(mainly intermolecular) at the phase boundary. 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 proceeds not by one, but by several mechanisms simultaneously. So, exclusion separation can be complicated by adsorption effects, adsorption - distribution, 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
The matrices used in HPLC are inorganic compounds such as 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:
- Molding of polysilicic acid microspheres;
- Drying silica gel particles;
- 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.
HPLC detectors
- UV
- Diode matrix
- Fluorescent
- Electrochemical
- Refractometric
- Mass selective
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See what “High Performance Liquid Chromatography” is in other dictionaries:
high performance liquid chromatography- - [A.S. Goldberg. English-Russian energy dictionary. 2006] Topics: energy in general EN high performance liquid chromatography HPLC ... Technical Translator's Handbook
The term high performance liquid chromatography The term in English high performance liquid chromatography Synonyms Abbreviations HPLC, HPLC Related terms adsorption, oligopeptide, proteomics, sorbent, fullerene, endohedral, chromatography... ...
Liquid chromatography, in which, to increase the efficiency of separation, the solvent (eluent) under pressure (more than 3x107 Pa) is pumped through columns filled with sorbent with particles of small diameter (up to 1 μm), and perfusion filters are also used... ...
A type of chromatography in which the liquid (eluent) serves as the mobile phase, and the stationary one. sorbent, TV a carrier with a liquid or gel applied to its surface. Carry out in a column filled with sorbent (column chromatography) on a flat... ... Natural science. encyclopedic Dictionary
- [κρώμα (υrum) color] a process based on the unequal ability of individual components of the mixture (liquid or gaseous) to remain on the surface of the adsorbent both when absorbing them from the carrier flow and when ... ... Geological Encyclopedia
- (from other Greek ... Wikipedia
The term chromatography The term in English chromatography Synonyms Abbreviations Related terms high performance liquid chromatography, clathrate, laboratory on a chip, porometry, proteome, proteomics, sorbent, enzyme, fullerene, endohedral... ... Encyclopedic Dictionary of Nanotechnology
Liquid chromatography based on decomp. ability of separated ions to ion exchange with fixed. sorbent ions formed as a result of dissociation of the latter’s ionogenic groups. Cation exchangers are used to separate cations, for... ... Chemical Encyclopedia
HPLC- high performance liquid chromatography… Dictionary of Russian abbreviations
High-performance liquid chromatography (HPLC) is one of the effective methods for separating complex mixtures of substances, widely used both in analytical chemistry and chemical technology. The basis of chromatographic separation is the participation ... Wikipedia
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- Practical High Performance Liquid Chromatography, Veronica R. Mayer. We present to the reader the 5th edition of the book, which has been expanded with modern methods and equipment. Much has been improved in the book and a large number of references have been added. Those places in the text where...
Liquid chromatography This is a method for separating and analyzing complex mixtures of substances in which liquid is the mobile phase. It is applicable to the separation of a wider range of substances than the gas chromatography method. This is due to the fact that most substances are not volatile, many of them are unstable at high temperatures (especially high-molecular compounds) and decompose when converted to a gaseous state. The separation of substances by liquid chromatography is most often carried out at room temperature.
The features of all types of liquid chromatography are due to the fact that the mobile phase in it is a liquid, and the sorption of components from a gaseous and liquid eluent proceeds differently. If in gas chromatography the carrier gas performs only a transport function and is not sorbed by the stationary phase, then the liquid mobile phase in liquid chromatography is an active eluent, its molecules can be sorbed by the stationary phase. When passing through the column, the molecules of the components of the analyzed mixture that are in the eluent must displace the molecules of the eluent from the surface layer of the sorbent, which leads to a decrease in the energy of interaction between the molecules of the analyzed substance and the surface of the sorbent. Therefore, the values of retained volumes V R, proportional to the change in the free energy of the system, is smaller in liquid chromatography than in gas chromatography, and the range of linearity of the sorption isotherm in liquid chromatography is wider.
By using various eluents, one can change the retention parameters and selectivity of the chromatographic system. Selectivity in liquid chromatography, in contrast to gas chromatography, is determined not by one, but by two factors - the nature of the mobile (eluent) and stationary phases.
The liquid mobile phase has a higher density and viscosity than the gaseous phase, diffusion coefficients D and 3–4 orders of magnitude lower than in gas. This leads to a slowdown in mass transfer in liquid chromatography compared to gas chromatography. The Van Deemter equation due to the fact that the term IN does not play a role in liquid chromatography ( D and D G), the graphic dependence of the efficiency also changes N on the linear velocity of the flow of the mobile phase has the form shown in fig. 1.9. 
In the classical version of column liquid chromatography, a glass column 1–2 m high, filled with a sorbent with a particle size of 100 μm and eluent, is injected with the analyzed sample dissolved in the eluent, and the eluent is passed through, taking portions of the eluate at the outlet of the column. This variant of liquid chromatography is still used in laboratory practice, but since the eluent flow rate under the action of gravity is low, the analysis is lengthy.
A modern version of liquid chromatography, the so-called high-performance liquid chromatography HPLC, uses volumetric and surface-porous sorbents with a particle size of 5–10 µm, pressure pumps that provide pressure in the system up to 400 atm, and highly sensitive detectors. Fast mass transfer and high separation efficiency make it possible to use HPLC for the separation of molecules (liquid-adsorption and liquid-liquid partition chromatography), for the separation of ions (ion-exchange, ion, ion-pair chromatography), for the separation of macromolecules (size-exclusion chromatography).
1.3. SOLVENT RETENTION AND STRENGTH
In order for the analytes to be separated on the column, as mentioned above, the capacity factor k" must be greater than 0, i.e. the substances must be retained by the stationary phase, the sorbent. However, the capacity factor should not be too large to obtain an acceptable elution time. If a stationary phase is chosen for a given mixture of substances, which holds them, then further work on the development of an analysis procedure consists in choosing a solvent that would provide, in the ideal case, different for all components, but acceptable not very large k. This is achieved by changing the eluting strength of the solvent.
In the case of adsorption chromatography on silica gel or alumina, as a rule, the strength of a two-component solvent (for example, hexane with the addition of isopropanol) is increased by increasing the content of the polar component (isopropanol) in it, or reduced by decreasing the content of isopropanol. If the content of the polar component becomes too low (less than 0.1%), it should be replaced with a weaker eluting strength. The same is done, replacing either the polar or non-polar component with others, even if this system does not provide the desired selectivity with respect to the mixture components of interest. When selecting solvent systems, both the solubilities of the mixture components and the eluotropic series of solvents compiled by different authors are taken into account.
Approximately in the same way, the strength of the solvent is selected in the case of using grafted polar phases (nitrile, amino, diol, nitro, etc.), taking into account possible chemical reactions and excluding solvents dangerous for the phase (for example, aldehydes and ketones for the amino phase).
In the case of reverse phase chromatography, the strength of the solvent is increased by increasing the content of the organic component (methanol, acetonitrile or THF) in the eluent and reduced by adding more water. If the desired selectivity cannot be achieved, another organic component is used or attempts are made to change it with the help of various additives (acids, ion-pair reagents, etc.).
In separations by ion-exchange chromatography, the strength of the solvent is changed by increasing or decreasing the concentration of the buffer solution or by changing the pH, in some cases modification with organic substances is used.
However, especially in the case of complex natural and biological mixtures, it is often not possible to choose the solvent strength in such a way that all sample components are eluted within an acceptable time. Then one has to resort to gradient elution, i.e. use a solvent whose eluting strength during the analysis changes so that it constantly increases according to a predetermined program. With this technique, it is possible to achieve the elution of all components of complex mixtures in a relatively short period of time and their separation into components in the form of narrow peaks.
1.6.1. Adsorption liquid chromatography. Adsorption liquid chromatography, depending on the polarity of the stationary and mobile phases, is divided into normal phase (NPC) and reversed phase (RPC) chromatography. In NPC, a polar adsorbent and non-polar mobile phases are used; in OFC, a non-polar adsorbent and polar mobile phases are used, but in both cases, the choice of the mobile phase is often more important than the choice of the stationary one. The stationary phase must hold the substances to be separated. The mobile phase, i.e. the solvent, must provide different column capacities and efficient separations in a reasonable time.
As a stationary phase in adsorption liquid chromatography, polar and non-polar finely dispersed porous materials with a specific surface area of more than 50 m 2 /g are used. Polar adsorbents (SiO 2 ,Al 2 O 3 , florisil, etc.) have weakly acidic groups on the surface, capable of retaining substances with basic properties. These adsorbents are mainly used for the separation of non-polar and medium polar compounds. Their drawback is high sensitivity to the water content in the eluents used. To eliminate this 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 soot, diatomite, kieselguhr) are non-selective to polar molecules. To obtain non-polar adsorbents, non-polar groups are often grafted onto the surface, for example, of silica gel, for example, alkylsilyl - SiR 3, where R - alkyl groups are C 2 - C 22.
The mobile phase 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 to the materials of all parts of the chromatograph, safe, cheap, suitable for the detector.
The mobile phases used in liquid chromatography differ in their eluting strength. The eluting power of a solvent shows how many times the sorption energy of a given eluent on a given adsorbent is greater than the sorption energy of the eluent chosen as a standard, for example, n-heptane. Weak solvents are poorly adsorbed by the stationary phase, so the distribution coefficients of sorbed substances (sorbate) are high. Conversely, strong solvents adsorb well, so the sorbate partition coefficients are low. The stronger the solvent, the higher the solubility of the analyzed sample in it, the stronger the solvent-sorbate interaction.
To ensure high separation efficiency on the column, it is necessary to select a mobile phase that has the optimal polarity for the mixture to be separated under the selected separation conditions. Typically, the stationary phase is selected first, which has a polarity close to that of the components to be separated. Then the mobile phase is selected, ensuring that the capacitance coefficient k" was in the range from 2 to 5. If the polarity of the mobile phase is too close to the polarity of the stationary phase, the retention time of the components will be too short, and if the polarities of the mobile and stationary phases differ very much, the retention time becomes too long.
When selecting mobile phases, they are guided by the so-called eluotropic series, based on the use of polarity indices Snider R", which subdivides all solvents into strong (polar) and weak (weakly polar and non-polar). The polarity scale is based on the solubility of substances used as mobile phases in dioxane, nitromethane and ethanol.
Table 1.2 shows the values of the polarity index and elution strength (with respect to SiO 2 ) of a number of solvents most commonly used in liquid chromatography as mobile phases. The short-wavelength transparency limits of these solvents are also indicated here, which facilitates the selection of conditions for detecting mixture components.
Table 1.2
Characteristics of solvents used in liquid chromatography
|
Solvent |
Polarity index |
Eluting power (SiO 2) |
Shortwave Transparency Limit |
|
Fluoralkane | |||
|
Cyclohexane | |||
|
n-Hexane | |||
|
Carbon tetrachloride | |||
|
Diisopropyl ether | |||
|
diethyl ether | |||
|
dichloromethane | |||
|
Tetrahydrofuran | |||
|
Chloroform | |||
|
Acetic acid | |||
|
Acetonitrile | |||
|
Nitromethane | |||
Liquid chromatography often uses not individual solvents, but their mixtures. Often, minor additions of another solvent, especially water, greatly increase the eluting strength of the eluent.
