Lecture 2. Analysis of pharmaceuticals by spectrophotometric methods in the visible and UV regions..pptx

Lecture 2. Analysis of pharmaceuticals by spectrophotometric methods in the visible and UV regions..pptx

• 1.
  LECTURE 2. ANALYSIS OFPHARMACEUTICALS BY SPECTROPHOTOMETRIC METHODS IN THE VISIBLE AND UV REGIONS. Lecturer: Lyazzat Sagyndykova, master degree of medicine science
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  Out list 1. Instrumentcharacteristics. 2. Measurement of optical density. 3. Derivative spectrophotometry. 4. Beer-Lambert Law. 5. Quantitative determination.
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  Characteristics of ElectromagneticRadiation 1. Wave Properties: An electromagnetic wave can be represented as two variable fields perpendicular to each other and to the direction of wave propagation. Several parameters can characterize an electromagnetic wave: wavelength (λ), frequency (ν), or wave number ( 𝒗), amplitude (a), speed (c), intensity (I), and power (P). Fig. 1
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  Wavelength λ isthe distance between two wave peaks. The main units of wavelength measurement in the UV and visible range are nanometers (1 nm = 10⁻⁹ m), while in the IR range, micrometers are used (1 μm = 10³ nm). Wavelength depends on the refractive index of the medium through which the radiation propagates, as it is directly related to the speed of wave propagation. The speed of radiation propagation varies in different media. Frequency (ν) is the number of oscillations per second and is expressed as the ratio of the speed of radiation propagation (speed of light) to the wavelength: 𝒗 = 𝑪 𝝀 (1) The speed of light and wavelength should be considered for the same medium in which the radiation propagates. Frequency is measured in inverse seconds (s⁻¹) or hertz (1 Hz = 1 s⁻¹). The frequency depends only on the nature of the radiation source, while the speed of electromagnetic wave propagation, and consequently the wavelength, also depend on the properties of the medium.
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  Wave number (𝒗 ) indicates how many wavelengths are present in 1 cm of radiation path in a vacuum and is determined by the relationship 𝐯 = 𝟏 𝛌 , where λ is the wavelength in vacuum. The dimension of wave numbers is cm⁻¹. Frequency is related to wave number by the relationship 𝑣 = 𝑐 𝑣. Amplitude (a) is the maximum value of the electric field vector. Intensity (I) is the energy of radiation per second per unit solid angle; it is proportional to the square of the amplitude. Power (P) is the energy carried by radiation through a certain surface per unit time.
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  2. Corpuscular Properties:Radiation consists of a stream of discrete particles (photons) moving at the speed of light. A photon is a material particle with a specific mass and momentum, deviating from a straight path under the influence of gravity. Each photon possesses energy related to its mass and frequency or wavelength by the relationships: 𝑬 = 𝒎𝒄² = ℏ𝜈 or 𝐄 = 𝐡𝒗 𝛌 (2) Thus, each photon can be characterized by frequency or energy as needed.
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  Spectroscopic analysis methodsare based on the ability of atoms and molecules of a substance to emit, absorb, or scatter electromagnetic radiation, i.e., on the interaction of the substance with electromagnetic radiation. Fig. 2. The spectrum of Light
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  Absorption wavelength range, nm (1) Spectralcolor (absorbed light) (2) Solution color (visible) (3) 400 - 435 Violet Greenish yellow 435 - 480 Blue Yellow 480 -490 Greenish blue Orange 490 -500 Bluish green Red 500 -560 Green Purple 560 - 580 Yellowish green Violet 580-595 Yellow Blue 595-605 Orange Greenish blue 605-730 Red Bluish green 730-760 Purple Green Table 1. Light absorption and solution color Based on the provided color solution table (3), it is possible to approximately (!) determine the absorption range (1).
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  Spectral ranges Spectral rangesWalelength Interaction with substance X-ray 0.01 – 10 nm K-, L- electrons Far ultraviolet (UV) 10 - 200 nm Middle electrons Near ultraviolet (UV) 200 – 400 nm Valence electrons Visible (View) 400 – 750 nm Valence electrons Near infrared 750 – 2500 nm (0,75 – 2,5 µm) Overtones and midrange molecular vibrations Mid infrared 2500 – 50000 nm (2.5 – 50 µm) Molecular Vibrations and Rotations Far infrared (IR) 50 – 1000 µm Molecular rotations Microwave 0.1 -100 cm Molecular rotations Radio wave 1 – 1000 m ЯМР
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  Fig. 3
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  Fig. 4. Fig. 5 Ifa substance is subjected to electromagnetic radiation, light can be absorbed by the substance, pass through it, reflect off it, scatter, or induce photoluminescence (glowing). The term photoluminescence encompasses several effects, such as fluorescence, phosphorescence, and combination light scattering (Raman effect).
