Spectrophotometery
The purpose of this lab experiment is to understand the uses of
spectrophotometry. This experiment concentrates on the uses of the
spectrophotometer, and using this instrument to specifically determine the
absorption spectrum of a cobalt chloride solution, a galactose solution, and to
determine an unknown concentration of a galactose solution. Modern biologists
frequently use the measurement of light absorption to determine concentration of
chemicals. The technique is called spectrophotometry. However, why is light
absorbed? Light may be simply scattered by particles, but this is extremely
important to the measurement of truly absorbed light. Light is the part of
electromagnetic radiation to which the human eye is sensitive. Light is energy,
and when absorbed by a chemical it results in a change in energy levels of the
chemical. The energy of light depends on its wavelengths. Longer wavelengths,
such as infrared, have less energy than shorter wavelengths, such as
ultraviolet. A molecule will absorb light energy when a wavelength exactly
matches the energy difference between two energy states of the molecule. A
spectrophotometer makes use of the transmission of light through a specific
solution to determine the concentration of a solute within a solution. It is
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. The design of a
single beam spectrophotometer involves a light source, a prism, a sample holder,
and a photocell. Connected to each are the appropriate electrical or mechanical
to control the illuminating intensity, the wavelengths, and for conversion of
energy received to readable data that can be recorded, which is known as a
voltage fluctuation. This voltage fluctuation is displayed digitally and
recorded for further analysis. The whole idea of spectrophotometery determining
the concentration of a compound is based upon Beer’s Law. Beer Law, or

Beer-Lambert Law is the relationship between absorbance and concentration of an
absorbing specimen. Applying Beer’s Law can be used to determine a solutes
absorption peek, and to plot the absorption spectrum on what is known as a

Beer’s Law plot or curve. The procedure to find the absorption spectrum of
cobalt chloride uses a Beer’s Law and spectrophotometry. The spectrophotometer
used specifically to this experiment is the spcectronic 20. As with any
experiment, a control is needed to base the experiment on. In this case,
distilled water will be used as the control. A cuvette, which is a small plastic
container used in the instrument to hold the solution, is filled with distilled
water. The cuvette is then place in the sample holder. At this time the machine
should be set at 400nm, and at this wavelength the transmittance should read

100%. If the instrument does not read 100%T, the instrument needs to be blanked,
or adjusted accordingly, until 100%T is achieved. Once the control is achieved,
the absorption level of cobalt chloride can know be determined. Taking a clean
cuvette, adding around 6ml of cobalt chloride to it, it is placed in the machine
and the wavelength is kept at 400nm. According to the data the spectrophotometer
reads, the optical density of cobalt chloride at a wavelength of 400nm is

0.03100. Once this information is recorded, the cuvette of cobalt chloride is
removed. The machine is then set to 410nm, and the control cuvette of distilled
water is placed back in the machine. The machine is then re-blanked, being sure
that it reads 100%T. Remove the cuvette of distilled water and place the cuvette
of cobalt chloride in the machine. At 410nm, the optical density of cobalt
chloride is 0.043000, and this information is recorded. The above procedure is
repeated continually, at wavelength settings that are at 10nm intervals. For
example, next would be 420nm, then 430nm, and so on. It is very important to be
sure to re-blank the spectrophotometer after every change of wavelength. Cobalt
chloride, at a wavelength of 510nm, has an optical density of 0.51200. As can be
seen in figures 1-1 and 1-2, this is the wavelength at which cobalt chloride
absorbs maximally. The next procedure involved in the experiment is to develop a
standard curve, using Beer’s Law, for galactose. Six test tubes are used for
the first part of the experiment. Each test tube should be clearly labeled 1
through 6 on the top of each tube, using a wax pencil. Also, one large beaker
should be filled with distilled water and sitting on a hot plate in preparation
for boiling. Six different concentrations of galactose are going to be needed,
corresponding to the six test tubes. The concentrations will consists of
galactose standard, distilled water, dinitrosalicylic acid, or all