Chromatographic methods of analysis: advantages and disadvantages

Chromatographic analysis today is the most widely used method for the study of various objects. These can be samples taken in the environment, at work, in the laboratory and so on. This method was proposed in 1903 by the Russian scientist M. S. Tsvet. His research became the basis for the development of all types of chromatography that exist today and are used to separate not only colored but also unstained compounds in all kinds of environments. Conducting chromatographic analysis is possible in various ways, using in each particular case its own techniques and calculation methods. It is based on differences in the adsorption or any other properties of the compounds, which contributes to their distribution between the solid sorbent and the liquid (or gas) passing through it.

chromatographic columns

Basic concepts

Chromatographic method of analysis refers to such a method of separation and determination of substances, which is based on the distribution of several components of the sample between two phases, one of which is mobile and the other is fixed.

A stationary (stationary) phase is usually a solid porous substance (usually referred to as a sorbent) or a film of a liquid that is deposited on a solid substance.

The mobile phase is a liquid or gaseous substance flowing through the stationary phase, sometimes under pressure. All components of the analyzed mixture (called sorbates) along with the mobile phase move along the stationary phase. As a rule, it is placed in a glass (metal) tube - column.

The speed of movement of the components along the column depends on the degree of their interaction with the surface of the sorbent. This leads to the fact that some components will remain in the upper part of the column, distributed in the sorbent volume, others in the lower part, and some will not stay at all in it and leave with the mobile phase.

Classification of chromatographic methods of analysis

These methods of research of substances are so diverse that there is no single classification of them. Usually they are divided by the following features:

  • the aggregate state of the sorbent and the mobile phase;
  • the mechanism of binding substances and sorbent;
  • analysis technique;
  • method of movement of the sample through the column;
  • purpose of the analysis.

According to the aggregate state of the phases

According to this feature, chromatographic analysis methods are divided into:

  • gas chromatography if the mobile phase is steam or gas;
  • liquid chromatography when the mobile phase is in a liquid state.

The first is usually used to separate volatile thermally stable compounds with a molecular weight of up to 300. The second is suitable for separating organic and inorganic components that have a molecular weight of up to 2000, even if they are thermally unstable.

separation of chlorophylls a and c

By the nature of the interaction of the substance with the stationary phase

According to the mechanism of action of a sorbent with a substance, chromatographic methods of analysis can be:

  • adsorption, if the separation is based on differences in the affinity of the components of the sample to the surface of the adsorbent;
  • distributive, if the separation of substances based on the differences in their solubility in the mobile phase and the sorbent;
  • ion exchange if the separation is based on differences in the ability of components to perform ion exchange;
  • penetrating, if the separation of substances occurs due to differences in the size and shape of molecules, as well as charges.

Also chromatographic methods of analysis are classified according to this method into sedimentary, redox, complex-forming and others.

According to the experiment technique

According to the method of processing the process chromatography is:

  • column, in which the separation is carried out in columns filled with sorbent.
  • planar, which can be carried out on paper or on plates (thin layer chromatography).
paper chromatography

You can also add to this list methods of chromatographic analysis carried out using capillaries. The inner diameter of such tubes is not more than 1 mm. In comparison with other types of chromatography, this allows you to increase the speed of analysis and makes it possible to study with expensive gases or sorbents. Also, the small size of the column allows you to combine this study with mass spectrometry. However, a significant drawback of the chromatographic method of analysis of this type is the difficulty of introducing the sample into the capillary.

On the movement of matter

This feature is also called the method of chromatography. There are:

  • Eluent chromatography. Best suited for solving analytical problems. It consists in the fact that the mixture of substances introduced into a continuously moving stream of eluent is sorbed better than the mobile phase. During the progress of the eluent along the column with sorbed components, they begin to move along the sorbent at different speeds and leave the column in separate fractions (zones). The advantages of the developmental method are a more complete separation of substances, continuous regeneration of the sorbent and good reproducibility of retention parameters.
  • Displacement chromatography. It consists in the fact that a divided sample is introduced into the mobile phase, and then the propellant, which has the highest sorption capacity, is started to pass. During its progress along the column, it displaces previously sorbed substances from the sorbent in order of increasing their sorption capacity.
  • Front chromatography. During the study, the analyzed mixture is also passed through a layer of sorbent.And in the course of filling the column with components, they gradually come out in order of increasing their ability to sorption.

By purpose of work

Classification of chromatographic methods of analysis, depending on the purpose of the process is as follows:

  • analytical chromatography - is a separate method, including the separation of the sample into components, as well as their qualitative and quantitative analysis;
  • preparative chromatography is used exclusively to separate the mixture of substances.

