Laboratory Equipment: Spectrophotometry

Background and Use

"It's the white house with green shutters."
"That coffee looks awfully strong."

The interaction of light with matter allows us to use sight to identify and quantify matter. Scientific instruments extend the useful wavelengths of electromagnetic radiation beyond the visible range and make quantification more reliable. This is the function of the spectrophotometer, one of the fundamental instruments of bench biology. The spectrophotometer passes light of a known wavelength through a sample and measures the amount of light reaching a photocell on the other side.

Illustration of how a spectrophotometer works.
Spectrophotometry light sourceDiffraction grating on refracting prism.Intensity of incident light.  To correct for absorbance of light by the container, solvent, etc. the instrument is set to 100%T using a blank identical to the sample, but lacking the compond of interest.Held in a quartz, glass or plastic container called a curvette.  Path length is usually 1 cm.Intesity of unaborbed light passing through the sample.

Purple Blue Aqua Green Yellow Red Spectrophotometer control nob

Select one of the samples and vary the concentration. Graph the relationship between %T and concentration. Then graph the relationship between absorbance and concentration. Write a sentence for each curve, describing its shape.

Select one of the samples and vary the wavelength of light. Plot absorbance against wavelength. Repeat with a different sample. Describe the relationship between the color of the sample and the color of light that passes through it most easily.

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