Chapter 3
Spectroscopic Techniques
Spectroscopy is a kind of instrumental analytical techniques used for identification as well quantification purposes (determination the unknown amount) of analyte solutions. The instrument that is used in spectroscopy is called spectrophotometer.
Procedure
In Spectroscopic techniques monochromatic light (single wave length light) is always passedfromanalyte solutions. Some timesthe analyte solutions absorb or emitthose radiations. The magnitude ofthose absorbed or emitted radiations byanalyte solutions is measured with the help of instruments likespectrophotometer, Spectroflourometer, Flourometer, atomic absorptionspectrophotometer, flame photometer etc.
Classification of Spectroscopic techniques
Spectroscopic techniquesare broadly categoriesed into two classes that is quantitative Spectroscopic techniques and qualitativeSpectroscopic techniques.
Quantitative Spectroscopic techniques like UV-Visible spectroscopy, atomic absorption spectroscopy are used to know the unknown amount of analyte, while the qualitativeSpectroscopic techniques like FTIR and NMR are used for identification as well as structure elucidation of analyte.
Quantitative Spectroscopic techniques arefurther sub categoriesed into two classes i.e. Absorption Spectroscopy and Emission Spectroscopy.
In case of absorption spectroscopy we measure the magnitude of radiationsabsorbedby the analyte solutions. The analyte specie may be molecular in nature or atomic, so based on the nature of analyte specie it is further sub classified into Molecular absorption spectroscopy and Atomic absorption spectroscopy (AAS).
While in case of emission spectroscopy we measure the magnitude of intensity of emitted radiations. It is also further sub classified into Molecular emission spectroscopy and Atomic emission spectroscopy based on the nature of analyte solutions.
Quantitative aspects of absorption spectroscopy
The Quantitative aspects of absorption spectroscopy are based onrelating the magnitude of absorbed radiations with the concentration of analyte species using Beer’s Lambert Law principle i.e.
A = bC
Where & b are constant so, A C(absorption is directly proportion to concentration of analyte specie)
The Quantitative determination of analyte can be done by using Standard Calibration Curve Method. In this method the standard solutions of different concentrations are prepared and for each standard solution the magnitude of absorption is measured with the help spectrophometer. In a similar way under the similar conditions the magnitude of absorption for analyte solution is also measured. By relating the magnitude of absorption of analyte solution with the magnitude of absorption with standard solution the unknown concentration of analyte is determined. This method is called standard calibration method.
In the above example the unknown concentration of analyte is now 6ppm that was found with the help of standard calibration curve.
While in case of emission spectroscopy it is based on relating the magnitude of emitted intensity of the radiations to the concentration of analyte species using the relationship as given below
Where
(IF)is the emitted fluorescent light
(φ)is quantum yield (the ratio of the number of photons emitted to the number of
photons absorbed),
(Io)is the intensity of the incident light,
(c)is the concentration of the solute,
(k)is the molar absorbance,
(l) is the path length of the cell.
Or
IF = Ko x Io x C
Where K0 = Constant = (φ) (k) (l)
So it shows thatIFC (Emitted Intensity is directly proportion to concentration of analyte specie)
As we know EMR or spectrum consists of different wavelengths or energy regions. For example x-rays radiations are occurring in the wavelength region of 0.1nm to 10nm, while the ultraviolet radiations are occurring in the wavelength region from 180nm to 380nm, while the visible radiations are occurring in the wavelength region from 380nm to 780nm, while the infrared radiations are occurring in the wavelength region from 780nm to 4000nm, while the radio frequency radiations are occurring in the wavelength region from 10cm to 1m.
The type of spectroscopy in that radiations in x-rays region interact with matter is called x-rays spectroscopy for example XRD and XRF used for elemental analysis.
The type of spectroscopy in that radiations in ultraviolet and visible region interact with matter is called UV-Visible spectroscopy is used for quantitive analysis.
The type of spectroscopy in that the radiations interact in infra red region with matter is called infra red or IR spectroscopy is used for functional group identification purposes.
The type of spectroscopy in that radiations interact in radiofrequency region with matter is called nuclear magnetic resonance or NMR spectroscopy is used for structure elucidation of organic compound.
