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What factors affect the activity of urease? Essay

What are the kinetics of the urea-urease reaction?

Aim:

To investigate closely the factors that can affect the activity of urease. The effect of concentration and temperature can be studied over a period of time leading to the order and activation enthalpy of the reaction.

Introduction:

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UREASE

(NH2)2CO + 3H2O ———-> 2NH3 (aq) + CO2 (g)

Urease is an enzyme found in microorganisms, invertebrates, and higher plants. It catalyses the hydrolysis of urea to ammonia and carbon dioxide. In some bacteria and invertebrates this reaction is the final step in the breakdown of complex nitrogenous compounds before ammonia is excreted.

Method:

Quench with known volume and concentration of HCl to stop the reaction. Some of the acid will react with the ammonia produced by Urease to determine the amount of HCl used up. Contents of the flask are titrated with standard Sodium Carbonate solution using methyl orange indicator. From the results of this titration, the amount of HCl that did not react with the ammonia can be found by substituting the amount of HCl used to neutralise ammonia can be found.

Background Knowledge:

An enzyme, such as Urease, is a protein that acts as a catalyst, speeding up the rate at which a biochemical reaction proceeds but not altering the direction or nature of the reaction. An enzyme catalyses virtually every metabolic reaction that takes place within a living organism. Enzyme molecules have a special feature in that they possess an active site. The active site is a region to which another molecule or molecules can bind. This molecule is the substrate of the enzyme. The shape of the active site allows the substrate to fit perfectly and to be held in place by temporary bonds, which form between the substrate and some of the R groups of the enzyme’s amino acids. This combined structure is termed the enzyme-substrate complex. Each type of enzyme will usually act on only one type of substrate molecule. This is because the shape of the active site will only allow one shape of molecule to fit in it. The enzyme is said to be specific for this substrate.

There are two types of enzyme inhibitors: Competitive and Non-Competitive inhibition. If an inhibitor molecule binds only briefly to the active site of the enzyme, there is competition between it and the substrate for the active site. This means it is more likely that the substrate will collide with a filled active site and so this is known as competitive inhibition. It is said to be reversible (not permanent) because it can be reversed by increasing the concentration of the substrate.

Sometimes, the inhibitor can remain permanently bonded with the active site and therefore cause a permanent block to the substrate. No competition occurs as it does not matter how much substrate is present. This is non-competitive irreversible inhibition.

Other factors affecting enzyme activity:

Substrate concentration

As substrate concentration increases, the initial rate of reaction also increases as collisions between enzyme molecules and reactants become more frequent. When the enzymes begin to approach the maximum rate at which they can combine with reactants and release products, the effects of increasing substrate concentration diminish. At this point the enzyme is saturated and the reaction remains at the saturation level.

pH level

A change in pH can also effect enzyme activity. Each enzyme has an optimal pH range that help maintain its normal configuration in an environment which it operates. Pepsins is a enzyme found in the stomach and functions at pH 2. At pH 2 the tertiary structure of pepsin is not altered and will catalyze the reaction. Trypsin is and enzyme found in the small intestine and prefers a pH of about 8. The tertiary structure of a protein depends on interactions such as hydrogen bonding, between R groups. A change in pH can alter the ionization of these side chains and disrupt the normal configuration and in some case denature the enzyme. A denatured protein cannot combine with a substrate.

Temperature

As temperature rises, the enzyme and substrate molecules move faster. Moreover, when they do collide, they do so with more energy. This makes it easier for bonds to be broken so that the reaction can occur. As temperature continues to increase, the speed of movement of the substrate and the enzyme molecules also continues to increase (increase in kinetic energy). However, above a certain temperature the structure of the enzyme molecules vibrates so energetically that some of the bonds holding the enzyme molecule in its precise shape begin to break. The enzyme molecule begins to lose its shape and activity and is said to be denatured. This is often irreversible.

