Methods of analysis in the technology of organic substances. Identification of organic compounds: elemental and functional analysis, solubility studies

ORGANIC SUBSTANCE ANALYSIS

(outdated organizational analysis), qualities. and quantities. determination of the composition of the org. in-in and establishment of their structure.

When determining qualities. composition of org. they use a variety of methods based on chemistry. p-tions, accompanied by the formation of products with characteristic properties (color, smell, melting temperatures, etc.), and on physical measurements. and physical-chemical (chromatographic, spectral, etc.) characteristics of the identified compounds.

With quantities, analysis of org. the quantity of the reagent entering the distribution with the determined org is established. conn., or measure diff. physical and physical-chemical characteristics associated with the number of compounds being determined.

O.v. A. includes elemental analysis, structural-group (including functional and stereospecific), molecular analysis, phase analysis And structural analysis.

Historically, methods for elemental analysis of org. were the first to be developed. in-in (A. Lavoisier, late 18th century), based on their oxidation and gravimetric, titrimetric. or gasometric determination of the formed simple compounds. individual elements. First elemental methods microchemical analysis(microanalysis) was developed by F. Pregl in the beginning. 20th century From the 2nd half. 20th century For elemental analysis, automatic methods are widely used. analyzers based on combustion of the analyzed sample org. in-va and gas chromato-graphic. separation and determination of combustion products. The analyzer is equipped with a computer and automatic sample introduction system.

Isotope analysis of org. the purpose is to determine the content of individual isotopes in them, as well as to determine the ratio of the same org. compounds containing different or combinations thereof. For this purpose, mass spectrometry or multiple gas-liquid chromatography is most often used (for example, when separating ordinary and deuterated forms of methane or benzene). Naib. chromatography-mass spectrometry is effective.

Most functional analysis methods are based on interaction. individual functions group org. conn. with suitable reagents. Such districts can be selective or limitedly selective, that is, they are characteristic, respectively. only for one or several. functional groups.

Most often, solutions associated with the formation or disappearance of substances, bases, oxidizing agents, reducing agents, water, gases, and, less commonly, sediments and colored substances are used. The resulting compounds and bases determine acid-base titration in aquatic or non-aqueous environments. In a non-aqueous environment, separate potentiometric titration of compounds and bases of different strengths when present together.

In the case of oxidation-reduction. solutions, the speed of which is low, back titration is usually used, i.e., the excess of the reagent is titrated. On the formation or absorption of water in the districts of the org. conn. based on the definition of plural. functional groups using Fischer's reagent(see also Aquametry).

Methods based on flows, which are accompanied by the release or absorption of gas, are rarely used, since measuring volume or pressure usually requires bulky equipment.

Gravimetric measurements are based on the formation of sediments. methods for determining a small number of functions. groups. Slightly soluble compounds used in these cases are, as a rule, metallic. carbonic and sulfonic acids, salts org. bases, complex connections. (including chelate ones).

Formation of colored compounds. often quite specific and allows you to selectively determine the function. photometric groups methods. Solutions have become widespread (especially in microanalysis), leading to the formation of fluorescent compounds, since the sensitivity of determining the function. The group in this case is quite large.

A special type of functional. analysis consider methods based on preliminary. interaction of the substance being determined with reagents and determination of the resulting product. For example, aromatic after nitration can be determined polarographically, and the relationship between the amino group and naphthalene sulfonyl chloride can be determined fluorometrically.

Below are examples of the most. frequently used functional methods. analysis.

The determination of active hydrogen in alcohols, amines, amides, carbonic and sulfonic compounds, mercaptans and sulfonamides is based on their interactions. with Grignard reagents (usually methyl magnesium iodide; see Cerevitin method)or with LiAlH 4 and measuring the volume of methane or hydrogen released, respectively. Active in acetylene and its homologues is determined by the solution with salts Ag(I), Hg(I) or Cu(I) with the last, titrimetric. determination of the separated ones.

Connections with unsaturated carbon-carbon bonds are most often brominated, iodinated or hydrogenated. In the first two cases, unreacted Br 2 or I 2 is determined iodometrically, and during hydrogenation the volume of absorbed H 2 is measured. The number of double bonds can be determined by the addition of mercury salts to the last. titration of the released substance.

When determining hydroxyl groups, they most often use acetic, phthalic or pyromellitic anhydride, the excess of which is titrated. You can use acid chlorides. Hydroxy groups in phenols are usually titrated with base solutions in a non-aqueous medium. Phenols are easily brominated and combined with diazonium salts, therefore they are titrated with solutions of Br 2 or diazonium salts, or a bromide-bromate mixture is added to the solution under study, the excess is determined iodometrically (see also Falin's reaction).

Carbohydrates can be determined by oxidation with sodium periodate and subsequent. titration of excess oxidizing agent or formed compounds. Numerous have been developed. variations of this method (see, for example, Malaprada reaction).

To determine org. peroxy compounds (including peroxy acids) most often use their interaction. with KI and subsequent titration of the released I 2 with Na 2 S 2 O 3 solution.

The analysis of alkoxy compounds consists of interaction. of the analyzed substance with hydroiodic acid to form alkyl iodides (see. Zeisel method). The latter are determined by different methods - gravimetrically (in the form of AgI) or titrimetrically (acid-base titration). Carbon compounds can be determined similarly. To identify C 1 -C 4 -alkoxy groups, the resulting alkyl iodides are converted into quaternary ammonium compounds, which are analyzed by thin-layer or paper chromatography.

The definition of epoxy groups is based on their reaction with hydrogen chloride to form chlorohydrins; upon completion of the solution, the excess HCl is titrated with alkali solution.

For the determination of carbonyl compounds. (aldehydes and ketones) max. oximation is often used, i.e. their conversion into interaction. with hydroxylamine hydrochloride; The HCl released as a result of the reaction is titrated with an alkali solution (the end point of the titration is set using an indicator or potentiometrically). Exists big number modifications of this method. Aldehydes can also be determined by the solution with Na bisulfite followed by. acid-base titration. Less commonly used are aldehydes with Ag + ions, reaction with hydrazines and the formation of Schiff bases.

Quinones are reduced with Ti(III) chloride or V(II) sulfate; the excess of the reducing agent is determined titrimetrically. Quinones can also be determined iodometrically.

To determine carbon compounds and their salts, max. Acid-base titration is often used in non-aqueous media.

A large number of methods have been developed for the analysis of carbon derivatives. Anhydrides after their hydrolysis to a solution are titrated with alkali solutions. In the case of analyzing a mixture of an acid and its anhydride, the sum of both substances is determined by acid-base titration, and then the anhydride is mixed with morpholine or aniline and the released acids are titrated. In the latter case, you can also determine the excess base by titration with HCl solution. Acid halides or their mixtures with compounds are determined in the same way. In this case, instead of solutions with amines, interactions are often used. acid halide with alcohol and last. separate titration of free carbonic acid and the released halogen-rich acid with alkali solution.

Definition esters carbon compounds are based on their hydrolysis with alkali solution, the excess is titrated with a solution. Small amounts of esters are usually determined spectrophotometrically in the form of Fe(III) salts of hydroxamic acids formed during interaction. esters with hydroxylamine.

To determine nitrogen-containing org. a large number of methods have been proposed. Compounds capable of reduction (nitro-, nitroso-, ) are determined titanium- or vanadatometrically: an excess solution of Ti(III) or V(II) salt is added and the unreacted reducing agent is titrated with a solution of Fe(III) salt.

Titration of ramie solution (usually HClO 4) in a non-aqueous medium is widely used in determination. This method often allows you to separately determine org. and non-org. bases in mixtures, as well as org. bases of varying strength when present together. Amines can be determined, like hydroxy derivatives, by the ratio of their acylation. To determine primary aromatics. amines are often titrated with solution in an acidic medium, accompanied by the formation of a diazo compound. A similar titration of secondary amines leads to their N-nitrosation and is also used in the analysis. During microanalysis of primary aromatics. amines, the resulting diazo compounds are usually combined with the corresponding azo components and the resulting dye is determined spectrophotometrically. In the case of analyzing mixtures of primary, secondary and tertiary amines, titration with HClO 4 solution is most often used in a non-aqueous medium of the initial mixture (all amines are titrated), the mixture after acetylation with acetic anhydride (only tertiary amines are titrated) and the mixture after treatment with acetylacetone or salicylic aldehyde ( the sum of secondary and tertiary amines is titrated).

To determine aryldiazonium salts with a solution of the analyzed substance, titrate weighed portions of the azo component (3-methyl-1-phenyl-5-pyrazolone, m-phenylenediamine, etc.) or add a solution of the azo component to the analyzed solution, the excess to The swarm is titrated with NaNO 2 solution in an acidic environment. In the case of the analysis of diazo compounds, it is also possible to use gasometry. analysis based on the decomposition of the studied compound. with the release of N 2, the volume of which is measured. Sometimes, as in the case of the analysis of amines, diazo compounds are determined by the combination with the last. spectrophotometric rich. determination of the resulting dye.

Hydrazines are usually titrated iodometrically. In the case of thiols, interaction can also be used. them with silver salts or acid-base titration. Org. sulfides are oxidized with a bromide-bromate mixture, the excess is determined titrimetrically.

Widespread for quality. and quantities. functional Selective and fairly sensitive methods of IR spectroscopy and NMR have also been analyzed.

The emergence of stereospecific analysis of org. in-in the 2nd half. 20th century associated with the development of chromatographic methods. To separate enantiomers, most often a preliminary reaction is carried out between the analyzed substances and optically active reagents to form diastereomers, which are then separated by gas-liquid or high-performance liquid chromatography on columns with optically active stationary phases.

Molecular analysis of org. in-in founded ch. arr. on the use of chromatography and others. spectral methods, which make it possible to establish the structure of the org. connections.

Phase analysis, allowing qualitative and quantitative analysis of crystalline. org forms connection, carried out using radiography And electronography. X-ray, structural analysis allows installation with high precision structural function org. v-va, determine the lengths of bonds between atoms and the angles between bonds.

The analysis methods listed above are based on direct definition analyzed substances or derivatives obtained from them. In O. v. A. Indirect methods are also often used. So, for example, carbon compounds can be isolated from the analyzed mixture in the form of sparingly soluble silver or other salts and then using the atomic absorption method. spectroscopy or X-ray fluorescence analysis to determine the amount of the corresponding metal; Based on the results of such an analysis, the content of carbon dioxide can be calculated. In liquid chromatography, the use of indirect detection of the separated substances is effective, in which an active component is added to the mobile phase, forming easily detectable compounds with the separation products or with the substances being chromatographed.

The methods of analysis and the equipment used depend on the specific task of O. v. a.: determination of the main ingredient of the mixture, org. or non-org. impurities in org. wow, org. impurities in inorganic in-ve or analysis of a complex multi-component mixture of in-in.

Methods O. century. A. widely used in the development of industrial technology. produced by org. products and in the production process itself for the development of methods for analyzing raw materials, auxiliary. in-in, in-between. products at different stages of production, to control production. process, finished products, Wastewater and gas emissions, for the identification of impurities in intermediate and final products, as well as for the development of analytes. techniques that ensure the necessary kinetic. research. In all cases, it is necessary to choose the optimal one. options for analysis methods and their combinations in accordance with the requirements for speed, reproducibility, accuracy, etc.

When developing an analyte. parts of normative and technical. documentation for raw materials, auxiliary. materials and finished products first of all establish the minimum necessary and sufficient number of analytes. indicators. Such indicators include melting point, pH, basic content. substances in the product, which are determined by a direct method (usually titrimetrically using potentiometry) or indirectly, by subtracting from the mass of the entire product the mass of impurities determined by chromatography. (most often), electrochemical. or spectrophotometric. methods. When using the function analysis to determine the main items usually choose a method that involves determining this item by function. group formed at the last stage of its receipt. If necessary, when the analyzed substance is obtained by multi-stage synthesis, it is determined according to different functions. groups. Analyst. the methods chosen for the analysis of raw materials and finished products must have Ch. arr. good reproducibility and accuracy.

Analytical methods used in production control must be rapid and continuous (for example, redox metry, pH metry, ). The basis of methods for monitoring production processes is org. in-in often lies the definition of a vanishing function. group, i.e., a group undergoing transformation at a given stage of production, which makes it possible to accurately record the end of the corresponding stage. In this case, thin-layer, gas-liquid, high-performance liquid chromatography, spectrophotometry, and electrochemical are widely used. methods, flow-injection. analysis.

For analysis there will be intervals. titrimetry is most often used for manufactured products, and for reaction analysis. mixtures-complex chromatographic. and spectral methods, including gas chromatography-mass spectrometry, a combination of gas chromatography with Fourier transform infrared spectroscopy.

Object analysis has become very important environment. When developing appropriate methods for analyzing basic the requirements for them are high sensitivity and correct identification of the substances being determined. These requirements are met by gas chromatography-mass spectrometry using two or more stationary phases.

In clinical analysis (analysis of blood, urine, tissues and other objects for the content of drugs, metabolites, steroids, amino acids, etc.) important is not only the sensitivity, accuracy and rapidity of the analysis, but also the reproducibility of its results. When the latter requirement is critical, gas chromatography-mass spectrometry under standard conditions, as well as high-throughput flow injection, are used. analysis and a variety of enzyme methods with high selectivity.

Lit.: Guben Weil, Methods organic chemistry, vol. 2, Methods of analysis, trans. with him. 4th ed., M.. 1963; Cheronis N. D., Ma T. S., Micro- and semi-micro methods of organic functional analysis, trans. from English, M., 1973; Siggia S.. Hannah J. G., Quantitative organic analysis by functional groups, trans. from English, M.; 1983. B. I. Kolokolov.

Chemical encyclopedia. - M.: Soviet Encyclopedia. Ed. I. L. Knunyants. 1988 .

See what "ORGANIC SUBSTANCES ANALYSIS" is in other dictionaries:

Water analysis is a method for studying the properties and qualities of water. It is used to determine the amount of various substances in water that is in contact with humans for industrial and domestic purposes, or for scientific purposes. Contents 1 Types of water for ... ... Wikipedia

Soil analysis is a set of operations performed to determine the composition, physical mechanical, physical chemical, chemical, agrochemical and biological properties of the soil. Carry out mechanical (granulometric), chemical, ... ... Wikipedia

WATER ANALYSIS- is carried out to determine the quality of water and determine the possibility of using it to supply fish ponds. A.v. is held four times a year: in spring (during the spring flood), in mid-summer (July), in autumn (during the autumn... ... Pond fish farming

analysis- ANALYSIS (from the Greek analysis, decomposition, dismemberment) is the procedure for real or mental division of an object, phenomenon or process, as well as their relationships into components, elements, properties, functions and subsystems. The procedure... ... Encyclopedia of Epistemology and Philosophy of Science

Identification (detection) of components analyzed in and approximate quantities, assessment of their content in waters and materials. As components it can be atoms and ions, isotopes of elements and individual nuclides, molecules, functional. groups and radicals... Chemical encyclopedia

Determination of content (mass, concentration, etc.) or quantities. ratios of components in the analyzed sample. Determined components may be. atoms, molecules, isotopes, functional. groups, phases, etc. (see Elemental analysis, Molecular ... ... Chemical encyclopedia

The study of organic matter begins with its isolation and purification.

