DR SHAKE’S LAB PROTOCOLS DR SHAKE 2022-23
LABORATORY RESEARCH TEAM RECOMMENDED ROLES Project Leader A leader should be appointed according to the lab activity. Each member should take turn to become one. The leader must coordinate all tasks during the lab research. Information manager A report manager shall compile all informations from members, into a complete lab report and distribute fair task for all to write. The manager shall upload materials in the e-portfolio. Result analyst An analyst shall calculate and prepare suitable data presentation. The analyst must interpret the findings. The findings must be discussed in the group. Technical manager A technical manager shall ensure all lab equipment are in place. The technical skills activity must be recorded and edited accordingly.
THE PROTOCOL enzyme ● Kinetic ● Stress marker protein ● Lowry method for protein estimation carbohydrate ● Phenol-Sulfuric acid method for Total Carbohydrate estimation lipid ● Determination the percentage of lipid ● Identification of saturated or unsaturated fats vitamin ● Determination of Vitamin C concentration
UNDERSTAND SOME BASIC CONCEPTS IN BIOCHEMISTRY LAB 1. BEER’S LAW SIMULATION 2. MOLARITY SIMULATION 3. HOW TO MAKE A SOLUTION 4. REACTANTS, PRODUCTS AND LEFTOVERS SIMULATION 5. CONCENTRATION AND REACTION RATE SIMULATION 6. USING A STANDARD CURVE INTERACTIVE 7. INTRODUCTION TO SPECTROPHOTOMETER - VIDEO 8. COLORIMETRIC TITRATION - VIRTUAL LAB
ENZYMES
STANDARD REFERENCE 1. Prepare starch solutions from the stock solution (1.0 mg/ml) into dilutions of : 0.01, 0.025, 0.05, 0.1, 0.3, 0.5, 0.7, and 1.0 mg/ml 2. Iodine solution is prepared by adding 5 g potassium iodide to 100 ml water. The dissolved potassium iodide is added with 1 g of iodine and is allowed to dissolve. [prepared by the lab] 2. Prepare a standard curve of Absorbance (@ 590 nm) vs. Concentration of a starch/iodine mixture. Use the following table as guide.
Standard reference : Preparation Plot a standard curve: Absorbance vs Starch concentration
STRESS MARKER Q: Does salivary amylase is a fit indicator for stress activity?
•STRESS •Salivary amylase increases •Enzyme Assay •The higher concentration of enzyme, the more starch is broken down to maltose. Therefore, more maltose are produced •STARCH DIGESTION • More maltose • more enzyme the higher stress level WHAT IS AN EXAMPLE OF A STRESSFUL ACTIVITY?
I. Salivary Amylase Activity 5 ml of 0.1mg/ml starch Add 1 ml of salivary amylase Incubate mixture at 35°C for 10 min Place in an ice bath Add 1 ml iodine Read absorbance at 590 nm 1. Rinse your mouth 2. Collect 1-2 ml of saliva (before stress activity) in a test tube. Label it 3. Use the saliva for the following assay* 4. Perform an activity or two that considered as stressful for 5-10 min 5. Rinse your mouth again 6. Collect 1-2 ml of saliva (after stress activity) in a test tube. Label it. 7. Use the saliva for the following assay* 8. Compare before-and-after salivary amylase activity *Assay
KINETIC OF AMYLASE Q: What is the Vmax and k m value?
II. SUBSTRATE CONCENTRATION EFFECT USING AMYLASE (INDUSTRIAL)
DATA ANALYSIS 1. Calculate starch concentration for each sample after hydrolysis (SF ) through use of the standard curve. The initial starch concentration (S0 ) is already known. [S] = (So ) – (SF ) 2. The velocity (rate of digestion) of the reaction for each sample can be calculated as: 3. V = ∆ S/ ∆ t = (S0 - SF ) / 10 minutes 4. Prepare a table showing rate of hydrolysis (V) at different the starch concentrations. 5. Plot a Michaelis-Menten graph. 6. Prepare a graph of 1/starch concentration (x-axis) versus 1/rate of digestion (y-axis). This type of reciprocal graph displaying enzyme kinetics is a Lineweaver-Burke plot. 7. State the value of V max and Michaelis constant K m from your graph. 8. The y-intercept of the Lineweaver Burke plot is the reciprocal of the maximum velocity of the reaction (Vmax ). The x-intercept is the negative reciprocal of the Michaelis constant. (Km ).
