Spectrophotometer (Lilach Cary)
Author
Lilach Cary, Albert Einstein Academy
Principles
In biology the spectrophotometer measures the concentration of organic molecules in solution by shining polarized light through a sample and measuring the absorbance or transmittance of light.
Standards
Communicate scientific and technical information about why the molecular-level structure is important in the functioning of designed materials
Apply scientific principles and evidence to provide an explanation about the effects of changing the temperature or concentration of the reacting particles on the rate at which a reaction occurs.
Materials needed
cuvettes or small test tubes
sample that needs to be analyzed
distilled water to create a blank
test tube rack
Procedure
Before you measure a solution in an experimental cuvette, first insert the blank cuvette into the machine. The spectrophotometer will register an absorbance value for the blank.
Now adjust the lower right hand dial to manually set the meter back to zero absorbance.
Take the blank out and now you are ready to read the absorbance of the material in solution without having your data affected by the absorbance of the cuvette or the solvent.
Also be aware that fingerprints on the cuvettes can affect your readings, so all cuvettes need to be wiped clean with a Kimwipe before inserting them into the spectrophotometer.
Explanation
A spectrophotometer is an instrument that measures either the transmittance of light through a solution or the absorbance of light by a solution. Light can either pass through an object or be stopped by an object which is in its path. Transmittance is an arithmetic scale that is divided into one hundred equal parts, so transmittance can be expressed as a percent. A value of 100% transmittance means that all the light has passed through the material in the cuvette and none was absorbed. A value of 0% transmittance means that no light passed through the sample in the cuvette. Absorbance is a logarithmic scale with unequal divisions. A value of 0.0 absorbance means that no light has been absorbed by the materials and all the light has been transmitted. A reading all the way at the other end of the scale means that all the light has been absorbed by the material.
The spectrophotometer can be used to assess how much protein is in solution by measuring how much light is absorbed by the protein. You can do this by "subtracting" the absorbance due to the cuvette and the solvent in which the protein was dissolved. In order to do this you would prepare a second cuvette with just the solution, for instance, water. This is called the "blank." In general, the blank is a cuvette which contains everything that is in the sample cuvette, except the one material whose absorbance you are trying to measure.
Questions
1. Explain what an Absorption Spectrum is.
The amount of light absorbed at a given wavelength.
2. Why is it important to perform a procedure to determine the Absorption Spectrum before beginning a lab?
This needs to be done to determine which wavelength of light is absorbed best by a given material.
A Standard Curve is used to determine how much of a test tube containing an unknown amount of a substance is in a solution by comparing it with different known concentrations of the substance and constructing a curve on graph paper.
Everyday examples of the principles illustrated
Light traveling through different liquids like water
Clarity of water--measuring amount of particles in a water sample
Photos
3. Explain what a Standard Curve is.
