Kingdom of Saud Arabia King Abdulaziz University Girls Collage of Science Biochemistry Department Metabolism (1) BIOC 211 Practical Manual Name: Name: Computer Computer No.: No.: Section: Section: 1 Contents Lab # 1 Experiment Page Expressing concentration of solutions 2 13 3 Calorimetric determination of glucose by the 3,5dinitrosalicylic acid method Estimation of carbohydrate by Anthrone method 4 Quantitative estimation of pentoses 19 5 Determination of Vitamin C in foods by iodometric assay 22 6 Assay of tissue glycogen 25 7 Estimation of total lipid by colorimetric method 28 8 Estimation of cholesterol by liberman-burchard method 31 9 Determination of saponification number 34 2 2 17 Experiment 1 Expressing Concentration Of Solutions In chemistry, concentration is the measure of how much of a given substance there is mixed with another substance, but most frequently the concept is limited to homogeneous solutions, where it refers to the amount of solute in the solvent. To concentrate a solution, one must add more solute (e.g. alcohol), or reduce the amount of solvent (e.g. water). By contrast, to dilute a solution, one must add more solvent, or reduce the amount of solute. Unless two substances are fully miscible there exists a concentration at which no further solute will dissolve in a solution. At this point, the solution is said to be saturated. Qualitative description These glasses containing red dye demonstrate qualitative changes in concentration. The solutions on the left are more dilute, compared to the more concentrated solutions on the right. concentration is described in a qualitative way, through the use of adjectives such as "dilute" for solutions of relatively low concentration and of others like "concentrated" for solutions of relatively high concentration Quantitative notation: 3 There are a number of different ways to quantitatively express concentration; the most common are listed below. They are based on mass, volume, or both. Molarity Molarity is probably the most commonly used unit of concentration. It is the number of moles of solute per liter of solution. 1. Solid material molarity = moles of solute volume of solution (liter) moles of solute= weigh gm molecular weight M = weigh gm . 1000 . molecular weight volume of solution (ml) 2. Liquid material: Molarity of liquid = Percentage x Specific gravity x 1000 x volume (ml) Molecular weight x volume of solution (ml) Example: What is the molarity of a solution made when water is added to 11 g CaCl2 to make 100 mL of solution? Normality Normality is equal to the gram equivalent weight of a solute per liter of solution. A gram equivalent weight or equivalent is a measure of the reactive capacity of a given molecule. Normality is the only concentration unit that is reaction dependent. Example: 1 M sulfuric acid (H2SO4) is 2 N for acid-base reactions because each mole of sulfuric acid provides 2 moles of H+ ions. On the other hand, 1 M sulfuric acid is 1 N for sulfate precipitation, since 1 mole of sulfuric acid provides 1 mole of sulfate ions. 4 For example, hydrochloric acid (HCl) is a monoprotic acid and thus has 1 mol = 1 gram equivalent. One liter of 1 M aqueous solution of HCl acid contains 36.5 grams HCl. It is called 1 N (one normal) solution of HCl. It is given by the following formula: 1. Solid material: Gram equivalent = weight Equivalent weight . Equivalent weight = molecular weight valence N = weight Equivalent weight . . 1000 .. volume of solution (ml) 2. Liquid material: Normality of liquid = Percentage x Specific gravity x 1000 x volume (ml) Equivalent weight x volume of solution (ml) Table of measures units Units Symbol Uses Convert to other unit Liter L volume 1L = 1000ml=106µl. I.E (1Lx 1000 = 1000ml, 1Lx1000000= 106µl) and (1dLx100=100 ml). Gram Gm mass 1gmx 1000= 1000 mg= 106µg 1mg/1000= mg, 1mgx1000= 1000µg 5 1- Acids and Bases Acids are compounds that contain hydrogen and can dissolve in water to release hydrogen ions into solution. For example, hydrochloric acid (HCl) dissolves in water as follows: H2O H+(aq) HCl Cl-(aq) + Base is substances that dissolve in water to release hydroxide ions (OH-) into solution. For example, a typical base according to the Arrhenius definition is sodium hydroxide (NaOH): H2O Na+(aq) NaOH + OH-(aq) Arrhenius's theory explains why all acids have similar properties to each other (and, conversely, why