When separating multi-component mixtures, one mobile phase as eluent may not separate all sample components in a reasonable time. In this case, a stepwise or gradient elution method is used, in which increasingly stronger eluents are used sequentially during chromatography, which makes it possible to elute highly retained substances in less time.
In liquid chromatography, there are some empirical rules that are very useful when choosing an eluent:
sorption of a compound, as a rule, increases with an increase in the number of double bonds and OH groups in it;
sorption decreases in a number of organic compounds: acidsalcoholsaldehydesketonesestersunsaturated hydrocarbonssaturated hydrocarbons;
to separate substances of different polarity and separate substances of different classes, normal-phase chromatography is used: the analyzed sample is dissolved and eluted with a non-polar eluent, compounds of different classes leave the column with a polar adsorbent for different times, while the retention time of compounds with different functional groups increases upon transition from non-polar compounds to weakly polar ones. For very polar molecules, retention times are so long that analysis is not possible when using a non-polar eluent. To reduce the retention time of polar components, one passes to polar eluents;
in the reversed-phase variant, the stationary phase (non-polar adsorbent) adsorbs the non-polar component more strongly from polar eluents, for example, from water;
By reducing the polarity of the eluent by adding a less polar solvent, the retention of the components can be reduced.
1.6.2. Partition liquid chromatography. In partition or liquid-liquid chromatography, the separation of the components of the analyzed sample is due to differences in the coefficients of their distribution between two liquid phases that do not mix with each other, one of which is stationary and is located on the surface or in the pores of a solid immovable carrier, and the second is mobile.
In terms of the nature of the interaction forces that determine the different distribution between the two phases of substances that differ in their chemical structure, distribution chromatography is similar to adsorption chromatography, i.e., here the separation is also based on the difference in the forces of intermolecular interaction between the components of the analyzed sample with the stationary and mobile liquid phases.
Depending on the performance technique, partition chromatography, like adsorption chromatography, can be column or planar (paper or thin layer).
As solid carriers, substances are used that are indifferent to the mobile solvent and the components of the analyzed sample, but capable of retaining the stationary phase on the surface and in the pores. Most often, polar substances (cellulose, silica gel, starch) are used as carriers. They are applied to the stationary phase - a polar solvent, most often water or alcohol. In this case, less polar or non-polar substances (alcohols, amines, ketones, hydrocarbons, etc.) are used as mobile phases. This type of partition chromatography is called normal-phase. It is used to separate polar substances.
The second version of partition chromatography differs in that non-polar carriers (rubber, fluoroplastic, hydrophobized silica gel) are used as the stationary solid phase, non-polar solvents (hydrocarbons) as the stationary liquid phase, and polar solvents (alcohols, aldehydes) as the mobile liquid phase. , ketones, etc., often water). This variant of partition chromatography is called reverse phase and is used to separate non-polar substances.
To achieve optimal separation of the components of the analyzed sample, the selection of the mobile phase is very important. Solvents (mobile and stationary liquid phases) should be selected so that the distribution coefficients of the mixture components differ significantly. The liquid phases are subject to the following requirements:
1) the solvents used should dissolve the substances to be separated well, and their solubility in the stationary phase should be greater than in the mobile one;
2) the solvents used as mobile and stationary phases must be mutually saturated, i.e. the composition of the solvent must be constant during passage through the column;
3) the interaction of solvents used as the mobile phase with the stationary phase should be minimal.
Most often, in partition liquid chromatography, not individual substances are used as mobile liquid phases, but their mixtures in various ratios. This allows you to control the polarity of the mobile phase, change the ratio of the polarities of the mobile and stationary phases, and achieve optimal conditions for separating the components of a particular mixture under analysis.
A significant disadvantage of this chromatographic method is the rather rapid washing off of the deposited stationary liquid phase from the carrier. To eliminate it, the solvent used as the mobile phase is saturated with the substance used as the stationary liquid phase, or the stationary liquid phase is stabilized by grafting it onto a carrier.
A variation of partition liquid chromatography is the widely used HPLC method.
The most common chromatographic systems are systems that have a modular assembly principle. Pumps, degassers, detectors, autosamplers, column ovens, fraction collectors, chromatography system controls, and recorders are available as separate modules. A wide range of modules allows you to flexibly solve various analytical problems, quickly change the system configuration if necessary, with minimal costs. At the same time, monomodular (integrated) LCs are also produced, the main advantage of which is the miniaturization of individual blocks and the compactness of the device.
Depending on the method of elution, liquid chromatographs are divided into isocratic and gradient.
Diagram of an isocratic chromatograph
The mobile phase from the container (1) through the inlet filter (9) is supplied by a high-pressure precision pump (2) to the sample input system (3) - a manual injector or an autosampler, where the sample is also injected. Further, through the in-line filter (8), the sample with the current of the mobile phase enters the separation element (elements) (4) - through the pre-column into the separation column. Then, the eluate enters the detector (5) and is removed into the drain tank (7). When the eluate flows through the measuring circuit of the detector, the chromatogram is registered and the data is transmitted to an analog recorder (recorder) (6) or another system for collecting and processing chromatographic data (integrator or computer). Depending on the design of the functional modules, the system can be controlled from the keyboard of the control module (usually a pump or system controller), from the keyboards of each of the system modules, or by a control program from a personal computer.
In the case of gradient elution, two fundamentally different types of liquid chromatographs are used. They differ in the point of formation of the mobile phase composition gradient.
Scheme of a gradient chromatograph with the formation of a gradient of the composition of the mobile phase on the low pressure line.
The mobile phase from the tanks (1) through the inlet filters (9) and the gradient programmer (10) is supplied by a high-pressure precision pump (2) to the sample injection system (3) - a manual injector or an autosampler, where the sample is also injected. The operation of the gradient programmer valves is controlled either by the system control module (pump or controller) or by a PC control program. Systems of this type form a binary, three-dimensional and four-dimensional gradient. The form of the gradient processing function depends on the specific control module or control program, as well as the functionality of the controlled and control modules. Further, through the in-line filter (8), the sample with the current of the mobile phase enters the separation element (elements) (4) - through the pre-column into the separation column. Then, the eluate enters the detector (5) and is removed to the drain tank (7). When the eluate flows through the measuring circuit of the detector, the chromatogram is registered and the data is transmitted to an analog recorder (recorder) (6) or another system for collecting and processing chromatographic data (integrator or computer). Depending on the design of the functional modules, the system can be controlled from the keyboard of the control module (usually a pump or system controller), or by a control program from a personal computer. In the case of controlling the control module, it is possible to independently control the detector from its own keyboard.
Despite the apparent attractiveness of such systems (they use only one high-pressure precision pump), these systems have a number of disadvantages, among which the main one, perhaps, is the severe need for thorough degassing of the mobile phase components even before the low-pressure mixer (gradient programmer chamber). It is carried out with the help of special flow degassers. Because of this fact, their cost becomes comparable to another type of gradient systems - systems with the formation of a gradient composition of the mobile phase on the high pressure line.
The fundamental difference between systems with the formation of a mobile phase gradient composition in the high pressure line is the mixing of components in the high pressure line, naturally, with this approach, the number of precision pumps is determined by the number of tanks for mixing the mobile phase. With this approach, the requirements for the thoroughness of the degassing of the components are significantly reduced.
Scheme of a gradient chromatograph with the formation of a gradient of the composition of the mobile phase on the high pressure line.
The mobile phase from the tanks (1) through the inlet filters (9) is supplied by high-pressure precision pumps (2 and 11) through a static or dynamic flow mixer (10) to the sample injection system (3) - a manual injector or an autosampler, where the sample is also injected. The operation of controlled pumps is controlled either by the system's control module (master pump or controller) or by a PC control program. In this case, all pumps are controlled. Systems of this type form a binary or three-dimensional gradient. The form of the gradient processing function depends on the specific control module or control program, as well as the functionality of the controlled and control modules. Further, through the in-line filter (8), the sample with the current of the mobile phase enters the separation element (elements) (4) - through the pre-column into the separation column. Then the eluate enters the detector (5) and is removed into the drain tank (7). When the eluate flows through the measuring circuit of the detector, the chromatogram is registered and the data is transmitted to an analog recorder (recorder) (6) or another system for collecting and processing chromatographic data (integrator or computer). Depending on the design of the functional modules, the system can be controlled from the keyboard of the control module (usually a pump or system controller), or by a control program from a personal computer. In the case of controlling the control module, it is possible to independently control the detector from its own keyboard.
The proposed schemes are rather simplified. The systems can 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. In the diagrams, the manometric modules are also not shown separately. As a rule, these devices are built into pump units. These units can combine multiple pumps, a pump with a gradient programmer, and a common system controller. The structure of the system depends on its configuration and each specific manufacturer.
Such a radical complication of the technical support of the chromatographic process leads to the emergence of a number of requirements for the properties of the mobile phase, which are absent in classical column and planar chromatography. The liquid phase must be suitable for detection (be transparent in a given region of the spectrum or have a low refractive index, a certain electrical conductivity or permittivity, etc.), inert to the materials of the parts of the chromatographic tract, not form gas bubbles in the pump valves and the detector cell, not have mechanical impurities.
In liquid chromatography many types of pumps are used. Low pressure LC often uses peristaltic pumps (Figure 1).
Fig.1 MasterFlex programmable peristaltic pump.
In HPLC, high pressure pumps are used to ensure the flow of the mobile phase through the column with the specified parameters.
The most important technical characteristics of HPLC pumps are: flow range; maximum working pressure; flow reproducibility; solvent supply pulsation range.
By the nature of the solvent supply, pumps can be of constant supply (flow) and constant pressure. Basically, in analytical work, a constant flow mode is used, when filling columns, a constant pressure mode is used.
According to the principle of operation, HPLC pumps are divided into syringe and on plunger reciprocating .
Syringe pumps
The main distinguishing feature of these pumps is their cyclical operation, and therefore the chromatographs in which these pumps are used also differ in cyclical operation.
Rice. 2. Principal arrangement of a syringe pump for HPLC.
Rice. 2A. Syringe pump.
The control unit BU supplies voltage to the motor D, which determines the speed and direction of its rotation. The rotation of the engine with the help of the gearbox P is converted into the movement of the piston P inside the cylinder D. The pump is operated in 2 cycles. In the filling cycle, valve K2 is closed, K1 is open, the solvent flows from the reservoir to cylinder C. In the supply mode, valve K1 is closed, and through valve K2 the mobile phase enters the dosing device.
Pumps of this type are characterized by the almost complete absence of pulsations in the flow of the mobile phase during operation.
Pump Disadvantages:
a) high consumption of time and solvent for washing when changing the solvent;
b) the volume of PF limited by the volume of the syringe, and hence the limited separation time;
c) suspension of separation during filling of the pump;
d) large dimensions and weight while providing high flow and pressure (you need a powerful engine and a large piston force with its large area).
Plunger reciprocating pumps.
Rice. 3. Principal device of a plunger pump.
Operating principle.
The engine D through the gearbox R reciprocates the plunger P, moving in the working head of the pump. Valves K1 and K2 open when the pump is in the suction and delivery phase respectively. The amount of volumetric feed is determined by three parameters: plunger diameter (usually 3.13; 5.0; 7.0 mm), its amplitude (12-18 mm) and frequency (which depends on the speed of rotation of the engine and gearbox).
