A spectrophotometer is an instrument designed to detect the amount of radiant light energy absorbed by molecules. To do this, the instrument must have five basic components: a light source; a prism or diffraction grating; an aperture or slit; a detector (a photoelectric tube); and a digital meter to display the output of the phototube. The arrangement of these parts is shown below.
When light is reflected from a diffraction grating, it is split into its component colors or wavelengths, which then diverge. Sections of the projected spectrum can be either blocked or allowed to pass through the slit so that only one wavelength will pass to the other sections of the spectrophotometer (The position of the grating is adjustable so that the region of the spectrum projected on the slit can be changed.). Light that passes through the slit travels to the phototube, where it creates an electric current proportional to the number of photons striking the phototube. If a digital meter is attached to the phototube, the electric current output can be measured and recorded. The scale is usually calibrated in two ways: percent transmittance, which runs on a scale from 0 to 100; and absorbance, or optical density units, which runs from 0 to 2.
Before the light-absorbing properties of a solution can be measured, two adjustments on the spectrophotometer are necessary. First, the diffraction grating must be adjusted so that the desired wavelength of light passes through the slit. This is usually the wavelength of light that is most absorbed by the compound under consideration. Secondly, the output of the phototube must be adjusted or calibrated to correct the drift in the electronic circuits and dirt or contaminating the material in the light path between the source and the detector.
Because all solutions of chemical compounds absorb light of specific wavelengths, spectrophotometry can be useful in identifying compounds. Furthermore, because the amount of light absorbed is proportional to the concentration of a compound, spectrophotometry is also useful in determining concentrations.
Beer's Law explains the relationship between absorbance, at a given wavelength and concentration.
A=ebC Where: A=absorbance e=molar exticntion coefficient b=length of the light path C=concentraion of the solute
A=ebC
Where:
A=absorbance
e=molar exticntion coefficient
b=length of the light path
C=concentraion of the solute
Note that the relationship between absorbance and concentration is linear. As concentration increase the absorbance also increases. This relationship allows one to convert an absorbance value into a concentration.
Molar Extinction Coefficent Example
Chlorophyll a has the following molar extinction coefficients:
eM662 = 8.4 x104 M-1 cm-1
A solution of plant pigment has an absorbance of 0.632 ODUs at 662nm. To calculate the concentration of chlorophyll a and B carotene in your sample you would use the following equation:
0.632=(8.4 x 104M-1 cm-1)(1cm)(X M)
X=7.5 x 10-6
A. Turn on the spectrophotometer (left-hand knob on the front of the instrument (A)) and allow it to warm up for at least 15 min.
B. Adjust the wavelength to the appropriate value. The knob on the right top (C)of the instrument controls the wavelength, which is indicated at the left of the digital display.
C. With the sample holder empty and the lid closed, adjust the Zero Adjust Knob (A) until the instrument reads 0% on the transmittance scale. Be sure that the display function is set to transmittance, if not push the "Mode" button until the display is set to transmittance.
D. Carefully insert the appropriate blank tube (cuvette) into the sample holder (E) and close the cover. Be sure you are using a cuvette with white markings. The cuvette's outside surface must be dry and clean, including free of fingerprints!! Use a Kimwipe to clean the cuvette before inserting. The white markings should line up with the notch on the sample holder. It is important to line up the markings. The cuvettes will be scratched otherwise.
E. Adjust the 100% Adjust Knob (B), on the right front of the instrument, until the display reads 100% on the transmittance scale.
F. Remove the blank cuvette and immediately insert the sample cuvette as described in step d above. Do not change any instrument setting! Switch the display to read absorbance by pushing the "Mode"(D) button.
G. Record the value indicated on the absorbance scale.
H. Repeat this procedure for additional cuvettes or wavelengths as required. Always adjust the blank transmittance to 100% before inserting and reading a new set of cuvettes.
Now Complete the Pre-Lab Quiz