ASCORBIC ACID Faurs and vegetables are important sources of ascorbic acid. The most satisfactory chemical methods of estimation are based on the reduction of 2,6-dichlorophenol indophenol by ascorbic acid and those based on the reaction of dehydroascorbic acid with 2,4-dinitrophenylhydrazine. 2,6-Dichlorophenol-Indophenol Visual Titration Method'-* ‘The dye,which is blue in alkaline solution and red inacid solution, is re- duced by ascorbic acid t0 a colourless form. ‘The reaction is quantitative and practically specific for ascorbic acid in solutions in the pH range 1-3.5. REAGENTS 1.3% Metaphosphoric acid (HPO, or pellets of HPO, in glass distilled water. 2. Ascorbic acid standard: Weigh accurately 100 mg of Lascorbie acid and make up to 100 ml with 3% HPO,. Dilute 10 ml to 100 ml with 3% HPO, (1 ml=0.1 mg of ascorbic acid). 3. Dye solution: Dissolve 50 mg of the sodium salt of 2,6-dichlorophenol- indophenol in approximately 150 ml of hot glass distilled water containing 42 mg of sodium bicarbonate. Cool and dilute with glass distilled water to 200 ml, Store in a refrigerator and standardize every day. PROCEDURE Standardization of Dye ‘Take 5 ml of standard ascorbic acid solution and add 5 ml of HPO,, Fill a microburette with the dye. Titrate with the dye solution toa pink colour which should persist for 15 sec. Determine the dye factor, ise. mg of ascorbic acid per mi of the dye, using the formula: repare by dissolving the sticks 05 Dye factor Preparation of Sample Fruit juices: Take 10 to 20 ml of sample and make up to 100 ml with 3% HPO,. Filter or centrifuge. Solid or semi-solid food: “Take 10 g of sample, blend with 3°% HPO, and make up to 100 ml with HPO,. Filter or centrifuge.Asay of Extract ‘Take an aliquot (2-10 ml) of the HPO, extract of the sample and titrate with the standard dye to a pink end-point which should persist for at least 15 sec. Titeate rapidly and make a preliminary determination of the titre. In the next determination, add most of the dye required and then titrate accurately. The aliquot of sample taken should be such that the titre should not exceed 3 to 5 mil. Elimination of Interference due to Sulphur Dioxide Sulphur dioxide, when present in sample, reduces the indophenol dye and thus interferes in ascorbic acid analysis. If the sample contains SO,, eliminate the interference by following the formaldehyde condensation procedure given below. To 10 ml of the filtrate in a test tube, add 1 ml of 40% formaldehyde and 0.1 ml of HCl, keep for 10 min and titrate as before. CaLcunaTion Calculate the ascorbic acid content of the sample from the following formula: mg of ascorbic acid__ Titre x Dye factor x Volume made up x 100 per 100 g or ml Aliquot of extract We or volume of sample taken for estimation “ taken for estimation References 1. Methods of Vitemin Atzey, The Association of Vitamin Qnemists, lntcracicoce Publishers New York, snd edn, p. 287 (1966). 2. Johnson, B.C, Mulads of Vitamin Deerminetoe, Burgess Publishing Co., Minnea polis, p. 98 (r948). ‘The Direct Colorimetric Determination ‘The direct colorimetric method is based on measurement of the extent to which a 2,6-dichlorophenol-indophenol solution is decolorized by ascorbic acid in sample extracts and in standard ascorbic acid solutions. Since inter- fering substances reduce the dye slowly, rapid determination would be measu- ring mainly the ascorbic acid. Reacenrs 1, 2% Metaphosphoric acid in glass distilled water. 2. Dye solution: Dissolve 100 mg of 2,6-dichlorophenol-indophenol_ dye and 84 mg of sodium bicarbonate in hot (85-95° C) distilled water, cool and make up to 100 ml. Filter and dilute 25 ml to 500 mi with distilled water. 