Ultraviolet and Absorption Methods: Theory

THEORY

1.  ELECTROMAGNETIC SPECTRUM

It has been established beyond any reasonable doubt that the absorption and the emission of energy in the electromagnetic spectrum take place in distinct separate pockets or photons. The relationship

existing between the energy of a photon and the frequency matching its propagation may be expressed as follows :

                   E = hν         ...(a)

where,         E = Energy (in ergs),

v= Frequency (in cycles sec^{– 1}), and

h = Universal constant termed as Planck’s constant (6.6256 × 10 ^{– 27} erg sec).

However, the relationship between wavelength and frequency may be expressed as follows :

ν = c/λ ...(b)

where, λ = Wavelength (in cms),

c = Velocity of propagation of radiant energy in vacuum (which is nothing but the speed of light in vacuum ; and is equivalent to 2.9979 × 10 ¹⁰ cm sec^{– 1}).

The radiant power of a beam is designated by its intensity of radiation, which in turn is directly proportional to the number of photons per second that are propagated in the beam.

Monochromatic Beam : A beam that carries radiation of only one distinctly separate wave length is known as monochromatic.

Polychromatic or Heterochromatic : A beam that carries radiation of several wavelengths is termed as polychromatic or heterochromatic.

2.  SCHEMATIC REPRESENTATION OF ELECTROMAGNETIC SPECTRUM

Figure 21.1, provides a schematic representation of electromagnetic spectrum, whereby the beam of a white light from an incandescent solid (e.g., the filament of an electric bulb consisting of numerous separate waves of different wavelengths) is passed through a prism thereby giving rise to a continuous spectrum wherein each colour corresponds to waves of a particular individual wavelength.

A few salient points from Figure 21.1 are enumerated below :

(a) The visible spectrum constitutes a small portion of the complete electromagnetic radiation spec-trum that extends from the ultra-short wave gamma rays at one end to that of the radio-waves at the other (400-700 nm),

(b) The wave length scale is nonlinear,

(c) γ-Rays Region : Mossbauer Spectroscopy (due to absorption) and γ-Ray Spectroscopy (due to emission) are used as analytical means.

(d) Inner-shell Electrons : X-Ray absorption spectroscopy (due to absorption) and X-Ray Fluorescence spectroscopy (XRF) (due to emission) are employed as analytical means.

(e) From Vacuum-UV to Infra-Red Region : UV-VIS, IR-spectroscopy, spectrophotometry, atomic absorption spectroscopy (AAS) (due to absorption) and atomic emission spectroscopy (AES, ESS, ICP) ; atomic fluorescence spectroscopy (AFS) (due to emission) are used as analytical techniques.

(f) Microwave Region : Microwave spectroscopy and electron spin resonance (ESR) (due to absorption) are employed as analytical methods.

(g) Radiowave Region : Nuclear Magnetic Resonance (NMR) (due to absorption) is used as analytical method.

3.  MOLAR ABSORPTIVITY

Usually, a molecule exists in the state of lowest energy the ground state. However, absorption of light of the right frequency (in the UV-region) raises a molecule to an excited state i.e., a state of higher energy. Considering the example for ethylene two situations arise, namely :

(a) Ground State : Here, both π electrons are in the π orbital. This configuration is designated as π², where the superscript represents the number of electrons in that orbital.

(b) Excited State : Here, an electron is in the π orbital while the other in the π* orbital (having an opposite spin). Thus, the resulting configuration ππ* is obviously less stable due to the fact that :

(i) only one electron helps to hold the atom together, and

(ii) the other electron tends to force them apart.

The molar absorptivity is mostly controlled by two vital factors, namely :

(i)          polarity of the excited state, and (ii) probability of the electronic transition. So as to materialize an interaction, a photon should evidently strike a molecule very closely within the space of the molecular di-mensions. The probability of the electronic transition, designated as ‘g’, shall be responsible for the target hits that may ultimately lead to absorption. However, the molar absorptivity may be expressed as follows :

                   .......................(c)

where, N_A = Avogadro Number,

A = Cross-sectional target area*

1/3 = Statistical factor (to permit random orientation),

g = Probability of the electronic transition

By inserting numerical constants and integration Eq. (c) we have :

log (Po/P) bC = ∈ = (0.87 × 10 ²⁰) g A                           ...(d)

where, ∈ = Molar absorptivity

Absorption with ∈ > 10⁴ is considered high-intensity absorption.

