YEREVAN STATE MEDICAL UNIVERSITY AFTER M. HERATSI

DEPARTMENT OF PHARMACY

PHARMACEUTICAL CHEMISTRY

DRUG ANALYSIS SPECTROSCOPIC METHODS

Manual

for pharmacy students

YEREVAN 2020

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SPECTROSCOPIC METHODS OF ANALYSIS

Characteristics of electromagnetic specter

Among different physical methods of organic molecule structure identification electromagnetic

radiation with wide frequency range interaction with a substance has important role: started from

radio waves until -rays (whole electromagnetic specter).

Light wave consists of electric and magnetic fields perpendicular to each other, which amplitude

is changed according to sinusoid while the beam spreads (figure 1).

Figure 1: Electromagnetic light wave in yz plane of electric field: vector E and in xz

plane of magnetic field: vector H.

Wave energy is expressed by Plank’s equation:

E=hc/=h,

where h-Plank’s constant (6.63x10-34 J*sec)

c- light speed (3*108m/sec)

-wave length

-frequency.

Electromagnetic radiation can be characterized by either wave or energetic parameters. Wave

parameter could be expressed by wave length (A0, nm, mmk, mk, cm, m) and vibration

frequency: (sec-1), which are connected to each other by the following formula:

=c/

Wave number is often used (it is also called frequency). It is measured by cm-1:

ύ=1/λ

Energy transition between two energy levels is measured by electron-volts (ev) or calories (cal).

Connection between wave length and wave number is expressed by the following relation:

(nm)=107/ύ(cm-1).

When the light with I0 intensity falls on the tested substance, some of it reflects (Ir) from that

substance surface, the other portion is scattered by the substance particles (Is), and the third part

(Ia) is absorbed by the molecules. Radiation with residual intensity (I) comes out from the

substance (figure 2).

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Figure 2: Light beam intensity (I0) decrease in result of absorption (Ia), refraction (Ir)

and Scattering (Is).

Thus,

I0 = I + Ir + Is + Ia.

It is necessary to distinguish external (non-quantum) and internal (quantum) effects.

Ir by its value and direction is conditioned only by the macroscopic properties of the sample,

and doesn’t contain any information about its intermolecular structure.

Is is conditioned by substance particles diameter and light wavelength relation and reaches the

maximum value when the mentioned two parameters are comparable values. Scattered radiation

can be emitted from very small particles of the substance (Tindal scatter) as well as from its

molecules (Rayleigh scattering). In case of Raleigh phenomenon, excitation light results in

electrons compulsory vibration in the molecule. These vibrating electronic dipoles are a secondary

source of radiation, and the light emitted from them has the same frequency as the absorbed light.

This type of scattering, as well as reflection, is external effects of light and substance interaction.

Ia is the light portion which is absorbed by the molecules (internal effects). If absorption occurs,

it means that the molecule has been excited or has turned to an excited state.

The probability of this or that process is due to the nature of molecule. A change of molecule's

energy state is expressed by Bor’s equation:

E=Ef -Ei= h,

where E is a change of system energy.

Ef and Ei are the system energies in final (f) and initial (i) states.

If the final energy is higher than initial energy (Ef>Ei), E is positive and it corresponds to

beam absorption. If E is negative (Ef<Ei) energy is emitted. In the first case we have absorption

spectra, and in the second one emission spectra.

Thus, as noted, part of the radiation is absorbed by the substance under the influence of

electromagnetic radiation. In this case, light energy is transformed into another type of energy

(caused by exciting beam wavelength).

The entire specter of electromagnetic rays is presented in Table1, which is expressed in both

energy (ev) and wave parameters.

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Table 1:

Characteristics of electromagnetic specter

Radiation Wavelength (cm) Radiation

energy

(EV)

Processes - taking place

during emission and

absorption

-rays 10-11-10-8 107 Energy state alteration of

nuclei (-resonance

spectroscopy)

X-rays 10-8-10-6 105 Energy state alteration of

atom’s inner electrons

(Roentgen spectroscopy)

Ultraviolet and visible 10-6-10-4 10 Energy state alteration of outer

electrons (electronic specters)

Infrared 10-4-10-2 10-1 Vibration of atoms in

molecule (vibrational spectra)

Microwaves 10-1-10 10-3 Vibration of atoms in crystal

lattice, alteration of rotational

energetic state

Radio waves >100 <10-6 Alteration of nuclei and

electrons spins energetic state

(NMR, EPR spectroscopy)

Electromagnetic specter is spread from 10-10cm -rays to 105cm radio waves. Thus, wavelength

is changed by 15 orders in the electromagnetic specter. Energy according to specter is also altered

by 15 orders and is changed from 107ev (for rigid rays) to 10-8ev (for radio waves).

The high energy region of the specter begins with 10-11 wavelengths -rays which have 107ev

and more energy. This radiation leads to alteration of nuclei’s energetic state and gives information

about nuclei forces interactions (-resonance spectroscopy region).

The energy of 100,000ev order (wave length is 10-8cm) corresponds to Roentgen rays which

alters inner electrons’ energetic state closed to atom’s nuclei. The study of this interaction allows

to detect inner electrons bond energy (Roentgen spectral study region).

The energy with dozen ev order (wave length is greater than 10-6cm) responsible for UV and

visible region and corresponds to energy alteration of valent electrons (electronic spectroscopy

region). The whole UV region is divided into three main parts;

1. near UV region 300-400nm (it relates to visible region),

2. farther UV region 200-300nm,

3. vacuum UV 10-200nm.

Basic measurements by spectrophotometer is possible to conduct in near region and in farther

region with a special device. The vacuum UV region has no analytical usage.

Next region is the infrared region which is spread from 10-4 to 10-2cm wave lengths. The energy

of this region corresponds to energetic transitions within atoms’ vibrational levels in molecules

and is a component of ev decimal part (vibration spectroscopy region).

The microwave region relates to the infrared region. Microwave absorption is connected with

the atom’s rotational energetic state alteration and atom’s vibrations in crystal lattice (microwave

spectroscopy region).

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The final is the radio spectroscopy region (nuclear magnetic resonance- NMR; proton magnetic

resonance- PMR; and electron magnetic resonance- EMR region). These beams form alteration of

nuclei and electrons’ spins energetic state.

It is impossible to investigate an entire electromagnetic specter with only one device. The

methods used in spectroscopy are significantly changed passing from one region to another but the

main principles are the same.

In the absorption spectroscopy devices are used, which consist of the following structural

elements:

• light (radiation) source,

• monochromator which gives chance to separate monochromatic radiation,

• output gap,

• sample carrier or holder (in UV and visible spectroscopy: cuvette, in atom absorption

spectroscopy: flame, etc.),

• receiving-recording system, detector.

A radiation source should emit polychromatic light with a required intensity. However,

practically there is no universal light source that can emit radiation from the investigating whole

region (from vacuum UV to farther IR region). For example, the incandescent bulb is suitable only

for visible and near infrared regions and is absolutely useless for UV and farther IR regions.

A monochromator is required in spectroscopic methods, as for study either certain narrow

region of beam is taken (non-monochromatic light): in case of colorimetry, photocolorimetry or

beams of one wavelength (monochromatic light) in all spectroscopic methods. Before,

monochromator role played prisms, and currently they are replaced by optical nets which are of

course made of those substances which do not absorb in studied region (glass, quartz, potassium

bromide, etc.). The monochromator breaks the light into separate waves or waves bunch, which

passing through the narrow output gap pass through the tested substance.

A sample carrier as well as the entire optical system must be permeable for the beams of the

investigated region. The glass, for example, is not suitable for both IR and UV rays as it absorbs

in these regions.

A receiver, also called detector, transforms the received light signal to the measured electrical

tension.

From the mentioned it is clear that the studied whole region is impossible to study with the help

of one device. Additionally, there are quite different ways to prepare samples for implementation

spectroscopy in different regions.

It is important to consider about light sources in spectrometers:

● Any radiation source has a certain heating time: this means that continuous radiation is

received in a few minutes, after which radiation long-term stability is observed.

● As any incandescent bulb, the radiation sources can be worn out in spectrometers, which in

time leads to I0 intensity decrease.

● Light sources with the constant distribution of energy are emitted in a wide range of waves,

but the radiation intensities in this case are not equal, i.e. I0 is conditioned by wavelength.

Besides, detector sensitivity is conditioned by wave frequency, which, in its turn, is the base for

two types of spectrometers creation: single beam and double beam.

Single beam spectrometer

As it is seen from the name, there is only one light beam in these devices, which falls on the

receiver. These types of devices are intended only for quantitative analysis, in which the light

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intensity I0 is initially measured at the beginning of experiment only for one wavelength (or, if it

is necessary, for certain periods).

Figure 3: Principle of quantitative analysis (I0 and I detection).

As it is seen from the schematic diagram of the device, at first the light intensity is measured

without sample in the absorbing compartment (in practice, the absorbing compartment is not

empty, but contains a "zero" solution, for example, in the UV and visible spectroscopy it is the

solvent in which tested sample was dissolved).

Double beam spectrometer

These devices contain more complex optics and are more expensive than single beam

spectrometers. They are specially designed to record spectra. The necessity of these type of

devices is conditioned by dependence of light source I0 intensity from radiated wave length. As

for getting the specter it is necessary to pass rays with different wavelength through the sample,

for each of them it is necessary to record its I0 value, thus it is more expedient that the light passes

through the sample and comparing compartment. This can be performed with a rotating mirror: in

that case detector reacts at the same frequency, consequently accepting I and I0.

Figure 4: Schematic structure of double beam spectrometer.

Thus, only double beam spectrometers are used to record spectra, which, of course, give

possibility also perform quantitative detection. In such devices, absorbing compartments can be

found both before and after the monochromator, as shown in the figure.

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Absorption lаws

Spectroscopy methods of analysis are based on specific absorption of electromagnetic radiation

by tested substance and are used for light-absorbing substances: structure investigation,

identification, and quantitative analysis. Spectrophotometry is a spectroscopy analysis method,

which is based on monochrome radiation absorption by substance. Measurements, which are used

for detection of absorbed electromagnetic radiation, are based on two laws. The first law was

formulated by Lambert and then worked out by Bouguer in 1729. This law expresses the

connection between substance’s absorption ability and layer thickness. Parallel flux intensity of

light monochrome radiation passing through a homogeneous absorbing environment is decreased

by exponential law:

I=Ioe-kd,

where

Io is the intensity of falling monochromatic radiation,

I- intensity of the transmitted monochromatic radiation,

d- absorption layer thickness,

k- absorption coefficient which is individual for each wavelength.

This law can be expressed by logarithm form:

I=I010-k1

d,

where k1 is an inverse value of substance such property passing through which radiation bunch

decreases 10 times. Usually Bouguer-Lambert law is used as follows:

D=lglo/l=k1d,

where k1=0.4343k; D is an optical density which characterizes substance’s absorbing property;

d is an absorption layer thickness.

All substances follow Bouguer’s law.

The second law was formulated by Beer in 1862, which expresses the connection between

substance’s absorbing ability and its concentration in solution.

k=χc,

where c is solution concentration,

χ is an absorption coefficient of a solution, concentration of which is 1.

χ value is a physical constant typical for each substance and can be used for identification. χ

value base on optical density (D) measurement gives chance to detect given substance quantity in

solution of unknown concentration.

Thus, according to Bouguer-Lambert-Beer’s combined law has the following form:

I=Ioe-χcd.

Logarithm form is also used:

D=lglo/l=χ1cd, χ1=0.4343χ,

where χ1 and χ are absorption coefficients.

Bouguer-Lambert-Beer’s combined law is true only for monochromatic radiation and can’t be

used in spectrophotometric method.

In contrast to Bouguer-Lambert’s law, Beer’s law isn’t universal. Deflections from Beer’s law

are due to intermolecular interactions in the solution. Beer’s law practical usage for the given

substance is based on the correlation between optical density and concentration. When solution

follows Beer’s law, that correlation is linear for different wave lengths. Deflections from Beers’

law are usually in the infrared region of specter. It is conditioned by concentric solutions, which

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are used for absorption spectra measurements, where intermolecular interactions are strong

enough.

Absorption spectra expression forms

Spectral data are registered as a correlation of absorption index from wave length, thus, are

expressed by means of two changeable data’s-intensity and wave length factors. These two factors

expressing methods depend on working conditions, studied region and further usage of obtained

data. Wave length parameter can be expressed in the optical specter’s whole region by inverse

centimeters (cm-1) or depending on studied region: by angstroms (A0), nanometers (nm),

millimicrometers (mmk) for visible and UV regions, micrometer (mk) for infrared region. Rarely

frequency is used which is expressed by inverse seconds (sec-1).

Intensity index can be expressed by the following ways:

I/Io----transparency (by the unit parts),

I/Iox100%---transparency, % (by percent),

Io-I/ Io----absorption (by the unit parts),

Io-I/ Iox100---absorption, % (by percent),

D=lgIo/I---optical density.

In some cases, lnlo/l is used in the infrared region.

In the mentioned methods, layer thickness and concentration could be expressed by any units;

thickness-mm, cm; concentration-g/l, mg/l, mg/ml, mol/l.

Concentration (c) can be expressed by mol/l or g/100ml units. Depending on this, molar or

specific absorption coefficients are detected. The molar absorption coefficient (ε) or extinction is

the optical density of substance one molar solution with 10mm layer thickness and specific

absorption coefficient (E1cm 1%) is optical density of 1g substance containing solution in 100ml in

the same layer thickness conditions. In that case Bouguer-Lambert-Beer’s law is written as

follows:

D= εcd or ε=D/cd or D=E c d

Specific absorption coefficient can be converted to the molar absorption coefficient by using

the following formula:

ε= E1sm 1% x M/10,

where M is substance molar mass.

Intensity coefficient can be expressed in the ordinate axis as ε or lgε. The last expression is used

in UV region, where substance absorption intensity is changed by several orders.

If the absorption curve width is comparative to device’s spectral hole width, so the absorption

curve form is distorted and the absorption molar coefficient value doesn’t correspond to the real

value and is called absorption “seeming” factor ε0.

As shown in the graphics (figure 5), the form of absorption layer depends on the ordinate

system choice, where registration is conducted. For comparing absorption layers, it is desirable to

observe built graphics in the same coordinate system. Moreover, only using ε, lgε and intensity

factors shape of absorption layer doesn’t depend on concentration.

Absorption specter can be characterized by the following values:

1. absorption maximum wave length and that maximum intensity,

2. absorption minimum wave length and intensity in that part,

3. absorption layer curve: inflection (wing) by corresponding wave length and intensity in that

part.

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Figure 5: Electronic spectra of fenantrene absorption in different coordinate systems.

Absorption electronic spectra characteristics according to organic molecules structure

In the 1000-10.000 Å region absorption is conditioned by electronic state alterations in a

molecule. Consequently, in UV and visible regions, absorption spectra are called electronic.

