OPTICAL nMETHODS OF THE ANALYSIS
CLASSIFICATION OF METHODS nOF THE ANALYSIS ON CHEMICAL, PHYSICAL-CHEMICAL, PHYSICAL
Analytical chemistry is the study of the nseparation, identification, and quantification of the chemical components of nnatural and artificial materials. Qualitative analysis gives an indication of nthe identity of the chemical species in the sample and quantitative analysis ndetermines the amount of one or more of these components. The separation of ncomponents is often performed prior to analysis.
Analytical methods can be separated into nclassical and instrumental. Classical methods (also known as wet chemistry nmethods) use separations such as precipitation, extraction, and distillatioand qualitative analysis by color, odor, or melting point. Quantitative nanalysis is achieved by measurement of weight or volume. Instrumental methods nuse an apparatus to measure physical quantities of the analyte such as light nabsorption, fluorescence, or conductivity. The separation of materials is naccomplished using chromatography, electrophoresis or Field Flow Fractionatiomethods.
Analytical chemistry is also focused oimprovements in experimental design, chemometrics, and the creation of new nmeasurement tools to provide better chemical information. Analytical chemistry nhas applications in forensics, bioanalysis, clinical analysis, environmental nanalysis, and materials analysis.
Classical methods
The presence of copper in this nqualitative analysis is indicated by the bluish-green color of the flame.
Although modern analytical chemistry is dominated by sophisticated ninstrumentation, the roots of analytical chemistry and some of the principles nused in modern instruments are from traditional techniques many of which are nstill used today. These techniques also tend to form the backbone of most nundergraduate analytical chemistry educational labs.
Qualitative analysis
A qualitative analysis determines the presence or absence of a nparticular compound, but not the mass or concentration. That is it is not nrelated to quantity.
Chemical ntests
For nmore details on this topic.
There are numerous qualitative chemical tests, for example, the acid ntest for gold and the Kastle-Meyer test for the presence of blood.
Flame ntest
For nmore details on this topic, see Flame test.
Inorganic qualitative analysis generally refers to a systematic scheme nto confirm the presence of certain, usually aqueous, ions or elements by nperforming a series of reactions that eliminate ranges of possibilities and nthen confirms suspected ions with a confirming test. Sometimes small carbocontaining ions are included in such schemes. With modern instrumentation these ntests are rarely used but can be useful for educational purposes and in field nwork or other situations where access to state-of-the-art instruments are not navailable or expedient.
Gravimetric nanalysis
For nmore details on this topic, see Gravimetric analysis.
Gravimetric analysis involves determining the amount of material present nby weighing the sample before and/or after some transformation. A commoexample used in undergraduate education is the determination of the amount of nwater in a hydrate by heating the sample to remove the water such that the ndifference in weight is due to the loss of water.
Volumetric nanalysis
For nmore details on this topic, see Titration.
Titration involves the addition of a reactant to a solution being nanalyzed until some equivalence point is reached. Often the amount of material nin the solution being analyzed may be determined. Most familiar to those who nhave taken college chemistry is the acid-base titration involving a color nchanging indicator. There are many other types of titrations, for example npotentiometric titrations. These titrations may use different types of nindicators to reach some equivalence point.
Instrumental nmethods
Block diagram of an analytical instrument showing the stimulus and nmeasurement of response
Spectroscopy
For nmore details on this topic, see Spectroscopy.
Spectroscopy measures the interaction of the molecules with nelectromagnetic radiation. Spectroscopy consists of many different applications nsuch as atomic absorption spectroscopy, atomic emission spectroscopy, nultraviolet-visible spectroscopy, x-ray fluorescence spectroscopy, infrared nspectroscopy, Raman spectroscopy, dual polarisation interferometry, nuclear nmagnetic resonance spectroscopy, photoemission spectroscopy, Mössbauer nspectroscopy and so on.
Mass spectrometry
For nmore details on this topic, see Mass spectrometry.
An accelerator mass spectrometer nused for radiocarbon dating and other analysis.
Mass spectrometry measures mass-to-charge ratio of molecules using nelectric and magnetic fields. There are several ionization methods: electroimpact, chemical ionization, electrospray, fast atom bombardment, matrix nassisted laser desorption ionization, and others. Also, mass spectrometry is ncategorized by approaches of mass analyzers: magnetic-sector, quadrupole mass nanalyzer, quadrupole ion trap, time-of-flight, Fourier transform ion cyclotroresonance, and so on.
Electrochemical analysis
For nmore details on this topic, see Electroanalytical method.
Electroanalytical methods measure the potential (volts) and/or current n(amps) in an electrochemical cell containing the analyte. These methods can be ncategorized according to which aspects of the cell are controlled and which are nmeasured. The three main categories are potentiometry (the difference ielectrode potentials is measured), coulometry (the cell’s current is measured nover time), and voltammetry (the cell’s current is measured while actively naltering the cell’s potential).
Thermal analysis
Further ninformation: Calorimetry, thermal analysis
Calorimetry nand thermogravimetric analysis measure the interaction of a material and heat.
Separation
Separation of black ink on a thin layer nchromatography plate.
Further ninformation: Separation process, Chromatography, electrophoresis
Separatioprocesses are used to decrease the complexity of material mixtures. nChromatography and electrophoresis are representative of this field.
Hybrid techniques
Combinations of the above techniques produce a “hybrid” or n”hyphenated” technique. Several examples are in popular use today and nnew hybrid techniques are under development. For example, gas nchromatography-mass spectrometry, gas chromatography-infrared spectroscopy, nliquid chromatography-mass spectrometry, liquid chromatography-NMR spectroscopy, nliquid chromagraphy-infrared spectroscopy and capillary electrophoresis-mass nspectrometry.
Hyphenated separation techniques refer to a combination of two (or more) ntechniques to detect and separate chemicals from solutions. Most often the nother technique is some form of chromatography. Hyphenated techniques are nwidely used in chemistry and biochemistry. A slash is sometimes used instead of nhyphen, especially if the name of one of the methods contains a hyphen itself.
Microscopy
Fluorescence microscope image of ntwo 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 nand nanomaterials is an important and attractive approach in analytical nscience. Also, hybridization with other traditional analytical tools is nrevolutionizing analytical science. Microscopy can be categorized into three ndifferent fields: optical microscopy, electron microscopy, and scanning probe nmicroscopy. Recently, this field is rapidly progressing because of the rapid ndevelopment of the computer and camera industries.
Lab-on-a-chip
A glass microreactor
Further ninformation: microfluidics, lab-on-a-chip
Devices that integrate (multiple) laboratory functions on a single chip nof only millimeters to a few square centimeters in size and that are capable of nhandling extremely small fluid volumes down to less than pico liters.
CLASSIFICATION OF PHYSICAL-CHEMICAL nMETHODS OF THE ANALYSIS: OPTICAL, ELECTROCHEMICAL, CHROMATOGRAPHIC, KINETIC.
PCMA are divided on:
§ nOptical nmethods are based on measurement of optical properties of substances.
§ nChromatographic nmethods are based on usage of ability of different substances to selective nsorption.
§ nElectrochemical nmethods are based on measurement of electrochemical properties of substances.
§ nRadiometric nmethods are based on measurement of radioactive properties of substances.
§ nThermal nmethods are based on measurement of heat effects of substances.
§ nMass spectrometric nmethods are based on studying of the ionized fragments (“splinters”) nof substances.
