ATOMIC-ABSORPTION AND EMISSION ANALYSIS

June 3, 2024
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ATOMIC-ABSORPTION AND EMISSION ANALYSIS. EXTRACTION AND EXTRACTION-PHOTOMETRIC ANALYSIS

FLAME EMISSION ANALYSIS: THEORETICAL BASES, PRINCIPAL SCHEMA, QUALITATIVE AND QUANTITATIVE ANALYSIS, USAGE

Flame emission analysis is based on measurement of radiation by excited metal in flame.

Me*  =  Me + hn

§        This analysis is the part of emission spectroscopy, in which as excitation source is used flame of different types.

In flame emission spectrometry, the sample solution is nebulized (converted into a fine aerosol) and introduced into the flame where it is desolvated, vaporized, and atomized, all in rapid succession. Subsequently, atoms and molecules are raised to excited states via thermal collisions with the constituents of the partially burned flame gases. Upon their return to a lower or ground electronic state, the excited atoms and molecules emit radiation characteristic of the sample components. The emitted radiation passes through a monochromator that isolates the specific wavelength for the desired analysis. A photodetector measures the radiant power of the selected radiation, which is then amplified and sent to a readout device, meter, recorder, or microcomputer system.

Combustion .flames provide a means of converting analytes in solution to atoms in the vapor phase freed of their chemical surroundings. These free atoms are then transformed into excited electronic states by one of two methods: absorption of additional thermal energy from the flame or absorption of radiant energy from an external source of radiation.

In the first method, known as flame emission spectroscopy (FES), the energy from the flame also supplies the energy necessary to move the electrons of the free atoms from the ground state to excited states. The intensity of radiation emitted by these excited atoms returning to the ground state provides the basis for analytical determinations in FES.

 

Schema of processes in the flame

Salt solution   ®   aerosol liquid-gas ® aerosol solid matter-gas  ® vapour of salt ® salt dissociation ®   Me

Characteristic of some types of flame

§        Lighting gas – air                   1700 – 1840 °С

§        Propyl hydride – air             1925 °С

§        Acetylene – air                       2125 – 2397 °С

§        Hydrogen – air                      2000 – 2045 °С

§        Lighting gas– oxygen            2370 °С

§        Acetylene – oxygen               3100 – 3137 °С

§        Oxalonitrile – oxygen           4380 °С

Schema of transformation of metal atoms in the flame

The intensity, I, of an emission line is proportional to the number of atoms (concentration), populating the excited state

Reason for rejection from linearity:

§        viscosity and interfacial tension of solution

§        self-absorption

§        ionization

§        Formation slightly dissociated, nonvolatile compounds in flame

The main part of flame photometer:

§        System of gas delivering and air (compressor)

§        flame

§        optical filter or monochromator

§        System of photo cell and amplifier

Instruments:

§        Flame photometer

§        Flame spectrophotometer

 

 INSTRUMENTATION FOR FLAME SPECTROMETRIC METHODS

The basic components of flame spectrometric instruments are discussed in this section. These components provide the following functions required in each method: deliver the analyte to the flame, induce the spectral transitions (absorption or emission) necessary for the determination of the analyte, isolate the spectral lines required for the analysis, detect the increase or decrease in intensity of radiation at the isolated lines(s), and record these intensity data.

 

 Pretreatment of Sample

Flame FES requires that the analy1e be dissolved in a solution in order to undergo nebulization (see the next section). The wet chemistry necessary to dissolve the sample in a matrix suitable for either flame method is often an important component of the analytical process. The analyst must be aware of substances that interfere with the emission measurement. When these substances are in the sample, they must be removed or masked (complexed). Reagents used to dissolve samples must not contain substances that lead to interference problems.

Sample Delivery

The device that introduces the sample into the flame or plasma plays a major role in determining the accuracy of the analysis. The most popular sampling method is nebulization of a liquid sample to provide a steady flow of aerosol into a flame. An introduction system for liquid samples consists of three components: a nebulizer that breaks up the liquid into small’ droplets, an aerosol. modifier that removes large droplets from the stream, allowing only droplets smaller than a certain size to pass, and the flame or atomizer that converts the anaIyte into free atoms.

 

 Nebulization

Pneumatic nebulization is the technique used in most atomic spectroscopy determinations. The sample solution is introduced through an orifice into a high- velocity gas jet, usually the oxidant. The sample stream may intersect the gas stream in either a parallel or perpendicular manner. Liquid is drawn through the sample capillary by the pressure differential generated by the high-velocity gas stream passing over the sample orifice. The liquid stream begins to oscillate, producing filaments. Finally, these filaments collapse to form a cloud of droplets in the aerosol modifier or spray chamber.

In the spray chamber the larger droplets are removed from the sample stream by mixer paddles or broken up into smaller droplets by impact beads or wall surfaces. The final aerosol, now a fine mist, is combined with the oxidizer/fuel mixture and carried into the burner.

