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Absorption spectrophotometry is a way for determining the concentration of a substance in

solution. How does it work? Lets say when a dissolved substance is put in the machine, it
will absorb light of specific wavelengths characteristic of that substance. When the light of
those wavelengths are passed through, a fraction of the light will be absorbed into the sample.
A spectrophotometer is a machine that is used for the measurement of transmittance of
solutions, transparent solids like glass, meaning it can determine the difference in the amount
of light entering and leaving the sample. This difference in the original and transmitted light
is known as absorbance. In this case, spectrophotometry is used to measure the concentration
of protein.

The biuret assay use biuret reagent to detect the presence of peptide bonds. When the peptide
bonds are confirmed present in the solution, the coordination complexes associated with a
copper ion are violet in colour. The intensity of the colour is affected by the protein intensity
so the higher the intensity, the higher the protein.

Materials and Apparatus

10 mg/ml stock solution of Bovive Serum Albumin (BSA), deionized water, test tubes, stand,
pipette, biuret reagent, spectrophotometer, red apple, green apple


(a) Preparation of biuret reagent

300ml 10% NaOH was added and stirred to 500 ml of solution containing 0.3% copper
sulphate pentahydrate and 1.2 % sodium potassium tartarate, then diluted to 1 liter.

(b) Protein preparation

1. 2 sets of test tubes with the numbers 1 to 6 labeled on were prepared and the bovine
serum albumin stock solution was also prepared according to the concentration list


conc. (ml) stock
(mg/ml (ml)
1 0 1.0 0
2 1 0.9 0.1
3 2 0.8 0.1
4 3 0.7 0.3
5 4 0.6 0.4
6 5 0.5 0.5
7 6 0.4 0.6
2. Duplicate test tubes for the red and green apples were prepared and 1 ml of each of
them were carefully pipetted into each tubes.
3. 2 ml of biuret reagent was added to every tube, which were the 14 tubes for the stand
curve and duplicate tubes for the red and green apples.
4. The tubes were covered with parafilm and briefly vortexed to ensure that the protein
standards and the biuret reagent were thoroughly mixed.
5. The tubes were allowed to stand for 15 minutes.
6. The spectrophotometer was switched on and the wavelength was adjusted to 550 nm.
(c) Determine protein concentration
1. 1 ml of solution was transferred from tube 1 into a cuvette and it was gently wiped
with a paper towel to remove any sign fingerprints and dust.
2. The absorbance was set to zero. This tube will be served as blank.
3. The absorbance of the other standards and samples were measured.
4. The absorbance of each standards and samples were recorded.
5. A graph of standard curve using the absorbance value of protein standards was plotted
and the absorbance values of the protein samples were interpolated.


Solution of green and red apples

Tube BSA H2O BSA Biuret Absorbance Concentratio

conc. (ml) stock n
(mg/ml (ml) Of Samples

1 0 1.0 0 0.000 -
2 1 0.9 0.1 0.088 -
3 2 0.8 0.1 Add 0.173 -
4 3 0.7 0.3 2 ml of 0.215 -
5 4 0.6 0.4 Biuret. 0.265 -
6 5 0.5 0.5 Incubate 0.359 -
7 6 0.4 0.6 For 0.528 -
8 Red Apple (1 ml) 15 0.656 8.3
9 Green Apple (1 ml) minutes 0.812 10.4

Mass of red apple: 4.0390 g

Mass of green apple: 9.2110 g


Errors were made during this experiment, especially on the part of determining the
absorbance of the test tubes. However, a linear graph was successfully made. This graph was
made as a base for determining the value of the ceoncentration of both apples due to the fact
that absorbance is directly proportional to concentration of solute.

From the results above, it can be seen that green apple has a higher amount of protein value
than the red apple hence the higher concentration of the sample. This in turns cause the graph
with red apple as a sample has a higher absorbance than the one with the green apple as the

Using spectropohotometry, it is discovered that the higher the absorbance, the higher the
concentration of the sample. This experiment has also proven that green apple has a higher
absorbance value than red apple. It means that the green apple has a higher protein value than
red apple ever has.


1. Allen, D., Cooksey, C., & Tsai, B. (2010, October 5). Spectrophotometry. Retrieved
from https://www.nist.gov/pml/div685/grp03/spectrophotometry.cfm