§ nKinetic methods nare based on measurement of dependence of speed of reaction from concentratioof substance
Advantage nof PCMA
§ nHigh sensitivity – a low limit of ndetection (10-9 mg) nand definition
§ nHigh selectivity
§ nRapid analysis methods
§ nAutomation and computerization is npossibility
§ nAnalysis is possibility on distance
§ nPossibility of the analysis without ndestruction of the sample
§ nPossibility of the local analysis
Lacks nof PCMA
§ nDefinition error is near ± 5 % n(on occasion to 20 %), whereas – 0,01-0,005 % for gravimetry and 0,1-0,05 % for ntitrimetry
§ nReproducibility of results iseparate methods is worse, than in classical methods of the analysis
§ nIt is necessary of usage of nstandards and standard solutions, graduation of equipment and plotting of ncalibration charts
§ nComplexity of used equipment, its nhigh cost, high cost of standard substances
CLASSIFICATION OF OPTICAL nMETHODS OF THE ANALYSIS:
A nmajor part of modern Instrumental Analytical Chemistry, focuses on the study of nthe energy exchange between electromagnetic radiation and matter. These ninteractions are visible to the naked eye, when the radiations concerned fall nwithin the visible spectrum.
А) On investigated objects
§ nThe nuclear nspectral analysis
§ nThe nmolecular spectral analysis
B) On the nature of interaction of nelectromagnetic radiation with substance
1. nAbsorption analysis
§ nAtomic-absorptioanalysis – Atomic absorption spectroscopy (AAS) is a spectroanalytical nprocedure for the quantitative determination of chemical elements employing the nabsorption of optical radiation (light) by free atoms in the gaseous state.
§ nMolecular-absorptioanalysis – Molecular absorption spectroscopy in the ultraviolet (UV) and nvisible (
§ nTurbidimetric nanalysis – Turbidimetry (the name being derived from turbidity) is the process nof measuring the loss of intensity of transmitted light due to scattering neffect particles suspended in it. Light is passed through a filter creating a nlight of known wavelength which is then passed through a cuvette containing a nsolution.
2. nThe emissive spectral analysis
§ nflame nphotometry – Flame nphotometry, more properly called flame atomic emission spectrometry, is a fast, nsimple, and sensitive analytical method for the determination of trace metal nions in solution.
§ nfluorescence nanalysis – Luminescence is emission of light by a substance not resulting from nheat; it is thus a form of cold body radiation.
§ nThe nspectral analysis with usage of effect of combinational dispersion of light
3. nOther methods
§ nnephelometric nmethod – Nephelometry is the measurement of scattered light. This technique requires a special measuring ninstrument, where the detector is set at an angle to the incident light beam.
Optical narrangements of nephelometry and turbidimetry
§ nrefractometric nanalysis – Refractometry is the method of measuring substances’ refractive nindex (one of their fundamental physical properties) in order to, for example, nassess their composition or purity. A refractometer is the instrument used to nmeasure refractive index (“RI”)
Components nin a typical reflectometer used to measure analytes on urine dipstick
§ npolarimetric nanalysis – Polarimetry is the measurement and interpretation of the npolarization of transverse waves, most notably electromagnetic waves, such as nradio or light waves.
Aautomatic digital polarimeter
§ ninterferometric nanalysis – Interferometry is a family of techniques in which waves, usually nelectromagnetic, are superimposed in order to extract information about the nwaves.
Three namplitude-splitting interferometers: Fizeau, Mach–Zehnder, and Fabry Perot
C) On electromagnetic spectral range nwhich use in analysis:
§ nSpectroscopy n(spectrophotometry) in UV and visible spectrum
§ nIR – nSpectroscopy – Infrared spectroscopy (IR spectroscopy) is the spectroscopy that ndeals with the infrared region of the electromagnetic spectrum, that is light nwith a longer wavelength and lower frequency than visible light.
§ nX-ray nspectroscopy – X-ray spectroscopy is a gathering name for several spectroscopic ntechniques for characterization of materials by using x-ray excitation.
§ nMicrowave nspectroscopy – The interaction of microwaves with matter can be detected by nobserving the attenuation or phase shift of a microwave field as it passes nthrough matter.
D) By the nature of energy jump
§ nElectronic nspectrum
§ nVibrational nspectrum
§ nRotational nspectrum
Spectrum (method) |
The characteristic of energy of quantum |
Process |
Radio-frequency (NMR, EPR)
Microwave
The optical UV The visible
Infra-red (IR)
X-ray
Gamma radiation (nuclear-physical) |
101-10–
10-1-10–
200-400 nm 400-750 nm
10-13000 cm-1
10-8-10–
10-10-10– |
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 |
MOLECULAR–ABSORPTION METHOD: A PRINCIPLE, AN ORIGIN nOF SPECTRUM IN UV, VISIBLE AND IR OF SPECTRAL RANGE.
Overview of nSpectroscopy
The focus of nthis chapter is photon spectroscopy, using ultraviolet, visible, and infrared nradiation. Because these techniques use a common set of optical devices for ndispersing and focusing the radiation, they often are identified as optical nspectroscopies.
For nconvenience we will usually use the simpler term “spectroscopy” in place of nphoton spectroscopy or optical spectroscopy; however, it should be understood nthat we are considering only a limited part of a much broader area of nanalytical methods. Before we examine specific spectroscopic methods, however, nwe first review the properties of electromagnetic radiation.
Electromagnetic nRadiation
Electromagnetic nradiation, or light, is a form of energy whose behavior is described by the nproperties of both waves and particles. The optical properties of nelectromagnetic radiation, such as diffraction, are explained best by ndescribing light as a wave.
Many of the ninteractions between electromagnetic radiation and matter, such as absorptioand emission, however, are better described by treating light as a particle, or nphoton. The exact nature of electromagnetic radiation remains unclear, as it nhas since the development of quantum mechanics in the first quarter of the ntwentieth century. Nevertheless, the dual models of wave and particle behavior nprovide a useful description for electromagnetic radiation.
Wave nProperties of Electromagnetic Radiation
Electromagnetic nradiation consists of oscillating electric and magnetic fields that propagate nthrough space along a linear path and with a constant velocity (Figure). In a nvacuum, electromagnetic radiation travels at the speed of light, c, which nis 2.99792 108 m/s. Electromagnetic radiation moves through a medium nother than a vacuum with a velocity, v, less than that of the speed of nlight in a vacuum. The difference between v and c is small enough n(< 0.1%) that the speed of light to three significant figures, 3.00 n108 m/s, is sufficiently accurate for most purposes.
Oscillations nin the electric and magnetic fields are perpendicular to each other, and to the ndirection of the wave’s propagation. Figure shows an example of plane-polarized nelectromagnetic radiation consisting of an oscillating electric field and aoscillating magnetic field, each of which is constrained to a single plane.
Normally, nelectromagnetic radiation is unpolarized, with oscillating electric and magnetic fields in all possible planes noriented perpendicular to the direction of propagation.
The ninteraction of electromagnetic radiation with matter can be explained using neither the electric field or the magnetic field. For this reason, only the nelectric field component is shown in Figure. The oscillating electric field is ndescribed by a sine wave of the form
where E is nthe magnitude of the electric field at time t, Ae is the electric nfield’s maximum amplitude,is the frequency, or the number of noscillations in the electric field per unit time, and F is a phase angle naccounting for the fact that the electric field’s magnitude need not be zero at nt = 0. An identical equation can be written for the magnetic field, M
where Am nis the magnetic field’s maximum amplitude.
