ة و التطبیقیةمجلة إبن الھیثم للعلوم الصرف 2012 السنة 25 المجلد 1 العدد Ibn A l-Haitham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 Determination of Ascorbic Acid (Vitamin C) by Direct Injection Enthalpimetry (DIE) Technique A. M. Abdullah Departme nt of Chemistry, College of Science, Sulaimani Unive rsity Email: ahmadmeer56@yahoo.com Abstract The ascorbic acid content of juices of some fruits and p harmaceutical tablets of Vitamin C was determined by a homemade app aratus of DIE technique using a thermocouple as heat sensor. T he method is simp le, sp eed, low cost and the different typ es of turbid, colored samples can be analy zed without any p roblem. The results were of a valu able accuracy and p recision, and the recovery of results was with accep table values. Keywords: DIE determination of Vitamin C, Pot assium Dichromate. Introduction The Vitamin C p lay s an imp ortant role in controllin g infections and the body ’s resp onse to stress. It is also found to be a powerful antioxidant that can neutralize harmful l free radicals; helps make collagen, a tissue need ed for h ealth bones, teeth, gums and b lood vessels [1, 2, and 3]. The term Vitamin C refers to both ascorbic acid and dehydro-ascorbic acid, since both comp ounds exhibit anti scorbutic activity [4]. Ascorbic acid, Vitamin C is an important nutrient that is p resent in many foods. Various reports show that fruits are excellent sources of Vitamin C. Ascorbic acid is sy nthesized for use as a drug, which occurs as white or slightly y ellow crystals or p owder or tablets with an acidic taste. Numerous methods have been reported for determination of ascorbic acid in verity sample of different fruits, vegetables, p harmaceutical p reparations, and commer cial samples these methods include: 1. Titrimetric M ethod: Dep ending on reduction of ascorbic acid by iodine [5] or 2,6- Dichlorop henolindophenol [6,7], these methods allowed for determination of 0.01M Ascorbic Acid. 2. Sp ectrop hotmetric M ethods M any sp ectrop hotometric methods dep end on the reduction of Fe (II) to Fe (III) with ascorbic acid, followed by the comp lexation of reduced Fe (II) with different ة و التطبیقیةمجلة إبن الھیثم للعلوم الصرف 2012 السنة 25 المجلد 1 العدد Ibn A l-Haitham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 reagents [8.11]. Two reagents, nitrosobenzene and P-nitro notrosobenzene have been st udied for analysis of ascorbic acid by its hyp o chromic effect on reagents [12]. 3. Chromatographic methods: Esp ecially HPLC technique [13], used for determination of ascorbic acid in foods, biolo gical sa mples, and p harmaceutical p reparations the method was used for determination of 10 -4 M ascorbic acid. 4. Electrochemical methods: Including p otentiometric [14] and amp erometric titrations [15] and differential p ulse p olarograhy [16] These methods depended also on the oxidation p rop erty of ascorbic acid. Amp erometric flow-injection methods using immobilized enzy me reactions [15] or p hotochemical reduction of methy lene blue [17] has also been recommended for p H=3.8. The methy lene blue method allows the determination of ascorbic acid in the range of 5-90 mg/L. 5. Ot her M ethods like, fluirimetric method [18], p hoto-oxidation of ascorb ic acid [19] , and flow-injection method [20], were used the p hoto-oxidation of ascorbic acid sensitized with thionine blue was invest igated by Perez-Ruriz, et.al [19] for its determination (8*10 -7 – 5*10 -5 M ) in p harmaceuticals. DIE techni que : DIE is an analytical method in which a reactant (excess amount) is injected into a colorimetric vessel containin g another reagent (usually the analyte). The enthalpy change of the insuring reaction is measured and directly related to the amount of the limitin g reagent or analyte. The main advantages of the