Molecule has quantum energetic levels and its energy consists of electronic, vibrational and

rotational states energies’ sum:

E=Eelec. + Evibr. + Erot.

Radiation absorption leads to alteration of that states’ energies, in which result electronic,

vibrational and rotational spectra are formed (figure 6). Moreover, each electronic level has its

vibrational and rotational levels.

10-1-10-3 kcal/mol energy corresponds to rotational spectra, thus they are in farther infrared and

microwave regions. Vibrational spectra are expressed in middle and near infrared regions, and

electronic spectra are formed due to transitions between electronic states and are observed in

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ultraviolet and visible regions. In a result of such energy absorption, beside electronic transitions

simultaneously alterations are carried out also in vibrational and rotational states.

Figure 6: Diatom molecules energetic levels’ scheme:

E-electronic levels, ν-vibrational levels, and j-rotational levels.

In the organic molecules, UV and visible beams absorption is conditioned by single and

multiple bonds’ valent electrons (σ, π electrons) and heteroatom’s unshared electron pair electrons

(n-electrons) transitions. These electrons have different energies and for that reason they are

excited under different wave lengths influences. In the organic molecules electrons’ energetic

levels order is presented in the figure 7.

anti-bonding σ*-orbital

anti-bonding π*-orbital

non-bonding n-orbital

bonding π-orbital

bonding σ-orbital

Figure 7: Electronic transitions in molecules.

As it is shown in the scheme, bonding π-orbital’s energy is higher than bonding σ-orbital’s

energy. For the anti-bonding orbitals, the relation is reversed: σ*- orbital’s energy is higher than

π*-orbital’s energy.

Electrons transition from the bonding orbital to the appropriate anti-bonding orbital is

mentioned by N―›V transition. These transitions are σ―›σ* and π―›π*.

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σ―›σ* transitions require the highest energy (figure 7), therefore, their absorption layer is in

the vacuum UV region (<170nm). π electrons excitation demands less energy and absorption for

π―›π* transition is in longer wavelength region of the specter. Electrons located on n-levels could

pass to the anti-bonding π* and σ* orbital. n―›σ* and n―›π* transitions are mentioned by N―›Q.

The intensity of n―›π* transition absorption layer usually is quite less than other transition curves

intensities. In organic molecules which don’t contain π and n electrons, the only transition is

σ―›σ*. In saturated compounds, the presence of an atom with an unshared electron couple leads

to the n―›σ* transition located in the longer-wave region than the σ―›σ* transition. n―›π*

transitions occur in those compounds where a heteroatom is linked by multiple bonds to another

atom. In simple non conjugated systems these transitions are the longest-waves. In case of

conjugation bonding, the π orbital with high energy could have more energy than n-orbital and

thus, π―›π* transition curve will be a longer wave.

Polyatomic molecules which include electrons in different states and different transitions (from

general state to excited states) could occur under radiation.

Absorption curve displacement is seen due to intermolecular and intramolecular interactions as

a result of energy alterations of general and excited states. However, it could occur as a result from

energy alteration of the general state or energy alteration of both states. If in result of general and

excited states’ energy alteration difference between them isn’t changed so that appropriate curve

isn’t displaced, though, in this case can be alteration of electron density distribution.

Absorption curves are characterized by wave length and absorption intensity within the electron

spectrum. The absorption curve’s wave length, which is responsible for the electron’s transition,

corresponds to that electron’s transition energy.

In electronic spectroscopy, absorption curve intensity is measured by the molar coefficient

value in the curve maximum (εmax or lgεmax). The absorption curve also can be characterized by

intensity integral -A:

A=∫ευdυ

Where ευ is the molar absorption coefficient in the case of υ frequency and υ1 and υ2 are wave

numbers. The intensity integral is measured by the absorption curve area within the ε―υ

coordinates.

The absorption curve intensity is detected by the possibility of transition.

The transition possibility between m and n states is detected by the transition moment:

Mmn=∫ψ*mMψndτ,

where ψ-wave function, τ-dependence on time.

Several rules exist, which determine conditions that inhibit absorption within the spectrum.

They are called rules of choice:

1. Rule of choice according to spine

Transitions are prohibited during which electron spine alteration is carried out. However,

sometimes due to spine-spine and spine-orbital interactions absorption curves responsible for those

transitions appear in the spectrum with less intensity (≤10-6). In heavy metal containing molecules,

spine-orbital interactions are significant and their appropriate curves are appeared with expressed

intensity.

Transitions that occur without electron spine alteration are called singlet-singlet. Transitions

that occur with electron spine alteration are called singlet-triplet.

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2. Rule of choice according to symmetry

The transition moment is a vector quantity and could be divided into X, Y, and Z parts. If

molecule’s general and excited states’ symmetry is so, that all these integrals equal 0, then the

transition is inhibited according to symmetry. When even one integral is differed from 0, transition

is allowed.

3. Rule of choice according to local symmetry

Compounds that include unsaturated groups like C=O, C=S, C=N, N=N, N=O yield absorption

curves with singlet-singlet n―›π* transitions. The intensity of this absorption curve isn’t high. If

n electrons are on the non-hybridized p-orbital, then the transition moment is equal to 0 and the

n―›π* transition is inhibited. In this case, transition is prohibited according to local symmetry. If

electrons are located on hybrid orbitals, then the transition moment does not equal 0 and its

quantity is detected by atoms inserted into hybrid orbitals. Hybrid nsp orbitals are located on the

bottom of np orbitals according to their energy, therefore absorption curves responsible for np―›π*

transitions will be in the longer wave regions than absorption curves of the nsp―›π* transition.

4. Inhibition rules where more than one electron excitement occurs.

Electron transition is well illustrated in the formaldehyde molecule which contains σ, π and n

electrons.

The formaldehyde molecule has a planar structure in the general state and 12 electrons are

distributed in the 6 following orbitals:

1. C-H group symmetric and non symmetric bonding σ-orbitals,

2. C=O group bonding σ-orbital,

3. C=O group bonding π-orbital,

4. bonding orbital of oxygen atom’s unshared electron couple.

One of the unshared electron couples of an oxygen atom is in a p-orbital (np) and the second is

in a hybrid sp-orbital (nsp).

High energy occupied orbitals will be non bonding p-orbitals, then bonding π-orbital, non

bonding sp, and bonding σ-orbitals.

The reduced-energy non occupied orbital of formaldehyde molecule will be an anti-bonding π-

orbital (π*), and the following is an anti-bonding σ-orbital C=O (σ*):

Figure 8: Scheme of formaldehyde electronic transitions energetic levels.

Longer wave curves in the formaldehyde spectrum λmax=295nm (εmax=10) belong to the

transition from the non-bonding p-orbital to the anti-bonding π* orbital, referred to the n―› π*

transition. The corresponding curve intensity is less studied because this transition is inhibited

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according to local symmetry. In the shorter wave region, formaldehyde also has 2 intense

absorption curves in the UV region. The first curve is 185 nm and the second is 155 nm and belong

to transitions, respectively np―›σ* and π―›π*.

Chromophors are the functional groups which provide absorption in the UV and visible regions.

Auxochromes are the functional groups (-OH, -OR, -NH2), which form complexes with

chromophors leading to bato-chrome, hypso-chrome shifts, and hyper-chrome, and hypo-chrome

effects.

Bato-chrome shift or “red shift’’ is the curve’s shift to the long wave’s side. For example, alkyl

groups give such kind of displacements, which are located near the chromophors.

Hypso-chrome shift or “blue shift’’ occurs when the curve is shifted to the short wave’s side.

Hyper chrome effect leads to an increase in absorption intensity.

Hypo chrome effect leads to a decrease in absorption intensity.

Factors influencing on the chromophore’s absorption ability

pH, solvent or neighboring molecules polarity, and neighboring chromophores relative

orientation influence on the chromophores’ absorption ability. These factors are based on

adsorption spectroscopy which is used for the macromolecule’s characterization.

pH- influence. Solution pH detects chromophores ionization degree and form. pH- influence

on tyrosine specters is presented in the Figure 9.

Figure 9: Thyrosine absorption specter at pH 6 and pH 13 conditions.

Increasing OH- group dissociation of phenolic residue both λmax- and ε- increase.

Polarity influence. In case of polar chromophores (especially, if molecule contains O, N or S

atoms) in the hydroxyl group (H2O, alcohols) containing polar solvents λmax- is observed in case

of the shorter waves than in non-polar solvents.

Solvent polarity influence on tyrosine specter is presented in the Figure 10.

It is shown λmax-shift in less polar solvent.

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Figure 10: Solvent’s polarity influence on tyrosine absorption specter

solvents: water (unceasing curve) and

20%- ethylenglycol (dotted line curve).

Orientation influence. λmax- and ε- values depend on geometric features of the molecule. It is

known nucleic acids hipochromy, i.e. nucleotide absorption coefficient value decrease, when free

nucleotides are included in polynucleotides content, in which nucleotides bases sequentially

binding with each other are located close.

ε- value further decrease are noticed in polynucleotides helix, where nucleotide residues have

more streamlined ordering. This is shown in figure 11.

Figure 11: Optical density decrease characterizing hypochromy, DNA absorption

specters for mono-chain (1), bi-chain (2) forms and free nucleotides (3) case.

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In result of large number of important biological compounds and macromolecules research,

structures of which are studied in various conditions, a number of experimental facts were

collected which can be called absorption spectroscopy applicable rules used in biochemistry. There

are:

1. If aminoacids (tryptophan, tyrosine, phenylalanine and histidin) are in the less polar

environment, λmax- and ε- are increased. Therefore, if λ max and ε greater value is noticed in the

specter of amino acid (from the protein composition) in polar solution, than in free amino acid

specter in case of the same solvent, so the amino acid is in the protein inner part (it is “hidden”)

and covered with non-polar amino acids. If the protein specter is sensitive toward solvent polarity

changes, thus the amino acid for which there is noticed λmax- and ε- change must be located in

the protein surface.

2. In case of aminoacids λmax- and ε- are always increasing, when titrated group (eg, tyrosine

OH- and cystein SH-) are charged. Therefore, if it is not noticed change in the any of these amino

acids specter, and the pH- is such that free amino acids ionization should take place, so it is hidden

in the protein non-polar part. If the amino acid groups ionization pK- value, which is detected by

spectral change at the pH-change, is such as free aminoacids in the solution, the amino acid is in

the protein surface. If pK- value, detected by specter change at changing pH is sigtificantly

different, so the amino acid is probably in very polar environment (e.g. tirozine surrounded by

carboxylic groups).

Electronic absorption spectra of different organic compounds:

Aliphatic compounds

Saturated compounds. In saturated compounds (alkanes and cyclic alkanes) containing only

simple bonds, the σ―› σ* transition is possible and appropriate curves are in the vacuum UV

region. For example, methane has an absorption curve in 125nm and ethane in 135nm. In the

spectrum of saturated compounds containing heteroatoms with unshared electron couples, long

wave curves belong to the n―›σ* transition. In this absorption region, other curves can be appear

occurring as a result of a σ―› σ* transition.

Table 2:

Unsaturated compounds. Unsaturated carbohydrates with isolated double bonds have

intensive absorption curves due to a π―› π* transition in the 165-200nm region. Ethylene absorbs

in the 165nm region. Alkyl substitutes in ethylene carbon atoms lead to a π―› π* transition curve

dislocation to the long wave side and appropriate absorption is occurrs in 175-200 nm. Cyclic

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unsaturated carbohydrates have a spectrum similar to alkenes. Thus, π―› π* transition curves of

cyclic compounds appear in 183 nm. Alkenes and cyclic alkenes absorption intensity’s change

from ε=6500 to ε=12000.

In acetylene carbohydrates with C≡C isolated bonds, the π―› π* transition curve occurs. The

acetylene absorption curve is in 173nm, alkyl acetylene has absorption in λmax=187nm and

dialkylacetilene is characterized by an absorption curve with λmax =190nm.

Table 2:

Double bonds lead to absorption curve dislocation to the long wave side and increase intensity.

Polyene spectrums are characterized by vibration structures of the absorption curve. Distance

between vibration peaks is equal to 1500-1600cm-1 which corresponds to an alteration of the C=C

bond vibration state in the excited electronic state.

Figure 12: Figure 13:

In the polyene chain, the C=C bond substitution by C≡C bond practically does not change the

absorption curve location but does lead to a decrease in intensity.

Cyclic dienes result in absorption in longer waves than linear compounds. Their absorption

intensity is low.

Compounds, that contain conjugated triple bonds C≡C (polyenes), have several absorption

curves with vibration structures. Diacetylene has absorption in the 210-250nm region with average

intensity. In the polyene spectra, expect average intensity curves in the 340-390 nm region.

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Absorption curves with higher intensities also appear in 200-280 nm region (ε>100000), as well

curves with vibration structures. Distance between vibration peaks of polyacetylene is about

2000cm-1.

Carbonyl compounds

Carbonyl compounds are aldehydes, ketones, carbonic acids and their esters, chloranhydrides,

anhydrides and amides. All of these compounds contain heteroatom binding by multiple bonds.

As mentioned above in these groups, three types of transitions are possible: π―› π*, n―› π* and

n―›σ*.

Absorption responsible for the n―›π* transition is more likely for carbonyl compounds. The

n―› π* transition curve has the following characteristics:

1. The molar absorption coefficient is not great (for C =O, ε≤100, C=N, ε≤2000).

2. Solvent polarity increase lead to dislocation of the n―› π* transition curve maximum to the

short-wave side (blue shift) due to a decrease in general state energy and an increase in excited

state energy.

3. In an acidic environment, the n―›π* transition curve disappears due to a blockade of

heteroatom’s unshared electron couple.

4. Usually less energy corresponds to the n―›π* transition and the curve is in the longer wave

within the spectrum.

Saturated carbonyl compounds. As mentioned above, formaldehyde has 3 absorption

curves with maximums of 295, 185, 155 nm. These curves correspond to n―›π*, n―›σ* and

π―›π* transitions, respectively. Short wave curves (λmax=155 nm), are more intensive. n―›π*

transition curves are inhibited according to local symmetry and differ by small intensity (ε295=10).

Saturated aldehydes and ketones have 2 absorptions within the vacuum UV region. One of

these is in 170-200 nm regions and the second is in 150-170nm region.

Longer wave curves belong to the n―›σ* transition (acetaldehyde λmax=182 nm, lgεmax=4.01

and acetone λmax=195 nm, lgεmax=3.96) whereas short wave curves (acetaldehyde λmax=167 nm,

lgεmax=4.3) belong to the π―›π* transition. Saturated aldehydes and ketones belong to the n―›π*

transition curve in the 270-290nm region. The transition of formaldehyde to acetaldehyde and

acetone results in n―›π* transition curve hypsochrome shift.