TABLE TO GUIDE S0 S f ΔS (S0 - Sf ) V=ΔS/t 1/S0 1/V Plot the Lineweaver-Burk plot using 1/V vs 1/S0
CARBOHYDRATES Phenol-Sulfuric acid method for Total Carbohydrate estimation
CARBOHYDRATE: EXAMPLES HIGH GLYCEMIC INDEX ▪ Sugar ▪ Flour ▪ Rice ▪ White potatoes ▪ Some fruits: bananas, grapes, cherries, watermelon ▪ Raisins ▪ Breakfast cereals ▪ Bread ▪ Soda ▪ Cookies and crackers LOW GLYCEMIC INDEX ▪ Sweet potatoes ▪ Vegetables: leafy greens, asparagus, cauliflower ▪ Quinoa ▪ Legumes, including lentils, chickpeas ▪ Skim milk ▪ Reduced-fat yogurt ▪ Sesame seeds, peanuts, flax seeds
PRINCIPLE ▪ In a hot acidic medium, glucose is dehydrated to hydroxymethyl furfural. ▪ This forms a yellow-orange colored product with phenol and has absorption maximum at 490 nm
MATERIALS 1. Phenol 5%: Redistilled (reagent grade) phenol (50g) dissolved in water and diluted to one liter. 2. Sulfuric acid 96% reagent grade 3. Standard Glucose Stock: 100 mg in 100mL of water (what is the final concentration) 4. Working standard : 10 mL of stock diluted to 100mL with distilled water (what is the final concentration)
SAMPLE PREPARATION/EXTRACTION 1. Grind 1g sample and 80% ethanol. Re-extract 2-3 times with ethanol 2. Bring the volume to 50 ml with ethanol 3. Reflux for 1 hour in boiling water bath 4. Filter and bring volume to 100 ml with ethanol 5. Neutralize it with solid sodium carbonate until the effervescence ceases. 6. Make up the volume to 100 mL and centrifuge.
TOTAL CARBOHYDRATE ESTIMATION 1. Pipette out 0.2, 0.4, 0.6, 0.8 and 1 mL from the working standard into 5 different test tubes. Make up the volume in each tube with 1ml water. 2. Prepare a separate test tube with 1mL of water for Blank. 3. Pipette out 0.1 ml of the sample solution in a separate test tube. Make up the volume in each tube to 1 mL with water. 4. Add 1 mL of phenol solution into ALL test tubes [work in fume hood] 5. Add 5 mL of 96% sulfuric acid and shake well. [caution : corrosive - work in fume hood] 6. After 10 min, shake the content in the tubes and place in a water bath at 25-30°C for 20 min. 7. Read the color at 490 nm.
ESTIMATION ▪ Calculate the amount of total carbohydrate present in the sample solution using the standard graph 1. Plot data from test tubes (blank, 0.2, 0.4, 0.6, 0.8 and 1 mL ) against absorbance [this is your Standard graph] 2. Estimate amount of total carbohydrate from the standard graph Sample’s absorbance Estimated concentration/amount
CALCULATION ▪ Absorbance corresponds to 0.1mL of the test = C mg of glucose ▪ 100mL of the sample solution contains: = C mg X100mL of glucose 0.1ml = X mg glucose in 1 g sample ▪ % of total carbohydrate = X mg x 100% 1000mg ▪ Carbohydrates: 1 gram = 4 calories, what is your sample calorie?
