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Practical 7 Ascorbic Acid Estimation Nutritional Biochemistry

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Practical 7 Ascorbic Acid Estimation Nutritional Biochemistry Methods of Estimation PRACTICAL 7 METHODS OF ESTIMATION of Ascorbic Acid (Vitamin C) OF ASCORBIC ACID (VITAMIN C) Structure 7.1 Introduction 7.2 Ascorbic Acid – Basic Concepts 7.3 Methods used for Estimation of Vitamin C 7.3.1 Titrimetric Method : 2, 6 Dichlorophenol Indophenol Method 7.3.2 Colorimetric Method: 2, 4 Dinitrophenylhydrazine Method Experiment 1: Titrimetric Method: 2, 6 Dichlorophenol Indophenol Method for Estimation of Vitamin C in a Given Solution Experiment 2: Titrimetric Method: 2, 6 Dichlorophenol Indophenol Method for Estimation of Vitamin C in Lemon Juice Experiment 3: Titrimetric Method: 2, 6 Dichlorophenol Indophenol Method for Estimation of Vitamin C in Chillies Experiment 4: Colorimetric Method: 2, 4 Dinitrophenylhydrazine Method for Estimation of Vitamin C in a Given Solution 7.1 INTRODUCTION Ascorbic acid is also known as vitamin C which is a water soluble vitamin. As you already know, vitamins are a group of small molecular compounds that are essential nutrients in many multi-cellular organisms, and humans in particular. The name “vitamin” is a contraction of “vital amine”, and came about because many of the first vitamins to be discovered were members of this class of organic compounds. And although many of the subsequently discovered vitamins were not amines, the name was retained. In this practical we will learn about and practically carry out the methods of estimating vitamin C, also known as ascorbic acid. Objectives After going through this practical and undertaking the experiments given herewith, you will be able to: describe the various methods of estimation of ascorbic acid, and estimate and compare the ascorbic acid content of different kinds of related vegetables or fruits. 7.2 ASCORBIC ACID – BASIC CONCEPTS Ascorbic acid is a water-soluble vitamin, whose structure is shown in Figure 7.1. You would have noticed that its structure resembles glucose. O C HO C O C H C C H CH2 OH Figure 7.1: Structure of vitamin C (ascorbic acid) 1750 Nutritional As the name suggests, ascorbic acid is acidic in nature and a pure solution of this white Biochemistry crystalline substance has a pH of about 3. Ascorbic acid also have two possible enantiomers – the L and the D form. The D form of ascorbic acid is generally inactive as anti-scorbutic agents and naturally occurring ascorbic acid occurs in L form. Ascorbic acid is stable in solid form and in acidic solutions but is easily destroyed in alkaline solutions. Oxidative destruction of ascorbic acid is accelerated by increasing the pH. Some mammals like rat can synthesize the vitamin from glucose by the uronic acid pathway. However, primates like humans and some other animals like guinea pig, bats, fish, birds cannot synthesize this vitamin. They lack the enzyme L gulonolactone oxidase which prevents its synthesis from glucose. Since these species from the animal kingdom cannot produce ascorbic acid, they have to obtain it entirely through the diet. Hence, its importance. The vitamin exists in the body largely in the reduced form with reversible equilibrium with a relatively small amount of dehydro ascorbic acid. Both forms are physiologically and metabolically active. We are already familiar with the functions of vitamin C. A brief review of its functions is presented next. Functions Vitamin C is easily oxidized, and the majority of its functions in vivo rely on this property. It plays a key role in the body’s synthesis of collagen and nor epinephrine by keeping the enzymes responsible for these processes in their active reduced form. Vitamin C may also play a role in detoxifying byproducts of respiration. Occasionally - during respiration, O2 is incompletely reduced to superoxide ion (O2 ) instead of being reduced completely to its -2 oxidation state (as in H2O). Normally an enzyme called - 2+ superoxide dismutase converts O2 to H2O2 and O2, but in the presence of Fe , the hydrogen peroxide may be converted into the highly-reactive hydroxyl radical (OH). The hydroxyl radical can initiate unwanted and deleterious chemistry within a cell when it removes a hydrogen atom (H) from an organic compound to form H2O and a new, potentially more reactive free radical. Ascorbic acid can donate a hydrogen atom to a free radical, and thus stop these reactions from occurring. The role of ascorbic acid as an antioxidant, therefore, is of prime importance. What would be the consequences of lack of vitamin C in the diet? Certainly we know of the consequences. Let us recall together. Deficiency diseases due to vitamin C Vitamin C deficiency in human’s results in the disease called scurvy, whose symptoms include hemorrhaging (especially in the gums), joint pain and exhaustion. In its final stages a profound exhaustion, diarrhoea, and then pulmonary and kidney failure