Reflection of Lab Report 4

Reflection by Nurzahidah binti Zamat

In this experiment, I learn that we have different way to measure the protein concentration in food sample. We obtain that, braised fish have high protein concentration even though it is cooked, but it does not change the protein concentration in it. After that, I learn that bean also have high protein concentration, and each of them are different.

Reflection by Aqmarollah bin Mohd Nasip

By doing this experiement, I had done both lowry and biuret test. I also know that there are several other types to determine protein concentration such as UV absorbance and dye-binding method, (Bradford method, Bio-Rad protein assay). However, we do not run these type of tests but only lowry and biuret. After this, I can explain the difference between these types of tests. This is a good exposure to me because before this I never do this kind of experiment.

Reflection by Afifah Syafikah binti Azizan

The major benefit of working in a team for me was that it facilitated learning and enhanced my understanding of the project. I have definitely learned more about the topic from other students than I would have if I had completed this assignment on my own. We discussed each entry in depth and that helped me understand the complexity and the number of protein in every samples. My team members and I felt free to share our ideas and knowledge each other, and that definitely made my learning process more interactive, interesting and enjoyable.
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Lab Report 4: Protein

INTRODUCTION
In the experiment to determine the protein concentration, Biuret assay and Lowry assay are used. The biuret test is a chemical test used to detect the presence of peptide bonds. In the presence of peptides, a copper(II) ion forms violet-coloured coordination complexes in an alkaline solution. The Biuret reaction can be used to assess the concentration of proteins because peptide bonds occur with same frequency per amino acid in the peptide. The intensity of the colour and hence the absorption at 540 nm, is directly proportional to the protein concentration, according to the Beer-Lambert law. Meanwhile, Lowry protein assay is a biochemical assay for determining the total level of protein in a solution. The total protein concentration is exhibited by a colour change of the sample solution in proportion to protein concentration, which can then be measured using  colourimetric techniques.

MATERIALS

Protein standard
Biuret reagent
Lowry reagent 1 and Lowry reagent 2
8 Test samples:
a. Animal samples: chicken, fish, beef, boiled fish
b. Plant proteins: Soybean, red bean, peanut, dhal bean 

PROCEDURE

1. Preparation of Protein Standard

1. Solutions of gelatin at 1, 2, 3, 4, 5, and 6 mg/mL in water from the gelatin stock solution (10 mg/ml) for Biuret assay was prepared.
2. Solutions of gelatin at 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mg/mL in water from the gelatin stock solution (1 mg/ml) for Lowry method was prepared.

2. Preparation of Test Samples

a) Animal Protein:
1. 10 gram of protein samples was weighted.
2. The sample was macerated into smaller size.
3. Sample was blend in Phosphate Buffer saline at 1:10 ratio.
4. The sample was shake/ stir/ for 15 minutes.
5. Sample was filtered by kitchen filter.
6. The supernatant was collected.
7. Sample was filtered again using Whatman filter No 1.
8. The supernatant was collected.

b) Plant Protein
1. 10 gram of protein samples was weighted.
2. The sample was crush and grind into a fine paste or powder using mortar and pestle.
3. The powder of sample was dissolve in Phosphate Buffer saline at 1:10 ratio.
4. The sample was shake/ stir/ for 15 minutes.
5. Sample was filtered by kitchen filter.
6. The supernatant was collected.
7. Sample was filtered again using Whatman filter No 1.
8. The supernatant was collected.

3. Protein Assay

a) Biuret assay:
1. All 8 test sample extracts was obtained from the other groups.
2. 0.50 mL of each protein (standard and test samples) was mixed with 2.50 mL of Biuret reagent.
3. The absorbance of the samples was measured at 540 nm after 10 minutes.
4. Standard curve was plotted.
5. The protein content of the test sample was estimate using the standard curve.
(Note: All series should include a zero protein (water) tube (reagent blank).

