Liquid chromatography principle of the method. High performance liquid chromatography of natural and waste water pollutants

Introduction

Chapter 1. Basic concepts and classification of liquid chromatography methods

1.1 Apparatus for liquid chromatography

Chapter 2. The essence of HPLC

2.1 Application

Chapter 3. Examples of the use of HPLC in the analysis of environmental objects

Chapter 4. Equipment for HPLC

Literature

Application

Introduction

Chromatographic methods are often indispensable for identifying and quantifying organic substances with a similar structure. At the same time, the most widely used for routine analysis of environmental pollutants are gas and high performance liquid chromatography. Gas chromatographic analysis of organic pollutants in drinking and waste water was first based on the use of packed columns, later quartz capillary columns also became widespread. The inner diameter of the capillary columns is usually 0.20-0.75 mm, the length is 30-105 m. Optimal results in the analysis of contaminants in water are achieved most often when using capillary columns with different film thicknesses made of methylphenylsilicones containing phenyl groups of 5 and 50% ... The sample introduction system often becomes a vulnerability in chromatographic techniques using capillary columns. Sample introduction systems can be divided into two groups: universal and selective. Versatile applications include split and splitless injection systems, “cold” column injection, and temperature programmed evaporation. With selective injection, blowing with intermediate trapping, headspace analysis, etc. is used. When using universal injection systems, the entire sample is supplied to the column; with selective injection, only a certain fraction is injected. The results obtained with selective injection are much more accurate, since the fraction entering the column contains only volatile substances, and the technique can be fully automated.

Gas chromatographic detectors used in the monitoring of pollutants are often divided into universal detectors that respond to each component in the mobile phase, and selective detectors that respond to the presence of a certain group of substances with similar chemical characteristics in the mobile phase. The universal ones include flame ionization, atomic emission, mass spectrometric detectors and infrared spectrometry. Selective detectors used in water analysis are electron capture (selective to substances containing halogen atoms), thermoionic (selective to nitrogen and phosphorus-containing compounds), photoionization (selective to aromatic hydrocarbons), electrolytic conductivity detector (selective to compounds, containing atoms of halogens, sulfur and nitrogen). The minimum detectable amounts of substances are from nanograms to picograms per second.

High performance liquid chromatography(HPLC) is an ideal method for the determination of a large number of thermally labile compounds that cannot be analyzed by gas chromatography. Modern agrochemicals, including methyl carbonates and organophosphate insecticides, and other non-volatile substances, often become the objects of analysis by liquid chromatography. High performance liquid chromatography is gaining popularity among other methods used in environmental monitoring, also because it has bright prospects in terms of the automation of sample preparation.

CHAPTER 1. BASIC CONCEPTS AND CLASSIFICATION OF LIQUID CHROMATOGRAPHY METHODS

Liquid chromatography is classified into several classes depending on the type of stationary phase support. Simple hardware design of paper and thin-layer chromatography has led to the widespread use of these methods in analytical practice. However, the great possibilities of liquid column chromatography stimulated the improvement of equipment for this classical method and led to the rapid introduction of HPLC. Passing the eluent through the column under high pressure made it possible to dramatically increase the analysis rate and significantly increase the separation efficiency due to the use of a finely dispersed sorbent. The HPLC method currently makes it possible to isolate, quantitatively and qualitatively analyze complex mixtures of organic compounds.

According to the mechanism of interaction of the substance to be separated (eluate) with the stationary phase, adsorption, distribution, ion-exchange, size-exclusion, ion-pair, ligand-exchange and affinity chromatography are distinguished.

Adsorption chromatography... Separation by adsorption chromatography is carried out as a result of the interaction of the substance to be separated with an adsorbent, such as alumina or silica gel, which have active polar centers on the surface. The solvent (eluent) is a non-polar liquid. The sorption mechanism consists in a specific interaction between the polar surface of the sorbent and the polar (or capable of polarization) regions of the molecules of the analyzed component (Fig. 1).

Rice. 1. Adsorption liquid chromatography.

Partition chromatography... In the distribution variant of liquid chromatography, the separation of a mixture of substances is carried out due to the difference in their distribution coefficients between two immiscible phases - the eluent (mobile phase) and the phase on the sorbent (stationary phase).

At normal phase In a variant of distribution liquid chromatography, a non-polar eluent and polar groups are used, grafted to the surface of the sorbent (most often, silica gel). Substituted alkylchlorosilanes containing polar groups, such as nitrile, amino groups, etc., are used as modifiers of the silica gel surface (grafted phases) (Fig. 2). The use of grafted phases makes it possible to finely control the sorption properties of the stationary phase surface and to achieve high separation efficiency.

Rice. 2. Partition chromatography with a grafted phase (normal-phase variant).

Reversed phase liquid chromatography is based on the distribution of mixture components between the polar eluent and non-polar groups (long alkyl chains) grafted to the sorbent surface (Fig. 3).

Rice. 3. Grafted phase partition chromatography (reversed-phase version).

A less widely used version of liquid chromatography with supported phases is when a liquid stationary phase is deposited on a stationary support.

Exclusive (gel-penetrating) chromatography is a variant of liquid chromatography, in which the separation of substances occurs due to the distribution of molecules between the solvent in the pores of the sorbent and the solvent flowing between its particles.

Affine chromatography is based on specific interactions of the separated proteins (antibodies) with substances (antigens) grafted onto the surface of the sorbent (synthetic resin), selectively forming complexes (conjugates) with proteins.

Ion exchange, ion pair, ligand exchange chromatography are used mainly in inorganic analysis.

Basic parameters of chromatographic separation.

The main parameters of chromatographic separation are the retention volume and retention time of the mixture component (Fig. 4).

The retention time tR is the time elapsed from the moment the sample is injected into the column until the maximum of the corresponding peak emerges. By multiplying the retention time by the eluent volumetric velocity F, we obtain the retention volume VR:

Corrected retention time - the time elapsed from the moment of the appearance of the maximum peak of the unsorbed component to the peak of the corresponding compound:

tR "= tR - t0;

The reduced or corrected retention volume is the retention volume corrected for the column dead volume V0, i.e. the retention volume of the unsorbed component:

VR "= VR - V0;

The retention characteristic is also the capacity coefficient k ", defined as the ratio of the mass of the substance in the stationary phase to the mass of the substance in the mobile phase: k" = mn / mp;

The k "value is easy to determine from the chromatogram:

The most important parameters of chromatographic separation are its efficiency and selectivity.

The efficiency of the column, measured by the height of theoretical plates (HETT) and inversely proportional to their number (N), the higher the narrower the peak of the substance exiting at the same retention time. The efficiency value can be calculated from the chromatogram using the following formula:

N = 5.54 . (tR / 1/2) 2 ,

where tR- retention time,

w 1/2 - peak width at half height

Knowing the number of theoretical plates per column, the column length L and the average sorbent grain diameter dc, it is easy to obtain the values of the height equivalent to the theoretical plate (HETT) and the reduced height (PVETT):

VETT = L / N PVETT = VETT / d c

These characteristics allow comparing the efficiency of different types of columns, assessing the quality of the sorbent and the quality of filling the columns.

The selectivity of the separation of two substances is determined by the equation:

When considering the separation of a mixture of two components, an important parameter is also the degree of separation RS:

;

Peaks are considered allowed if the RS value is greater than or equal to 1.5.

The main chromatographic parameters are linked by the following equation for resolution:

;

The factors determining the separation selectivity are:

1) the chemical nature of the sorbent;

2) the composition of the solvent and its modifiers;

3) the chemical structure and properties of the components of the mixture to be separated;

4) column temperature

1.1 Apparatus for liquid chromatography

In modern liquid chromatography, devices of varying degrees of complexity are used - from the simplest systems to high-class chromatographs equipped with various additional devices.

In fig. 4. 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.

Rice. 4. Block diagram of a liquid chromatograph.

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

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

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

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

The HPLC columns, which are most commonly used in the analysis of environmental pollutants, are 25 cm long and 4.6 mm internal diameter, filled with spherical silica gel particles 5-10 µm in size with grafted octadecyl groups. In recent years, columns with smaller internal diameters, filled with smaller particles, have appeared. The use of such columns leads to a decrease in the consumption of solvents and the duration of the analysis, an increase in the sensitivity and efficiency of separation, and also facilitates the problem of connecting the columns to spectral detectors. Columns with an internal diameter of 3.1 mm are equipped with a safety cartridge (precolumn) to increase the service life and improve the reproducibility of analyzes.

As detectors in modern HPLC instruments, a UV detector on a diode matrix, fluorescence, and electrochemical are usually used.

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

In practice, the most widespread is "reversed phase" (distribution) chromatography, in which the stationary phase is not polar, but the mobile phase is polar (that is, the reverse of the "direct phase" chromatography).

In most laboratories in the world, a group of 16 priority PAHs are analyzed by HPLC or CMS.

CHAPTER 2. ESSENCE OF HPLC

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

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 in the hydraulic system of the chromatograph.