1. Precipitation

Precipitation– separation of one of the compounds of a gas or liquid mixture of substances into a precipitate, crystalline or amorphous. The method is based on changing the solvation conditions. The effect of solvation can be greatly reduced and the solid substance can be isolated in its pure form using several methods.

One of them is that the final (often called target) product is converted into a salt-like compound (simple or complex salt), if only it is capable of acid-base interaction or complex formation. For example, amines can be converted to substituted ammonium salts:

(CH 3) 2 NH + HCl -> [(CH 3) 2 NH 2 ] + Cl – ,

and carboxylic, sulfonic, phosphonic and other acids - into salts by the action of corresponding alkalis:

CH 3 COOH + NaOH -> CH 3 COO – Na + + H 2 O;

2CH 3 SO 2 OH + Ba(OH) 2 -> Ba 2+ (CH 3 SO 2 O) 2 – + H 2 O;

CH 3 P(OH) 2 O + 2AgOH -> Ag(CH 3 PO 3) 2– + 2H 2 O.

Salts as ionic compounds dissolve only in polar solvents (H 2 O, ROH, RCOOH, etc.). The better such solvents enter into donor-acceptor interactions with the cations and anions of the salt, the greater the energy released during solvation, and the higher solubility. In non-polar solvents, such as hydrocarbons, petroleum ether (light gasoline), CHCl 3, CCl 4, etc., salts do not dissolve and crystallize (salt out) when these or similar solvents are added to a solution of salt-like compounds. The corresponding bases or acids can be easily isolated from salts in pure form.

Aldehydes and ketones of non-aromatic nature, adding sodium hydrosulfite, crystallize from aqueous solutions in the form of poorly soluble compounds.

For example, acetone (CH 3) 2 CO from aqueous solutions crystallizes with sodium hydrosulfite NaHSO 3 in the form of a slightly soluble hydrosulfite derivative:

Aldehydes easily condense with hydroxylamine, releasing a water molecule:

The products formed in this process are called oximes They are liquids or solids. Oximes have a weakly acidic character, manifested in the fact that the hydrogen of the hydroxyl group can be replaced by a metal, and at the same time they have a weakly basic character, since oximes combine with acids, forming salts such as ammonium salts.

When boiled with dilute acids, hydrolysis occurs, releasing the aldehyde and forming a hydroxylamine salt:

Thus, hydroxylamine is an important reagent that makes it possible to isolate aldehydes in the form of oximes from mixtures with other substances with which hydroxylamine does not react. Oximes can also be used to purify aldehydes.

Like hydroxylamine, hydrazine H 2 N–NH 2 reacts with aldehydes; but since there are two NH 2 groups in the hydrazine molecule, it can react with two aldehyde molecules. As a result, phenylhydrazine C 6 H 5 –NH–NH 2 is usually used, i.e. the product of replacing one hydrogen atom in a hydrazine molecule with a phenyl group C 6 H 5:

The reaction products of aldehydes with phenylhydrazine are called phenylhydrazones.Phenylhydrazones are liquid and solid and crystallize well. When boiled with dilute acids, like oximes, they undergo hydrolysis, as a result of which free aldehyde and phenylhydrazine salt are formed:

Thus, phenylhydrazine, like hydroxylamine, can serve to isolate and purify aldehydes.

Sometimes another hydrazine derivative is used for this purpose, in which the hydrogen atom is replaced not by a phenyl group, but by an H 2 N–CO group. This hydrazine derivative is called semicarbazide NH 2 –NH–CO–NH 2. The condensation products of aldehydes with semicarbazide are called semicarbazones:

Ketones also readily condense with hydroxylamine to form ketoximes:

With phenylhydrazine, ketones give phenylhydrazones:

and with semicarbazide - semicarbazones:

Therefore, hydroxylamine, phenylhydrazine and semicarbazide are used for isolating ketones from mixtures and for their purification to the same extent as for isolating and purifying aldehydes. It is, of course, impossible to separate aldehydes from ketones in this way.

Alkynes with a terminal triple bond react with an ammonia solution of Ag 2 O and are released in the form of silver alkinides, for example:

2(OH) – + HC=CH -> Ag–C=C–Ag + 4NH 3 + 2H 2 O.

The starting aldehydes, ketones, and alkynes can be easily isolated from poorly soluble substitution products in their pure form.

2. Crystallization

Crystallization methods separation of mixtures and deep purification of substances are based on the difference in the composition of the phases formed during partial crystallization of the melt, solution, and gas phase. An important characteristic of these methods is the equilibrium, or thermodynamic, separation coefficient, equal to the ratio of the concentrations of the components in the equilibrium phases - solid and liquid (or gas):

Where x And y– mole fractions of the component in the solid and liquid (or gas) phases, respectively. If x<< 1, т.е. разделяемый компонент является примесью, k 0 = x / y. In real conditions, equilibrium is usually not achieved; the degree of separation during single crystallization is called the effective separation coefficient k, which is always less k 0 .

There are several crystallization methods.

When separating mixtures using the method directional crystallization the container with the initial solution slowly moves from the heating zone to the cooling zone. Crystallization occurs at the boundary of the zones, the front of which moves at the speed of movement of the container.

It is used to separate components with similar properties. zone melting ingots cleaned of impurities in an elongated container moving slowly along one or more heaters. A section of the ingot in the heating zone melts and crystallizes again at the exit from it. This method provides a high degree of purification, but is low-productive, therefore it is used mainly for cleaning semiconductor materials (Ge, Si, etc.).

Counterflow column crystallization is produced in a column, in the upper part of which there is a cooling zone where crystals are formed, and in the lower part there is a heating zone where the crystals melt. The crystals in the column move under the influence of gravity or using, for example, a screw in the direction opposite to the movement of the liquid. Method characterized by high productivity and high yield of purified products. It is used in the production of pure naphthalene, benzoic acid, caprolactam, fatty acid fractions, etc.

To separate mixtures, dry and purify substances in a solid-gas system, they are used sublimation (sublimation) And desublimation.

Sublimation is characterized by a large difference in equilibrium conditions for different substances, which makes it possible to separate multicomponent systems, in particular, when obtaining substances of high purity.

3. Extraction

Extraction- a separation method based on the selective extraction of one or more components of the analyzed mixture using organic solvents - extractants. As a rule, extraction is understood as the process of distributing a dissolved substance between two immiscible liquid phases, although in general one of the phases may be solid (extraction from solids) or gaseous. Therefore, a more accurate name for the method is liquid-liquid extraction, or simply liquid-liquid extraction Usually in analytical chemistry the extraction of substances from an aqueous solution using organic solvents is used.

The distribution of substance X between the aqueous and organic phases under equilibrium conditions obeys the distribution equilibrium law. The constant of this equilibrium, expressed as the ratio between the concentrations of substances in two phases:

K= [X] org / [X] aq,

at a given temperature there is a constant value that depends only on the nature of the substance and both solvents. This value is called distribution constant It can be approximately estimated by the ratio of the solubility of the substance in each of the solvents.

The phase into which the extracted component has passed after liquid extraction is called extract; phase depleted of this component - raffinate.

In industry, the most common is countercurrent multi-stage extraction. The required number of separation stages is usually 5–10, and for difficult-to-separate compounds – up to 50–60. The process includes a number of standard and special operations. The first includes the extraction itself, washing the extract (to reduce the content in impurities and removal of mechanically entrapped source solution) and re-extraction, i.e. reverse transfer of the extracted compound into the aqueous phase for the purpose of its further processing in an aqueous solution or repeated extraction purification. Special operations are associated, for example, with a change in the oxidation state of the separated components.

Single-stage liquid-liquid extraction, effective only at very high distribution constants K, are used primarily for analytical purposes.

Liquid extraction devices – extractors– can be with continuous (columns) or stepped (mixers-settlers) phase contact.

Since during extraction it is necessary to intensively mix two immiscible liquids, the following types of columns are mainly used: pulsating (with reciprocating movement of the liquid), vibrating (with a vibrating package of plates), rotary-disk (with a package of disks rotating on a common shaft), etc. d.

Each stage of the mixer-settler has a mixing and settling chamber. Mixing can be mechanical (mixers) or pulsating; multi-stage is achieved by connecting the required number of sections into a cascade. Sections can be assembled in a common housing (box extractors). Mixer-settlers have an advantage over columns in processes with a small number of stages or with very large flows of liquids. Centrifugal devices are promising for processing large flows.

The advantages of liquid-liquid extraction are low energy costs (there are no phase transitions requiring external energy supply); possibility of obtaining highly pure substances; possibility of complete automation of the process.

Liquid-liquid extraction is used, for example, to isolate light aromatic hydrocarbons from petroleum feedstocks.

Extraction of a substance with a solvent from the solid phase often used in organic chemistry to extract natural compounds from biological objects: chlorophyll from green leaves, caffeine from coffee or tea mass, alkaloids from plant materials, etc.

4. Distillation and rectification

Distillation and rectification are the most important methods for separating and purifying liquid mixtures, based on the difference in the composition of the liquid and the vapor formed from it.

The distribution of mixture components between liquid and vapor is determined by the value of relative volatility α:

αik= (yi/ xi) : (yk / xk),

Where xi And xk,yi And yk– mole fractions of components i And k respectively, in a liquid and the vapor formed from it.

For a solution consisting of two components,

Where x And y– mole fractions of the volatile component in liquid and vapor, respectively.

Distillation(distillation) is carried out by partial evaporation of the liquid and subsequent condensation of steam. As a result of distillation, the distilled fraction is distillate– is enriched with a more volatile (low-boiling) component, and the non-distilled liquid – VAT residue– less volatile (high-boiling). Distillation is called simple if one fraction is distilled from the initial mixture, and fractional (fractional) if several fractions are distilled. If it is necessary to reduce the temperature of the process, distillation is used with water vapor or an inert gas bubbling through a layer of liquid.

There are conventional and molecular distillation. Conventional distillation are carried out at such pressures when the free path of molecules is many times less than the distance between the surfaces of liquid evaporation and vapor condensation. Molecular distillation carried out at very low pressure (10 –3 – 10 –4 mm Hg), when the distance between the surfaces of liquid evaporation and vapor condensation is commensurate with the free path of the molecules.

Conventional distillation is used to purify liquids from low-volatile impurities and to separate mixtures of components that differ significantly in relative volatility. Molecular distillation is used to separate and purify mixtures of low-volatile and thermally unstable substances, for example, when isolating vitamins from fish oil and vegetable oils.

If the relative volatility α is low (low-boiling components), then the separation of mixtures is carried out by rectification. Rectification– separation of liquid mixtures into practically pure components or fractions that differ in boiling points. For rectification, column devices are usually used, in which part of the condensate (reflux) is returned for irrigation to the upper part of the column. In this case, repeated contact is carried out between the flows of the liquid and vapor phases. The driving force of rectification is the difference between the actual and equilibrium concentrations of the components in the vapor phase, corresponding to given composition of the liquid phase. The vapor-liquid system strives to achieve an equilibrium state, as a result of which the vapor, upon contact with the liquid, is enriched with highly volatile (low-boiling) components, and the liquid - with low-volatile (high-boiling) components. Since the liquid and steam move towards each other (countercurrent), with sufficient at the height of the column in its upper part, an almost pure, highly volatile component can be obtained.

Rectification can be carried out at atmospheric or elevated pressure, as well as under vacuum conditions. At reduced pressure, the boiling point decreases and the relative volatility of the components increases, which reduces the height of the distillation column and allows the separation of mixtures of thermally unstable substances.

By design, distillation apparatuses are divided into packed, disc-shaped And rotary film.

Rectification is widely used in industry for the production of gasoline, kerosene (oil rectification), oxygen and nitrogen (low-temperature air rectification), and for the isolation and deep purification of individual substances (ethanol, benzene, etc.).

Since organic substances are generally thermally unstable, for their deep purification, as a rule, packed distillation columns, operating in a vacuum. Sometimes to obtain especially pure organic matter They use rotary film columns that have a very low hydraulic resistance and a short residence time of the product in them. As a rule, rectification in this case is carried out in a vacuum.

Rectification is widely used in laboratory practice for deep purification of substances. Note that distillation and rectification serve at the same time to determine the boiling point of the substance under study, and, therefore, make it possible to verify the degree of purity of the latter (constancy of the boiling point). For this purpose they use also special devices - ebulliometers.

5.Chromatography

Chromatography is a method of separation, analysis and physico-chemical study of substances. It is based on the difference in the speed of movement of the concentration zones of the components under study, which move in the flow of the mobile phase (eluent) along the stationary layer, and the compounds under study are distributed between both phases.

All the various methods of chromatography, which were started by M.S. Tsvet in 1903, are based on adsorption from the gas or liquid phase on a solid or liquid interface.

In organic chemistry, the following types of chromatography are widely used for the separation, purification and identification of substances: column (adsorption); paper (distribution), thin-layer (on a special plate), gas, liquid and gas-liquid.

In these types of chromatography, two phases come into contact - one stationary, adsorbing and desorbing the substance being determined, and the other mobile, acting as a carrier of this substance.

Typically, the stationary phase is a sorbent with a developed surface; mobile phase – gas (gas chromatography) or liquid (liquid chromatography).The flow of the mobile phase is filtered through the sorbent layer or moves along this layer.B gas-liquid chromatography The mobile phase is a gas, and the stationary phase is a liquid, usually deposited on a solid carrier.

Gel permeation chromatography is a variant of liquid chromatography, where the stationary phase is a gel. (The method allows the separation of high molecular weight compounds and biopolymers over a wide range of molecular weights.) The difference in the equilibrium or kinetic distribution of components between the mobile and stationary phases is a necessary condition for their chromatographic separation.

Depending on the purpose of the chromatographic process, analytical and preparative chromatography are distinguished. Analytical is intended to determine the qualitative and quantitative composition of the mixture under study.