Table 4:

Carbonyl group’s n―›π* transitions absorption maximum values in different

environments

Compound λmax nm εmax Solvent

HCHO

CH3CHO

CH3COCH3

CH3COOH

CH3COCI

CH3COOC2H5

CH3CONH2

295

290

279

204

235

204

214

10

16,6

14,8

41

53

60

-

Vapors

Heptan

Hexan

Alcohol

Hexan

Water

››

17

This occurs due to the inductive effect of the alkyl group that increases carbonyl groups’ excited

π* level energy while energy of non-bonding n-electrons is not changed. Aldehyde group hydrogen

substitution by OR, NR2, Hal groups results in n―› π* transition curve hypsochrome shift, which

is also due to an increase in excited π* state energy and saving n-electrons’ energy.

Figure 14:

Unsaturated carbonyl compounds. Conjugation of short bonds in carbonyl groups leads to

π―›π* and n―›π* transitions dislocated to the long wave side in comparison with isolated

chromophore groups. α, and β unsaturated aldehydes and ketones are characterized by intensive

curve of the π―›π* transition (ε≈10000) in the 220-200nm region and by low intensity curve of

the n―› π* transition (ε<100) around 320nm. In case of solvent polarity increases, the π―› π*

transition curve undergoes a red shift and the n―› π* curve undergoes a blue shift so low intensity

that long wave curve sometimes occur like a wing. In the spectra of α, and β unsaturated acids and

their analogs, batochrome shift of the absorption curve is also occurs.

Figure 15: UV-specter of mesityl oxide in 1) heptane, 2) ethanol, 3) water.

Aromatic compounds

Benzene has 3 absorption curves due to π-electron transition. Shorter wave curves belong to

the π―›π* allowed transition which is a maximum of 180 nm (ε180=50000). The next curve is

inhibited according to symmetry and the intensity is ε≈7000.

18

The typical absorption for benzene, “benzene absorption curve”, is located in the 230-260nm

regions. It is inhibited according to symmetry, has less intensity (ε≈200), and expressed vibration

structure.

Substituent insertion in the benzene ring leads to spectrum dislocation due to substituent nature.

If the substituent possesses only inductive effects (-N+R3), then a change to the benzene spectrum

is slight. A change during substitution is due to a conjugation effect and is determined by the

interaction force of the substituent and the benzene ring π electron. It is not determined by the

substituent nature that is, substituent is electro donor or electro acceptor.

Alkyl substituents and halogens result in a slight curve shift to the long wave side and increase

in intensity. The vibration structure of benzene ring is unchanged.

Figure 16:

Insertion of polar groups with an unshared electron couple in the benzene ring (OH, OR, NH2,

NR2) leads to expressed batochrome shift. The benzene ring intensity is increased about 10 times.

In an aniline molecule, vibration structure almost disappears. This change of spectrum is due to an

interaction of heteroatom’s unshared electron couple and benzene ring electrons. In case of

unshared electrons pair blockage (e.g. salt formation of amino group), the absorption specter

becomes similar to the specter of benzene alkyl homologs.

Multiple bonds containing substituents lead to an increase of the absorption curve intensity and

dislocation to the long wave side. If the substituent is a carbonyl group, n―›π* transition curves

are seen. They are well visible in the benzaldehyde and acetophene spectra in non-polar solvents.

In polar solvents, the n―›π* transition weak curves are covered by the benzene absorption curve.

The n―›π* transition curve is not detected in benzoic acid and its derivatives.

19

Figure 17: Figure 18:

The n―›π* transition curve is expressed clearly in the spectra of aromatic nitrozo compounds.

In the nitrobenzene spectrum, intensity is also low and belongs to the nitro group n―›π* transition.

Figure 19: Figure 20:

20

Figure 21: Figure 22:

The other part of the nitrobenzene specter is strongly altered in comparison to specter of other

mono substituted benzenes. A strong change in specter also is seen for the phenyldiazonium cation.

For nitro and carbonyl compounds, intermolecular charge movements may occur and nitrobenzene

(λmax=252 nm, ε=8660) short wave absorption is considered a curve of charge transition. In case

of accumulation, conjugated multiple bonds in substituent wide intensive absorption curves occur.

Figure 23:

If two substituents are in the benzene ring, the spectrum of the compound is due to the

substituents’ nature and position to each other. The greatest change of spectrum is noticed in those

bisubstituted benzene compounds which contain both electron donor and electron acceptor

substituents, however, spectrum nature is due to their position. Ortho- and meta- isomers have

similar spectra but the para-isomer spectrum differs sharply and as a rule has one intensity

absorption curve.

21

Addition of different substituents in the benzene ring or their alteration could bring significant

changes in the spectrum.

Figure 24: Figure 25:

Figure 26:

Heterocyclic compounds

Saturated heterocyclic compounds which contain one or more heteroatoms with unshared

electron couplets have absorption curves responsible for the n―› σ* transition. The absorption

curves of oxygen- and nitrogen-containing compounds are located in the vacuum UV region.

Sulfur containing compounds have absorption curves in the UV region.

Five-membered unsaturated heterocyclic compounds. These compounds have 2 absorption

curves: an intensive short-wave curve in the 200-210 nm regions and a less intensive curve in long

wave region. Furan long wave curve has less intensity (ε≈1) and thiophene has greater intensity

(ε≈4500) (table 5).

22

Table 5:

Non saturated five-membered heterocyclic compounds’ absorption

Compound λmaxnm εmax λmax nm εmax Solvent

Furan

Thiophen

Pyrol

Imidazol

Pyrazol

1,2,3-Triazol

Thiazol

200

-

210

210

210

210

-

10000

-

15000

5000

5000

3980

-

252

235

350

250

250

-

240

1

4500

300

60

60

-

4000

Hexane

››

››

Alcohol

››

››

››

In polar solvents, furan and pirole long wave curves have vibration structure. Insertion of a

substituent containing an unshared electron couple or multiple bonds leads to a batochrome shift.

Condensated five-membered heterocyclic benzene rings containing compounds also have 2

absorption curves with vibration structure.

Table 6:

Absorption of furan derivatives

Compound λmax, nm εmax λmax, nm εmax Solvent

Furan

O

CHO

O

COOCH3

O

COCH3

O

CH=CHCOOH

200

278

245

275

303

10000

15000

17000

13900

50000

252

-

-

-

-

1

-

-

-

-

Hexane

Water

Alcohol

Water

Hexane

23

Figure 27:

Figure 28: Absorption spectra in heptane: 1) indole, 2) carbazole

Six-membered aromatic heterocyclic compounds. Spectra of aromatic monoazicyclic

compounds (pyridine, chinoline, akridine) differ slightly from their respective carbohydrate

spectra. The difference is that long wave absorption curve intensity is increased and vibration

structure planarization is seen within the spectra.

Table 7:

Compound λmax նմ εmax λmax nm εmax Solvent

Pyridin

Quinoline

Acridine

195

275

252

7500

4500

170000

250

311

347

2000

6300

8000

Hexane

››

Alcohol

24

Figure 29: Figure 30:

=CH group substitution by =N- in aromatic compounds leads to n―›π* transition. n―›π*

absorption does not occur in the pyridine molecule; it appears like wing in the spectrum. The

addition of nitrogen atoms in the cycle dislocates n―›π* to the long wave side and for diazo cyclic

compounds, it is distinctly different. In the symmetric tetrazine molecule, it is seen in visible

region.

Figure 31: Figure 32:

Electronic spectra usage for the detection and identification of organic compound

structures

Electronic spectra in the 200-800nm interval are used widely for organic compound structure

identification and detection because measurement below 190 nm demands complicated devices.

As it is known, aliphatic and alicyclic carbohydrates and their derivatives, alcohols, ethers and

amines, don’t absorb in the 800-200 nm region. Mono-olefins and mono-acetylenes absorption

also is within this limit. The absorption curve end of the chlorine alkyl and unconjugated carbonic

acids is in the 200-250 nm regions. Thus, these compounds are not studied by common UV

spectroscopy.

25

Electronic spectra are used for those organic compounds which contain conjugated multiple

bonds, non conjugated compounds with heteroatoms, n―›π* transition curves above 200 nm, and

for some halogen derivatives.

In the study of organic compound absorption, we are guided by the absorption molar coefficient

and wave length maximum. Absorption low intensity curves (lgε≤2) are due to carbonyl and

thiocarbonyl groups in aldehydes and ketones, nitro- and nitrozo- groups. Absorption curves in the

250-300 nm lgε=2÷3 region is due to common benzene compounds. Those curves usually have

vibrational structure.

Intensive absorption curves (λmax>200 nm and lgε≥4) characterize compounds with conjugated

bonds. Absorption curve position is due to molecular structure and the chromophore groups’

position to each other. If chromophores are linked directly, then considerable changes are noticed

in the specter in comparison to those compounds with separated chromophores.

If chromophores are linked by one methyl group the interaction between them is decreased

significantly and a change in specter is not great. This is also true when intermolecular interactions

do not occur (for example, keto-enol tautomerization).

If two chromophores are separated from each other by two or more methyl groups, then the

specter appears as an overlap of compounds spectra containing an isolated choromophores specter.

In some cases, if chromophores are divided by saturated groups, the specter is changed. This

may be due to chromophores’ close proximity, causing their π- electrons to interact with each

other. Intermolecular interaction of this type occurs in carbonyl compounds. For example, for

ketone carbonyl group absorption, the curve is dislocated to 305 nm and the intensity is great which

is due to the carbonyl group location near to the double bond.

1. Conjugated and non conjugated compounds easily differ with respect to UV spectra.

2. Multiple systems’ structures in unsaturated carbohydrates and carbonyl compounds are

determined by the absorption curve maximum value.

3. Conjugation breaking connected with steric factors leads to a change in the absorption

specter. For instance, large volume substituent insertion at ortho-position of diphenyl compounds

dislocates molecular co-planarity and the absorption specter becomes similar to the appropriate

substituted benzene specter.

The same is noticed in ortho-substituted anilines where substituents break nitrogen n-

electrons’ interaction with benzene ring π-electrons.

4. UV-spectroscopy could be used for identification of cis- and trans- isomers if they include

conjugated multiple bonds, however, as a rule, the intensity of the π―›π* transition long wave

absorption curve for the trans isomer is always higher than the cis-isomer.

For polyene-chains containing compounds, a configuration change even for one multiple bond

is expressed on the absorption specter. For instance, trans-lycopene has complete absorption

curves with λmax=504 nm (ε=170000) and 470 nm (ε=186000), absorption curves of neo-lycopene

A are in λmax=500 nm (ε=100000) and λmax=470 nm (ε=122000).

26

It is necessary to compare the spectra of tested substance with the substances of known spectra

for studying organic compound’s structure by using absorption spectra.

Since UV spectra are mainly found in conjugated unsaturated bonding systems, the spectra of

substances containing such multiple bonding systems can be used as a model compound. It should

be considered that when comparing the absorbtions of tested substance and model sunstance it is

not enough to use only the wavelength and intensity, the whole range should be compared, and only

in case of the entire absorption layer is matched conjugated system identification can be carried out.

UV spectroscopy method is used for both drug identification and quantitative analysis. In this

case the calculations are performed using Bouguer-Lambert-Beer’s law. The electronic spectra of

drugs are obtained by using very dilute solutions (10-2-10-5M). As solvents those substances can be

used that do not form absorption in the studied region and do not interact with the dissolved

substane. In the UV and visible region spectroscopy, most frequently used solvents are saturated

hydrocarbons, water, alcohols, simple ethers and acids.

It should be considered that the solvents influence on the tested substance absorption specter.

The observed changes are conditioned by the nature and properties of solvent. If the non-polar

compound is dissolved in non-polar solvent, the specter is similar to the gas phase specter. In the

polar solvents, the specter of non-polar compounds is modified by the dipole-induced dipole

interaction. More significant specter changes are observed dissolving the polar substance in polar

solvent. They are conditioned by strong dipole-dipole interactions. In the protonated solvents

hydrogen bonds are also formed, which moreover modify the absorption spectra of substances.

The usage of UV-spectroscopy method in molecular biology and biophysics

UV-spectroscopy can also be used for detecting macromolecules structural parameters (e.g., α-

spiral degree). By UV-spectroscopy, it is possible to control protein molecules interaction and

detect conditions of that process, kinetics.

By UV-spectroscopy method spiral-coil transitions in proteins or denaturation and renaturation

in DNA double chain are detected in case of pH, temperature, ionic forces change. hidden

chromophore inside the protein in case of denaturation (macromolecules conformation

modification), becomes accessible towards solvent, which is accompanied by hyperchromism.

One of the applications of UV-spectroscopy is proteins’ spectroscopic titration. In many studies

of protein structure, there is a need for protons dissociation pK detection of amino acids ionized

terminal groups, as this value indicates amino acids location in proton. It can be often detected by

spectroscopic method, as parallel with dissociation any of the chromophores specter is changed

(e.g., in case of tyrosine). Consider hypothetic protein containing tyrosine. Let's assume that 5

tyrosine residues are contained. If all of these are located on the protein surface and are ionized

increasing the pH, thus the specter responsible for tyrosine residue at high pH conditions will be

close to free tyrosine specter, which is expressed in Figure 12. In other words, D295 (λmax for ionic

forms) dependence from pH is the A absorption layer in the figure. When, on the contrary, three

tyrosine residues are located inside in non-polar section, the obtained absorption layer corresponds

to the B absorbing layer shown in the figure. By the way, in case of very high pH, D295 increase is

observed in the absorption layer. It shows that internal tyrosine residues have become available

for the solvent, that is, the protein has been opened (transformed). If three internal tyrosine residues

were found in the polar region, the absorption layer would be similar to the C absorption layer

27

(figure 12), which shows that these three residues have pK value, which occupying the

intermediate position, is differed from the other two states.

Figure 33: Tyrosine titration absorption layers, which were obtained at λ295 wavelength.

Protein contains 5 tyrosine residues: A-all 5 residues are on the surface; B-two residues are

on the surface, three in non-polar region and therefore are not titrated; C-three internal

residues are in the polar region and are available for the solvent.

28

LUMINESCENCE METHODS OF DRUGS ANALYSIS

Many organic and inorganic substances are able to automatically emit light under influences of

different factors. This process is called luminescence. According to the character of luminescence

formation reason, the following types of luminescence are differentiated, which are represented in

the table 8.

Table 8:

Types of luminescence

Luminescence type Excitation mechanism Example

Radioluminescence

stimulation by gamma-radiation

of high-energy particles or

radioactive processes

self-lighting numbers in the

clock display

Electroluminescence stimulation by electric field lighting of gas discharged

lamps

Chemiluminescence stimulation by chemical reactions

energy

white phosphorus oxidation in

the air

Bioluminescence biochemical luminescent fishes, bacteria

Triboluminescence stimulation by mechanical

influence crushing of sugar crystals

Crystal luminescence crystallization arsenic oxides

Thermoluminescence stimulation by radiation (heating) Lamps, luminophores of TV

Photoluminescence light absorption in UV/visible or

IR-region

fluorescence and

phosphorescence

Two types of luminescence have great importance in the chemical analysis:

chemiluminescence and photoluminescence.

