SBL1023

TECHNIQUES IN BIOLOGICAL AND

BIOCHEMISTRY LABORATORY

SPECTROPHOTOMETRY: PROTEIN ANALYSIS

NAME: NUR IZZATIE BINTI KHAIZAAR

MATRICS NUMBER: E20161013729
GROUP: A
LECTURERS NAME: ASSC. PROF DR. SHAKINAZ BINTI DESA
 SPECTROPHOTOMETRY: PROTEIN ANALYSIS

INTRODUCTION
Protein is one of classes of food that very important in our body. To know how
much protein needed to our body, there are many ways to measure the concentration
of protein. To measure the concentration of protein is also known as quantitative
analysis. There are five ways to determine the concentration of protein such as Biuret
test, Lowry (Folin) protein assay, Bradford protein assay, Spectrophotometry based
on UV light absorption and BCA assay. For this experiment, spectrophotometry was
chosen because this method is very quick and easy. This is because this method has
no chemical reaction to be carried out. This method[1] can measure how much a
chemical substance absorbs light by measuring the intensity of light as a beam of light
passes through sample solution. The main principle is each solution absorbs or
transmits light over a certain range of wavelength.
Other method that have to be applied in this experiment is Biuret protein assay.
This is because biuret reagent can be detect the presence of peptide bond. When
peptide bond present in an alkaline solution, the protein reacts with copper (II) ions to
form a violet coloured complex that called as biuret. Nitrogen atoms of peptide bond
will form coordination with copper (II) ions. Therefore, protein intensity affects the
intensity of the colour which means that, the colour will be more intense with higher
concentration of protein.

OBJECTIVE
To determine the protein concentration of sample A (fresh milk) and sample B
(chocolate milk).
METHODOLOGY
1. Nine test tube are prepared and labelled. Every test tube are added with
different volume of Gelatin, and water except for test tube eight and nine. Table
1.0 below shows that the volume of Gelatin and water needed for every test
tube.
Table 1.0

Test Gelatin, Water, Protein

tube mL mL concentration,
mg/mL

1 0 1.0 0

2 0.1 0.9 1

3 0.2 0.8 2

4 0.3 0.7 3
2 mL of 15 minutes
5 0.4 0.6 4 Biuret incubation

6 0.5 0.5 5

7 0.6 0.4 6

8 1 mL of sample A X2

9 1 mL of sample B X1

2. Sample A and sample B are prepared for test tube eight and nine.
3. Both samples are prepared by adding 5 mL of distilled water and the volume of
both type of milks are taken for 10L by using micropipette.
4. Sample A for test tube eight and sample B for test tube nine. 1 mL of every
sample are taken to be tested.
5. All test tubes are added with 2 mL of Biuret and incubated for 15 minutes.
6. The spectrophotometer is switched on and adjusted the wavelength to 550nm.
7. 1 mL of solution from every test tube are transferred into cuvette and gently the
cuvette wiped with tissue to remove fingerprint and dust.
8. The absorbance is set to zero by using solution from test tube 1. It served as
blank.
9. The absorbance of every test tube are measured and recorded by using
spectrophotometer without blank the instrument again.
10. A graph of standard curve is plotted by using the absorbance value of protein
standards and interpolate the absorbance values of the protein samples.
RESULTS

Test Gelatin, Water, Protein Absorbance

tube mL mL concentration,
mg/mL

1 0 1.0 0 0.000

2 0.1 0.9 1 0.049

3 0.2 0.8 2 0.104

4 0.3 0.7 3 0.161

2 mL 15 minutes
5 0.4 0.6 4 of incubation 0.286
Biuret
6 0.5 0.5 5 0.347

7 0.6 0.4 6 0.415

8 1 mL of sample A X2 0.209

9 1 mL of sample B X1 0.052

Diagram 2.0

The diagram 2.0 shows that solution for every test tube after added with biuret
reagent.
 Diagram 3.0

The diagram 3.0 shows that 1 mL solution of every test tube are transferred into
cuvette to measure the absorbance by using spectrophotometer. From left is taken
form test tube 1 and so on.
 Graph 1.0

Graph protein concentration against absorbance

0.45
0.415
0.4

0.35 0.347
Absorbance at 550 nm

0.3
0.286
0.25

0.2

0.161
0.15

0.1 0.104

0.05 0.049

0
1 X1 2 3 X2 4 5 6

Protein concentration, mg/mL

Graph 1.0 shows that the concentration for sample A, X2 and sample B X1 can be
determine by using graph.

The value of X2 of sample A = 3.20 mg/mL

The value of X1 of sample B = 1.15 mg/mL
DISCUSSION
Based on this experiment, we used to find protein concentration by using biuret
reagent and the spectrophotometer. We chose the fresh milk as sample A and
chocolate milk as sample B. Based on the nutritional information[2] of fresh milk, protein
contain 6.4g per serving 200 mL meanwhile for chocolate milk, the protein contain
5.6g per serving 200mL. However, we had to prepare of both milk to be our sample.
The preparation of our samples because the milk is too saturated if we wanted to
measure the protein concentration. This is the spectrophotometer cannot measure the
saturated concentration. So, that is the reason we should diluted both of milk by taking
1 mL of the milk, added with 5 mL of distilled water. Then, after mixing them, we took
1 mL of every samples to proceed with the next step by adding biuret reagent and
incubated them for 15 minutes.
The way to use the spectrophotometer, we should measure the first test tube,
which has no protein so that absorbance will be recorded as 0. Then, continue
measured the absorbance with other test tubes without press the button zero. The
absorbance recorded shows that the higher the concentration of protein, the higher
the absorbance reading. Therefore, to measure the absorbance of both sample, we
used to plot a graph between the protein concentrations against absorbance at 550
nm. This is because, we knew that the value of absorbance for both samples. To know
the protein concentrations for both samples, we used to match the new line in the
graph as shown in graph 1.0. The sample A, X2 we got the value of protein
concentration is 3.20 mg/mL meanwhile for sample B, X1 we got 1.15 mg/mL. From
this result, we understood that the protein concentrations of sample A (fresh milk) is
higher than sample B (chocolate milk). This result also matched with the nutritional
information of both milk. This experiment is successfully worked.
REFERENCES
1. Archived on 3 December 2017
https://chem.libretexts.org/Core/Physical_and_Theoretical_Chemistry/Kinetics
/Reaction_Rates/Experimental_Determination_of_Kinetcs/Spectrophotometry

2.
3. Lecture note, Protein (The Analysis)
4. Laboratory Manual SBL1023