Chromatography is usually carried out using special instruments - chromatographs, the main parts of which are a chromatographic column and a detector. At the moment of sample introduction, the analyzed mixture is located at the beginning of the chromatographic column. Under the influence of the flow of the mobile phase, the components of the mixture begin to move along the column at different speeds, and well-sorbed components move along the sorbent layer more slowly. Detector at the outlet from the column automatically continuously determines the concentrations of separated compounds in the mobile phase. The detector signal is usually recorded by a recorder. The resulting diagram is called chromatogram.

Preparative chromatography includes the development and application of chromatographic methods and equipment to obtain highly pure substances containing no more than 0.1% impurities.

A feature of preparative chromatography is the use of chromatographic columns with a large internal diameter and special devices for isolating and collecting components. In laboratories, 0.1–10 grams of a substance are isolated on columns with a diameter of 8–15 mm; in semi-industrial installations with columns with a diameter of 10–20 cm, several kilograms. Unique industrial devices with columns with a diameter of 0.5 m have been created to produce several tons of the substance annually.

Substance losses in preparative columns are small, which allows the widespread use of preparative chromatography for the separation of small quantities of complex synthetic and natural mixtures. Preparative gas chromatography used to produce highly pure hydrocarbons, alcohols, carboxylic acids and others organic compounds, including chlorine-containing ones; liquid– for the production of drugs, polymers with a narrow molecular weight distribution, amino acids, proteins, etc.

Some studies claim that the cost of high-purity products obtained chromatographically is lower than those purified by distillation. Therefore, it is advisable to use chromatography for the fine purification of substances previously separated by rectification.

2.Elemental qualitative analysis

Qualitative elemental analysis is a set of methods that make it possible to determine what elements an organic compound consists of. To determine the elemental composition, an organic substance is first converted into inorganic compounds by oxidation or mineralization (alloying with alkali metals), which are then examined by conventional analytical methods.

The enormous achievement of A.L. Lavoisier as an analytical chemist was the creation elemental analysis of organic substances(the so-called CH analysis). By this time, numerous methods for gravimetric analysis of inorganic substances (metals, minerals, etc.) already existed, but they were not yet able to analyze organic substances in this way. Analytical chemistry at that time she was clearly “limping on one leg”; Unfortunately, the relative lag in the analysis of organic compounds and especially the lag in the theory of such analysis is felt even today.

Having taken up the problems of organic analysis, A.L. Lavoisier, first of all, showed that all organic substances contain oxygen and hydrogen, many contain nitrogen, and some contain sulfur, phosphorus or other elements. Now it was necessary to create universal methods quantitative determination of these elements, primarily methods for the precise determination of carbon and hydrogen. To achieve this goal, A. L. Lavoisier proposed burning samples of the substance under study and determining the amount of carbon dioxide released (Fig. 1). In doing so, he was based on two of his observations: 1) carbon dioxide is formed during the combustion of any organic substance; 2) the starting substances do not contain carbon dioxide; it is formed from the carbon that is part of any organic substance. The first objects of analysis were highly volatile organic substances - individual compounds such as ethanol.

Rice. 1. The first device of A. L. Lavoisier for the analysis of organic

substances by combustion method

To ensure the purity of the experiment, the high temperature was provided not by any fuel, but by solar rays focused on the sample by a huge lens. The sample was burned in a hermetically sealed installation (under a glass bell) in a known amount of oxygen, the released carbon dioxide was absorbed and weighed. The mass of water was determined indirect method.

For the elemental analysis of low-volatile compounds, A. L. Lavoisier later proposed more complex methods. In these methods, one of the sources of oxygen necessary for sample oxidation was metal oxides with which the burnt sample was mixed in advance (for example, lead(IV) oxide). This approach was later used in many methods of elemental analysis of organic substances, and usually gave good results. However, the methods of CH analysis according to Lavoisier were too time-consuming, and also did not allow the hydrogen content to be determined accurately enough: direct weighing of the resulting water was not carried out.

The CH analysis method was improved in 1814 by the great Swedish chemist Jens Jakob Berzelius. Now the sample was burned not under a glass bell, but in a horizontal tube heated from the outside, through which air or oxygen was passed. Salts were added to the sample, facilitating the combustion process. The released water absorbed solid calcium chloride and weighed. The French researcher J. Dumas supplemented this technique with the volumetric determination of released nitrogen (CHN analysis). The Lavoisier-Berzelius technique was once again improved by J. Liebig, who achieved quantitative and selective absorption of carbon dioxide in a ball absorber he invented (Fig. 2.).

Rice. 2. Yu. Liebig's apparatus for burning organic substances

This made it possible to sharply reduce the complexity and labor intensity of CH analysis, and most importantly, to increase its accuracy. Thus, Yu. Liebig, half a century after A.L. Lavoisier, completed the development of gravimetric analysis of organic substances, begun by the great French scientist. Applying his methods, Yu. By the 1840s, Liebig had figured out the exact composition of many organic compounds (for example, alkaloids) and proved (together with F. Wöhler) the existence of isomers. These techniques remained virtually unchanged for many years, their accuracy and versatility ensured the rapid development of organic chemistry in the second half of the 19th century. Further improvements in the field of elemental analysis of organic substances (microanalysis) appeared only at the beginning of the 20th century. The corresponding research of F. Pregl was awarded the Nobel Prize (1923).

It is interesting that both A.L. Lavoisier and J. Liebig sought to confirm the results of a quantitative analysis of any individual substance by counter-synthesis of the same substance, paying attention to the quantitative ratios of the reagents during the synthesis. A.L. Lavoisier noted that chemistry generally has two ways to determine the composition of a substance: synthesis and analysis, and one should not consider oneself satisfied until one succeeds in using both of these methods for testing. This remark is especially important for researchers of complex organic substances. Their reliable identification and identification of the structure of compounds today, as in the time of Lavoisier, require the correct combination of analytical and synthetic methods.

Detection of carbon and hydrogen.

The method is based on the oxidation reaction of organic matter with copper (II) oxide powder.

As a result of oxidation, the carbon included in the analyzed substance forms carbon (IV) oxide, and hydrogen forms water. Carbon is determined qualitatively by the formation of a white precipitate of barium carbonate upon interaction of carbon (IV) oxide with barite water. Hydrogen is detected by the formation of crystalline hydrate Cu8O4-5H20, blue in color.

Execution method.

Copper (II) oxide powder is placed in test tube 1 (Fig. 2.1) at a height of 10 mm, an equal amount of organic matter is added and mixed thoroughly. A small lump of cotton wool is placed in the upper part of test tube 1, onto which a thin layer of white powder without aqueous copper (II) sulfate is poured. Test tube 1 is closed with a stopper with a gas outlet tube 2 so that one end of it almost touches the cotton wool, and the other is immersed in test tube 3 with 1 ml of barite water. Carefully heat in the burner flame first the upper layer of the mixture of the substance with copper (II) oxide, then the lower

Rice. 3 Discovery of carbon and hydrogen

In the presence of carbon, turbidity of barite water is observed due to the formation of barium carbonate precipitate. After a precipitate appears, test tube 3 is removed, and test tube 1 is continued to be heated until water vapor reaches aqueous copper (II) sulfate. In the presence of water, a change in the color of copper (II) sulfate crystals is observed due to the formation of crystalline hydrate CuSO4*5H2O

Halogen detection. Beilyitein's test.

The method for detecting chlorine, bromine and iodine atoms in organic compounds is based on the ability of copper (II) oxide to decompose halogen-containing organic compounds at high temperatures to form copper (II) halides.

The analyzed sample is applied to the end of a pre-calcined copper wire and heated in a non-luminous burner flame. If there are halogens in the sample, the resulting copper (II) halides are reduced to copper (I) halides, which, when evaporated, color the flame blue-green (CuC1, CuBr) or green (OD) color. Organofluorine compounds do not color the flame of copper (I) fluoride is non-volatile. The reaction is non-selective due to the fact that nitriles, urea, thiourea, individual pyridine derivatives, carboxylic acids, acetylacetone, etc. interfere with the determination. If available alkali and alkaline earth metals, the flame is viewed through a blue filter.

Nitrogen detection, sulfur and halogens. "Lassaigne's Test"

The method is based on the fusion of organic matter with sodium metal. When fused, nitrogen turns into sodium cyanide, sulfur into sodium sulfide, chlorine, bromine, iodine into the corresponding sodium halides.

Fusion technique.

A. Solids.

Several grains of the test substance (5-10 mg) are placed in a dry (attention!) refractory test tube and a small piece (the size of a grain of rice) of sodium metal is added. The mixture is carefully heated in a burner flame, uniformly heating the test tube, until a homogeneous alloy is formed. It is necessary to ensure that the sodium melts with the substance. When fused, the substance decomposes. Fusion is often accompanied by a small flash of sodium and blackening of the contents of the test tube from the resulting carbon particles. The test tube is cooled to room temperature and 5-6 drops of ethyl alcohol are added to eliminate residual sodium metal. After making sure that the remaining sodium has reacted (the hissing stops when a drop of alcohol is added), 1-1.5 ml of water is poured into the test tube and the solution is heated to a boil. The water-alcohol solution is filtered and used to detect sulfur, nitrogen and halogens.

B. Liquid substances.

A refractory test tube is vertically fixed on an asbestos mesh. Metallic sodium is placed in the test tube and heated until it melts. When sodium vapor appears, the test substance is introduced dropwise. Heating is intensified after the substance is charred. After the contents of the test tube are cooled to room temperature, it is subjected to the above analysis.

B. Highly volatile and sublimating substances.

The mixture of sodium and the test substance is covered with a layer of soda lime about 1 cm thick and then subjected to the above analysis.

Nitrogen detection. Nitrogen is qualitatively detected by the formation of Prussian blue (blue color).

Method of determination. Place 5 drops of the filtrate obtained after fusing the substance with sodium into a test tube, and add 1 drop of an alcohol solution of phenolphthalein. The appearance of a crimson-red color indicates an alkaline environment (if the color does not appear, add 1-2 drops of a 5% aqueous solution of sodium hydroxide to the test tube). Subsequently, add 1-2 drops of a 10% aqueous solution of iron (II) sulfate , usually containing an admixture of iron (III) sulfate, a dirty green precipitate is formed. Using a pipette, apply 1 drop of cloudy liquid from a test tube onto a piece of filter paper. As soon as the drop is absorbed by the paper, 1 drop of a 5% solution of hydrochloric acid is applied to it. If available nitrogen, a blue spot of Prussian blue appears.

Detection of sulfur.

Sulfur is qualitatively detected by the formation of a dark brown precipitate of lead (II) sulfide, as well as a red-violet complex with a solution of sodium nitroprusside.

Method of determination. The opposite corners of a piece of filter paper measuring 3x3 cm are moistened with the filtrate obtained by fusing the substance with metallic sodium (Fig. 4).

Rice. 4. Carrying out a seu test on a square piece of paper.

A drop of a 1% solution of lead (II) acetate is applied to one of the wet spots, retreating 3-4 mm from its border.

A dark brown color appears at the contact boundary due to the formation of lead (II) sulfide.

A drop of sodium nitroprusside solution is applied to the border of another spot. At the border of the “leaks” an intense red-violet color appears, gradually changing color.

Detection of sulfur and nitrogen when present together.

In a number of organic compounds containing nitrogen and sulfur, the discovery of nitrogen is hindered by the presence of sulfur. In this case, a slightly modified method for determining nitrogen and sulfur is used, based on the fact that when an aqueous solution containing sodium sulfide and sodium cyanide is applied to filter paper, the latter is distributed along the periphery of the wet spot. This technique requires certain operating skills, which makes its application difficult.

Method of determination. Apply the filtrate drop by drop into the center of a 3x3 cm filter paper until a colorless wet spot with a diameter of about 2 cm is formed.

Rice. 5. Detection of sulfur and nitrogen in the joint presence. 1 - a drop of a solution of iron (II) sulfate; 2 - a drop of a solution of lead acetate; 3 - drop of sodium nitroprusside solution

1 drop of a 5% solution of iron (II) sulfate is applied to the center of the spot (Fig. 5). After the drop is absorbed, 1 drop of a 5% solution of hydrochloric acid is applied to the center. In the presence of nitrogen, a blue Prussian blue spot appears. Then, 1 drop of a 1% solution of lead (II) acetate is applied along the periphery of the wet spot, and 1 drop of sodium nitroprusside solution is applied on the opposite side of the spot. If sulfur is present, in the first case, a dark brown spot will appear at the place of contact of the “leaks”, in the second case, a spot of red-violet color. The reaction equations are given above.

Fluoride ion is detected by the discoloration or yellow discoloration of alizarine zirconium indicator paper after acidification of the Lassaigne sample with acetic acid.

Detection of halogens using silver nitrate. Halogens are detected in the form of halide ions by the formation of flocculent precipitates of silver halides of various colors: silver chloride is a white precipitate that darkens in the light; silver bromide - pale yellow; silver iodide is an intense yellow precipitate.

Method of determination. To 5-6 drops of the filtrate obtained after fusing the organic substance with sodium, add 2-3 drops of diluted nitric acid. If the substance contains sulfur and nitrogen, the solution is boiled for 1-2 minutes to remove hydrogen sulfide and hydrocyanic acid, which interfere with the determination of halogens Then add 1-2 drops of 1% solution of silver nitrate. The appearance of a white precipitate indicates the presence of chlorine, pale yellow - bromine, yellow - iodine.

If it is necessary to clarify whether bromine or iodine is present, the following reactions must be carried out:

1. To 3-5 drops of the filtrate obtained after fusing the substance with sodium, add 1-2 drops of dilute sulfuric acid, 1 drop of a 5% solution of sodium nitrite or a 1% solution of iron (III) chloride and 1 ml of chloroform.

When shaken in the presence of iodine, the chloroform layer turns purple.

2. To 3-5 drops of the filtrate obtained after fusing the substance with sodium, add 2-3 drops of diluted hydrochloric acid, 1-2 drops of a 5% solution of chloramine and 1 ml of chloroform.

In the presence of bromine, the chloroform layer turns yellow-brown.

B. Discovery of halogens using Stepanov’s method. It is based on the transformation of a covalently bonded halogen in an organic compound into an ionic state by the action of sodium metal in an alcohol solution.

Detection of phosphorus. One method for detecting phosphorus is based on the oxidation of organic matter with magnesium oxide. Organically bound phosphorus is converted into phosphate ion, which is then detected by reaction with molybdenum liquid.