The temperature, which an enzyme catalyses a reaction at the maximum rate is called the optimum temperature. Most human enzymes have an optimum temperature of around 40?C.

Enzyme concentration

Increasing enzyme concentration increases the rate of reaction by increasing the possibility of collision with substrate as more active sites are available. Therefore increasing the rate at which substrate is converted to product.

Making Standard Solution(s)

What concentration of Na2CO3 is needed?

Possible range of HCl in titration flask

1) If urea is not reacted

– Therefore, no NH3 is produced.

– Therefore, HCl is unreacted.

So, 50 cm3 of 1M HCl

= 50/1000 x 1 = 0.05 mol HCl

In Titration/

2 HCl ; Na2CO3 = 2 NaCl ; CO2 ; H2O

Therefore, 2:1 mol ratio

Therefore, mol Na2CO3 = 0.025 moles

SAY MAX IN BURETTE = 50 cm3

� 0.025 = 50/1000 x conc.

� Conc. of Na2CO3 = (0.025x 1000)/50

THEREFORE, Standard solution of Na2CO3 = 0.5M

Preparing a standard solution of Sodium Carbonate

Based on my calculations, I have come to the conclusion that a 1dm3 solution of 0.5M Sodium Carbonate will have to be made up.

– Molar Mass of Na2CO3 = (2×23) + (1×12) + (3×16) = 106g

– Therefore, for a 1 Molar solution 106 grams of Sodium Carbonate is needed to dissolve in 1dm3.

– Therefore, to make a 0.5 Molar solution, 53 grams of Sodium Carbonate is needed to dissolve in 1 dm3 of water.

– A magnetic stirrer was used to try to get the Sodium Carbonate solution to dissolve.

Starting with 2M solution

How much solution is needed?

-100 cm3 at each dilution

Concentration used

Ratio

2M

100cm3 of 2M solution

1.0M

50cm3 2M + 50 cm3 water

0.5M

25cm3 2M + 75 cm3 water

0.4M

20cm3 2M + 80cm3 water

0.25M

12.5 cm3 2M + 87.5cm3 water

0.1M

5cm3 2M + 95cm3 water

Therefore, the Total Volume needed = 212.5 cm3 of 2M

NB/ The 2M solution was only used in my preliminary work.

Preparing my solutions of Urease

E.G. making a 1% solution of Urease

A 1% solution means 1 gram in 100cm3.

Therefore, in a 250cm3 solution, 2.50 grams of Urease needs to be measured out on electronic weighing scales.

Once this is achieved, the powder should be placed into a 250cm3 volumetric flask and filled up to the bottom of the meniscus with the addition of distilled water.

The Urease solution is very difficult to dissolve and needs intensive stirring using a magnetic stirrer.

Calculations

2M Urea – 80cm3

+ 1% Urease – 20cm3

Total – 100cm3

In 80cm3 of 2M Urea,

No. of moles = 2 x 80/1000 = 0.16

Therefore, max. amount of ammonia made = 0.32 moles

Titration/

NH3 (aq) + HCl (aq) � NH4Cl (aq) + H2O (l)

Therefore, we need 0.32m of HCl.

Therefore, in 100cm3 acid we need 0.32 mol.

C = n/v = 0.32/ 100/1000

Therefore, we need to use HCl of 3.2 molar concentration.

Equipment ; Chemicals:

5+ Beakers

Urease powder

Urea powder

Hydrochloric acid

Sodium Carbonate powder

Equipment for possible titration

Distilled Water

Bunsen Burner

Gauze

Tripod

Conical flask

Volumetric flask

Measuring cylinder

Pipette

Teat pipette

Electronic Weighing Scales

Safety Goggles

White tile

Methyl Orange Indicator

Stop watch

Preliminary Work 1

Aim:

To have an insight into roughly how much Sodium Carbonate titrated into a conical flask containing the urea-urease reaction before it turns neutral and to investigate whether I am using suitable concentrations of urea and urease.

