Spectroscopy or simply spectrometry is a main branch of analytical chemistry which deals with the study of concentration of analyte as the function of amount of radiation absorbed whenever electromagnetic radiation from suitable source is directed at it.
All the chemical species interact by electromagnetic radiation, and in the course, reducing the intensity or the power of the radiated beam. Measurement can be brought on by the infrared, visible and ultraviolet areas of the spectrometer.
Definition and general principle of spectrometry:
Spectroscopy is that stream of chemistry that is based on the measurement of decrease in the power of the radiation (that is, attenuation) brought about via the analyte whenever electromagnetic radiation is made to pass via the analyte.
Electromagnetic radiation is a kind of energy which is transmitted via space at huge velocities. This is a form of energy which is propagated as the transverse waves that vibrate perpendicularly to the direction of propagation and this passes on a wave motion to the radiation. Wave parameters employed to further illustrate the propagation comprise frequency, velocity, wave length and amplitude. Wave number is the reciprocal of wavelength that is the number of waves in a unit. The arithmetical relationship is:
λ = C/V
λ = Wavelength
C = Velocity of light
V = Wave number or frequency
Electromagnetic radiation method is a certain amount of energy, the unit of which is termed as photon. This is associated to frequency by:
E = hv = hc/λ
E = Energy (photon)
h = Plank's constant
The electromagnetic spectrum can be randomly broken down into various regions according to the wave-length.
Regions of Electromagnetic Spectrum:
1) Ultraviolet region that extends from around 10 to 380 nm, however the most analytical helpful region is from 200 - 300 nm, termed as near ultraviolet region.
2) Beneath 200 nm. Here, air plays considerable roles and therefore the instrument operates under vacuum. Therefore the region is termed as vacuum ultraviolet.
3) Visible region. This is a very small wavelength region which can be seen via human eyes. The region begins from 380 nm to around 780 nm. The light in that appears in colors.
4) Infrared region. This extends from around 0.78µm (780nm) to around 300 nm however the range often used in analysis from 2.5nm to 25nm.
Absorption of Radiation:
Whenever radiation passes via a transparent layer of materials (that is, solids, liquid or gas), a few radiation is absorbed via the atom or molecule in the materials. There are three fundamental methods through which molecules can absorb the radiations. All includes bringing molecules to higher internal energy level. These are (a) Rotational transition (b) Vibration transition and (c) Electronic transition.
The molecule at normal room temperature is taken to be at lowest electronic energy state (Eo). On absorbing a photon of energy, it moves to the higher energy state termed as excited state.
The absorption of electromagnetic radiation through some species 'M' is considered to experience a two-step procedure:
i) M + hv → M*
ii) M* → M + heat
The primary step includes absorbing radiation and the species is transformed to an excited species (M*) the life time of M* is extremely short, after which it experiences the second step termed as relaxation that results in products of heat and the original metal 'M'.
The absorption of radiation can be employed either for the qualitative or quantitative analysis.
1) Qualitative techniques:
When the absorption of light occurs in the visible region, then object transmits or reflects only a part of the light. Whenever polychromatic light (that is, white light), which includes the whole spectrum of wavelength in the visible region is passed via an object, it absorbs some wavelengths and leaving the unabsorbed wavelength to be transmitted. The transmitted (or unabsorbed) wavelengths are seen as colors. The table below illustrates the absorbed and unabsorbed colour of various wavelengths.
Table: Absorption of light in the visible region
Absorption spectroscopy as well gives helpful tools for qualitative analysis. The radiation whose wavelengths are in the ultra-violet and infrared areas is particularly helpful in this regard.
Recognition of pure compounds comprises comparing the spectral features of unknown sample by those of pure compounds. A close match is accepted to be a good proof of chemical identity, specifically if the spectrum of the unknown includes a number of sharp and well-defined peaks.
Absorption in the infrared region is more helpful for the qualitative purposes as wealth of fine structure that exists in the spectral of numerous compounds.
2) Quantitative techniques:
The absorption measurement comprises reduction of power (that is, attenuation) experienced via the beam of radiation as it passes via the solution. This can be associated quantitatively to the concentration of analyte in the solution.
