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 only difference is in the use of liquid as a stationary phase. Due to the high density of liquid mobile phases and the high resistance of columns, gas and liquid chromatography differ greatly in instrumentation.

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

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

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 during movement with the mobile phase along the column. The zone separation of the components achieved in this case depends on the interaction with both the solvent and the adsorbent.

The most widely used in HPLC are silica gel adsorbents with different volumes, surface areas and pore diameters. 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 high pressures characteristic of HPLC;

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

Detectors for HPLC

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

Detectors:

UV detector on a diode matrix. A “matrix” of photodiodes (more than two hundred of them) constantly registers signals in the UV and visible regions of the spectrum, thus providing recording of UV-B spectra in scanning mode. This allows you to continuously record, at high sensitivity, undistorted spectra of components quickly passing through a special cell.

Compared to single wavelength detection, which does not provide information about peak purity, the ability to compare full spectra of a diode array provides a much higher degree of confidence in the identification result.

Fluorescence detector. The great popularity of fluorescent detectors is due to their very high selectivity and sensitivity, and the fact that many environmental pollutants fluoresce (eg 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 a mixture on a column due to the slow progress of the PF takes a lot of time. To speed up the process, chromatography is carried out under pressure. This method is called high-performance liquid chromatography (HPLC)

Modernization of equipment used in classical liquid column chromatography has made it one of the most 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 with 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 reverse-phase high-performance liquid chromatography (RPHPLC).

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

Detectors. The output of an individual component from the column is recorded using a detector. For registration, you can use a change in any analytical signal coming from the mobile phase and associated with the nature and quantity of the mixture component. Liquid chromatography uses analytical signals such as light absorption or light emission of the output solution (photometric and fluorimetric detectors), refractive index (refractometric detectors), potential and electrical conductivity (electrochemical detectors), etc.

The continuously detected signal is recorded by a recorder. A chromatogram is a sequence of detector signals recorded on a recorder tape, generated when individual components of a mixture leave the column. If the mixture is separated, individual peaks are visible on the external chromatogram. The position of the peak in the chromatogram is used for the purpose of identifying the substance, the height or area of ​​the peak - for the purpose of quantitative determination.

Application

HPLC is most widely used in the following areas of chemical analysis (objects of analysis where HPLC has virtually no competition are highlighted):

· Food quality control - tonics and flavoring additives, aldehydes, ketones, vitamins, sugars, dyes, preservatives, hormonal drugs, antibiotics, triazine, carbamate and other pesticides, mycotoxins, nitrosamines, polycyclic aromatic hydrocarbons, etc.

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

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

· Pharmaceutical industry - steroid hormones, almost 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 diagnosing diseases, determining the rate of elimination 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 applied, 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 (almost all soluble compounds in the form of ions and complex compounds).

· control of the quality and safety of food, alcoholic and non-alcoholic drinks, 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, alkali and alkaline earth metals) in liquid effluents, air emissions and solid waste from enterprises and in living organisms;

· monitoring of processes of organic synthesis, oil and coal refining, biochemical and microbiological production;

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 microquantities of ultrapure substances.



Introduction.

The rapid development of liquid chromatography in the last 10 years is mainly due to the intensive development of theoretical foundations and the practical use of its highly effective version, as well as the creation and industrial production of the necessary sorbents and equipment.

A distinctive feature of high-performance liquid chromatography (HPLC) is the use of sorbents with a grain size of 3-10 microns, which ensures rapid mass transfer with very high separation efficiency.

Currently, HPLC has taken first place among instrumental methods in terms of development rates, even surpassing gas chromatography. The most important advantage of HPLC compared to gas chromatography is the ability to study almost any object without any restrictions on their physicochemical properties, for example, boiling points or molecular weight.

Today, HPLC is a well-designed instrumental method that is widely used in a wide variety of fields of science and technology. Its importance is especially great in such critical areas as biochemistry, molecular biology, environmental pollution control, as well as in the chemical, petrochemical, food and pharmaceutical industries.

since it is necessary to take into account a number of very specific requirements due to the following features of the methodology.

A. HPLC columns are packed with very small particle diameter media. As a result, at such solvent volumetric velocities as are necessary for rapid sample separation, high pressure is created on the column.

b. Detectors used in HPLC are sensitive to fluctuations in eluent flow and pressure (noise). Moreover, when using concentration detectors, even higher stability of the eluent volumetric velocity is required.

V. The process of chromatographic separation is accompanied by a number of antagonistic effects, for example, dispersion of the sample in the mobile phase leads to mixing of the separated components and reduces the maximum concentration of the substance in the eluted peak (in the detector). Dispersion is observed in all areas of the system from the sample injection point to the detector.

d. Solvents acting as a mobile phase can often cause corrosion of equipment. This primarily applies to solvents used in reverse phase chromatography, which is preferred in biochemical HPLC applications.

The specifics of HPLC as an instrumental technique must be taken into account during the development, creation and operation of these systems. It took more than ten years of search and research to create commercial samples of chromatographic systems and their components that are sufficiently reliable, simple and safe to operate with an acceptable ratio between price and technical characteristics. The recent trends towards reducing columns (both length and diameter) force new demands on instruments.

1.1. EFFICIENCYANDSELECTIVITY

Chromatography is a method of separating the components of a mixture based on the difference in their equilibrium distribution between two "immiscible phases, one of which is stationary and the other is mobile. The components of the sample move along the column when they are in the mobile phase, and remain in place when they are in the stationary phase. The greater the affinity of the component for the stationary phase and the less for the mobile phase, the slower it moves through the column and the longer it is retained in it. Due to the difference in the affinity of the components of the mixture for the stationary and mobile phases, the main goal of chromatography is achieved - separation. an acceptable period of time of the mixture into individual bands (peaks) of the components as they move along the column with the mobile phase.

From these general concepts, it is clear that chromatographic separation is possible only if the components of the sample, entering the column when the sample is introduced, firstly, are dissolved in the mobile phase and, secondly, interact (retained) with the stationary phase . If, when introducing a sample, any components are not in the form of a solution, they will be filtered and will not participate in the chromatographic process. Likewise, components that do not interact with the stationary phase will pass through the column with the mobile phase without separating into their components.

Let us accept the condition that some two components are soluble in the mobile phase and interact with the stationary phase, i.e. the chroiatographic process can proceed without disturbances. In this case, after passing the mixture through the column, you can obtain chromatograms of the form a, b or V(Fig. 1.1). These chromatograms illustrate chromatographic separations that differ in efficiency (A and b) with equal selectivity and selectivity (b And V) with equal efficiency.

The narrower the peak obtained at the same retention time, the higher the column efficiency. Column efficiency is measured by the number of theoretical plates (NPT) N: the higher the efficiency

Rice. 1.2. Chromatographic peak parameters and calculation of the number of theoretical plates:

t R - peak retention time; h - peak height; Wj/j - peak width at half its height

Rice. 1.1. Type of chromatogram depending on the efficiency and selectivity of the column:

A- normal selectivity, reduced efficiency (fewer theoretical plates); b - usual selectivity and efficiency; V - normal efficiency, increased selectivity (higher ratio of component retention times)

efficiency, the greater the FTT, the smaller the broadening of the peak of the initially narrow band as it passes through the column, and the narrower the peak at the exit of the column. PTT characterizes the number of steps in establishing equilibrium between the mobile and stationary phases.

Knowing the number of theoretical plates per column and the length of the column L (µm), as well as the average sorbent grain diameter d c (µm), it is easy to obtain the values ​​of the height equivalent to a theoretical plate (HETT), as well as the reduced height equivalent to a theoretical plate (RHETT):

BETT = L/ N

PVETT =B3TT/d c .

Having the values ​​of FTT, HETT and PHETT, one can easily compare the efficiency of columns of different types, different lengths, filled with sorbents of different nature and granularity. By comparing the PTT of two columns of the same length, their efficiency is compared. When comparing HETP, columns with sorbents of the same grain size and different lengths are compared. Finally, the PVETT value allows for any two columns to evaluate the quality of the sorbent, firstly, and the quality of filling the columns, and secondly, regardless of the length of the columns, the granulation of the sorbents of its nature.

Column selectivity plays a large role in achieving chromatographic separation.

The selectivity of a column depends on many factors, and the skill of the experimenter is largely determined by the ability to influence the selectivity of separation. For this, three very important factors are in the hands of the chromatographer: the choice of the chemical nature of the sorbent, the choice of the composition of the solvent and its modifiers, and taking into account the chemical structure and properties of the separated components. Sometimes a change in the temperature of the column, which changes the distribution coefficients of substances between the mobile and stationary phases, has a noticeable effect on selectivity.

When considering and evaluating the separation of two components in a chromatogram, resolution is an important parameter. R s, which relates the output times and peak widths of both separated components

Resolution as a parameter characterizing peak separation increases as selectivity increases, reflected by an increase in the numerator, and efficiency increases, reflected by a decrease in the denominator value due to a decrease in the width of the peaks. Therefore, the rapid progress of liquid chromatography led to a change in the concept of “high pressure liquid chromatography” - it was replaced by “high resolution liquid chromatography” (while the abbreviated form of the term in English was preserved HPLC as the most correctly characterizing the direction of development of modern liquid chromatography).

Thus, column washout is reduced and efficiency is increased when a finer sorbent is used, more uniform in composition (narrow fraction), more densely and uniformly packed in the column, using thinner graft phase layers, less viscous solvents and optimal flow rates.

However, along with the blurring of the chromatographic zone band during the separation process in the column, it can also be washed out in the device for introducing the sample, in the connecting capillaries injector - column and column - detector, in the detector cell and in some auxiliary devices (microfilters for trapping mechanical particles from samples installed after the injector, pre-columns, coil reactors, etc.) - The greater the extra-column volume compared to the retained volume of the peak, the greater the erosion. It also matters where the dead volume is located: the narrower the chromatographic signal, the greater the blurring of the dead volume. Therefore, special attention should be paid to the design of that part of the chromatograph where the chromatographic zone is the narrowest (the injector and devices from the injector to the column) - here extra-column erosion is most dangerous and has the greatest impact. Although it is believed that in well-designed chromatographs the sources of additional extra-column dilution should be reduced to a minimum, nevertheless, each new device, each modification of the chromatograph must necessarily end with testing on a column and comparison of the resulting chromatogram with the passport one. If peak distortion or a sharp decrease in efficiency is observed, capillaries and other devices newly introduced into the system should be carefully inspected.