Aelectromagnetic wave, therefore, is characterized by several fundamental nproperties, including its velocity, amplitude, frequency, phase angle, npolarization, and direction of propagation.4 Other properties, which are based non these fundamental properties, also are useful for characterizing the wave nbehavior of electromagnetic radiation. The wavelength of aelectromagnetic wave, l, is defined as the distance between successive maxima, nor successive minima (see Figure above). For ultraviolet and visible nelectromagnetic radiation the wavelength is usually expressed ianometers n(nm, 10–9 m), and the wavelength for infrared radiation is given imicrons (mm, 10–6 m). Unlike frequency, wavelength depends on the nelectromagnetic wave’s velocity, where
Thus, for nelectromagnetic radiation of frequency, n, the wavelength in vacuum is longer nthan in other media. Another unit used to describe the wave properties of nelectromagnetic radiation is the wavenumber, –n, which is the reciprocal nof wavelength
Wavenumbers nare frequently used to characterize infrared radiation, with the units given ireciprocal centimeter (cm–1).
Particle nProperties of Electromagnetic Radiation
When a sample nabsorbs electromagnetic radiation it undergoes a change in energy. The ninteraction between the sample and the electromagnetic radiation is easiest to nunderstand if we assume that electromagnetic radiation consists of a beam of nenergetic particles called photons.
When a photois absorbed by a sample, it is “destroyed,” and its energy acquired by the nsample. The energy of a photon, in joules, is related to its frequency, nwavelength, or wavenumber by the following equations
where h is Planck’s constant, which has a value of 6.626 × n10–34 J · s.
The nElectromagnetic Spectrum
The frequency nand wavelength of electromagnetic radiation vary over many orders of magnitude. nFor convenience, electromagnetic radiation is divided into different regions nbased on the type of atomic or molecular transition that gives rise to the nabsorption or emission of photons (Figure).
The nboundaries describing the electromagnetic spectrum are not rigid, and aoverlap between spectral regions is possible.
Measuring nPhotons as a Signal
In the nprevious section we defined several characteristic properties of nelectromagnetic radiation, including its energy, velocity, amplitude, nfrequency, phase angle, polarization, and direction of propagation. nSpectroscopy is possible only if the photon’s interaction with the sample leads nto a change in one or more of these characteristic properties.
Spectroscopy nis conveniently divided into two broad classes. In one class of techniques, nenergy is transferred between a photon of electromagnetic radiation and the nanalyte (Table 10.1).
In absorptiospectroscopy the energy carried by a photon is absorbed by the analyte, npromoting the analyte from a lower-energy state to a higher-energy, or excited, nstate (Figure).
The source of nthe energetic state depends on the photon’s energy. The electromagnetic nspectrum in Figure, for example, shows that absorbing a photon of visible light ncauses a valence electron in the analyte to move to a higher-energy level. Whean analyte absorbs infrared radiation, on the other hand, one of its chemical nbonds experiences a change in vibrational energy.
The intensity nof photons passing through a sample containing the analyte is attenuated nbecause of absorption. The measurement of this attenuation, which we call absorbance, nserves as our signal. Note that the energy levels in Figure have nwell-defined values (i.e., they are quantized). Absorption only occurs when the nphoton’s energy matches the difference in energy, E, between two nenergy levels. A plot of absorbance as a function of the photon’s energy is ncalled an absorbance spectrum (Figure).
Ultraviolet/visible nabsorption spectrum for bromothymol blue.
Emission of na photon occurs when an analyte in a higher-energy state returns to a nlower-energy state (Figure).
Simplified nenergy level diagram showing emission of a photon.
The nhigher-energy state can be achieved in several ways, including thermal energy, nradiant energy from a photon, or by a chemical reaction. Emission following the nabsorption of a photon is also called photoluminescence, and that nfollowing a chemical reaction is called chemiluminescence.
A typical emissiospectrum is shown in Figure.
Photoluminescent nspectra for methyltetrahydrofolate and the enzyme methyltransferase. Whemethyltetrahydrofolate and methyltransferase are mixed, the enzyme is no longer nphotoluminescent, but the photoluminescence of methyltetrahydrofolate is nenhanced.
In the second nbroad class of spectroscopy, the electromagnetic radiation undergoes a change nin amplitude, phase angle, polarization, or direction of propagation as a nresult of its refraction, reflection, scattering, diffraction, or dispersion by nthe sample. Several representative spectroscopic techniques are listed in Table n10.2.
Absorbance of nElectromagnetic Radiation
In absorptiospectroscopy a beam of electromagnetic radiation passes through a sample.
Much of the nradiation is transmitted without a loss in intensity. At selected frequencies, nhowever, the radiation’s intensity is attenuated. This process of attenuatiois called absorption. Two general requirements must be met if an analyte is to nabsorb electromagnetic radiation. The first requirement is that there must be a nmechanism by which the radiation’s electric field or magnetic field interacts nwith the analyte. For ultraviolet and visible radiation, this interactioinvolves the electronic energy of valence electrons. A chemical bond’s nvibrational energy is altered by the absorbance of infrared radiation. A more ndetailed treatment of this interaction, and its importance in determining the nintensity of absorption, is found in the suggested readings listed at the end nof the chapter.
The second nrequirement is that the energy of the electromagnetic radiation must exactly nequal the difference in energy, DE, between two of the analytes quantized nenergy states. Figure 10.4 shows a simplified view of the absorption of a nphoton. The figure is useful because it emphasizes that the photon’s energy nmust match the difference in energy between a lower-energy state and a nhigher-energy state. What is missing, however, is information about the types nof energetic states involved, which transitions between states are likely to noccur, and the appearance of the resulting spectrum.
We can use nthe energy level diagram in Figure to explain an absorbance spectrum. The thick nlines labeled E0 and E1 represent the nanalyte’s ground (lowest) electronic state and its first electronic excited nstate. Superimposed on each electronic energy level is a series of lines nrepresenting vibrational energy levels.
Infrared Spectra nfor Molecules and Polyatomic Ions
The energy of ninfrared radiation is sufficient to produce a change in the vibrational energy nof a molecule or polyatomic ion. As shown in Figure above, vibrational energy nlevels are quantized; that is, a molecule may have only certain, discrete vibrational nenergies. The energy for allowed vibrational modes, Ev, is
where v is nthe vibrational quantum number, which may take values of 0, 1, 2, . . ., and n0 nis the bond’s fundamental vibrational frequency. Values for n0 are ndetermined by the bond’s strength and the mass at each end of the bond and are ncharacteristic of the type of bond. For example, a carbon–carbon single bond n(C—C) absorbs infrared radiation at a lower energy than a carbon–carbon double nbond (C=C) because a C—C bond is weaker than a C=C bond.
At room ntemperature most molecules are in their ground vibrational state (v = n0). A transition from the ground vibrational state to the first vibrational nexcited state (v = 1) requires the absorption of a photon with an energy nof hn0.
Transitions nin which Dv is n±1 give rise to the fundamental absorption lines. Weaker absorption lines, ncalled overtones, are due to transitions in which Dv is ±2 or ±3. nThe number of possible normal vibrational modes for a linear molecule is 3N – n5, and for a nonlinear molecule is 3N – 6, where N is the number nof atoms in the molecule. Not surprisingly, infrared spectra often show a nconsiderable number of absorption bands. Even a relatively simple molecule, such nas benzene (C6H6), for example, has 30 possible normal nmodes of vibration, although not all of these vibrational modes give rise to aabsorption. A typical IR spectrum is shown in Figure.
Fourier ntransform infrared (FT–IR) spectrum of polyvinylchloride
UV/Vis nSpectra for Molecules and Ions
When a nmolecule or ion absorbs ultraviolet or visible radiation it undergoes a change nin its valence electron configuration.
The valence nelectrons in organic molecules, and inorganic anions such as CO32–, noccupy 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 nenough in energy that the transition of an electron from an occupied to aunoccupied orbital is possible.