3. Standard ascorbic acid solution: Accurately weigh 100 mg of ascorbic acid and make up to 100 ml with 2% HPO,. Dilute 4 ml of this solution to 100 ml with 2% HPO, (1 ml = 40 yg of ascorbic acid).PROCEDURE Preparation of the sample: Prepare the sample as in visual dye titration method, but using 2% HPO, If the sample is solid or semi-solid, to get a represen- tative sample, it would be advisable to blend 50 to 100 g of the sample with an equal weight of 6% HPO, and make up an aliquot of the macerate to 100 ml. ‘Standard curve: To dry cuvettes or test tubes, pipette the requisite volume of standard ascorbic acid solution—1, 2, 2.5, 3, 4 and 5 ml and make up to 5 ml with the requisite amount of 2% HPO,. Add 10 ml of dye with a rapid delivery pipette, shake and take the reading within 15 to 20 sec. Set the ins- trument to 100% transmission using a blank consisting of 5 ml of 2% HPO, solution and 10 ml of water. Measure the red colour at 518 nm or a wavelength nearest to the required wavelength using a suitable filter, Plot absorbence against concentration. Sample: Place 5 ml of the extract (or less made to 5 ml with HPO,) ina dry cuvette or test tube, add 10 ml of dye and measure as in standard. CatcuraTion Note the concentration of ascorbic acid from the standard curve and cal- culate the ascorbic acid content in the sample as given below: Ascorbic acid | Volume content _™ made up * 10 mg of ascorbic acid per = —_Somtent___madeup © 100 g or ml of sample ml of solution j9 Wt oF volume taken for estimation of sample taken Reference 1 Loci, JH and J.D. Posting, Ind. Eg. Chum, Ano. Edn, 14, 846 (1942) Xylene Extraction Method ‘The method is similar to that of direct colorimetric method, except that the excess of dye is taken up in xylene and the colour measured. ‘The method is particularly suitable in such fresh materials and stored products where there are considerable interfering substances. It enables the determination of true ascorbic acid. Racers 1. Acetate buffer—pH 4: Mix 1 litre of 50% sodium acetate (CH,COONa, 3H,O), with 1 litre of glacial acetic acid. 2. Dye: Dissolve 125 mg of 2,6-dichlorophenol-indophenol in warm dis- tilled water, cool, make up to 100 ml and filter. Dilute 18 ml to 100 ml withwater (1 ml of dye should be equal to 0.1 mg of ascorbic acid). The stock solution of dye may be stored in refrigerator for about a week. 3. Metaphosphoric acid: Dissolve 30 g of sticks or pellets of HPO, in distilled water and dilute to 1000 ml 4. Xylene: Use redistilled xylene. Xylene used in the method may be re- covered by shaking with a 20% solution of NaOH to neutralize acetic acid, followed by distillation. 5. Standard ascorbic acid solution: Weigh exactly 100 mg of ascorbic acid and make up to 100 ml with 3% HPO, Dilute 10 ml to 100 ml (1 ml =0.1 mg of ascorbic acid). 6. 3% peroxide: Dilute 30% hydrogen peroxide 10 times. 7. Formaldehyde. Exrnacrion Macerate 100 g of sample with 100 g of 3% HPO, ina blender. Weigh a portion of the macerate containing 10 to 15 mg of ascorbic acid (10 to 20 g of macerate), teansfer to a 100-ml volumetric flask, make up to mark with 3% HPOg, and filter. SranpAarD Curve Pipette into 50-ml stoppered conical flasks 0.0, 0.5, 0.75, 1, 1.5 and 2 ml of standard ascorbic acid solution and make up to 2 ml with 3% HPO,. Add 2 ml of acetate buffer, 3 ml of dye and 15 ml of xylene in rapid succession. Stopper the flasks and shake vigorously for 10 sec to extract the excess of dye into the xylene. Allow to separate. Pipette out the water layer below the xylene layer and discard. To the xylene layer, add a few crystals of anhydrous Na,SO, to remove traces of moisture, Measure the colour at 520 nm setting the instrument to 100% transmittance using xylene as blank. Plot absorb- ence against concentration to get the standard curve, Procepure Basie method: Take 2 mi of filtrate in a stoppered conical flask, add 2 ml of buffer, 3 ml of the dye and 15 ml of xylene in rapid succession. Stopper and shake for 10 to 15 sec. Pipette out the water layer, add anhydrous Na,SO, to the xylene and measure the colour as in the case of the standard curve. Since ascorbic acid is quite unstable after addition of buffer (pH 4) and as other reducing substances may react with the dye, Lufler, dye and xylene should be added in