Method:

I set up the apparatus needed for a titration experiment as shown in the diagram below:

Using a measuring cylinder and teat pipette I accurately measured out 80cm3 of 2 Molar Urea solution and 20cm3 of 1% Urease solution from my already made up standard solutions. The Urease solution had to made up fresh as it apparently becomes inactive after 2-3 days, so I didn’t want to srisk it.

Before the experiment starts, run the acid through the burette to remove any air bubbles. This acid will then be run out of the burette and replaced, ready to begin the actual titration. Also before beginning the titration the Burette must be cleaned by passing Hydrochloric acid through it; this is so that any chemicals from previous experiments are not still in the burette. Once the burette has been washed then it must be filled up with 1 Molar Hydrochloric acid by using a glass funnel to the 0 cm3 mark. To increase accuracy the bottom of the meniscus of the acid must be touching the line indicating 0 cm3. This can be achieved by positioning your eyes level with the bottom of the meniscus.

Pour the 80cm3 of 2M Urea solution and the 20cm3 of the 1% Urease into a 100ml beaker, starting the stopwatch as soon as both reactants have been added. Every six minutes I pipetted out 20cm3 of the solution into a 100ml conical flask. I added a few drops of methyl orange indicator into the conical flask. This turned the solution orange. I kept my hand on the tap and only turned it on slightly as I didn’t know exactly how much Hydrochloric acid would need to be added to neutralise the reaction, whilst also swirling the conical flask. When the solution turned completely from orange to pink I knew that the solution had been completely neutralised. At this point I turned off the tap on the burette and recorded the volume of acid titrated. The experiment was carried out at room temperature.

Conclusion to Preliminary

The reaction was far too fast and was really over within the first few minutes. Therefore, it makes it very difficult to measure the rate of the reaction. I also became clear to me that 30 minutes for each concentration and temperature was too long and therefore I need to carry out each experiment for only five minutes, taking samples of the reaction every minute. I will have to use smaller concentrations of both urea and urease to prolong the reaction.

Method of actual Experimental Procedure

Before the experiment starts, run the acid through the burette to remove any air bubbles. This acid will then be run out of the burette and replaced, ready to begin the actual titration. Also before beginning the titration the Burette must be cleaned by passing Hydrochloric acid through it; this is so that any chemicals from previous experiments are not still in the burette. Once the burette has been washed then it must be filled up with Sodium Carbonate by using a funnel to the 0 cm3 mark. To increase accuracy the bottom of the meniscus of the acid must be touching the line indicating 0 cm3.

Once I prepared my standard solutions, I began setting up my apparatus as shown in the diagram above. However, this time I only made up a 1 Molar solution of Urea and diluted it down to make my other concentrations. Using two measuring cylinders I measured out exactly 80cm3 of 1M Urea solution and 20cm3 of the 1% Urea solution. I also prepared a conical flask with 50cm3 of 1M Hydrochloric acid (that was chilled after being left overnight each night in a fridge) and a burette containing the Sodium Carbonate solution that will be titrated into the conical flask later in the experiment. I then poured both solutions, one by one, into a 150ml beaker and started the stop watch as soon as both reactants were added. I decided to use a bulb pipette to pipette out 10ml of the reaction into the conical flask containing the Hydrochloric acid solution. At the exact moment that the 10ml of the reaction was added, I also added a few drops of methyl orange indicator. The solution now turned orange. I turned on the tap on the burette and the Sodium Carbonate began to run into the flask. When the solution showed a hint of colour change, I slowly closed the tap on the burette and read off the value of the amount of Sodium Carbonate added. According to my research the solution should change colour from orange to pink. I repeated each experiment for each concentration of urea and urease three times over or until two results are within 5% of each other. Any anomalies will be noted. I will carry out the same procedure for every urea and urease concentration but it must be noted that when varying the urea concentration, the urease concentration will be kept constant throughout and vice versa.

