Analytical chemistry is the study of the separation, identification, and quantification of the chemical components of natural and artificial materials. Qualitative analysis gives an indication of the identity of the chemical species in the sample and quantitative analysis determines the amount of one or more of these components. The separation of components is often performed prior to analysis.

Analytical methods can be separated into classical and instrumental. Classical methods (also known as wet chemistry methods) use separations such as precipitation, extraction, and distillation and qualitative analysis by color, odor, or melting point. Quantitative analysis is achieved by measurement of weight or volume. Instrumental methods use an apparatus to measure physical quantities of the analyte such as light absorption, fluorescence, or conductivity. The separation of materials is accomplished using chromatography, electrophoresis or Field Flow Fractionation methods.

Analytical chemistry is also focused on improvements in experimental design, chemometrics, and the creation of new measurement tools to provide better chemical information. Analytical chemistry has applications in forensics, bioanalysis, clinical analysis, environmental analysis, and materials analysis.

Classical methods

 The presence of copper in this qualitative analysis is indicated by the bluish-green color of the flame.

Although modern analytical chemistry is dominated by sophisticated instrumentation, the roots of analytical chemistry and some of the principles used in modern instruments are from traditional techniques many of which are still used today. These techniques also tend to form the backbone of most undergraduate analytical chemistry educational labs.

Qualitative analysis

A qualitative analysis determines the presence or absence of a particular compound, but not the mass or concentration. That is it is not related to quantity.

Chemical tests

For more details on this topic.

There are numerous qualitative chemical tests, for example, the acid test for gold and the Kastle-Meyer test for the presence of blood.

Flame test

For more details on this topic, see Flame test.

Inorganic qualitative analysis generally refers to a systematic scheme to confirm the presence of certain, usually aqueous, ions or elements by performing a series of reactions that eliminate ranges of possibilities and then confirms suspected ions with a confirming test. Sometimes small carbon containing ions are included in such schemes. With modern instrumentation these tests are rarely used but can be useful for educational purposes and in field work or other situations where access to state-of-the-art instruments are not available or expedient.

Gravimetric analysis

For more details on this topic, see Gravimetric analysis.

Gravimetric analysis involves determining the amount of material present by weighing the sample before and/or after some transformation. A common example used in undergraduate education is the determination of the amount of water in a hydrate by heating the sample to remove the water such that the difference in weight is due to the loss of water.

Volumetric analysis

For more details on this topic, see Titration.

Titration involves the addition of a reactant to a solution being analyzed until some equivalence point is reached. Often the amount of material in the solution being analyzed may be determined. Most familiar to those who have taken college chemistry is the acid-base titration involving a color changing indicator. There are many other types of titrations, for example potentiometric titrations. These titrations may use different types of indicators to reach some equivalence point.

Instrumental methods

Block diagram of an analytical instrument showing the stimulus and measurement of response


For more details on this topic, see Spectroscopy.

Spectroscopy measures the interaction of the molecules with electromagnetic radiation. Spectroscopy consists of many different applications such as atomic absorption spectroscopy, atomic emission spectroscopy, ultraviolet-visible spectroscopy, x-ray fluorescence spectroscopy, infrared spectroscopy, Raman spectroscopy, dual polarisation interferometry, nuclear magnetic resonance spectroscopy, photoemission spectroscopy, Mössbauer spectroscopy and so on.

Mass spectrometry

For more details on this topic, see Mass spectrometry.

 An accelerator mass spectrometer used for radiocarbon dating and other analysis.

Mass spectrometry measures mass-to-charge ratio of molecules using electric and magnetic fields. There are several ionization methods: electron impact, chemical ionization, electrospray, fast atom bombardment, matrix assisted laser desorption ionization, and others. Also, mass spectrometry is categorized by approaches of mass analyzers: magnetic-sector, quadrupole mass analyzer, quadrupole ion trap, time-of-flight, Fourier transform ion cyclotron resonance, and so on.

Electrochemical analysis

For more details on this topic, see Electroanalytical method.

Electroanalytical methods measure the potential (volts) and/or current (amps) in an electrochemical cell containing the analyte. These methods can be categorized according to which aspects of the cell are controlled and which are measured. The three main categories are potentiometry (the difference in electrode potentials is measured), coulometry (the cell's current is measured over time), and voltammetry (the cell's current is measured while actively altering the cell's potential).

Thermal analysis

Further information: Calorimetry, thermal analysis

Calorimetry and thermogravimetric analysis measure the interaction of a material and heat.


 Separation of black ink on a thin layer chromatography plate.

Further information: Separation process, Chromatography, electrophoresis

Separation processes are used to decrease the complexity of material mixtures. Chromatography and electrophoresis are representative of this field.

Hybrid techniques

Combinations of the above techniques produce a "hybrid" or "hyphenated" technique. Several examples are in popular use today and new hybrid techniques are under development. For example, gas chromatography-mass spectrometry, gas chromatography-infrared spectroscopy, liquid chromatography-mass spectrometry, liquid chromatography-NMR spectroscopy, liquid chromagraphy-infrared spectroscopy and capillary electrophoresis-mass spectrometry.

Hyphenated separation techniques refer to a combination of two (or more) techniques to detect and separate chemicals from solutions. Most often the other technique is some form of chromatography. Hyphenated techniques are widely used in chemistry and biochemistry. A slash is sometimes used instead of hyphen, especially if the name of one of the methods contains a hyphen itself.


 Fluorescence microscope image of two mouse cell nuclei in prophase (scale bar is 5 µm).

For more details on this topic, see Microscopy.

The visualization of single molecules, single cells, biological tissues and nanomaterials is an important and attractive approach in analytical science. Also, hybridization with other traditional analytical tools is revolutionizing analytical science. Microscopy can be categorized into three different fields: optical microscopy, electron microscopy, and scanning probe microscopy. Recently, this field is rapidly progressing because of the rapid development of the computer and camera industries.


 A glass microreactor

Further information: microfluidics, lab-on-a-chip

Devices that integrate (multiple) laboratory functions on a single chip of only millimeters to a few square centimeters in size and that are capable of handling extremely small fluid volumes down to less than pico liters.



PCMA are divided on:

§        Optical methods are based on measurement of optical properties of substances.

§        Chromatographic methods are based on usage of ability of different substances to selective sorption.

§        Electrochemical methods are based on measurement of electrochemical properties of substances.

§        Radiometric methods are based on measurement of radioactive properties of substances.

§        Thermal methods are based on measurement of heat effects of substances.

§        Mass spectrometric methods are based on studying of the ionized fragments ("splinters") of substances.

§        Kinetic methods are based on measurement of dependence of speed of reaction from concentration of substance


Advantage of PCMA

§        High sensitivity - a low limit of detection (10-9 mg) and definition

§        High selectivity

§        Rapid analysis methods

§        Automation and computerization is possibility

§        Analysis is possibility on distance

§        Possibility of the analysis without destruction of the sample

§        Possibility of the local analysis


Lacks of PCMA

§        Definition error is near ± 5 % (on occasion to 20 %), whereas - 0,01-0,005 % for gravimetry and 0,1-0,05 % for titrimetry

§        Reproducibility of results in separate methods is worse, than in classical methods of the analysis

§        It is necessary of usage of standards and standard solutions, graduation of equipment and plotting of calibration charts

§        Complexity of used equipment, its high cost, high cost of standard substances



         A major part of modern Instrumental Analytical Chemistry, focuses on the study of the energy exchange between electromagnetic radiation and matter. These interactions are visible to the naked eye, when the radiations concerned fall within the visible spectrum.

