Facebook Pixel
ib chemistry hl notes

Denaturation on Temperature

UPDATED ON - 24 OCT 2019



Table of contents




The aim of this investigation was to investigate how the rate of denaturation of egg white proteins is dependent on temperature and to experimentally determine the Activation Energy of the denaturation process.

Comment: Research questions are clearly stated and the purpose is well focused.


The original idea for this project came from a lesson on boiling temperature and vapour pressure when we learned why it takes longer for an egg to hard boil at high
altitude (due to the lower boiling temperature of water). This topic stimulated many thoughts. How is the time it takes to boil an egg dependant on temperature? Can the time taken to exactly hard boil an egg be predicted over all temperatures? Below what temperature do eggs cease to hard boil?

Comment: The student clearly describes the whole process that resulted in his/her engagement in this investigation.

I decided that the investigation would concentrate on determining one important parameter which is the Activation Energy barrier to egg protein denaturation. If this
can be determined then predictions of the egg’s behaviour during boiling at a range of temperatures can be made and then tested.

Comment: The choice of research is well explained.



This project has two main theoretical bases, the principles of kinetics and the process of the nature of protein denaturation, which I will describe below.  

Part A:  Kinetics and the Arrhenius Equation 

The way temperature affects the rate of a reaction is explained in Figure 1 below


Only collisions with more energy than that of Activation Energy  (the minimum energy that must be surpassed in order for a chemical reaction to take place)  will
cause a reaction. Therefore, in the graph above, the shaded area represents those collisions.   

According to theory, as temperature increases, the molecule velocities increase, and therefore, both the frequency of collision between molecules is greater and a greater proportion of collisions cause a reaction. In figure 1, this is apparent. At the lower temperature, T1, the fraction of molecules reacting is less than of T2, (shaded area on the graph). The rate of reaction is proportional to the number of molecules with more energy than Ea and increases exponentially with temperature. 

The relationship between the reaction rate and the temperature is expressed by the Arrhenius equation which relates the rate constant of a reaction k to the absolute temperature T: 

                                                               k = Ae-(Ea/R.T)

where k= rate is constant, Ea= Activation energy, T= Reaction Temperature, R= Gas constant and A = Arrhenius constant  which is a factor that relates to the orientation of collision; only molecules colliding in the correct orientation with sufficient energy react.   

Note that the Arrhenius equation is an exponential function and only applies when the activation energy lies within the exponential decay part of the curve to the right

Hand side of the Boltzman distribution graph in Figure 1.

Comment: The student provides a good support for the chosen approach.
Comment: The student establishes the scientific context for the investigation through a discussion on its significance.

Part B: Proteins & Denaturation 

Proteins are formed by a combination of amino acids containing often 50 to 1000 amino acid residues).  All proteins, independent of their nature (shape, complexity Etc…) have structures, which are divided into four categories: primary, secondary, tertiary and quaternary.  

The primary structure is mainly concerned with protein polypeptide chains (subunits) and with its amino acid sequence. In the secondary structure, there are different types of energetically stable three-dimensional structures of the polypeptide chain (also referred to as confirmations). For some proteins, their polypeptide chain might form a β-pleated sheet and for others, it might follow the spiral a-helix conformation. The tertiary structure is the overall three-dimensional appearance of the protein which is held together by strong intermolecular forces (e.g. Hydrogen bonding). For example, a globular protein such as in egg white is approximately spherical and folding is extensive to obtain a compact tertiary structure. Lastly, the interaction of various polypeptide chains in a non-covalent way to pattern the protein molecule is said to present the quaternary structure.  

Denaturation is when the biological activity of a protein is lost and disruption in the secondary, tertiary and quaternary structure of a protein occurs due to changes in temperature, pH, ionic strength, or due to an addition of organic solvents. For instance, when the egg white is exposed to heat, it thickens and changes color. At that point, denaturation has occurred and all its structure has been disrupted, except for
its primary structure, and an alternative energetically stable three-dimensional The structure is formed.  It is the energy barrier to this process of permanently disrupting the three-dimensional structure of the egg protein that is the focus of this investigation.