Pumps of this type provide a constant volumetric flow of the mobile phase for a long time. Maximum working pressure 300-500 atm, flow rate 0.01-10 ml/min. Volume feed repeatability -0.5%. The main drawback is that the solvent is fed into the system in the form of a series of successive pulses, so there are pressure and flow pulsations (Fig. 4). This is the main reason for the increased noise and desensitization of almost all detectors used in LC, especially electrochemical.
Fig.4. Plunger pump pulsation.
Ways to deal with pulsations.
1. Application of damping devices.
These are spiral tubes of a special profile made of stainless steel, included in series or in parallel in the system between the pump and the dispenser.
Rice. 5. Spiral damper.
The damper is untwisted with an increase in pressure in it (acceleration of the pump). With a decrease in pressure, it twists, its volume decreases, it squeezes out part of the solvent, maintaining a constant flow rate and reducing pulsations. Such a damper works well at a pressure of 50 atm and above.
At a pressure of 5-30 atm, it smooths out pulsations better air damper, made from a column (Fig. 6.). The air in the plugged column (6x200 mm) is compressed and the pulsations are extinguished. The air in it dissolves in 24 hours.
Rice. 6. Air damper.
2. The use of electronic devices.
When using an electronic pressure transducer, the readings from the transducer can be used to control the operation of the pump. When the pressure drops, the engine speed increases and compensates for the decrease in pressure. It is also possible to compensate for leaks in the valves and partially in the cuffs. The use of an electronic damper (BPZh-80, KhPZh-1, etc.) makes it possible to reduce pressure pulsations to 1 atm at a pressure of 100-150 kgf/cm2.
1.6.3. Ion-exchange, ion, ion-pair chromatography. The methods of ion-exchange, ion and ion-pair chromatography are based on the dynamic process of replacing ions associated with the stationary phase with eluent ions entering the column. The main goal of the chromatographic process is the separation of inorganic or organic ions of the same sign. Retention in these types of chromatography is determined by the change in the free energy of the ion exchange reaction. The ratio of the concentrations of exchanging ions in the solution and in the sorbent phase is characterized by ion-exchange equilibrium. Ion exchange consists in the fact that some substances (ion exchangers), when immersed in an electrolyte solution, absorb cations or anions from it, releasing an equivalent amount of other ions with a charge of the same sign into the solution. Between the cation exchanger and the solution there is an exchange of cations, between the anion exchanger and the solution there is an exchange of anions.
Cation exchangers are most often specially synthesized insoluble polymeric substances containing acidic ionogenic groups in their structure: -SO 3 H; –COOH; -OH; –PO 3 H 2 ; –AsO 3 H 2 .
The chemical formulas of cation exchangers can be schematically represented as R-SO 3 H; R-SO 3 Na. In the first case, the cation exchanger is in the H-form, in the second, in the Na-form. R is a polymer matrix.
Cation exchange reactions are written as ordinary heterogeneous chemical reactions:
RN + Na + RNa + H +
Anion exchangers contain basic ionogenic groups in their structure: –N(CH 3) 3 + ; =NH 2 + ; =NH + etc. Their chemical formulas can be represented as RNH 3 OH and RNH 3 Cl or ROH, RCl. In the first case, the anion exchanger is in the OH form, in the second, in the Cl form. The anion exchange reaction can be written as follows:
R–OH+Cl – RCl+OH –
Amphoteric ion exchangers are known that contain both acidic and basic groups in their structure. Ion exchangers that have in their composition the same type (for example, SO 3 H) acidic (basic) groups are called monofunctional; ion exchangers containing heterogeneous (for example, - SO 3 H, - OH) acid (basic) groups - polyfunctional.
Monofunctional ion exchangers are obtained by a polymerization reaction. The polycondensation reaction makes it possible to obtain polyfunctional ion exchangers. In order for the resulting ion exchangers to have sufficiently high performance characteristics, they must be insoluble, but swellable in the appropriate solvent and have a sufficiently large number of ionogenic groups capable of exchanging with the ionogenic groups of the analyzed sample. This can be achieved if the resulting polymer chains are sufficiently branched and connected to each other by "cross-linking bridges". For example, in the preparation of polymerization type cation exchangers based on styrene, divinylbenzene is most often used as a crosslinking agent, the introduction of which in an amount of up to 16% ensures the production of ion exchangers with various degrees of swelling and, therefore, allows one to control the ion exchanger porosity. The degree of swelling of the ion exchanger, expressed in milliliters/gram, is the volume of 1 g of air-dry ion exchanger packed into the column.
The ion exchanger absorbs, as a rule, one of the counterions - ions in the mobile phase, i.e. it exhibits a certain selectivity. Series of affinity, or selectivity, of ions with respect to ion exchangers of various types have been experimentally established. For example, at low solution concentrations on strongly acidic cation exchangers, ions with the same charge are sorbed in the following sequence:
Li + Na + K + Rb + Cs +
Mg 2+ Ca 2+ Sr 2+ Ba 2+ .
For ions with different charges, the sorbability increases with increasing charge:
Na+Ca2+ However, changing the conditions for carrying out the ion exchange reaction can lead to series inversion. Affinity series have also been established for anion exchangers. For example, the sorbability of anions on strongly basic anion exchangers increases in the series: F - OH - Cl - Br - NO 3 - J - SCN - ClO 4 - . Ion exchangers containing strongly acidic or strongly basic groups in their structure enter into ion exchange reactions with any ions in solution that have charges of the same sign as the sign of the counterion. Such ion exchangers are called universal. The process of ion exchange between the analyte and the ion exchanger can be carried out in one of three ways: static, dynamic (ion exchange filter method) and chromatographic. Static method
ion exchange is that a sample of the ion exchanger is brought into contact with a certain volume of solution and stirred or shaken for a certain time until equilibrium is established. This is a fast and simple method of ion exchange, used to concentrate ions from dilute solutions, remove unwanted impurities, but it does not provide complete absorption of ions, since ion exchange is a non-equilibrium process, and therefore does not guarantee complete separation of ions. When carrying out ion exchange in a dynamic way
a solution is passed through the column with an ion exchanger, which, as it moves along the column, comes into contact with new granules of the ion exchanger. This process provides a more complete exchange than the static method, since the exchange products are removed by the solution flow. They can concentrate ions from dilute solutions and separate ions that differ greatly in properties, for example, differently charged ions (separate cations from anions), but the separation of ions of the same charge sign is almost impossible. Quantitative separation of such ions is possible only with repeated repetition of sorption-desorption elementary acts under dynamic conditions, i.e. chromatographic method
. When working with this method, high layers of ion exchanger are used, and the mixture to be separated is introduced into this layer in an amount much less than the capacity of the column, due to which repeated repetition of elementary acts of ion exchange is ensured. According to the analysis technique, ion-exchange chromatography is similar to molecular chromatography and can be carried out according to the eluent (developing), frontal, and displacement options. The difference between molecular and ion-exchange chromatography is that in molecular chromatography, the separated components of the mixture are eluted from the column with a pure eluent, while in ion-exchange chromatography, an electrolyte solution is used as an eluent. In this case, the exchanged ion of the eluent should be sorbed less selectively than any of the ions of the mixture being separated. When carrying out developing ion-exchange chromatography, which is used most often, a column filled with an ion exchanger is first washed with an electrolyte solution until the ion exchanger completely replaces all its ions with the ions contained in the eluent. Then, a small volume of the analyte solution containing separable ions in an amount of about 1% of the capacity of the ion exchanger is injected into the column. Next, the column is washed with an eluent solution, taking fractions of the eluate and analyzing them. A mixture of Cl - , Br - , J - ions can be separated on a highly basic anion exchange resin (cross-linked polystyrene containing groups of quaternary ammonium bases N (CH 3) 3 +), for example, AB-17, which has a range of selectivity (selectivity): NO 3 - Cl – Br – J – . As a result, a NaNO 3 solution is used as an eluent. First, this solution is passed through the ion exchanger until it is completely saturated with NO 3 - ions. When the mixture to be separated is introduced into the column, ions Cl – , Br – , J – are absorbed by the anion exchanger, displacing NO 3 – ions. During subsequent washing of the column with NaNO 3 solution, the ions Cl – , Br – , J – in the upper layers of the anion exchanger are gradually again replaced by NO 3 – ions. Cl - ions will be displaced the fastest of all, J - ions will stay in the column the longest. The difference in the selectivity of the ion exchanger to the ions of the mixture leads to the fact that separate zones of adsorbed ions Cl – , Br – and J – are formed in the column, moving through the column at different speeds. As you move along the column, the distance between zones increases. In each zone there is only one of the anions of the mixture being separated and the anion of the eluent, in the interval between the zones there is only an anion of the eluent. Thus, fractions containing individual components of the mixture to be separated will appear in the eluent at the column outlet. To solve practical problems, the conditions for ion separation are varied by selecting a suitable mobile phase (composition, concentration, pH, ionic strength) or by changing the porosity of the polymer matrix of the ion exchanger, i.e., the number of interchain bonds in the matrix, and creating ion exchange sieves that are permeable to some ions and capable of their exchange and impenetrable to others. It is also possible to change the nature and mutual arrangement of ionogenic groups, as well as to obtain sorbents capable of selective chemical reactions due to complex formation. High selectivity is possessed, for example, by complexing ion exchangers containing in their structure chelating groups of organic reagents dimethylglyoxime, dithizone, 8-hydroxyquinoline, etc., as well as crown ethers. The greatest application in ion exchange, ion, and ion pair chromatography is found in synthetic macro- and micronet organic ion exchangers having a large exchange capacity (3–7 mmol/g), as well as inorganic ion-exchange materials. Micromesh ion exchangers are capable of exchanging ions only in the swollen state, while macromesh ones are capable of exchanging ions in the swollen and unswollen states. Another structural type of ion exchangers are surface-film ion exchangers, the solid core of which is made of a non-porous copolymer of styrene and divinylbenzene, glass or silica gel and is surrounded by a thin film of the ion exchanger. The total diameter of such a particle is about 40 µm, and the thickness of the ion exchanger film is 1 µm. The disadvantage of such ion exchangers is the relatively large particle diameter and low exchange capacity due to the low specific surface area, as a result of which it is necessary to work with small samples and, accordingly, to 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, space-porous polystyrene ion-exchangers, volume-porous silicas with a granule diameter of about 10 μm, and practically non-swelling surface-porous and surface-modified copolymers of styrene and divinylbenzene with ionogenic sulfo and amino groups are used. In ion-pair chromatography, "brush" sorbents are used - silica gels with grafted reversed phases C 2, C 8, C 18, which are easily converted into a cation exchanger upon absorption of ionic surfactants from the mobile phase, for example, alkyl sulfates or salts of quaternary ammonium bases. When carrying out chromatographic separation using ion exchangers, aqueous solutions of salts are most often used as the mobile phase. This is due to the fact that water has excellent dissolving and ionizing properties, due to which the molecules of the analyzed sample instantly dissociate into ions, the ion exchange groups of the ion exchanger are hydrated and also go into a fully or partially dissociated form. This ensures a rapid exchange of counterions. The eluting strength of the mobile phase is mainly influenced by pH, ionic strength, the nature of the buffer solution, the content of the organic solvent or surfactant (ion-pair chromatography). The pH value is chosen depending on the nature of the ionogenic groups, the ions to be separated, and the matrix. It is possible to work with strongly acidic and strongly basic ion exchangers at pH = 2–12, with weakly acidic ones at pH = 5–12, and with weakly basic ones at pH = 2–6. Silica-based sorbents cannot be used at pH 9. The ionic strength of the mobile phase affects the capacity of the ion exchanger. With an increase in ionic strength, the sorption of ions usually decreases, since the eluting strength of the mobile phase increases. Therefore, at the beginning of separation, the mobile phase should have a low ionic strength (0.05–0.1), and the final value of this characteristic should not exceed 2. In gradient elution, buffers with increasing ionic strength are often used. For the selective elution of ions absorbed by the ion exchanger, water, buffer solutions (phosphate, acetate, borate, hydrocarbonate, etc.) with a certain pH value and ionic strength, solutions of mineral (hydrochloric, nitrogen, sulfuric, phosphoric) and organic (phenol, citric, lactic, tartaric, oxalic, EDTA) acids. The choice of eluent is facilitated by the fact that the limiting coefficients of distribution of most elements between aqueous (water-organic) solutions of many complexants and standard-type ion exchangers are determined and presented in tables. 