Adsorption chromatography is carried out as a result of the interaction of a substance with adsorbents, such as silica gel or alumina, 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 division into zones during the movement with the mobile phase along the column. The separation of the zones of the components 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 are most widely used in HPLC. Alumina 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, in comparison with aluminum oxide, has a wider range of porosity, surface area and pore diameter; a significantly higher catalytic activity of aluminum oxide leads to distortion of the analysis results due to the decomposition of sample components or their irreversible chemisorption.

HPLC detectors

High performance liquid chromatography (HPLC) is used to detect polar nonvolatile substances that, for whatever reason, cannot be converted into a form convenient for gas chromatography, even in the form of derivatives. These substances include, in particular, 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 spectral regions, thus providing the recording of UV-B spectra in the scanning mode. This makes it possible to continuously record at high sensitivity undistorted spectra of components quickly passing through a special cell.

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

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, ketone aldehydes, benzidines.

Chromatographic separation of the mixture on the column due to the slow progress of the PP 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 most promising and modern methods of analysis. High performance liquid chromatography is a convenient method for separation, preparative isolation, and qualitative and quantitative analysis of nonvolatile thermolabile compounds with both low and high molecular weights.

Depending on the type of sorbent used in this method, 2 chromatography options 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 reversed-phase high-performance liquid chromatography (HPLC).

When the eluent passes to the eluent, equilibrium under the conditions of HPLC is established many times faster than under the conditions of polar sorbents and non-aqueous PPs. As a result of this, as well as the convenience of working with aqueous and aqueous-alcoholic eluents, Off-HPLC has gained great popularity at the present time. Most HPLC analyzes are performed using this method.

Detectors. The registration of the exit from the column of a separate component is performed using a detector. For registration, you can use a change in any analytical signal coming from the mobile phase and associated with the nature and amount of the mixture component. In liquid chromatography, analytical signals are used such as light absorption or light emission of the output solution (photometric and fluorometric detectors), refractive index (refractometric detectors), potential and electrical conductivity (electrochemical detectors), etc.

The continuously detected signal is recorded by a recorder. A chromatogram is a sequence of detector signals recorded on the recorder tape, generated when the individual components of the mixture leave 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 identification purposes, the height or area of the peak is used for quantification purposes.

2.1 Application

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

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

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

Forensic science - drugs, organic explosives and dyes, strong pharmaceuticals.

Pharmaceutical industry - steroid hormones, almost all organic synthesis products, 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, determination of the rate of elimination of drugs from the body for the purpose of their individual dosage.

Agriculture - determination of nitrate and phosphate in soils to determine the required amount of applied 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 (almost all soluble compounds in the form of ions and complex compounds).

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

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

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

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

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

analysis of the quality of soil 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 trace amount of ultrapure substance.

CHAPTER 3. EXAMPLES OF USING HPLC IN ANALYSIS OF ENVIRONMENTAL OBJECTS

HPLC - a method for monitoring PAHs in environmental objects

For polycyclic aromatic hydrocarbons (PAHs), ecotoxicants of the 1st hazard class, extremely low levels of maximum permissible concentrations (MPC) in natural objects have been established. Determination of PAHs at the MPC level and below is one of the very complex analytical tasks and high-tech methods of analysis (GC-MS, GC, HPLC) are used to solve them. When choosing a method for monitoring, to the main considered characteristics - sensitivity and selectivity, rapidity and economy are added, since monitoring involves a serial analysis. The HPLC option on short, small bore columns meets these requirements to a large extent. Using this method, the authors have developed and certified methods for monitoring benzo [a] pyrene in three natural environments: aerosol, snow cover and surface waters. The techniques are characterized by: simple unified sample preparation, including the extraction of PAHs with organic solvents and concentration of the extract, direct introduction of the concentrated extract into the chromatographic column, the use of multi-wavelength photometric detection in the UV region of the spectrum, identification of PAH peaks in chromatograms using two parameters, retention time and spectral ratio ... The total error does not exceed 10% when determining benzo [a] pyrene in aerosol in the concentration range from 0.3 to 450 ng / m 3, in surface waters in the concentration range from 10 to 1000 ng / L, in the snow cover in the range of surface density from 0.5 up to 50 μg / m 2. For the case of simultaneous determination of priority PAHs (up to 12 compounds) and registration of inhomogeneous peaks of analytes, it is proposed to re-separate the extract with a change in the selectivity of the mobile phase, detection wavelength, and column temperature, taking into account the individual properties of the determined PAH.

1 ... Ambient air quality. Mass concentration of benzo [a] pyrene. HPLC measurement technique. Certificate of attestation MVI No. 01-2000.

2 ... The quality of surface and treated waste water. Mass concentration of benzo [a] pyrene. HPLC measurement technique. Certificate of attestation MVI No. 01-2001.

3 ... Snow quality. Mass concentration of benzo [a] pyrene. HPLC measurement technique. Certificate of attestation MVI No. 02-2001.

Removal of aniline from aqueous solutions using waste alumothermal reduction of rolled copper scale

The problem of removing hydrocarbons from wastewater is an urgent task. In many chemical, petrochemical and other industries, aniline and its derivatives are formed, which are toxic substances. Aniline is a highly toxic substance, maximum concentration limit is 0.1 mg / m 3. Aniline and its derivatives are soluble in water and therefore cannot be removed by gravity settling.

One of the best methods for purifying wastewater from organic pollutants is the use of inorganic and organic adsorbents capable of regeneration (aluminosilicates, modified clays, wood, fibers, etc.) and incapable of regeneration (activated carbon, macroporous polymer materials, etc.). ).

Regenerated adsorbents can remove organic substances of different polarities from water. The search for effective adsorbents is an urgent task.

This report presents the results of a study in the field of application of rolled copper scale of the Yerevan Cable Plant (OPMOERKZ) as aniline sorbents.

Chromatographic studies were carried out on an HPLC chromatograph / high-performance liquid chromatography / systems (Waters 486 - detector, Waters 600S - controller, Waters 626 - Pump), on a 250 x 4 mm column filled with the sorbents under study, the mobile phase rate 1 ml / m / mobile phase are the solvents we investigate /, the detector is UV-254. UV spectroscopic analysis was carried out on a Specord-50 spectrophotometer, the spectra were obtained using the ASPECT PLUS computer program.

Precisely weighed portions of sorbents were added to certain volumes of aniline in water, the initial concentrations of which were varied. The mixture was thoroughly shaken for 6 hours. Then the sample was left to settle. The adsorption is completed practically within 48 hours. The amount of precipitated aniline was determined by UV spectrophotometric and refractometric analysis.

First, the adsorption properties of OPMOErKZ were investigated when aniline was removed from a solution in carbon tetrachloride. It turned out that aniline absorbs sorbent 3 best of all (table).

Measurements were also carried out for aqueous solutions of aniline in concentrations of 0.01-0.0001 mol / l. The table shows data for a 0.01 M solution.

Aniline absorption by various sorbents from a 0.01 M aqueous solution of aniline at 20 ° C

Previously, it was found that adsorption increases within the specified concentration range and linearly depends on the refractive index. The amount of aniline was determined from the graphical relationship "refractive index - molar concentration" and corrected by the data of both liquid chromatography and UV spectral analysis.

Sorbent 3 is the most active for aqueous solutions. The amount of adsorbed pollutant was calculated as the difference between the total amount of pollutant added to the initial solution and its remainder in the final solution.

Methods for the determination of PAHs in environmental objects

Typically, gas chromatography (GC) and high performance liquid chromatography (HPLC) methods are used to determine PAHs. separation of the main 16 PAHs, sufficient for quantitative analysis, is achieved by using either capillary columns in gas chromatography, or high-performance columns used in HPLC. It should be remembered that a column that separates well the calibration mixtures of sixteen PAHs does not guarantee that they will also separate well against the background of accompanying organic compounds in the test samples.

In order to simplify the analysis, as well as to achieve high quality of the obtained results, most of the analytical procedures contain the stage of preliminary isolation (separation) of PAHs from other groups of related compounds in the samples. Low pressure liquid-solid or liquid-liquid liquid chromatography techniques are most commonly used for this purpose using adsorption mechanisms such as silica gel or alumina, sometimes mixed mechanisms are used, such as adsorption and elimination using Sephadex.

The use of preliminary purification of samples makes it possible to avoid the influence of:

Completely non-polar compounds such as aliphatic hydrocarbons;

Moderately to highly polar compounds such as phthalans, phenols, polyhydric alcohols, acids;

High molecular weight compounds such as resins.

High performance liquid chromatography (HPLC) uses mainly two types of detectors: a fluorometric detector or a spectrophotometric detector with a photodiode array. The detection limit for PAHs in fluorometric detection is very low, which makes this method particularly suitable for the determination of trace amounts of polyaromatic compounds. However, classical fluorometric detectors provide practically no information on the structure of the compound under study. Modern designs make it possible to record fluorescence spectra that are characteristic of individual compounds, but they have not yet become widespread in the practice of routine measurements. A spectrophotometric detector with a photodiode ruler (PDL) makes it possible to register absorption spectra in the UV and visible spectral range, these spectra can be used for identification. Similar information can be obtained using fast scanning detectors.