A typical distribution range of droplet diameters is shown. Droplets larger than about 20 μm are trapped in the spray chamber and flow to waste. The distribution of drop sizes is a function of the solvent as well as the components of the sampling system. In AAS only a small percentage (usually 2% or 3%) of the nebulized analyte solution reaches the burner.

Atomization

The atomization step must convert the analyte within the aerosol into free analyte atoms in the ground state for FES analysis. Very small sample volumes (5-100 ILL) or solid samples can be handled by flameless electrothermal methods.

Flame Atomizers. The sequence of events involved in converting a metallic element, M, from a dissolved salt, MX, in the sample solution to free M atoms in the flame is depicted. After the aerosol droplets containing 1v.IX enter the flame, the solvent is evaporated, leaving small particles of dry, solid 1v.IX. Next, solid 1v.IX is converted to NIX vapor. Finally, a portion of the MX molecules are dissociated to give neutral free atoms. The efficiency with which the flame produces neutral analyte atoms is of equal importance in all the flame techniques.

If the events proceed vertically from the top down in Figure 9.4, the efficiency of free- atom production is high. Processes that branch horizontally interfere with the production of free analyte atoms. These processes include: excitation and emission of radiation by MX(g) molecules, reaction of M(g) atoms with flame components at high temperatures to produce molecules and ions that also absorb and emit radiation, and formation of M+x ions, which, in addition to reducing the efficiency of free-atom production,. complicate the analysis by adding lines to the spectrum. The flame remains the most generally useful atomizer for atomic spectroscopy despite the developments in electrothermal atomization. A satisfactory flame source must provide the temperature and fuel/oxidant ratio required for a given analysis. The maximum operating temperature of the flame is determined by the identities of the fuel and oxidant, whereas the exact flame temperature is fixed by the fuel/oxidant ratio. In addition, the spectrum of the flame itself should not interfere with the emission or absorption lines of the analytes. Components of the flame gases limit the usable range to wavelengths longer than 210 nm.

Ionization Interference

At elevated flame and furnace temperatures, atoms with low ionization potentials become ionized. Any ionization reduces the population of both the ground state and the excited state of neutral free atoms, thus lowering the sensitivity of the determination. This problem is readily overcome by adding an excess (ca. 100- fold) of a more easily ionized element such as K, Cs, or Sr to suppress ionization in both sample and calibration solutions. The more easily ionized atoms produce a large concentration of electrons in the vapor. These electrons, by mass action, suppress the ionization of analyte atoms. Thus, suppressant should be added to samples that contain variable amounts of alkali metals analyzed by acetylene/air flames to stabilize free-electron concentrations. The addition of suppressants is even more important in analyses that require the hotter acetylene/nitrous oxide flames.

 

Technique of analysis

§        Preparation of sample to analysis (dissolution)

§        Introduction of sample solution into the flame

§        Selection of analytical spectral line of atoms of investigated element

§        Measurement of intensity spectral line

§        Calculation of investigated element (substance) concentration in the sample

Quantitative flame emission analysis

It is based on measurement of dependence intensity from concentration metal atoms in solution

Methods of quantitative analysis:

§                   A method of calibration chart

 

§                   Method of additives

§                   Comparison method (sometimes method of limiting solutions)

           

§                   or as method of limiting solutions:

Advantages of flame emission analysis

§        High sensitivity of flame photometry: for Sodium – 0.001 µg/mL, for Potassium and another alkali metals – 0.01 µg/mL, for another metals – 0.1 µg/mL

§        High accuracy (error of method – 1-3 %)

§        High reproducibility of results

§        It is necessary to define many metals

APPLICATIONS

Most applications of FES have been the determination of trace metals, especially in liquid samples. It should be remembered that FES offers a simple, inexpensive, and sensitive method for detecting common metals, including the alkali and alkaline earths, as well as several transition metals such as Fe, Mn, Cu, and Zn. FES has been extended to include a number of nonmetals: H, B, C, N, P, As, O, S, Se, Te, halogens, and noble gases. FES detectors for P and S are commercially available for use in gas chromatography.

FES has found wide application in agricultural and environmental analysis, industrial analyses of ferrous metals and alloys as well as glasses and ceramic materials, and clinical analyses of body fluids. FES can be easily automated to handle a large number of samples. Array detectors interfaced to a microcomputer system permit simultaneous analyses of several elements in a single sample.

 

ATOMIC ABSORPTION SPECTROSCOPY: THEORETICAL BASES, PRINCIPAL SCHEMA, QUALITATIVE AND QUANTITATIVE ANALYSIS, USAGE

Atomic absorption, along with atomic emission, was first used by Guystav Kirchhoff and Robert Bunsen in 1859 and 1860, as a means for the qualitative identification of atoms. Although atomic emission continued to develop as an analytical technique, progress in atomic absorption languished for almost a century. Modern atomic absorption spectroscopy was introduced in 1955 as a result of the independent work of A. Walsh and C. T. J. Alkemade.18 Commercial instruments were in place by the early 1960s, and the importance of atomic absorption as an analytical technique was soon evident.