1.6.4. Size exclusion chromatography. Size exclusion chromatography is a type of liquid chromatography in which the separation of components is based on the distribution of molecules according to their size between the solvent in the pores of the sorbent and the solvent flowing between its particles. During separation, small molecules enter the polymer network, in the pores of which the solvent serves as a stationary phase, and are retained there. Large molecules cannot penetrate the polymer network and are washed out of the column by the mobile phase. The largest molecules are eluted first, then the medium ones, and finally the small ones. Size exclusion chromatography is subdivided into gel permeation and gel filtration. In gel permeation chromatography, separation occurs on polymers that swell in organic solvents. The gel filtration version of size exclusion chromatography involves the use of water-swellable polymers as stationary phases. The 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 size of the pores of the stationary phase. In this type of liquid chromatography, the partition coefficient D for the smallest molecules of the analyzed sample, which move in the chromatographic column at the lowest speed, penetrating into the grid of the stationary phase, it is equal to 1, since the mobile phase and the solvent in the pores of the stationary phase have the same composition. In this case, the main equation of column chromatography takes the form Large molecules that do not enter the pores of the stationary phase are eluted from the column along with the mobile phase. For them D= 0, a V R =V m. Such a range of distribution coefficient values (from 0 to 1) is typical only for size exclusion chromatography. All molecules of the analyzed multicomponent substance should be washed out of the column by passing a small volume of solvent from V m before V m +V s and the separation is completed before the solvent peak exits. Therefore, in this type of chromatography, it is necessary to use sufficiently long columns with a large free volume. V m and a large number of pores in the sorbent. The resolution of chromatographic peaks in size exclusion separations can be improved by using gradient elution with mixed solvents. Each sorbent used in size exclusion chromatography is characterized by a certain pore volume and, therefore, has a certain area of separable molecular weights and a certain calibration curve. In this case, the calibration curve characterizing the dependence of the retained volume on the molecular weight or size of the molecules, as a rule, has a complex form. Stationary phases in size exclusion chromatography are selected based on specific analytical tasks. Initially, it is established which solvent system can be used for analysis (aqueous or water-organic). Depending on this, the type of sorbent is determined. If water-soluble samples are to be separated, for example water-swellable cross-linked dextrans (Sephadex) or polyacrylamides (Biogel P) are used as stationary phases. The separation of substances soluble in organic solvents can be carried out on polystyrenes with various degrees of crosslinking, which swell in organic solvents (styrogel, poragel, biobid C). Such swollen gels are generally not pressure stable and allow very low mobile phase flow rates, which increases analysis time. In order 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 surface silanization or selection of an eluent of the appropriate polarity. Substances that can be used as mobile phases in size exclusion chromatography are: completely dissolve the analyzed sample; wet the sorbent well; counteract adsorption of sample components on the sorbent; have low viscosity and toxicity. 1.6.5. Planar chromatography. Planar chromatography includes thin layer and paper chromatography. These types of liquid chromatography are simple in technique, express, do not require expensive equipment, which is their undeniable advantage. The separation of a mixture of substances by these methods can be performed using various chromatographic systems. Therefore, adsorption, distribution, normal and reversed phase, ion-exchange, etc., paper and thin-layer chromatography are distinguished. At present, thin layer chromatography is the most widely used. Paper and thin layer chromatography are similar in technique. Cellulose paper fiber is used as a stationary phase in paper chromatography, and various sorbents (Al 2 O 3 , silica gel, etc.) applied in a uniform thin (100-300 μm) layer on a glass, metal or plastic substrate (carrier) in thin-layer chromatography . The adsorbent layer on the support may or may not be fixed. Chromatographic separation in planar methods, as well as on a column, is due to the transfer of the components of the analyte by the mobile phase along the layer of the stationary phase at different rates in accordance with the distribution coefficients of the substances to be separated. In both cases, liquid-solid sorbent chromatographic systems (adsorption separation mechanism), liquid-liquid-solid carrier (distribution, ion-exchange and other mechanisms) are used. Various solvents or their mixtures, organic or inorganic acids are used as mobile phases. Practical obtaining planar chromatograms consists in the following. On a strip of chromatographic paper or on a thin layer of sorbent, a starting line is marked with a pencil at a distance of 1 cm from the bottom edge of the strip or plate. A micropipette is used to apply a sample to the start line in the form of a spot with a diameter of not more than 2–3 mm. Then the edge of the strip or plate is lowered into the vessel with the mobile phase, located in a sealed chamber. As the mobile phase rises along the strip or plate and the multiple elementary acts of sorption-desorption, distribution between two liquid phases, ion exchange, etc., which are common in chromatography, occur, the components of the analyzed mixture are separated. The process is usually continued until the solvent has passed from the start line 10 cm. After that, the strip or plate is removed from the chamber and dried. If the components of the analyte are colored, they give the corresponding color spots on the chromatogram. To detect unstained components of the analyte, the chromatogram must be developed. The development of a chromatogram and the detection of sample components can be carried out by various methods and depend on the composition of the analyzed mixtures. The manifestation can be done: -Using UV light. The method is applicable for the detection of substances capable of emitting their own radiation (luminescence) in the visible wavelength range under the action of UV radiation; by means of reagents-developers. For example, the presence of amino acids in the analyzed mixture can be detected using ninhydrin. The dried chromatogram is immersed in a 0.2% solution of ninhydrin in acetone, then dried. Spots corresponding to various components of the mixture acquire a visual and, as a rule, specific color for each substance; - using iodine. In this case, the detected chromatogram is introduced into a vessel, at the bottom of which there are iodine crystals. Iodine vapors are adsorbed on the spots more strongly, due to which the spots are visualized. Iodine is a non-specific developer reagent. Using specific reagents, it is possible not only to determine the number of components of a mixture, but also to identify the separated substances by the color of the spots. Paper and thin layer chromatography is most often carried out in the so-called ascending variant described above. Quite often, to improve the quality of chromatograms, it is necessary to use more complex variants of planar chromatography, for example, top-down, circular, two-dimensional. In paper or thin layer down chromatography, the analyte is applied to the starting line of the plate or paper strip at the top, and the eluent is fed from the top instead of the bottom. The positive effect of improved separation is due to the contribution of the gravity forces of the components to the separation process. Both upstream and downstream chromatography can be performed in one and two-dimensional versions. In contrast to the one-dimensional flat-bed separation process described above, in two-dimensional chromatographic separation, the separation of the analyzed sample is first carried out in one solvent, then separation is carried out in the direction perpendicular to the first, using another solvent, rotating the first chromatogram by 90 ° C. In circular chromatography, the analyte is applied as a drop in the middle of a plate or sheet of chromatographic paper. One or more solvents are also added dropwise here. This leads to the fact that the resulting chromatogram is a set of radial spots. The position of the spots (zones) that form the separated components of the analyte on a flat chromatogram is characterized by the values of the relative speed of movement of the components in a thin layer R fi. Experimental value R fi defined as the ratio of distance L i, passed i-th component, to the distance L passed by the solvent from the starting line to the front line (Fig. 1.10): Magnitude R fi depends on the nature of the relevant component of the analyzed sample, the nature of the stationary phase, its thickness, the nature and quality of the mobile phase, the method of application of the sample and other factors, but always R fi
1. 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 spot diameter is identical to the height or area of the chromatographic peak and, therefore, to some extent reflects the quantitative content of the substance. Quantitative determination of the composition of the analyzed sample in the simplest case can be assessed visually by the intensity of the intrinsic color of the spots or the intensity of the fluorescent glow of the resulting spots during UV detection. For these purposes, the elution of chromatographic spots is widely used. In this case, the spot obtained on the chromatogram is carefully cut out or scraped off, treated with a suitable solvent, and the resulting solution is examined by the appropriate physicochemical method. You can also use the gravimetric method, in which the corresponding spot is cut out from the chromatogram and weighed. The amount of substance is determined by the difference in the weights of clean paper of the same area and paper with the substance. Paper
(BH
) And thin layer chromatography
(TLC
) according to the separation mechanism belong to partition chromatography
. In the HD method, the carrier is a special chromatographic paper
with certain properties. Stationary phase
is water adsorbed on the surface and pores of paper (up to 20%), mobile - organic solvent, miscible or immiscible with water, water or electrolyte solutions. Mechanism
quite complicated on paper. In the stationary phase, the substance can be retained not only due to dissolution in the water adsorbed by the paper, but also be adsorbed
cellulose directly. Drawn on paper shared components
pass into the mobile phase and move through the capillaries of the paper at different speeds in accordance with interfacial distribution coefficient
each of them. At first chromatography
some of the substance from the paper passes into mobile phase
and move on. When the organic solvent reaches the area of the paper that does not contain the solute, redistribution
: from the organic phase, the substance passes into the aqueous phase, sorbed on paper. Since the components have different affinity for the sorbent
, when the eluent moves, separation occurs: some substances are delayed at the beginning of the path, others move further. Here they combine thermodynamic
(establishment of an equilibrium distribution of substances between the phases) and kinetic
(moving components at different speeds) separation aspects. As a result, each component is concentrated on a specific area of the paper sheet: zones of individual components
on chromatogram
. The use of chromatography on paper has a number of significant disadvantages: the separation process depends on the composition and properties of the paper, the change in the water content in the pores of the paper with changes in storage conditions, a very low chromatography speed (up to several days), and low reproducibility of the results. These shortcomings seriously affect the spread of paper chromatography as a chromatographic method. 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 provided by the movement mobile phase
(solvent) through the sorbent under the action of capillary forces
.