LIPIDS to determine the percentage of lipid in YOUR DAILY snacks to differentiate between saturated and unsaturated fats presence in the selected snacks
PRINCIPLES ▪ Fats/Lipid can be classified as saturated or unsaturated. The nature of the extracted fats will be different. ▪ Some will form a white solid and some will form a liquid. It can be used to determine which of the food types have saturated and which have unsaturated fats. ▪ Acetone , OC(CH3 )2 , is the solvent used to remove the fats from the foods. This colorless, flammable liquid is the simplest example of the ketones. ▪ Acetone is the most commonly used solvent, which promotes protein precipitation and simultaneously dissolves nonpolar molecules like lipids. ▪ Acetone is miscible (forming a homogeneous mixture) with water. Its bonding makes it a polar molecule. ▪ Fats dissolve in the acetone, because acetone is also a non polar solvent ▪ When the acetone is evaporated, what is left behind is the extracted fat.
ESTIMATION OF LIPID PERCENTAGE 1. Crush your snack finely using mortar and pestle, and weigh about 1 g for the test 2. Label and weigh a clean test tube/beaker for this sample. Record the mass of the test tube and label. 3. Add the sample of crushed snack into the labeled test tube/beaker and weigh again. Record this value. 4. Add about 5 mL of acetone to the test tube/beaker. WARNING: WEAR GOGGLE AND USE the fume hood as the fumes will be noxious. 5. Swirl gently to get the snack sample soaked in the acetone for 5 minutes to allow the fat to be extracted into the solvent. Centrifuge if possible. 6. Weigh and label a Petri dish. 7. Carefully pipet the acetone into a clean labeled petri dish. MAKE SURE all of the solid particles remain in the beaker. 8. Add another 5 mL sample of acetone to the test tube/beaker. 9. Again swirl for 5 minutes. Again decant the acetone to the petri dish. And again MAKE SURE that all the solid particles remain in the test tube/beaker. (You may repeat 3x) 10. Weigh the test tube/beaker with the remaining solids particles. Record. Note: This is the mass of the food sample WITHOUT the fat. 11. Allow the petri dish to evaporate in the fume hood for about 30 minutes or until acetone is evaporated. 12. Weigh the Petri dish again.
CALCULATION MY DAILY Snack Weight of empty test tube/ beaker, g Weight of test tube/ beaker with crushed snack, g Calculate the weight of snack, g Weight of test tube/beaker with snack particles after fat extraction, g Weight of extracted fat, g = weight of snack – weight of snack after fat extraction. % fat extracted Weight of Petri dish Weight of Petri dish with acetone extraction (after acetone evaporation) Weight of extracted fat in acetone Observation: Saturated or unsaturated fat? Snack 1 Snack 2 Snack 3
PROTEINS Lowry method for protein estimation
ESTIMATION OF PROTEIN ▪ Protein can be estimated by different methods as described by Lowry and also by estimating the total nitrogen content. ▪ No method is 100% sensitive. ▪ Hydrolyzing the protein and estimating the amino acids alone will give the exact quantification. ▪ The method is sensitive enough to give a moderately constant value and hence largely followed.
PRINCIPLE The blue color developed by the reduction of the phosphomolybdic-phosphotungstic components in the Folin-Ciocalteau reagent by the amino acids tyrosine and tryptophan present in the protein. The color developed by the reaction of protein with the alkaline cupric tartrate are measured in the Lowry’s method. BLUE
MATERIALS 1. 2% Sodium Carbonate in 0.1N Sodium Hydroxide (Reagent A) 2. 0.5% Copper sulphate (CuSO4.5H2O) in 1% potassium sodium tartrate (Reagent B) 3. Alkaline Copper Solution: Mix 50mL of A and 1 mL of B prior to use (Reagent C) 4. Folin-Ciocalteu Reagent (Reagent D) Protein Solution (Stock Standard) Weigh accurately 50mg of bovine serum albumin (Fraction V) and dissolve in distilled water and make up to 50 mL in a standard flask. Working Standard Dilute 10 mL of the stock solution to 50 mL with distilled water in a standard flask. 1 mL of this solution contains 200 mg protein. Phosphate buffer, pH 7.4. Add 11.3g dry anhydrous disodium hydrogen phosphate and 2.7g dry anhydrous potassium dihydrogen phosphate in one liter volumetric flask and make up to the mark with water. Check the pH and store at 4°C.