Off-column washout and its misjudgment can lead to a significant (over 50%) loss of efficiency, especially in cases where relatively old chromatographs are attempted to be used for high-speed HPLC, microcolumn HPLC, and other variants of modern HPLC that require microinjectors, connecting capillaries with internal with a diameter of 0.05-0.15 mm of minimum length, columns with a capacity of 10-1000 µl, detectors with microcuvettes with a capacity of 0.03-1 µl and with high speed, high-speed recorders and integrators.

1.2. SOLVENT RETENTION AND STRENGTH

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

In the case of adsorption chromatography on silica gel or aluminum oxide, 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), or decreased by decreasing the isopropanol content. If the containing polar component becomes too small (less than 0.1%), it should be replaced with a weaker elution force. 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 components of interest in the mixture. When selecting solvent systems, both the solubility of the components of the mixture and the eluotropic series of solvents compiled by different authors are taken into account.

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

In the case of reverse phase chromatography, the solvent strength is increased by increasing the content of the organic component in the eluent (methanol, acetonitrile or THF) and decreased by adding more water. If it is not possible to achieve the desired selectivity, they use another organic component or try to change it using various additives (acids, ion-pair reagents, etc.).

In separations using ion exchange chromatography, the strength of the solvent is changed by increasing or decreasing the concentration of the buffer solution or 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 select the strength of the solvent in such a way that all sample components elute within an acceptable time. Then you have to resort to gradient elution, i.e., use a solvent whose elution strength changes during the analysis process so that it constantly increases according to a predetermined program. This technique makes it 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.

1.3. SORBENT PARTICLE SIZE, PERMEABILITY AND EFFICIENCY

Considering the erosion in the column, we indicated that the efficiency of the column (HETT) depends on the size of the sorbent particles. To a large extent, the rapid development of HPLC over the past 10-12 years was due, firstly, to the development of methods for producing sorbents with particle sizes from 3 to 10 microns and with a narrow fractional composition, providing high efficiency with good permeability, and secondly, the development methods for filling columns with these sorbents and, thirdly, the development and serial production of liquid chromatographs with high-pressure pumps, injectors and detectors with small-volume cuvettes capable of recording small-volume peaks.

For well-packed slurry-packed columns, the reduced equivalent theoretical plate height (LPHE) can be 2 regardless of whether 3, 5, 10, or 20 μm particles are used for packing. In this case, we will receive respectively columns (with a standard length of 250 mm) with an efficiency of 41670, 25000, 12500 and 6250 t.t. It seems natural to select the most efficient column packed with 3 µm particles. However, this efficiency will come at the cost of very high pressure operation and relatively low separation speed, since the existing pump will most likely be able to pump solvent through such a column at a high volumetric velocity. Here we are faced with the question of the relationship between the particle size of the sorbent, the efficiency and permeability of the columns.

If we express the column resistance factor from here - a dimensionless quantity, we obtain the following equation:

The resistance factor for columns packed with microparticles of the same type using the same method varies slightly and is the following values:

Particle type "... Irregular Spherical

form form

Dry packaging. . . . . 1000-2000 800-1200

Suspension packaging. . . 700-1500 500-700

The column inlet pressure is proportional to the linear flow velocity, column drag factor, solvent viscosity, and column length and inversely proportional to the square of the particle diameter.

Applying this relationship to the above-described columns with particles with diameters of 3, 5, 10 and 20 µm and assuming constant linear flow rate, column resistance factor and solvent viscosity, we obtain an inlet pressure ratio of 44:16:4:1 for columns of equal length. Thus, if for a reverse-phase sorbent with a particle size of 10 μm when using methanol solvent systems - . water (70:30) usually on a standard column at a solvent flow rate of 1 ml/min, the pressure at the entrance to the column is 5 MPa, then for particles of 5 μm - 20 MPa and for 3 μm - 55 MPa. When using silica gel and a less viscous solvent system, hexane - isopropanol (100:2), the values ​​will be significantly lower: 1, 4 and 11 MPa, respectively. If in the case of a reversed-phase sorbent the use of particles with a size of 3 μm is very problematic, and 5 μm is possible, but not on all devices, then for a normal-phase sorbent there are no problems with pressure. It should be noted that modern high-speed HPLC typically uses a higher solvent flow rate than in the example above, so the pressure requirements increase even more.

However, in cases where a certain number of theoretical plates is required for separation and it is desirable to carry out rate analysis, the picture changes somewhat. Since the lengths of columns with sorbents with grain sizes of 3, 5, 10 microns, with equal efficiency, will be 7.5, respectively; 12.5 and 25 cm, then the pressure ratio at the inlet to the columns will change to 3:2:1. Accordingly, the duration of analysis on such columns of equal efficiency will be in the ratio 0.3:0.5:1, i.e., when moving from 10 to 5 and 3 microns, the duration of analysis will be reduced by 2 and 3.3 times. This faster analysis comes at the cost of proportionately higher pressure at the column inlet.

The data presented are valid for those cases where sorbents of different grain sizes have the same particle size distribution curves, the columns are packed in the same way and have the same column resistance factor. It should be borne in mind that the difficulty of obtaining narrow fractions of the sorbent increases as the particle size decreases and that. Fractions from different manufacturers have different fractional compositions. Therefore, the column resistance factor will vary depending on the grain size, sorbent type, column packing method, etc.

CLASSIFICATION OF HPLC METHODS BY SEPARATION MECHANISM

Most separations carried out by HPLC are based on a mixed mechanism of interaction of substances with a sorbent, providing greater or lesser retention of components in the column. Separation mechanisms in a more or less pure form are quite rare in practice, for example, adsorption when using absolutely anhydrous silica gel and anhydrous hexane to separate aromatic hydrocarbons.

With a mixed retention mechanism for substances of different structures and molecular weights, it is possible to evaluate the contribution to retention of adsorption, distribution, exclusion and other mechanisms. However, for a better understanding and understanding of the separation mechanisms in HPLC, it is advisable to consider separations with a predominance of one or another mechanism as relating to a certain type of chromatography, for example, ion exchange chromatography.

2.1.1 ADSORPTION CHROMATOGRAPHY

Separation by 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 during movement with the mobile phase along the column. The zone separation of the components achieved in this case depends on the interaction with both the solvent and the adsorbent.

Sorption on the surface of an adsorbent containing hydroxyl groups is based on a specific interaction between the polar surface of the adsorbent and polar (or polarizable) groups or sections of molecules. Such interactions include dipole-dipole interaction between permanent or induced dipoles, the formation of a hydrogen bond, up to the formation of r-complexes or charge transfer complexes. A possible and quite frequent occurrence in practical work is the manifestation of chemisorption, which can lead to a significant increase in retention time, a sharp decrease in efficiency, the appearance of decomposition products or irreversible sorption of the substance.

Adsorption isotherms of substances have a linear, convex or concave shape. With a linear adsorption isotherm, the peak of the substance is symmetrical and the retention time does not depend on the sample size. Most often, adsorption isotherms of substances are nonlinear and have a convex shape, which leads to some asymmetry of the peak with the formation of a tail.

The most widely used in HPLC are silica gel adsorbents with different pore volumes, surface areas, and pore diameters. Aluminum oxide is used much less frequently and other adsorbents, widely used in classical column and thin-layer chromatography, are used extremely rarely. The main reason for this is the insufficient mechanical strength of most other adsorbents, which does not allow them to be packaged or used at high pressures characteristic of HPLC.

The polar groups that cause adsorption and are located on the surface of silica gel and aluminum oxide are similar in properties. Therefore, usually the order of elution of mixtures of substances and the eluotropic series of solvents are the same for them. However, the difference in the chemical structure of silica gel and aluminum oxide sometimes leads to differences in selectivity - then preference is given to one or another adsorbent that is more suitable for a given specific task. For example, alumina provides greater selectivity for the separation of certain polynuclear aromatic hydrocarbons.

The preference usually given to silica gel compared to aluminum oxide is explained by a wider choice of silica gels in terms of porosity, surface and pore diameter, as well as the significantly higher catalytic activity of aluminum oxide, which often leads to distortion of analysis results due to the decomposition of sample components or their irreversible chemisorption .

2.1.2 Disadvantages of adsorption chromatography that limit its use

The popularity of adsorption chromatography gradually fell as the HPLC method developed; it was increasingly replaced and continues to be replaced by other options, such as reverse-phase and normal-phase HPLC on sorbents with a grafted phase. What are the disadvantages of adsorption chromatography that led to this?

First of all, this is the long duration of the processes of equilibrating adsorbents with solvents containing water in trace quantities, the difficulty of preparing such solvents with a certain and reproducible humidity. This results in poor reproducibility of retention, resolution, and selectivity parameters. For the same reason, it is impossible to use gradient elution - the return to the initial state is so long that it significantly exceeds the time gained by using a gradient.

Significant disadvantages of adsorbents, especially aluminum oxide, associated with frequent cases of rearrangements of compounds sensitive to catalysis, their decomposition, and irreversible sorption, are also well known and have been repeatedly noted in the literature. Irreversibly sorbed substances, accumulating at the initial section of the column, change the nature of the sorbent and can lead to an increase in the resistance of the column or even to its complete clogging. The last drawback can be eliminated by using a pre-column, which By- As resistance and clogging increases, it is replaced with a new one* or refilled with a new sorbent. However, irreversible sorption, which also occurs in this case, results in a chromatogram in which sample components sensitive to sorption or catalytic decomposition are completely or partially absent.

2.2. DISTRIBUTION CHROMATOGRAPHY

Partition chromatography is a variant of HPLC in which the separation of a mixture into components is carried out due to the difference in their distribution coefficients between two immiscible phases: a solvent (mobile phase) and a phase on the sorbent (stationary phase). Historically, the first were sorbents of this type, which were obtained by applying liquid phases (oxydipropionitrile, paraffin oil, etc.) onto porous supports, similar to how sorbents were and are prepared for gas-liquid chromatography (GLC). However, the disadvantages of such sorbents were immediately revealed, the main of which was the relatively rapid rinsing of the phase from the carrier. Due to this, the amount of phase in the column gradually decreased, the retention times also decreased, and adsorption centers not covered by the phase appeared in the initial section of the column, causing the formation of peak tails. This drawback was combated by saturating the solvent with the applied phase before it entered the column. Entrainment was also reduced when more viscous and less soluble polymer phases were used, but in this case, due to the difficulty of diffusion from thick polymer films, column efficiency was markedly reduced.