Chemiluminescence is the light radiation in result of chemical reaction (often oxidation). The

molecule is excited, that is, absorbs energy. When this energy is released, the molecule either itself

emits light or transmits its energy to another molecule, which then emits light. Synthetic chemicals

have the chemiluminescence property. Typical luminogens are, for example, acridine ether,

luminol, lofin, etc.

Photoluminescence is the emitted light after a certain time of absorbed electromagnetic

radiation by atom or molecule. The emitted light is a result of transition states energies difference

(from excited singlet state to the stable electronic level).

Bioluminescence is a special type of chemiluminescence that occurs in living organisms, such

as bacteria. In this case enzymes (eg, luciferases) and substrates (eg, luciferins) are essential.

Luminescence also can be classified according to light back emitting presence. It can be stopped

immediately after stopping excitation; this process is called fluorescence; and it can be continued

during some time after stimulation stopping; this process is called phosphorescence. The average

duration of fluorescence is 10-8sec; phosphorescence is 10-3sec. In the latter case the average length

of duration can be extended from several seconds to several hours. This difference in durations has

a simple physical explanation. In the case of fluorescence there is a single-singlet transition and in

the case of phosphorescence singlet-triplet transition exists. In case when from the singlet state

29

fluorescence reversion is conducted very easily and fast, the reversion from the triplet state to the

ground state is difficult because in this case spin change should be carried out again.

In the chemical analysis usually fluorescence is used, that is why method is called fluorimetry.

Fluorescence methods of analysis are based on substance light emitting intensity detection after

processing by UV/visible radiations (photoluminescence), wavelength is 200-800nm.

Fluorimetry is an emission spectroscopy method that is used both in identity and quantitative

analysis.

The advantages of this method are:

● provides information on the surrounding molecular environment,

● can be used to control dynamic processes during nanoseconds such as protein motion or their

complexes changes study,

● can be used to investigate the binding processes of drugs with proteins and receptors,

● it is highly sensitive method and can be used in subnanomoles quantities, up to unique

molecules detection.

This method is applied in a limited number of compounds, is conditioned by UV absorbed

specter and is very sensitive toward temperature changes.

Theoretical bases of fluorescence

Usually exciting compound returns the absorbed energy excess by non-radiation deactivation

way. In this case, the excitation energy is transformed into the kinetic or vibrational energy (heat

emission). It is supposed also that in case of non-fluorescence substances deactivation is carried

out due to pre-dissociation processes, which is possible because the excitation energy is enough

for the destruction of corresponding chemical bond. On the other hand, if the structure of the

compound does not allow for such processes performance (e.g. substances with rigid structure),

some of the energy excess may be released in the radiation form. Thus, photoluminescence is

possible only in case of those organic compounds which contain π-bond electrons. This is

explained by the fact that, on the one hand, there is small amount of energy required for such

electrons excitation, on the other hand, these compounds have such a strong system of bond which

limits the possibility of non-radiation deactivation. Some metals ions (uranium), many complexes

and organic compounds (mainly aromatic) possess fluorescence ability. Almost all the aromatic

compounds (in the presence of appropriate substituents) are able to cause fluorescence. Thus,

flourofor /luminescence/ groups are aromatic rings, conjugated systems (with coupled double

bonds), electron-donor groups connected to aromatic ring, but electron-acceptor groups, contrary,

decrease or completely eliminate luminescence.

At the room temperature majority of that molecules are in lowest vibrational level of the basic

state. In result of quant’s (h0) absorption substance molecules are excited passing to the excited

B state (first or second excited state) and after some time returns to the ground A state losing

energy excess in quant form. However, some part of the energy is radiated in thermal radiation hT

quant form, which leads to the molecule certain stabilization in lowest excited C level, then h

quant is radiated in the visible or UV region of specter, which is conditioned by passing to the

ground state.

30

Figure 34: Fluorescence formation mechanism.

Fluorescence radiation frequency is always lower (longer wavelength, red or batochrome shift)

than exciting radiation frequency because of thermal radiation. This principle is called Stock’s law.

As a result of excitation molecule can reach to any vibrational level which are characterized as

subtypes of individual excitation states. If the molecule absorbing energy, reaches to upper

vibrational level of any excited state, then it very quickly loses vibrational energy excess due to

collisions and again turns to the lowest vibrational level of excited state (Figure 35). Thus, in

molecules, additional internal transitions are carried out from the upper vibrational levels to the

lowest vibrational level of first excitation state close to energy. As a result, however, the molecule

does not reach yet to the vibrational level of the ground state. These transitions are conducted

without radiation (non-radiation deactivation), the duration is 10-12. Then, the molecule irradiates

its energy excess in the form of light, passing from the first excited state to vibrational levels of

ground state.

Figure 35. Energy state of light absortion and fluorescence

In case of atoms excitation, such deviation is not observed (atomofluorescence spectroscopy),

as vibrational levels are absent in atoms.

Fluorescence main characteristics are quantum and energetic yields, and fluorescence

specter as well. Quantum yield is the number of molecules radiated quant’s (Nr) ratio to the

number of absorbed quants (Na). Energetic yield is the ratio of radiated Er and absorbed Ea energies.

31

Quantum Bq and energetic Be yields depend on radiation and absorption light frequency and wave

length.

Bq=Nr/Na; Be=Er/Ea; Be=υr/υa Bq=λa/λr K

Thus, fluorescence generally depends on exciting radiation wave length, but fluorescence

specter doesn’t depend on exciting radiation wave length and is a substance characteristic.

Substance fluorescence and absorption spectra are connected with each other by some laws.

First is Stock’s law, according to which fluorescence spectrum is in longer wave length

region, than absorption spectrum λrλa.

Second is the mirror symmetry law, according to which absorption and fluorescence spectra

built in frequency scale are symmetric to each other (Figure 36).

Since energy intervals between vibrational levels are energy intervals of lower electronic

levels, it leads to the formation of compound’s radiation absorption and emission symmetric

spectra. The transition from the ground state lowest vibrational level to the lowest vibrational level

of first excited state, the 0-0 transition, is the same from the energy point of view in case of both

absorption and emission. Therefore, both spectra, absorbing and fluorescence, have 0-0 transition

curve. In case of all other transitions, absorption needs greater energy than any transition at

fluorescence radiation. The emission layers are dispersed to the lower energy level toward

absorption layers of the mirror-symmetric 0-0 (batochrom shift) transition (Figure 36).

Figure 36. Excitation and emission mirror spectrum

Thus, all the absorption and fluorescence spectra theoretically must be covered in the

wavelength that corresponds to the 0-0 transition. Actually, 0-0 transitions are rarely match in case

of absorption and emission. This is explained by the fact that in the range of wavelengts where the

absorption and emission spectra are overlapped, a partial reabsorption of emitted light is carried

out, which is conditioned by absorbing substance concentration and layer thickness.

In the first excited state, the absorption of energy affects on the molecule's shape in small

amount. This means that the vibration levels distribution are similar in the main and first excited

states. Therefore, the energetic ranges between the emission radiation layers are similar to the

ranges of absorption layers. Due to this the fluorescence specter is often a mirror image of the

absorption specter.

Since fluorescence radiation is always conditioned by the transitions from the lowest vibrational

level of the first excited state, so the external form of the radiation specter is not conditioned by

excitation light wavelength.

As molecules can pass to the different vibrational levels of stable (not excited) state by emitting,

so the released photons will have different energies and hence different frequencies. Thus, by

examining different frequencies of the emitted light, the structure of different vibration levels can

be detected.

32

Substance emission specter is obtained by measuring light emissions in different frequency

conditions in case of absorbing light certain constant wave.

Excitation specter is detected by measuring various emission spectra in different wave length

conditions. Excitation specter depends on:

• -bonds conjugation degree; electrons quantity, which participate in the conjugation.

Double bonds addition decreases excitation radiation energy.

• Aromatic rings quantity. In case of ring addition radiation wave length increases and energy

decreases.

Fluorescence methods usage in drug analysis

Nowdays fluorescence methods are widely used in biochemistry, biophysics, cellular and

molecular biology as well as in clinical chemistry for diagnostic purposes. Fluorimetry is

remarkable with high sensitivity (fluororescence photometry may be up to four times sensitive to

absorbtion photometry), and with a large range of linearity, it is performed without substance

degradation and a large amount of tested substance is not necessary for measurement.

Fluorescence analysis methods are carried out by direct and indirect ways. In direct method

fluorescence intensity is measured directly. In indirect fluorescence analysis fluorescence serves

as an indicator indicating substance detection end. Method of direct fluorescence analysis is based

on Vavilov’s law. According to this law solution’s fluorescence intensity is in linear correlation

with concentration in low concentrations region (10-7-10-4 mol/dm3).

If = КС.

Increasing the solution’s concentration, the linear correlation is broken in the result of

fluorescence quenching or fluorescence intensity decrease.

Here, error percent of analytical detection is high, and usually measurements are not

conducted in concentric concentrations range.

Fluorescence intensity is changed depending on:

●substance concentration,

●quantum and energetic yields,

●exciting radiation wavelength,

●solution temperature,

●foreign mixtures presence in solution,

●solution pH value,

●solution nature.

Under these factors either fluorescence enhancement or fluorescence quenching or decrease

can be carred out. The latter is performed because the radiating energy is transferred to other

molecules in solution and fluorescence is not caused. Temperature quenching occurs when the

temperature increases. Concentration quenching is performed in case of concentric solutions.

Foreign mixtures presence in solution also can lead to fluorescence quenching. Heavy metal

ions, halogen ions, oxygen, as well as large number of unsaturated and aromatic compounds are

strongly quenching substances and interfere analysis. However, this phenomenon is also used in

substances analysis, in this case, primarily, pyrocatechine, hydroquinone and other phenols as well

as aniline, nitrobenzene and other similar compounds are used as quenching substances.

Most of the luminescence substances have typical pH range, in which fluorescence occurs.

33

By fluorescence method those substances can be detected, which:

• have their own fluorescence (vitamin B1 detection),

• form fluorescent compounds with different reagents (Al3+ detection by its complex with

salicylic-o-aminophenol),

• don’t have own fluorescence, but interacting with other fluorescence substances lead to

fluorescence quench (Zn2+ is a fluorescence quench for rodamine (C)-thiocianate).

As a reagent, the substance can be used for the non-fluorescence cation, which fluorescence

specter is changed as a result of interaction with tested substance. Zn compounds fluorescence blue

with salicylicdehyde semicarbazone, when the reagent itself has yellowish-green fluorescence.

One of the varieties of fluorescence method is extraction-fluorescence analysis, in result of

which the formed compound is extracted from the water solution by an organic solvent, and then

fluorescence intensity of extract is measured. By this method, Al content is detected by obtaining

its oxyquinolate, which is then extracted with chloroform. Fluorescence intensity of chloroform

extract is measured by fluorimeter.

As an indirect fluorescence method titration with fluorescent indicators is widely used.

As a result of interaction with corresponding reagents, even non-fluorescence substances can

turn into light-emitting products which can be detected by fluorimetric method. For example,

fluorescent indicators are used for metals detection. These indicators form complexes with metals

and change fluorescence properties.

Oxido-reductive fluorescent indicators change fluorescence intensity or color depending on

system’s oxido-reductive potential. For example, during titration of cerium sulfate by Fe3+ in

presence of siloxane indicator (Si6H6O3)n solution’s fluorescence disappears in case of titrant’s

small amount of excess.

Fluorescence indicators are widely used in acid-base titration methods. Acid-base fluorescent

indicators possess different fluorescence in ionized and non-ionized forms and are characterized

by pH certain interval of fluorescence color transition. Fluorescence indicators compared with

usual indicators have color transition narrower interval, which increases analysis precision.

Fluorescent indicators can be used for titration of turbid or dark-colored environments. Titration

process is carried out in darkness by luminescence lamp brightening titrating solution, where

indicator is added.

Table 9:

Fluorescence indicators

Name Structure pH range Color of fluorescence

anthranilic acid COOH

NH2

1,5-3,0 colorless-sky blue

salicylic acid OH

COOH

2,5-4,0 colorless-blue

acridine CH

N 5,2-6,6 green-violet

luminol

NH2

CO

CO

NH

NH 6,0-7,0 colorless-blue

In chemistry and pharmacy fluorescence method is used for analysis of metals spots, organic

(aromatic) compounds, vitamin D, B1. By this method by means of luminescence lamp quality of

food, water, and presence of pathogen organisms can be detected as well.

34

Devices used in fluorimetry

The principle of fluorimetry is that the substance is excited by the beam released from the light

source, after which the fluorescence radiation intensity is measured as a function of exciting beam

wavelength and the emission. In contrast to absorbtion spectroscopy, in this method two

wavelengths: excited and emitted are always considered.

Fluorescence measurement is conducted by photoelectrofluorimeter (figure 37), which consists

of:

• exciting light source (mercury-quartz or xenon lamp),

• light filter,

• cuvette, in which tested solution is filled,

• fluorescent radiation receiver (photoelement),

• amplifier,

• measuring device.

Figure 37. Structure of fluorimeter

Fluorescence radiation released in all directions is recorded at 900 degrees over the falling beam.

The distance between receiver (detector) and cuvette should be as small as possible.

For conducting fluorimetric analysis calibration graph is used. Standard solutions

fluorescence intensity is detected for the calibration graph building. Comparison of analyte and

standard solutions fluorescence intensities is used as well.

35

INFRARED (IR)-SPECTROSCOPY

Infrared spectroscopy usage in pharmaceutical analysis

The phenomenon of substances interaction with infrared (IR) radiation has been revealed by U.

Ebn and I. Festing in 1861. Currently, infrared spectroscopy is one of the main methods for

studying various chemical substances, including drugs.

The method was incorporated in SPh X in 1968, where it was recommended as a means of

quality control for 3 pharmaceuticals (fluorotane, oxalicin and methicillin sodium salts), and data

on certain aspects of IR-spectroscopy were included in the pharmacopoeia "physicochemical,

chemical and biological studies general methods" section.

After SPh X edition, the number of drugs for which study this method was rcommended has

significantly increased, which can be explained by annually published pharmacopeia articles.

The IR-spectroscopy is included in all modern pharmacopeias. For example, international

pharmacopeia (Geneva, 1990) offers this method for analyzing half of the drugs included therein.