Four types of transitions betweequantized energy levels account for molecular UV/Vis spectra. The approximate nwavelength ranges for these absorptions, as well as a partial list of bonds, nfunctional groups, or molecules that give rise to these transitions is shown iTable 10.5. Of these transitions, the most important are the n®p* and p ® p*, because they involve functional groups that are ncharacteristic of the analyte and wavelengths that are easily accessible. The nbonds and functional groups that give rise to the absorption of ultraviolet and nvisible radiation are called chromophores.
Many transition metal ions, such as Cu2+ nand Co2+, form solutions that are colored because the metal ioabsorbs visible light. The transitions giving rise to this absorption are due nto valence electrons in the metal ion’s d-orbitals. For a free metal ion, the nfive d-orbitals are of equal energy. In the presence of a complexing ligand or nsolvent molecule, however, the d-orbitals split into two or more groups that differ ienergy. For example, in the octahedral complex Cu(H2O)62+ nthe six water molecules perturb the d-orbitals into two groups as shown iFigure.
The resulting d–d transitions for ntransition metal ions are relatively weak. A more important source of UV/Vis nabsorption for inorganic metal–ligand complexes is charge transfer, in which nabsorbing a photon produces an excited state species that can be described iterms of the transfer of an electron from the metal, M, to the ligand, L.
Charge-transfer absorption is important nbecause it produces very large absorbances, providing for a much more sensitive nanalytical method. One important example of a charge-transfer complex is that nof o-phenanthroline with Fe2+, the UV/Vis spectrum for which is nshown in Figure.
UV/Vis spectrum for Fe(o-phenanthroline)32+.
Charge-transfer absorption in which the nelectron moves from the ligand to the metal also is possible.
Comparing the IR spectrum in Figure above nto the UV/Vis spectrum in Figure
above, we note that UV/Vis absorption bands are often significantly nbroader than those for IR absorption.
When a species absorbs UV/Vis nradiation, the transition between electronic energy levels may also include a ntransition between vibrational energy levels. The result is a number of closely nspaced absorption bands that merge together to form a single broad absorptioband.
UV/Vis Spectra for Atoms
The energy of ultraviolet and visible nelectromagnetic radiation is sufficient to cause a change in an atom’s valence nelectron configuration. Sodium, for example, with a valence shell electroconfiguration of [Ne] 3s1, has a single valence electron in its 3s atomic orbital. nUnoccupied, higher energy atomic orbitals also exist. Figure 10.18 shows a npartial energy level diagram for sodium’s occupied and unoccupied valence shell natomic orbitals.
Valence shell energy diagram for sodium
This configuration of atomic orbitals, nwhich shows a splitting of the p orbitals into two levels with slightly different nenergies, may differ from that encountered in earlier courses. The reasons for nthis splitting, however, are beyond the level of this text, and unimportant ithis context.
Absorption of a photon is accompanied nby the excitation of an electron from a lower-energy atomic orbital to aorbital of higher energy. Not all possible transitions between atomic orbitals nare allowed.
For sodium the only allowed transitions nare those in which there is a change of ±1 in the orbital quantum number (l); thus ntransitions from s®p orbitals are nallowed, but transitions from s®d orbitals are forbidden. The wavelengths of electromagnetic radiatiothat must be absorbed to cause several allowed transitions are shown in Figure nabove.
The atomic absorption spectrum for Na nis shown in Figure and is typical of that found for most atoms.
Atomic absorption spectrum for sodium
The most obvious feature of this nspectrum is that it consists of a few, discrete absorption lines corresponding nto transitions between the ground state (the 3s atomic orbital) and the 3p and 4p atomic orbitals. nAbsorption from excited states, such as that from the 3p atomic orbital to nthe 4s or 3d atomic orbital, which are included in the energy level diagram in Figure nabove, are too weak to detect. Since the lifetime of an excited state is short, ntypically 10–7–10–8 s, an atom in the excited state is nlikely to return to the ground state before it has an opportunity to absorb a nphoton.
Another feature of the spectrum showin Figure is the narrow width of the absorption lines, which is a consequence nof the fixed difference in energy between the ground and excited states. nNatural line widths for atomic absorption, which are governed by the nuncertainty principle, are approximately 10–5 nm. Other ncontributions to broadening increase this line width to approximately 10–3 nnm.
THE FUNDAMENTAL LAW OF LIGHT ABSORPTION
First nlaw of light absorption
§ nEach thin layer of constant thickness nof a homogeneous environment absorbs an identical part of incident radiation
or:
§ nThe part of the light which is absorbed by a homogeneous nenvironment, is directly proportional to a thickness of an absorbing layer:
Second nlaw of light absorption
The part of the absorbed nradiation is proportional to number of absorbing particles in volume of a nsolution, that is concentration
Bouguer-Lambert-Beer nlaw
Reduction of intensity nof light which has passed through a layer of light-absorbing substance is nproportional concentration of this substance and a thickness of a layer
Quantitative characteristics of absorption
1. Transmittance – nthe ratio of the radiant npower passing through a sample to that from the radiation’s source (T).
The attenuation of electromagnetic nradiation as it passes through a sample is described quantitatively by two nseparate, but related terms: transmittance and absorbance. Transmittance is defined as the nratio of the electromagnetic radiation’s power exiting the sample, PT, to that incident non the sample from the source, P0,
Multiplying the transmittance by 100 ngives the percent transmittance (%T), which varies between 100% (no absorption) and 0% n(complete absorption).
All methods of detection, whether the nhuman eye or a modern photoelectric transducer, measure the transmittance of nelectromagnetic radiation.
Attenuation of radiation as it passes nthrough the sample leads to a transmittance of less than 1. As described, nequation 10.1 does not distinguish between the different ways in which the nattenuation of radiation occurs. Besides absorption by the analyte, several nadditional phenomena contribute to the net attenuation of radiation, including nreflection and absorption by the sample container, absorption by components of nthe sample matrix other than the analyte, and the scattering of radiation. To ncompensate for this loss of the electromagnetic radiation’s power, we use a nmethod blank. The radiation’s power exiting from the method blank is taken to nbe P0.
Diagram of Beer–Lambert nabsorption of a beam of light as it travels through a cuvette of width ℓ.
Optical ndensity А (Absorbance)
An alternative method for expressing the nattenuation of electromagnetic radiation is absorbance, A, which is defined as
An alternative method for expressing nthe attenuation of electromagnetic radiation is absorbance, A, which is defined nas
Absorbance is the more common unit for nexpressing the attenuation of radiation because, as shown in the next section, nit is a linear function of the analyte’s concentration.
Bouguer-Lambert-Beer nlaw
So:
§ nThe absorbance of a solution is nproportional to concentration of light-absorbing substance and a thickness of a nlayer
Or
§ nThe relationship between a sample’s nabsorbance and the concentration of the absorbing species
where: nA – optical density (absorbance), ε – the molar absorptivity, C – nconcentration (molarity)
Additivity nof optical densities
Beer’s law can be extended to samples containing nseveral absorbing components provided that there are no interactions betweethe components. Individual absorbances, Ai, are additive. For na two-component mixture of X and Y, the total nabsorbance, Atot, is
So
A = l(e1С1 n+ e2С2 n+ …ekСk)
permeate |
e (the molar absorptivity) |
Iro (ІІІ) rhodanate |
103 |
Complex Ti with H2O2 |
103 |
Complex Ti with chromotrope acid |
105 |
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 calibratiocurve of absorbance versus the concentration of analyte in a series of standard nsolutions should be a straight line with an intercept of 0 and a slope of ab or eb. In many cases, nhowever, calibration curves are found to be nonlinear (Figure 10.22).
Calibratiocurves showing positive and negative deviations from Beer’s law.
