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Spectrophotometry
| Question | Answer |
|---|---|
| What is Spectroscopy? | It is the branch of physical science involved with the measurement of the spectra, has had a profound role in the conversion of biology from the purely descriptive to more quantitative measurements |
| Why measurements of the absorbance of light by substances in solution are used in biochemistry? | Because many compounds of interest to biochemist absorb light in the ultraviolet, visible, or near infrared region. |
| What is a typical application of spectroscopy in biological research? | It is the measurement of the concentration of a substance (i.e. protein or nucleic acid) present in a solution based on the amount of visible or ultraviolet light absorbance by the substance. |
| What is a spectrophotometer? | It is an instrument that is designed to measure the absorbance of light. |
| In what consists the spectrophotometer? | It consists of a light source (usually white light or ultraviolet light) which is focused on a prism or other light-dispersive device. |
| what is the function of the prism? | The prism is designed to separate the white light into separate bands of radiant energy. Each different wavelength is selectively focused through a a narrow slit. This beam of light, called the incident beam, passes through the sample to be measure |
| What happens after the beam of light traverse the sample? | It emerges as the transmitted beam and strikes a photoelectric cell. If the substance has absorbed any of the incident light, the transmitted light will be reduced in the total energy content. |
| What happens when the transmitted light strikes the photoelectric cell? | It generates an electric current that is proportional to the intensity of the light striking it. |
| What does the Beer's law state? | The concentration of a light-absorbing solute is directly proportional to the absorbance over a given range of concentrations. a straight line is generated after plotting absorbance vs sample concentration |
| What is the use of the blank? | The blank contains the solvent where the biological molecules will be dissolve in order to avoid that the solvent itself absorbs light. |
| What is an absorption spectrum? | It shows the wavelengths of maximum absorption for the substance being studied |
| Why should a concentration curve be made at the Amax wavelength for a given compound? | A concentration curve should be made at the maximum wavelength for a given compound because the sample is most sensitive to concentration changes; therefore, the change in concentration will be measured more accurately. |
| How can a concentration curve be used to determine the molarity of a solution of unknown molarity? | by using the absorbance of the known solutions at the maximum wavelength in conjunction with the concentrations to obtain a function describing the relationship among the variable. That functions can then be used to find the concentration of the unknown. |
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