When choosing an analytical technique for the separation, identification and quantitative analysis of the mentioned PAHs, the following conditions should be taken into account:

The level of the determined contents in the test samples;

The number of related substances;

Analytical procedure used (measurement technique);

Capabilities of serial equipment.

Development of a method for the determination of alkaline earth elements and magnesium by ionic high-performance liquid chromatography

The development and improvement of methods that allow solving the problems of water analysis is an important problem in analytical chemistry. The development of high-performance liquid chromatography of high pressure stimulated the development of a new direction in ion-exchange chromatography, the so-called ion chromatography. The synthesis of sorbents for ion chromatography is difficult, since there are many requirements for them. Due to the lack of commercially available highly effective cation exchangers, a dynamically modified reversed phase was used, for which a modifier was synthesized: N-hexadecyl-N-decanoyl-paraminobenoylsulfonic acid ethyl-diisopropylammonium (DHDASK), where a hydrophobic amine containing an SO 3 - group, capable of cation exchange. After passing the modifier solution, the absorption at l = 260 nm reached 6.4 units of optical density (° E) with a plateau. The calculated ion exchange capacity is 15.65 μmol. Since the cations of alkaline earth elements and magnesium do not absorb in the UV region of the spectrum, indirect UV detection was used using the synthesized UV absorbing eluent 1,4-dipyridinium butane bromide (DPB bromide). Since halogen ions destroy the steel parts of the column, the bromide ion of 1,4-dipyridiniumbutane was replaced with acetate ion. When the column is washed with the eluent, the modifier counterion, ethyldiisopropylammonium, is replaced by the UV-absorbing 1,4-dipyridiniumbutane ion. Separation of cations was carried out at the optimal wavelength l = 260 nm on a scale of 0.4 A in the “scale folding” mode; the polarity of the recorder was reversed. Separation of all studied cations was achieved by introducing a complexing additive, oxalic acid. The detection limits for Mg 2+, Ca 2+, Sr 2+, Ba 2+ are 8 μg / L; 16 μg / L; 34 μg / L; 72 μg / L, respectively. Under the selected conditions, tap water was analyzed, the content of Ca 2+ in which is 10.6 +1.9 mg-ion / l, Mg 2+ -2.5 + mg-ion / l. The reproducibility error does not exceed -2.2% for Ca 2+ and 1.4% for Mg 2+.

Analysis of cadmium complexes in the environment

To study the mechanisms of migration of heavy metals in the biosphere, data on the chemical forms of the existence of metals in nature are needed. Difficulties in the analysis of compounds of one of the most toxic metals - cadmium - are associated with the fact that it forms fragile complexes, and when trying to isolate them, natural equilibria are distorted. In this work, cadmium compounds in soil and plants were investigated using a technique based on chromatographic separation of extracts with subsequent identification of the components by chemical analysis. This approach made it possible not only to identify the chemical forms of cadmium, but also to trace their transformations in environmental objects.

OH-groups of carbohydrates and polyphenols (including flavonoids), C = O, phosphates, NH 2, NO 2, SH-groups are coordinated with cadmium in objects of the biosphere. For the purposes of this study, a set of model ligands representing these classes of compounds was compiled. The interaction of model ligands with water-soluble cadmium salts was investigated by UV spectroscopy and HPLC.

To isolate cadmium compounds, we used extraction with specially selected (not forming complexes with Cd) solvents. So it is possible to separate cadmium from all heavy metals, except for its close chemical analogue - zinc. Cadmium and zinc containing peaks in the chromatograms of the obtained extracts were detected by binding metals in the form of their dithizonates. For separation from zinc, the difference in the stability of the Cd and Zn complexes at pH 6-8 was used. The isolated Cd compounds were identified by HPLC with a change in pH during elution. An analysis of the compounds of cadmium with components of soils and plant tissues was carried out, and the substances produced by plants in response to an increase in the intake of cadmium from the soil were identified. It has been shown that flavonoids, in particular tricine, are protective agents in cereals, alkoxy derivatives of cysteine in legumes, and both polyphenols and thiols in crucifers.

CHAPTER 4. HPLC EQUIPMENT

SERIES ACCELA

The new ACCELA ultra-high-performance liquid chromatograph is capable of operating in the widest range of flow rates and pressures, providing both typical HPLC separation on conventional columns and ultra-fast and efficient separation on columns with a sorbent particle size of less than 2 μm at ultra-high pressures (over 1000 atm.).

The system includes a quarterly gradient inlet pump capable of pressures in excess of 1000 bar and with a hold volume of only 65 µl for high speed chromatographic separation. Autosampler ACCELA is capable of operating in a 30 second sample injection cycle and provides the highest injection reproducibility. Diode Array Detector Accela PDA with a minimized flow cell volume (2 μL) is optimized for high-speed chromatography mode, uses the patented LightPipe technology and maintains the symmetrical peak shape, which results from the use of a flawless chromatographic system and columns.

The system integrates perfectly with mass spectrometers to create the most powerful and best HPLC / MS systems available in the world.

UHP columns with 1.9 μm grain size available from Thermo Electron for any application

SERIES TSP

The modular design principle of HPLC devices allows the customer to flexibly complete equipment for solving any analytical tasks, and if they change, it can be quickly and economically modified. The wide range of modules includes pumps ranging from isocratic to four-component gradient, from microcolumn to semi-preparative, all available detectors, sample injection systems from handheld injectors to autosamplers with any sample manipulation capability, powerful software for processing measurement results and controlling all system modules. All modules are certified according to CSA, TUF / GS, FCC (EMI), VDE (EMI), ISO-9000, they are compact, have a modern design, are easy to operate, equipped with a built-in display and a self-diagnostic system, allow you to create and save task methods parameters. They meet the criteria of "Good Laboratory Practice" (GLP) and are listed in the Register of Measuring Instruments of the Russian Federation. Measurement reports are issued in accordance with the Pharmacopoeias of England, USA, Germany and France.

TSP modular systems are characterized by the highest reliability and operational stability.

The combination of modules provides the analyst with all the advantages of an integral system on the one hand and the flexibility of a modular system on the other. In whatever field of application of High Efficiency Liquid Chromatography (HPLC) -pharmacology, biotechnology, environmental analysis, clinical analysis,

Indoor air: control and cleaning methods. Control of the source of harmful substances and the environment. Gas analyzers: application and their modern types for monitoring the composition of the gas mixture - universal photometric liquid and tape.

Monitoring as a system for monitoring and controlling the environment. Methods for monitoring pollutants in environmental objects.

Separation of anions by single column ion chromatography. Image of the structure of an ion exchange resin particle. Examples of the use of ion exchange chromatography in the analysis of environmental objects. Features of beer analysis by ion chromatography.

General characteristics of organochlorine compounds, their main physical and chemical properties and applications, negative impact on the environment, the body of animals, fish and humans. Organochlorine pesticides in food and methods for their determination.

Fundamentals of planar (thin-layer) chromatography: the state and prospects of using modern instrumental methods for the analysis of pesticides, organochlorine pesticides in water, food, feed and tobacco products by chromatography in a thin layer.

Methods available for taking indoor air samples for analysis. The principle of operation of colorimetric tubes. Discoloration of a particular reagent upon contact with a particular contaminant. Detection of volatile organic compounds.

Theoretical foundations of fluometry (luminescence), areas of its application in the analysis of environmental objects and modern research equipment. Extraordinary sensitivity and speed of luminescence analysis. Excitation energy supply problems.

The development of chemical analytical equipment not only does not eliminate the problem of the quality of the measurements performed, but, on the contrary, makes ever higher demands in all aspects of the measurements.

General information about the industrial facility. Climatic conditions of the area. Technological chain. Sources of pollution and disturbance of the natural environment. Pollution of natural waters. Surface water quality observation points. Water sampling and analysis methods.

A wide range of organic compounds introduced into the environment in the course of human economic activity leads to the fact that these substances have become the main pollutants that determine the nature of technogenic pollution of the hydrosphere.

Characteristics of spectroscopic methods of analysis. The essence of extraction-photometric methods. Examples of using the method for the determination of heavy metals in natural waters. Method for detecting bromide ions, nitrate ions. Modern equipment.

The concept and purpose of gas chromatography, the parameters of its retention. Retention time and retention volume. Equations in gas chromatography. Additional devices for gas chromatography. Air pollution control in emergency situations.

The concept and characteristics of the method of mass spectrometry. Dual focusing mass spectrometers in inductively coupled plasma mass spectrometry. The use of chromatography-mass-spectrometry in the identification of environmental pollutants, equipment.

Methods for assessing the pollution of gas streams. Basic requirements for gas sampling and analysis and measurement methods. Methods for assessing parametric pollution. Methods for assessing the pollution of the aquatic environment, soil, soil and vegetation. Identification of changes.