Deviations from linearity are divided ninto three categories: fundamental, chemical, and instrumental.
Fundamental Limitations to Beers Law
Beer’s law is a limiting law that is nvalid only for low concentrations of analyte. There are two contributions to nthis fundamental limitation to Beer’s law. At higher concentrations the nindividual particles of analyte no longer behave independently of one another. nThe resulting interaction between particles of analyte may change the value of e. A second contribution is that the nabsorptivity, a, and molar absorptivity, e, depend on the sample’s refractive index. Since the refractive index nvaries with the analyte’s concentration, the values of a and e will change. For sufficiently low nconcentrations of analyte, the refractive index remains essentially constant, nand the calibration curve is linear.
Instrumental Limitations to Beer’s Law
There are two principal instrumental nlimitations to Beer’s law. The first limitation is that Beer’s law is strictly nvalid for purely monochromatic radiation; that is, for radiation consisting of nonly one wavelength.
However, even the best wavelength nselector passes radiation with a small, but finite effective bandwidth. Using npolychromatic radiation always gives a negative deviation from Beer’s law, but nis minimized if the value of e is essentially constant over the wavelength range passed by the nwavelength selector.
For this reason, as shown in Figure n10.23, it is preferable to make absorbance measurements at a broad absorptiopeak.
Effect nof wavelength on the linearity of a Beer’s law calibration curve
In addition, deviations from Beer’s law nare less serious if the effective bandwidth from the source is less than one ntenth of the natural bandwidth of the absorbing species. When measurements must nbe made on a slope, linearity is improved by using a narrower effective nbandwidth.
Stray radiation is the second ncontribution to instrumental deviations from Beer’s law. Stray radiation arises nfrom imperfections within the wavelength selector that allows extraneous light nto “leak” into the instrument. Stray radiation adds an additional contribution, nPstray, to the radiant npower reaching the detector; thus
For small concentrations of analyte, Pstray is significantly nsmaller than P0 and PT, and the absorbance is unaffected by the stray radiation. At higher nconcentrations of analyte, however, Pstray is no longer significantly smaller nthan PT and the absorbance is smaller than expected. The result is a negative ndeviation from Beer’s law.
Physical nLimitations to Beer’s Law
§ nNOT monochromaticity of light:
A n= el×l×С.
§ nNOT parallelism of light.
§ nTemperature.
§ nNOT identical value of refraction of nsolutions.
§ nNOT proportionality of a photocurrent nand intensity of a light
Chemical nLimitations to Beer’s Law
§ nDilution of solution (thamore of reagent excess, it is less deviation from the law);
§ nрН of medium: nstate of metal ion
stability of ncomplex ions
§ ncompetitive reactions n(for ligand)
§ ncompetitive reactions n(complexing agent)
§ npolymerization and dissociatioreactions
§ nox-red reactions
Chemical deviations from Beer’s law caoccur when the absorbing species is involved in an equilibrium reaction. nConsider, as an example, an analysis for the weak acid, HA. To construct a nBeer’s law calibration curve, several standards containing known total nconcentrations of HA, Ctot, are prepared and the absorbance of each is measured nat the same wavelength. Since HA is a weak acid, it exists in equilibrium with nits conjugate weak base, A–
If nboth HA and A– absorb at the selected wavelength, then Beers law is nwritten as
where CHA and CA are the equilibrium concentrations of nHA and A–. Since the weak acid’s total concentration, Ctot, is
the nconcentrations of HA and A– can be written as
where naHA is the fractioof weak acid present as HA. Substituting equations gives
Because values of aHA may depend on the concentration of nHA, equation may not be linear. A Beer’s law calibration curve of A versus Ctot will be linear if none of two conditions is met. If the wavelength is chosen such that eHA and eA are equal, theequation simplifies to
and a linear Beer’s law calibratiocurve is realized. Alternatively, if aHA is held constant for all standards, then equatiowill be a straight line at all wavelengths.
Because HA is a weak acid, values of aHA change with pH. nTo 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 calibratiocurve will show a positive or negative deviation from Beer’s law if the nstandards are not buffered to the same pH.
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OPTIMUM CONDITIONS OF nPHOTOMETRIC DEFINITION
Components of a single-beam nspectrophotometer
A, exciter lamp; B, entrance nslit; C, monochromator; D, exit slit; E, cuvet; F, photodetector; G, LED ndisplay.
§ nMolecular–absorption method nis based on measurement of absorption by molecules (or ions) substances of nelectromagnetic radiation of an optical range:
§ nColorimetry nin which visible light was absorbed by a sample. The concentration of analyte nwas determined visually by comparing the sample’s color to that of a set of nstandards using Nessler tubes (as described at the beginning of this chapter), nor by using an instrument called a colorimeter.
§ nPhotocolorimetry – nin which polychromatic light was absorbed by a sample
§ nSpectrophotometry – in which monochromatic light was absorbed nby a sample
§ nUV – Spectrum (100-200 to 380-400 nnanometers)
§ nVisible spectrum (380-400 to 780-800 nnanometers)
Instrumentation
Frequently an analyst must select, from nseveral instruments of different design, the one instrument best suited for a nparticular analysis. In this section we examine some of the different types of ninstruments used for molecular absorption spectroscopy, emphasizing their nadvantages and limitations. Methods of sample introduction are also covered ithis section.
Instrument Designs for Molecular UV/Vis nAbsorption
The simplest instrument currently used nfor molecular UV/Vis absorption is the filter photometer shown in Figure, nwhich uses an absorption or interference filter to isolate a band of radiation.
The filter is placed between the source nand sample to prevent the sample from decomposing when exposed to high-energy nradiation. A filter photometer has a single optical path between the source and ndetector and is called a single-beam instrument.
The instrument is calibrated to 0% T while using a nshutter to block the source radiation from the detector. After removing the nshutter, the instrument is calibrated to 100% T using an appropriate blank. The blank nis then replaced with the sample, and its transmittance is measured. Since the nsource’s incident power and the sensitivity of the detector vary with nwavelength, the photometer must be recalibrated whenever the filter is changed. nIn comparison with other spectroscopic instruments, photometers have the nadvantage of being relatively inexpensive, rugged, and easy to maintain. nAnother advantage of a photometer is its portability, making it a useful ninstrument for conducting spectroscopic analyses in the field. A disadvantage nof a photometer is that it cannot be used to obtain an absorption spectrum.
Instruments using monochromators for nwavelength selection are called spectrometers. In absorbance spectroscopy, nwhere the transmittance is a ratio of two radiant powers, the instrument is ncalled a spectrophotometer. The simplest spectrophotometer is a single-beam ninstrument equipped with a fixedwavelength monochromator, the block diagram for nwhich is shown in Figure.
Photo courtesy of Fisher Scientific
Single-beam spectrophotometers are ncalibrated and used in the same manner as a photometer. One common example of a nsingle-beam spectrophotometer is the Spectronic-20 manufactured by Milton-Roy. nThe Spectronic-20 can be used from 340 to 625 nm (950 nm with a red-sensitive ndetector), and has a fixed effective bandwidth of 20 nm. Because its effective nbandwidth is fairly large, this instrument is more appropriate for a nquantitative analysis than for a qualitative analysis. Battery-powered, nhand-held single-beam spectrophotometers are available, which are easily ntransported and can be used for on-site analyses.
Other single-beam spectrophotometers nare available with effective bandwidths of 2–8 nm. Fixed-wavelength single-beam nspectrophotometers are not practical for recording spectra since manually nadjusting the wavelength and recalibrating the spectrophotometer is awkward and ntime-consuming. In addition, the accuracy of a single-beam spectrophotometer is nlimited by the stability of its source and detector over time.
