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Introduction to spectrophotometry lab

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The spectrophotometer is an essential tool for biologists and chemists in analyzing chemical and biological samples. Gaining familiarity with its operating protocols and understanding what its outputs mean are very important in the development of lab technique for students of cell biology. This experiment will help laboratory students gain experience in using the spectrophotometer. Basic Laws of Light Absorption – For a uniform absorbing medium (solution: solvent and solute molecules that absorb light) the proportion of light radiation passing through it is called the transmittance, T, and the proportion of light absorbed by molecules in the medium is absorbance, Abs. Transmittance is defined as:

T = I/Io where: Io= intensity of the incident radiation entering the medium. I = intensity of the transmitted radiation leaving the medium. T is usually expressed as percent transmittance, %T:

%T = I/Io x 100
The Beer-Lambert law (or Beer’s law) is the linear relationship between absorbance and concentration of an absorbing species. The general Beer-Lambert law is usually written as: A = a() * b * c
where A is the measured absorbance, a() is a wavelength-dependent absorptivity coefficient, b is the path length, and c is the analyte concentration. When working in concentration units of molarity, the Beer-Lambert law is written as: A = * b * c
where is the wavelength-dependent molar absorptivity coefficient with units of M-1 cm-1.

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