UV visible spectroscopy. Acceptance of the WRC for publication in the ebs spbgetu "leti". Study of UV spectra
GOU VPO Irkutsk State Medical University
ROSZDRAVA RF
Tyzhigirova V.V., Filippova S.Yu.
APPLICATION OF IR- and UV- SPECTROSCOPIC
METHODS IN PHARMACEUTICAL ANALYSIS
Pharmaceutical Chemistry Study Guide for Students
Faculty of Pharmacy
Senior Lecturer, Department of Pharmaceutical and Toxicological Chemistry, ISMU, Ph.D. Tyzhigirova V.V., Assistant of the Department of Pharmaceutical and Toxicological Chemistry, ISMU, Ph.D. Filippova S.Yu.
Reviewers:
Head Department of Pharmacognosy with a course of botany at ISMU, Doctor of Pharmaceutical Sciences, Professor Fedoseeva G.M., Professor of the Department of Chemical Technology at ISTU, Doctor of Chemistry Shaglaeva N.S.
Published by the decision of the CKMS ISMU (Minutes No. from) Introduction This manual has been prepared for students of the Faculty of Pharmacy in order to master the analysis of drugs by IR and UV spectroscopic methods.
Modern regulatory documents for the analysis of drugs suggest the widespread use of these methods. IR spectroscopy is the main method in the testing of medicinal substances for authenticity. UV spectrophotometry is used to assess the quality of both medicinal substances and preparations made from them in terms of authenticity, good quality and quantitative content. In addition, the method is widely used in assessing the quality of solid dosage forms in terms of "Dissolution" and "Uniformity of dosage".
The manual summarizes the basics of the methods, their capabilities and limitations. The material on the application of methods in the analysis of drugs for various purposes is presented. The material presented is accompanied by specific examples on the use of methods in pharmaceutical analysis. At the end of the manual for self-control of mastering the material, control questions, test tasks, situational tasks with explanations are given. A list of tasks for independent work of students and a standard for solving one of them are proposed.
The manual is compiled in accordance with the standard program in pharmaceutical chemistry (2001) and is intended for self-preparation of students for a cycle of studies on the analysis of drugs by spectrophotometric methods.
1. Characteristics of spectroscopic methods of analysis To spectroscopic methods of analysis include physical methods based on the interaction of electromagnetic radiation with matter.
Electromagnetic radiation has a dual nature: wave and corpuscular, therefore it can be characterized by wave and energy parameters. Wave parameters include:
wavelength - the distance traveled by the wave during one complete oscillation. Wavelengths are usually expressed in nanometers nm 110 m or in micrometers μm 110 m;
9 frequency - the number of times per second when the electromagnetic field reaches its maximum value. The frequency is measured in hertz;
wavenumber - the number of wavelengths that fit into a unit of length: 1. The wavenumber is measured in inverse centimeters cm 1.
The corpuscular nature of light is characterized by the energy of quanta of electromagnetic radiation. In SI units, energy is measured in joules.
is described by the Planck equation:
- change in the energy of the elementary system as a result of absorption of a photon with energy h;
c - speed of light (3 1010 cm s-1).
When light quanta are absorbed, the internal energy of the particle increases, which is made up of the energy of motion of electrons EE, the vibrational energy of atoms of the EV molecule and the energy of rotation.The value of these energies decreases in the order: EE EV ER, and their numerical values are related as: 103: 102: 1.
As can be seen from the presented relationship, depending on the magnitude of the energy of electromagnetic radiation in the molecule, various energy transitions are possible. If, in accordance with equation (1), we take into account that the wavelength and radiation energy are related inversely proportional, then certain areas can be distinguished in the electromagnetic spectrum (Table 1).
the corresponding processes of energy transitions The interaction of electromagnetic radiation with matter in the optical (ultraviolet, visible, infrared) region forms the basis of the spectrophotometric method, which is widely used in pharmaceutical analysis.
The absorption of electromagnetic radiation in the UV, visible and IR regions of the spectrum is quantitatively described by the Bouguer-LambertBer law, which expresses the dependence of the intensity of the monochromatic light flux passing through the layer of absorbing substance (I) on the intensity of the light flux incident on it (I concentration of the absorbing substance (with ), the thickness of the absorbing layer (L) and on the molar absorption coefficient (), which characterizes the absorbing substance:
To measure the degree of absorption of electromagnetic radiation, devices have been designed that make it possible to determine not the intensity of the electromagnetic flux, but its attenuation due to the absorption of the analyte. And to characterize the degree of absorption of electromagnetic radiation, such photometric quantities as transmission and optical density are introduced.
Transmission (T) is the ratio of the intensity of the light flux that has passed through the layer of absorbing substance to the intensity of the incident light flux:
Based on formulas (2) and (3), you can write:
The transmission ranges from 0 to 1 and is usually expressed as a percentage (%) from 0 to 100.
The inconvenience of calculations led to the introduction of another photometric quantity - optical density (D) as the decimal logarithm of the reciprocal of the transmission:
practically measured in the range from 0 to 2. Formula (5) clearly shows that the absorption of electromagnetic radiation by a substance does not depend on the intensity of the light flux, but depends on the nature of the substance and is directly proportional to the concentration of the substance and the thickness of the absorbing layer.
From the formula (5) it can be seen that on the basis of the measured optical density it is possible to calculate the absorption index by the formula:
Concentration (C) can be expressed in moles per 1 liter or in grams per 100 ml of solution and, depending on this, the molar or specific absorption index is calculated using formula (6):
- the molar absorption index is the optical density of a one-molar solution of a substance with an absorbing layer thickness of 10 mm.
optical density of 1% solution at the thickness of the absorbing layer, see.
The absorption coefficient in the UV region can reach high values (up to 105 L cm-1 mol-1). In the IR region, the value has insignificant values and is usually not determined.
3. Characteristics of spectrophotometers Regardless of the spectral region, instruments for measuring transmission or absorption consist of 5 main components:
1 - energy radiation source; 2 - a dispersing device that allows you to select a limited region of wavelengths; 3 - cuvettes for sample and solvent; 4 - detector converting radiation energy into a measured signal; 5 - signal indicator with a scale.
The source of radiation in the UV region is a hydrogen or deuterium lamp. In a hydrogen lamp, a hydrogen glow occurs during a discharge, and an almost continuous radiation appears in the 200 nm region.
Infrared radiation is received from an inert solid that has been electrically heated to a very high temperature. So, for example, a silicon carbide rod, called a globar, when heated to 1500 0 С between two electrodes emits energy in the range of 1 - 40 microns.
A monochromator is a dispersing device that decomposes radiation into its constituent waves of different lengths. The most versatile monochromators in the UV region are prisms made of quartz or glass. For IR spectroscopy, prisms made of halides of alkali or alkaline earth metals are used. A system of lenses, mirrors and slits is connected to the dispersing element, which directs radiation with the required wavelength from the monochromator to the detector of the device.
Detectors - in the UV region, photocells are usually used to convert light energy into electrical energy.
Infrared radiation is detected by the rise in temperature of the blackened material placed in the flow path.
The measuring scale of the spectrophotometer is graduated in percentage of transmission T (I 1 0 0) and in values of optical density D (log I), and the scale of wavelengths or wave numbers is in nanometers or inverse centimeters, respectively.
Spectrophotometers are a combination of the key components discussed above and vary in complexity and performance. Spectrophotometers are available in single and double beam types.
The most commonly used two-beam devices, in which the luminous flux is divided into two - the main and the comparison flux. With this method of measurement, most of the random noise from the source and detector is compensated for, which provides a lower determination error.
The fundamental difference between UV and IR spectrometers lies in the different arrangement of the cuvettes: between the dispersing device and the photodetector in UV spectrophotometers or between the radiation source and the dispersing device in IR spectrometers. This is due to the fact that in the UV region absorption can reach large values, which makes it possible to accurately measure the absorption of a monochromatic light flux. In the IR region, absorption takes on insignificant values, which complicates its direct measurement. Therefore, to register IR spectra, the so-called inverted design of devices is used, in which the entire radiation spectrum that has passed through the substance is recorded. Then the IR spectrum will have high transmission values in the entire region except for the region where absorption has occurred. Therefore, the scale of the recording device in IR spectrometers is calibrated for transmission. UV spectrophotometers are calibrated for both transmission and optical density.
4. Characteristics of absorption spectra The most important characteristic of electromagnetic radiation is its spectrum. The absorption spectra in the UV and IR regions are of different nature and are characterized as electronic and vibrational spectra, respectively.
If an organic molecule interacts with radiation in the UV region of the spectrum, then at a certain frequency a quantum of energy will be absorbed, accompanied by the transition of valence electrons from the ground to an excited level.
Therefore, the physical nature of absorption bands in the UV region is associated with electronic transitions: when a molecule absorbs electromagnetic radiation in the UV region, a transition occurs between the electronic levels of the molecule.
Different electronic transitions require different energies, so the absorption bands are located at different wavelengths.
The types of electronic transitions from the ground state from bonding and orbitals and from nonbonding n orbitals to an excited state to antibonding and orbitals are presented in Table 2.
Table 2. Types of electronic transitions The presence in the structure of single bonds (–C – C–) and isolated chromophore groups (–CH = N; –N = N–; –N = O, etc.) causes absorption in the far UV region ( 100-200 nm.). However, absorption in the far UV region (up to 200 nm) has no analytical value, since modern spectrophotometers operate in the spectral range starting from 180-200 nm. For the purposes of spectrophotometric analysis, electronic transitions of conjugated bonds are used. Conjugation of sublevels, the transitions of electrons on which require a much longer wavelength region of the spectrum and has a high intensity.
The position and intensity of absorption bands are greatly influenced by electron donor (-NH2, -OH, -SH) and electron-withdrawing (-N = O, -NO2, etc.) substituents, which play the role of auxochromes. They enter into p, and, conjugate with the -electronic system of the chromophore and cause a shift in the electron density in it, thereby reducing the energy of the corresponding transitions. The absorption bands are shifted to longer wavelengths (the so-called bathochromic effect). In addition, the delocalization of electrons increases the intensity of the absorption bands (the so-called hyperchromic effect of the substituent).
Thus, in the UV region, molecules that have in their structure conjugated chromophore groups absorb. The longer the conjugation system, the longer the wavelength region of the spectrum the substance absorbs.
