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Visible Spectroscopy

The visible spectroscopy has played a very big role in the study of all kinds of natural products from plant and animal sources and these investigations have involved the steps of isolation, characterisation, synthesis and bio-synthesis. These further helped in the biological mode of action. The visible spectroscopy has been of value in each of these stages for a wide range of biochemical materials.

The visible spectroscopy helped in the detailed study of carotenoids and other related substances. These also helped in amino acids, proteins and enzyme studies along with all kinds of heterocyclic compounds including nucleotides and nucleic acids. These visible spectroscopy also helped in the studies of vitamins and coenzymes. Apart from these the study of all kinds of antibiotics and medicinal substances requires detailed molecular structure fragmentation report to understand the level of potency and visible spectroscopy helps in such things. The analytical part of all kinds of compounds needs a specific route of study and this is helped in a big way by using visible spectroscopy reports. 

 

Visible Spectroscopy Definition

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For any visible spectroscopy to work proper it requires electromagnetic radiation of high energy. The visible region corresponds to 800 to 400 nm ultra violet region to 400 to 200 nm.

The UV region extends from 1000 to 4000 A or 100 to 400 nm and it needs to be noted that UV measurements are reported directly in nm and the corresponding range in per cm are used to calculate the relevant molecular analysis. 

On passing electromagnetic radiation in ultra violet and visible regions through a compound with multiple bonds, a portion of the radiation is normally absorbed by the compound. The amount of absorption depends on the wavelength of the radiation and the structure of the molecule. 

The absorption of radiation is due to the subtraction of energy from the radiation beam when electrons in orbitals of lower energy are excited into orbitals of higher energy. As this is an electron excitation phenomenon the ultra violet is sometimes called electronic spectroscopy. 

The ultra violet spectrum or visible spectra records the wavelength of an absorption maximum and also the strength of the absorption. This is given out by Beer Lambert law. The molar absorptivity is a constant for any organic compound at a given wavelength and these are never considered as dimensionless but is correctly expressed in units of 10-2 m2 per mol. 

The infra-red spectrum uses a strong and weak medium to define the intensity of different bands but however in discussing the visible spectroscopy the value of molar absorptivity is reported to know how intense the absorption was.

The value of molar absorptivity varies from 100 to 104 and the range of 104 is considered to be a strong absorption and anything less than 103 as weak. The intensity of the peak is a measure of transition probability and the peaks with low molar absorptivity values often arise from transition which are not used.

The foundations of quantitative chemical analysis can be traced back to development of trimetric analysis in which titration end points depended on the change in colour of the species being analysed or of that of a specially added chemical indicator.

These colour changes arise due to molecular and structural changes in the substances being examined leading to changes in the ability to absorb light in the visible region of the electromagnetic spectrum. 

In various ways absorption spectroscopy along with visible spectroscopy or within the visible range has been an important tool to the analysts. 
Many important and sensitive colour tests have been developed for the detection and determination of a wide range of chemical species both inorganic and organic in nature. 

Today the visible spectroscopy is applied to many determination which are developed and can be considered to be useful in particularly for biochemical analysis and is of vital importance in clinical laboratory attached to most modern hospitals where the components of blood and other various samples like urine and stones are determined and monitored for better treatment and recovery.

The visible spectroscopy is an essential tool in identifying and quantifying a broad range of chemical and biological substance. The equipment for these purpose range from very simple colour comparison to bigger computer controlled automatic scanning instruments which cover the whole of the visible spectroscopy region of electromagnetic spectrum. 

Sometimes the materials are transparent to a particular wavelength of light for certain reasons and it is useful in relating visible spectroscopy to such analysis. Any optical substance which are clear and hence do not absorb visible light and hence are not considered for visible spectroscopy. Anything which needs to be studied has to absorb some amount of visible light which helps in analysing the sample better.

Molecular compounds also have vibrational absorptions and are not commonly used in IR spectroscopy. This is mainly due the fact that these materials like window lenses etc. do not absorb IR light. 

