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Materials and Methods

For
the
experiment,
two
instruments were used in order to test the
accuracy and precision of the operators.
In order to test for the accuracy
of the operators, the red and blue
micropipettors with its white and blue
tip,
made
from
polypropylene,
respectively, were used to transfer 1000
mL of distilled water and the required
volumes of bromophenol blue (uL) to
the microcentrifuge tubes. One member
only did the transfer of the set volumes
of 0.5, 1.0, 1.5, 2.0 and 2.5 L of
Bromophenol blue and 1000 mL of
distilled water. Once the liquid has been
transferred to the microcentrifuge tube, it
was then vortexed to mix the contents of
the tube until the dye immersed in the
solution.
After the tubes have been
prepared, the spectrophotometer was
warmed up and set to a wavelengthmeasured 540nm. With distilled water
(blank solution), the spectrophotometer
was set to zero. Once calibrated, the
absorbance of the dye solutions was read
by the spectrophotometer starting from
the least concentrated to the most
concentrated.
In order to test for the precision
of operators, again, the red and blue
micropipettors, with its white and blue
tips, respectively, were utilized to
transfer the required volume of 2.5 L to
the microcentrifuge tubes. All the
members took part in this process as the
precision of each student in utilizing the
micropipettor
was
taken
into
consideration for this portion of the
experiment. Each member, using the
blue micropipettor with its blue tip

transferred
1000
mL
to
the
microcentrifuge tube, then afterwards, a
volume of 2.5 L of Bromophenol blue
was placed in each of the five tubes.
Once the solution has been prepared, the
tubes were then vortexed in order to mix
the contents of the tube and immerse the
dye into the solution, the tube was
placed to the microcentrifuge. After the
tubes have been prepared, the tubes,
together with the reagent blank was
placed in the spectrophotometer, which
read the absorbance of the dye solutions
starting from the least concentrated to
the most concentrated.
Sample Preparation
1000 mL of distilled water was
transferred to an empty microcentrifuge tube
using the blue micropipettor and 0.5 L of
Bromophenol blue was transferred to the
same microcentrifuge tube using the red
micropipettor. Once the tube has been
prepared, the tube was vortexed for 5
seconds to mix the contents of the solution.
The spectrophotometer, set to the
wavelength of 540 nm was calibrated and
set to zero using the blank solution. Once
calibrated, the absorbance of the tube
containing water and Bromophenol blue was
read.

Results and Discussion

Table 1. Data table of average


absorbance (A) read per L of
Bromophenol blue
The average absorbance read by
the spectrophotometer per volume of
Bromophenol blue per group were
compared, and as the data table presents,
it can be concluded that as more volume
of Bromophenol blue is added, the
absorbance read increases. As seen in
table 1, the numbers marked red show an
inconsistency with the results as the
absorbance values read by the
spectrophotometer is fluctuating thus
providing an inaccurate and non-precise
data. This can be seen in the data of
group 5 and 6. A result of not properly
mixing the Bromophenol blue or errors
with transferring the right amount of
colorant can account for the fluctuations
with the absorbance levels, thus may
result in the error of the data.
As for group 1, the absorbance
level of 0.013 A is very far from the
absorbance level of 0.031 A yet only
having a large difference with the
amount of volume added, thus shows the
non-precision of data taken. As for the
numbers colored in purple, under group
3, the values highlighted show the most
precise results our of all the 8 groups. As
for accuracy, group 8, having the
absorbance of 0.185 A, is the most
accurate as its value is closest to to set
absorbnce of 0.183 A.
The succeeding linear graphs
show
the
relationship
between
concentration of Bromophenol blue and
absorbance (nm). Using the formula
C1V1=C2V2, concentration 2 can be
solved for. C1 is measured as (1.25%
(g/w)/100) is multiplied to the volume of
Bromophenol blue added to the

microcentrifuge tube (V1) then is


divided by the total volume of distilled
water and Bromophenol blue added to
the microcentrifuge tube (V2), thus
solving for C2. Once the x data
(absorbance,
nm)
and
y
data
(concentration, g/ L) have been
tabulated, the points were plotted in a
linear graph. After plotting these points,
a linear regression graph was drawn
using the values of the slope (m), y
intercept (b), and a. In order to generate
a linear graph, the volume of
Bromophenol blue was converted to
concentration, as previously stated in
order to show the linear relationship
between concentration and absorbance.
Thus, the linear equation is as follows,
y=mx+b.