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  Classification of opticalspectroscopy methods (metrics) Spectral methods Molecular spectroscopy Absorption UV/Vis spectroscopy IR spectroscopy Emission Luminescence spectroscopy Raman spectroscopy Atomic spectroscopy Atomic absorption Atomic emissions Molecular absorption methods
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  Spectroscopy involves themeasurement and interpretation of spectra that arise from the interaction of electromagnetic radiation with matter, and it applies the obtained results to solve various practical tasks (studying structure, composition, properties). Spectroscopy is commonly understood as qualitative analysis (identification of substances, functional or elemental analysis). Spectrometry studies quantitative relationships, such as signal concentration, signal mass; in other words, it refers to quantitative analysis. Since quantitative analysis is not possible without identification, the concept of spectrometry carries a broader meaning, encompassing both qualitative and quantitative components simultaneously.
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  Fig.6. Energy leveldiagrams with electronic transitions
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  Electronic transitions Transition type Description Stripe structureEffect of polar solvent Acidic environment Band position in the spectrum, lgεmax σ→σ* Far UV 100-200 nm, logε max = 0-2 (in the working area) n→σ* Average UV: 190-250 nm !π→π*! Visible in most solvents Shifts to the red (bathochromic) region No affect Mid and near UV: 130- 300 nm, logεmax = 3-4 !n→π*! Distinct in non- polar solvents, smeared in polar ones Distinct in non-polar solvents, smeared in polar ones Disappears Near UV and visible region: 250-500 nm, logεmax =1-2
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  Chromophores It’s a Greekterm i.e., Chroma “colour” and phoros “bearer”. It is defined as any isolated covalently bound group that exhibits distinctive electromagnetic radiation absorption in the UV or visible area. The chromophore-containing compound is chromagen. A chromophore is an atom or group that contributes to the color of a chemical. Molecules absorb some visible spectrum wavelengths while reflecting others. The wavelength reflected by the molecule is what we experience as color.
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  The energy differencebetween two distinct chemical orbitals in a chromophore falls within the visible spectrum region. Then, the chromophore was exposed to the visual spectrum, which activated the electrons from their ground state. As a result, when exposed to light, a chromophore changes conformation. If more than one chromophore is required to impart the color in a compound then it is known as dependent chromophores. For example: Acetone having one ketone group is colorless while diacetyl having two ketone groups is yellow.
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  There are twokinds of chromophores: 1. Chromophores that can only π contain electrons. They only go through π -π * transitions, such as the ethylenic group (C=C) and the acetylenic group (C≡C). 2. Chromophores that contain π as well as n (nonbonding) electrons. This chromophore has a single pair of electrons. As a result, they are responsible for two types of transitions, namely n-π * and π -π *, such as nitrite group (−NO2), nitrate group (−NO3), nitroso group (−NO), azo group (−N=N-), azomethine group (-CONH2), carbonyl group (>C=O), and quinoid structure (quinone).
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  Auxochromes An auxochrome isa group of atoms that can get attached to a chromophore, thereby increasing the colourfulness of the chromophore. Therefore, it is a modifier of a chromophore. An auxochrome itself cannot cause the development of colour. It can increase the ability of chromophore to absorb the wavelengths from visible range of light. Therefore, an auxochrome can be defined as a functional group in a molecule. An auxochrome can increase the colour of any organic compound. Auxochromic groups in organic compound molecules can be • electron-donating (-OH, -NH2, -SH, -OCH3, -NHCH3, -N(CH3)2, -NHC6H5, -O-) or • electron-accepting (-NH3 +, -SO2NH2, -COO-, -COOH, -COOCH3, -COCH3, -CHO, -NO2, -NO). Electron-accepting groups are sometimes referred to as antiauxochromic.
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  Auxochromes For example, benzeneis a colourless compound, but nitrobenzene is a yellow- coloured compound (nitrobenzene contains a nitro group attached to benzene). Here, the nitro group is a chromophore for benzene molecule. When a hydroxyl group gets attached to the para position of nitrobenzene, it appears in a dark yellow colour (the intensity of nitrobenzene is increased due to the auxochrome group). Benzene a colourless compound Nitrobenzene a yellow-coloured compound 4-nitrophenol a dark yellow coloured compund
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  Auxochrome groups havethe following effects on chromophores: • Bathochromic displacement. The absorption of the chromophore is shifted towards longer wavelengths. • Hypsochromic displacement. The absorption of the chromophore is shifted towards shorter wavelengths. • Hypsochromic effect. ϵmax increases, presenting the band with greater intensity. • Hypochromic effect. ϵmax decreases, decreasing the intensity of absorption.