In this course we will be discussing the type of spectroscopy in that the radiations interact in UV-visible regions only i.e. absorption spectroscopy.
UV-Visible Molecular absorption spectroscopy
The type of spectroscopy in that ultraviolet and visible region radiations interact with molecular species and absorbed is called UV-Visible molecular absorptionspectroscopy. The energy of radiations in this region is used for excitation of electron in different molecular levels. The changes depend on the probability of electronic transitions between the individual energy states of the molecule.
The probability of electronic transitions in a molecule depends on the presence of multiple bonds in the molecule and on the kind, number and positions of the substituent groups.
Spectral transitions of electrons associated with absorption of radiation correspond to transitions from binding orbitals to anti-bonding orbitals of higher energy state. The energy of the respective transitions decreases in the following order:7t
σ → σ*
n → σ*
π → π*
n → π*
Aromatic π → aromatic π*
The sigma to sigma star transitions (o--~ o*) may take place in the far ultraviolet region of radiation, which is generally not recorded in spectrophotometers. Other transitions occur in the near ultraviolet and visible regions. The n to Π* transitions are characterized by high intensity which varies depending on the number and kind of multiple bonds in the molecule. An increase in the number of conjugated bonds results in a reduction of the distance between the Π to Π* levels, an increase in the probability of transition, and increase of intensity of the spectrum recorded.
The color of a molecule is an effect of the presence of chromophoric groups. A chromophore may be a group of atoms containing easily excitable pi electrons, including the most important groups for the visible region: the azo group -N=N- and the p-quinonoid system. Nitro group etc
The features of the absorption spectra change if the so-called auxoehromes(e.g., -NH2,-NR2, -SH, -OH, -OR) are introduced into the molecules. The presence of free electron pairs in the auxochromic group that interact with lone pair electrons of the chromophoric group (e.g., the free electron pair at nitrogen in the-NH2 group) leads to a state of conjugation which may result in formation of a new absorption band in the spectrum
An action of a substituent or a solvent may give rise to a shift of absorption band towardslonger wavelengths is called the bathochromic effect, or towards shorter wavelengths is called thehypsochromic effect. An increase or a decrease of band intensity is referred to as thehyperchromic or the hypsochromic effect, respectively.
Absorption laws
Spectrophotometric measurements are generally made on solutions, either in water or inorganic solvents, contained in a measuring cell which is placed in the path of a beam ofmonochromatic radiation of chosen wavelength.
From the total radiation of intensity Io that impinges upon a layer of solution, one fraction of the beam Ia is absorbed on passing through the solution, another fraction It is transmitted, and still another fraction Ir is reflected by the cell walls and scattered:
lo= Ia + l, + lr
The amount of radiation absorbed depends on the thickness of the absorbing layer and on the concentration of the solution. In the formula derived by the Lambert & Beer for the absorption of radiations by solution they took into account both the thickness of the medium layer and concentration.When a parallel beam of monochromatic radiation of intensity impinges upon a layer of solution a part of the radiant energy is absorbed. The fraction of radiation absorbed increases exponentially with linear increase of the layer thickness:
Similarly the absorption of radiation also increase with increase in concentration of analyte species, so they expressed a relationship in that they showed the relationship among the absorption, thickness of the layer of the medium as well as concentration of analyte solution. This relationship is called Beer Lambert Law, It is given as below
Where E is a constant called the molar absorptivity (or absorption coefficient), c is the concentration of absorbing species (M, in moles per litre), and l is the layer thickness (in cm).
The equation is a mathematical expression of a fundamental law of spectrophotometry, the Bouguer-Lambert-Beer law, which states that absorption of radiation depends on the total number of absorbing centres, i.e., on the product of concentration and layer thickness of the solution.
In spectrophotometric measurements the thickness of the sample layer is usually identical to that of the reference solution, and only Beer's law, which relates the absorbance with the concentration of the sample solution, is of practical significance.
From a practical point of view it is desirable that the solution should follow Beer's law for the concentration range corresponding to absorbances not exceeding 1 (unity).
Deviations from Beer's law
Deviations from Beer's law may also result from either chemical reasons connected with the sample, or physical ones connected with the instruments involved.