Byseparation mechanism
distinguish partition, adsorption and ion exchange chromatography
. The separation of the components occurs in these cases either as a result of their different distribution coefficient between the two liquid phases ( partition chromatography
), or due to different adsorbability of compounds by the sorbent ( adsorption chromatography
). The adsorption method is based on different degrees of sorption-desorption of the separated components on the stationary phase. Adsorption
carried out at the expense van der Waals forces
, which is the basis physical adsorption
,
polymolecular
(formation of several adsorbate layers on the surface of the adsorbent) and chemisorption
(chemical interaction of adsorbent and adsorbate). In the case of using such sorbents for TLC as alumina
or silica gel
play a role in separation distribution
, so adsorption
on the developed active surface of the sorbent (150–750 m2/g). Distribution
components of the mixture occurs between water on the surface of the carrier (such adsorbents
, How alumina
,
starch
,
cellulose
,
diatomaceous earth
- And water
form stationary phase
), and the solvent moving through this stationary phase ( mobile phase
). The component of the mixture that is more readily soluble in water moves more slowly than the one that is more readily soluble in the mobile phase. Adsorption
manifested in the fact that between carrier
, for example, aluminum oxide, and the components of the mixture are set adsorption equilibria
- for each component its own, the result of which is different travel speed
mixture components. Two extreme cases can be distinguished: a) the concentration of the substance on the adsorbent is zero. The substance completely dissolves in the mobile phase and is carried away by it (moves along with solvent front
). b) the substance is adsorbed completely, does not interact with the solvent and remains at the start. In practice, with the skillful selection of solvent and adsorbent distribution
compounds are located between these extreme cases, and the substance gradually carried over
from one sorbent layer to another due to simultaneously occurring processes sorption
And desorption
. The solvent passing through the sorbent is called eluent
, the process of moving a substance together with an eluent elution
.
As the liquid moves on the plate, the mixture of substances separates due to the action of forces adsorption
,
distribution
,
ion exchange
or a combination of all of these factors. As a result, separate chromatographic zones
mixture components, i.e. it turns out chromatogram
. Correct selection sorbent
And eluent
determines the efficiency of mixture separation. The mobility of the test substance depends on its affinity for the sorbent and eluting force
(polarity) of the eluent. As the polarity of the compound increases, so does its affinity for the polar sorbent. By increasing the degree of adsorption silica gel
organic compounds are arranged in a row: hydrocarbons<алкилгалогенидыарены<нитросоединения<простые
эфиры <сложные эфиры<альдегиды<спирты<амины<карбоновые
кислоты. В свою очередь for silica gel
eluents can be arranged in ascending order of "polarity" ( eluting power
) and form a series of solvents ( eluotropic series
) in accordance with experimental data: alkanes> benzene> chloroform> diethyl ether> ethyl acetate> alcohols С 2 -С 4> water> acetone> acetic acid> methanol. Thus, a polar compound, alcohol, is adsorbed quite strongly on silica gel and therefore moves weakly under the action of such a nonpolar solvent as hexane, and remains near the start line. In turn, the non-polar aromatic hydrocarbon biphenyl is noticeably more mobile in hexane, but even here, to achieve R
f
about 0.5, a more polar aprotic eluent, methylene chloride, is needed. eluent strength
regulate using mixtures of solvents - neighbors in eluotropic series
with different polarity. Currently, TLC mainly uses the following sorbents
: for separation lipophilic substances
silica gel
,
alumina
,
acetylated cellulose
,
polyamides
; to separate hydrophilic substances
cellulose
,
cellulose ion exchangers
,
diatomaceous earth
,
polyamides
. The most important characteristic of a sorbent is its activity
, i.e. ability absorb
(hold) the components of the mixture to be separated. Abroad, a number of firms produce silica gel
,
diatomaceous earth
And alumina
with the addition of 5% gypsum, which is used to fix the sorbent layer in the self-manufacturing of plates. The most common sorbent is silica gel
- hydrated silicic acid, formed by the action of mineral acids on Na 2 SiO 3 and drying 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 sorbs water on the surface and forms hydrogen bonds. Alumina
is a weakly basic adsorbent and is used mainly for the separation of weakly basic and neutral compounds. The disadvantage of plates on aluminum oxide is the obligatory activation of the surface before use in a drying cabinet at a high temperature (100-150 o C) and the low adsorption capacity of the layer compared to silica gel. diatomaceous earth
- adsorbent obtained from natural minerals - diatomaceous earths. The sorbent has hydrophilic properties and a lower adsorption capacity of the layer compared to silica gel. Cellulose:
cellulose-coated thin-layer plates are very effective in separating complex organic molecules. The adsorbent is mainly cellulose balls with a diameter of up to 50 microns, fixed on the carrier with starch. As in paper chromatography, the rise of the solvent front is very slow. Chromatographic analysis
is carried out on industrial plates of Czech production " Silufol
»
(« Silufol
"") of aluminum foil, sometimes reinforced with cardboard, and " Siluplast
» made of plastic coated with a layer of sorbents - silica gel LS 5-40 with starch or gypsum as a binder (up to 10%), or aluminum oxide with or without fluorescent indicators. Records " Silufol
» have a high elution rate, however, they are characterized by low separating power and low sensitivity. During storage, they are sensitive to conditions (humidity, temperature, aggressive media, etc.). Individual firms supply chromatographic plates
with a layer of sorbent of different (usually up to 0.25 mm), but strictly constant thickness (silica gel, cellulose, ion-exchange resin), on glass and substrates made of aluminum foil, plastic, impregnated fiberglass. Plates « Sorbfil
» (TU 26-11-17-89) are produced in Russia on a polymer base (polyethylene terephthalate, grade P) or aluminum substrate (grade AF) with a working layer applied microfractionated silica gel sorbent
grades STX-1A and STX-1VE (produced in the USSR as fractionated silica gel KSKG) with a thickness of 90-120 microns (up to 200 microns), fixed with a special binder - silicasol
. When using silicic acid (silicazole) sol as a binder, which transforms into silica gel after heating, the resulting TLC plates consist of two components: a silica gel layer and a substrate. The thickness uniformity of the sorbent layer on one plate is ±5 µm. Designation example: "Sorbfil-PTSKh-AF-V-UF (10x10)" - high-performance TLC plates on an aluminum substrate, with a phosphor, 10x10 cm. If a glass substrate (grade C) is used, then such plates are reusable and chemically resistant. Their chemical resistance is determined by the chemical resistance of silica gel. As a result, TLC plates can be repeatedly treated with aggressive reagents, for example, with a hot chromium mixture, which removes restrictions on the use of correlating reagents for spot detection and sorbent modification, and allows multiple (up to 30 times or more) plate regeneration with a chromium mixture. Glass plates can be cut to the desired size. The mechanical strength of the sorbent layer can be controlled, providing, on the one hand, transportation and multiple processing of the plates and, on the other hand, the possibility of extracting adsorbent layers with separated substances for subsequent washing out of individual compounds from the sorbent and their further study by instrumental methods (IR and UV spectrometry). , X-ray diffraction methods, NMR, etc.). The plates differ in the size of the fractions (particle distribution) of the silica gel that makes up the layer. On analytical plates (grade A) the fraction is 5-17 microns, on highly efficient (grade B) - 8-12 microns. A narrower distribution increases the efficiency of the plates, i.e. the spots of the substances to be separated become more compact (smaller in size) and therefore are better separated when the eluent front passes a shorter distance. On Russian wafers, analytical and high-performance layers do not differ very much, in contrast to wafers from Merck (Germany). High performance plates should be used if substances cannot be separated on analytical plates. Plates of all modifications are produced with a phosphor (UV grade) with 254 nm excitation. The shelf life is not limited, the plates " Sorbfil
» widely tested in the analysis of amino acid derivatives, pesticides, lipids, antibiotics. The TLC method is carried out qualitative identification
components. quantitation
for TLC is also possible, this requires applying the exact amount of substance and additional densitometric studies
with a clear fixation of the intensity of the spots. The most common is semiquantitative method
. It is based on visual comparison
the size and intensity of the spot of a component with the corresponding characteristics of a series of spots of the same substance of different concentrations ( standard reference solutions
). When using a sample in the amount of 1–5 μg, such a simple method provides an accuracy of determining the component content of about 5–10%. Often, in order to determine the components in a sample, it is necessary to carry out sample preparation to obtain a mixture containing the analyzed compounds. Sample preparation is based on the extraction of drugs from the sample with organic solvents ( n-hexane, petroleum ether, diethyl ether, chloroform), purification of the extract and subsequent chromatography in a thin layer of alumina or silica gel. There are several variants of TLC and BC, differing in the way solvent supply
. Depending on the direction of movement of the mobile phase, there are: A)ascending chromatography
the mobile phase is poured onto the bottom of the separation chamber, the paper (plate) is placed vertically; b)descending chromatography
the mobile phase is fed from above and moves down along the sorbent layer of the plate or paper; 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 deposited. The most common is upward elution
(chromatography). Front
eluent
while moving from bottom to top. The choice of solvent (mobile phase) is determined by the nature of the sorbent and the properties of the substances to be separated. Chromatographic separation
BC and TLC methods are carried out in separation chamber
with screwed lid. A quantitative measure of the rate of transfer of a substance using a specific adsorbent and solvent is R value
f
(from English. retention
factor
– delay coefficient, this parameter is analogous to retention time). Position zones of the chromatographed component
set to size coefficient
R
f
equal to the ratio of the velocity of its zone to the velocity of the solvent front. Magnitude R
f
is always less than unity and does not depend on the length of the chromatogram. By the amount R
f
influenced by various factors. So, at low temperatures, substances move more slowly; solvent contamination, adsorbent inhomogeneity, foreign ions in the analyzed solution can change the value R
f
up to 10%. In the selected system, the analytes must have different values R
f
and distributed over the entire length of the chromatogram. It is desirable that the values R
f
lay in the range of 0.05-0.85. In practice, the value R f
calculated as the ratio of the distance l
traveled by the substance to the distance L
passed by the solvent: R
f
=
l/l
(6.1
)
Usually for calculation choose spot center
(Fig. 1). Magnitude R
f
depends on many factors such as chromatographic paper
(its porosity, density, thickness, degree of hydration) and sorbent
(grain size, nature of groups on the surface, layer thickness, its moisture content, nature of the substance, composition of the mobile phase), experimental conditions (temperature, chromatography time, etc.). With the constancy of all chromatography parameters, the value R
f
determined only by the individual properties of each component. Rice. 1. Determination of values on the chromatogram RF
for components A And IN, their degree of separation Rs
and the number of theoretical plates N
. The efficiency of BC and TLC also depends on selectivity and sensitivity
reactions used to detect the components of the analyzed mixture. Usually, reagents are used that form colored compounds with the components to be determined - developers. For a more reliable identification of shared components
apply " witnesses
» -solutions standard substances
(in the same solvent as the sample) that are expected to be present in the sample. Standard Substance
applied to the starting line next to the analyzed sample and chromatographed under the same conditions. In practice, a relative value is often used: R
f
rel
=
R
f
x
/
R
f
stand
(6.2)
Where R
f
stand
also calculated by formula (6.1). Efficiency
chromatographic separation
characterize number of equivalent theoretical plates
and them height
. Thus, in the TLC method, the number of equivalent theoretical plates N
A for component A the mixture to be separated is calculated by the formula: N
A
= 16 (l
OA
/
a
(A
))
2
(6.3)
Values l
OA
And A
(A
)
determined as shown in Fig. 6.1. Then the height of the equivalent theoretical plate N
A
is: H
A
=
l
OA
/N
=
a
(A
)
2
/ 16
l
OA
.
