Question

1. Use the Beer-Lambert law to explain how a colorimetric assay works in the spectrophotometer. In your answer, define the following terms; chromophore, extinction coefficient, absorbance, transmission and reagent blank.

2. Most of the spectrophotometers in the teaching lab emit light of wavelengths between 400-700nm. However some spectrophotometers also emit light between 200-400nm. Why is this extended range useful? Provide a specific example.

3. How can you use a spectrophotometer to perform a qualitative analysis of a substance?

Answer 1

1. The procedure based on absorption of visible light is called colorimetry. The colorimeter is based on Beer-Lambert's law. According to the law, the absorption of light transmitted through the medium is directly proportional to the medium concentration. A beam of light with a specific wavelength is passed through a solution via a series of lenses in a colorimeter. The colored light is navigated to the measuring device. This analyzes the color compared to an existing standard. A microprocessor then measures the absorbance or percent transmittance. Higher the concentration of the solution, the more light will be absorbed. The amount of light absorbed can be calculated by measuring the difference between the amount of light at its origin and that after passing the solution. Transmission ranges from 0% for an opaque sample to 100% for a transparent sample. The results of known samples are plotted to compare and calculate the concentration of unknown samples.

If the substance absorbs light, specific peaks of absorbance will be exhibited. These are caused by the presence of chromophore in the substance. A chromophore is the chemical structure that absorbs light. The purine ring structure of ATP is an example of chromophore.

The extinction coefficient is the measure of the rate of transmitted light via scattering and absorption for a medium.

Transmittance is the fraction of incident light that is transmitted, or it is the amount of light that passes through the substance. Absorbance explains the other side of transmittance, that is, what amount of the light the sample absorbed.

A reagent blank is used to denote a small positive error in test results that comes from the reagents themselves. It is used to cancel out the absorbance of all the reagents in the sample except the component whose absorbance is to be measured.

2. Most of the spectrophotometers in the teaching lab emit light of wavelengths between 400-700nm. However, some spectrophotometers also emit light between 200-400nm. This means they can use light in the visible and adjacent ranges (UV range). The UV extends from 100–400 nm and the visible spectrum from 400–700 nm. Ultraviolet-visible spectroscopy is very versatile and able to detect nearly every molecule. This is suitable for analytes that can be dissolved in solvents like water, ethanol and hexane, such as transition metal ions, highly conjugated organic compounds, and biological macromolecules.

3. Spectrophotometry can be used in both qualitative and quantitative analysis of compounds. In qualitative analyses, Spectrophotometers record spectra of compounds by scanning broad wavelength regions to determine the intensity of the colour of the compound at each wavelength. They could be used to monitor the appearance of a product or disappearance of a reactant.