ZCfICALT METHODS OF ESTIMATION pe OF ASCORBIC ACID ung ature bite Acid — Basic Concepts ne cor 2 Methods use 13 31 Titrimet 732 Colorimel .d for Estimation of Vitamin Cc j¢ Method : 2, 6 Dichlorophenol Indophenol Method. ‘ric Method: 2,4 Dinitrophenylhydrazine Method ant: Titrimetric Method: 2, 6 Dichlorophenol Indophenol Method for sapsriment imation of Vitamin C in a Given Solution FArimetrie Method: 2, 6 Dichlorophenol Indophenol Method for Estimation of Vitamin C in Lemon Juice seinen’: Titrimetic Method 2,6 Dichlorephenal Indophenol Method for Estimation of Vitamin C in Chillies agernet 4:Colorimetrie Method: 2,4 Dinitropheny hydrazine Method for Estimation of Vitamin C in a Given Solution EEE a1 INTRODUCTION acid is also known as vitamin C which is a water soluble vitamin. As you vitamins are a group of small molecular compounds that are essential ens in many multi-cellular organisms, and humans in particular, The name “vitamin” eecontaction of “vital amine”, and came about beeause many of the fist vitamins to t discovered were members of this class of organic compounds. And although many Fite subsequently discovered vitamins were not amines, the name was retained. In hs practical we will learn about and practically carry out the methods of estimating ymin C, also known as ascorbic acid. paperiment 2: Ascorbic aeady know, Objectives Mier going through this practical and undertaking the experiments given herewith, you ill be able to: zs | 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. 2 ASCORBIC ACID - BASIC CONCEPTS Kcorbic acid is a water-soluble vitamin, whose structure is shown in Figure 7.1. You ®uld have noticed that its structure resembles glucase. rructure of vitamin C (ascorbic acid)Nutritional Biochemistry AAs the name suggests, ascorbie acid is acidic in nature and a pure solution ofthis white 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, Ascor acid is stable in solid form and in acidic solutions but is easily destrayed 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 eannot synthesize this vitamin, They lack the enzyme L gulonolactone oxidase ‘hich prevents its synthesis irom glucose. Since these species from the animal kingdom ceannot produce ascorbie acid, they have to obtain it entirely through the diet. Henee, ts 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. Funetions ‘Vitamin Cis easily oxidized, and the majority ofits functions in vivo rely on this propery. Itplays a key role in the body’s synthesis of collagen and nor epinephrine by keepingthe 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, O, is incompletely reduced to superoxide ion (O,. instead of being ~ reduced completely to its -2 oxidation state (as in H,O). Normally an enzyme called superoxide dismutase converts 0, to H,O, and O,, 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 acell when it removes a hydrogen atom (H) from an organic compound to form H,O and a new, potentially more reactive free radical. Ascorbic acid can donate a hydrogen atom free radical, and thus stop these reactions from occurring. The role of ascorbic acid 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. liver and kidney) generally are als eats and most seeds do not contain siPe and preeeioant determinant we ored and prepared of Vitam joy much vitamin Cig ry Pepto to sear in C Content in foods is how the quired by our Let’s find out body to keep it fit and healthy and away intake of vitamin C ( ney and stave off scuryy #eon what the optimum dail Intakes of Indians as recommended by wR) a daily intake of 40 mg for adult has UES to be, Vigorous nin. According tothe Recommended the Indian Council of Medical Research been suggested, about the structure, properties, We now move on to : the study o of vitamin C in the laboratory, spits basic understandin, frenentsof vitamin C, nature, food sources and soyed for the estimation f the methods Which are Titrimetric methods - The'2, 6 di titrimetric method used for irophenyl hydrazine method is a common of ascorbic acid in Solutions and foods, NS get to know about these methods in preater details, Point is reached the next drop of dye gi icating the end point. The dye in this titration burléss in the reduced form. Also the dye is pink in ition, ives a pink colour to the solution coloured in the oxidized form and acidic solution and blue in alk i i i i less substance in presence of Xitie solution the pink dye is reduced to a colourles e 0 bic acid The soltciioains colourless as more dye