In the term of chemistry spectroscopy is specifically refers to the study of the interaction between matter and electromagnetic radiation Electromagnetic radiation comprised of gamma rays X rays ultraviolet UV visible light vis infrared light as well as longer wavelength microwaves television waves and radio waves The forming of these waves are coming from the vibrations of electric and magnetic fields that pass through the space In fact spectroscopy is originated from the research of the dispersion of visible light according to its wavelength by a prism This concept was then further extended deeply into many branches after conducting variety experiments including the interaction with radiative energy as a function of its wavelength or frequency Actually the light energy is made up of many frequencies and wavelengths The studies of spectroscopy will mainly measure the change in absorption or emission of an analyte to different wavelength in order to determine its interaction with each tested wavelengths of electromagnetic radiation especially for visible light and ultraviolet light The absorbance of a chemical substance can be determined by measuring the intensity of light in a straight beam light that passed through the sample solution which is known as the spectrophotometry method The basic principle behind it is that each compound will be capable to absorb or transmit light over a certain range of wavelength In this aspect the amount of a chemical substance can be figured out through the obtained measurement of absorbance Absorption of infrared radiation relates to vibrational or rotational excitations of molecules Absorption of visible and ultraviolet light will result in the excitation of electrons changing the electron distribution in the molecules or ions of the absorbing material As a consequence of light absorption in the beam of light that emerges from the sample has a diminished intensity symbolized by I
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Spectrophotometry is considered as one of the most useful methods of quantitative analysis that widely applied in various fields such as chemistry physics biochemistry material and chemical engineering The extent to which radiation is absorbed is related to the nature and concentration of absorbing material present in a sample as well as the wavelength of the radiation employed Cuvette is a small transparent vessel that used for putting the analyte in a spectrophotometer There are two major classes of the spectrophotometer which is single beam and double beam A double beam spectrophotometer contained two light paths where one path with reference sample and the other path with the test sample comparing the light intensity between these two light paths Conversely a single beam instruments will only able to measure the relative light intensity of the beam before and after a test sample is inserted which is simpler and more compact to the double beam spectrophotometer Depending on the range of wavelength of the light source it can be further classified into two different types which is UV visible spectrophotometer and IR spectrophotometer When a beam of monochromatic light is passing through the sample solution the intensity of the light beam that enters the solution is called the incident intensity and is given the symbol I0 The incident intensity is essentially referred to the number of photons per second that can enter the sample solution Since some photons or light will be leave the sample that entered it there will be the loss of transmitted light that can be detected by the detector in spectrophotometer The fraction of light that actually passes the sample is represented by the ratio I I0 which is called the transmittance t
For the convenience in comprehension the quantity of transmittance are generally expressed as a percentage To give an illustration a certain solution with 10 transmittance will only allow 40 nm wavelength passes through the cuvette when 400 nm wavelength is given and the rest will be absorbed by the sample In the experimental conditions the transmittance of solution containing a light absorbing material are all possibly related to the Beer's Law log I I0 log T A abC In the equation listed above T is the transmittance that expressed as a decimal value 10 transmittance will correspond to t 0 10 and A is known as absorbance without unit C is the concentration of the analyte b is the length of the light path through the absorbing solution and a is the absorptivity a value which depends on both the nature of the light absorbing substance as well as the wavelength of light For instance the molar absorptivity or the molar extinction has units of L mol 1cm 1 when b is expressed in cm and C in mol L units For the ease to memorize it the Beer s Law is frequently written as A abc a is actually in symbol of epsilon ε The graph of absorbance versus concentration is called a calibration curve It is frequently used to correlate absorbance with the molar concentration of the absorbing species the transition metal ion As a result the concentration of unknown solution can be measured by measuring its absorbance in this graph As a matter of fact Beer's Lambert Law does not work on over concentrated sample This is due to the fact that the calibration graph will not be able to obtain a straight line since the absorbance of the analyte become unexpectable In this experiment the Cr3 is acted as the analyte or sample solution Firstly five Cr3 solutions with desired concentration will be measured by using the dilution formula M1V1 M2V2 After that an unknown will be given by the lecturer The graph will be plotted after obtaining all the result that getting from the spectrophotometer The higher the concentration or density of the sample the more the amount of light absorbed
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