The UV absorption spectrum is expressed as a graphical dependence of the optical density (D) or molar absorption coefficient () on the wavelength () of the incident light.
Instead of D or, their logarithms are often used. The wavelength can be expressed in different units - nm or microns. The construction of the spectrum in various coordinates will affect its nature, therefore, it requires regulation in regulatory documents.
The UV spectrum is characterized as electronic, but the excitation of electrons will change the energy of vibrational motion of atoms and the energy of rotational motion of the molecule, therefore, a number of lines appear in the spectrum, which merge to form broad absorption bands (Fig. 1).
The absorption bands in the UV spectrum, as a rule, are characterized by the location of the max and the intensity, expressed in terms of the specific absorption index (E1cm).
The absorption bands in the UV region have a tendency to broadening; therefore, UV spectra are not very selective. However, they provide reliable information on the presence of a system of conjugated bonds in the structure of the substance being determined.
a chromophore system including a double bond –C = C– conjugated with a carbonyl group –C = O, and the enol hydroxyl located at the end of the conjugation chain plays the role of an auxochrome.
the characteristic absorption maximum max = 243 nm and the value of the specific absorption index E1cm = 543, which are used to determine its authenticity.
Rice. 1. UV spectrum of 0.001% solution of ascorbic acid The bands associated with the excitation of vibrational energy levels are located in the spectral region from about 300 to 4000-5000 cm-1, which corresponds to the energy of infrared radiation quanta (3-60 kJ / mol).
The energy of infrared radiation is insufficient for the implementation of electronic transitions; under the influence of infrared radiation only vibrational and rotational transitions are possible.
As a result, the physical nature of the absorption bands in the IR region is associated with the vibrations of atoms in the molecule: when a molecule absorbs electromagnetic radiation in the IR region, a transition occurs between the vibrational energy levels of one electronic state. In this case, the rotational energy levels also change, therefore the IR spectra are vibrational - rotational.
oscillatory movements. Normal vibrations are usually subdivided into stretching vibrations, characterized by the movement of atoms along the bond axes, and deformation vibrations, at which the bond angles change, while the bond lengths practically do not change.
During normal vibration, all the nuclei of the molecule vibrate with the same frequency and phase, although the amplitudes of their vibrations can differ significantly. Therefore, in the normal vibrational state in a molecule, the centers of gravity of positive and negative charges coincide and, therefore, the molecule will be generally non-polar, although each chemical bond can be polarized.
Upon absorption of infrared radiation, the amplitude of atomic vibrations into vibrational quantum levels. In this case, the oscillatory process is accompanied by a general change in the dipole of the molecule.
Thus, in the IR region, molecules are absorbed, in which the electric moment of the dipole changes upon the excitation of the vibrational motions of the atoms.
The vibration frequency depends on the mass of atoms in the molecule and the forces acting between them. And the number of vibrational states of a molecule is largely determined by the number of atoms and, consequently, by the number of bonds formed by them.
The absorption spectrum in the IR region is expressed as a graphical dependence of the transmission (T) on the frequency (), expressed in reciprocal centimeters.
The IR spectrum is characterized by a series of closely spaced absorption bands, which are described by their position in the spectrum and relative intensities: strong, medium, weak (Fig. 2).
In the spectra, characteristic bands and the area of "fingerprints" are distinguished. The absorption bands corresponding to the vibrations of single bonds C – C, C – N, C – O fall into the range of 1300 - 400 cm – 1. As a result of the fact that the C, N, and O atoms are close in mass and are connected by bonds that are approximately the same in energy, the assignment of the bands to separate groups and bonds is impossible. However, the entire set of bands in this region of the spectrum is a characteristic of the nuclear skeleton of the molecule as a whole. This area is called the “fingerprint” area.
If the bonds and atomic masses in the atomic grouping differ greatly from the parameters of the rest of the molecule, then the vibrations are observed in a narrow frequency range and are manifested in the spectra of all compounds containing this grouping. Such vibrations are called characteristic (group), and they appear in the range 4000 - 1300 cm-1. Thus, vibrations of groups containing a light hydrogen atom (C – H, O – H, N – H, etc.) and vibrations of groups with multiple bonds (C = C, C = C, C = N, C = O , N = N, etc.). As can be seen, the characteristic vibrations correspond to the atoms that make up the functional groups. The position of the characteristic bands in the spectrum is practically independent of the carbon skeleton to which the group is bound and provides valuable information on the general structure of the molecule.
For structural analysis of substances by their vibrational spectra, there are special correlation tables.
Table 3. Characteristic absorption maxima of some Bond atoms with characteristic frequency IR spectra of even relatively simple compounds consist of a huge number of sharp maxima and minima. However, it is precisely this multitude of peaks that determines, in part, the specificity of the spectrum. Thus, in the IR spectrum of ascorbic acid (Fig. 2), an intense corresponding double C = C bond is observed; absorption band in the region of the unsaturated ring - lactone. In addition, a series of characteristic absorption bands is observed in the range of 3500 - 3200 cm 1, caused by stretching vibrations of alcohol and en-diol hydroxyl OH groups. In the area of "fingerprints" absorption bands are expressed, which characterize single C – C and C – O bonds.
Interpretation of IR spectra is rather complicated, therefore, the IR spectrum of a standard sample of ascorbic acid is obtained in parallel. The spectrum of the analyte must have coincidence of the absorption bands in position and relative intensities with the standard spectrum.
5. Preparation of a sample for photometric determinations by preparing a solution of the appropriate concentration. Since the spectrophotometric method is highly sensitive, solutions with a very low concentration of 10-6 - 10-8 g / ml are photometric.
To reduce the error at the stage of taking a micro sample, it is increased to macro, and then a dilution technique is used.
reasonable choice of solvent for spectrophotometric determinations. First of all, it must be transparent in the measured region of the spectrum, for which its transmission limit is taken into account (Table 4).
used in photometry solvents cause the ionization of the substance, which leads to a redistribution of the electron density in the conjugation chain and, consequently, a change in the pattern of the spectrum. With acid ionization, an additional lone pair of electrons appears in the molecule, which leads to intensity. Basic ionization (protonization) can often lead to the opposite effect, since the lone electron pair binds to the proton, which leads to a decrease in the effect of the substituent.
An illustrative example of the influence of the nature of the solvent on the spectrum pattern is the position of the absorption band in the spectrum of folic acid (max = 320 nm in an acid solution, max = 365 nm in an alkali solution). Folic acid has both acidic and basic functional groups in its structure, which allow the use of acid and alkali solutions as solvents for spectrophotometric determinations:
The largest bathochromic shift of the absorption band in the spectrum of folic acid is observed in a sodium hydroxide solution, since the dissolution of a substance in an alkali solution is accompanied by acid ionization. Moreover, the main contribution to the conjugation is made by the anionic oxygen atom at C 4 of the heterocyclic system - pterin.
The preparation of the analyzed sample in IR spectroscopy is associated with additional difficulties due to the fact that most solvents are not transparent in the IR region, and therefore the choice of a solvent requires special care. In this case, one should take into account not only its transparency in the infrared region of the spectrum, but also the possibility of influencing the absorbing system. So, for example, water is generally excluded, and not only because of strong absorption, but also due to the effect on the materials from which the cuvettes and the optical part of the devices are made. Of all the solvents, the most suitable are carbon tetrachloride and carbon disulfide, the use of which also has limitations: the first is used in the range up to 7.6 μm, the second in the range of 7.6 - 15 μm. To reduce the absorption of radiation by the solvent, it is necessary to use narrow cuvettes with a thickness of 0.1 - mm. At the same time, it is necessary to increase the concentration of solutions up to - 4.5%, so that the transmittance in measurements in the IR region takes optimal values.
The most frequently analyzed sample for IR spectrometry is prepared by obtaining tablets, when the analyzed sample is crushed, mixed with spectroscopically pure potassium bromide and pressed; or by obtaining a paste, when the test sample is triturated with vaseline or other mineral oil transparent in the IR region, and then the resulting paste is squeezed between two plates of sodium chloride.
6. Comparative characteristics of absorption methods The most important characteristics of any method, including photometric. are its sensitivity and accuracy.
Quantitatively, the sensitivity of spectrophotometric determinations can be characterized by the sensitivity coefficient S, which determines how much the optical density of a solution changes with a very small change in the concentration of the analyte.
Mathematically, it is expressed by the first derivative of optical density with respect to concentration:
Thus, the sensitivity is proportional to the molar absorption index, and the larger it is, the smaller the amount of substance, other things being equal, can be determined.
The molar absorption coefficient in the UV and visible regions of the spectrum is tens of times higher than in the IR range. The thickness of the absorbing layer used in measurements is 1 cm for the UV region of the spectrum and 0.5–5.0 cm for the visible region; for the IR region, see. Therefore, the sensitivity of photometric determination in the UV and visible ranges is much higher than in the IR range, and for the UV range it is 10-4-10-6 of the molar mass of the analyte.
The error in the spectrophotometric determination of the concentration (C) can be characterized by expressing it as a function of the optical density and the thickness of the absorbing layer:
Thus, the error in determining the concentration of C will be the smaller, the larger and l, which is typical for the UV and visible spectral regions.
Based on the foregoing, it follows that for the purposes of quantitative analysis, spectrophotometry in the UV and visible regions of the spectrum has advantages over IR spectroscopy. At the same time, as it was shown in the characterization of spectra, IR spectroscopy is a more selective and informative method and therefore is widely used for the purposes of qualitative analysis.
7. Application of spectrophotometry in pharmaceutical analysis IR spectroscopy in pharmaceutical analysis is most widely used to determine authenticity. This is due to the high specificity of the vibrational spectrum.
The identification of a medicinal substance can be carried out by comparing the IR spectrum of the investigated substance with the analogous spectrum of its standard sample or with the figure of the standard spectrum given in the pharmacopoeial monograph.
In practice, when interpreting the spectra, the position of the absorption bands and their intensity (strong, medium, weak) are determined.
It is recommended to start the comparison of IR spectra with an analysis of the characteristic bands, which usually show up well in the spectra, and only if they coincide, the low-frequency region is compared. The coincidence of the spectral curve of the test substance with the pattern of the standard spectrum indicates the identity of the two substances. The absence of bands in the spectrum of the investigated substance, which are observed in the spectrum of the standard sample, unambiguously indicates that these substances are different. The presence of a larger number of bands in the spectrum of the investigated substance, in comparison with the spectrum of the standard, can be explained both by the contamination of the investigated substance and by the difference between the two substances.