rapid succession. Formaldehyde modification. YE the material has undergone extensive heat treat- ment or long storage, apply the formaldehyde condensation procedure to correct for the non-ascorbic acid reducing substances as given below.Conical Flask No. 2 Conical Flask No. 2 Total Ascorbic Acid (A) Nosascorbie Acid Reducing Substances (B) (Basic method) (Formaldehyde condensation) 2.0 ml sample 2.0 ml sample 2.0 ml buffer 2.0 ml buffer 1.0 ml water 1.0 ml formaldehyde (40%) 3.0 ml dye Allow to stand for 10 min. Then add 15.0 ml xylene 3 ml of dye and 15 ml of xylene. ‘Add in rapid succession, Stopper and shake. stopper, and shake, ‘Measure the colour in the xylene layer as given in the basic method. Peraxide modification, YE the product has been sulphite treated or if it has been stored for a considerable period in a metal container, use peroxide modi- fication to correct for interference by sulphite or iron. Follow the procedure given below. Conical Flask No. 1 Conical Flark No. 2 Total Ascorbie Acid Non-ascorbie acid re- 4) acing substances (©) asic: method) (Peroxide modifica tion) Sample 20 ml 2.0 mi Baffer 2.0 ml 2.0 ml 3% Hydrogen peroxide 2.0 mi ‘Water 2.0 ml 3 Dye 3.0 mi 3.0 ml Xylene 15.0 ml 15.0 ml ‘Add in rapid succession, stopper and shake. Measure the colour in the xylene layer as given in the basic method. Cancuarion Find the ascorbic acid concentration from the standard curve and calculate as below. Ascorbic acid, Volume Total ascorhic acid in sample made up mg per 100 gor ml “Tal of solut Wr or volume of taken * sample taken Calculate non-ascorbic acid reducing substances in the formaldehyde or x 100hydrogen peroxide modification procedure as given in the case of total ascor- bie acid. ‘True ascorbic _ Total ascorbic _Non-ascorbie acid reduc- acid = acid (A) ‘cing substances (B or C) Reference 1, Robinton, W.B. and E. Stot, J. Bio, Chom. 160, 217 (1945). Ascorbic Acid, Dehydroascorbic Acid and Diketogulonic Acid ‘The reaction of the oxidation products of ascorbic acid with 2,4-dinitropheayl- hydrazine to form osazone has been made use of by Roc and his co-workers!-* to estimate ascorbic acid as well as its oxidation products—dehydroascorbic acid and 2,3-diketogulonic acid. 2,4-Dinitrophenylhydrazine forms osazone with dehydroascorbic acid and diketogulonic acid which dissolves in conc H,SO, forming an intense red solution, and can be measured colorimetrically at 520 nm. Ascorbic acid can be estimated by oxidation with bromine to de- hydroascorbic acid and subsequent formation of osazone with the dinitro- phenylhydrazine. The method has been described in detail by the Associa tion of Vitamin Chemists.5 Reacents 1. 9 N H,SO,: Dilute 250 ml H,SO, to 1 litre with water, 2, 2%, 2,4-Dinitrophenylhydrazine: Dissolve 2 g of 2,4-dinitrophenyl- hydrazine in 100 ml of 9 N H,SO,. Store in amber coloured bottle in a refrigerator. Filter before use. ‘The solution is stable for 2 weeks. 3. 20%, Metaphosphoric acid: Dissolve 100 g of HPO, in 500 ml of dis- tilled water. Store in refrigerator. Stable for 2 weeks. Preferably prepare fresh. Prepare 5 or 10% solutions of HPO, from the stock solution by dilution with water. 4. 2% Thiourca in 5%, metaphosphoric acid: Dissolve 10 g of thiourca in 500 ml of 5% HPO,. 5. 85% H,SO,: Dilute 900 ml of H,SO, to 1000 mi with water. 6. Bromine. 7. Ascorbic acid standard: Dissolve 100 mg ascorbic acid in 100 ml of 5%, HPO, (1 ml = 1 mg of ascorbic acid). 8, Hydrogen sulphide (Kips). 9. Carbon dioxide (Kipps). 10. Stannous chloride. Procapuns 1. Total ascorbic acid: Blend 100 g of sample and 100 g of 10% HPO, ina blender toa uniform slurry. Weigh a portion of the slurry estimated to contaia1 to 2 mg of ascorbic acid. Transfer to ~ 100-ml volumetric Mask and dilute to volume with 5% HPO,. Mix and filter or centrifuge to get « cleat liquid. To the filtrate, add 1 or 2 drops of bromine and shake gently until the solution becomes yellow. If the solution does not become yellow, add another drop and shake. Remove the excess of bromine by passing air or nitrogen (saturated with water) through the solution until it is colourless. Measure the volume and add thiourea to give a concentration of 1% (0.4 g for 40 ml of the extract). Proceed with osazone formation as given later. 