EXPERIMENT 5 : EXPERIMENT USING LIPID

ABSTRACT

This experiment is carried on in order to evaluate one of several triglycerides to determine the saponification number. This experiment also been carried out to make soap by the same process called the saponification, using the modern ingredient. The experiment to determine the saponification had been carried out in the fume hood. After that, it had been titrate by using 0.5M of HCL. The saponification number is used as an indicator of fatty acid chain length in triglycerides. The value is simply a measurement of the mg of KOH required to complete the hydrolysis of one gram of fat oil. For making the soap, sunflower oil had been used. The oil had been boil with 6.0M of NaOH. In the process of making soap, vegetable fat, which is triglyceride, is hydrolyzed by the action of strong base, such as sodium hydroxide, and heat. The resulting products are soap and glycerol.

INTRODUCTION

Lipids are molecules that contain hydrocarbons and make up the building blocks of the structure and function of living cells. For examples, fats, oils, waxes, certain vitamins, hormone and most of the non-protein membrane of cells. Besides that, lipids are a large and diverse group of naturally occurring organic compounds that are related by their solubility in non-polar organic solvents and general insolubility in water. The tri-esters of fatty acids with glycerol (1,2,3-trihydroxypropane) compose the class of lipids known as fats and oils. These triglycerides are found in both plants and animals, and compose one of the major food groups of our diet. Triglycerides that are solid or semisolid at room temperature are classified as fats, and occur predominantly in animals. Those triglycerides that are liquid are called oils and originate chiefly in plants, although triglycerides from fish are also largely oils. The process of saponification, by heating a triglyceride in aqueous potassium hydroxide (KOH) the fatty acyl esters can be cleaved off (hydrolysis) leaving behind glycerol and the potassium salt of the fatty acid. So, the triglycerides that contain high fatty acids number will have a lower saponification number that triglycerides with low fatty acids number.

PROCEDURE

1. Saponification of triglyceride

2. Application : making soap

RESULT

1. saponification of triglyceride

2. Application : making soap

DISCUSSION

1. Saponification of triglyceride

The saponification number (sap) measures the bonded and unbonded acids present in an oil or fat. It defines the exact amount of potassium hydrate in mg necessary to emulsify 1g of fat or oil.  The smaller the molar mass of the fat, the higher the saponification value. For saponification of triglycerides experiment to determine the saponification number of triglycerides  , we were using sunflower oil as a sample for our group. Different group used different oil which is palm oil and corn oil. Then at the end of the experiment we obtained the final results for each sample. Based on the results obtained, sunflower oil has the highest saponification number compared to corn oil and palm oil. So it shown that, sunflower oil has shorter fatty acids.

Theory said that triglycerides containing long fatty acids will have a lower saponification number than triglycerides with shorter fatty acids. Since 1 gram of lipid containing long chains will have less chains in total than 1 gram of lipid containing short chains. Actually, we should get the highher saponification number for palm oil followed by corn oil and the lowest is sunflower oil.  Low fatty acid fats like coconut oil or palm kernel fat have high saponification numbers of 250, whereas most vegetable oils have a saponification number of approximately 190 It means that, palm oil have the shorter fatty acid chain than corn oil and sunflower oil. For the palm oil and corn oil, the saponification number should higher than sunflower oil because they have shorter fatty acid chain than sunflower oil. A few error occured during the experiment that effect the results obtained that are, there is no standard colour of solution when turns colourless. the pinkish colour might still there . So, this affect the titration process. it then, affect the volume of  HCL  used.

2. Application : making soap

Soap is made by the saponification reaction. It is an exothermic chemical reaction which happens when fatty acids react with base. This process involves boiling the fats together with the base. For this experiment, we boiled sunflower oil with 6M NaOH. The hydrolysis of the oil occurs, thus producing glycerol and crude soap. Then, sodium chloride is added into it once the saponification reaction completed. Addition of sodium chloride causes the soap to precipitate. The crude soap acquired from the process consists of sodium chloride, sodium hydroxide and glycerol. The crude soap curds is then boiled in water and re-precipitated with salt in order to remove the impurities. After that, we add some color into it and the result is as the picture below.