Method of determination. Several grains of the substance (5-10 mg) are mixed with double the amount of magnesium oxide and ashed in a porcelain crucible, first with moderate and then with strong heating. After cooling, the ash is dissolved in concentrated nitric acid, 0.5 ml of the resulting solution is transferred to a test tube, added 0.5 ml of molybdenum liquid and heat.

The appearance of a yellow precipitate of ammonium phosphomolybdate indicates the presence of phosphorus in the organic matter

3. Qualitative analysis by functional groups

Based on selective reactions of functional groups (See presentation on the topic).

In this case, selective reactions of precipitation, complexation, decomposition with the release of characteristic reaction products, and others are used. Examples of such reactions are presented in the presentation.

What is interesting is that it is possible to use the formation of organic compounds, known as organic analytical reagents, for group detection and identification. For example, dimethylglyoxime analogs interact with nickel and palladium, and nitroso-naphthols and nitrosophenols with cobalt, iron and palladium. These reactions can be used for detection and identification (See presentation on topic).

4. Identification.

Determination of the degree of purity of organic substances

The most common method for determining the purity of a substance is to measure boiling point during distillation and rectification, most often used for the purification of organic substances. To do this, the liquid is placed in a distillation flask (a round-bottomed flask with an outlet tube soldered to the neck), which is closed with a stopper with a thermometer inserted into it and connected to a refrigerator. The thermometer ball should be slightly higher holes in the side tube through which steam comes out. The thermometer ball, being immersed in the steam of a boiling liquid, takes on the temperature of this steam, which can be read on the thermometer scale. If the boiling point of the liquid is above 50 ° C, it is necessary to cover the upper part of the flask with thermal insulation. At the same time, it is necessary to using an aneroid barometer, record the atmospheric pressure and, if necessary, make a correction. If a chemically pure product is distilled, the boiling point remains constant throughout the entire distillation time. If a contaminated substance is distilled, the temperature during distillation rises as more is removed low boiling impurity.

Another commonly used method for determining the purity of a substance is to determine melting point For this purpose, a small amount of the test substance is placed in a capillary tube sealed at one end, which is attached to the thermometer so that the substance is at the same level as the thermometer ball. The thermometer with a tube with the substance attached to it is immersed in some high-boiling liquid, for example glycerin, and slowly heat over low heat, observing the substance and the increase in temperature. If the substance is pure, the moment of melting is easy to notice, because the substance melts sharply and the contents of the tube immediately become transparent. At this moment, the thermometer reading is noted. Contaminated substances usually melt at a lower temperature and over a wide range.

To control the purity of a substance, you can measure density.To determine the density of liquids or solids, they most often use pycnometer The latter, in its simplest form, is a cone equipped with a ground glass stopper with a thin internal capillary, the presence of which helps to more accurately maintain constant volume when filling a pycnometer. The volume of the latter, including the capillary, is found by weighing it with water.

Pycnometric determination of the density of a liquid comes down to simply weighing it in a pycnometer. Knowing the mass and volume, it is easy to find the desired density of the liquid. In the case of a solid substance, first weigh the pycnometer partially filled with it, which gives the mass of the sample taken for research. After this, the pycnometer is supplemented with water (or whatever - another liquid with a known density and not interacting with the substance under study) and weighed again. The difference between both weighings makes it possible to determine the volume of the part of the pycnometer not filled with the substance, and then the volume of the substance taken for research. Knowing the mass and volume, it is easy to find the desired density of the substance.

Very often, to assess the degree of purity of organic matter, they measure refractive index. The refractive index value is usually given for the yellow line in the spectrum of sodium with wavelength D= 589.3 nm (line D).

Typically, the refractive index is determined using refractometer.The advantage of this method for determining the degree of purity of an organic substance is that only a few drops of the test compound are required to measure the refractive index. This manual presents the considered physical properties of the most important organic substances. Note also that the universal method for determining the degree of purity of an organic substance is chromatography This method allows not only to show how pure a given substance is, but also to indicate what specific impurities it contains and in what quantities.

The basics of the definition were developed by F. Pregl. A 3-5 mg sample of the substance is burned at a temperature of 900 °C in a stream of oxygen purified from hydrogen, water and carbon dioxide. Hydrogen is purified from oxygen by passing the gas over a platinum catalyst at 800 °C. Complete removal of carbon dioxide and water is carried out by passing through anhydrous magnesium perchlorate ( anhydrone) and through asbestos impregnated with molten caustic soda ( ascarite).

After the combustion tube, tubes with absorbers are installed: anhydrone and ascarite. The weight gain of the first absorber corresponds to the amount of water from which the hydrogen content in a sample of the substance is calculated; the weight gain of the second absorber gives the amount of carbon dioxide, which is used to determine the carbon content in the analyzed substance.

Halogens and sulfur can be determined by decomposing the substance according to Carius. Halogens in the form of silver halide - by gravitation or by titrating excess silver nitrate. Sulfur is determined in the form of barium sulfate. After combustion of the sample, gases are passed over a layer of copper metal, where nitrogen oxides are reduced to free nitrogen. Nitrogen is determined by the volumetric method by the amount of unabsorbed gas.

Determination of molecular weight

To determine the molecular weight of a compound, cryoscopy methods based on Raoult's law are often used. To do this, determine the freezing point of the solvent and then the solution. The difference is directly proportional to the number of molecules of the substance dissolved in a given mass of solvent. Molecular mass is determined by the formula:

Where R- weight of the substance; P - solvent weight; K - cryoscopic constant;

Similarly, in the ebullioscopic method, molecular weight is determined through the difference between the boiling points of a pure solvent and a solution.

For high molecular weight compounds the methods described above are completely inapplicable. In this case, three methods are used: viscometric, osmotic and sedimentation, which, in turn, are not applicable to substances with normal molecular weight.

Currently, mass spectrometry is most often used to determine the molecular mass of an unknown substance.

Methods for isolating individual substances

To isolate compounds, the following physical methods are used: various types of distillation - fractional at atmospheric pressure, in vacuum, in high vacuum, molecular distillation, crystallization, extraction, chromatography. In addition, there are many special methods that take into account the specifics of the functional group.

· Molecular distillation . For substances that decompose at boiling point even in high vacuum, “molecular distillation” is used. Its principle is that under strong vacuum (10 -5 -10 -8 mm Hg) from the heated surface of the molten substance to be distilled, the molecules pass into the gas phase at a temperature much lower than the boiling point of the given compound. The vapor of the substance then condenses on the cold surface. This makes it possible to purify substances with a relatively large molecular weight and fragile structure.

· Steam distillation . As is known, a substance boils at a temperature when its vapor pressure is equal to atmospheric pressure. If you heat two immiscible liquids, they will boil at a temperature when the total vapor pressure of both liquids equals atmospheric pressure. Water is used as the second liquid. Thus, distillation of this mixture of liquids can be carried out below 100°C. The amount of both substances in the distillate is determined by the ratio of the product of the vapor pressure of each substance and its molecular weight.

· Crystallization . Recrystallization is used to purify solids. The method is based on the fact that for most compounds, when their solutions are cooled, the solubility of the substance decreases.

· Extraction . A separation method based on the difference in the distribution coefficients of a substance between two immiscible liquids.

Chromatography

Ø Chromatography – separation method based on different speeds of movement of concentration zones of the components of the mixture under study in the flow of the mobile phase ( eluent) relative to the stationary phase.

§ By tasks to be solved allocate preparative(quantitative separation of substances) and analytical chromatography (detection of substances and quantitative and qualitative characteristics of mixtures).

§ According to the principles of separation chromatography is divided into adsorption, distribution, ion exchange And sieve.

v Adsorption chromatography . The substances to be separated must differ in their affinity for the solid adsorbent, which is the stationary phase. Aluminum oxide and silica gel are usually used as adsorbents. Activated carbon, barium sulfate, magnesium silicate, and polyamides are used much less frequently.

The ability of substances to be adsorbed on a polar adsorbent is largely determined by their polarity. Based on their ability to be adsorbed, substances with different functional groups can be arranged in the following sequence:

RH< ROCH 3 < R-NO 2 < R-N(CH 3) 2 < R-COOCH 3 < R-NH 2 < R-OH < R-CONH 2 < R-COOH.

In terms of polarity, and therefore in terms of elution ability, eluent solvents form the following series:

H 2 O > CH 3 OH > C 2 H 5 OH > CH 3 COCH 3 > CH 3 COOC 2 H 5 > C 2 H 5 OC 2 H 5 > CHCl 3 > CCl 4 > cyclohexane > n-hexane

Elution is carried out either with one eluent (a mixture of eluents), or sequentially with several eluents, moving from less polar to more polar, or with a mixture of two solvents (successively increasing the concentration of the more polar one).

· Main variants of adsorption chromatography .

§ Column adsorption chromatography. The adsorbent is placed in a column. The substance to be separated is first applied on top, and then the eluent is passed through, which moves under the influence of gravity or is pumped under pressure by a special pump.

The separation of substances is monitored either by physicochemical methods (UV detection, refractometry) or by analytical chromatographic methods.

§ Thin layer chromatography (TLC). The sorbent is placed in a thin layer on a glass, aluminum or plastic substrate.

The sorbent layer can be loose or fixed using special chemicals (starch, gypsum). A sample of the substance is applied to the bottom of the plate, which is then placed in a box with an eluent. The solvent rises along the plate due to capillary forces ( ascending chromatography), producing separation. In the case of difficult-to-separate substances, resort to two-dimensional chromatography, when the substance is first eluted in one direction, and then the elution is carried out in a perpendicular direction.

IN modern conditions Typically, commercially produced preparative or analytical TLC plates are used.

· Identification and characterization of substances. Colored compounds are observed directly during chromatography. Colorless substances must be “identified” - converted into colored compounds.

Depending on the sorbent, fixative and the nature of the separated substance, various methods of “detection” are used, for example, carbohydrates are charred at high temperatures, including after spraying with sulfuric acid solutions, amino acids give colored products after treatment with a ninhydrin solution. Based on the color intensity of the separated compounds with specific reagents, their content in the mixture is judged.

To characterize substances, the term “ chromatographic mobility", which is denoted as Rf, is the ratio of the range of the substance zone to the range of the eluent.

v Partition chromatography . This version of chromatography is based on the distribution of substances between the mobile phase (gas, liquid) and the stationary phase - liquid held on a solid inert carrier. The most widely used are partition chromatography on paper and gas-liquid chromatography.

§ Paper chromatography. The basis of paper chromatography is the distribution of a mixture of separated substances between water adsorbed on paper and a solvent saturated with water. Using this method, the separation and identification of amino acids and monosaccharides was successfully carried out. Currently, this option of chromatography has lost its relevance.

§ Gas-liquid chromatography. This is partition chromatography between a stationary liquid phase supported on a carrier and a gas (usually helium, nitrogen or hydrogen).

To characterize the separated substances, use “ retention time" This is the time from the moment the mixture is introduced into the column until it exits the column and passes the substance through an appropriate detector, for example a detector that records changes in thermal conductivity. This option is one of the most widely used chromatographic methods, especially for analytical purposes.

v Ion exchange chromatography . The method is based on the distribution of charged substances (ions) between the mobile and stationary phases depending on their affinity for the ionic centers of the stationary phase.

According to the nature of the ion exchanger there are cationic And anionic chromatography. Water, solutions of acids and alkalis, and buffer solutions are widely used as eluents. The most common ion exchange materials are cation exchangers and anion exchangers based on cross-linked polymers containing ionogenic functional groups, as well as modified cellulose.

v Sieve chromatography (gel chromatography) . Distinctive feature Gel chromatography is that in gels formed by three-dimensional “cross-linked” macromolecules, there are pores of certain sizes, into which the smaller molecules of the separated molecules enter and do not enter the larger ones. Therefore, unlike adsorption chromatography, in gel chromatography the larger molecules pass through the column first, and the small ones last. The separation column is filled with grains of lyophilic or hydrophilic gel. Examples of such chromatographic materials are modified natural gelling agents - agar, dextrins, sephadexes (cross-linked dextrans) and synthetic sieves based on polyacrylamide or “cross-linked” polystyrene.

Berzelius J. (1779 1884) – Swedish chemist. Scientific research cover all the main problems of first chemistry half of the 19th century century.

Wehler F. (1800-1882) – German chemist. Works in inorganic and organic chemistry. Along with J. Liebig, he established the isomerism of salts of explosive acid.

3 Gmelin L. (1788-1853) - German chemist. He published reference books on experimental data, which went through several editions.

Liebig J. (1803-1873) – German chemist. Creator of the theory of radicals, founder of agrochemistry. Studied organic acids.

Butlerov A. (1828-1886) - Russian chemist, creator of the theory of the structure of organic compounds. Predicted isomerism of many compounds.

Gerard S. (1816-1856) – French chemist. Worked for Yu. Liebig, listened to lectures by J. Dumas. Created a theory of types. Many Russian chemists studied with C. Gerard.

Schorlemmer K. (1834 – 1892) – German organic chemist. He worked in the field of alkanes and has works on the history of chemistry.

Lossen V. (1838 - 1905) - German chemist. The main works are related to the study of alkaloids, discovered the rearrangement of hydroxamic acids.

Carius L. (1829-1875) – German chemist. Developed a method for determining sulfur, halogens and other elements in organic compounds (1860)

Beilstein F. (1838-1906) – Russian organic chemist. Main works in the field of synthesis of aromatic compounds. Initiator and first compiler of the multi-volume reference book on organic compounds (Handbuch der organische Chemie), known as the Belstein Handbook.

Pregl F. (1869-1930) – German chemist. Founder of microanalysis of organic compounds. Nobel Prize 1923

Raoul F. (1830-1901) – French chemist. The main direction of research is the study of solutions.