À) On investigated objects

§        The nuclear spectral analysis

§        The molecular spectral analysis

B) On the nature of interaction of electromagnetic radiation with substance

1. Absorption analysis

§        Atomic-absorption analysis - Atomic absorption spectroscopy (AAS) is a spectroanalytical procedure for the quantitative determination of chemical elements employing the absorption of optical radiation (light) by free atoms in the gaseous state.

§        Molecular-absorption analysis - Molecular absorption spectroscopy in the ultraviolet (UV) and visible (VIS) is concerned with the measured absorption of radiation in its passage through a gas, a liquid or a solid.

§        Turbidimetric analysis - Turbidimetry (the name being derived from turbidity) is the process of measuring the loss of intensity of transmitted light due to scattering effect particles suspended in it. Light is passed through a filter creating a light of known wavelength which is then passed through a cuvette containing a solution.

2. The emissive spectral analysis

§        flame photometry - Flame photometry, more properly called flame atomic emission spectrometry, is a fast, simple, and sensitive analytical method for the determination of trace metal ions in solution.

§        fluorescence analysis - Luminescence is emission of light by a substance not resulting from heat; it is thus a form of cold body radiation.

§        The spectral analysis with usage of effect of combinational dispersion of light

3. Other methods

§        nephelometric method - Nephelometry is the measurement of scattered light.  This technique requires a special measuring instrument, where the detector is set at an angle to the incident light beam.


Optical arrangements of nephelometry and turbidimetry


§        refractometric analysis - Refractometry is the method of measuring substances' refractive index (one of their fundamental physical properties) in order to, for example, assess their composition or purity. A refractometer is the instrument used to measure refractive index ("RI")


Components in a typical reflectometer used to measure analytes on urine dipstick


§        polarimetric analysis - Polarimetry is the measurement and interpretation of the polarization of transverse waves, most notably electromagnetic waves, such as radio or light waves.


An automatic digital polarimeter

§        interferometric analysis - Interferometry is a family of techniques in which waves, usually electromagnetic, are superimposed in order to extract information about the waves.

File:Three amplitude-splitting interferometers.svg

Three amplitude-splitting interferometers: Fizeau, Mach–Zehnder, and Fabry Perot

C) On electromagnetic spectral range which use in analysis:

§        Spectroscopy (spectrophotometry) in UV and visible spectrum

§        IR - Spectroscopy - Infrared spectroscopy (IR spectroscopy) is the spectroscopy that deals with the infrared region of the electromagnetic spectrum, that is light with a longer wavelength and lower frequency than visible light.

§        X-ray spectroscopy - X-ray spectroscopy is a gathering name for several spectroscopic techniques for characterization of materials by using x-ray excitation.

§        Microwave spectroscopy - The interaction of microwaves with matter can be detected by observing the attenuation or phase shift of a microwave field as it passes through matter.

D) By the nature of energy jump

§        Electronic spectrum

§        Vibrational spectrum

§        Rotational spectrum

Spectrum (method)

The characteristic of energy of quantum


Radio-frequency  (NMR, EPR)




The optical


The visible


Infra-red (IR)





Gamma radiation (nuclear-physical)

101-10-1 meters



10-1-10-3 meters



200-400 nm

400-750 nm


10-13000 cm-1



10-8-10-10 m



10-10-10-13 m

Change of electron spin and nuclear spin


Change of rotational conditions


Change of valence electron conditions


Change of vibrational conditions


Change of a condition of internal electrons


Nuclear reactions




Overview of Spectroscopy

The focus of this chapter is photon spectroscopy, using ultraviolet, visible, and infrared radiation. Because these techniques use a common set of optical devices for dispersing and focusing the radiation, they often are identified as optical spectroscopies.

For convenience we will usually use the simpler term “spectroscopy” in place of photon spectroscopy or optical spectroscopy; however, it should be understood that we are considering only a limited part of a much broader area of analytical methods. Before we examine specific spectroscopic methods, however, we first review the properties of electromagnetic radiation.


Electromagnetic Radiation

Electromagnetic radiation, or light, is a form of energy whose behavior is described by the properties of both waves and particles. The optical properties of electromagnetic radiation, such as diffraction, are explained best by describing light as a wave.

Many of the interactions between electromagnetic radiation and matter, such as absorption and emission, however, are better described by treating light as a particle, or photon. The exact nature of electromagnetic radiation remains unclear, as it has since the development of quantum mechanics in the first quarter of the twentieth century. Nevertheless, the dual models of wave and particle behavior provide a useful description for electromagnetic radiation.


Wave Properties of Electromagnetic Radiation

Electromagnetic radiation consists of oscillating electric and magnetic fields that propagate through space along a linear path and with a constant velocity (Figure). In a vacuum, electromagnetic radiation travels at the speed of light, c, which is 2.99792  108 m/s. Electromagnetic radiation moves through a medium other than a vacuum with a velocity, v, less than that of the speed of light in a vacuum. The difference between v and c is small enough (< 0.1%) that the speed of light to three significant figures, 3.00  108 m/s, is sufficiently accurate for most purposes.

Oscillations in the electric and magnetic fields are perpendicular to each other, and to the direction of the wave’s propagation. Figure shows an example of plane-polarized electromagnetic radiation consisting of an oscillating electric field and an oscillating magnetic field, each of which is constrained to a single plane.


Normally, electromagnetic radiation is unpolarized, with oscillating electric  and magnetic fields in all possible planes oriented perpendicular to the direction of propagation.


The interaction of electromagnetic radiation with matter can be explained using either the electric field or the magnetic field. For this reason, only the electric field component is shown in Figure. The oscillating electric field is described by a sine wave of the form

where E is the magnitude of the electric field at time t, Ae is the electric field’s maximum amplitude, n is the frequency, or the number of oscillations in the electric field per unit time, and F is a phase angle accounting for the fact that the electric field’s magnitude need not be zero at t = 0. An identical equation can be written for the magnetic field, M

where Am is the magnetic field’s maximum amplitude.

An electromagnetic wave, therefore, is characterized by several fundamental properties, including its velocity, amplitude, frequency, phase angle, polarization, and direction of propagation.4 Other properties, which are based on these fundamental properties, also are useful for characterizing the wave behavior of electromagnetic radiation. The wavelength of an electromagnetic wave, l, is defined as the distance between successive maxima, or successive minima (see Figure above). For ultraviolet and visible electromagnetic radiation the wavelength is usually expressed in nanometers (nm, 10–9 m), and the wavelength for infrared radiation is given in microns (mm, 10–6 m). Unlike frequency, wavelength depends on the electromagnetic wave’s velocity, where

Thus, for electromagnetic radiation of frequency, n, the wavelength in vacuum is longer than in other media. Another unit used to describe the wave properties of electromagnetic radiation is the wavenumber, –n, which is the reciprocal of wavelength

Wavenumbers are frequently used to characterize infrared radiation, with the units given in reciprocal centimeter (cm–1).