Determination of thousandths of a percent of the substance content in pure metals by optical methods of analysis using adsorption methods by spectrophotometry, photocolorimetry and colorimetry. Sale of chemical analytical equipment through websites.

Purpose and basic principles for the implementation of conductometric analysis methods. Varieties of methods used and features of their application. Examples of the use of conductometry in the analysis of environmental objects and the equipment required for this.

With regard to natural waters, the problems of quantitative determination and separation into anthropogenic and natural components of hydrocarbons (CH) are considered.

Sorption methods of water purification are now more and more widely used, and one of the most frequently used sorbent is activated carbon.

The main types of chromatography. Application of chromatographic methods in environmental monitoring. Application of chromatography in the analysis of environmental objects. Modern hardware design. Methods for the development of chromatograms and the work of a chromatograph.

Monitoring of natural waters by physicochemical methods: planar (thin-layer chromatography) and its application for water lysis. Separation of a mixture of substances in a flat layer of sorbent and solvent. Intensity of luminescence of petroleum products on a fluorometer.

(OFS 42-0096-09)

High performance liquid chromatography (HPLC) is a column chromatography technique in which the mobile phase (PF) is a liquid

bone moving through a chromatographic column filled with non-

the mobile phase (sorbent). HPLC columns have high hydraulic pressures at the inlet to the column, so HPLC is sometimes referred to as

vyvayut "high pressure liquid chromatography".

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

HPLC options: adsorption, distribution, ion-exchange,

exclusive, chiral, etc.

In adsorption chromatography, the separation of substances occurs due to their different ability to adsorb and desorb with

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

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

(usually chemically grafted to the surface of an immobile support) and

mobile phases.

According to the polarity of PP and NF, HPLC is divided into normal-phase and vol-

expanded phase.

Normal-phase chromatography is called a variant 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 developed

personal additions). In the reversed phase chromatography, the

use non-polar chemically modified sorbents (for example,

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

methanol, acetonitrile).

In ion-exchange chromatography, molecules of substances in a mixture, dissociation

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

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

mi sorbent groups.

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

chromatography, molecules of substances are separated by size due to their different ability to penetrate into the pores of the stationary phase. In this case, the first of the co-

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

and the latter are substances with small sizes of molecules.

Separation often proceeds not one by one, but several mechanisms simultaneously.

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

of zygomatic analytes. For the analysis, use the appropriate instruments - liquid chromatographs.

The composition of a liquid chromatograph usually includes the following basics -

ny nodes:

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

with individual solvents that are part of the mobile phase

zy) and the PF degassing system;

– pumping system;

– mobile phase mixer (if necessary);

– sample injection system (injector);

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

- detector;

– data collection and processing system.

Pumping system

The pumps provide PF supply 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. In front of the pumping system, it is sometimes installed

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

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

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

PF components in the mixer can occur both at low pressure

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

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

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

shalo 20 MPa. Pressure pulsations are minimized by special damping

ferrous systems included in the design of the pumps. Working parts on

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

Mixers

By their design, mixers can be static or dynamic

mental.

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

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

sewing.

Injectors

Injectors can be versatile for sample injection from

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

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

directly in front of the chromatographic column. The design of the injector allows changing the direction of the PF flow and performing preliminary injection of the sample into a loop of a certain volume (usually from 10 to 100 μL).

This volume is indicated on the hinge label. The injector design allows replacement of the loop. For the introduction of the analyzed solution into the unknown

the tomato injector is a manual micro-syringe with a volume that is

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

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

dosing accuracy and reproducibility and, therefore, impairs the accuracy

ness and reproducibility of chromatographic analysis.

Chromatographic column

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

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

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), inner diameter - from 2 to 10 mm (usually 4.6 mm). Columns with an inner diameter less than 2 mm are used in micro-column chromium

tography. Capillary columns with internal diameters are also used.

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

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

columns (guard columns) performing various auxiliary functions

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

temperature, however, to increase the separation efficiency and

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

tirovanie columns at temperatures not higher than 60 C. At higher temperatures, the destruction of the sorbent and a change in the composition of the PF is possible.

Stationary phase (sorbent)

Usually used as sorbents:

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

small phase chromatography. The holding mechanism in this case

tea - usually adsorption;

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

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

4. Chemically modified sorbents (sorbents with grafted

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

noah and stationary phases;

5. Chemically modified chiral sorbents, for example,

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

technical modification. Sorbent particles can be spherical or non-

regular shape and varied porosity.

The most commonly used grafted phases are:

– octyl groups(sorbent octylsilane or C8);

– 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, the analysis is performed on non-polar grafted phases in

reversed-phase mode using C18 sorbent.

In some cases, it is more advisable to apply normal

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

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

Sorbent particles can have spherical or irregular shapes and various porosities. 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 provided by the high surface area of the sorbent particles (which is a consequence of their microscopic

size and the 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, the PP with the components dissolved in it after the chromatographic column

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

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

or physicochemical parameter of the PF versus time.

The most common are spectrophotometric de-

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

density in ultraviolet, visible and often near infrared

spectral regions 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

It can carry out detection at any wavelength in its working range.

zone. Multi-wavelength detectors are also used, allowing

to provide detection at several wavelengths simultaneously.

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

to obtain the optical spectrum of the FS at any time instant (without scanning), which greatly simplifies the qualitative analysis of the separated components.

ponents.

The sensitivity of fluorescent detectors is about 1000 times higher than the sensitivity of spectrophotometric ones. In this case, either intrinsic fluorescence or the fluorescence of the corresponding derivatives is used, if the substance to be determined itself does not fluoresce. Modern

fluorescent detectors allow not only obtaining chromatographic

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

connections.

Refractometric detectors are used to analyze samples that do not absorb in the UV and visible spectral regions (e.g. carbohydrates).

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

Also used are electrochemical detectors (conductometric

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

detectors, light scattering detectors, radioactivity and some other

Mobile phase

V As PP, a variety of solvents can be used - both individual and their mixtures.

V normal phase chromatography usually uses liquid carbon

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

solvents with small additions of polar organic compounds,

which regulate the eluting power of the PF.

In reversed-phase chromatography, the PF contains polar

ganic solvents (usually acetonitrile and methanol) and water. For opt-

separations often use aqueous solutions with a certain

the pH, in particular buffer solutions. Additives are used inorganic

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

example, chiral modifiers for the separation of enantiomers into achiral-

sorbent).

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

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

reach - of three or more. The composition of the PP is indicated as the volume ratio of the solvents included in it. In some cases, mass

the 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. Limit of transparency or optical density when determining

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

found on the packaging.

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

specifically for liquid chromatography (including water).

PP and analyzed solutions should not contain insoluble

particles and gas bubbles. Water obtained in laboratory conditions

aqueous solutions, premixed with water, organic solutions

The instruments, 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 with respect to the given solvent or solution.

Data collection and processing system

The modern data processing system is an interface

personal computer connected with the chromatograph with installed

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 indicated

In a private monograph, the sizes of co-

columns, type of sorbent with an indication of the particle size, column temperature (if necessary, thermostating), volume of injected sample (loop volume),

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

ION EXCHANGE AND IONIC HPLC

Ion exchange chromatography is used for the analysis of both organic

(heterocyclic bases, amino acids, proteins, etc.), and inor-

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

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

pami sorbent. Anionites or cation-

you. These sorbents are mainly either polymeric ionic

exchange resins (usually copolymers of styrene and divinylbenzene with grafted

ionic groups), or silica gels with grafted ion-exchange groups. Sorbents with groups - (СН2) 3 N + X– are used to separate anions, and sorbents with groups - (СН2) SO3 - Н + are used to separate cations.

Usually, polymer resins are used to separate anions, and

leaching of cations - modified silica gels.

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

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

chemical solvents - acetonitrile, methanol, ethanol, tetrahydrofuran.

Ion chromatography- a variant of ion exchange chromatography, in

which to determine the concentration of ions of the analyte is

uses a conductometric detector. For highly sensitive op-

Determination of changes in the conductivity passing through the PF detector, the background conductivity of the PF should be low.

There are two main options for ion chromatography.

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

the PF using a second ion-exchange column located between the ana-

lytic column and detector. In this column, neutralization occurs

PF and analyzed compounds enter the detector cell in the deionic

water. The detected ions are the only ions

providing PF conductivity. The disadvantage of the suppressor column is the need to regenerate it at fairly short intervals.

me. The suppressor column can be replaced by a continuously acting

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

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

opposite to the direction of the PF flow.

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

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

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

skic acids - benzoic, salicylic or isophthalic.

EXCLUSIVE HPLC

Size exclusion chromatography (size exclusion chromatography) is a special type 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-

lecules and partly on their shape and polarity. For separation, use a

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

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

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

you are copolymers of styrene and divinylbenzene (these are hydro-

phobic sorbents used with non-polar mobile phases) and

hydrophilic gels made from sulfonated divinylbenzene or polyacrylamide resins.

