Spectrophotometry

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SPECTROPHOTOMETRY
Herman, Harmon Chris T.

1Prof. Meynard Austria, of Chemical Engineering, Chemistry and Biotechnology, Mapua Institute of Technology, Chm171L/A1, School of Chemical Engineering, Chemistry and Biotechnology, Mapua Institute of Technology, Experiment # 4 [pic]

ABSTRACT

The objectives of this experiment are to examine the components of a simple spectrophotometer- the Jenway 6100 & Perkin Elmer Lambda 40. As well as to determine the absorption spectrum of a solution and to prepare a Beer’s Law Plot. In the spectrometer used, the light source is imaged upon the sample. A fraction of the light is transmitted or reflected from the sample. The light from the sample is imaged upon the entrance slit of the monochromator. The monochromator separates the wavelengths of light and focuses each of them onto the photo detector sequentially. The absorption spectrum is determined to select the optimal wavelength for analyzing the cobalt (II) nitrate. The optimal wavelength for measuring absorbance is that wavelength that is most absorbed by the compound. The Beer’s Law Plot showed and differentiate the proportionality of the Concentration vs Absorbance graph and the Wavelength vs Absorbance graph. The first graph is linear while the latter one is characterized by the wavelength at which the absorbance is the at the greatest.

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INTRODUCTION
A spectrophotometer or colorimeter makes use of the transmission of light through a solution to determine the concentration of a solute within the solution. A spectrophotometer differs from a colorimeter in the manner in which light is separated into its component wavelengths. A spectrophotometer uses a prism to separate light and a colorimeter uses filters. Both are based on a simple design of passing light of a known wavelength through a sample and measuring the amount of light energy that is transmitted. This is accomplished by placing a photocell on the other side of the sample. All molecules absorb radiant energy at one wavelength of another. Those that absorb energy from within the visible spectrum are known as pigments. Proteins and nucleic acids absorb light in the ultraviolet range. The following figure demonstrates the radiant energy spectrum with an indication of molecules which absorb in various regions of that spectrum. The design of the single beam spectrophotometer involves a light source, a prism, a sample holder and a photocell. Connected to each are the appropriate electrical or mechanical systems to control the illuminating intensity, the wavelength, and for conversion of energy received at the photocell into a voltage fluctuation. The voltage fluctuation is then displayed on a meter scale, is displayed digitally, or is recorded via connection to a computer for later investigation. Spectrophotometers are useful because of the relation of intensity of color in a sample and its relation to the amount of solute within the sample. For example, if you use a solution of red food coloring in water, and measure the amount of blue light absorbed when it passes through the solution, a measureable voltage fluctuation can be induced in a photocell on the opposite side. If now the solution of red dye is diluted in half by the addition of water, the color will be approximately 1/2 as intense and the voltage generated on the photocell will be approximately half as great. Thus, there is a relationship between the voltage and the amount of dye in the sample. Given the geometry of a spectrophotometer, what is actually measured at the photocell is the amount of light energy which arrives at the cell. The voltage meter is reading the amount of light TRANSMITTED to the photocell. Light transmission is not a linear function, but is rather an exponential function. That is why the solution was APPROXIMATELY half as intense when viewed in its diluted form. We can however monitor the transmission level and convert it to a percentage of the amount transmitted...
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