EXTRACTION OF PROTEIN FROM SAMPLE 1. Weigh 100mg of the sample and grind well with a pestle and mortar in 5-10mL of the buffer or 1 gm of sample in 50 ml of buffer 2. Centrifuge and use the supernatant for protein estimation
ESTIMATION OF PROTEIN 1. Pipette out 0.2, 0.4, 0.6, 0.8 and 1 mL from working standard and place it into 5 different test tubes. 2. Pipette out 0.1mL of the sample extract in separate test tube and label it. 3. Make up the volume to 1 mL in all the test tubes by adding it with distilled water. 4. Prepare a test tube with 1 mL of water and label it blank. 5. Add 5 mL of Reagent C to each tube including the blank. Mix well and allow it to stand for 10min. 6. Then add 0.5mL of Reagent D, mix well and incubate at room temp. in the dark for 30 min. Blue color is developed. 7. Take the reading at 660 nm. 8. Draw a standard graph and calculate the amount of protein in the sample. 9. Calculate the amount of protein mg per g sample or per 100g sample.
ESTIMATION USING STANDARD REFERENCE ▪ Calculate the amount of protein present in the sample solution using the standard graph 1. Plot data from test tubes (blank, 0.2, 0.4, 0.6, 0.8 and 1 mL ) against absorbance [this is your Standard reference graph] 2. Estimate amount of protein from the standard graph Sample absorbance Estimated concentration/amount Proteins: 1 gram = 4 calories, what is your sample calorie?
VITAMIN C Redox Titration Using Iodine Solution
PRINCIPLES As the iodine is added during the titration, the ascorbic acid is oxidized to dehydroascorbic acid, while the iodine is reduced to iodide ions. ascorbic acid + I2 → 2 I− + dehydroascorbic acid Due to this reaction, the iodine formed is immediately reduced to iodide as long as there is any ascorbic acid present. Once all the ascorbic acid has been oxidized, the excess iodine is free to react with the starch indicator, forming the blue-black starch-iodine complex. This is the endpoint of the titration
REDOX TITRATION Iodine solution: (0.005 mol/L). 1. Weigh 2 g of potassium iodide into a 100 mL beaker. 2. Weigh 1.3 g of iodine and add it into the same beaker. 3. Add a few mL of distilled water and swirl for a few minutes until iodine is dissolved. 4. Transfer iodine solution to a 1 L volumetric flask, making sure to rinse all traces of solution into the volumetric flask using distilled water. 5. Make the solution up to the 1 L mark with distilled water. Starch indicator solution: (0.5%). Weigh 0.25 g of soluble starch and add it to 50 mL of near boiling water in a 100 • burette and stand • 100 mL or 200 mL volumetric flask • 20 mL pipette • 10 mL and 100 mL measuring cylinders • 250 mL conical flasks
SAMPLE PREPARATION • Vitamin C tablets: Dissolve a single tablet in 200 mL of distilled water. • Fresh fruit juice: Strain the juice through cheesecloth to remove seeds and pulp which may block pipettes. • Packaged fruit juice: This may also need to be strained through cheesecloth if it contains a lot of pulp or seeds. • Fruits and vegetables: • Cut a 100 g sample into small pieces and grind in a mortar and pestle. • Add 10 mL portions of distilled water several times while grinding the sample, each time decanting off the liquid extract into a 100 mL volumetric flask. • Finally, strain the ground fruit/vegetable pulp through cheesecloth, rinsing the pulp with a few 10 mL portions of water and collecting all filtrate and washings in the volumetric flask. • Make the extracted solution up to 100 mL with distilled water. • Alternatively the 100 g sample of fruit or vegetable may be blended in a food processor together with about 50 mL of distilled water. • After blending, strain the pulp through cheesecloth, washing it with a few 10mL portions of distilled water, and make the extracted solution up to 100 mL in a volumetric flask.
