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Spectrophotometry

QuestionAnswer
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.
Created by: daysivp