DEPARTMENT OF BIOLOGY

FACULTY OF SCIENCE AND MATHEMATICS

SULTAN IDRIS EDUCATION UNIVERSITY

SBL 1023

TECHNIQUE IN BIOLOGY AND BIOCHEMISTRY LABORATORY

EXPERIMENT 3 : SPECTROPHOTOMETRY (PROTEIN ANALYSIS)

NAME: NOR HAIZATUL BINTI SABIDI

NO MATRIC: E20161015640

GROUP : B

LECTURE NAME : PROFESOR MADYA DR. SHAKINAZ BINTI DESA

Title

Experiment 3 : Spectrophotometry (Protein Analysis)

Introduction

.A spectrophotometer is an instrument that measures the amount of photons (the

intensity of light) absorbed after it passes through sample solution. With the
spectrophotometer, the amount of a known chemical substance (concentrations) can also
be determined by measuring the intensity of light detected. Depending on the range of
wavelength of light source, it can be classified into two different types:

 UV-visible spectrophotometer: uses light over the ultraviolet range (185 - 400 nm)
and visible range (400 - 700 nm) of electromagnetic radiation spectrum.
 IR spectrophotometer: uses light over the infrared range (700 - 15000 nm) of
electromagnetic radiation spectrum.

In visible spectrophotometry, the absorption or the transmission of a certain substance can

be determined by the observed color. For instance, a solution sample that absorbs light
over all visible ranges (i.e., transmits none of visible wavelengths) appears black in theory.
On the other hand, if all visible wavelengths are transmitted (i.e., absorbs nothing), the
solution sample appears white. If a solution sample absorbs red light (~700 nm), it appears
green because green is the complementary color of red. Visible spectrophotometers, in
practice, use a prism to narrow down a certain range of wavelength (to filter out other
wavelengths) so that the particular beam of light is passed through a solution sample.

There are many ways to measure protein concentration; one of the common protocol is by
measuring the absorbance of a coloured product formed by the protein and an organic
molecule. Different methods can yield somewhat different results for the same protein as
there is no absolute photometric protein concentration assay. All methods have advantages
and disadvantages and a few aspects will be taken into consideration: 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.

Biuret protein assay use biuret reagent that detect the presence of peptide bonds.
When the peptide bonds present in an alkaline solution, the coordination complexes
associated with a cooper ion (Cu2+) are violet in colour. Nitrogen atoms of the peptide bonds
form a coordination bond with metal ion. The quantity of the complexes formed is
proportional to the number of peptide bonds. Thus protein intensity affect the intensity of the
colour, where the colour will be more intense with more protein.

Objective

1. To determine the concentration of protein

2. To determine whether the sample protein is same with nutrition facts of sample.
Materials

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

2. Deionized water (dH2O)
3. Test tubes and stand
4. Micropipette
5. Biuret reagent
6. Spectrophotometer
7. Protein samples

Methods

A. Preparation of biuret reagent.

300ml of 10% (w/v) NaOH is added to 500 ml of a solution containing 0.3% copper
sulfatepentahydrate and 1.2% sodium potassium tartarate. The solution then is diluted
to 1 liter.

B. Proteion preparation.
1. 2 sets of test tubes with the numbers 1 to 6 is prepared and the bovine serum
albumin (BSA) stock solution (10mg/ml) is prepared 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 0.2
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 (1ml)

2. Duplicate test tubes for protein samples is prepared and 1ml of the protein
samples is pippetted carefully into each tubes.
3. 2ml of biuret reagent is added to every tube: the 14 tubes for the standard
curve and the duplicate tubes for protein samples.
4. The tubes is covered with parafilm and the samples are briefly vortex to
ensure that the protein standards/samples and the biuret reagent are thoroughly
mixed.
5. The tubes are allowed to stand at 15 minutes.
6. The spectrophotometer is switched on and the wavelength is adjusted to
550nm.
 C. Determine protein concentration.
1. 1ml of solution was transferred from tube 1 into a cuvette and the cuvette was
gently wiped with a paper towel to remove fingerprints and dust.
2. The absorbance was set to ‘zero’. This tube will serve as ‘blank’.
3. The absorbance of the other standards and sample proteins was measured used
the step as in (1).
4. The absorbance of each standards and sample was recorded.
5. A graph of standard curve was plotted using the absorbance value of protein
standards and interpolate with the concentration of protein sample.

Results

Diagram 1. Protein standards and sample protein

Cuvettes BSA Concentration (mg/ml) Absorbance value

1 0 0
2 1 0.180
3 2 0.277
4 3 0.391
5 4 0.526
6 5 0.619
7 6 0.684
8 Protein shaklee 0.914
Table 1. Result for the experiment
Discussion

In this experiment, we are used protein Shaklee for our sample. We want to verify
either the concentration protein of the sample that we observe is same like nutrition facts of
sample. However, we used two method in this experiment to determine the concentration of
protein which are spectrometry test and biuret reagent. We used biuret reagent to detect the
presence of peptide bond. It was treated with copper sulphate solution. Biuret test in presence
of alkali, protein reacts with copper (II) ions that are blue colour to form a violet coloured
complex called biuret.

Based on the diagram above, we can see the different color of solution in each of the
cuvette. In cuvette 7, the color of solution is in violet color. Coloured in cuvette 7 is more
intense than test tube 1. The BSA concentration of cuvette 7 is 6 mg/ml with 0.4 ml of
distilled water while BSA concentration of cuvette 1 is 0 mg/ml with 1.0 ml of distilled
water. The concentration of BSA in cuvette 7 is higher compare to cuvette 1. So we can
conclude that, the higher the concentration of BSA stock solution the colour of protein
become more intense.The absorbane value for protein Shaklee is higher compare to other
sample. So we know that, the concentration of protein in that sample is more. But the
objective of this experiment does not reach because there are some error occur during this
experiment.

There are some recommendation while doing this experiment. First, ensure that the
spectrophotometer down to zero point by the reagent blank since spectrophotometric
measurements are affected by many factors, such as the type of solvent used, temperature,
wavelength of light at which the measurements are made and presence of impurities in the
sample. The spectrophotometer always be zeroed against the solvent because want to
eliminate absorbance due to the solvent. Another potential problem is light scattering is due
to suspended paticles. The particulates will deflect light rays and cause an artificial increase
in absorbance. To avoid light scattering, it is common practice to centrifuge the sample
before measuring absorbance to remove particulates.

Conclusion

In the conclusion, the higher the concentration of sample the higher the absorbance
value.
Reference

 Chemistry Libretexts. Spectrophotometry. 22 July 2015. From

https://chem.libretexts.org/Core/Physical_and_Theoretical_Chemistry/Kinetics/Reacti
on_Rates/Experimental_Determination_of_Kinetcs/Spectrophotometry

 Wikipedia. Spectrophotometry. 25 December 2017. From

https://en.wikipedia.org/wiki/Spectrophotometry

Reflection

This experiment was my first experience. I learned how to use the spectrophotometer
to determine the concentration of proteins. In my experiment, I would like to compare the
sample protein with nutrition facts either the information is true or just a fake. But the worst
thing, I cannot indentify it maybe because of my lack knowledge to investigate the
concentration of protein sample. In my future, I will try to prove whether the information
given is true or not so that people more confident to eat the protein.