BP701T-INSTRUMENTAL METHODS OF ANALYSIS (Theory)
UV Visible spectroscopy – The absorption spectroscopy uses electromagnetic radiations between 200nm to 800nm and is subdivided into the ultraviolet (UV 200-400nm) and visible (VIS, 400-800 nm) regions, UV visible spectroscopy are all about the recording of the absorption of radiations in the ultraviolet and visible regions of the electromagnetic spectrum. 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. The information provided by this spectroscopy when combined with the information provided by NMR and IR spectral data leads to valuable structural proposals.
Chromophores - The term chromophore was previously used to denote a functional group which is responsible for a colour in a given compound. For example- Nitro group is a chromophore because its presence in a compound gives yellow colour to the compound. But nowadays the term chromophore is used in a broader sense which can be defined as “Any group which exhibit absorption of electromagnetic radiation in a visible or ultra-visible region “. It may or may not impart any colour to the compound. Some of the important chromophores are: ethylene, acetylene, carbonyls, acids, esters and nitrile groups etc.
A carbonyl group is an important chromophore, though the absorption of light by an isolated group does not give rise to any colour in the UV spectroscopy.
Types of chromophores:
So far known chromophores are basically two types.
(i) Chromophores in which the groups have π electrons undergo π-π* transitions. For examples: ethylene, acetylene etc.
(ii) Chromophores having both π- electrons and n (non-bonding) electrons undergo two types of transitions. i.e., π-π* and n-π*, for examples: - carbonyls, nitriles, azo compounds and nitro compounds.
Identification of chromophores:
There is no any particular set rule for the identification of a chromophore. The change in position as well as the intensity of the absorption depends upon a large number of factors. Following points may be useful.
1. Spectrum having a band near 300 mµ may possess two or three conjugated units.
2. Absorption bands near 270-350 mµ with very low intensity ɛmax 10-100 are because of n-π* transitions of carbonyl group.
3. Simple conjugated chromophores likes dienesor,α β –unsaturated ketones have εmax values, i.e., from 10,000 to 20,000.
4. The absorption with ɛmax value between1, 000-10,000 reveals the presence of an aromatic system. If aromatic nucleus is substituted with groups which can extends the chromophore, the absorption take place at still higher value of extinction coefficients.
Auxochromes – It is a group which itself does not act as a chromophore but when attached to a chromophore, it shifts the adsorption towards longer wavelength along with an increase in the intensity of absorption. Some commonly known auxochromic groups are: -OH, -NH2, -OR, -NHR, and –NR2. For example: When the auxochrome –NH2 group is attached to benzene ring. Its absorption changes from λ max 225 (ɛmax 203) to λmax 280 (εmax1430)
All auxochromes have one or more non-bonding pairs of electrons. If an auxochromes is attached to a chromophore, it helps is extending the conjugation by sharing of non-bonding pair of electrons as shown below.
CH2 = CH – NR2 ------------- > CH2-CH-NH2
The extended conjugation has been responsible for bathochromic effect of auxochromes.
Spectral Measurements - The UV-Vis spectra are usually measured in very dilute solutions and the most important criterion in the choice of solvent is that the solvent must be transparent within the wavelength range being examined. In below table lists some common solvents with their lower wavelength cut off limits. Below these limits, the solvents show excessive absorbance and should not be used to determine UV spectrum of a sample.
Table: Common solvents with their cut-off limits.
S. No. Solvent Cut-off wavelength(nm)
1 Acetonitrile 190
2 Water 191
3 Cyclohexane 195
4 Hexane 201
5 Methanol 203
6 95% ethanol 304
7 1,4-dioxane 215
8 Ether 215
9 Dichloromethane 220
10 Chloroform 237
11 Carbon tetrachloride 257
12 Benzene 280
Beer and Lambert’s law – in spectroscopy, a relation concerning the absorption of radiant energy by an absorbing medium. Formulated by German mathematician and chemist August Beer in 1852, it states that the absorptive capacity of a dissolved substance is directly proportional to its concentration in a solution. The relationship can be expressed as A = εlc where A is absorbance, ε is the molar extinction coefficient (which depends on the nature of the chemical and the wavelength of the light used), l is the length of the path light must travel in the solution in centimetres, and c is the concentration of a given solution.
The term fluorometry is used to measure the intensity of fluorescence (Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence) and it is superior to spectrophotometry in terms of sensitivity and specificity. In general, the sensitivity of fluorescence is 10–1000-fold higher in comparison to absorbance measurements. Fluorometry was introduced in immunological assays to improve immunoassay sensitivity.
Singlet state: when all electrons in the molecule are spinning and paired. It is called a singlet because there is only one possible orientation in space.
When we look at excited singlet state, one of the paired electrons from the ground state moves to an excited state but does not change spin. When something happens to the molecule like a collision with another molecule, the electron in the excited state could have a spin inversion. Now, we see an excited triplet state. The problem with this spin flipping, now the electron cannot return to the ground state until its spin is flipped again. Otherwise, Pauli exclusion principle that all electrons must have a different set of quantum numbers would be violated. Now that we understand this aspect, let’s look at how this relates to fluorescence and phosphorescence.
Double State: Doublet State: free radical due to odd electron, molecules with one unpaired electron are in doublet state.
Triplet state: One set of electron spins is unpaired. It is called a triplet because there are three possible orientations in space with respect to the axis
Internal conversion is a non-radiative and isoenergetic transition between two electronic states of the same spin multiplicity. Internal conversion occurs because of the overlap of vibrational and electronic energy states.
External conversion: the energy transfer between molecules through molecular collisions.
Quenching - quenching is referred to the process that reduces intensity of fluorescence of substance. This is due to the internal and external factor like pH, concentration, temperature, viscosity, heavy metals, and other chemicals. Quenching agents - Chloride, Disulphide, Nitric oxide.
Instrumentation - The basic components of fluorescence spectrometers are a white light source, excitation monochromator, sample chamber, emission monochromator, and detector. Two general types of instruments exist: filter fluorometers that use filters to isolate the incident light and fluorescent light and spectrofluorometers that use a diffraction grating monochromator to isolate the incident light and fluorescent light.
Both types use the following scheme: the light from an excitation source passes through a filter or monochromator, and strikes the sample. A proportion of the incident light is absorbed by the sample, and some of the molecules in the sample fluoresce. The fluorescent light is emitted in all directions. Some of this fluorescent light passes through a second filter or monochromator and reaches a detector, which is usually placed at 90° to the incident light beam to minimize the risk of transmitted or reflected incident light reaching the detector.
Application of fluorescence - Fluorescence spectroscopy is used in biochemical, medical, pharmacy and chemical research fields for analysing organic compounds. It has also been used in differentiating malignant skin tumours from benign.