High performance liquid chromatography. Course work: High performance liquid chromatography of pollutants in natural and waste waters. See what "" is in other dictionaries

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 allows the isolation, quantitative and qualitative analysis of 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:

sediment chromatography

· 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

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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

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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 grafted 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.

ion exchange chromatography,

ionites.

cation exchangers

anion exchangers.

amphoteric ion exchangers

ampholytes

Ionite

Cationite

Anion exchangers

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.

ion pair chromatography

ligand exchange chromatography

different abilities of the separated compounds to form complexes with transition metal cations

size exclusion chromatography

differences in molecular sizes

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Rice. Scheme of gel permeation chromatography

affinity 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.
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.)

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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.

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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.).

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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:

retention times of non-sorbing (t0), separated components (tR1, tR2, tR3, etc.); width of the peak bases (tw1, tw2, etc.).

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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

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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

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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

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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.)

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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

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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.)

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Rice. Zeolites

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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.
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.

In 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.

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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 stopping the pump and are therefore less convenient than the loop pipettes developed by Reodyne.

Columns 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 in this case is impossible.

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

Self-test questions

What is liquid chromatography? Name its types and areas of application. List about basic chromatographic quantities and their definition What types of liquid chromatography exist depending on the mechanism of retention of the separated substances by the stationary phase of LC? What types of chromatography exist depending on the method of moving the substance? What substances are used as adsorbents? What is the difference? What serves as the liquid mobile phase - the eluent? Requirements for solvents. What is the difference between partition chromatography and adsorption chromatography? List the main parts of a liquid chromatograph circuit and their purpose.

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

(OFS 42-0096-09)

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

bone moving through a chromatography column filled with inaccessible

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

called "high pressure liquid chromatography".

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

current HPLC options: adsorption, distribution, ion exchange,

exclusive, chiral, etc.

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

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

In distribution HPLC, separation occurs due to differences in the distribution coefficients of the substances being separated between the stationary

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

mobile phases.

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

extended-phase.

Normal-phase is a variant of chromatography in which

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

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

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

use non-polar chemically modified sorbents (for example,

non-polar alkyl radical C18) and polar mobile phases (e.g.

methanol, acetonitrile).

In ion exchange chromatography, molecules of substances in a mixture, dissociate

in solution into cations and anions are separated when moving through

sorbent (cation exchanger or anion exchanger) due to their different rates of exchange with ion-

mi groups of sorbent.

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

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

The columns yield the largest molecules (with the highest molecular weight) capable of penetrating into the minimum number of pores of the stationary phase,

and the last to emerge are substances with small molecular sizes.

Often separation occurs not through one, but through several mechanisms simultaneously.

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

zooform analytes. To carry out the analysis, appropriate instruments are used - liquid chromatographs.

The composition of a liquid chromatograph usually includes the following basic elements:

Nodes:

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

ties with individual solvents included in the mobile phase

3) and the PF degassing system;

– pumping system;

– mobile phase mixer (if necessary);

– sample introduction system (injector);

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

– detector;

– data collection and processing system.

Pumping system

Pumps supply PF to the column at a given constant speed. The composition of the mobile phase can be constant or variable.

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

and in the second - gradient. They are sometimes installed in front of the pumping system

filters with a pore diameter of 0.45 microns for filtration of the mobile phase. Modern

A liquid chromatograph pump system consists of one or more computer-controlled pumps. This allows you to change the

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

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

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

however, a more accurate ratio of components is achieved by pre-

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

shal 20 MPa. Pressure pulsations are minimized by special dampers

ferrous systems included in the pump design. Working parts on-

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

Faucets

By design, mixers can be static or dynamic

mic.

In the mixer, a single mobile phase is formed from the

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

sewing.

Injectors

Injectors can be universal for introducing samples from

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

ema. Both types of injectors can be automatic ("autoinjectors" or "autosamplers"). The injector for introducing the sample (solution) is located

directly in front of the chromatography column. The design of the injector allows you to change the direction of the PF flow and pre-introduce a sample into a loop of a certain volume (usually from 10 to 100 μl).

