Pabitra Kumar Mani; Assoc. Prof.; BCKV;

Class - 16

Spectroscopy

ACSS-501ACSS-501

Spectrophotometry property is mainly concerned with the following regions of the spectrum:

ultraviolet, 185-400 nm; visible 400-760 nm; and infrared, 0.76- 15 µm.

Colorimetry is concerned with the visible region of the spectrum.

EMR behaves as a particle and as a wave (the dual nature of light); and the wavelength of such a particle, a photon, is related to energy by the equation

where h is the Planck’s constant (6.63 X10−34 Js), c is the speed of light in vacuum (2.998 X108 ms−1), E is the energy of the photon and is the wavelength in nm.

Plane-polarized electromagnetic radiation showing the electric field, and the direction of propagation.

Electric field component of plane-polarized electromagnetic radiation.

We can illustrate the process by way of a simple calculation using Eqn. Assume that a photon of energy 8.254 X10−19 joules interacts with the electron cloud of a particular molecule and causes promotion of an electron from the ground to an excited state. This is illustrated in Fig.The diff. in the molecular energy levels, E2 −E1, in the molecule corresponds exactly to the photon energy.

Converting this energy into wavelength reveals that this excitation process occurred at a wavelength of 240 nm. This is an electronic transition and is in the ultraviolet part of the spectrum. If this were the only transition that the molecule was capable of undergoing, it would yield a sharp single spectral line.

Photon capture by a molecule.

The origin of spectra, absorption of radiation by atoms, ions & molecules

When a photon interacts with an electron cloud of matter, it does so in a specific and discrete manner. This is in contrast to the physical attenuation of energy by a filter that is continuous. These discrete absorption processes are quantised and the energies associated with them relate to the type of transition involved

Molecular spectra are not solely derived from single electronic transitions between the ground and excited states. Quantised transitions do occur between vibrational states within each electronic state and between rotational sublevels. As we have seen, the wavelength of each absorption is dependent on the diff.between the energy levels. Some transitions require less energy and consequently appear at longer wavelengths.

The electromagnetic spectrum showing the colors of the visible spectrum.

THE ORIGINS OF ABSORPTION SPECTRA

The absorption of radiation is due to the fact that molecules contain electrons which can be raised to higher energy levels by the absorption of energy. The requisite energy can in some cases be supplied by radiation of visible wavelengths, thus producing an absorption spectrum in the visible region, but in other cases the higher energy associated with UV radiatn is reqd.

In addition to the change in electronic energy which follows the absorption of radiation, there are also changes associated with variation in the vibrational energy of the atoms in the molecule and changes in rotational energy. This means that many different amounts of energy will be absorbed depending upon the varying vibrational levels to which the electrons may be raised and the result is that we do not observe a sharp absorption line, but instead a comparatively broad absorption band.

Electrons in a molecule can be classified in 3 different types.

1. Electrons in a single covalent bond (σ-bond): these are tightly bound and radiation of high energy (short wavelength) is required to excite them.

2. Electrons attached to atoms such as chlorine, oxygen or nitrogen as 'lonepairs': these non-bonding electrons can be excited at a lower energy (longer wavelength) than tightly bound bonding electrons.

3. Electrons in double or triple bonds (π-orbitals) which can be excited relatively easily. In molecules containing a series of alternating double bonds (conjugated systems), the π-electrons are delocalised and require less energy for excitation so that the absorption rises to higher wavelengths.

A diagram showing the various kinds of electronic excitation that may occur in organic molecules is shown above. Of the six transitions outlined, only the two lowest energy ones (left-most, colored blue) are achieved by the energies available in the 200 to 800 nm spectrum. As a rule, energetically favored electron promotion will be from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO), and the resulting species is called an excited state

Attenuation of a beam of radiation by an absorbing solution.

Reflection and scattering losses with a soln contained in a typical glass cell.