Q 18:a) Calculate the percent transmittance of a solution if its absorbance is 0.352.(5 marks)
Solution
As we know
A = 2 – log % T
So re arranging the above equation
log % T = 2 – A = 2 – 0.352 = 1.648
Now taking anti log of 1.64
% T = 1.64/ log = 44.46%
a)Calculate the absorbance of a solution if its percent transmittance is 55%.
Solution
As we know
A = 2 – log % T
So substituting the values in the above expression as
A = 2 – log % T = 2 – log 55 = 2 – 1.740 = 0.259
Spectrophotometric apparatus
The instrument that has been used for measurement of radiation has been absorbed by molecular specie is called UV-Visible Spectrophotometer. It consist of the following components, radiation source, monochromator, cuvette, and detector with the data treatment system
1. Radiation sources
In most cases spectrophotometers are equipped with two independent radiation sources:
UV and VIS. The UV source is usually a deuterium- or xenon lamp that emits radiation inthe range of 180-400 nm or 190-750 nm, respectively. The sources emitting visible light are tungsten- and halogen lamps. A feature of thehalogen lamps is their wider spectral range, higher radiation intensity, and longer lifetime. Inmodem spectrophotometers the exchange between the UV and VIS proceeds automatically.The increasing use of lasers as high intensity sources of monochromatic radiation isobserved.
2. Monochromator
The principal element of the spectrophotometer is the monochromator which serves fordispersion of the radiation emitted by the source and isolation of a beam of monochromaticradiation of definite wavelength. The monochromator comprises a system of slits, acollimator, a light-dispersing element, and lenses or mirrors to focus the dispersed radiation.The dispersing system is the essential part of the monochromator. The degree ofmonochromatization is an important feature of the dispersing element.Beams of monochromatic radiation or radiation of wavelength comprised within aspecified narrow range are isolated by means of filters, prisms, or diffraction gratings. Thebeams of radiation of a limited range of wavelength are separated from the continuousspectra by means of properly selected colour filters.
Modem spectrophotometers are equipped with diffraction gratings, whose dispersion isindependent of the kind of material used and the wavelength of radiation applied. Gratings of
1,800 and 2,400 grooves/mm are used for the UV region, and those with 600 and 1,200grooves/mm are applicable for the visible light. The separation of the grooves, denoted as thegrating constant, is the parameter characteristic for the given grating. The high precision offorming the grooves and the regularity of their separations are characteristic for holographic
diffraction gratings having up to 6,000 grooves/mm. The substitution of diffraction gratingsfor prisms enabled researchers to increase the spectral resolution and to extend themeasuring range from 1 to 4 in the absorbance scale.To record a diffraction spectrum in a required wavelength range it is necessary to changethe position of the grating to isolate the beam of a given wavelength. The manual method ofchanging the position, used in former instruments, has been replaced by mechanical systems.
3. Measuring cuvettes/ sample holders
Measuring cuvettes, in which sample solutions are placed, are made of various materialsdepending on the range of radiation used in the measurement. Measurements in the UV areperformed with the use of quartz cuvettes. Synthetic quartz, which is less contaminated withtraces of metals, has better optical properties. Measurements in the VIS range are made usingquartz, glass, or plastic cuvettes.The cuvette should provide maximum transmission of radiation and definite, preciselyknown thickness of the light-absorbing layer. Cuvettes of different thicknesses within therange 5 ~tm - 10 cm are produced. Small cuvettes capable of accepting samples of volumesdown to 100 ~tl are also available. Small volume cuvettes that enable multiple passage of thebeam of radiation are of special interest.The cuvette material should be resistant to the action of chemicals. The cuvettes areplaced in measuring chambers in special holders that provide accurate and reproduciblelocation of the sample in the path of the radiation beam.Cuvettes of special design are used for measurements over wide ranges of temperatureand pressure or under conditions of permanent flow.
4. Detectors
After traversing the measuring cuvette the radiation impinges on the detector. The roleof the detector is to convert the energy of the incoming electromagnetic radiation intoelectrical energy. The signal transformation should be linear, which means that the electricalsignal generated should be proportional to the optical signal received. This condition issuccessfully fulfilled by photocells, photomultipliers, photoresistors and photodiodes.
