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 con

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.

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 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 #4: 0.6ml galactose, 1.4ml water, 2ml dinitrosalicylic acid

Cobalt chloride, at

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