Comment: Relevant scientific concepts are correctly considered.



Comment: The methodology allows the use of concepts and techniques appropriate to the Diploma level.


A common procedure (Hill, G & Holman, J (2001))2 to determine the Ea, is by measuring the time of reaction (in this case of the final time of denaturation of egg proteins determined as the time when the film of egg white between the two microscope slides became opaque) at various different reaction temperatures using the Arrhenius equation:


Comment: The methodology could be easily repeated by others.

k = Ae-(Ea/R.T)       Ink = InA-(Ea/R.T)

and since k is proportional to 1/Time: 

Ink = -InTime + a constant  ⇒   InTime = (Ea/R.T) –InA + a constant 

Now, we can plot a graph, InTime versus 1/Temperature (in Kelvin) and calculate the gradient. Since we recognize the gas constant(R=8.3145 JK-1mol-1), we can determine the Activation energy:  

Gradient = Ea/R  ⇒   Ea = R x Gradient




The focus of the experimental work was to measure how long it took the egg white and egg yolk to denature over a range of temperatures. The development of a The suitable procedure was far more time consuming than originally anticipated since it,

It proved difficult to experimentally determine exactly when the egg sample had ‘boiled’ (denatured).

Comment: There is a consideration of limitations in the methodology.

In the end, some procedures yielded results and these, experiments are described below. The final successful experiments only focused on egg whites.

Comment: The student presents a brief discussion on the development of the the method including  obstacles found during this process. This shows personal input and initiative.


The procedure was as below: 

1. The egg white was separated from the egg yolk in a small beaker and a 500ml beaker was filled with tap water to heat over a flame.
2. With a syringe, a drop of egg white was put on the center of a preweighed microscope glass slide and then using another clean preweighed microscope
glass slide, I pressed them together (with egg white in between) and wiped up the sides of the slides. They were weighed again.
3. Afterward, the diameter of the circular shaped liquid egg white pressed between the two slides was measured.
4. Then, at different temperatures of the heated water slides were added to the water and were closely observed, as the stopwatch was running.

5. When I noticed the denature of the egg white, I stopped the stopwatch and simultaneously placed the two slides in room temperature water to cool down.

6. In each experiment, recorded was the time the egg white took to denature temperature it was at.


Comment: The methodology allows the collection of data that are both sufficient and relevant.
Comment: The methodology employed has taken the most relevant variables into account. 



Egg white results                                                                                                                                 

Diameter (+/- 0.1 

Mass of egg white
(+/- 0.005 g)

Temperature of
water (+/- 0.5 oC)


Time of 
denaturation (+/-
0.5 sec)

Comment: Sufficient quantitative data have been
collected. Uncertainties have been
recorded although those for time are not
consistent with the cited precision of the data


2.5 by 5.0  0.01  25.0 Never denatured
2.5 by 5.0  0.01  30.0 Never denatured
2.5 by 4.5  0.01  35.0 Never denatured. Not
even after 15 min. 
2.5 by 5.0  0.01  40.0 Never denatured. Not
even after 10 min. 
2.5 by 5.0  0.02 45.0  Never denatured. Not
even after 5 min. 
2.5 by 5.0  0.01  50.0 Never denatured. Not
even after 5 min. 
2.5 by 5.5  0.01  55.0 Never denatured. Not
even after 5 min. 
2.5 by 5.0  0.01  60.0  Never denatured. Not
even after 5 min. 
2.5 by 5.0  0.01  62.5  49.9 sec.
2.5 by 5.0  0.01  62.5  49.7 sec.
2.5 by 5.0  0.01  65.0  32.8 sec.
2.5 by 4.5  0.01  67.5 21.0 sec
2.5 by 5.5  0.01  70.0  15.9 sec.
2.5 by 5.5  0.01  75.0  11.0 sec.
2.5 by 5.0 0.01  80.0  8.0 sec.
2.5 by 5.0  0.01  81.0  7.6 sec.
2.5 by 5.0  0.01  82.5  7.0 sec.
2.5 by 5.0  0.01  84.0  6.4 sec.
2.5 by 5.0  0.01  85.0  6.0 sec.
2.5 by 5.0  0.01  86.0  5.5 sec.
2.5 by 5.5  0.01  87.5  4.9 sec.
2.5 by 5.0  0.01  89.0  4.2 sec.
2.5 by 5.0  0.09      90.0       4.0 sec.
2.5 by 5.0  0.01  91.0  3.8 sec.
2.5 by 5.5  0.02 92.5  3.5 sec.
2.5 by 5.0  0.01  94.0  3.3 sec.
2.5 by 5.5  0.01  95.0  3.0 sec.
2.5 by 5.0  0.01  97.5  2.4 sec.
2.5 by 5.0  0.01  97.5  2.5 sec.
2.5 by 5.0  0.01  100.0  2.1 sec.
2.5 by 5.0  0.01  100.0  2.2 sec. 






















