Transcript

1 FEDERAL AGENCY FOR EDUCATION STATE EDUCATIONAL INSTITUTION OF HIGHER PROFESSIONAL EDUCATION “VORONEZH STATE UNIVERSITY” PHYSICAL AND CHEMICAL METHODS OF ANALYSIS OF ORGANIC COMPOUNDS Guidelines for universities Publishing and Printing Center of Voronezh State University 2008

2 Approved by the scientific and methodological council of the Faculty of Chemistry on February 7, 2008, protocol 3 Compiled by: S.I. Karpov, V.F. Selemenev, M.V. Matveeva, N.A. Belanova Reviewer Dr. Chem.. Sciences, Professor G.V. Shatalin The guidelines present the theoretical foundations for the qualitative and quantitative determination of organic substances using physicochemical methods of analysis: chromatography (GLC, HPLC, TLC), spectral methods (spectrophotometry, IR spectroscopy); Some theoretical aspects of chromatography concerning the basic parameters of retention and separation efficiency of the components of the analyzed mixture are considered. The main attention is paid to the description of the implementation of laboratory work devoted to the consideration of techniques and methods of identification, qualitative and quantitative analysis of organic substances using GLC, HPLC, TLC, spectrophotometry (UV, visible), and IR spectroscopy. The educational and methodological manual is intended for 5th year students of the evening department of the Faculty of Chemistry and is compiled in accordance with the program of the special course “Physico-chemical methods of analysis of organic compounds”, taught at the Department of Analytical Chemistry of Voronezh State University. For specialty: Chemistry 2

3 CONTENTS Introduction Chromatographic methods of analysis Classification of chromatographic methods Column chromatography Theoretical basis gas chromatography Theoretical foundations of high-performance liquid chromatography (HPLC) Retention parameters and main characteristics of the separation of substances in column gas and liquid chromatography Plane chromatography Stages of the chromatographic process, materials and reagents used in plane chromatography Basic characteristics of the separation of substances in plane chromatography Spectral methods of analysis Spectral band parameters absorption Molecular absorption spectroscopy in the visible and UV regions of electromagnetic radiation Characteristics of spectrophotometric determination Optimal conditions for photometric determination Quantitative Analysis absorption methods Infrared spectroscopy Some characteristics of molecular spectra Vibrations of a diatomic molecule Group frequencies and interpretation of the spectrum Practical part Work 1. Applying a stationary liquid phase to a solid carrier and filling the column Work 2. Determining the optimal flow rate of the carrier gas Work 3. Determining the impurity content in toluene Work 4. Identification of organic compounds using Kovacs indices Work 5. Determination of trace amounts of acetone in tap water Work 6. Obtaining sorption isotherms of alcohols using the Gluckauf method

4 Work 7. Qualitative and quantitative determination of salicylic acid impurities in acetylsalicylic acid (aspirin) by the reverse-phase method HPLC Operation 8. Separation and identification of dicarboxylic acids by TLC in aqueous-organic mobile phases Work 9. Determination of the content of impurities in drug preparations using TLC data Work 10. Qualitative and quantitative determination of flavonoids by TLC Work 11. Spectrophotometric determination of the content of nicotinic acid in the drug Work 12 Spectrophotometric determination of the content of cyanocobalamin for injection (vitamin B12) Work 13. Determination of the authenticity of substances from the IR spectra of samples dispersed in potassium bromide Work 14. Identification of substances from the IR spectra of samples in the form of a suspension in petrolatum oil Work 15. Quantitative analysis of a mixture of xylene isomers by IR spectra List of used literature

5 INTRODUCTION The use of physical phenomena occupies one of the leading places in the analysis of chemical systems. Today, everyone who is involved in chemistry or studies the composition of a substance must be well versed in physicochemical methods of analysis. There are a number of methods used in analytical chemistry. Chromatographic and spectral methods are used in most research laboratories for production quality control. It should be noted that there is great interest and practical use these methods in various areas of human activity and the occurrence of chromatographic and optical processes in nature. It is enough just to list the areas of application: analysis of environmental pollution, analysis of food, drugs, clinical analysis, toxicological and forensic applications, etc. The place of chromatography in the field of molecular analysis of organic compounds. Chromatography prevails over other separation methods without replacing them. This is evidenced by data from a survey conducted in the United States on the use of various analytical instruments in 3000 research centers. Chromatographic instruments occupy one of the first places both in terms of the degree of use and the growing need for them. However, any chromatographic analysis is often associated with other physicochemical methods of analysis. Optical methods allow for qualitative and quantitative determination of a substance. For a comprehensive analysis of a substance for authenticity and the presence of impurities, quantitative determination involves the use of various physicochemical methods. To characterize any chemical compound, it is necessary to know its optical properties, ability to distribute and adsorb on various materials, and the possibility of its isolation. It should be emphasized that chromatographic and optical methods (spectrophotometry (UV, visible), IR spectroscopy, etc.) do not compete with each other, but harmoniously complement each other. 1. CHROMATOGRAPHIC METHODS OF ANALYSIS In 2003, 100 years have passed since the discovery of one of the most fruitful methods for studying the composition of complex multicomponent mixtures of substances, chromatography. This discovery belongs to the Russian botanist M.S. Tsvet, who for the first time did not limit himself to simply observing the phenomena of adsorption of plant pigments on powdered adsorbents, but realized that in these simple experiments a veil of the unknown opened before him, behind which there were truly boundless possibilities for studying the composition and properties of a wide variety of substances. 5

6 For the first time, the terms “chromatographic method” and “chromatogram” appear in two articles by M.S. Colors in 1906, as for the term “chromatography”, we find it in publications of the same year. “Chromatography (from the Greek chromatos color) is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase), while the other (mobile phase) moves in a certain direction” (IUPAC terminology, 1993 G. ). However, chromatography is not only a “physical separation method”. Chromatography can be defined as the science of methods of separation, as well as qualitative and quantitative determination of the components of liquid and gaseous mixtures, based on their various sorption (adsorption, distribution, etc.) under dynamic conditions. In the simplest case, dynamic conditions are created when the analyzed mixture of components (mobile phase) moves through a sorbent layer (stationary phase). The stationary phase (SP) in chromatography can be solid and liquid sorbents. Mobile phase (MP) is a gas or liquid passing through a chromatographic column. Classification of chromatographic methods 1. According to the state of aggregation of the phases. Gas chromatography mobile phase (MP) is a gas; gas-solid phase (stationary phase (SP) solid), gas-liquid chromatography (stationary phase liquid). Liquid chromatography mobile phase liquid; liquid solid phase chromatography (stationary phase solid sorbent), liquid liquid chromatography (stationary phase liquid). 2. According to the shape of the stationary phase. Column chromatography (CC). Planar chromatography stationary phase applied to a plane (chrome paper (BC)), thin layer chromatography (TLC). 3. According to the sorption mechanism. Adsorption absorption by a solid sorbent due to intermolecular interaction forces. Distributive differences in solubility in the mobile and stationary phases. Ion exchange differences in the electrostatic interaction of ions with ionogenic groups of sorbents. Sedimentary difference in the solubility of the separated substances. Ligand exchange difference in the ability to form coordination compounds with the analyte component. 6

7 Exclusive separation based on differences in the sizes and shapes of molecules. 4. By methods of carrying out the chromatographic process. Frontal, displacement, eluent Column chromatography Theoretical foundations of gas chromatography Gas chromatography (GC) is a method for separating volatile compounds. The mobile phase in gas chromatography is gas or vapor. Depending on the state of the stationary phase, gas chromatography is divided into gas adsorption, when the stationary phase is a solid adsorbent, and gas-liquid, when the stationary phase is a liquid, or rather a liquid film on the surface of solid sorbent particles. Gas chromatographic methods can be used to analyze gaseous, liquid and solid substances with a molecular weight of less than 400 that meet certain requirements: volatility, thermal stability, inertness. Gas chromatography is one of the most modern methods multicomponent analysis. Its advantages: speed, high accuracy, sensitivity, automation. GC refers to instrumental methods of analysis, since to determine the composition of the gas phase, not only a chromatographic system is required, but also a rather complex thermostatting and detection system. The block diagram of the chromatograph is shown in Fig. Fig. 1.1 Fig T thermostatic zones 1. Carrier gas supply system (mobile phase). Most often this is a gas cylinder with inert gases helium, argon, nitrogen. 2. Dispenser-sample injection system. It is a thermostated evaporator into which a specified exact volume of the test mixture is injected using a microsyringe, syringe or other calibrated device. Liquid substances, evaporating, pass into the gaseous phase, are captured by the flow of carrier gas and enter the column (3). 7

8 3. Chromatographic column is a glass or metal tube with a diameter of 2 to 4 mm and a length of 0.5 to 10 m, filled with a sorbent (packed column). Along with packed ones, micropacked (diameter 0.8-1.5 mm) and capillary (diameter 0.1-0.8 mm) columns up to 100 m long are used. The components of the mixture are separated in the column. Since the sorbability of substances is greatly influenced by temperature, the columns are thermostated. 4. Detector is a device designed to detect changes in the composition of the gas passing through the column. The detector readings are usually converted into an electrical signal and transmitted to a recording device. The most commonly used thermal conductivity detector (katarometer) and flame ionization detector (FID), thermal ionization detector (TID), and electron capture detector (ECD). To record stable, reproducible results, the detector is thermostated. 5. Recorder is a device that records or records the electrical signal received from the detector. Most often, a recorder or an integrator is used as a recorder in modern modifications of computer devices. The GC method is used to carry out qualitative and quantitative analysis, which is discussed in more detail in the works Theoretical foundations of high-performance liquid chromatography (HPLC) High-performance liquid chromatography (HPLC) column or planar liquid chromatography, in which sorbents with a particle size of 3-10 microns are used, as a result of which the efficiency sharply increases chromatographic separation. Based on the polarity of the contacting phases, liquid chromatography (both column and planar) is conventionally divided into normal phase chromatography (NPC) and reverse phase chromatography (RPC). Normal phase chromatography is a liquid chromatography in which the stationary phase is more polar than the mobile phase. This type of chromatography includes liquid adsorption chromatography with silica gel and aluminum oxide as NF. Also referred to as NFC is the distribution version 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). Reversed phase chromatography is a liquid chromatography in which the stationary phase is less polar than the mobile phase. This is a variant of partition chromatography, which uses sorbents with grafted nonpolar (usually long alkyl or alkyl)

9 silyl) groups and a polar solvent (for example, water-methanol, water-acetonitrile mixtures). In HPLC, about 70% of all analytical separations are carried out using reverse phase chromatography. Operation in the OFC mode is characterized by the use of a non-polar sorbent and a polar eluent. The sorbents are silica gels with grafted alkylsilyl groups of various lengths (from C 2 to C 22) with a direct alkyl group or with phenyl and diphenyl groups. Mobile phases (acetonitrile, water, alcohols and their mixtures) used in OFC allow detection in a wide UV range and easily dissolve almost everything critical connections, included in the composition of biological objects, medicinal substances, etc. RP HPLC is widely used in determining the purity of drugs, this is what the work is devoted to: Retention parameters and the main characteristics of the separation of substances in column gas and liquid chromatography Chromatogram (Fig. 1.2) curve, displaying the dependence of the concentration of a substance in the PF flow at the outlet of the column on time from the start of the process (output curve). The eluent (developing) method is often used. The output curve is presented in the form of a peak (for one substance). Experimentally measured in gas and liquid chromatography are the parameters presented in Fig. a) b) Fig. Substance retention parameters (a) and chromatographic peak parameters (b) in column chromatography t m is the transit time of the unsorbed component (dead time). t R full time retention of components is the time from the moment of insertion 9

10 samples until the maximum concentration zone of the corresponding substance appears at the column outlet. t" Ri = t Ri t m. (1) corrected (reduced) retention time. Peak width (W) length of the segment formed by the zero line and two tangents at the points of inflection of the peak between two points of intersection of the tangents at the point of inflection with the zero line. Height The peak is considered to be either the value of h or h". The retained volume V R is proportional to the retention time t R: V R = t U, where U is the volumetric velocity of the PF. Corrected (reduced) volume V" R retention R V" R = V R V m, where V m is the volume of the mobile phase required to elute the unretained substance, or dead volume. The retention factor (or capacity coefficient) k i is the ratio of the amounts of component i in the stationary (m i, s) and mobile (m i, m) phases, which is associated with the retention characteristics k i =t R "/t m Hence or k i t R m =. 10 t t t Ri = (1+k i)t m. (2) This is the basic equation characterizing retention in chromatography. As can be seen from equations (1, 2), the retention factor can be determined from the chromatogram data. In the practice of gas and liquid chromatography, the retention of two compounds consistently recorded on the chromatogram are characterized by the separation factor (α): " " " V R t (2) R l (2) R k (2) (2) α = = = = " " " V t l k. (3) R (1 ) R (1) The separation factor α is sometimes called selectivity. The numerical value of α is always greater than one. However, α does not describe the actual separation of two chromatographic peaks. There are two parameters, the distance between the peaks and their width. These determine whether the two chromatographic peak The distance between the peaks can be expressed as the difference in retention time (Δt R), and the width of the peak at its base W is defined as the distance between the touches m R (1) (1)

11 related to the guide peaks (Fig. 1.2b). The resolution (R S) of two peaks is defined as " " 2(tr t (2) R) Δt (1) R RS = =, (4) (W1 + W2) (W0.5(1) + W0.5(2) ) where W 0.5 is the peak width at half maximum; R S dimensionless quantity; Δt R and W must be expressed in the same units. The resolution is equal to one if the distance between two peaks is equal to the average peak width. When R S >1 the peaks should be resolved. However, full resolution may not be achieved if the width of the peak at the base is large, i.e., the blurring effects are large. The degree of peak attenuation determines the efficiency of the column. Efficiency in chromatography is the ability of a system to “prevent” (limit) the blurring of zones of separated substances. Efficiency is expressed by the number of theoretical plates N or the height equivalent to a theoretical plate (HETT). The theoretical plate (T.T.) is a section of the sorbent layer on which the distribution of a substance between two phases ends with the establishment of equilibrium. The number of theoretical plates can be calculated using the formula: 2 2 t N 5.54 R = W or 16 tr N, (5) 0.5 W where t R is the total retention time or, equivalent to this value, the total retention distance of the substance, the segment of the time axis of the chromatogram corresponding retention time. W and W 0.5 are the width of the peak at the base and at half its height, respectively (Fig. 1.2b). HETT is the height of the sorbent layer (column) required to establish equilibrium: H= L/ N, (6) where L is the length of the sorbent layer. The more N and less H, the higher the efficiency of the column. HETT depends on the flow rate of the mobile phase (U). This dependence can be represented as a curve in H U coordinates, which makes it possible to determine the minimum HETT for a given chromatographic system at a certain optimal flow rate. eleven