DIE method are not necessary to standardize the reagents p rovided, it is added in sufficient excess to make the reaction at least 99% comp lete [21]and the sp eed with which individual determinations may be carried out. The addition of excess reagent favors fast kinetics and a “temperature p ulse” may be recorded in less than one minute. ة و التطبیقیةمجلة إبن الھیثم للعلوم الصرف 2012 السنة 25 المجلد 1 العدد Ibn A l-Haitham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 Any rap id process involves a heat of re action (∆H) > [1 K.Cal /mo l] is suitable to use in direct injection enthalpimetry Fig.-1- (DIE) whether exothermic o r endothermic. This technique is successfully applied on different typ es of reactions. The redox reaction of ascorbic acid with p otassium dichromate in acidic mediu m which is e xothermic r eaction is the base for this work. 3C6H8O6 + Cr2O7 2- +8H + 2Cr 3+ + 3C6H6O6 + 7H2O An acidified solution of ascorbic acid p laced in an isolated vessel and excess amount of st andard K2Cr2O7 injected rapidly to it, then the ∆T was measured, through a 0.5 minute from the injection. Experime ntal Reagents: All the chemicals used were either of the an aly tical grade or the highest p urity available, unless otherwise st ated. Usual distilled water, p rotected from carbo n dioxide was used for all p reparations.  Diluted 5M H2SO4 {2ml used for each injection) was p repared from the concentrated acid (98% fro m M erck).  Standard p otassium dichromate (0.5 M ) was p repared from the solid salt K2Cr2O7 (BDH).  A stock solution of ascorbic acid (2M ) was p repared from solid reagent (M erck), freshly used for p reparation of the calibration curv e.  Tablets of vitamin C from different p harmaceutical factories and juices fro m different fruits were analyzed. App aratus: The homemade isolated sy stem was p rep ared as shown in Fig. -2- which consists of a block of a Sty rofoam and a p last ic beaker (50ml) was fi xed inside it. A Sty rofoam cover was made also and two small holes were made through it, one for p assing the sensor and other for p assing small tube for injection. Recommended Procedure: ة و التطبیقیةمجلة إبن الھیثم للعلوم الصرف 2012 السنة 25 المجلد 1 العدد Ibn A l-Haitham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 A- Vitamin C tablets: A t ablet of vitamin C was weigh ed and dissolved in distilled water in a p last ic beaker and acidified with 2ml 5M H2SO4, then the volume was comp leted to about 25ml and the p lastic beaker was p laced inside the block of Sty rofoam in its p osition, then covered, the thermometric sensor was p ut inside the solution and also the tube of injection, when the thermal equilibriu m was recorded, 2ml of 0.5M K2Cr2O7 solution was injected rap idly by a sy ringe. The output signal (ΔT ) was recorded as a peak height (Fig.-2-). The (ΔT) measured by the temp erature difference before injection (when a thermal equilibriu m reached) and after 30 sec. following the injection. The value of (ΔT ) is p rop ortional and to the amount of ascorbic acid p resent. This p rocess was repeated for five tablets individually and the average value of (ΔT ) was reduced. B- Calibration Curve: Prep aration of the calibration curve was p erformed by taking a series of (0.5, 1, 2, 3, 4 and 5ml) p ortion of st andard ascorbic acid solution (1000pp m). The same st eps p erformed as above for the tablets to read out the (ΔT ) obtained. The calibr ation curve (f igure -3-) obtained by drawing the (ΔT ) obtained against the amount of ascorb ic acid in(0.5-5 mg), as shown in (figure -3-). The amount or p ercentage of the ascorbic acid in the different vitamin C tablets from different p harmaceutical p reparations were obtained from the calibration curve. The results were described in (table -1- ) C- Juices of different fruits: Some fresh ripe orange, lemon and gr apefruit were weighed accurately and the fruit were p eeled and cut into two transversely and then squeezed to dischar ge their ju ice into a p re-washed beaker, sufficient juice was obt ained for each kind. A 25ml of a liquate of each juice after acidification with H2SO4 solution was p ut in the p last ic beaker and the ascorbic acid content was determined as p erformed for vitamin C tablets. Results and Discussion The sample matrix in p harmaceutical formulation is comp lex du e to p resence of a variety of comp lexs such as active substances and a lar ge nu mber of additives which act as supp orting material, b inders or st abilizers. Also the fruit juice samples are comp lex and contain many other constituents. The DIE technique depending only on the (ΔH) obtained from the reaction of the ascorbic acid with the reagent, so the method does not suffer from effect of color or turbidity like in titrimetric or sp ectroscop ic methods or ion ic st rength of the ة و التطبیقیةمجلة إبن الھیثم للعلوم الصرف 2012 السنة 25 المجلد 1 العدد Ibn A l-Haitham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 sample mixture lik e in electrochemical methods of analysis, Possible interferences affect on the results of these methods: Sulfuric Acid Concentration: Effect of sulfuric acid studied and it was found that the concentration of 5M is suitable for these measurements. Although the p rocess of mixin g of sulfur ic acid with aqueous solution of the p harmaceutical samp les and juice is an exothermic, but durin g the preparation of sample solution for injection, the reagent was not injected until the thermal equilibrium was obt ained as described in the recommended p rocedure. K2Cr2O7 Concentration: In DIE technique t he main condition is t hat t he amount of the reagent injected in each injection must be in excess, and the addition must be rap id as a p ulse so that a maximum (ΔH) obtained in a certain small limited time (0.5 minutes) and it was found that the concentration of (0.5M ) is more suitable for this p urp ose sp ecially for the range of ascorbic acid amount which we work on it. The main advantage of DIE technique as described b efore is that the st andardization of reagents not required, this p rovides a fast, simp le p rocedure for app lication. De termination of Ascorbic Acid % in diffe rent fruits and pharmaceutical preparations: The ascorbic acid content in juices of three different fruits (orange, lemon and grapefruit) were successfully determined by the p rop osed DIE method, results are shown in table-1- which is the average valu e of the five samples of each fruit. The standard deviation of the five measurements for each juice was found between (0.4 – 0.6) which indicate the reproducibility of the results, and also the content of ascorbic acid which obtain ed for these fruits are agr eed with the average contents of ascorbic acid in these fruits. In order to est ablish the validity of the new DIE method for determination of ascorbic acid, differ ent types of vitamin C were analyzed, the same type of these tablets were also analyzed by the reference titrimetric method of “ British p harmacop eias” [15], results of the two method as shown in table-2- show a good agreement. Also t he ascorbic acid contents of t he juices which found are with t he accep table range of these typ es of the fruits. The accuracy of the prop osed DIE method was also obtained by p erforming a standard addition method by addition of d ifferent amounts (25, 50, 75 and 100 mg) of p ure solid ascorbic acid to the solution of vitamin C tablet (250 mg from Alta p harma). The recoveries which obtained as shown in (table-3- ) are with acceptable ran ge: The results obtained from each tablet are the average of four measurements for each tablets, the