Particle Properties of Electromagnetic Radiation

When a sample absorbs electromagnetic radiation it undergoes a change in energy. The interaction between the sample and the electromagnetic radiation is easiest to understand if we assume that electromagnetic radiation consists of a beam of energetic particles called photons.

When a photon is absorbed by a sample, it is “destroyed,” and its energy acquired by the sample. The energy of a photon, in joules, is related to its frequency, wavelength, or wavenumber by the following equations

where h is Planck’s constant, which has a value of 6.626 × 10–34 J · s.


The Electromagnetic Spectrum

The frequency and wavelength of electromagnetic radiation vary over many orders of magnitude. For convenience, electromagnetic radiation is divided into different regions based on the type of atomic or molecular transition that gives rise to the absorption or emission of photons (Figure).

The boundaries describing the electromagnetic spectrum are not rigid, and an overlap between spectral regions is possible.

Measuring Photons as a Signal

In the previous section we defined several characteristic properties of electromagnetic radiation, including its energy, velocity, amplitude, frequency, phase angle, polarization, and direction of propagation. Spectroscopy is possible only if the photon’s interaction with the sample leads to a change in one or more of these characteristic properties.

Spectroscopy is conveniently divided into two broad classes. In one class of techniques, energy is transferred between a photon of electromagnetic radiation and the analyte (Table 10.1).


In absorption spectroscopy the energy carried by a photon is absorbed by the analyte, promoting the analyte from a lower-energy state to a higher-energy, or excited, state (Figure).

The source of the energetic state depends on the photon’s energy. The electromagnetic spectrum in Figure, for example, shows that absorbing a photon of visible light causes a valence electron in the analyte to move to a higher-energy level. When an analyte absorbs infrared radiation, on the other hand, one of its chemical bonds experiences a change in vibrational energy.

The intensity of photons passing through a sample containing the analyte is attenuated because of absorption. The measurement of this attenuation, which we call absorbance, serves as our signal. Note that the energy levels in Figure have well-defined values (i.e., they are quantized). Absorption only occurs when the photon’s energy matches the difference in energy, E, between two energy levels. A plot of absorbance as a function of the photon’s energy is called an absorbance spectrum (Figure).

Ultraviolet/visible absorption spectrum for bromothymol blue.


Emission of a photon occurs when an analyte in a higher-energy state returns to a lower-energy state (Figure).

Simplified energy level diagram showing emission of a photon.


The higher-energy state can be achieved in several ways, including thermal energy, radiant energy from a photon, or by a chemical reaction. Emission following the absorption of a photon is also called photoluminescence, and that following a chemical reaction is called chemiluminescence.

A typical emission spectrum is shown in Figure.

Photoluminescent spectra for methyltetrahydrofolate and the enzyme methyltransferase. When methyltetrahydrofolate and methyltransferase are mixed, the enzyme is no longer photoluminescent, but the photoluminescence of methyltetrahydrofolate is enhanced.


In the second broad class of spectroscopy, the electromagnetic radiation undergoes a change in amplitude, phase angle, polarization, or direction of propagation as a result of its refraction, reflection, scattering, diffraction, or dispersion by the sample. Several representative spectroscopic techniques are listed in Table 10.2.


Absorbance of Electromagnetic Radiation

In absorption spectroscopy a beam of electromagnetic radiation passes through a sample.

Much of the radiation is transmitted without a loss in intensity. At selected frequencies, however, the radiation’s intensity is attenuated. This process of attenuation is called absorption. Two general requirements must be met if an analyte is to absorb electromagnetic radiation. The first requirement is that there must be a mechanism by which the radiation’s electric field or magnetic field interacts with the analyte. For ultraviolet and visible radiation, this interaction involves the electronic energy of valence electrons. A chemical bond’s vibrational energy is altered by the absorbance of infrared radiation. A more detailed treatment of this interaction, and its importance in determining the intensity of absorption, is found in the suggested readings listed at the end of the chapter.

The second requirement is that the energy of the electromagnetic radiation must exactly equal the difference in energy, DE, between two of the analytes quantized energy states. Figure 10.4 shows a simplified view of the absorption of a photon. The figure is useful because it emphasizes that the photon’s energy must match the difference in energy between a lower-energy state and a higher-energy state. What is missing, however, is information about the types of energetic states involved, which transitions between states are likely to occur, and the appearance of the resulting spectrum.

We can use the energy level diagram in Figure to explain an absorbance spectrum. The thick lines labeled E0 and E1 represent the analyte’s ground (lowest) electronic state and its first electronic excited state. Superimposed on each electronic energy level is a series of lines representing vibrational energy levels.

Infrared Spectra for Molecules and Polyatomic Ions

The energy of infrared radiation is sufficient to produce a change in the vibrational energy of a molecule or polyatomic ion. As shown in Figure above, vibrational energy levels are quantized; that is, a molecule may have only certain, discrete vibrational energies. The energy for allowed vibrational modes, Ev, is

where v is the vibrational quantum number, which may take values of 0, 1, 2, . . ., and n0 is the bond’s fundamental vibrational frequency. Values for n0 are determined by the bond’s strength and the mass at each end of the bond and are characteristic of the type of bond. For example, a carbon–carbon single bond (C—C) absorbs infrared radiation at a lower energy than a carbon–carbon double bond (C=C) because a C—C bond is weaker than a C=C bond.

At room temperature most molecules are in their ground vibrational state (v = 0). A transition from the ground vibrational state to the first vibrational excited state (v = 1) requires the absorption of a photon with an energy of hn0.

Transitions in which Dv is ±1 give rise to the fundamental absorption lines. Weaker absorption lines, called overtones, are due to transitions in which Dv is ±2 or ±3. The number of possible normal vibrational modes for a linear molecule is 3N – 5, and for a nonlinear molecule is 3N – 6, where N is the number of atoms in the molecule. Not surprisingly, infrared spectra often show a considerable number of absorption bands. Even a relatively simple molecule, such as benzene (C6H6), for example, has 30 possible normal modes of vibration, although not all of these vibrational modes give rise to an absorption. A typical IR spectrum is shown in Figure.

Fourier transform infrared (FT–IR) spectrum of polyvinylchloride


UV/Vis Spectra for Molecules and Ions

When a molecule or ion absorbs ultraviolet or visible radiation it undergoes a change in its valence electron configuration.

The valence electrons in organic molecules, and inorganic anions such as CO32–, occupy quantized sigma bonding, s, pi bonding, p, and nonbonding, n, molecular orbitals.

Unoccupied sigma antibonding, s*, and pi antibonding, p*, molecular orbitals often lie close enough in energy that the transition of an electron from an occupied to an unoccupied orbital is possible.

Four types of transitions between quantized energy levels account for molecular UV/Vis spectra. The approximate wavelength ranges for these absorptions, as well as a partial list of bonds, functional groups, or molecules that give rise to these transitions is shown in Table 10.5. Of these transitions, the most important are the n®p* and p ® p*, because they involve functional groups that are characteristic of the analyte and wavelengths that are easily accessible. The bonds and functional groups that give rise to the absorption of ultraviolet and visible radiation are called chromophores.