Varying the temperature of the reaction

The set up of the apparatus was identical to the previous experiment. I have decided to react my 0.25M concentration of Urea with my 1% solution of Urease. The five different temperatures I will be using are: 10�C, 20�C (room temp.), 30�C, 40�C and 50�C. The temperature should be raised by heating or cooled by adding ice. I carried out the exact same procedure as stated above (pages 8/9) with the exception that, for example, for the 50�C experiment, I heated my 20cm3 of 1% Urease solution using a Bunsen Burner but turned it off as it reached around 45�C, knowing that it would continue to rise anyway. Therefore, it is fair to say that the exact temperature would have been roughly +/- 2�C of this value. I then placed the heated solution into a beaker containing my measured 80cm3 of Urea. I immediately started the stopwatch when both reactants reacted. Once again every minute I took a sample of 10ml of this solution using a bulb pipette and placed it in a conical flask containing my 50ml of 1M Hydrochloric acid. I will then add a few drops of methyl orange indicator to the solution. Finally, I will titrate my 0.5M solution of Sodium Carbonate and will turn the tap off the burette at the first sign of colour change. The volume of alkali added will be recorded. I will each temperature three times over. I will carry out the same procedure for each experiment, except for the 10�C, as I will have to add ice to the urease solution to cool it down.

Diagram of Experimental Procedure

Prediction:

I predict that as both the temperature and the concentration increase then so does the rate of the reaction increase.

As the temperature increases, molecules have more kinetic energy (vibrate more often per second) with which to react. They collide more often and are more likely to collide with enough energy to overcome the activation energy needed. This is the energy that is needed to start breaking the bonds in the colliding molecules so that the collision can lead to a reaction. Figure 4 in Chemical Ideas page 224 shows that when you increase the temperature by 10�C, from 300K to 310K.

E.g. The activation enthalpy of a reaction is +50kJ mol-1.

Figure 5 (Chemical Ideas, pg. 225) shows the number of collisions with energy greater than 50kJ mol-1, for the reaction at 300K. It’s given by the shaded area underneath the curve.

However, the optimum temperature at which most enzymes work is approximately 40�C or roughly body temperature and I believe this to be true for Urease also. From my background knowledge though, I know

that as the temperature increases above 50�C, then the enzyme begins to denature. This involves the enzyme’s active site changing shape/ becoming distorted. The substrate is no longer able to fit in the enzyme’s active site and the enzyme-substrate complex is no longer achieved.

In this investigation, both the urea and urease concentrations will be varied. Firstly, from my own reading I know that as the urea (substrate) concentration increases, the rate also increases because there will be more substrate molecules to collide with the urease molecules (enzyme), therefore increasing the possibility/chance of a successful collision.

At higher substrate concentrations, enzyme molecules become more saturated with substrate molecules, so there are fewer free enzyme molecules, which are available to combine with substrate molecules. This is why I predict that a graph plotting the rate of a reaction versus substrate concentration will flatten off at a certain level, which is unknown to me. At this point, the reaction remains at the saturation level.

Secondly, as the enzyme concentration increases then the rate of the reaction also increases. The increase in enzyme concentrations means that there are more active sites available to react with the substrate molecules. Initially, again from my research and outside reading, I predict that initially the graph-plotting rate versus enzyme concentration will be linear, before eventually beginning to level off. This is because as the enzyme concentration increases, there are more enzyme molecules available to catalyse the reaction. At very high enzyme concentrations, the substrate concentration becomes rate limiting, thereby the reaction begins to level off and therefore slows down.

Choosing the right Indicator

There are a large variety of indicators to choose from in this investigation. However, different indicators change colour at different pH ranges. The following are possible indicators to be used:

In this investigation, the standard solution of Sodium Carbonate that I made up had a pH of approximately 11.6 (according to my research on the internet). This was titrated to neutralise the Hydrochloric acid in the conical flask, which had a pH of approximately 2. The Methyl Orange indicator will turn the solution orange, which in turn should turn pink as the solution begins to be neutralised.