Taking into account the informative value of IR specters (informative method), for a long time this

method is one of the most important methods for analyzing substances. IR-spectra decoding is

practically much simpler and faster (especially by computerized method) than theoretical analysis

of corresponding UV and visible spectra. The existence of many patterns and rules make it possible

to make quite reliable predictions about the molecular structure of the investigated substance. IR-

spectra can be obtained not only for individual substances, but also for a number of ready-made

drugs. It is conditioned that the excipients included in the composition of a medicinal product (e.g.,

tablets) should have no effect on the specter of the main (effecting) substance. This condition is

usually possible if the percentage of excipients is not too high, usually less than 60-70%. IR-

spectroscopy is a compulsory method for standard substances quality control, additionally, it is

currently included in pharmacopeial article (PhA) of many drugs. The method can be used to prove

the absence of the drug substances derivatives (the same sequence) of similar structure (mixtures

detection) in the pharmacopeia analysis. For example, in the PhA of estocin belonging to aryl

aliphatic acids ethers group it is necessary that in the IR-specter which is obtained in the 3300-

3500 cm-1 region in vaseline oil should be absent absorption layer typical to free hydroxyl group

(in contrast to aminazine and metacin). In mixtures extra absorption layers appearance, shapes of

individual absorption layers, absorption layers intensity and sharpness are checked.

Thus, IR-spectroscopy is used in pharmacy:

● to detect the structure of new substances that have been obtained through chemical synthesis

or from natural objects (animal or plant raw materials, microorganisms products), while examining

metabolites' structures,

● to detect drugs identity,

● to detect drugs purity,

● for drugs quantitative analysis,

● to control technological process of drugs production.

36

Theoretical bases of IR-spectroscopy

IR-spectroscopy is an investigation method for substances which is based on absorption of IR-

radiation, in result of which promotes vibrational and rotational vibrations in molecules.

In most of the molecules, there is a change in the atomic vibrational energetic states, so the IR-

spectra are called vibrational or molecular.

Thus, the term “vibrational spectroscopy” is used to describe infrared and Raman (Combination

scattering) spectroscopic methods. The physical nature of these spectra is different. IR-absorption

spectra are conditioned by transitions between molecul’s vibrational levels, and combination

scattering spectra։ by change of molecul’s polarization in result of vibration. Raman and infrared

spectroscopic methods give similar information and each method can be used to accomplish the

other method.

It is known, that atoms in molecules are never in ground state, but there are vibrated around

somewhat medium position, as a result atoms arrangement to each other is periodically changed.

IR-rays increase the vibrations because of which some of the radiation energy is transmitted to the

molecule, resulting in IR-rays intensity decrease arising from substance. Intensity loss ΔJ=J0-J is

the function from wavelength like in electron spectra.

Vibrational spectroscopy

Types of vibration

Thus, in 2.5 to 50 mkm wavelengths region, in molecule atoms vibrational and general molecule

rotational motions are excited. Spectra recorded in this region give information about substance’s

molecular structure. As we know, the infrared region is the largest part of the electromagnetic

specter, wavelength of which varies from 1mkm-1mm. It can be divided into three main regions:

Near-infrared (overtone region) 0.8-2.5 μm (12 500-4000 cm-1),

Middle-infrared (vibration-rotation region) 2.5-50 μm (4000-200 cm"1), the energy required

to induce the vibrations of atoms in the molecules generally corresponds to a wavelength energy

of 1-15 cm or a wavelength of 200 to 4000 cm -1,

Far-infrared (rotation region) 50-1000 μm (200-10 cm'1) The near and far words describe

being close to the visible region.

The main region of interest for analytical purposes is from 2.5 to 25 mkm (i. e. wavenumber

4000 to 400 cm-1 Middle-infrared region). Normal optical materials such as glass or quartz absorb

strongly in the infrared, so instruments for carrying our measurements in this region differ from

those used for the electronic (UV/visible) region. As mentioned Infrared spectra originate from the

different modes of vibration and rotation of a molecule. At wavelengths below 25 pm the radiation

has sufficient energy to cause changes in the vibrational energy levels of the molecule, and these

are accompanied by changes in the rotational energy levels. The pure rotational spectra of

molecules occur in the far-infrared region.

Atoms inside the molecule are motional due to their atomic bonds. They vibrate by certain

(resonance) frequency, which is conditioned by atomic mass and bond strength. Because of atoms’

small sizes their resonance frequency is about 1013 cm -1, and the frequency of the IR-rays is

1013cm-1, comparable to that value, so it is possible to transfer energy between the molecule and

the IR radiation. Since molecular vibrational levels are quantified, the result is that the transition

energy between them and, hence, the vibration frequencies can have only certain definite values.

37

Absorbing the light quantum, the molecule can reach to higher vibrational level, usually from the

ground vibrational state to exited state.

Infrared radiation absorption causes vibrations that are conditioned by the change of the bonds

length or angle between the atoms. It means that, depending on the absorbed radiation frequency

some bonds start to periodically stretch and shorten or the angle between the bonds are changed.

Thus, the main types of vibration are valence and deformation vibrations.

The vibrations that lead to change in length of the bonds between joined atoms and are not

accompanied by the deviation of the internuclear axis are called valence. In other words, the

valence vibrations are called the atoms’ nuclei vibrations along the bond. This type of vibrations

is mentioned with ν (ν C = C, ν C = O).

Two balls system joined by spring can serve as anapproximate schematic model of valence

vibration (the balls replace the atoms and spring to the chemical bond). In the case of pulling or

pressing the spring, the balls begin to vibrate around the position of balance performing harmonic

vibrations, which vibration frequency is expressed by Hook’s equation:

where։

ν- is vibration frequency;

c- is light speed;

F-is strength constant or rigidity that characterizes bond strength or the force that returns the

balls to the balance position.

mr- is the atoms’ given mass that is calculated by the following formula:

where m1 and m2 are separate atoms’ masses.

The given mass is also mentioned with μ and can be detected by the following formula։

μ=A1A2/1000L(A1+A2),

where A1 and A2 are atoms’ relative atomic masses,

L is Avogadro's constant (NA).

Usually there is an agreement between the calculated and tested values of the given wave

number. As an example we can discuss C-O bond in methanol, in which case F = 5x102 N/m, μ =

6,85mu (same atomic mass constant = 1,66x10-27 kg) and light speed c=2,998x1010 cm/s.

Investigated C-O bond specter in methanol will be 1034 cm-1 detecting by the formula. However,

this simple calculation does not consider any possible effects from other atoms in the molecule.

The frequencies of valence vibrations are detected by atomic mass and bond strength (energy).

The larger is the mass, the smaller is the frequency, for example:

The stronger is the bond and larger rigidity, the higher is vibration frequency։

Valence vibrations can be symmetric (νs) if two bonds simultaneously extend or shorten, and

the pair of atoms simultaneously attract each other or disattract, and asymmetric, if one of the

bonds get shorter, the other elongates (νas), in that case different atoms pairs attraction and

disattraction is not performed simultaneously.

38

C

HH

C

H H

Figure 38: Symmetric valence vibrations.

C

H H

C

H H

Figure 39: Asymmetric valence vibrations.

Asymmetric (non-symmetric) vibrations frequencies are often higher than symmetric vibrations

frequency.

Next type of vibration is deformational vibrations which are conditioned by change of common

atom having bonds valence angle. In such vibration case atoms’ internuclear axis is deflected: the

vibrations are mentioned with δ.

Figure 40: Deformational vibrations.

Deformational vibrations can be manifestated as in the plane, such as scissors-like and

pendulum-like vibrations, as well as outside the plane։ rotational and fan-like vibrations.

Figure 41: Types of deformation vibrations.

Less energy is required for deformational than for valence vibrations, hence low frequency.

Thus, the valence vibrations are in high frequency region։ 4000-1400 cm-1, and deformational

vibrations are in low frequency region։ <1400 cm-1.

Rule of choice in IR-spectroscopy

IR-radiation absorption occurs only in case of interaction between the changing dipol moment

due to molecul’s vibration and vibrating vector of the electromagnetic field. One simple rule gives

chance to detect when this interaction occurs and therefore absorption.

"Molecule’s dipole moment in vibration one extremume should be differed from the molecul’s

dipole moment in the other extremume of that vibration."

Thus, in infrared specter occur only those vibrations (are active), which take place with the

change of molecul’s dipole moment. Thus, in the IR specter, the vibrations that are not

accompanied by the change of dipole moment are prohibited. Since symmetric molecules do not

have a constant dipole moment, and this dipole moment does not occur as a result of the vibration

due to the symmetric distribution of the charge, so vibrational excitation is not possible in such

molecules. Therefore, IR-spectra can not be obtained from the following compounds։

39

● Inert gases,

● Salts without covalent bonds (NaCl, KBr),

● Metals,

● Diatomic molecules of the same atom (CI2, N2, O2).

Infrared absorption spectra can be used to detect pure compounds, or to detect and identify

mixtures. The IR-spectroscopy has practical use in analysing organic molecules, although this

method can also be used to detect inorganic salts with covalent bonds, such as KMnO4. The other

reason why this method is most applicable for organic compounds is that the main solvent of

inorganic compounds is water that strongly absorbs under 1.5μm region. Moreover, inorganic

compounds often have wide absorption layers, whereas organic substances can have many

narrower layers.

Transparent compounds in IR region also have some applications in infrared spectroscopy.

Firstly, gases։ oxygen, nitrogen, and inert gases serve to clean (to spray) spectrometers, because

water and carbon dioxide in the air absorb IR-rays. Secondly, IR-permeable substances are used

as sample carriers, as well as to prepare spectrometer’s optical components. For this purpose

alkaline metals halides are used.

IR-specters, their role and usage for substances structure detection

In described examples, we spoke about very simple molecules consisting of two atoms

vibrations of which can be considered as harmonic and the vibration frequencies are conditioned

only by vibrating atoms’ masses and the type of bond between them. However, in more complex

molecules, the atomic vibrations are interconnected and can effect on each other. Each complex

molecule is able to vibrate variously. Moreover, by increasing the number of atoms in the

molecule, the number of possible vibrations increases, in other words, the more the atoms in the

molecules, the better the vibration types. The vibration type is detected by the molecular structure,

and is typical to it.

Molecules’ spectra are different vibrations complexes, each of which appears in the narrow

(small) range of the frequency. The total number of absorptional layers in the specter is conditioned

by the number of vibrating atoms and is detected by the formula։ 3N-6 main vibration for nonlinear

molecules, and 3N-5 main vibration in case of linear molecules, where N is the number of atoms

in the molecule. However, in the actual specter number of absorbtion leyers are not always equal

to that number. They can be decreased, as some of the layers are not visible in IR-specter because

of molecule's symmetry degree. The number of absorption layers decrease due to the fact that

different vibrations can have the same frequency in the symmetric molecules and as a result, one

absorption layer appears instead of 2-3 vibration line in the specter.

Polyatom molecul’s vibrations can be subdivided into two main types։ local (localized), in >

1500 cm-1 region, with separate bonds or functional groups, and molecular (skeletal) vibrations in

the 800-1500 cm-1 region. Many localized vibrations serve for identification of separate functional

groups in molecule. Thus, studying substances of different chemical structure (model compounds)

and IR-radiation interaction, it was discovered that many functional groups, such as> CH2, -CH3,

-COOH, -CH2OH, -OH, -NH2, -NO,> CO, etc., as well as some bonds, such as C-H, C=C, C=O

are characterized by a certain frequency, which is slightly differed in different compounds as their

interaction with other parts of molecule, particularly with molecule's skeleton, is not so significant.

Therefore, their absorptions are always (with some minor deviations) in the same region. Such

40

localized frequencies are called characteristic or group frequency. That is why the IR-

spectroscopy is mainly used to detect functional groups in the molecule.

Group frequencies are mentioned with ν and for the basic classes of organic compounds are

shown in the tables (Apendix 1). This gives possibility to detect compound’s structural units by

using tabular data. These vibrational forms are useful for the confirmation of compound structure,

particularly for estimating the structure of unknown substance. From the position of the spectral

lines towards each other, it is possible to conclude about the functional groups configuration.

Molecule’s skeleton vibrations absorption specter is relatively in low energy region։ <1500 cm-

1 (wavelength>6.7mkm). These spectral lines are often overlapped and makes localized vibrations

identification difficult below 1500cm-1. In this region, there are often appear non main vibrations։

overtones and combined lines.

In molecule like any kind of energy state, the vibrational energy of atoms is also quanted and

vibrational quantum numbers are only expressed by complete numbers։ ν=0, 1, 2, 3 .... In contrast

with excitation energy of rotational motion, the excitation energy of atomic vibrations usually

significantly exceeds molecules’ thermal energy at room temperature. It means that practically in

normal conditions atoms vibrations by termal method generally are not excited. That is, all the

atoms are of their lowest vibrational energy level, and observed IR-radiation absorption is fulfilled

due to transition of higher energetic level than ν=0 level. From this level, theoretically, transitions

to all upraised levels are possible, but parallel to the ν increase the possibility of these transitions,

and consequently the intensity drasticly decreases. According to initial and final states of

vibrational quantum numbers, there are the following types of transitions։

ν= 0 -ν= 1՝ main vibration,

ν= 0 -ν= 2՝ first overtone,

ν= 0 -ν= 3՝ second overtone.

As it is noticed, the most intensive absorption corresponds to transition from ν=0 to ν=1, and it

is mentioned as main vibration. The overtones are significantly less intensive, and according to

their larger energy, are in short-wave region in the specter compared to the main vibration. For the

first overtone excitation, approximately double energy of the main vibration is required and for the

second overtone excitation - triple energy.

Combined lines (component frequencies) appear as a result of two or more vibrations overlap.

Sometimes, very high intensity lines appear in spectra, which are formed as a result of Fermi

resonance. These absorption layers of resonance interaction occur when the overtone of the given

frequency and any other main frequency have the same values, that is, they coinside. In certain

cases, these component frequencies are used for diagnostic purposes, but generally they don’t have

practical use.

Thus, the IR-specter of compound consists of two regions։

● Higher than 1500cm-1 region, where localized absorption lines of functional groups are

available,

● Lower than 1500cm-1 region, where many absorption lines characterize the whole molecule.

This region is known as "fingerprint" for obvious reasons, and it is used for substance identity by

comparing with the reference.

Thus, for detection of pure compound, the specter of unknown substance is compared with other

limited number of possible spectra. When a coincidence is observed between the spectra, the

detection ends. This process is particularly valued to detect structural isomers (but not optical

isomers).

41

The specter of the compounds mixture is basically the sum of the individual compounds on

condition that the substances are not associated or dissociated, are not polymerized or form new

compound. For detecting the mixture in drug substance’s specter can be compared with the pure

compound’s specter։ mixtures will cause additional absorption layers that are observed in the

specter. The most convenient case will be when the existing mixtures have typical groups, which

will be abcent in the main substance.

Vibrational spectroscopy։

● is molecular-specific method which gives possibility to obtain information about functional

groups, their types, interactions, and positions in molecule,

● is a selective methot towards the isomers due to "fingerprint",

● gives possibility to perform quantitative analysis without destroying or damaging the structure

of the tested substance, even in case of very unstable compounds,

● is a method which allows to work within 0.1% and 100% concentrations, it is also suitable

for detection of sample microquantities after appropriate concentrating.

