Colorimeter
A spectrophotometer or colorimeter makes use of the transmission of light through a solution to
determine the concentration of a solute within the solution. A spectrophtometer differs from a
colorimeter in the manner in which light is separated into its component wavelengths. A
spectrophotometer uses a prism to separate light and a colorimeter uses filters.
Both are based on a simple design of passing light of a known wavelength through a sample and
measuring the amount of light energy that is transmitted. This is accomplished by placing a
photocell on the other side of the sample. All molecules absorb radiant energy at one wavelength
of another. Those that absorb energy from within the visible spectrum are known as pigments.
Proteins and nucleic acids absorb light in the ultraviolet range. The following figure
demonstrates the radiant energy spectrum with an indication of molecules which absorb in
various regions of that spectrum.
The design of the single beam spectrophotometer involves a light source, a prism, a sample
holder and a photocell. Connected to each are the appropriate electrical or mechanical systems to
control the illuminating intensity, the wavelength, and for conversion of energy received at the
photocell into a voltage fluctuation. The voltage fluctuation is then displayed on a meter scale, is
displayed digitally, or is recorded via connection to a computer for later investigation.
Spectrophotometers are useful because of the relation of intensity of color in a sample and its
relation to the amount of solute within the sample. For example, if you use a solution of red food
coloring in water, and measure the amount of blue light absorbed when it passes through the
solution, a measureable voltage fluctuation can be induced in a photocell on the opposite side. If
now the solution of red dye is diluted in half by the addition of water, the color will be
approximately 1/2 as intense and the voltage generated on the photocell will be approximately
half as great. Thus, there is a relationship between the voltage and the amount of dye in the
sample.
Given the geometry of a spectrophotometer, what is actually measured at the photocell is the
amount of light energy which arrives at the cell. The voltage meter is reading the amount of light
TRANSMITTED to the photocell. Light transmission is not a linear function, but is rather an
exponential function. That is why the solution was APPROXIMATELY half as intense when
viewed in its diluted form.
We can however monitor the transmission level and convert it to a percentage of the amount
transmitted when no dye is present. Thus, if 1/2 the light is transmitted, we can say that the
solution has a 50% Transmittance. Note that it is always relative to a solution containing no dye.
Transmittance is the relative percent of light passed through the sample.
What makes all of this easy to use, however, is the conversion of that information from a percent
transmittance to an inverse log function known as the Absorbance (or Optical Density).
The Beer-Lambert Law
Definiton
Absorbance: The negative log of the transmittance.
A = - log T EQUATION G.1
This value is more useful in spectrophotometry than transmittance, because of plot of absorbance
vs concentration yields a straight line. A plot of transmittance vs concentration is an exponential.
The - log calculates the inverse of transmittance, so that absorbance increases with increasing
concentration. Transmittance would decrease as we increased the amount of red dye in our
example. The relationship of Absorbance to concentration was shown by two biochemists to
follow the equation for a straight line, y = mx +b, where m is the slope of the line and b is the y
intercept. If the measurement is made in such a way that b = 0 (that is, a solution containing no
dye has no absorbance), and if we substitute Absorbance for y, concentration for x, and variant
for m, we arrive at the formulation of the Beer-Lambert Law:
A = C
where A = absorbance
C = concentration
= the extinction coefficient
Note that variant is equal to the slope of the straight line which will result from a plot of
absorbance (y axis) vs concentration (x axis).
To use a spectrophotometer it is necessary to establish a known series of dilutions containing
known quantities of a solute. One of these will contain no solute and is known as the blank . It is
used to adjust the instrument to read 100% transmittance or 0 absorbance. In use, a 0%
transmittance value (infinite absorbance) is established by placing a curtain between the light
source and the photocell. Electronic control is then exerted so that the meter will read 0%
Transmittance on its scale. The blank sample (containing no solute or dye) is inserted, the curtain
opened and the meter readjusted to read 100% transmittance. All other measures are then made
by merely inserting the samples into the light path and measuring the % transmittance. Most
spectrophotometers have a built in means of direct conversion of this reading to absorbance.
After recording the absorbance for a series of standards, a plot is made of the absorbance value
(y axis) vs the concentration (x axis). The slope of the line is the extinction coefficient.
Note that this may be computed directly by rearrangement of the Beer-Lambert law to
= A/C EQUATION G.2
This value can be calculated for each reading and the average taken as the value of variant.
Remember that this value is a constant. Thus, once calculated, it can subsequently be used to
determine an unknown concentration by one more rearrangement of the Beer-Lambert law
C = A/ EQUATION G.3
Any measured value of A can be readily converted to a corresponding concentration merely by
dividing the absorbance by .
The use of the Beer-Lambert law is easy to visualize with red food coloring. It is not as easy to
visualize, but none the less, just as accurate to measure wavelengths of light which are not
visible. Either infra red or ultraviolet can be used. UV is more useful to biologists since many
molecules (all proteins and nucleic acids) absorb ultraviolet light. The only changes that need to
be made is the use of quartz cuvettes instead of glass tubes. Glass absorbs UV light and thus is
inappropriate for use in a UV spectrophotometer. An instrument capable of using visible light
(usually with a tungsten or halogen lamp source) and UV light is known as a UV/Vis
Spectrophotometer.