 

This method is based on absorption by free metal atoms resonance emission at it travels through atomic vapour

Schema of process in the flame at atomic absorption determination

§        Absorption resonance emission by free metal atoms

Me + hn  =  Me*

nfrequency (on Bohr):

Instrumentation

Atomic absorption spectrophotometers (Figure) are designed using either the single-beam or double-beam optics described earlier for molecular absorption spectrophotometers. There are, however, several important differences that are considered in this section.

 

Main parts of atomic absorption spectrophotometer:

§        monochromatic light source (hollow cathode lamp);

Me + Ar+(e-) ® Me* ® Me + hnreson.

The source for atomic absorption is a hollow cathode lamp consisting of a cathode and anode enclosed within a glass tube filled with a low pressure of Ne or Ar (Figure).

Schematic diagram of a hollow cathode lamp showing mechanism by which atomic emission is obtained

 

When a potential is applied across the electrodes, the filler gas is ionized. The positively charged ions collide with the negatively charged cathode, dislodging, or “sputtering,” atoms from the cathode’s surface. Some of the sputtered atoms are in the excited state and emit radiation characteristic of the metal from which the cathode was manufactured. By fashioning the cathode from the metallic analyte, a hollow cathode lamp provides emission lines that correspond to the analyte’s absorption spectrum.

The sensitivity of an atomic absorption line is often described by its characteristic concentration, which is the concentration of analyte giving an absorbance of 0.00436 (corresponding to a percent transmittance of 99%).

Usually the wavelength providing the best sensitivity is used, although a less sensitive wavelength may be more appropriate for a high concentration of analyte.

A less sensitive wavelength also may be appropriate when significant interferences occur at the most sensitive wavelength. For example, atomizing a sample produces atoms of not only the analyte, but also of other components present in the sample’s matrix. The presence of other atoms in the flame does not result in an interference unless the absorbance lines for the analyte and the potential interferant are within approximately 0.01 nm. When this is a problem, an interference may be avoided by selecting another wavelength at which the analyte, but not the interferant, absorbs.

The emission spectrum from a hollow cathode lamp includes, besides emission lines for the analyte, additional emission lines for impurities present in the metallic cathode and the filler gas. These additional lines serve as a potential source of stray radiation that may lead to an instrumental deviation from Beer’s law. Normally the monochromator’s slit width is set as wide as possible, improving the throughput of radiation, while being narrow enough to eliminate this source of stray radiation.

§        atomizer  (graphite electrochemical atomizer or flame);

 

Atomization The most important difference between a spectrophotometer for atomic absorption and one for molecular absorption is the need to convert the analyte into a free atom. The process of converting an analyte in solid, liquid, or solution form to a free gaseous atom is called atomization. In most cases the sample containing the analyte undergoes some form of sample preparation that leaves the analyte in an organic or aqueous solution. For this reason, only the introduction of solution samples is considered in this text. Two general methods of atomization are used: flame atomization and electrothermal atomization. A few elements are atomized using other methods.

Flame Atomizers

In flame atomization the sample is first converted into a fine mist consisting of small droplets of solution. This is accomplished using a nebulizer assembly similar to that shown in the inset to Figure.

Flame atomization assembly equipped with spray chamber and slot burner. The inset shows the nebulizer assembly.

 

The sample is aspirated into a spray chamber by passing a high-pressure stream consisting of one or more combustion gases, past the end of a capillary tube immersed in the sample. The impact of the sample with the glass impact bead produces an aerosol mist. The aerosol mist mixes with the combustion gases in the spray chamber before passing to the burner where the flame’s thermal energy desolvates the aerosol mist to a dry aerosol of small, solid particles. Subsequently, thermal energy volatilizes the particles, producing a vapor consisting of molecular species, ionic species, and free atoms.

Thermal energy in flame atomization is provided by the combustion of a fuel–oxidant mixture. Common fuels and oxidants and their normal temperature ranges are listed in Table 10.9.

Of these, the air–acetylene and nitrous oxide-acetylene flames are used most frequently. Normally, the fuel and oxidant are mixed in an approximately stoichiometric ratio; however, a fuel-rich mixture may be desirable for atoms that are easily oxidized. The most common design for the burner is the slot burner shown in Figure. This burner provides a long path length for monitoring absorbance and a stable flame.

The burner is mounted on an adjustable stage that allows the entire burner assembly to move horizontally and vertically. Horizontal adjustment is necessary to ensure that the flame is aligned with the instrument’s optical path. Vertical adjustments are needed to adjust the height within the flame from which absorbance is monitored. This is important because two competing processes affect the concentration of free atoms in the flame. An increased residence time in the flame results in a greater atomization efficiency; thus, the production of free atoms increases with height. On the other hand, longer residence times may lead to the formation of metal oxides that absorb at a wavelength different from that of the atom. For easily oxidized metals, such as Cr, the concentration of free atoms is greatest just above the burner head. For metals, such as Ag, which are difficult to oxidize, the concentration of free atoms increases steadily with height (Figure).