QUESTION 

1.    What is the relationship between saponification and phase (liquid / solid) of a triglyceride?

In saponification, boiling triglycerides (oils) react with concentrated alkali (potassium hydroxide) to produce glycerol and salts of fatty acids. Triglycerides exist in the organic phase while the hydroxides exist in the aqueous phase. By increasing agitation, the reactants will happen to be in the same phase. Therefore, the oils can be hydrolyzed to form glycerol and fatty acids. The acids then react with potassium hydroxide to form potassium salts. Thus, glycerol and fatty acid salts that have appreciable solubility in both phases can be formed. The reagents will increase in concentration in the phase which is most favored by the other as the reaction progresses.

2. Why do triglycerides with longer fatty acids have a lower saponification number than those with shorter fatty acids?

It is because the longer the fatty acid chains, the fewer the number of carboxylic functional groups per unit mass of the fat. It results in a higher molecular weight. Therefore, less acid is liberated per gram of fat hydrolyzed. As less KOH is needed to hydrolyze the ester bonds, saponification number is lower.

3. Why is the difference in the molar amount of HCl used to neutralize the control and the amount of HCl used to neutralize the sample equivalent to the molar amount of KOH used to saponify the test sample?

      More HCl is used to neutralize the control as compared to the neutralization of the sample. It is because in blank test, all KOH reacts with HCl to produce a neutralized solution. On the other hand, in sample test, some KOH is used to hydrolyze the fatty acids while some is neutralized by HCl. Therefore, more HCl is utilized in the blank test than in the sample test.

4. Why do soaps disperse grease?

Soaps have hydrocarbon and ionic ends. The hydrocarbon ends are hydrophobic and non-polar. The ionic ends are hydrophilic and polar. When both oil and water present and mix with the soap. The hydrophobic part of the soap mixes with the oil, while the hydrophilic part of the soap mixes with the water. The attraction of the polar end of the soap is strong enough to pull the grease molecules into the water. Therefore, the grease molecules associated with the non-polar ends are pulled into the water along with the soap molecules. The soap and grease combine and arrange in the form of micelles. Finally the micelles will disperse in the water.   

CONCLUSION

The smaller the molar mass of the fat, the higher the saponification value,and shorter fatty acids. Sunflower oil has the highest saponification number compared to corn oil and palm oil. Soap is one of the product produced by undergo the saponification reaction. It undergo an exothermic chemical reaction which happens when fatty acids react with base. 

REFERENCES

            Chempro (2013), Chempro Edible Oil Refining ISO TUV Austria. Retrived from website,   

            on May 20th http://www.chempro.in/fattyacid.htm

            Florin Switzerland (2013), Saponification value. Retrived from website, on May 20th,

http://www.florinag.ch/index.php?sid=3ahd81hE5Q0ynses55dCxQ57BGRGh4j3lkdebwj3&c5p=495&c5l=en

Anonymous. Soap and saponification: preparation and chemical structure. Retrieved from

http://chemistry.about.com/library/weekly/blsapon.htm at 19th of May 2013.           

Anonymous. Estimation of saponification value of fats/oils. Virtual Amrita Laboratories Universalizing Education. Retrieved on May 17, 2013 from http://amrita.vlab.co.in/?sub=3&brch=63&sim=688&cnt=1

David, A. K. 2000. The science of soaps and detergents. Retrieved on May 17, 2013 from http://www.chymist.com/Soap%20and%20detergent.pdf

Experiment 4: Vitamin C

TITLE

Experiment on Vitamin C

OBJECTIVES

1. To measure the amount of Vitamin C content in many different types of food.

2. To measure the amount of Vitamin C content in types of food in different conditions of the food        

    is served.

ABSTRACT

This experiment is carried on in order to measure the amount of Vitamin C content in several types of food. This experiment also been carried out by measuring Vitamin C content in several types of food condition for example, when the food is steamed, boiled and when the food is served fresh. Titration method is used when, iodine is used to test the Vitamin c content where, the volume of iodine used to titrate the Vitamin C sample from foods been taken. The volume is taken just after, the vitamin C sample turns to blue black colour and not further turns to its original colour. In this experiment, Red Apple sample of food is used. This experiment been carried out in three different conditions of red apple that are in fresh, steam and also boil condition. The result shows fresh sample has highest content of Vitamin C, followed by steamed red apple sample and the lowest content of Vitamin C is sample of red apple when the sample is been boiled. This experiment shows that, fresh red apple juice has higher level of Vitamin C compared to processed red apple juice.