The limitations of fixed-wavelength, nsingle-beam spectrophotometers are minimized by using the double-beam in-time nspectrophotometer as shown in Figure.
Block ndiagram for a double-beam in-time scanning spectrophotometer with photo of a ntypical instrument
A chopper, similar to that shown in the ninsert, controls the radiation’s path, alternating it between the sample, the nblank, and a shutter. The signal processor uses the chopper’s known speed of nrotation to resolve the signal reaching the detector into that due to the ntransmission of the blank (P0) and the sample (PT). By including an opaque surface as a nshutter it is possible to continuously adjust the 0% T response of the ndetector.
The effective bandwidth of a ndouble-beam spectrophotometer is controlled by means of adjustable slits at the nentrance and exit of the monochromator. Effective bandwidths of between 0.2 nm nand 3.0 nm are common. A scanning monochromator allows for the automated nrecording of spectra. Double-beam instruments are more versatile thasingle-beam instruments, being useful for both quantitative and qualitative analyses; nthey are, however, more expensive.
The instrument designs considered thus nfar use a single detector and can only monitor one wavelength at a time. A nlinear photodiode array consists of multiple detectors, or channels, allowing nan entire spectrum to be recorded in as little as 0.1 s.
Source radiation passing through the nsample is dispersed by a grating. The linear photodiode array is situated at nthe grating’s focal plane, with each diode recording the radiant power over a nnarrow range of wavelengths.
The sample compartment for the ninstruments in Figures above provides a light-tight environment that prevents nthe loss of radiation, as well as the addition of stray radiation. Samples are nnormally in the liquid or solution state and are placed in cells constructed nwith UV/Vis-transparent materials, such as quartz, glass, and plastic (Figure).
Typical ncells used in UV/Vis spectroscopy
Quartz or fused-silica cells are nrequired when working at wavelengths of less than 300 nm where other materials nshow a significant absorption. The most common cell has a pathlength of
In some circumstances it is desirable nto monitor a system without physically removing a sample for analysis. This is noften the case, for example, with the on-line monitoring of industrial nproduction lines or waste lines, for physiological monitoring, and for nmonitoring environmental systems. With the use of a fiber-optic probe it is npossible to analyze samples in situ. A simple example of a remote-sensing, nfiber-optic probe is shown in Figure a nand consists of two bundles of fiber-optic cable.
Example of fiber-optic probes
One bundle transmits radiation from the nsource to the sample cell, which is designed to allow for the easy flow of nsample through the cell. Radiation from the source passes through the solution, nwhere it is reflected back by a mirror. The second bundle of fiber-optic cable ntransmits the nonabsorbed radiation to the wavelength selector. In aalternative design (Figure b), the sample cell is a membrane containing a nreagent phase capable of reacting with the analyte. When the analyte diffuses nacross the membrane, it reacts with the reagent phase, producing a product that nabsorbs UV or visible radiation. Nonabsorbed radiation from the source is nreflected or scattered back to the detector. Fiber-optic probes that show nchemical selectivity are called optrodes.
Choice nof optimum conditions of spectrophotometry:
§ nChoice absorption filters (iphotometry)
§ nChoice of absorbance
Аoptimal= n0.435
(less nerror)
А n= 0.6 – 0.7
§ n!!!! Not probably to measure nabsorbance
2 n< А < 0.03
§ nChoice of thickness of a layer – not nmore 5 сm
А n= e l nC
§ nWay of transformation of a defined ncomponent in photometric compound
Choice of optimal wavelenght (lmах)
Sensitivity nof photometric definition
А n= e l nC
Cmin n= Аmin / e l
§ nА = 0.01
§ nl = 1 cм
§ ne = 1000
then Сmin = 10-5 mol/L
Basic nComponents of Spectroscopic Instrumentation
The ninstruments used in spectroscopy consist of several common components, including na source of energy that can be input to the sample, a means for isolating a nnarrow range of wavelengths, a detector for measuring the signal, and a signal processor nto display the signal in a form convenient for the analyst. In this section we nintroduce the basic components used to construct spectroscopic instruments. A nmore detailed discussion of these components can be found in the suggested nend-of-chapter readings. Specific instrument designs are considered in later nsections.
Sources of nEnergy
All forms of nspectroscopy require a source of energy. In absorption and scattering spectroscopy nthis energy is supplied by photons. Emission and luminescence spectroscopy use nthermal, radiant (photon), or chemical energy to promote the analyte to a less nstable, higher energy state.
Sources of nElectromagnetic Radiation
A source of nelectromagnetic radiation must provide an output that is both intense and nstable in the desired region of the electromagnetic spectrum. Sources of nelectromagnetic radiation are classified as either continuum or line sources. A ncontinuum source emits radiation over a wide range of wavelengths, with na relatively smooth variation in intensity as a function of wavelength (Figure). n
Emissiospectrum from a typical continuum source
Line sources, non the other hand, emit radiation at a few selected, narrow nwavelength ranges (Figure).
Table nprovides a list of the most common sources of electromagnetic radiation.
Sources of nThermal Energy
The most ncommon sources of thermal energy are flames and plasmas. Flame sources use the ncombustion of a fuel and an oxidant such as acetylene and air, to achieve ntemperatures of 2000–3400 K. Plasmas, which are hot, ionized gases, provide ntemperatures of 6000–10,000 K.
Chemical nSources of Energy
Exothermic nreactions also may serve as a source of energy. In chemiluminescence the nanalyte is raised to a higher-energy state by means of a chemical reaction, nemitting characteristic radiation when it returns to a lower-energy state. Whethe chemical reaction results from a biological or enzymatic reaction, the nemission of radiation is called bioluminescence. Commercially available “light nsticks” and the flash of light from a firefly are examples of chemiluminescence nand bioluminescence, respectively.
Wavelength nSelection
In Nessler’s noriginal colorimetric method for ammonia, described at the beginning of the nchapter, no attempt was made to narrow the wavelength range of visible light passing nthrough the sample. If more than one component in the sample contributes to the nabsorption of radiation, however, then a quantitative analysis using Nessler’s original nmethod becomes impossible. For this reason we usually try to select a single wavelength nwhere the analyte is the only absorbing species. Unfortunately, we cannot isolate na single wavelength of radiation from a continuum source. Instead, a wavelength nselector passes a narrow band of radiation (Figure) characterized by a nominal nwavelength, an effective bandwidth, and a maximum throughput of radiation. n
Band of nradiation exiting wavelength selector showing the nominal wavelength and neffective bandpass.
The effective nbandwidth is defined as the width of the radiation at half the maximum nthroughput.
The ideal nwavelength selector has a high throughput of radiation and a narrow effective nbandwidth. A high throughput is desirable because more photons pass through the nwavelength selector, giving a stronger signal with less background noise. A nnarrow effective bandwidth provides a higher resolution, with spectral nfeatures separated by more than twice the effective bandwidth being resolved. nGenerally these two features of a wavelength selector are in opposition (Figure). n
Conditions favoring na higher throughput of radiation usually provide less resolution. Decreasing nthe effective bandwidth improves resolution, but at the cost of a noisier nsignal. For a qualitative analysis, resolution is generally more important thathe throughput of radiation; thus, smaller effective bandwidths are desirable. nIn a quantitative analysis a higher throughput of radiation is usually ndesirable.