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

go first. Molecules with a diameter much smaller than the pore size of the sor-

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

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

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

In size exclusion chromatography to characterize the retention of

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

Mobile phase. The choice of PP depends on the type of sorbent. Exclusive-

ny chromatography is generally divided into gel filtration and gel

penetration chromatography.

The method of gel filtration chromatography is used to separate

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

In gel permeation chromatography, hydrophobic sor-

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

rahydrofuran). This method is used to analyze compounds with low solubility.

rim in the water.

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

Viscometric and flow-through laser detectors are also used.

These detectors, in combination with a refractometer or other concentration

detector allows you to continuously determine the molecular weight of the

limer in PF.

ULTRA EFFICIENT LIQUID CHROMATOGRAPHY

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

in comparison with classical HPLC.

A feature of ultra-performance liquid chromatography is

The use of sorbents with a particle size of 1.5 to 2 microns is used. Dimensions chro-

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 be from 1 to 50 μl.

Chromatographic equipment used in classical

riante HPLC, usually specially adapted for this type of chromatography

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

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 a liquid applied to its surface or gel.

Distinguish columnar and thin layer liquid chromatography. In the column version, a portion of the mixture of substances to be separated is passed through a column filled with a stationary phase in an eluent flow that moves under pressure or under the action of gravity. In thin layer chromatography, the eluent moves under the action of capillary forces along a flat sorbent layer applied to a glass plate or metal foil, along a porous polymer film or along a strip of special chromatographic paper. A method of thin-layer liquid chromatography under pressure has also been developed, when the eluent is pumped through a sorbent layer sandwiched between the plates.

There are such types of liquid chromatography as analytical(for the analysis of mixtures of substances) and preparative(to isolate pure components).

Distinguish liquid chromatography (LC) in its classical version, carried out at atmospheric pressure, and high speed) carried out at high blood pressure... High performance liquid chromatography (HPLC) uses columns up to 5 mm in diameter, tightly packed with a sorbent with small particles (3-10 microns). To pump the eluent through the column, apply a pressure of up to 3.107 Pa. This type of chromatography is called high pressure chromatography... Passing the eluent through the column under high pressure makes it possible to dramatically increase the analysis rate and significantly increase the separation efficiency due to the use of a finely dispersed sorbent.

HPLC options are microcolumn chromatography on small diameter columns 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 physicochemical 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 products of chemical and petrochemical synthesis, intermediates, dyes, fuels, lubricants, oil, waste water;

· Study of isotherms of sorption 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, the quality of monomers is analyzed using liquid chromatography, the molecular weight distribution and the distribution by types of functionality of oligomers and polymers are studied, which is necessary for product control.

Liquid chromatography is also used in perfumery, food industry, for the analysis of environmental pollution, in forensic science.

The method of high performance liquid chromatography (HPLC) was developed and introduced in the mid 70s of the XX century. Then the first liquid chromatographs appeared.

Liquid chromatography is the optimal method for the analysis of chemically and thermally unstable molecules, high-molecular 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 the mechanism of retention of the separated substances by the stationary phase LC distinguish between:

sediment chromatography

· adsorption chromatography , in which the separation is carried out as a result of the interaction of the substance to be separated with adsorbent such as aluminum oxide or silica gel, having on the surface active polar centers. Solvent(eluent) - non-polar liquid.

Rice. Scheme of separation of a mixture of substances by adsorption chromatography

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The sorption mechanism consists in a specific interaction between the polar surface of the sorbent and the polar (or capable of polarization) regions of the molecules of the analyzed component (Fig.). Interaction occurs due to donor-acceptor interaction or the formation of hydrogen bonds.

Rice. Adsorption liquid chromatography diagram

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Rice. ... Grafted phase partition chromatography (normal phase variant).

http: // www. chemnet. ru / rus / teaching / oil / spezprakt-chr. html

At normal-phase In the variant of partition liquid chromatography, substituted alkylchlorosilanes containing polar groups, such as nitrile, amino groups, etc., are used as modifiers of the silica gel surface (grafted phases) (Fig.). The use of grafted phases makes it possible to finely control the sorption properties of the stationary phase surface and to achieve high separation efficiency.

Reversed phase liquid chromatography is based on the distribution of the mixture components between the polar eluent and non-polar groups (long alkyl chains) grafted to the sorbent surface (Fig.). Less commonly, a variant of liquid chromatography with supported phases is used, when a liquid stationary phase is applied to a stationary support.

Rice. ... Grafted phase partition chromatography (reversed-phase version). http: // www. chemnet. ru / rus / teaching / oil / spezprakt-chr. html

Partition liquid chromatography includes extraction liquid chromatography, in which the stationary phase is an organic extractant deposited on a solid support, and the mobile phase is an aqueous solution of the compounds to be separated. As extractants used, for example, phenols, trialkyl phosphates, amines, quaternary ammonium bases, as well as sulfur-containing organophosphorus compounds. 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 processing of spent nuclear fuel.

ion exchange chromatography,

ion exchangers.

cation exchangers

anionites.

amphoteric ion exchangers

ampholytes

Ionite

Cation exchanger

Anionites

R-H + Na + + Cl - → R-Na + H + + Cl - (cation exchange)

R-OH + Na + + Сl - → R-Сl + Na + + OH - (anion exchange)

Ion exchangers must meet the following requirements: be chemically stable in various environments, mechanically strong in a dry and especially in a swollen state, have a high absorption capacity and the ability to be well regenerated.

In ion-exchange (ion) chromatography, separated anions (cations) are detected as acids (corresponding bases) with a highly sensitive conductometric detector, where high-performance columns are filled with a surface-active ion exchanger with a small capacity.

ion pair chromatography

ligand exchange chromatography

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

size exclusion chromatography

differences in molecular size

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Rice. Gel Permeation Chromatography Scheme

affinity chromatography

Rice. Affinity Chromatography Scheme

http: // www. chemnet. ru / rus / teaching / oil / spezprakt-chr. html

Then it is removed from the polymer by passing a solution of a compound with an even greater affinity for the macromolecule. Such chromatography is especially effective in biotechnology and biomedicine for the isolation of enzymes, proteins, hormones.

Depending on on the way the substance is transported the following liquid chromatography options are distinguished: expressive, frontal and displacement.
Most often used expressive a variant in which a portion of the mixture to be separated is introduced into the column in the flow of the eluent. The yield of the mixture components from the column is recorded on the chromatogram as peaks. (rice.)

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Rice. Scheme of the developing variant of chromatography

Height or peak area characterizes concentration of components, a held volumes – qualitative composition of the mixture... Components are usually identified 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, only the substance that is least sorbed in the column can be obtained in pure form.

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Rice. Frontal chromatography scheme

The chromatogram in this case represents steps, the heights of which are proportional to the concentrations of the components; the retention volumes are determined by the retention time of the components. When differentiating such a chromatogram, a picture is obtained that is similar to that obtained in the developing version.

V displacement In a variant, 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, adjoining fractions of the substances to be separated are obtained. The order of the release of the components is determined by the force of their interaction with the sorbent surface (Fig.).

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Rice. Displacement chromatography scheme

3. Basic chromatographic quantities and their determination.

When separating substances using liquid chromatography, the developing, frontal and displacement variants can be used, as indicated above. Most often, a developing version is used, in which a portion of the mixture to be separated is introduced into the column in the flow of the eluent. The yield of the mixture components from the column is recorded on the chromatogram as peaks. From the chromatogram (Fig.) Determine:

retention times of non-absorbable (t0), separated components (tR1, tR2, tR3, etc.); the width of the base of the peaks (tw1, tw2, etc.).

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b) corrected component holding volume ,

where t "R - corrected component retention time;

c) column capacity ratio to a given component ;

d) column efficiency characterized by number of equivalent theoretical plates

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f) permission https://pandia.ru/text/80/271/images/image024_9.gif "width =" 203 height = 51 "height =" 51 ">

Capacitance factor k " has a significant effect on the value R S: on change 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 constant of adsorption equilibrium (Henry's constant).

Selectivity coefficient α is determined by the difference in the adsorption equilibrium constants of the two separated components. With increasing α (from 1 to ~ 5) R S sharply increases, with a further increase in α - changes little. Column selectivity 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 to be 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 to be 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 the packing of the column, the viscosity of the eluent and the rate of mass transfer. Column lengthening does not always lead to improved separation, since the column resistance increases, the eluent pressure at the inlet and the time of the experiment increase, and the sensitivity and accuracy of the analysis decreases due to the broadening of the peak of the analyzed component. If, then the peaks of the two substances on the chromatogram are almost completely separated. With growth R S the separation time increases. At R S < 1 - the separation is unsatisfactory. In preparative chromatography, in connection with the introduction of relatively large amounts of separated substances, the column is operated with overload. This decreases the capacitance ratio, increases the height equivalent to the theoretical plate, which leads to a decrease in resolution.

4. Adsorbents

Chromatographic separation of the mixture will be effective if the adsorbent and solvent (eluent) are correctly selected.

The adsorbent should not chemically interact with the separated components, exhibit a catalytic effect on the solvent. It is also necessary that the adsorbent is selective with respect to the components of the mixture. A properly selected desiccant must have the maximum absorbency.