b) Lowry assay:
1. Obtain all 8 test sample extracts from the other groups
2. 0.25 mL of each protein (standard and test samples) was mixed with 2.5 mL of Lowry reagent 1.
3. The mixture was incubated at room temperature for 10 minutes.
4. 0.25 mL of Lowry reagent 2 was added and mix well immediately.
5. The mixture was incubated at room temperature for 30 minutes.
6. The absorbance was measured at 750 nm.
7. The standard curve was plotted.
8. The protein content of the test sample was estimated using the standard curve.
(Note: All series should include a zero protein (water) tube (reagent blank)

RESULT

Protein Standard
Protein Number
(Biuret)
B1
0.439
B2
0.509
B3
0.512
B4
0.542
B5
0.769
B6
0.995

Protein Number
(Lowry)
L1
0.132
L2
0.106
L3
0.406
L4
0.256
L5
0.115
L6
0.081














Protein Number for Samples

Sample
Biuret
Lowry
Beef
0.488
0.944
Fish
1.020
1.320
Braise Fish
1.138
0.406
Chicken
0.552
1.328
Soybean
0.447
0.377
Red bean
0.645
0.926
Peanut
0.476
1.219
Dal bean
0.746
0.214
Green bean
0.983
1.945




DISCUSSION

From this experiment, we used Biuret Assay and Lowry Assay to determine the protein content in the samples. In Biuret Assay, we used samples combine with the biuret reagent. Biuret reagent contains copper ions in a basic solution. The copper ions will complex with the amide groups in the proteins to create a blue colour that will be measured using a spectrophotometer. Purpose of Biuret Assay is to prepare the standard curve, to determine the protein content in the sample and to analyse data from standard curve and unknown concentration of the samples.

 To determine the protein concentration, we construct a standard curve after performing the Biuret reaction on a series of prepared solutions of gelatin at 1, 2, 3, 4, 5 and 6 mg/mL in water. The absorbance readings was used to plot the graph of absorbance functioning as protein concentration called the standard curve for assay. This graph can be used to determine the other protein concentration in food samples.
From the graph, the highest protein concentration is braised fish, followed by fish, green bean, and the lowest is soy bean. The Lowry assay is based on the reaction of cupric ions with peptide bonds under alkaline conditions (the Biuret test). Protein samples are mixed with an alkaline solution containing copper sulphate (Cu2+ ions) which react with peptide bonds to produce Cu+ ions. As the amount of Mo(IV) and Mo(V) complex is dependent on the amount of Cu+ ions which is, itself, dependent on the amount of protein in the unknown sample, the colour produced is a direct reflection of protein concentration and, with the use of standards, can allow protein concentration to be determined.

 Lowry Assay is one of the method that is used to  determine the total level of protein in a solution. A standard curve is constructed to determine how much protein is represented  by a particular absorbance reading. This is obtained by the Lowry reaction on a series of prepared solutions of gelatin at 0.1,0.2,0.3,0,4.0,5 and 0.6 mg/ml in water. The curve then is used to convert  the absorbance reading for protein samples into protein concentration in the samples.

 According to the graph that has been constructed, we able to find the protein concentration in the samples. From the graph, we found that the green bean (1.945 mg/ml) has the highest protein concentration followed by  chicken (1.328 mg/ml), fish (1.320 mg/ml), peanut (1.219 mg/ml), and the lowest is dal beal (0.124 mg/ml)


CONCLUSION

 In conclusion, both test can be used to determine the protein concentration. In biuret test, we obtain braised fish to have higher protein concentration while in Lowry test, green bean have the highest protein concentration.

 QUESTIONS

 1. Describe three alternative methods of determining protein concentration


UV absorbance :
Protein concentrations can be determined directly by ultraviolet spectroscopy because
of the presence of tyrosine and tryptophan which absorb at 280 nm. Because the levels of these two amino acids vary greatly from protein to protein, the UV absorbance per milligram protein is highly variable.

BCA Protein Assay :
The BCA Protein Assay is an alternative to the Lowry assay. The key component in this assay is bicinchoninic acid (BCA) that reacts with cuprous ions to generate an intense purple colour at 562 nm. Cuprous ions are produced by the reduction of cupric ions by proteins in alkaline solutions.

