Liquid adsorption chromatography on a column

The separation of a mixture of substances in an adsorption column occurs as a result of their difference in sorbability on a given adsorbent (in accordance with the law of adsorption substitution established by M. S. Tsvet).

Adsorbents are porous bodies with a highly developed inner surface that hold liquids with the help of intermolecular and surface phenomena. These can be polar and non-polar inorganic and organic compounds. Polar adsorbents include silica gel (dried gelatinous silicon dioxide), aluminum oxide, calcium carbonate, cellulose, starch, etc. Non-polar sorbents - activated carbon, rubber powder and many others obtained synthetically.

The adsorbents are subject to the following requirements: S they must not enter into chemical reactions with the mobile phase and the substances to be separated; S must have mechanical strength; S grains of the adsorbent must be of the same degree of dispersion.

When choosing the conditions for the chromatographic process, the properties of the adsorbent and adsorbed substances are taken into account.

In the classical version of liquid column chromatography (LCC), an eluent (PF) is passed through a chromatographic column, which is a glass tube 0.5–5 cm in diameter and 20–100 cm long, filled with a sorbent (NP). The eluent moves under the influence of gravity. The speed of its movement can be adjusted by the crane at the bottom of the column. The mixture to be analyzed is placed at the top of the column. As the sample moves through the column, the components separate. At certain intervals, fractions of the eluent released from the column are taken, which are analyzed by any method that allows measuring the concentrations of analytes.

Column adsorption chromatography is currently used mainly not as an independent method of analysis, but as a method of preliminary (sometimes final) separation of complex mixtures into simpler ones, i.e. to prepare for analysis by other methods (including chromatographic). For example, a mixture of tocopherols is separated on an alumina column, the eluent is passed, and the α-tocopherol fraction is collected for subsequent photometric determination.

Chromatographic separation of the mixture on the column due to the slow advance of the PF takes a long time. To speed up the process, chromatography is carried out under pressure. This method is called High Performance Liquid Chromatography (HPLC)

Modernization of the equipment used in classical liquid column chromatography has made it one of the promising and modern methods of analysis. High performance liquid chromatography is a convenient method for the separation, preparative isolation, and qualitative and quantitative analysis of non-volatile, thermolabile compounds of both low and high molecular weight.

Depending on the type of sorbent used, this method uses 2 chromatography options: on a polar sorbent using a non-polar eluent (direct phase option) and on a non-polar sorbent using a polar eluent - the so-called reversed-phase high-performance liquid chromatography (RP HPLC).

When the eluent passes to the eluent, the equilibrium under RPHLC conditions is established many times faster than under the conditions of polar sorbents and non-aqueous PFs. As a result of this, as well as the convenience of working with water and water-alcohol eluents, RPHLC has now gained great popularity. Most HPLC analyzes are carried out using this method.

Instrumentation for HPLC

A set of modern equipment for HPLC, as a rule, consists of two pumps 3,4 (Fig. 7.1.1.1), controlled by a microprocessor 5, and supplying eluent according to a specific program. Pumps create pressure up to 40 MPa. The sample is injected through a special device (injector) 7 directly into the eluent flow. After passing through the chromatographic column 8, the substances are detected by a highly sensitive flow detector 9, the signal of which is recorded and processed by the microcomputer 11. If necessary, fractions are automatically selected at the time of the peak output.

Columns for HPLC are made of stainless steel with an inner diameter of 2 - 6 mm and a length of 10-25 cm. The columns are filled with a sorbent (NF). Silica gel, alumina, or modified sorbents are used as NF. Silica gel is usually modified by chemically introducing various functional groups into its surface.

Detectors. Registration of the output from the column of a separate component is performed using a detector. For registration, you can use the change in any analytical signal coming from the mobile phase and related to the nature and amount of the mixture component. Liquid chromatography uses such analytical signals as light absorption or light emission of the exiting solution (photometric and fluorimetric detectors), refractive index (refractometric detectors), potential and electrical conductivity (electrochemical detectors), etc.

The continuously detected signal is recorded by the recorder. A chromatogram is a sequence of detector signals recorded on a tape recorder, which are generated when individual components of a mixture leave the column. In the case of separation of the mixture, separate peaks are visible on the external chromatogram. The position of the peak on the chromatogram is used for the purposes of identifying the substance, the height or area of ​​the peak is used for the purposes of quantitative determination.

Qualitative Analysis

The most important characteristics of the chromatogram - the retention time tR and the retention volume associated with it - reflect the nature of the substances, their ability to sorption on the material of the stationary phase and, therefore, under constant chromatography conditions, they are a means of identifying the substance. For a given column with a certain flow rate and temperature, the retention time of each compound is constant (Figure 7.1.1.2), where t.R(A) is the retention time of component A of the analyzed mixture from the moment it is injected into the column until the maximum peak appears at the column outlet, 1K( ss) - retention time of the internal standard (substance initially absent in the analyzed mixture), h - peak height (mm), ab - peak width at half its height, mm.

To identify a substance by chromatogram, standard samples or pure substances are usually used. Compare the retention time of the unknown IR* component with the IRCT retention time of the known substances. But more reliable identification by measuring the relative retention time

In this case, a known substance (internal standard) is first introduced into the column and its retention time tR(Bc) is measured, then the test mixture is chromatographically separated (chromatographed), to which the internal standard is preliminarily added. The relative retention time is determined by formula (7.1.1.1).

Quantitative Analysis

This analysis is based on the dependence of the peak height h or its area S on the amount of substance. For narrow peaks, measurement h is preferable, for wide blurry peaks - S. The peak area is measured in different ways: by multiplying the peak height (h) by its width (ai / 2), measured at half its height (Fig. 7.2.3); planning; using an integrator. Modern chromatographs are equipped with electrical or electronic integrators.

Three methods are mainly used to determine the content of substances in a sample: the absolute calibration method, the internal normalization method, and the internal standard method.

The absolute calibration method is based on a preliminary determination of the relationship between the amount of the introduced substance and the area or height of the peak on the chromatogram. A known amount of the calibration mixture is introduced into the chromatogram and the areas or heights of the resulting peaks are determined. Build a graph of the area or height of the peak from the amount of injected substance. The test sample is analyzed, the area or height of the peak of the component to be determined is measured, and its amount is calculated based on the calibration curve.

This method provides information only on the relative content of the component in the mixture, but does not allow determining its absolute value.

The internal standard method is based on the comparison of a selected peak parameter of an analyte with the same parameter of a standard substance introduced into the sample in a known amount. A known amount of such a standard substance is introduced into the test sample, the peak of which is sufficiently well separated from the peaks of the components of the test mixture

The last two methods require the introduction of correction factors characterizing the sensitivity of the detectors used to the analyzed substances. For different types of detectors and different substances, the sensitivity coefficient is determined experimentally.

Liquid adsorption chromatography also uses the analysis of fractions of solutions collected at the moment the substance exits the column. The analysis can be carried out by various physicochemical methods.

Liquid adsorption chromatography is used primarily for the separation of organic substances. This method is very successful in studying the composition of oil, hydrocarbons, effectively separating trans- and cis-isomers, alkaloids, etc. HPLC can be used to determine dyes, organic acids, amino acids, sugars, pesticide and herbicide impurities, medicinal substances and other contaminants in food products.

Liquid chromatography This is a method for separating and analyzing complex mixtures of substances in which liquid is the mobile phase. It is applicable to the separation of a wider range of substances than the gas chromatography method. This is due to the fact that most substances are not volatile, many of them are unstable at high temperatures (especially high-molecular compounds) and decompose when converted to a gaseous state. The separation of substances by liquid chromatography is most often carried out at room temperature.

The features of all types of liquid chromatography are due to the fact that the mobile phase in it is a liquid, and the sorption of components from a gaseous and liquid eluent proceeds differently. If in gas chromatography the carrier gas performs only a transport function and is not sorbed by the stationary phase, then the liquid mobile phase in liquid chromatography is an active eluent, its molecules can be sorbed by the stationary phase. When passing through the column, the molecules of the components of the analyzed mixture that are in the eluent must displace the molecules of the eluent from the surface layer of the sorbent, which leads to a decrease in the energy of interaction between the molecules of the analyzed substance and the surface of the sorbent. Therefore, the values ​​of retained volumes V R, proportional to the change in the free energy of the system, is smaller in liquid chromatography than in gas chromatography, and the range of linearity of the sorption isotherm in liquid chromatography is wider.

By using various eluents, one can change the retention parameters and selectivity of the chromatographic system. Selectivity in liquid chromatography, in contrast to gas chromatography, is determined not by one, but by two factors - the nature of the mobile (eluent) and stationary phases.

The liquid mobile phase has a higher density and viscosity than the gaseous phase, diffusion coefficients D well 3–4 orders of magnitude lower than in gas. This leads to a slowdown in mass transfer in liquid chromatography compared to gas chromatography. The Van Deemter equation due to the fact that the term AT does not play a role in liquid chromatography ( D well  D G), the graphic dependence of the efficiency also changes H on the linear velocity of the flow of the mobile phase has the form shown in fig. 1.9.

In the classical version of column liquid chromatography, a glass column 1–2 m high, filled with a sorbent with a particle size of 100 μm and eluent, is injected with the analyzed sample dissolved in the eluent, and the eluent is passed through, taking portions of the eluate at the outlet of the column. This variant of liquid chromatography is still used in laboratory practice, but since the eluent flow rate under the action of gravity is low, the analysis is lengthy.

The modern version of liquid chromatography, the so-called high-performance liquid chromatography HPLC, uses volumetric and surface-porous sorbents with a particle size of 5–10 μm, pressure pumps that provide pressure in the system up to 400 atm, and highly sensitive detectors. Fast mass transfer and high separation efficiency make it possible to use HPLC for the separation of molecules (liquid-adsorption and liquid-liquid partition chromatography), for the separation of ions (ion-exchange, ion, ion-pair chromatography), for the separation of macromolecules (size-exclusion chromatography).

1.3. SOLVENT RETENTION AND STRENGTH

In order for the analytes to be separated on the column, as mentioned above, the capacity factor k" must be greater than 0, i.e. the substances must be retained by the stationary phase, the sorbent. However, the capacity factor should not be too large to obtain an acceptable elution time. If a stationary phase is chosen for a given mixture of substances, which holds them, then further work on the development of an analysis procedure consists in choosing a solvent that would provide, in the ideal case, different for all components, but acceptable not very large k. This is achieved by changing the eluting strength of the solvent.

In the case of adsorption chromatography on silica gel or alumina, as a rule, the strength of a two-component solvent (for example, hexane with the addition of isopropanol) is increased by increasing the content of the polar component (isopropanol) in it, or reduced by decreasing the content of isopropanol. If the content of the polar component becomes too low (less than 0.1%), it should be replaced with a weaker eluting strength. The same is done, replacing either the polar or non-polar component with others, even if this system does not provide the desired selectivity with respect to the mixture components of interest. When selecting solvent systems, both the solubilities of the mixture components and the eluotropic series of solvents compiled by different authors are taken into account.

Approximately in the same way, the strength of the solvent is selected in the case of using grafted polar phases (nitrile, amino, diol, nitro, etc.), taking into account possible chemical reactions and excluding solvents dangerous for the phase (for example, aldehydes and ketones for the amino phase).

In the case of reverse phase chromatography, the strength of the solvent is increased by increasing the content of the organic component (methanol, acetonitrile or THF) in the eluent and reduced by adding more water. If the desired selectivity cannot be achieved, another organic component is used or attempts are made to change it with the help of various additives (acids, ion-pair reagents, etc.).

In separations by ion-exchange chromatography, the strength of the solvent is changed by increasing or decreasing the concentration of the buffer solution or by changing the pH, in some cases modification with organic substances is used.

However, especially in the case of complex natural and biological mixtures, it is often not possible to choose the solvent strength in such a way that all sample components are eluted within an acceptable time. Then one has to resort to gradient elution, i.e. use a solvent whose eluting strength during the analysis changes so that it constantly increases according to a predetermined program. With this technique, it is possible to achieve the elution of all components of complex mixtures in a relatively short period of time and their separation into components in the form of narrow peaks.