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  Beer-Lambert Law The dependenceof the intensity of monochromatic light flux passing through the analyzed solution is determined by the combined Beer-Lambert-Bouguer law (or Beer-Lambert Law): where I or I0 is the intensity of the transmitted or incident light, respectively; k is the absorption coefficient, a constant of proportionality; C is the concentration of the dissolved substance; l is the thickness of the absorbing layer. 𝑰 = 𝑰𝟎 ∙ 𝟏𝟎−𝒌𝑪𝒍 (1) Fig. 6
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  The dependence oflight absorption on the thickness of the layer of the analyzed solution was discovered in 1729 by the French physicist-optician and navigator Bouguer, who was engaged in studying light absorption by the atmosphere and colored glasses. More than 30 years later, Lambert gave it a modern mathematical interpretation. Beer subsequently verified the validity of this law for solutions of various substance concentrations. The Beer-Lambert law states that there is a linear relationship between the concentration and the absorbance of the solution, which enables the concentration of a solution to be calculated by measuring its absorbance. The fundamental law of light absorption is valid only for the absorption of monochromatic light flux with a constant wavelength (λ=const).
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  The magnitude kis a specific physical constant for each substance; it depends on the nature of the dissolved substance, the solvent, temperature, light wavelength, and is independent of the concentration of the dissolved substance and the thickness of the absorbing layer. Depending on the method of expressing substance concentration, the absorption coefficient in formula (1) can have two values: molar absorption coefficient (ε) and specific absorption coefficient (𝑬𝟏 𝒄𝒎 𝟏% ). The molar absorption coefficient (𝜺 = 𝑫 𝑪∙𝒍 ) represents the optical density of a solution with a substance concentration of 1 mole/L and an absorbing layer thickness of 1 cm. The specific absorption coefficient (𝑬𝟏 𝒄𝒎 𝟏% ) is the optical density of a 1% solution with a thickness of the absorbing layer of 1 cm.
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  They are commonlyreferred to by a single term - extinction coefficients ε and 𝑬𝟏 𝒄𝒎 𝟏% . The relationship between the molar and specific absorption coefficients is determined by the following relations: 𝑬𝟏 𝒄𝒎 𝟏% = 𝟏𝟎 𝑴.𝒎 ∙ 𝜺 or 𝜺 = 𝑬𝟏 𝒄𝒎 𝟏% ∙ 𝑴.𝒎 𝟏𝟎 (2), where M.m is the molecular mass. In the practice of pharmaceutical analysis, the specific absorption coefficient 𝑬𝟏 𝒄𝒎 𝟏% is most commonly used. The sensitivity of the method for a specific substance is determined by the value of 𝑬𝟏 𝒄𝒎 𝟏% : the higher the numerical value of 𝑬𝟏 𝒄𝒎 𝟏% ​, the higher the sensitivity. Specific absorption coefficient values are calculated from experimental data of a series of solutions with different concentrations (in %) of a specific substance (𝑬𝟏 𝒄𝒎 𝟏% = 𝑫 𝑪∙𝒍 ).
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  Values of specificabsorption coefficients for medicinal substances are usually provided in reference guides on spectroscopy, indicated when describing spectra, in pharmacopoeias, and in pharmacopoeial articles in periodic literature. № Substance λmax 𝑬𝟏 𝒄𝒎 𝟏% Solvent 1. Adrenalin 280 150 0,01mol/l HCl 2. Anaprilin 217 293 1350 220 0.1 mol/l H2SO4 3. Novocaine 290 680 Water 4. Ergocalciferol 265 460–504 Absolute ethyl alcohol Table 1. Absorption maxima and specific coefficient values in the UV spectra of some medicinal substances
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  The intensity ofthe transmitted radiation flux (equation 1) in logarithmic form is as follows: 𝒍𝒈 𝑰𝟎 𝑰 = 𝑬𝟏 𝒄𝒎 𝟏% ∙ 𝑪 ∙ 𝒍 (𝟑) The quantity (𝒍𝒈 𝑰𝟎 𝑰 ​) is called optical density and is denoted by the letter D: 𝑫 = 𝑬𝟏 𝒄𝒎 𝟏% ∙ 𝑪 ∙ 𝒍 (𝟒) The ratio of the intensity of monochromatic radiation flux transmitted through the investigated object to the intensity of the incident flux is called transmittance and is denoted by the letter T: 𝑻 = 𝑰𝟎 𝑰 𝟓 . ​Optical density D and transmittance (T) are related by the equation: 𝑫 = −𝒍𝒈𝑻 (𝟔)
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  Usually, T isexpressed as a percentage, then 𝑫 = 𝟐 − 𝒍𝒈𝑻 . The values of optical density and transmittance depend on the wavelength and concentration of the substance in the solution. Unlike optical density, transmittance depends exponentially on concentration, 𝑻′ = 𝒆−𝒌𝒄𝒍 so it is relatively rarely used in analytical measurements and calculations.
