There are many different methods for analyzing the composition and studying the properties of various compounds and mixtures of substances. One such method is chromatography. The authorship in the invention and application of the method belongs to the Russian botanist M. S. Tsvet, who at the beginning of the 20th century carried out the separation of plant pigments.
Definition and fundamentals of the method
Chromatography is a physicochemical method for separating mixtures and determining their components, based on the distribution between the mobile and stationary phases of the substances that make up the mixture (sample). The stationary phase is a porous solid substance - a sorbent. It can also be a liquid film deposited on a solid surface. The mobile phase - the eluent - must move along the stationary phase or flow through it, being filtered by the sorbent.
The essence of chromatography is that different components of a mixture are necessarily characterized by different properties, such as molecular weight, solubility, adsorbability, and so on. Therefore, the rate of interaction of the components of the mobile phase - sorbates - with the stationaryis not the same. This leads to a difference in the velocities of the molecules of the mixture relative to the stationary phase, as a result of which the components are separated and concentrated in different zones of the sorbent. Some of them leave the sorbent together with the mobile phase - these are the so-called unretained components.
A special advantage of chromatography is that it allows you to quickly separate complex mixtures of substances, including those with similar properties.
Methods for classifying types of chromatography
The methods used in the analysis can be classified according to various criteria. The main set of such criteria is as follows:
- aggregate state of stationary and mobile phases;
- physical and chemical nature of the interaction of the sorbent and sorbates;
- how to introduce eluent and move it;
- method of placement of the stationary phase, i.e. chromatography technique;
- chromatography targets.
In addition, methods can be based on the different nature of the sorption process, on the technical conditions of the chromatographic separation (for example, low or high pressure).
Let's take a closer look at the above main criteria and the most widely used types of chromatography associated with them.
Eluent and sorbent state of aggregation
On this basis, chromatography is divided into liquid and gas. Method names reflect the state of the mobile phase.
Liquid chromatography is a technique usedin the processes of separation of mixtures of macromolecular compounds, including biologically important ones. Depending on the state of aggregation of the sorbent, it is divided into liquid-liquid and liquid-solid phase.
Gas chromatography is of the following types:
- Gas adsorption (gas-solid-phase), which uses a solid sorbent, such as coal, silica gel, zeolites or porous polymers. An inert gas (argon, helium), nitrogen, carbon dioxide acts as an eluent - a carrier of the mixture to be separated. The separation of the volatile components of the mixture is carried out due to the different degree of their adsorption.
- Gas-liquid. The stationary phase in this case consists of a liquid film deposited on a solid inert base. Sample components are separated according to their adsorbability or solubility.
Gas chromatography is widely used for the analysis of mixtures of organic compounds (using their decomposition products or derivatives in gaseous form).
Interaction between sorbent and sorbates
According to this criterion, such types are distinguished as:
- Adsorption chromatography, through which mixtures are separated due to differences in the degree of adsorption of substances by an immobile sorbent.
- Distribution. With its help, separation is carried out on the basis of different solubility of the components of the mixture. Dissolution occurs either in the mobile and stationary phases (in liquid chromatography), or only in the stationary phase (in gas-liquidchromatography).
- Sedimentary. This chromatography method is based on the different solubility of the formed precipitates of the substances to be separated.
- Exclusion, or gel chromatography. It is based on the difference in the size of molecules, due to which their ability to penetrate into the pores of the sorbent, the so-called gel matrix, varies.
- Affine. This specific method, which is based on a special type of biochemical interaction of separated impurities with a ligand that forms a complex compound with an inert carrier in the stationary phase. This method is effective in separating mixtures of protein-enzymes and is common in biochemistry.
- Ion exchange. As a sample separation factor, this method uses the difference in the ability of the components of the mixture to ion exchange with the stationary phase (ion exchanger). During the process, the ions of the stationary phase are replaced by ions of substances in the composition of the eluent, while due to the different affinity of the latter to the ion exchanger, a difference arises in the speed of their movement, and thus the mixture is separated. For the stationary phase, ion exchange resins are most often used - special synthetic polymers.
Ion-exchange chromatography has two options - anionic (retains negative ions) and cationic (retains positive ions, respectively). This method is used extremely widely: in the separation of electrolytes, rare earth and transuranium elements, in water purification, in the analysis of drugs.
The difference in methods of technique
There are two main ways in which the sample moves relative to the stationary phase:
- Column chromatography carries out the separation process in a special device - a chromatographic column - a tube, in the inner cavity of which an immovable sorbent is placed. According to the method of filling, the columns are divided into two types: packed (the so-called "packed") and capillary, in which a layer of a solid sorbent or a liquid film of the stationary phase is applied to the surface of the inner wall. Packed columns can have different shapes: straight, U-shaped, spiral. Capillary columns are helical.
- Planar (planar) chromatography. In this case, special paper or a plate (metal, glass, or plastic) can be used as a carrier for the stationary phase, on which a thin layer of sorbent is deposited. In this case, the chromatography method is referred to as paper or thin-layer chromatography, respectively.
Unlike the column method, where chromatographic columns are used repeatedly, in planar chromatography, any carrier with a sorbent layer can be used only once. The separation process occurs when a plate or sheet of paper is immersed in a container with eluent.