The visible spectroscopy can be carried out for molecules which are absorbing light both in visible range and beyond for assigning chemical structures to such compounds which differ only by location of a carbon- carbon double bond or of carbonyl group.

The visible spectroscopy helps in predicting the wavelengths at which maximum absorption can occur by simply studying the component chemical group in the molecule. 

The visible spectroscopy was not confined to only visible range and it quickly spread to other regions of the electromagnetic spectrum. The progress was delayed until photography and instrumental methods were developed to detect non-visible photons as well. Modern spectroscopy covers virtually the entire electromagnetic spectrum.

Visible Spectroscopy Principle

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The visible spectroscopy uses ultra violet radiation and visible light to help determine chemical structure. The ultra violet region of the electromagnetic spectrum corresponds to wavelengths of 200 to 400 nm. The visible region is adjacent with wavelengths 400 to 800 nm. These two regions are analysed by same spectrometer usually called UV or UV vis spectrometer.

The radiation of ultra violet and visible wavelengths is absorbed by compounds with pi electrons which upon absorption of the energy are raised to an excited state.

The visible spectroscopy branch deals with transition between the electronic energy levels of a molecule brought about by absorption of ultra violet or visible radiation. If a sample electronic spectrum is studied in its gaseous state it shows rotational vibrational structure but these are rendered useless in liquid and solution samples. This is mainly due to the fact that electronic spectra in solution are usually broad.

The visible spectroscopy is based on the principle that identical nuclei in different chemical compounds exist at different energy levels due to existence of nuclear quadrupole moment. These also give rise to resonance peaks at different frequencies. This becomes a guide to the nature of bonding and molecular structure.

The visible spectroscopy is studied in nuclear quadrupole resonance spectroscopy section under this visible range where the transition occurs between 108 and 109 Hz.

The basic operating principle of visible spectroscopy is based on measurement of light absorption due to electronic transition in a sample. Since the wavelength of light required for electronic transition is typically in UV and visible range of electromagnetic radiation spectrum.

Visible Spectroscopy Instrumentation

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In the visible spectroscopy the light from a suitable source is passed through a prism or grating monochromator and then passed through the sample before reaching the detector. This arrangement which is opposite of that used in infrared spectrometry reduces exposure of the sample to light of wavelengths that might cause photo decomposition.

Since light source, output and detector response will be wavelength dependent such instruments are only used for single wavelength studies where a reference sample is measured separately from the test sample. 

Double beam spectrometers help facilitate the measurement of absorption spectra as the light from the source is split into two beams equal in spectral range after the original ray passes through the monochromator. 

As the two beams pass through the test sample and reference sample and the spectrum obtained is thus compensated for changes in some of the system variables. In most of the instruments the two beams are recombined before detection by a single detector and thus the beam can be distinguished for even beam splitting. 

Visible Spectroscopy Instrumentation

Visible Spectroscopy Applications

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The visible spectroscopy is considered as one of the best determining methods to distinguish impurity in organic molecules. The appearance of additional peaks due to impurities in any sample can be compared with the standard data and can also be compared against absorbance of specific wavelength. 

These can be utilised for identifying the impurity in any sample of organic molecules. The use of visible spectroscopy is also used for determination compounds quantitatively. These are determined on Beer Lambert based calculations.

The visible spectroscopy is very helpful in identifying and understand structures of organic molecules. These also help in getting the right picture of saturation or unsaturation along with hetero atoms in the molecules. 

The visible spectroscopy can help identify and characterise compounds which absorb UV radiation and the identification is carried out by comparing the spectrum of unknown samples with that of known samples.

The visible spectroscopy also helps in getting the correct dissociation constant of all kinds of acids and bases by plotting the graph between absorbance and wavelengths of various pH limits.

The visible spectroscopy can help in getting the correct kinetic readings of molecules. The visible spectroscopy also helps in determining the molecular weight which is considered to be one of the most common and easy of all determining tests.

The visible spectroscopy helps in getting / detect HPLC by comparative study between analyte and proportional concentration. The visible spectroscopy also help in getting the right pharmaceutical combination for specific dosage and treatment.

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