AN UPDATE ON FUNDAMENTALS OF UV- VISIBLE SPECTROSCOPY

ABOUT AUTHORS:
Prashanta Kr. Deb^1*, Kaushik Nath Bhoumik²,
¹Dept. of Pharmacy, Tripura University; Suryamaninagar –799022, Agartala, Tripura (W), India.
²Regional Institute of Pharmaceutical Science & Technology, Agartala–799005, Tripura (W), India.
^*shaandeb2010@gmail.com

INTRODUCTION
Spectroscopy is the measurement and interpretation of Electromagnetic radiation (EMR) absorbed or emitted when the molecules or atoms or ions of a sample move from one energy state to another energy state. This change may be ground state to exited state or vice-versa. EMR is made up of discrete particles called photons. EMR has got both wave characteristic as well as particle characteristics. This means that it can travel in vacuum also.

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The energy of an EMR can be given by the following equation…

E=hν

Where,
E= Energy of radiation
h= Plank’s constant (6.624 x 10^{-34 jSec)}
ν= frequency of radiation

Frequency = c/λ or velocity of light in vacuum/ wavelength

Hence, E = hc/ λ = hcv

Where, v = wave number

Therefore the energy of a radiation depends upon frequency and wavelength of the radiation. Frequency is defined as the number of complete wavelength units passing through a given point in unit time. Frequency is measured in Hz or cps.

Wavelength is the distance between two successive maxima or minima or distance between two successive through or peas. It can be measured in meters, cm, mm, nm or Å.

Electromagnetic Spectrum
The electromagnetic spectrum is the range of all possible frequencies of electromagnetic radiation. The "electromagnetic spectrum" of an object is the characteristic distribution of electromagnetic radiation emitted or absorbed by that particular object. The electromagnetic spectrum extends from low frequencies used for modern radio communication to gamma radiation at the short-wavelength (high-frequency) end, thereby covering wavelengths from thousands of kilometers down to a fraction of the size of an atom. It is for this reason that the electromagnetic spectrum is highly studied for spectroscopic purposes to characterize matter.  The limit for long wavelength is the size of the universe itself, while it is thought that the short wavelength limit is in the vicinity of the Planck length, although in principle the spectrum is infinite and continuous.

Regions of the spectrum
The types of electromagnetic radiation are broadly classified into the following classes.

1. Gamma radiation
2. X-ray radiation
3. Ultraviolet radiation
4. Visible radiation
5. Infrared radiation
6. Microwave radiation
7. Radio waves

Spectrum of Electromagnetic Radiation
Region	Wavelength (Angstroms)	Wavelength (centimeters)	Frequency (Hz)	Energy (eV)
Radio	> 109	> 10	< 3 x 109	< 10-5
Microwave	109 - 106	10 - 0.01	3 x 109 - 3 x 1012	10-5 - 0.01
Infrared	106 - 7000	0.01 - 7 x 10-5	3 x 1012 - 4.3 x 1014	0.01 - 2
Visible	7000 - 4000	7 x 10-5 - 4 x 10-5	4.3 x 1014 - 7.5 x 1014	2 - 3
Ultraviolet	4000 - 10	4 x 10-5 - 10-7	7.5 x 1014 - 3 x 1017	3 - 103
X-Rays	10 - 0.1	10-7 - 10-9	3 x 1017 - 3 x 1019	103 - 105
Gamma Rays	< 0.1	< 10-9	> 3 x 1019	> 105

This classification goes in the increasing order of wavelength, which is characteristic of the type of radiation. While, in general, the classification scheme is accurate, in reality there is often some overlap between neighboring types of electromagnetic energy. The distinction between X-rays and gamma rays is partly based on sources: the photons generated from nuclear decay or other nuclear and sub nuclear/particle process, are always termed gamma rays, whereas X-rays are generated by electronic transitions involving highly energetic inner atomic electrons. In general, nuclear transitions are much more energetic than electronic transitions, so gamma-rays are more energetic than X-rays, but exceptions exist. The convention that EM radiation that is known to come from the nucleus, is always called "gamma ray" radiation is the only convention that is universally respected, however. Many astronomical gamma ray sources (such as gamma ray bursts are known to be too energetic (in both intensity and wavelength) to be of nuclear origin. Quite often, in high energy physics and in medical radiotherapy, very high energy EMR (in the >10 MeV region) which is of higher energy than any nuclear gamma ray, is not referred to as either X-ray or gamma-ray, but instead by the generic term of "high energy photons."