• Transmission• Reflection • Refraction• Scattering• Luminescence• Chiro-optical phenomena

When light impinges on a cuvette containing our molecule of interest (solute) in a soln (solvent), other optical processes do or can occur:

THEORY OF SPECTROPHOTOMETRY AND COLORIMETRY

When light (monochromatic or heterogeneous) falls upon a homogeneous medium, a portion of the incident light is reflected, a portion is absorbed within the medium, and the remainder is transmitted. If the intensity of the incident light is expressed by Io, that of the absorbed light by Ia, that of the transmitted light by It and that of the reflected light by Ir , then:

For air-glass interfaces arising from the use of glass cells, it may be stated that about 4 per cent of the incident light is reflected. Ir is usually eliminated by the use of a control, such as a comparison cell, hence:

Lambert's Law. This law States that “when monochromatic light passes througha transparent medium, the rate of decrease in intensity with the thickness of the medium is proportional to the intensity of the light”. (= the intensity of the emitted light decreases exponentially as the thickness of the absorbing medium increases arithmetically). We may express the law by the differential eqn:

I is the intensity of the incident light of wavelength λ, l (ell) is the thickness of the medium, and k is a proportionality factor. Integrating equation (1) and putting I = Io when l = 0, we obtain:

or, stated in other terms,

Eqn........ 1

Eqn..... 2

where Io is the intensity of the incident light falling upon an absorbing medium of thickness(l ), It is the intensity of the transmitted light, and k is a constant for the wavelength and the absorbing medium used. By changing from natural to common logarithms we obtain:

where K = k / 2.3026 and is usually termed the absorption coefficient. The absorption coefficient is generally defined as the reciprocal of the thickness (l cm) required to reduce the light to 1/10 of its intensity. This follows from equation (3), since:

Eqn....... 3

The ratio It/Io is the fraction of the incident light transmitted by a thickness (l)

of the medium and is termed the transmittance T.

Its reciprocal Io/It is the opacity, and the absorbance A of the medium

(formerly called the optical density ) is given by:*

Beer's Law.The intensity of a beam of monochromatic light decreases exponentially as the concn. of the absorbing substance increases arithmetically. This may be written in the form:

where c is the concentration, and k/ and K/ are constants. Combining equations (3) and (4), we have:

This is the fundamental equation of colorimetry and spectrophotometry, and is often spoken of as the Beer-Lambert Law

Eqn......4

The value of a will clearly depend upon the method of expression of the

concn. If c is expressed in mole L-1 and l in cm then a is given the symbol

E and is called the molar absorption coefficient or molar absorptivity (formerly the molar extinction coefficient).

It will be apparent that there is a relationship between the absorbance

A, the transmittance T, and the molar absorption coefficient, since:

Application of Beer's Law.

Consider the case of two solutions of a coloured substance with concn c1and c2. These are placed in an instrument in which the thickness of the layers can be altered and measured easily, and which also allows a comparison of the transmitted light . When the two layers have the same colour intensity:

Here l1, and l2, are the lengths of the columns of solns with concn. cl and c2, respectively when the system is optically balanced. Hence, under these conditions, and when Beer's law holds:

A colorimeter can, therefore, be employed in a dual capacity: (a)to investigate the validity of Beer's Law by varying c1, and c2, and noting whether equation applies, and(b) for the determination of an unknown concn c2 of a coloured solution by comparison with a solution of known concn c1.It must be emphasised that eqn (5) is valid only if Beer's Law is obeyed over the concn. range employed and the instrument has no optical defects

Eqn. …… (5)

• Linearity is observed in the low concentration ranges(<0.01), but may not be at higher concentrations.

• This deviation at higher concentrations is due to intermolecular interactions.

• As the concentration increases, the strength of interaction increases and causes deviations from linearity.

• The absorptivity not really constant and independent of concentration but e is related to the refractive index (h ) of the solution :

• At low concentrations the refractive index is essentially constant-so e constant and linearity is observed.

Real Limitations

2. Refractive index deviation A = bC [ n / (n2 + 2)2] where n is refractive index

3. Instrumental deviation ; difficult to select single wavelength beam

max

The effect of polychromatic radiation on Beer’s law.

Slope of the best straight line through the data points in thecalibration plot is 1.65. Plot intercept is 0.008.

