spectroscopy

Principle of UV-VISIBLE spectroscopy

UV-VISIBLE spectroscopy obeys the Beer-Lambert law, which states that: when a beam of monochromatic light is passed through a solution of an absorbing substance, the rate of decrease of intensity of radiation with thickness of the absorbing solution is proportional to the incident radiation as well as the concentration of the solution.
The expression of Beer-Lambert law is-
A = log (I0/I) = Ecl
Where, A = absorbance
I0 = intensity of light incident upon sample cell
I = intensity of light leaving sample cell
C = molar concentration of solute
L = length of sample cell (cm.)
E = molar absorptivity

From the Beer-Lambert law it is clear that greater the number of molecules capable of absorbing light of a given wavelength, the greater the extent of light absorption. This is the basic principle of UV-VISIBLE spectroscopy.
Instrumentation and working of UV-VISIBLE spectroscopy
Instrumentation and working of the UV-VISIBLE spectrometers can be studied simultaneously. Most of the modern UV-VISIBLE spectrometers consist of the following parts-

Light Source- Tungsten filament lamps and Hydrogen-Deuterium lamps are most widely used and suitable light source as they cover the whole UV-VISIBLE region. Tungsten filament lamps are rich in red radiations; more specifically they emit the radiations of 375 nm, while the intensity of Hydrogen-Deuterium lamps falls below 375 nm.

Monochromator- 

Monochromators generally composed of prisms and slits. The most of the spectrophotometers are double beam spectrophotometers. The radiation emitted from the primary source is dispersed with the help of rotating prisms. The various wavelengths of the light source which are separated by the prism are then selected by the slits such the rotation of the prism results in a series of continuously increasing wavelength to pass through the slits for recording purpose. The beam selected by the slit is monochromatic and further divided into two beams with the help of another prism.

Sample and reference cells- One of the two divided beams is passed through the sample solution and second beam is passé through the reference solution. Both sample and reference solution are contained in the cells. These cells are made of either silica or quartz. Glass can't be used for the cells as it also absorbs light in the UV-VISIBLE region.

Detector- 

Generally two photocells serve the purpose of detector in UV-VISIBLE spectroscopy. One of the photocell receives the beam from sample cell and second detector receives the beam from the reference. The intensity of the radiation from the reference cell is stronger than the beam of sample cell. This results in the generation of pulsating or alternating currents in the photocells.

Amplifier- 

The alternating current generated in the photocells is transferred to the amplifier. The amplifier is coupled to a small servometer. Generally current generated in the photocells is of very low intensity, the main purpose of amplifier is to amplify the signals many times so we can get clear and recordable signals.

Recording devices- 

Most of the time amplifier is coupled to a pen recorder which is connected to the computer. Computer stores all the data generated and produces the spectrum of the desired compound.

Applications of UV-VISIBLE spectroscopy

1. Detection of functional groups- UV-VISIBLE spectroscopy is used to detect the presence or absence of chromophore in the compound. This is technique is not useful for the detection of chromophore in complex compounds. The absence of a band at a particular band can be seen as an evidence for the absence of a particular group. If the spectrum of a compound comes out to be transparent above 200 nm than it confirms the absence of –
a) Conjugation b) A carbonyl group c) Benzene or aromatic compound d) Bromo or iodo atoms.

2. Detection of extent of conjugation- The extent of conjugation in the polyenes can be detected with the help of UV-VISIBLE spectroscopy. With the increase in double bonds the absorption shifts towards the longer wavelength. If the double bond is increased by 8 in the polyenes then that polyene appears visible to the human eye as the absorption comes in the visible region.

3. Identification of an unknown compound- An unknown compound can be identified with the help of UV-VISIBLE spectroscopy. The spectrum of unknown compound is compared with the spectrum of a reference compound and if both the spectrums coincide then it confirms the identification of the unknown substance.

4. Determination of configurations of geometrical isomers- It is observed that cis-alkenes absorb at different wavelength than the trans-alkenes. The two isomers can be distinguished with each other when one of the isomers has non-coplanar structure due to steric hindrances. The cis-isomer suffers distortion and absorbs at lower wavelength as compared to trans-isomer.