characterize scurvy, which result in death. So then what are the rich dietary sources of vitamin C? Let us recall and list all the vitamin C-rich food sources. Dietary Sources of Vitamin C Fruits (particularly citrus fruits), vegetables are the best sources of ascorbic acid. Amla is the richest source of ascorbic acid. The amount of ascorbic acid in plants varies greatly, depending on such factors as the variety, weather and maturity. Organ meats (e.g., liver and kidney) generally are also good sources of vitamin C. However, muscle meats and most seeds do not contain significant amounts of ascorbic acid. 176 Remember, the most significant determinant of vitamin C content in foods is how the Methods of Estimation food is stored and prepared. of Ascorbic Acid (Vitamin C) Next, how much vitamin C is required by our body to keep it fit and healthy and away from the symptoms to scurvy? Let’s find out. Requirements and Recommended Dietary Intake A very small daily intake of vitamin C (10-15 mg/day for an adult) is required to avoid deficiency and stave off scurvy. However, there has been, and continues to be, vigorous debate on what the optimum daily intake of vitamin C. According to the Recommended Dietary Intakes of Indians as recommended by the Indian Council of Medical Research (ICMR), a daily intake of 40 mg for adult has been suggested. With this basic understanding about the structure, properties, nature, food sources and requirements of vitamin C, we now move on to the study of the methods which are employed for the estimation of vitamin C in the laboratory. 7.3 METHODS USED FOR ESTIMATION OF VITAMIN C The most commonly used methods to determine ascorbic acid in foods are: 1. Titrimetric methods - The 2, 6 dichlorophenol indophenol method is the common titrimetric method used for determination of ascorbic acid. 2. Colorimetric methods - The 2, 4 dinitrophenyl hydrazine method is a common colorimetric method used for estimation of ascorbic acid in solutions and foods. Let us get to know about these methods in greater details. 7.3.1 Titrimetric Method : 2, 6 Dichlorophenol Indophenol Method The titrimetric method is a redox titration method which depends on the reduction of the blue dye 2,6 dichlorophenolindophenol to a colourless leuco compound by ascorbic acid in solution or in extracts made out of foodstuff. Ascorbic acid, as you have already studied, is a strong reducing agent because of which it reduces the dye 2,6 dichlorophenolindophenol and itself gets converted to dehydro ascorbic acid. After equivalence point is reached the next drop of dye gives a pink colour to the solution indicating the end point. The dye in this titration is coloured in the oxidized form and colourless in the reduced form. Also the dye is pink in acidic solution and blue in alkaline solution. In acidic solution the pink dye is reduced to a colourless substance in presence of ascorbic acid. The solution remains colourless as more dye is added until all the ascorbic acid has reacted. As soon as the next drop of dye solution is added the solution becomes light pink due to excess dye indicating that the end point of the titration has reached. The solution of ascorbic acid or its extracts are prepared in metaphosphoric acid or glacial acetic acid or a mixture of the two. The acid prevents the oxidation of ascorbic acid due to oxygen in the atmosphere which is faster in presence of metal ions like Fe3+ and Cu2+. Metaphosphoric acid can also form complexes with metal ions which reduces the catalytic oxidation of ascorbic acid. In foods, metaphosphoric acid releases the ascorbic acid from proteins and inactivates ascorbic acid oxidase. The reaction involved in this method is highlighted in Figure 7.2 1770 OH Nutritional C O C O Cl Biochemistry Cl HO C O C O O O C C + N H C H C H C H C O CH OH CH2 OH 2 L - Ascorbic acid 2,6 dichloro phenol L - dehydro ascorbic ac indophenol Figure 7.2: Conversion of ascorbic acid to dehydro ascorbic acid A detailed step by step procedure for titrimetric method is given in experiment 1, 2 and 3 in this practical. We shall follow this method when we conduct these experiments in the laboratory. Now let us get to know about the other method of estimating the vitamin C content i.e. the colorimetric method. 7.3.2 Colorimetric Method: 2, 4 Dinitrophenylhydrazine Method A detail step by step procedure is enumerated in experiment 4 later in this practical. We shall use this method in estimating vitamin C in a given sample in the laboratory. Here, let us look at the principle involved in the calorimetric method. Reaction O C O C COOH O HO C O C C O C O O C C O C H C H C H C Oxal H H C C H C CH2 OH CH2 OH CH2 OH L - Ascorbic acid L - dehydro Diketogulonic acid ascorbic acid O H H H C ON N N H 2 N H C NO O 2 NO2 ON2 N N C NO2 H C NO2 Dinitrophenylhydrazine OH C H CH2 OH Osazone Figure 7.3: Osazone formation from ascorbic acid Principle Ascorbic acid is oxidized to dehydro ascorbic acid by copper(Cu++) ions.
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