standard deviations of t he four rep eated measurements for each tablet was between (0.3 – 0.5), this also indicates t he good p recision of the results obtained. Conclusion ة و التطبیقیةمجلة إبن الھیثم للعلوم الصرف 2012 السنة 25 المجلد 1 العدد Ibn A l-Haitham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 The p rop osed DIE method is very simple, sp eed, p recise and accurate. The main advantage of this method is that it does not suffer from the effect of interferences whi ch p resent in the sample matrix (juice or p harmaceutical p reparations), the method also can b e successfully applied for different samples containin g ascorbic acid with acceptable accuracy and p recision. The sensitivity of the method can be greatly imp roved by using a more sensitive heat sensor than thermocouple which used, like a thermistor or any other sensitive heat sensor. Re ferences 1. Ohio st ate university, dept of human nutrition and OSU Extension (2004). Vitamin C (Ascorbic Acid), HYG-5552-05. 2. Carr, A.C. and B, Frei, (1999). Toward a new recommended d ietary allowance fo r vitamin C based on antioxidant and health effects in humans. Am. J. Clin. Nutr., 69:1086-1107. 3. Hwang, M .Y., (1999). How much vitamin C do y ou need ? The Journal of American M edical Association., 281 (15): 1640. 4. G.K.M c Evoy ; AHFS Drug (2005). information “American So ciety of health Syst em p harmacists, USA, P. 3536 5- Izuagic ,A.A. and Izua gic, F.O. (2007). Journal of A gricu lture and biological Science, 3(5), 367 – 369). Cited from A. M . Pisoschi, A. F. Danet, and S. Kalinowski, (2008) Ascorbic Acid Determination in Co mmercial Fruit Juice Samp les by Cy clic Voltammetry . Journal of Automated M ethods and M anagement in Chemist ry , Hindawi Volume 2008, Art icle ID 937651, 8 p ages. doi:10:1155/2008/937651. 6- Rrdy , L. and Svehla, G. (1963). chemist – Analyst , 52, 24. Cited from Ana-M . Hossu, and V. M agearu, (2004) Determination of vitamin C in pharmaceutical p roducts with p hysico-chemical and bioanaly tical technics. Roum. B iotechnol. Lett ., Vol. 9(1): 1497-1504. 7- Hughes ,D.E. (1983). Titrimetric determin ation of ascorbic acid with 2,6- dichlorophenol indophenol in co mmercial liquid diets. Journal of pharmaceutical Sciences, 72(2), 126. 8- Sick i,B.I. ; M imnough, E.G. and Gram, T.E. (1977). Effects of dietary ascorbic acid supp lementation on hepatic drug-metabolizing enzy mes in the guinea p ig. Biochem. Pharmacol., 26 : 2037. 9- Ary a, S.P. and M ahajan, M . (1977). p roc, Nat. Acad. Sci. India, 67, 39. C ited from Ana-M . Hossu, and V. M agearu, (2004) Determination of vitamin C in p harmaceutical products with p hy sico-chemical and bioanalytical technics. Roum. Biotechnol. Lett., Vol. 9(1):1497-1504. 10- Okamura, M . (1981) A specific method for determination of tot al ascorbic acids in urine by the α,α-dipy ridyl method. Clin. Chim, Acta, 115, 393. 11- Okamura, M . (1980). An improved method for determination of l-ascorbic acid and l- dehydroascorbic acid in blood p lasma. Clin. Chim, Acta, 103, 259. ة و التطبیقیةمجلة إبن الھیثم للعلوم الصرف 2012 السنة 25 المجلد 1 العدد Ibn A l-Haitham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 12- Ursic, S.; Luteratt i, S. and Lijubas, D.(2001). Partial multifactorial d esign in modellin g of UV-sp ectrometric assay s of ascorbic acid with nitrosobenzene and p - nitro-nitrosobenzene Journal: Fresenius Journal of Analytical Chemistry . J. Anal. Chem., 369-719. 13- Rase, R.C. and Nahrwold, D.L. ((1981). Quantitative analysis of ascorbic acid and dehydroascorbic acid by high-p erformance liqu id chromatography . Analytical Biochemist ry ., 114, 140. 