5. Determination of the purity of a substance- Purity of a substance can also be determined with the help of UV-VISIBLE spectroscopy. The absorption of the sample solution is compared with the absorption of the reference solution. The intensity of the absorption can be used for the relative calculation of the purity of the sample substance.                                           ---------------------------

Atomic Absorption Spectroscopy

Meaning of Atomic Absorption Spectroscopy

Atomic Absorption Spectroscopy was invented by Alan Walsh in 1950’s for the qualitative determination of trace metals in liquids. The superiority of the technique over other is based on the fact that by this technique 50-60 elements can be determined without any interference from trace to big quantities.All these elements can be detected here which fail to yield satisfactory result in flame photometry. Thus, it is a successful instruments for detection and estimation of metals and non-metals both types of pollution from factories.The technique has also proved very helpful to both aqueous and non-aqueous solutions.

Principle of Atomic Absorption Spectroscopy:

When a solution having a mixture of metallic species is introduced into the flame, the solvent evaporates and vapour of metallic species is obtained. Some of metal atoms can be raised to an energy level sufficiently high to emit characteristics radiation of metal-a phenomenon that is used in flame photometry. Here a large amount of metal atoms remain in non-emitting ground state.These ground state atoms of a particular element are receptive of light radiation of their own specific resonance wavelength. In this way, when a light of this wavelength passes through a flame, a part of light will be absorbed and this absorption will be proportional to the intensity of atoms in the flame.So in atomic absorption spectroscopy the amount of light absorbed is determined because the absorption is proportional to the concentration of the element.

Advantages of Atomic Absorption over Flame Photometry:

(1) It does not suffer from spectral interference, which occurs in flame emission spectroscopy.
(2) It is independent of flame temperature.
(3) By atomic absorption technique, traces of one element can easily be determined in presence of high concentration of other elements.
(4) It has proved very successful in the analysis of bronze and copper alloys and in the determination of metals like platinum, gold etc.
Disadvantages of Atomic Absorption Spectroscopy:

Some of the disadvantages are summarized as follows:

(1) This technique has not proved very successful for the estimation of elements like V, Si, Mo, Ti and A1 because these elements give oxides in the flame.
(2) In aqueous solution, the anion affects the signal to a noticeable degree.
(3) A separate lamp is needed for the determination of each element. Attempts are being made to overcome this difficulty by using a continuous source.

Fig.1.Scheme representation of atomic absorption spectroscopy.

Applications of Atomic Absorption Spectroscopy:

(i) Quantitative Analysis:
As we know that each element has its own characteristic emission spectrum, hence the intensity of the lines is compared with standard and the concentration can be easily evaluated from the graph (Fig. 3).
Suppose the intensity of unknown element is C, then the concentration is evaluated by drawing a perpendicular on the line (calibration curve) and from the point it cuts the curve. A perpendicular is drawn on the x-axis. The value from (0 to 0) will give the concentration of unknown in moles per litre.
In atomic adsorption spectroscopy, the same method is followed for determining the concentration of the element in an unknown solution.
(ii) Method of Standard Addition:
If calibration graph is linear, the sample concentration can be calculated by adding known amount of the test element to the sample. This gives a section of calibration graph above the unknown sample concentration and the resulting straight line can be extrapolated back to zero signal intensity.
The concentration scale is determined by standard additions and unknown concentration is given by the point at which extrapolated line crosses concentration axis.

*Typical atomic absorption calibration curve.
(iii) Quantitative Analysis:
Generally first a curve is plotted between absorbance valve vs. concentration of standard samples of the element. A linear curve is obtained.
From this curve, the concentration of unknown is evaluated by knowing absorbance value only from the following equation:
A = S × C
or Absorbance = Slope × Concentration
As it is very sensitive technique hence it gives more accurate results than many analytical methods.                                         ------------------

Nuclear Magnetic Resonance (NMR)

Definition of NMR:

(1) Nuclear magnetic resonance is defined as a condition when the frequency of the rotating magnetic field becomes equal to the frequency of the processing nucleus.

 (2) If ratio frequency energy and a, magnetic field are simultaneously applied to the nucleus, a condition as given by the equation v = үH0/2π is met. The system at this condition is said to be in resonance [v — frequency of radiation associated with transition from one state to the other; ү = proportionality constant and H0 = magnetic field]’.

Principle of NMR:

The principle of nuclear magnetic resonance is based on the spins of atomic nuclei. The magnetic measurements depend upon the spin of unpaired electron whereas nuclear magnetic resonance measures magnetic effect caused by the spin of protons and neutrons. Both these nucleons have intrinsic angular momenta or spins and hence act as elementary magnet.The existence of nuclear magnetism was revealed in the hyper fine structure of spectral lines. If the nucleus with a certain magnetic moment is placed in the magnetic field, we can observe the phenomenon of space quantization and for each allowed direction there will be a slightly different energy level.