Figure 42: IR-specter of organic compound

The absorption intensity in IR-spectroscopy is usually expressed by optical density (D):

D=log J0/J = εlc

or often with a light flow transparency (T, by percents).

In this case, the absorption regions are expressed as minimums in the received specter. The

position of the absorption leyers is either indicated by wavelength λ (μm) or by wave number ύ

(cm-1):

ύ=1/ λ.

Of these two values, the preference is given to the wave number, as, in contrast with wavelength,

it is directly proportional to transitions’ excitation energy. In this case, the same regions in the

specter corresponds to the difference of similar energies, which makes it easier to distinguish

between main absorptions overtones and combined lines.

The absorption layers, in their turn, according to intensity are evaluated as strong, medium and

weak.

42

Quantitative analysis in IR-spectroscopy. Beer’s law

Thus, infrared spectra record using either absorption (D) or transparency (T) by percents, or

both, as in UV and visible electronic spectra. Here, Lambert-Beer’s law also is applied, which,

however, may have some deviations in case of concentric samples։

D=εcl= log1/T=logIo/I.

In case of the compound’s mixture (in the case where the absorption layers cover each other)

the obtained absorption in case of the certain wavelenght (or frequency) is the sum of mixture’s

individual components absorption at that wavelength։

Aobtained=A1+A2+A3=ε1c1l+ε2c2l+ε3c3l։

In case where the absorption layers do not overlap each other, quantitative detection is

conducted samely as for individual compounds.

Substances quantitative detection in IR-spectroscopy, especially in the past, had disadvantages

and limitations. This is conditioned by the following reasons։

● The molar absorption coefficients (molar absorptions) are usually ten times smaller than the

electronic reagion constants. Thus, the infrared method is usually less sensitive.

● The most suitable is T=55% to T=20% range (D=0,26 to D=0,70) for quantitative

measurements։ the accuracy of measurement outside of these values dramatically decreases.

● The error of old spectrometers is 1% in the range of mentioned T values.

Although nothing can correct the method disadvantage conditioned by low molar absorption,

nevertheless, the design and improvement of modern device, the implimentation of retio recording

and FT-IR type of devices, overcome limitations due to accuracy and instrumental factors. As a

result, quantitative infrared methods are currently mostly used, and are often for quality control

and substances structure identification.

Devices used in IR-spectroscopy

Tested sample radiation is on the base of substance’s IR specter by IR radiation of constantly

changed frequency.

IR spectrometer is like UV and visible spectrometers structure, but the structure of this device

is more complicated.

The IR radiation sources are heat, such as Nernst’s shtift or ceramic axis from carborundum

(SiC, carbide silica, globar), which is heated by electric current. Nernst’s shtift is a mixture of

zirconium, torium, and anthrium oxides.

All optical materials of the device (lenses, cuvettes and former prisms) must be permeable for

the IR-radiation. Alkali halide crystals (LiF, 2000-3800cm-1, NaCl։ 700-2000cm-1 and KBr։ 400-

700cm-1) were used in the old type of prismal monochromators, currently the water-soluble and

hygroscopic substances have been replaced by diffractional net.

In order to record specters, double beam spectrometers are mainly used here (Figure 43). On

these devices, a solvent-free cuvette is inserted on the path of the second beam, or KBr tin plate

without the sample. By the help of prisms or diffractional nets the light flow is devided into two

identical rays, the one passes through the cuvette filled with the sample, and the other with the

cuvette of comparing solution. Radiation enters into the monochromator, which allows to devide

the radiation to a very certain frequencies and smoothly alter it.

43

Figure 43: Scheme of double beam spectrometer.

The detector records the difference between the intensities of two light streams (main and

comparative ray) flowed from the monochromator. Since IR radiation is thermal radiation, the

detector should accurately measure temperature changes and convert them into electrical impulse,

which is enhanced and regulated by autorecording potentiometer.

To obtain correct result, the device should be placed on a solid, stable, non-vibrating surface,

which is especially important for recording the specter. The device should be kept away from

heating devices and water source (humidity). The latter is especially important because water

makes difficult to obtain the IR spectra absorbing the IR radiation.

Implementation of the IR-spectroscopic method involves the following stages։

1. Preparation of tested sample,

2. Specter registration by device,

3. Interpretation of data։ specter analysis.

Preparation of tested sample

In fact, spectrometric studies can be conducted in all aggregate states։ solid, liquid, gas and in

any consistencies, in types of membranes or solutions. Preparation of samples within IR methods,

significantly differs from the methods used in UV and visible spectroscopy. This is usually can be

explained by substances high molar absorption. The amount of substance required for analysis is

measured by mg. In case of gases, the thickness of substance layer should be big, and thin in case

of pure solutions and solids, since the density of absorbing substances is greater in this case. Taking

into account the hygroscopicity of the cuvettes used in IR-spectroscopy they are not suitable for

the analysis of water solutions.

Analysis of liquids and solutions by IR-spectroscopy

Liquid samples are studied in cuvettes, which are placed on the pathway of light. For obvious

reasons, water is used as a solvent only in combination scattering spectroscopy. This method also

allows working with glass cuvettes. Usually it is not difficult to prepare organic liquids samples,

as most of them do not break the salt-cuvettes. In IR-spectroscopy, complete or collected cuvettes

are used. Two flat parallel plates form windows, in which liquid sample is placed. These plates are

combined with steel tin plates. The thickness of this cuvettes layer can be changed. Usually, the

thickness of cuvette working layer is from 10μm to 1mm. Another advantage of the collecting

cuvettes is that they can also measure viscous liquids, paste-like substances, which after

measurement, cuvettes can be detached and cleaned. In IR-spectroscopy cuvettes with the

changeable layer thickness are ireplacible։ to compensate solvent absorption; quantitative

analyzes, and differential studies implementation. By accurately changing the layer thickness it is

44

possible to obtain important details in the compound specter. The layer thickness is regulated by

screw and the sample into the cuvette is added by a special syringe.

In most cases, during fluids analysis there comes nessecity to dilute them. As it has repeatedly

mentioned water is not used in this case, but beside water the other solvents also can absorb in IR-

region. For this reason, it is preferable to use those kind of substances as a solvent, which don’t

have large molecular sizes and symmetric structure, thus in IR-spectroscopy CS2, CCl4 are greatly

important.

Analysis of solid substances in IR-spectroscopy

Generally, for analyzing solid substaces it is commonly used to obtain solid substaces solutions,

but in IR-spectroscopy solid substaces investigate by obtaining their homogeneous thin layer

ribbon-like shape which is fixed to the sample holder. The sample holder in this case is a tin plate

with rectangular hole on which the tested sample ribbon is placed, from above it is closed by a

magnet plate, which also has an open rectangular window. The magnet provides the tin plate’s

fixation in vertical position.

Two methods are used to dilute solid samples։

● compression method by KBr,

● suspensions obtain by fluid paraffin bases.

The cost of the tested substance in this case is very small - a few milligrams. This quantity can

also be reduced by using special equipment.

Compression method by KBr

This method of solid and powdered substances is widely used in IR-spectroscopy. It is quite

time-consuming and labor-intensive, as it is necessary to weigh and grind the sample thoroughly,

as well as to clean the pressing device. 1-2mg of the tested substance is thoroughly rubbed by 200-

300mg hyperpure KBr by hand in mortar made of agate. The particles sizes of the substance should

be smaller than the length of the studied wave for obtaining high-quality spectra. For homogeneous

grinding and distributing the sample, vibrational mills can be used, by which several samples can

be prepared at the same time. Since the KBr is permeable from 43,000 to 400cm-1 region, and does

not cause absorption, so in the sample prepared by this method (in contrast, for example solvents

usage) is recorded only tested rubbed substance specter.

The rubbed mass is beeing compressed by hydravlic press machine for getting tablets-like

briquettes. In case of 10 tonnes of force KBr becomes plastic and mixing with the sample "solid

solution" is obtained. In order to remove moisture from tablet, compression is carried out in

vacuum. Additionally, until the mixting KBr is dried in drying chamber at 40C0. The water

contained in KBr is expressed immediately by absorbtion in the specter.

For standard measurements, tablets are prepared with 13 mm diameter, and in microanalysis –

tablets up to 2 mm diameter. It should be remembered that the crystal lettuce of the sample can be

changed during the compression, becides if the sample’s and KBr's refraction indexes are

significantly differed (KBr n=1.53), thus the asymmetric distortion of spectral lines can occur, to

avoid it other refraction indexes having substances are used։ KJ (n=1.62), AgCl (n=1.98), KRs

(n=2.37).

45

Methods for preparing samples with paraffin oil

In this case, in paraffin oil studied substance suspension is obtained. Approximately 2 mg of

substance is rubbed in mortar with a few drops of liquid paraffin oil (high molecular liquid

paraffins mixture). The oil contributes to the decrease of light reflections from the surface of

substance crystalline microparticles. Its refraction index should be as close as possible to the tested

substance refraction index. Obtained "milk porridge" is placed between the walls of the collecting

cuvette and then transparency specter is recorded in the passing light. Paraffin absorption lines

which refer to the CH2 and CH3 groups vibrations cover these groups vibrations in tested sample

as accurate compensation can not be performed in this case. This method is used in case of those

samples which interact with KBr or for which there is no other solvent than water.

Analysis of gases by IR-spectroscopy

Since oxygen and nitrogen do not absorb in IR-region, this method is ideal to identify gases։

CO, CO2, CH4, N2O, NH3, SO2, HCl or some other solvents’ vapours in the atmosphere. Of

course, there are some difficulties due to that for gases thicker layer is required. Additionally, the

gases absorption spectra are more complex than those of solid and liquid substances absorption

spectra.

Combination scattering or Raman-spectroscopy

Non-elastic scattering of light was discovered in 1928 by an indian physicist Raman and Raman

- measurements were widely used in 1930s than infrared measurements. This type of spectroscopy

is acomplishing to IR-spectroscopy, but the Raman effect is significantly differed from the other

methodes of molecular spectroscopy by light quants absorption and vibration excition type. Here

it is not about absorbed or emitted, but about scattered radiation spectroscopy.

The difference between these new frequencies (Raman lines) and the original frequency is

conditioned by the molecular structure, is characteristic to the radiated molecule, and numerically

identical with molecule’s certain vibrational and rotational frequencies. Obtained combination

scattering spectra (Raman spectra) can be concern to the tested substance molecules vibrations.

Depending on the spectral lines’ intensity, frequency and form it is possible to conclude about the

sample identity or structure. In result of excitation different conditions in the combination

scattering spectra such vibrations can be studied which are not active in IR-region. Of course, there

are vibrations that are not active both in IR and Raman spectra. The simultaneous use of these two

methods gives possibility more thorough study compound’s structure.

Theoretical bases of combination scatering spectroscopy

Combination scattering lines appear in specter when monochromatic light perpendicular falls

to study direction and the spectral separation of scattered light occurs.

Under visible and UV-rays impact electronic orbitals of molecules are deviated from their

equilibrium state. It means that the electrons begin to vibrate at the same frequency as the

frequency of the falling light. As a result they become the source of radiation for Rayleigh

scattering, which beams have the same frequency as the falling light beam. The entire energy

which was absorbed by the primary rays is again released in the secondary radiation type, and

46

energy change in this case is not observed. As a result, electrons are not excited, but remain in

their original configuration area. The precondition for this phenomenon is that the wavelength of

the given length is far from the wavelength by which influence electronic transitions are carried

out in the molecule.

Thus, the prevailing part of the scattered light has the same frequency as the falling light.

However, the more easy electrons move in the molecule, i.e. the higher is their polarization ability,

the stronger the electronic orbitals are deformated. As in case of such deformations, it is about

valence electronic orbitals by which chemical bond formation is conditioned, and hence the

intraatomar space and bond’s angle, so their geometrical structure change leads to the change of

that chemical bonds’ length or angle change between atoms. As a result, this leads to atomic and

respectively molecular vibrations. In case when in IR-spectroscopy these vibrations were excited

directly by IR light rays, in Raman spectroscopy they are mediated by deformations of electronic

orbitals.

The energy required for that excitiation is transmitted from the beam of the falling light. In this

case, along with the Rayleigh scattering, spectral lines are formed, which are deviated from the

initial excitation line by the measure of certain frequencies. Diferencies of the excitation line and

that spectral lines are Raman frequencies, which are detected on the base of IR-spectroscopy.

Combination scattering deviation in the specter from the Rayleigh line as in IR is expressed by

wave number cm-1.

The combination scattering specter consists of weaker lines than Rayleigh’s and either in higher

or lower energy regions (compared to the Rayleigh line). Here, there are differed Stock’s in lower

frequency, and anti Stock’s in higher frequencies appeared lines.

Stock’s lines appear in the non-elastic collision of the photon and molecule when the latter

absorbs energy in a single vibrational or rotational quantum measure։ ΔE. In this case, photon loses

appropriate amount of energy։ according to ΔE=hգv equation, the frequency deviates to the

measure of ν value.

As opposed to, anti-Stock’s lines are formed when the molecule is in excited rotational or

vibrational state. Moreover, photon absorbs this rotational or vibrational energy such that the

combination scattering spectral line frequency is greater than excitation wave line frequency by

the measure of ν value. The molecule in this case is less excited or is on zero state.

Figure 44: Scheme of energetic levels for Stock’s and Anti-Stock’s lines.

47

Since most of the molecules are in main vibrational state at room temperature, so the anti Stocks

lines which correspond to transitions of lower energy levels than excited state usually have less

probability. Consequently, combination scattering typical specter usually contains along with one

Rayleigh’s line Stokes lines with high-intensity and anti Stocks lines with low-intensity. As the

temperature increases, the number of excited molecules can increase, so the probability of anti

Stocks lines increases with temperature.

Rules of choice in Raman-spectroscopy

Like in IR-spectroscopy, there are rules of choice in this case. Interacting with the light, the

electrons pass to vibration state which size is conditioned by molecul’s polarization degree. In the

result of the polarization change which is caused by electrical dipole moment change, molecules

can interact with light.

Combination-active are molecules those vibrations and rotations at which vibrational and

rotational motion molecul’s polarization is changed. IR-active are molecules those vibrations and

rotations during which vibrational and rotational motion molecul’s diple momentum is changed.

Most of the molecular vibrations are mainly infrared and Raman active. However, in the case

of molecules with the center of symmetry, the principle of mutual exception is applied, which is

detected by an alternative prohibition rule, according to which in case of symmetry center having

molecules symmetric vibrations toward the symmetry are IR active and are combination-active

and vice versa, asymmetric vibrations are active in IR spectra, which are inactive in CS spectra.