Comment: The processing is easy to follow

In order to find the activation energy, I need to calculate ln Time and 1/Temperature values for the reaction temperatures where denaturation occurred:

Temperature(k) Time(sec.)  ln Time 

1/ Temp.(k-1)

Comment: Tables are presented unambiguously.
298.0 -----  -----   
303.0 -----  -----   
308.0 -----  -----   
313.0 -----  -----   
318.0 -----  -----   
323.0 -----  -----   
328.0 -----  -----   
333.0 -----  -----   
335.5 49.7  3.906 


Comment: Appreciation of decimal places
evidenced in this table.
338.0  32.8  3.490     


340.5  21.0  3.045 


343.0  15.9  2.766  


348.0  11.0  2.398 


353.0  8.0  2.079 


354.0  7.6  2.028 


355.5  7.0  1.946  


357.0  6.4  1.856 


358.0  6.0  1.792 


   359.0      6.0  1.705 


360.5  4.9  1.580


362.0  4.2  1.435 


363.0  4.0  1.386 


364.0  3.8   1.335 


365.5   3.5  1.253 


367.0 3.3  1.194 


368.0  3.0  1.099 


370.5  2.5  0.916 


373.0  2.2  0.788  2.681x10-3 


Graph 1.  The plot of ln Time against 1/Temperature

Comment: Graphs are presented unambiguously.



Calculation to determine Activation energy, Ea. 

The gradient from Excel derived a linear equation 

= 9.6164 × 103    =   9616
Gradient= Ea/R   so 

                                                 Ea= 9616x 8.314 
                                                 Ea= 79974 J mol-1 

                                            Ea= 80kJ mol-1

 The two data points corresponding to the lowest reaction temperatures at 335.5 and
338.0 K does not appear to conform to the linear plot.

Comment:The student takes reliability into account.

I have removed these two Points as anomalous in the graph below and recalculated Ea.

Comment: Processing pays due consideration to anomalies.


Graph 2.  The plot of ln Time against 1/Temperature with discarded data points


The gradient from Excel derived a linear equation 

= 8.6521 × 103    =   8652
Gradient= Ea/R   so 

                                                              Ea=  8652x 8.314 
                                                              Ea=  71932.73 J mol-1 

                                                       Ea= 72kJ mol-1

Comment: Processing correctly uses chemical concepts and graphical analysis to determine Ea.

By cutting the data back further to a maximum 1/Temp value of 2.766 x 10-3 which represents the closely spaced data points the graph becomes

Comment: The student shows evidence of a good understanding of graphical analysis.


Graph 3.  The plot of ln Time against 1/Temperature with further discarded data points



Comment: Evidence supporting the student has considered the impact of uncertainties on results (line of best fit)

and                                   Ea= 8753x 8.314  

                                         Ea= 72772.44 J mol-1 

                                         Ea =73kJ mol-1

Comment: Correct use of significant figures.


The calculated Es results are tabulated below along with the R2 correlation value that relates to how good the linear fit was in the graphs (with 1 being a perfect fit)

Comment: The processing involves correctly constructed lines of best fit and makes use of R2 for evaluating uncertainties.


