Добавил:
ivanov666
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Optical methods of analysis. Educational aid
.pdf
11
According to the location of the peak on the axis (λ, ν, υ), a qualitative analysis is carried out, and according to the magnitude of the peak, a quantitative
analysis is carried out.
Distinguish between atomic and molecular absorption. When light is
absorbed by a substance containing atoms of the same type (for example,
mercury or sodium vapor), only the energy of UV-radiation is absorbed.
This is enough to transfer the electron to a higher level, since the atom has
only one component of internal energy: E
int
= Eel. In this case, valence
electrons, which characterize a certain element, participate in absorption.
Therefore, the spectrum of an atom contains a limited number of peaks that
are used to identify elements (qualitative analysis). The content of the substance is determined by the magnitude of the peak (quantitative analysis).
The method of atomic absorption spectroscopy is widely used in analytical laboratories (despite the high cost of the device – atomic absorption
spectrometer), has high selectivity, sensitivity, analysis speed, simplicity and
convenience of analysis. You can determine up to 70 components in a few
minutes (at the level of 10-4–10
-10
%).
Atomic absorption of light by a substance consisting of molecules is
carried out after the preliminary atomization of the substance – a process in
which the molecules break down into atoms and ions that exist in a gaseous
state. Atomization is carried out in a flame, an electric arc, a spark. Absorption spectra arise when an atomic plasma (steam) absorbs energy corresponding to the energy of characteristic electronic transitions from the ground state
to the excited state.
The absorption of light by atomic vapor is described by the equation:
In=I0 e
-kl
,
where I0 is the intensity of the incident light; In is the intensity of the transmitted light; l is the flame length (atomic vapor layer thickness); k – absorption coefficient per unit length, proportional to the concentration of electrons
located at the lower level E0.
Molecular absorption spectra are a series of peaks that may be symmetrical or unsymmetrical. The spectrum has the following characteristics:
the number of maxima (or peaks), their position on the wavelength (or frequency) scale, the height of the maximum, and the shape of the peak.
We can see on Fig. 2.2 a view of the spectral curve in different regions of
the spectrum.
12
The appearance of absorption bands, due to the discrete nature of the energy states of absorbing particles and the quantum nature of electromagnetic
radiation, is associated with the absorption of a certain amount of energy, i.e.
with an increase in the internal energy of the molecules. This changes the magnitude or direction of the electric dipole moment of the molecule.
The mechanism of interaction of electromagnetic radiation with matter
in each region of the spectrum has its own character, which causes a different
contribution of individual components to the increase in internal energy.
Fig. 2.2. View of the spectral curve in the UV-, visible and IR-regions
of the spectrum
In the IR-region, a relatively small radiation energy is absorbed from 3
to 60 kJ/mol (0.7–14 kcal/mol), which is enough only to change E
oscillatory
and E
rotational
.
Each molecule is characterized by normal vibrations, in which the position of the center of gravity does not change. Under normal conditions,
the molecule is in the lowest electronic and vibrational states.
Energy vibrational states are characterized by directions, frequencies
and amplitudes of motion of atoms in a molecule. Depending on whether
the distances between atoms or bond angles change during the excitation of
vibrations, they are designated as stretching n or bending d vibrations. They
are characteristic of certain groups of atoms in molecules.
Normal vibrations are divided into two classes:
– skeletal vibrations affecting all the atoms that make up the molecule
to the same extent; their frequencies fall in the region 1400–700 cm-1;
– vibrations of characteristic groups, which refer to the vibrations of
individual parts of the molecule. For example, С–Н at υ = 2800 cm-1, С=С at
υ =1650 cm-1, >С=О at υ =1700 cm-1, –OH at υ = 3500 cm-1.
13
The rotational and vibrational absorption spectra have the form of narrow bands, they are individual for the analytes and serve to identify the components. The magnitude of the peak determines the amount of substance.
The method of IR-spectroscopy is widely used to determine organic
compounds containing a large number of functional groups.
In the visible region, the absorbed energy (> 60 kJ/mol) is sufficient not
only to increase Е
osc
and Е
rot
, but also to increase the energy of valence (op-
tical) electrons.