Other atoms show concentration profiles that maximize at a characteristic height.

The most common means for introducing samples into a flame atomizer is continuous aspiration, in which the sample is continuously passed through the burner while monitoring the absorbance. Continuous aspiration is sample intensive, typically requiring 2–5 mL of sample. Flame microsampling provides a means for introducing a discrete sample of fixed volume and is useful when the volume of sample is limited or when the sample’s matrix is incompatible with the flame atomizer. For example, the continuous aspiration of a sample containing a high concentration of dissolved solids, such as sea water, may result in the build-up of solid deposits on the burner head. These deposits partially obstruct the flame, lowering the absorbance. Flame microsampling is accomplished using a micropipet to place 50–250 mL of sample in a Teflon funnel connected to the nebulizer, or by dipping the nebulizer tubing into the sample for a short time.

Dip sampling is usually accomplished with an automatic sampler. The signal for flame microsampling is a transitory peak whose height or area is proportional to the amount of analyte that is injected.

The principal advantage of flame atomization is the reproducibility with which the sample is introduced into the spectrophotometer. A significant disadvantage to flame atomizers is that the efficiency of atomization may be quite poor. This may occur for two reasons. First, the majority of the aerosol mist produced during nebulization consists of droplets that are too large to be carried to the flame by the combustion gases. Consequently, as much as 95% of the sample never reaches the flame.

A second reason for poor atomization efficiency is that the large volume of combustion gases significantly dilutes the sample. Together, these contributions to the efficiency of atomization reduce sensitivity since the analyte’s concentration in the flame may be only 2.5 *10–6 of that in solution.

 

Electrothermal Atomizers

A significant improvement in sensitivity is achieved by using resistive heating in place of a flame. A typical electrothermal atomizer, also known as a graphite furnace, consists of a cylindrical graphite tube approximately 1–3 cm in length, and 3–8 mm in diameter (Figure).

Diagram of an electrothermal analyzer

The graphite tube is housed in an assembly that seals the ends of the tube with optically transparent windows.

The assembly also allows for the passage of a continuous stream of inert gas, protecting the graphite tube from oxidation, and removing the gaseous products produced during atomization. A power supply is used to pass a current through the graphite tube, resulting in resistive heating.

Samples between 5 and 50 mL are injected into the graphite tube through a small-diameter hole located at the top of the tube. Atomization is achieved in three stages. In the first stage the sample is dried using a current that raises the temperature of the graphite tube to about 110 °C. Desolvation leaves the sample as a solid residue. In the second stage, which is called ashing, the temperature is increased to 350–1200 °C. At these temperatures, any organic material in the sample is converted to CO2 and H2O, and volatile inorganic materials are vaporized. These gases are removed by the inert gas flow. In the final stage the sample is atomized by rapidly increasing the temperature to 2000–3000 °C. The result is a transient absorbance peak whose height or area is proportional to the absolute amount of analyte injected into the graphite tube. The three stages are complete in approximately 45–90 s, with most of this time used for drying and ashing the sample.

Electrothermal atomization provides a significant improvement in sensitivity by trapping the gaseous analyte in the small volume of the graphite tube. The analyte’s concentration in the resulting vapor phase may be as much as 1000 times greater than that produced by flame atomization. The improvement in sensitivity, and the resulting improvement in detection limits, is offset by a significant decrease in precision. Atomization efficiency is strongly influenced by the sample’s contact with the graphite tube, which is difficult to control reproducibly.

Miscellaneous Atomization Methods

A few elements may be atomized by a chemical reaction that produces a volatile product. Elements such as As, Se, Sb, Bi, Ge, Sn, Te, and Pb form volatile hydrides when reacted with NaBH4 in acid. An inert gas carries the volatile hydrides to either a flame or to a heated quartz observation tube situated in the optical path. Mercury is determined by the cold-vapor method in which it is reduced to elemental mercury with SnCl2. The volatile Hg is carried by an inert gas to an unheated observation tube situated in the instrument’s optical path.

§        monochromatizater;

§        photo multiplier.

 

Preparing the Sample

Flame and electrothermal atomization require that the sample be in a liquid or solution form. Samples in solid form are prepared for analysis by dissolving in an appropriate solvent. When the sample is not soluble, it may be digested, either on a hot plate or by microwave, using HNO3, H2SO4, or HClO4. Alternatively, the analyte may be extracted via a Soxhlet extraction. Liquid samples may be analyzed directly or may be diluted or extracted if the matrix is incompatible with the method of atomization. Serum samples, for instance, may be difficult to aspirate when using flame atomization and may produce unacceptably high background absorbances when using electrothermal atomization. A liquid–liquid extraction using an organic solvent containing a chelating agent is frequently used to concentrate analytes. Dilute solutions of Cd2+, Co2+, Cu2+, Fe3+, Pb2+, Ni2+, and Zn2+, for example, can be concentrated by extracting with a solution of ammonium pyrrolidine dithiocarbamate in methyl isobutyl ketone.