Dye-binding method, (Bradford method, Bio-Rad protein assay):
In this assay, the dye Coomassie Blue G-250 is dissolved in an acidic solution causing it to absorb at 465 nm (reddish brown). When the dye (negatively charged) binds to the positively charged protein molecule the absorbance undergoes a shift to 595 nm (blue). This shift in absorption maximum is proportional to protein concentration over a broad range.

2. What is an “appropriate blank” and why?

Solution that will be a suitable blank on the exact details of the method we are using.


So, use of blank ensures that all errors of measurements that may be introduced into absorption spectrum from cuvette material, solvent, temperature fluctuations, gases in the atmosphere are taken into account.

 REFERENCES

1. Ronald E. wrolstad, Terry E. Acree, Eric A. Decker, et al. (2005). Handbook of Food Analytical Chemistry: Water, Proteins, Enzymes, Lipids and Carbohydrates. John Wiley & Sons, Inc. Hoboken. New Jersey.

2. http://bio.classes.ucsc.edu/bio20L/MANUAL/Lab%203.pdf
3. https://socratic.org/questions/what-is-the-purpose-of-blank-solution-in-spectrophotometer 

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Reflection of Project 4

Reflection by Nurzahidah binti Zamat

In this project, I learn how to make soaps. Before this, we learn how to make soap back then in school, theoretical and practical, but at that time I did not go to school. Therefore, this is the first time I make soap in the lab. Our group choose palm oil to make our soap, because we always used palm oil in our kitchen. Before doing this experiment, I search on the internet how to make DIY soap, and their soap looks amazing. But in our experiment, I never expected that soap does not look the same like on the internet. We also prepared perfume, water colour to decorate our soap, but it turns out to be not as easy as what I expected. But still, this is a great experience to make something that we used in our daily life. 

Reflection by Aqmarollah bin Mohd Nasip

Setelah menjalankan eksperimen ini, saya mampu untuk membuat sabun sendiri walaupun tidak sama dengan sabun di pasaran tetapi yang penting saya dapat mengetahui langkah-langkah untuk membuat sabun tersebut. Saya dapat membuat sabun tersebut mengikut bentuk dan warna saya sendiri serta dapat menambah wangian ke dalam sabun tersebut. Selepas ini, saya berharap banyak lagi produk dapat dihasilkan melalui eksperimen dan projek yang dilakukan.

Reflection by Afifah Syafikah binti Azizan

From this experiment, I can enhance my knowledge about preparation of soap. I very excited when I know that soap can produce from lipid oil. As I thought that soap produced from the mother of soap. I just can’t wait to know the result of the soap and I feeling so excited about it.  Furthermore, I can make the soap using my own idea. First about the coloured, I can the mix the colour or make the colour rainbow as I desired. Second about the shape, I can design the shape of love, star, flower and leave. Finally, I can make soap with the scent of its own like scent of flower for example lavender, lily and roses. 
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Project 4: Making Soap

INTRODUCTION

A soap is a salt of a compound, known as a fatty acid. A soap molecule has a long hydrocarbon chain with a carboxylic acid group on one end, which has ionic bond with metal ion, usually sodium or potassium. The hydrocarbon end is non polar which is highly soluble in non-polar substances and the ionic end is soluble in water. The structure of the soap molecule is represented below:


The cleaning action of soaps because of their ability to emulsify or disperse water-insoluble materials and hold them in the suspension of water. This ability is seen from the molecular structure of soaps. When soap is added to water that contains oil or other water-insoluble materials, the soap or detergent molecules surround the oil droplets. The oil is, dissolved in the alkyl groups of the soap molecules while the ionic end allows it to be dissolved in water. As a result, the oil droplets are to be dispersed throughout the water and can be washed away. A number of things affect the soap-making process and the quality of this soap produced. The characteristics of this soap depend on the quality of oil, and the amounts of the caustic soda and water used to make it. The speed of the reaction between the oil and the caustic soda is influenced by free fatty acid content of the oil, the heat of the components before mixing, and how vigorously the mixing is to be done. Free fatty acid contents, vigorous mixing, and heat, speed up the given soap-making process.

