Biol 1440, Principles of Biology I

Introduction to Spectrophotometry

We are going to use spectrophotometric techniques to measure the concentration of solutes in solution. To do this, we will measure the amount of light that is absorbed by the solutes in solution in a cuvette in the spectrophotometer. Spectrophotometry takes advantage of the dual nature of light. Namely, light has:

  1. a particle nature which gives rise to the photoelectric effect (used in the spectrophotometer)

  2. a wave nature which gives rise to the visible spectrum of light

A spectrophotometer measures the intensity of a light beam after it is directed through and emerges from a solution. As an example, let's look at how a solution of copper sulfate (CuS04) absorbs light.

 spec1.jpg (6955 bytes)

The red part of the spectrum has been almost complete absorbed by CuSO4 and blue light has been transmitted. Thus, CuSO4 absorbs little blue light and therefore appears blue.

In spectrophotometry, we can gain greater sensitivity by directing red light through the solution because CuSO4 absorbs strongest at the red end of the visible spectrum. But to do this, we have to isolate the red wavelengths!

SPECTRUM OF VISIBLE LIGHT
How do you isolate the red wavelengths of light? In a spectrophotometer, a light source gives off white light which strikes a prism, separating the light into its component wavelengths:

spec2.jpg (6992 bytes)
                                                                                   
   l = Wavelength, nm=nanometers

Now, all we have to do is isolate the red wavelengths and pass them through the CuSO4 solution and measure the amount of red light absorbed. The spectrophotometer will actually measure the amount of light transmitted by the CuSO4 solution.

The important point to note here is that, colored compounds absorb light differently depending on the l of incident light.

IMPORTANT:
Why do solutes absorb photic (light) energy within characteristic wavelength bands? Atoms and molecules absorb light energy in such a manner that an alteration in atomic or molecular energy state occurs. In light absorption changes can occur in atomic or molecular energy states in one of three ways:

  1. by the boosting of e- to higher energy levels

  2. by increasing the rate at which atoms within a molecule vibrate relative to one another

  3. by increasing the rotational speed of molecules in the solution

DESIGN OF A SPECTROPHOTOMETER

spec3.jpg (11453 bytes)

Io is the incident light and represents 100% of the light striking the cuvette. I is the transmitted light. This is the light which has not been absorbed by the solution in the cuvette and will strike the phototube. The photons of light which do strike the phototube will be converted into electrical energy. This current which has been produced is very small and must be amplified before it can be efficiently detected by the galvanometer. The deflection of the needle on the galvanometer is proportional to the amount of light which originally struck the phototube and is thus an accurate measurement of the amount of light which has passed through (been transmitted by) the sample.

THE BLANK
In order to effectively use a spectrophotometer we must first zero the machine, we do this using "the blank." The blank contains everything except the compound of interest which absorbs light. Thus, by zeroing the machine using "the blank," any measured absorbance is due to the presence of the solute of interest.

ABSORPTION SPECTRUM
Different compounds having dissimilar atomic and molecular interactions have characteristic absorption phenomena and absorption spectra which differ. The point (wavelength) at which any given solute exhibits maximum absorption of light (the peaks on the curves on the figure below) is defined as that compounds particular
lmax.

In Lab 1, you will learn the fundamentals of operating a spectrophotometer and you will use this instrument to measure the absorption of light by a compound, betacyanin. Learning this technique will help you in future labs.