Many transition metal ions, such as Cu2+ and Co2+, form solutions that are colored because the metal ion absorbs visible light. The transitions giving rise to this absorption are due to valence electrons in the metal ion’s d-orbitals. For a free metal ion, the five d-orbitals are of equal energy. In the presence of a complexing ligand or solvent molecule, however, the d-orbitals split into two or more groups that differ in energy. For example, in the octahedral complex Cu(H2O)62+ the six water molecules perturb the d-orbitals into two groups as shown in Figure.

The resulting dd transitions for transition metal ions are relatively weak. A more important source of UV/Vis absorption for inorganic metal–ligand complexes is charge transfer, in which absorbing a photon produces an excited state species that can be described in terms of the transfer of an electron from the metal, M, to the ligand, L.

Charge-transfer absorption is important because it produces very large absorbances, providing for a much more sensitive analytical method. One important example of a charge-transfer complex is that of o-phenanthroline with Fe2+, the UV/Vis spectrum for which is shown in Figure.

UV/Vis spectrum for Fe(o-phenanthroline)32+.


Charge-transfer absorption in which the electron moves from the ligand to the metal also is possible.

Comparing the IR spectrum in Figure above to the UV/Vis spectrum in Figure

above, we note that UV/Vis absorption bands are often significantly broader than those for IR absorption.

When a species absorbs UV/Vis radiation, the transition between electronic energy levels may also include a transition between vibrational energy levels. The result is a number of closely spaced absorption bands that merge together to form a single broad absorption band.

UV/Vis Spectra for Atoms

The energy of ultraviolet and visible electromagnetic radiation is sufficient to cause a change in an atom’s valence electron configuration. Sodium, for example, with a valence shell electron configuration of [Ne] 3s1, has a single valence electron in its 3s atomic orbital. Unoccupied, higher energy atomic orbitals also exist. Figure 10.18 shows a partial energy level diagram for sodium’s occupied and unoccupied valence shell atomic orbitals.

Valence shell energy diagram for sodium


This configuration of atomic orbitals, which shows a splitting of the p orbitals into two levels with slightly different energies, may differ from that encountered in earlier courses. The reasons for this splitting, however, are beyond the level of this text, and unimportant in this context.

Absorption of a photon is accompanied by the excitation of an electron from a lower-energy atomic orbital to an orbital of higher energy. Not all possible transitions between atomic orbitals are allowed.

For sodium the only allowed transitions are those in which there is a change of ±1 in the orbital quantum number (l); thus transitions from s®p orbitals are allowed, but transitions from s®d orbitals are forbidden. The wavelengths of electromagnetic radiation that must be absorbed to cause several allowed transitions are shown in Figure above.

The atomic absorption spectrum for Na is shown in Figure and is typical of that found for most atoms.

Atomic absorption spectrum for sodium


The most obvious feature of this spectrum is that it consists of a few, discrete absorption lines corresponding to transitions between the ground state (the 3s atomic orbital) and the 3p and 4p atomic orbitals. Absorption from excited states, such as that from the 3p atomic orbital to the 4s or 3d atomic orbital, which are included in the energy level diagram in Figure above, are too weak to detect. Since the lifetime of an excited state is short, typically 10–7–10–8 s, an atom in the excited state is likely to return to the ground state before it has an opportunity to absorb a photon.

Another feature of the spectrum shown in Figure is the narrow width of the absorption lines, which is a consequence of the fixed difference in energy between the ground and excited states. Natural line widths for atomic absorption, which are governed by the uncertainty principle, are approximately 10–5 nm. Other contributions to broadening increase this line width to approximately 10–3 nm.



First law of light absorption

§        Each thin layer of constant thickness of a homogeneous environment absorbs an identical part of incident radiation


§        The part of the light  which is absorbed by a homogeneous environment, is directly proportional to a thickness of an absorbing layer:


Second law of light absorption

The part of the absorbed radiation is proportional to number of absorbing particles in volume of a solution, that is concentration

Bouguer-Lambert-Beer law

Reduction of intensity of light which has passed through a layer of light-absorbing substance is proportional concentration of this substance and a thickness of a layer


Quantitative characteristics of absorption

1. Transmittance - the ratio of the radiant power passing through a sample to that from the radiation’s source (T).

The attenuation of electromagnetic radiation as it passes through a sample is described quantitatively by two separate, but related terms: transmittance and absorbance. Transmittance is defined as the ratio of the electromagnetic radiation’s power exiting the sample, PT, to that incident on the sample from the source, P0,


Multiplying the transmittance by 100 gives the percent transmittance (%T), which varies between 100% (no absorption) and 0% (complete absorption).

All methods of detection, whether the human eye or a modern photoelectric transducer, measure the transmittance of electromagnetic radiation.

Attenuation of radiation as it passes through the sample leads to a transmittance of less than 1. As described, equation 10.1 does not distinguish between the different ways in which the attenuation of radiation occurs. Besides absorption by the analyte, several additional phenomena contribute to the net attenuation of radiation, including reflection and absorption by the sample container, absorption by components of the sample matrix other than the analyte, and the scattering of radiation. To compensate for this loss of the electromagnetic radiation’s power, we use a method blank. The radiation’s power exiting from the method blank is taken to be P0.


Diagram of Beer–Lambert absorption of a beam of light as it travels through a cuvette of width ℓ.


Optical density À (Absorbance)

An alternative method for expressing the attenuation of electromagnetic radiation is absorbance, A, which is defined as

An alternative method for expressing the attenuation of electromagnetic radiation is absorbance, A, which is defined as

Absorbance is the more common unit for expressing the attenuation of radiation because, as shown in the next section, it is a linear function of the analyte’s concentration.

Bouguer-Lambert-Beer law


§        The absorbance of a solution is proportional to concentration of light-absorbing substance and a thickness of a layer


§        The relationship between a sample’s absorbance and the concentration of the absorbing species

where: A – optical density (absorbance), ε – the molar absorptivity, C – concentration (molarity)


Additivity of optical densities

Beer’s law can be extended to samples containing several absorbing components provided that there are no interactions between the components. Individual absorbances, Ai, are additive. For a two-component mixture of X and Y, the total  absorbance, Atot, is


A = l(e1Ñ1 + e2Ñ2 + …ekÑk)


e (the molar absorptivity)

Iron (²²²) rhodanate


Complex Ti with H2O2


Complex Ti with chromotrope acid


Complex Cu with ammonia

5 ×102

Complex Cu with dithizon

5 ×104

Complex Al with aluminon

1,7 ×104

Complex Al with 2-stilbazole

3,5 ×104


Limitations to Beer’s Law

According to Beer’s law, a calibration curve of absorbance versus the concentration of analyte in a series of standard solutions should be a straight line with an intercept of 0 and a slope of ab or eb. In many cases, however, calibration curves are found to be nonlinear (Figure 10.22).

Calibration curves showing positive and negative deviations from Beer’s law.


Deviations from linearity are divided into three categories: fundamental, chemical, and instrumental.