The region of the spectrum in which a particular observed electromagnetic radiation falls, is reference frame-dependent (due to the Doppler shift for light), so EM radiation that one observer would say is in one region of the spectrum could appear to an observer moving at a substantial fraction of the speed of light with respect to the first to be in another part of the spectrum.

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Ultraviolet and Visible Spectroscopy Range
This absorption spectroscopy uses electromagnetic radiations between 190 nm to 800 nm and is divided into the ultraviolet (UV, 190-400 nm) and visible (VIS, 400-800 nm) regions. Since the absorption of ultraviolet or visible radiation by a molecule leads transition among electronic energy levels of the molecule, it is also often called as electronic spectroscopy.

Wave length and Color Relationship
For a particular type of light the wavelength for it will have a characteristic value which will determine the energy of the light. Visible light has wavelengths ranging from 400 nm (violet) to 780 nm (red). The colors we see with our eyes in our day to day lives have wavelengths between these 400-780 nm. The wavelengths at which different colors are observed are shown below:

Violet	400-420 nm
Indigo	420-440 nm
Blue	440-490 nm
Green	490-570 nm
Yellow	570-585 nm
Orange	585-620 nm
Red	620-780 nm

White light is a combination of all the colors of light. Thus if you pass white light through a clear glass prism, it will split up to form the different visible colors of light.

Interaction of EMR with matter and its effects

Region of the spectrum	Main interactions with matter
Radio	Collective oscillation of charge carriers in bulk material (plasma oscillation). An example would be the oscillation of the electrons in an antenna.
Microwave through far infrared	Plasma oscillation, molecular rotation
Near infrared	Molecular vibration, plasma oscillation (in metals only)
Visible	Molecular electron excitation (including pigment molecules found in the human retina), plasma oscillations (in metals only)
Ultraviolet	Excitation of molecular and atomic valence electrons, including ejection of the electrons (photoelectric effect)
X-rays	Excitation and ejection of core atomic electrons, Compton scattering (for low atomic numbers)
Gamma rays	Energetic ejection of core electrons in heavy elements, Compton scattering (for all atomic numbers), excitation of atomic nuclei, including dissociation of nuclei
High-energy gamma rays	Creation of particle-antiparticle pairs. At very high energies a single photon can create a shower of high-energy particles and antiparticles upon interaction with matter.
Technique	Type of Electromagnetic Radiation	Type of Matter Observed	Type of Interaction
Ultraviolet-Visible Spectroscopy (UV-Vis Spectroscopy)	Ultraviolet and Visible spectrum	Electrons and electronic excitations	Absorbance
Infrared Spectroscopy (IR Spectroscopy)	Infrared spectrum	Molecular rotations, molecular vibrations, bonds between atoms	Absorbance (or transmittance)
Fluorescence Spectroscopy	Ultraviolet and Visible spectrum	Electrons and electronic excitations	Emission
Nuclear Magnetic Resonance Spectroscopy (NMR Spectroscopy)	Radio waves	Nucleus	Resonance or Coherance
Flame emission spectroscopy (Flame photometry)	Ultraviolet and Visible spectrum	Atoms	Emission
X-Ray Diffraction Crystallography	X-rays	Electron denisities	Diffraction or Scattering
Atomic Absorption and Emission Spectroscopy	Ultraviolet and Visible spectrum	Atoms	Absorption or Emission
Ultraviolet-Visible Spectroscopy (UV-Vis Spectroscopy)	Ultraviolet and Visible spectrum	Electrons and electronic excitations	Absorbance

CHROMOPHORE
The energy of radiation being absorbed during excitation of electrons from ground state to excited state primarily depends on the nuclei that hold the electrons together in a bond. The group of atoms or molecules which are covalently unsaturated containing electrons responsible for the absorption is called chromophore. E.g: C=C, C=O, C=N. Most of the simple un-conjugated chromophores give rise to high energy transitions of little use.

For example, alkanes contain only single bonds (C-C, C-H etc.) with only possible σ -- σ^* type electronic transitions. These transitions absorb radiations shorter than wavelengths that are experimentally accessible in usually available spectrophotometers. In saturated molecules with heteroatom bearing non-bonding pairs of electrons, n – σ^*transitions become available. These are also high energy transitions. In unsaturated compounds, π --- π* transitions become possible. Alkenes and alkynes absorb ~ 170 nm but the presence of substituents significantly affects their position. The carbonyl compounds and imines can also undergo n ---π* transitions in addition to π-- π*. Amongst these, the most studied transitions are n--- π* as these absorb at relatively longer wavelength 280-300 nm. These are low intensity (ε 10-100) transitions.

