Spectroscopy and Chromatography Techniques
Vibrational Spectra (IR Spectra)
IR spectroscopy is concerned with the study of absorption of IR radiation (800nm- 1mm), which causes vibrational transition in the molecule. When a gaseous molecule absorbs IR radiation, it produces transition between the vibrational energy levels. The resultant spectrum is called vibrational spectra. The essential requirement for a molecule to produce an IR spectrum is that the dipole moment of the molecule must change during vibration. Thus, a vibrational spectrum is given by hetero nuclear diatomic molecules. Polyatomic molecules with and without a dipole moment also produce an IR spectrum. Homo nuclear diatomic molecules such as O2, N2, H2 etc. will not produce an IR spectrum, since they have zero dipole moment. But molecules like HCl, HBr, NO2, H2O, CO2, NH3, CH3Cl and SO2 can produce IR spectra and they are IR active molecules. In the case of polyatomic molecules without a dipole moment, the exciting radiations can induce an oscillating dipole moment. Hence, they are also IR active.
When a gaseous molecule having a dipole moment vibrates, it generates an electric field which can interact with the electrical component of IR radiation. During this interaction, energy can be emitted or absorbed in the form of radiation of a definite frequency.
Instrumentation of IR spectrometer
Theory of IR spectra
In order to explain the theory of IR spectra, consider the vibration of a hetero nuclear diatomic molecule. It is similar to the vibration of a simple harmonic oscillator.
Vibrational energy of a simple harmonic oscillator = Vibrational energy of a diatomic molecule
Evib=(v+ 1⁄ 2)hω
Reduced mass (μ) = m1m2/m1+m2
When the molecule is in the vibrational ground level (v =0), E vib= ⁄ hω. This is the lowest vibrational energy level and it is called zero point energy.
Let us consider the vibrational transition from the lowest vibrational (v1) to the next higher vibrational energy level (v).
Chromatography
This method is generally used for the separation of complex mixtures which cannot be separated by other methods. It is also useful for the separation and purification of organic compounds when they are available in very small quantities.
General principle of chromatography: It is based on the principle of selective distribution of a mixture of compounds between two phases, viz., a stationary phase and a mobile phase.
The stationary phase is the fixed phase.
The stationary phase is a solid or a liquid. If the stationary phase is a solid, then the principle is adsorption and such type of chromatography is called adsorption chromatography. Example: column chromatography.
If the stationary phase is a liquid, then the principle is partition and such type of chromatography is called partition chromatography. Example: Gas liquid chromatography.
The mobile phase is the moving phase. It can be a liquid or gas. The components to be separated are carried by the mobile phase through the stationary phase.
Classification of chromatography based on the mechanism of separation:
a) Adsorption chromatography: In this chromatography, the stationary phase is solid and the mobile phase is a liquid or gas. Here, separation occurs due to the difference in the adsorption coefficients of the components.
b) Partition chromatography: In this chromatography, the stationary phase is a liquid supported on an inert solid and the mobile phase is a liquid or gas. Here, separation occurs due to the difference in the partition coefficients of the components.
c) Ion exchange chromatography: In this chromatography, the stationary phase is an ion exchanger and the separation of the mixture is based on the ion exchange principle and is applicable for ionic species.
Classification of chromatography based on the mobile phase:
a) Liquid chromatography: If the mobile phase is liquid and the stationary phase is solid, then the chromatography is called LSC (Liquid Solid Chromatography).
If the mobile phase is liquid and the stationary phase is liquid supported on solid, then the chromatography is called LLC (Liquid Liquid Chromatography)
b) Gas chromatography: If the mobile phase is gas and the stationary phase is solid, then the chromatography is called GSC (Gas Solid Chromatography).
If the mobile phase is gas and the stationary phase is liquid, then the chromatography is called GLC (Gas Liquid Chromatography)
Column chromatography
In column chromatography, the stationary phase is held in a narrow tube and is packed with solid adsorbents like Al2O3, silica gel, MgO etc. This can function as the stationary phase.
Thin Layer Chromatography (TLC)
Thin layer chromatography was developed by Izmailov and Shraiber. It is better than column chromatography. This method is used for determining the number of components in a mixture. This method will give an idea about the identity of components in the mixture. This will give an idea about the purity of compounds.
Applications
It is used for finding the purity of compounds.
To determine the appropriate solvent for column chromatographic separation.
It is used for the identification of a compound.
It is used for monitoring column chromatographic separation.
High Pressure Liquid Chromatography (HPLC)
HPLC is widely used for analytical separation because of its high sensitivity, accurate quantitative determination and its ability to separate non-volatile species.
HPLC Apparatus
i) Solvent delivery system
ii) Sample injection system iii) Chromatographic column iv) Detector
v) Recorder
Applications
Used in pharmaceutical biological studies.
Used in the analysis of water-soluble and fat-soluble vitamins.
Used in the analysis and separation of amino acids & proteins.
Used in the separation of lipids & steroids.
Gas chromatography (GC)
Gas chromatography is based on the principle of selective distribution of components of a mixture between two phases, viz., a stationary phase and a mobile phase. In gas chromatography, separation occurs between a gas mixture and the stationary phase. The mobile phase is a mixture of a vaporized sample and an inert carrier gas. Usually, the carrier gas used is nitrogen. Sometimes Ar, He, H2 can also be used as the carrier gas depending on the nature of the mixture. The stationary phase may be a solid or liquid. So there are two types of gas chromatography:
1) Gas Solid Chromatography (GSC) 2) Gas liquid Chromatography (GLC)
Applications
Used for qualitative analysis. Used to test the purity of compounds. The presence of impurities will be revealed by the appearance of an extra peak. Widely used for the study of air pollution.