Thus, the IR spectrum of the test sample should have complete coincidence of the absorption bands with the absorption bands of the standard spectrum in position and relative intensity.
pharmaceutical analysis can consider the IR spectra of structurally similar steroid compounds: cortisone acetate, hydrocortisone acetate and prednisolone (Fig. 6 - 8).
The most typical for all three substances is the region 1600 - cm 1, which contains stretching vibrations of the C = C group at C 4 of medium intensity (1606 - 1626 cm 1), stretching vibrations of the C = O groups at C 3 and C 11 (1656 - 1684 cm 1), groups C = O at C 20 (1706 - 1733 cm 1). All spectra exhibit maxima in the region from 3200 to 3500 cm 1, which correspond to vibrations of the free hydroxyl group.
Cortisone acetate and hydrocortisone acetate are esters, which manifests itself in their IR spectra in the form of characteristic bands in the region of 1219 - 1279 cm 1. These absorption bands are absent in the spectrum of prednisolone. But for the IR spectrum of prednisolone, as 3-keto-1, pregnadiene, there is a band of strong intensity of stretching vibrations of the C = C bond at C 1 (1595 cm 1).
identification of steroids of similar structure by the position of the main bands in the spectrum and their relative intensities.
In pharmaceutical analysis, for the purpose of quantifying difficulties in achieving comparable accuracy. These include the need to measure in a very narrow cuvette, the length of which is difficult to reproduce; high probability of overlapping absorption bands; a small width of the absorption band at the maximum, which leads to deviations from the basic law of light absorption.
round of steroid compounds. So, in fig. 6 - 8 show the IR spectra of cortisone acetate, hydrocortisone acetate and prednisolone.
7.2. Uses of UV Spectrophotometry in Analysis UV spectroscopy in pharmaceutical analysis is used for a variety of purposes.
structure, it is advisable to use UV spectrophotometry in order to use such spectral characteristics as the position and intensity of absorption bands.
Determination of authenticity by the UV spectrophotometric method can be carried out in various ways.
One of them is based on constructing a spectral curve and determining on it the characteristic, so-called analytical wavelengths at which the maximum (max), minimum (min) are observed, not strictly defined values of max and min are regulated, but their permissible intervals. This circumstance is explained by the permissible error in the calibration of the wavelength scale on various instruments.
Since the UV spectrum has one, two, rarely three broad bands to use. However, the conditions of determination (solvent, concentration of the working solution) are strictly regulated in the FS, and the spectral curve should be plotted in the coordinates or D, regulated in the FS.
absorption at a given analytical wavelength, expressed through the specific absorption index E 1%. The essence of the definition is to measure the optical density of the analyzed sample at max and is compared with the value of the specific absorption index, which, in turn, is determined by the standard sample for the analyzed medicinal substance and is given in the FS in the form of an acceptable interval.
spectrophotometry in order to determine the authenticity of substances having a system of conjugated bonds in the structure is mandatory, but due to its low selectivity it is considered as an additional method in the test unit. So, substances with the same type of system of conjugated bonds are characterized by absorption in the same region of the spectrum.
An illustrative example of this is the spectral characteristics of steroid compounds: prednisolone, cortisone acetate and hydrocortisone acetate (Fig. 3 - 5).
As seen from Fig. 3-5, in the structure of these substances there is a chromophore system of the same type, which arises as a result of conjugation of the carbonyl group at C 3 and a double bond at C 4. Therefore, these substances absorb in the same spectral region at wavelengths of 238 - 242 nm. In general, the absorption due to the chromophore system of 4-en-3-one bonds is analytical for steroid compounds and can be considered as a group-wide test for this class of substances.
Ergocalciferol and retinol acetate, belonging to the same group of alicyclic vitamins, differ in the number of conjugated double bonds and therefore absorb in different regions of the spectrum.
Ergocalciferol has in its structure a system of three conjugated double bonds. Conjugation of double -C = C- bonds causes the absorption of ergocalciferol at 265 nm with a specific index of 480 - 485.
Retinol acetate is also based on an alicyclic structure:
However, unlike ergocalciferol, retinol has a pentaene conjugation chain. An increase in the number of conjugated bonds leads to a decrease in the energy of electronic transitions and, as a consequence, to a shift of the absorption band to longer wavelengths with an increase in its intensity. Retinol acetate has a pronounced absorption maximum in the longer wavelength, compared to ergocalciferol, spectral region at 326 nm, and the specific absorption index takes on a value of 1550.
Photometric characteristics of other medicinal substances from the class of vitamins, alkaloids, steroid hormones and antibiotics used for analytical purposes are shown in Tables 5-9.
specific impurities in medicinal substances.
absorption (), the smaller the amount of substance can be determined.
The application of the method to determine the impurity is justified only in terms of the absorption coefficient. Such an impurity is called light-absorbing.
Determination of impurities by the spectrophotometric method is reduced to two cases. If an impurity absorbs in a spectral region different from the absorption region of a drug, then the presence of an impurity is judged by the appearance of an additional absorption band in the spectrum. An example of hydrotartrate:
Conjugation of the aromatic ring with two –OH groups located in the ortho – position relative to each other causes the absorption of epinephrine in the UV region at a wavelength of 279 nm. Adrenolone, a product of adrenaline oxidation, has a quinoid structure, which causes absorption in the longer wavelength region of the spectrum at 310 nm.
The impurity can absorb in the region of the spectrum characteristic of the drug. In this case, the presence of an impurity is judged by the increase in optical density at the analytical wavelength.
The use of this technique is possible subject to the law of additivity, according to which the optical density of the sum of substances is equal to the sum of the optical densities of individual substances, provided that these substances are independently absorbed: DADBDAB Since the absolute values of optical density are poorly reproduced, the relative value is determined - the ratio of optical densities at different analytical wavelength :.
For example, this technique is used to determine the absorbing impurities in cyanocobalamin. Determine the optical density of the drug solution at 278 nm, 361 nm and 548 nm.
Then the ratios of optical densities are calculated, which should be included in the intervals given in the FS:
Table 5. Photometric characteristics of some medicinal products Table.6. Photometric characteristics of steroid hormones acetate Table 7. Photometric characteristics of some phenylamines Table 8. Photometric characteristics of some medicinal Drotaverine hydrochloride 0.1 mol / l Table 9. Photometric characteristics of some medicinal Benzylpenicillin Water Nitrophenylalkylamine derivatives are widely used. The application of the method is based on the existence of a directly proportional dependence of the absorption value on the concentration of the substance in the analyzed solution:
spectrophotometric method:
graphic according to the calibration schedule;
comparative against a standard sample;
calculated according to the specific absorption index (E1% 1cm).
The first method is the most rational for serial analyzes. Its essence boils down to the following: a series of dilutions of a standard sample is prepared in the concentration range at which the Bouguer – Lambert – Beer law is observed. The optical density of the standard sample solutions is measured and a calibration graph is plotted. Then a solution of the analyzed sample is prepared at a concentration approximately corresponding to the middle of the calibration graph, and its optical density (DX) is measured on the same device, the CX value, g / ml is determined (Fig. 6).
Rice. 6. Calibration graph of the analyzed sample and the method of its dilution:
The second way to quantify is as follows:
in parallel, solutions of the analyzed and standard samples of approximately the same concentration (CX and SSO) are prepared and their optical density (DX and DCO) is measured under equal conditions (max, l.) In accordance with the basic law of light absorption, you can write:
Considering that and l are the same, combining both equations, we get:
DCO CCO DCO
Further, in the calculation formula, the size of the macros of the standard and analyzed samples and the method of their dilution are taken into account:This method is more accurate, therefore it is widely used when performing single analyzes.
If there are no standard samples in the laboratory, calculations in quantitative analysis can be made using the known value of E1% 1cm according to the formula:
However, this method of analysis is the least preferable, since in this case the role of errors due to the individual characteristics of the devices increases.
To ensure the required accuracy of the analysis, it is necessary to scientifically substantiate the conditions for quantitative determination.
Choice of analytical wavelength. For this purpose, a spectral curve of the dependence of the optical density on the wavelength of the standard sample solution is constructed. Analytical wavelengths corresponding to the wavelengths of maximum absorption are determined on the spectral curve. Of all the absorption bands available in the spectrum, for the purposes of quantitative analysis, the one that is characterized by light absorption provides the highest detection sensitivity. On the other hand, shallow maxima are more preferable, since in this case, the error in setting the wavelength is less affected.
obedience to the Bouguer – Lambert – Beer law. For this, a series of dilutions of the standard sample is prepared and the values of their optical densities are measured at the selected analytical wavelength. Based on the data obtained, a calibration graph is built - a graphical dependence of optical density on concentration. The absorption of electromagnetic radiation by a substance obeys the basic law of light absorption at the concentration range at which the graph is a straight line coming out of the origin (Fig. 7).
Rice. 7. Calibration graph Cn - Cm - concentration range, in which observance of the Bouguer – Lambert – Beer law is observed. The error of determination greatly increases in the absence of a direct proportional dependence of the absorption value on the concentration.
This can be clearly demonstrated using Figure 8.
Rice. 8. Calibration graph:
1 - if the Bouguer – Lambert – Beer law is observed, 2 - if the Bouguer – Lambert – Beer law is not observed. Lambert – Beer exceeds the error C1 when the law is fulfilled.
Selecting the working range of optical density (D). It was found that the relative error in measuring the optical density takes on minimum values at D = 0.434. Therefore, they try to work in the range of values of optical density from 0.3 to 0.8, in which the device is calibrated with the greatest accuracy. Since the optical density is directly proportional to the concentration of the substance in the analyzed sample and the thickness of the absorbing layer, it is these parameters that should be varied to select the optimal values of the optical density. At the same time, the concentration is chosen in such a way that its value fits within the interval at which the observance of the Bouguer – Lambert – Beer law is observed.
Selection of a standard sample (CO). Spectrophotometry is a relative method and, therefore, requires the use of reference materials, which can be state standard samples (GSO) or working standard samples (RSO). When performing the analysis of substances, the GSO is used, and when analyzing medicinal products, the use of the RSO is allowed.