12 1.3. Plane chromatography Stages of the chromatographic process, materials and reagents used in planar chromatography (PC). Planar chromatography includes paper chromatography (PC), in which special paper is used as a sorbent, and thin layer chromatography (TLC), in which the processes of separating a mixture of substances are carried out in thin layers of sorbent deposited on an inert solid substrate or in films of porous polymer material, as well as electrochromatography. The TLC method forms the basis of screening tests in chemical, industrial, clinical, pharmaceutical, biochemical and biological laboratories. The method was proposed in 1938 by domestic scientists N.A. Izmailov and M.S. Schreiber. However, the wide possibilities of the method were discovered later thanks to the work of J. Kirchner and E. Stahl. TLC analysis includes the following stages: sample selection and preparation for analysis; plate pre-treatment; preparation of the chromatographic chamber; sample application; chromatographic separation of substances; removing the eluent from the plate; detection of components, identification of substances and semi-quantitative analysis. The stationary phases used in TLC are the same materials used in HPLC for separations based on adsorption, partition (normal or reverse phase), ion exchange or exclusion. The sorbent (silica gel, aluminum oxide, cellulose, polyamides, kieselguhr) in the form of finely ground particles 20 microns in size is applied in a thin layer (µm) onto a glass, metal or polymer plate. In this case, with the development of the chromatogram and its length of 12 cm, about 200 separations are achieved. One of the important tasks facing the researcher is the correct choice of the mobile phase (MP). In normal-phase chromatography (see also section 1.2.2), as in the column version, the elution capacity increases with increasing polarity of the solvent. In this case, solvents are sorbed to a lesser extent by the stationary phase, therefore the distribution coefficients of sorbed substances between the PF and NF are high. In the reversed-phase version, the elution force decreases with increasing solvent polarity. The mobile phase, rising along the sorbent layer due to the action of capillary forces, interacts with the gas phase. Therefore, pre- 12

13 Therefore, before the start of the chromatography process, the chamber and sorbent layer are saturated with a solvent in the vapor phase, i.e., a state of equilibrium between the mobile phase and the gas phase is achieved. In a typical chamber, saturation is achieved in approximately 5–10 minutes for a solvent with a boiling point below C. It takes several hours to saturate the chamber with a high-boiling solvent. Preliminary saturation of the sorbent layer with any pure solvent increases the speed of movement of the solvent front across the layer and reduces the chromatographic mobility Rf of the analyzed substances. Both normal and reversed phases are subject to preliminary saturation. When separating substances in normal (polar) phases, it is preferable to use the polar components of multicomponent eluents to saturate the sorbent layer, and non-polar components in the RP phase. According to the chromatographic methods, linear, circular and anticircular TLC are distinguished. The most widely used is linear chromatography. In this case, samples are applied to the starting line parallel to one of the sides of the paper or plate (see works 8-10). The latter are placed vertically in a chromatographic chamber, at the bottom of which the eluent is poured, and ascending planar chromatography is carried out (Fig. 1.3a). The linear development of chromatograms can also be carried out horizontally with the eluent supplied from one or both sides (Fig. 1.3b). Descending vertical TLC and HD can also be used. In a circular HRP, samples are applied at a certain distance from the center of the plate along the circumference, and the eluent is fed to the center (Fig. 1.3c). In an anti-circle PC, samples are applied in a circle around the periphery of the plate and the eluent is fed towards the center of the plate (Fig. 1.3d). Fig. Options for chromatography in PC: a linear vertical; b linear horizontal; in a circular g anti-circling When applying samples to a plate, a number of requirements must be met to obtain reproducible results. Initially, mark the plate, marking the starting line. It is essential that the distance of the sample application line from the edge or center of the plate (usually 1–2 cm) and the line of immersion of the plate in the eluent (about 0.5 cm) be constant in the case of linear chromatography. Width 13

14 starting zone on the plate should be as minimal as possible, for TLC 2 3 mm, for HPTLC 1 mm. To apply samples, glass or platinum-iridium capillaries, micropipettes, syringes, and special dosing devices are used. For TLC, sample volumes are 0.5–3.0 μl, for HPTLC ~ 200 nl. To maintain the activity of the adsorbent layer, it is recommended to cover the adsorbent above the application line with a glass plate during sample application and apply the sample as quickly as possible. When carrying out identification, this procedure is most simply performed if the substances being separated have their own color. Identification of uncolored compounds can be carried out using specific chemical reagents or instrumental methods. Identification by recording the absorption of substances in the UV region or their own fluorescence is based on the introduction into the sorbent layer of fluorescent indicators (luminophores), which, when irradiated with UV light, are excited at a wavelength at which the detected substances absorb. They become clearly visible in the form of dark zones on the greenish luminous background of the sorbent. When detecting using chemical reagents, universal reagents are used ( sulfuric acid, KMnO 4, K 2 Cr 2 O 7, phosphomolybdic acid (PMA)) and specific compounds of individual classes. Thus, ninhydrin is used to visualize amino groups, iron (III) chloride for phenols, and complexing reagents for visualizing metal ions. Sprayers are used to spray the plates. In this case, the accuracy of quantitative determinations depends on the quality of detection. After visualization of the separated substances, the chromatograms are processed. Basic characteristics of the separation of substances in plane chromatography Sorption properties systems in TLC are characterized by the relative speed of movement (chromatographic mobility) R f, which is calculated from experimental data using the equation: l Rf =, (7) L where l is the distance from the starting line to the center of the zone: L is the distance traveled by the solvent during the same time. The most common approach to qualitative analysis is based on R f values. Chromatographic mobility is a sensitive characteristic of a substance, but it significantly depends on the determination conditions. This difficulty is overcome by conducting the experiment under strictly fixed standard conditions, which regulate the size of the plates, the thickness of the sorbent layer, the sample volume, and the length of the solution front path.

15 reader and other factors. If standard conditions are met, reproducible R f values ​​are obtained, which can be used for analytical purposes when compared with tabulated values, if they are obtained under the same experimental conditions. The most reliable is the bystander method, when individual substances corresponding to the expected components of the mixture are applied to the starting line next to the sample. The influence of various factors on all substances will be the same, therefore the coincidence of the R f component of the sample and one of the witnesses provides grounds for identifying substances, taking into account possible overlaps. A discrepancy in R f is interpreted more unambiguously: it indicates the absence of the corresponding component in the sample. Within the meaning of the definition, R f as a property characteristic of a given system should not depend on concentration and other factors. Experience shows, however, that reproducibility and consistency of R f values ​​are not always sufficient, especially when analyzing inorganic ions. R f is affected by the quality and activity of the sorbent, its humidity, layer thickness, solvent quality and other factors that are not always sufficiently controllable. In practice, the relative value relative mobility R f, rel is often used: R f, rel R f, x =, (8) R where R f, x and R f, st are the mobility of the analyte and standard substances, respectively. A standard substance (witness) in the same solvent is applied to the starting line next to the sample being analyzed and is thus chromatographed under the same conditions. As in other variants of chromatography, the separation efficiency in TLC is determined by the number of theoretical plates (N) and the height equivalent to the theoretical HETT plate (H), which can be calculated using the equations: 2 l I N = 16 w H f, st LR = 16 w f 2 , (9) 2 L w = =, (10) N 16 R L where w is the width of the zone in the direction of movement of the eluent. The H value characterizes the blurring of the chromatographic zone, N the efficiency of the chromatographic plate. f 15

16 sorbent is minimal, therefore, the concentration of the substance will be maximum and the sensitivity of the analysis will increase. Reducing the grain diameter in a thin layer leads to an increase in the duration of the analysis and increases diffuse erosion. Quantitative determinations in TLC can be made either directly on the plate or after removing the substance from the plate. In a direct determination, the spot area is measured using one method or another on a plate (for example, using millimeter tracing paper) and the amount of the substance is found using a pre-constructed calibration graph. Direct spectrophotometry of the plate using photodensitometers is also used. For quantitative calculations, also the prevalence of the eluent finish w 2 Δ X L w 1 start line l The resolution RS (resolution) of two chromatographic zones is determined by the distance between their centers (ΔХ), related to the arithmetic mean of their widths (w 1) and (w 2) (Fig. 1.4): R S 2ΔX = w + w 1 2. (11) The separation coefficient in a thin layer K f is related to the number of theoretical plates and mobilities R f by the equation K f R f, x1 R f, x2 = n, (12) R R f, x1 where R f, x1, R are the mobility of neighboring components of the mixture. f,x2 Theoretical analysis shows that with small values ​​of R and a decrease in the duration of the analysis, the zone of the substance is blurred by f,x1 Fig. Parameters of retention of substances in TLC f, x2 16

17 A calibration graph is constructed using the optical density at the center of the spot. The most accurate method is considered to be one in which the substance, after separation, is removed from the plate and analyzed by spectrophotometric or other method. Removal of the substance from the plate is usually done mechanically, although sometimes washing with a suitable solvent is used. 17

18 2. SPECTRAL METHODS OF ANALYSIS Among physical methods in the study of organic compounds, along with chromatographic ones, spectral methods are most widely used. The greatest information can be obtained by studying the interactions of matter with electromagnetic radiation in a wide range of frequencies, starting with radio waves and ending with γ-rays. In this case, a change in the energy of the molecules occurs, which is determined by the relation Δ E = E1 E2 = hν, (13) where Δ E is the change in the energy of the system; 1 2 energy of the system in various states; h Planck's constant; ν radiation frequency. When a molecule is placed in an electromagnetic field, absorption occurs only if Bohr's condition (13) is satisfied. When transitioning from state E 1 to E 2, the molecule absorbs energy; when returning from state E 2 to E 1, it emits it with the same frequency. The electromagnetic spectrum covers a huge range of wavelengths or energies. The main spectral regions used in spectral analysis: Wavelength interval Spectrum section, 1 nm, or m γ-radiation nm, or m X-ray radiation nm, or m Ultraviolet radiation nm, or m Visible light nm, or 7, m Infrared radiation m Microwaves, or microwave λ > 1 m Radio waves 1 nm = 10 9 m. Molecular spectral analysis involves qualitative and quantitative determination of the composition of the sample using absorption and emission spectra. The energy of a molecule, to a first approximation, can be divided into three components associated with the rotation of the molecules as a whole, the vibrations of the atoms that form the molecule, and the movement of electrons in the molecule. Molecular spectra are very complex, located in different wavelength (frequency) regions and are divided into electronic vibrational, vibrational-rotational and rotational. They are usually located in the area of ​​cm 1 (0.10-1.25 microns); , cm 1 (1.25 40 µm); 2, cm 1 (μm), respectively, and characteristic 18

19 include electronic transitions in molecules, as well as vibrational transitions with a change in the vibrational and rotational states of the molecule. Molecular absorption spectroscopy methods are based on measuring the decrease in the intensity of electromagnetic radiation passing through the analyzed sample. Depending on the wavelength of the incident light, spectrophotometry is distinguished in the ultraviolet (UV), visible (visible) and infrared (IR) regions of electromagnetic radiation. Spectral parameters of the absorption band The absorption band (Fig. 2.1) is characterized by the following values: ν max frequency value at the maximum of the band ( characterizes the position of the band in the IR spectrum); I λ peak intensity (at maximum), i.e. the value corresponding to the maximum energy absorption, rel. units: ν 2 ν 1 Q = I(ν) Δν integral intensity corresponding to the area of ​​the figure limited by the absorption band within ν 1 ν 2, cm 1; Δν 1/2 half-width of the band (width of the absorption maximum at half the maximum height). I λ I 1/2 Δν1/2 ν1 νmax ν2 ν,cm -1 Fig Contour of the absorption band When the structure of the molecule changes in the spectrum, not only a shift in v max is observed, but also a change in the value Δν 1/2. Physical meaning spectral quantities: νmax frequency of light when transitioning from one level to another, cm 1; Q integrated intensity, 19

20 is proportional to the probability of a given transition. The larger Q, the more likely it is for electrons to transition from one level to another. The dependence of the intensity of light transmitted through a substance (with a certain wavelength) on the concentration of the substance in the sample (if the concentration of the substance is expressed by the number of moles in dm 3 (mol/l)) and the thickness of the layer is described by a mathematical expression established experimentally: di = -εcidl ( 14) or after integration from zero to l as I k λ lc λ = I 0 e λ, (15 a) formulated as Bouguer-Lambert Beer’s law, where I λ and I 0λ intensity of transmitted and incident radiation, rel. units; k λ absorption index at a given wavelength (absorbency of the substance); c molar concentration of the substance, mol/l; l thickness of the sample layer, cm. The subscript λ is usually omitted, assuming that determinations are made at a given wavelength. Writing expression (15) in logarithmic form, we obtain: ln(i o /I) = kcl. (15b) When switching to decimal logarithms, equation (15a) takes the form I = I εlc, (16) where ε is the light absorption index (molar extinction coefficient), calculated per unit concentration of the substance and per unit layer thickness (a constant independent of intensity incident light and the concentration of the substance, but depending on the wavelength of the incident light). The relationship between the constants k and ε is ε = 0.4343 k. Bouguer Lambert Beer's law, written in the form of equation (16), is inconvenient to use in analytical chemistry, since there is no convenient way to measure I and I 0 on the one hand, and the expression has a power-law dependence on the concentration of the substance. To take into account light losses due to reflection and scattering, compare the intensity of light transmitted through the test solution (I) with the intensity of light transmitted through a cuvette with a solvent (I 0). The ratio of the light flux passing through a substance to the flux incident on the substance I/I 0 is called transmittance (or simply transmittance): 20

21 T I = 100% (17) I 0 The value of the ratio of the radiation flux absorbed by a given substance to the radiation flux incident on it (I 0 I)/I 0 = 1 T is called the absorption coefficient (or absorption), and the inverse value logarithm of transmittance, optical density of the substance. Thus, A = log T /100 = log I / I0 = log I0/ I, (18a) A = εlc. (18b) When solutions obey the absorption law, it is observed directly linear dependence optical density on the concentration of the substance in solution at a constant value of l. This proportionality is strictly observed only for monochromatic radiation (at a certain wavelength). If concentration c is expressed by the number of molecules n in 1 dm 3, then the absorption index k is called the molecular index, referred to one molecule and denoted by γ-. If the concentration c is expressed by the number of gram-moles in 1 liter of solution, then the absorption coefficient k is called the molar absorption coefficient and is denoted by ε; its dimension is l cm 1 -mol 1. The relationship between the coefficients γ and ε is written as follows: γn = cε, ε/γ = n/c = 6, / or ε = γ, γ = l, ε. If a substance does not have a constant, precisely known composition and the molar mass cannot be accurately indicated for it, then in such cases it is customary to use the concentration C, which is expressed in mg/ml or in% (1 mg/ml 0.1%), then the absorption index k is called the specific absorption coefficient and is designated E. Its dimension is % 1 cm 1. The basic law of light absorption in this case should be written as A = ElC. (18c) The law of additivity is an important addition to Bouguer-Lambert Beer’s law. The essence of the law lies in the independence of the absorption of an individual substance from the presence of other substances that have their own absorption or are indifferent to electromagnetic radiation. The mathematical notation can be represented as follows: 21