1.6.1. Adsorption liquid chromatography. Adsorption liquid chromatography, depending on the polarity of the stationary and mobile phases, is divided into normal phase (NPC) and reversed phase (RPC) chromatography. In NPC, a polar adsorbent and non-polar mobile phases are used; in OFC, a non-polar adsorbent and polar mobile phases are used, but in both cases, the choice of the mobile phase is often more important than the choice of the stationary one. The stationary phase must hold the substances to be separated. The mobile phase, i.e. the solvent, must provide different column capacities and efficient separations in a reasonable time.

As a stationary phase in adsorption liquid chromatography, polar and non-polar finely dispersed porous materials with a specific surface area of ​​more than 50 m 2 /g are used. Polar adsorbents (SiO 2 ,Al 2 O 3 , florisil, etc.) have weakly acidic groups on the surface, capable of retaining substances with basic properties. These adsorbents are mainly used for the separation of non-polar and medium polar compounds. Their drawback is high sensitivity to the water content in the eluents used. To eliminate this drawback, polar sorbents are treated with amines, diols, and other reagents, resulting in surface grafting of these reagents, surface modification, and a change in selectivity with respect to the analytes.

Non-polar adsorbents (graphitized soot, diatomite, kieselguhr) are non-selective to polar molecules. To obtain non-polar adsorbents, non-polar groups are often grafted onto the surface, for example, of silica gel, for example, alkylsilyl - SiR 3, where R - alkyl groups are C 2 - C 22.

The mobile phase should completely dissolve the analyzed sample, have a low viscosity (so that the diffusion coefficients are large enough), it is desirable that it is possible to separate the separated components from it. It must be inert to the materials of all parts of the chromatograph, safe, cheap, suitable for the detector.

The mobile phases used in liquid chromatography differ in their eluting strength. The eluting power of a solvent shows how many times the sorption energy of a given eluent on a given adsorbent is greater than the sorption energy of the eluent chosen as a standard, for example, n-heptane. Weak solvents are poorly adsorbed by the stationary phase, so the distribution coefficients of sorbed substances (sorbate) are high. Conversely, strong solvents adsorb well, so the sorbate partition coefficients are low. The stronger the solvent, the higher the solubility of the analyzed sample in it, the stronger the solvent-sorbate interaction.

To ensure high separation efficiency on the column, it is necessary to select a mobile phase that has the optimal polarity for the mixture to be separated under the selected separation conditions. Typically, the stationary phase is selected first, which has a polarity close to that of the components to be separated. Then the mobile phase is selected, ensuring that the capacitance coefficient k" turned out to be in the range from 2 to 5. If the polarity of the mobile phase is too close to the polarity of the stationary phase, the retention time of the components will be too short, and if the polarities of the mobile and stationary phases differ very much, the retention time becomes too long.

When selecting mobile phases, they are guided by the so-called eluotropic series, based on the use of polarity indices Snider R", which subdivides all solvents into strong (polar) and weak (weakly polar and non-polar). The polarity scale is based on the solubility of substances used as mobile phases in dioxane, nitromethane and ethanol.

Table 1.2 shows the values ​​of the polarity index and elution strength (with respect to SiO 2) of a number of solvents most commonly used in liquid chromatography as mobile phases. The short-wavelength transparency limits of these solvents are also indicated here, which facilitates the selection of conditions for detecting mixture components.

Table 1.2

Characteristics of solvents used in liquid chromatography

Solvent	Polarity index	Eluting power (SiO 2)	Shortwave Transparency Limit
Fluoralkane
Cyclohexane
n-Hexane
Carbon tetrachloride
Diisopropyl ether
diethyl ether
dichloromethane
Tetrahydrofuran
Chloroform
Acetic acid
Acetonitrile
Nitromethane

Liquid chromatography often uses not individual solvents, but their mixtures. Often, minor additions of another solvent, especially water, greatly increase the eluting strength of the eluent.

When separating multi-component mixtures, one mobile phase as eluent may not separate all sample components in a reasonable time. In this case, a stepwise or gradient elution method is used, in which increasingly stronger eluents are used sequentially during chromatography, which makes it possible to elute highly retained substances in less time.

In liquid chromatography, there are some empirical rules that are very useful when choosing an eluent:

 sorption of a compound, as a rule, increases with an increase in the number of double bonds and OH groups in it;

 sorption decreases in a number of organic compounds: acidsalcoholsaldehydesketonesestersunsaturated hydrocarbonssaturated hydrocarbons;

 to separate substances of different polarity and separate substances of different classes, normal-phase chromatography is used: the analyzed sample is dissolved and eluted with a non-polar eluent, compounds of different classes leave the column with a polar adsorbent for different times, while the retention time of compounds with different functional groups increases during the transition from non-polar compounds to weakly polar ones. For highly polar molecules, retention times are so long that analysis is not possible when using a non-polar eluent. To reduce the retention time of polar components, one passes to polar eluents;

 in the reversed-phase version, the stationary phase (non-polar adsorbent) adsorbs the non-polar component more strongly from polar eluents, for example, from water;

 By reducing the polarity of the eluent by adding a less polar solvent, the retention of the components can be reduced.

1.6.2. Partition liquid chromatography. In partition or liquid-liquid chromatography, the separation of the components of the analyzed sample is due to differences in the coefficients of their distribution between two liquid phases that do not mix with each other, one of which is stationary and is located on the surface or in the pores of a solid immovable carrier, and the second is mobile.

In terms of the nature of the interaction forces that determine the different distribution between the two phases of substances that differ in their chemical structure, partition chromatography is similar to adsorption chromatography, i.e., here the separation is also based on the difference in the forces of intermolecular interaction between the components of the analyzed sample with the stationary and mobile liquid phases.

Depending on the performance technique, partition chromatography, like adsorption chromatography, can be column or planar (paper or thin layer).

As solid carriers, substances are used that are indifferent to the mobile solvent and the components of the analyzed sample, but capable of retaining the stationary phase on the surface and in the pores. Most often, polar substances (cellulose, silica gel, starch) are used as carriers. They are applied to the stationary phase - a polar solvent, most often water or alcohol. In this case, less polar or non-polar substances (alcohols, amines, ketones, hydrocarbons, etc.) are used as mobile phases. This type of partition chromatography is called normal-phase. It is used to separate polar substances.

The second variant of partition chromatography differs in that non-polar carriers (rubber, PTFE, hydrophobized silica gel) are used as the stationary solid phase, non-polar solvents (hydrocarbons) as the stationary liquid phase, and polar solvents (alcohols, aldehydes) as the mobile liquid phase. , ketones, etc., often water). This variant of partition chromatography is called reverse phase and is used to separate non-polar substances.

To achieve optimal separation of the components of the analyzed sample, the selection of the mobile phase is very important. Solvents (mobile and stationary liquid phases) should be selected so that the distribution coefficients of the mixture components differ significantly. The liquid phases are subject to the following requirements:

1) the solvents used should dissolve the substances to be separated well, and their solubility in the stationary phase should be greater than in the mobile one;

2) the solvents used as mobile and stationary phases must be mutually saturated, i.e. the composition of the solvent must be constant during passage through the column;

3) the interaction of solvents used as the mobile phase with the stationary phase should be minimal.

Most often, in partition liquid chromatography, not individual substances are used as mobile liquid phases, but their mixtures in various ratios. This makes it possible to regulate the polarity of the mobile phase, change the ratio of the polarities of the mobile and stationary phases, and achieve optimal conditions for separating the components of a particular mixture being analyzed.

A significant disadvantage of this chromatographic method is the rather rapid washing off of the deposited stationary liquid phase from the carrier. To eliminate it, the solvent used as the mobile phase is saturated with the substance used as the stationary liquid phase, or the stationary liquid phase is stabilized by grafting it onto a carrier.

A variation of partition liquid chromatography is the widely used HPLC method.

The most common chromatographic systems are systems that have a modular assembly principle. Pumps, degassers, detectors, dispensers (autosamplers), column ovens, fraction collectors, chromatography system controls and recorders are available as separate modules. A wide range of modules allows you to flexibly solve various analytical problems, quickly change the system configuration if necessary, with minimal costs. At the same time, monomodular (integrated) LCs are also produced, the main advantage of which is the miniaturization of individual blocks and the compactness of the device.

Depending on the method of elution, liquid chromatographs are divided into isocratic and gradient.

Diagram of an isocratic chromatograph

The mobile phase from the container (1) through the inlet filter (9) is supplied by a high-pressure precision pump (2) to the sample input system (3) - a manual injector or an autosampler, where the sample is also injected. Further, through the in-line filter (8), the sample with the current of the mobile phase enters the separation element (elements) (4) - through the pre-column into the separation column. Then, the eluate enters the detector (5) and is removed into the drain tank (7). When the eluate flows through the measuring circuit of the detector, the chromatogram is registered and the data is transmitted to an analog recorder (recorder) (6) or another system for collecting and processing chromatographic data (integrator or computer). Depending on the design of the functional modules, the system can be controlled from the keyboard of the control module (usually a pump or system controller), from the keyboards of each of the system modules, or by a control program from a personal computer.

In the case of gradient elution, two fundamentally different types of liquid chromatographs are used. They differ in the point of formation of the mobile phase composition gradient.

Scheme of a gradient chromatograph with the formation of a gradient of the composition of the mobile phase on the low pressure line.

The mobile phase from the tanks (1) through the inlet filters (9) and the gradient programmer (10) is supplied by a high-pressure precision pump (2) to the sample injection system (3) - a manual injector or an autosampler, where the sample is also injected. The operation of the gradient programmer valves is controlled either by the system control module (pump or controller) or by a PC control program. Systems of this type form a binary, three-dimensional and four-dimensional gradient. The form of the gradient processing function depends on the specific control module or control program, as well as the functionality of the controlled and control modules. Further, through the in-line filter (8), the sample with the current of the mobile phase enters the separation element (elements) (4) - through the pre-column into the separation column. Then, the eluate enters the detector (5) and is removed to the drain tank (7). When the eluate flows through the measuring circuit of the detector, the chromatogram is registered and the data is transmitted to an analog recorder (recorder) (6) or another system for collecting and processing chromatographic data (integrator or computer). Depending on the design of the functional modules, the system can be controlled from the keyboard of the control module (usually a pump or system controller), or by a control program from a personal computer. In the case of controlling the control module, it is possible to independently control the detector from its own keyboard.

Despite the apparent attractiveness of such systems (they use only one high-pressure precision pump), these systems have a number of disadvantages, among which the main one, perhaps, is the severe need for thorough degassing of the mobile phase components even before the low-pressure mixer (gradient programmer chamber). It is carried out with the help of special flow degassers. Because of this fact, their cost becomes comparable to another type of gradient systems - systems with the formation of a gradient composition of the mobile phase on the high pressure line.

The fundamental difference between systems with the formation of a mobile phase gradient composition in the high pressure line is the mixing of components in the high pressure line, naturally, with this approach, the number of precision pumps is determined by the number of tanks for mixing the mobile phase. With this approach, the requirements for the thoroughness of the degassing of the components are significantly reduced.

Scheme of a gradient chromatograph with the formation of a gradient of the composition of the mobile phase on the high pressure line.

The mobile phase from the tanks (1) through the inlet filters (9) is supplied by precision high-pressure pumps (2 and 11) through a static or dynamic flow mixer (10) to the sample injection system (3) - a manual injector or an autosampler, where the sample is also injected. The operation of controlled pumps is controlled either by the system's control module (master pump or controller) or by a PC control program. In this case, all pumps are controlled. Systems of this type form a binary or three-dimensional gradient. The form of the gradient processing function depends on the specific control module or control program, as well as the functionality of the controlled and control modules. Further, through the in-line filter (8), the sample with the current of the mobile phase enters the separation element (elements) (4) - through the pre-column into the separation column. Then the eluate enters the detector (5) and is removed into the drain tank (7). When the eluate flows through the measuring circuit of the detector, the chromatogram is registered and the data is transmitted to an analog recorder (recorder) (6) or another system for collecting and processing chromatographic data (integrator or computer). Depending on the design of the functional modules, the system can be controlled from the keyboard of the control module (usually a pump or system controller), or by a control program from a personal computer. In the case of controlling the control module, it is possible to independently control the detector from its own keyboard.

