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 three. The
procedures for filling each test tube with the right concentrations are as
follows: Tube #1: 2ml distilled water, 2ml dinitrosalicylic Tube #2: 0.2ml
galactose, 1.8ml water, 2ml dinitrosalicylic acid Tube #3: 0.4ml galactose,
1.6ml water, 2ml dinitrosalicylic acid Tube #4: 0.6ml galactose, 1.4ml water,
2ml dinitrosalicylic acid Tube #5: 0.8ml galactose, 1.2ml water, 2ml
dinitrosalicylic acid Tube#6: 1.0ml galactose, 1.0ml water, 2ml dinitrosalicylic
acid Boil the water on the hot plate, and place all 6 test tubes in the water
for five minutes. After boiling for 5 minutes, remove the test tubes and add 7ml
of distilled water to each tube in order to cool and dilute the solution so the
machine can read it. Set the machine to the wavelength of 540nm, which is the
wavelength at which galactose absorbs maximally. Using test tube #1 as the
blank, pour about 6 to 7ml of the concentration onto a cuvette and blank the
machine, being sure it reads 100%T. Take the remaining 5 test tubes and add 6 to
7ml of each concentration into separate cuvetts. According to the
spectrophotometer, the first concentration has an optical density of 0.10300.
The sixth cuvette, having the highest concentration, has an optical density of
0.63800. As can be clearly seen by figures 2-1 and 2-2, this experiment proves
the higher the concentration of a solute in a solution, the greater the optical
density. The procedure to determine the unknown galactose concentration is
basically the same as before. 2ml of the unknown galactose solution are added to
2ml of dinitrosalicylic acid. It is then place in boiling water for 5 minutes.
Once removed, 7ml of distilled water are added to the test tube. In order to
standardize the spectrophotometer, a blank is needed, which would be the same
blank used in the preceding experiment. Next the machine is blanked at a
wavelength of 540nm. Next transfer around 6ml of the unknown galactose solution
into a clean cuvette and read the optical density. According to the machine, the
optical density of the unknown galactose solution at 540nm, is 0.335.
Spectrophotometry is important in science today because it shows the peak at
which a chemical absorbs the most light. The absorption spectrum can be used
almost like fingerprints, identifying unknown chemicals. It is also important
because it can determine unknown concentrations in solutions, say for example,
the amount of cocaine in a persons blood. For these reasons, it is possible to
see why spectrophotometry is one of the most widely used techniques in biology.