Preparation of the analyzed sample. Measurement of UV absorption is carried out in solutions. Due to the high sensitivity of the spectrophotometric method, the working concentration of the CX solution has low values. Therefore, in the method of spectrophotometric determination, the scientifically grounded size of the macro weighed and the volumetric glassware used for its dilution should be regulated.
Selection of reference solution. Photometric determinations in any region of the spectrum assume the use of reference solutions - these are solvents or solutions containing all the components of the analyzed sample, except for the analyte. Photometric devices are designed in such a way that the use of cuvettes with a reference solution allows the optical density scale to be brought to zero and thereby level the absorption due to the walls of the cuvette, solvent and other reagents used to prepare the analyzed sample.
Due to its high sensitivity, UV spectrophotometry is widely used in dosage uniformity testing of solid dosage drugs. This test is mandatory when the active substance is contained in a dose of 0.05 g or less. Estimating this amount requires highly sensitive methods. One of them is UV spectrophotometry.
The high sensitivity of the method also makes it possible to estimate the amount of active substance released from the dosage form into the dissolving medium. Therefore, UV spectrophotometry is often used in the determination of the "Dissolution" test, adopted by the State Pharmacopoeia for solid drugs.
Thus, one of the advantages of UV spectrophotometry is its versatility, which makes it possible to use the method for solving various analytical problems.
1. The phenomenon underlying spectroscopic methods of analysis.
2. Classification of spectroscopic methods of analysis. The principle of classification.
3. The nature of absorption in the UV and IR regions of the spectrum.
4. The basic law of light absorption.
5. Basic photometric quantities.
6. Characteristics of the main units of spectrophotometers.
Fundamental difference between UV spectrophotometers and IR spectrometers.
7. Characteristics of absorption spectra in the UV and IR regions of the spectrum.
8. Comparative characteristics of the applicability of UV and IR spectroscopy for solving pharmaceutical problems.
9. Features of sample preparation for spectrophotometric determinations in the UV and IR regions of the spectrum.
10. Application of UV spectrophotometry to determine the authenticity of medicinal substances.
11. Possibilities of using UV spectrophotometry for the determination of impurities. Determination methods.
12. Application of UV spectrophotometry in quantitative analysis.
Choice of conditions for quantitation. Methods for calculating the analysis results.
13. Application of IR spectroscopy in pharmaceutical analysis.
1. The spectrophotometric method is based on a) selective absorption of electromagnetic radiation by the analyte b) emission of electromagnetic radiation by excited atoms or molecules c) reflection of electromagnetic radiation by the analyte 2. Absorption of electromagnetic radiation by a substance depends on a) the intensity of the light flux b) the nature of the substance c) the thickness of the absorbing layer d) the content of the substance in the analyzed solution 3. Set the correspondence to the electromagnetic radiation 4. The absorption spectrum 1) in the UV region is a) a graphical dependence of the optical density (D) or molar absorption coefficient () on the wavelength () of the incident light b) the graphical dependence of the transmission (T) on the frequency (), expressed in inverse centimeters 5. The picture of the spectrum 1) in the UV region depends on a) the mass of atoms and the forces acting between them b) the number of atoms and the number of bonds formed between them c) presence in the structure of the system of conjugated bonds 6. The absorption bands in the spectrum 1) in the UV region are characterized by a) the location of the analytical wavelengths max, min b) the position in the analytical range of the spectrum of the entire set of absorption bands c) the absorption intensity, expressed through the specific absorption index (E1cm) d) the relative intensity, characterized by as a small, medium and high degree 7. Set the correspondence 1) the area of 1300 - 400 cm 1 a) characteristics of the nuclear skeleton 2) the area of 4000 - 1300 cm 1 of the molecule as a whole 8. More selective and informative for the purposes of determining the authenticity of drugs is a) spectrophotometry in the UV-region b) spectrophotometry in the IR-region 9. Identification of a medicinal substance by IR-spectra can be carried out a) by coincidence of absorption bands and relative intensity with the spectrum of a standard sample b) by coincidence of absorption bands and relative intensity with the spectrum pattern, given in FS c) according to the position and intensity of the analytical wavelengths, reg lamented in FS 10. When testing for the authenticity of medicinal substances, the UV-spectrophotometric method is considered as a) main b) additional Determination of the authenticity of medicinal substances UV-11.
spectrophotometric method can be carried out a) according to the spectral curve b) according to the calibration graph c) according to the specific absorption index at analytical wavelength 12. The detection sensitivity is higher, and the measurement error of the absorption is less a) in the UV region b) in the IR region 13. In the quantitative analysis of medicinal substances, a) spectrophotometry in the UV region is used b) spectrophotometry in the IR region 14.
spectrophotometric determination assumes a) taking a macro portion of a medicinal substance, followed by its dissolution and dilution with an appropriate solvent using volumetric flasks b) rubbing the medicinal substance with liquid paraffin or other liquid and placing the resulting suspension between two plates of potassium bromide c) rubbing the medicinal substance with potassium bromide and subsequent pressing 15. The choice of the concentration of the analyte solution in UV spectrophotometric determinations is carried out a) according to the spectral curve b) according to the calibration graph c) based on the concentration of the standard solution 16. In the method for the quantitative determination of medicinal substances by the UV spectrophotometric method, a) the size of the macro portion b) volumetric glassware for diluting the weighed portion c) concentration of the analyte solution d) concentration of the standard solution or the method of its preparation e) analytical wavelength f) solution with Equations 17. In the longer-wavelength part of the spectrum, there are absorption bands
S NH N S NH N
18. It is possible to distinguish medicinal substances using the method a) spectrophotometry in the UV region b) spectrophotometry in the infrared region 19. For two derivatives of 5 - nitrofuran absorption bands in the UV region of the spectrum a) allow to distinguish medicinal substances b) do not allow to distinguish medicinal substances 20. The use of the UV spectrophotometric method in glucose analysis is justified for the purpose of a) determination of the authenticity of glucose b) determination of the impurity of hydroxymethylfurfural c) quantitative determination of glucose 21. For two drugs from the class of antibiotics, a) the “fingerprint” area in IR is more specific Spectrum b) characteristic absorption bands of the IR spectrum 2. b, c, d 9. a, b 10. b 11. a, c 12. a 13. a 14. a 15. b, c 16. a, b, d, e, f 17. b 18. b 19. a 20. b 21. b 1. The UV spectrum of a 0.002% solution of dibazol in 95% alcohol in the region from 225 nm to 300 nm has maxima at wavelengths of 244 ± 2 nm ;275 ± 1 nm; 281 ± 1 nm and minima at 230 ± 2 nm;
253 ± 2 nm; 279 ± 1 nm.
How to prepare an alcoholic solution of dibazol and obtain its spectrum?
2. The specific absorption index of furacilin in an alcohol solution at = 365 nm is 850 - 875. To determine the specific index, the analyst prepared a 0.0005% furacilin solution.
hydrochloric acid at = 243 nm has a specific absorption index E11cm = 542.5. To determine the indicator, the analyst prepared a 0.001% solution of ascorbic acid according to the following procedure: about 0.05 g (exact weighed) of ascorbic acid was placed in a 100 ml volumetric flask and dissolved in a 0.001 M solution of hydrochloric acid, brought the volume of the solution to the mark. 2 ml of the resulting solution was diluted with a solvent in a volumetric flask with a capacity of 100 ml, resulting in a 0.001% solution. Check the correctness of the calculation of the concentration of the solution and evaluate the method of preparation of the solution from the standpoint of metrology.
4. The analyst prepared a 0.001% solution of papaverine hydrochloride using a 0.1 M solution of hydrochloric acid as a solvent. Measured on the device the optical density of the prepared solution at = 310 nm in a cuvette with a layer thickness of 1 cm relative to the solvent. The optical density of the solution was D = 0.23. Then, using the formula, he calculated the specific absorption rate:
In accordance with ND, the specific indicator should be 211 - 220.
Based on the data obtained, the analyst made a conclusion about the non-compliance of the medicinal substance with the requirements of ND in terms of E11cm. Evaluate the analyst's actions.
analytical chemical reactions. With concentrated sulfuric acid, a bright yellow oxonium salt is obtained. The reaction with a solution of silver nitrate in nitric acid is confirmed by the melting point. When preparing a new draft FSP, it was decided to use the spectral characteristics of diphenhydramine instead of analytical reactions. The following change was made to the "Test for Authenticity" section: The UV spectrum of a 0.05% solution of diphenhydramine in 95% alcohol in the region from 230 nm to 280 nm has maxima at wavelengths of 253 ± 2 nm; 258 ± 2 nm; 264 ± 2 nm and wavelength minimums Is your decision correct?
6. When developing a new draft ND for acid ascorbic spectral characteristics of the substance, obtained by UV and IR spectroscopy.
spectroscopy and analytical chemical reactions. The IR spectrum of novocaine, obtained in tablets with potassium bromide in the range from 4000 to 600 cm 1, should have full coincidence of absorption bands with absorption bands of the attached spectrum.
Analytical chemical reactions confirm the presence of a primary aromatic amino group and a chlorine ion in the structure of novocaine.
novocaine for authenticity.
8. The admixture of adrenolone in the drug substance epinephrine hydrotartrate is determined spectrophotometrically. In hydrogen chloride at = 310 nm in a cuvette with a layer thickness of 10 mm should not exceed 0.2.
The analyst prepared a 0.2% solution of a medicinal substance and measured its optical density, observing the conditions specified in the ND. The optical density of the analyte was 0.26. When the analysis was repeated, similar results were obtained. Based on the data obtained, the analyst made a conclusion about the non-compliance of the medicinal substance with the requirements of the ND for the content of adrenolone impurity.
9. In the draft FSP for tablets of acetylsalicylic acid 0.5 g in the section "Test for authenticity", along with analytical reactions, spectral characteristics were included by the spectrophotometric method. The same method is recommended for Dissolution test determination and quantitative analysis.
10. The quantitative determination of the riboflavin substance, according to the FS, is carried out by the spectrophotometric method according to the method:
about 0.07 g of riboflavin (accurately weighed) is placed in a volumetric flask with a capacity of 500 ml, add 5 ml of water and mix until the sample is completely moistened. Add dropwise (not more than 5 ml) 1 M sodium hydroxide solution and stir until the sample is completely dissolved. Immediately add 100 ml of water and 2.5 ml of glacial acetic acid, stir and dilute the volume of the solution to the mark with water. Transfer 20 ml of this solution into a 200 ml volumetric flask, add 3.5 ml of 0.1 M sodium acetate solution and bring the volume of the solution to the mark with water. Measure the optical density of the resulting solution at = 444 nm in a cuvette with a layer thickness of 10 mm.