(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 division Rs
: Rs
=
l /
(a
(A)
/
2
+
a
(B)
/
2)=
2
l /
(a
(A)
+
a
(B))
(6.5)
Where
l
distance between component spot centers 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 spots of the components are separated A And IN on the chromatogram. Conditions chromatography
are chosen so that the value Rs
different from zero and one, the optimal value Rs
is 0.3
0.7. For rate separation selectivity
two components 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. (mainly intermolecular) at the phase boundary. 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 proceeds not by one, but by several mechanisms simultaneously. So, exclusion separation can be complicated by adsorption effects, adsorption - distribution, 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. The stationary phase is more polar than the mobile phase, therefore the nonpolar solvent predominates in the eluent: 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: The matrices used in HPLC are inorganic compounds such as 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: Preparation of silica gel for HPLC: Sorbent particles: 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. Normal phase HPLC: Reversed phase HPLC: 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. Wikimedia Foundation. 2010 . high performance liquid chromatography- - [A.S. Goldberg. English-Russian energy dictionary. 2006] Topics: energy in general EN high performance liquid chromatography HPLC ... Technical Translator's Handbook high performance liquid chromatography- The term high performance liquid chromatography The term in English high performance liquid chromatography Synonyms Abbreviations HPLC, HPLC Related terms adsorption, oligopeptide, proteomics, sorbent, fullerene, endohedral, chromatography... ... HIGH PERFORMANCE LIQUID CHROMATOGRAPHY- liquid chromatography, in which, to increase the efficiency of separation, the solvent (eluent) under pressure (more than 3x107 Pa) is pumped through columns filled with sorbent with particles of small diameter (up to 1 μm), and perfusion filters are also used... ... LIQUID CHROMATOGRAPHY- a type of chromatography in which the liquid (eluent) serves as the mobile phase, and the stationary one. sorbent, TV a carrier with a liquid or gel applied to its surface. Carry out in a column filled with sorbent (column chromatography) on a flat... ... Natural science. encyclopedic Dictionary Chromatography- [κρώμα (υrum) color] a process based on the unequal ability of individual components of the mixture (liquid or gaseous) to remain on the surface of the adsorbent both when absorbing them from the carrier flow and when ... ... Geological Encyclopedia Chromatography- (from other Greek ... Wikipedia chromatography- Term chromatography Term in English chromatography Synonyms Abbreviations Related terms high performance liquid chromatography, clathrate, laboratory on a chip, porometry, proteome, proteomics, sorbent, enzyme, fullerene, endohedral... ... Encyclopedic Dictionary of Nanotechnology ION EXCHANGE CHROMATOGRAPHY- liquid chromatography based on decomp. ability of separated ions to ion exchange with fixed. sorbent ions formed as a result of dissociation of the latter’s ionogenic groups. Cation exchangers are used to separate cations, for... ... Chemical Encyclopedia HPLC- high performance liquid chromatography… Dictionary of Russian abbreviations HPLC- High-performance liquid chromatography (HPLC) is one of the effective methods for separating complex mixtures of substances, widely used both in analytical chemistry and chemical technology. The basis of chromatographic separation is the participation ... Wikipedia In high performance liquid chromatography (HPLC), the nature of the processes occurring in the chromatographic column is generally identical to the processes in gas chromatography. The difference is only in the use of a liquid as a stationary phase. Due to the high density of liquid mobile phases and the high resistance of the columns, gas and liquid chromatography differ greatly in instrumentation. In HPLC, pure solvents or their mixtures are usually used as mobile phases. To create a stream of pure solvent (or mixtures of solvents), called eluent in liquid chromatography, pumps are used, which are part of the chromatograph hydraulic system. Adsorption chromatography is carried out as a result of the interaction of a substance with adsorbents, such as silica gel or aluminum oxide, which have active centers on the surface. The difference in the ability to interact with the adsorption centers of different sample molecules leads to their separation into zones in the process of moving with the mobile phase through the column. The division of the component zones achieved in this case depends on the interaction with both the solvent and the adsorbent. Silica gel adsorbents with different volumes, surfaces, and pore diameters find the greatest application in HPLC. Aluminum oxide and other adsorbents are used much less frequently. The main reason for this: Insufficient mechanical strength, which does not allow packaging and use at elevated pressures typical for HPLC; silica gel compared to aluminum oxide has a wider range of porosity, surface and pore diameter; a significantly higher catalytic activity of aluminum oxide leads to a distortion of the analysis results due to the decomposition of the sample components or their irreversible chemisorption. HPLC detectors High performance liquid chromatography (HPLC) is used to detect polar non-volatile substances, which for some reason cannot be converted into a form convenient for gas chromatography, even in the form of derivatives. Such substances, in particular, include sulfonic acids, water-soluble dyes and some pesticides, such as phenyl-urea derivatives. Detectors: UV - diode array detector. The "matrix" of photodiodes (there are more than two hundred of them) constantly registers signals in the UV and visible region of the spectrum, thus ensuring the recording of UV-B spectra in the scanning mode. This makes it possible to continuously record, at high sensitivity, undistorted spectra of components rapidly passing through a special cell. Compared to single-wavelength detection, which does not provide information about the "purity" of the peak, the ability to compare the full spectra of the diode array provides an identification result with a much greater degree of certainty. Fluorescent detector. The great popularity of fluorescent detectors is due to the very high selectivity and sensitivity, and the fact that many environmental pollutants fluoresce (for example, polyaromatic hydrocarbons). An electrochemical detector is used to detect substances that are easily oxidized or reduced: phenols, mercaptans, amines, aromatic nitro and halogen derivatives, aldehydes, ketones, benzidines. Chromatographic separation of the mixture on the column due to the slow advance of the PF takes a long time. To speed up the process, chromatography is carried out under pressure. This method is called high performance liquid chromatography (HPLC). Modernization of the equipment used in classical liquid column chromatography has made it one of the promising and modern methods of analysis. High performance liquid chromatography is a convenient method for separating, preparatively isolating, and performing qualitative and quantitative analysis of non-volatile thermolabile compounds of both low and high molecular weight. Depending on the type of sorbent used in this method, 2 variants of chromatography are used: on a polar sorbent using a non-polar eluent (direct phase option) and on a non-polar sorbent using a polar eluent - the so-called reverse-phase high-performance liquid chromatography (RPHLC). When the eluent passes to the eluent, the equilibrium under RPHLC conditions is established many times faster than under the conditions of polar sorbents and non-aqueous PFs. As a result of this, as well as the convenience of working with water and water-alcohol eluents, RPHLC has now gained great popularity. Most HPLC analyzes are carried out using this method. Detectors. Registration of the output from the column of a separate component is performed using a detector. For registration, you can use the change in any analytical signal coming from the mobile phase and related to the nature and amount of the mixture component. Liquid chromatography uses such analytical signals as light absorption or light emission of the exiting solution (photometric and fluorimetric detectors), refractive index (refractometric detectors), potential and electrical conductivity (electrochemical detectors), etc. The continuously detected signal is recorded by the recorder. The chromatogram is a sequence of detector signals recorded on the recorder tape, generated when individual components of the mixture exit the column. In the case of separation of the mixture, individual peaks are visible on the external chromatogram. The position of the peak on the chromatogram is used for the purpose of identification of the substance, the height or area of the peak - for the purpose of quantitative determination. Application HPLC finds the widest application in the following areas of chemical analysis (objects of analysis where HPLC has practically no competition are highlighted): · Food quality control - tonic and flavor additives, aldehydes, ketones, vitamins, sugars, dyes, preservatives, hormones, antibiotics, triazine, carbamate and other pesticides, mycotoxins, nitrosoamines, polycyclic aromatic hydrocarbons, etc. · Environmental protection - phenols, organic nitro compounds, mono- and polycyclic aromatic hydrocarbons, a number of pesticides, major anions and cations. · Criminalistics - drugs, organic explosives and dyes, potent pharmaceuticals. · Pharmaceutical industry - steroid hormones, practically all products of organic synthesis, antibiotics, polymer preparations, vitamins, protein preparations. Medicine - the listed biochemical and medicinal substances and their metabolites in biological fluids (amino acids, purines and pyrimidines, steroid hormones, lipids) in the diagnosis of diseases, determining the rate of excretion of drugs from the body for the purpose of their individual dosage. · Agriculture - determination of nitrate and phosphate in soils to determine the required amount of fertilizers, determination of the nutritional value of feed (amino acids and vitamins), analysis of pesticides in soil, water and agricultural products. Biochemistry, bioorganic chemistry, genetic engineering, biotechnology - sugars, lipids, steroids, proteins, amino acids, nucleosides and their derivatives, vitamins, peptides, oligonucleotides, porphyrins, etc. · Organic chemistry - all stable products of organic synthesis, dyes, thermolabile compounds, non-volatile compounds; inorganic chemistry (practically all soluble compounds in the form of ions and complex compounds). · Quality control and safety of food products, alcoholic and non-alcoholic beverages, drinking water, household chemicals, perfumes at all stages of their production; determination of the nature of pollution at the site of a man-made disaster or emergency; detection and analysis of narcotic, potent, poisonous and explosive substances; determination of the presence of harmful substances (polycyclic and other aromatic hydrocarbons, phenols, pesticides, organic dyes, ions of heavy, alkaline and alkaline earth metals) in liquid effluents, air emissions and solid waste from enterprises and in living organisms; · monitoring of processes of organic synthesis, oil and coal processing, biochemical and microbiological productions; analysis of soil quality for fertilization, the presence of pesticides and herbicides in soil, water and products, as well as the nutritional value of feed; complex research analytical tasks; obtaining a micro amount of ultrapure substance.