 

Standardizing the Method

Because Beer’s law also applies to atomic absorption, we might expect atomic absorption calibration curves to be linear. In practice, however, most atomic absorption calibration curves are nonlinear, or linear for only a limited range of concentrations. Nonlinearity in atomic absorption is a consequence of instrumental limitations, including stray radiation from the hollow cathode lamp and a nonconstant molar absorptivity due to the narrow width of the absorption line. Accurate quantitative work, therefore, often requires a suitable means for computing the calibration curve from a set of standards. Nonlinear calibration curves may be fit using quadratic and cubic equations, although neither works well over a broad range of concentrations.

When possible, a quantitative analysis is best conducted using external standards.

Unfortunately, matrix interferences are a frequent problem, particularly when using electrothermal atomization. For this reason the method of standard additions is often used. One limitation to this method of standardization, however, is the requirement that there be a linear relationship between absorbance and concentration.

 

Quantitative analysis

Bouguer-Lambert-Beer law:

§        Reduction of intensity of resonant light in atomic absorption spectroscopy is proportional atom number into atomic vapour (concentration of this metal in solution)

Reasons of absorbance nonlinearity and metal ions concentration in solution

§        physical:

– Instability of work of different equipment part

         Not monochromatic emission lines

         Metal atomic ionization

§        chemical:

anionic effect

cationic effect

Methods of quantitative analysis in atomic absorption:

§        method of calibration chart

§        Method of additives

Advantages of atomic absorption analysis:

§        Very high selectivity

§        Wide spectrum of investigated ions (nearly 70)

§        Low determined minimum (10-5 – 10-6 %)

§        High reproducibility (error ~5% to 10 %)

Lacks of ААА in comparison with flame emission analysis:

§        Low sensitivity

§        Complexity of equipment

§        Presence of nonselective absorption (because dispersion of light and molecular absorption)

 

Taking into account nonselective absorption:

§        automatic

§        usage of polarized light

 


Atomic absorption using either flame or electrothermal atomization is widely used for the analysis of trace metals in a variety of sample matrices. Using the atomic absorption analysis for zinc as an example, procedures have been developed for its determination in samples as diverse as water and wastewater, air, blood, urine, muscle tissue, hair, milk, breakfast cereals, shampoos, alloys, industrial plating baths, gasoline, oil, sediments, and rocks.

Developing a quantitative atomic absorption method requires several considerations, including choosing a method of atomization, selecting the wavelength and slit width, preparing the sample for analysis, minimizing spectral and chemical interferences, and selecting a method of standardization. Each of these topics is considered in this section.

 

MOLECULAR PHOTOLUMINESCENCE SPECTROSCOPY: THEORETICAL BASES, PRINCIPAL SCHEMA, QUALITATIVE AND QUANTITATIVE ANALYSIS, USAGE

Luminescence is excess of temperature radiation of substance in the event that this excessive emission owns final duratioearly 10-10 с and more.

 

Types of luminescence at nature of initiation energy:

§        Triboluminescence is an optical phenomenon in which light is generated when asymmetrical crystalline bonds in a material are broken when that material is scratched, crushed, or rubbed. The phenomenon is not fully understood, but appears to be caused by the separation and reunification of electrical charges.

 

§        Cathodoluminescence is an optical and electrical phenomenon whereby a beam of electrons is generated by an electron gun (e.g. cathode ray tube) and then impacts on a luminescent material such as a phosphor, causing the material to emit visible light.

§        Chemiluminescence (sometimes “chemoluminescence“) is the emission of light with limited emission of heat (luminescence), as the result of a chemical reaction. Given reactants A and B, with an excited intermediate ◊,

[A] + [B] → [◊] → [Products] + light

§        For example, if [A] is luminol and [B] is hydrogen peroxide in the presence of a suitable catalyst we have:

luminol + H2O2 → 3-APA[◊] → 3-APA + light

where:

§        3-APA is 3-aminophthalate

§        3-APA[◊] is the excited state fluorescing

as it decays to a lower energy level.

A chemoluminescent reaction carried out in an erlenmeyer flask producing a large amount of light.

§        Electroluminescence (EL) is an optical phenomenon and electrical phenomenon in which a material emits light in response to an electric current passed through it, or to a strong electric field. This is distinct from light emission resulting from heat (incandescence) from the action of chemicals (chemiluminescence), the action of sound (sonoluminescence), or other mechanical action (mechanoluminescence).