This is very important conclusion which proves that these two methods complete each other. If the

Raman and infrared spectra of the molecule have the same peaks at the same frequencies, the

molecule can not have a symmetry center.

Table 10:

Different types of vibrations IR and combination activity in CO2 molecule

In case of symmetric valence vibration, the molecule stretches and compresses, i.e. electrons

concentration is changed in unit volume, consequently the molecule polarization changes.

Therefore, such vibration is a combination-active. But in the same case the dipole moment does

not change and it is IR inactive. It is clear that in case of asymmetric vibration the image is the

opposite.

The frequency of Raman specter refers to the force constant and the given mass by the same

expression as in case of infrared spectroscopy.

In combination scattering spectroscopy, mercury lamps are used from which specter 435.8nm

length having wave is separated as for the excitation monochromatic light is necessary which

48

wavelength is far from the sample absorption specter. Currantly, in modern devices laser light

sources are used that allow for getting more monochromatic light. Combination scattering is

usually seen at 900 or 1800 angles.

Considering the excitation wave length (visible region), in this case, water can be used as a

solvent, as well as glass and quartz cuvettes which was impossible in IR-spectroscopy.

Raman spectroscopy has the following advantages over infrared spectroscopy.

1. Water is an excellent solvent for Raman spectroscopy whereas it cannot be used in infrared

studies.

2. Preparing the sample in this case is easy.

3. Glass and quartz cells can be used in Raman spectroscopy.

4. Raman spectra are usually simpler than the corresponding infrared spectra, so overlapping

bands are much less common in Raman spectroscopy.

5. Totally symmetric modes of vibration can be studied by the Raman effect, whereas they are

not observed in infrared spectroscopy.

6. Because of the nature of the Raman effect, one instrument can be used to cover the entire

range of molecular vibration frequencies.

7. The intensity of a Raman line is directly proportional to concentration, whereas Beer’s law

has to be applied in infrared spectroscopy. Thus, quantitative analysis is often more convenient in

Raman spectroscopy and often more accurate.

8. As the combination scattering of transparent polymers is relatively weak, this method can

be used to analyze wrapped substances which has great practical importance in modern

pharmaceutical analysis. By this way, for instance, tablets composition can be detected without

breaking blister’s wholeness of wrap.

Combination scattering spectroscopy can be used to detect molecular structure. Disadvantages

of this method are։

● Low intensity of spectral lines with only 1% of total scattered light. The intensity of

combination scattering lines is detected by altering the molecule polarization.

● The sensitivity of the analysis is not so big.

● Any type of resonance interaction, such as fluorescence, is able to overlap combination

scattering lines. The only chance to correct this disadvantage is the use of such an excitation wave

which is far from the fluorescence causing wave. It was not possible or hard to conduct when

mercury lamps were used, which are lighting in fluorescent region, but it could be used by applying

laser light sources.

49

APPENDIX 1.

Group frequencies characterizing functional groups in IR-specter

Group frequencies typical to alkanes, alkenes and aromatic compounds

50

Absorption frequiencies typical to hydroxyl group in alcohols, phenols and acids

51

Absorption frequiencies typical to amines, imines and their salts

52

Absorption frequiencies typical to carbonyl group

53

IR-spectra

54

55

56

57

ATOMIC ABSORPTION AND FLAME EMISSION SPECTROSCOPY

As it is known, atoms and molecules can be found in discrete energetic states, and transitions

between these states are possible due to energy absorption or emission. Moreover, the only

radiation is absorbed or released, the energy of which (E) corresponds to the energy difference

between two levels (ΔE). Only certain energy levels are possible for atoms, each of which is

conditioned by the electrons’ specific orientation in the atom. Thus, if the electronic, vibrational

and rotational states can be altered in the molecules, the energy change of an atom occurs only

through electrons transitions between the orbitals of different energies. Because of the limited

number of electrons, the energy exchange with the environment cannot be realized continuously

and is carried out in case of a very definite (ΔE) values, to which correspond certain absorption

and emission wave frequencies. These frequencies, in some context, are atoms “own” frequencies.

The atoms interaction with electromagnetic radiation can be approximately described by

comparing it with the resonance of the mechanical oscillator, thus the atom absorbs only the

frequencies ν1, ν2, ν3 which correspond to its own frequency from continuous light specter. Atom

excited with the waves of same frequency can emit to the lower-energy orbitals through electrons

transition. Atomic spectra are linear spectra. As external valent electrons transitions are carried

out in atoms, thus atoms’ absorption and emission spectra are in the UV and visible region of

electromagnetic spectrum. In atoms both transitions emission and absorption are used in

spectroscopic methods.

Along with the light energy, thermal energy can also cause excited state in the atom. It means

that depending on the external environmental temperature different energy levels have different

levels of placement and absorption will occur as far as the low energy levels of electrons are placed

and the high energy levels of electrons are less saturated. The Boltzmann equation is true for two

states, E0 - ground state and E1 - excited state, in thermal balance

N1/N0 = (g1/g0)e-ΔE/kT

where,

N1 -is the number of atoms in the excited state,

N0 -is the number of atoms in the ground state,

g1/g0 is the ratio of static weights for the ground and excitation states;

ΔE -is the excitation energy, equal to hν,

k- is Boltzmann’s constant,

T-temperature is Kelvin.

The general description of the method

In the base of this method lies the known phenomenon that in the presence of metal vapors, the

colorless flame is colored (for example, in case of sodium it becomes yellow). If the emitting light

of this flame divides by means of monochromator, the corresponding element will be detected in

respect to the presence of open lines in the specter. In the absence of metal, the source of the beam

(flame) emits continuous, even specter (flame background), and in case of metal presence, the

specter becomes linear, the reason of which, as it was mentioned, are transitions of the metal atom

valent electrons between different energetic levels that occur by heat or light excitation. Thus,

when a solution containing metal salt (or another metal-containing compound) is introduced into

58

the flame (for example, in the acetylene flame) then the vapor containing metal atoms is formed.

Some of the metal atoms in this gaseous state may rise the energetic level and the energy absorbed

during the thermal excitation atom releases in the form of typical emission specter. This

phenomenon lies in the base of flame emission spectroscopy (FES) or flame photometry.

However, even in vapors state, most of the atoms are in ground (not excited) state and is able

to absorb light beams. If the atom's excitation is carried out under the influence of light, thus, as

mentioned earlier, the atom absorbs waves of certain energy amount, and in this case absorption

spectra are formed. The absorption takes place as much as the atoms in the ground state are present

in vapor. This is the basic principle of atomic absorption spectroscopy (AAS). Another method

based on light energy emission which was absorbed by atoms is atomic fluorescence

spectroscopy (AFS).

Atomic absorption is possible only when metal atoms are present in the absorbing compartment,

that is, the testing sample, for example the salt of metal, must convert to the atomic state. This

happens under high temperature condition. It is known that at temperatures higher than 2000oC,

almost all known chemical compounds are in the atomic state, that is, except of some exceptions,

there are no ions and molecules at this temperature. The further increase in temperature will lead

to the heat excitation of atoms, which will continue until all the atoms convert to the ionized state

by plasma formation (Figure 45). In this state, atoms can’t absorb the corresponding light and the

atomic absorption phenomenon will no longer be observed. Thus, the temperature should be high

enough to provide substance’s full and rapid transition to the atomic state, on the other hand, not

so high that atoms can no longer absorb the light beams. Thus, ideally at AAS, the atoms must be

only in ground state in vapor, in case of FES, the temperature should be higher so that more atoms

are found to be in excited state, and the emission specter is more expressed (but in this case also

there is a T0 limit, from which atoms lose their energy and plasma is formed, which is also not

advisable).

Substance’s transfer to the atomic state can be achieved by using the appropriate flame. In the

gas state, the import process of the metal atoms can be presented as follows. When the solution of

metal-containing compound is inserted into the flame, the following stages are rapidly occurred:

Figure 45: Atomization stages of metal containing compounds.

59

1. solvent evaporation by the formation of a solid residue;

2. solid residue evaporation, which is carried out by dissociation of the ground state atoms in

the substance;

3. some atoms excitation by flame heat energy by transition to higher energy levels and

absorbed energy radiation (FES);

4. atoms light excitation and emission in the flame (AAS and AFS).

In the flame, the dominant part of the atoms is in its ground (E0- the lowest energy level) state.

When the light of the corresponding frequency passes through the flame, even minimal amount of

energy absorption leads to the atoms transition to the first excited state - E1 energy level, also

called resonance energy level.

Figure 46: Simplified energy level diagram

Statistically, at the atom’s excitation placement possibility of this energy level is the highest.

More noticeable absorption of energy leads to atom’s transitions to E2, E3 states.

The amount of absorbed energy ΔE is detected by Bohr’s equation:

ΔE= E1 - E0 = hν = hc/λ

where,

c - is the light speed;

h – Plank’s constant;

ν - is the frequency;

and λ - is the wavelength of the absorbed beam.

Transition from E1 to E0 corresponds to beam emission of ν frequency.

After short time (about 10-8 s), after being in excited metastable conditions, the atoms drop back

down to their ground state by releasing their typical wavelengths. Emission specter includes not

only transitions from excited state to ground state (e.g. E3 to E0, E2 to E0), but also transitions such

as E3 to E2, E3 to E1 (figure 46). Thus, the emission specter of the given element can be difficult

enough. Theoretically, radiation absorption is also possible from the excited states, such as E1 to

E2, E2 to E3 transitions, but as in practice, the ratio between excited and ground state atoms is very

small, so the absorption specter of the certain element usually consists of only the transitions of

ground state to higher energy states, and is simpler in its nature than the emission specter.

According to Boltzmann equation, N1/N0 ratio depends on excitation energy (ΔE) and

temperature (T). In the case of increasing temperature and lowering ΔE (when dealing with

transitions with longer wave frequencies regions), N1/N0 ratio increases. The calculation shows

that only a small fraction of atoms is excited, even in the most favorable conditions, i.e., when the

temperature is high and the excitation energy is low. Alkaline and alkaline-earth metals, the

60

energies difference between their electron orbitals, is relatively small, absorb and emit in the

visible region of the specter (this is the reason they give a certain color to colorless flame);

therefore, they are excited at lower temperatures. This also can be explained the usage limitations

of the flame emission spectroscopy, it is used mainly for substances containing alkaline and, in

some cases, alkaline-earth metals identity and quantitative analysis (NaCl, KJ) in injection and

dialysis liquids and for other metallic mixtures detection as well in inorganic salts. On the other

hand, flamephotometry is a clear, affordable and selective method with relatively simple devices

that allows quantitative detection of the mentioned metals (Na, K, Li, Ca, Ba) with high accuracy.

Table 11 shows the dependence of N1/N0 ratio on temperature and the appropriate wavelengths

for some metals.

Table 11

Since the absorption spectra of many elements are simple in their nature compare to emission

spectra, thus atomic absorption spectroscopy is less prone to interelement interferences than flame

emission spectroscopy. Moreover, considering the high ratio of the number of atoms in the ground

and excited states, atomic adsorption spectroscopy is more sensitive than flame emission

spectroscopy (in case of heat excitation fewer atoms transit to the excited state than in case of light

excitation). However, in this ratio, the wavelength of resonance line is important (crucial) factor,

and in flame emission spectroscopy the elements which resonance lines have relatively low energy

values are more sensitive than those which resonance lines are associated with higher energy

values. Thus, sodium’s emission line at 589 nm shows high sensitivity in flame emission

spectroscopy, whereas zinc’s emission line is relatively less sensitive at 213.9 nm.

Atomic absorption spectroscopy is used to detect presence of heavy metals in different samples

(biological, nutritional) and drugs. This method is more sensitive than flame photometry; it is

highly selective method for drugs quality control. Disadvantages of the method are:

- it is mostly used for metals detection;

- for detection of each element, certain cathode lamp is required, as atoms absorb and emit in

very narrow range. For instance, for identification of zinc (which absorbs and emits at 214 nm)

cathodic lamp covered with zinc is used (cathode lamp is covered by its material for each metal).

The disadvantage is that the lamp should be changed for detecting each element, and only one

metal can be detected at the same time. Modern devices are equipped with up to 12 cathode lamps.

61

Linear spectra

If the atom had only one excited state, we would have received only one absorption spectral

line, in fact, the atomic spectra consist of many lines, spaces between which decrease along with

the wavelength decrease, the absorption intensity also decreases until the moment when in the

specter no line is seen: full absorption is carried out. This point is called spectral series boundary

or continuous environment of ionization.

Figure 47: Sodium atomic specter.

All the electronic transitions and, consequently, the number of spectral lines for the certain

element depends on its external electrons’ distribution. Atoms containing less electrons in external

orbital, such as alkali metals, have a minimum number of spectral lines and vice versa, the atoms

of complex external electron structure, especially elements of the supporting groups, have rather

complex absorption spectra.

For example, Na has an electron in its outer orbital in ground state, which, in case of heat or

light excitation, passes to a higher energy orbital, as shown in Figure 48. Subsequently, the

electron passes to a lower energy level, in which result sodium emits yellow light at 589.3 nm

wavelength, conditioned by 3p-3s transition, which is the most intensive line of sodium specter. In

the Na specter, there are two more lines, at 330.2 and 819.5 nm wavelengths, which are

conditioned by transitions from other levels, but for Na “yellow” line of its specter is

characteristic.

Figure 48: Sodium valent electrons transitions.

62

Besides, as we have seen already, different energies are required for different atoms excitation,

and therefore different wavelengths and passing from the alkali metals to heavy metals resonance

line shifts to the shorter wavelengths side, up to vacuum UV zone (metal-like elements, elements

of d subgroup). Theoretically, any element can be detected by atomic absorption method, but in

practice, there are limitations which are conditioned by used device.

Thus, the absorption of metals is in visible and UV-region and the absorption of the metal-like

elements is carried out mainly in vacuum UV-region, so AAS is used primarily for metal elements

identity and quantitative analysis. For detecting metal’s identity, only one characteristic spectral

line is sufficient and it is not mandatory to realize registration of element entire linear specter. Any

line is selective and is typical for a particular element, though some coincidence might occur with

the other elements line. All the lines of the atomic specter are resonance and, depending on their

transition possibility are differed by their absorption intensity. For simple atomic structures,

alkaline and alkaline-earth metals, the most intensive, hence the easiest excited line corresponds

to the transition from the ground state to the first excited state, E0- E1. This transition usually

requires less energy, so this line has long wavelength. The most intensive line among a number of

atoms corresponds to transition from ground to higher energy levels. Generally, for atoms

detection as a characteristic line, the most effective line of the specter is used, but in some cases,

it is possible to use less intensive line to avoid coincidences. Usually, the characteristics of the

main resonance lines are given by the atomic absorption device as well.