It turned out to be logical to graft the liquid phase onto the carrier through chemical bonds in such a way that its removal becomes physically impossible, i.e., to turn the carrier and the phase into one - into the so-called grafted-phase sorbent.

Subsequent efforts of researchers were aimed at searching for reagents whose grafting would proceed fairly quickly and completely, and the bonds formed would be as stable as possible. Such reagents were alkylchlorosilanes and their derivatives, which made it possible, using a similar technology, to obtain graft-phase sorbents of various types and with different polar and non-polar groups on the surface.

The successful application of the latter type of sorbents for HPLC has contributed to the growth of their production by a wide variety of manufacturers. Each company produced such sorbents, as a rule, based on its own type of silica gel and using its own technology, which usually constitutes production “know-how”. As a result, a large number of sorbents, chemically called exactly the same (for example, octadecylsilane-grafted silica gel), have very different chromatographic characteristics. This is due to the fact that silica gel can have wider or narrower pores, a different surface, porosity, its surface before grafting can be hydroxylated or not, mono-, di- or trichlorosilanes can be grafted, grafting conditions can give monomeric, polymeric or mixed layer phase, different methods are used to remove residual reagents, additional deactivation of silanol and other active groups may or may not be used.

The complexity of the technology for grafting reagents and preparing raw materials and materials, its multi-stage nature, leads to the fact that even batches of sorbents obtained using the same technology from one manufacturing company can have slightly different chromatographic characteristics. This is especially true in cases where such sorbents are used for the analysis of multicomponent mixtures containing substances that differ markedly in the number and position of functional groups, and the type of functionality.

Taking into account the above, one should always strive to ensure that when using the analysis technique described in the literature, the same sorbent and the same operating conditions are used. In this case, the likelihood that the work will not be reproduced is minimal. If this is not possible, but a sorbent from another company with a similar grafted phase is taken, you need to be prepared for the fact that it will take a long time to rework the technique. At the same time, there is a possibility (and it should be taken into account) that with this sorbent, even after long development, the required separation may not be achieved. The presence in the literature of many described separation techniques on old sorbents that have been produced for a long time stimulates their further production and use for this reason. However, in cases where it is necessary to move on to the development of original methods, especially in relation to substances prone to decomposition, chemisorption, rearrangements, it is advisable to start working on sorbents that have been developed recently and produced using new, improved versions of the technology. New sorbents have a more uniform fractional composition, more uniform and complete surface coverage with the grafted phase, and more advanced final stages of sorbent processing.

2.3. ION EXCHANGE CHROMATOGRAPHY

In ion exchange chromatography, the separation of mixture components is achieved through the reversible interaction of ionizing substances with the ionic groups of the sorbent. Preservation of the electrical neutrality of the sorbent is ensured by the presence of counterions capable of ion exchange located in close proximity to the surface. The ion of the introduced sample, interacting with the fixed charge of the sorbent, exchanges with the counterion. Substances with different affinities for fixed charges are separated on anion exchangers or cation exchangers. Anion exchangers have positively charged groups on the surface and sorb anions from the mobile phase. Cation exchangers accordingly contain groups with a -negative charge that interact with cations.

As a mobile phase, aqueous solutions of salts of acids, bases and solvents such as liquid ammonia are used, i.e., solvent systems with a high dielectric constant e and a greater tendency to ionize compounds. They usually work with buffer solutions that allow the pH value to be adjusted.

During chromatographic separation, ions of the analyte compete with ions contained in the eluent, tending to interact with oppositely charged groups of the sorbent. It follows that ion exchange chromatography can be used to separate any compounds that can be ionized in some way. It is possible to analyze even neutral sugar molecules in the form of their complexes with borate ion:

Sugar + VO 3 2 - = Sugar -VO 3 2 -.

Ion exchange chromatography is indispensable for the separation of highly polar substances, which cannot be analyzed by GLC without conversion to derivatives. These compounds include amino acids, peptides, and sugars.

Ion exchange chromatography is widely used in medicine, biology, biochemistry, for environmental monitoring, in the analysis of the content of drugs and their metabolites in the blood and urine, pesticides in food raw materials, as well as for the separation of inorganic compounds, including radioisotopes, lanthanides, actinides, etc. The analysis of biopolymers (proteins, nucleic acids, etc.), which usually took hours or days, is carried out using ion exchange chromatography in 20-40 minutes with better separation. The use of ion exchange chromatography in biology has made it possible to observe samples directly in biological media, reducing the possibility of rearrangement or isomerization, which can lead to incorrect interpretation of the final result. It is interesting to use this method to monitor changes occurring in biological fluids. The use of porous weak anion exchangers based on silica gel allowed the separation of peptides. V

The ion exchange mechanism can be represented in the form of the following equations:

for anion exchange

X-+R+Y- h ->■ Y-+R+X-.

for cation exchange |

X+ + R-Y+ h=* Y++R-X+.

In the first case, the X~ sample ion competes with the mobile phase ion Y~ for the R+ ion centers of the ion exchanger, and in the second case, the X+ sample cations compete with the Y+ ions of the mobile phase for the R~ ionic centers.

Naturally, sample ions that weakly interact with the ion exchanger will be weakly retained on the column during this competition and will be the first to be washed out from it, and, conversely, more strongly retained ions will be the last to elute from the column. Typically, BTqpH4Hbie interactions of a nonionic nature occur due to adsorption or hydrogen bonds of the sample with the nonionic part of the matrix or due to the limited solubility of the sample in the mobile phase. It is difficult to isolate “classical” ion exchange chromatography in its “pure” form, and therefore some chromatographers proceed from empirical rather than theoretical principles in ion exchange chromatography.

The separation of specific substances depends primarily on the choice of the most suitable sorbent and mobile phase. Ion exchange resins and silica gels with grafted ionogenic groups are used as stationary phases in ion exchange chromatography.

2.4. SECTION EXCLUSION CHROMATOGRAPHY

Exclusion chromatography is an option! liquid chromatography, in which separation occurs due to the distribution of molecules between the solvent located inside the pores of the sorbent and the solvent flowing " between its particles.

Unlike other HPLC options, where separation coming Due to the different interactions of the components with the surface of the sorbent, the role of the solid filler in size exclusion chromatography is only to form pores of a certain size, and the stationary phase is the solvent that fills these pores. Therefore, the use of the term “sorbent” to these fillers is to a certain extent arbitrary.

A fundamental feature of the method is the ability to separate molecules according to their size in solution in the range of almost any molecular weight - from 10 2 to 10 8, which makes it indispensable for the study of synthetic and biopolymers.

Traditionally, the process carried out in organic solvents is still often called gel permeation chromatography, and in aqueous systems - gel filtration chromatography. In this book, a single term is adopted for both options, which comes from the English “Size Exclusion” - exclusion by size - and most fully reflects the mechanism of the process.

A detailed analysis of existing ideas about the very complex theory of the size exclusion chromatography process is carried out in monographs.

Total volume of solvent in the column Vt (this is often called the total volume of the column, since Vd does not take part in the chromatographic process) is the sum of the volumes of the mobile and stationary phases.

The retention of molecules in an exclusion column is determined by the probability of their diffusion into the pores and depends on the ratio of the sizes of molecules and pores, which is shown schematically in Fig. 2.15. Distribution coefficient Ka, as in other variants of chromatography, it is the ratio of the concentrations of a substance in the stationary and mobile phases.

Since the mobile and stationary phases have the same composition, then Kd a substance for which both phases are equally accessible is equal to unity. This situation is realized for molecules C of the smallest sizes (including solvent molecules), which penetrate into all pores (see Fig. 2.15) and therefore move through the column most slowly. Their retention volume is equal to the total volume of the solvent Vt-

Rice. 2.15. Model of molecular separation by measure in size exclusion chromatography

All molecules whose size is larger than the size of the sorbent pores cannot enter them (complete exclusion) and pass through the channels between the particles. They elute from the column with the same retention volume equal to the volume of the mobile phase V 0 - The partition coefficient for these molecules is zero.

Molecules of intermediate size, capable of penetrating only some of the pores, are retained in the column according to their size. The distribution coefficient of these molecules varies from zero to one and characterizes the fraction of the pore volume accessible to molecules of a given size. Their retained volume is determined by the sum of V o and the accessible portion of the pore volume.

QUALITATIVE ANALYSIS

A chromatographer entering the field of high-performance liquid chromatography must become familiar with the fundamentals of qualitative analysis. Qualitative analysis is used to identify a known product, obtained in a new way or found in a mixture with other products." It is necessary when isolating various components from complex biological and chemical mixtures, which is especially important in medicine, forensics, ecology, for monitoring the presence of some medicinal chemical products and their metabolites in bioml.ter.ials..„. "Familiarity with the basics of qualitative" analysis will help to avoid common mistakes, for example/distinguishing impurities in a sample from impurities in a solvent or checking the purity of a substance at more than one wavelength. spectrophotometer, but on different ones, etc.

Before proceeding with the analysis, it is necessary to determine whether the entire sample is eluted from the column by a given solvent system or not. To be sure of complete elution, it is necessary to collect all the liquid flowing from the column, evaporate the solvent, weigh the residue and find the degree of sample recovery.

Identification of components in HPLC can be done in three ways: 1) using retention information; 2) examine the zones obtained during separation in a liquid chromatograph column using spectral or chemical analysis methods; 3) directly connect the spectrum analyzer to the column.

Retention volume is used to record peaks in chromatography. V R or retention time t R. Both quantities are characteristic of a substance in a given chromatographic system. Since the retention time of the substance being separated consists of the interaction time in the column and the time of passage of the empty sections of the tube, it varies from instrument to instrument. It is convenient to have a substance not retained by a given column, taking it as a standard whose retention time and volume t 0 , V o. Chromatography of the substance and the standard must be carried out under the same conditions (pressure and flow rate). When identified by retention data, known individual substances that may be present in the samples are separated in the same chromatographic system and values ​​are obtained for them t R. Comparing these values t R with the retention time of the unknown peak, it can be found that they either coincide, in which case it is likely that the peaks correspond to the same substance, or t R known substance does not correspond t R unknown zone. Then an approximate estimate of the values ​​is still possible t R substances that are not available for direct measurement of the degree of their retention. Let's consider both options.