 

§        The amount of light given off is proportional to the dose of radiation received. In thermoluminescence dating, this can be used to date buried objects that have been heated in the past (eg pottery) since the dose received from radioactive elements in the soil, cosmic rays etc is proportional to age. This phenomenon has been used for Thermoluminescent dosimeters, to measure the radiation dose received by a chip of suitable material that is carried around by a person or placed with an object.

 

Photoluminescence is divided into two categories: fluorescence and phosphorescence.

Absorption of an ultraviolet or visible photon promotes a valence electron from its ground state to an excited state with conservation of the electron’s spin. For example, a pair of electrons occupying the same electronic ground state have opposite spins (Figure a) and are said to be in a singlet spin state.

Difference between singlet and triplet states

 

Absorbing a photon promotes one of the electrons to a singlet excited state (Figure b). Emission of a photon from a singlet excited state to a singlet ground state, or between any two energy levels with the same spin, is called fluorescence. The probability of a fluorescent transition is very high, and the average lifetime of the electron in the excited state is only 10–5–10–8 s. Fluorescence, therefore, decays rapidly after the excitation source is removed. In some cases an electron in a singlet excited state is transformed to a triplet excited state (Figure c) in which its spin is no longer paired with that of the ground state. Emission between a triplet excited state and a singlet ground state, or between any two energy levels that differ in their respective spin states, is called phosphorescence. Because the average lifetime for phosphorescence ranges from 10–4 to 104 s, phosphorescence may continue for some time after removing the excitation source.

The use of molecular fluorescence for qualitative analysis and semiquantitative analysis can be traced to the early to mid-1800s, with more accurate quantitative methods appearing in the 1920s. Instrumentation for fluorescence spectroscopy using filters and monochromators for wavelength selection appeared in, respectively, the 1930s and 1950s. Although the discovery of phosphorescence preceded that of fluorescence by almost 200 years, qualitative and quantitative applications of molecular phosphorescence did not receive much attention until after the development of fluorescence instrumentation.

Molecular Fluorescence and Phosphorescence Spectra

To appreciate the origin of molecular fluorescence and phosphorescence, we must consider what happens to a molecule following the absorption of a photon. Let’s assume that the molecule initially occupies the lowest vibrational energy level of its electronic ground state. The ground state, which is shown in Figure, is a singlet state labeled S0.

Energy level diagram for a molecule showing pathways for deactivation of an excited

state: vr is vibrational relaxation; ic is internal conversion; ec is external conversion, and iscis intersystem crossing. The lowest vibrational energy level for each electronic state is indicated by the thicker line.

 

Absorption of a photon of correct energy excites the molecule to one of several vibrational energy levels in the first excited electronic state, S1, or the second electronic excited state, S2, both of which are singlet states. Relaxation to the ground state from these excited states occurs by a number of mechanisms that are either radiationless, in that no photons are emitted, or involve the emission of a photon. These relaxation mechanisms are shown in Figure.

The most likely pathway by which a molecule relaxes back to its ground state is that which gives the shortest lifetime for the excited state.

Radiationless Deactivation

One form of radiationless deactivation is vibrational relaxation, in which a molecule in an excited vibrational energy level loses energy as it moves to a lower vibrational energy level in the same electronic state.

Vibrational relaxation is very rapid, with the molecule’s average lifetime in an excited vibrational energy level being 10–12 s or less. As a consequence, molecules that are excited to different vibrational energy levels of the same excited electronic state quickly return to the lowest vibrational energy level of this excited state.

Another form of radiationless relaxation is internal conversion, in which a molecule in the ground vibrational level of an excited electronic state passes directly into a high vibrational energy level of a lower energy electronic state of the same spin state. By a combination of internal conversions and vibrational relaxations, a molecule in an excited electronic state may return to the ground electronic state without emitting a photon. A related form of radiationless relaxation is external conversion in which excess energy is transferred to the solvent or another component in the sample matrix.

A final form of radiationless relaxation is an intersystem crossing in which a molecule in the ground vibrational energy level of an excited electronic state passes into a high vibrational energy level of a lower energy electronic energy state with a different spin state. For example, an intersystem crossing is shown in Figure between a singlet excited state, S1, and a triplet excited state, T1.

Fluorescence

Fluorescence occurs when a molecule in the lowest vibrational energy level of an excited electronic state returns to a lower energy electronic state by emitting a photon. Since molecules return to their ground state by the fastest mechanism, fluorescence is only observed if it is a more efficient means of relaxation than the combination of internal conversion and vibrational relaxation. A quantitative expression of the efficiency of fluorescence is the fluorescent quantum yield, Ff, which is the fraction of excited molecules returning to the ground state by fluorescence.

Quantum yields range from 1, when every molecule in an excited state undergoes fluorescence, to 0 when fluorescence does not occur.

The intensity of fluorescence, If, is proportional to the amount of the radiation from the excitation source that is absorbed and the quantum yield for fluorescence Fluorescence is generally observed with molecules where the lowest energy absorption is a p → p* transition, although some n → p* transitions show weak fluorescence.

