Principles of Spectrophotometry

Principles of Spectrophotometry
A spectrophotometer consists of two instruments, namely a spectrometer for producing

light of any selected color (wavelength), and a photometer for measuring the intensity of
light. The instruments are arranged so that liquid in a cuvette can be placed between the
spectrometer beam and the photometer. The amount of light passing through the tube is
measured by the photometer. The photometer delivers a voltage signal to a display
device, normally a galvanometer. The signal changes as the amount of light absorbed by
the liquid changes.
If development of color is linked to the concentration of a substance in solution then that
concentration can be measured by determining the etent of absorption of light at the
appropriate wavelength. !or eample hemoglobin appears red because the hemoglobin
absorbs blue and green light rays much more effectively than red. The degree of
absorbance of blue or green light is proportional to the concentration of hemoglobin.
"hen monochromatic light (light of a specific wavelength) passes through a solution
there is usually a quantitative relationship (#eer$s law) between the solute concentration
and the intensity of the transmitted light, that is,
where I sub % is the intensity of transmitted light using the pure solvent, I is the intensity
of the transmitted light when the colored compound is added, c is concentration of the
colored compound, l is the distance the light passes through the solution, and k is a
constant. If the light path l is a constant, as is the case with a spectrophotometer, #eer$s
law may be written,
where k is a new constant and T is the transmittance of the solution. There is a
logarithmic relationship between transmittance and the concentration of the colored
compound. Thus,
The &.'. is directly proportional to the concentration of the colored compound. (ost
spectrophotometers have a scale that reads both in &.'. (absorbance) units, which is a
logarithmic scale, and in ) transmittance, which is an arithmetic scale. As suggested by
the above relationships, the absorbance scale is the most useful for colorimetric assays.
Using a Spectronic 20 spectrophotometer
The *pectronic +% spectrometer is widely used in teaching laboratories. The specific
instructions will differ with other models, but the principles remain.
,. The instrument must have been warm for at least ,- min. prior to use. The power
switch doubles as the .eroing control.
+. /se the wavelength knob to set the desired wavelength. 0treme wavelengths, in
the ultraviolet or infrared ranges, require special filters, light sources, and1or
sample holders (cuvettes).
2. "ith the sample cover closed, use the .ero control to ad3ust the meter needle to
4%4 on the ) transmittance scale (with no sample in the instrument the light path
is blocked, so the photometer reads no light at all).
5. "ipe the tube containing the reference solution with a lab wipe and place it into
the sample holder. 6lose the cover and use the light control knob to set the meter
needle to 4%4 on the absorbance scale.
-. 7emove the reference tube, wipe off the first sample or standard tube, insert it and
close the cover. 7ead and record the absorbance, not the transmittance.
8. 7emove the sample tube, read3ust the .ero, and recalibrate if necessary before
checking the net sample.
"hy use a reference solution9 6an$t you 3ust use a water blank9 A proper reference
solution contains color reagent plus sample buffer. The difference between the reference
and a sample is that the concentration of the assayable substance in the reference solution
is .ero. The reference tube transmits as much light as is possible with the assay solution
you are using. A sample tube with any concentration of the assayable substance absorbs
more light than the reference, transmitting less light to the photometer. In order to obtain
the best readability and accuracy, the scale is set to read .ero absorbance (,%%)
transmission) with the reference in place. :ow you can use the full scale of the
spectrophotometer. If you use a water blank as a reference, you might find that the assay
solution alone absorbs so much light relative to distilled water that the usable scale is
compressed, and the accuracy is very poor.

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Principles of Spectrophotometry

A spectrophotometer consists of two instruments, namely a spectrometer for producing

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