D is the optical density of the test solution;
a - weighed amount of riboflavin in g;
328 - specific absorption index at 444 nm.
riboflavin by specific absorption index. Check the correctness of the calculation of the sample.
Quantitative determination of 1% dibazol solution for 11.
injections are carried out in accordance with the ND spectrophotometric method according to the following procedure:
2 ml of the drug is placed in a volumetric flask with a capacity of 100 ml, the volume of the solution is brought up to the mark with 95% alcohol and mixed.
with a capacity of 50 ml, add 30 ml of 95% alcohol, 1 ml of 0.1 M sodium hydroxide solution, bring the volume of the solution with alcohol to the resulting solution on a spectrophotometer at = 244 nm in a cuvette with a layer thickness of 10 mm. Alcohol 95% is used as a reference solution. In parallel, the optical density of the standard sample solution (RSO) of dibazol is measured.
1 ml of PCO solution contains about 0.00002 g of dibazol.
Check the calculations of the weighed portion of the drug dibazol.
12.In accordance with the FSP, the quantitative determination of 20 mg picamilon tablets is carried out by the UV spectrophotometric method according to the following procedure: about 0.08 g (exact weighed portion) of the powder of crushed tablets is quantitatively transferred with water into a volumetric flask with a capacity of 500 ml, the volume of the solution is brought to the mark with water, stirred and filtered through a paper filter (red tape).
The optical density of the resulting solution is measured on a spectrophotometer at the absorption maximum at a wavelength of ± 2 nm in a cuvette with a layer thickness of 10 mm. In parallel, the optical density of a solution of a standard sample of picamilon is measured. Water is used as a reference solution.
Is the quantitation method correct?
1. To prepare a 0.002% alcohol solution of dibazol, dissolve 0.2 g of dibazol in a volumetric flask with a capacity of ml in 95% alcohol, bring the volume of the solution to the mark. You get a 0.2% solution, which must be diluted 100 times. For this, 1 ml of the prepared solution is placed in a volumetric flask with a capacity of ml and adjusted to the mark with alcohol.
Then the optical density of a 0.002% solution of dibazole in a cuvette with a layer thickness of 10 mm relative to the solvent in the region from 225 nm to 300 nm after 5 nm is measured on a spectrophotometer, and near the maxima and minima after 1 nm. The obtained values are used to construct a spectral curve of the dependence of the optical density (D) on the wavelength ().
The wavelengths corresponding to the maximum and minimum absorptions are marked on the spectral curve. They must correspond to the wavelengths given in the ND.
The task is greatly facilitated when working on modern spectrophotometers with an automatic device for recording spectra.
2. The specific absorption index is the absorption of a 1% solution with a layer thickness of 1 cm. This indicator is calculated by the formula:
E1 cm of furacilin is 850 - 875. This means that its 1% solution has an optical density D = 850 - 875. This solution density is almost impossible to measure on a spectrophotometer, since its scale is graduated from 0 to 2. Moreover, the smallest calibration error is in the area of 0.3 - 0.8. And the optical density optimal for measurement is D = 0.43. Therefore, prepare a test solution of such a concentration that its optical density is close to the value of 0.43.
Thus, the analyst's calculations are correct.
3. The concentration of the solution of ascorbic acid to determine the specific absorption index E11cm is calculated by the formula:
The analyst prepared a 0.001% solution instead of a 0.0008% solution. This is quite acceptable, since the prepared solution will have an optical density:
This density is included in the recommended measurement range of optical densities of 0.3 - 0.8. Consequently, the metrology analyst did not prepare the solution accurately enough, taking a sample of the substance equal to 0.05 g. To ensure the accuracy of weighing, it is better to take a sample of the substance as much as possible, in the extreme case, equal to 0.1 g.
From it, you should first prepare a 0.1% solution using a 100 ml volumetric flask, and then dilute it 100 times to obtain a solution with the required concentration of 0.001%. To do this, you can take 2 ml of a 0.1% solution and a volumetric flask with a capacity of 200 ml.
4. The analyst made an unfounded conclusion. He prepared a solution of low concentration (0.001%). When measuring the optical density (D) of such a solution, a significant error was made, since D = 0.23 does not correspond to the optimal value of D = 0.43.
The inaccuracy of measuring the optical density was reflected in the calculations of the specific absorption index.
prepare a new solution with a concentration of 0.002%, measure its absorption and only then make a conclusion.
5. The decision to establish the authenticity of diphenhydramine only on the basis of its UV spectrum is unreasonable.
The UV spectrum of diphenhydramine characterizes only aromatic rings in the structure of the substance:
Similar chromophore groups are found in a number of medicinal substances (ephedrine g / chl, atropine sulfate, etc.).
Therefore, the UV spectra of a substance do not provide reliable information about its authenticity.
The identity test must be supplemented with analytical chemical reactions confirming other structural fragments of diphenhydramine, in particular the ether bond and the chlorine ion.
spectrophotometric methods are rational in assessing the authenticity of diphenhydramine.
6. Methods of UV and IR spectroscopy ensure the reliability of testing ascorbic acid for authenticity. Therefore, analytical chemical reactions can be excluded. The accepted ND reactions, as a rule, confirm the presence of the ascorbic en-diol group in the structure of the acid, which determines the reducing properties of the substance.
However, the structure of the substance also contains primary and secondary alcohol groups, an internal ester group, which are not evaluated by a chemical method.
Only the IR spectrum of ascorbic acid provides complete information on the structure of a substance by the presence of characteristic absorption bands of enol and alcohol OH - groups, a double bond in the ring and a lactone group, as well as a set of absorption bands in the "fingerprint" region. Reliability is ensured by comparing the spectrum of ascorbic acid with the spectrum of its standard sample or spectrum pattern.
The UV spectrum of ascorbic acid reflects the presence of only conjugated double bonds in the structure. Therefore, UV spectroscopy is a complementary method, and IR spectroscopy is the main method in testing for authenticity.
Thus, the analyst's proposal on the use of a set of UV and IR spectroscopy methods in testing ascorbic acid for authenticity is justified.
7. The complex of testing novocaine for authenticity using IR spectroscopy and the chemical method is scientifically grounded and rational. IR spectroscopy is a specific method of functional analysis that makes it possible to detect all functional groups in the structure of novocaine: primary aromatic amino group, ester group, substituted ammonium cation, by the presence of characteristic absorption bands in the IR spectrum in the range of 3500 - 1300 cm 1. Skeletal vibration region ( below 1300 cm 1) is characterized by many absorption bands and is purely individual for novocaine.
An analytical reaction proves the presence of a chlorine ion, the azo dye is a group for aromatic amines and allows the drug to be classified as a local anesthetic.
8. When determining the impurity of adrenolone by the spectrophotometric method, it must be borne in mind that the method for determining the impurity is due to the fact that the absolute value of the optical density is poorly reproduced on different devices. Therefore, it is advisable to determine the ratio of optical densities at different wavelengths () and normalize the relative value, which is more or less constant and is better reproduced on different devices.
To make a reasonable conclusion about the content of adrenolone impurity, the analyst must be confident in the correctness of the readings of the spectrophotometer. Therefore, instruments in the laboratory must be verified by the bodies of the metrological service. If the instruments are verified, then the optical density of the test solution can be measured on different spectrophotometers and the obtained values can be compared. With the reproducibility of the value D = 0.26 on different devices, it can be confidently asserted that adrenaline hydrotartrate does not meet the requirements of the ND for the content of adrenolone impurities.
9. The choice of UV spectrophotometric method for testing acetylsalicylic acid tablets for authenticity and determining the "Dissolution" test is scientifically based. The UV spectrum of acetylsalicylic acid complements analytical reactions for authenticity, since the absorption band in the spectrum indicates the aromatic nature of the substance.
It is quite logical to use the method when determining the "Dissolution" test, which shows the amount of substance that has passed into the dissolving medium from the dosage form in 45 minutes at 37O C. For testing, a "Rotating basket" device is used. One tablet is placed in a basket and immersed in a dissolution medium - an acetate buffer solution with a pH of 4.5 and a volume of 700 ml. After 45 minutes, a sample is taken and the content of acetylsalicylic acid is determined.
Since the amount of active substance in the dissolution medium will be approximately:
a highly sensitive method will be required to determine it in the sample.
This method is UV spectrophotometry.
For quantitative purposes, it is better to use a titrimetric method that is absolute and does not require comparison with a standard sample. The dosage of tablets of acetylsalicylic acid, equal to 0.5 g, allows you to use this method.
highly sensitive methods. Therefore, the determination of the substances of medicinal substances, as a rule, is carried out by means of titration.
However, the riboflavin substance does not have analytical reactions that meet the requirements of titrimetry. For this reason, for the quantitative determination of riboflavin, a spectrophotometric rather than chemical method is chosen, since riboflavin intensively absorbs in the UV and visible regions of the spectrum.
quantitative targets is reasonable. However, the method for determining the specific absorption index given in the FS requires improvement, since it is accompanied by a significant error.
More correct and accurate is the method of comparison with a standard sample of the GSO category.
The calculation of the weighed portion of riboflavin is carried out according to the specific absorption index E11cm = 328.
percentage, taking into account the optimal value of optical density D = 0.43:
increase, then use the dilution technique. According to the FS method, the weighed portion is increased 5000 times and thus obtained, the weighed portion of riboflavin is calculated correctly. However, from the point of view of metrology, it is better to increase the micro weight by 8000 times and get a weight equal to 0.1 g.
11. The choice of spectrophotometric method for quantitative scientifically sound. The content of the active substance in the preparation is low; therefore, its determination requires a highly sensitive method, which is UV spectrophotometry. In addition, by chemical structure, dibazole belongs to the heteroaromatic series and actively absorbs UV radiation, which makes it possible to use the method for quantitative purposes.
The spectrophotometric method is relative and requires comparison with a standard sample. The solution to be analyzed and the standard sample solution are prepared with approximately the same concentration.
The concentration of the standard sample solution is indicated in ND. This is the basis for calculating the sample weight. In our case, the concentration of the working standard sample solution is 0.00002 g / ml.
The test solution must be prepared with the same dibazol content.
