Introducing Spectrophotometry with
Enzymes
Elana Abernathy
BIOL 1406-81026
Abstract
This experiment teaches one how to use a spectrophotometer, a tool used to determine the presence and
measure of a dissolved chemical’s concentration, and a micropipette, a tool used to measure and transfer
extremely small amount of chemicals. The spectrophotometer and micropipette are later used to practice
running an enzyme assay which will be done in later experiments. First, the scientist must calibrate the
spectrophotometer by using a blank solution. Then, the spectrophotometer is used to test two blue-dye
solutions to determine the wavelength of maximum absorbance, found to be 633.8nm of .034841 absorbance.
This data is collected using the graph shown on the monitor when using the spectrophotometer and then
setting the tool to capture the data. After this is completed, the absorbance and concentration of the solutions
is measured the same way with the addition of a new graph. This graph, concentration on the x-axis and
absorbance on the y-axis, should obtain a straight line when the collection of the data is completed. Using this,
one can see which sample had the highest concentration and highest absorbance of the blue-dye solutions,
which where both sample two. After learning to accurately use a spectrophotometer, the practice of the
enzyme essay is ready to start. To prepare, three micro-tubes are prepared with the micropipette, the
spectrophotometer is calibrated, and the “Absorbance vs. Time” setting is selected for collection mode. Once
the one-hundred and twenty second timer is starts, micro-tubes two and three are mixed and are transferred
to a clean cuvette. The data is collected after twenty seconds of the mixing, then the linear fit’s slope is used to
find the value if the enzyme reaction rate, .002576, completing the experiment.
Introduction
To determine the presence and measure the concentration of dissolved chemicals, biologists use
a tool called a spectrophotometer. The tool has a light inside that shines through a filter and
passes through the sample onto a light-sensitive phototube that produces an electric current
proportional to the amount of light that lands on it. How the spectrophotometer works is based
on the principle that different atoms and molecules each absorb a unique pattern of light’s
wavelength. An oxidized guaiacol solution, as used in this experiment, appears a certain way due
to the amount of light absorbed in the substance based on the darkness of its color. The darker
the solution, the greater its absorbance (amount of light absorbed by a solution). It makes sense,
then, that a tube of a clear substance is the blank used to calibrate the spectrophotometer. Beer’s
Law is used in this experiment as the amount of light absorbing particles in solution is directly
proportional to the absorbance reading, forming the desired straight line in the second part of the
experiment. Along with the introduction of the spectrophotometer, the micropipette is introduced
in the practice of the enzyme assay as the common volume unit for this work is a microliter (μl).
To better visualize the small size, it can be pointed out that one microliter is 1/1000 of a
millimeter. The enzyme assay practice looks at factors that affect the reaction rate of the enzyme
peroxidase, which is extracted from turnips. This enzyme oxidizes guaiacol, as mentioned
before, and changes its color to brown therefore allowing one to follow the reaction with the
spectrophotometer. I hypothesized that as the solutions get darker the concentration also
increases, which will lead the absorbance will increase as well.
Materials and Methods
The first experiment in this procedure was to find the wavelength of maximum absorbance of the
blue dye. To find this value, the spectrophotometer was calibrated using the blank-filled cuvette
and two test tubes are prepared. Test tube one was filled with 9.9 ml dH₂O + 0.1 ml blue dye and
test tube two filled with 9.0 ml dH₂O = 1.0 ml blue dye. Test tube one was used to measure the
maximum absorbance of wavelength by collecting data in “Absorbance vs. Wavelength” mode
and observing the chromatogram produced. Beer’s Law was practiced in the second experiment
of this procedure, as the “Absorbance vs. Concentration” mode was observed. A blank cuvette
was used to calibrate the tool at first, then test tubes one and two were inserted into the
spectrophotometer and their data was collected and shown using “Linear Fit”. This resulted in a
straight line, as stated in the introduction. At last, the final experiment of the procedure was to
practice an enzyme assay, which must be completed precisely. First, the same steps were used to
determine the maximum wavelength of absorption of oxidized guaiacol solution as they were
used to measure the blue-dye solutions. After this, three micro-tubes were prepared using the
micropipette and the spectrophotometer was set up for the enzyme run, calibrated and set for the
“Absorbance vs. Time” collection mode. One-hundred twenty seconds were given to complete
the run, including mixing the substances and placing the mixture into a cuvette then collecting
the data. After all data is collected, the “Linear Fit” tool is used to show the slope of the
produced line, which is also the value of the enzyme reaction rate.
Results
No graphs were saved from this procedure, so the results will be in written words. After
completing the first experiment, which was finding the wavelength of maximum absorbance, it
was shown that the maximum absorbance for the blue-dye solution was 633.8nm of .034841
absorbance. This value was found by observing the maximum value, or point, on the graph made
on the chromatogram. Relating to Beer’s Law, the second experiment, which was to determine
which solution had the highest concentration and absorbance reading, results were shown when
the linear graph concluded that the solution in cuvette two had the higher value in both factors.
To be reminded, this solution was 9.0 ml dH₂O = 1.0 ml blue dye. Moving on to the final part of
the procedure, the maximum wavelength of absorption for oxidized guaiacol was found to be
400nm for .1974 absorbance which then leads to the end of the procedure, the value of enzyme
peroxidase reaction rate. This was determined after the practice enzyme assay as the value of the
slope in the linear fit, .002576, concluding the experiment.
Discussion and Conclusions
After such a complex procedure, one can grow to understand the concept of absorbance and the
basic principles of spectrophotometry. One may also learn how to operate a spectrophotometer
and SpectroVIS, the program used in these experiments to read the spectrophotometer. The
practice enzyme assay was also beneficial as it prepared for real enzyme assays in future
procedures. Most importantly, from this experiment one can better comprehend how the change
in absorbance with time can create a graph to find the enzyme reaction rate by looking at the
data’s slope, as the data is represented as a line using linear fit tool. Improvements that could be
done in the future mostly deals with the timing of the practice enzyme run, as it can be
inconsistent even with the timer used. The micropipette could also use more practice before
making measurements substantial to the experiment as they need to be exact. Overlooking this,
though, the hypothesis proved to be correct throughout the experiment as the darker and more
concentrated solutions absorbed more light than the clearer ones.
References:
1. Perez-Ramos, Sara, Couvillon, Stacie; Laboratory Manual for Biol 1406, Molecular and
Cellular Aspects of Life, Lab 5