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Instrumentations associated with the UV visible Spectroscopy:
The UV-Vis spectrum can by recorded via the following types of absorbance instruments: a. Single beam spectrometer b. Double beam spectrometer c. Simultaneous spectrometer
Light source (mostly tungsten lamps), small holder and detector are common to all the three type of spectrometers. However, a filter may be used, in addition, to select one wavelength at a time. This filter is often termed as the monochromator. Single beam spectrometer (shown in Figure 3 ) includes a monochromator between the light source and specimen. The specimen is analysed individually for all wavelengths. Double beam spectrometer ( Figure 4 ) uses a single light source, monochromator, a splitter and a series of mirrors, to direct the beam towards the reference and the sample under investigation. Whereas, a simultaneous spectrometer ( Figure 5 ) uses an array of diodes for simultaneous detection of absorbance at all wavelengths. This is the fastest and most efficient of the three.
1.2 Instrumentation: The basic components of a spectrometer include: light source (UV and visible), monochromator (wavelength selector), sample stage, and detector. A tungsten filament, continuous over UV region is generally used as light source. Detector is usually a photodiode or CCD. Photodiodes go with monochromators to filter light of a particular wavelength, to be fed to the detector. While monitoring the absorbance in UV spectrum, the visible lamp must be turned off, and vice-versa. Figure 6 includes schematic UV-Vis-NIR Spectrometer.
Instrumental components
1. UV Source : The power of radiating source should not vary in its operating wavelength range. Continuous UV spectrum is produced by electrically exciting deuterium or hydrogen at low pressures. The mechanism for generation of UV light includes creating an excited molecular species, that breaks into two atomic species and a UV photon. The emission wavelengths of both deuterium and hydrogen lamps are in 160 to 375 nm range. The material of the cUVettes needs to selected such that it does not absorb the light incident, because this will result in errors in obtained absorption spectrum. Thus, quartz is usually used.
The linear photodiode array is an example of a multichannel photon detector. These detectors can simultaneously measure all elements of a beam of dispersed radiation. A linear photodiode array consists of several small silicon photodiodes created on a single silicon chip. The number of photodiodes can vary between 64 to 4096 sensor elements on a chip, however, 1024 photodiodes is most common. For each diode, there is also a storage capacitor and a switch. The individual diodecapacitor circuits can be sequentially scanned. Charge- Coupled Devices (CCDs) are like diode array detectors, but instead of diodes, they consist of an array of photocapacitors. Reference beam intensity, should suffer little or no absorption, and termed I0 whereas that of sample beam is called I. The spectrophotometer automatically examines all wavelength components in a short time. This technique is good to evaluate the concentration as well as molecular structure or structural changes. It may also be used to examine the vibrational and conformational energy levels alterations before and after an interaction with a substrate, or a molecule.
Applications of UV Visible Spectroscopy
1. Detections of impurities : it is the one of the best method to determine the impurity in organic molecules additional peaks can be observed due to impurity in the samples and it can be compared with that of standard raw material. By also measuring the absorbance of specific wavelength the impurities can be detected. 2. Quantitative analysis : it can be used for the quantitative analysis of compound that absorbs UV radiation. It is determined by using beer’s law which is given as:
A = log10/IT log1/T = -logT = abc Ʃbc
where Ʃ is extinctions coefficient c is concentrations b is the length of the cells that is used in UV spect.
3. Qualitative analysis: UV absorptions spect. Can be characterized those types of compound which absorbs UV radiation. Identifications is done by the comparing the absorptions spectrum with the spectra of known compound. 4. Chemical kinetics : kinetics of the reactions can also be studies using UV spec. the UV radiations is passed through the reactions cell and the absorbance changes can be observed 5. Detections of functional groups: this techniques used by the presence or absence of functional group in the compound absence of bond at particular wavelength regarded as evidence for absence of particular group. 6. Quantitative analysis of pharmaceutical substances: many drugs are either in the form of raw material or in the form of formulations. They can be assayed b the making a suitable solutions of the drugs in solvent and measuring the absorbance of specific wavelength. Diazepam tab can be analyzed by the 0.5% H 2 SO 4 in methanol at the wavelength 284nm.