In the first case, a preliminary study of the sample is obviously necessary to postulate the presence of specific substances in it. When working with simple mixtures, it is not difficult to determine whether the degree of retention of the zones of the sample and known substances coincides or not, i.e. the values tB same or different. In the case of complex mixtures, several substances may elute with similar values t R, and the zones actually obtained during chromatographic separation overlap. As a result, obtaining accurate values t R becomes impossible for different zones. Reliability of identification increases with increasing resolution, more careful control of separation conditions, and repeated measurement of values t R and averaging the found values. In this case, the chromatographic separation of known and unknown substances must alternate. When separating complex mixtures, the value t R substances can change under the influence of the matrix of the sample itself. This effect is possible at the beginning of the chromatogram and when peaks overlap; It is also possible to tighten the zones, as has already been mentioned.

In such cases, the standard should be added to the sample in a 1:1 ratio. If the substances are identical, the value t R the starting material does not change, and only one peak is obtained in the chromatogram. If you have a device with a cyclic chromatography system, then for reliable identification it is advisable to pass the mixture through the column several times.

Information on retention rates can also be found in the literature, but the value of this information is limited. Since columns from even the same batch give poor reproducibility, literature values ​​do not always correspond to the true value t R on this column. For adsorption chromatography, however, it is possible to predict t R based on literature data. Another difficulty associated with the use of literary meanings t R, - the difficulty of finding them in the specialized literature, although bibliographic reviews published in the Jornal of chromatography have an updated index by type of substance.

In the second case, when the retention times of known compounds and sample zones do not coincide, it is possible to predict the retention time of the unknown component. Predictions of relative retention based on structure data in steric exclusion chromatography are quite reliable. They are less accurate in adsorption and partition chromatography, and especially when working on a chemically bound phase. For ion and ion-pair chromatography of substances with known p Ka Only approximate determinations of values ​​are possible tR. It is always easier to predict relative retention or *x values ​​than absolute values k". Relative values t R easier to assess for related compounds or derivatives, such as substituted alkylcarboxylic acids or benzene derivatives.

When isocratically separating homologues or oligomers, the following pattern is sometimes observed:

\ gk" = A + Bn,

Where A And IN- constants for a number of selected samples and for a given chromatographic system (on the same column, with the same mobile and stationary phases); P- the number of identical structural units in the sample molecule.

Introduction of a functional group / into the sample molecule will lead to a change k" in the first equation by some constant coefficient a/ in a given chromatographic system. It is possible to obtain group constants a/ for various substituent groups/, the values ​​of which will increase with increasing polarity of functional groups in all types of chromatography, except reverse phase, where the values ​​of the constants will decrease with increasing polarity.

Some group constants a/ for various substituent groups/ are given in table. 9.1.

In adsorption chromatography, the first equation is not always applicable, since it is valid provided that all isomers have the same value k", which is not always observed. It is possible, however, to plot the logfe" of the same compounds on one column versus the logfe" of the same compounds on a different column or versus corresponding characteristics in thin layer chromatography, for example, log[(l- Rf) IRf].

Capacity coefficient values ​​can be used when comparing retention data k", because unlike him t R the speed of the mobile phase and the geometric features of the column are not affected.

Chemically bound phase separations are similar to partition chromatography separations with similar phases, and therefore steady state extraction data can be used to predict retention times.

In ion exchange chromatography, three factors influence the degree of retention: the degree of ionization of acids and bases, the charge of the ionized molecule, and the ability of the substance from the aqueous mobile phase used in ion exchange chromatography to migrate into the organic phase. The latter depends on the molecular weight of the compound and its hydrophobicity. Therefore, stronger acids or bases are more strongly retained during anion-exchange or cation-exchange separation. When decreasing pK a of an individual acid included in the sample, the retention increases when a number of acids are separated due to anion exchange, and with an increase in p/C o, the retention of bases increases when they are separated due to cation exchange.

Thus, the coincidence of the retention times of a known substance with the observed one makes it possible to assume their identity. The reliability of identification increases if the chromatograms of a known substance and an unknown component are compared under different conditions. If substances behave identically in adsorption and reverse phase or ion exchange and size exclusion chromatography, the reliability of identification increases. If the reliability of identification with equal relative retention is 90%, then when studying the behavior of the same substances under significantly different conditions, the reliability of identification is already 99%.

A valuable characteristic of a substance used in identification is the ratio of the signals obtained for a given substance on two different detectors. The analyzed substance, after leaving the column, passes first through the first detector, then through the second, and the signals coming from the detectors are recorded simultaneously using a multi-pen recorder or on two recorders. Typically, a series connection of an ultraviolet detector (more sensitive, but selective) with a refractometer, or an ultraviolet with a fluorescence detector, or two ultraviolet detectors operating at different wavelengths is used. The relative response, i.e. the ratio of the refractometer signal to the photometer signal, is a characteristic of the substance, provided that both detectors operate in their linear range; this is tested by administering different amounts of the same substance. Qualitative information can be obtained by working with photometric detectors equipped with a stop flow device that allows the spectrum of the peak emerging from the column to be recorded while it is in the flow cell, comparing it with the spectrum of a known compound.

Modern, still expensive, spectrophotometers with a diode array are of significant interest in identification.

A completely unknown substance cannot be identified using high-performance liquid chromatography alone; other methods are also necessary.

QUANTITATIVE ANALYSIS

Quantitative liquid chromatography is a well-developed analytical method that is not inferior in accuracy to quantitative gas chromatography and significantly exceeds the accuracy of TLC or electrophoresis. Unfortunately, in HPLC there is no detector that would have close sensitivity for compounds of different chemical structures (like a katharometer in GLC). Therefore, to obtain quantitative results, calibration of the device is mandatory.

Quantitative analysis consists of the following stages: 1) chromatographic separation; 2) measurement of peak areas or heights; 3) calculation of the quantitative composition of the mixture based on chromatographic data; 4) interpretation of the results obtained, i.e. statistical processing. Let's consider all these stages.

4.1. CHRMATOGRAPHIC SEPARATION

Errors may be made during sample collection. It is especially important to avoid error and to obtain an adequate representative sample of heterogeneous solids, volatile or unstable substances, and agricultural products and biomaterials. Heterogeneous samples, for example food products, are thoroughly mixed and quartered. By performing this operation many times, sample homogeneity is achieved.

Errors and losses of substances can be made at the stage of extraction, isolation, purification, etc.

Samples must be completely dissolved and their solutions prepared to an accuracy of ±0.1%. It is advisable to dissolve the sample in the mobile phase, which will eliminate the possibility of its precipitation after introduction into the chromatograph. If dissolution in the mobile phase is not possible, then a solvent miscible with it should be used and sample volumes (less than 25 µl) should be introduced into the chromatograph.

Significant errors can occur during sample injection due to sample fractionation, leakage, and peak smearing. The blurring of peaks causes the formation of tails, leading to partial overlap of peaks and, as a consequence, to errors in detection. Loop valve devices are preferable to syringes for sample introduction in quantitative analysis due to higher accuracy and less operator dependency.

When chromatographic separation of substances, complications can also arise that lead to distortion of data: quantitative analysis. There may be decomposition or conversion of the sample during the chromatographic process or irreversible adsorption of the substance onto the column. It is important to ensure the absence of these undesirable phenomena and, if necessary, regenerate the column or replace it. Peak overlap and tailing can also be reduced by varying the chromatography conditions.

Peaks with false or unclear shapes, as well as peaks whose release time is close to to, since their separation may not be sufficient. Typically, peaks with a value d"^0.5 are used. The highest efficiency of the column is achieved by introducing 10~ 5 -10~ 6 g of dissolved substance per 1 g of sorbent. When introducing large quantities of sample, the dependence of the peak height on the load may turn out to be nonlinear and a quantitative assessment is required by peak areas.

Errors associated with detection or amplification lead to significant distortion of the results of chromatographic separation. Each detector is characterized by specificity, linearity and sensitivity. Selectivity testing is especially important when analyzing trace impurities. The response of UV detectors can vary by a factor of 104 to substances with similar functional groups. It is necessary to calibrate the detector response for each analyte. Naturally, substances that do not absorb in the UV region will not give a signal to the recorder when used as a photometer detector. When using a refractometer, negative peaks may appear. In addition, this detector must be thermostated, which is not required for the UV detector.

The linearity of the detector determines the size of the injected sample. It is important to remember that column flow rate, column and detector temperatures, and detector design all affect the accuracy of the quantitative analysis. Errors in the transmission of an electrical signal to an output device (recorder), integrator or computer can arise due to noise induction, lack of grounding, voltage fluctuations in the network, etc.

4.2. MEASUREMENT OF PEAK AREA OR HEIGHT

Peak height h (Fig. 10.1) is the distance from the top of the peak to the baseline; it is measured linearly or by counting the number of divisions on a recorder. Some electronic integrators and computers provide information on peak heights. The position of the baseline of the shifted peaks is found by interpolating the ordinate values ​​corresponding to the beginning and end of the peak (peak 1 and 3 see fig. 10.1). To improve accuracy, it is necessary to have a flat, stable baseline. In the case of unsplit peaks, the baseline is drawn between the start and end of the peak rather than replaced by a zero line. Because peak heights are less dependent on the influence of adjacent overlapping peaks, peak height estimation is more accurate and is almost always used in trace trace analysis.

Peak area can be determined in various ways. Let's look at some of them.

1. The planimetric method involves tracing the peak with a hand-held planimeter, which is a device that mechanically determines the area of ​​the peak. The method is accurate, but labor-intensive and poorly reproducible. This method is not recommended.

2. Paper silhouette method - the peak is cut out and weighed. The method is highly reproducible, but labor-intensive, and the chromatogram is destroyed. Its applicability depends on the uniformity of the chart strip. The method also cannot be widely recommended.

4. The triangulation method consists of constructing a triangle by drawing tangents to the sides of the peak. The top of the triangle is higher than the top of the peak. Increasing the area formed by this extended vertex will be consistent throughout the chromatogram and will not greatly affect the accuracy. In addition, some area lost when drawing tangents will be compensated. The base of the triangle is determined by the intersection of the tangents with the base line, and the area is determined by the product of 7 g of the base and the height. This method is the best for determining the areas of asymmetric peaks. However, the reproducibility when constructing tangents by different operators is different and therefore; low.

5. The disk integrator method is based on an electromechanical device attached to a recorder. The pen attached to the integrator moves along a strip at the bottom of the tape at a speed proportional to the movement of the recorder pen.

As with manual measurements, the peak should remain on the recorder scale, but adjustments to compensate for baseline shifts and incomplete separation of adjacent peaks reduce reliability and increase analysis time.