The proposed schemes are rather simplified. Additional devices can be included in the systems - a column thermostat, post-column derivatization systems, sample preparation and concentration systems, a solvent recycler, membrane systems for suppressing background electrical conductivity (for ion chromatography), additional protective systems (filters, columns), etc. In the diagrams, the manometric modules are also not shown separately. As a rule, these devices are built into pump units. These units can combine multiple pumps, a pump with a gradient programmer, and a common system controller. The structure of the system depends on its configuration and each specific manufacturer.

Such a radical complication of the technical support of the chromatographic process leads to the emergence of a number of requirements for the properties of the mobile phase, which are absent in classical column and planar chromatography. The liquid phase must be suitable for detection (be transparent in a given region of the spectrum or have a low refractive index, a certain electrical conductivity or permittivity, etc.), inert to the materials of the parts of the chromatographic tract, not form gas bubbles in the pump valves and the detector cell, not have mechanical impurities.

In liquid chromatography many types of pumps are used. Low pressure LC often uses peristaltic pumps (Figure 1).

Fig.1 MasterFlex programmable peristaltic pump.

In HPLC, high pressure pumps are used to ensure the flow of the mobile phase through the column with the specified parameters.

The most important technical characteristics of HPLC pumps are: flow range; maximum working pressure; flow reproducibility; solvent supply pulsation range.

By the nature of the solvent supply, pumps can be of constant supply (flow) and constant pressure. Basically, in analytical work, a constant flow mode is used, when filling columns, a constant pressure mode is used.

According to the principle of operation, HPLC pumps are divided into syringe and on plunger reciprocating .

Syringe pumps

The main distinguishing feature of these pumps is their cyclical operation, and therefore the chromatographs in which these pumps are used also differ in cyclical operation.

Rice. 2. Principal arrangement of a syringe pump for HPLC.

Rice. 2A. Syringe pump.

The control unit BU supplies voltage to the motor D, which determines the speed and direction of its rotation. The rotation of the engine with the help of the gearbox P is converted into the movement of the piston P inside the cylinder D. The pump is operated in 2 cycles. In the filling cycle, valve K2 is closed, K1 is open, the solvent flows from the reservoir to cylinder C. In the supply mode, valve K1 is closed, and through valve K2 the mobile phase enters the dosing device.

Pumps of this type are characterized by the almost complete absence of pulsations in the flow of the mobile phase during operation.

Pump Disadvantages:

a) high consumption of time and solvent for washing when changing the solvent;

b) the volume of PF limited by the volume of the syringe, and hence the limited separation time;

c) suspension of separation during filling of the pump;

d) large dimensions and weight while providing high flow and pressure (you need a powerful engine and a large piston force with its large area).

Plunger reciprocating pumps.

Rice. 3. Principal device of a plunger pump.

Operating principle.

The engine D through the gearbox R reciprocates the plunger P, moving in the working head of the pump. Valves K1 and K2 open when the pump is in the suction and delivery phase respectively. The amount of volumetric feed is determined by three parameters: plunger diameter (usually 3.13; 5.0; 7.0 mm), its amplitude (12-18 mm) and frequency (which depends on the speed of rotation of the engine and gearbox).

Pumps of this type provide a constant volumetric flow of the mobile phase for a long time. Maximum working pressure 300-500 atm, flow rate 0.01-10 ml/min. Volume feed repeatability -0.5%. The main drawback is that the solvent is fed into the system in the form of a series of successive pulses, so there are pressure and flow pulsations (Fig. 4). This is the main reason for the increased noise and desensitization of almost all detectors used in LC, especially electrochemical.

Fig.4. Plunger pump pulsation.

Ways to deal with pulsations.

1. Application of damping devices.

These are spiral tubes of a special profile made of stainless steel, included in series or in parallel in the system between the pump and the dispenser.

Rice. 5. Spiral damper.

The damper is untwisted with an increase in pressure in it (acceleration of the pump). When the pressure drops, it twists, its volume decreases, it squeezes out part of the solvent, maintaining a constant flow rate and reducing pulsations. Such a damper works well at a pressure of 50 atm and above.

At a pressure of 5-30 atm, it smooths out pulsations better air damper, made from a column (Fig. 6.). The air in the plugged column (6x200 mm) is compressed and the pulsations are extinguished. The air in it dissolves in 24 hours.

Rice. 6. Air damper.

2. The use of electronic devices.

When using an electronic pressure transducer, the readings from the transducer can be used to control the operation of the pump. When the pressure drops, the engine speed increases and compensates for the decrease in pressure. It is also possible to compensate for leaks in the valves and partially in the cuffs. The use of an electronic damper (BPZh-80, KhPZh-1, etc.) makes it possible to reduce pressure pulsations to 1 atm at a pressure of 100-150 kgf/cm2.

1.6.3. Ion-exchange, ion, ion-pair chromatography. The methods of ion-exchange, ion and ion-pair chromatography are based on the dynamic process of replacing ions associated with the stationary phase with eluent ions entering the column. The main goal of the chromatographic process is the separation of inorganic or organic ions of the same sign. Retention in these types of chromatography is determined by the change in the free energy of the ion exchange reaction. The ratio of the concentrations of exchanging ions in the solution and in the sorbent phase is characterized by ion-exchange equilibrium. Ion exchange consists in the fact that some substances (ion exchangers), when immersed in an electrolyte solution, absorb cations or anions from it, releasing an equivalent amount of other ions with a charge of the same sign into the solution. Between the cation exchanger and the solution there is an exchange of cations, between the anion exchanger and the solution there is an exchange of anions.

Cation exchangers are most often specially synthesized insoluble polymeric substances containing acidic ionogenic groups in their structure: -SO 3 H; –COOH; -OH; –PO 3 H 2 ; –AsO 3 H 2 .

The chemical formulas of cation exchangers can be schematically represented as R-SO 3 H; R-SO 3 Na. In the first case, the cation exchanger is in the H-form, in the second, in the Na-form. R is a polymer matrix.

Cation exchange reactions are written as ordinary heterogeneous chemical reactions:

RN + Na + RNa + H +

Anion exchangers contain basic ionogenic groups in their structure: –N(CH 3) 3 + ; =NH 2 + ; =NH + etc. Their chemical formulas can be represented as RNH 3 OH and RNH 3 Cl or ROH, RCl. In the first case, the anion exchanger is in the OH form, in the second, in the Cl form. The anion exchange reaction can be written as follows:

R–OH+Cl – RCl+OH –

Amphoteric ion exchangers are known that contain both acidic and basic groups in their structure. Ion exchangers that have in their composition the same type (for example, SO 3 H) acidic (basic) groups are called monofunctional; ion exchangers containing heterogeneous (for example, - SO 3 H, - OH) acidic (basic) groups - polyfunctional.

Monofunctional ion exchangers are obtained by a polymerization reaction. The polycondensation reaction makes it possible to obtain polyfunctional ion exchangers. In order for the resulting ion exchangers to have sufficiently high performance characteristics, they must be insoluble, but swellable in the appropriate solvent and have a sufficiently large number of ionogenic groups capable of exchanging with the ionogenic groups of the analyzed sample. This can be achieved if the resulting polymer chains are sufficiently branched and connected to each other by "cross-linking bridges". For example, in the preparation of polymerization type cation exchangers based on styrene, divinylbenzene is most often used as a crosslinking agent, the introduction of which in an amount of up to 16% ensures the production of ion exchangers with various degrees of swelling and, therefore, allows one to control the ion exchanger porosity. The degree of swelling of the ion exchanger, expressed in milliliters/gram, is the volume of 1 g of air-dry ion exchanger packed into the column.

The ion exchanger absorbs, as a rule, one of the counterions - ions in the mobile phase, i.e. it exhibits a certain selectivity. Series of affinity, or selectivity, of ions with respect to ion exchangers of various types have been experimentally established. For example, at low solution concentrations on strongly acidic cation exchangers, ions with the same charge are sorbed in the following sequence:

Li +  Na +  K +  Rb +  Cs +

Mg 2+  Ca 2+  Sr 2+  Ba 2+ .

For ions with different charges, the sorbability increases with increasing charge:

Na+Ca2+

However, changing the conditions for carrying out the ion exchange reaction can lead to series inversion. Affinity series have also been established for anion exchangers. For example, the sorbability of anions on strongly basic anion exchangers increases in the series:

F -  OH -  Cl -  Br -  NO 3 -  J -  SCN -  ClO 4 - .

Ion exchangers containing strongly acidic or strongly basic groups in their structure enter into ion exchange reactions with any ions in solution that have charges of the same sign as the sign of the counterion. Such ion exchangers are called universal.

The process of ion exchange between the analyte and the ion exchanger can be carried out in one of three ways: static, dynamic (ion exchange filter method) and chromatographic.

Static method ion exchange is that a sample of the ion exchanger is brought into contact with a certain volume of solution and stirred or shaken for a certain time until equilibrium is established. This is a fast and simple method of ion exchange, used to concentrate ions from dilute solutions, remove unwanted impurities, but it does not provide complete absorption of ions, since ion exchange is a non-equilibrium process, and therefore does not guarantee complete separation of ions.

When carrying out ion exchange in a dynamic way a solution is passed through the column with an ion exchanger, which, as it moves along the column, comes into contact with new granules of the ion exchanger. This process provides a more complete exchange than the static method, since the exchange products are removed by the solution flow. They can concentrate ions from dilute solutions and separate ions that differ greatly in properties, for example, differently charged ions (separate cations from anions), but the separation of ions of the same charge sign is almost impossible. Quantitative separation of such ions is possible only with repeated repetition of sorption-desorption elementary acts under dynamic conditions, i.e. chromatographic method . When working with this method, high layers of ion exchanger are used, and the mixture to be separated is introduced into this layer in an amount much less than the capacity of the column, due to which repeated repetition of elementary acts of ion exchange is ensured.

According to the analysis technique, ion-exchange chromatography is similar to molecular chromatography and can be carried out according to the eluent (developing), frontal, and displacement options. The difference between molecular and ion-exchange chromatography is that in molecular chromatography, the separated components of the mixture are eluted from the column with a pure eluent, while in ion-exchange chromatography, an electrolyte solution is used as an eluent. In this case, the exchanged ion of the eluent should be sorbed less selectively than any of the ions of the mixture being separated.

When carrying out developing ion-exchange chromatography, which is used most often, the column filled with an ion exchanger is first washed with an electrolyte solution until the ion exchanger completely replaces all its ions with the ions contained in the eluent. Then, a small volume of the analyte solution is introduced into the column, which contains separable ions in an amount of about 1% of the capacity of the ion exchanger. Next, the column is washed with an eluent solution, taking fractions of the eluate and analyzing them.

A mixture of Cl - , Br - , J - ions can be separated on a highly basic anion exchange resin (cross-linked polystyrene containing groups of quaternary ammonium bases N (CH 3) 3 +), for example, AB-17, which has a range of selectivity (selectivity): NO 3 - Cl – Br – J – . As a result, a NaNO 3 solution is used as an eluent. First, this solution is passed through the ion exchanger until it is completely saturated with NO 3 - ions. When the mixture to be separated is introduced into the column, ions Cl – , Br – , J – are absorbed by the anion exchanger, displacing NO 3 – ions. During subsequent washing of the column with NaNO 3 solution, the ions Cl – , Br – , J – in the upper layers of the anion exchanger are gradually again replaced by NO 3 – ions. Cl - ions will be displaced the fastest of all, J - ions will stay in the column the longest. The difference in the selectivity of the ion exchanger to the ions of the mixture leads to the fact that separate zones of adsorbed ions Cl - , Br - and J - are formed in the column, moving along the column at different speeds. As you move along the column, the distance between zones increases. In each zone there is only one of the anions of the mixture being separated and the anion of the eluent, in the interval between the zones there is only an anion of the eluent. Thus, fractions containing individual components of the mixture to be separated will appear in the eluent at the column outlet.

