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 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.