CHAPTER 3: MATERIALS AND METHODS Materials and Methods. The from different husks, fruits, and vegetables peels were estimated quantitatively by the following volumetric procedure s such as Bertrand s, Benedict s and Lane-Eynon methods. Preparation of Reagents: Take a 1 litter of distilled water and dissolve 69.28g of CuSO 4.5H 2 O to make Fehling -A solution. Dissolve 346g sodium potassium tartarate & 100g of NaoH in a 1 litter of distilled water to prepare Fehling -B solution. In the preparation of methylene blue indicator 1g dissolved in 100mL then make up to the mark in a standard flask. 100gm of ferric ammonium sulphate is dissolved in 109 ml of concentrated H 2 SO 4 and diluted to 1000mL. to make Ferric alum. In the preparation of 500mL Benedict s reagent 100g of sodium citrate, 62.5g of potassium thiocyanate and 75g of sodium carbonate were dissolved in the 300mL of hot water. Then take a 9g of copper sulphate pentahydrate were dissolved in 50mL of distilled water and both the solution are cooled and mixed with constant stirring by adding 2.5mL of 2.5% potassium ferrocyanide. KMnO 4 solution(0.1n) Oxalic acid (AR) 44
Prepare a standard glucose and unknown reducing solutions of the range from 0.2-0.8g and made up to 100ml each. Bertrand s method: Is based on the reducing action of on alkaline solution of tartarate complex with cupric ion; the cuprous oxide formed is dissolved in warm acid solution of ferric alum. The ferric alum is reduced to FeSO 4 which is titrated against standardized KMnO 4 ; Cu equivalence is compared with the table to get the amount of reducing. This is based on the alkaline solution of tartarate complex of cupric ion 172 Procedure and calculation Standardization of KMnO 4 The KMnO 4 can be standardized by using oxalic acid in the following steps. Take a 500mL of standard flask and add a exactly weighed oxalic acid crystals of 3.2-3.4g and made up to the mark. X= oxalic acid crystals weight in g. N= Normality of oxalic acid N= X g (100)/63(500) = (N). Burette: Standard KMnO 4 solution. Conical flask: Oxalic acid (20mL) + 10mL of 1M H 2 SO 4 and boiled for 2 min. End Point: Appearance of pink color. 45
Calculation: V1= 20mL (Volume of oxalic acid) V2= Titrate value (volume of KMnO 4 consumed) N1= (x) N (Normality of oxalic acid) Normality of KMnO4 = x(n) X 1000/V2 = (Y) N Unknown solution was made up to 100mL In a beaker 20mL of unknown solution in addition of 20mL of both felhing s solution (A&B). and heat the contents for 15min, allowed to cool in room temperature followed by titration by repeated washing. Then take cuprous oxide in to conical flask and titrate against the ferric alum(20ml) to dissolve cuprousoxide after that titrate against standard KMnO 4. Amount of = 6.35 X Normality of KMnO 4 /0.1= mg () X mg = Y mg (glucose) To compared the glucose equivalence table Amount of reducing = Y mg of glucose factor X 100/1000 X unknown reducing (ml) = Z g of glucose. 46
Table 8: Glucose and Equivalence 10 20.2 33 63.9 56 105.2 79 143.9 11 22.1 34 65.4 57 107.1 80 145.8 12 24.0 35 66.3 58 109.0 81 147.4 13 25.9 36 69.8 59 111.6 82 149.0 14 27.3 37 72.2 60 112.4 83 150.2 15 29.2 38 73.6 61 114.0 84 152.1 16 31.2 39 75.4 62 115.8 85 153.6 17 34.0 40 77.1 63 116.2 86 155.1 18 35.9 41 79.0 64 119.1 87 156.6 19 37.8 42 79.9 65 120.7 88 157.9 20 39.8 43 81.2 66 122.8 89 159.7 21 41.9 44 84.7 67 123.7 90 161.8 22 42.9 45 85.2 68 125.6 91 162.6 23 44.8 46 86.2 69 127.5 92 164.8 24 47.2 47 89.8 70 129.1 93 166.1 25 48.9 48 90.9 71 130.8 94 167.3 26 50.4 49 92.6 72 132.6 95 169.2 27 53.1 50 94.4 73 134.1 96 169.6 28 54.2 51 96.8 74 135.9 97 172.7 29 56.6 52 98.2 75 137.5 98 174.2 30 59.0 53 100.1 76 139.2 99 175.8 31 59.9 54 101.8 77 141.4 100 176.2 32 61.8 55 103.2 78 142.2 47
Benedict s method 173 Procedure: Standardization of reagent: Conical flask: 5mL of Benedict s reagent + 2g of sodium carbonate +Porcelain beads then the content were heated at 60-70 C. Burette: Standard glucose solution End Point: Blue to Chalky white precipitate. Quantitative Estimation of Sugar Unknown sample: Made up to 100mL distilled water Burette: Unknown sample solution. Conical flask: Same as mentioned in the standardization procedure. End point: Chalky white precipitate. Calculation: Standard value=x g of glucose in 100mL Therefore 1mL contain x/100 g (Standard glucose) Titrate value =..ml Standard value= (x) X (y) g Unknown value is the tire value obtained for unknown reducing Amount of reducing = StandardvalueX100/Unknown value =..g 48
Lane-Eynon Method 174-175 Procedure: Burette: Sugar solution Conical flask: Fehling s solution, heat at 60-70 C. Indicator: Methylene blue. End point: Pale blue color to Red Calculation: 0.005 g of glucose =1mL of Fehling solution Unknown value =Amount of reducing Sugar consumed=titrevalue Amount of Fehling s solution taken = ml Therefore Amount of reducing = StandardvalueX100/Unknown value Standard procedure followed for entire samples of husks, fruits and vegetable peels. The hydrolysis of sample was carried out at constant stirring using 50mL of 0.2N sulphuric acid temperature in a hot plate, Equipped with a temperature controller, and continuously shaken during the operation 176.Initially, 50mL of 0.2N sulphuric acid solution(20mesh) sample were put into the beaker and kept under hot plate temperature controller was adjusted. Then the reaction was expected to be at constant temperature (Isothermal) but before the temperature was achieved, reaction has occurred. Then it neutralized (ph7) by adding calcium carbonate and activated carbon followed by filtration. 49