1.

Tittle

Experiment 3: Spectrophotometry (protein analysis in different type of apples).

2. Objectives

-To use the Spectrophotometer correctly.

-To analysis the amount of protein in a sample.
-To compare the amount of protein in two different apples.

3. Introduction

Proteins are one of the most important nutrients for our body. There are many foods that contain
a lot of protein like egg, fish and others. But, in this experiment we try to see the amount of fruit
that stored in the apple. We also compare it in two different apple between red apple and green
apple. Protein is importance character for our body because they help by catalyzing reaction in
our body, transport molecule like O2 and CO2, they are also keep us healthy by enhancing our
immune system and they are helping in transmitting messages from cell to cell (Waikato, 2011).

They are various ways to determine the concentration of protein. But it depends on the
specificity, sensitivity, the measurable range of concentration, the accuracy, the nature of the
protein to be examined, the presence of materials interfering with the measurement, and the time
required for the measurement.

For example, biuret protein assay that use biuret reagent that detect the presence of
peptide bond has the shortest time to analyze the protein contain in one of the sample. But, it also
has disadvantageous; it cannot determine the specific concentration of protein in the sample. In
this test; the more intense the changing o color in the biuret reagent, the more peptide bond
presence. (Lab 3: Spectrophotometry, 2017).
 Figure 3.1: Biuret reagent is a blue liquid that changes to purple when proteins are present (Lab
3: Spectrophotometry, 2017)

The more accurate method to determine the concentration of protein in a sample is to use
spectrophotometer. It can determine the concentration of protein in a sample, but it has
disadvantages too. The advantages of this method are we have to extract the sample first into
liquid form which can overshadow the sample.

4. Equipment and materials

-Stock solution of Bovive Serum Albumin (BSA): 10mg/ml

-Deionized water (dH2O)
-Test tubes and stand
-Pipette
-Biuret reagent
-Spectrophotometer
-Protein samples
5. Procedure/ methodology
(A) Preparation of biuret reagent
300ml of 10% (w/v) NaOH was added to 500 ml of a solution containing 0.3% copper sulfate
pentahydrate and 1.2% sodium potassium tartarate while being stirred, the it was diluted to 1
liter.

(B) Protein preparation

1. 9 test tube was prepared.
2. the bovine serum albumin (BSA) stock solution (10 mg/ml) according to the
concentration listed below:

Tube BSA conc. H2O (ml) BSA stock Biuret

(mg/ml) (ml) reagent
(ml)

1 0 1.0 0
2 1 0.9 0.1
3 2 0.8 0.1
4 3 0.7 0.3 2.0
5 4 0.6 0.4
6 5 0.5 0.5
7 6 0.4 0.6
8 Sample A (1 ml)
9 Sample B (1 ml)
Table 5: Substances needed for each test tube

3. Test tube 1-7 can be made until two sets to increase the precision of the result.
4. The test tube has been cover with parafilm and it was briefly vortex to ensure that the
protein standards/ samples and the biuret reagent are thoroughly mixed.
5. The test tube was allowed to stand at 15 minutes.
6. The spectrophotometer was switched on and the wavelength was adjusted to 550nm.
(C) Determine protein concentration
1. 1ml of solution was transferred from tube 1 into a cuvette and the cuvette was gently wipe
with a paper towel to remove fingerprint and dust.
2. The absorbance was set to zero. It will serve as ‘blank’.
3. The other standard and sample and protein have been measured by using step 1. But the
instrument was not ‘blank’ again.
4. The absorbance of each standard and samples has been recorded.
5. The graph of the absorbance vs concentration has been made.

6. Result

Test tube BSA concentration (mg/ml) Absorbance Concentration of the

sample (mg/ml)
(from the graph)
1 0 0.000 -
2 1 0.088 -
3 2 0.173 -
4 3 0.215 -
5 4 0.265 -
6 5 0.359 -
7 6 0.528 -
8 Red apple 0.656 8.3
9 Green apple 0.812 10.4
Table 6: Result for the experiment
 1
y = 0.0792x - 0.0051
0.9
0.8
0.7 y-axis= absorbance
0.6
x-axis= concentration
0.5
0.4 Red line= red apple
0.3
Green line= green
0.2 apple
0.1
0
-0.1 0 2 4 6 8 10 12

Graph 6: graph absorbance vs concentration

7. Discussion

As we can see from graph 6, the data that we get is not accurate. But, we still manage to make a
straight line graph which is necessary to find the concentration of protein for the green apple and
red apple. 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 result of the experiment, we can see that the green apple has more protein than
red apple. 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 sample.

From this experiment, we already achieve the entire objective.

8. Conclusion

For the conclusion, green apple has more protein than red apple. This is also a proof for a
vegetarian can get protein without eating meat or fish.
9. References

Lab 3: Spectrophotometry. (2017). SBL 1023 LAB MANUAL. Faculty science and mathematic.

Waikato, T. U. (2011, June 10). Role of proteins in the body. Retrieved 12 4, 2017, from Science Learning
Hub: https://www.sciencelearn.org.nz/resources/209-role-of-proteins-in-the-body