MATERIALS
60mL of 6M NaOH solution
17.5g of fat (coconut oil, corn oil, palm oil, margarine, butter)
75mL of distilled water
300mL of sodium chloride (NaCl) solution
100mL graduated cylinder
400mL beaker
250mL beaker
stirring rod
color and fragrance
PROCEDURE

 1. 40mL of 6M NaOH and 17.5g fat was placed in 250mL beaker.
2. The mixture was heated slowly while stirring constantly for 20 minutes until all the water evaporated.
3. Then the remaining NaOH was added carefully, then boiled until all the water has boiled off.
4. When the crude soap cools, 12.5mL of distilled water was added and 50mL of hot and saturated NaCl solution was added. 
5. The mixture was stir, and the lump was breaked.
6. The mixture was filtered using wire screen to trap soap small particles.
7. The soap was press between two filtered paper to removed as many water as possible. 
RESULT
Corn oil
Sunflower oil
Butter
Margarine
Palm oil
DISCUSSION
Chemically, soap is a salt of a fatty acid. Soaps are mainly used as surfactants for washing, bathing, and cleaning but they are also used in textile spinning and are important components of lubricants. When we were conducting this experiment, some precautions steps had been taken such as we always wear eye protection (goggles) and rubber gloves. Clothing also should consist of long sleeve shirt, long pants and socks and shoes. Beside that, avoid breathing the dust released and fumes created when adding sodium hydroxide to water. Moreover, do not use aluminum pans, utensils, or foil with soap recipes that require the use of sodium hydroxide since these chemicals will react with aluminium and always add sodium hydroxide slowly to water.
This project is an application to our experiment saponification. In this project, we had make our own soap by following the procedures given to us. We were given the opportunity to make our own shape of soap, put any fragrance and colour to the soap. The soap is being pressed between two sheets of paper to allow water removal and being left for two days. After two days, the papers were removed and the soap was obtained.

CONCLUSION
In this experiment, we are able to make our own soap by using sodium hydroxide and fat. This reaction is exothermic and the white suspension formed is made up of soap and fat. The process of formation of soap is called saponification.

QUESTION

1. Why do soaps disperse grease?

Soaps can disperse grease with water because part of the soap is soluble with grease and part of it is soluble with water. Soap combines with both grease and water and washes away in a shower. Combine the 2 and you get rid of the both. Part of the soap molecule is similar to grease and combines, with grease,another part of the soap molecule easily combines with water. Soap is a surfactant and combines with both grease and water.

REFERENCES

1. Bryan Wright Chem 102 Lab Section 28. Project 17-SOAP Lab Report, February 25, 2010.
2. http://www.laney.edu/wp/cheli-fossum/files/2012/01/13-Saponification.pdf
3. http://amrita.olabs.edu.in/?sub=73&brch=3&sim=119&cnt=2 
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Reflection Lab Report 3

Reflection by Nurzahidah binti Zamat

 In this experiment, I knew a new term which is saponification number and how to calculate its value. Every sample gave different reading based on the number of fatty acid in it. In this experiment, we used reflux, therefore I can improve my skill by using this technique as we seldomly used this technique in any experiment.
Reflection by Aqmarollah bin Mohd Nasip


Untuk eksperimen kali ini, saya dapat mengetahui maksud istilah “saponification number” dan tahu untuk mengira nilai tersebut. Daripada lima sampel yang diberi, kami dapat mengetahui nilai “saponification number” untuk setiap satu sampel tersebut. Setiap sampel sepatutnya memberi bacaan yang berbeza mengikut nombor asid lemak yang ada dalam sampel tersebut. Saya juga dapat memahirkan diri untuk membuat eksperimen berkaitan “reflux” kerana eksperimen ini melibatkan teknik tersebut.