To solve practical problems, the conditions for ion separation are varied by selecting a suitable mobile phase (composition, concentration, pH, ionic strength) or by changing the porosity of the polymer matrix of the ion exchanger, i.e., the number of interchain bonds in the matrix, and creating ion exchange sieves that are permeable to some ions and capable of their exchange and impenetrable to others. It is also possible to change the nature and mutual arrangement of ionogenic groups, as well as to obtain sorbents capable of selective chemical reactions due to complex formation. High selectivity is possessed, for example, by complexing ion exchangers containing in their structure chelating groups of organic reagents dimethylglyoxime, dithizone, 8-hydroxyquinoline, etc., as well as crown ethers.

The greatest application in ion exchange, ion, and ion pair chromatography is found in synthetic macro- and micronet organic ion exchangers having a large exchange capacity (3–7 mmol/g), as well as inorganic ion-exchange materials. Micromesh ion exchangers are capable of exchanging ions only in the swollen state, while macromesh ones are capable of exchanging ions in the swollen and unswollen states. Another structural type of ion exchangers are surface-film ion exchangers, the solid core of which is made of a non-porous copolymer of styrene and divinylbenzene, glass or silica gel and is surrounded by a thin film of the ion exchanger. The total diameter of such a particle is about 40 µm, and the thickness of the ion exchanger film is 1 µm. The disadvantage of such ion exchangers is the relatively large particle diameter and low exchange capacity due to the low specific surface area, as a result of which it is necessary to work with small samples and, accordingly, use highly sensitive detectors. In addition, such ion exchangers are quickly poisoned and are not capable of regeneration.

In high-performance ion-exchange and ion chromatography, volumetric-porous polystyrene ion exchangers, volumetric-porous silicas with a granule diameter of about 10 μm, and practically non-swelling surface-porous and surface-modified copolymers of styrene and divinylbenzene with ionogenic sulfo and amino groups are used.

In ion-pair chromatography, "brush" sorbents are used - silica gels with grafted reversed phases C 2, C 8, C 18, which are easily converted into a cation exchanger upon absorption of ionic surfactants from the mobile phase, for example, alkyl sulfates or salts of quaternary ammonium bases.

When carrying out chromatographic separation using ion exchangers, aqueous solutions of salts are most often used as the mobile phase. This is due to the fact that water has excellent dissolving and ionizing properties, due to which the molecules of the analyzed sample instantly dissociate into ions, the ion exchange groups of the ion exchanger are hydrated and also go into a fully or partially dissociated form. This ensures a rapid exchange of counterions. The eluting strength of the mobile phase is mainly influenced by pH, ionic strength, the nature of the buffer solution, the content of the organic solvent or surfactant (ion-pair chromatography).

The pH value is chosen depending on the nature of the ionogenic groups, the ions to be separated, and the matrix. It is possible to work with strongly acidic and strongly basic ion exchangers at pH = 2–12, with weakly acidic ones at pH = 5–12, and with weakly basic ones at pH = 2–6. Silica-based sorbents cannot be used at pH 9. The ionic strength of the mobile phase affects the capacity of the ion exchanger. With an increase in ionic strength, the sorption of ions usually decreases, since the eluting strength of the mobile phase increases. Therefore, at the beginning of separation, the mobile phase should have a low ionic strength (0.05–0.1), and the final value of this characteristic should not exceed 2. In gradient elution, buffers with increasing ionic strength are often used.

For the selective elution of ions absorbed by the ion exchanger, you can use water, buffer solutions (phosphate, acetate, borate, hydrocarbonate, etc.) with a certain pH value and ionic strength, solutions of mineral (hydrochloric, nitrogen, sulfuric, phosphoric) and organic (phenol, citric, lactic, tartaric, oxalic, EDTA) acids. The choice of eluent is facilitated by the fact that the limiting coefficients of distribution of most elements between aqueous (water-organic) solutions of many complexants and standard-type ion exchangers are determined and presented in tables.

1.6.4. Size exclusion chromatography. Size exclusion chromatography is a type of liquid chromatography in which the separation of components is based on the distribution of molecules according to their size between the solvent in the pores of the sorbent and the solvent flowing between its particles. During separation, small molecules enter the polymer network, in the pores of which the solvent serves as a stationary phase, and are retained there. Large molecules cannot penetrate the polymer network and are washed out of the column by the mobile phase. The largest molecules are eluted first, then the medium ones, and finally the small ones.

Size exclusion chromatography is subdivided into gel permeation and gel filtration. In gel permeation chromatography, separation occurs on polymers that swell in organic solvents. The gel filtration version of size exclusion chromatography involves the use of water-swellable polymers as stationary phases.

The retention time of the components of the analyzed sample in the size exclusion column depends on the size of their molecules and diffusion into the pores of the sorbent, as well as on the size of the pores of the stationary phase.

In this type of liquid chromatography, the partition coefficient D for the smallest molecules of the analyzed sample, which move in the chromatographic column at the lowest speed, penetrating into the grid of the stationary phase, it is equal to 1, since the mobile phase and the solvent in the pores of the stationary phase have the same composition. In this case, the main equation of column chromatography takes the form

Large molecules that do not enter the pores of the stationary phase are eluted from the column along with the mobile phase. For them D= 0, a V R =V m. Such a range of distribution coefficient values ​​(from 0 to 1) is typical only for size exclusion chromatography.

All molecules of the analyzed multicomponent substance should be washed out of the column by passing a small volume of solvent from V m before V m +V s and the separation is completed before the solvent peak exits. Therefore, in this type of chromatography, it is necessary to use sufficiently long columns with a large free volume. V m and a large number of pores in the sorbent.

The resolution of chromatographic peaks in size exclusion separations can be improved by using gradient elution with mixed solvents.

Each sorbent used in size exclusion chromatography is characterized by a certain pore volume and, therefore, has a certain area of ​​​​separable molecular weights and a certain calibration curve. In this case, the calibration curve characterizing the dependence of the retained volume on the molecular weight or size of the molecules, as a rule, has a complex form.

Stationary phases in size exclusion chromatography are selected based on specific analytical tasks. Initially, it is established which solvent system can be used for analysis (aqueous or water-organic). Depending on this, the type of sorbent is determined. If water-soluble samples are to be separated, for example water-swellable cross-linked dextrans (Sephadex) or polyacrylamides (Biogel P) are used as stationary phases. The separation of substances soluble in organic solvents can be carried out on polystyrenes with various degrees of crosslinking, which swell in organic solvents (styrogel, poragel, biobid C). Such swollen gels are generally not pressure stable and allow very low mobile phase flow rates, which increases analysis time. To implement a highly efficient version of size exclusion chromatography, it is necessary to use stationary phases with rigid matrices - silica gels, the disadvantage of which - high adsorption activity - is eliminated by silanization of the surface or selection of an eluent of the appropriate polarity.

Substances that can be used as mobile phases in size exclusion chromatography are:

 completely dissolve the analyzed sample;

 wet the sorbent well;

 counteract adsorption of sample components on the sorbent;

 have low viscosity and toxicity.

1.6.5. Planar chromatography. Planar chromatography includes thin layer and paper chromatography. These types of liquid chromatography are simple in technique, express, do not require expensive equipment, which is their undeniable advantage.

The separation of a mixture of substances by these methods can be performed using various chromatographic systems. Therefore, adsorption, distribution, normal and reversed phase, ion-exchange, etc., paper and thin-layer chromatography are distinguished. At present, thin layer chromatography is the most widely used.

Paper and thin layer chromatography are similar in technique. Cellulose paper fiber is used as a stationary phase in paper chromatography, in thin-layer chromatography - various sorbents (Al 2 O 3 , silica gel, etc.) deposited in a uniform thin (100-300 μm) layer on a glass, metal or plastic substrate (carrier) . The adsorbent layer on the support may or may not be fixed.

Chromatographic separation in planar methods, as well as on a column, is due to the transfer of the components of the analyte by the mobile phase along the layer of the stationary phase at different rates in accordance with the distribution coefficients of the substances to be separated. In both cases, liquid-solid sorbent chromatographic systems (adsorption separation mechanism), liquid-liquid-solid carrier (distribution, ion-exchange and other mechanisms) are used.

Various solvents or their mixtures, organic or inorganic acids are used as mobile phases.

Practical obtaining planar chromatograms consists in the following.

On a strip of chromatographic paper or on a thin layer of sorbent, a starting line is marked with a pencil at a distance of 1 cm from the bottom edge of the strip or plate. A micropipette is used to apply a sample to the start line in the form of a spot with a diameter of not more than 2–3 mm. Then the edge of the strip or plate is lowered into the vessel with the mobile phase, located in a sealed chamber. As the mobile phase rises along the strip or plate and the multiple elementary acts of sorption-desorption, distribution between two liquid phases, ion exchange, etc., which are common in chromatography, occur, the components of the analyzed mixture are separated. The process is usually continued until the solvent has passed from the start line 10 cm. After that, the strip or plate is removed from the chamber and dried. If the components of the analyte are colored, they give the corresponding color spots on the chromatogram. To detect unstained components of the analyte, the chromatogram must be developed. The development of a chromatogram and the detection of sample components can be carried out by various methods and depend on the composition of the analyzed mixtures. The manifestation can be done:

-Using UV light. The method is applicable for the detection of substances capable of emitting their own radiation (luminescence) in the visible wavelength range under the action of UV radiation;

 by means of reagents-developers. For example, the presence of amino acids in the analyzed mixture can be detected using ninhydrin. The dried chromatogram is immersed in a 0.2% solution of ninhydrin in acetone, then dried. The spots corresponding to the various components of the mixture acquire a visual and, as a rule, specific color for each substance;

- using iodine. In this case, the detected chromatogram is introduced into a vessel, at the bottom of which there are iodine crystals. Iodine vapors are adsorbed on the spots more strongly, due to which the spots are visualized. Iodine is a non-specific developer reagent. Using specific reagents, it is possible not only to determine the number of components of a mixture, but also to identify the separated substances by the color of the spots.

Paper and thin layer chromatography is most often carried out in the so-called ascending variant described above. Quite often, to improve the quality of chromatograms, it is necessary to use more complex variants of planar chromatography, for example, top-down, circular, two-dimensional. In paper or thin layer down chromatography, the analyte is applied to the starting line of the plate or paper strip at the top, and the eluent is fed from the top instead of the bottom. The positive effect of improved separation is due to the contribution of the gravity forces of the components to the separation process.

Both upstream and downstream chromatography can be performed in one and two-dimensional versions. In contrast to the one-dimensional flat-bed separation process described above, in two-dimensional chromatographic separation, the separation of the analyzed sample is first carried out in one solvent, then separation is carried out in the direction perpendicular to the first, using another solvent, rotating the first chromatogram by 90 ° C.

In circular chromatography, the analyte is applied as a drop in the middle of a plate or sheet of chromatographic paper. One or more solvents are also added dropwise here. This leads to the fact that the resulting chromatogram is a set of radial spots.

The position of the spots (zones) that form the separated components of the analyte on a flat chromatogram is characterized by the values ​​of the relative speed of movement of the components in a thin layer R fi. Experimental value R fi defined as the ratio of distance L i, passed i-th component, to the distance L passed by the solvent from the starting line to the front line (Fig. 1.10):

Value R fi depends on the nature of the relevant component of the analyzed sample, the nature of the stationary phase, its thickness, the nature and quality of the mobile phase, the method of application of the sample and other factors, but always R fi 1.

Value R fi is actually identical to the retention time of a substance or its retention volume, which characterizes the rate of passage of a substance through a chromatographic column, and can be used for qualitative identification of the components of the analyzed sample, and the spot diameter is identical to the height or area of ​​the chromatographic peak and, therefore, to some extent reflects the quantitative content of the substance.