INTRODUCTION

Vitamins are organic compounds that have important biological functions. For instance, in humans they enable a variety of enzymes in the body to function. The human body cannot synthesize vitamin molecules, so they must be obtained in the diet. If a particular vitamin is lacking in the diet, a deficiency disease will result. Vitamin C, also called ascorbic acid is a water-soluble vitamin that occurs naturally in many fruits and vegetables, such as citrus fruits, green peppers, tomatoes, and parsley. It is not stable to heat, so cooking fruits and vegetables destroys much of their vitamin C content. It is also easily oxidized (converted to a non-useful form) by certain compounds, such as oxygen in the air. Therefore, the vitamin C content in an orange will be reduced if the orange has been cut in half and left exposed to the air for a period of time. Although fruit juice is a good source of vitamin C, it is not the healthiest way to obtain the vitamin, since fruit juices are very high in sugar/simple carbohydrates. Starch-iodine is used because Vitamin c can react with iodine. When iodine is added, it will first react with the ascorbic acid in the food sample, producing a colorless product (dehydroascorbic acid). When the ascorbic acid runs out, the iodine will react with the starch in the food sample. At this point, participants will be able to recognize a highly visible color change. This color change is the endpoint of the reaction. Therefore, the more iodine added, the more Vitamin C the food sample contained.

PROCEDURE

Part A: Measuring Vitamin C Using Starch-Iodine Test

Preparing vitamin C extract:

                                 i.            The food material is chopped into small pieces and is placed into blender.

                               ii.            100 mL of distilled water is added to the blender.

                              iii.            The material is blended using the highest speed.

                             iv.            The ground extract is strained.

                               v.            30 mL of strained extract is measured into a 250 mL Erlenmeyer flask.

1.       Comparing cooked food and raw food’s vitamin C:

                                 i.            The raw, steamed and boiled food are used to determine the amount vitamin C.

Part B:

Application: Magic Writing

RESULT

Part A: Measuring Vitamin C Using Starch-iodine Test

CALCULATION

Standard:

1 mg/ml ascorbic acid   = 72.5 ml iodine

Fresh Apple:

Fresh :-       26.20 ml iodine

Vitamin C in 30 g fresh apple = 26.20 ml/72.5 ml

                                            = 0.361 mg/ml of Vitamin C

Vitamin C in 100 g fresh apple = (100 g/30 g) x 0.361 mg/ml

                                            = 1.203 mg/ml of Vitamin C

Steamed Apple :

Steamed :-       22.45 ml iodine

Vitamin C in 30 g steamed apple = 22.45 ml/72.5 ml

                                                  = 0.310 mg/ml of Vitamin C

Vitamin C in 100 g steamed apple = (100 g/30 g) x 0.310 mg/ml

                                                    = 1.033 mg/ml of Vitamin C

Boiled Apple :

Boiled :-       20.15 ml iodine

Vitamin C in 30 g boiled apple = 20.15 ml/72.5 ml

                                               = 0.278 mg/ml of Vitamin C

Vitamin C in 100 g boiled apple = (100 g/30 g) x 0.278 mg/ml

                                                    = 0.927 mg/ml of Vitamin C

DISCUSSION

Part A: 

Measuring Vitamin C Using Starch-iodine Test

(I) On Fresh, Raw Apple

Based on the experiment that had been carry out, the result show that the amount of vitamin C in fresh apple is higher than in apple that had been boiled and steamed.

There are dietary fibre content skins and core of apples. About 10% of an apple is made up of carbohydrate and 4% of an apple is made up from a variety of vitamins and minerals and the rest of the apple, more than 80%, is water. For a medium-sized eating apple contains about 40 calories while one kilogram of fresh apples provides approximately 500 kcal of energy. Without the peel and core of apples from the diet halves the amount of vitamin C and dietary fiber consumed but makes very little difference to the sugar intake.

Consumption of vitamin C-rich foods in their fresh or raw form is the best way to maximize vitamin C intake. Furthermore, vitamin C is highly sensitive to air, water, and temperature. About 25% of the vitamin C in vegetables can be lost simply by blanching which is boiling or steaming the food for a few minutes. This same degree of loss occurs in the freezing of vegetables and fruits. By cooking vegetables and fruits for longer periods of time within 10-20 minutes can result in a loss of over one half the total vitamin C content. That’s why fresh apple have high amount of vitamin c than steam and boiled apple. 

