Light is either absorbed, reflected, or transmitted. The color of a substance depends on the wavelengths that it reflects. A substance that appears blue absorbs orange and reflects blue light.
The energy of light must be equal to the energy gap between the HOMO and LUMO.
Dyes typically contain conjugated double and single bonds. This results in a lower energy gap between the HOMO and LUMO, and therefore, a lower required energy of light is needed to excite an electron. Lower energy light has a longer wavelength, making most dyes able to absorb light in the visible range.
A spectrophotometer measures transmittance, or the amount of light that passes through a substance. This is related to the absorbance, or the amount of light absorbed by the substance. The spectrophotometer produces a plot of absorbance versus wavelength called an absorbance spectrum.
The concentration of a sample affects the absorbance through the Beer-Lambert law. Absorbance is dependent on the concentration times the molar attenuation coefficient and the path length. Therefore, as concentration increases, so does absorbance. A standard curve of a sample’s absorbance versus known concentrations can derive a linear equation that can be used to find the concentration of an unknown sample based on the absorbance.
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