This volume is indicated on the loop label. The injector design allows for loop replacement. To introduce the test solution into a non-automatic

Tomatic injector uses a manual microsyringe with a volume of significantly

significantly exceeding the volume of the loop. Excess of injected solution, not

located in the loop is reset, and an exact and always equal volume of sample is injected into the column. Manually incompletely filling the loop reduces the accuracy

accuracy and reproducibility of dosing and, therefore, impairs the accuracy

accuracy and reproducibility of chromatographic analysis.

Chromatographic column

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

lined on both sides with filters with a pore diameter of 2–5 microns. Analytical length

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

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

tography. Capillary columns with an internal diameter of

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

Short tubes can be installed in front of the analytical column.

columns (pre-columns) performing various auxiliary functions

(more often – protection of the analytical column). Usually the analysis is carried out with

at normal temperature, however, to increase the efficiency of separation and con-

To shorten the duration of analysis, thermostats can be used.

testing of columns at temperatures not higher than 60 C. At higher temperatures, destruction of the sorbent and changes in the composition of the PF are possible.

Stationary phase (sorbent)

The following are usually used as sorbents:

1. Silica gel, aluminum oxide, porous graphite are used in normal

small-phase chromatography. The retention mechanism in this case is

tea - usually adsorption;

2. Resins or polymers with acidic or basic groups. Area of application: ion exchange chromatography;

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

4. Chemically modified sorbents (sorbents with grafted fa-

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

new and stationary phases;

5. Chemically modified chiral sorbents, for example those produced

aqueous celluloses and amyloses, proteins and peptides, cyclodextrins,

used for the separation of enantiomers (chiral chromatography

Sorbents with grafted phases can have varying degrees of chemical

ical modification. Sorbent particles can be spherical or non-spherical

correct shape and varied porosity.

The most commonly used grafted phases are:

– octyl groups(octylsilane or C8 sorbent);

– octadecyl groups(sorbent octadecylsilane

(ODS) or C18);

– phenyl groups(phenylsilane sorbent);

– cyanopropyl groups(CN sorbent);

– aminopropyl groups(NH2 sorbent);

– diol groups (sorbent diol).

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

reverse phase mode using C18 sorbent.

In some cases, it is more appropriate to use normal

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

Sorbents with grafted phases are chemically stable at pH values from 2.0 to 8.0, unless otherwise specifically stated by the manufacturer.

Sorbent particles can have spherical or irregular shapes and varied porosity. The particle size of the sorbent in analytical HPLC is usually 3–10 µm, in preparative HPLC – up to 50 µm or more.

Monolithic sorbents are also used.

High separation efficiency is ensured by the high surface area of sorbent particles (which is a consequence of their microscopic

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

Detectors

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

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

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

logical or physicochemical parameter of PF as a function of time.

The most common are spectrophotometric de-

tectors (including diode-matrix) that record changes in optical

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

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

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

The traditionally used spectrophotometric detector allows

allows detection at any wavelength in its operating range

zone. Multi-wave detectors are also used, allowing

Perform detection at several wavelengths simultaneously.

Using a diode array detector, you can not only detect several wavelengths at once, but also almost instantly

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

ponents.

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

variable fluorescent detectors allow not only to obtain chromato-

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

zirovable connections.

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

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

Electrochemical detectors are also used (conductometric

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

detectors, light scattering detectors, radioactivity detectors and some other

Mobile phase

IN A variety of solvents can be used as PF - both individual and mixtures thereof.

IN normal-phase chromatography usually uses liquid carbon

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

solvents with small additions of polar organic compounds,

which regulate the elution strength of the PF.

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

organic solvents (usually acetonitrile and methanol) and water. For optical

separation systems often use aqueous solutions with a certain

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

chemical and organic acids, bases and salts and other compounds (for example,

example, chiral modifiers for separating enantiomers into achiral-

nom sorbent).

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

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

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

owl ratio, which should be specially stipulated.

When using a UV spectrophotometric detector, the PF should not have pronounced absorption at the wavelength selected for detection. Transparency limit or optical density when determined

The specific wavelength of a solvent from a particular manufacturer is often indicated

is on the packaging.

