1. Required readings:
· Text: Any biochemistry textbook.
Eg. McKee & McKee “Biochemistry: The Molecular Basis of Life”
· Laboratory notes
OBJECTIVES: At the conclusion of this unit the student will be expected to:
1. Pre-lab discussion on the program for the session and familiarization with the spectrophotometer.
2. Prepare a standard curve for inorganic phosphate using the spectrophotometer.
3. Determine the concentration (unknown) of a solution of inorganic phosphate.
4. Determine the absorption spectrum of p-nitrophenol
5. Determine the molar extinction coefficient for p-nitrophenol and learn what it means and how it can be used.
6. Post-lab discussion on the results from the laboratory session and what follow up is needed.
Prepare a laboratory report for assessment (15%).
LAB 3: SPECTROPHOTOMETRY FOR THE QUALITATIVE AND QUANTITATIVE MEASUREMENT OF BIOCHEMICAL COMPOUNDS
To apply the principles of spectrophotometry to the measurement of biochemical compounds.
If white light is passed through a solution containing a coloured compound, certain wavelengths of light are selectively absorbed. The resultant colour observed is that of the transmitted light. Measuring light absorption aids in both the identification and quantification of substances, i.e. the absorption spectrum is characteristic of a particular substance and the amount of absorption by that substance at a given wavelength is a function of the concentration of the substance.
The concentration of a biochemical compound can be determined by measuring the light absorbed by a solution using a spectrophotometer. This instrument consists of two parts: a spectrometer and photometer. By means of a radiant light source and monochromator, the spectrometer emits discrete light frequencies usually in the region from 200 to 800 nm. The photometer consists of a photoelectric cell, which converts light to an electric signal, and a galvanometer which records the potentials induced in the photoelectric cell. These potentials are transcribed on a scale, which reads out as per cent transmittance or absorbance.
Consider a ray of light of initial intensity Io passing through a solution in a transparent vessel: some of the light is absorbed, so that the intensity of the transmitted light I is less than Io. The ratio of I to Io is known as the transmittance and depends on the path length of the light through the solution.
This is expressed in Lambert’s Law which states that: “When a ray of monochromatic light passes through an absorbing medium, its intensity decreases exponentially as the length of the medium increases”.
A similar law by Beer relates the transmittance to the concentration of the solution: “When a ray of monochromatic light passes through an absorbing medium, the intensity decreases exponentially as the concentration of the absorbing medium increases”. These two laws are conveniently combined in the Beer‑Lambert Law to give the expression:
log10 Io/I = e c l
where l is the light path length; c is the concentration of the solution; and e is the molar extinction coefficient or molar absorptivity.