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 - a sorbent. It can also be a liquid film deposited on a hard 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 motionless is 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 uncontrollable components.
A particular advantage of chromatography is that it allows you to quickly separate complex mixtures of substances, including those with similar properties.
Methods for classifying chromatographic species
The methods used in the analysis can be classified according to various criteria. The main set of such criteria is as follows:
- state of aggregation of the stationary and mobile phases;
- physicochemical nature of the interaction of the sorbent and sorbates;
- the method of introducing the eluent and its movement;
- a method for placing a stationary phase, i.e. a chromatography technique;
- chromatographic goals.
In addition, the methods can be based on the different nature of the sorption process, on the technical conditions of chromatographic separation (for example, low or high pressure).
Let us consider in more detail the above basic criteria and the most widely used types of chromatography associated with them.
Aggregate state of eluent and sorbent
On this basis, chromatography is divided into liquid and gas. The names of the methods reflect the state of the mobile phase.
Liquid chromatography is a method used in the separation processes of mixtures of high molecular weight 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), in which a solid sorbent is used, for example coal, silica gel, zeolites or porous polymers. An inert gas (argon, helium), nitrogen, carbon dioxide acts as an eluent - a carrier of the separated mixture. The separation of the volatile components of the mixture is carried out due to the varying degrees of their adsorption.
- Gas-liquid. The stationary phase in this case consists of a liquid film deposited on a solid inert base. The components of the sample are separated according to their adsorbability or solubility.
The method of gas chromatography is widely used for the analysis of mixtures of organic compounds (using products of their decay or derivatives in gaseous form).
The interaction of the sorbent and sorbates
By this criterion, such types are distinguished as:
- Adsorption chromatography, by which the separation of mixtures is carried out due to differences in the degree of adsorption of substances by a stationary sorbent.
- Distribution. With its help, separation is carried out on the basis of different solubilities 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-liquid chromatography).
- Sedimentary. The basis of this chromatography method is the different solubility of the precipitates of the separated substances.
- Size exclusion, or gel chromatography. It is based on the difference in the size of the molecules, due to which their ability to penetrate into the pores of the sorbent, the so-called gel matrix, varies.
- Affinity. This specific method, which is based on a special type of biochemical interaction of shared impurities with a ligand, which forms a complex compound with an inert carrier in the stationary phase. This method is effective in the separation of protein-enzyme mixtures and is common in biochemistry.
- Ion exchange. As a factor for sample separation, this method uses the difference in the ability of the mixture components to ion exchange with a 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, and due to the different affinity of the latter to the ion exchanger, a difference in the speed of their movement occurs, and thus the mixture is separated. For the stationary phase, ion-exchange resins are most commonly 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 transuranic elements, in water treatment, in the analysis of drugs.
The difference in methods
There are two main ways in which a sample moves relative to a stationary phase:
- Column chromatography performs the separation process in a special device — a chromatographic column — a tube, in which the stationary sorbent is placed in the internal cavity. According to the filling method, the columns are divided into two types: packed (so-called “stuffed”) and capillary, in which a layer of solid sorbent or a liquid film of a stationary phase is applied to the surface of the inner wall. Packed columns can have various shapes: straight, U-shaped, spiral. Capillary columns have a spiral shape.
- Plane (planar) chromatography. In this case, special paper or a plate - metal, glass, plastic - on which a thin layer of sorbent is applied, can be used as a carrier for the stationary phase. The chromatography method is hereinafter referred to as paper or thin layer.
In contrast to the column method, where chromatographic columns are used repeatedly, in plane 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 an eluent.
Entering and moving the eluent
The nature of the movement of chromatographic zones formed during separation of the mixture depends on this factor. The following eluent feeding methods are distinguished:
- Frontal. This method is the simplest in implementation technique. The mobile phase is directly the sample itself, continuously fed into a column filled with a sorbent. In this case, the least retained component adsorbed worse than others moves along the sorbent faster than others. As a result, only this first component can be isolated in pure form, followed by zones containing mixtures of components. The distribution of the sample is as follows: A; A + B; A + B + C and so on. Frontal chromatography is therefore not used for the separation of mixtures, but it is effective in various purification processes, provided that the emitted substance has low retention.