The operation of photocells and photomultipliers is based on the external photoelectriceffect. Photons impinging on the surface of a photosensitive cathode (photocathode) knockout electrons which are then accelerated in the electrical field between the cathode and theanode and give rise to electric current in the outer circuit. The spectral sensitivity of aphotocell depends on the material of the photocathode. The photocathode usually consists ofthree layers: a conductive layer (made, e.g., of silver), a semiconductive layer (bimetallic oroxide layer) and a thin absorptive surface layer (a metal from the alkali metal group, usuallyCs). A photocathode of the composition, Ag, Cs-Sb alloy, Cs (blue photocell), isphotosensitive in the wavelength range above 650 nm; for longer wavelengths the redphotocell with Ag, Cs-O-Cs, Cs is used. The response time of the photocell (the timeconstant) is of the order of 10 -s s.
Photomultipliers are equipped with several supplementary diodes (dynodes) to which theelectrons emitted from the photocathode are directed. The electrons impinging on thedynodes give rise to the emission of secondary electrons from the successive dynodes andthey thus amplify the signal generated by a factor of up to 108.
In the photoresistors and photodiodes use is made of the internal photoelectricphenomenon and of specific properties of semiconducting materials. Photons impinging onthe photosensitive element generate an electrical current, which flows through thephotoconductor and is amplified by the effect of a small applied voltage. The increase of thecurrent intensity is proportional to the intensity of photons that strike the photosensitiveelement.The microcrystalline layer of lead(H) sulphide deposited on a dielectric (glass or quartz)plate may serve as an example of a photoresistor applied in the wavelength range above 700nm. Photodiodes are made of two or three layers of semiconducting materials containingsuitable admixtures. Silicon photodiodes are used in the UV-VIS range. Modern spectrophotometers are equipped with multichannel detecting devices thatcontain a large number of photodiodes (a photodiode array) and enable simultaneous
detection over the whole range of the spectrum. Details of the design and the advantages ofusing such detectors in spectrophotometric measurements have been presented .
5. Data recording and processing
The application of microprocessors and the rapid development of computer techniqueshas made it possible to automate the analytical operations from the step of sampling up tofull processing of the data obtained. In modern spectrophotometers, microprocessors areapplied to control many operations that were formerly operated manually.The functions now realized by microprocessors include the control of the optical system(lamp and analytical wavelength selection), selection of the kind of data collected (e.g.,absorbance, concentration), zero-adjustment, autocalibration and control of measurement parameters. The microprocessor determines the equation of the regression curve andprovides statistical processing of the results. It can also be programmed to measure theabsorbance, the % transmittance at a selected wavelength, or the concentration based on therelationship (linear or non-linear) established between the measured absorbance and theconcentration.
The advanced spectrophotometers are coupled with computers that facilitate therecording of results and the processing of the data obtained. Appropriate software enablesthe presentation of results on the display, smoothing of the obtained spectrum, calculation ofpeak heights with respect to the base-line, and mathematical processing of the results thatprovides the possibility of, e.g., resolving signals owing to individual components of the sample analysed. The development ofthe computer techniques has facilitated the identification of the structures of chemicalcompounds by enabling quick and easy access to catalogues of UV-VIS spectra.
The data recorded and the results obtained can be stored in the computer memory. Thisgives the possibility of comparing the obtained results and evaluating their quality by rapidcomparison with greater numbers of data. A critical evaluation of the obtained results alwaysremains the task of the analyst.
Spectrophotometric technique
If the value of the molar absorptivity, e, for the wavelength used in measurement ofabsorbance of the given system is known, it is possible to determine directly theconcentration of the analyte by means of an equation based on Beer's law. The value of e isdetermined from the measurement of absorbance of several solutions containing preciselyknown amounts of the analyte under conditions identical to those used in the measurement ofthe sample solution.
In analytical practice, the concentration of the given analyte is, in most cases, determinedby the standard curve technique. The technique is based on the determination of therelationship between the absorbance and the analyte concentration under the measuringconditions. The relationship is given in terms of the regression equation, or graphically in theform of a standard curve. For systems that obey Beer's law this curve is a straight line.
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