The method of UV- and visible spectrophotometry is based on the ability of substances to selectively (i.e., at strictly defined wavelengths) absorb
electromagnetic energy in the ultraviolet and visible region of the spectrum –
from 185 to 750 nm. The resulting absorption spectrum has the form of
a wide band with a maximum, since each electronic state corresponds to a set
of vibrational and rotational energy states. The magnitude of the peak is used
to judge the amount of the substance. The substance is identified by the wavelength, corresponding to the peak maximum.
In the UV-region, the absorption spectrum also has the form of a wide
band, since the radiation energy is spent on increasing E
osc
and E
rot
and on
excitation of electrons located at deeper energy levels of the atom. This corresponds to a large number of electronic transitions, as well as electrons involved in the formation of bonds in organic molecules.
For example, in the UV-spectrum of diazepam (benzodiazepine) there
are several absorption bands, which is explained by the presence of several
chromophore groups (Fig. 2.3).
Fig. 2.3. UV-spectrum of diazepam (solution preparation: 2 mg in 200 ml
of H2SO4 solution in 99.5 % ethanol (3:1000)
14
The inherent characteristic light absorption of the analyte arises due
to its electronic excitation. In this case, a transition occurs from the ground
(unexcited) electronic state to one of the excited states. At room temperature,
matter is usually in the ground electronic state. The substance absorbs the energy of incident light (i.e., a light beam passing through a cuvette with an
analyzed solution) at a certain wavelength. At the same time, it is energetically excited and passes into a higher lying (in energy) electronic state,
the lifetime of which is very short. The energy of such electronic transitions
corresponds to the energy of electromagnetic radiation.
The nature of the ground and excited electronic states of various chemical compounds is specific, therefore, individual compounds absorb light at
different wavelengths that are characteristic of them. If related compounds
contain the same structural fragments – chromophores (for example,
the same heterocycles, functional groups, ions, etc.), then bands located approximately in the same region are observed in their electronic absorption
spectra. The position of these characteristic bands changes when the solvent
is changed. A chemical compound, depending on its nature, may have not
one, but several bands in the electronic absorption spectrum.
Absorption spectrophotometry in the ultraviolet and visible regions of
the spectrum is used in the analysis. For this you need:
– compare the absorption spectra of the test solution and the standard
sample solution; in the indicated region of the spectrum, the positions of
the maxima, minima, shoulders, and inflection points should coincide;
– indicate the position of highs, lows, shoulders and inflection points;
the difference between the observed and indicated wavelengths at the absorption maxima and minima should not normally exceed ±2 nm.
However, the absorption band is characterized not only by the position
(wavelength of the maximum λ
max
or minimum λ
min
), but also by its intensity.
2 . 2 . T h e d e v i c e a n d p r i n c i p l e o f o p e r a t i o n o f
the s p e c t r o p h o t o m e t e r
Spectrophotometers designed for measurements in the ultraviolet and
visible regions of the spectrum consist of an optical system that emits monochromatic radiation in the region from 190 to 780, ensures its passage through
the sample and equipment for measuring optical density. Instruments for obtaining the spectrum consist of five main nodes, regardless of the region of
the spectrum in which the study of the absorption of light by a substance
takes place:
1) a stable source of radiation;
2) devices for selecting a limited region of wavelengths – a mono-
chromator;
3) transparent cuvettes for sample and solvent;
4) a detector or transducer that converts radiation energy into a meas-
urable signal, usually electrical;
5) signal recorder.
Fig. 2.4 shows a schematic diagram of a spectrophotometer.
A beam of light from a radiation source 1 (for example, an incandescent
lamp for the visible part of the spectrum or a gas-discharge hydrogen lamp
for the ultraviolet part of the spectrum) passes through a monochromator 2
(prism or diffraction grating), with which light of a certain wavelength is
selected, which then passes through a glass or quartz cuvette 3 of a certain
thickness, filled with the analyzed solution. In this case, a part of the light
energy corresponding to the wavelength of the electronic excitation of the analyzed substance is selectively absorbed by this substance. The light passing
through the cuvette falls on the detector (transducer) 4 and then on the recording device 5.