all bases are similar): because all acids release H+ into solution (and all bases release OH-). Neutralization As you can see from the equations, acids release H+ into solution and bases release OH-. If we were to mix an acid and base together, the H+ ion would combine with the OH- ion to make the molecule H2O, or plain water: H+(aq) + OH-(aq) H2O The neutralization reaction of an acid with a base will always produce water and a salt, as shown below: Acid Base Water 6 Salt HCl + NaOH H2O + NaCl 2- pH pH is the concentration of hydrogen ions present. Acids increase the concentration of hydrogen ions, while bases decrease the concentration of hydrogen ions (by accepting them). The acidity or basicity of something, therefore, can be measured by its hydrogen ion concentration. The pH scale is described by the formula: pH = -log [H+] Note: concentration is commonly abbreviated by using square brackets, thus [H+] = hydrogen ion concentration. When measuring pH, [H+] is in units of moles of H+ per liter of solution. The pH scale ranges from 0 to 14. Substances with a pH between 0 and less than 7 are acids (pH and [H+] are inversely related - lower pH means higher [H+]). Substances with a pH greater than 7 and up to 14 are bases (higher pH means lower [H+]). Right in the middle, at pH = 7, are neutral substances, for example, pure water. The relationship between [H+] and pH is shown in the table below alongside some common examples of acids and bases in everyday life. 7 Acids Neutral Bases [H+] pH Example 1 X 100 0 HCl 1 x 10-1 1 Stomach acid 1 x 10-2 2 Lemon juice 1 x 10-3 3 Vinegar 1 x 10-4 4 Soda 1 x 10-5 5 Rainwater 1 x 10-6 6 Milk 1 x 10-7 7 Pure water 1 x 10-8 8 Egg whites 1 x 10-9 9 Baking soda 1 x 10-10 10 Tums® antacid 1 x 10-11 11 Ammonia 1 x 10-12 12 Mineral lime - Ca(OH)2 1 x 10-13 13 Drano® 1 x 10-14 14 NaOH 8 - pH indicator A pH indicator is a halochromic chemical compound that is added in small amounts to a solution so that the pH (acidity or basicity) of the solution can be determined visually. Normally, the indicator causes the color of the solution to change depending on the pH. - pH meter PH can measured by: 1. pH meter 2. PH strips 3. titration pH meter: A pH meter is an electronic instrument used to measure the pH (acidity or alkalinity) of a liquid (though special probes are sometimes used to measure the pH of semi-solid substances). A typical pH meter consists of a special measuring probe (a glass electrode) connected to an electronic meter that measures and displays the pH reading. 3- Buffer A buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. Buffer solutions are used as a means of 9 keeping pH at a nearly constant value in a wide variety of chemical applications. Many life forms thrive only in a relatively small pH range; an example of a buffer solution is blood. Method : 1. Prepare 20 ml of HCL (0.5 M)? 2. Prepare 20 ml of NaOH (2 N)? Homework: What is the molarity and normality of a solution made when water is added to 10 gm of H 2SO4 to make 100 mL of solution? Results 10 Experiment 2 Spectrophotometer A spectrophotometer is one of the scientific instruments commonly found in many research and industrial laboratories. Spectrophotometers are used for research in physics, molecular biology, chemistry, and biochemistry labs. The spectrophotometer is measure the amount of light of a specificed wavelength which passes through a medium. The amount of light absorbed by a medium is proportional to the concentration of the absorbing material or solute present. Thus the concentration of a colored solute in a solution may be determined in the lab by measuring the absorbency of light at a given wavelength. Wavelength (often abbreviated as lambda) is measured in nm. The spectrophotometer allows selection of a wavelength pass through the solution. At the spectrophotometer, you should have two cuvettes in a plastic rack. Solutions which are to be read are poured into cuvettes which are inserted into the machine. One should be marked "B"for the blank and one "S" for your sample Here is a summary of the steps of operation of a spectrophotometer: 1- Power >>>>>>>>Turn on power. 2-Warmup>>>>>>Allow about 5 minutes when first turned on. 11 3-Wavelength>>>>>Select appropriate wavelength. 