Distinguish polar (hydrophilic) and non-polar (hydrophobic) adsorbents... It should be remembered that the adsorptive affinity of polar substances for polar sorbents is much higher than that of 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 and in non-polar solvents... Neutral alumina 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 a unit. If necessary, you can prepare alumina with different moisture content by mixing freshly prepared alumina with water (Brockmann scale).

Dependence of the activity of aluminum oxide on moisture content

For example, Al2O3 with an activity of 1.5-2 is used to separate hydrocarbons; for the separation of alcohols and ketones - 2-3.5.

Specific surface area of aluminum oxide 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. silicagel. /

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 rate of mass transfer and improves the efficiency of the column. Modern analytical columns are 10-25cm long. They are filled with silica gel with a particle size of 5 microns and allow the separation of 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 a flow rate of the eluent of 0.5-2.0 ml / min, a pressure of (1-3) · 107Pa is required. Silica gel can withstand such a pressure drop, while polymer sorbent granules are more elastic and deformable. Recently, mechanically strong polymer sorbents with a macroporous structure with a dense network have been developed, which are close to silica gels in their efficiency. The shape of the sorbent particles with a size of 10 μm and above does not have a large effect on the efficiency of the column, however, spherical sorbents are preferred, which provide a more permeable packing. (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. Sorbents with a pore diameter of 6-25 nm are used for liquid chromatography. The separation of liquid chromatography is carried out mainly on silica gels modified by the reaction of alkyl and arylchlorosilanes or alkyl ethoxysilanes with silanol surface groups. With the help of 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. Modified silica gel structure

Silica gels used in chromatography for the separation of 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 its water content: the less water it contains, the more activity (Brockmann scale).

Dependence of the activity of silica gel on the moisture content

The specific surface area of silica gels is 500-600 m2 / g.

Activated carbons are a form of carbon that becomes extremely porous during processing and acquires a very large surface area 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 effect 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 greater proportion of micropores (up to 2 nm), based on coal - a greater 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 molecules and 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 types of zeolites (A, X, Y, M) with different crystal structures. Depending on the cation, zeolites are designated as follows: KA, NaA, CaM, NaX, KY, CaY. Feature of zeolites is that pores of crystals have sizes of the order of 0.4-1 nm, commensurate with 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 a substance are not adsorbed. By selecting zeolites with different pore sizes, it is possible to clearly separate mixtures of different substances.

The specific surface area 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 adsorbed poorly, while unsaturated ones (have double bonds) are better adsorbed. Functional groups enhance the adsorption capacity of a substance.

5. Eluents

When choosing a solvent (eluent), it is necessary to take into account the nature of the adsorbent and the properties of the substances in the mixture to be separated. Eluents should dissolve well all components of the chromatographed mixture, have a 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).

Normal phase chromatography usually uses hydrocarbons (hexane, heptane, isooctane, cyclohexane) with the addition of small amounts of chloroform CHCl3, isopropanol iso-C3H7OH, diisopropyl ether; in reverse phase chromatography - a mixture of water with acetonitrile CH3CN, methanol CH3OH, ethanol C2H5OH, dioxane, tetrahydrofuran, dimethylformamide. To isolate the individual components of the mixture, separated during chromatography, they are often sequentially washed out (eluted). For this purpose, solvents with different desorption capacities are used. The solvents are arranged in decreasing order of desorption capacity in polar adsorbents - eluotropic Trappe series... If the components of the mixture to be separated have close values k "( column capacity ratio with respect to a given component), then chromatographic with one eluent. If the individual components of the mixture are strongly retained by the sorbent, a series of eluents of increasing strength is used.

Eluotropic range of solvents

6. Equipment for liquid chromatography

In modern liquid chromatography, devices 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. presents a block diagram of a liquid chromatograph 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 exclude 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 primarily determined by the operating pressure in the system. For operation in the range of 10-500 MPa, plunger (syringe) type pumps are used. Their disadvantage is the need for periodic stops for filling 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 bubble formation 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, which make it possible to smooth out the flow pulsation within 1–2% and provide volumetric velocities from 0.1 to 25 ml / min at pressures up to ~ 3.107 Pa. In microcolumn chromatography, the 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 components of the eluent to the mixing chamber, leaving the total flow rate constant. To inject a sample into a column under high pressure, without stopping the flow, use special microdosing taps connected to a loop of a known volume for the solution sample under investigation. Dosing systems with automatic sampling and sample injection using microdosing valves or syringes have been developed.

Injector provides mixture sample injection components to be separated into a column with a sufficiently high reproducibility. Simple stop-flow sampling systems require a pump stop and are therefore less convenient than Reodyne loop dispensers.

Loudspeakers for HPLC are most often made of polished stainless steel tube 10-25 cm in length and 3-5 mm in inner diameter.

Rice. Chromatography columns for liquid chromatography

Also use glass columns placed in a metal casing; in microcolumn chromatography - printed metal columns with an inner diameter of 1.0-1.5mm, printed glass microcolumns with a diameter of 70-150 microns and hollow capillary columns with a diameter of 10-100 microns; in preparative chromatography - columns with a diameter of 2 to 10 cm and more. For uniform and dense filling of columns with sorbent, a suspension packing method is used. The suspension is prepared from a sorbent and a suitable organic liquid, which is supplied under a pressure of up to 5 × 107 Pa to the column. To determine the separated components leaving the column use detectors. Temperature constancy provided thermostat.

Detectors for liquid chromatography have a flow cell in which there is a continuous measurement of any property of the flowing eluent. They must be very sensitive. To increase the detector sensitivity, derivatization of the mixture components after the column is sometimes used. To do this, with the flow of the eluent, such reagents are introduced which, 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 a 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 optical density of the solvent 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 of connections, which may or may not contain chromophore groups. On the other hand, the use of refractometers is limited to isocratic systems (with a constant eluent composition), so that the use of a solvent gradient is not possible in this case.

Differential "href =" / text / category / differentcial / "rel =" bookmark "> differential amplifier and recorder. integrator, which allows you to calculate the relative areas of the resulting peaks. Complex chromatographic systems use interface unit connecting the chromatograph with personal computer, which carries out not only the collection and processing of information, but also controls the device, calculates the quantitative characteristics and, in some cases, the qualitative composition of mixtures. Microprocessor provides automatic sample injection, change by a given program of eluent composition with gradient elution, maintaining column temperature.

Bruker ". Rice. Liquid chromatograph Jasco

Self-test questions

What is Liquid Chromatography? Name its types, areas of application. List about The main chromatographic quantities and their determination 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 way the substance is transported? What substances are used as adsorbents? What is the difference? What serves as a liquid mobile phase - an eluent? Requirements for solvents. What is the difference between partition chromatography and adsorption chromatography? List the main parts of the liquid chromatograph circuit, their purpose.

List of used literature

1 "Liquid chromatography in medicine"

Http: // journal. issep. rssi. ru / articles / pdf / 0011_035.pdf

2 "Introduction to the methods of high performance liquid chromatography"

Http: // www. chemnet. ru / rus / teaching / oil / spezprakt-chr. html

3 "Liquid chromatography"

Http: // e-science. ru / index /? id = 1540

4 "Chromatography"

Http: // belchem. narod. ru / chromatography1.html

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 in the hydraulic system of the chromatograph.

Silica gel adsorbents with different volumes, surfaces, and pore diameters are most widely used in HPLC. Alumina 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, in comparison with aluminum oxide, has a wider range of porosity, surface area and pore diameter; a significantly higher catalytic activity of aluminum oxide leads to distortion of the analysis results due to the decomposition of sample components or their irreversible chemisorption.

HPLC detectors

Detectors:

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).

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

Detectors. The registration of the exit from the column of a separate component is performed using a detector. For registration, you can use a change in any analytical signal coming from the mobile phase and associated with the nature and amount of the mixture component. In liquid chromatography, analytical signals are used such as light absorption or light emission of the output solution (photometric and fluorometric detectors), refractive index (refractometric detectors), potential and electrical conductivity (electrochemical detectors), etc.

Introduction.

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

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

At present, HPLC has come out on top in terms of development rates among instrumental methods, surpassing even gas chromatography. The most important advantage of HPLC over gas chromatography is the ability to study almost any object without any restrictions on their physicochemical properties, for example, on boiling points or molecular weight.

Today, HPLC is a well-defined instrumental method that is widely used in various fields of science and technology. It is especially important in such critical areas as biochemistry, molecular biology, environmental pollution control, as well as in the chemical, petrochemical, food and pharmaceutical industries.

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

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

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

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

d. Solvents acting as the mobile phase are often corrosive to equipment. This primarily applies to solvents used in reverse phase chromatography, which is preferred in biochemical HPLC applications.

The specificity of HPLC as an instrumental technique must be taken into account in the development, creation, and operation of these systems. It took more than ten years of searching and research to create commercial samples of chromatographic systems and their components, which are sufficiently reliable, simple and safe to work with an acceptable ratio between price and technical characteristics. Recent trends towards a decrease in columns (both in length and diameter) are forcing new requirements for instruments.