Introduction and transfer of eluent
This factor determines the nature of the movement of the chromatographic zones along the sorbent layer, which are formed during the separation of the mixture. There are the following eluent delivery methods:
- Front. This method is the simplestexecution technique. The mobile phase is directly the sample itself, which is continuously fed into the column filled with the sorbent. In this case, the least retained component, adsorbed worse than others, moves along the sorbent faster than the others. As a result, only this first component can be isolated in pure form, followed by zones containing mixtures of components. The sample distribution looks like this: A; A+B; A+B+C and so on. Frontal chromatography is therefore not useful for separating mixtures, but it is effective in various purification processes, provided that the substance to be isolated has low retention.
- The displacement method differs in that after entering the mixture to be separated, an eluent with a special displacer is fed into the column - a substance characterized by a greater sorbability than any of the components of the mixture. It displaces the most retained component, which displaces the next one, and so on. The sample moves along the column at the speed of the displacer and forms adjacent zones of concentration. With this type of chromatography, each component can be obtained individually in liquid form at the outlet of the column.
- The eluent (developing) method is the most common. In contrast to the displacement method, the eluent (carrier) in this case has a lower sorbability than the sample components. It is continuously passed through the sorbent layer, washing it. Periodically, in portions (pulses), the mixture to be separated is introduced into the eluent flow, after which the pure eluent is fed again. When washing out (elution), the components are separated,moreover, their concentration zones are separated by eluent zones.
Eluent chromatography makes it possible to almost completely separate the analyzed mixture of substances, and the mixture can be multicomponent. Also, the advantages of this method are the isolation of the components from each other and the simplicity of the quantitative analysis of the mixture. The disadvantages include a high consumption of the eluent and a low concentration of the sample components in it after separation at the column outlet. The eluent method is widely used in both gas and liquid chromatography.
Chromatographic processes depending on the purposes
The difference in chromatography goals makes it possible to distinguish methods such as analytical, preparative and industrial.
By means of analytical chromatography, qualitative and quantitative analysis of mixtures is carried out. When analyzing the sample components, when leaving the chromatograph column, they go to the detector - a device that is sensitive to changes in the concentration of a substance in the eluent. The time elapsed from the moment the sample is introduced into the column until the maximum peak concentration of the substance on the detector is called the retention time. Provided that the column temperature and the eluent rate are constant, this value is constant for each substance and serves as the basis for a qualitative analysis of the mixture. Quantitative analysis is carried out by measuring the area of individual peaks in the chromatogram. As a rule, the eluent method is used in analytical chromatography.
Preparative chromatography aims to isolate pure substances from a mixture. Preparative columns have a much largerdiameter than analytical.
Industrial chromatography is used, firstly, to obtain large quantities of pure substances needed in a particular production. Secondly, it is an important part of modern control and regulation systems for technological processes.
Industrial chromatograph has a scale of concentration of one or another component and is equipped with a sensor, as well as control and registration systems. Samples are delivered to such chromatographs automatically with a certain frequency.
Multifunction Chromatography Equipment
Modern chromatographs are complex high-tech devices capable of being used in a variety of fields and for various purposes. These devices make it possible to analyze complex multicomponent mixtures. They are equipped with a wide range of detectors: thermal conductometric, optical, ionization, mass spectrometric and so on.
In addition, modern chromatography uses automatic control systems for the analysis and processing of chromatograms. Control can be performed from a computer or directly from the device.
An example of such a device is the multifunctional gas chromatograph "Crystal 5000". It has a set of four replaceable detectors, a column thermostat, electronic pressure and flow control systems, and gas valve controls. To solve various problems, the device hasthe ability to install both packed and capillary columns.
The chromatograph is controlled using a full-featured keyboard and control display or (in another modification) from a personal computer. This new generation device can be effectively used in production and in various research laboratories: medical, forensic, environmental.
High pressure chromatography
Carrying out liquid column chromatography is characterized by a rather long duration of the process. To accelerate the movement of the liquid eluent, the flow of the mobile phase into the column under pressure is used. This modern and very promising method is called the high performance liquid chromatography (HPLC) method.
The HPLC liquid chromatograph's pumping system delivers eluent at a constant rate. The developed inlet pressure can reach 40 MPa. Computer control makes it possible to change the composition of the mobile phase according to a given program (this method of elution is called gradient).
HPLC can be used various methods based on the nature of the interaction of the sorbent and sorbate: distribution, adsorption, size exclusion, ion-exchange chromatography. The most common type of HPLC is the reversed-phase method, based on the hydrophobic interaction of a polar (aqueous) mobile phase and a non-polar sorbent, such as silica gel.
The method is widely used for separation, analysis,quality control of non-volatile, thermally unstable substances that cannot be converted into a gaseous state. These are agrochemicals, medicines, food components and other complex substances.
The importance of chromatography studies
Different types of chromatography are widely used in various fields:
- inorganic chemistry;
- petrochemicals and mining;
- biochemistry;
- medicine and pharmaceuticals;
- food industry;
- ecology;
- criminology.
This list is incomplete, but reflects the coverage of industries that cannot do without chromatographic methods of analysis, separation and purification of substances. In all areas of application of chromatography, from scientific laboratories to industrial production, the role of these methods is even more increasing as modern technologies for information processing, management and control of complex processes are introduced.
