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Date of Experiment: October 18, 2017

Title: Data Analysis

Aim:

To analyze the fluorimetry experiment performed by two groups of students and to
determine which group produced the most accurate results.

Abstract:

A data analysis was conducted based on the results produced by two groups of students in
order to determine the most accurate results. By doing this, a calibration graph for each group was
devised with a best fit line drawn, using the, triplicate results, plot of absorbance (OD units) against
the substrate concentration (mM). Consequently, it was found that group A produced the most
accurate results in comparison to group B, as group A produced standard deviations that showed
precision in their method.

Introduction:
Fluorometry is an analytical method for detecting and measuring fluorescence in
compounds such as protein and nucleotides, which are usually colored using fluorescent dyes or
naturally contains fluorophores. To determine the presence of compounds, a specific instrument
called the fluorimeter or spectrophotometer is used. The fluorimeter is a device used to measure
the fluorescence (the visible or invisible radiation) of a substance, by measuring its intensity and
wavelength distribution of emission spectrum after an incident radiation with a shorter wavelength
has passed through the substance which causes excitation of the molecules. Fluorescence, in
essence, is a property whereby a molecule emits light at a specific wavelength when irradiated by
a light of shorter wavelength. Therefore, the use of a fluorimeter or spectrophotometer is able to
measure the emittance of light.
The device is able to do this because of its strategically placed compartments. A lamp
provided the source of light, from which beams of light are able to strike the diffraction grating,
which operates like a prism. The diffraction grating separates the light into its component
wavelengths; therefore, it is rotated so that only specific wavelengths of light reaches the exit slit.
Thenceforth, light interacts with the sample and from this point, the detector is able to measure the
DATA ANALYSIS| Page |1

transmittance and absorbance of light, of the sample. Transmittance is the amount of light that
passes through the sample, completely, and strikes the detector; whereas, absorbance is the
measurement of the light that is absorbed by the sample. Furthermore, the detector senses the light
being transmitted through the sample and converts it to a digital representation.
With this functionality of the spectrophotometer, the Beer-Lambert’s law can be applied to
the analysis of the samples. The law is written as A=E lc, where A is the absorbance, E is the

extinction coefficient, l is the length of the path through which the light traveled, and c is the
concentration of the sample. From this it can be seen where the concentration of the sample is
directly proportional to the absorbance, therefore, as the concentration increases so should the
absorbance. Because of this proportionality, the fluorescence of a sample can be measured and the
concentration of an unknown sample can be determined.
Therefore, fluorimetry, is a useful technique to measure the light intensity of a sample
containing fluorophores or fluorescent dyes, by measuring its absorbance. From this, the
proportionality of the concentration and the absorbance can be evaluated and be used to measure
the concentration of an unknown sample.

Methodology:
A fluorometry assay conducted by two groups of students, group A and group B, was
analyzed to determine which group produced the better results. Each group carried out the test in
a triplicate procedure, using these results, tables containing the triplicate absorbances and the
average absorbances were constructed in order to determine the better results. Additionally, a
calibration curve featuring a best-fit line was devised for each group and the standard deviations
were further determined, from which the group with the more precise results, and thus the more
accurate/better results, can be determined.
DATA ANALYSIS| Page |2

Results:
Table 1. showing the absorbance in OD units 500nm obtained by group A in their investigation on
the amount of protein produced using horse radish peroxidase with luminol. Furthermore, the table
contains the triplicate absorbance at various substrate concentration, the average absorbance along
with the standard deviation

0 mM 20 mM 40 mM 60 mM 80 mM
Trial 1/ OD units 500nm Blank 0.253 0.479 0.729 0.900
Trial 2/ OD units 500nm Blank 0.215 0.459 0.704 0.980
Trial 3/ OD units 500nm Blank 0.250 0.461 0.777 1.080
Average/ OD units 500nm - 0.239 0.466 0.737 0.987
Standard deviation/ OD units 500nm - ± 0.021 ± 0.011 ± 0.041 ± 0.090
Standard deviation
_
S. D.= ∑ (x- x )2
n-1
n
_ = 3
x = average absorbance
________________________
S. D20mM = (0.253-0.239)2 + (0.215-0.239)2 + (0.250-0.239)2
3-1
________________
S. D20mM = (0.014)2 + (- 0.024)2 + (0.011)2
2
= ± 0.021

________________________
S. D40mM = (0.479-0.466)2 + (0.459-0.466)2 + (0.461-0.466)2
3-1
________________
S. D40mM = (0.013)2 + (- 0.007)2 + (- 0.005)2
2
= ± 0.011

