Chromatography

Thin-layer chromatography is used to separate parts of a plant extract, illustrating the experiment with plant pigments which gave chromatography its name

Chromatography is a lab technique that helps scientists separate, identify, and purify the different parts of a mixture. It works by using differences in the speed at which the mixed substances move through special media, or chemical substances. It uses a stationary phase (a solid) and a mobile phase (a liquid or a gas). The fluid mobile phase flows through the stationary phase. Different substances in the mixture move at different speeds because they interact differently with each phase. This causes them to separate so scientists can study them one by one. This method is very common in chemistry, biology, and many industries. It allows scientists to take apart complex mixtures and study each piece closely, even if there are only tiny amounts.

Chromatography was first invented in the early 1900s by a Russian scientist named Mikhail Tsvet. He used it to separate plant pigments like chlorophyll (the green color in leaves) and carotenoids (orange and yellow colors). His work helped scientists understand how to separate and study the parts of a mixture. Since then, chromatography has grown into a large group of techniques, each designed for different kinds of substances. Some of the main types include paper chromatography, thin-layer chromatography (TLC), liquid chromatography (LC), gas chromatography (GC), and high-performance liquid chromatography (HPLC). Each method uses different materials and takes advantage of how substances behave, such as how big they are, how easily they evaporate, how they stick to surfaces, or how they react to charges. These differences help separate the parts of a mixture so scientists can study them more easily.

Gas chromatography (GC) is a method used to study substances that can easily turn into gas, like those found in forensic science, environmental testing, perfumes, and fuel quality checks. It works best with things that are light and can be heated without breaking down. High-performance liquid chromatography (HPLC) is used for substances that do not evaporate easily or can be damaged by heat, like medicines, proteins, and plastics. It separates these materials in a liquid form. There are also special types of chromatography for different jobs. Size-exclusion chromatography (SEC) separates molecules based on how big or small they are. Ion-exchange chromatography (IEC) separates them by their electric charge. Affinity chromatography separates them based on how they interact with other molecules, like how a key fits a lock. Some advanced methods combine two tools in one. For example, GC-MS mixes gas chromatography with mass spectrometry to separate and identify substances. LC-MS does the same with liquid chromatography. These combinations help scientists get even more detailed and accurate results.

Chromatography is not just used to study small samples in the lab, it is also very important for making and purifying large amounts of important substances. In medicine and biomanufacturing, it helps scientists clean and collect things like drug ingredients, peptides, and monoclonal antibodies with very high purity. In biology, chromatography is used to separate and study enzymes, DNA and RNA, and metabolites (small chemicals made by the body). This helps scientists learn how these molecules work and what they do. Chromatography is also used in many real-world jobs, like medical testing, food safety, checking pollution, and finding illegal drugs. As technology has improved, chromatography has become faster, more accurate, and easier to use. Today’s chromatography systems often use computers, tiny columns, and smart detectors to do quick and reliable testing. This helps scientists get results faster and with better detail.

Flat-plane chromatography

The stationary phase is a flat plane, such as paper, or a substance on glass.

Paper chromatography

Main article: Paper chromatography

Chromatography jar

Paper chromatography is an old technique for separating and identifying mixtures that are (or can be) coloured. It has been largely replaced by thin layer chromatography, but is still a powerful teaching tool. Double-way paper chromatography, also called 'two-dimensional chromatography', uses two solvents and rotates the paper 90° in between. It is useful for separating complex mixtures of compounds having similar polarity, for example, amino acids. The compounds used initially ensures you to know whether the colour is pure, (one substance only) or mixtures (multiple substances.)

Thin layer chromatography

Main article: Thin layer chromatography

Thin layer chromatography (TLC) is a common laboratory technique similar to paper chromatography. Instead of a stationary phase of paper, it uses a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat substrate. Compared to paper, it has the advantage of faster runs, better separations, and the choice between different adsorbents. For even better resolution and to allow for quantification, high-performance TLC can be used.

Column chromatography

Column chromatography separates compounds using many chemical actions between the chemical being tested and the chromatography column (a rod with a blending of special chemicals). The column is run using either gravity or a pump.

The mixed substance to be tested is added in a small amount and is slowed by certain chemical or physical activity with the chemicals in the chromatography column. The amount of slowing depends on the type of chemicals in the substance being tested and the different phases. The time at which a certain chemical elutes (comes out of the end of the column) is called the "retention time" and there is thought to be only one for one chemical.

High-performance liquid chromatography results

The most common stationary phase for column chromatography is silica gel, followed by alumina. Cellulose powder has been used in the past. The mobile phase is either a pure solvent or a mixture of solvents. It is chosen to make the time and amount of solvent used as little as possible, while still clearly separating the chemicals being tested.

HPLC

High-performance liquid chromatography (HPLC) is also sometimes referred to as high-pressure liquid chromatography. It is column chromatography run under pressure to raise the speed of the process.

Common solvents used in HPLC are mixes of water or various organic liquids (the most common are methanol, ethanol or acetonitrile).