Wavelength nSelection Using Filters
The simplest nmethod for isolating a narrow band of radiation is to use an absorption or ninterference filter. Absorption filters work by selectively absorbing nradiation from a narrow region of the electromagnetic spectrum. Interference nfilters use constructive and destructive interference to isolate a narrow range nof wavelengths. A simple example of an absorption filter is a piece of colored nglass. A purple filter, for example, removes the complementary color green from n500–560 nm. Commercially available absorption filters provide effective nbandwidths from 30–250 nm. The maximum throughput for the smallest effective nbandpasses, however, may be only 10% of the source’s emission intensity over nthat range of wavelengths. Interference filters are more expensive thaabsorption filters, but have narrower effective bandwidths, typically 10–20 nm, nwith maximum throughputs of at least 40%.
Wavelength nSelection Using Monochromators
One nlimitation of an absorption or interference filter is that they do not allow nfor a continuous selection of wavelength.
If nmeasurements need to be made at two wavelengths, then the filter must be changed nin between measurements. A further limitation is that filters are available for nonly selected nominal ranges of wavelengths. An alternative approach to nwavelength selection, which provides for a continuous variation of wavelength, nis the monochromator.
The nconstruction of a typical monochromator is shown in Figure.
Radiation from nthe source enters the monochromator through an entrance slit. The radiation is ncollected by a collimating mirror, which reflects a parallel beam of radiation to na diffraction grating. The diffraction grating is an optically reflecting nsurface with a large number of parallel grooves (see inset to Figure above). nDiffraction by the grating disperses the radiation in space, where a second nmirror focuses the radiation onto a planar surface containing an exit slit. Isome monochromators a prism is used in place of the diffraction grating.
Radiatioexits the monochromator and passes to the detector. As shown in Figure above, a npolychromatic source of radiation at the entrance slit is converted at the nexit slit to a monochromatic source of finite effective bandwidth. The nchoice of which wavelength exits the monochromator is determined by rotating nthe diffraction grating. A narrower exit slit provides a smaller effective nbandwidth and better resolution, but allows a smaller throughput of radiation.
Monochromators nare classified as either fixed-wavelength or scanning. In a fixed-wavelength nmonochromator, the wavelength is selected by manually rotating the grating. nNormally, a fixed-wavelength monochromator is only used for quantitative analyses nwhere measurements are made at one or two wavelengths. A scanning monochromator nincludes a drive mechanism that continuously rotates the grating, allowing nsuccessive wavelengths to exit from the monochromator. Scanning monochromators nare used to acquire spectra and, when operated in a fixed wavelength mode, for nquantitative analysis.
Interferometers n
An interferometer nprovides an alternative approach for wavelength selection. Instead of nfiltering or dispersing the electromagnetic radiation, an interferometer nsimultaneously allows source radiation of all wavelengths to reach the detector n(Figure).
Block diagram of an interferometer
Radiation from nthe source is focused on a beam splitter that transmits half of the radiation to na fixed mirror, while reflecting the other half to a movable mirror. The nradiation recombines at the beam splitter, where constructive and destructive ninterference determines, for each wavelength, the intensity of light reaching nthe detector.
As the moving nmirror changes position, the wavelengths of light experiencing maximum nconstructive interference and maximum destructive interference also changes. nThe signal at the detector shows intensity as a function of the moving mirror’s nposition, expressed in units of distance or time. The result is called ainterferogram, or a time domain spectrum. The time domain spectrum is converted nmathematically, by a process called a Fourier transform, to the normal spectrum n(also called a frequency domain spectrum) of intensity as a function of the nradiation’s energy.
In comparisowith a monochromator, interferometers provide two significant advantages. The nfirst advantage, which is termed Jacquinot’s advantage, results from the higher nthroughput of source radiation. Since an interferometer does not use slits and nhas fewer optical components from which radiation can be scattered and lost, nthe throughput of radiation reaching the detector is 80–200 times greater thathat achieved with a monochromator. The result is an improved signal-to-noise nratio. The second advantage, which is called Fellgett’s advantage, reflects na savings in the time needed to obtain a spectrum. Since all frequencies are nmonitored simultaneously, an entire spectrum can be recorded in approximately 1 ns, as compared to 10–15 min with a scanning monochromator.
Detectors
The first ndetector for optical spectroscopy was the human eye, which, of course, is limited nboth by its accuracy and its limited sensitivity to electromagnetic radiation.
Moderdetectors use a sensitive transducer to convert a signal consisting of nphotons into an easily measured electrical signal. Ideally the detector’s nsignal, S, should be a linear function of the electromagnetic nradiation’s power, P,
where k is nthe detector’s sensitivity, and D is the detector’s dark current, or nthe background electric current when all radiation from the source is blocked nfrom the detector.
PhotoTransducers
Two general nclasses of transducers are used for optical spectroscopy, several examples of nwhich are listed in Table 10.4.
Phototubes nand photomultipliers contain a photosensitive surface that absorbs radiation ithe ultraviolet, visible, and near infrared (IR), producing an electric current nproportional to the number of photons reaching the transducer. Other photodetectors use a semiconductor as the photosensitive surface. When the nsemiconductor absorbs photons, valence electrons move to the semiconductor’s nconduction band, producing a measurable current. One advantage of the Si nphotodiode is that it is easily miniaturized. Groups of photodiodes may be ngathered together in a linear array containing from 64 to 4096 individual nphotodiodes. With a width of
By placing a photodiode narray along the monochromator’s focal plane, it is possible to monitor nsimultaneously an entire range of wavelengths.
Thermal nTransducers
Infrared nradiation generally does not have sufficient energy to produce a measurable ncurrent when using a photon transducer. A thermal transducer, therefore, is nused for infrared spectroscopy. The absorption of infrared photons by a thermal ntransducer increases its temperature, changing one or more of its ncharacteristic properties. The pneumatic transducer, for example, consists of a nsmall tube filled with xenon gas equipped with an IR-transparent window at one nend, and a flexible membrane at the other end. A blackened surface in the tube nabsorbs photons, increasing the temperature and, therefore, the pressure of the ngas. The greater pressure in the tube causes the flexible membrane to move iand out, and this displacement is monitored to produce an electrical signal.
Accuracy of nphotometric definition depends from:
§ nSpecific features of photometric nreaction or photometric compounds
§ nCharacteristics of the used device n(usually makes 1 – 2 % relative)
METHODS OF QUANTITATIVE ANALYSIS.
Quantitative Applications
The determination of an analyte’s nconcentration based on its absorption of ultraviolet or visible radiation is none of the most frequently encountered quantitative analytical methods. One nreason for its popularity is that many organic and inorganic compounds have nstrong absorption bands in the UV/Vis region of the electromagnetic spectrum. nIn addition, analytes that do not absorb UV/Vis radiation, or that absorb such nradiation only weakly, frequently can be chemically coupled to a species that ndoes. For example, nonabsorbing solutions of Pb2+ can be reacted nwith dithizone to form the red Pb–dithizonate complex. An additional advantage nto UV/Vis absorption is that in most cases it is relatively easy to adjust nexperimental and instrumental conditions so that Beer’s law is obeyed.
Quantitative analyses based on the nabsorption of infrared radiation, although important, are less frequently nencountered than those for UV/Vis absorption. One reason is the greater ntendency for instrumental deviations from Beer’s law when using infrared nradiation. Since infrared absorption bands are relatively narrow, deviations due nto the lack of monochromatic radiation are more pronounced. In addition, infrared nsources are less intense than sources of UV/Vis radiation, making stray radiatiomore of a problem. Differences in pathlength for samples and standards wheusing thin liquid films or KBr pellets are a problem, although an internal standard ncan be used to correct for any difference in pathlength. Finally, establishing a n100% T (A = 0) baseline is often difficult since the optical properties of NaCl nsample cells may change significantly with wavelength due to contamination and ndegradation. This problem can be minimized by determining absorbance relative to na baseline established for the absorption band.