Chemical shift

The most important molecular parameter determined by NMR is the chemical shift. The chemical shift is defined as a measure of the resonance frequency of the nuclei in a given chemical environment.The magnitude of the chemical shift is proportional to the strength of applied field and is caused by the circulations of surrounding electrons about the protons.

The chemical shift parameter is defined

δ = (Hr – Hs)/Hr × 106 ppmwhere Hr and Hs are field strengths corresponding to resonance for a particular nucleus in the sample (Hs) and reference (Hr).

Factors which influence Chemical shift:

Actually the chemical shift parameter is a function of electron density around the nucleus as the electrons are directly involved in the diamagnetic shielding which acts to attenuate the applied magnetic field.

Hence following factors are responsible for influencing its value:

(a) Specific solvent,

(b) Bulk diamagnetic susceptibility effect,

(c) Temperature (only when change in temperature causes changes in some type of association equilibrium or changes in amplitude of torsional vibrations),

(d) Electron density,

(e) Inductive effect,

(f) Vander Waal deshielding, and

(g) Hydrogen bonding.

Applications of N.M.R. Spectroscopy:

(1) Quantitative Analysis:

The area of peak is directly proportional to the number of nuclei responsible for that peak. Thus the concentration of species can be determined directly by making use of signal area per proton. The signal area per proton can easily be calculated by use of a known concentration of an internal standard.Similarly, (he concentration of new species formed during the reaction can also be calculated from the spectrum of parent compound.

(2) Qualitative Analysis:

The qualitative analysis of the compound can easily be made by knowing:

(i) Chemical shift values of hydrogen containing groups,

(ii) The presence of particular functional group,

(iii) The relative position of these groups and

(iv) The relative number of nuclei in these groups.

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Electron Paramagnetic resonance (EPR)

Introduction

Electron Paramagnetic Resonance (EPR), also called Electron Spin Resonance (ESR), is a branch of magnetic resonance spectroscopy which utilizes microwave radiation to probe species with unpaired electrons, such as radicals, radical cations, and triplets in the presence of an externally applied static magnetic field.

In many ways, the physical properties for the basic EPR theory and methods are analogous to Nuclear Magnetic Resonance (NMR). The most obvious difference is that the direct probing of electron spin properties in EPR is opposed to nuclear spins in NMR. Although limited to substances with unpaired electron spins, EPR spectroscopy has a variety of applications, from studying the kinetics and mechanisms of highly reactive radical intermediates to obtaining information about the interactions between paramagnetic metal clusters in biological enzymes. EPR can even be used to study the materials with conducting electrons in the semiconductor industry.

Comparison between EPR and NMR

EPR is fundamentally similar to the more widely familiar method of NMR spectroscopy, with several important distinctions. While both spectroscopies deal with the interaction of electromagnetic radiation with magnetic moments of particles, there are many differences between the two spectroscopies:

EPR focuses on the interactions between an external magnetic field and the unpaired electrons of whatever system it is localized to, as opposed to the nuclei of individual atoms.

The electromagnetic radiation used in NMR typically is confined to the radio frequency range between 300 and 1000 MHz, whereas EPR is typically performed using microwaves in the 3 - 400 GHz range. 

In EPR, the frequency is typically held constant, while the magnetic field strength is varied. This is the reverse of how NMR experiments are typically performed, where the magnetic field is held constant while the radio frequency is varied.

Due to the short relaxation times of electron spins in comparison to nuclei, EPR experiments must often be performed at very low temperatures, often below 10 K, and sometimes as low as 2 K. This typically requires the use of liquid helium as a coolant.

EPR spectroscopy is inherently roughly 1,000 times more sensitive than NMR spectroscopy due to the higher frequency of electromagnetic radiation used in EPR in comparison to NMR. 

The electron paramagnetic resonance (EPR) differs from NMR principally because in that the frequencies of electron resonance occur in microwave region for magnetic fields of the order of several thousand gauss. Therefore EPR spectrometer uses such components as Klystrons, wave guides and resonance cavities for the sample.EPR method is applicable whenever the compound displays at least one unpaired electron, i.e., in free radicals, crystalline and amorphous solids subjected to irradiation or containing transition element ions and rare earths had some chelates. The other different examples are metals, odd molecules, graphite’s and impurities in semiconductors.