4- Zero>>>>> With sample holder empty and closed, adjust meter needle to 0%T (or infinite A) using zero control knob. 5- Blank >>>>Fill tube half full with water. Place in sample holder and close cover. Adjust meter needle to 100%T (or 0 A) using light control knob. 6- Standard>>>>Measure absorbance (or %T) of known solution. Fill tube half full with sample of known concentration. Place in sample holder and close cover. Read absorbance value (or %T) from meter. Repeat this step if making a calibration curve or verifying proportionality (Beer's Law). 7- Sample>>>>>Measure absorbance (or %T) of solution with unknown concentration as in previous step. Light source slit condenser cuvette filter Lens 12 photocell galvanometer 1- Serial dilution A serial dilution is the stepwise dilution of a substance in solution. Usually the dilution factor at each step is constant, resulting in a geometric progression of the concentration in a logarithmic fashion. A ten-fold serial dilution could be 1 M, 0.1 M, 0.01 M, 0.001 M... Serial dilutions are used to accurately create highly diluted solutions as well as solutions for experiments resulting in concentration curves with a logarithmic scale Serial dilutions are widely used in experimental sciences, including biochemistry, pharmacology, microbiology, and physics, as well as in homeopathy. Dilution factor= Final volume Initial volume 2- Standard curve A standard curve is a quantitative research tool, a method of plotting assay data that is used to determine the concentration of a substance, particularly proteins and DNA. It can be used in many biological experiments. 13 For example a standard curve for protein concentration is often created using known concentrations of bovine serum albumin. The assay procedure may measure absorbance, optical density, luminescence, fluorescence, radioactivity, or something else. EXPERIMENT 3___ _______ Calorimetric Determination of Glucose by the 3,5dinitrosalicylic acid Method. Principle: Several reagents have been employed which assay sugars by using their reducing properties. This method tests for the presence of free carbonyl group (C=O), the so-called reducing sugars. This involves the oxidation of the aldehyde functional group present in, for example, glucose and the ketone functional group in fructose. Simultaneously, 3,5-dinitrosalicylic acid (DNS) is reduced to 3-amino-5-nitrosalicylic acid under alkaline conditions, as illustrated in the equation below: The chemistry of the reaction is complicated since standard curves do not always go through the origin and different sugars give different color yields. The method is therefore not suitable for the determination of a complex mixture of reducing sugar. Materials: 1. Standard Glucose Solution: 14 0.1g anhydrous glucose is dissolved in distilled water and then raised the volume to 100 ml with distilled water. 2. Dinitro salicylic acid reagent: a. Solution "a" is prepared by dissolving 300g of sodium potassium tartarate in about 500 ml distilled water. b. Solution "b" is prepared by dissolving 10 g of 3,5-dinitrosalicylic acid in 200 ml of 2N NaOH solution. c. The dinitrosalycilate reagent is prepared by mixing solutions a & b and raising the final volume to 1 litre with distilled water. Procedure: 1. Pipette in duplicate the following reagents into a series of dry-clean and labelled test tubes and as indicated in the following table, take Section A. SECTION A SECTION B ml. H2O bbbbbB BB Tube No. ml. Stand. Glucose. ml. H2O ml. Dinitrosalicylic reagent 1 0.0 1.0 2.0 7.0 2 0.2 0.8 2.0 7.0 3 0.4 0.6 2.0 7.0 4 0.6 0.4 2.0 7.0 5 0.8 0.2 2.0 7.0 6 1.0 0.0 2.0 7.0 2. After replacing the above mentioned solutions as in section A in the labelled tubes, shake well and then place them in a boiling water bath for 5 minutes. 3. Cool the tubes thoroughly and then add 7.0 ml of distilled water to each tube as indicated in section B of the previous table, Read the extinction (Optical density) of the colored solutions at 540 nm using the solution in tube 1 as a blank (control). Note: All the tubes must be cooled to room temperature before reading since the extinction is sensitive to temperature change. 15 4. Record the readings in section B, and plot the relationship between the optical density and the concentration of glucose solution. See whether there is a linear relationship between the concentrations of glucose solutions and their corresponding optical densities. 