Spectrophotometery Essays - Spectroscopy, Spectrophotometry, Cuvette

Spectrophotometery The purpose of this lab experiment is to understand the uses of spectrophotometry. This experiment concentrates on the uses of the spectrophotometer, and using this instrument to specifically determine the absorption spectrum of a cobalt chloride solution, a galactose solution, and to determine an unknown concentration of a galactose solution. Modern biologists frequently use the measurement of light absorption to determine concentration of chemicals. The technique is called spectrophotometry. However, why is light absorbed? Light may be simply scattered by particles, but this is extremely important to the measurement of truly absorbed light. Light is the part of electromagnetic radiation to which the human eye is sensitive. Light is energy, and when absorbed by a chemical it results in a change in energy levels of the chemical. The energy of light depends on its wavelengths. Longer wavelengths, such as infrared, have less energy than shorter wavelengths, such as ultraviolet. A molecule will absorb light energy when a wavelength exactly matches the energy difference between two energy states of the molecule. A spectrophotometer makes use of the transmission of light through a specific solution to determine the concentration of a solute within a solution. It is 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. The design of a single beam spectrophotometer involves a light source, a prism, a sample holder, and a photocell. Connected to each are the appropriate electrical or mechanical to control the illuminating intensity, the wavelengths, and for conversion of energy received to readable data that can be recorded, which is known as a voltage fluctuation. This voltage fluctuation is displayed digitally and recorded for further analysis. The whole idea of spectrophotometery determining the concentration of a compound is based upon Beer's Law. Beer Law, or Beer-Lambert Law is the relationship between absorbance and concentration of an absorbing specimen. Applying Beer's Law can be used to determine a solutes absorption peek, and to plot the absorption spectrum on what is known as a Beer's Law plot or curve. The procedure to find the absorption spectrum of cobalt chloride uses a Beer's Law and spectrophotometry. The spectrophotometer used specifically to this experiment is the spcectronic 20. As with any experiment, a control is needed to base the experiment on. In this case, distilled water will be used as the control. A cuvette, which is a small plastic container used in the instrument to hold the solution, is filled with distilled water. The cuvette is then place in the sample holder. At this time the machine should be set at 400nm, and at this wavelength the transmittance should read 100%. If the instrument does not read 100%T, the instrument needs to be blanked, or adjusted accordingly, until 100%T is achieved. Once the control is achieved, the absorption level of cobalt chloride can know be determined. Taking a clean cuvette, adding around 6ml of cobalt chloride to it, it is placed in the machine and the wavelength is kept at 400nm. According to the data the spectrophotometer reads, the optical density of cobalt chloride at a wavelength of 400nm is 0.03100. Once this information is recorded, the cuvette of cobalt chloride is removed. The machine is then set to 410nm, and the control cuvette of distilled water is placed back in the machine. The machine is then re-blanked, being sure that it reads 100%T. Remove the cuvette of distilled water and place the cuvette of cobalt chloride in the machine. At 410nm, the optical density of cobalt chloride is 0.043000, and this information is recorded. The above procedure is repeated continually, at wavelength settings that are at 10nm intervals. For example, next would be 420nm, then 430nm, and so on. It is very important to be sure to re-blank the spectrophotometer after every change of wavelength. Cobalt chloride, at a wavelength of 510nm, has an optical density of 0.51200. As can be seen in figures 1-1 and 1-2, this is the wavelength at which cobalt chloride absorbs maximally. The next procedure involved in the experiment is to develop a standard curve, using Beer's Law, for galactose. Six test tubes are used for the first part of the experiment. Each test tube should be clearly labeled 1 through 6 on the top of each tube, using a wax pencil. Also, one large beaker should be filled with distilled water and sitting on a hot plate in preparation for boiling. Six different concentrations of galactose are going to be needed, corresponding to the six test tubes. The