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  The values ofoptical density and transmittance depend on the wavelength (λ) (Fig. 7) and the concentration of the substance being determined in the solution. The parameter T characterizes the ability of the solution to transmit light. Transmittance is measured in percentages (%) or fractions (from 0 to 1). Figure 7. Dependence of optical density and transmittance on wavelength.
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  4. Additivity Law(rule) – K. Firovdt (1873) Essence of the law: "Independence of the absorption of an individual substance from the presence of other substances with their own absorption or indifference to electromagnetic radiation." Law: "If a solution contains n light-absorbing components that do not chemically interact with each other, then, provided the fundamental law of light absorption is observed, the optical density of such a solution will be equal to the sum of the partial optical densities of all light-absorbing components present in the solution." This demonstrates the principle (rule) of additivity of optical densities: 𝑨 = 𝜺𝟏 ⋅ 𝒄𝟏 ⋅ 𝒍 + 𝜺𝟐𝒄𝟐 ⋅ 𝒍 + ⋯ 𝜺𝒏 ⋅ 𝒄𝒏 ⋅ 𝒍 = 𝒍 𝒊=𝟏 𝑵 𝜺𝒊 ⋅ 𝒄𝒊 All quantitative methods of spectrophotometric analysis of multicomponent systems for the simultaneous determination of their components are based on the principle of additivity.
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  Classification of SpectroscopicMethods Spectroscopic methods can be classified based on several criteria. 1. Optical Phenomena: Spectroscopy can be categorized according to the type of optical phenomena observed, distinguishing emission, absorption, and scattering spectroscopy. Emission spectroscopy further includes emission and fluorescence spectroscopy. 2. Energy Range: Based on the energy ranges of electromagnetic radiation, spectroscopy is classified into various types: gamma-ray spectroscopy, X-ray spectroscopy, optical spectroscopy (UV and visible spectroscopy, IR spectroscopy), and radiofrequency spectroscopy (microwave and radiofrequency spectroscopy). 3. Objects of Study: Spectroscopy can also be subdivided based on the objects under investigation:
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  Nuclear • α-Spectroscopy • β-Spectroscopy •γ-Spectroscopy Atomic • Atomic Emission Spectroscopy • Atomic Fluorescence Spectroscopy • Atomic Absorption Spectroscopy • X-ray Fluorescence Spectroscopy • Electron Paramagnetic Resonance (EPR) Spectroscopy • Nuclear Magnetic Resonance (NMR) Spectroscopy Molecular • Electronic Molecular Absorption Spectroscopy • Infrared (IR) Spectroscopy • Combustion or Raman Spectroscopy • Microwave Spectroscopy • Fluorescence Spectroscopy
• 33.
  Spectrophotometry is amethod of quantitative substance determination based on the absorption of monochromatic radiation by molecules in the near UV, visible, and near IR regions of the spectrum within the range of 190–1000 nm. Photocolorimetry is a method of quantitative substance determination based on the absorption of polychromatic radiation in the visible part of the spectrum within the interval of 380–780 nm by molecules. Spectrophotometry and photocolorimetry are often combined under the term photometry.
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  Causes of Deviationfrom the Beer-Lambert-Bouguer Law The behavior of absorbing systems adheres to the Beer-Lambert-Bouguer Law only under the following conditions: 1. Monochromaticity of the light flux; 2. Absence of chemical changes in the absorbing system; 3. Constant refractive index. When these conditions are violated, the molar absorptivity changes, and the calibration curve becomes distorted. If the value of the molar absorptivity decreases, a negative deviation from the law is observed, and if it increases, a positive deviation occurs. Positive deviation results when a small change in concentration produces a greater change in absorbance. Negative deviation results when a large change in concentration produces a smaller change in absorbance.
