When white light passes through or is reflected by a colored substance, a characteristic portion of the mixed wavelengths is absorbed. The remaining light will then assume the complementary color to the wavelength(s) absorbed. Absorption of nm light renders a substance yellow, and absorption of nm light makes it red. Green is unique in that it can be created by absorption close to 400 nm as well as absorption near 800 nm.

Spectrophotometer: A spectrophotometer is a photometer (a device for measuring light intensity) that can measure intensity as a function of the color, or more specifically, the wavelength of light. Spectrophotometers deal with visible light. The light source shines through the sample. The sample absorbs light. The detector detects how much light the sample has absorbed. The detector then converts how much light the sample absorbed into a number

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Io Ii Io/IiA = log(Io/Ii) 100%= %= %= %= %= %= %=0.00#DIV/0! Beer ’ s Law: A = log(Io/Ii) = Ebc But what is Absorbance?? Abs itself is the log(Io/Ii), where Io = light transmitted through a blank It is always set to 100% Ii = light transmitted through the sample It can be from 0 – 100%

So, let’s review dilutions: Direct Dilutions Serial Dilutions Precision: how close values are too each other vs. Accuracy: how close values are to true value Quick way to determine precision is the coefficient of variation or %CV = (stdev/mean) x 100 Lower %CV is better precision (<5% OK)

You will formulate a hypothesis to answer this question. What is the optimal wavelength to measure the absorbance of water colored with blue, red, yellow, and green food coloring? (You will be given solutions of different colors). Record dilutions and absorbencies. Find the optimal wavelength.

Next, you will choose one colored solution to perform serial and direct dilutions. You will know the appropriate wavelength based on your previous experiment. You will be asked to perform 1:10, 1:100, 1:1000 and 1:10,000 dilutions using serial and direct methods. You will do these in TRIPLICATE (3 times). Which of the dilution methods are more accurate? Precise? Use the spectrophotometer to find out.