To this mixture, six drops of phenolphthalein solution was added to each of the test tubes. The tubes were then shaken until all displayed a pinkish purple shade of colour. At this point, timing began for the twelfth test tube as no lipase was to be added.
Next, the first tube was taken and to this, 1cm3 of lipase solution was added. The moment that all of the contents of this 1cm3 graduated pipette were expelled, the stopclock commenced its timing. Immediately after this, the tube was tapped rhythmically, continuously, until a change in colour from pink to absolute white was observed. At this point, the timing was stopped. This process was then carried out to the rest of the test tubes.
The whole process of this experiment was carried out three days later.
For those who wish to perform the experiment themselves, here is the condensed form:
- Place all of the test tubes into their racks.
- Put on your safety goggles.
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(Remembering to measure from the bottom of the meniscus when measuring the liquids), Add 5cm3 of milk to each tube using a 5cm3 graduated pipette.
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Add 7cm3 of sodium carbonate solution to each tube using a 10cm3 graduated pipette.
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Add 3% bile salts to all of the test tubes starting off with 1cm3 in the first tube and then decreasing in 0.1cm3 decrements from then on. This should continue until you get to the twelfth tube. In this test tube, a full 1cm3 should be added.
- Add six drops of phenolphthalein solution to each tube using a dropping pipette.
- Shake all the tubes.
- Begin timing the twelfth test tube.
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Take the first test tube and add 1cm3 of lipase solution using a 1cm3 graduated pipette.
- Immediately start timing using the stopclock. Begin tapping the tube rhythmically.
- Stop the stopclock and recorded the result when the colour change of pink to white has occurred.
- Repeat the lipase adding and timing to each of the rest of the test tubes in turn, all except the final tube, which is to have no lipase added.
Results
Experiment 1
Experiment 2
Average results
Standard deviation of the average results (omitting no lipase)
344.2957 seconds
Conclusion
From the results, I believe that it is clear to see that there is a clear correlation between the amount of bile salts added, the concentration if you will, and the time in which lipase can hydrolyse fat. I would also go as far as to say that the bile salts are the sole cause of an increase in reaction hydrolisation rates.
In the tube with no bile salts, the hydrolisation of the fat occurred very slowly indeed. Up until that point, the times were only increasing by –at the maximum- two hundred seconds per 0.1cm3 decrease of bile alts added. When no bile salts were added, the time needed to hydrolyse all of the fat increased by about 340 seconds. This, coupled with the fact that the higher the concentration of bile salts added, the faster the reaction occurred, indicates that the correlation is in fact positive, and that the bile salts have almost catalytic properties. This is due to the fact that bile salts emulsify fats. Bile salts are secreted from the liver, where they are stored in the gall bladder (in most mammals), before passing through the bile duct and on into the intestine when food is passing through. They are steroids with detergent properties with their central purpose in digestion being to emulsify lipids in food and, more precisely in terms of this investigation, fats. It emulsifies the large fat droplets into many smaller droplets so that the lipase can ‘break’ the fat down, hydrolysing it into the fatty acids and glycerol fore-mentioned, that can then be absorbed through the wall of the intestine. The fact the bile salts create smaller droplets of fat, aids the efficiency of the lipase because it then creates a larger surface area of fat for the enzyme to act upon. This makes the whole process work faster. Therefore, what the results indicate is that an increase in the concentration of bile salts results in a much faster emulsification of the fat, which then in turn allows the lipids to act upon these smaller fat droplets faster. This is due to the fact the bile salts create a larger surface area for the lipase to act upon. An increase in bile salt concentration means that the smaller droplets are created faster and also many more are formed.
The test tube that contained no lipase indicates that the bile salts alone cannot hydrolyse the fat. During the first experiment, no colour change had occurred after 6780 seconds. This was a massive 1 hour and 53 minutes. Because of this, it was decided that no change would occur and the timer was stopped. The same occurred in the second experiment so it was decided to stop at this point also. The test tube that was absent of bile salts was also slow to react as mentioned earlier. This shows that the two substances share a relationship and together, they can hydrolyse fats quicker. This relationship is present in the mammalian body, and it is clear to see why this relationship is present in all mammals. It obviously works.
Evaluation
Generally, I believe that the investigation was accurate and I think this is reflected by the fact that there are no glaring anomalies to be found in the table of results or the graphs. The fact that the graphs look so similar, almost identical in fact, shows that the procedures and method I followed were accurate and also the same each time.
The fact that the measurements of liquid were take by looking at the bottom of the meniscus shows that my results were accurate and also, as this was what I did throughout, reliable. The method I followed was kept identical in both experiments and the rhythmical tapping of the test tubes was also identical.
The fact that both experiments were conducted on different days allows me to rule out any external factors such as temperature. Had the results from both experiments been very different then I would have taken these factors into account.
The test tube that contained no lipase indicates that the bile salts alone cannot hydrolyse the fat. During the first experiment, no colour change had occurred after 6780 seconds. This was a massive 1 hour and 53 minutes. Because of this, it was decided that no change would occur and the timer was stopped. I don’t think that this had any detrimental affects on the results, as this was only a control test tube anyway. What this tube showed is that, the bile salts had no hydrolysing properties. This was an important factor to investigate because the bile salts contained acids and the hydrolisation was to be highlighted by a drop in pH. The fact this control made no change shows that the results are reliable.
There are however, limitations to the investigation. The fact that one needed to look for a colour change to show that the reaction had occurred has one major limitation. It is very subjective as it is very difficult to define when the colour has become absolutely white or not.
A further limitation to this investigation comes about from the fact that all of the test tubes were prepared at the same time. The problem is that whilst the adding of lipase and timing of the first test tube was occurring, the bile salts in the other test tubes would have more time to emulsify the fats present before they were timed. This would certainly affect the results in a negative way and if this was the case then it is expected that the later tubes would have had quicker hydrolisation times. The opposite was in fact the case. This shows that my results are still valid.
If I were to conduct he experiment again, then I would prepare and hydrolyse each of the test tubes one at a time, so that the above problem does not arise again. The only reason it did was because this experiment was confined to a limited space of time.
If I were to conduct further investigations, I would also stop the tapping motion that was used to speed up the reactions. There is a chance that one tube may have been tapped more than another and this may have affected the results. However, if one is to look at the graph, there are no blatant anomalies. So again this problem can be ruled out as having detrimental affects to the experiment. Although it is a problem that would be resolved if I had more time.
To rule out the subjectivity of identifying when the mixture becomes all white, I would use universal indicator solution instead of the phenolphthalein solution. This would allow me to have more control over the observation of any pH changes that occur.
In my plan, I predicted that an increase in the concentration of bile salts would result in a faster and more pronounced emulsification of the fat, leading to a faster action of the lipase on the small fat droplets. This was proved and the prediction resulted from observing the processes that occur in the mammalian body. Again my results reflect this assumption. Therefore I can assume that this investigation was accurate, reliable and more importantly, valid.