1.1. EFFICIENCYANDSELECTIVITY

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

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

Let us accept the condition that some two components are soluble in the mobile phase and interact with the stationary phase, that is, the chroiathographic process can proceed without disturbances. In this case, after passing the mixture through the column, one can obtain chromatograms of the form a, b or v(fig. 1.1). These chromatograms illustrate chromatographic separations that differ in efficiency. (a and b) with equal selectivity and selectivity (b and v) with equal efficiency.

The more efficient the column is, the narrower the peak is obtained with the same retention time. Column efficiency is measured by the number of theoretical plates (PTT) N: the higher the ef-

Rice. 1.2. Parameters of the chromatographic peak and calculation of the number of theoretical plates:

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

Rice. 1.1. Chromatogram type depending on the efficiency and selectivity of the column:

a- conventional selectivity, reduced efficiency (less theoretical plates); b - conventional selectivity and efficiency; v - conventional efficiency, increased selectivity (greater ratio of retention times of components)

efficiency, the larger the FTT, the less the expansion of the peak of the initially narrow band as it passes through the column, the narrower the peak at the exit from the column. PTT characterizes the number of stages for establishing equilibrium between the mobile and stationary phases.

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

HETT = L/ N

PVETT = B3TT / d c.

Having the values of PTT, HETT, and PVETT, one can easily compare the efficiency of columns of different types, different lengths, filled with sorbents of different nature and grain size. By comparing the CTT of two columns of the same length, the performance is compared. When comparing HETT, columns with sorbents of the same grain size and different lengths are compared. Finally, the PVETT value makes it possible for any two columns to assess the quality of the sorbent, firstly, and the quality of filling the columns, and secondly, regardless of the length of the columns, the granulation of sorbent by its nature.

Column selectivity plays an important role in achieving chromatographic separation.

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

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

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

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

However, along with the blurring of the chromatographic zone band during the separation process in the column, it can also be blurred in the device for introducing the sample, in the connecting capillaries injector - column and column - detector, in the detector cell and in some auxiliary devices (microfilters for capturing mechanical particles from samples installed after the injector, guard columns, coil reactors, etc.) - The more the extra-column volume compared to the retention peak volume, the greater the smearing. It also matters where the dead volume is located: the narrower the chromatographic call, the greater the blurring will give the dead volume. Therefore, special attention should be paid to the design of that part of the chromatograph where the chromatographic zone is the narrowest (injector and devices from the injector to the column) - here extracolumn erosion is the most dangerous and has the strongest effect. Although it is believed that in well-designed chromatographs, the sources of additional extracolumn dilution should be minimized, nevertheless, each new device, each alteration of the chromatograph must necessarily end with testing on the column and comparing the obtained chromatogram with the passport one. If peak distortion is observed, a sharp decrease in efficiency, the newly introduced capillaries and other devices should be carefully inspected.

Off-column washout and miscalculation can lead to significant (more than 50%) loss of efficiency, especially in cases where relatively long-term chromatographs are attempted to be used for high-speed HPLC, microcolumn HPLC and other modern HPLC options that require microinjectors, interconnecting capillaries with internal diameter 0.05-0.15 mm minimum length, columns with a capacity of 10-1000 μl, detectors with micro cuvettes with a capacity of 0.03-1 μl and with high speed, high-speed recorders and integrators.

1.2. RETENTION AND STRENGTH OF SOLVENT

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 held by the stationary phase, the sorbent. However, the capacitance factor should not be too high in order to obtain an acceptable elution time. If a stationary phase is chosen for a given mixture of substances, which retains them, then further work on the development of an analytical method consists in choosing a solvent that would provide, in the ideal case, different for all components, but acceptably 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 the two-component solvent (for example, hexane with the addition of isopropanol) is increased by increasing the content of the polar component (isopropanol), or decreased by decreasing the content of isopropanol. If the containing polar component becomes too small (less than 0.1%), it should be replaced with a weaker one in eluting strength. The same is done, replacing either the polar or non-polar component with others, and in the event that the given system does not provide the desired selectivity with respect to the mixture components of interest. When selecting solvent systems, both the solubility of the mixture components and the eluotropic series of solvents compiled by different authors are taken into account.

In approximately 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, 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 constituent (methanol, acetonitrile or THF) in the eluent and decreased by adding more water. If it is not possible to achieve the desired selectivity, use a different organic component or try to change it using different 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 changing the pH; in some cases, modification with organic substances is used.

However, especially in the case of complex natural and biological mixtures, it is often not possible to select the strength of the solvent in such a way that all components of the sample are eluted within a reasonable time. Then it is necessary to resort to gradient elution, that is, to use a solvent, the elution force of which changes during the analysis so that it constantly increases according to a predetermined program. This technique makes it possible to achieve the elution of all components of complex mixtures in a relatively short period of time and their separation into components in the form of narrow peaks.

1.3. SIZE OF SORBENT PARTICLES, PERMEABILITY AND EFFICIENCY

When considering column smearing, we indicated that the column efficiency (HETP) depends on the particle size of the sorbent. To a large extent, the rapid development of HPLC over the past 10-12 years was caused, firstly, by the development of methods for obtaining sorbents with a particle size of 3 to 10 μm and with a narrow fractional composition, providing high efficiency with good permeability, and, secondly, by the development of methods of filling columns with these sorbents; and, thirdly, the development and serial production of liquid chromatographs with high-pressure pumps, injectors and detectors with small-volume cuvettes capable of registering small-volume peaks.

For well packed slurry columns, the theoretical plate equivalent height (PVETT) can be 2 regardless of whether 3, 5, 10, or 20 µm particles are used for packing. In this case, we will obtain, respectively, columns (with a standard length of 250 mm) with an efficiency of 41670, 25000, 12500 and 6250 tt. It seems natural to choose the most efficient column packed with 3 µm particles. However, this efficiency will come at the expense of very high pressures and relatively low separation rates, as an existing pump is likely to be able to pump solvent through such a column at a high space velocity. Here we are just faced with the question of the relationship between the size of the sorbent particles, the efficiency and permeability of the columns.

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

The resistance factor for columns packed with microparticles of the same type using the same method changes insignificantly and amounts to the following values:

Particle type ".... Irregular Spherical

form form

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

Suspension packaging. ... ... 700-1500 500-700

Column inlet pressure is proportional to linear flow rate, column resistance factor, solvent viscosity, and column length, and is inversely proportional to the square of the particle diameter.

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

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

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

CLASSIFICATION OF HPLC METHODS BY SEPARATION MECHANISM

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

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

2.1.1 ADSORPTION CHROMATOGRAPHY

Separation by adsorption chromatography is carried out as a result of the interaction of a substance with adsorbents, such as silica gel or alumina, 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 division into zones during the movement with the mobile phase along the column. The separation of the zones of the components achieved in this case depends on the interaction with both the solvent and the adsorbent.

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

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

Silica gel adsorbents with different pore volumes, surfaces, and pore diameters are most widely used in HPLC. Alumina is used much less frequently and, extremely rarely, other adsorbents widely used in classical column and thin layer chromatography. The main reason for this is the insufficient mechanical strength of most other adsorbents, which does not allow them to be packaged and used at elevated pressures typical for high pressure dryers.

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

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

2.1.2 Disadvantages of adsorption chromatography limiting its use

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

First of all, this is the long duration of the processes of equilibration of adsorbents with solvents containing water in trace amounts, the difficulty of preparing such solvents with a certain and reproducible moisture content. This results in poor reproducibility of retention parameters, resolution, selectivity. For the same reason, it is impossible to use gradient elution - the return to the initial state is so long that it significantly outweighs the gain in time due to the use of a gradient.

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

2.2. DISTRIBUTION CHROMATOGRAPHY

Partition chromatography is a variant of HPLC, in which the separation of a mixture into components is carried out due to the difference in their distribution coefficients between two immiscible phases: a solvent (mobile phase) and a phase on a sorbent (stationary phase). Historically, the first were sorbents of this type, which were obtained by the deposition of liquid phases (oxydipropionitrile, paraffin oil, etc.) on porous carriers, similar to how sorbents for gas-liquid chromatography (GLC) were prepared and prepared. However, the drawbacks of such sorbents were immediately discovered, the main of which was the relatively fast washing off of the phase from the carrier. Due to this, the amount of phase in the column gradually decreased, the retention times also decreased, and adsorption centers not covered by the phase appeared at the initial section of the column, causing the formation of peak tails. This drawback was fought by saturating the solvent with the deposited phase even before it entered the column. The carryover also decreased when more viscous and less soluble polymer phases were used, however, in this case, due to diffusion from thick polymer films, the efficiency of the columns was markedly reduced.

It turned out to be logical to graft the liquid phase with chemical bonds to the carrier in such a way that its entrainment becomes physically impossible, that is, to transform the carrier and the phase into one whole - into the so-called graft-phase sorbent.