Equation of straight line:

To find an unknown concentration for a sample, subtract theintercept from the absorbance reading and divide the result by the slope. Here the equation would be

Compilation of spectrophotometric nomenclature

aquated Cu(II) = pale blue whereas when it is complexed with NH3 it is a darker blue.

Crystal Field Theory: In the absence of an external electrical or magnetic field, the energies of the 5d orbitals are identical. When a complex forms between the metal ion and water (or some other ligand), the d-orbitals are no longer degenerate (not the same energy). Therefore, absorption of radiation of energy involves a transition from one of the lower energy to one of the higher energy d-orbitals.

Many complexes of metals with organic ligands absorb in the visible part of the spectrum and are important in quantitative analysis. The colours arise from(i)d d transitions within the metal ion (these usually produce absorptions of low intensity) and (ii) n π* and π π* transitions within the ligand.

(iii)Another type of transition referred to as ‘charge-transfer’ may also be operative in which an electron is transferred between an orbital in the ligand and an unfilled orbital of the metal or vice versa. These give rise to more intense absorption bands which are of analytical importance.

TURBIDITIMETRY

Nephelometry & Turbidity: Units• Basic unit of turbidity is Nephelometric Turbidity Unit (NTU)• NTU has replaced previous unit of ‘Jackson candle turbidity

units’ (JTU)• Formazin suspensions are used as a standard for calibration• Formazin particles have uniform size and shape

PhototubeMeasuringScattered Light

Small amounts of some insoluble compounds may be prepared in a state of aggregation such that moderately stable suspensions are obtained. The optical properties of each suspension will vary with the concn. of the dispersed phase. When Iight is passed through the suspension, part of the incident radiant energy is dissipated by absorption, reflection, and refraction, while the remainder is transmitted.

Measurement of the intensity of the transmitted light as a function of the concn. of the dispersed phase is the basis of turbidimetric analysis.

When the suspension is viewed at right angles to the direction of the incident light the system appears opalescent due to the reflection of light from the particles of the suspension (Tyndall effect). The light is reflected irregularly and diffusely, and consequently the term 'scattered light' is used to account for this opalescence or cloudiness.

The measurement of the intensity of the scattered light (at right angles to the direction of the incident light) as a function of the concn of the dispersed phase is the basis of nephelometric analysis

The construction of calibration curves is recommended in nephelometricand turbidimetric determinations, since the relationship between the opticalproperties of the suspension and the concn of the disperse phase is, at best, semi-empirical. If the cloudiness or turbidity is to be reproducible, the utmost care must be taken in its preparation. The precipitate must be veryfine, so as not to settle rapidly.

The following conditions should be carefully controlled :

1. the concns. of the two ions which combine to produce the precipitateas well as the ratio of the concns in the solns which are mixed;

2. the amounts of other salts and substances present, especially protective colloids (gelatin, gum arabic, dextrin, etc.);3. the temperature.

In Turbidimetry, the amount of light passing through a solution is measured. The higher the turbidity, the smaller the quantity of light transmitted (i.e. more light is absorbed). Any spectrophotometer or photometer can be used as a turbidimeter, without modification. Since property concerns visible light the measurement is commonly carried out at 420 nm.

In nephelometry, on the other hand, the detecting cell is placed at right angles to the light source, to measure light scattered by particles. The intensity of the scattered light serves as a measure of the turbidity. The instrument is called a ‘Nephelometer’ or a ‘Nephelometric Turbidimeter’. A spectrophotometer can be used, however, a special attachment is required for nephelometry.

Significance of Turbidity Measurements

Turbidity is an important factor for many uses of water:

• Potability: The turbidity is connected with potability of water directly, indirectly or aesthetically. High turbidity is generally associated with polluted water. Turbidity meast. is very imp. in deciding the quality of water samples.

In fact, the turbidity is taken as an indicator for efficiency and performance of coagulation, flocculation, sedimentation and filtration processes of water treatment plants.• Use in industry: The turbidity of water being used in industry is also an important consideration in quality control for industries manufacturing chemicals, cosmetics, food products, beverages, etc.• Ecology: many aquatic animals and plants need clear water to survive. Turbidity is therefore damaging to ecology.