Quantitative determination of the composition of the analyzed sample in the simplest case can be assessed visually by the intensity of the intrinsic color of the spots or the intensity of the fluorescent glow of the spots obtained during UV detection. For these purposes, the elution of chromatographic spots is widely used. In this case, the spot obtained on the chromatogram is carefully cut out or scraped off, treated with a suitable solvent, and the resulting solution is examined by the appropriate physicochemical method. You can also use the gravimetric method, in which the corresponding spot is cut out from the chromatogram and weighed. The amount of substance is determined by the difference in the weights of clean paper of the same area and paper with the substance.

Paper (BH ) and thin layer chromatography (TLC ) according to the separation mechanism belong to partition chromatography . In the HD method, the carrier is a special chromatographic paper with certain properties. Stationary phase is water adsorbed on the surface and pores of paper (up to 20%), mobile - organic solvent, miscible or immiscible with water, water or electrolyte solutions.

Mechanism quite complicated on paper. In the stationary phase, the substance can be retained not only due to dissolution in the water adsorbed by the paper, but also be adsorbed cellulose directly. Drawn on paper shared components pass into the mobile phase and move through the capillaries of the paper at different speeds in accordance with interfacial distribution coefficient each of them. At the beginning chromatography some of the substance from the paper passes into mobile phase and move on. When the organic solvent reaches the area of ​​the paper that does not contain the solute, redistribution : from the organic phase, the substance passes into the aqueous phase, sorbed on paper. Since the components have different affinity for the sorbent , when the eluent moves, separation occurs: some substances are delayed at the beginning of the path, others move further. Here are combined thermodynamic (establishment of an equilibrium distribution of substances between the phases) and kinetic (moving components at different speeds) separation aspects. As a result, each component is concentrated on a specific area of ​​the paper sheet: zones of individual components on the chromatogram . The use of chromatography on paper has a number of significant disadvantages: the dependence of the separation process on the composition and properties of the paper, the change in the water content in the pores of the paper with changes in storage conditions, a very low chromatography speed (up to several days), and low reproducibility of the results. These shortcomings seriously affect the spread of paper chromatography as a chromatographic method.

AT TLC method the process of separating a mixture of substances is carried out in a thin layer sorbent deposited on an inert solid substrate, and provided by the movement mobile phase (solvent) through the sorbent under the action of capillary forces . Byseparation mechanism distinguish partition, adsorption and ion exchange chromatography . The separation of the components occurs in these cases either as a result of their different distribution coefficient between the two liquid phases ( partition chromatography ), or due to different adsorbability of compounds by the sorbent ( adsorption chromatography ). The adsorption method is based on different degrees of sorption-desorption of the separated components on the stationary phase. Adsorption carried out at the expense van der Waals forces , which is the basis physical adsorption , polymolecular (formation of several adsorbate layers on the surface of the adsorbent) and chemisorption (chemical interaction of adsorbent and adsorbate).

In the case of using such sorbents for TLC as alumina or silica gel play a role in separation distribution , and adsorption on the developed active surface of the sorbent (150–750 m2/g). Distribution components of the mixture occurs between water on the surface of the carrier (such adsorbents , as alumina , starch , cellulose , diatomaceous earth - and water form stationary phase ), and the solvent moving through this stationary phase ( mobile phase ). The component of the mixture that is more readily soluble in water moves more slowly than the one that is more readily soluble in the mobile phase.

Adsorption manifested in the fact that between carrier , for example, aluminum oxide, and the components of the mixture are set adsorption equilibria - for each component its own, the result of which is different travel speed mixture components. Two extreme cases can be distinguished:

a) the concentration of the substance on the adsorbent is zero. The substance completely dissolves in the mobile phase and is carried away by it (moves along with solvent front ).

b) the substance is adsorbed completely, does not interact with the solvent and remains at the start.

In practice, with the skillful selection of solvent and adsorbent distribution compounds are located between these extreme cases, and the substance gradually carried over from one sorbent layer to another due to simultaneously occurring processes sorption and desorption .

The solvent passing through the sorbent is called eluent , the process of moving a substance together with an eluent  elution . As the liquid moves on the plate, the mixture of substances separates due to the action of forces adsorption , distribution , ion exchange or a combination of all of these factors. As a result, separate chromatographic zones mixture components, i.e. it turns out chromatogram .

Correct selection sorbent and eluent determines the efficiency of mixture separation. The mobility of the test substance depends on its affinity for the sorbent and eluting force (polarity) of the eluent. As the polarity of the compound increases, so does its affinity for the polar sorbent. By increasing the degree of adsorption silica gel organic compounds are arranged in a row: hydrocarbons<алкилгалогенидыарены<нитросоединения<простые эфиры <сложные эфиры<альдегиды<спирты<амины<карбоновые кислоты. В свою очередь for silica gel eluents can be arranged in ascending order of "polarity" ( eluting power ) and form a series of solvents ( eluotropic series ) in accordance with experimental data: alkanes> benzene> chloroform> diethyl ether> ethyl acetate> alcohols С 2 -С 4> water> acetone> acetic acid> methanol. Thus, a polar compound, alcohol, is adsorbed quite strongly on silica gel and, therefore, moves weakly under the action of such a nonpolar solvent as hexane, and remains near the start line. In turn, the non-polar aromatic hydrocarbon biphenyl is noticeably more mobile in hexane, but even here, to achieve R f about 0.5, a more polar aprotic eluent, methylene chloride, is needed. eluent strength regulate using mixtures of solvents - neighbors in eluotropic series with different polarity.

Currently, TLC mainly uses the following sorbents : for separation lipophilic substances  silica gel , alumina , acetylated cellulose , polyamides ; to separate hydrophilic substances  cellulose , cellulose ion exchangers , diatomaceous earth , polyamides . The most important characteristic of a sorbent is its activity , i.e. ability absorb (hold) the components of the mixture to be separated. Abroad, a number of firms produce silica gel , diatomaceous earth and alumina with the addition of 5% gypsum, which is used to fix the sorbent layer in the self-manufacturing of plates.

The most common sorbent is silica gel - hydrated silicic acid, formed by the action of mineral acids on Na 2 SiO 3 and drying of the resulting sol. After grinding the sol, a fraction of a certain grain size is used (indicated on the plate, usually 5-20 microns). silica gel is an polar sorbent with OH groups as active sites. It easily sorbs water on the surface and forms hydrogen bonds.

Alumina is a weakly basic adsorbent and is used mainly for the separation of weakly basic and neutral compounds. The disadvantage of plates on aluminum oxide is the obligatory activation of the surface before use in a drying cabinet at a high temperature (100-150 o C) and the low adsorption capacity of the layer compared to silica gel.

diatomaceous earth - adsorbent obtained from natural minerals - diatomaceous earths. The sorbent has hydrophilic properties and a lower adsorption capacity of the layer compared to silica gel.

Cellulose: cellulose-coated thin-layer plates are very effective in separating complex organic molecules. The adsorbent is mainly cellulose balls with a diameter of up to 50 microns, fixed on the carrier with starch. As in paper chromatography, the rise of the solvent front is very slow.

Chromatographic analysis is carried out on industrial plates of Czech production " Silufol » (« Silufol "") of aluminum foil, sometimes reinforced with cardboard, and " Siluplast » made of plastic coated with a layer of sorbents - silica gel LS 5-40 with starch or gypsum as a binder (up to 10%), or aluminum oxide with or without fluorescent indicators. Records " Silufol » have a high elution rate, however, they are characterized by low separating power and low sensitivity. During storage, they are sensitive to conditions (humidity, temperature, aggressive media, etc.). Individual firms supply chromatographic plates with a layer of sorbent of different (usually up to 0.25 mm), but strictly constant thickness (silica gel, cellulose, ion-exchange resin), on glass and substrates made of aluminum foil, plastic, impregnated fiberglass.

Plates « Sorbfil » (TU 26-11-17-89) are produced in Russia on a polymer base (polyethylene terephthalate, grade P) or aluminum substrate (grade AF) with a working layer applied microfractionated silica gel sorbent grades STX-1A and STX-1VE (produced in the USSR as fractionated silica gel KSKG) with a thickness of 90-120 microns (up to 200 microns), fixed with a special binder - silicasol . When using silicic acid (silicazole) sol as a binder, which transforms into silica gel after heating, the resulting TLC plates consist of two components: a silica gel layer and a substrate. The thickness uniformity of the sorbent layer on one plate is ±5 µm. Designation example: "Sorbfil-PTSKh-AF-V-UF (10x10)" - high-performance TLC plates on an aluminum substrate, with a phosphor, 10x10 cm.

If a glass substrate (grade C) is used, then such plates are reusable and chemically resistant. Their chemical resistance is determined by the chemical resistance of silica gel. As a result, TLC plates can be repeatedly treated with aggressive reagents, for example, with a hot chromium mixture, which removes restrictions on the use of correlating reagents for spot detection and sorbent modification, and allows multiple (up to 30 times or more) plate regeneration with a chromium mixture. Glass plates can be cut to the desired size. The mechanical strength of the sorbent layer can be controlled, providing, on the one hand, transportation and multiple processing of the plates and, on the other hand, the possibility of extracting adsorbent layers with separated substances for subsequent washing out of individual compounds from the sorbent and their further study by instrumental methods (IR and UV spectrometry). , X-ray diffraction methods, NMR, etc.).

The plates differ in the size of the fractions (particle distribution) of the silica gel that makes up the layer. On analytical plates (grade A) the fraction is 5-17 microns, on highly efficient (grade B) - 8-12 microns. A narrower distribution increases the efficiency of the plates, i.e. the spots of the substances to be separated become more compact (smaller in size) and therefore are better separated when the eluent front passes a shorter distance. On Russian wafers, analytical and high-performance layers do not differ very much, in contrast to wafers from Merck (Germany). High performance plates should be used if substances cannot be separated on analytical plates. Plates of all modifications are produced with a phosphor (UV grade) with 254 nm excitation. The shelf life is not limited, the plates " Sorbfil » widely tested in the analysis of amino acid derivatives, pesticides, lipids, antibiotics.

The TLC method is carried out qualitative identification components. quantitation for TLC is also possible, this requires applying the exact amount of substance and additional densitometric studies with a clear fixation of the intensity of the spots. The most common is semiquantitative method . It is based on visual comparison the size and intensity of the spot of a component with the corresponding characteristics of a series of spots of the same substance of different concentrations ( standard reference solutions ). When using a sample in the amount of 1–5 μg, such a simple method provides an accuracy of determining the component content of about 5–10%. Often, in order to determine the components in a sample, it is necessary to carry out sample preparation to obtain a mixture containing the analyzed compounds. Sample preparation is based on the extraction of drugs from the sample with organic solvents ( n-hexane, petroleum ether, diethyl ether, chloroform), purification of the extract and subsequent chromatography in a thin layer of alumina or silica gel.

There are several variants of TLC and BC, differing in the way solvent supply . Depending on the direction of movement of the mobile phase, there are:

a)ascending chromatography  the mobile phase is poured onto the bottom of the separation chamber, the paper (plate) is placed vertically;

b)descending chromatography  the mobile phase is fed from above and moves down along the sorbent layer of the plate or paper;

in)radial chromatography  horizontal advance of the solvent front: the mobile phase is brought to the center of the paper disk (plate), where the mixture to be separated is deposited.

The most common is upward elution (chromatography). Front eluent while moving from bottom to top. The choice of solvent (mobile phase) is determined by the nature of the sorbent and the properties of the substances to be separated.

Chromatographic separation BC and TLC methods are carried out in separation chamber with screwed lid. A quantitative measure of the rate of transfer of a substance using a specific adsorbent and solvent is R value f (from English. retention factor – delay coefficient, this parameter is analogous to retention time). Position zones of the chromatographed component set to size coefficient R f equal to the ratio of the velocity of its zone to the velocity of the solvent front. Value R f is always less than unity and does not depend on the length of the chromatogram. By the amount R f influenced by various factors. So, at low temperatures, substances move more slowly; solvent contamination, adsorbent inhomogeneity, foreign ions in the analyzed solution can change the value R f up to 10%. In the selected system, the analytes must have different values R f and distributed over the entire length of the chromatogram. It is desirable that the values R f lay in the range of 0.05-0.85.

