SPECTROPHOTOMETRY White light contains all wavelengths of light in the visible region of the EM. A substance appears colored because it absorbs light.

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Presentation transcript:

SPECTROPHOTOMETRY White light contains all wavelengths of light in the visible region of the EM. A substance appears colored because it absorbs light at one or more wavelengths in the visible region and transmits the remaining wavelengths. The human eye, a type of EM radiation detector, sees the transmitted light.

COMPLEMENTARY COLORS The color of the light transmitted is complementary to the color of light absorbed. From the color wheel, you can see that a substance that absorbs blue wavelengths transmits orange wavelengths, and so appears orange to the human eye. What color does a red object absorb?

SPECTROPHOTOMETRY A spectrophotometer uses this concept to determine the relative absorbance or transmittance of wavelengths of light by a solute in solution. A wavelength of light is specified, and then beamed through a small test tube (cuvette) of solution to determine the relative amount transmitted or absorbed.

SPECTROPHOTOMETRY

SPEC 20D+ Display: λnm • % • MODE Cuvette chamber Wavelength adjustment On/Off; Transmission adjustment Absorbance adjustment

CALIBRATING THE SPEC Set the WAVELENGTH (large knob on top) to 500nm Set MODE to transmittance With the sample compartment empty and the cover closed, adjust the ZERO knob (on the left) until transmittance reads 0%

RUNNING A BLANK Fill the BLANK cuvette, the reference trial. Wipe off the outside with a Kim-Wipe and insert into the sample holder Adjust the light control knob (RIGHT) until the transmittance registers 100%

RUNNING A SAMPLE Create the test sample (S + E). Quickly wipe the cuvette. Insert into the Spec. Close the cover. Read & record % transmittance. Transmission below 30% indicates a too concentrated sample. Dilute with DI water. Recalibrate to 100% transmittance with the blank between trials.