Liquid chromatography Liquid chromatography is a type of chromatography in which mobile phase, called the eluent, is liquid. Stationary phase May be solid sorbent, solid carrier with liquid deposited on its surface or gel. Distinguish columnar And thin-layer liquid chromatography. In the column version, a portion of the separated mixture of substances is passed through a column filled with a stationary phase in an eluent stream that moves under pressure or under the influence of gravity. In thin layer chromatography, the eluent moves under the action of capillary forces along a flat layer of sorbent deposited on a glass plate or metal foil, along a porous polymer film, or along a strip of special chromatography paper. A method of thin-layer liquid chromatography under pressure has also been developed, in which the eluent is pumped through a layer of sorbent sandwiched between plates. There are such types of liquid chromatography as analytical(for analysis of mixtures of substances) and preparative(to isolate pure components). Distinguish liquid chromatography (LC) in its classical version, carried out with atmospheric pressure, And high speed), carried out at high blood pressure. High-performance liquid chromatography (HPLC) uses columns with a diameter of up to 5 mm, tightly packed with a sorbent with small particles (3-10 µm). To pump the eluent through the column, apply pressure up to 3.107 Pa. This type of chromatography is called high pressure chromatography. Passing the eluent through a column under high pressure allows you to dramatically increase the speed of analysis and significantly increase the efficiency of separation due to the use of finely dispersed sorbent. HPLC options are microcolumn chromatography on columns of small diameter filled with sorbent and capillary chromatography on hollow and sorbent-filled capillary columns. The HPLC method currently makes it possible to isolate, quantitatively and qualitatively analyze complex mixtures of organic compounds. Liquid chromatography is the most important physical and chemical research method in chemistry, biology, biochemistry, medicine, and biotechnology. It is used for: · study of metabolic processes in living organisms of drugs; · diagnostics in medicine; · analysis of chemical and petrochemical synthesis products, intermediates, dyes, fuels, lubricants, oil, wastewater; · study of sorption isotherms from solution, kinetics and selectivity of chemical processes; · discharge · analysis and separation of mixtures, their purification and isolation of many biological substances from them, such as amino acids, proteins, enzymes, viruses, nucleic acids, carbohydrates, lipids, hormones. In the chemistry of macromolecular compounds and in the production of polymers, liquid chromatography is used to analyze the quality of monomers, study the molecular weight distribution and distribution by type of functionality of oligomers and polymers, which is necessary for product control. Liquid chromatography is also used in perfumery, the food industry, for the analysis of environmental pollution, and in forensic science. The high-performance liquid chromatography (HPLC) method was developed and introduced in the mid-70s of the 20th century. Then the first liquid chromatographs appeared. Liquid chromatography is the optimal method for analyzing chemically and thermally unstable molecules, high molecular weight substances with reduced volatility. This can be explained by the special role of the mobile phase in LC, in contrast to gas chromatography: the eluent performs not only a transport function. 2. Basic concepts and classification of liquid chromatography methods. By mechanism of retention of separated substances by the stationary phase LC distinguish: · adsorption chromatography ,
in which separation is carried out as a result of the interaction of the substance being separated with adsorbent such as aluminum oxide or silica gel, having on the surface active polar centers. Solvent(eluent) - non-polar liquid. Rice. Scheme for separating a mixture of substances using adsorption chromatography http://www. xumuk. ru/biologhim/bio/img014.jpg The mechanism of sorption consists of a specific interaction between the polar surface of the sorbent and the polar (or capable of polarization) sections of the molecules of the analyzed component (Fig.). The interaction occurs due to donor-acceptor interaction or the formation of hydrogen bonds. Rice. Scheme of adsorption liquid chromatography https://pandia.ru/text/80/271/images/image006_11.jpg" width="219" height="200"> Rice. . Partition chromatography with grafted phase (normal phase version). http://www. chemnet. ru/rus/teaching/oil/spezprakt-chr. html At normal phase In the partition liquid chromatography version, substituted alkylchlorosilanes containing polar groups, such as nitrile, amino group, etc., are used as silica gel surface modifiers (grafted phases) (Fig.). The use of bonded phases makes it possible to finely control the sorption properties of the surface of the stationary phase and achieve high separation efficiency. Reversed phase liquid chromatography is based on the distribution of mixture components between a polar eluent and non-polar groups (long alkyl chains) grafted onto the surface of the sorbent (Fig.). Less commonly used is a variant of liquid chromatography with deposited phases, when a liquid stationary phase is deposited on a stationary carrier. Rice. . Partition chromatography with grafted phase (reversed phase version). http://www. chemnet. ru/rus/teaching/oil/spezprakt-chr. html Partition liquid chromatography also includes extraction liquid chromatography, in which the stationary phase is an organic extractant deposited on a solid carrier, and the mobile phase is an aqueous solution of the compounds being separated. As extractants, for example, phenols, trialkyl phosphates, amines, quaternary ammonium bases, as well as sulfur-containing organophosphorus compounds are used. Extraction liquid chromatography is used to separate and concentrate inorganic compounds, for example, alkali metal ions, actinides and other elements with similar properties, in the processes of processing spent nuclear fuel. R-H + Na+ + Cl - → R-Na + H+ + Cl - (cation exchange) R-OH + Na+ + Cl - → R-Cl + Na+ + OH - (anion exchange) Ion exchangers must meet the following requirements: be chemically stable in various environments, mechanically strong in a dry and especially swollen state, have high absorption capacity and the ability to regenerate well. In ion exchange (ion) chromatography, the separated anions (cations) are detected as acids (corresponding bases) by a highly sensitive conductometric detector, where high-efficiency columns are packed with a low-capacity surfactant ion resin. https://pandia.ru/text/80/271/images/image009_7.jpg" align="right" width="429" height="319"> Rice. Scheme of gel permeation chromatography Rice. Affinity chromatography scheme http://www. chemnet. ru/rus/teaching/oil/spezprakt-chr. html It is then removed from the polymer by passing a solution of a compound that has an even greater affinity for the macromolecule. Such chromatography is especially effective in biotechnology and biomedicine for isolating enzymes, proteins, and hormones. depending on the method of movement of matter The following types of liquid chromatography are distinguished: developmental, frontal And repressive. https://pandia.ru/text/80/271/images/image012_4.jpg" width="291" height="165"> Rice. Scheme of developing chromatography variant Height or peak area characterizes concentration of components, A held volumes – high-quality mixture composition. Identification of components is usually carried out by coincidence of retention times with standard substances; chemical or physicochemical methods are also used. At frontal In the variant (Fig.), a mixture of the substances to be separated is continuously passed through the column, which plays the role of a mobile phase. As a result, it is possible to obtain in pure form only the substance that is least sorbed in the column. https://pandia.ru/text/80/271/images/image014_2.jpg" width="279" height="145"> Rice. Scheme of the frontal chromatography option The chromatogram in this case represents steps, the heights of which are proportional to the concentrations of the components; the retained volumes are determined by the retention time of the components. When differentiating such a chromatogram, a picture is obtained similar to that obtained in the developing version. IN repressive In this case, the components of the mixture introduced into the column are displaced by the eluent, which is adsorbed more strongly than any component. As a result, fractions of the separated substances adjacent to each other are obtained. The order of release of the components is determined by the strength of their interaction with the surface of the sorbent (Fig.). https://pandia.ru/text/80/271/images/image016_3.jpg" width="320" height="175"> Rice. Scheme of displacement chromatography 3. Basic chromatographic quantities and their determination. When separating substances using liquid chromatography, developmental, frontal and displacement options can be used, as indicated above. Most often, a developing option is used, in which a portion of the mixture to be separated is introduced into the column in the eluent flow. The output of the mixture components from the column is recorded on the chromatogram in the form of peaks. From the chromatogram (Fig.) determine: https://pandia.ru/text/80/271/images/image018_12.gif" width="61" height="24 src=">; b) corrected component retention volume ,
Where t"R - corrected component retention time; c) coefficient of column capacitance in relation to a given component d) column efficiency characterized number of equivalent theoretical plates https://pandia.ru/text/80/271/images/image022_8.gif" width="129" height="51 src=">; f) permission https://pandia.ru/text/80/271/images/image024_9.gif" width="203 height=51" height="51"> Capacity factor k"
has a significant impact on the value R S: when changing k" from 0 to 10 (optimal limits) R S increases greatly. Meaning k" is determined by the doubled surface of the sorbent and its amount in the column, as well as the adsorption equilibrium constant (Henry’s constant). Selectivity coefficient α is determined by the difference in the adsorption equilibrium constants of the two separated components. As α increases (from 1 to ~5) R S increases sharply, with a further increase in α - changes little. The selectivity of a column depends on factors such as the chemical structure of the sorbent surface, the composition of the eluent, the temperature of the column, and the structure of the compounds being separated. Since the sorption of chromatographed substances in liquid chromatography is determined by the pairwise interaction of the three main components of the system - the sorbent, the substances being separated and the eluent, changing the composition of the eluent is a convenient way to optimize the separation process. Column efficiency depends on the particle size and pore structure of the adsorbent, on the uniformity of column packing, the viscosity of the eluent and the mass transfer rate. Lengthening the column does not always lead to improved separation, since the resistance of the column increases, the eluent pressure at the inlet and the time of the experiment increase, and the sensitivity and accuracy of the analysis decrease due to the broadening of the peak of the analyzed component. If , then the peaks of the two substances in the chromatogram are separated almost completely. With growth R S separation time increases. At R S <
1 - separation is unsatisfactory. In preparative chromatography, due to the introduction of relatively large quantities of separated substances, the column operates with overload. In this case, the capacitance coefficient decreases, the height equivalent to the theoretical plate increases, which leads to a decrease in resolution. 4. Adsorbents Chromatographic separation of a mixture will be effective if the adsorbent and solvent (eluent) are correctly selected. The adsorbent should not chemically interact with the separated components or exhibit a catalytic effect on the solvent. It is also necessary that the adsorbent be selective with respect to the components of the mixture. A correctly selected adsorbent should have maximum absorption capacity. Distinguish polar (hydrophilic) And non-polar (hydrophobic) adsorbents. It should be remembered that the adsorption affinity of polar substances for polar sorbents is much higher than for non-polar ones. Aluminum oxide, activated carbons, silica gel, zeolites, cellulose and some minerals are used as adsorbents. Aluminium oxideAl2O3 – amphoteric adsorbent.