5. Use the already prepared standard curve for the determination of the unknown concentration of the glucose solution provided and tissue extract form exp.6 or any other unknown reducing sugar sample. Name: No. Experiment 3: Results Sheet The concentration of standard glucose solution : mg/ml - After conducting your test, fill the following table : Tube Concentration (Mg/ml) No. Absorbance (At 540 nm) 1 2 3 4 5 6 7 - Plot the standard curve of the absorbance (y- axis) against the concentration ( x-axis ) - Use this plot to estimate the concentration of your unknown glucose sample. - Express your results in mg/dl , mg% , g/ml and g/l 16 Name: No. Experiment 3: Results Sheet 17 EXPERIMENT 4 ___ _______ Est imation of carbohydrate by Anthrone Method. The anthrone reaction is the basis of a rapid and convenient method for the determination of carbohydrates, either free or present polysaccharides. Principle : Carbohydrates are dehydrated by concentrated H2SO4 to form furfural.Furfural condenses with anthrone to form a blue-green colored complex solution shows an absorption maximum at 620nm, which is measured colorimetrically. note that some carbohydrates may give other colors. The extinction depends on the compound investigated, but is constant for a particular molecule. Materials : 1. Anthrone reagent (0.2% in conc. H2SO4). 2. Glucose (10mg/100ml). Procedure : 1. Pipette out into a series of test tubes different volumes of glucose solution and make up the volume to 1ml with water. 2. Add 4ml of anthrone reagent to each tube. 3. mix well. 4. Cover the tubes with marbles on top to prevent loss of water by evaporation. 5. Keep the tubes in a boiling water bath for 10 minutes. 6. cool to room temperature. 7. Measure the optical density at 620 nm using a blank tube containing 1ml water and 4ml reagent. 8. Draw the standard cure and determine the concentration of unknown glucose solution 18 in Name: No. Experiment 4: Results Sheet 19 EXPERIMENT 5__ _ _______ Quantitative Estimation of Pentoses Principle: When pentoses are heated with conc. HCl , furfural is formed which condenses with orcinol in the presence of ferric ions to give a blue-green color. CH3 OH HO orcinol (3,5-dihidroxytoluene) Materials : 1- Orcinol reagent. (Dissolve 1.5 g of orcinol in 500 ml of conc. HCl and add 20 drops of a 100g/l solution of FeCl3.) or 1.5 g Orcinol + (0.5 g FeCl3 + 500 ml conc. HCl) Procedure: Carry the experiment in two test tubes one for the standard and the other for the unknown.In each tube place the following: 1- 7.5 ml Orcinol reagent. 2- 2.5 ml sample. Shake well. 3- Heat for 25 minutes in a boiling water bath with a marple on top of each tube (use a glass stopper). 4- Cool to room temperature in cold water. 5- Read at 665 nm. 20 Calculation: Standard : Ribose 2mg/ml (0.2%) in water. Concentration of unknown = Absorbance of unk x Conc of std Absorbance of std 21 Name: No. Experiment 5: Results Sheet Concentration of standard pentose solution : Calculations: Ast.= Aun.= 22 mg/ml Experiment 6 Determination of Vitamin C in Foods By Iodometric Assay Definition: Vit. C is a water soluble vitamin that is necessary for normal growth and development. Alternative Names: Ascorbic acid Food Sources: Green peppers, Citrus fruits, Strawberries, Tommatos and white potato. Function: 1- Promote healthy teeth and gums 2- Helps for absorption of iron 3- Promote wound healing Recommended daily allowance (RDAs) of vit. C is = 70mg/day. Side Effects: Deficiency → Scurvy Increased intake → Diarrhea Principle: Vit. C can be assayed by direct titration with iodine. Procedure: 1- measure 10ml of juice 2- add 10 drops of starch 3- titer with 0.1 N iodine → blue color 23 Calculation: 1 L of 1 N iodine ≡ 88.06 ascorbic acid 1 ml of 0.1 N iodine = 88.06/ 10 x 1000= 0.008806 g% ascorbic acid ≡ Z x 0.008806 x 100/ 10ml (volume of juice) Z = end point of titration 24 Results 25 EXPERIMENT 7___ _______ T he assay of t issue glycogen Principle: Glycogen is released from the tissue by heating with strong alkali and precipitated on the addition of ethanol. Sodium sulphate is added as a co precipitant to give a quantitative yield of glycogen. The polysaccharide is then hydrolyzed in acid and the glucose released is estimated. Materials: 1. Heart, liver, and muscle from a freshly killed rat. 2.potassium hydroxide (300 g/l) 3. Calibrated centrifuge tubes (10 ml). 30 4. Boiling water bath. 24 5. Saturated Na2 S04. 20 ml 6. Ethanol (95% v/v). 