In practice, the value R f calculated as the ratio of the distance l traveled by the substance to the distance L passed by the solvent:

R f = l/l (6.1 )

Usually for calculation choose spot center (Fig. 1). Value R f depends on many factors such as chromatographic paper (its porosity, density, thickness, degree of hydration) and sorbent (grain size, nature of groups on the surface, layer thickness, its moisture content, nature of the substance, composition of the mobile phase), experimental conditions (temperature, chromatography time, etc.). With the constancy of all chromatography parameters, the value R f determined only by the individual properties of each component.

Rice. 1. Determination of values ​​on the chromatogram RF for components BUT and AT,

their degree of separation Rs and the number of theoretical plates N .

The efficiency of BC and TLC also depends on selectivity and sensitivity reactions used to detect the components of the analyzed mixture. Usually, reagents are used that form colored compounds with the components to be determined - developers. For a more reliable identification of shared components apply " witnesses » -solutions standard substances (in the same solvent as the sample) that are expected to be present in the sample. Standard Substance applied to the starting line next to the analyzed sample and chromatographed under the same conditions. In practice, a relative value is often used:

R f rel = R f x / R f stand (6.2)

where R f stand also calculated by formula (6.1). Efficiency chromatographic separation characterize number of equivalent theoretical plates and them height . Thus, in the TLC method, the number of equivalent theoretical plates N BUT for component BUT the mixture to be separated is calculated by the formula:

N A = 16 (l OA / a (A )) 2 (6.3)

Values l OA and a (BUT ) determined as shown in Fig. 6.1. Then the height of the equivalent theoretical plate H BUT is:

H A = l OA /N = a (A ) 2 / 16 l OA . (6.4)

Separation is practically possible if R f (BUT)  R f (AT)  0,1 .

To characterize the separation of two components BUT and AT use degree (criterion) of division Rs :

Rs =  l / (a (A) / 2 + a (B) / 2)= 2  l / (a (A) + a (B)) (6.5)

where  l  distance between component spot centers BUT and AT;

a (BUT) and a (AT)  spot diameters BUT and AT on the chromatogram (Fig. 6.1). The more Rs , the more clearly the spots of the components are separated BUT and AT on the chromatogram. Conditions chromatography are chosen so that the value Rs different from zero and one, the optimal value Rs is 0.3  0.7. For rate separation selectivity two components BUT and AT use separation factor α :

α = l B / l A (6.6)

If α = 1, then the components BUT and AT are not separated.

In high performance liquid chromatography (HPLC), the nature of the processes occurring in the chromatographic column is generally identical to the processes in gas chromatography. The difference is only in the use of a liquid as a stationary phase. Due to the high density of liquid mobile phases and the high resistance of the columns, gas and liquid chromatography differ greatly in instrumentation.

In HPLC, pure solvents or their mixtures are usually used as mobile phases.

To create a stream of pure solvent (or mixtures of solvents), called eluent in liquid chromatography, pumps are used that are part of the chromatograph hydraulic system.

Adsorption chromatography is carried out as a result of the interaction of a substance with adsorbents, such as silica gel or aluminum oxide, which have active centers on the surface. The difference in the ability to interact with the adsorption centers of different sample molecules leads to their separation into zones in the process of moving with the mobile phase through the column. The division of the component zones achieved in this case depends on the interaction with both the solvent and the adsorbent.

Silica gel adsorbents with different volumes, surfaces, and pore diameters find the greatest application in HPLC. Aluminum oxide and other adsorbents are used much less frequently. The main reason for this:

Insufficient mechanical strength, which does not allow packaging and use at elevated pressures typical for HPLC;

silica gel compared to aluminum oxide has a wider range of porosity, surface and pore diameter; a significantly higher catalytic activity of aluminum oxide leads to a distortion of the analysis results due to the decomposition of the sample components or their irreversible chemisorption.

HPLC detectors

High performance liquid chromatography (HPLC) is used to detect polar non-volatile substances, which for some reason cannot be converted into a form convenient for gas chromatography, even in the form of derivatives. Such substances, in particular, include sulfonic acids, water-soluble dyes and some pesticides, such as phenyl-urea derivatives.

Detectors:

UV - diode array detector. The "matrix" of photodiodes (there are more than two hundred of them) constantly registers signals in the UV and visible region of the spectrum, thus ensuring the recording of UV-B spectra in the scanning mode. This makes it possible to continuously record, at high sensitivity, undistorted spectra of components rapidly passing through a special cell.

Compared to single-wavelength detection, which does not provide information about the "purity" of the peak, the ability to compare the full spectra of the diode array provides an identification result with a much greater degree of certainty.

Fluorescent detector. The great popularity of fluorescent detectors is due to the very high selectivity and sensitivity, and the fact that many environmental pollutants fluoresce (for example, polyaromatic hydrocarbons).

An electrochemical detector is used to detect substances that are easily oxidized or reduced: phenols, mercaptans, amines, aromatic nitro and halogen derivatives, aldehydes, ketones, benzidines.

Chromatographic separation of the mixture on the column due to the slow advance of the PF takes a long time. To speed up the process, chromatography is carried out under pressure. This method is called high performance liquid chromatography (HPLC).

Modernization of the equipment used in classical liquid column chromatography has made it one of the promising and modern methods of analysis. High performance liquid chromatography is a convenient method for separating, preparatively isolating, and performing qualitative and quantitative analysis of non-volatile, thermolabile compounds of both low and high molecular weight.

Depending on the type of sorbent used in this method, 2 variants of chromatography are used: on a polar sorbent using a non-polar eluent (direct phase option) and on a non-polar sorbent using a polar eluent - the so-called reverse-phase high-performance liquid chromatography (RPHLC).

When the eluent passes to the eluent, the equilibrium under RPHLC conditions is established many times faster than under the conditions of polar sorbents and non-aqueous PFs. As a result of this, as well as the convenience of working with water and water-alcohol eluents, RPHLC has now gained great popularity. Most HPLC analyzes are carried out using this method.

Detectors. Registration of the output from the column of a separate component is performed using a detector. For registration, you can use the change in any analytical signal coming from the mobile phase and related to the nature and amount of the mixture component. Liquid chromatography uses such analytical signals as light absorption or light emission of the exiting solution (photometric and fluorimetric detectors), refractive index (refractometric detectors), potential and electrical conductivity (electrochemical detectors), etc.

The continuously detected signal is recorded by the recorder. The chromatogram is a sequence of detector signals recorded on the recorder tape, generated when individual components of the mixture exit the column. In the case of separation of the mixture, individual peaks are visible on the external chromatogram. The position of the peak on the chromatogram is used for the purpose of identification of the substance, the height or area of ​​the peak - for the purpose of quantitative determination.

Application

HPLC finds the widest application in the following areas of chemical analysis (objects of analysis where HPLC has practically no competition are highlighted):

· Food quality control - tonic and flavor additives, aldehydes, ketones, vitamins, sugars, dyes, preservatives, hormones, antibiotics, triazine, carbamate and other pesticides, mycotoxins, nitrosoamines, polycyclic aromatic hydrocarbons, etc.

· Environmental protection - phenols, organic nitro compounds, mono- and polycyclic aromatic hydrocarbons, a number of pesticides, major anions and cations.

· Criminalistics - drugs, organic explosives and dyes, potent pharmaceuticals.

· Pharmaceutical industry - steroid hormones, practically all products of organic synthesis, antibiotics, polymer preparations, vitamins, protein preparations.

Medicine - the listed biochemical and medicinal substances and their metabolites in biological fluids (amino acids, purines and pyrimidines, steroid hormones, lipids) in the diagnosis of diseases, determining the rate of excretion of drugs from the body for the purpose of their individual dosage.

· Agriculture - determination of nitrate and phosphate in soils to determine the required amount of fertilizers, determination of the nutritional value of feed (amino acids and vitamins), analysis of pesticides in soil, water and agricultural products.

Biochemistry, bioorganic chemistry, genetic engineering, biotechnology - sugars, lipids, steroids, proteins, amino acids, nucleosides and their derivatives, vitamins, peptides, oligonucleotides, porphyrins, etc.

· Organic chemistry - all stable products of organic synthesis, dyes, thermolabile compounds, non-volatile compounds; inorganic chemistry (practically all soluble compounds in the form of ions and complex compounds).

· Quality control and safety of food products, alcoholic and non-alcoholic beverages, drinking water, household chemicals, perfumes at all stages of their production;

determination of the nature of pollution at the site of a man-made disaster or emergency;

detection and analysis of narcotic, potent, poisonous and explosive substances;

determination of the presence of harmful substances (polycyclic and other aromatic hydrocarbons, phenols, pesticides, organic dyes, ions of heavy, alkaline and alkaline earth metals) in liquid effluents, air emissions and solid waste from enterprises and in living organisms;

· monitoring of organic synthesis processes, oil and coal processing, biochemical and microbiological productions;

analysis of soil quality for fertilization, the presence of pesticides and herbicides in soil, water and products, as well as the nutritional value of feed; complex research analytical tasks; obtaining a micro amount of ultrapure substance.



(OFS 42-0096-09)

High performance liquid chromatography (HPLC) is a column chromatography method in which the mobile phase (MP) is liquid

bone moving through a chromatographic column filled with unsupported

the visual phase (sorbent). HPLC columns are characterized by high hydraulic pressure at the column inlet, therefore HPLC is sometimes called

called "High Pressure Liquid Chromatography".

Depending on the mechanism of separation of substances, the following are distinguished:

general HPLC options: adsorption, distribution, ion-exchange,

exclusive, chiral, etc.

In adsorption chromatography, the separation of substances occurs due to their different ability to be adsorbed and desorbed with increasing

surface of the adsorbent with a developed surface, for example, silica gel.

In partition HPLC, separation occurs due to the difference in the distribution coefficients of the substances to be separated between the immobile

(usually chemically grafted onto the surface of a fixed carrier) and

mobile phases.

By polarity, PF and NF HPLC are divided into normal-phase and ob-

phase-rotation.

Normal-phase is called a variant of chromatography, in which

use a polar sorbent (for example, silica gel or silica gel with added

twisted NH2 - or CN-groups) and non-polar PF (for example, hexane with different

personal supplements). In the reversed-phase variant of chromatography,

use non-polar chemically modified sorbents (for example,

non-polar alkyl radical C18 ) and polar mobile phases (for example,

methanol, acetonitrile).

In ion-exchange chromatography, the molecules of the substances of the mixture, dissociation

in solution into cations and anions, are separated when moving through

sorbent (cation exchanger or anion exchanger) due to their different exchange rates with ionic

mi groups of the sorbent.

In exclusion (sieve, gel-penetrating, gel-filtration)

Chromatography molecules of substances are separated by size due to their different ability to penetrate into the pores of the stationary phase. At the same time, the first of

the largest molecules (with the highest molecular weight) that can penetrate into the minimum number of pores of the stationary phase come out of the columns,

and the substances with small molecular sizes come out last.

Often the separation proceeds not by one, but by several mechanisms at the same time.

The HPLC method can be used to control the quality of any nega-

similar analytes. For analysis, appropriate instruments are used - liquid chromatographs.

The composition of a liquid chromatograph usually includes the following basic

nodes:

– PF preparation unit, including a container with a mobile phase (or a container

sti with individual solvents that are part of the mobile phase

zy) and PF degassing system;

– pumping system;

– mobile phase mixer (if necessary);

– sample injection system (injector);

– chromatographic column (can be installed in a thermostat);

– detector;

– data collection and processing system.