The amount of radiation absorbed via the sample is found out by what is recognized as Beer's law that states whenever a monochromatic radiation passes via absorbing specie, the power of the beam is gradually reduced as more energy is absorbed via the particle. The decrease in power based on the concentration of the absorber and the length of the path transverse via the beam.
Fig: Quantitative techniques
log Po/P = εbc = A
Po = Incident ray
P = Transmitted ray
? = Molar absorptivity or extinction coefficient
b = Path length
c = Concentration
A = Absorbance
Limitations of Beers law:
There are a few observed factors which limit the application of Beers law. These comprise, linear relationship between the absorptive and concentration. The linear relationship doesn't for all time take place as are result of the given observations:
1) The Beers explains successfully only diluted solution. Therefore, at high concentration (above 0.01F), there is a deviation from the linearity nature of the relationship.
2) Chemical deviation: Chemical causes non-linearity to take place whenever non-symmetrical chemical equilibrium is operational. This is brought regarding a result of related dissociation or reaction of absorbing species by the solvent.
3) Instrumental deviation: Beers law is just obeyed whenever monochromatic light is employed. However the use of truly monochromatic light is rarely practical, the alternative polychromatic light will cause deviation from the law.
General Principle of instrumentation:
Spectrometer, the name of the instrument used in spectrometry is built on 4.
Fig: Principle of instrumentation
1) A source of constant radiation over the wavelength from the source spectrum.
2) A Monochromator for choosing a narrow band of wavelength from the source spectrum.
3) A detector for transforming radiant energy to the electrical energy.
4) Read out device to interpret the response of the detector.
The source should encompass a readily detectable output of radiation over the wavelength for which the device is designed.
a) For visible region: the generally employed source is tungsten filament incandescent lamps whose behavior is identical to that of the black-body radiator.
Sources of this type emit constantly radiation that is more characteristic of the temperature of the emitting surface than that of materials of that it is composed.
b) For ultraviolet region: A low pressure hydrogen or deuterium discharge tube is commonly used as a source. Ultraviolet sources should encompass a quartz window as glass is not transparent to the UV radiation.
c) For infrared region: A Nernst glower is employed as a source. This comprise of a rod made up of mixture of extraordinary earth oxides.
This is a tool that disperses radiation to its component wavelength. It comprises of system of lenses, mirrors and slits that direct radiation of the deserved wavelength from the Monochromator in the direction of the detectors of the instrument.
There are three kinds of Monochromator: Prism, grating and double Monochromator.
a) Prism Monochromator: It uses a 60-deg prism for the dispersion.
b) Grating Monochromator: Dispersion of UV, visible and infrared radiation is brought about via the passage of a beam via a transmission grating or through reflection from a reflection grating.
c) Double Monochromator: Most of the modern Monochromator include two disperses element; two prisms, two gratings or a prism and a grating for efficient performance.
The sample container or else termed as cell should be transparent in the wavelength region being measured. There are different materials which can be employed for the cell construction. These comprise NaCl, KBr, Ti and Br. The cell for utilization in visible and as ultraviolet spectrometers is generally square curvet of 0.1m thickness. Though for infrared, short path length is needed, although it is often hard to produce.
Detectors will as well differ with the wavelength area to be measured. To be helpful, a radiation detector should respond over a wide wavelength range. It must in addition, be sensitive to low levels of radiation power, respond quickly to the radiation, generate an electrical signal which can be amplified, and encompass a relatively low noise level. The signal generated should be directly proportional to the power of beam hitting it.
G = K1P + Kn
G = Electrical response of the detection
K1 = Sensitivity of detector
Kn = Current constant (or Dark current)
Various detectors comprise generally employed are:
a) Phototube generally employed for UV and visible region.
b) Photomultiplier tube is very sensitive than phototube; employed for visible and UV region. Others comprise photocell, photo conductive cell, thermocouple or Bolometer and also pneumatic cell.
Usually the design of different spectrometers is similar however there are some variations based on the maker.
Kinds of spectrophotometer known comprise:
a) Single-beam spectrometer
b) Double beam spectrometer
c) Gilford spectrometer
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