Safety

When handling the chemicals, eye protection, safety goggles and a lab coat must be worn especially when handling hydrochloric acid. Hazardous safety symbols must be placed on the solution/powder(s) to warn users of the dangers when handling the chemicals. These precautions must be kept worn until the experiment has been completed and the benches have been wiped down in case of spillages and sprinkles.

�

Throughout the entire experiment, these precautions must be worn until the entire experiment each day is complete.

Risk Assessment

Urease

If in contact with eyes, flush eyes with water for at least 15 minutes and seek medical attention. In case of contact with the skin, immediately flush with plenty of water. If ingested seek medical attention immediately. Should be treated as potentially hazardous.

Urea

Contact may cause eye irritation including stinging, watering and redness. Contact with the skin may cause redness, itching and pain. Urea powder may cause irritation of the nose, throat and respiratory tract. These hazards do not really apply to the weak solutions of urea that I will be using.

Sodium Carbonate

Eye contact may produce notable irritation. Upon contact with the eyes, allow the normal tearing action of the eye to flush the material from the eyes. Due to the hyponicity of the solution, prolonged skin contact can wash away oils, leaving the skin dry and prone to cracking. In the event of ingestion, recommend that the person drink at least eight ounces of water to wash out the urea. These hazards are unlikely given the weak solution of Sodium Carbonate that I will be using.

Hydrochloric acid

Hydrochloric acid is highly corrosive. In case of contact, it rapidly causes burns of the mucous membranes, the eyes and the skin. Contact with the eyes, should be rinsed immediately under cold water and followed by treatment from a doctor.

�

These symbols must be placed in the laboratory at all times to warn of the potential dangers when handling the chemicals in my investigation

How will I produce accurate and reliable results?

As an enzyme, Urease becomes inactive after roughly 2-3 days. To overcome this problem I will make up a fresh, new solution of urease each day. When investigating the effect of temperature on Urease’s activity, I will store my Urease sample in the fridge every morning before varying the temperature.

It is very difficult to stop the Urea-Urease mixture from reacting in the flask when beginning the titration. However, this should be overcome by the use of a buffer solution, which in this case will be Hydrochloric acid.

I will replace/wash each beaker, conical flask and burette after every experiment. This prevents any chemicals from mixing/reacting and will also alter the volumes of each reactant that has been added, which could affect the rate of reaction.

I will repeat each temperature and concentration twice for each investigation or even possibly a third time depending on whether the two results are within 5% of each other. I am also using a wide range of concentrations and temperatures to vary (exactly five temperatures and five concentrations). This will ensure that my results are comparable and therefore reliable, especially given that I will be repeating my results.

-Having a white tile underneath the flask will make it clearer to see when the solution changes colour.

-Read accurately the readings from the burette and avoid rounding to be accurate.

-When measuring the volume in the volumetric flask, make sure that the flask is placed on a level surface and that your eye level is in line with the graduation mark on the flask.

Finding the order of the reaction:

Changing the concentration of substances taking part in a reaction usually changes the rate of the reaction. A rate equation shows this effect mathematically. Orders of reaction are a part of the rate equation.

For a general equation in which A and B are the reactants:

A + B � products

the general rate equation is:

Rate = k [A]m [B]n

m and n are the powers to which the concentration needs to be raised: they usually have values of 0, 1 and 2. m and n are called the order of the reaction, with respect to A and B.

e.g. rate = k [H2O2] [Catalase]

In this case ‘m’ and ‘n’ are both equal to 1. We say the reaction is first order with respect to H2O2 and first order with respect to catalase. The overall order of the reaction is given by (m+n), so in this case the reaction is overall second order.

NB/ Only one concentration should be varied at any one time and the temperature must be kept constant throughout.