In the future, the efforts of the researchers were directed to the search for reagents, the grafting of which would proceed fairly quickly and completely, and the formed bonds were as stable as possible. Alkylchlorosilanes and their derivatives became such reagents, which made it possible to obtain graft-phase sorbents of different types and with different both polar and non-polar groups on the surface using a similar technology.

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

The complexity of the technology of grafting reagents and preparation of raw materials and materials, its multistage nature lead to the fact that even batches of sorbents obtained using the same technology from the same manufacturing company may have slightly different chromatographic characteristics. This is especially true when such sorbents are used for the analysis of multicomponent mixtures containing substances that differ markedly in the number and position of functional groups, in terms of functionality.

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

2.3. ION EXCHANGE CHROMATOGRAPHY

In ion-exchange chromatography, the separation of mixture components is achieved due to the reversible interaction of ionizable substances with the ionic groups of the sorbent. The retention of the electroneutrality of the sorbent is ensured by the presence of counterions capable of ion exchange, located in the immediate vicinity of the surface. The ion of the introduced sample, interacting with a fixed charge of the sorbent, exchanges with a counterion. Substances with different affinity "to fixed charges are separated on anionites or on cationites. Anionites have positively charged groups on the surface and sorb anions from the mobile phase. Cationites, respectively, contain groups with a negative charge interacting with cations.

As the mobile phase, use is made of aqueous solutions of salts of acids, bases and solvents such as liquid ammonia, ie, solvent systems with a high dielectric constant e and a great tendency to ionize compounds. Usually, they work with buffer solutions that allow adjusting the pH value.

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

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

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

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

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

for anion exchange

X- + R + Y- h ->■ Y- + R + X-.

for cation exchange |

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

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

Naturally, the ions of the sample, weakly interacting with the ion exchanger, with this competition will be weakly retained on the column and are the first to be washed out from it, and, conversely, the more strongly retained ions will be the last to elute from the column. Usually, BTqpH4Hbie interactions of non-ionic nature arise due to adsorption or hydrogen bonds of the sample with the non-ionic part of the matrix or due to the limited solubility of the sample in the mobile phase. It is difficult to isolate "classical" ion-exchange chromatography in a "pure" form, and therefore some chromatographers proceed from empirical rather than theoretical principles in ion-exchange chromatography.

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

2.4. EXCLUSIVE CHROMATOGRAPHY

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

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

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

Traditionally, the process carried out in organic solvents is still often called gel permeation chromatography, and in aqueous systems - gel filtration chromatography. In this book, a single term is adopted for both options, which comes from the English "Size Exclusion" - an exception by size - and to the fullest extent reflects the mechanism of the process.

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

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

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

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

Rice. 2.15. Model for the separation of molecules by measure in size exclusion chromatography

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

Molecules of intermediate size, capable of penetrating only some part of the pores, are retained in the column in accordance with their size. The distribution coefficient of these molecules varies from zero to one and characterizes the fraction of pore volume available for molecules of a given size. Their retention volume is determined by the sum of Y about and the available portion of the pore volume.

QUALITATIVE ANALYSIS

A chromatographer starting out in high performance liquid chromatography should be familiar with the basics of qualitative analysis. Qualitative analysis is used to identify a known product obtained in a new way or in a mixture with other products. "It is necessary for the isolation of various components from complex biological, chemical mixtures, which is especially important in medicine, forensic science, ecology, to control the location some medicinal chemical products and their metabolites in biomaterials .. „." Familiarity with the basics of qualitative "analysis will help to avoid typical mistakes, for example / to distinguish impurities in a sample from impurities in a solvent or to check the purity of a substance at more than one wavelength spectrophotometer, and on different, etc.

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

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

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

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

In such cases, add the standard to the sample in a 1: 1 ratio. If the substances are identical, the value t R the starting material does not change, and only one peak is obtained on the chromatogram. If there is an instrument with a cyclic chromatography system, then for reliability of identification it is desirable to pass the mixture through the column several times.

Retention information can also be found in the literature, but the value of this information is limited. Since columns of even one batch give poor reproducibility, the literary values do not always correspond to the true value. t R on this column. For adsorption chromatography, however, it is possible to predict t R based on literature data. Another difficulty associated with the use of literary meanings t R, - the complexity of their search in the specialized literature, although the bibliographic reviews published in the Jornal of chromatography have an updated index of the types of substances.

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

With isocratic separation of homologues or oligomers, the following pattern is sometimes observed:

\ gk" = A + Bn,

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

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

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

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

When comparing the data on retention of substances, the values of the capacity factor can be used k", since on him, unlike t R the speed of the mobile phase and the geometric features of the column are not affected.

Separation on chemically bound phase is similar to partition chromatography separation with similar phases, and therefore equilibrium extraction data can be used to predict retention times.

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

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

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

Modern, still expensive, diode array spectrophotometers are of considerable interest in identification.

A completely unknown substance cannot be identified with high performance liquid chromatography alone, and other methods are needed.

QUANTITATIVE ANALYSIS

Quantitative liquid chromatography is a well-developed analytical method that is not inferior in accuracy to quantitative gas chromatography and significantly exceeds the accuracy of TLC or electrophoresis. Therefore, in order to obtain quantitative results, the instrument must be calibrated.

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

4.1. CHMATOGRAPHIC SEPARATION

Sampling can be error-prone. It is especially important to avoid error and to obtain an adequate representative sample of inhomogeneous solid samples, volatile or unstable substances, as well as agricultural products and biomaterials. Inhomogeneous samples, such as food products, are thoroughly mixed and quartered. By carrying out this operation many times, the homogeneity of the sample is achieved.

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

Samples should be completely dissolved and their solutions prepared with an accuracy of ± 0.1%. It is desirable to dissolve the sample in the mobile phase, which excludes the possibility of precipitation after its introduction into the chromatograph. If dissolution in the mobile phase is impossible, then a solvent miscible with it should be used and sample volumes (less than 25 μl) should be introduced into the chromatograph.

Significant inaccuracies can occur during sample injection due to its fractionation, leakage and peak smearing. Peak blurring causes the formation of tails, leading to partial overlapping of the peaks, and as a consequence, to errors in detection. For quantitative injection, loop valve devices are preferred over syringes because of the higher accuracy and less operator dependency.

In the chromatographic separation of substances, complications can also arise that lead to data distortion: quantitative analysis. Decomposition or transformation of the sample during the chromatographic process or irreversible adsorption of the substance on this column is possible. It is important to ensure that these undesirable phenomena are absent and, if necessary, regenerate or replace the column. Peak overlap and tailing can also be reduced by changing the chromatographic conditions.

You cannot use spurious or fuzzy peaks in quantitative analysis, as well as peaks whose release time is close to to, as there may be insufficient separation. Typically, peaks with a value of ^ 0.5 are used. The highest column efficiency is achieved with the introduction of 10 ~ 5 -10 ~ 6 g of solute per gram of sorbent. When injecting large amounts of sample, the dependence of the peak height on the load may be non-linear and quantification is required by peak areas.

Errors associated with detection or amplification lead to a significant distortion of the results of chromatographic separation. Each detector is characterized by specificity, linearity and sensitivity. The selectivity test is especially important when analyzing trace impurities. The response of UV detectors can change to substances with similar functional groups by a factor of 10 4. It is necessary to calibrate the detector response for each analyte. Naturally, substances that do not absorb in the UV region will not give a signal to the recorder when using a photometer as a detector. Negative peaks may occur when using a refractometer. In addition, this detector needs to be thermostated, which is not required for a UV detector.

The linearity of the detector determines the size of the injected sample. It should be remembered that column flow rate, column and detector temperature, and design will affect the accuracy of quantitation. Errors in the transmission of an electrical signal to an output device (recorder), an integrator or to a computer can occur due to noise pickup, lack of grounding, voltage fluctuations in the network, etc.

4.2. MEASURING AREA OR PEAK HEIGHT

Peak height h (Fig. 10.1) the distance from the top of the peak to the baseline is called, it is measured linearly or the number of divisions is counted on the recorder. Some electronic integrators and computers provide information on peak heights. The position of the baseline of the displaced peaks is found by interpolating the ordinate values corresponding to the beginning and end of the peak (peak 1 and 3 see fig. 10.1). To improve accuracy, you must have a gentle, stable baseline. In the case of unseparated peaks, the baseline is drawn between the beginning and end of the peak, rather than being replaced by a zero line. Since the peak height is less dependent on the influence of adjacent overlapping peaks, the peak height estimate is more accurate and is almost always used for trace analysis.

The peak area can be determined in various ways. Let's take a look at some of them.

1. The planimetric method consists in the fact that the peak is surrounded by a hand-held planimeter, which is a device that mechanically determines the area of the peak. The method is accurate, but laborious and poorly reproducible. This method is undesirable.

2. Method of paper silhouettes - the peak is cut out and weighed. The method is well reproducible, but laborious, and the chromatogram is destroyed. Its applicability depends on the uniformity of the chart tape. The method also cannot be widely recommended.

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

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

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

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

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

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