• 35.
  Causes of deviationfrom the fundamental law of light absorption can be apparent or true. Apparent causes can be physical (instrumental) or chemical. Apparent causes, resulting from non-monochromaticity of the light flux, light scattering, and random emissions, improper slit width are referred to as instrumental. Those caused by chemical interactions are termed chemical. Chemical effects such as association, dissociation polymerisation, complex formation, etc. as a result of the variation in the concentration. True causes are associated with changes in the refractive index (n). Since we are investigating solutions with relatively low concentrations, small changes in the refractive index (n) can be neglected.
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  1) Based onLight Source Types of Spectrophotometer
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  Single beam spectrophotometer Inthis, a fraction of light from the diverging devices is wholly passed from the sample solution. A beam of light from the light source falls onto the collimator convex lens and moves to the diaphragm. The diaphragm ensures 100% transmittance and allows the light to fall onto the monochromator device. A dispersion medium or monochromator device allows the transmittance of a single source of light onto the focusing convex lens. The focusing convex lens transmits light of a particular wavelength from the sample to the photocell detector. A photocell detects the portion of light transmitted or absorbed and gives the reading on the display meter.
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  Double beam spectrophotometer In this,a fraction of light coming from the monochromator device parts into two beams. One falls onto the reference sample and the other onto the test sample. Its mechanism is more or less similar to a single beam spectrophotometer but differs because the dual mirrors divide a single beam of light into two. One beam of light passes from the test sample to the photocell, and the other passes from the reference sample to another photocell. A photocell detects the amount of light transmitted or absorbed and gives the reading on the display meter.
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  Types of Spectrophotometer 2)Based on Light Wavelength 2.1 Ultraviolet spectrophotometer It uses cuvettes made of quartz and hydrogen or deuterium lamps as a light source. The hydrogen lamp emits continuous or discontinuous spectral UV- light ranging between 200-450 nm. This device determines the absorbance or transmittance for the fluids and even solutions. 2.2 Visible spectrophotometer It uses plastic and glass cuvettes and a tungsten halogen light source. The tungsten lamp consists of a tungsten filament, emitting a visible spectral range between 330-900 nm. The tungsten lamp has a long life of 1200 h. This device can measure the change in colour intensity according to the change in the concentration of moderately diluted solutions. 2.3 Infrared spectrophotometer It makes the use of Nernst glowers as a conductive device having a long life. This kind of spectrophotometer helps in studying the vibrations of different molecules at a specific wavelength. Near and mid-IR-rays cause rotational and harmonic vibrations.
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  INSTRUMENTATION • Source oflight. • Monochromator. • Sample soliotion in cuvette. • Photo detector. • Readout device.
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  How does aspectrophotometer work?
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  Mechanism A spectrophotometer includesthe following sequential events: • Firstly, a light source falls onto the monochromator (Dispersion device). • Then, the monochromator will produce a single source of light that falls onto the focusing wavelength selector. • The focusing convex lens will pass a fraction of the monochromatic light source from the sample solution to the photocell detector. • A photocell detector converts the light energy into electrical energy, and an amplifier transmits this electrical signal to the internal circuit. • Finally, an internal circuit inside a spectrophotometer gives out a final output on a digital meter.
• 47.
  1. Light Source Dependingon the spectral range, the following light sources are used: • In the UV range, deuterium lamps (180-350 nm) or hydrogen lamps (100-400 nm) are employed. • In the visible and near-infrared (NIR) regions of the spectrum, incandescent lamps (W) (320-1000 nm) are used. • Xenon lamps (100-800 nm) or Nernst lamps (400 – far-infrared) are occasionally used. • Mercury lamps are utilized for instrument calibration adjustments. Part of the UV and Visible radiation source is Tungsten lamp. UV radiation source is Deuterium or Hydrogen lamp. Range of wavelength 200-400 nm.
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  2) Entrance Slit Thanksto the slit, the radiation is parallelized, reducing background radiation. The narrower the slit, the less background radiation there is.