Why is beta-carotene yellow……..?
Beta-carotene has the sort of delocalization that we've just been looking at, but on a much greater scale with 11 carbon-carbon double bonds conjugated together. The diagram shows the structure of beta-carotene with the alternating double and single bonds.

The more delocalisation there is, the smaller the gap between the highest energy pi bonding orbital and the lowest energy pi anti-bonding orbital. To promote an electron therefore takes less energy in beta-carotene than in the cases we've looked at so far - because the gap between the levels is less. It should be noted that less energy means a lower frequency of light gets absorbed - and that's equivalent to a longer wavelength.

Beta-carotene absorbs throughout the ultra-violet region into the violet - but particularly strongly in the visible region between about 400 and 500 nm with a peak about 470 nm. That’s why it shows colour. These properties like colour of organic compound, chromophoric and auxochromic effects on colour changes and finding what type of transaction is going into the molecules due to E.M.R we can do qualitative and quantitative estimation.

AUXOCHROME
The co-ordinatively saturated or unsaturated substituents that themselves do not absorb ultraviolet radiations but their presence shifts the absorption maximum to longer wavelength are called auxochromes. The substituents like methyl, hydroxyl, alkoxy, halogen, amino group etc. are some examples of auxochromes.

Electronic transitions
The absorption of UV or visible radiation corresponds to the excitation of outer electrons. There are three types of electronic transition which can be considered;

1. Transitions involving p, s, and n electrons
2. Transitions involving charge-transfer electrons
3. Transitions involving d and f electrons (not covered in this Unit)

When an atom or molecule absorbs energy, electrons are promoted from their ground state to an excited state. In a molecule, the atoms can rotate and vibrate with respect to each other. These vibrations and rotations also have discrete energy levels, which can be considered as being packed on top of each electronic level.

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 p, s, and n electrons are;

σ- σ^*  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 σ- σ^* transitions) shows an absorbance maximum at 125 nm. Absorption maxima due to σ- σ^* transitions are not seen in typical UV-Vis. spectra (200 - 700 nm)

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

n--- π* and π --- π*Transitions
Most absorption spectroscopy of organic compounds is based on transitions of n or π electrons to the π*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--- π* transitions are relatively low, and range from 10 to100 L mol^-1 cm^-1. π --- π*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 π --- π*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 π --- π*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--- π* transitions but is overshadowed by the blue shift resulting from solvation of lone pairs.

Allowed transitions:  these are transitions having molar coefficient 104 or more. These are generally designated as π→ π transitions. For example in 1,3-butadiene which exhibits absorption at 217nm has  €max value 21000 represents an allowed transition.

Forbidden transitions: these are transitions for which €max is generally less than 104. E.g: transition of saturated aldehyde showing weak absorption near 290nm and having €max 100 has been a forbidden transition.

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Different bands in UV-visible Spectroscopy

K- Band
The band due to π→ π* transitions in a compound with conjugated π system is usually intense (€max.>10000) and is frequently referred to as the k-band. The examples of the compounds in which k-band appears are butadiene, Mesityl oxide. Benzene itself displays three absorption bands at 184,204 and 256nm and of these the band at 204nm is often designated as k-band.

R- Band
The n→π* transition  in compounds with single chromatographic groups i.e., carbonyl or nitro are forbidden with € value less than 100 are called R bands.
Ex.  Acetone, acroline, methyl vinyl ketone, acet aldehyde, acetophenone.

B- Bands
These bands are observed in aromatic compounds and hetero aromatic compounds. Here B refers to Benzenoid bands.
Ex. Benzene, toluene, acetophenone, benzoic acid, naphthalene, styrene.

E- Bands
Such band originates due to electronic transition in the benzenoid system of the ethylinic part enclosed in cyclic conjugation. Here E refers to Ethylene. These are further classified as E1 and E2. 
Ex. Benzene, naphthalene, anthracene.

Effect of solvent on absorption maxima
Highly pure, non-polar solvents such as saturated hydrocarbons do not interact with solute molecules either in the ground or excited state and the absorption spectrum of a compound in these solvents is similar to the one in a pure gaseous state. However, polar solvents such as water, alcohols etc. may stabilize or destabilize the molecular orbitals of a molecule either in the ground state or in excited state and the spectrum of a compound in these solvents may significantly vary from the one recorded in a hydrocarbon solvent.