Figure 1. Linear Regression Graph of


Absorbance x Concentration
Figure 1 shows the linear
regression graph of the absorbance x
concentration.

It can be depicted from the


graphs that there is a linear relationship
between concentration and absorbance
such as an increase in absorbance (A)
leads to an increase in concentration (g/
L). This direct relationship can be
explained using the Beers-Law. The
Beers law can be represented by the
formula A=ebc; a as the absorbance, b as
the path length, e is the molar
absorptivity or extinction component and
c as the concentration. The linear graphs
in figure 1 show linear relationship, as
seen in the graphs of group 2, 3, 4 and 8.
Group 1 and 7 shows linearity in its
graph but as clearly shown, there is a
portion in the graph which shows a
number of outliers from the straight line

Asides measuring the slope, y


intercept and a value using the x and y
points, the correlation coefficient r, or
the Pearson Product Moment Sample
Coefficient of Correlation are also
measured. As stated by Mendenhall et al
(2012), this is the measure of strength of
the linear relationship between two
variables, and that the largest possible
value for r is 1. The r values of groups
2,3,4 and 8 are also close to the
correlation coefficient, 1, which means
that the x and y points generated the
best-fitting line, regression line, and
shows the direct relationship of the two
variables
(concentration
and
absorbance). Group 1 and 7s r values on
the other hand are further from 1 due to
the outliers in its
regression line.

thus any point not plotted in the linear


line is called an outlier. Group 5 and 6
both show errors in its graph as group 5
made use of the volume values as its y
values instead of the concentration of
Bromophenol blue thus having a
different graph among all other groups.
As for group 6, a huge error has been
made with the absorbance reading as
there is a negative absorbance reading.
Absorbance cannot be less than 0 and
should not go beyond 1 if absorbance
does become negative or go beyond one,
this is due to the wrong calibration of the
spectrophotometer or a problem with the
solution that is being read by the
spectrophotometer.

With the data presented and the


equations indicated per linear graph, it
can be concluded that as concentration
increases, the absorbance of the solution
increases, and as the absorbance
increases, the %T decreases as
transmittance is the fraction of radiant
energy that having entered a layer of
absorbing material reaches it father
boundary.
Figure 2. Bar Graph showing the standard
deviation of each student in relation to the
average absorbance

Standard deviation determines


how far a value (+/-) is from the mean
(x) of its data set. Figure 2 illustrating
the bar graph that shows the level of the
average
absorbance
read
by
spectrophotometer per each student per
group including the standard deviation
from the mean or the average
absorbance. The bars, on top of the cells
represent the standard deviation from
the mean. An accurate data would
present only a small deviation, while a
precise data will have bars almost the
same length.
It can be seen that the bars/lines
representing the standard deviation in
group 3, 4, 5, 6, 7 and 8 go beyond the
boundary or range of the standard
deviation. The usual accepted percent
error is supposedly 10% but based on the
interpretations, based on the computed
standard deviations, it can be said that
the percent error in most groups greatly
exceed 10%; this of which is seen in the
groups mentioned above. With the data
presented in figure 2, it can be concluded
that only group 1 and 2 were precise
with their measured absorbance levels
per student.
Conclusion
Data should be accurate and precise
but it can be concluded that some groups
were not precise in transferring the L of
Bromophenol blue and water to tube errors
and had inaccurate results. For this reason,
human errors and errors with the operators
(such as in calibration) can be the reason
why such errors occurred during the
experiment.
Fankhauser, D.B. (2007). Spectrophotomer use.
Retrieved from:
http://biology.clc.uc.edu/fankhauser/Labs/Microbiolog
y/Growth_Curve/Spectrophotometer.htm

Friedl, S. (n.d). Evaluating data : precision, accuracy


and error. Retrieved from:
http://study.com/academy/lesson/evaluating-dataprecision-accuracy-error.html