Fundamental Limitations to Beers Law

Beer’s law is a limiting law that is valid only for low concentrations of analyte. There are two contributions to this fundamental limitation to Beer’s law. At higher concentrations the individual particles of analyte no longer behave independently of one another. The resulting interaction between particles of analyte may change the value of e. A second contribution is that the absorptivity, a, and molar absorptivity, e, depend on the sample’s refractive index. Since the refractive index varies with the analyte’s concentration, the values of a and e will change. For sufficiently low concentrations of analyte, the refractive index remains essentially constant, and the calibration curve is linear.


Instrumental Limitations to Beer’s Law

There are two principal instrumental limitations to Beer’s law. The first limitation is that Beer’s law is strictly valid for purely monochromatic radiation; that is, for radiation consisting of only one wavelength.

However, even the best wavelength selector passes radiation with a small, but finite effective bandwidth. Using polychromatic radiation always gives a negative deviation from Beer’s law, but is minimized if the value of e is essentially constant over the wavelength range passed by the wavelength selector.

For this reason, as shown in Figure 10.23, it is preferable to make absorbance measurements at a broad absorption peak.

Effect of wavelength on the linearity of a Beer’s law calibration curve


In addition, deviations from Beer’s law are less serious if the effective bandwidth from the source is less than one tenth of the natural bandwidth of the absorbing species. When measurements must be made on a slope, linearity is improved by using a narrower effective bandwidth.

Stray radiation is the second contribution to instrumental deviations from Beer’s law. Stray radiation arises from imperfections within the wavelength selector that allows extraneous light to “leak” into the instrument. Stray radiation adds an additional contribution, Pstray, to the radiant power reaching the detector; thus

For small concentrations of analyte, Pstray is significantly smaller than P0 and PT, and the absorbance is unaffected by the stray radiation. At higher concentrations of analyte, however, Pstray is no longer significantly smaller than PT and the absorbance is smaller than expected. The result is a negative deviation from Beer’s law.


Physical Limitations to Beer’s Law

§        NOT monochromaticity of light:

A = el×l×Ñ.

§        NOT parallelism of light.

§        Temperature.

§        NOT identical value of refraction of solutions.

§        NOT proportionality of a photocurrent and intensity of a light


Chemical Limitations to Beer’s Law

§        Dilution of solution (than more of reagent excess, it is less deviation from the law);

§        ðÍ of medium: state of metal ion

                             stability of complex ions

§        competitive reactions (for ligand)

§        competitive reactions (complexing agent)

§        polymerization and dissociation reactions

§        ox-red reactions

Chemical deviations from Beer’s law can occur when the absorbing species is involved in an equilibrium reaction. Consider, as an example, an analysis for the weak acid, HA. To construct a Beer’s law calibration curve, several standards containing known total concentrations of HA, Ctot, are prepared and the absorbance of each is measured at the same wavelength. Since HA is a weak acid, it exists in equilibrium with its conjugate weak base, A

If both HA and A absorb at the selected wavelength, then Beers law is written as

where CHA and CA are the equilibrium concentrations of HA and A. Since the weak acid’s total concentration, Ctot, is

the concentrations of HA and A can be written as

where aHA is the fraction of weak acid present as HA. Substituting equations gives

Because values of aHA may depend on the concentration of HA, equation may not be linear. A Beer’s law calibration curve of A versus Ctot will be linear if one of two conditions is met. If the wavelength is chosen such that eHA and eA are equal, then equation simplifies to

and a linear Beer’s law calibration curve is realized. Alternatively, if aHA is held constant for all standards, then equation will be a straight line at all wavelengths.

Because HA is a weak acid, values of aHA change with pH. To maintain a constant value for aHA, therefore, we need to buffer each standard solution to the same pH.

Depending on the relative values of eHA and eA, the calibration curve will show a positive or negative deviation from Beer’s law if the standards are not buffered to the same pH.



Components of a single-beam spectrophotometer

A, exciter lamp; B, entrance slit; C, monochromator; D, exit slit; E, cuvet; F, photodetector; G, LED display.


§        Molecular–absorption method is based on measurement of absorption by molecules (or ions) substances of electromagnetic radiation of an optical range:

§        Colorimetry in which visible light was absorbed by a sample. The concentration of analyte was determined visually by comparing the sample’s color to that of a set of standards using Nessler tubes (as described at the beginning of this chapter), or by using an instrument called a colorimeter.

§        Photocolorimetry - in which polychromatic light was absorbed by a sample

§        Spectrophotometry  - in which monochromatic light was absorbed by a sample

§        UV - Spectrum (100-200 to 380-400 nanometers)

§        Visible spectrum (380-400 to 780-800 nanometers)



Frequently an analyst must select, from several instruments of different design, the one instrument best suited for a particular analysis. In this section we examine some of the different types of instruments used for molecular absorption spectroscopy, emphasizing their advantages and limitations. Methods of sample introduction are also covered in this section.

Instrument Designs for Molecular UV/Vis Absorption

The simplest instrument currently used for molecular UV/Vis absorption is the filter photometer shown in Figure, which uses an absorption or interference filter to isolate a band of radiation.


The filter is placed between the source and sample to prevent the sample from decomposing when exposed to high-energy radiation. A filter photometer has a single optical path between the source and detector and is called a single-beam instrument.

The instrument is calibrated to 0% T while using a shutter to block the source radiation from the detector. After removing the shutter, the instrument is calibrated to 100% T using an appropriate blank. The blank is then replaced with the sample, and its transmittance is measured. Since the source’s incident power and the sensitivity of the detector vary with wavelength, the photometer must be recalibrated whenever the filter is changed. In comparison with other spectroscopic instruments, photometers have the advantage of being relatively inexpensive, rugged, and easy to maintain. Another advantage of a photometer is its portability, making it a useful instrument for conducting spectroscopic analyses in the field. A disadvantage of a photometer is that it cannot be used to obtain an absorption spectrum.

Instruments using monochromators for wavelength selection are called spectrometers. In absorbance spectroscopy, where the transmittance is a ratio of two radiant powers, the instrument is called a spectrophotometer. The simplest spectrophotometer is a single-beam instrument equipped with a fixedwavelength monochromator, the block diagram for which is shown in Figure.

Photo courtesy of Fisher Scientific


Single-beam spectrophotometers are calibrated and used in the same manner as a photometer. One common example of a single-beam spectrophotometer is the Spectronic-20 manufactured by Milton-Roy. The Spectronic-20 can be used from 340 to 625 nm (950 nm with a red-sensitive detector), and has a fixed effective bandwidth of 20 nm. Because its effective bandwidth is fairly large, this instrument is more appropriate for a quantitative analysis than for a qualitative analysis. Battery-powered, hand-held single-beam spectrophotometers are available, which are easily transported and can be used for on-site analyses.

Other single-beam spectrophotometers are available with effective bandwidths of 2–8 nm. Fixed-wavelength single-beam spectrophotometers are not practical for recording spectra since manually adjusting the wavelength and recalibrating the spectrophotometer is awkward and time-consuming. In addition, the accuracy of a single-beam spectrophotometer is limited by the stability of its source and detector over time.

The limitations of fixed-wavelength, single-beam spectrophotometers are minimized by using the double-beam in-time spectrophotometer as shown in Figure.


Block diagram for a double-beam in-time scanning spectrophotometer with photo of a typical instrument



A chopper, similar to that shown in the insert, controls the radiation’s path, alternating it between the sample, the blank, and a shutter. The signal processor uses the chopper’s known speed of rotation to resolve the signal reaching the detector into that due to the transmission of the blank (P0) and the sample (PT). By including an opaque surface as a shutter it is possible to continuously adjust the 0% T response of the detector.