- The displacement method is characterized in that after the shared mixture is introduced into the column, an eluent is supplied with a special displacer - a substance characterized by greater sorbability than any of the components of the mixture. It displaces the most retained component, it displaces the next and so on. The sample moves along the column with the speed of the displacer and forms concentration zones adjacent to each other. Using this type of chromatography, each component can be obtained individually in liquid form at the column exit.
- The eluent (manifest) method is the most common. In contrast to the displacement method, the eluent (carrier) in this case has less sorbability than the components of the sample. It is continuously passed through a layer of sorbent, washing it. From time to time, in portions (pulses), a separable mixture is introduced into the eluent stream, after which pure eluent is again fed. When washing (eluting), the components are separated, and their concentration zones are separated by the eluent zones.
Eluent chromatography allows for almost complete separation of 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 eluent and a low concentration of sample components in it after separation at the outlet of the column. The eluent method is widely used in both gas and liquid chromatography.
Chromatographic processes depending on the goals
The difference in the goals of chromatography allows us to distinguish methods such as analytical, preparative and industrial.
Through analytical chromatography, a qualitative and quantitative analysis of the mixtures is carried out. When analyzing the components of the sample when leaving the column of the chromatograph are fed to the detector - a device that is sensitive to changes in the concentration of the substance in the eluent. The time elapsed from the moment the sample is fed into the column to the maximum peak in the concentration of the substance on the detector is called the retention time. Given the constant temperature of the column and the speed of the eluent, 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, analytical chromatography uses the eluent method.
Preparative chromatography aims to isolate pure substances from the mixture. Preparative columns have a much larger diameter than analytical ones.
Industrial chromatography is used, firstly, to obtain large quantities of pure substances required in a particular production. Secondly, this is an important part of modern systems of control and regulation of technological processes.
An industrial chromatograph has a concentration scale for one or another component and is equipped with a sensor, as well as control and recording systems. Samples for such chromatographs are automatically received at regular intervals.
Multifunction chromatography equipment
Modern chromatographs are complex high-tech devices that can be used in various fields and for various purposes. These instruments allow the analysis of complex multicomponent mixtures. They are equipped with a wide range of detectors: thermoconductometric, optical, ionization, mass spectrometric and so on.
In addition, modern chromatography uses automatic control systems for the analysis and processing of chromatograms. Management can be done from a computer or directly from the device.
An example of such a device is the Crystal 5000 multifunctional gas chromatograph. 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 has the ability to install both packed and capillary columns.
The chromatograph is controlled using a fully functional keyboard and control display, or (in another version) 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
Conducting liquid column chromatography is characterized by a rather long process. To accelerate the movement of the liquid eluent, a mobile phase is applied to the column under pressure. This modern and very promising method is called the method of high performance liquid chromatography (HPLC).
The pump system, which is part of the liquid chromatograph for HPLC, provides the eluent at a constant speed. 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 applied various methods based on the nature of the interaction of the sorbent and sorbate: distribution, adsorption, exclusion, ion exchange chromatography. The most common type of HPLC is the reverse phase method, based on the hydrophobic interaction of the polar (aqueous) mobile phase and a non-polar sorbent, for example silica gel.
The method is widely used for the separation, analysis, and quality control of non-volatile, thermally unstable substances that cannot be converted to a gas state. These are agrochemicals, drugs, food components and other complex substances.
The Importance of Chromatographic Studies
Various types of chromatography are widely used in various fields:
- inorganic chemistry;
- petrochemicals and mining;
- biochemistry;
- medicine and pharmaceuticals;
- food industry;
- ecology;
- forensics.
This list is not complete, but reflects the coverage of industries that cannot do without chromatographic methods of analysis, separation and purification of substances. In all areas of chromatography application, from scientific laboratories to industrial production, the role of these methods increases even more with the introduction of modern information processing technologies, management and control of complex processes.