Types of the electronic transition

Transition Wavelength (nm) log Examples

* < 200 >3 Saturated hydrocarbon

n * 160~260 2~3 Alkenes, alkynes, aromatics

E * 200~500 ~ 4 H2O,CH3OH, CH3Cl CH3NH2

n * 250-600 1~2 Carbonyl, nitro, nitrate, carboxyl

(note) forbidden transition ; * , *

James D. Ingle, Jr., Stanley R. Crouch, Spectrochemical Analysis, Prentice-Hall, NJ,1988, p. 335.

*

*

n

ELUMO

HOMO

Unoccupied levels (antibonding)

bonding

non-bonding

Frontierorbital

Occupied level

Characteristics of electronic transitions.

* Transitions

An electron in a bonding s orbital is excited to the corresponding antibonding orbital. The energy required is large. For example, methane (which has only C-H bonds, and can only undergo * transitions) shows an absorbance maximum at 125 nm. Absorption maxima due to * transitions are not seen in typical UV-Vis. spectra (200 - 700 nm)

n * Transitions

Saturated compounds containing atoms with lone pairs (non-bonding electrons) are capable of n * transitions. These transitions usually need less energy than * transitions. They can be initiated by light whose wavelength is in the range 150 - 250 nm. The number of organic functional groups with n * peaks in the UV region is small.

n * and * Transitions

Most absorption spectroscopy of organic compounds is based on transitions of n or electrons to the * excited state. This is because the absorption peaks for these transitions fall in an experimentally convenient region of the spectrum (200 - 700 nm). These transitions need an unsaturated group in the molecule to provide the p electrons.

Molar absorbtivities from n * transitions are relatively low, and range from 10 to100 L mol-1 cm-1 . * transitions normally give molar absorbtivities between 1000 and 10,000 L mol-1 cm-1 .

The solvent in which the absorbing species is dissolved also has an effect on the spectrum of the species. Peaks resulting from n * transitions are shifted to shorter wavelengths (blue shift) with increasing solvent polarity. This arises from increased solvation of the lone pair, which lowers the energy of the n orbital. Often (but not always), the reverse (i.e. red shift) is seen for * transitions. This is caused by attractive polarisation forces between the solvent and the absorber, which lower the energy levels of both the excited and unexcited states. This effect is greater for the excited state, and so the energy difference between the excited and unexcited states is slightly reduced - resulting in a small red shift. This effect also influences n * transitions but is overshadowed by the blue shift resulting from solvation of lone pairs.

The part of the molecule involved in these absorption processes is known as the chromophore. The spectra arising from different chromophores are the ‘fingerprints’ that allow us to identify and quantify specific molecules.

These chromophores are the basic building blocks of spectra and are associated with molecular structure and the types of transition between molecular orbitals. There are three types of ground state molecular orbitals—sigma (σ) bonding, pi (π) bonding and nonbonding (n)—and two types of excited state— sigma star (σ*) antibonding, and pistar (π*) antibonding—from which transitions are observed in the UV region. (Fig. 7).

Absorbing species containing p, s, and n electrons

Absorption of ultraviolet and visible radiation in organic molecules is restricted to certain functional groups (chromophores) that contain valence electrons of low excitation energy. The spectrum of a molecule containing these chromophores is complex. This is because the superposition of rotational and vibrational transitions on the electronic transitions gives a combination of overlapping lines. This appears as a continuous absorption band.

Possible electronic transitions of , , and n electrons are;

we have only considered the absorption of energy by electronic and molecular transitions. When we make spectral measurements, it is necessary to consider other optical processes. This is particularly imp. for soln. spectrophotometry.

Idealised energy transitions for a diatomic molecule.

Molecular spectra are not solely derived from single electronic transitions betweenthe ground and excited states. Quantised transitions do occur between vibrationalstates within each electronic state and between rotational sublevels. As we haveseen, the wavelength of each absorption is dependent on the difference between the energy levels. Some transitions require less energy and consequently appear at longer wavelengths.

Considering a simplified model of a diatomic molecule, we might expect that our spectra would be derived from the three transitions between the ground and first excited state ( Fig.). In practice, of course, even the simplest diatomic molecules have many energy levels resulting in complex spectra.