The effective bandwidth of a double-beam spectrophotometer is controlled by means of adjustable slits at the entrance and exit of the monochromator. Effective bandwidths of between 0.2 nm and 3.0 nm are common. A scanning monochromator allows for the automated recording of spectra. Double-beam instruments are more versatile than single-beam instruments, being useful for both quantitative and qualitative analyses; they are, however, more expensive.

The instrument designs considered thus far use a single detector and can only monitor one wavelength at a time. A linear photodiode array consists of multiple detectors, or channels, allowing an entire spectrum to be recorded in as little as 0.1 s.

Source radiation passing through the sample is dispersed by a grating. The linear photodiode array is situated at the grating’s focal plane, with each diode recording the radiant power over a narrow range of wavelengths.

The sample compartment for the instruments in Figures above provides a light-tight environment that prevents the loss of radiation, as well as the addition of stray radiation. Samples are normally in the liquid or solution state and are placed in cells constructed with UV/Vis-transparent materials, such as quartz, glass, and plastic (Figure).

Typical cells used in UV/Vis spectroscopy


Quartz or fused-silica cells are required when working at wavelengths of less than 300 nm where other materials show a significant absorption. The most common cell has a pathlength of 1 cm, although cells with shorter (³ 1 mm) and longer pathlengths (£ 10 cm) are available. Cells with a longer pathlength are useful for the analysis of very dilute solutions or for gaseous samples. The highest quality cells are constructed in a rectangular shape, allowing the radiation to strike the cell at a 90° angle, where losses to reflection are minimal. These cells, which are usually available in matched pairs having identical optical properties, are the cells of choice for double-beam instruments. Cylindrical test tubes are often used as a sample cell for simple, single-beam instruments, although differences in the cell’s pathlength and optical properties add an additional source of error to the analysis.

In some circumstances it is desirable to monitor a system without physically removing a sample for analysis. This is often the case, for example, with the on-line monitoring of industrial production lines or waste lines, for physiological monitoring, and for monitoring environmental systems. With the use of a fiber-optic probe it is possible to analyze samples in situ. A simple example of a remote-sensing, fiber-optic probe is shown in Figure a and consists of two bundles of fiber-optic cable.

Example of fiber-optic probes


One bundle transmits radiation from the source to the sample cell, which is designed to allow for the easy flow of sample through the cell. Radiation from the source passes through the solution, where it is reflected back by a mirror. The second bundle of fiber-optic cable transmits the nonabsorbed radiation to the wavelength selector. In an alternative design (Figure b), the sample cell is a membrane containing a reagent phase capable of reacting with the analyte. When the analyte diffuses across the membrane, it reacts with the reagent phase, producing a product that absorbs UV or visible radiation. Nonabsorbed radiation from the source is reflected or scattered back to the detector. Fiber-optic probes that show chemical selectivity are called optrodes.

Choice of optimum conditions of spectrophotometry:

§        Choice absorption filters (in photometry)

§        Choice of absorbance

Àoptimal= 0.435

(less error)

À = 0.6 – 0.7

§        !!!! Not probably to measure absorbance

2 < À < 0.03

§        Choice of thickness of a layer - not more 5 ñm

À = e l C

§        Way of transformation of a defined component in photometric compound


Choice of optimal wavelenght (lmàõ)

Sensitivity of photometric definition

À = e l C

Cmin = Àmin / e l

§        À = 0.01

§        l = 1 cì

§        e = 1000

then  Ñmin = 10-5 mol/L


Basic Components of Spectroscopic Instrumentation

The instruments used in spectroscopy consist of several common components, including a source of energy that can be input to the sample, a means for isolating a narrow range of wavelengths, a detector for measuring the signal, and a signal processor to display the signal in a form convenient for the analyst. In this section we introduce the basic components used to construct spectroscopic instruments. A more detailed discussion of these components can be found in the suggested end-of-chapter readings. Specific instrument designs are considered in later sections.


Sources of Energy

All forms of spectroscopy require a source of energy. In absorption and scattering spectroscopy this energy is supplied by photons. Emission and luminescence spectroscopy use thermal, radiant (photon), or chemical energy to promote the analyte to a less stable, higher energy state.

Sources of Electromagnetic Radiation

A source of electromagnetic radiation must provide an output that is both intense and stable in the desired region of the electromagnetic spectrum. Sources of electromagnetic radiation are classified as either continuum or line sources. A continuum source emits radiation over a wide range of wavelengths, with a relatively smooth variation in intensity as a function of wavelength (Figure).

Emission spectrum from a typical continuum source

Line sources, on the other hand, emit radiation at a few selected, narrow wavelength ranges (Figure).


Table provides a list of the most common sources of electromagnetic radiation.

Sources of Thermal Energy

The most common sources of thermal energy are flames and plasmas. Flame sources use the combustion of a fuel and an oxidant such as acetylene and air, to achieve temperatures of 2000–3400 K. Plasmas, which are hot, ionized gases, provide temperatures of 6000–10,000 K.

Chemical Sources of Energy

Exothermic reactions also may serve as a source of energy. In chemiluminescence the analyte is raised to a higher-energy state by means of a chemical reaction, emitting characteristic radiation when it returns to a lower-energy state. When the chemical reaction results from a biological or enzymatic reaction, the emission of radiation is called bioluminescence. Commercially available “light sticks” and the flash of light from a firefly are examples of chemiluminescence and bioluminescence, respectively.

Wavelength Selection

In Nessler’s original colorimetric method for ammonia, described at the beginning of the chapter, no attempt was made to narrow the wavelength range of visible light passing through the sample. If more than one component in the sample contributes to the absorption of radiation, however, then a quantitative analysis using Nessler’s original method becomes impossible. For this reason we usually try to select a single wavelength where the analyte is the only absorbing species. Unfortunately, we cannot isolate a single wavelength of radiation from a continuum source. Instead, a wavelength selector passes a narrow band of radiation (Figure) characterized by a nominal wavelength, an effective bandwidth, and a maximum throughput of radiation.

Band of radiation exiting wavelength selector showing the nominal wavelength and effective bandpass.


The effective bandwidth is defined as the width of the radiation at half the maximum throughput.

The ideal wavelength selector has a high throughput of radiation and a narrow effective bandwidth. A high throughput is desirable because more photons pass through the wavelength selector, giving a stronger signal with less background noise. A narrow effective bandwidth provides a higher resolution, with spectral features separated by more than twice the effective bandwidth being resolved. Generally these two features of a wavelength selector are in opposition (Figure).


Conditions favoring a higher throughput of radiation usually provide less resolution. Decreasing the effective bandwidth improves resolution, but at the cost of a noisier signal. For a qualitative analysis, resolution is generally more important than the throughput of radiation; thus, smaller effective bandwidths are desirable. In a quantitative analysis a higher throughput of radiation is usually desirable.