Pumping system

The pumps supply the PF to the column at a given constant rate. The composition of the mobile phase may be constant or variable.

during the analysis. In the first case, the process is called isocratic,

and in the second - gradient. Sometimes installed in front of the pumping system

filters with a pore diameter of 0.45 µm for filtering the mobile phase. Modern

The variable pumping system of a liquid chromatograph consists of one or more pumps controlled by a computer. This allows you to change the

becoming PF according to a specific program with gradient elution. Sme-

the mixing of the PF components in the mixer can occur both at low pressure

ion (before pumps) and at high pressure (after pumps). The mixer can be used for PF preparation and isocratic elution,

however, a more accurate ratio of components is achieved with preliminary

mixing of PF components for an isocratic process. Analytical HPLC pumps make it possible to maintain a constant flow rate of PF into the column in the range from 0.1 to 10 ml/min at a column inlet pressure of up to 50 MPa. It is advisable, however, that this value does not exceed

shalo 20 MPa. Pressure pulsations are minimized by special damping

ferrule systems included in the design of pumps. Working parts on-

pumps are made of corrosion-resistant materials, which allows the use of aggressive components in the composition of the PF.

Faucets

By design, mixers can be static or dynamic.

mic.

In the mixer, a single mobile phase is formed from

specific solvents supplied by pumps, if the required mixture has not been prepared in advance. Mixing of solvents usually occurs spontaneously, but systems with forced mixing are sometimes used.

sewing.

Injectors

Injectors can be universal for introducing samples from

1 µl to 2 ml or discrete for sample injection of only a certain volume

ema. Both types of injectors can be automatic ("auto-injectors" or "auto-samplers"). The injector for entering the sample (solution) is located not -

just before the chromatographic column. The design of the injector makes it possible to change the direction of the PF flow and to preliminarily introduce a sample into a loop of a certain volume (usually from 10 to 100 μl).

This volume is indicated on the loop label. The design of the injector allows the replacement of the loop. To introduce the analyzed solution into non-av-

tomatic injector uses a manual microsyringe with a volume significantly

greatly exceeding the volume of the loop. The excess of the injected solution, not

in the loop is discarded and the exact and always the same volume of sample is injected into the column. Manual incomplete filling of the loop reduces the accuracy

dosing accuracy and reproducibility and, consequently, degrades the accuracy

and reproducibility of chromatographic analysis.

Chromatography column

Chromatographic columns are usually stainless steel, glass or plastic tubes filled with sorbent and closed.

on both sides with filters with a pore diameter of 2–5 µm. The length of the analytical

column, depending on the mechanism of chromatographic separation, can be in the range from 5 to 60 cm or more (usually it is

10-25 cm), inner diameter - from 2 to 10 mm (usually 4.6 mm). Columns with an inner diameter of less than 2 mm are used in microcolumn chromium

tography. Capillary columns with internal diameters are also used.

rum about 0.3-0.7 mm. Columns for preparative chromatography have an internal diameter of up to 50 mm or more.

Before the analytical column, short cables can be installed.

columns (pre-columns) performing various auxiliary functions

(more often - protection of the analytical column). Typically, the analysis is carried out at

at room temperature, however, to increase the separation efficiency and

shortening the duration of the analysis, a thermostat can be used

column tiring at temperatures not exceeding 60 C. At higher temperatures, sorbent degradation and a change in the PF composition are possible.

Stationary phase (sorbent)

Commonly used sorbents are:

1. Silica gel, alumina, porous graphite are used in normal

small phase chromatography. The retention mechanism in this case

tea - usually adsorption;

2. Resins or polymers with acidic or basic groups. Scope - ion-exchange chromatography;

3. Porous silica gel or polymers (size exclusion chromatography);

4. Chemically modified sorbents (sorbents with grafted fa-

zami), prepared most often on the basis of silica gel. The retention mechanism in most cases is the distribution between mobile

noah and stationary phases;

5. Chemically modified chiral sorbents, for example,

aqueous celluloses and amyloses, proteins and peptides, cyclodextrins,

used to separate enantiomers (chiral chromatography)

Bonded phase sorbents can have varying degrees of chemical

chesky modification. The sorbent particles may be spherical or non-spherical.

regular shape and varied porosity.

The most commonly used bonded phases are:

– octyl groups(sorbent octylsilane or C8);

– octadecyl groups(sorbent octadecylsilane

(ODS) or C18);

– phenyl groups(sorbent phenylsilane);

– cyanopropyl groups(sorbent CN);

– aminopropyl groups(NH2 sorbent);

– diol groups (sorbent diol).

Most often, the analysis is performed on non-polar bonded phases in

reverse-phase mode using C18 sorbent.

In some cases, it is more appropriate to use normal

phase chromatography. In this case, silica gel or polar bonded phases (“CN”, “NH2”, “diol”) are used in combination with non-polar solutes.

Bonded phase sorbents are chemically stable at pH values ​​from 2.0 to 8.0 unless otherwise specified by the manufacturer.

The sorbent particles may have a spherical or irregular shape and a variety of porosity. The particle size of the sorbent in analytical HPLC is usually 3–10 µm, in preparative HPLC, up to 50 µm or more.

Monolithic sorbents are also used.

The high separation efficiency is provided by the high surface area of ​​the sorbent particles (which is a consequence of their microscopy).

the presence of pores), as well as the uniformity of the composition of the sorbent and its dense and uniform packing.

Detectors

Various detection methods are used. In the general case, PF with components dissolved in it after a chromatographic column

ki falls into the detector cell, where one or another of its properties is continuously measured (absorption in the UV or visible region of the spectrum, fluorescence,

refractive index, electrical conductivity, etc.). The resulting chromatogram is a graph of the dependence of some physical

or physico-chemical parameter of the PF on time.

The most common are spectrophotometric

detectors (including diode-matrix), registering a change in the optical

density in the ultraviolet, visible and often in the near infrared

other regions of the spectrum from 190 to 800 or 900 nm. The chromatogram in this case

tea is the dependence of the optical density of the PF on time.

The traditionally used spectrophotometric detector allows

allows detection at any wavelength in its operating range

zone. Multiwave detectors are also used, which allow conducting

perform detection at several wavelengths simultaneously.

With the help of a diode array detector, it is possible not only to carry out detection at several wavelengths at once, but also almost instantly

it is possible (without scanning) to obtain the optical spectrum of the PF at any time, which greatly simplifies the qualitative analysis of the separated components

components.

The sensitivity of fluorescent detectors is approximately 1000 times higher than that of spectrophotometric ones. In this case, either its own fluorescence or the fluorescence of the corresponding derivatives is used, if the analyte itself does not fluoresce. Modern

Interchangeable fluorescent detectors make it possible not only to obtain chromato-

grams, but also to record the excitation and fluorescence spectra of the analy-

zyable connections.

Refractometric detectors are used to analyze samples that do not absorb in the UV and visible regions of the spectrum (for example, carbohydrates).

(refractometers). The disadvantages of these detectors are their low (compared to spectrophotometric detectors) sensitivity and significant temperature dependence of the signal intensity (the detector must be thermostated).

Electrochemical detectors are also used (conductometric

sky, amperometric, etc.), mass spectrometric and Fourier-IR

detectors, detectors of light scattering, radioactivity and some other

mobile phase

AT A variety of solvents, both individual and their mixtures, can be used as PF.

AT normal-phase Chromatography usually uses liquid carbon

hydrocarbons (hexane, cyclohexane, heptane) and other relatively non-polar

solvents with small additions of polar organic compounds,

which regulate the eluting strength of the PF.

In reversed-phase chromatography, the composition of the PF includes polar or-

organic solvents (usually acetonitrile and methanol) and water. For opti-

Separation studies often use aqueous solutions with a certain sign

pH value, in particular buffer solutions. Inorganic additives are used

calic and organic acids, bases and salts and other compounds (on-

example, chiral modifiers to separate enantiomers into achiral-

nom sorbent).

The control of the pH value must be carried out separately for the aqueous component, and not for its mixture with an organic solvent.

PF can consist of one solvent, often two, if necessary

dimity - from three or more. The composition of PF is indicated as the volume ratio of its constituent solvents. In some cases, the mass

ratio, which must be specially stipulated.

When using a UV spectrophotometric detector, the PF should not have a pronounced absorption at the wavelength chosen for detection. The limit of transparency or optical density when determining

the specified wavelength of the solvent of a particular manufacturer is often indicated

is on the package.

Chromatographic analysis is greatly influenced by the degree of purity of the PF, so it is preferable to use solvents produced

nye specifically for liquid chromatography (including water).

PF and analyzed solutions should not contain undissolved

particles and gas bubbles. Water obtained in the laboratory

aqueous solutions pre-mixed with water organic solvents

The solvents, as well as the analyzed solutions, must be subjected to fine filtration and degassing. Filtering is usually used for this purpose.

under vacuum through a membrane filter inert with respect to this solvent or solution with a pore size of 0.45 μm.

Data collection and processing system

A modern data processing system is a conjugated

personal computer connected with the chromatograph with software installed

software that allows you to register and process chrono-

matogram, as well as manage the operation of the chromatograph and monitor the main

mi parameters of the chromatographic system.

List of chromatographic conditions to be specified

In a private monograph, the dimensions of the co-

columns, type of sorbent with indication of particle size, column temperature (if temperature control is necessary), volume of injected sample (loop volume),

PF status and method of its preparation, PF feed rate, detector and detection conditions, description of the gradient mode (if used), chromatography time.

ION EXCHANGE AND ION HPLC

Ion exchange chromatography is used for analysis as an organic

skikh (heterocyclic bases, amino acids, proteins, etc.), and non-or-

ganic (various cations and anions) compounds. Separation of components

components of the analyzed mixture in ion-exchange chromatography is based on the reversible interaction of the ions of the analyzed substances with ionic groups.

pami sorbent. Anion exchangers or cation exchangers are used as sorbents.

you. These sorbents are mainly either polymeric ion-

exchange resins (usually copolymers of styrene and divinylbenzene with graft

ionic groups), or silica gels with grafted ion exchange groups. Sorbents with -(CH2)3 N+ X– groups are used to separate anions, and sorbents with -(CH2)SO3 – H+ groups are used to separate cations.

Typically, polymer resins are used to separate anions, and to separate e-

cations are modified silica gels.

As PF in ion-exchange chromatography, aqueous solutions of acids, bases and salts are used. Buffer races are usually used

solutions that allow you to maintain certain pH values. It is also possible to use small additives of water-miscible organic

cal solvents - acetonitrile, methanol, ethanol, tetrahydrofuran.

Ion chromatography- a variant of ion-exchange chromatography, in

which, to determine the concentration of ions of the analyte, is used

using a conductometric detector. For highly sensitive op-

To determine changes in the electrical conductivity passing through the PF detector, the background electrical conductivity of the PF must be low.

There are two main variants of ion chromatography.

The first of them is based on the suppression of the electrical conductivity of the electrolytic

that PF using the second ion-exchange column located between the anal-

lytic column and detector. In this column, neutralization takes place

PF and the analyzed compounds enter the detector cell in deionization

zirovannoy water. The detected ions are the only ions

ensuring the conductivity of the PF. The disadvantage of the suppressor column is the need for its regeneration at fairly short intervals.

me. The suppression column can be replaced by a continuously operating

membrane suppressor, in which the composition of the membrane is continuously

is the flow of regenerating solution moving in the direction

opposite to the direction of the PF flow.

The second version of ion chromatography is single-column ion chromatography.

matography. In this variant, a PF with a very low electrical conductivity is used.

water content. Weak organic compounds are widely used as electrolytes.

sky acids - benzoic, salicylic or isophthalic.

SIZE HPLC

Size exclusion chromatography (gel chromatography) is a special version of HPLC based on the separation of molecules according to their size. Distribution

molecules between the stationary and mobile phases is based on the size of the mo-

molecules and partly on their shape and polarity. For separation, use

porous sorbents - polymers, silica gel, porous glasses and polysaccharides.

The particle size of the sorbents is 5–10 µm.