(II) On Steamed Apple

While, for steam apple we had steamed it in oven for 20 minutes under 55⁰C. So, the results of vitamin C that we had obtained from the experiment for 1mL of juice was 0.31g/ml. The vitamin C content of food may be reduced by prolonged storage and by cooking. But by steaming or microwaving may lessen cooking losses. For steam fruit, most of the vitamin C retain in the fruit. Other than eating it raw, the fruit can be steam to avoid losing too much vitamin C.Fortunately, many of the best food sources of vitamin C, such as fruits and vegetables, are usually eaten raw.

(III) On Boiled, Cooked Apple

When boiling is applied to the fruit, the high temperature affects the amount of vitamin C content. It is

because vitamin C, which is sensitive to heat, is easily destroyed by excessive heat. The concentration of

vitamin C is decreased after cooking. As vitamin C is a type of water soluble, boiling may cause great loss of 

vitamin C not only thermal degradation, but also by leaching into surrounding water. Boiling causes more loss 

of vitamin C than steaming.            

In order to prepare the food in the most nutritious way, it is suggested to eat the fruits and vegetables raw 

because eating raw keeps the vitamins intact. For the exceptions, the food may undergo steaming before 

eating. The more water kept in the fruits or vegetables, the better for the nutrition. 

Part B:

Application: Magic Writing

After adding the iodine solution, the writing appears glowing. However this condition only last for awhile as we are forgetting to dilute the iodine solution. This condition happened due to the reaction between iodine and the vitamin C (from the lemon juice). When this reaction happens, both the iodine and the vitamin C turned into different chemicals called dehydroascorbic acid. As the amount of iodine is more than the amount of vitamin C that was on the paper, all of the vitamin C is destroyed and the iodine remains. 

CONCLUSION

Fresh apple retains the most vitamin C. It is followed by steamed apple. The vitamin C in boiled apple is 

damaged.

REFERENCES

Anonymous, Hands on Science lesson. Retrieved on 9 May 2013 from, http://www.life.illinois.edu/boast1/sciencelessons/vitaminc.htm

Anonymous. Vitamin C content of fruit juice. Retrieved on 9 May 2013 from,
http://carbon.indstate.edu/inlow/LabManuals/Vitamin%20C.pdf

Jennipher, W. The most nutritious ways to prepare your veggies. Retrieved on May 13, 2013 from http://www.shape.com/healthy-eating/cooking-ideas/most-nutritious-ways-prepare-your-veggies

Yuan, G.-F., Sun, B., Yuan, J. and Wang, Q.-M. 2009. Effects of different cooking methods on health promoting compounds of broccoli. Journal of Zhejiang University Science B 10(8): 580-588.

Lorna McAusland (2009). Apple Facts. Retrieved from 

http://www.ifr.ac.uk/info/society/spotlight/apples.htm.  on May 12, 2013.

Richard E. Barrans Jr. Iodine and vitamin C test. Retrieved on May 14, 2013 from http://www.newton.dep.anl.gov/askasci/chem03/chem03023.htm

George Mateljan (2013). Vitamin C. Retrieved form

            http://www.whfoods.com/genpage.php?tname=nutrient&dbid=109.  on May 12, 2013.

Experiment 3 : Enzyme Kinetics

TITLE

Experiment 3: Enzyme Kinetics Experiment

OBJECTIVES

1. To determine the effects of substrate concentration, pH, and temperature on enzyme activity.

INTRODUCTION

Enzymes are protein molecule that acts as biological catalysts. Without changing of the overall process, they increase the rate of reactions. Enzymes are long chains of amino acids bound together by peptide bonds. Besides that, they are seen in all living cells and controlling the metabolic processes in which they converted nutrients into energy and new cells. Other than that, enzymes also help in the breakdown of food materials into its simplest form. The reactants of enzyme catalyzed reactions are termed substrates and each enzyme is quite specific in character, acting on a particular substrates to produce a particular products. The central approach for studying the mechanism of an enzyme-catalyzed reaction is to determine the rate of the reaction and its changes in response with the changes in parameters such as substrate concentration, enzyme concentration, pH, temperature and known as enzyme kinetics. The substrate concentration, [S] is one of the important parameter that affecting the rate of a reaction that catalyzed by an enzyme. However, studying the effects of substrate concentration is elaborated by the fact that during the course of an in vitro reaction, [S] changes due to the conversion of substrate to product. One simplest method to study enzyme kinetics is to measure the initial rate of the reaction designated V₀ ( Initial velocity), when [S] is much greater than the concentration of enzyme, [E]. The effect on V₀, when the enzyme concentration becomes constant is shown in Figure 1.