Wavelength Selection Using Filters

The simplest method for isolating a narrow band of radiation is to use an absorption or interference filter. Absorption filters work by selectively absorbing radiation from a narrow region of the electromagnetic spectrum. Interference filters use constructive and destructive interference to isolate a narrow range of wavelengths. A simple example of an absorption filter is a piece of colored glass. A purple filter, for example, removes the complementary color green from 500–560 nm. Commercially available absorption filters provide effective bandwidths from 30–250 nm. The maximum throughput for the smallest effective bandpasses, however, may be only 10% of the source’s emission intensity over that range of wavelengths. Interference filters are more expensive than absorption filters, but have narrower effective bandwidths, typically 10–20 nm, with maximum throughputs of at least 40%.

Wavelength Selection Using Monochromators

One limitation of an absorption or interference filter is that they do not allow for a continuous selection of wavelength.

If measurements need to be made at two wavelengths, then the filter must be changed in between measurements. A further limitation is that filters are available for only selected nominal ranges of wavelengths. An alternative approach to wavelength selection, which provides for a continuous variation of wavelength, is the monochromator.

The construction of a typical monochromator is shown in Figure.


Radiation from the source enters the monochromator through an entrance slit. The radiation is collected by a collimating mirror, which reflects a parallel beam of radiation to a diffraction grating. The diffraction grating is an optically reflecting surface with a large number of parallel grooves (see inset to Figure above). Diffraction by the grating disperses the radiation in space, where a second mirror focuses the radiation onto a planar surface containing an exit slit. In some monochromators a prism is used in place of the diffraction grating.

Radiation exits the monochromator and passes to the detector. As shown in Figure above, a polychromatic source of radiation at the entrance slit is converted at the exit slit to a monochromatic source of finite effective bandwidth. The choice of which wavelength exits the monochromator is determined by rotating the diffraction grating. A narrower exit slit provides a smaller effective bandwidth and better resolution, but allows a smaller throughput of radiation.

Monochromators are classified as either fixed-wavelength or scanning. In a fixed-wavelength monochromator, the wavelength is selected by manually rotating the grating. Normally, a fixed-wavelength monochromator is only used for quantitative analyses where measurements are made at one or two wavelengths. A scanning monochromator includes a drive mechanism that continuously rotates the grating, allowing successive wavelengths to exit from the monochromator. Scanning monochromators are used to acquire spectra and, when operated in a fixed wavelength mode, for quantitative analysis.


An interferometer provides an alternative approach for wavelength selection. Instead of filtering or dispersing the electromagnetic radiation, an interferometer simultaneously allows source radiation of all wavelengths to reach the detector (Figure).

Block diagram of an interferometer


Radiation from the source is focused on a beam splitter that transmits half of the radiation to a fixed mirror, while reflecting the other half to a movable mirror. The radiation recombines at the beam splitter, where constructive and destructive interference determines, for each wavelength, the intensity of light reaching the detector.

As the moving mirror changes position, the wavelengths of light experiencing maximum constructive interference and maximum destructive interference also changes. The signal at the detector shows intensity as a function of the moving mirror’s position, expressed in units of distance or time. The result is called an interferogram, or a time domain spectrum. The time domain spectrum is converted mathematically, by a process called a Fourier transform, to the normal spectrum (also called a frequency domain spectrum) of intensity as a function of the radiation’s energy.

In comparison with a monochromator, interferometers provide two significant advantages. The first advantage, which is termed Jacquinot’s advantage, results from the higher throughput of source radiation. Since an interferometer does not use slits and has fewer optical components from which radiation can be scattered and lost, the throughput of radiation reaching the detector is 80–200 times greater than that achieved with a monochromator. The result is an improved signal-to-noise ratio. The second advantage, which is called Fellgett’s advantage, reflects a savings in the time needed to obtain a spectrum. Since all frequencies are monitored simultaneously, an entire spectrum can be recorded in approximately 1 s, as compared to 10–15 min with a scanning monochromator.


The first detector for optical spectroscopy was the human eye, which, of course, is limited both by its accuracy and its limited sensitivity to electromagnetic radiation.

Modern detectors use a sensitive transducer to convert a signal consisting of photons into an easily measured electrical signal. Ideally the detector’s signal, S, should be a linear function of the electromagnetic radiation’s power, P,

where k is the detector’s sensitivity, and D is the detector’s dark current, or the background electric current when all radiation from the source is blocked from the detector.

Photon Transducers

Two general classes of transducers are used for optical spectroscopy, several examples of which are listed in Table 10.4.


Phototubes and photomultipliers contain a photosensitive surface that absorbs radiation in the ultraviolet, visible, and near infrared (IR), producing an electric current proportional to the number of photons reaching the transducer. Other photon detectors use a semiconductor as the photosensitive surface. When the semiconductor absorbs photons, valence electrons move to the semiconductor’s conduction band, producing a measurable current. One advantage of the Si photodiode is that it is easily miniaturized. Groups of photodiodes may be gathered together in a linear array containing from 64 to 4096 individual photodiodes. With a width of 25 mm per diode, for example, a linear array of 2048 photodiodes requires only 51.2 mm of linear space.

By placing a photodiode array along the monochromator’s focal plane, it is possible to monitor simultaneously an entire range of wavelengths.

Thermal Transducers

Infrared radiation generally does not have sufficient energy to produce a measurable current when using a photon transducer. A thermal transducer, therefore, is used for infrared spectroscopy. The absorption of infrared photons by a thermal transducer increases its temperature, changing one or more of its characteristic properties. The pneumatic transducer, for example, consists of a small tube filled with xenon gas equipped with an IR-transparent window at one end, and a flexible membrane at the other end. A blackened surface in the tube absorbs photons, increasing the temperature and, therefore, the pressure of the gas. The greater pressure in the tube causes the flexible membrane to move in and out, and this displacement is monitored to produce an electrical signal.


Accuracy of photometric definition depends from:

§        Specific features of photometric reaction or photometric compounds

§        Characteristics of the used device (usually makes 1 - 2 % relative)



Quantitative Applications

The determination of an analyte’s concentration based on its absorption of ultraviolet or visible radiation is one of the most frequently encountered quantitative analytical methods. One reason for its popularity is that many organic and inorganic compounds have strong absorption bands in the UV/Vis region of the electromagnetic spectrum. In addition, analytes that do not absorb UV/Vis radiation, or that absorb such radiation only weakly, frequently can be chemically coupled to a species that does. For example, nonabsorbing solutions of Pb2+ can be reacted with dithizone to form the red Pb–dithizonate complex. An additional advantage to UV/Vis absorption is that in most cases it is relatively easy to adjust experimental and instrumental conditions so that Beer’s law is obeyed.

Quantitative analyses based on the absorption of infrared radiation, although important, are less frequently encountered than those for UV/Vis absorption. One reason is the greater tendency for instrumental deviations from Beer’s law when using infrared radiation. Since infrared absorption bands are relatively narrow, deviations due to the lack of monochromatic radiation are more pronounced. In addition, infrared sources are less intense than sources of UV/Vis radiation, making stray radiation more of a problem. Differences in pathlength for samples and standards when using thin liquid films or KBr pellets are a problem, although an internal standard can be used to correct for any difference in pathlength. Finally, establishing a 100% T (A = 0) baseline is often difficult since the optical properties of NaCl sample cells may change significantly with wavelength due to contamination and degradation. This problem can be minimized by determining absorbance relative to a baseline established for the absorption band.