PROTOCOL 1. Pipette a 20 mL aliquot of the sample solution into a 250 mL conical flask 2. Add about 150 mL of distilled water and 1 mL of starch indicator solution 3. Titrate the sample with 0.005 mol L−1 iodine solution. 4. The endpoint of the titration is identified as the first permanent trace of a dark blue-black color due to the starch-iodine complex 5. Repeat the titration with further aliquots of sample solution until you obtain concordant results (titers agreeing within 0.1 mL).
CALCULATION ▪ Calculate the average volume of iodine solution used from your concordant titer ▪ Calculate the moles of iodine reacting ▪ Calculate the concentration in mol/L of ascorbic acid in the solution obtained from fruit/vegetable/juice. ▪ Calculate the concentration, in mg/100mL or mg/100g of ascorbic acid, in the sample of fruit/vegetable/juice Suggested reference : http://www.chemcollective.org/chem/ubc/exp09/index.php
EXAMPLE 1. Calculate the moles of iodine (I2) obtained in the redox reaction given by equation. 2. Consider iodine (I2 ) to be in excess in this reaction. 1. 2mM of KIO3 = 0.002 M 2. Iodate used (average) = 9.03 mL= 0.00903 L 3. Number of moles of iodate = 0.00903 x 0.002= 1.806 x10-6 moles 4. From the equation, IO3 -+ 5I-+ 6H+ 3I2 + 3H2O 5. 3 x (1.806 x10-6 )= 5.418 x10-6 moles of I2 . 3. Calculate the number of moles of ascorbic acid that react with the moles of iodine obtained above. From the equation : C6H8O6 + I2 C6H6O6 + 2H+ + 2 I1. 1 mol I2 = 1 mol ascorbic acid 2. Therefore, 5.418 x 10-6 I2 will react with 5.418 x 10-6 ascorbic acid
Calculate the moles of ascorbic acid that reacted as follows: a. Calculate the molarity of ascorbic acid (Vitamin C) in the diluted orange juice. Moles of ascorbic acid = as calculated in step 3 = 5.418 x 10-6 Total volume of solution = 10 ( original sample of orange juice) + 75 (distilled water) + 2.5 (HCl) + 2.5 ( KI) + 4 (starch indicator) = 94 mL Molarity of dilute orange juice = (5.418 x 10-6 )/(94/1000)= 5.76 x 10-5 M b. Calculate the molarity of ascorbic acid (Vitamin C) in the original sample of orange juice. 5.418 x 10-6 / (10/1000) = 5.418 x10-4 M c. Calculate the concentration in mg/mL of ascorbic acid (Vitamin C) in the original sample of orange juice. 1 mol of ascorbic acid (C6H8O6 ) = 176.08 g 5.418 x 10-6 = 0.000954 g = 0.954 mg Therefore, molarity in mg/ml = 0.954/10 = 0.0954 mg/ml
ALL CONTENTS ARE ADAPTED FROM OPEN EDUCATIONAL RESOURCES Dr Shake 2022-23
REPORTING GUIDE USING IMRaD CLO3 - Carry out laboratory technical skills in solving complex problems ▪ Report mode choice : Written - Video docu - Audio - Combination ▪ Upload the report to your e-portfolio ▪ You may add short videos/photos/narrations - you decide ▪ Individual lab skills - 10% ▪ Group report - 20%
ENZYMES Introduction ▪ Briefly, describe the concepts you have learned. ▪ What are the objectives of the experiment? ▪ What is the hypothesis in the experiment (if any) Methodology ▪ Briefly, describe the methods/principles that you have applied in the experiment. ▪ INDIVIDUAL: Describe the skill(s) that have you acquired in this lab? (include the evidence, if any) Results ▪ What is the data you have collected? ▪ How did you analyze the data? ▪ What are the results? ▪ Did you achieved the objectives? Discussion ▪ Predict why you have obtained such results ▪ What could possibly have happened (use Biochemistry/Biology/Chemistry/Physics/Math/etc concepts) ▪ What have you solved? ▪ What does the results tell you about? ▪ How are you going to use the results in your PrBL?