14- Hu., X. and Leng, Z. (1995). Ana l. Lett. 28: 2263. Cited from A.H. Sah eed and K.M . M ahmood (2008). M .Sc. Thesis, chem. Dep t., Salahaddin Univ., Iraq. 15- Greenway, G. M . and Onrgomo, P. (1990). Determination of L-ascorbic acid in fruit and vegetable ju ices by flow injection with immobilised ascorbate oxidase. Analyst . , 115, 1297. 16- Koz ar, S.; beijak, A.; Trifunovic, J.E.; Kni ewald, G. and Z. Frensenius, G. (1988). Anal. Chem. 329, 760. Cited from J. A. Rodrigues, I. M . Valente, L. M . Gonçalves, J. G. Pacheco and A. A. Barros (2010). Polarographic determination of vitamin C after derivatization with o-p heny lenediamine, Vol. 75, Issue 7, pp . 731–741. Collect. Czech. Chem. Commun., 75(7), 731-741. do i:10.1135/cccc2010026. 17- E.Lean , L. (1996). Amperometric flow-injection method for t he assay of l-ascorbic acid based on the photochemical reduction of M ethy lene Blue. Talanta, 43:1275. 18- Perez-Ruiz, T. ; M artinez-Lozano, C. ; Tomas V. and Fenol J. (2001). Fluorimetric determination of total ascorbic acid by a stopp ed-flow mixing technique. Analyst ., 126:1436. 19- Perez-Ruiz, T. ; M artinez-Lozano, C. ; Tomas, V. and Sidrach, C. (1997). Flow injection Fluorimetric Determination of Ascorbic Ac id Based on its Phot ooxidation by Thionine Blue. Analy st ., 122:239. 20- Saheed, A.H. and M ahmood, K.M . (2008). M .Sc. Thesis, chem. Dep t., Salahadd in Univ., Iraq. 21- Boric, L.S. and Bark, S.M . (1969) (Thermometric Titrimetry ), 1st Edition, Pergamon Press Ltd. Headington Hill Hall, O xford. London W. 1. Table(1):Determination of ascorbic acid in different jui ces Juice Sample pp m Ascorbic Acid Orange 580 Lemon 300 Grup e fruit 450 Table(2): Comparison of the results obtained for determinati on of ascorbic acid content in different pharmaceutical preparations by DIE method and refe rence method Vitamin C Amount Amount found by E% Amount found by E% ة و التطبیقیةمجلة إبن الھیثم للعلوم الصرف 2012 السنة 25 المجلد 1 العدد Ibn A l-Haitham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 tablets form nominal (mg/tablet) DIE method (mg/tablet) titrimetric method (mg/tablet) Alta pharma. Germany 250 246.7 -1.32 247.1 -1.16 German Vit. Germany -p ol (LTD) 180 184.0 -2.22 183.2 -1.77 Cal-C-Vita “Bayer” 1000 985.6 -1.44 988.1 -1.19 AL SHAHAB labs. “ Sy ria” 500 507.7 -+1.54 506.5 +1.30 Table (3):Recovery experiments for ascorbic acid to Vitamin C tablets mg Ascorbic Acid in Vitamin C tablets mg p ure A.A. added mg p ure A.A. p resent mg p ure A.A. found Recovery % 250 25 275 272.2 98.98 250 50 300 296.4 98.80 250 75 325 330.1 101.50 250 100 350 358.5 102.43 ة و التطبیقیةمجلة إبن الھیثم للعلوم الصرف 2012 السنة 25 المجلد 1 العدد Ibn A l-Haitham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 Fig.(1):DIE The rmogram 1. A Sty rofoam block 2. Plastic beaker 3. A Sty rofoam cover 4. Small sy ringe for injection 5. Thermocouple sensor 6. Temp erature reading Fig.( 2): The arrangement for the Injection Process ة و التطبیقیةمجلة إبن الھیثم للعلوم الصرف 2012 السنة 25 المجلد 1 العدد Ibn A l-Haitham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 y = 3.4029x + 5.84 R2 = 0.9945 0 5 10 15 20 25 30 0 1 2 3 4 5 6 7 Wt. ( m g) Ascor b ic Acid ∆ T Fig.(3): A Calibration Curve between the (ΔT) obtained and the amount of A.A. ة و التطبیقیةمجلة إبن الھیثم للعلوم الصرف 2012 السنة 25 المجلد 1 العدد Ibn A l-Haitham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 بوساطة تقنیة تقدیر المحتوى الحراري عند الحقن المباشر ) C )فیتامین ك تقدیرحامض األسكوربی DIRECT INJ ECTION ENTH ALPIMET RY (DIE) احمد عبد اهللا العلوم جامعة السلیمانیةقسم الكیمیاء كلیة الخالصة حبـوب فیتـامین (في عصائر مختلف الفواكه والمستحـضرات الـصیدالنیة ) cفیتامین (قدرت نسبة حامض االسكوربیك c( بتقنیة تقدیر المحتوى الحراري عند الحقن المباشر بوساطة جهاز مصنوع محلیا في المختبر مع استخدام Thermo couple . الطریقة بسیطة ،وسریعة ،ورخیصة التكلفة وكذلك یمكن تقدیر محالیل ملونة وعكرة من دون ایة مشكل .أظهرت النتائج انها ذو دقة وتوافق قیمة وكذلك ذو نسبة استعادة مقبولة ایضا