The method is more accurate than manual measurement methods, especially for asymmetric peaks, and offers a speed advantage. In addition, it provides a permanent quantitative record of the analysis.

6. Methods using electronic integrators that determine peak area and print information about that area and retention times may include baseline shift correction and determine the area of ​​only partially resolved peaks. The main advantages are accuracy, speed, independence of action from the operation of the recorder. Integrators have memory and can be programmed for a specific analysis using a pre-installed program. The advantages of the integrator include its ability to use correction factors for the detector response when recalculating the original data on peak areas, compensating for differences in the sensitivity of the detector to different substances. Such systems save time, improve analytical accuracy, and are useful for routine analytical analysis.

7. In liquid chromatography, computers are widely used to measure peak areas. They print a complete message, including the names of the substances, peak areas, retention times, detector response correction factors, and abundance (wt %) for the various sample components.

Liquid chromatography is a method for separating and analyzing complex mixtures of substances in which a liquid serves as the mobile phase. It is applicable to the separation of a wider range of substances than gas chromatography. 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 into 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 liquid, and the sorption of components from 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 located 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 of the molecules of the analyte with 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 different eluents, the retention parameters and selectivity of the chromatographic system can be changed. Selectivity in liquid chromatography, unlike 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 and 3–4 orders of magnitude lower than in gas. This leads to slower mass transfer in liquid chromatography compared to gas chromatography. The Van Deemter equation due to the fact that the term IN does not play a role in liquid chromatography ( D and  D G), the graphical dependence of efficiency also changes N from the linear flow rate of the mobile phase has the form shown in Fig. 1.9.

In the classic version of column liquid chromatography, the analyzed sample dissolved in the eluent is introduced into a glass column 1–2 m high, filled with a sorbent with a particle size of 100 μm and an eluent, and the eluent is passed through, selecting portions of the eluate at the exit of the column. This version of liquid chromatography is still used in laboratory practice, but since the rate of passage of the eluent under the influence 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 superficially porous sorbents with a particle size of 5–10 microns, injection pumps providing system pressure 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 analyzed substances to be separated on the column, as mentioned above, the capacity coefficient k" must be greater than 0, i.e., the substances must be retained by the stationary phase, the sorbent. However, the capacity coefficient should not be too large in order to obtain an acceptable elution time. If for a given mixture of substances a stationary phase is selected that retains them, then further work on the development of an analysis technique consists in choosing a solvent that would ideally provide different but acceptably not very large k ". This is achieved by changing the elution strength of the solvent.

In the case of adsorption chromatography on silica gel or aluminum oxide, 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), or decreased by decreasing the isopropanol content. If the content of the polar component becomes too low (less than 0.1%), it should be replaced with a weaker elution force. The same is done, replacing either a polar or a non-polar component with others, even if this system does not provide the desired selectivity with respect to the components of interest in the mixture. When selecting solvent systems, both the solubility of the mixture components and the eluotropic series of solvents compiled by different authors are taken into account.

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

In the case of reverse phase chromatography, the solvent strength is increased by increasing the content of the organic component in the eluent (methanol, acetonitrile or THF) and decreased by adding more water. If it is not possible to achieve the desired selectivity, they use another organic component or try to change it using various additives (acids, ion-pair reagents, etc.).

In separations using ion exchange chromatography, the strength of the solvent is changed by increasing or decreasing the concentration of the buffer solution or 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 select the solvent strength so that all sample components elute within an acceptable time frame. Then you have to resort to gradient elution, i.e. use a solvent whose elution strength changes during the analysis process so that it constantly increases according to a predetermined program. This technique makes it 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 reverse-phase (RPC) chromatography. In NPC, a polar adsorbent and non-polar mobile phases are used, in OPC, a non-polar adsorbent and polar mobile phases are used, but in both options, the choice of the mobile phase is often more important than the choice of the stationary phase. The stationary phase must retain the substances being separated. The mobile phase, i.e., the solvent, must provide varying column capacities and efficient separation within an acceptable time.

Polar and non-polar finely dispersed porous materials with a specific surface area of ​​more than 50 m 2 /g are used as a stationary phase in adsorption liquid chromatography. Polar adsorbents (SiO 2 , Al 2 O 3 , florisil, etc.) have weak acid groups on the surface that can retain substances with basic properties. These adsorbents are used mainly for the separation of non-polar and moderately polar compounds. Their disadvantage is their high sensitivity to the water content in the eluents used. To eliminate this disadvantage, 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 carbon black, diatomaceous earth, kieselguhr) are non-selective towards polar molecules. To obtain nonpolar adsorbents, nonpolar groups are often grafted onto the surface of, for example, silica gel, for example, alkylsilyl SiR 3, where Ralkyl groups are C 2 C 22.

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

Mobile phases used in liquid chromatography vary in their elution strength. The elution force 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, therefore the distribution coefficients of sorbed substances (sorbate) are high. On the contrary, strong solvents are adsorbed well, so the sorbate partition coefficients are low. The stronger the solvent, the higher the solubility of the analyzed sample in it, and the stronger the solvent-sorbate interaction.

To ensure high separation efficiency on a column, it is necessary to select a mobile phase that has a polarity that is optimal for the mixture to be separated under the selected separation conditions. Typically, a stationary phase is first selected that has a polarity close to that of the components being separated. Then the mobile phase is selected, ensuring that the capacity 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 are very different, the retention time will become too long.

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

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

Table 1.2

Characteristics of solvents used in liquid chromatography

Solvent	Polarity index	Elution force (SiO 2)	Short-wavelength transparency limit
Fluoroalkan
Cyclohexane
n-Hexane
Carbon tetrachloride
Diisopropyl ether
Diethyl ether
Dichloromethane
Tetrahydrofuran
Chloroform
Acetic acid
Acetonitrile
Nitromethane

In liquid chromatography, mixtures of them rather than individual solvents are often used. Often minor additions of another solvent, especially water, will significantly increase the eluent strength of the eluent.

When separating multicomponent mixtures, a single mobile phase as eluent may not separate all sample components in an acceptable time. In this case, a stepwise or gradient elution method is used, in which increasingly stronger eluents are sequentially used during the chromatography process, 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 at 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 very polar molecules the retention times are so long that analysis is not possible with a non-polar eluent. To reduce the retention time of polar components, switch to polar eluents;

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

- By reducing the polarity of the eluent by adding a less polar solvent, the retention of 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 immiscible liquid phases, one of which is stationary and located on the surface or in the pores of a solid stationary 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, too, the separation is based on the difference in the forces of intermolecular interaction between the components of the analyzed sample and the stationary and mobile liquid phases.

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

Substances that are indifferent to the mobile solvent and components of the analyzed sample, but capable of retaining the stationary phase on the surface and in the pores, are used as solid carriers. Most often, polar substances (cellulose, silica gel, starch) are used as carriers. A stationary phase—a polar solvent, most often water or alcohol—is applied to them. 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 version of partition chromatography differs in that non-polar carriers (rubber, fluoroplastic, hydrophobized silica gel) are used as a stationary solid phase, non-polar solvents (hydrocarbons) are used as a stationary liquid phase, and polar solvents (alcohols, aldehydes) are used as a mobile liquid phase , ketones, etc., often water). This version of partition chromatography is called reversed 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) must be selected so that the distribution coefficients of the mixture components differ quite significantly. The following requirements apply to liquid phases: requirements:

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

2) 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 a mobile phase with the stationary phase should be minimal.

Most often, in partition liquid chromatography, not individual substances, but their mixtures in various ratios are used as mobile liquid phases. 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 the separation of the components of a particular mixture being analyzed.

A significant disadvantage of this chromatographic method is that the applied stationary liquid phase is quickly washed off from the carrier. To eliminate it, the solvent used as the mobile phase is saturated with a substance used as the stationary liquid phase, or the stationary liquid phase is stabilized by grafting it to the 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, degassing devices, detectors, dispensers (autosamplers), column thermostats, fraction collectors, chromatographic system control units and recording devices are available as separate modules. A wide selection of modules allows you to flexibly solve various analytical problems and 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 units and the compactness of the device.

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

Schematic of an isocratic chromatograph

The mobile phase from the container (1) through the inlet filter (9) is supplied by a precision high-pressure pump (2) to the sample injection system (3) - a manual injector or autosampler, and the sample is also introduced there. Next, through an in-line filter (8), the sample with a 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 container (7). When the eluate flows through the measuring circuit of the detector, the chromatogram is recorded and data is transferred 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 at which the gradient of the mobile phase composition is formed.

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

The mobile phase from containers (1) through input filters (9) and a gradient programmer (10) is supplied by a precision high-pressure pump (2) to the sample injection system (3) - a manual injector or autosampler, and the sample is also introduced there. 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. Next, through an in-line filter (8), the sample with a 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 container (7). When the eluate flows through the measuring circuit of the detector, the chromatogram is recorded and data is transferred 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 carried out by a control program from a personal computer. In the case of control by a 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 precision high-pressure pump), these systems have a number of disadvantages, among which the main one, perhaps, is the strict need for thorough degassing of the mobile phase components even before the low-pressure mixer (gradient programmer chamber). It is carried out using special flow-through degassers. Because of this fact, their cost becomes comparable to another type of gradient systems - systems with the formation of a mobile phase gradient composition on a high-pressure line.

The fundamental difference between systems with the formation of a mobile phase gradient composition on a 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 thorough degassing of components are significantly reduced.

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

The mobile phase from containers (1) through input filters (9) is supplied by precision high-pressure pumps (2 and 11) through a static or dynamic flow mixer (10) into the sample input system (3) - a manual injector or autosampler, and the sample is also introduced there. The operation of controlled pumps is controlled either by the system control module (master pump or controller) or by a PC control program. In this case, all pumps are controllable. 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. Next, through an in-line filter (8), the sample with a current of the mobile phase enters the separation element (elements) (4) - through the pre-column into the separation column. The eluate then enters the detector (5) and is removed into the drain container (7). When the eluate flows through the measuring circuit of the detector, the chromatogram is recorded and data is transferred 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 carried out by a control program from a personal computer. In the case of control by a control module, it is possible to independently control the detector from its own keyboard.

The proposed schemes are quite simplified. The systems may include additional devices - a column thermostat, post-column derivatization systems, sample preparation and sample concentration systems, a solvent recycler, membrane systems for suppressing background electrical conductivity (for ion chromatography), additional protective systems (filters, columns), etc. The diagrams also do not show manometric modules separately. As a rule, these devices are built into pump units. These units can combine several 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 that 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 dielectric constant, etc.), inert to the materials of the chromatographic tract parts, not form gas bubbles in the pump valves and detector cell, not have mechanical impurities.

