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Accuracy of Absorbance

GROUP 9 4BIO8
POLICINA RELOPEZ
PUA PHEE RAMOS
Absorbance

• amount of light absorbed by a sample

• used in relating the amount of light absorbed to the concentration of


the absorbing molecule

• inversely related to transmittance:


• The more a particular wavelength of light is absorbed by a
substance, the less it is transmitted
Accuracy

▶ How close or near a measured value is to the standard value

▶ Obtained ‘accurate’ values may not be close to each other

▶ Focuses more on the quality of the data


Methodology
Testing Accuracy In using a Micropipettor (Procedure 1)
A. Testing Accuracy of Operators
1. The spectrophotometer was set at 540 nm.
2. 1 ml of distilled water was placed in each five microcentrifuge
tubes.
3. The following amount of bromphenol blue that was added to each
sucessive tube are 0.5, 1.0, 1.5, 2.0, 2.5 uL.
4. Each tube was subjected to the vortex until the dye is evenly
distributed in the solution.
5. The spectrophotometer was then set to zero with distilled water.
6. The absorbance of the dye solution was noted starting with the
least concentrated to the most concentrated
7. The readings were recorded in Table 1-2.
Principles
▶ Maximum Transmittance = 3, 99.9% ; 2, 99% ; 1, 90%
▶ Plastic cuvette
▶ Not glass because glass will absorb the light from the prism.

▶ Beer-Lambert’s Law (Beer Law) - Formula of the absorbance.


▶ A=log10 (incident intensity / transmitted intensity)

▶ Wrong placement of the cuvette in the spectrophotometer


▶(thick side and thin side)
▶ Can be one of the ways to prevent the lowering of the accurate results in
the experiment.
* The accuracy will be determined in the average of the trials done in the experiment,
while the precision in the standard deviation; wherein the results can be:
Accurate but not precise; Precise but not accurate.
Precision is independent of accuracy. The result can be very precise but
inaccurate.
Accuracy of operator

Bromphenol
Absorbance (540 nm)
blue volume
(µL) Trial 1 Trial 2 Trial 3 Average Stddev

0.5 0.144 0.441 0.446 0.344 0.173

1.0 0.613 0.606 0.682 0.634 0.042

1.5 0.761 0.767 0.696 0.741 0.039

2.0 1.015 0.906 0.863 0.928 0.078

2.5 1.057 1.046 1.118 1.034 0.039