250 ml 7. Volumetric flasks (100 ml). 24 8. Test tubes calibrated at 10 ml. 100 ml 9. HCl (1.2 mol/l.). 100 ml 10. Marbles. 11. Phenol red indicator solution. 12 ml 12. NaOH (0.5 mol/l). 250 ml 13. Reagents for the estimation of glucose (Experiment 1). Procedure: Isolation of glycogen:Accurately weigh the complete heart and muscle and about 1.5 g of liver. Place the tissues into a calibrated centrifuge tube containing 2 ml of KOH (300 g/l) and heat in a boiling water bath for 20 min with occasional shaking. Cool the tubes in ice, add 0.2 ml of saturated Na2 SO4, and mix thoroughly. Precipitate the glycogen by adding 5 26 ml of ethanol (95% v/v), stand on ice for 5 min, and remove the precipitate by centrifugation. Discard the supernatant and dissolve the precipitated glycogen in about 5 ml of water with gentle warming, then dilute with distilled water to the 10 ml calibration mark and mix thoroughly. In the case of the fed animals, transfer the liver sample quantitatively to a 100 ml volumetric flask and make up to the mark with water. Hydrolysis and estimation of glycogen:Pipette duplicate 1 ml samples of the glycogen solutions into test tubes calibrated at 10 ml, add 1 ml of HCl (1.2 mol/l), place a marble on top of each tube, and heat in a boiling water bath for 2 h. At the end of this period, add 1 drop of phenol red indicator and neutralize carefully with NaOH (0.5 mol/l) until the indicator changes from yellow through orange to a pink color. Dilute to 5 ml with distilled water and determine the glucose content by the 3.5 dinitrosalisylic acid method (Experiment 1 ).Then use the standard curve you obtained to estimate the concentration of glucose per100 g sample. 27 Name: No. Experiment 7: Results Sheet Calculate the amount of glycogen in the liver sample, using the standard curve you plotted in experiment 1. 28 EXPERIMENT 8____ _______ Estimation of total lipids by colorimetric method. Principle: Lipids react with sulfuric acid to form carbonium ions which subsequently react with the vanillin phosphate ester to yield a purple complex that is measured photometrically at 540 nm. The intensity of the colour is proportional to the Total lipids concentration. Materials : 1. Vanillin reagent, 0.04M. Dissolve 6.1 g of vanillin in water and dilute to 1 liter. This solution is stable for about 2 months in a brown bottle at room temperature. 2. . Phosphovanillin reagent. Add 350 ml of the vanillin reagent and 50 ml of water to a flask. Add with constant stirring, 600 ml of concentrated (85%) phosphoric acid. This solution is also stable for about 2 months in a brown bottle at room temperature. 3. Sulfuric acid, concentrated, reagent grade. 4. Standard solution. A good U.S.P. grade of olive oil may be used as a standard. In two tarred 100 ml volumetric flasks add approximately 0.5 and 1 ml of the olive oil and weigh again to obtain the exact weight of oil added. (It is time consuming to try to weigh out exactly 500 mg , or any other definite weight, of the oil; the approximate amounts are added. and the exact weight determined.) The above standards should be about 500 and 1,000 mg/dl. Dissolve the oil in absolute ethanol and dilute to the mark with the ethanol. This solution is stable for about month in the refrigerator. 5. Standard solution of cholesterol (1g/100 ml acetone) 29 Procedure: In separate tubes add 20 l of water (blank), 20 l of samples, and 20 l of standards. To each tube add 0.2 ml of concentrated sulfuric acid. Mix well, preferably on a vortex mixer. Place all tubes in boiling water bath for 10 min, remove, and cool in water to room temperature. To each tube add 10 ml of the phosphovanillin reagent and mix well. Incubate at 370C in a water bath for 15 min. Cool and read standards and samples against blank at 540 nm. Calculations : Concentration of unknown = Absorbance of unk x Conc of std Absorbance of std 30 Name: Experiment 8: No. Results Sheet Concentration of standard cholesterol solution : mg/ml Concentration of standard olive oil solution : mg/ml 1) Ast. Cholesterol = Aun cholesterol = 2) Ast. Olive Oil = Aun Olive Oil = 31 EXPERIMENT 9 ___ Estim ation of Cholesterol Liberman – Bu rchard Reaction Principle : Cholesterol is readily soluble in acetone, while most complex lipids are insoluble in this solvent. Blood or serum is extracted with an alcohol-acetone mixture which removes cholesterol and other lipids and precipitates protein. The organic solvent is removed by evapotation on a boiling water bath and dry residue dissolved in chloroform. The cholesteror is then determined colorimetrically using the Liebermann-Burchard reaction. Acetic anhydride reacts with cholesterol in a chloroform solution to produce a characteristic blue-green color. The exact nature of the chromophore is not known but the reaction probably includes esterification of the hydroxyl group in the 3 position as well as other rearrangement in the molecule.The cholesterol is determined colorimetrically using the Libermann – Burchard reaction. Materials : 1. Serum or blood. 