In liquid chromatography Many types of pumps are used. For low-pressure LC, peristaltic pumps are often used (Fig. 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 include: flow range; maximum working pressure; flow reproducibility; solvent supply pulsation range.

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

Based on their operating principle, HPLC pumps are divided into: syringe and on plunger reciprocating .

Syringe pumps

The main distinctive feature of these pumps is the cyclical nature of their operation, and therefore the chromatographs in which these pumps are used are also distinguished by their cyclical operation.

Rice. 2. Basic design of a syringe pump for HPLC.

Rice. 2A. Syringe pump.

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

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

Disadvantages of the pump:

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

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

c) suspension of separation while filling the pump;

d) large dimensions and weight while ensuring high flow and pressure (a powerful engine and a large piston force with its large area are needed).

Reciprocating plunger pumps.

Rice. 3. Basic design of a plunger pump.

Operating principle.

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

Pumps of this type provide a constant volumetric supply of the mobile phase for a long time. Maximum operating pressure 300-500 atm, flow rate 0.01-10 ml/min. Volumetric feed repeatability -0.5%. The main disadvantage is that the solvent is supplied to 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 reduced sensitivity of almost all detectors used in LC, especially electrochemical ones.

Fig.4. Plunger pump pulsations.

Ways to deal with pulsations.

1. Application of damping devices.

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

Rice. 5. Spiral damper.

The damper unwinds as the pressure in it increases (pump acceleration). When the pressure drops, it curls, its volume decreases, it squeezes out part of the solvent, maintaining a constant flow rate and reducing pulsations. This damper works well at pressures of 50 atm and above.

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

Rice. 6. Air damper.

2. Application of electronic devices.

When using an electronic pressure sensor, you can use the sensor readings 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 concentrations of exchanged ions in solution and in the sorbent phase is characterized by ion exchange equilibrium. Ion exchange is that some substances (ion exchangers), when immersed in an electrolyte solution, absorb cations or anions from it, releasing into the solution an equivalent amount of other ions with a charge of the same sign. An exchange of cations occurs between the cation exchanger and the solution, and an exchange of anions occurs between the anion exchanger and the solution.

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

The chemical formulas of cation exchangers can be schematically depicted 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 – polymer matrix.

Cation exchange reactions are written as ordinary heterogeneous chemical reactions:

RNa +Na + RNa+H +

Anion exchangers contain in their structure basic ionogenic groups: –N(CH 3) 3 + ; =NH 2 + ; =NH + etc. Their chemical formulas can be depicted 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 case, 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 containing the same type (for example, SO 3 H) acidic (basic) groups are called monofunctional; ion exchangers containing different types (for example, SO 3 H, OH) acidic (basic) groups are polyfunctional.

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

The ion exchanger absorbs, as a rule, one of the counterions - ions present in the mobile phase, i.e. it exhibits a certain selectivity. The affinity series, or selectivity, of ions with respect to different types of ion exchangers has 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, sorption increases with increasing charge:

Na+ Ca 2+

However, changing the conditions of the ion exchange reaction can lead to the reversal of the series. Affinity series have also been established for anion exchangers. For example, the sorbability of anions on strong basic anion exchangers increases in the following order:

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 with 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 mixed or shaken for a certain time until equilibrium is established. This is a quick and simple method of ion exchange, used to concentrate ions from dilute solutions, removing unnecessary impurities, but it does not ensure complete absorption of ions, since ion exchange is a nonequilibrium process, and as a result does not guarantee complete separation of ions.

When performing ion exchange in a dynamic way a solution is passed through the column with the ion exchanger, which, as it moves along the column, comes into contact with new ion exchanger granules. This process provides a more complete exchange than the static method, since the exchange products are removed by the flow of solution. They can concentrate ions from dilute solutions and separate ions that differ greatly in properties, for example, ions of different charges (separate cations from anions), but separating 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 significantly less than the capacity of the column, due to which multiple repetitions of elementary acts of ion exchange are ensured.

In terms of analysis techniques, ion exchange chromatography is similar to molecular chromatography and can be carried out using 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 development ion exchange chromatography, which is used most often, a column filled with an ion exchanger is first washed with an electrolyte solution until all its ions are completely replaced in the ion exchanger by the ions contained in the eluent. Then a small volume of a solution of the analyte, containing the separated ions in an amount of about 1% of the ion exchanger capacity, is introduced into the column. Next, the column is washed with the eluent solution, selecting eluate fractions and analyzing them.

A mixture of Cl – , Br – , J – ions can be separated on a highly basic anion exchanger (cross-linked polystyrene containing groups of quaternary ammonium bases – N (CH 3) 3 +), for example, AB-17, which has a number 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 completely saturated with NO 3 – ions. When the mixture to be separated is introduced into the column, the Cl – , Br – , J – ions are absorbed by the anion exchanger, displacing NO 3 – ions. When the column is subsequently washed with a NaNO 3 solution, the Cl – , Br – , J – ions in the upper layers of the anion exchanger are gradually replaced again by NO 3 – ions. Cl – ions will be displaced the fastest, and J – ions will remain in the column the longest. The difference in the selectivity of the ion exchanger to the ions of the mixture leads to the formation of separate zones of sorbed Cl – , Br – and J – ions 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 eluent anion; in the interval between the zones there is only the eluent anion. Thus, fractions will appear in the eluent at the outlet of the column, which contain individual components of the mixture being separated.

To solve practical problems, the conditions for ion separation are varied by selecting a suitable mobile phase (composition, concentration, pH, ionic strength) or 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 exchanging them and impenetrable to others. It is also possible to change the nature and relative arrangement of ionogenic groups, as well as obtain sorbents capable of selective chemical reactions due to complex formation. For example, complex-forming ion exchangers containing in their structure chelating groups of organic reagents dimethylglyoxime, dithizone, 8-hydroxyquinoline, etc., as well as crown ethers, have high selectivity.

The greatest use in ion exchange, ion and ion-pair chromatography is found in synthetic macro- and micromesh organic ion exchangers with 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 ion exchangers are capable of exchanging ions only 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 ion exchanger. The total diameter of such a particle is about 40 µm, 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 microns, as well as 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 reverse phases C 2, C 8, C 18, which are easily converted into a cation exchanger when absorbing 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 transform into a completely or partially dissociated form. This ensures rapid exchange of counterions. The elution strength of the mobile phase is mainly influenced by pH, ionic strength, the nature of the buffer solution, and the content of organic solvent or surfactant (ion-pair chromatography).

The pH value is selected depending on the nature of the ionogenic groups, the separated ions and the matrix. You can work with strongly acidic and strongly basic ion exchangers at pH = 2–12, with weakly acidic ones at pH = 5–12, 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. As the ionic strength increases, the sorption of ions usually decreases, as the elution force 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 selective elution of ions absorbed by an ion exchanger, you can use water, buffer solutions (phosphate, acetate, borate, hydrocarbonate, etc.) with a certain pH value and ionic strength, mineral solutions (hydrochloric, nitrogen, sulfur, phosphorus) and organic (phenol, citric, lactic, tartaric, oxalic, EDTA) acids. The choice of eluent is facilitated by the fact that the limiting distribution coefficients of most elements between aqueous (aqueous-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 located in the pores of the sorbent and the solvent flowing between its particles. During the separation process, 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-sized ones, and finally the small ones.

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

The duration of retention 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 pore size of the stationary phase.

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

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

All molecules of the multicomponent substance being analyzed 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 ends before the solvent peak is released. Therefore, in this type of chromatography it is necessary to use fairly 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 region of separable molecular weights and a certain calibration curve. In this case, the calibration graph characterizing the dependence of the retained volume on the molecular weight or molecular size, as a rule, has a complex appearance.

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 aqueous-organic). Depending on this, the type of sorbent is determined. If it is necessary to separate water-soluble samples, for example, water-swellable cross-linked dextrans (Sephadex) or polyacrylamides (Biogel R) are used as stationary phases. The separation of substances soluble in organic solvents can be carried out on polystyrenes with varying degrees of cross-linking, swelling in organic solvents (styrogel, poragel, biobid C). Such swollen gels are typically pressure unstable 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 that is appropriate in polarity.

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

- completely dissolve the analyzed sample;

 wet the sorbent well;

- counteract the adsorption of sample components on the sorbent;

 have low viscosity and toxicity.

1.6.5. Plane chromatography. Plane chromatography includes thin layer chromatography and paper chromatography. These types of liquid chromatography are simple in technique, rapid, and 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 reverse-phase, ion-exchange, etc. paper and thin-layer chromatography are distinguished. Currently, thin layer chromatography is most widely used.

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

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 stationary phase layer at different rates in accordance with the distribution coefficients of the substances being separated. In both cases, chromatographic systems are used: liquid - solid sorbent (adsorption separation mechanism), liquid - liquid - solid carrier (distribution, ion exchange and other mechanisms).

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

The practical production of planar chromatograms is as follows.

On a strip of chromatographic paper or on a thin layer of sorbent, mark a starting line with a pencil at a distance of 1 cm from the bottom edge of the strip or plate. Using a micropipette, apply the sample to the starting line in the form of a spot with a diameter of no more than 2–3 mm. The edge of the strip or plate is then lowered into a vessel containing the mobile phase located in a sealed chamber. As the mobile phase rises along the strip or plate and 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 passes from the starting line 10 cm. After this, the strip or plate is removed from the chamber and dried. If the components of the analyte are colored, they give corresponding colored spots on the chromatogram. To detect uncolored 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. Manifestation can be carried out:

- using UV lighting. The method is applicable for the detection of substances capable of emitting their own radiation (luminesce) in the visible wavelength range under the influence of UV radiation;

- through developing reagents. 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. Spots corresponding to various components of the mixture acquire a visual and, as a rule, color specific to 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 vapor is more strongly adsorbed on the spots, making the spots visible. Iodine is a nonspecific developer reagent. Using specific reagents, it is possible not only to determine the number of components of the mixture, but also to identify the separated substances by the color of the spots.

Paper and thin layer chromatography are most often carried out in the so-called ascending version described above. Quite often, to improve the quality of chromatograms, it is necessary to use more complex variants of planar chromatography, for example, descending, circular, two-dimensional. When carrying out descending paper or thin layer chromatography, the analyte is applied to the starting line of a plate or paper strip located on top, and the eluent is supplied not from below, but from above. The positive effect of improving separation is due to the contribution of gravity of the components to the separation process.