In this experiment,

Temperature and rate constants

Rate = k [A]m[B]n

If the temperature increases by 10�C then the rate of many reactions is roughly doubled. Since the concentrations are not changed, the rate equation

Rate = k [A]m[B]n

Shows that it must be the value of k, the rate constant, which has doubled.

Generalising this

By doing experiments involving a reaction between A and B, you would find that the rate of the reaction was related to the concentrations of A and B in this way:

This is called the rate equation for the reaction.

The concentrations of A and B have to be raised to some power to show how they affect the rate of the reaction. These powers are called the orders of reaction with respect to A and B.

For UK A’ level purposes, the orders of reaction you are likely to meet will be 0, 1 or 2. But other values are possible including fractional ones like 1.53, for example.

If the order of reaction with respect to A is 0 (zero), this means that the concentration of A doesn’t affect the rate of reaction. Mathematically, any number raised to the power of zero (x0) is equal to 1. That means that that particular term disappears from the rate equation.

The overall order of the reaction is found by adding up the individual orders. For example, if the reaction is first order with respect to both A and B (a = 1 and b = 1), the overall order is 2. We call this an overall second order reaction.

Some examples

Each of these examples involves a reaction between A and B, and each rate equation comes from doing some experiments to find out how the concentrations of A and B affect the rate of reaction.

Example 1:

In this case, the order of reaction with respect to both A and B is 1. The overall order of reaction is 2 – found by adding up the individual orders.

Activation enthalpy

Activation enthalpy is the minimum kinetic energy required by a pair of colliding particles before a reaction will occur. It is the energy that must be supplied to enable bonds in the reactants to stretch and break as new bonds form in the product.

Temperature in ?C

Temperature in kelvin (273+temp.)

1/temp

Rate

Log of rate

10

283K

0.0035335

20

293K

0.0034129

30

303K

0.0033003

38

311K

0.0032154

50

323K

0.0030959

Analysis

From all the results I have gathered, I can give the judgement that the prediction I made was accurate and correct. As the concentration of both the enzyme and the substrate increase, then so does the rate of reaction increase. The same can be said for increasing the temperature. However, both these statements are not as straightforward as they may seem. From my graph(s), I can see that the rate of reaction was fastest at around 40�C. This is Urease’s optimum activity temperature. As I mentioned in my background knowledge, as the temperature rises the enzyme and substrate molecules move faster. They collide more often and with more energy and are therefore more likely to collide with enough energy to overcome the activation enthalpy. Beyond 40�C, then the reaction begins to slow down. The enzyme molecules begin to change shape in heated conditions. Their active sites are affected and the enzyme is said to be denatured. My results and graphs also indicate that as the enzyme concentration increases there are more active sites available to react with the substrate molecules. However, once the enzyme concentration reaches a certain level, then the rate no longer increases and begins to level off, as the substrate concentration becomes a limiting factor.

As substrate concentration increases, the initial rate of reaction also increases. There are more substrate molecules to collide with the enzyme molecules, therefore increasing the likelihood of a successful collision. However, as the enzyme molecules begin to become fully saturated with substrate molecules then the reaction begins to slow down. This stage is known as the saturation level of an enzyme-catalysed reaction.

However, it must be noted that some of graphs produced perverse trends.

E.G. my graph to investigate the trend produced by varying the temperature of an enzyme-controlled reaction produced the following trend:

This scatter graph has produced the inverse trend to the ‘hill-top’ that I had expected and researched in my reading on the factors that affect enzyme-catalysed reactions. There is, however, an explanation as to why this has occurred. This graph has plotted temperature against the volume of Sodium Carbonate titrated into the conical flask.

If, however, I plot the temperature versus the amount of Sodium Carbonate that remained in the burette after the titration (Vo-VT), then the trend/pattern of my graph is very similar to the prediction I made.