π → π^*Transitions
In case of π → π^*transitions, the excited states are more polar than the ground state and the dipole-dipole interactions with solvent molecules lower the energy of the excited state more than that of the ground state. Therefore a polar solvent decreases the energy of π → π^*transition and absorption maximum appears ~10-20 nm red shifted in going from hexane to ethanol solvent.

n→π*Transitions
In case of n→π* transitions, the polar solvents form hydrogen bonds with the ground state of polar molecules more readily than with their excited states. Therefore, in polar solvents the energies of electronic transitions are increased. For example, the figure 5 shows that the absorption maximum of acetone in hexane appears at 279 nm which in water is shifted to 264 nm, with a blue shift of 15 nm.

Choice of Solvent for UV visible region:   Criterion for solvent:
1. A good solvent should not absorb ultraviolet radiation in the same region as the substance whose spectrum is being determined.
2. Usually solvents which do not contain conjugated systems are most suitable for this purpose, although they vary as to the shortest wavelength at which they remain transparent to ultraviolet radiation.
3. The solvents most commonly used are water, 95% ethanol, and n hexane.
4. The ability of a solvent to influence the wavelength of ultraviolet light which will be absorbed.
5. Polar solvents may not form hydrogen bonds as readily with excited states as with ground states of polar solvents. Transitions of the π → π^*type are shifted to shorter wavelengths by polar solvents.
6. On the other hand, in some cases the excited states may form n→π stronger hydrogen bonds than the corresponding ground state. In such cases, a polar solvent would shift absorption to longer wavelength, since the energy of the electronic transition would be decreased. Transitions of the type are shifted to longer wavelengths by polar solvents.

    SOLVENT                                     TRANSPERANCY (nm)

1.     Water	180
2.     Ethanol	220
3.     Hexane	200
4.     Cyclohexane	200
5.     Benzene	280
6.     Carbon tetrachloride	260
7.     Acetone	210
8.     Dioxane	330
9.     Cellosolve	320
10.   Diethyl ether	210

Theory of UV-Visible Spectroscopy
Most organic molecules and functional groups are transparent in the portions of the electromagnetic spectrum that we call the ultraviolet (UV) and visible (VIS) regions - that is, the regions where wavelengths range from 190 nm to 800 nm. Consequently, absorption spectroscopy is of limited utility in this range of wavelengths. However, in some cases we can derive useful information from these regions of the spectrum. That information, when combined with the detail provided by infrared and nuclear magnetic resonance (NMR) spectra, can lead to valuable structural proposals.

Absorption of Photon
Absorption of light energy involves a transfer of energy from the radiation field to an absorber resulting in transition of the absorber from a lower to a higher energy level. Absorption of the light may lead to excitation of electrons from one energy level to another (subject to other conditions). The absorption of light gives rise to complementary color in the absorbing substance. In a molecule the energy spacing between ground and excited states is determined by the bonding strength between the nuclei and the electrons. Therefore, the characteristic energy of a transition and the wavelength of radiation absorbed are properties of a group of atoms. The group of atoms causing such absorption is called a chromophore. Different substances absorb different wavelengths of light. Therefore, the wavelength of maximum absorption by a substance is one of the characteristic properties of that substance. The most probable transition often occurs from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO). The following figures schematically show some such transitions.

The absorption of light is usually experimentally measured in terms of transmittance (T) or absorbance (A). Transmittance is defined as T = I / I₀, where I is the light intensity after it passes through the sample and I0 is the initial light intensity. The relation between A and T is:

A=-log₁₀T=-log₁₀ (I/I₀)

Laws Involved in Absorption of Radiation
The two laws related to the absorption of radiation are:

1. Beer’s Law(related to Conc. Of absorbing species)
2. Lambert’s law(related to thickness/path length of absorbing species)
3. These two laws are applicable under the following condition:

I= I_a + I_t

Where, 

I= Intensity of incident light,
I_a= Intensity of absorbed light,
I_t = Intensity of transmitted light and

Lambert’s law States that“When a beam of light is allowed to pass through a transparent medium, the rate of decrease of intensity with the thickness of medium is directly proportional to the intensity of light.”

Mathematically,the Lambert’s law may be expressed as follows.

- dI / dt  α I

-dI / dt = KI          . . . . . . . . . .(1)

Where
I = intensity of incident light
t = thickness of the medium
K= proportionality constant

By integration of equation (1), and putting I=I₀ when t=0,

I₀/ I_t = kt    or    I_t= I₀ e^-kt

Where,I₀ = intensity of incident light
I_t  = intensity of transmitted light
k = constant which depends upon wavelength and absorbing medium used.