CARBOHYDRATES Introduction ▪ Briefly, describe the concepts you have learned. ▪ What are the objectives of the experiment? ▪ What is the hypothesis in the experiment (if any) Methodology ▪ Briefly, describe the methods/principles that you have applied in the experiment. ▪ INDIVIDUAL: Describe the skill(s) that have you acquired in this lab? (include the evidence, if any) ▪ Why did you choose to analyze the sample(s)? Results ▪ What is the data you have collected? ▪ How did you analyze the data? ▪ What are the results? ▪ Did you achieved the objectives? Discussion ▪ Predict why you have obtained such results ▪ What could possibly have happened (use Biochemistry/Biology/Chemistry/Physics/Math/etc concepts) ▪ What have you solved? ▪ How are you going to use the results in your PrBL - Redesigning Me?
LIPIDS Introduction ▪ Briefly, describe the concepts you have learned. ▪ What are the objectives of the experiment? ▪ What is the hypothesis in the experiment (if any) Methodology ▪ Briefly, describe the methods/principles that you have applied in the experiment. ▪ What are the snack samples that you use? describe the snack (s) ▪ INDIVIDUAL: Describe the skill(s) that have you acquired in this lab? (include the evidence, if any) Results ▪ What is the data you have collected? ▪ How did you analyze the data? ▪ What are the results? ▪ Did you achieved the objectives? Discussion ▪ Predict why you have obtained such results ▪ What could possibly have happened (use Biochemistry/Biology/Chemistry/Physics/Math/etc concepts) ▪ What have you solved? ▪ How are you going to use the results in your PrBL - Redesigning Me?
PROTEINS Introduction ▪ Briefly, describe the concepts you have learned. ▪ What are the objectives of the experiment? ▪ What is the hypothesis in the experiment (if any) Methodology ▪ Briefly, describe the methods/principles that you have applied in the experiment. ▪ What are the snack samples that you use? ▪ Describe the snack (s) ▪ INDIVIDUAL: Describe the skill(s) that have you acquired in this lab? (include the evidence, if any) Results ▪ What is the data you have collected? ▪ How did you analyze the data? ▪ What are the results? ▪ Did you achieved the objectives? Discussion ▪ Predict why you have obtained such results ▪ What could possibly have happened (use Biochemistry/Biology/Chemistry/Physics/Math/etc concepts) ▪ What have you solved? ▪ How are you going to use the results in your PrBL - Redesigning Me?
VITAMINS Introduction ▪ Briefly, describe the concepts you have learned. ▪ What are the objectives of the experiment? ▪ What is the hypothesis in the experiment (if any) Methodology ▪ Briefly, describe the methods/principles that you have applied in the experiment. ▪ What are the samples that you use? describe the snack (s) ▪ INDIVIDUAL: Describe the skill(s) that have you acquired in this lab? (include the evidence, if any) Results ▪ What is the data you have collected? ▪ How did you analyze the data? ▪ What are the results? ▪ Did you achieved the objectives? Discussion ▪ Predict why you have obtained such results ▪ What could possibly have happened (use Biochemistry/Biology/Chemistry/Physics/Math/etc concepts) ▪ What have you solved? ▪ How are you going to use the results in your PrBL - Redesigning Me?
RUBRICS Individual skills (10%) Find the score reference in the Psychomotor skills
RUBRICS Group (20%) Introduction ▪ 10 marks Methodology ▪ 10 marks Results ▪ 10 marks Discussion ▪ 10 marks TOTAL = 50 marks % = 20%