The chromatographic analysis is greatly influenced by the degree of purity of the PF, therefore it is preferable to use solvents produced

specially designed for liquid chromatography (including water).

PF and analyzed solutions should not contain undissolved

of particles and gas bubbles. Water obtained in laboratory conditions

aqueous solutions, organic solutions pre-mixed with water

The media, as well as the analyzed solutions, must be subjected to fine filtration and degassing. Filtration is usually used for these purposes.

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

Data collection and processing system

A modern data processing system is a con- nected

a personal computer connected to a chromatograph with installed software

software that allows you to register and process chro-

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

parameters of the chromatographic system.

List of chromatographic conditions to be specified

The private pharmacopoeial monograph must indicate the dimensions of the co-

columns, type of sorbent indicating particle size, column temperature (if thermostatting is necessary), volume of injected sample (loop volume),

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

ION EXCHANGE AND ION HPLC

Ion exchange chromatography is used for the analysis of both organic

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

ganic (various cations and anions) compounds. Composite separation

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

memory of the sorbent. Anion exchangers or cation exchangers are used as sorbents.

You. These sorbents are mainly either polymer ion-

exchange resins (usually copolymers of styrene and divinylbenzene with

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

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

cation removal – modified silica gels.

Aqueous solutions of acids, bases and salts are used as PFs in ion exchange chromatography. Usually buffers are used

solutions that allow you to maintain certain pH values. It is also possible to use small organic additives that mix with water.

ski solvents - acetonitrile, methanol, ethanol, tetrahydrofuran.

Ion chromatography- a variant of ion exchange chromatography, in

in which, to determine the concentration of ions of the analyte, we use

uses a conductometric detector. For highly sensitive op-

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

There are two main variants of ion chromatography.

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

PF using a second ion exchange column located between the

lytic column and detector. Neutralization occurs in this column

tion of the PF and the analyzed compounds enter the detector cell in deionization

distilled water. The detected ions are the only ions

ensuring conductivity of the PF. The disadvantage of the suppression column is the need for its regeneration after fairly short periods of time.

me. The suppression column can be replaced continuously

a common membrane suppressor in which the composition of the membrane is continuously

is renewed by a flow of regenerating solution moving in the direction

opposite to the direction of PF flow.

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

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

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

Chinese acids - benzoic, salicylic or isophthalic.

SECURE HPLC

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

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

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

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

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

The advantages of porous glasses and silica gel are the rapid diffusion of PF and molecules of the analyzed substance into the pores, stability in various conditions (even at high temperatures). Polymer sorbene-

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

phobic sorbents used with non-polar mobile phases) and

hydrophilic gels obtained from sulfonated divinylbenzene or polyacrylamide resins.

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

zoy first. Molecules with a diameter significantly smaller than the pore size of the sorbent

benta freely penetrate into it, remain in the stationary phase for the longest time and are the last to elute. Medium-sized molecules penetrate into the pores of the sorbent depending on their size and partly depending on their shape. They elute with different retention times between

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

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

In size exclusion chromatography, to characterize retention,

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

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

Chromatography is generally divided into gel filtration and gel chromatography.

permeation chromatography.

The gel filtration chromatography method is used to separate

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

In gel permeation chromatography, hydrophobic sorbs are used.

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

ragidofuran). This method is used for the analysis of compounds that are poorly soluble

rims in the water.

Detectors. Differential refractometric detectors, as well as spectrophotometric detectors (including in the IR spectral region) are used as detectors in size exclusion chromatography.

Viscometer and flow laser detectors are also used.

These detectors, in combination with a refractometer or other concentration

detector make it possible to continuously determine the molecular mass

limer in PF.

ULTRA-PERFORMANCE LIQUID CHROMATOGRAPHY

Ultraperformance liquid chromatography is a variant of liquid chromatography that is more efficient

sity compared to classical HPLC.

A feature of ultra-performance liquid chromatography is

The use of sorbents with particle sizes from 1.5 to 2 microns is possible. Dimensions chrome

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

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

Chromatographic equipment used in classical va-

riant HPLC, usually specially adapted for this type of chromatogra-

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