!!! The nmethod can be applied, if:
§ nStructure of standard and ninvestigated solutions are similar
§ nThe interval of concentration ocalibration chart should cover of defined concentration
2. Comparison method (a method on one nstandard)
!! The method can be used if:
§ nDependence structure – property is nstrictly rectilinear and passes through the beginning of co-ordinates
§ nConcentration of standard and ninvestigated solutions values of analytical signals as much as possible similar nand minimum different
§ nStructure of standard and ninvestigated solutions are as much as possible similar
3. Method of molar or specific n(concentration on % w/w) absorptivity
!! The method can be used if:
§ nStrict linearity of dependence nstructure – an analytical signal is observed
§ nThe analytical device maintains nrequirements of metrological checking
4. Method of additives
!!! The method can be applied, if:
§ nIt is necessary to consider stirring ninfluence of extraneous components of sample on analytical signal of defined nsubstance
Usage nof UV – spectroscopy and nspectrophotometry in visible spectrum:
§ nIdentification and establishment of identity of drugs
§ nQuantitative definition of substance contain
§ nCleanliness check
§ nThe express control of the forged drugs
§ nResearch of new substances structure
The applications of Beer’s law for the nquantitative analysis of samples in environmental chemistry, clinical nchemistry, industrial chemistry and forensic chemistry are numerous.
Environmental Applications
Methods for the analysis of waters and nwastewaters relying on the absorption of UV/Vis radiation are among some of the nmost frequently employed analytical methods. Many of these methods are outlined nin Table, and a few are described later in more detail.
Although the quantitative analysis of nmetals in water and wastewater is accomplished primarily by atomic absorptioor atomic emission spectroscopy, many metals also can be analyzed following the nformation of a colored metal–ligand complex. One advantage to these nspectroscopic methods is that they are easily adapted to the field analysis of nsamples using a filter photometer. One ligand used in the analysis of several nmetals is diphenylthiocarbazone, also known as dithizone. Dithizone is ninsoluble in water, but when a solution of dithizone in CHCl3 is nshaken with an aqueous solution containing an appropriate metal ion, a colored nmetal–dithizonate complex forms that is soluble in CHCl3. The nselectivity of dithizone is controlled by adjusting the pH of the aqueous nsample. For example, Cd2+ is extracted from solutions that are made nstrongly basic with NaOH, Pb2+ from solutions that are made basic nwith an ammoniacal buffer, and Hg2+ from solutions that are slightly nacidic.
When chlorine is added to water that nportion available for disinfection is called the chlorine residual. Two forms nof the chlorine residual are recognized. The free chlorine residual includes Cl2, nHOCl, and OCl–. The combined chlorine residual, which forms from the nreaction of NH3 with HOCl, consists of monochloroamine, NH2Cl, ndichlororamine, NHCl2, and trichloroamine, NCl3. Since nthe free chlorine residual is more efficient at disinfection, analytical nmethods have been developed to determine the concentration of both forms of nresidual chlorine. One such method is the leuco crystal violet method. Free nresidual chlorine is determined by adding leuco crystal violet to the sample, nwhich instantaneously oxidizes giving a bluish color that is monitored at 592 nnm. Completing the analysis in less than 5 min prevents a possible interference nfrom the combined chlorine residual. The total chlorine residual (free + ncombined) is determined by reacting a separate sample with iodide, which reacts nwith both chlorine residuals to form HOI. When the reaction is complete, leuco ncrystal violet is added and oxidized by HOI, giving the same bluish colored nproduct. The combined chlorine residual is determined by difference.
The concentration of fluoride idrinking water may be determined indirectly by its ability to form a complex nwith zirconium. In the presence of the dye SPADNS, solutions of zirconium form na reddish colored compound, called a “lake,” that absorbs at 570 nm. Whefluoride is added, the formation of the stable ZrF62– complex ncauses a portion of the lake to dissociate, decreasing the absorbance. A plot of nabsorbance versus the concentration of fluoride, therefore, has a negative nslope.
Spectroscopic methods also are used idetermining organic constituents in water. For example, the combined nconcentrations of phenol, and ortho- and metasubstituted phenols are determined nby using steam distillation to separate the phenols from nonvolatile nimpurities. The distillate is reacted with 4-aminoantipyrine at pH 7.9 ± 0.1 in the presence of K3Fe(CN)6, nforming a colored antipyrine dye.
The dye is extracted into CHCl3, nand the absorbance is monitored at 460 nm. A calibration curve is prepared nusing only the unsubstituted phenol, C6H5OH. Because the nmolar absorptivities of substituted phenols are generally less than that for phenol, nthe reported concentration represents the minimum concentration of phenolic compounds.
Molecular absorption also can be used nfor the analysis of environmentally significant airborne pollutants. In many ncases the analysis is carried out by collecting the sample in water, converting nthe analyte to an aqueous form that can be analyzed by methods such as those ndescribed in Table. For example, the concentration of NO2 can be ndetermined by oxidizing NO2 to NO3–. The nconcentration of NO3– is then determined by reducing to nNO2
– with Cd and reacting the NO2
– with sulfanilamide
and N-(1-naphthyl)-ethylenediamine to form a nbrightly colored azo dye. Another important application is the determination of nSO2, which is determined by collecting the sample in an aqueous nsolution of HgCl42– where it reacts to form Hg(SO3)22–. nAddition of p-rosaniline and formaldehyde results in the formation of a bright purple ncomplex that is monitored at 569 nm. Infrared absorption has proved useful for nthe analysis of organic vapors, including HCN, SO2, nitrobenzene, methyl nmercaptan, and vinyl chloride. Frequently, these analyses are accomplished using nportable, dedicated infrared photometers.
Clinical Applications
UV/Vis molecular absorption is one of nthe most commonly employed techniques for the analysis of clinical samples, nseveral examples of which are listed in Table.
Industrial Analysis
UV/Vis molecular absorption is used for nthe analysis of a diverse array of industrial samples, including npharmaceuticals, food, paint, glass, and metals. In many cases the methods are nsimilar to those described in Tables above. For example, the iron content of nfood can be determined by bringing the iron into solution and analyzing using nthe o-phenanthroline method listed in Table.
Many pharmaceutical compounds contaichromophores that make them suitable for analysis by UV/Vis absorption. nProducts that have been analyzed in this fashion include antibiotics, hormones, nvitamins, and analgesics. One example of the use of UV absorption is idetermining the purity of aspirin tablets, for which the active ingredient is nacetylsalicylic acid. Salicylic acid, which is produced by the hydrolysis of nacetylsalicylic acid, is an undesirable impurity in aspirin tablets, and should nnot be present at more than 0.01% w/w. Samples can be screened for unacceptable nlevels of salicylic acid by monitoring the absorbance at a wavelength of 312 nnm. Acetylsalicylic acid absorbs at 280 nm, but absorbs poorly at 312 nm. nConditions for preparing the sample are chosen such that an absorbance of ngreater than 0.02 signifies an unacceptable level of salicylic acid.
Forensic Applications
UV/Vis molecular absorption is nroutinely used in the analysis of narcotics and for drug testing. One ninteresting forensic application is the determination of blood alcohol using nthe Breathalyzer test. In this test a 52.5-mL breath sample is bubbled through nan acidified solution of K2Cr2O7. Any ethanol present nin the breath sample is oxidized by the dichromate, producing acetic acid and nCr3+ as products. The concentration of ethanol in the breath sample nis determined from the decrease in absorbance at 440 nm where the dichromate nion absorbs.
A blood alcohol content of 0.10%, which nis the legal limit in most states, corresponds to 0.025 mg of ethanol in the nbreath sample.