Advantages of the method

Chromatographic analysis has the following advantages over other methods of separation and research of substances:

  1. Due to the fact that this method of separation has a dynamic nature, characterized by repeated repetition of sorption-desorption, its efficiency is significantly higher than that of static sorption or extraction.
  2. The use of various types of interaction of sorbates and sorbents (physical and chemisorption) allows to expand the range of substances for selective separation.
  3. Some methods provide for the imposition of gravitational, magnetic and other fields on the emitted substances, which expands the possibilities of chromatography by changing the conditions of separation of components.
  4. This method is hybrid because it combines the separation and definition of several components at once.
  5. Chromatography makes it possible to solve several problems simultaneously. Thus, analytical tasks include separation, identification and determination of substances, and preparative ones - their purification, isolation and concentration.
chromatographic analysis

Chromatography features

High-performance and highly selective preparative chromatography is indispensable for the separation of complex samples containing a large number of individual compounds with similar physico-chemical parameters (oil, various drugs, extracts from plants, biological fluids, and others). Thus, gas chromatographic analysis methods are widely used for the purification of chemicals or the isolation of individual compounds in preparative chemistry. For ion extraction, ion exchange chromatography is suitable, based on differences in the ability of ions from a solution to exchange processes with an ion exchanger.

Modern chromatographic methods allow the determination of gaseous, liquid and solid substances. Selection of conditions for the analysis focuses on the nature and composition of the analyzed sample.Gas adsorption and gas-liquid chromatography allow the study of volatile substances resistant to heat. Thus, gas adsorption chromatography is widely used for the analysis of gas mixtures and low-boiling hydrocarbons that do not have active functional groups. Gas-liquid chromatography is important in petrochemistry, analysis of pesticides and fertilizers, drugs.

Liquid chromatographic analysis of transformer oil allows timely detection of defects or the nature and extent of damage to the transformer. His condition is assessed by comparing the data obtained during the analysis with the permissible values, as well as the rate of change in the content of gases in the oil. So, the high content of CO and CO2usually signals abnormal cellulose insulation. But the presence of furan derivatives suggests aging of paper insulation. Thus, chromatographic analysis of gases contributes to the safe and long-term operation of the equipment.

Gas chromatography

It is one of the most common varieties of the method, due to the fact that various techniques with a full theoretical justification have been developed for it, as well as reliable and relatively inexpensive instrumentation.The mobile phase (carrier gas) are gases or their mixtures, as well as substances that are gases under the conditions in which the analysis is performed. The stationary phase is solid sorbents (gas adsorption method) or a liquid on the surface of an inert carrier (gas-liquid method).

gas chromatography

For gas chromatography, you can additionally name a number of advantages:

  • high speed of the process;
  • microprobe analysis capability;
  • automatic recording of results with the availability of appropriate equipment;
  • the possibility of isolating components not only in the laboratory, but also on an industrial scale.

Paper chromatography

As the stationary phase, filter or special chromatographic paper is used. The latter is a cellulose filter paper of high purity and with some special properties. It absorbs the solvent at different capillary lifting speeds, depending on the density of the paper.

The main equipment are special chambers or vessels, trays placed on racks, pipettes, spray guns, chromatogram lamps, measuring devices, and also planimeters and densitometers used for quantitative determinations.

This method is best suited for the analysis of various organic substances containing different functional groups from alcohols to steroids, from amines to indoles, from vitamins to antibiotics.

Ion exchange chromatography

This method is based on the exchange of ions between the swollen ion exchanger and the mobile phase. Ion-exchange separation of a mixture of ions is characterized by the difference in their charges and the ionic strength of the solution. In the volume of the ionite grains, the separation process also depends on the rate of diffusion of ions, which is determined by the density of the ion exchanger.

Ionite is selected by a parameter called affinity, which is proportional to the charge of the ion and inversely proportional to the radius of the hydrated ion. The choice of an ion exchanger is carried out using tables with the given characteristics of the types of ion exchangers produced. Their main characteristics are grain size and shape, exchange capacity, acid-base properties, swelling, density.

ion exchange in a chromatographic column

Separation of inorganic substances is carried out on inorganic ion exchangers (zeolites, aluminum hydroxides) or resins (styrene with divinylbenzene). Thus, the chromatographic method of analyzing water for the presence of various ions in it is often used, for example, to determine its hardness.

Gel chromatography

The essence of the method is that the analyzed solution is slowly filtered through columns filled with gel. Sometimes it is called gel filtration. Individual particles of the gel are composed of plastic linear molecules of substances with the highest molecular weight, connected by cross-links. Such a mesh structure contributes to the swelling of the gel in water and the appearance of pores of different diameters in it. Pore ​​sizes depend on the nature of the polymer, the temperature of the medium and the nature of the solvent.

Separation is based on the ability of smaller molecules to penetrate deeper into the pores and stay there for a longer time. Therefore, at first larger molecules leave the column, and then those that are smaller.

gel chromatography

This method carries out two types of separation: group and fractionation. The former is characterized by the separation of a mixture of components according to their molecular weight. For the second, in terms of the speed and intensity of diffusion of particles inside the gel. The most commonly used gel chromatography in biochemistry, in organic synthesis and chemistry of polymers to determine the molecular masses. As an example, chromatographic analysis of proteins and peptides in plasma is possible.In comparison with mass spectrometric methods, tracking protein-peptide homeostasis by the distribution of proteins and their degradation products by gel chromatography is much more accessible.

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