Figure 1

PROCEDURE

The Standard Reference

Test tube	8 ml starch of x mg/ml	Water (ml)	Iodine (ml)	Absorbance at 590nm
1	0	9	1
2	0.01	1	1
3	0.025	1	1
4	0.05	1	1
5	0.1	1	1
6	0.3	1	1
7	0.5	1	1
8	0.7	1	1
9	1.0	1	1

The Standard Graph

 The Effect of Substrate Concentration

 Experiment of starch hydrolysis in different substrate concentration had been prepared as the following table:

Test tube	8 ml starch of x mg/ml	Water (ml)	Amylase (ml)	Incubate each sample at 350C for 10 minutes	Iodine (ml)	Place all test tubes in an ice bath. Measure the absorbance at 590nm
1	0	8	1		1
2	0.01	0	1		1
3	0.025	0	1		1
4	0.05	0	1		1
5	0.1	0	1		1
6	0.3	0	1		1
7	0.5	0	1		1
8	0.7	0	1		1
9	1.0	0	1		1

The Effect of Temperature

Prepare as the following for the experiment of different temperature:

Test tube	8 ml starch of x mg/ml	Water (ml)	Amylase (ml)	Incubate each sample at 20, 28, 35, 400C for 10 minutes	Iodine (ml)	Place all test tubes in an ice bath. Measure the absorbance at 590nm
1	0	8	1		1
2	0.01	0	1		1
3	0.025	0	1		1
4	0.05	0	1		1
5	0.1	0	1		1
6	0.3	0	1		1
7	0.5	0	1		1
8	0.7	0	1		1
9	1.0	0	1		1

The Effect of pH

Prepare the following for the experiment using different pH:

Test tube	Starch of 0.5 mg/ml	2 ml buffer of pH x	Amylase (ml)	Incubate each sample at 350C for 10 minutes	Iodine (ml)	Place all test tubes in an ice bath. Measure the absorbance at 590nm
1	5	4	1		1
2	5	5	1		1
3	5	6	1		1
4	5	7	1		1
5	5	8	1		1
6	5	9	1		1
7	5	10	1		1
Blank	5	3 ml of dH2O			1

RESULT
Result for substrate concentration

Test tube	So ( 8 ml starch of x mg/ml)	SF	S (So – SF )	V = S/ 20 minute
1	0	0.015	-0.015	-7.5×10-4
2	0.01	0.045	-0.035	-1.75×10-3
3	0.025	0.040	-0.015	-7.5×10-4
4	0.05	0.050	0	0
5	0.1	0.050	0.050	2.5×10-3
6	0.3	0.030	0.270	1.35×10-2
7	0.5	0.050	0.450	2.25×10-2
8	0.7	0.060	0.640	3.2×10-2
9	1.0	0.100	0.900	4.5×10-2

Graph Rate of Hydrolysis (V) at different Starch Concetration (Substrate)

Result for Temperature 

Graph of 1/V against 1/[S]

Result for pH

Test Tube	pH	Absorbance (mg/ml)
1	4	0.447
2	5	0.391
3	6	0.876
4	7	0.373
5	8	0.301
6	9	0.344
7	10	0.202
8	3ml of H2O	0.186

By using,

ΔS /V = (S₀ – S_f) / 20 min 

Test Tube	pH	S0(mg/ml)	Sf (mg/ml)	ΔS / V (mg/ml min)
1	4	5	0.097	0.2452
2	5	5	0.081	0.2459
3	6	5	0.023	0.2488
4	7	5	0.076	0.2462
5	8	5	0.052	0.2474
6	9	5	0.064	0.2468
7	10	5	0.025	0.2487
8	3ml of H2O	5	0.018	0.2491