________________________
S. D60mM = (0.729-0.737)2 + (0.704-0.737)2 + (0.777-0.737)2
3-1
________________
S. D60mM = (-0.008)2 + (- 0.033)2 + (0.040)2
2
= ± 0.041
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________________________
S. D80mM = (0.900-0.987)2 + (0.980-0.987)2 + (1.080-0.987)2
3-1
________________
S. D80mM = (-0.087)2 + (- 0.007)2 + (0.093)2
2
= ± 0.090
DATA ANALYSIS| Page |4

Table 2. showing the absorbance in OD units 500nm obtained by group B in their investigation on
the amount of protein produced using horse radish peroxidase with luminol. Furthermore, the table
contains the triplicate absorbance at various substrate concentration, the average absorbance along
with the standard deviation

0 mM 20 mM 40 mM 60 mM 80 mM
Trial 1/ OD units 500nm Blank 0.213 0.330 0.620 0.540
Trial 2/ OD units 500nm Blank 0.135 0.423 0.748 0.750
Trial 3/ OD units 500nm Blank 0.161 0.326 0.859 0.769
Average/ OD units 500nm - 0.170 0.360 0.742 0.686
Standard deviation/ OD units 500nm - ± 0.040 ± 0.055 ± 0.120 ± 0.127
Standard deviation
_
S. D.= ∑ (x- x )2
n-1
n
_ = 3
x = average absorbance
________________________
S. D20mM = (0.213-0.170)2 + (0.135-0.170)2 + (0.161-0.170)2
3-1
________________
S. D20mM = (0.043)2 + (- 0.035)2 + (-0.009)2
2
= ± 0.040

________________________
S. D40mM = (0.330-0.360)2 + (0.423-0.360)2 + (0.326-0.360)2
3-1
________________
S. D40mM = (-0.030)2 + (0.063)2 + (- 0.034)2
2
= ± 0.055

________________________
S. D60mM = (0.620-0.742)2 + (0.748-0.742)2 + (0.859-0.742)2
3-1
________________
S. D60mM = (-0.122)2 + ( 0.006)2 + (0.152)2
2
= ± 0.120
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________________________
S. D80mM = (0.540-0.686)2 + (0.750-0.686)2 + (0.769-0.686)2
3-1
________________
S. D80mM = (-0.146)2 + (0.064)2 + (0.083)2
2
= ± 0.127
DATA ANALYSIS| Page |6

Discussion:
From the graphs plotted, it was seen that concentration and absorbance were directly
proportional, which is in agreement with the Beer-lambert’s law incorporated in fluorometry.
When the results were compared, it was found that group A had the results which were in better
agreement with the Beer-lambert’s law as it gave a straight line, with outliers relatively close to
the line; whereas group B gave line with outliners well beyond the line. All the trials done by group
A was consistently increasing in absorbances as the concentrations increased. For group B,
however only trial two followed the law even though the difference between the 60mM and 80mM
concentrations had absorbances, 0.748 and 0.750 OD units respectively, which differed by only
0.002 OD units. For trials 1 and 2, it was noted that there were consistent increases up to 60mM,
but, at 80mM there were decreases in the absorbances, each having 0.540 OD units and 0.769 OD
units, for the trials 1 and 3 respectively. This deviation, can furthermore be explained by Beer-
Lambert Law where the direct proportionality of concentration to absorbance begins to taper off
or becomes indirectly proportional due to increases in concentration. This can be related to group
B’s results by considering that the concentrations of the enzyme/protein in the standards/samples
may have been more concentrated due to inaccuracies in their preparation. Fluctuations may have
also resulted from there being smudges on the cuvette window or the cuvette not being dried, as
well as at 80mM the samples may not have been given enough incubation time to form coloured
complexes.
The graph for group A straight line of best fit was obtained with the outliers being in close
proximity to the line. The graphical representation shows the agreement with the Beer-Lambert’s
law, as well as, the precision of their results; the line, also, cuts through one point on the graph.
For group B as a best-fit line was also obtained from the scatter plot of the triplicate results. The
line did not pass through any of the points; furthermore, the points that were further away from the
line shows that as concentration increases the absorbance of the sample begins to taper off or
decreases after a time. The proximity of the points to the line shows that there was not much
precision in the results, hence, accuracy is reduced.
Based on the graphs and standard deviation of the results obtained it can be said that group
A had more accurate results, thus having the best results.
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Conclusion:
From the analysis of the results of fluorimetry obtained by two groups, A and B, through the use
of graphs and calculation of standard deviation, it was found that group A had more accurate
results.

