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You are on page 1of 6

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.

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

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.

Absorbance x Concentration

Figure 1 shows the linear

regression graph of the absorbance x

concentration.

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

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.

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.

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

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

and error. Retrieved from:

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

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