Estimation of serum protein using biurette method

Introduction:

The colors we see as they are; for example, coal as black or the leaves as green is actually the
part of the visible spectrum which is reflected off their surfaces after a part of the spectrum is
absorbed. This essentially means that the colors we see are the complementary colors or
wavelengths of which are absorbed and the color of a species is due to its ability to absorb light.
Further, with the increase of concentration, the absorbance of a solution also increases. This is
denoted by the Beer Lambert law, where the linear relationship between concentration and
absorbance was instated. The equation for the law in terms of intensities is given below.

Beer lambert law in terms of intensities

(Chemistry LibreTexts, 2017)

The device used to measure the transmittance and absorbance of light passing through a liquid
sample is known as a colorimeter. This was one of the earliest analysis techniques to be
developed. The principle of this device is based on the Beer Lambert law discussed previously.
A beam of light with a specific wavelength is passed through the solution. This transmitted light
is then compared with a standard solution with zero absorbance to give the relative absorbance
of the solution. By using standard calibration curves, concentrations of unknown solutions can
be found. This practical is based on finding the concentration of an unknown solution using
standard calibration curves.

The colorimeter was used to find the serum protein level in a sample. To do this, since cupric
ions form chelates with peptide bonds of proteins in an alkaline medium, the intensity of the
violet color is used to find the the amount of proteins in the specimen.

Aim:

The aim of this practical was to gain competency in measuring the serum protein level of a give
sample.

Objective:

The objective of this practical was to determine the serum protein level using a graph drawn
using the data obtained from the colorimetric readings.
Safety:

Since chemical substances which are irritants and electrical equipment are used, safety
precautions must be followed. The chemicals (CuSO4) should not be touched with bare hands
and PPE including the lab coat, gloves and appropriate shoes must be worn prior to starting the
practical.

Materials:

 CuSO4 salt
 Distilled water
 Watch glass
 Wash bottle
 Test tubes
 Stirring rod
 Pipette
 Colorimeter
 Beaker
 Potassium Iodide
 Sodium potassium Tartrate
 Sodium hydroxide
 Bovine albumin
 Unknown plasma serum sample
 Electronic balance

Methodology:

1. The glassware needed were thoroughly washed and brought into the working
area.
2. 3g of Copper sulfate was measured using an electronic balance and was
transferred into a beaker using a spatula and wash bottle to make the initial
solution.
3. Water was then added to the 500 ml mark and the Copper sulfate was dissolved
in it to make a solution using a glass rod.
 4. 9g of sodium potassium tartrate was measured using the electronic balance and
was added to the previously prepared solution of copper sulfate.
5. 5g of KI was also measured using the balance and was added to the copper
sulfate solution.
6. 24g of sodium hydroxide pellets were measured using the analytical balance
which was then transferred into the beaker to which water was added upto the
100ml mark and was stirred with a glass rod to make another solution.
7. The first solution of copper sulfate, KI and Sodium potassium tartrate was then
added to a Schott bottle.
8. The previously prepared sodium hydroxide solution was also added to the Schott
bottle and water was added upto the 1000ml mark.

Figure 1.0, Solution of KI, NaOH and sodium potassium tartrate

Preparation of blank solution

9. 5g of KI and 9g of sodium potassium tartrate were measured using an analytical

balance and was added to a beaker.

10. Water was added to the beaker up to the 500 ml mark and was stirred using the
glass rod until KI and sodium potassium tartrate were dissolved.

11. 24g of sodium hydroxide pellets were measured using the analytical balance
which was then transferred into the beaker to which water was added upto the
100ml mark and was stirred with a glass rod to make another solution.
 12. The first solution of copper sulfate, KI and Sodium potassium tartrate was then
added to a Schott bottle.
13. The previously prepared sodium hydroxide solution was also added to the Schott
bottle and water was added upto the 1000ml mark.

Figure 2.0 – NaOH, KI and potassium tartrate solution

Preparation of the protein standard.

14. 6g of bovine albumin was measured and added to a beaker.

15. Water was added upto the 100 ml mark and was mixed well.
16. Reagents were added to 3 test tubes as shown in table 1.0.

Table 1.0
Reagents added to test tubes
(Laboratory manual)

Test (ml) Standard (ml) Blank (ml)

Biuret reagent 5 5 5

Serum 0.1 - -

Std. Protein - 0.1 -

 solution
Distilled water - - 0.1

17. After the preparation of the solutions, the test tubes were named accordingly and
were kept aside for 30 mins.

Figure 3.0 – Prepared solutions kept aside for 30 min

18. After the time was up, the solutions were inserted to colorimeter cuvettes using a
dropper up to the mark according to the type of solution. (Blank, standard and the
test.
19. The wavelength of the colorimeter was set to 540 nm.
20. The outer surfaces of the cuvettes were cleaned using a tissue and the
absorbance readings were obtained for blank, standard and test solutions
separately. First the blank was inserted to the colorimeter and was set to zero.
Then the test cuvette was inserted and the reading was noted down. Then again
the blank was inserted and the colorimeter was set to zero. Then the standard
was inserted and the reading was taken.

21. This was repeated two more times to obtain an average value.

Results

Table 2.0 – Results

Reading 1 Reading 2 Reading 3

Test 0.50 0.60 0.50

Standard 0.27 0.27 0.28

Blank 0.00 0.00 0.00

Serum protein (mg%) = (AbsT/Abss)* [Conc. Std]

0.53
x 60 g /dl
0.273

= 116.48 g/dl
Discussion:

Colorimetry is one of the earliest analysis techniques to be developed. Therefore, colorimeters

often have a limited range of wavelengths to select from to set the filter for analysis of samples.
Unlike the UV spectrophotometer which has a wide range of filters to select from, the filters of
the colorimeter were limited to 420, 490, 540, 590 and 650 nm. Though the ideal wavelength
filter for CuSO4 is around 635 nm, since the device is unable to set to that filter, 540 nm filter
was used instead.

When preparing the copper sulfate solution, as the copper sulfate is being measured it can be
condensed with air and humidity causing it to become heavier and this can cause an error when
measuring and preparing the solution.

Since sodium hydroxide is available in pellets it was difficult to exactly measure 24g of sodium
hydroxide and there may have been an error since the exact amount was not able to be
measured.

The blank solution did not give the proper zero absorbance value. This means that there have
been errors in preparing the blank solution or there may have been an issue in the colorimeter.

Also according to Johnson and Swanson (1952), an ideal biurette solution should not react with
any other substances except protein. The biurette solution which was prepared in the lab also
did not react with any other substance therefore we can conclude that it was an ideal solution.

Also the serum protein concentration obtained according to the colorimetric readings were way
high (116 g/dl) than the given reference range which is given as 3.5-5.5 g/dl. This is almost
more than 10 times the given reference range. The reasons for this might be due to the errors
mentioned above or it can be due to the fact that the patient is suffering from a bone marrow
disease such as multiple myeloma.

Conclusion:

The concentration of the serum protein in the given sample was found to be 116 mg/dl.
Reference:

Chemistry LibreTexts. (2017). The Beer-Lambert Law. [online] Available at:

https://chem.libretexts.org/Core/Physical_and_Theoretical_Chemistry/Spectroscopy/
Electronic_Spectroscopy/Electronic_Spectroscopy_Basics/The_Beer-Lambert_Law

Johnson, B. and Swanson, A. (1952). Milk Serum Proteins. I. A Quantitative Biuret Test for Milk
Serum Proteins. Journal of Dairy Science, 35(10), pp.823-828.

Group members – Dilumi, Kalani, Hashini,

Ruwanthika