Microplate reader spectrophotometer

The Beer-Lambert Law A=abc

• Now let us look at the Beer-Lambert law and explore it's significance.

• A is absorbance (no units)

• “a” is the molar absorbtivity with units of L mol-1 cm-1

• “b” is the path length of the sample - that is, the path length of the cuvette

(cm)

• “c” is the concentration of the compound in solution, expressed in mol L-1

Light Source: Must be able to generate many wavelengths. The light goes through a monochromator excitation filter to make the light beam a single wavelength.

Absorbance or transmittance can be measured with a microplate reader.

• Sample: The sample is in a microplate which is transparent to the light. This means only the sample interacts with the single wavelength of light.

• Detector: The detector is a photoelectric cell. When light strikes the detector, a flow of electrons (a current) is created in the detector. The current generated is proportional to the light intensity.

• The detector functions as a result of the light transmitted through the emission filter.

Example: The absorbance maxima of DNA is at 260 nm. This is what we would set our reader at.

The absorbance maxima of this substance is 300nm. This is the wavelength that the microplate reader should be set at. You should know this ahead of time!

The Absorbance Maxima for Food Coloring

The Whole Process

Using a Spectrophotometer

Light Source: Must be able to generate many wavelengths. The light goes through a monochromator excitation filter to make the light beam a single wavelength.

Absorbance or transmittance can be measured with a spectrophotometer.

• Sample: The sample is in a cuvette which is transparent to the light. This means only the sample interacts with the single wavelength of light.

• Detector: The detector is a photoelectric cell. When light strikes the detector, a flow of electrons (a current) is created in the detector. The current generated is proportional to the light intensity.

• The detector functions as a result of the light transmitted through the emission filter. Readings are absorbance or transmittance.

Example: The absorbance maxima of DNA is at 260 nm. This is what we would set our reader at.

The Absorbance Maxima for Food Coloring