DISCUSSION 

The Effect of substrate Concentration

As the concentration of substrate increases, the rate of reaction also increases until the point saturation occurs. It means as you increase the concentration, rate keeps increasing and then one point comes when the maximum rate is achieved and there is no free enzyme to bind with substrate and all the active sites of enzyme are bound to the substrate. So after that point, increasing the concentration wont have any effect. What is the maximum for each enzyme is usually given by Km value (michealis menten graph or the other one called sumting like Lineweaver burke plot). The Km value is the rate constant or it can be explained as how much substrate concentration is required by an enzyme to reach to the half of maximum rate or velocity of enzyme. Each enzyme has different Km values. So I hope that answers ur quesiton- wherever the Vmax occurs and it intersects the curve drawn for substrate concentraion and velocity (or rate of reaction), that point is the saturation point or maximum substrate concentration to have maximum rate of the reaction.

Based on our experiment, we got negative value for rate of hydrolysis (V). According to the theory we are not supposed to get negative value for (V). In other words it states that, when the substrate concentration change and while enzyme concentration is keeps constant, the rate of reaction will increase. This is because there are a few error that occurred during we ran the experiment. 

The Effect of Temperature

           Based on the plotted graph, the line for 10°C has the Km value of 0.30. The line for 28°C has the Km value of 0.14. The line for 35°C has Km value of 0.07 while the Km value for 40°C is 0.10. The lower the Km value, the higher the affinity to the substrate. As a result, the rate of reaction is greater. According to the three different temperatures applied, temperature of 35°C is the optimum temperature for the enzyme to react as its Km value is the lowest among all. On the other hand, the lines share the same Vmax value, which is 0.003. Although temperatures change, the active site does not change. The substrates can still bind with the enzyme. The only difference is the rate of reaction. The amylase reacts the best at temperature of 35°C.

The Effect of pH

pH can give several effect on structure and activity of an enzyme. For example, pH can have an effect of the state of ionization of acidic or basic amino acids. Acidic amino acids have carboxyl functional groups in their side chains. Basic amino acids have amine functional groups in their side chains. If the state of ionization of amino acids in a protein is altered then the ionic bonds that help to determine the 3-D shape of the protein can be altered. This can lead to altered protein recognition or an enzyme might become inactive.

Changes in pH may not only affect the shape of an enzyme but it may also change the shape or charge properties of the substrate so that either the substrate cannot bind to the active site or it cannot undergo catalysis.

The most favorable pH value - the point where the enzyme is most active - is known as the optimum pH.

The velocity of pH is increases from pH of 4 up to pH 6. But, starting from pH 7, the velocity decreases from 0.2488 for pH 6 to 0.2462 for pH 7. The velocity increase until pH 8 with value of 0.2474. Starting from pH 9, the velocity decreases again but after pH 9, the velocity keep increased until pH 10 and so with test tube blank test tube.

Based on this result of experiment, the optimum pH that is with highest velocity is sample pH 6 with velocity 0.2488. The lowest velocity is pH 1.

There is fluctuation of value of absorbance that give the fluctuation of value of velocity might because of the sample is contaminated. The other factor might be the time for taking reading of absorbance for every of pH sample is not fixed. Besides that, the process of placing all test tubes in ice bath might affect the result because, while taking reading of absorbance, the water vapour from condensation of the sample is still there, so, it might affect the absorbance reading.

CONCLUSION

Based on our experiment, some of the results are not the same as the theory. For the substrate concentration, as the concentration increase the rate of enzymatic reaction. However, the obtained result is different from the expected. As for the temperature, the rate of reaction increase gradually as the temperature increase. For the effect of pH, the most favorable pH value for amylase is pH 6.   

REFERENCE

Wise Geek (2013). What Is Substrate Concentration? Retrieved on May 1, 2013 from

            http://www.wisegeek.com/what-is-substrate-concentration.htm

Analyzing Enzyme Kinetic Data with a Graphing Calculator. Retrieved on 1 May, 2013 from

http://dwb.unl.edu/calculators/pdf/Enzyme-Calc.pdf

Worthington Biochemical Corporation (2013). Introduction to enzyme. Retrieved on April 28,

            2013 from http://www.worthington-biochem.com/introbiochem/effectsph.html

Anonymous, Effect of pH on enzyme. Retrieved on April 28, 2013

from http://academic.brooklyn.cuny.edu/biology/bio4fv/page/ph_and_.htm