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  3) Monochromator (mono– single, chroma – colour, ator – donating Agent) – the main part of the instrument; the key characteristic defining the capabilities of the instrument, its degree of monochromatization. A monochromator is a device that allows the extraction of a light beam of a specific wavelength or a narrow range of wavelengths from a directed light beam. Monochromators can use optical filters or monochromators (prisms, diffraction gratings). Types of Monochromators Optical Filters Diffraction Grating Monochromator Prism Monochromator
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  Principle • A dispersiveelement disperse the polychromatic light into several bands of single wavelength and then a slit is used which stops the unwanted bands of light, allowing only the desired monochromatic light to pass through its exit point. • By fixing the slit and rotating the dispersive element, the direction of the dispersed light is turned so that the colour of the resulting monochromatic light changes. 10/29/2020 50
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  1- Prism Monochromator Whenelectromagnetic radiation passes through a prism, it is refracted because the index of refraction of the prism material is different from that of air. Shorter wavelengths are refracted more than longer wavelengths. By rotation of the prism, different wavelengths of the spectrum can be made to pass through an exit slit and through the sample. 10/29/2020 ANALYTICAL II CHEMISTRY PRESENTATION 51
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  Features of PrismMonochromators 10/29/2020 ANALYTICAL II CHEMISTRY PRESENTATION 52 A prism works satisfactorily in the ultraviolet and visible regions and can also be used in the infrared region. Because of its nonlinear dispersion, it works more effectively for the shorter wavelengths. Glass prisms and lenses can be used in the visible region. Quartz or fused silica must be used in the ultraviolet region. The entire monochromatic compartment must be kept dry.
• 53.
  Diffraction Grating Monochromator 10/29/2020ANALYTICAL II CHEMISTRY PRESENTATION 53 The dispersive element in grating monochromator is a reflecting diffraction grating. It provides a constant dispersion for all wavelengths and a low dependence on temperature. However, they produce relatively large amounts of scattered light and require the use of filters to block higher order light. Diffraction gratings are often used in modern instruments due to their superior dispersion properties. The most popular design for grating is the Czerny-Turner monochromator
• 54.
  Czerny-Turner Monochromator The inputlight is focused onto the input slit and therefore divergent after the slit. It is collimated by a curved mirror and hits a diffraction grating, which deflects different wavelength components in slightly different directions. A second curved mirror translates different beam directions into different positions on the exit slit, so that only light in a narrow wavelength region can get through that slit. 10/29/2020 ANALYTICAL II CHEMISTRY PRESENTATION 54
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  Prism vs DiffractionGrating Monochromator 10/29/2020 ANALYTICAL II CHEMISTRY PRESENTATION 55 PRISM MONOCHROMATOR DIFFRACTION GRATING MONOCHROMATOR Exploits differences in the material refractive index according to wavelength Wavelength dependency of dispersion is Variable, high for UV and low for visible High temperature dependency for dispersion Low Polarization Low stray light Exploits diffraction from a reflective surface with a regular grating structure Wavelength dependency of dispersion is High and approximately constant. Low temperature dependency for dispersion High polarization High stray light
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  Optical filters Optical Filtersare used to selectively transmit wavelength or range of wavelengths while rejecting the remainders. They are of following two categories • Absorptive optical filters • Dichroic optical filters 10/29/2020 ANALYTICAL II CHEMISTRY PRESENTATION 56
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  Absorptive and DichroicOptical filters 10/29/2020 ANALYTICAL II CHEMISTRY PRESENTATION 57 ABSORPTIVE OPTICAL FILTER DICHROIC OPTICAL FILTER Absorptive filters have a coating of different organic and inorganic materials that absorb certain wavelengths of light, thus allowing the desired wavelengths to pass through. Since they absorb light energy, the temperature of these filters increases during operation. Dichroic filters are more complicated in their operation. They consist of a series of optical coatings with precise thicknesses that are designed to reflect unwanted wavelengths and transmit the desired wavelength range.
• 58.
  Types of opticalfilters: 10/29/2020 ANALYTICAL II CHEMISTRY PRESENTATION 58 • Long-Pass Filter: A long-pass configuration transmits longer wavelengths above a specified range while attenuating shorter wavelengths. These filters are commonly used with dichroic mirrors and emission filters. • Short-Pass Filter: A short-pass configuration transmits shorter wavelengths over an active range while attenuating longer wavelengths. • Bandpass Filter: Short-pass and long-pass filters can be combined to form a bandpass filter, which features lower transmittance values and rejects any wavelengths outside a predetermined interval.
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  • Sample Holder Acuvette is a sample holding tube that can be made of plastic, glass, fibre etc. A cuvette with a blank solution helps in calibrating the spectrophotometer by giving a zero reference number. The calibration of the spectrophotometer is necessary to check the accuracy of the light source.
• 60.