(Fig.) On it mixtures can be separated substances in polar, so in non-polar solvents. Neutral aluminum oxide is usually used for chromatography from non-aqueous solutions of saturated hydrocarbons, aldehydes, alcohols, phenols, ketones and ethers. Rice. Aluminum Oxide for Chromatography http://images. /542857_w200_h200_product5.jpg The activity of Al2O3 depends on its moisture content. Anhydrous aluminum oxide has the highest activity. It is conventionally taken as one. If necessary, alumina with different moisture contents can be prepared by mixing freshly prepared alumina with water (Brockman scale). Dependence of aluminum oxide activity on moisture content For example, Al2O3 with an activity of 1.5-2 is used for the separation of hydrocarbons; for the separation of alcohols and ketones – 2-3.5. The specific surface of aluminum oxide is 230-380 m2/g. silica gel(hydroxylated or chemically modified) is a dried gelatinous silicon dioxide, which is obtained from supersaturated solutions of silicic acids ( n SiO2 m H2O) at pH > 5-6. (fig.) Solid hydrophilic sorbent. Rice. silica gel http://www. silica gel. / http://silikagel. ru/images/askg. gif The particle size of silica gel in analytical columns is 3-10 microns, in preparative columns - 20-70 microns. The small particle size increases the mass transfer rate and improves column efficiency. Modern analytical columns are 10-25 cm long. They are filled with silica gel with a particle size of 5 microns and allow you to separate complex mixtures of 20-30 components. As the particle size decreases to 3-5 microns, the efficiency of the column increases, but its resistance also increases. So, to achieve an eluent flow rate of 0.5-2.0 ml/min, a pressure of (1-3)·107 Pa is required. Silica gel can withstand such a pressure difference, while the granules of polymer sorbents are more elastic and deformable. Recently, mechanically strong polymer sorbents with a macroporous structure with a dense network have been developed, which in their effectiveness are close to silica gels. The shape of sorbent particles with a size of 10 µm and above does not have a big impact on the efficiency of the column, however, spherical sorbents are preferred, which provide more permeable packaging. (Fig.) Rice. Spherical silica gel http://images. /6450630_w200_h200_silicagelksmg. gif http:///N6_2011/U7/silikagel-2.jpeg The internal structure of a silica gel particle is a system of communicating channels. For liquid chromatography, sorbents with a pore diameter of 6-25 nm are used. Liquid chromatography separation is carried out mainly on silica gels modified by the reaction of alkyl and aryl chlorosilanes or alkyl ethoxy silanes with silanol groups on the surface. Using such reactions, C8H17-, C18H37- or C6H5- groups are grafted (to obtain sorbents with a hydrophobized surface), nitrile, hydroxyl groups, etc. (Fig.) Rice. Structure of modified silica gel Silica gels used in chromatography for separating mixtures of petroleum products, higher fatty acids, their esters, aromatic amines, nitro derivatives organic compounds. silica gel – hydrophilic sorbent, easily wetted with water. Therefore, it cannot be used for sorption from aqueous solutions. The activity of silica gel depends on the water content in it: the less water it contains, the greater the activity (Brockmann scale). Dependence of silica gel activity on moisture content The specific surface area of silica gels is 500-600 m2/g. Activated carbons are a form of carbon that, when processed, becomes extremely porous and acquires a very large surface area available for adsorption or chemical reactions. (Fig.) They have a specific surface area of 1300-1700 m2/g. Rice. Activated carbon http://e-catalog. rusbiz. ru/user_images/ru/prod_picture/58035161249b9016f64372.jpg The main influence on the pore structure of activated carbons is exerted by the starting materials for their production. Activated carbons based on coconut shells are characterized by a larger proportion of micropores (up to 2 nm), while those based on coal are characterized by a larger proportion of mesopores (2-50 nm). A large proportion of macropores is characteristic of wood-based activated carbons (more than 50 nm). Micropores are particularly well suited for the adsorption of small sized molecules, while mesopores are particularly well suited for the adsorption of larger organic molecules. Zeolites (molecular sieves)– porous crystalline aluminosilicates of alkali and alkaline earth metals of natural and synthetic origin. (rice.) https://pandia.ru/text/80/271/images/image036_2.jpg" width="211 height=211" height="211"> Rice. Zeolites http://www. zeolite spb. ru/_img/_36mm. jpg http://kntgroup. ru/thumb. php? file=/uploads/produkts/6.jpg&x_width=250 There are four known types of zeolites (A, X, Y, M), having different crystal structures. Depending on the cation, zeolites are designated as follows: KA, NaA, CaM, NaX, KY, CaY. Feature of zeolites is that the pores of the crystals have sizes of the order of 0.4-1 nm, comparable to the size of molecules many liquid or gaseous substances. If the molecules of a substance are able to penetrate into these pores, then adsorption occurs in the pores of zeolite crystals. Larger molecules of the substance are not adsorbed. By selecting zeolites with different pore sizes, mixtures of different substances can be clearly separated. The specific surface of zeolites is 750-800 m2/g. When choosing an adsorbent, it is necessary to take into account the structure of substances and their solubility. For example, saturated hydrocarbons are poorly adsorbed, while unsaturated hydrocarbons (have double bonds) are adsorbed better. Functional groups enhance the adsorption ability of a substance. 5. Eluents When choosing a solvent (eluent), you need to take into account the nature of the adsorbent and the properties of the substances in the mixture to be separated. Eluents must dissolve well all components of the chromatographed mixture, have low viscosity, provide the required level of selectivity, be cheap, non-toxic, inert, and compatible with detection methods (for example, benzene cannot be used as an eluent with a UV detector). In normal-phase chromatography, hydrocarbons (hexane, heptane, isooctane, cyclohexane) are usually used with the addition of small amounts of chloroform CHCl3, iso-propanol iso-C3H7OH, diisopropyl ether; in reverse phase chromatography - a mixture of water with acetonitrile CH3CN, methanol CH3OH, ethanol C2H5OH, dioxane, tetrahydrofuran, dimethylformamide. To isolate individual components of a mixture separated during chromatography, they are often washed out (eluted) successively. For this purpose, solvents with different desorption abilities are used. Solvents are arranged in descending order of desorbing ability in polar adsorbents - eluotropic Trappe series. If the components of the mixture being separated have similar values k" ( coefficient of column capacity relative to a given component), then chromatograph with one eluent. If individual components of the mixture are strongly retained by the sorbent, a series of eluents of increasing strength are used. Eluotropic series of solvents 6. Equipment for liquid chromatography In modern liquid chromatography, instruments of varying degrees of complexity are used - from the simplest systems to high-class chromatographs. On fig. a block diagram of a liquid chromatograph is presented, containing the minimum required set of components, in one form or another, present in any chromatographic system. https://pandia.ru/text/80/271/images/image038_2.jpg" width="361" height="254 src="> Rice. Diagram of a liquid chromatograph: 1 - reservoir for the mobile phase, 2 - pump, 3 - injector, 4 - column, 5 - thermostat, 6 - detectors, 7 - recording system, 8 - computer. Storage tank for the mobile phase, must have sufficient capacity for analysis and solvent degassing device to prevent the formation of bubbles of gases dissolved in the eluent in the column and detector. Pump intended to create a constant flow of solvent. Its design is determined primarily by the operating pressure in the system. To operate in the range of 10-500 MPa, plunger (syringe) type pumps are used. Their disadvantage is the need for periodic stops to fill with eluent. For simple systems with low operating pressures of 1-5 MPa, inexpensive peristaltic pumps are used. Eluents enter the pump through a filter that retains dust particles (more than 0.2 microns). Sometimes a small current of helium is passed through the eluents to remove dissolved air and prevent the formation of bubbles in the detector (especially in the case of aqueous and polar eluents). In analytical chromatographs, piston pumps with a feedback system are used to supply the eluent to the column, making it possible to smooth out flow pulsations within 1-2% and provide volumetric flow rates from 0.1 to 25 ml/min at pressures up to ~ 3.107 Pa. In microcolumn chromatography, volumetric flow rates of the eluent are much lower - 10-1000 µl/min. In the case of gradient elution, several pumps are used, which are controlled by a programmer and supply 2-3 eluent components into the mixing chamber, leaving the overall flow rate constant. To introduce a sample into a column under high pressure without stopping the flow, special microdosing taps are used, connected to a loop of known volume for the solution sample being studied. Dosing systems have been developed with automatic sampling and injection of samples using microdosing taps or syringes. Injector provides injection of mixture sample separated components into a column with fairly high reproducibility. Simple "stop-flow" sample injection systems require the pump to be stopped and are therefore less convenient than Reodyne's loop pipettes. speakers for HPLC they are most often made from a stainless steel polished tube 10-25 cm long and an internal diameter of 3-5 mm. Rice. Chromatography columns for liquid chromatography Also used glass speakers, placed in a metal casing; in microcolumn chromatography - packed metal speakers with an internal diameter of 1.0-1.5mm, packed glass microcolumns with a diameter of 70-150 microns and hollow capillary columns diameter 10-100 microns; in preparative chromatography - columns with a diameter of 2 to 10 cm or more. To uniformly and densely fill the columns with sorbent, the suspension packing method is used. A suspension is prepared from a sorbent and a suitable organic liquid, which is supplied under pressure up to 5·107 Pa into the column. To determine the separated components leaving a column use detectors. Consistency of temperature provided thermostat. Detectors for liquid chromatography they have a flow cell in which a continuous measurement of some property of the flowing eluent occurs. They must be very sensitive. To increase the sensitivity of the detector, derivatization of the components of the mixture after the column is sometimes used. To do this, reagents are introduced with the eluent flow that, interacting with the separated substances, form derivatives with more pronounced properties, for example, they absorb more strongly in the UV or visible region of the spectrum or have greater fluorescent ability. Sometimes derivatization is carried out before chromatographic analysis and the derivatives are separated rather than the starting materials. Most popular types detectors general purpose are refractometers, measuring refractive index, And spectrophotometric detectors, defining solvent optical density at a fixed wavelength (usually in the ultraviolet region). TO advantages of refractometers(And disadvantages of spectrophotometers) should be attributed low sensitivity to the type being determined connections, which may not contain chromophore groups. On the other hand, the use of refractometers is limited to isocratic systems (with a constant eluent composition), so the use of a solvent gradient is not possible in this case. Differential "href="/text/category/differentcial/" rel="bookmark">differential amplifier and recorder. It is also desirable to have integrator, which allows you to calculate the relative areas of the resulting peaks. In complex chromatographic systems it is used interface block, connecting the chromatograph to personal computer, which not only collects and processes information, but also controls the device, calculates quantitative characteristics and, in some cases, the qualitative composition of mixtures. Microprocessor provides automatic sample injection, change by specified eluent composition program with gradient elution, maintaining column temperature. Bruker". Rice. Liquid chromatograph Jasco Questions for self-examination List of used literature 1 "Liquid chromatography in medicine" http://journal. issep. rssi. ru/articles/pdf/0011_035.pdf 2 “Introduction to high-performance liquid chromatography methods” http://www. chemnet. ru/rus/teaching/oil/spezprakt-chr. html 3 "Liquid chromatography" http://e-science. ru/index/?id=1540 4 "Chromatography" http://belchem. people. ru/chromatography1.html
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sediment chromatography, based on the different solubility of precipitates that are formed during the interaction of the components of the analyzed mixture with the precipitant. The advantage of the method is that the resulting zones along the sorbent have sharp boundaries, contain sediments of only one substance and are often separated by zones of pure sorbent. However, this method has not yet found widespread use.
ion exchange chromatography, which is based on the reversible stoichiometric exchange of ions contained in the analyzed solution with mobile ions included in the composition ionites. Depending on the sign of the charge of the ionizing groups, ion exchangers are divided into cation exchangers And anion exchangers. There are also amphoteric ion exchangers –ampholytes, which can simultaneously exchange both cations and anions. Ion exchange chromatography is used only for the separation of charged particles. The separation is based on the ability of the ion exchange resin to hold different ions with different strengths. Ionite consists of a polymer matrix and active groups associated with it, which are capable of ion exchange. Cationite has acidic or slightly acidic properties, since it contains groups: - SO3H, –CH2SO3H, - COOH, - PO3H2 and others in which hydrogen ions are mobile. Anion exchangers have basic or weakly basic properties and contain groups: = NH2, - NH2, –NR3+, -OH and others. The separation of ions is regulated by selecting the optimal pH values of the eluent and its ionic strength. Schematically, ion exchange can be represented by the reactions:
ion pair chromatography, which can be considered as a combination of adsorption and ion exchange chromatography. The method is based on the extraction of ionic substances - their transfer from the aqueous phase to the organic phase in the form of ion pairs. To do this, a counterion is added to the mobile phase, which is capable of selectively reacting with the analyzed components, turning them into complex compounds with the formation of an ion pair. The main advantages of this option are that acidic, basic and neutral substances can be analyzed simultaneously.
ligand exchange chromatography based on different abilities of the separated compounds to form complexes with transition metal cations– Cu+2, Ni+2, Zn+2, Cd+2, Co+2, etc. - and fixing groups (ligands) of the stationary phase. Part of the coordination sphere of metal ions is occupied by water molecules or other weak ligands, which can be displaced by molecules of the compounds being separated. This type of chromatography is used to separate optical isomers.
size exclusion chromatography(sieve, gel permeation, gel filtration), in which the separation is based on differences in molecular sizes.
affinity chromatography(biospecific), based on the fact that many biologically active macromolecules, for example, enzymes, can specifically bind to a certain reagent. The reagent is fixed to a carrier (often agarose), then washed with the mixture to be analyzed. Only the desired macromolecule is retained on the polymer (Fig.).
Most often used manifestive a variant in which a portion of the mixture to be separated is introduced into the column in the eluent flow. The output of the mixture components from the column is recorded on the chromatogram in the form of peaks. (rice.)retention times of non-sorbing (t0), separated components (tR1, tR2, tR3, etc.); width of the peak bases (tw1, tw2, etc.).
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A modern liquid chromatograph includes: containers for eluents, high-pressure pumps, a dispenser, a chromatographic column, a detector, a recording device, a control system and mathematical processing of results.