4. LAWS OF PHOTOMETRY

The ‘Laws of Protometry’ has been discussed.

5.  SPECTRAL PRESENTATION

Absorption spectra may be presented in a number of fashions as depicted in Figure 21.2, namely :

(a) Wavelength Vs Absorbance,

(b) Wavelength Vs Molar Absorptivity, and

(c) Wavelength Vs Transmittance.

A few important features related to spectral presentation are enumerated below :

(a)       In order to simplify the conversion of spectra in qualitative identification the spectral data should be plotted either as log A or as log ∈ Vs wavelength, thereby giving rise to the following expres-sion :

log A = log ∈ + log b + log c   ...(e)

where,    b = Cell-length, and  

c = Sample concentration.      

From Eq. (e), one may observe that the resulting curve is independent of both cell-length and sample concentration,

(b) The identity and nonconformity of sample may be ascertained by simply carrying out the compari-son of spectral presentation both up or down the ordinate scale,

(c) In order to obtain both reproducible and fairly consistent accurate plot the ordinate in absorbance values must be plotted on graph paper having 1 mm equivalent to 0.005 absorbance,

(d) Most importantly all relevant informations pertaining to : solvent employed, concentrations used, the band pass and ultimately the Model/Make of the Spectrophotometer,

(e) Choice of Solvents : For instance :

Water-common solvent for a number of inorganic substances,

Ethanol (96% w/v)-good choice as fairly polar solvent,

Cyclohexane-common solvent for a number of aromatic compounds.

6.  STRUCTURAL FEATURES

While discussing the structural features special emphasis shall be laid only to those molecules that are capable of absorption within the wavelength region from 185 to 800 mµ.

A few salient structural features are enumerated below :

(i) Compounds having single bonds involving σ-valency electrons usually display absorption spectra below 150 mµ. Such spectra will be observed only in interaction with other types.

(ii) Excitation help in promoting a p-orbital electron into an antibonding σ orbit thereby giving rise to an n → σ* transition, for example : ethers, sulphides, amines, and alkyl halides.

(iii) Unshared p-electrons exist besides σ-electrons in saturated compounds having covalent bonds and heteroatoms, for instance : N, S, O, Cl, Br, I,

(iv) Unsaturated compounds give rise to the absorption spectra by the displacement of π-electrons.

(v) Molecules that have single chromophores (i.e., absorbing groups)-normally undergo transitions almost very close to their respective wavelengths,

(vi) Interestingly, a molecule containing only a single chromophore of a particular species shall absorb light of approximately the same wavelength as that of a molecule having two or more insulated chromophores, however, the intensity of the absorption shall be directly proportional to the number of the latter type of chromophore present in the compound.

Examples : 

(a) meta-orientation about an aromatic ring, and

(b) interposition of a single methylene (= CH₂) moiety.

The above two instances are sufficient to insulate chromophores from each other totally,

(vii) Hyperconjugation—is usually observed when slight interaction takes place with alkyl radicals attached to chromophores.

(vii)In fact, four different types of absorption bands have so far gained cognizance in the spectra of organic compounds, which are namely : K-bands ; R-bands ; B-bands ; and E-bands.

These bands will be discussed briefly here with regard to the structural features.

(a) K-bands : They normally arise from π-π structures and result from π → π* transitions.

These are invariably characterized by high molar absorptivity.

Examples :

(i) A diene : C = C—C = C to C⁺—C = C—C^– ; where K-band is due to the resonance transi-tion,

(ii) Vinyl benzene or acetophenone : i.e., aromatic compounds having chromophoric substitu-tion.