22 A = ε (19) ilc. i To assess the degree of absorption of the analyte, the intensity of radiation transmitted through the test solution is compared with the intensity of radiation transmitted through the solution, the absorption of which is assumed to be zero for the reference solution. As reference solutions, a solvent is usually used, on the basis of which a solution is prepared containing all components, with the exception of the substance being determined. In this case, it is very important to maintain a constant composition of the solvent and avoid changing the position of the absorption maximum, as well as the molar absorption coefficient of the substance depending on the composition of the solution Molecular absorption spectroscopy in the visible and UV region of electromagnetic radiation Characteristics of spectrophotometric determination Absorption spectroscopy in the visible and UV regions is one of the most useful methods of quantitative analysis for chemists. The most important advantages of the spectrophotometric and photometric methods are the following. 1. Wide range of applications. Numerous inorganic and organic substances absorb in the visible and UV regions, which makes their quantitative determination possible. In addition, many non-absorbing compounds can be determined after they have been converted to absorbing compounds by appropriate chemical reaction. 2. High sensitivity. Molar absorption coefficients usually lie in the range; therefore, as a rule, it is possible to determine concentrations in the range M; lower limit sometimes it is possible to reach 10 6 or even 10 7 M by appropriate changes in the procedure. 3. Quite high selectivity. Under the right conditions, it is possible to find a range of wavelengths in which the analyte is the only absorbing component in the sample. Moreover, absorption band overlap can sometimes be eliminated by making additional measurements at other wavelengths. 4. High precision. The relative error when determining concentration by spectrophotometric and photometric methods usually lies in the range of 1–3%. Using special techniques, errors can often be reduced to a few tenths of a percent. 22

23 5. Simplicity and convenience. Spectrophotometric and photometric measurements on modern devices they are performed easily and quickly. Moreover, the method can often be automated to perform serial analyses. Therefore, absorption analysis is widely used for chemical determinations in the continuous monitoring of air and water pollution, as well as industrial processes. Optimal conditions for photometric determination. Choice of wavelength. It is recommended to measure optical density at the wavelength corresponding to the maximum absorption, since here the maximum change in optical density per unit concentration is observed, therefore, one can expect strict compliance with the Bouguer-Lambert Beer law and less error due to inaccuracy in reproducing the wavelength set on the device. If there are several bands in the spectrum, the choice is made on the most intense one, since working in the maximum region allows for greater detection sensitivity. Flat maxima are preferable, since in this case the error in establishing the wavelength is less affected than in the case of sharp or steeply decreasing sections of the curve. When choosing the optimal wavelength in photometric analysis, they are also guided by the greatest difference in absorption of the analytical form and the starting reagents (for colored compounds) (Fig. 2.2). Thickness of the light-absorbing layer. The Bouguer-Lambert Beer law equation shows that the greater the layer thickness (l), the greater the optical density, and, therefore, the greater, other things being equal, the detection sensitivity. However, in practice it is impossible to infinitely increase the layer thickness (l): losses due to light scattering increase, especially when working with solutions. Cuvettes with a layer thickness greater than five centimeters are not used for photometry. op op op Fig The principle of choosing the optimal wavelength for photometric determination: 1 absorption of the initial reagent; 2 absorption of analytical form 23

24 Optical density (or transmittance). The measuring devices of photometric instruments are designed in such a way that the absolute error T usually has a constant value over the entire range of T values. In Fig. 2.3 shows that for the same error T, the absolute error c increases significantly with increasing solution concentration (c 2 > c 1, although T 2 = T 1). The relative error c/c will decrease with increasing concentration and increase with increasing absolute error c. At what values ​​of T will the relative error s/s be minimal? It is mathematically shown that s/s is a function of the value of T (Fig. 2.4). The relative error in determining the concentration passes through a minimum at T = 0.398 (A = 0.435). Calculations and experiments have shown that measurements of solutions with A > 2.0 and A< 0,03, характеризуются большими погрешностями. Отсюда концентрация определяемого вещества должна быть такова, чтобы оптическая плотность раствора находилась в пределах 0,03 < А < 2,00. Например, концентрация определяется: c =. Если молярный коэффици- 0, 435 ε λ l ент поглощения равен 10 3, то при толщине светопоглощающего слоя l = 1 см 0435, 4 c = = 435, 10 М l ΔT 1 ΔT 2 Δc 1 Δc 2 Рис Зависимость Т от с 24

25 Δc/c Fig Dependence of the relative error on the transmission of the solution Photometric response. Many organic and inorganic substances absorb in the visible and UV regions, which makes their determination possible. In addition, many non-absorbing compounds can be determined after they have been converted into absorbing compounds by an appropriate (photometric) chemical reaction. Colored compounds in solution are obtained mainly as a result of oxidation-reduction and complexation reactions, which have the following requirements. 1. The analytical reagent must be introduced in sufficient quantity to convert all the analyte into the analytical form. 2. Only those reactions that proceed at high speed should be selected, therefore, the equilibrium state is achieved in a short time. 3. The compounds under study must be stable over time, insensitive to light and sufficiently intensely colored. 4. If the colored compound is complex, then it must have a constant composition and a small dissociation constant (i.e., be quite stable). To determine the optimal photometric conditions, each system requires a special physical and chemical study to establish the required pH of the solution, the concentration of the reagent, the stability of the resulting complex, the influence of competing reactions and the presence of foreign ions on the stability of complex ions, etc. Sensitivity of the method. In general, the sensitivity of photometric analysis is determined by the formula: c min = A min /ε l. Setting A min = 0.01, at which it is still possible to conduct analysis, and with l = 1 cm, ε = .398

26 (typical of many colored compounds) is obtained with min = 001, = M. l Quantitative analysis by absorption methods Calibration graph method. Based on the construction of a calibration graph in A c coordinates. To do this, at a certain wavelength, the optical densities of a series of standard solutions, as well as the analyzed solution, are measured, then the concentration of the substance with x is determined from the calibration graph. Typically, calibration graphs are a straight line from the origin. In case of deviations from the Bouguer-Lambert Beer law, that is, in case of violation of the linear dependence A(c), the number of points on the graph should be increased. However, a linear relationship increases the accuracy of the determination. The main limitations of the method are related to the difficulties of preparing standard solutions and taking into account the influence of the so-called third components, that is, components that are in the sample, are not themselves determined, but influence the result. Molar absorption coefficient method. If the average value ε λ is known in advance, determined for several standard solutions under completely identical conditions, then, knowing the thickness of the cuvette layer, the concentration Aλ can be calculated using the formula: c = x. ε λ l A ​​limitation of the method is the mandatory subordination of the system in the concentration range under study to Beer’s law. Additive method. This method is used when analyzing solutions of complex composition, since it automatically allows one to take into account the influence of third components. First, the optical density A x of the analyzed solution with concentration c x is determined. Then a known amount of the analyte component (with st) is added to the analyzed solution and the optical density A x+st is measured again. Since A x = εl with x and A x+st = εl (with x + with st), then A x c x =, A x+ st cx + cst A x cx = cst. (20) Ax+ st Ax The concentration of the analyte in the additive method can also be found from the graph in coordinates A x+st = f(with st) (Fig. 2.5). 26

27 Fig Determination of concentration by the addition method The graph represents a straight line, the extrapolation of which to the intersection with the abscissa axis gives a segment equal to -c x. Indeed, at A x+st = 0 from equation (20) with x = - c st. Determination of a mixture of light-absorbing substances. The spectrophotometric method allows the determination of several light-absorbing substances in one solution without prior separation. Of great practical importance is the analysis of a mixture of two colored substances in a special case of such a system. In accordance with the law of additivity of light absorption for such a mixture of substances, for example A and B, we can write: A λ = l(ε 1 A,λ c 1 A + εb,λ c 1 B), A λ = l(ε 2 A, λ c 2 A + εb,λ c 2 B). Solving this system of equations for l = 1 gives: Aλ ε 1 B,λ -A 2 λ ε 2 B,λ1 c A =, εa,λ ε 1 B,λ -ε 2 A,λ ε 2 B,λ1 Aλ ε 2 B,λ -A 1 λε 1 B,λ2 c A =. (21) ε ε -ε ε A,λ B,λ A,λ B,λ The wavelengths λ 1 and λ 2 at which optical density measurements should be carried out are selected according to the absorption spectra of substances A and B. The spectral regions are of particular interest , in which one of the substances does not absorb light, while the other has intense light absorption. If, for example, ε B,λ = 0, then instead of (21) we will have: A A ε A ε c = λ1 λ 2 Α, λ1 λ1 Α, λ 2 ; c =, A ε B ε ε Α, λ 1 Α, λ B, λ 1 2 This case is realized, for example, in the determination of phenylalanine and tryptophan. In the wavelength region of 279 nm, only tryptophan absorbs,

28 and it can be determined by the optical density of the solution at this wavelength. At 257 nm, both components absorb light. Method of differential photometry. Absorption spectroscopy is a difference spectroscopy, since the absorption of the solvent, reagents, impurities, cuvettes, etc. is always subtracted from the absorption of a solution. Differential spectroscopy is a method of determination when a solution of the analyte with a known concentration is used as a reference solution. With the differential measurement method, the device is zeroed using absorbing solutions with constant optical density. Depending on the adjustment method, a distinction is made between the high absorption method, the low absorption method and the extreme precision method. In essence, the differential measurement method comes down to stretching the measuring scale of the device. In the high absorption method, adjustment to 100% transmittance is carried out using a reference solution with a lower concentration than the one being tested. This method makes it possible to measure the transmission of highly absorbing solutions and thus determine relatively large concentrations of substances. But in such cases, highly concentrated solutions often do not obey the Bouguer-Lambert Beer law. Therefore, it is recommended to use a two-way differential method for measuring optical density when constructing a calibration graph, choosing not the first solution of a series of standards as a reference solution, but the one for which the product εc is maximum. In the low absorption method, the device is first set to zero, but instead of a shutter, a solution with a higher concentration than the test solution is used. The method is applicable for solutions with optical density less than 0.1. In the extreme accuracy method, adjustment to T = 0 and T = 100% is carried out using two solutions. The concentration in one of them is greater, and in the other less, than in the test solution. With the differential measurement method, the reproducibility of measurements increases. Infrared spectroscopy Some characteristics of molecular spectra If a molecule absorbs or emits relatively small energy quanta (one or two orders of magnitude less than for excitation electronic spectrum), the vibrational spectrum of the molecule is observed. Change in the dipole moment of the molecule at the moment of excitation of the vibration - 28

29 body state is a necessary condition absorption or emission of energy. The presence of dipole moment changes during vibration depends on the symmetry of the system. In a diatomic molecule, the only possible vibration is the movement of atoms along the bond axis. In molecules such as O 2, C1 2, etc., the dipole moment is zero; vibrations of these molecules are not accompanied by absorption of IR radiation. Such vibrations are called inactive in the IR spectrum. In molecules such as CO, HC1 and others, the centers of positive and negative atoms do not always coincide, therefore the electronic distribution changes when absorbing infrared radiation, which leads to a change in the dipole moment of the molecule. Such vibrations are called active in the IR region. They can interact with electromagnetic radiation, absorbing energy and leading to the appearance of an absorption band in the spectrum. 1 2 Fig Vibrations of triatomic molecules: a symmetrical stretching vibrations in nonlinear (1) and linear (2) molecules (ν s); b asymmetric vibrations in nonlinear (1) and linear (2) molecules (ν as); c bending vibrations in a nonlinear molecule (δ); d degenerate vibration in a linear molecule Infrared radiation imparts to the molecule, which is in the ground (lowest) electronic state, the energy necessary for transitions between rotational and vibrational energy levels. When a molecule absorbs a particular energy quantum, light of a certain (characteristic) frequency is absorbed, which is usually associated with functional groups and atoms in the molecule. The beam passing through the sample is attenuated in the absorption region. By recording the intensity of the transmitted radiation, a curve is obtained on which absorption maxima are visible. The vibrational spectra of molecules are rich in bands, each of which corresponds to the excitation of a vibrational state of a certain 29

Lecture 6 Chromatographic methods of analysis Lecture plan 1. Concepts and terms of chromatography. 2. Classification of chromatographic methods of analysis. Chromatographic equipment. 3. Types of chromatography: gas,

MINISTRY OF HEALTH OF THE RUSSIAN FEDERATION GENERAL PHARMACOPOEIAL ARTICLE Spectrophotometry in OFS.1.2.1.1.0003.15 ultraviolet and Instead of OFS GF X, OFS GF XI, visible areas OFS 42-0042-07 GF XII,

MINISTRY OF HEALTH OF THE RUSSIAN FEDERATION GENERAL PHARMACOPOEIAL ARTICLE Thin layer chromatography OFS.1.2.1.2.0003.15 Instead of Art. State Fund XI, issue 1 Chromatographic process occurring during movement

Discovery of chromatography (1903) MIKHAIL SEMENOVICH TSVET (1872-1919) Main stages in the development of chromatography 1903 Discovery of chromatography (Tsvet M.S.) 1938 Thin layer or planar chromatography (Izmailov

PRACTICAL LESSON 6 on the discipline PHYSICAL AND CHEMICAL METHODS OF ANALYSIS OF NUCLEAR MATERIALS SPECTROPHOTOMETRY Photocolorimetric analysis (molecular absorption spectroscopy) refers to optical

Physico-chemical analysis Photometric analysis Optical analysis methods Atomic adsorption analysis based on the absorption of light energy by atoms of the analyzed substances. Molecular adsorption

8. Questions 1. Define chromatography. 2. What features of chromatography make it possible to achieve better separation of substances with similar properties compared to other separation methods. 3. List

LECTURE 7 CHROMATOGRAPHY AS A METHOD OF SEPARATION, IDENTIFICATION AND QUANTIFICATION Basic concepts and definitions Various classifications of chromatographic methods Chemisorption chromatography

MINISTRY OF HEALTH OF THE RUSSIAN FEDERATION GENERAL PHARMACOPOEIAL MOUNTING Chromatography on paper OFS.1.2.1.2.0002.15 Instead of Art. State Fund XI, issue 1 Chromatographic process occurring on a filter sheet

MINISTRY OF HEALTH OF THE RUSSIAN FEDERATION GENERAL PHARMACOPOEIAN ARTICLE Gas chromatography GPM.1.2.1.2.0004.15 Instead of Art. State Fund XI Gas chromatography is a method for separating volatile compounds based

FEDERAL AGENCY FOR EDUCATION State educational institution higher vocational education"Ural State University named after. A.M. Gorky" IONC "Ecology and Nature Management"

general characteristics and classification of instrumental analysis methods Instrumental analysis methods are based on the dependence physical properties substance from its nature, and the analytical signal represents

Lecture 3. Absorption spectroscopy. Photocolorimetry and spectrophotometry. Spectral methods of analysis and research are based on interaction electromagnetic waves with substance. Radiation is directed

ANALYSIS OF THE ABSORPTION SPECTRUM OF A COLORED SUBSTANCE Levin S.S. Kuban State University of Technology Krasnodar, Russia The property of molecules and atoms to absorb light of a certain wavelength, characteristic

Laboratory work 7b Chromatographic determination of the composition of the gas phase of soils. Chromatography (from Greek chroma, genitive case chromatos color, paint) - a physicochemical method of separation and analysis

1. Explanatory note 1.1. Requirements for students The student must have the following initial competencies: basic mathematical and natural sciences; possess independent skills

Gas chromatography 1 Requirements for substances 1. Volatility 2. Thermal stability (the substance must evaporate without decomposition) 3. Inertness Gas chromatograph diagram 1 2 3 4 5 1. Cylinder with carrier gas

DETERMINATION OF AMMONIUM CONTENT IN WATER. Why do you need to know the ammonium content in drinking water and pool water? The presence of ammonium ion indicates the presence of organic matter of animal origin in the water.