1. A method of calibration chart

!!! The method can be applied, if:

§        Structure of standard and investigated solutions are similar

§        The interval of concentration on calibration chart should cover of defined concentration

2. Comparison method (a method on one standard)

!! The method can be used if:

§        Dependence structure - property is strictly rectilinear and passes through the beginning of co-ordinates

§        Concentration of standard and investigated solutions values of analytical signals as much as possible similar and minimum different

§        Structure of standard and investigated solutions are as much as possible similar


3. Method of molar or specific (concentration on % w/w) absorptivity

!! The method can be used if:

§        Strict linearity of dependence structure - an analytical signal is observed

§        The analytical device maintains requirements of metrological checking


4. Method of additives

!!! The method can be applied, if:

§        It is necessary to consider stirring influence of extraneous components of sample on analytical signal of defined substance


Usage of UV  – spectroscopy and spectrophotometry in visible spectrum:

§        Identification and establishment of identity of drugs

§        Quantitative definition of substance contain

§        Cleanliness check

§        The express control of the forged drugs

§        Research of new substances structure


The applications of Beer’s law for the quantitative analysis of samples in environmental chemistry, clinical chemistry, industrial chemistry and forensic chemistry are numerous.

Environmental Applications

Methods for the analysis of waters and wastewaters relying on the absorption of UV/Vis radiation are among some of the most frequently employed analytical methods. Many of these methods are outlined in Table, and a few are described later in more detail.

Although the quantitative analysis of metals in water and wastewater is accomplished primarily by atomic absorption or atomic emission spectroscopy, many metals also can be analyzed following the formation of a colored metal–ligand complex. One advantage to these spectroscopic methods is that they are easily adapted to the field analysis of samples using a filter photometer. One ligand used in the analysis of several metals is diphenylthiocarbazone, also known as dithizone. Dithizone is insoluble in water, but when a solution of dithizone in CHCl3 is shaken with an aqueous solution containing an appropriate metal ion, a colored metal–dithizonate complex forms that is soluble in CHCl3. The selectivity of dithizone is controlled by adjusting the pH of the aqueous sample. For example, Cd2+ is extracted from solutions that are made strongly basic with NaOH, Pb2+ from solutions that are made basic with an ammoniacal buffer, and Hg2+ from solutions that are slightly acidic.

When chlorine is added to water that portion available for disinfection is called the chlorine residual. Two forms of the chlorine residual are recognized. The free chlorine residual includes Cl2, HOCl, and OCl. The combined chlorine residual, which forms from the reaction of NH3 with HOCl, consists of monochloroamine, NH2Cl, dichlororamine, NHCl2, and trichloroamine, NCl3. Since the free chlorine residual is more efficient at disinfection, analytical methods have been developed to determine the concentration of both forms of residual chlorine. One such method is the leuco crystal violet method. Free residual chlorine is determined by adding leuco crystal violet to the sample, which instantaneously oxidizes giving a bluish color that is monitored at 592 nm. Completing the analysis in less than 5 min prevents a possible interference from the combined chlorine residual. The total chlorine residual (free + combined) is determined by reacting a separate sample with iodide, which reacts with both chlorine residuals to form HOI. When the reaction is complete, leuco crystal violet is added and oxidized by HOI, giving the same bluish colored product. The combined chlorine residual is determined by difference.

The concentration of fluoride in drinking water may be determined indirectly by its ability to form a complex with zirconium. In the presence of the dye SPADNS, solutions of zirconium form a reddish colored compound, called a “lake,” that absorbs at 570 nm. When fluoride is added, the formation of the stable ZrF62– complex causes a portion of the lake to dissociate, decreasing the absorbance. A plot of absorbance versus the concentration of fluoride, therefore, has a negative slope.

Spectroscopic methods also are used in determining organic constituents in water. For example, the combined concentrations of phenol, and ortho- and metasubstituted phenols are determined by using steam distillation to separate the phenols from nonvolatile impurities. The distillate is reacted with 4-aminoantipyrine at pH 7.9 ± 0.1 in the presence of K3Fe(CN)6, forming a colored antipyrine dye.

The dye is extracted into CHCl3, and the absorbance is monitored at 460 nm. A calibration curve is prepared using only the unsubstituted phenol, C6H5OH. Because the molar absorptivities of substituted phenols are generally less than that for phenol, the reported concentration represents the minimum concentration of phenolic compounds.

Molecular absorption also can be used for the analysis of environmentally significant airborne pollutants. In many cases the analysis is carried out by collecting the sample in water, converting the analyte to an aqueous form that can be analyzed by methods such as those described in Table. For example, the concentration of NO2 can be determined by oxidizing NO2 to NO3. The concentration of NO3 is then determined by reducing to NO2

– with Cd and reacting the NO2

– with sulfanilamide

and N-(1-naphthyl)-ethylenediamine to form a brightly colored azo dye. Another important application is the determination of SO2, which is determined by collecting the sample in an aqueous solution of HgCl42– where it reacts to form Hg(SO3)22–. Addition of p-rosaniline and formaldehyde results in the formation of a bright purple complex that is monitored at 569 nm. Infrared absorption has proved useful for the analysis of organic vapors, including HCN, SO2, nitrobenzene, methyl mercaptan, and vinyl chloride. Frequently, these analyses are accomplished using portable, dedicated infrared photometers.


Clinical Applications

UV/Vis molecular absorption is one of the most commonly employed techniques for the analysis of clinical samples, several examples of which are listed in Table.


Industrial Analysis

UV/Vis molecular absorption is used for the analysis of a diverse array of industrial samples, including pharmaceuticals, food, paint, glass, and metals. In many cases the methods are similar to those described in Tables above. For example, the iron content of food can be determined by bringing the iron into solution and analyzing using the o-phenanthroline method listed in Table.

Many pharmaceutical compounds contain chromophores that make them suitable for analysis by UV/Vis absorption. Products that have been analyzed in this fashion include antibiotics, hormones, vitamins, and analgesics. One example of the use of UV absorption is in determining the purity of aspirin tablets, for which the active ingredient is acetylsalicylic acid. Salicylic acid, which is produced by the hydrolysis of acetylsalicylic acid, is an undesirable impurity in aspirin tablets, and should not be present at more than 0.01% w/w. Samples can be screened for unacceptable levels of salicylic acid by monitoring the absorbance at a wavelength of 312 nm. Acetylsalicylic acid absorbs at 280 nm, but absorbs poorly at 312 nm. Conditions for preparing the sample are chosen such that an absorbance of greater than 0.02 signifies an unacceptable level of salicylic acid.


Forensic Applications

UV/Vis molecular absorption is routinely used in the analysis of narcotics and for drug testing. One interesting forensic application is the determination of blood alcohol using the Breathalyzer test. In this test a 52.5-mL breath sample is bubbled through an acidified solution of K2Cr2O7. Any ethanol present in the breath sample is oxidized by the dichromate, producing acetic acid and Cr3+ as products. The concentration of ethanol in the breath sample is determined from the decrease in absorbance at 440 nm where the dichromate ion absorbs.

A blood alcohol content of 0.10%, which is the legal limit in most states, corresponds to 0.025 mg of ethanol in the breath sample.