By changing the above equation from natural log, we get,

I_t = I₀ e^-Kt          . . . . . . . . . .(2)

WhereK = k/ 2.303

           So,     I_t = I₀ e^{-0.4343 kt}   

                    I_t = I₀10^-Kt          . . . . . . . . . .(3)

Beer’s law may be stated as follows:   
“Intensity of incident light decreases exponentially as the concentration of absorbing medium increases arithmetically.”

The above sentence is very similar to Lambert’s law. So,

 I_t = I₀ e^{-k' c}
I_t = I₀ 10^{-0.4343 k' c}
I_t = I₀ 10^{K' c}       . . . . . . . . . .(4)     

Wherek' and K'= proportionality constants
c = concentration

By combining equation (3) and (4), we get,

I_t = I₀ 10^-act     

I₀ / I_t = 10^act

Where,K and K' = a or ε
c = concentration
t or b = thickness of the medium
log I₀ / I_t = εbc              . . . . . . . . . .(5)

Where ε = absorptivity, a constant dependent upon the λ of the incident radiation and nature of absorbing material. The value of ε will depend upon the method of expression of concentration.

The ratio I₀ / I_t is termed as transmittance T, and the ratio log I₀ / I_t is termed as absorbance A. formerly, absorbance was termed as optical density D or extinction coefficient E. the ratio I₀ / I_t is termed as opacity. Thus,

                 A = log I₀ / I_t          . . . . . . . . . .(6)

From equation (5) and (6),

                 A = εbc                 . . . . . . . . . .(7)

Thus, absorbance is the product of absorptivity, optical path length and the concentration of the solution.

The term E^1%_{1 cm} or A^1%_{1 cm} refers to the to the absorbance of 1 cm layer of the solution whose concentration is 1 % at a specified λ.

According to equation (7),

                A = log I₀ / I_t

Transmittance T is a ratio of intensity of transmitted light to that of the incident light.

T = I₀ / I_t

The more general equation can be written as follows:

A = log I₀ / I_t = log 1/ T = – log T = abc = εbc

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Deviations from Beer-Lambert’s Law
As per the Beer’s law discussed above, there is a direct proportionality between the absorbance and concentration. A plot of absorbance versus concentration is expected to be a straight line passing through origin. However, this is not always true; there are certain limitations. The law does not hold for all species under every condition. Many a times instead of a straight line, a curvature in the plot may be observed as shown in Fig. The upward curvature, curve (a), is known as positive deviation and the downward curvature, curve(c), as negative deviation.

Beer-Lambert law plots; the curvatures show deviations from the law

Some of the factors responsible for the deviation from Beer’s law are as follows.

Presence of Electrolytes
The presence of small amounts of colourless electrolytes which do not react chemically with the coloured components does not affect the light absorption as a rule. However, large amounts of electrolytes may affect the absorption spectrum qualitatively as well as quantitatively. This is due to the physical interaction between the ions of the electrolyte and the coloured ions or molecules. This interaction results in a deformation of the later, thereby causing a change in its light absorption property.

Hydrogen Ion Concentration
There are a number of substances whose ionic state in solution is greatly influenced by the presence of hydrogen ions. In some cases, two absorbing species are in equilibrium and have a common value of absorptivity at a certain wavelength. For example, in case of bromothymol blue the absorption spectra at different pH values are different. However, at wavelength of 501 nm, we see that all species have same molar absorptivity (see Fig. 2.9). Therefore, no matter to what extent does one species change into the other, there is no change in the total absorption. Such a wavelength is known as isosbestic point. At this wavelength the Beer’s law holds, though the measurements have low sensitivity. However, such wavelengths should be avoided for the quantitative work.

The absorption spectra for bromothymol blue at different pH values showing the isosbestic point at 501 nm

Complexation, Association or Dissociation: Some salts have a tendency to form complexes whose colours are different from thoseof the simple compounds.  The degree of complex formation increases with increase in concentration, therefore, Beer’s law does not hold at high concentrations. Similar discrepancies are found when the absorbing solute dissociates or associates in solution because the nature of the species in solution depends on the concentration.

Non-monochromatic Nature of the Radiation: In order for the Beer’s law to hold, it is necessary that monochromatic light is used.However, most spectrophotometers and all filter photometers, discussed later employa finite group of frequencies. The wider the bandwidth of radiation passed by the filteror other dispersing devices, the greater will be the apparent deviation of a system from adherence to Beer’s law.

Concentration of the Analyte: As per Beer and Lambert’s law, the plot of absorbance versus the concentration ofabsorbing substance should be a straight line when e and b are constant. The pathlength can always be held constant but there are some factors which affect e and it isfound that at high concentration, e is not constant. Therefore, at higher concentrations(>10_3 mol dm_3) there may be deviation from the law