Most unsubstituted, nonheterocyclic aromatic compounds show favorable fluorescence quantum yields, although substitution to the aromatic ring can have a significant effect on Ff. For example, the presence of an electron-withdrawing group, such as —NO2, decreases Ff, whereas adding an electron-donating group, such as —OH, increases Ff. Fluorescence also increases for aromatic ring systems and for aromatic molecules with rigid planar structures.

A molecule’s fluorescence quantum yield is also influenced by external variables such as temperature and solvent. Increasing temperature generally decreases Ff because more frequent collisions between the molecule and the solvent increases external conversion. Decreasing the solvent’s viscosity decreases Ff for similar reasons.

For an analyte with acidic or basic functional groups, a change in pH may change the analyte’s structure and, therefore, its fluorescent properties. Changes in both the wavelength and intensity of fluorescence may be affected.

As shown in Figure abov, fluorescence may return the molecule to any of several vibrational energy levels in the ground electronic state. Fluorescence, therefore, occurs over a range of wavelengths. Because the change in energy for fluorescent emission is generally less than that for absorption, a molecule’s fluorescence spectrum is shifted to higher wavelengths than its absorption spectrum.

 

Phosphorescence A molecule in the lowest vibrational energy level of an excited triplet electronic state normally relaxes to the ground state by an intersystem crossing to a singlet state or by external conversion. Phosphorescence is observed when relaxation occurs by the emission of a photon. As shown in Figure, phosphorescence occurs over a range of wavelengths, all of which are at a lower energy than the molecule’s absorption band. The intensity of phosphorescence, Ip, is given by an equation similar to equation for fluorescence

Ip = 2.303kFpP0ebC

where Fp is the quantum yield for phosphorescence.

Phosphorescence is most favorable for molecules that have n→p* transitions, which have a higher probability for an intersystem crossing than do p → p* transitions.

For example, phosphorescence is observed with aromatic molecules containing carbonyl groups or heteroatoms. Aromatic compounds containing halide atoms also have a higher efficiency for phosphorescence. In general, an increase in phosphorescence corresponds to a decrease in fluorescence.

Since the average lifetime for phosphorescence is very long, ranging from 10–4 to 104 s, the quantum yield for phosphorescence is usually quite small. An improvement in Fp is realized by decreasing the efficiency of external conversion. This may be accomplished in several ways, including lowering the temperature, using a more viscous solvent, depositing the sample on a solid substrate, or trapping the molecule in solution.

Excitation Versus Emission Spectra

Photoluminescence spectra are recorded by measuring the intensity of emitted radiation as a function of either the excitation wavelength or the emission wavelength. An excitation spectrum is obtained by monitoring emission at a fixed wavelength while varying the excitation wavelength.

Figure shows the excitation spectrum for the hypothetical system described by the energy level diagram in Figure above. When corrected for variations in source intensity and detector response, a sample’s excitation spectrum is nearly identical to its absorbance spectrum. The excitation spectrum provides a convenient means for selecting the best excitation wavelength for a quantitative or qualitative analysis.

Stokes – Lommel’ law: always emission spectrum and its maximum accents in part larger wavelength in comparison with absorbance spectrum and its maximum.

Example of molecular excitation and emission spectra

 

In an emission spectrum a fixed wavelength is used to excite the molecules, and the intensity of emitted radiation is monitored as a function of wavelength. Although a molecule has only a single excitation spectrum, it has two emission spectra, one for fluorescence and one for phosphorescence.

§        Rule of mirror symmetry: absorbance spectrum and fluorescence (construct in frequency diagram) nearly symmetry rather straight line, which pass through point of their crossing. 

 

§        Distance between maximums of absorption spectrum and luminescence spectrum is named Stokes displacement.

 

§        Luminescing substances are characterised by size Stokes displacement. Than more its value, substances a luminescent method especially reliably define.

 

Instrumentation

The basic design of instrumentation for monitoring molecular fluorescence and molecular phosphorescence is similar to that found for other spectroscopies. The most significant differences are discussed in the following sections.

Molecular Fluorescence A typical instrumental block diagram for molecular fluorescence is shown in Figure. In contrast to instruments for absorption spectroscopy, the optical paths for the source and detector are usually positioned at an angle of 90°.

Block diagram for molecular fluorescence spectrometer

 

Two basic instrumental designs are used for measuring molecular fluorescence.

In a fluorometer the excitation and emission wavelengths are selected with absorption or interference filters. The excitation source for a fluorometer is usually a lowpressure mercury vapor lamp that provides intense emission lines distributed throughout the ultraviolet and visible region (254, 312, 365, 405, 436, 546, 577, 691, and 773 nm). When a monochromator is used to select the excitation and emission wavelengths, the instrument is called a spectrofluorometer. With a monochromator, the excitation source is usually a high-pressure Xe arc lamp, which has a continuum emission spectrum. Either instrumental design is appropriate for quantitative work, although only a spectrofluorometer can be used to record an excitation or emission spectrum.