2. Alcohol-acetone mixture (1:1). 3. Chloroform. 4. Acetic anhydride-sulphuric acid mixture (30:1 mix just before use, Care!). 5. Stock cholesterol solution (2 mg/ml in chloroform). 6. Working cholesterol solution. (Dilute the above solution one in five with chloroform to give a solution of 0.4 mg/ml.) Procedure : 32 25 ml 21 500 ml 930 ml 250 ml 11 1- Place 10 ml of the alcohol-acetone solvent in a centrifuge tube and 0.2 ml of serum or blood. 2- Immerse the tube in a boiling water bath with shaking until the solvent begins to boil. 3- Remove the tube and continue shaking the mixture for a further 5 min. 4- Cool to room temperature and centrifuge. 5- Decant the supernatant fluid into a test tube and evaporate to dryness on a boiling water bath. 6- Cool and dissolve the residue in 2 ml of chloroform. 7- Add 2 ml of acetic anhydride-sulphuric acid mixture to all tube and thoroughly mix. 8- Leave the tubes in the dark at room temperature and read the extinction at 680 nm. 9- Carry the experiment in a test tube one for the standard: a-0.2 ml of standard then 2 ml of chloroform. b-Add 2 ml of acetic anhydride-sulfuric acid mixture and thoroughly mix. c- Leave the tube in the dark at room temperature and read the extinction at 680 nm. Calculation: Determine the concentration of the unknown sample according to the following equation: Concentration of unknown = Absorbance of unk x Conc of std Absorbance of std 33 Name: No. Experiment 9: Results Sheet Calculate the concentration of cholesterol in your sample. 34 EXPERIMENT 10____ _______ Determination of Saponification Number. Principle: On refluxing with alkali, triacylglycerols (fatty acid esters) are hydrolyzed to give glycerol and potassium salts of fatty acids (soap).Such process is known as, Saponification . The saponification equation is shown below: O CH2 CH O O C O R C R Saponification O CH2 O C R + 3 KOH CH2 OH CH H e+ at Water OH CH2 OH + 3 RCOONa Soap Fat The saponification value is the number of milligrams of KOH required to neutralize the fatty acids resulting from the complete hydrolysis of 1g of fat. The saponification value gives an indication of the nature of the fatty acids constituent of fat and thus, depends on the the average molecular weight of the fatty acids constituent of fat. The greater the molecular weight (the longer the carbon chain), the smaller the number of fatty acids is liberated per gram of fat hydrolyzed and therefore, the smaller the saponification number and vice versa. Materials: 1- Fats and oils (olive oil, coconut oil, sesame oil, and butter) 2- Fat solvent (equal volumes of 95% ethanol and ether) 3-Alcholic KOH (0.5 mol/liter) 35 4-Boiling water bath. 5-Phenolphethalein. 6-Hydrochloric acid (0.5 mol/liter) 7-Burettes (10 ml and 25 ml) 8-Conical flasks (250ml) Procedure: 1- Accurately take 0.5 ml of oil in a 250 ml conical flask (flask 1). 2- Add 10 ml of alcoholic KOH in flask 1 and in another flask (flask 2) add 10 ml of KOH only. 3- Heat flask 1 on a boiling water bath for 10 min. 4- Leave to cool to room temperature. 5- Add 10 drops of Ph. Ph. as indicator in each flask 6- Titrate with 0.5 mol/liter HCl until the pink color disappears. 7- Record your readings as T ml (flask 1) for test and B ml (flask 2) for blank. Calculations: The difference between the blank and the test reading gives the number of milliliters of KOHrequired to saponify 1g fat. You can use this formula to calculate the saponification value: 1ml (0.5 N HCl ) = 28.05 mg KOH ( B-T ) = S saponification value (S) = ( B-T ) x 28.05 = Wt. of fat (1g 36 mg KOH/1g Name: No. Experiment 10: Results Sheet 1- Calculate the Saponification value -for your test oil. 2- Record the results your friends have obtained for other oils. 1- your results: 37 38 39