Absorption for one line is also measured for quantitative analysis. Here, also Lambert-Ber’s

combined law is worked, in this case, the intensity of emission source is directly conditioned by

the number of absorbing atoms. In atomic absorption spectroscopy, as in the molecular absorption,

the optical density D is detected by the logarithmic ratio of falling light intensity (I0) and passed

light intensity (Ip).

D = logI0/Ip = KLN0,

where,

N0 is the atoms concentration in flame (number of atoms per milliliter),

L- length of the pathway passed through flame (cm),

K- absorption coefficient constant.

This is a linear function for small values of optical density.

Graded graphs are used for quantitative analysis by these methods, which are build testing a

few standard samples and "zero" solution.

Sensitivity and detection limit of the method

The analytical sensitivity of the element analysis method is usually expressed by two

measurable indicators: characteristic concentration and detection limit. Characteristic

concentration is the amount of sensitivity which is the mass of the analyzed element that gives

D=0.0044 absorption signal, which corresponds to 1% absorption. Detecting minimum limit (as

in other analytical methods) is the concentration of the element in the solution which forms 3 times

more intensive absorption from the main line noise (in this case from the absorption line of zero

solution). It is noteworthy to mention that it is impossible precise detection near to this limit, as

the error possibility in this case is 33.3%. For this reason, laboratory tests usually are carried out

for concentrations exceeding minimum detection limit 6-10 times.

63

Used devices

Devices used for flame emission, atomic absorption and atomic fluorescence spectroscopy have

the following main structural components:

Figure 49: The schematic structure of atomic absorption, atomic fluorescence and flame

emission spectrometers

The flame-atomization providing component (in flame method) is present in all methods’

devices (AAS, FES, AFS) and represents a mixture of burning and oxidant gases, which transforms

metal containing sample into atoms in gaseous state. In these methods a nebulizer-burner system

is required. In contrast to UV and visible spectroscopic methods, where the absorbing compartment

is a quartz cuvette, the absorbing compartment in AAS and FES is a flame of the burner where the

atoms of the tested sample are present. The sample is introduced into the burner by injection way

only in form of solution after which, with the help of nebulizer which provides sample’s

transformation to fine aerosol, passes into the flame.

Figure 50: The structure of nebuliser-burner.

64

The aerosol drops of the solution should have diameter maximum of several μm for effective

atomization of the sample. Larger drops should be removed, as for their evaporation and salts

decomposition more time is required, in which result complete atomization may not occur. Thus,

system’s sensitivity is evaluated by its ability to convert the solution to the desired particle state

by large drops sedimentation. The yield of ready aerosol can be 5-15%. As a nebulizing

environment oxidant gas (for instance, air) can be used, which is given under 2-3 bar pressure.

Since, according to Lambert-Ber's law, the absorption is directly proportional to the passed light

pathway in the absorbing compartment, the burners are made in the form of long path. Usually,

the burner path has a length of 10 cm and a width of 0.5 cm. For the light passing through the

length of the path it is required that the burner should be placed directly on the pathway of the

beam.

Table 12:

Flame temperatures with various fuels

Fuel gas Temperature (K)

Air Nitrous oxide

Acetylene 2400 3200

Hydrogen 2300 2900

Propane 2200 3000

An essential requirement of flame emission and atomic absorption spectroscopy is a flame

temperature greater than 2000K. As an oxidant gas usually, air, nitrous oxide (NO) or oxygen

diluted with either nitrogen or argon are used. As a fuel gas, methane, propane, acetylene, hydrogen

are used. In the table 13 some of gas mixtures characteristics are presented. The acetylene-air

mixture is the most frequently used gaseous mixture in the atomic absorption spectroscopy which

temperature is t0=2300°C. This flame is absolutely permeable in the quite wide range of the specter

and gives some absorption only at wavelength less than 230nm.

Table 13:

Flames characteristics of various gas mixtures.

Fuel Oxidizer

Temperature

of flame, 0C

Burning speed

sm•sec-1

Nature

of flame Analytic usage

CH4 air 1800 55 laminar Alkaline metals detection

C2H8 air 2300 260 laminar

Absorbing chamber for

atomic absorption

spectroscopy

H2 O2 2600 3700 turbulent Used for heavy metals

detection

C2H8 N2O 2800 120 laminar

Absorbing chamber for

atomic absorption

spectroscopy

65

The usage of acetylene-air mixture is convenient because it has wide ranges of applications,

thus, for example, in standard conditions, it works by principle of weak oxidizer (in the air excess).

Under strong oxidation conditions it can be used for some noble metals, Au, Ir, Pd, Pt, Rh detection

with high sensitivity and without decomposition of sample. In case of alkaline-earth metals, in

contrast, it is convenient to use flame with weak reducing properties (acetylene excess).

The other mixture used for absorption chamber in atomic absorption is the mixture of acetylene-

nitrogen oxide gases (t0=2800oC), which is used for the elements which are difficult to detect with

acetylene-air mixture. This mentioned flame has expressed reducing properties and is used for

those elements detection that have great affinity to the oxygen: aluminum, bor, silicium.

The disadvantages of the atomization-flame method are: at first, the sample should be in liquid

form in form of solution and its viscosity should not be too high. Samples such as blood, blood

serum, and oils should be diluted beforehand. Additional procedures are required for this, as well

as the waste of time and ssubstances. To obtain the analytic signal, a certain minimum sample

volume (0.5-1mL) is required. According to data, only 5-15% of the inserted sample reaches the

flame and the sensitivity of this method sometimes does not enough to detect very small quantities.

And finally, the flame has certain self-absorption, which disturbs measuring in further UV-region.

Beside flame method, there are also other methods of atomization the sample, using the non-

flammable chamber for which burner is not required. These methods are:

• usage of graphite tube heaters,

• hydride compounds and cold vapor method.

The graphite tube has a length of 30 mm and an internal diameter of 6 mm and is found in the

cooling chamber on the pathway of AAS beam filled with any inert gas, argon or nitrogen, which

protects the graphite from burning. A few mcl of sample is put in the chamber and it is heated up

to 3000oC. In such tube evaporation and atomization of the sample occurs at the same time.

Moreover, it should be noted that, in contrast to flame method, here the efficiency of samples

imsertion is 50-90%.

On the other hand, this method allows to import samples not only in form of a solution (even in

viscous), but also in solid form, in form of a powder or suspension. The method difference is that

the tube temperature remains high about 10sec during which the atomization is carried out, then

the tube is cooled up to room temperature, while the flame temperature always remains constant.

This helps to abolish the existing substances, such as solvent during the analysis.

Figure 51: The working principle of graphite tube heater.

66

However, the greatest advantage of this method is its sensitivity, which exceeds flame method

100-1000 times, its detection limit is within picograms limit, and the sample volume can be 10-50

mcl in this case. However, in contrast to flame method, this method is slower, 2-3 minutes (10sec

in flame method) are needed for analysis, and for the next analysis the heater should be cooled up

to room temperature with the help of water. The other, the more serious disadvantage is that there

may be chemical interferences in the graphite tube, which may become noticeable inhibition

reason of signal. It is not possible to eliminate them correcting the background as in flame method,

but require complex procedures.

Next non-flame method is based on the use of hydrid compounds and mercury cold vapors. In

this method, the phenomenon is used, that some elements of IV, V and VI major groups form

volatile hydrides with hydrogen. The great advantage of this method is that the analyzed element

is released from other components containing in the sample prior to atomization in the gasous

hydride form, that is why, there is almost no spectral interferences occur in case of method correct

usage.

Thus, hydride generating elements such as arsenic, selenium, tellurium, bismuth, tin (stannum),

antimony can be released and detected with high sensitivity and almost without deviations. In the

reaction tube in the result of reaction with the reducer sodium bortetrahydride gasous hydrides are

formed, which pass through up to 800-1000oC heated quartz tube along with the formed hydrogen

vapors by argon flow. There, at relatively low temperatures, atomization is occurred. In case of

flame, due to the substance conversion to gaseous state dilution is carried out by 105-106, whereas

in hydride method dilution factor does not exceed 500. In this case, the sensitivity of method

increases 103 times compared to flame method. In hydride method detection absolute limit is

smaller than that of graphite tube usage, but the amount of sample wasted is greater about 0.5ml,

the relative standard deviation is 1-2%. On the other hand, the sample preparation process here is

more laborious than in graphite tube method, the sample should be fully dissolved, and the

detecting element should be converted to required oxidation degree.

By the same method mercury also can be detected which is already in atomic vapor state at

room temperature, and it should only be transported by inert gas flow to absorbing chamber.

Figure 52: The scheme of hydride compounds and mercury cold vapors method.

Taking into account the high toxicity of mercury, it should be detected everywhere, even in

drinking water, where its quantity must not exceed 0.002mg/l. Mercury shows low sensitivity in

flame and, in case of graphite tubes usage, a problem occurs when the mercury is released from

matrix as it evaporates at low temperatures and is practically impossible to perform. Thus, this

method is the most preferable for the mercury detection, since the problem is only to reduce

67

mercury to free metal with the help of suitable reducer. Mercury is the only metal which has

considerable amount of vapor pressure at room temperature, that provides detection standard

limits approximately 0.1 mcg/l (100 billion portion). Tin chloride or sodium borhydride are used

as reducer. During the latter's usage any of the mercury, organic or inorganic compound

decomposition happens, in case of tin chloride, pre-mineralization or hydrolysis of organic

compound is required.

Monochromator: in flame emition method it is necessary to isolate emitted light, and in atomic

absorption method monochromator serves to isolate emitted light from the cathode lamp.

Detector: is available in all types of devices, atomic absorption, atomic fluorescence and flame

emission spectrometers: it is light-sensitive chamber. The detector is connected to the

corresponding recorder by which the absorption, emission or fluorescence spectra are obtained.

Source of Resonance Line: it is also required source of resonance line (light) in atomic

absorption and atomic fluorescence spectroscopy. Moreover, these sources of light are usually

changed (automatically or mechanically) for each element detection. As already mentioned, the

source of light is a cathode lamp that is covered by corresponding element. The source of light in

atomic absorption spectrometer is in the same direction as a detector, and in atomic fluorescence

spectrometer it is located under straight angle towards detector from the right side as shown in

Figure 49.

The cathode lamp is a glass cylindric tube which under pressure is filled with any inert gas,

neon or argon.

Figure 53: Cathode lamp structure.

There are anode and cathode inside the lamp. The cathode is small cylinder filled with an

element of interest or made of this substance. Anode is a tube made of tungsten (wolfram) or

nickel. When 100-400V voltage is given between the anode and cathode, inert gas ionization is

carried out; the ions touched by the cathode are discharged, simultaneously the cathode metal

atoms are detached and excited. Transiting from the excited state to ground state, the atoms emit

waves with corresponding frequency. In AAS also combined lamps are used, in which mixtures

of more than two elements are present. The advantage is that with these lamps several elements

can be detected at the same time. The disadvantage is that the intensity of the specter for each

element is less than in case of individual lamps usage and this is more noticeable in lamps

containing more than 4 components. Generally, this method is not so justified. Nowadays, there

are used devices that have several lamps simultaneously gathered into a narrow bunch of 3 mm

68

diameter through special optics that passes through an absorbing chamber, and then is split by

polycromator to separate beams.

Atomic absorption spectroscopy corresponding method choice criteria

By choosing one of the mentioned three methods is conditioned by a number of factors for

solving certain analytical problem. Here, it should be considered not only the sensitivity of method,

but also the time required for analysis and the waste of sample. In table 14 each method criteria

are shown which can serve as a support to orientate in that matter.

Table 14:

Different methods of atomic absorption spectroscopy

By flame

• high accuracy

• high speed

• detection limit in million parts range

By graphite tube heaters

• detection limit from milliard to trillion parts range

• work with microsamples

• Possibility of solid sample dosage

By hydrides and mercury

cold vapor

• the best detection limits for Hg, As, Bi, Sb, Se, Sn, Te

• interference relative absence

Besides, sometimes the automatization possibility of method should be considered. In fact,

flame, graphite tube and hydides (cold) vapors methodes are handful for automatization.

Interferences of atomic absorption spectroscopy

Since the atomic absorption spectroscopy is relative method, i.e. quantitative detection is carried

out only based on standards, so any behavior of the sample differing from the standard can be a

reason of interferences. Depending on the caused reasons interferences are:

• chemical,

• physical,

• ionized,

• background,

• spectral.

Chemical interference is the formation of any compound which can interfere quantitative

atomization of the element. When aerosol drops dry, chemical reactions can occur, for example, if

in the solution where calcium is detected Na+, Cl-, SO42- ions are contained, first of all CaSO4 is

formed in the flame, which is transformed into CaO under heat influence, which ialmost doesn’t

undergo atomization, but if CaCl2 is used for calibration, CaO does not occur. Thus, under the

same Ca quantity conditions in the standard and tested sample’s flame, lesser calcium ions are

formed due to sulphate ions. The universal method to avoid such disorders is the addition of

another cation with excess, which with sulphate forms less saluble salt than tested element. In the

given example barium can be used: CaCl2 is formed and the entire is sedimented in BaSO4

sediment form.

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In phosphates presence, hardly melting calcium phosphate can be formed, which can be

dissociated to atoms with great difficulty. In this case, complex forming ethylenediamine tetra

acetic acid is added which prevents the formation of calcium phosphate, on the other hand calcium

is easily undegoes atomization.

To avoid such interferences, they are also used with a higher heat and strong recovery

properties, with a mixture of acetylene/nitrogen oxide that can even atomize even difficult

compounds.

Physical interferences include all the disorders in which the number of atoms is detected by

the physical properties of the sample. In any case, this is due to the ability of the spray to produce

aerosols, which, in turn, depends on the following physical properties of the sample:

• density,

• viscosity,

• surface tension.

If the sample and the standard have different physical properties, the number of aerosols in the

unit times are formed, causing different quantities atoms formation. In order to avoid such

interferences, the samples are diluted, but this is possible when the concentration of the detected

element is high enough. If the components influencing the physical properties of the sample, thus

the standard can also be prepared by adding them. If it is not possible to match the physical

properties of the sample and the standard, then adds the method of adding, constantly increasing

the standard concentration, builds the graph, and submits the data.

There are also special hydraulic, high pressure nebulizers in which physical interferences are

not occur.

Ionization interferences. Many metals alkaline and alkaline-earth are ionized in high-

temperature conditions losing electrons. Since ionized atoms have quite different spectra, so it

can’t be detected by atomic absorption method. Thus, the total loss of atoms and disorders reoccur.

There are two principal approaches to avoid such interferences.

At first, a lower temperature flame can be used: air-hydrogen, in which, for example, sodium is

almost not ionized, whereas it is 20% ionized in the air-acetylene mixture. On the other hand, the

loss of electron is a reversible process, and the balance can be shifted to the left, adding to the

sample easy ionizable element (mainly potassium or cesium), due to which excess of electrons is

formed.

Spectral interferences. These types of disorders are not typical for the atomic absorption

method, but may occur when multi-element lamps used.