Fig. 2.4. Scheme of the spectrophotometer: 1 – radiation source;
2 – monochromator; 3 – cuvette with the test sample; 31 – comparison
cuvette; 4 – detector; 5 – registrar
In photometry, light filters (colored glasses) with a significant transmission width of 30–70 nm serve as a monochromator. Therefore, such light
is called pseudomonochromatic.
15
In spectrophotometry, the use of a prism or a diffraction grating as
Range
wavelengths,
nm
Type of
radiation
Radiation
source
Material for the
manufacture of
optical equip-
ment
Detector
1100–760
IR
Heated body,
globar
Halides metals:
LiF, LiCl,
NaCl
Thermocouple
Thermal
resistance
760–400
Visible
light
Incandescent
lamp
Glass
Photocell
400–200
UV
Hydrogen or
deuterium
lamps
Quartz
Photocell
a monochromator makes it possible to obtain a light beam with a small transmission width of 0.2–5 nm.
To study the spectra in different wavelength ranges, certain equipment
is required: a radiation source, optical equipment, a cuvette, a detector (radiation receiver), which are presented in Table 2.1.
Thus, in a spectrophotometer, a beam of light from an excitation source
(an incandescent lamp for the visible region, a gas-discharge hydrogen or
deuterium lamp for the UV-region) passes through a glass (in the visible region) or quartz (in the UV-region) cuvettes of a fixed thickness filled with
a solution test substance. In this case, part of the light energy corresponding
to the wavelength of the intrinsic (characteristic) electronic excitation of
the analyzed substance is selectively absorbed by it.
The light passing through the cell with the solution is directed to the entrance slit of the spectrophotometer, in which it is decomposed into a spectrum.
Table 2.1
Main components and materials used in spectroscopy
Spectrophotometers commonly used in analytical practice provide
a sufficiently high degree of monochromatization due to the use of special
dispersive elements – prisms and diffraction gratings. After decomposition
into a spectrum, the electromagnetic energy of light is recorded automatically
or by points in the form of a spectral curve. The latter is the dependence of
the optical density A (absorption) on the wavelength λ.
16
3 . P H O T O M E T R Y A N D S P E C T R O P H O T O M E T R Y
3 . 1 . B a s i c l a w o f l i g h t a b s o r p t i o n
The absorption method is based on measuring the attenuation of
the power (intensity) of a radiation flux as it passes through an absorbing
medium. When monochromatic (one wavelength) radiation passes through
a solution, the radiation flux is weakened due to the absorption of light energy by the particles of this substance. Fig. 3.1 shows the process of attenuation of radiation I0 during the passage of light through a solution with concentration c of the absorbing substance and layer thickness l. Transmitted
light intensity I < I0.
Fig. 3.1. Attenuation of radiation I0 by a solution with concentration c
of the absorbing substance and layer thickness l: I < I0
The decrease in intensity obeys the combined Bouguer–Lambert–Beer
law, according to which the intensity of radiation when passing through
a substance decreases as a power law depending on the concentration c and
the size of the layer of substance l:
I=I0・10
- εlc
,
where I0 is the intensity of the incident radiation; I is the intensity of the transmitted radiation; ε – the molar absorption coefficient characteristic of a given
substance depends on the wavelength, temperature, and pressure.
When deriving the law of light absorption, we consider the absorption
of photon energy by a layer of matter with an area S and an infinitely small
thickness dx, containing dn absorbing particles. The change in intensity due
17
18
to absorption by this layer dIx is proportional to the number of particles in it,
the relative change in intensity I/I0 when radiation is absorbed by the entire
volume V=S・l is proportional to the concentration of the substance c=n/V.
The value -lg I/I0 = εlc=A is called the optical density. Obviously,
the optical density increases in proportion to the concentration of the absorbing substance and the thickness of the layer through which the flow passes:
А = εlc.
The value I/I0 = 10
- εlc
is called the transmission T, which characterizes
the part of the incident radiation that has passed through the solution, expressed as a percentage. Optical density and transmission are related by:
А = - lg Т.
The intensity of light absorption at a selected wavelength is determined
by the value of optical density (extinguishing, extinction) – A or the value of
the molar coefficient of extinction (extinction) – ε. Optical density is a dimensionless quantity. The molar extinction coefficient is expressed in
units – l·mol
-1
·cm
-1
. The molar extinction coefficient is numerically equal to
the optical density of the solution at a dissolved light-absorbing substance
concentration of 1 mol/l and a light-absorbing layer thickness of 1 cm.