(b) R-bands : They usually arise from n → π* transitions. They seldom display very noticeable results in aliphatic compounds, but marked and pronounced bathochromic shifts (i.e., shifting of absorption towards longer wavelengths—as in extended open-chain-conjugated systems) do take place when—SH, —OH and —NH₂ replace hydrogen atom in unsaturated groups. Thus, R-bands help in the confirmation of a particular structure whereby additional bands are obtained by appropriate modifications in the electronic-structure of the parent compound.

(c) B-bands : These are rather weak-type  of absorption bands. They are characteristic of both heteroatomic and aromatic molecules and may also consist of fine vibrational sub-bands.

(d) E-bands : They usually result from oscillations of electrons in aromatic-ring systems,

(ix) Conjugated Systems :

It is quite evident that the conjugated systems might fail to display the expected conjugated bands due to the following two reasons, namely :

(a) Orbitals of adjacent multiple bonds are at right angles instead of being parallel, and

(b) Resonating dipolar structures cannot be envisaged.

The resulting spectrum may seem to appear as a mere superimposition of the spectra of the indi-vidual chromophoric groups.

Examples : Allene and ketene systems

Polyphenyls (e.g., m-terphenyl)

(x) Steric Hindrance : The attachment of bulky functional entities to ring systems offering steric-hindrance may ultimately prevent the coplanarity of two resonating structures either completely or partially.

However, partial hindrance specifically leads to such characteristic bands pertaining to those parts of conjugated system.

7. ABSORPTION OF RADIANT ENERGY BY MOLECULES

In reality, the molecules are as energetic as the modern teenagers. They invariably rock, roll, twist, jerk, and bend, and if the music is of the right rhythm, choice, and frequency, the electrons within the molecule shall move from the ‘ground state’ to the ‘excited state’.

Explicitly, the total energy in a molecule is the sum of the energies associated with the translational, rotational, vibrational and electronic motions of the molecule/or electrons/or nuclei in the molecule. These four motion-related-energies are briefly explained below :

(a) Transational Energy : It is associated with the motion (velocity) of the molecule as a whole.

(b)       Rotational Energy : It is associated with the overall rotation of the molecule.

(c) Vibrational Energy : It is associated with the motion of atoms within the molecule.

(d) Electronic Energy : It is associated with the motion of electrons arounds the nuclei.

Electrons generally found in the conjugated double bonds invariably give rise to spectra in the UV and visible regions of the electromagnetic spectrum.

It is pertinent to mention here that an excited electron normally returns to the ground state in about 10^–9 to 10^–8 seconds. Consequently, energy must now be released to compensate for the energy absorbed by the system. In actual practice however, the following three situations arise, namely :

Firstly, if the electron returns directly to the ground state, the net effect would be evolution of heat. Secondly, if the electron returns to the ground state by passing through a second excited state, the net outcome would be release of energy in the form of heat and light.

Thirdly, if a large amount of energy is absorbed by certain substances, bonds may be ruptured and thereby giving rise to altogether new compounds.

For instance : ergosterol on being subjected to UV radiation yields cholecalciferols which are, in fact, altogether new substances.

In general, the changes incurred are usually minimal and for this very reason the UV-spectrophotometry is considered to be a non-destructive method of analysis.

However, the relative energies due to electrons (d), vibration (c), and rotation (b) are more or less in the order of 10,000 : 100 : 1 ; and the total energy for any one state at any material time may be depicted by the following expression :

E_Total = E_Electronic + E_Vibrational + E_Rotational

The diagrammatic representation of the potential energy of a diatomic molecule showing :

(i) Potential energy-nuclear separation curves, and

(ii)       Relationship between electronic transitions and absorption curves ; is illustrated in Figure 21.3.