Questions:
1. Which of the data set would be more suitable for determining the concentration of protein
in an unknown sample? Why?

The data obtained by group A is more suitable to determine the protein concentration in the
unknown sample. This is because, group A produced better results in terms of precision, based on
the standard deviation calculated. With the greater precision of group A’s results, there is a greater
chance of it being more accurate in comparison to group B. In addition, there was a steady linear
increase in absorbance as the concentration for each trial increased, which is synchronized with
the Beer-Lambert’s law where absorbance is directly proportional to the concentration. However,
in group B there were multiple anomalies observed.

2. What could account for the variation seen for both data set?

The variation in the absorbances for each group may have been due to slight variations in the
concentration of each standard, smudges on the cuvette window as well as unproperly dried
cuvettes (resulting in dilution of the substrate). Furthermore, any errors in solution making,
pipetting, massing, etc. gets propagated as more solutions are made.

3. Why should you not extrapolate the calibration curve beyond the data presented?

Data should not be extrapolated beyond the data presented because there were no true values
obtained at those points. Therefore, the line should not be extrapolated because it will not reflect
the true results.

4. Why do some colorimetric/fluorometric assays have non-linear calibration curves?

Many calibration curves are linear and can be fit with the basic equation y = m x + c, where m is
the slope and c is the y-intercept. However, not all curves are linear in some cases, the plot may
begin linear and plateaus after a certain concentration of substrate, especially reactions that are
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investigating the effects of substrate and enzyme concentration on the rate of the reaction. Since
these reactions usually utilize enzymatic reactions, there is always the probability that the enzyme
has been denatured or there were more/less enzymes present when compared to the substrate
concentration resulting in the maximum rate of reaction, thus a plateau or curve. Also, there is the
possibility of reagent depletion which limits the complex formation. In addition, the
colorimetric/fluorometry assays incorporates the Beer-Lambert’s law, which uses a specific range
of concentration, so if the concentration is too high, then the measured absorbance would not
correspond to the concentration causing a plateau.

5. What would be the major similarities between the fluorometric assay conducted here and
a colorimetric assay to determine protein concentration?

Major similarities include:

 The use of standards which may be similar in composition to those being tested at various
concentrations.
 A spectrophotometer is used to measure the absorbance of the complexes in both assays.
 A calibration curve of absorbance against concentration is deduced in both instances to
estimate the concentration of a substance in an unknown sample by comparing the
unknown to a set of standard samples of known concentration.
 Both assays use a specific wavelength of light to measure the absorbance.

6. Why are assays typically conducted in duplicate and triplicate?

The number of replicates you run for each test, determines if there is any variation in the
technique carried out. It is, usually, done in triplicate because it is the minimum number to
have a standard deviation. This way you are able to ensure that your technique gives the same
result each time you test the same sample. Furthermore, if the procedure is done only twice,
and two different results are obtained, it would be difficult to identify the error. Whereas with
the procedure performed thrice, and two results agree, it is easier to determine the one which
fell out of agreement. Therefore, the triplicate only ensure that you will be able to compare
your different samples between them.
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7. Explain the difference between a duplicate/triplicate and a replicate. Which is most useful
and why?
 A replicate is a repeated experiment performed at the same time that the target sample
is undergoing the test. That is, there is one sample, and three replicates of sample one,
so in all there are four samples undergoing the experiment at the same time.
 A duplicate, on the other hand, is a repeat of the entire experiment, so, if there are three
replicates, and the procedure should be duplicated. Then, two of the four samples, i.e.
sample one and its three replicates, can undergo the procedure which will result in
duplicated results since the samples a replicate of each other (having the same make-
up). A triplicate is similar to a duplicate; however, the procedure is done three times.
Usually a replicate is used to test for standard error and deviation, while duplicates are
used to test for reproducibility, though they can be used interchangeably.
 The most useful technique is the triplication of the experiment. This is because with
this, any parallax error that arise in the procedure maybe identified and accounted for
via the standard deviation.

References:
Calibration Curves | Protocol. (2013). Jove.com. Retrieved 21 October 2017, from
https://www.jove.com/science-education/10188/calibration-curves
Fluorescence Fundamentals | Thermo Fisher Scientific. (2011). Thermofisher.com. Retrieved 22
October 2017, from https://www.thermofisher.com/jm/en/home/references/molecular-probes-the-
handbook/introduction-to-fluorescence-techniques.html
Sanz-Medel, A., & Pereiro, R. Atomic absorption spectrometry. Retrieved 21 October 2017, from
https://www.chem.uci.edu/~dmitryf/manuals/Fundamentals/Fluorescence%20Spectroscopy.pdf