  Cuvette compartment materials MaterialSpectral area of use Various types of glass 300 nm – 2.5 µm Crystalline quartz 185 nm – 400 nm; 700 nm – 3.5 µm Fluorite CaF2 125 nm – 200 nm; 3 – 7 µm Rock salt NaCl 6 µm – 15 µm Silvin KCl 10 µm – 20 µm KBr 15 µm – 25 µm
• 61.
  5. Radiation Detector(Radiation Receiver) a) Visual detector (eye) – devices with visual radiation detection, where the detector is the eye, suitable for operation only in the visible spectrum (from 400 to 700 nm). b) Photographic plate – used in emission spectral analysis. c) Photocells (PC) – are most commonly used as radiation receivers in modern spectral instruments used for quantitative photometric analysis – photoelectroсolorimeters and spectrophotometers. PCs refers to the photoresistor that detects the range of light transmitted from the test sample and transforms it into an electrical signal.
• 62.
  Photocell detector showsthe following properties: • High sensitivity • Short response time • Long-term stability • An electrical signal that can be easily amplified. According to the operating principle, PCs are divided into: a) PCs with a barrier layer (valve-type); b) PCs with an external photoeffect; c) PCs with an internal photoeffect (photoresistors). Among the photocells with an external photoeffect, the most common ones are: a) Antimony-cesium (Sb-Cs) – 180–650 nm; b) Oxygen-cesium (O-Cs) – 600–1000 nm.
• 63.
  For measuring highoptical density values, • photomultipliers (PMTs), • photodiodes and phototriodes are used as detectors, providing greater sensitivity and stability. Principle of operation of PCs with an external photoeffect: PCs represent an evacuated (vacuum) or gas-filled bulb with two electrodes. In this case, the cathode is photosensitive. Electrons knocked out of the photosensitive cathode are directed towards the anode, resulting in the generation of an electric current in the external circuit.
• 64.
  Application of UV-visiblespectroscopy in pharmaceutical analysis: 1. Authentication test of medicinal substances 2. Purity test. 3. Determination of the quantitative content of medicinal substances. Spectrophotometric determination typically involves the following stages: 1. Dissolving the analyzed sample in a solution. 2. Formation of a colored compound. 3. Measurement of the absorption of the test solution – photometry. 4. Calculation of the content of the determined substance in the analyzed sample and its metrological evaluation.
• 65.
  Methods of determiningsubstance content in spectrophotometry Methods of determining substance content in spectrophotometry are divided into absolute and differential. In absolute methods, the reference solution contains all components of the analyzed solution except the one being determined (such a reference solution is sometimes called a zero solution). In differential photometry, the reference solution contains a precisely known amount of the component being determined.
• 66.
  Methods of absolutespectrophotometry The absolute methods include • calibration curve (CC) methods, • comparative methods, • method of additions, • calculation based on the molar absorption coefficient.
• 67.
  1. Calibration curve(CC) methods A series of solutions (5-10) of the standard sample (SO) of the investigated substance is prepared with gradually increasing concentrations. The optical density of each prepared solution is measured at λmax, and a graph of dependence D = f(C) is plotted (Fig. 7). Then, the optical density of the investigated solution Dₓ is measured, and the desired concentration Cₓ is determined graphically. The content of the medicinal substance in percentage (X) is determined by the formula: 𝑿 = 𝑪𝒙 ∙ 𝑷(𝑽) ∙ 𝟏𝟎𝟎 𝒎 , where m is the mass (volume) of the medicinal substance or dosage form taken for analysis, in grams (milliliters); Cₓ is the amount of substance found according to the calibration curve, in grams per milliliter or percent; P is the mass of the dosage form, in grams; or V is the volume of the dosage form, in milliliters.
• 68.
  This method israrely used for determining the content of medicinal substances. However, the calibration curve allows determining: • the range of concentrations of the analyzed substance where a linear relationship between optical density and concentration is maintained • the values of specific absorption characteristics of the analyzed substances. Figure 7. Calibration Curve
• 69.
  2. Method ofAdditions The method represents a variation of the comparative method. It is based on comparing the optical density D of the investigated solution with the same solution containing an addition of a known quantity of the substance being determined. This method allows for creating identical conditions for the photometric analysis of these solutions and is widely used for determining low concentrations in the presence of large amounts of foreign substances. It is particularly useful for mitigating the influence of extraneous components when studying complex objects. The desired concentration is determined by either a computational or graphical method. If Cₓ is the concentration of the investigated solution, Dₓ is the optical density of the investigated solution, Cₐ is the concentration of the addition in the investigated solution, and Dₓ₊ₐ is the density of the investigated solution with the addition, then: 𝑪𝒙 = 𝑪𝒂 ∙ 𝑫𝒙 𝑫𝒙−𝒂 − 𝑫𝒙
• 70.
