We explain what chromatography is, how it is used to separate mixtures, what its phases are, what types exist and examples.

Chromatography allows the components of a mixture to be separated and identified.

What is chromatography?

Chromatography is a mixture separation method complex, which is widely used in various branches of the science. It can be used to quantify, identify and separate the components of a mixture. To do this, it uses the principle of selective retention, which consists of the different behavior of the components of a mixture on a specific support (such as a paper, a gas, a liquid, a resin) and a liquid or gaseous phase that flows through the support.

In this way, chromatography employs various techniques that take advantage of the differences in the retention rate of each component, and can separate, identify and quantify them.

In many cases the key adsorption (different from the absorption, which refers to the diffusion of a component from one phase to another), a concept that refers to the process by which the particles are retained on a surface. According to the difference in adsorption rates on a support and the affinity for this support of the components of the mixture, they can be separated and then quantified or identified.

In general, all types of chromatography depend on a number of instruments, chemical compounds and determined technology. Due to this, it is important to know some concepts in order to understand the operation of chromatographic techniques:

  • Stationary phase. It is a substance that remains immobile while the chromatography is running.
  • Mobile phase. It is the substance that moves during chromatography. It can be a liquid or a gas. The sample containing the analyte is administered in the mobile phase.
  • Analytes. They are the substances that are going to be separated, quantified and / or identified using chromatography, that is, they are the substances that are going to be analyzed.
  • Shows. It is the mixture to be analyzed. It can be made up of one or more analytes, and other components that may not be of interest, from which the analytes will be separated.
  • Holding time. It is the time it takes for an analyte to pass from the column or system through which the mobile phase passes, to the detector (equipment that can give a detection signal using some property of the analyte).
  • Selectivity. It is the ability to differentiate each component in the mix.
  • Eluent It also refers to the mobile phase as it exits the chromatographic column.

The chromatographic method consists of inoculating a sample in a stationary phase or mobile phase (depending on the type of chromatographic technique). Then, if, for example, the mobile phase is the one that contains the sample, it passes through a certain stationary phase.

The separation of the analytes will depend on the affinity of each of the components for both the stationary phase and the mobile phase. Depending on their nature, some substances they will tend to move with the mobile phase and others to remain on the stationary phase.

Types of chromatography

Depending on the technology used, the nature of the support (stationary phase) and the mobile substance (mobile phase), the following types of chromatography can be differentiated:

  • Chromatography on paper. The stationary phase is made up of a filter paper strip. The sample to be analyzed is placed as a drop on one end of the paper. Then the paper strip is immersed in a container where the mobile phase is located, taking into account that the end where the sample is placed is at the bottom of the paper. The mobile phase rises by capillarity, dragging the sample with it and separating each component according to its affinity for the stationary phase. This type of chromatography is used mainly when each component of the sample has a color different, then you can see the display of colors on the paper to identify them.
  • Thin layer chromatography. The operation of this technique is similar to that of paper chromatography, but in this case the stationary phase is built by depositing a polar resin (almost always silica gel) on a glass or aluminum plate. A certain amount of the sample is placed 1cm from the lower edge of the plate. This plate is then immersed, keeping in mind that the end that contains the sample must be down, in a container that contains the mobile phase. The mobile phase rises by capillary action, separating the components of the sample.
  • Column chromatography. The stationary phase is placed inside a column that can be made of glass or stainless steel, among other materials. The mobile phase can be liquid or gaseous. The sample is placed at the top of the column and allowed to descend with the mobile phase using the gravity. Thus, column chromatography can be classified as:
    • Solid-liquid chromatography. The stationary phase is solid and the mobile is liquid.
    • Liquid-liquid chromatography. Both phases are liquid.
    • Liquid-gas chromatography. The stationary phase is liquid and the mobile phase is soda.
    • Solid-gas chromatography. The stationary phase is a solid and the mobile is gaseous.

On the other hand, taking into account the type of interaction of the analyte between the stationary and mobile phases, we have the following types of chromatography:

  • Adsorption chromatography. In this type of chromatography the stationary phase is a solid, while the mobile phase is a liquid. The substance that forms the stationary phase can be alumina (Al2O3), silica (SiO2) or ion exchange resins (matrices that have electrostatically active sites, due to which the analyte is retained in them by electrostatic interaction). The mobile phase can be made up of a solvent or a mixture of solvents. Some components of the mixture will be retained with greater force than others, in this way the separation occurs.
  • Partition chromatography. It occurs when the separation of the analytes from the mixture occurs due to differences in their solubilities or polarities between the stationary phase and the mobile phase, both phases being immiscible liquid. The technology of stationary phases has advanced and there are already varieties of liquids embedded in solids and resins that are used for this purpose. In this sense, there are two types of cormatography depending on the polarity of the stationary phase and the mobile phase:
    • In normal phase. The stationary phase is polar and the mobile phase is apolar.
    • In reverse phase. The stationary phase is apolar and the mobile phase is polar.
  • Ion exchange chromatography. When the stationary phase is solid and has ionizable functional groups, that is, charged, which are capable of exchanging their charge with the analyte. It can be classified into:
    • Cation exchange chromatography. The stationary phase contains negatively charged functional groups, therefore it retains cations (positively charged).
    • Anion exchange chromatography. The stationary phase contains positively charged functional groups, thus retaining (negatively charged) anions.
  • Size Exclusion Chromatography. The stationary phase is a porous material through which analytes elute, depending on their sizes. In this type of chromatography there is no type of physical or chemical interaction between the analytes and the stationary phase. Larger analytes elute first, that is, they are not retained in the stationary phase. While the smaller analytes are trapped in the pores of the stationary phase and leave it as the mobile (liquid) phase passes.

With the advance of knowledge and technology, chromatographic techniques were being perfected and each time it has been possible to separate, identify and quantify more accurately the substances present in a mixture. Two examples of advanced chromatography are HPLC (High Performance Liquid Chromatography) and GC (Gas Chromatography).

  • HPLC. It consists of a type of column chromatography, but whose mobile phase is pumped at high pressures through the stationary phase inside the column. The application of a high pressure reduces the diffusion of the analytes through the stationary phase, thus achieving better results, in addition to reducing working times.
  • GC. The mobile phase is a gas and the stationary phase can be a solid or a liquid. The sample volatilizes before injecting it into the chromatographic column, as it must be gaseous for the carrier gas to transport it.

Chromatography examples

To analyze blood, its components are separated through chromatography.

Some everyday examples of the application of chromatography are:

  • Spilled wine on a white tablecloth. An accident at dinner time will allow us to observe, when the wine dries by contact with the air, the various substances that compose it. Each one will dye the white of the fabric in a different tone or color, and they can be identified separately, which would normally be impossible.
  • Blood test. Chromatography of blood samples is often carried out to identify the substances contained in it, which are normally imperceptible since it is a very complex mixture. To do this, the color that the blood reflects on a support or subjected to a light specific.
  • Urine tests. Like blood, urine is a mixture of various compounds, some solids and other liquids, the presence or absence of which can reveal details about how the body works. Chromatographic separation can be carried out to detect unusual residues, such as blood, salts, glucose, or illegal substances.
  • Review of a crime scene. Something that we often see in films: researchers take fabrics, fibers, fabrics or other supports and observe the separation by adherence of the different substances spilled on them, such as semen or blood, even when with the naked eye they could pass unnoticed.
  • Sanitary checks of food. Assuming that specialists in food know the reaction of food components when subjected to a chromatographic spectrum, this technique can be used to detail in a sample if there is some type of improper substance in them, a product of microbial agents or some type of pollution, before he product go to the market and put in risk the Health from the people.
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