2: Dekydroascerbie acid ard diketogulonic acid: Weigh portion of the sample containing 5 to 10 yg of ascorbic acid per ml. Assuming that the sample contains 50 mg/100 g ascorbic acid and the final volume to be made up to is 250 mi, weigh 2.5 to 5.0 g of the sample, add 1.25 g of stannous chloride and 12.5 ml of HPO,, grind the sample and make up to 250 ml with 5% HPO, Filter and use the filtrate for osazone formation as described later. 3. Diketegulonie acid: Pipette 100 mf of the filtrate from 2 into a gas wash- ing bottle. Pass H,S for 15 min. To minimize odour, pass the extiaust Hy through NaOH solution. To 40 ml of the H,S-saturated solution, add 0.4 g of thiourea to give a concentration of 1%. Shake until dissolved and filter. Bubble CO, into the filtrate for 5 min. The H,S reduces dehydroascorbic acid to ascorbic acid which does not react with 2,4-dinitrophenylhydrazine. Proceed for osazone formation as described below. In heated andfor stored simples, the resulting solution is coloured and it is not possible to measure the diketogulonic acid. Formation of Osexone Solution 1 gives: Ascorbic acid + Dehydroascorbic acid + Diketoga- lonic acid Solution 2 gives: Dehydroascorbic acid + Diketogulonic acid Solution 3 gives: Diketogulonic acid For each of the above solutions, take 3 test tubes, mark as 1, 2and 3, and form osazone as described below. 1 2 3 Blank Sample Sample Sample 40 ml 4.0 ml 4,0 mi 2% 24-dinitrophenyl- hydrazine reagent oy 1.0 mi 1.0 ml + + 4 Incubate at 37°C 4 0.5° C for exacily 6 hr in a water bath Remove from the water bath and keep in an ice bath + 4 + 85% H,SO, 5.0 mi 5.0 mal 5.0 mi‘Aad 11,50, drop by drop with thorough mixing, and allow to cool 0 t + 4 29%, 2.4-dinitrophenyl- 1.0 ml . . hydrazine reagent } J 1 Remove the tubes from ice bath and allow to stand at room temperature for 30 min. Cououn Measunencensr Measure the colour st 520 nm setting the instrument to 100%, transmittance with the blank in place in each set. ‘Stanpano Cuave ‘To 50 ml of standard ascorbic acid solution (1.0 ml = 1.0 mg) in a conical flask, add 2 to 3 drops of bromine until the colour becomes yellow and transfer to a gas washing bottle. Pass water saturated air through the solution to re- move unreacted bromine. Pipette 10 ml of this solution into a 500-ml volume- tic flask, add 5.0 g of thiourea and make up to volume with 5% HPO, (1 ml = 20 pg). Pipette 5, 10, 20, 25, 30, 40, 50 and 60 ml of the above solution (1. ml = 20 yg) into separate 100-ml volumetric flasks and make up to mark with 5% HPO, solution containing 1% thiourea to give solutions containing 1,2, 4, 5, 6,8, 10 and 12 ug per ml respectively. Form osazone for each of the seven standards as in the sample, and measure the colour st 520 nm. Plot % transmittance as ordinate and concentration of ascorbic acid as abscissa on a semilogarithmic paper or absorbence against concentration on an ordinary graph paper. CALCULATION Read the concentration of ascorbic acid in each of the solutions 1, 2 and 3 from the standard curve, and calculate using the following expression. mg/i00 g — #8 #04 from the graph x Volume made up x 100 ml of sample taken x Wt of sample x 1000 Determine ascorbic acid, dehydroascorbic acid and diketogulonic acid using the following relationship: Diketogulonic acid = 3 Dehydroascorbie acid = 2-3 Ascorbic acid t2 References 1. Roe, JH, MB. Mill, MJ. Oxeling & CM. Dameron, Biol Chem.174,20% (1949)- a Roe, JH. de MJ. Osteling, I. Bil. Chm,.152, 511 (1544) 3. Rot, JH. J. Biel Chem, 116, 699 (1936). 4 Koc, JLH, & CA, Kuether, J- Biol. Chom, 147, $39 (1943)- §: Maths of Vitamis’ Assy, The Association of Viwnin Chemists, Interscience Publisher, New York, srd edn, p. $28 (1966).