forces. Therefore, it will be a lot harder for the non-polar molecules to move through the solvent, thus slowing them down. 

Video Animation of Normal Phase and Reversed Phase:

Explanation of HPLC Process:

A pump forces the mobile phase from the solvent reservoir through the system’s column to the detector. Depending on the column dimensions, temperature, particle size of the stationary phase, flow rate and composition of the mobile phase, operating pressures of up to 400 atmospheres can be generated. The injector can either be a single injection or an automated injector. It is able to inject liquid samples between 0.1 ml and 100 ml under high pressure conditions. Columns are usually made of polished stainless steel, made between 50 and 300 mm long and have an internal diameter between 2 and 5 mm. They are commonly filled with a stationary phase with a particle size of 3 to 10 µm. Ideally, the temperature of the mobile phase and column should be kept constant. The detector at the end detects the analytes as they elute from the chromatographic column. The most commonly used detectors is UV spectroscopy. From the detector, the data is sent to a computer system which creates a chromatograph with the detector signal on the vertical axis and retention time on the horizontal axis. The retention time is the time taken by a particular compound to go through the column to the detector. Based on the retention time, a chemist can identify the ID of a certain compound. 

Some of the important applications of HPLC include control drug stability and drug quality control in Pharmaceutics. It is also used to detect phenolic compounds in drinking water, quantify drugs, analyse the safety of food preservatives, and is also beginning to be used for antibiotics analysis in blood, thus playing an increasingly important role in the world of medicine. 


Note: In GC, the mobile phase is an inert gas (eg.helium) and the stationary phase is a high-boiling point liquid adsorbed into a solid.

Gas chromatography is not only able to separate mixtures but also identify how much there is of each chemical/compound. Substances in the mixture with greater affinity for the mobile phase reach the detector quicker. The mixture to be analysed is injected into the stream of carrier gas. The injector itself is hot enough so all the sample boils and enters the column as a gas alongside the helium. The temperature of the column is cooler than the injector oven so that some components of the mixture will condense at the beginning of the column. As it passes the column, the mixture is separated. Whilst moving through the column, there are three possible situations a molecule in the mixture may run into. It can either condense on the stationary phase, dissolves in the liquid on the stationary phase, or remain in the gas phase. Once it reaches the detector, the detector sends information to the computer system which produces a chromatogram. 

Similar to HPLC, retention time in GC is the time taken for a molecule to travel through the column to the detector. It is the time at which the sample is injected to the time at which its maximum peak height is displayed on the computer. Retention time depends on three factors: boiling point, the solubility of the molecule in the liquid phase, and the temperature of the column. A compound which has a boiling point higher than the temperature of the column will condense and spend most of its time as a liquid, therefore its retention time will be long. Similarly, a compound which is more soluble in the liquid phase will spend most of its time as a liquid and have a long retention time. Drastically increasing the temperature of the column will likely break the attractions of all the molecules in the liquid state and shorten all retention times. Drastically decreasing the temperature would then lengthen all retention times.

Most professionals don’t start with higher column temperatures as compounds will pass through quickly, resulting in clumped up peaks on the chromatogram and inaccurate data. It is recommended to start with cooler temperatures and increase it on regular time intervals. The area under the peaks on the resulting chromatogram are proportional to the amount of each compound and can be calculated to help quantify each compound. 


Most mass spectrometers ionise atoms and molecules to give off a positive ion, even for atoms that would normally form a negative ion. Once atoms/molecules go through the 1st stage which is ionisation, the ions are accelerated in stage 2 so that they have the same kinetic energy. It is also extremely important that there is a vacuum so that ions do not run into air molecules, slowing them down. The ions are then deflected in stage 3 according to their masses. The lighter they are or the greater the charge, the more it gets deflected. With these two factors, a mass to charge ratio is created. Ion streams with a smaller mass to charge ratio are deflected more than those with greater mass to charge ratios. However, some ion streams collide with the walls where they pick up electrons, get neutralised and are removed through the vacuum pump. In stage 4, the beam of ions that make it through hit the metal box in the detector, triggering a flow of electrons in the wire to the amplifier which is recorded as the current. The greater the number of arriving ions, the greater the current is.  

The amplifier signals are then sent to the computer system and a mass spectrum is produced. The vertical axis of the mass spectrum reads relative abundance and the horizontal axis is the mass to charge ratio. Relative abundance essentially is proportional to the current, so the greater the current, the more abundant the ion is.

Whilst mass spectrometry alone is more useful for identifying different isotopes of an element, it is embedded into other methods such as HPLC, making it a very important technique for separation. It is used in the following fields of study: proteomics, drug discovery, clinical testing, genomics, environment, and geology.



Applications in my Hometown:

There are many industries where these separation techniques are vital to the daily running of proceedings. For example, in Hong Kong, HPLC and Mass Spectrometry are used by Hong Kong Jockey Club to run tests on horse food. Horse racing is a popular spectacle with high stakes for those who choose to bet in Hong Kong so the club must ensure that competition is fair. These tests confirm that the food is healthy and that there are no performance-enhancing drugs. These same techniques are used by the Hong Kong government for inspections on the food delivered from China as it forms the bulk of Hong Kong’s food source. Ensuring that seven million people are eating healthy foods is a big task and it sure emphasises the importance of these separation techniques.


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