Spectrometry in the infrared region OFS.1.2.1.1.0002.15 Instead of GFKh Instead of Art. GF XI, issue 1 Instead of GF XII, part 1, OFS 42-0043-07 Infrared spectra (vibrational spectra) (IR spectra) arise due to

Moscow Institute of Physics and Technology ( State University) Department of Molecular and Biological Physics Physical research methods Lecture 9 Liquid chromatography Methods and technology.

Physicochemical methods of analysis Chromatography The chromatography method is based on the phenomenon of sorption Sorption is the process of absorption of gases, vapors and dissolved substances by solid or liquid sorbents Types

2 Methods of analysis: 1. Chemical methods. Chemical equilibrium and its use in analysis. Acid-base balance. The strength of acids and bases, patterns of their changes. Hammett function. Calculation

MINISTRY OF EDUCATION AND SCIENCE OF THE RUSSIAN NATIONAL RESEARCH TOMSK STATE UNIVERSITY FACULTY OF CHEMISTRY Annotated work program of the discipline Chromatographic methods of analysis Direction of training

46. ​​CHROMATOGRAPHIC SEPARATION METHODS Chromatographic methods are multi-stage separation methods in which the components of the sample are distributed between two phases, stationary and mobile. motionless

BELARUSIAN STATE UNIVERSITY FACULTY OF CHEMISTRY DEPARTMENT OF ANALYTICAL CHEMISTRY PROGRAM SPECIAL COURSE “CHROMATOGRAPHIC ANALYSIS” FOR STUDENTS OF THE 5TH COURSE OF THE SPECIALTY

Physico-chemical analysis Physico-chemical methods of analysis Physico-chemical methods of analysis (PCMA) are based on the dependence of the physical properties of a substance on its nature, and the analytical signal represents

ANNOTATION work program academic discipline“Introduction to chromatographic methods of analysis” in the field of preparation 04.03.01 Chemistry in the training profile “Analytical chemistry” 1. Goals of mastering the discipline

FEDERAL STATE BUDGETARY EDUCATIONAL INSTITUTION OF HIGHER EDUCATION "AMUR STATE MEDICAL ACADEMY" OF THE MINISTRY OF HEALTH OF THE RUSSIAN FEDERATION LIGHT ABSORPTION.

THE CONCEPT OF AN ANALYTICAL SIGNAL Information about the qualitative and quantitative composition The analytical chemist receives the analyzed object from the analytical signal. Analytical signal average of results

01/2016:20224 2.2.24. ABSORPTION SPECTROPHOTOMETRY IN THE INFRARED REGION Infrared spectrophotometers are used to record spectra in the region from 4000 cm -1 to 650 cm -1 (from 2.5 μm to 15.4 μm), and

MINISTRY OF HEALTH OF THE RUSSIAN FEDERATION PHARMACOPOEIAL ARTICLE Ketorolac trometamol FS.2.1.0022.15 Ketorolac trometamol Ketorolacum trometamolum Instead of GF XII, part 1, FS 42-0242-07 (1RS)-5-Benzoyl-2,3-dihydro- 1H-pyrrolysine-1 -carboxylate

CONTENTS Preface.................................................. 6 List of symbols and abbreviations.... ............... 9 Chapter 1 Atomic emission analysis.................................. 11 Physical Basics atomic

MINISTRY OF HEALTH OF THE RUSSIAN FEDERATION GENERAL PHARMACOPOEIAL ARTICLE Electrophoresis OFS.1.2.1.0021.15 Instead of Art. State Fund XI, issue 1 Electrophoresis is an analysis method based on the ability of charged particles to

Analytical chemistry 4th semester, Lecture 17. Module 3. Chromatography and other methods of analysis. Chromatography. Principle and classification of methods. 1. The principle of chromatographic separation. Stationary and mobile

MINISTRY OF HEALTH OF THE RUSSIAN FEDERATION GENERAL PHARMACOPOEIAL ARTICLE Raman spectrometry OFS.1.2.1.1.0009.15 Introduced for the first time Raman spectrometry is rapid (1 2 s) and non-destructive

Physicochemical methods of analysis 1 Physicochemical methods of analysis 2 Spectral Type of disturbance energy Electromagnetic radiation Measured property Wavelength and spectral line intensity in

FEDERAL AGENCY FOR EDUCATION STATE EDUCATIONAL INSTITUTION OF HIGHER PROFESSIONAL EDUCATION “SARATOV STATE UNIVERSITY named after. N.G. CHERNYSHEVSKY" V.I. Kochubey DEFINITION

Moscow Institute of Physics and Technology Department of Molecular and Biological Physics Physical research methods Lecture 9 Gas chromatography Techniques and experimental methods Dolgoprudny, April 3

Ministry of Education and Science Russian Federation FEDERAL STATE BUDGET EDUCATIONAL INSTITUTION OF HIGHER EDUCATION “SARATOV NATIONAL RESEARCH STATE UNIVERSITY”

1. List of competencies indicating the stages (levels) of their formation. PC-1: ability to use knowledge of theoretical, methodological, procedural and organizational foundations of forensic examination and criminology

04.07 Moscow Institute of Physics and Technology Department of Molecular and Biological Physics Physical research methods Lecture 8 Chromatography Dolgoprudny, April 6, 07 Plan. History of origin

Analytical methods for studying the state of the environment 1. The purpose and objectives of the discipline The goal of mastering the discipline “Analytical methods for studying the state of the environment” is to master the basics

Vodyankin Aleksey Yuryevich Department of ChTRE Physicochemical methods of analysis Method of analysis A fairly universal and theoretically based method for determining the composition regardless of the component being determined

Training program compiled on the basis of the educational standard OSVO 1-31 05 01 2013 and the curriculum of the educational institution G 31 153/school. 2013 COMPILER: V.A. Vinarsky, associate professor, candidate chemical sciences, Associate Professor RECOMMENDED

Work 4.20 Study of light absorption by solid and liquid bodies Equipment: photoelectric colorimeter-nephelometer FEK-60, a set of solid samples, a set of cuvettes with solutions of different concentrations.

Scientific and technological company SINTECO METHOD OF QUANTITATIVE CHEMICAL ANALYSIS OF COFFEE AND TEA FOR CAFFEINE CONTENT USING LIQUID CHROMATOGRAPHY METHOD. DZERZHINSK 1997 1 This document is distributed

PRACTICAL LESSONS 8 in the discipline PHYSICAL AND CHEMICAL METHODS OF ANALYSIS OF NUCLEAR MATERIALS LUMINESCENCE ANALYSIS 1 Luminescence intensity and phosphor concentration. If the luminescence intensity

Lecture 5 Electron spectroscopy. Spectroscopy in the visible and ultraviolet (UV) regions Lecture plan 1. Probabilities of transitions between electronic-vibronic-rotational states. Franck-Condon principle.

Methods for studying the composition of oils, gases and gas condensates Lecture 7 Existing methods studies of oils and non-products can be divided into: General methods analysis of oils and petroleum products: A) technical methods

Validation of analytical methods: practical application. Pisarev V.V., Ph.D., MBA, Deputy General Director of the Federal State Unitary Enterprise "State Scientific Center for Antibiotics", Moscow (www.pisarev.ru) Introduction

Moscow Institute of Physics and Technology (State University) Department of Molecular and Biological Physics Physical research methods Lecture 8 Detectors in chromatography Liquid chromatography

GOST R 51435-99 Apple juice, concentrated apple juice and drinks containing apple juice. Method for determining patulin content using high-performance liquid chromatography. OKS 67.160.20

Lecture 14 Interaction of light with matter Today: Tuesday, November 12, 2013 Lecture content: Dispersion of light Group velocity Elementary theory of dispersion Absorption of light Scattering of light 1. Dispersion

Dispersion of light. Thermal radiation Lecture 7 Postnikova Ekaterina Ivanovna Associate Professor of the Department of Experimental Physics Dispersion of light Dispersion of light dependence of the phase speed of light c (refractive index

Benefit from Agilent AdvanceBio SEC Size Exclusion Chromatography Columns for Biopharmaceutical Analysis Compare Columns from Multiple Manufacturers for Improved Data Quality Technical Overview

MINISTRY OF HEALTH OF THE RUSSIAN FEDERATION GENERAL PHARMACOPOEIAL MOUNTING Chromatography OFS.1.2.1.2.0001.15 Instead of Art. SP XI, issue 1 Chromatography is a method of separating mixtures of substances based

ANALYTICAL CHEMISTRY UDC 543.544 ADSORPTION CHROMATOGRAPHY IN BIOGAS ANALYSIS 1999 M.V. Nikolaev Research Institute of Chemistry, UNN. N.I. Lobachevsky L.P. Prokhorov Nizhny Novgorod aeration station Methodology developed

Methods for analyzing organic medicinal substances differ from methods for analyzing inorganic medicinal substances and have their own characteristics. Unlike inorganic compounds, most organic compounds are not electrolytes, so ionic reactions are not applicable to them. The exceptions are: organic acids and their salts (a):

And mineral acids, which dissociate into ions (b):

While the reactions between inorganic compounds, for the most part, occur instantly due to the exchange between ions; reactions of organic substances, as a rule, proceed slowly and can often be stopped at the formation of intermediate products, i.e., a whole series of transformations can be observed between the initial and final results. At the same time, all organic compounds are more or less unstable at high temperatures; when heated strongly, they burn completely.

In order to establish whether a given substance belongs to organic compounds. It is necessary, first of all, to discover the presence of carbon in it. Sometimes this does not present any difficulties, since many organic substances become carbonized when calcined, that is, they turn into coal, and thereby confirm the presence of carbon. But in a number of cases, organic substances do not become carbonized when ignited. For example, if you heat alcohol, it can evaporate, and if it catches fire, it burns without a trace. Therefore, the most reliable way to open carbon in an organic compound is to burn this compound with some kind of oxidizing agent.

The composition of a molecule of an organic substance can include, in addition to carbon and hydrogen, other inorganic elements, often halogens - Cl. Vg, F, I

As can be seen from the above formulas, the halogen in the molecules of bromoisal, diiodotyrosine and fluorothane is bonded directly to carbon (covalent bond). Such compounds do not dissociate into ions and therefore it is impossible to determine the halogen in the molecule by the usual analytical reactions (for example, with a solution of silver nitrate).

In this case, to confirm the presence of a halogen in the molecule, it must be converted into an ionogenic state. For this purpose, organic matter must first be destroyed. This process is called mineralization, which is carried out in various ways: combustion, oxidation, heating with hydroxides, fusion with alkali metals, etc. As a result of mineralization, simple inorganic substances are formed in the form of hydrohalic acids or their salts (halides), which dissociate and can be discovered by the usual analytical reactions of the ionic type .

Among the products of mineralization of organic matter, CO 2 and H 2 O are required, which serve as an indicator of the organic nature of the substance.

In the analysis of organic medicinal substances, the determination of relevant physical and chemical indicators, which can serve not only for identification, but also to confirm the purity of medicinal substances, is of great importance.

For example, for solid substances one of the characteristic indicators is the melting point, for liquid substances - the boiling point, density, and refractive index.

These indicators are quite definite only for pure substances. .

If there is one or another impurity in a medicinal substance, the melting point of solid substances decreases, and liquid substances The boiling point increases during distillation.

The refractive index, being a constant value for a pure substance, can deviate greatly in the presence of impurities. However, determining these indicators for organic medicinal substances is not enough. They give only an approximate preliminary idea of ​​the purity of the drug substance. For the reliability of the analysis, it is necessary to carry out a chemical analysis along with the determination of physical and chemical indicators.

A characteristic feature of organic medicinal substances is the presence in their molecules of so-called functional groups, i.e., reactive atoms or groups of atoms determined by chemical reactions.

Functional groups determine the approach to the analysis of organic medicinal substances, since they determine the properties of substances, determine the nature of identification reactions and methods for the quantitative determination of a particular medicinal substance. Knowing the detection reactions of individual functional groups, you can consciously approach the analysis of any medicinal substance of organic nature that is complex in structure.

There are a lot of functional groups (about 100) and the molecules of most medicinal substances are polyfunctional in nature, that is, they simultaneously contain several functional groups in the molecule.

Control questions to secure:

1. What is the main difference between medicinal substances of organic nature and medicinal substances of inorganic nature?

2. What is the main feature of the analysis of organic drugs in contrast to inorganic ones?

3. What physical and chemical indicators are used to authenticate organic medicinal products?

Mandatory:

1. Glushchenko N.N., Pletneva T.V., Popkov V.A. Pharmaceutical chemistry. M.: Academy, 2004.- 384 p. With. 151-154

2. State Pharmacopoeia of the Russian Federation / Publishing house “Scientific Center for Expertise of Medicinal Products”, 2008.-704 pp.: ill.

Additional:

1. State Pharmacopoeia 11th edition, issue. 1-M: Medicine, 1987. - 336 p.

2. State Pharmacopoeia 11th edition, issue. 2-M: Medicine, 1989. - 400 p.

3. Belikov V. G. Pharmaceutical chemistry. – 3rd ed., M., MEDpress-inform-2009. 616 pp.: ill.

Electronic resources:

1. Pharmaceutical library [ Electronic resource].

URL:http://pharmchemlib.ucoz.ru/load/farmacevticheskaja_biblioteka/farmacevticheskaja_tekhnologija/9

2. Pharmaceutical abstracts - Pharmaceutical educational portal[Electronic resource]. URL: http://pharm-eferatiki.ru/pharmtechnology/

3. Computer support of the lecture. Disk 1CD-RW.