is added untill the ascorbic has reacted. As soon as the next drop of dye solution is added the solution Ee bt pink due to excess dye indicating that the end point of the titration has reached. i it horic acid or ® solution of ascorbie acid or its extracts are Prepared in metaphosp 0 cei ade ‘mixture of the two. The acid prevents the oxidation atari, idue to oxygen in the atmosphere which is faster in presence) sceat aas si Cu. Metaphosphoric acid can also form: some it Beal git oe tee ar tear Satalytic oxidation of ascorbie acid. In foods, met ic acid from proteins and inactivates gscorbie acid oxidase, Teaction involved in this method is highlighted in Figure 7.2 t OF Ase Methods of Estimation ‘orbie Acid (Vitamin oe <pen Salcal “ey ‘eS one ee oe Hain ce $ aga aah L.-dehydro ascorbic acid Reduced dye | Jnloro phenol indophenol Figure 7.2: Conversion of ascorbic acid to dehydro ascorbic acid, L- Ascorbic acid 24 ‘A detailed step by step procedure for titrimetric method is given in experiment 1, 2 and 4 in this practical. We shall follow this method when we conduct these experiments in the laboratory, Now lets get to know about the other method of estimating the vitamin C content ic. the colorimetric method. 7.3.2 Colorimetric Method: 2,4 Dia ‘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, letus look at the principle involved in the calorimetric method. {trophenylhydrazine Method : Reaction ong ot | ene | : Hoe I E HoO-G—H I CHAOH Soe = Ascorbic ack L- dehydro —_iketogulonic acid ieriaconies L-Assorbis acid ascorbic acid = c 1 Dinitrophenylhydrazine ‘On aa rot con Osazone Figure 7.3: Osazone formation from ascorbic acid Principle * Ascorbic acid is oxidized to dehydro ascorbic avid by copper(Cu') ions. 2,4 dit | hydrazine reacts with the ketone group of dehydro ascorbie Sta n | sive osazone, as highlighted in figure 7:3. The depth of the yellowish orange © osazone produced upon acidification with sulphuric acid is directly proportion amount of ascorbic acid present and ically peat of Dp can be measured photometrically at As you carry out the procedure in the laboratory i i tl Qu will understand a better. So then lets get started and practically leam the two methods above. There are four activities inthis practical, “agiMETRIC METHOD: 2, 6 DICHLOROPHENOL ts MiENOL METHOD FOR ESTIMATION OF rn AMIN CINA GIVEN SOLUTION ate the ascorbic acid content in the given solution by 2, 6 dichlorophenol To stim: Brena} meso’ | principle ae theprinciple behind the trimetri method inthe space provided herewith along wane reaction involved) , $3 on peaction oe ac , oO ee Ret 5 Lo fren ple | t-drhytrn avoad?e 2" 9) cl, nage [Ascorbr oid 15 oxthized to dehyden oscoscl Se by Oppel’ 2) ii ‘Burette- 10 ml. {24 bites he panes! naheee Me wstn th ketene gaa Lo dehy bu tes 5 ml, 10 ml | BM ot undey ovldye tend ne oe ogoent» Fe de nt es ak lecial [ Yelorns wh enone? colo ed ayacene fo dire ed upper adldifice’ wth, | essuring eylinders | 52 1c on'§ 1s aint rend te tHe omevd GY OstorW C Piscaih Veen dete: wossb fro te meted abe at Gvon™ wy hthe dye solution. Titrate the solution against which persists for 30 seconds. SandanPrecautions 1. Rinse all glassware with 3% metaphosphoric acid before you beg and subsequently each time you wash your flasks, cylinders, etc, 2, Make up all volumes with 3 % metaphosphoric acid, nYour practical * Method of Calculation Clearly note down te observations andthe readings as indicated in the format below for standard and sample titration. 1. Standard titration: j) Strength of standard ascorbic acid solution = 1 mg/S ml ii) Volume of standard ascorbic acid solution = 5 ml iil) Volume of dye solution required We Burette reading (ml) Initial Final Difference Pilot. 0 Cine WA 2 6.8 wee 69 / 3 4.8 Boil bb Titer value= A=6:.2 ml iv) A) £2. ml of dye solution is reduced by 5 mL of standard ascorbic acid solution But 5 ml of standard ascorbic acid solution contains 1 mg of ascorbic acid +. (A) .8..ml of dye solution is reduced by 1 mg of ascorbic acid : 8) Flask No.) % . ¥) Given ascorbic acid solution is diluted to 100ml us vi) Volume of dilute sample ascorbic acid solution = 10 ml vil) Volume of dye solution required ~~ Burette reading (ml) Initial Final 20-4 wi) (A) f. ml of dye solution i feasts el mote: aa is al by:'mS [232 ¥ J0e ei Q lo M& ascorbic acid 12. my ¢ & ipeerved Value = D= 1-2-2. mg ascorbic acid i | gapected Value = -mg (Take from counselor) | pected Value error = Expected value — Observed value x 109 ‘ FI : 5