Results Tables

Urea concentration (moles/dm3)

Rate (V0-VT) cm3/ minute

0.102

4.2

0.25

12.1

0.40

23.6

0.60

27.4

1.00

47.7

First order

Results at room temperature, varying concentration of urea

0.1M Urea

Time (mins)

Vol. of added Na2CO3 (VT) in cm3

V0-VT (50-VT) in cm3

1

45.3

4.7

2

38.6

11.4

3

35.9

14.1

4

20.4

29.6

5

15.1

34.9

0.25M Urea

Time (mins)

Vol. of added in Na2CO3 (VT) in cm3

V0-VT (50-VT) in cm3

1

36.8

13.2

2

26.4

23.6

3

18.5

31.5

4

14.4

35.6

5

12.8

37.2

0.4M Urea

Time (mins)

Vol. of added in Na2CO3 (VT) in cm3

V0-VT (50-VT) in cm3

1

27.0

23.0

2

16.8

33.2

3

11.9

38.1

4

8.8

41.2

5

7.1

42.9

0.6M Urea

Time (mins)

Vol. of added in Na2CO3 (VT) in cm3

V0-VT (50-VT) in cm3

1

22.2

27.8

2

11.6

38.4

3

7.9

42.1

4

3.7

46.3

5

2.4

47.6

1.0M Urea

Time (mins)

Vol. of added in Na2CO3 (VT) in cm3

V0-VT (50-VT) in cm3

1

10.3

39.7

2

3.2

46.8

3

2.8

47.2

4

2.4

47.6

5

2.1

47.9

1M Urea – Varying Urease

Urease concentration (%)

Volume Na2CO3 titrated at the end of 5mins in cm3

V0-VT (50-VT) in cm3

1%

0.40

49.60

0.7%

8.35

41.65

0.5%

12.40

37.60

0.2%

27.40

22.60

0.1%

39.70

10.30

1% Urease

Time (mins)

Volume Na2CO3 titrated at the end of 5mins in cm3

V0-VT (50-VT) in cm3

1

12.20

37.80

2

3.90

46.10

3

1.80

48.20

4

0.75

49.25

5

0.40

49.60

0.7% Urease

Time (mins)

Volume Na2CO3 titrated at the end of 5mins in cm3

V0-VT (50-VT) in cm3

1

33.90

16.10

2

14.35

25.65

3

17.00

33.00

4

12.10

37.90

5

8.35

41.65

0.5% Urease

Time (mins)

Volume Na2CO3 titrated at the end of 5mins in cm3

V0-VT (50-VT) in cm3

1

38.50

11.50

2

29.45

20.55

3

21.75

28.25

4

15.95

34.05

5

12.40

37.60

0.2% Urease

Time (mins)

Volume Na2CO3 titrated at the end of 5mins in cm3

V0-VT (50-VT) in cm3

1

43.30

6.70

2

37.45

12.55

3

32.50

17.50

4

29.05

20.95

5

27.40

22.60

0.1% Urease

Time (mins)

Volume Na2CO3 titrated at the end of 5mins in cm3

V0-VT (50-VT) in cm3

1

48.15

1.85

2

45.50

4.50

3

43.05

6.95

4

41.10

8.90

5

39.70

10.30

Bibliography

www.btinternet.com/~chemistry.diagrams/ titration.htm -This website provided me with the diagram of the set-up of the apparatus.

www.geog.soton.ac.uk/school/safety/ prepared/palaeo/fusion.rtf – I used this site to help in my risk assessment. It helped me to be aware of some of the potential hazards that can occur whilst carrying out the experiment. This information allowed me to take the necessary safety precautions.

http://www.lakes-environmental.com/toxic/HYDROCHLORIC_ACID.HTML was used to examine the possible hazards when dealing with Hydrochloric acid. However, we will only be using a weak concentration of the acid, so the hazards will be minimised.

Chemical ideas book on pages 12-13 gave me an insight to working out the concentrations and how to go about the experiment.