CX = CCO = 0.00002 g / ml Then recalculation is made to the dibazol solution:
Since the sample is small, it is increased 1000 times and the dilution technique is used:
Thus, the weighed amount of the dibazol preparation is calculated correctly.
12. Picamilon is a heterocyclic drug substance of aminobutyric acids:
It is used as a nootropic agent in the form of tablets with a dosage of 20 mg. The structure of the substance contains a pyridine chromophore conjugated with an amide group and causing the absorption of picamilon in the UV region. Therefore, the choice of the spectrophotometric method for quantitative purposes is quite justified. In addition, the content of the active substance in the tablets is insignificant (20 mg), therefore, a highly sensitive method is required, such as UV spectrophotometry.
There is no doubt about the choice of water as a solvent, since the active substance, being a sodium salt of a carboxylic acid, dissolves in water.
Pre-treatment is provided for the purpose of separating auxiliary substances that are insoluble in water and interfere with the determination.
the standard sample ensures the correctness and accuracy of the analysis.
The disadvantage of the proposed technique is that the analyzed sample of the tablet mass is small (0.08 g). From the point of view of metrology, it is better to work with a sample of 0.1 g or more. The larger the sample, the smaller the weighing error. An increase in the sample size in this case is quite possible, since there is no need to save the analyzed material, since the sample is taken from 20 tablets ground into powder.
Provide a rationale for the use of UV spectrophotometric quantitation with full calculated reasoning. When completing the task, use the algorithm and an example of solving the problem.
1. Anaprilin solution 0.25% in CCO ampoules 0.00002 g ml 2. Nicotinic acid solution 1% in CCO ampoules 0.00001 g ml 4. Ointment hydrocortisone eye 0.5% - 3.0 CCO 0.00001 g ml 7. Rectal suppositories with diclofenac sodium 50 and 100 mg 9. Cortisone acetate tablets 0.025 g 10. Prednisolone tablets 0.001 g 11. Ethinylestradiol tablets 0.00001 g Average weight 0.056 g 12. Pregnine tablets 0.01 g Average weight 0.108 g 13. Pyridoxine tablets 0.002 g Average weight 0.205 g 14. Thiamine chloride tablets 0.002 g Average weight 0.212 g for the preparation of a method for quantitative analysis of drugs by UV spectrophotometric method 1. Justify the choice of method.
3. To resolve the issue of preliminary processing.
4. Draw up methods for preparing a solution of the test drug and a solution of a standard sample.
5. Draw up a technique for spectrophotometric analysis.
6. Draw up a calculation formula for the content of the active substance.
The object of analysis is an injection solution with a low content of a medicinal substance. The latter circumstance requires the use of the most sensitive method in quantitative analysis. Such methods include UV spectrophotometry. In addition, this method does not require laborious and time-consuming analytical operations.
The spectrophotometric method is possible if there is a system of conjugated bonds in its structure.
electromagnetic radiation in the UV region is due to the presence in the spectrophotometric method.
Calculation of the sample: the starting point for calculating the sample of the dosage form during its spectrophotometric analysis is which determination will be carried out.
In the condition of the problem, the concentration of a solution of a working standard sample (PCO) CCO 0.00005 g ml is given.
The analyzed solution is 0.1%, therefore, you can make up the proportion:
0.1 g of adrenaline - 100 ml of solution The calculated sample can be increased 100 times. This will allow you to use a 5 ml pipette to measure it, and a 100 ml volumetric flask for subsequent dilution.
carried out in a 0.1 M solution of hydrochloric acid. The choice of the solvent is due to ensuring the stability of the drug in its solution.
additional analytical operations to extract a substance from its dosage form are not required.
Methodology:
5 ml of a solution of epinephrine hydrochloride is placed in a volumetric flask with a capacity of 100 ml and the volume of the solution is brought up to the mark with a 0.1 M solution of hydrochloric acid. The optical density of the resulting solution is measured on a spectrophotometer at an analytical wavelength in a cuvette with a layer thickness of 10 mm. In parallel, the optical density of a solution of a working standard sample (RSO) of epinephrine hydrochloride is measured.
A 0.1 M solution of hydrochloric acid is used as a comparison solution.
Calculation of results.
DX; DCO - the values of the optical density of the test solution and the PCO solution of epinephrine hydrochloride, respectively.
medicinal substances. M .: "Medicine", 1978. - 248 p.
"Medicine", 1975. - 151 p.
Belikov V.G. Pharmaceutical chemistry. At 2 o'clock / V.G.
Belikov. - Pyatigorsk, 2003 .-- 720 p.
State Pharmacopoeia of the USSR. / Ministry of Health of the USSR. - 11th edition. - M .: Medicine, 1987. - Issue. 1.- 336 p.
State Pharmacopoeia of the USSR. / Ministry of Health of the USSR. - 11th edition. - M .: Medicine, 1989. - Issue. 2.- 400 p.
State Pharmacopoeia of the Russian Federation / 12 - edition. - "Publishing house" NTsESMP ", 2008. - 704 p.
Kazitsina L.A., Kupletskaya N.B. Application of UV -, IR -, NMR - and mass spectroscopy in organic chemistry. M., Ed. Moscow un - that, 1979 .-- 240 p.
Methods of analysis of drugs / N.P. Maksyutina and others - Kiev:
Health, 1984. - 224 p.
Fundamentals of Analytical Chemistry. In 2 books. 2. Methods of Yu.A. Zolotov. - 2nd ed. - M .: Higher. school; 2002 .-- 494 p.
Otto M. Modern methods of analytical chemistry. / M.
Otto. - M .: Technosphere, 2006 .-- 416 p.
Pharmaceutical Chemistry: Textbook / Ed. A.P.
Arzamastseva. - M .: GEOTAR - MED, 2004 .-- 640 p.
FSP 42 - 0035225102 Ascorbic acid.
Introduction
1. Characteristics of spectroscopic methods of analysis
2. The basic law of light absorption Photometric quantities ............... 3. Characteristics of spectrophotometers
4. Characterization of absorption spectra
5. Sample preparation for photometric determinations
6. Comparative characteristics of absorption methods
7. Application of spectrophotometry in pharmaceutical analysis .............. 7.1. The use of IR - spectroscopy in the analysis of drugs 7.2. Application of UV spectrophotometry in drug analysis
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Due to the simplicity of analytical operations and, in most cases, high sensitivity, the method has found wide application in pharmaceutical analysis.
UV spectrophotometry is used to establish the authenticity (identification), good quality, quantitative determination of both individual substances and components of dosage forms; tests according to the tests "Dissolution" and "Uniformity of dosage".
The method is used at such stages of studying medicinal substances and dosage forms as pharmacokinetics, bioavailability, stability studies and establishing shelf life.
Test for the authenticity of medicinal substances. This stage of pharmaceutical analysis is based on the following techniques:
a) finding in the spectrum λ max and λ min, which characterize the regions of maximum and minimum absorption;
b) calculation of the ratio of the values of optical densities of the test solution at different wavelengths;
c) the characteristic of the absorption intensity by the value of the specific indicator (E);
d) comparison of the spectrum of the analyte with the spectrum of a standard sample of the same substance.
In all cases, it is necessary to obtain a spectrum under the conditions specified in the ND - solvent, concentration, wavelength range, size (thickness) of the cuvette.
For case (a), λ max and λ min are found in the resulting spectrum, compared with the same characteristics given in the ND - if the substances are identical, both values should coincide (Table 7).
A convenient technique for authenticity testing is to determine the ratio of the absorbance values at two maxima. This reduces the influence of the instrument's variable characteristics on the test and eliminates the need to use a standard sample. This method is used in the case of sodium analysis of sodium para-aminosalicylate
Table 7
Characteristics of UV spectra used in the identification of certain medicinal substances in pharmacopoeial analysis
|
№ p / p |
Medicinal substance |
Concentration and solvent |
Characteristic used for identification |
|
Amlodipine besilate |
0.005% in 1% solution of 0.1 M HCl in methanol |
λ max = 360 ± 2nm; E = 113-121 |
|
|
Aminazin |
0.0005% in 0.01 M HCl |
λ max = 254 ± 2nm, 307 ± 2nm |
|
|
Anestezin |
0.0005% in 0.1 M NaOH |
λ max = 281 ± 2nm; λ min = 238 ± 2nm |
|
|
Verapamil hydrochloride |
0.002% in 0.01 M HCl |
D 229 = 0.61 - 0.64 D 278 = 0.23 - 0.24 |
|
|
Dexamethasone |
0.001% in 95% alcohol |
λ max = 240 ± 2nm; D 240nm / D 263nm = 1.9 - 2.1 |
|
|
0.002% in 95% alcohol |
λ max = 244 ± 2nm, 275 ± 2nm, 281 ± 2nm; λ min = 230 ± 2nm, 259 ± 2nm, 279 ± 2nm; the spectrum is shown |
||
|
Diphenhydramine |
0.05% in 95% alcohol |
λ max = 253 ± 2nm, 258 ± 2nm, 264 ± 2nm; λ min = 244 ± 2nm, 255 ± 2nm, 263 ± 2nm |
|
|
Drotaverine hydrochloride |
0.0015% in 0.1 M HCl |
λ max = 241 ± 2nm, 302 ± 2nm, 353 ± 2nm; λ min = 223 ± 2nm, 262 ± 2nm, 322 ± 2nm |
|
|
Zopiclone |
0.001% in 0.1 M HCl |
λ max = 303 ± 2nm; D 303 = 0.340-0.380 |
|
|
0.0006% solution of 2,4-dinitrophenylhydrazone camphor in 95% ethyl alcohol |
λ max = 231 ± 2nm, 265 ± 2nm; shoulder in the range from 273 nm to 277 nm |
||
|
Ascorbic acid |
0.001% in buffer solution with pH 7.0 |
λ max = 265 ± 2nm |
|
|
Nicotinic acid |
0.002% in 0.1 M NaOH |
λ max = 258 ± 2nm, 264 ± 2nm, 270 ± 2nm; λ min = 240 ± 2nm; in the area from 240nm to 256nm, two unidentified shoulders are observed |
|
|
Folic acid |
0.001% in 0.1 M NaOH |
Full coincidence with the GSO spectrum in the range from 230 to 380 nm |
|
|
Nitroxoline |
0.0005% solution in a mixture of 95% alcohol - buffer solution with pH 9.18 (98: 2) |
λ max = 249 ± 2nm, 341 ± 2nm, two shoulders in the range from 228nm to 238nm and from 258nm to 268nm |
|
|
Ofloxacin |
0.001% in 0.1 M HCl |
λ max = 226 ± 2nm, 295 ± 2nm; λ min = 265 ± 2nm |
|
|
Papaverine hydrochloride |
0.0025% in 0.01 M HCl |
λ max = 285 ± 3nm, 309 ± 2nm; λ min = 289 ± 2nm |
|
|
Piracetam |
1% aqueous solution |
Has no pronounced absorption maxima in the range from 230nm to 350nm |
|
|
Progesterone |
0.001% in 95% alcohol |
λ max = 241 ± 2nm; E = 518-545 |
|
|
Ranitidine hydrochloride |
0.01% aqueous solution |
λ max = 229 ± 2nm; 315 ± 2nm; D 229nm / D 315nm = 1.01 - 1.07 |
|
|
Sulfa-dimethoxin |
0.000015% in NaOH 0.000015% in HCl |
The spectrum of the alkaline solution of the preparation, taken against the acidic solution, has λ max = 253 ± 2nm, 268 ± 2nm; λ min = 260 ± 2nm; The spectrum of the acidic solution of the preparation, taken against the alkaline solution, has λ max = 288 ± 2nm |
|
|
Tamoxifen Citrate |
0.002% in methanol |
λ max = 237nm, 275nm |
|
|
Famotidine |
0.0025% in phosphate buffer |
Full coincidence with the RSO spectrum in the range from 230nm to 350nm |
|
|
Furazolidone |
0.0015% in DMF |
λ max = 260 ± 2nm, 367 ± 2nm; λ min = 302 ± 2nm |
|
|
Furacilin |
0.0006% in DMF |
λ max = 260 ± 2nm, 375 ± 2nm; λ min = 306 ± 2nm |
When testing for authenticity, it is often recommended to calculate Ev maximum absorption (for example, for chloramphenicol, epinephrine, progesterone) or compare the found value of optical density in a certain wavelength range with the values given in the ND. So the absorption spectrum of a solution of pyridoxine hydrochloride in a phosphate buffer solution (pH = 6.9) with a concentration of 0.5 mg / ml in the range from 230 to 250 nm has maxima at 254 and 324 nm, and the optical density at these maxima is equal to 0, respectively. 18 and 0.35.