  Graph 1  Graph 2  Graph 3 
Ea (kJ mol-1) 80  72  73 
R 0.9838  0.9975 


Comment: Evidence that the stu
dent understands
the impact of uncertainties on results.


The best value is from graph 2 and the value from Graph 3 gives some idea as to the amount of uncertainty arising from the plots.

 Comment: The processing presents a valid a comparison which duly considers uncertainties and shows a good grasp of graphical analysis.


My final experimental value for Ea of egg protein denaturation
= 72 ± 1 kJ mol-1

Comment: Uncertainties considered in the final value.


  Conclusion and Evaluation

Comment: The report has been easy to follow, concise and shows a logical sequence.

The initial aims of the investigation have been met. It has been seen that denaturation did not take place at 60ºC and below. Above this temperature, the rate of protein denaturation increases rapidly with temperature.I was able to calculate an Activation Energy for the activation energy of egg protein denaturation and it was

Comment: Subject-specific terminology is correctly used throughout the report.

        Ea = 72 ± 1 kJ mol-1

Comment: The report makes use of subject-specific notation.

I could not find an exact literature value for the Ea of egg protein (albumin). One articlestudied the effect at acidic pH’s (which will change the Ea because of acidic pH also denatures proteins) and gave the values as 36.7 and 50.0 kcal./mole which correspond to 150-200 kJ mol-1

Comment: Comparison with scientific literature made.

 My value is about a half or a third of this literature value. When I reflect on the The simplicity of the method I am impressed that this investigation has arrived at a value that is so sensible in size.  

It is also significant that the Arrhenius equation seems appropriate for the determination of egg protein denaturation as long as the temperature range for the measurements is kept within specifically defined limits. This is because the Arrhenius equation strictly applies to ideal gas reactions only although it has been widely used in the study of liquids and solution reactions where collision theory still holds and only the Arrhenius constant A is affected by the change of state. 

However, the denaturation reaction of proteins is not a collision reaction (it depends on the protein chains rotating and intermolecular forces breaking and reforming) and the theoretical basis of the equation no longer so obviously holds.


Comment: There are no clear suggestions of feasible extensions to this investigation.

There is no obvious reason why the plot of ln(Time) v 1/Temperature should have been so clearly linear.

Comment: There are no clear suggestions on relevant and feasible alternatives to improve the methodology.

It is maybe the most interesting finding of this investigation that the relationship in the Arrhenius Equation still seems appropriate.

Comment: The reflections d demonstrate a clear understanding of implications of the conclusion.


Comment: Student considers the limitations of the methodology.



1. http://www.webchem.net/notes/how_far/kinetics/rate_factors.htm, last accessed 3rd March 2012
2. Hill, G & Holman, J (2001). Chemistry in Context: Laboratory Manual and Study Guide, 5th Edition, pp 54-55, Surrey, Nelson
3. Investigations on proteins and polymers. VII. The denaturation of egg albumin, Robert J. Gibbs, M. Bier, F.F. Nord, Archives of Biochemistry and
Biophysics, Volume 35, Issue 1, January 1952, Pages 216–228, Last accessed at http://www.sciencedirect.com/science/article/pii/S0003986152800670 on 4th March 2012.


Further Bibliography
    Chemistry for the IB Diploma, G. Neuss, Oxford University Press 2007
4. http://chemistry.about.com/od/biochemistry/a/proteinstructur.htm, last accessed 26th February 2012








Subscribe today to get the latest IBDP news, tips and product updates.

By submitting this form, I agree to the data entered being used by Nail IB NSW for sending newsletters and promotional offers. Your data shall be kept until you unsubscribe. In accordance with current laws and regulations, you can unsubscribe at any time by clicking on the link in the promotional emails that we send to you. Subject to the conditions provided for by applicable legislation, you have rights in relation to your data. To find out more, see our data protection policy . You can exercise your rights at any time by writing to help.nailib@gmail.com.

Follow us:
Payments Secured By:
Payment Companies
© Copyright 2020 Nail IB Inc. All rights reserved.