Explanation of various features in Figures 21.3 :

(i) The mutual forces are also zero when the nuclei are at infinity ; but as the latter come closer to one another, forces of attraction start operating and the potential energy decreases,

(ii) The potential energy records an increase when the nuclei get very close to one another thereby causing repulsion,

(iii) The atoms, therefore, can vibrate about the minimum position RC at the vibrational level 0,

(iv) The electronic configuration of the molecule gives rise to different quantum of energy associated with it which may be indicated and represented by the horizontal lines in Figure 21.3 (0 → 6),

(v) At ambient temperature, the molecule is in the lowest ebb of the vibrational level of the ground state,

(vi) The corresponding electronic transition from the ground state to an excited state, is represented by the upper curve in Figure 21.3,

(vii) Rotational energy variations usually accompany electronic variations, however, they are compara-tively smaller in size and often yield a fine structure superimposed on the electronic-vibrational change,

(viii) The frequency of the absorption bands associated with the transition is put forward by the follow-ing expression :

h_ν = E_{Excited state}  – E_{Ground state}       ...(e)

where, h = Planck’s Constant,

v= Frequency, and E = Energy level.

In reality, their appearance as a pattern comes into being chiefly from transitions to the various vibrational levels of the excited state as shown in Figure 21.3.

8. FACTORS INFLUENCING ABSORPTION OF RADIANT ENERGY

There are various cardinal factors that govern measurement of absorption of radiant energy, namely :

(a) Absorbing groups (or Chromophores),

(b) Solvent effects,

(c) Effect of temperature, and

(d) Inorganic ions.

These vital factors would be discussed briefly with specific examples hereunder :

8.1. Absorbing Groups (or Chromophores)

A ‘chromophore’ is a group which when attached to a saturated hydrocarbon produces a molecule that absorbs a maximum of visible of UV energy at some specific wavelength.

A few typical examples having electronic absorption bands for various representive chromophores are provided in the following Table : 21 : 1 :

8.2. Solvent Effects

The absorption spectrum of a pharmaceutical substance depends partially upon the solvent that has been employed to solubilize the substance. A drug may absorb a miximum of radiant energy at a particular wavelength in one solvent but shall absorb practically little at the same wavelength in another solvent. These apparent changes in spectrum are exclusively due to various characteristic features, namely :

(a) Nature of the solvent,

(b) Nature of the absorption band, and

(c) Nature of the solute.

Some salient features of ‘Solvent Effects’ are enumerated below :

(i)          Absorption bands of many substances are relatively sharper and may also exhibit fine structure when measured in solvents of low dipole moment,

(ii) Interactions of solvent-solute are found to be much stronger in such substances where strong dipole forces are involved,                                                              

(ii)       Solvent effects do help in reorganizing electronic transitions of the type n—π* that essentially involve the nonbonding electrons of nitrogen and oxygen,

(iv) The nonbonding electrons of nitrogen and oxygen usually interact with polar solvents that ultimately give rise to a characteristic shift to shorter wavelengths.Example : The spectrum of Iodine in a nonpolar solvent like CHCl3 is found to be distinctly different (purple to the naked eye) when the same is compared in a polar solvent such as C2H5OH (brownish to the naked eye) in Figure 21.4.

(v) A  spectrum  normally  shows  appreciable changes with varying pH when an ionizable moiety is present in the molecule and thereby constitutes part of the chromophore structure.

8.3 Effect of Temperature

·              Low temperature offfer sharper absorption bands of many pharmaceutical substances than at room temperature,

·              Vibrational resolutions are definitely well-defined at low temperatures because of the following two reasons, namely :

(a) Fewer vibrational levels are occupied, and

(b) Degree of solute-solvent interaction is minimised,

·              Samples in highly rigid or viscous media (e.g., glass) is examined frequently in phosphorescence methods and also in some fluorescence methods.

8.4 Inorganic Ions

The ‘chromophoric entities’ present in the inorganic compounds are of two types, namely :

(a) Involving several atoms : such as : permanganate (MnO₄^–) and dichromate (Cr₂O₇^–) moieties, and

(b) Involving single atoms : Those having incomplete outer d-electron shells where closely spaced, unoccupied energy levels are available in abundance for instance : coordination compounds with

Rare Earths : e.g., Be, Sr, Ra, and Transition Elements : Cr, Mn, Ni, Pt, Ag, Pd, Cd, Hg, Au,

It is worth while to note that the absorption spectra for these elements are caused due to a charge-transfer-process whereby an electron gets transferred form one part of the ion to another.

Interestingly, inclusion of readily polarizable atoms do exert an effect likewise to lengthening a con-jugated chain. Examples :