The sample cells for molecular fluorescence are similar to those for optical molecular absorption. An analyte that is fluorescent can be monitored directly. For analytes that are not fluorescent, a suitable fluorescent probe molecule can be incorporated into the tip of the fiber-optic probe. The analyte’s reaction with the probe molecule leads to an increase or decrease in fluorescence.

 

Quantitative Applications Using Molecular Luminescence

Molecular fluorescence and, to a lesser extent, phosphorescence have been used for the direct or indirect quantitative analysis of analytes in a variety of matrices. A direct quantitative analysis is feasible when the analyte’s quantum yield for fluorescence or phosphorescence is favorable. When the analyte is not fluorescent or phosphorescent or when the quantum yield for fluorescence or phosphorescence is unfavorable, an indirect analysis may be feasible. One approach to an indirect analysis is to react the analyte with a reagent, forming a product with fluorescent properties. Another approach is to measure a decrease in fluorescence when the analyte is added to a solution containing a fluorescent molecule. A decrease in fluorescence is observed when the reaction between the analyte and the fluorescent species enhances radiationless deactivation, or produces a nonfluorescent product. The application of fluorescence and phosphorescence to inorganic and organic analytes is considered in this section.

 

Fluorescent analysis is divided on:

§        direct (immediate measure)

§        indirect (fluorescence with indicator and show on ending of process of substance definition, especially  at acid-base titration – acridine, luminol, salicylic acid, anthranilic acid).

 

Quantitative analysis in fluorometry

§        Method of direct fluorometric analysis (on Vavilov law):

Ф= К × С

§        for small concentration 10–7 – 10–4 mol/L

Methods of quantitative analysis:

§        A method of calibration chart

§        Comparison method

                   

Method of additives

 

Advantages of method is high sensitivity (~10-5 %).

Error of direct fluorometry is 5-7 %.

 

TURBIDIMETRY AND NEPHELOMETRY: ESSENCE AND USAGE

Turbidimetry and nephelometry are two related techniques in which an incident source of radiation is elastically scattered by a suspension of colloidal particles. In turbidimetry, the detector is placed in line with the radiation source, and thedecrease in the radiation’s transmitted power is measured.

In nephelometry, scattered radiation is measured at an angle of 90° to the radiation source. The similarity of the measurement of turbidimetry to absorbance, and of nephelometry to fluorescence, is evident in the block instrumental designs shown in Figure.

Block diagrams for (a) a turbidometer and (b) a nephelometer

Choosing between turbidimetry and nephelometry is determined by two principal factors. The most important consideration is the intensity of the transmitted or scattered radiation relative to the intensity of radiation from the source. When the solution contains a small concentration of scattering particles, the intensity of the transmitted radiation, IT, will be very similar to the intensity of the radiation source, I0. As we learned earlier in the section on molecular absorption, determining a small difference between two intense signals is subject to a substantial uncertainty. Thus, nephelometry is a more appropriate choice for samples containing few scattering particles. On the other hand, turbidimetry is a better choice for samples containing a high concentration of scattering particles.

The second consideration in choosing between turbidimetry and nephelometry is the size of the scattering particles. For nephelometry, the intensity of scattered radiation at 90° will be greatest if the particles are small enough that Rayleigh scattering is in effect. For larger particles, as shown in Figure, scattering intensity is diminished at 90°. When using an ultraviolet or visible source of radiation, the optimum particle size is 0.1–1 mm. The size of the scattering particles is less important for turbidimetry, in which the signal is the relative decrease in transmitted radiation.

In fact, turbidimetric measurements are still feasible even when the size of the scattering particles results in an increase in reflection and refraction (although a linear relationship between the signal and the concentration of scattering particles may no longer hold).

 

§        Nephelometry is a method in which the intensity of scattered radiation is measured at an angle of 90° to the source.

§        At light passage through disperse systems dispersion or light absorption by firm parts is observed.

§        Light intensity, which disperse by small particle of cloud, is described by Rayleigh equation:

§        In nephelometry, the relationship between the intensity of scattered radiation, IS, and the concentration (% w/v) of scattering particles is given as

if F, V, r, b is constant

 

Methods of quantitative analysis in nephelometry:

§        A method of calibration chart

§        Comparison method

Turbidimetry is a method in which the decrease in transmitted radiation due to scattering is measured.

§        Reduction of light intensively (because of dispersion and absorbance) is described by equation:

§        or in identical conditions:

Methods of quantitative analysis in turbidimetry:

§        A method of calibration chart.

For very dilute solution of suspension (less 100 mg/L)

 

Advantages: high sensitivity (in comparison with quality reactions)

Lacks: small precision of analyses, high error (more than 10 %).

 

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