  3. Comparative Method Thecomparative method is used for one-time analyses, provided that the fundamental law of light absorption is observed. To determine the substance content using this method, an aliquot of the investigated solution (Vₓ, ml) is taken, necessary reagents are added to form a light- absorbing compound, and the optical density is measured under selected conditions. Then, similarly to the investigated solution, 1-3 solutions with known concentrations of the determined substance are prepared, and their optical density is measured under the same conditions.
• 71.
  By comparing theoptical density values of the standard solution Dstd and the investigated solution Dx​​, the average value of the unknown concentration Cx of the determined substance is determined. If two standard solutions C1 and C2 are prepared in such a way that the optical density of the first solution D1 is less than the optical density of the investigated solution Dx​, and the optical density of the second solution D2 is greater than Dx ​, then the unknown concentration of the investigated solution is calculated using the formula: 𝑪𝒙 = 𝑪𝟏 + (𝑪𝟐 − 𝑪𝟏) ∙ (𝑫𝒙 − 𝑫𝟏) 𝑫𝟐 − 𝑫𝟏 This method provides more accurate results when the concentrations (or optical densities) are sufficiently close.
• 72.
  4. Calculation Method(Calculation based on ε) A series of solutions with a known concentration of the analyzed substance is prepared, and the average value of the molar (or specific) absorption coefficient is calculated based on the measured optical densities: 𝜺𝝀 = 𝑫𝒔𝒕 𝑪𝒔𝒕 ∙ 𝒍 Then, a solution of the investigated substance is prepared with the same reagents and under the same conditions, and its optical density is measured. The concentration of the substance is determined by the formula: 𝑪𝒙 = 𝑫𝒙 𝜺𝝀 ∙ 𝒍
• 73.
  The total contentof the substance in the solution (mx​, mg) is determined by the expression: 𝒎𝒙 = 𝑪𝒙 ∙ 𝑽𝒙 ∙ 𝑴𝒙 ∙ 𝑽𝒕𝒐𝒕𝒂𝒍 𝑽𝟏 where: • V1 is the volume of the aliquot part of the analyzed solution taken to prepare the photometric solution, in Vx ml; • Vx is the volume of the photometric solution, in ml; • Vtotal is the volume of the investigated solution, in ml; • Mx is the molar mass of the determined substance, in g/mol; • Cx is the molar concentration of the solution, found using the average molar absorption coefficient, in mol/L. The calculation method requires strict adherence to the fundamental law of light absorption.
• 74.
  Differential Spectrophotometry Method Thedifferential method is employed to enhance the accuracy of analysis when determining large quantities of substances. When there is a high concentration of dissolved substance, the fundamental law of light absorption may be disrupted, or the optical density values of colored solutions may exceed the scale limits of the instrument. Further dilution of the analyzed solution is sometimes undesirable due to the dissociation of compounds. In such cases, the differential method of concentration determination is used. The essence of the method lies in measuring the optical densities of the investigated and standard solutions not relative to a pure solvent with zero absorption but relative to a colored solution of the determined element with a concentration Co​, close to the concentration of the investigated solution. The determination can be carried out using the calibration curve method or the comparative method.
• 75.
  Depending on theconcentration of the determined component in the comparison solution, one can distinguish between one-sided and two- sided differential photometry. In one-sided photometry, Co can be either less (direct order of measurement) or greater than the concentration of the calibration solutions, and consequently, the sought concentration of the analyzed component. The differential method, depending on the ways of measuring the relative optical density of the investigated solution and calculating its concentration, can have several variations.
• 76.
  Derivative Spectrophotometry • While differentialspectrophotometry enhances the accuracy of spectrophotometric analysis results, derivative spectrophotometry offers selectivity and, in many cases, increased sensitivity. In derivative spectrophotometry, the analytical signal is not the optical density itself but its derivative 𝑑𝑛𝐴 𝑑𝜆𝑛. Currently, derivatives from the 1st to the 5th order are employed in derivative spectrophotometry.
• 77.
  Derivative spectrophotometry isa modern variant of the spectrophotometric analysis method that is increasingly gaining popularity, especially in the analysis of complex multicomponent systems. It often allows the simultaneous determination of multiple components in a single sample without the need for special mathematical techniques for spectrum processing (see "multicomponent system analysis"). It also facilitates the identification of substances based on their derivative absorption spectra.