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

When performing circular chromatography, the analyte is applied as a drop into the middle of a plate or sheet of chromatography paper. One or more solvents are also added dropwise here. This results in the resulting chromatogram being 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 relative speed of movement of the components in a thin layer R fi. Experimentally the value R fi defined as the ratio of the distance L i passed i-th component, to the distance L traversed by the solvent from the starting line to the front line (Fig. 1.10):

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

Magnitude 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 diameter of the spot is identical to the height or area of ​​the chromatographic peak and, therefore, to some extent reflects the quantitative content of the substance.

In the simplest case, the quantitative determination of the composition of the analyzed sample can be assessed visually by the intensity of the spots’ own color or the intensity of the fluorescent glow of the resulting spots during UV detection. For these purposes, 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 using the appropriate physicochemical method. You can also use the weight method, in which the corresponding spot is cut out from the chromatogram and weighed. The amount of a 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 BH method, the carrier is a special chromatography paper with certain properties. Stationary phase water is adsorbed on the surface and pores of the paper (up to 20%), mobile is an organic solvent, miscible or immiscible with water, water or electrolyte solutions.

Mechanism On paper it's quite complicated. In the stationary phase, a substance can be retained not only due to dissolution in water adsorbed by paper, but also adsorb directly from cellulose. Printed 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 first chromatography some of the substance from the paper goes into mobile phase and move on. When the organic solvent reaches a section of the paper that does not contain the solute, it occurs again. 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 retained at the beginning of the path, others move further. Here they combine thermodynamic (establishing an equilibrium distribution of substances between phases) and kinetic (movement of components at different speeds) aspects of separation. As a result, each component is concentrated on a specific area of ​​the paper sheet: zones of individual components on 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, changes in the water content in the pores of the paper when storage conditions change, a very low chromatography speed (up to several days), and low reproducibility of results. These shortcomings seriously affect the spread of paper chromatography as a chromatographic method.

IN 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 is provided by movement mobile phase (solvent) through the sorbent under the influence capillary forces . Byseparation mechanism differentiate partition, adsorption and ion exchange chromatography . The separation of 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 varying degrees of sorption-desorption of the separated components on the stationary phase. Adsorption carried out at the expense of van der Waals forces , which is the basis physical adsorption , polymolecular (formation of several layers of adsorbate 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 , so adsorption on the developed active surface of the sorbent (150–750 m 2 /g). Distribution components of the mixture occurs between water on the surface of the carrier (such adsorbents , How alumina , starch , cellulose , kieselguhr - And water form stationary phase ), and the solvent moving through this stationary phase ( mobile phase ). The component of the mixture that is more soluble in water moves more slowly than the one that is more soluble in the mobile phase.

Adsorption manifests itself in the fact that between carrier , for example, aluminum oxide, and the components of the mixture are established adsorption equilibria – each component has its own, the result of which is different moving speeds components of the mixture. 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 completely adsorbed, does not interact with the solvent and remains at the start.

In practice, with skillful selection of solvent and adsorbent distribution compounds are located between these extreme cases, and the substance gradually transferred 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 the eluent  by elution . As the liquid moves along the plate, the mixture of substances separates due to the action of forces adsorption , distribution , ion exchange or the combination of all of these factors. As a result, separate chromatographic zones components of the mixture, 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 elution force (polarity) of the eluent. As the polarity of a compound increases, its affinity for the polar sorbent also increases. 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 order of increasing “polarity” ( elution capacity ) and form a series of solvents ( eluotropic series ) in accordance with experimental data: alkanes>benzene>chloroform>diethyl ether>ethyl acetate>C 2 -C 4 alcohols>water>acetone>acetic acid>methanol. Thus, the polar compound, alcohol, is quite strongly adsorbed on silica gel and therefore moves weakly under the influence of a non-polar solvent such as hexane, and remains near the starting line. In turn, the nonpolar aromatic hydrocarbon biphenyl is noticeably more mobile in hexane, but even here to achieve R f about 0.5, a more polar aprotic eluent is required - methylene chloride. Eluent strength regulate using mixtures of solvents that are neighboring eluotropic series with different polarity.

Currently, the following are mainly used in TLC: sorbents : for division lipophilic substances  silica gel , alumina , acetylated cellulose , polyamides ; for separation hydrophilic substances  cellulose , cellulose ion exchangers , kieselguhr , polyamides . The most important characteristic of the sorbent is its activity , i.e. ability sorb (hold) the components of the mixture to be separated. Abroad, a number of companies produce silica gel , kieselguhr And alumina with the addition of 5% gypsum, which is used to secure the sorbent layer when making plates independently.

The most common sorbent is silica gel - hydrated silicic acid, formed by the action of mineral acids on Na 2 SiO 3 and drying 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 polar sorbent with OH groups as active centers. It easily adsorbs 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 aluminum oxide plates is the mandatory activation of the surface before use in an oven at high temperatures (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: thin-layer plates coated with cellulose are very effective for separating complex organic molecules. The adsorbent consists mainly of cellulose beads with a diameter of up to 50 microns, fixed to a carrier with starch. As in paper chromatography, the rise of the solvent front occurs very slowly.

Chromatographic analysis performed on industrial plates made in the Czech Republic " Silufol » (« Silufol ") made 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 and without the addition of fluorescent indicators. Records « Silufol » have a high elution rate, but are characterized by low separation ability and low sensitivity. During storage, they are sensitive to conditions (humidity, temperature, aggressive environments, etc.). Some companies supply chromatographic plates with a sorbent layer of varying (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 an aluminum substrate (grade AF) with an applied working layer 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 - silica sol . When using silicic acid sol (silica sol) as a binder, which after heating turns into silica gel, the resulting TLC plates consist of two components: a silica gel layer and a substrate. The uniformity of the thickness of the sorbent layer on one plate is ±5 µm. Example of designation: “Sorbfil-PTSH-AF-V-UV (10x10)” - high-performance TLC plates on an aluminum substrate, with a phosphor, 10x10 cm.

If you use a glass substrate (grade C), 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, a hot chromium mixture, which removes restrictions on the use of correlating reagents for detecting spots and modifying the sorbent, and allows for repeated (up to 30 times or more) regeneration of the plates with a chromium mixture. Glass plates can be cut to required sizes. The mechanical strength of the sorbent layer can be adjusted, providing, on the one hand, transportation and repeated processing of the plates and, on the other hand, the possibility of extraction of adsorbent layers with separated substances for subsequent leaching of individual compounds from the sorbent and their further study by instrumental methods (IR and UV spectrometry , X-ray structural 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 high-performance plates (grade B) - 8-12 microns. A narrower distribution increases the efficiency of the plates, i.e. the spots of the separated substances become more compact (smaller in size) and therefore are better separated when the eluent front passes a shorter distance. On Russian wafers, the analytical and high-performance layers do not differ very much, unlike wafers from Merck (Germany). High efficiency plates should be used if substances cannot be separated on the analytical plates. Plates of all modifications are produced with phosphor (UV grade) with excitation 254 nm. Shelf life is unlimited, plates " Sorbfil » widely tested in the analysis of amino acid derivatives, pesticides, lipids, antibiotics.

The TLC method is carried out quality identification components. quantitation for TLC is also possible, this requires applying the exact amount of substance and additional densitometric studies with a clear recording of the intensity of the spots. The most common is semi-quantitative method . It is based on visual comparison the size and intensity of a spot of a component with the corresponding characteristics of a series of spots of the same substance of varying concentrations ( standard reference solutions ). When using a sample in an amount of 1-5 μg, this simple method ensures an accuracy of determination of the component content of about 5-10%. Often, 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 aluminum oxide or silica gel.

There are several options for TLC and HD, 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;

V)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 applied.

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 being separated.

Chromatographic separation by BCh and TLC methods are carried out in separation chamber with a lapped lid. A quantitative measure of the rate of transfer of a substance when using a particular 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 by size coefficient R f , equal to the ratio of the speed of movement of its zone to the speed of movement of the solvent front. Magnitude R f is always less than one and does not depend on the length of the chromatogram. By the amount R f are influenced by various factors. Thus, at low temperatures, substances move more slowly; solvent contamination, inhomogeneity of the adsorbent, foreign ions in the analyzed solution can change the value R f up to 10%. In the chosen system, the analytes must have different values R f and distributed along the entire length of the chromatogram. It is desirable that the values R f was in the range of 0.05-0.85.

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

R f = l/L (6.1 )

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

Rice. 1. Determination of values ​​on the chromatogram Rf for components A And IN,

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

The efficiency of HD and TLC also depends on selectivity and sensitivity reactions used to detect the components of the analyzed mixture. Typically, reagents are used that form colored compounds - developers - with the components being determined. For more reliable identification of shared components apply " witnesses » solutions standard substances (in the same solvent as the sample), the presence of which is expected 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 using 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 A for component A the mixture to be separated is calculated using the formula:

N A = 16 (l O.A. / a (A )) 2 (6.3)

Values l O.A. And A (A ) determined as shown in Fig. 6.1. Then the height of the equivalent theoretical plate N A is:

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

Separation is practically possible if R f (A)  R f (IN)  0,1 .

To characterize the separation of two components A And IN use degree (criterion) of separation Rs :

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

Where  l  distance between component spot centers A And IN;

A (A) And A (IN)  spot diameters A And IN on the chromatogram (Fig. 6.1). The more Rs , the more clearly the component spots are separated A And IN on the chromatogram. Conditions chromatography selected so that the value Rs differed from zero and one, the optimal value Rs is 0.3  0.7. For rate separation selectivity two components A And IN use separation factor α :

α = l B / l A (6.6)

If α = 1, then the components A And IN are not separated.

9801 0

HPLC is a liquid column chromatography in which a variety of sorption mechanisms can be used. Essentially, HPLC is a modern form of classical liquid column chromatography. Some of the most significant quality characteristics of HPLC are listed below:
- high speed of the process, which made it possible to reduce the duration of separation from several hours and days to minutes;
- minimal degree of blurring of chromatographic zones, which makes it possible to separate compounds that only slightly differ in sorption constants;
- a high degree of mechanization and automation of information separation and processing, thanks to which column liquid chromatography has reached a new level of reproducibility and accuracy.

Intensive research in recent decades and a huge amount of accumulated experimental data allow today to talk about the classification of variants within the framework of the high-performance liquid chromatography method. Of course, the classification according to the sorption mechanism given above remains valid.