Some authenticity tests using UV spectrophotometry require the use of standard samples (RM) of medicinal substances. In this case, the RM sample should be prepared and simultaneously determined under the same conditions as the test substance. So, the UV spectrum of a 0.0005% solution of ethinitestradiol in ethyl alcohol should have maxima and minima at the same wavelengths as a CO solution of the same concentration, the corresponding absorption values calculated for dry matter at λ max = 281 nm should not differ more than than 3%. This technique provides more reliable results than when analyzing the spectrum of only one test compound.
UV spectrophotometry is also one of the components of a complex of spectral methods for studying new biologically active substances. Certain absorption bands in the spectrum may indicate the presence in the structure of this compound of certain functional groups, fragments of structures (chromophores). This explains the similarity of the spectra of substances containing a phenyl radical, for example, ephedrine, diphenhydramine, atropine, benzylpenicillin. They have three absorption maxima: 251, 257 and 263 nm (Fig. 7).
Medicinal substances containing a substituted aromatic radical - adrenaline, morphine, estradiol, chloramphenicol, etc. - have one maximum in the spectrum of about 260 nm, the conjugated enone system in medicinal substances from the group of corticosteroids - about 238 nm (Fig. 8).
For some medicinal substances (derivatives of barbituric acid, sulfonamides, phenols, some derivatives of purine, etc.), the nature of the spectrum may change depending on the pH of the solution (Fig. 9, 10, 11, 12, 14). In this case, λ max (bathochromic shift) changes, absorption increases (optical density increases), and a hyperchromic effect is observed. Caffeine does not exhibit acidic properties; therefore, its maximum absorption in acidic and alkaline media at the same wavelength is 272 nm (Fig. 13). That is, UV spectrophotometry can provide information about the specific properties of the test substance.
It is impossible to draw a single conclusion about the structure of a chemical compound using UV spectrophotometry, since the interpretation of the spectrum is difficult due to the presence of more than one chromophore in the molecule. Nevertheless, the method allows one to determine some groupings - chromophores and draw a conclusion about the nature and degree of conjugation (with lengthening of the conjugation chain, a shift of the absorption maximum to a longer wavelength region is observed, Fig. 11).
UV spectrophotometry is used to study the properties of organic compounds: the ability to form hydrogen bonds, determine the pK a of acids and bases, determine the composition and properties of complex compounds of medicinal substances, isomerism.
The cis and trans isomers have different spectra. The trans form usually absorbs more strongly and its absorption band is shifted to longer wavelengths; this fact can serve as evidence of a structural change in the course of the reaction.
However, UV spectra do not provide any information about the structure of the test substance, because they only make it possible to establish the presence of chromophores and heteroatoms.
In addition, UV spectrophotometry provides an excellent opportunity for the quantitative analysis of substances containing such groups.
When tested for goodness (purity) use the same characteristics as for identification. In the presence of impurities, λ max can change, additional maxima appear, and the absorption intensity can change.
Specific impurities present in medicinal substances, as a rule, have a close chemical structure with the substance under study. Therefore, of particular interest are cases when a drug and its specific impurity are absorbed at different wavelengths.
For example, λ max of adrenaline (Ι) is located at 278 nm, and its specific impurity, adrenolone (ΙΙ), has an absorption maximum at 310 nm.
According to the requirement of the Pharmacopoeia Monograph, in a 0.05% adrenaline solution prepared for testing for purity, the optical density at 310 nm should not exceed 0.1 (i.e., a strictly standardized adrenolone content is allowed in adrenaline).
Quantitation. The principle of quantitative determination by UV spectrophotometry is as follows: a weighed portion of the analyzed sample (substance, dosage form, etc.) is dissolved in a suitable solvent, if necessary, a dilution of the resulting solution is additionally prepared and its optical density is measured at the wavelength specified in the procedure. The concentration (content) of the analyte is found by one of the previously described methods (clause 1.2.3.4).
In accordance with modern requirements for tablets, dragees, dry medicinal products for injection and medicinal substances in capsules with an active ingredient content of 0.05 g or less, it is mandatory to test for dosage uniformity, i.e. the content of the substance in each individual dose. For such an assessment, especially in the case of the content of the active substance in mg or its fractions (clonidine tablets contain 0.075 and 0.15 mg of the active substance), a highly sensitive method is required. In most cases, this is what UV spectrophotometry is.
The study of the bioavailability of medicinal substances is relevant. Its definite characteristic is the “Dissolution” test (GF ΧΙ, issue 2, p. 154). UV spectrophotometry, which is usually characterized by high sensitivity, is one of the most commonly used methods for this purpose (Table 8).
Below are the methods for the analysis of some medicinal substances by the spectrophotometric method in the UV region, and Table 8 shows a number of examples of the use of the UV spectrophotometry method in pharmacopoeial analysis.
1The restoration of cells damaged by UV radiation is a complex process that has been improved since the inception of living things. In the early stages of life development, the presence of intense UV radiation, including short-wave radiation (due to the absence of atmospheric screens), led to the development of powerful, repairing UV damage, intracellular systems, which are excessive for most cells of multicellular animals under current conditions. At a sufficiently low irradiation intensity, the reparation processes in the cell have time to eliminate the resulting damage before their number exceeds a certain "critical" value, leading to the appearance of non-reparable disorders. Cellular systems manage to complete the genome repair in a time measured in several hours, but for individual, most active DNA sites, the repair of thymine dimers is much faster. The time during which external influences can change the integral response of a cell to an impulse damaging effect is associated with the repair processes. For UV-damaged skin keratinocytes, this time (judging by the results of inhibitory analysis) is tens of minutes. The damaging effect can be considered "impulsive" (single) if its duration is less than the time during which the fate of the damaged cell is determined. There is reason to believe that for some cell responses to UV damage, this time is measured in seconds. If, according to the DER criterion, the law of interchangeability of intensity and exposure time for UV-erythema is fulfilled in the range from fractions to hundreds of seconds, then erythema from a dose exceeding the DER increases with increasing intensity. Quantitatively, this is expressed in the fact that with an increase in the intensity of irradiation with the full spectrum of the PRK-2 lamp by 3 orders of magnitude, the slope of the dose dependence increases by more than 3 times.
Modern knowledge of cellular mechanisms allows us to assert that, in addition to cell repair, there is another variant of the physiological response of a cell to damage - apoptosis, which prevents the “pathological” cell death by the mechanism of necrosis. The program of cell elimination by the apoptosis mechanism is activated when complete repair is impossible. In this case, the damage should not exceed the threshold at which the apoptosis program breaks down. In the latter case, cell death occurs by the mechanism of necrosis with the formation of an inflammatory reaction. For UV radiation, the absorbed dose is traditionally measured by the energy incident on a unit surface of the irradiated object, that is, the surface dose density. This is possible because the most biologically active part of UV radiation is absorbed by the surface layers of the skin. In the interval between the doses of damage that lead the cell to "pure" apoptosis or necrosis, it is possible (at a dose of UVB radiation of about 350 J / m2) to implement a program of apoptosis with "altered morphology" or "proinflammatory apoptosis", which occurs when the apoptosis program is modified probably by the same damaging effect that caused apoptosis. Experimentally, pro-inflammatory apoptosis was discovered in (Caricchio R e.a., J Immunol. Dec 2003). The bimodality of the action of UV radiation on keratinocytes (non-monotonic dose dependence of apoptosis) has also been shown in other works. But the nature of these phenomena has not been established. The most probable model seems to be that the outcome of UV irradiation of skin keratinocytes is determined by the number (proportion) of UV-damaged mitochondria. These assumptions are confirmed by the peculiarities of the dose-dependent kinetics of the two-component UVB-erythema of the skin. Experiments on the culture of human keratinocytes show that UVC irradiation produces a significantly larger amount of photoproducts (CPDs and (6-4) PPs), and the approximately equal apoptogenic effect of UVC and UVB radiation is due to the fact that UVB irradiation activates not only mitochondrial, but also caspase-8 dependent apoptosis activation pathway (Takasawa R ea, PubMed - in process Oct 2005). The most important task is to study the relationship of UV-induced apoptosis with erythemogenesis, but the peculiarities of UV absorption in various layers of the skin should be taken into account. Establishing a relationship between UV-induced apoptosis and erythemogenesis will make it possible to develop a non-invasive method for diagnosing the parameters of the apoptosis system. At present, special attention should be paid to the development of diagnostic methods based on the analysis of the reactions of body systems developing in time to any (external) influence. The developed diagnostic method is characterized by low cost, non-invasiveness, absolute sterility and the ability to provide a physiological, strictly dosed testing effect on the skin and other integuments.