The optical density and extinction coefficient depend on the nature of
the absorbing substance and solvent, the wavelength of the absorbed light,
and the temperature.
Quantitative analysis is based on the Bouguer–Lambert–Beer law:
А = ε l С,
where А = – log (I/I0) = – log T – optical density; T – light transmission;
I0 and I are the intensity of the light flux directed at the absorbing solution
and passing through it; C is the concentration of the substance, mol/l; l is
the thickness of the light-absorbing layer; ε – molar coefficient of light absorption (extinction, extinction).
In the absence of the influence of other physico-chemical factors,
the measured optical density A is proportional to the concentration of
the substance in the solution C and the layer thickness l. To take into account
the loss of radiation due to reflection from the edge of the cuvette, absorption
by the walls of the cuvette and scattering when radiation passes through
the solution, the intensity is measured relative to a blank sample, for example, a solvent. In this case, the optical density is close to true.
19
The Bouguer–Lambert–Beer law is also applicable to solutions containing several absorbing substances, provided that there is no interaction between different compounds. Then, for a system consisting of n components,
the additivity law is valid:
А
total
= А1+А
2
+…+А
n
= ε1l1c1 + ε2 l2c2 + …+ εn l ncn.
That is, the general property of a multicomponent system is the sum of
the properties of individual components There are limitations to the applicability of the Bouguer–Lambert–Beer law. For the Bouguer–Lambert–Beer
law to be satisfied, the following conditions must be met:
1) monochromatic radiation – light of a certain wavelength (λ 1–20 nm);
2) a parallel beam of incident radiation;
3) dilute solutions;
4) solutions without complexing agents;
5) constancy of pH, T;
6) with a layer thickness of light absorption < 5 cm;
7) with a limited time for the formation of the color of the solution in
the study of absorption in the visible region of the spectrum.
3 . 2 . C o n d i t i o n s f o r c o n d u c t i n g q u a n t i t a t i v e
d e t e r m i n a t i o n s i n p h o t o m e t r y a n d
s p e c t r o p h o t o m e t r y
The photometry method is used for the quantitative analysis of onecomponent solutions. When this measure the optical density at the selected
wavelength in the visible region of the spectrum. The method is based on
the basic law of light absorption:
А = ε l c.
The equation shows that the main parameters of the photometric determination are the wavelength λ at which the optical density is measured, since
ε = f (λ), the value of the optical density A, the optical path length – the cuvette thickness l and the concentration of the colored solution c. In addition,
one should take into account the chemical factors associated with
20
the completeness and conditions of the spectrochemical reaction, the concentration of colored and other reagents, and their stability.
Each definition consists of two stages:
1) dissolution of the analyzed sample and appropriate processing of
the resulting solution – since most of the analyzed substances do not have
a color, it is necessary to introduce additional reagents, which, as a result of
chemical interaction with the analyzed substance, form intensely colored
compounds (in most cases, complex formation reactions are used);
2) measurement of optical density A or relative intensity of light absorption of colored solutions and determination of the concentration of
the analyte by one of the photometry methods.
Certain analysis conditions are selected depending on the properties of
the analyzed system and the characteristics of the device used.
1. Choice of filter. When determining in a solution of one light-absorb-
ing substance, the analytical wavelength is chosen at the maximum of the absorption band. This achieves the highest detection sensitivity. Measurement
of optical density on a photocolorimeter is carried out in approximately monochromatic light (transmission half-width 30–70 nm), for which light filters
are used. In this case, the color of the selected light filter is complementary
to the color of the photometric solution, i.e. the light filter must transmit light
in the wavelength range that is absorbed by the analyzed solution (Fig. 3.2).
Fig. 3.2. Absorption spectrum of the test substance
and the corresponding light filter
The optical density of the analyzed solution at c = const for a given
solution with a selected light filter should be maximum. Therefore, if
the color of the light filter or the wavelength interval in which the maximum
absorption of light occurs is unknown, the choice of the light filter is carried
Соседние файлы в предмете [НЕСОРТИРОВАННОЕ]