A common classification is based on the comparative polarity of the mobile and stationary phases. In this case, a distinction is made between normal and reverse phase chromatography.

Normal phase chromatography (NPC) is a variant of HPLC when the mobile phase is less polar than the stationary phase, and there is reason to believe that the main factor determining retention is the interaction of sorbates directly with the surface or volume of the sorbent.

Reversed phase chromatography (RPC) is a variant of HPLC where the mobile phase is more polar than the stationary phase, and retention is determined by direct contact of sorbate molecules with the surface or volume of the sorbent; in this case, ionized sorbates are not exchanged for mobile phase ions sorbed on the surface.

Ion exchange chromatography is an option in which sorption is carried out by exchanging sorbed ions of the mobile phase for ions of the substances being chromatographed; Ligand exchange chromatography can be defined in a completely analogous manner.

Chromatography on dynamically modified sorbents is a variant of HPLC in which the sorbate does not interact directly with the surface of the sorbent, but enters into association with molecules of the surface layers of the eluent.
Ion-pair chromatography is a variant of reverse-phase chromatography of ionized compounds in which a hydrophobic counterion is added to the mobile phase, which qualitatively changes the sorption characteristics of the system.

Size exclusion chromatography is a method of separating compounds by their molecular weights, based on the difference in the diffusion rate of molecules of different sizes in the pores of the stationary phase.

For HPLC, a very important characteristic is the size of the sorbents, usually 3-5 microns, now up to 1.8 microns. This allows complex mixtures of substances to be separated quickly and completely (average analysis time from 3 to 30 minutes).

The separation problem is solved using a chromatographic column, which is a tube filled with a sorbent. When performing an analysis, a liquid (eluent) of a certain composition is fed through a chromatographic column at a constant speed. A precisely measured dose of sample is injected into this stream. The components of a sample introduced into a chromatographic column, due to their different affinities for the column sorbent, move along it at different speeds and reach the detector sequentially at different times.

Thus, the chromatographic column is responsible for the selectivity and efficiency of separation of components. By selecting different types of columns, the degree of separation of the analytes can be controlled. Compounds are identified by their retention time. Quantitative determination of each of the components is calculated based on the magnitude of the analytical signal measured using a detector connected to the output of the chromatographic column.

Sorbents. The development of HPLC is largely associated with the creation of new generations of sorbents with good kinetic properties and diverse thermodynamic properties. The main material for sorbents in HPLC is silica gel. It is mechanically strong and has significant porosity, which gives a large exchange capacity with small column sizes. The most common particle size is 5-10 microns. The closer to the spherical shape of the particles, the lower the flow resistance, the higher the efficiency, especially if a very narrow fraction is screened out (for example, 7 +1 microns).

The specific surface of silica gel is 10-600 m /g. Silica gel can be modified with various chemical groups grafted to the surface (C-18, CN, NH2, SO3H), which allows the use of sorbents based on it to separate a wide variety of classes of compounds. The main disadvantage of silica gel is its low chemical resistance at pH< 2 и рН >9 (silica dissolves in alkalis and acids). Therefore, there is currently an intensive search for sorbents based on polymers that are stable at pH from 1 to 14, for example, based on polymethyl methacrylate, polystyrene, etc.

Sorbents for ion exchange chromatography. Due to the peculiarities of separation (in an acidic or alkaline environment), the main material is sorbent into polystyrene with divinylbenzene of varying degrees of cross-linking with SO3 -H+ (strongly acidic cation exchangers) or -COO-Naf (weakly acidic cation exchangers), -H2N+ (CH3) groups grafted to their surface 3Cl- (strong basic anion exchangers) or -N+HR2Cl- (weak basic anion exchangers).

Sorbents for gel permeation chromatography. The main type is styrene-DVB. Macroporous glasses, methyl methacrylate, and silica gel are also used. The same sorbents are used for ion exclusion chromatography.
Pumps. To ensure the flow of the mobile phase (MP) through the column with the specified parameters, high-pressure pumps are used. The most important technical characteristics of LC pumps include: flow range; maximum working pressure; flow reproducibility; solvent supply pulsation range.

Depending on the nature of the solvent supply, pumps can be of constant supply (flow) and constant pressure. Basically, during analytical work, a constant flow rate mode is used, and when filling columns, a constant pressure mode is used. Based on their operating principle, pumps are divided into syringe pumps and reciprocating plunger pumps.

Syringe pumps. Pumps of this type are characterized by an almost complete absence of pulsations in the flow of the mobile phase during operation. Disadvantages of the pump: a) high consumption of time and solvent for washing when changing the solvent; b) suspension of separation while filling the pump; c) large dimensions and weight while ensuring high flow and pressure (a powerful engine and a large piston force with its large area are needed).

Reciprocating plunger pumps. Pumps of this type provide a constant volumetric supply of the mobile phase for a long time. Maximum operating pressure 300-500 atm, flow rate 0.01-10 ml/min. Volume flow reproducibility is 0.5%. The main disadvantage is that the solvent is supplied to the system in the form of a series of successive pulses, so there are pressure and flow pulsations.

This is the main reason for the increased noise and reduced sensitivity of almost all detectors used in LC, especially electrochemical ones. Ways to combat pulsations: using double pumps or a double-plunger Bag-Lai pump, using damping devices and electronic devices.

The amount of volumetric feed is determined by three parameters: the diameter of the plunger (usually 3.13; 5.0; 7.0 mm), its amplitude (12-18 mm) and frequency (which depends on the rotation speed of the motor and gearbox).

Dispensers. The purpose of the dispenser is to transfer a sample at atmospheric pressure to the inlet of a column at a pressure of up to several atmospheres. It is important that there are no “dead” volumes in the dispenser that cannot be washed by the mobile phase and that there is no erosion of the sample during dosing. At first, LC dispensers were similar to gas dispensers with a puncture of the membrane. However, the membranes do not withstand more than 50-100 atm; their chemical resistance is insufficient; their pieces contaminate column filters and capillaries.

The liquid phase has a much lower diffusion rate than the gas phase. Therefore, you can dose by stopping the flow - the sample does not have time to erode in the dispenser. While the sample is being introduced into the dispenser, a special valve shuts off the solvent flow. The pressure at the inlet to the column decreases quickly; after a few seconds, the sample can be injected into the dispenser chamber with a conventional microsyringe. Next, the dispenser is locked, the solvent flow is turned on, and separation occurs.

The pressure that this tap holds is up to 500-800 atm. But when the flow stops, the equilibrium in the column is disturbed, which can lead to the appearance of “vacant” additional peaks.

Loop dispensers are the most widely used. When the dispenser is filled, inlets 1, 2 and the channel between them are under high pressure. Inputs 3-6, the channels between them and the dosing loop are under atmospheric pressure, which allows you to fill the loop using a syringe or pump. When the dispenser is turned, the flow of the mobile phase displaces the sample into the column. To reduce the error, the loop is washed with 5-10 times the sample volume. If the sample is small, it can be injected into the loop with a microsyringe. The loop volume is usually 5-50 µl.

ON THE. Voinov, T.G. Volova

(mainly intermolecular) at the phase interface. As an analysis method, HPLC is part of a group of methods that, due to the complexity of the objects under study, includes the preliminary separation of the original complex mixture into relatively simple ones. The resulting simple mixtures are then analyzed using conventional physicochemical methods or special methods developed for chromatography.

The HPLC method is widely used in such fields as chemistry, petrochemistry, biology, biotechnology, medicine, food industry, environmental protection, pharmaceutical production and many others.

According to the mechanism of separation of the analyzed or separated substances, HPLC is divided into adsorption, distribution, ion exchange, exclusion, ligand exchange and others.

It should be borne in mind that in practical work, separation often occurs not through one, but through several mechanisms simultaneously. Thus, exclusion separation can be complicated by adsorption effects, adsorption separation by distribution effects, and vice versa. Moreover, the greater the difference between substances in a sample in terms of degree of ionization, basicity or acidity, molecular weight, polarizability and other parameters, the greater the likelihood of a different separation mechanism for such substances.

Normal phase HPLC

The stationary phase is more polar than the mobile phase, therefore the nonpolar solvent predominates in the eluent:

• Hexane:isopropanol = 95:5 (for low-polarity substances)
• Chloroform:methanol = 95:5 (for mid-polar substances)
• Chloroform:methanol = 80:20 (for highly polar substances)

Reversed phase HPLC

The stationary phase is less polar than the mobile phase, so the eluent almost always contains water. In this case, it is always possible to ensure complete dissolution of the BAS in the mobile phase, it is almost always possible to use UV detection, almost all mobile phases are mutually miscible, gradient elution can be used, the column can be quickly re-equilibrated, the column can be regenerated.

Common eluents for reverse phase HPLC are:

• Acetonitrile:water
• Methanol:water
• Isopropanol:water

Matrices for HPLC

HPLC uses inorganic compounds as matrices, such as silicon oxide (silica gel) or alumina, or organic polymers, such as polystyrene (cross-linked with divinylbenzene) or polymethacrylate. Silica gel is, of course, now generally accepted.

Main characteristics of the matrix:

• Particle size (µm);
• Internal pore size (Å, nm).

Preparation of silica gel for HPLC:

1. Molding of polysilicic acid microspheres;
2. Drying silica gel particles;
3. Air separation.

Sorbent particles:

• Regular (spherical): higher pressure resistance, higher cost;
• Non-spherical: lower pressure resistance.

Pore ​​size in HPLC is one of the most important parameters. The smaller the pore size, the worse their permeability for molecules of eluted substances. And therefore, the worse the sorption capacity of sorbents. The larger the pores, the less, firstly, the mechanical stability of the sorbent particles, and, secondly, the smaller the sorption surface, therefore, the worse the efficiency.

Stationary phase vaccinations

Normal phase HPLC:

• Stationary phase with propylnitrile grafting (nitrile);
• Stationary phase with propylamine grafting (amine).

Reversed phase HPLC:

• Stationary phase with alkyl grafting;
• Stationary phase with alkylsilyl grafting.

End-capping is the protection of ungrafted areas of the sorbent by additional grafting with “small” molecules. Hydrophobic end-capping (C1, C2): higher selectivity, worse wettability; hydrophilic end-capping (diol): lower selectivity, higher wettability.

Detectors for HPLC

• UV
• Diode matrix
• Fluorescent
• Electrochemical
• Refractometric
• Mass selective

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