The advantages of porous glasses and silica gel are fast diffusion of PF and analyte molecules into pores, stability under various conditions (even at high temperatures). Polymeric sorbene-

you are copolymers of styrene and divinylbenzene (this is a hydro-

phobic sorbents used with non-polar mobile phases) and

hydrophilic gels obtained from sulfonated divinylbenzene or polyacrylamide resins.

Two limiting types of interaction of molecules with a porous stationary phase are possible. Molecules larger than the average diameter a pore do not penetrate into the sorbent at all and are eluted together with the mobile phase.

zoy first. Molecules with a diameter much smaller than the pore size of the sort

bents freely penetrate into it, remain in the stationary phase for the longest time and are eluted last. Molecules of medium size penetrate into the pores of the sorbent depending on their size and partially depending on their shape. They elute with different retention times between

our largest and smallest molecules. The separation of the components of the chromatographed sample occurs as a result of repeated ac-

the diffusion of the sample components into the pores of the sorbent, and vice versa.

In size exclusion chromatography, to characterize retention,

a retention volume equal to the product of the PF flow rate and the retention time is used.

mobile phase. The choice of PF depends on the type of sorbent. Exclusion-

chromatography is generally divided into gel filtration and gel chromatography.

permeation chromatography.

The gel filtration chromatography method is used to separate

of water-soluble compounds on hydrophilic sorbents. The mobile phases are aqueous buffer solutions with a given pH value.

Gel permeation chromatography uses hydrophobic

bents and non-polar organic solvents (toluene, dichloromethane, tetra-

hydrofuran). This method is used to analyze compounds that are slightly soluble

rimmed in water.

Detectors. As detectors in size exclusion chromatography, differential refractometric detectors are used, as well as spectrophotometric detectors (including those in the IR region of the spectrum).

Viscometric and flow laser detectors are also used.

These detectors, in combination with a refractometer or other concentration

detector allows you to continuously determine the molecular weight of

limer in PF.

ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY

Ultra performance liquid chromatography is a variant of liquid chromatography that is more efficient

stu compared with classical HPLC.

A feature of ultra-performance liquid chromatography is

the use of sorbents with a particle size of 1.5 to 2 microns. Chro-

matographic columns are usually 50 to 150 mm in length and 1

up to 4 mm in diameter. The volume of the injected sample can be from 1 to 50 µl.

Chromatographic equipment used in the classical

riante HPLC, usually specially adapted for this type of chromatography

Equipment designed for ultra performance liquid chromatography can also be used in the classic version of HPLC.

Chromatographic separation of the mixture on the column due to the slow advance of the PF takes a long time. To speed up the process, chromatography is carried out under pressure. This method is called High Performance Liquid Chromatography (HPLC)

Modernization of the equipment used in classical liquid column chromatography has made it one of the promising and modern methods of analysis. High performance liquid chromatography is a convenient method for the separation, preparative isolation, and qualitative and quantitative analysis of non-volatile, thermolabile compounds of both low and high molecular weight.

Depending on the type of sorbent used in this method, 2 variants of chromatography are used: on a polar sorbent using a non-polar eluent (direct phase option) and on a non-polar sorbent using a polar eluent - the so-called reverse-phase high-performance liquid chromatography (RPHLC).

When the eluent passes to the eluent, the equilibrium under RPHLC conditions is established many times faster than under the conditions of polar sorbents and non-aqueous PFs. As a result of this, as well as the convenience of working with water and water-alcohol eluents, RPHLC has now gained great popularity. Most HPLC analyzes are carried out using this method.

Instrumentation for HPLC

Columns for HPLC are made of stainless steel with an inner diameter of 2-6 mm and a length of 10-25 cm. The columns are filled with a sorbent (NF). Silica gel, alumina, or modified sorbents are used as NF. Silica gel is usually modified by chemically introducing various functional groups into its surface.

Detectors. Registration of the output from the column of a separate component is performed using a detector. For registration, you can use the change in any analytical signal coming from the mobile phase and related to the nature and amount of the mixture component. Liquid chromatography uses such analytical signals as light absorption or light emission of the exiting solution (photometric and fluorimetric detectors), refractive index (refractometric detectors), potential and electrical conductivity (electrochemical detectors), etc.

The continuously detected signal is recorded by the recorder. The chromatogram is a sequence of detector signals recorded on the recorder tape, generated when individual components of the mixture exit the column. In the case of separation of the mixture, separate peaks are visible on the external chromatogram. The position of the peak on the chromatogram is used for the purpose of identification of the substance, the height or area of ​​the peak - for the purpose of quantitative determination.

Qualitative Analysis

The most important characteristics of the chromatogram - the retention time t r and the retention volume associated with it - reflect the nature of the substances, their ability to sorption on the stationary phase material and, therefore, under constant chromatography conditions, they are a means of identifying the substance. For a given column with a certain flow rate and temperature, the retention time of each compound is constant (Fig. - retention time of the internal standard (substance initially absent in the analyzed mixture), h - peak height (mm), a 1/2 - peak width at half its height, mm.

To identify a substance by chromatogram, standard samples or pure substances are usually used. The retention time of the unknown component t Rx is compared with the retention time t RCT of the known substances. But more reliable identification by measuring the relative retention time

In this case, a known substance (internal standard) is first introduced into the column and its retention time t R(BC) is measured, then the test mixture is chromatographically separated (chromatographed), to which the internal standard is preliminarily added.

Quantitative Analysis

This analysis is based on the dependence of the peak height h or its area S on the amount of substance. For narrow peaks, measurement h is preferable, for wide blurry peaks - S. The peak area is measured in different ways: by multiplying the peak height (h) by its width (a 1/2), measured at half its height; planning; using an integrator. Modern chromatographs are equipped with electrical or electronic integrators.

Three methods are mainly used to determine the content of substances in a sample: the absolute calibration method, the internal normalization method, and the internal standard method.

The absolute calibration method is based on a preliminary determination of the relationship between the amount of the introduced substance and the area or height of the peak on the chromatogram. A known amount of the calibration mixture is introduced into the chromatogram and the areas or heights of the resulting peaks are determined. Build a graph of the area or height of the peak from the amount of injected substance. The test sample is analyzed, the area or height of the peak of the component to be determined is measured, and its amount is calculated based on the calibration curve.

The method of internal normalization is based on bringing to 100% the sum of the areas of all peaks in the chromatogram.

This method provides information only on the relative content of the component in the mixture, but does not allow determining its absolute value.

The internal standard method is based on the comparison of a selected peak parameter of an analyte with the same parameter of a standard substance introduced into the sample in a known amount. A known amount of such a standard substance is introduced into the test sample, the peak of which is sufficiently well separated from the peaks of the components of the test mixture.

The last two methods require the introduction of correction factors characterizing the sensitivity of the detectors used to the analyzed substances. For different types of detectors and different substances, the sensitivity coefficient is determined experimentally.

Liquid adsorption chromatography also uses the analysis of fractions of solutions collected at the moment the substance exits the column. The analysis can be carried out by various physicochemical methods.

Liquid adsorption chromatography is used primarily for the separation of organic substances. This method is very successful in studying the composition of oil, hydrocarbons, effectively separating trans- and cis-isomers, alkaloids, etc. HPLC can be used to determine dyes, organic acids, amino acids, sugars, pesticide and herbicide impurities, medicinal substances and other contaminants in food products.

Equipment for liquid chromatography.

In modern liquid chromatography, instruments of varying degrees of complexity are used - from the simplest systems to high-end chromatographs equipped with various additional devices. On fig. 1. shows a block diagram of a liquid chromatograph containing the minimum required set of components, in one form or another, present in any chromatographic system.

Rice. 1. Block diagram of a liquid chromatograph.

The pump (2) is designed to create a constant solvent flow. Its design is determined primarily by the operating pressure in the system. For operation in the range of 10-500 MPa, plunger (syringe) or piston type pumps are used. The disadvantage of the first is the need for periodic stops for filling with eluent, and the second is the great complexity of the design and, as a result, the high price. For simple systems with low operating pressures of 1-5 MPa, inexpensive peristaltic pumps are successfully used, but since it is difficult to achieve a constant pressure and flow rate, their use is limited to preparative tasks.

The injector (3) ensures that a sample of the mixture of separated components is injected into the column with a sufficiently high reproducibility. Simple "stop-flow" sample injection systems require the pump to be stopped and are therefore less convenient than Reodyne's loop pipettes.

The HPLC columns (4) are thick-walled stainless steel tubes capable of withstanding high pressure. An important role is played by the density and uniformity of packing the column with a sorbent. For low pressure liquid chromatography, thick-walled glass columns are successfully used. Temperature constancy is ensured by thermostat (5).

Detectors (6) for liquid chromatography have a flow cell in which some property of the flowing eluent is continuously measured. The most popular types of general purpose detectors are refractometers, which measure the refractive index, and spectrophotometric detectors, which measure the absorbance of a solvent at a fixed wavelength (usually in the ultraviolet region). The advantages of refractometers (and the disadvantages of spectrophotometers) include low sensitivity to the type of compound being determined, which may not contain chromophore groups. On the other hand, the use of refractometers is limited to isocratic systems (with a constant eluent composition), so the use of a solvent gradient is not possible in this case.

The recording system (7) in the simplest case consists of a differential amplifier and a recorder. It is also desirable to have an integrator that makes it possible to calculate the relative areas of the resulting peaks. In complex chromatographic systems, an interface block is used that connects the chromatograph to a personal computer (8), which not only collects and processes information, but also controls the instrument.

HPLC detectors

High performance liquid chromatography (HPLC) is used to detect polar non-volatile substances, which for some reason cannot be converted into a form convenient for gas chromatography, even in the form of derivatives. Such substances, in particular, include sulfonic acids, water-soluble dyes and some pesticides, such as phenyl-urea derivatives.

Detectors:

UV - diode array detector. The "matrix" of photodiodes (there are more than two hundred of them) constantly registers signals in the UV and visible region of the spectrum, thus ensuring the recording of UV-B spectra in the scanning mode. This makes it possible to continuously record, at high sensitivity, undistorted spectra of components rapidly passing through a special cell.

Compared to single-wavelength detection, which does not provide information about the "purity" of the peak, the ability to compare the full spectra of the diode array provides an identification result with a much greater degree of certainty.

Fluorescent detector. The great popularity of fluorescent detectors is due to the very high selectivity and sensitivity, and the fact that many environmental pollutants fluoresce (for example, polyaromatic hydrocarbons).

An electrochemical detector is used to detect substances that are easily oxidized or reduced: phenols, mercaptans, amines, aromatic nitro and halogen derivatives, aldehydes, ketones, benzidines.

Indicator tubes for the test determination of aromatic amines in the air of the working area

Aromatic amines have high toxic properties and are characterized by low maximum allowable concentrations in the air. The propensity for oxidative degradation of these pollutants in environmental objects limits the possibility of operational control of their content in places of local emissions. This determines the relevance of using analytical systems based on test determinations of substances to assess the contamination of various media. Test methods, which include analytical devices with direct detection of an analytical signal without additional sampling and sample preparation of analyzed samples, allow the most economical and objective obtaining of information about the state of the environment.

The aim of this work is to develop indicator tubes for the test determination of amino compounds in air based on a new, promising class of reagents for molecular organic analysis of chlorodinitro-substituted benz-2,1,3-oxadiazole and their N-oxides.

The color of the resulting derivatives depends on the nature of the compound being determined, and the observed bathochromic shift of the absorption bands is determined by the degree of substitution of the amino group and the presence of substituents. This makes it possible to use direct visual detection of an analytical signal as the basis of the test method. The limits of visual detection of toxicants reach 0.05 mg/m 3 .

The developed indicator tubes are used as effective chemisorption samplers (chemical dosimeters) for the analysis of air containing a mixture of amino compounds. The sampling rate was experimentally established by the degree of absorption of toxicants by the selective layer. The composition and amount of, for example, substituted anilines can be determined after elution of the chemisorption products from silica gel by HPLC. At the same time, a wide range of potential components of industrial ecosystems does not affect the results of the determinations.