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 concentrations will consists of galactose standard, distilled water, dinitrosalicylic acid, or all three. The procedures for filling each test tube with the right concentrations are as follows:
Tube #1: 2ml distilled water, 2ml dinitrosalicylic
Tube #2: 0.2ml galactose, 1.8ml water, 2ml dinitrosalicylic acid
Tube #3: 0.4ml galactose, 1.6ml water, 2ml dinitrosalicylic acid
Tube #4: 0.6ml galactose, 1.4ml water, 2ml dinitrosalicylic acid
Tube #5: 0.8ml galactose, 1.2ml water, 2ml dinitrosalicylic acid
Tube#6: 1.0ml galactose, 1.0ml water, 2ml dinitrosalicylic acid
Boil the water on the hot plate, and place all 6 test tubes in the water for five minutes. After boiling for 5 minutes, remove the test tubes and add 7ml of distilled water to each tube in order to cool and dilute the solution so the machine can read it. Set the machine to the wavelength of 540nm, which is the wavelength at which galactose absorbs maximally. Using test tube #1 as the blank, pour about 6 to 7ml of the concentration onto a cuvette and blank the machine, being sure it reads 100%T. Take the remaining 5 test tubes and add 6 to 7ml of each concentration into separate cuvetts. According to the spectrophotometer, the first concentration has an optical density of 0.10300. The sixth cuvette, having the highest concentration, has an optical density of 0.63800. As can be clearly seen by figures 2-1 and 2-2, this experiment proves the higher the concentration of a solute in a solution, the greater the optical density.
The procedure to determine the unknown galactose concentration is basically the same as before. 2ml of the unknown galactose solution are added to 2ml of dinitrosalicylic acid. It is then place in boiling water for 5 minutes. Once removed, 7ml of distilled water are added to the test tube. In order to standardize the spectrophotometer, a blank is needed, which would be the same blank used in the preceding experiment. Next the machine is blanked at a wavelength of 540nm. Next transfer around 6ml of the unknown galactose solution into a clean cuvette and read the optical density. According to the machine, the optical density of the unknown galactose solution at 540nm, is 0.335.
Spectrophotometry is important in science today because it shows the peak at which a chemical absorbs the most light. The absorption spectrum can be used almost like fingerprints, identifying unknown chemicals. It is also important because it can determine unknown concentrations in solutions, say for example, the amount of cocaine in a persons blood. For these reasons, it is possible to see why spectrophotometry is one of the most widely used techniques in biology.