Advancing Chemistry book provided me with a table of the possible choice of indicators to use. From the table I decided to use Methyl Orange.

http://www.biologymad.com/master.html?http://www.biologymad.com/Enzymes/enzymes.htm – provided me with my background knowledge and some excellent graphs along with simple scientific knowledge.

Evaluation

There were many limitations to this experiment that I tried my best to overcome. Firstly, it was very hard to control the temperature at which the reactions were taking place at. When varying the temperatures, it dropped quickly and varied from start to finish. In reality there was very little I could do to overcome this problem, which may have slightly affected the rate of the reactions. I did record the temperature at 0 and 5 minutes and the difference was never greater than 3.5�C. Another limitation related to temperature was the fact that the room temperature varied each day. It is difficult to know the extent of the affect this had on my results. I recorded the room temperature every day for two weeks and the range varied by 0.6�C but there was little else I could have done otherwise. Getting my Sodium Carbonate powder to dissolve was arguably the hardest challenge in the experiment. The process took a very long time and in reality I noticed a few, tiny bits of powder in the bottle of the solution. I used a magnetic stirrer for almost half an hour, combined with intensive stirring using a glass stirrer, to try to overcome this problem. From my research, I knew that my Urease solution became inactive after 1-2 days.

This meant that everyday I had to make up a new solution of Urease to avoid this problem. Another problem I experienced throughout the whole investigation was knowing when exactly the indicator has changed colour. My eyesight would have varied throughout each experiment and therefore the exact moment when I turned the tap off the burette would have varied. This meant that at certain times I may have added too much or not enough Sodium Carbonate to fully neutralise the Hydrochloric acid in the flask. I also had a problem with transferring the mixture from the beaker into the conical flask. This meant that the reactants were still reacting in the pipette so a buffer solution of Hydrochloric acid was used. However, the exact effects of it are unknown to me, though research indicated it was effective in stopping the reaction from taking place in the flask. The final limitation in this investigation was that there was a 30 second gap before titration is underway while I sample from beaker to conical flask. I tried to overcome this by being quicker in pippetting out 10ml of the reaction.

It is very difficult to know how exactly I could have improved the reliability and accuracy of my results. I suppose that having more accurate apparatus on hand would have been helpful, but the extent to which my results would have become more accurate is unknown and unavailable to me. I could also have carried out more repeats for each concentration and temperature and also used more temperatures and concentrations to investigate. This would have improved the accuracy of results and graphs.

Having a control sample of the end point of the colour of the indicator beside each experiment would have helped me to identify exactly when to stop titrating in Sodium Carbonate into the flask. Arguably I may have turned the tap off at different points in each experiment therefore adding too much or too little Sodium Carbonate. Otherwise using a pH meter would have provided me with quantitative data e.g. a solution of pH 7.2, rather than qualitative data e.g. using your naked eye to decide approximately when the solution changed colour. Facts would have been more accurate than judgement.

Another alternative procedure:

An aqueous solution of urea is stable at room temperature, but when the urease is added the pH of the solution goes up as ammonia is produced.

The experiment shows how you can measure the speed of the reaction quite accurately by titrating the ammonia, as it is produced, with acid so that the pH stays constant. The rate at which you add the acid gives you a measure of the rate of ammonia production.

Prepare a standard solution of urease. Filter the mixture into the conical flask, stopper it and keep the filtrate refrigerated between practical sessions. Using a pipette, transfer a certain volume of the known concentration of urea into a beaker. Set up the apparatus as shown in figure 9.

Stir the solution carefully using a magnetic stirrer. Adjust the pH of the mixture to 5 by the addition of drops of a known concentration of hydrochloric acid solution. Add a certain volume of urease solution using a graduated pipette. Shake the mixture and start the stop watch. Very carefully add the acid at a steady rate so that the pH remains at 5. Read the volume of acid added at regular intervals e.g. every 15 seconds. Stop taking readings after 5 minutes. Run a control experiment with distilled water in place of the urease.

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