Bibliographic reference
Bondyrev Yu.A. ANALYSIS OF THE POSSIBILITY OF USING UV RADIATION AS A TESTING DIAGNOSTIC EFFECT // Modern problems of science and education. - 2006. - No. 2 .;URL: http://science-education.ru/ru/article/view?id=183 (date accessed: 01/05/2020). We bring to your attention the journals published by the "Academy of Natural Sciences"
For atomic spectroscopy, it is necessary to destroy a substance into individual atoms, but for molecular spectroscopy it is impossible, therefore, absorption spectra in the UV, visible and IR ranges are usually studied at ordinary temperatures. Atoms and molecules obey the laws of quantum mechanics. They can be in states with different energies due to the transitions of electrons to higher levels, and for molecules also due to vibrations and rotations. The energy levels of each type of movement are discrete and characterized by quantum numbers. The energy of a diatomic molecule consists of electronic, vibrational and rotational,
E = E el + E count + E time.
E el >> E oscillation >> E rotation
The figure shows an example of the energy levels of a diatomic molecule. Two electronic states are shown - the main one and the first excited one. Each state has sublevels due to vibrational states, while there are sublevels due to rotational ones. There are many levels compared to atoms, between them there are many transitions that are close in frequency, they merge with each other and instead of lines, bands are observed. The atomic spectra are linear, the molecular ones are striped.
Molecular spectra are studied using two types of spectrometers - UV (combined with visible) and IR.
UV and visible spectroscopy
The electronic absorption spectra associated with the transition of electrons to higher energy levels are investigated. Spectra of organic molecules are observed containing double or triple bonds, or atoms with lone electron pairs (absorbing groups are called chromophores). An example in the table, which shows the wavelengths corresponding to the maximum of the UV spectrum band.
|
Chromophore |
molecule |
max (mmk) |
|
C 2 H 5 CH = C = CH 2 | ||
The detection of such bands in the spectra reveals the groups included in the molecule, which is important for qualitative analysis. Quantitative analysis is based on measuring the light absorption coefficient of the test solution at certain frequencies.
A UV spectrophotometer consists of a radiation source, a prism, a slit and a photocell. The source is a hydrogen lamp, that is, a direct current arc in a hydrogen atmosphere at low pressure, giving continuous radiation in a wide frequency range. Light travels through a prism and then through a slit that highlights a narrow range of wavelengths (frequencies). Then the light passes through a cuvette - a vessel with plane-parallel transparent walls, filled with a test solution and hits a photocell. The light absorption coefficient is the ratio of the intensities of the light rays incident on the sample and transmitted through it from the source. To correct for light absorption by the solvent, use a pure solvent reference sample. Light absorption is measured using a two- or one-beam scheme. In the first case, the luminous flux of the source is divided into 2 fluxes of equal intensity and one is passed through the test solution, the other through the standard one, then the intensities of the fluxes at the outlet are compared. With a single-beam scheme, both solutions are installed in turn.
The same device is used to record spectra in the visible region; an incandescent lamp is used as a source.
For all methods of molecular spectroscopy, the Bouguer-Lambert-Baire law is valid:
I = I 0 exp (-lc)
ln (I 0 / I) = lc
where is the molar absorption coefficient (l / mol cm), s is the concentration, l is the thickness of the cuvette, I 0 is the intensity of the incident flow, I is the intensity of the outgoing flow; the ratio I 0 / I is called transmission, and log (I o / I) is called optical density. If several absorbing substances are present in a solution, then the optical density of the solution is equal to the sum of the contributions of each of the components.
The Bouguer-Lambert-Beer law is strictly fulfilled for monochromatic radiation,
Sometimes photocolorimeters are used for measurements, in which a limited set of replaceable broadband glass light filters is used; these instruments are not spectral instruments.
Spectrophotometry in the UV and visible ranges is widely used in the analysis of substances; in particular, for the determination of colored compounds of a number of metals, as well as As, P, for the determination of certain functional groups of organic compounds, for example phenols and compounds with multiple chemical bonds.
To increase the selectivity of the determination, photometric reagents are used that selectively interact with the analyte to form a colored product. For example, when determining Fe, Mo, W, Nb, Co, etc., thiocyanates are used, and when determining copper, ammonia. Organic dyes are widely used as photometric reagents that form colored complexes with metal cations. The preliminary separation of the components is also used.
The advantages of this spectrophotometry are the relative simplicity of the apparatus and the extensive experience in application. The disadvantage is low selectivity.
The minimum concentration determined by the spectrophotometric method is not lower than 10 -7 M, that is, the sensitivity of the methods is average.
UV SPECTROSCOPY(UV spectroscopy, UVS), opt. spectroscopy, which includes obtaining, research and application of emission, absorption and reflection spectra in the ultraviolet region, ie, in the wavelength range of 10-400 nm (wave numbers 2.5 · 10 4 - 10 6 cm -1). UVC at wavelengths less than 185 nm is called. vacuum, since in this region UV radiation is so strongly absorbed by air (mainly oxygen) that it is necessary to use vacuum or spectral instruments filled with non-absorbing gas.
Technique for measuring UV spectra in the main. is the same as the spectra in the visible region (see Spectrophotometry). Spectral instruments for UFS are distinguished by the fact that instead of glass optics. parts use similar quartz (less often fluorite or sapphire), to-rye do not absorb UV radiation. Aluminum coatings are used to reflect UV radiation. The receivers are ordinary or low-gelatin photographic materials, as well as photoelectric. devices, ch. arr. photomultiplier tubes, photon counters, photodiodes, ionization chambers. To increase the sensitivity when using photographic materials, fluorescence caused by the UV radiation under investigation is sometimes recorded.
As a rule, when exposed to UV radiation, B-BO is not destroyed or changed, which makes it possible to obtain data on its chemical. composition and structure. In the UV region, electronic spectra appear, i.e., the position of the bands and lines is determined by the difference in energies of the decomp. electronic states of atoms and molecules. Here lie the resonance lines of neutral, singly and doubly ionized atoms, as well as spectral lines emitted by multiply ionized atoms in an excited state. In the near UV region, absorption bands of most semiconductors are concentrated, arising from direct transitions of electrons from the valence band to the conduction band.
There are also electronic vibrations in the UV region. bands of molecules (the vibrational structure manifests itself only at low m-ts; under normal conditions it leads to diffuse, that is, blurred, spectra), which is widely used in chemistry. analysis and research. The appearance of these bands is associated with the transitions of electrons between bonding and non-bonding n- and antibonding- and-orbitals (see Molecular spectra). This allows you to use UFS to study the electronic structure of molecules, the effect of substituents on chem. Holy Island aromatic. connections, for mouthnew type of chem. connections, determination of parameters of po-stay potentials. energies of excited states of molecules, etc. These studies are based on the assignment of absorption bands of UV spectra to certain electronic transitions. In this case, it is necessary to take into account the position and intensity of the stripes. Usually, the term "UV spectroscopy" refers to this particular area of spectroscopy.
For sat. hydrocarbons, only -transitions requiring high energies are possible, and the corresponding bands lie in the vacuum UV region, for example. in the case of methane and ethane, at 125 and 135 nm, respectively. For unsaturated. compounds are characterized by transitions that appear at wavelengths of 165-200 nm. The presence of conjugation, alkyl or other substituents (including those containing heteroatoms) leads to a shift of the bands to longer wavelengths (batochromic shift), for example. butadiene already absorbs at 217 nm. In carbonyl (as in thiocarbonyl) comp. in naib. In the long-wavelength region, there is a low-intensity band caused by a symmetry-forbidden transition. In the shorter wavelength region, bands of high intensity of n-transitions appear. So, in the spectrum of formaldehyde there are absorption maxima at 295 (weak), 185 and 155 nm.
The absorption bands of esters, amides, halogen-hydrides are shifted to the short-wavelength region, and the bands of thiocarbonyl compounds are shifted to the long-wavelength region in comparison with the absorption bands of the corresponding carbonyl compounds, for example: absorption maxima of CH 3 C (O) H, CH 3 C (O) NH 2 and CH 3 C (S) NH 2 are observed at 290, 214 and 358 nm, respectively. Due to hybridization of the lone pair of nitrogen electrons in compounds containing the C = N group, the intensity of the -transition band in them is higher than in carbonyl compounds. The spectra contain nitroeat. the position and intensity of the transition band depend on the nature of the atom adjacent to the nitro group. So, O-nitroeater. this low-intensity band is located in a shorter wavelength region than that of C-nitro compounds. In the spectrum of nitramines (N-NO 2), the transition band at Naib. intense.
Azo- and nitroso compounds are also characterized by -transitions. The bands of the UV spectrum of N- and O-nitroso compounds are shifted to shorter wavelengths in comparison with the bands of C-nitroso compounds. Conjugation of multiple bonds with nitrogen-containing chromophore groups such as NO 2, NO, N = N, N 3 causes a bathochromic shift of all absorption bands and an increase in their intensity.
The nature of the absorption spectrum depends on the relative position of the chromophores. If the chromophore groups are connected directly, then the spectrum shows strong changes in comparison with the spectra comp. with isolated chromophore groups. Relates. the arrangement of chromophores at multiple bonds makes it possible to distinguish between cis and trans isomers.
Bands in the spectra of aromatic. conn. associated with transitions -electrons aromatic. systems. The form of the spectrum is influenced by substituents: such as alkyl, halogens - insignificantly, groups with lone pairs of electrons (OH, OR, NH 2, NF 2) - strongly. If there is a carbonyl, nitro- or nitroso group, then the spectrum additionally exhibits β-transition bands. In the spectra of some substituted benzene, for example. nitrobenzene, it is possible to isolate bands with intramol. charge transfer (corresponding to transitions, for which there are advantages, a decrease
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