Reflection by Afifah Syafikah binti Azizan

It’s awesome to know more about lipids. From carry out this experiment, I got new knowledge which is fats and oils are triesters of glycerol, they react with water to form fatty acids and glycerin. When the reaction is carried out in a basic solution, salts of the fatty acids are produced instead of the fatty acids themselves. The salts of fatty acids are soaps and an individual molecule is characterized by an ionic end which is the salt part and a nonpolar end which is the hydrocarbon part. The ionic salt end is water-soluble and the nonpolar hydrocarbon end is water insoluble.Then I also know how to identify them based on their saponification number.
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Lab Report 3: Triglyceride

INTRODUCTION

Triglycerides are fat in the blood. They are used to give energy to our body. If it is excess, they are stored in different places, to be used later. They are important to life and are the main form of fat, called lipids in the body. They are the end product of digesting and breaking down fats in food. Some are made in the body from other energy sources such as carbohydrates. 

But high triglycerides might raise your risk of heart disease and may be sign of metabolic syndrome. Metabolic syndrome is the combination of high blood pressure, high blood sugar, too much fat around the waist, low HDL and high triglycerides. 

 Vegetable oils and animal fats are the main materials that are saponified. These greasy materials, triesters called triglycerides are mixtures derived from diverse fatty acids. triglycerides can be converted to soap in either one or two step process. 

MATERIALS

Triglyceride sample (coconut oil, corn oil, palm oil, margarine, butter) solvent (1:1 ethanol/ether), 0.5M KOH/ethanol solution, phenolphtalein, 0.5M HCl.

PROCEDURE

1. 1.0 g sample triglyceride was placed into a small beaker and dissolved in 4 ml solvent(1:1 ethanol /ether).

2. Dissolved triglyceride was transferred into a small distillation flask and beaker wshed twice with 1 ml solvent to collect all residual material. “wash” also added to the distillation flask.

3. 25 ml of 0.5 M KOH/ethanol solution was added.

4. Exact volume of the mixture was measured.

5. Second system as “control” was set up with 25 ml 0.5 M KOH/ethanol solution + 2 ml of 1:1 ethanol/ether solvent for a final volume identical to our test sample solution.

6. Reflux condenser was set up on each flask and boiling water bath was placed in for 30 minutes. Hydrolysis occur.

7. Flask allowed to cool. Three drops indicators (phenolphthalein) was added to both flasks and titrated with 0.5 ml HCl solution.

RESULT

Sample
B (ml)
T (ml)
Saponification number (mg KOH/1g)
Palm oil
23.00
22.00
28.06 mg KOH/1g
Sunflower
22.20
21.00
33.67mg KOH/1g
Corn oil
23.00
22.00
28.06mg KOH/1g
Margerin
23.00
20.00
84.17mg KOH/1g
Butter
28.00
25.00
84.17mg KOH/1g

DISCUSSION

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 palm oil as a sample for our group. Different group used different lipid which are sunflower oil, corn oil, margarine and butter. Then at the end of the experiment we obtained the final results for each sample. Based on the results obtained, sunflower oil has the higher saponification number between oil which is 33.67 mg KOH/1g compared to corn oil and palm oil which has the same value at 28.06 mg KOH/1g. So it shown that, sunflower oil has shorter fatty acids. However, margarine and butter has the highest saponification number at 84.17 mg KOH/1g. It is because Fats and oils can be characterized by their saponification numbers. One mole of fat requires three moles of potassium hydroxide for complete saponification. If a fat contains fatty acids of relatively high molecular weights, then one gram of the fat will consist of fewer moles. Thus, fats having greater percentages of high molecular weight fatty acids will have lower saponification numbers than fats having greater percentages of lower molecular weight 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 higher 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 occur 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.

CONCLUSION

 From this experiment, we are able to calculate the saponification number (mg KOH/1g) and can differentiated which fats or oil that containing high fatty acid number based on their saponification number.

 REFERENCES

 1. http://www.chemistryexplained.com/Di-Fa/Fats-and-Fatty-Acids.html#ixzz4gYl8E55s
2. https://en.m.wikipedia.org/wiki/Saponification
3. www.webmd.com/cholestrol-management/tc/high-triglycerides-overview

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