ة و التطبیقیةمجلة إبن الھیثم للعلوم الصرف 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for 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 Department of Chemistry, College of Science, Sulaimani University Email: ahmadmeer56@yahoo.com Abstract The ascorbic acid content of juices of some fruits and pharmaceutical tablets of Vitamin C was determined by a homemade apparatus of DIE technique using a thermocouple as heat sensor. The method is simple, speed, low cost and the different types of turbid, colored samples can be analyzed without any problem. The results were of a valuable accuracy and precision, and the recovery of results was with acceptable values. Keywords: DIE determination of Vitamin C, Potassium Dichromate. Introduction The Vitamin C plays an important role in controlling infections and the body’s response to stress. It is also found to be a powerful antioxidant that can neutralize harmful l free radicals; helps make collagen, a tissue needed for health bones, teeth, gums and blood vessels [1, 2, and 3]. The term Vitamin C refers to both ascorbic acid and dehydro-ascorbic acid, since both compounds exhibit anti scorbutic activity [4]. Ascorbic acid, Vitamin C is an important nutrient that is present in many foods. Various reports show that fruits are excellent sources of Vitamin C. Ascorbic acid is synthesized for use as a drug, which occurs as white or slightly yellow crystals or powder or tablets with an acidic taste. Numerous methods have been reported for determination of ascorbic acid in verity sample of different fruits, vegetables, pharmaceutical preparations, and commercial samples these methods include: 1. Titrimetric Method: Depending on reduction of ascorbic acid by iodine [5] or 2,6- Dichlorophenolindophenol [6,7], these methods allowed for determination of 0.01M Ascorbic Acid. 2. Spectrophotmetric Methods Many spectrophotometric methods depend on the reduction of Fe (II) to Fe (III) with ascorbic acid, followed by the complexation of reduced Fe (II) with different ة و التطبیقیةمجلة إبن الھیثم للعلوم الصرف 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 reagents [8.11]. Two reagents, nitrosobenzene and P-nitro notrosobenzene have been studied for analysis of ascorbic acid by its hypo chromic effect on reagents [12]. 3. Chromatographic methods: Especially HPLC technique [13], used for determination of ascorbic acid in foods, biological samples, and pharmaceutical preparations the method was used for determination of 10-4M ascorbic acid. 4. Electrochemical methods: Including potentiometric [14] and amperometric titrations [15] and differential pulse polarograhy [16] These methods depended also on the oxidation property of ascorbic acid. Amperometric flow-injection methods using immobilized enzyme reactions [15] or photochemical reduction of methylene blue [17] has also been recommended for pH=3.8. The methylene blue method allows the determination of ascorbic acid in the range of 5-90 mg/L. 5. Other Methods like, fluirimetric method [18], photo-oxidation of ascorbic acid [19], and flow-injection method [20], were used the photo-oxidation of ascorbic acid sensitized with thionine blue was investigated by Perez-Ruriz, et.al [19] for its determination (8*10 -7 – 5*10-5M) in pharmaceuticals. DIE technique: DIE is an analytical method in which a reactant (excess amount) is injected into a colorimetric vessel containing another reagent (usually the analyte). The enthalpy change of the insuring reaction is measured and directly related to the amount of the limiting reagent or analyte. The main advantages of the DIE method are not necessary to standardize the reagents provided, it is added in sufficient excess to make the reaction at least 99% complete [21]and the speed with which individual determinations may be carried out. The addition of excess reagent favors fast kinetics and a “temperature pulse” may be recorded in less than one minute. ة و التطبیقیةمجلة إبن الھیثم للعلوم الصرف 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 Any rapid process involves a heat of reaction (∆H) > [1 K.Cal /mol] is suitable to use in direct injection enthalpimetry Fig.-1- (DIE) whether exothermic or endothermic. This technique is successfully applied on different types of reactions. The redox reaction of ascorbic acid with potassium dichromate in acidic medium which is exothermic reaction is the base for this work. 3C6H8O6 + Cr2O7 2- +8H + 2Cr 3+ + 3C6H6O6 + 7H2O An acidified solution of ascorbic acid placed in an isolated vessel and excess amount of standard K2Cr2O7 injected rapidly to it, then the ∆T was measured, through a 0.5 minute from the injection. Experimental Reagents: All the chemicals used were either of the analytical grade or the highest purity available, unless otherwise stated. Usual distilled water, protected from carbon dioxide was used for all preparations.  Diluted 5M H2SO4 {2ml used for each injection) was prepared from the concentrated acid (98% from Merck).  Standard potassium dichromate (0.5 M) was prepared from the solid salt K2Cr2O7 (BDH).  A stock solution of ascorbic acid (2M) was prepared from solid reagent (M erck), freshly used for preparation of the calibration curve.  Tablets of vitamin C from different pharmaceutical factories and juices from different fruits were analyzed. Apparatus: The homemade isolated system was prepared as shown in Fig. -2- which consists of a block of a Styrofoam and a p lastic beaker (50ml) was fixed inside it. A Styrofoam cover was made also and two small holes were made through it, one for passing the sensor and other for passing small tube for injection. Recommended Procedure: ة و التطبیقیةمجلة إبن الھیثم للعلوم الصرف 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 A- Vitamin C tablets: A tablet of vitamin C was weighed and dissolved in distilled water in a plastic beaker and acidified with 2ml 5M H2SO4, then the volume was completed to about 25ml and the plastic beaker was p laced inside the block of Styrofoam in its position, then covered, the thermometric sensor was put inside the solution and also the tube of injection, when the thermal equilibrium was recorded, 2ml of 0.5M K2Cr2O7 solution was injected rapidly by a syringe. The output signal (ΔT) was recorded as a peak height (Fig.-2-). The (ΔT) measured by the temperature difference before injection (when a thermal equilibrium reached) and after 30 sec. following the injection. The value of (ΔT) is proportional and to the amount of ascorbic acid present. This process was repeated for five tablets individually and the average value of (ΔT) was reduced. B- Calibration Curve: Preparation of the calibration curve was performed by taking a series of (0.5, 1, 2, 3, 4 and 5ml) portion of standard ascorbic acid solution (1000ppm). The same steps performed as above for the tablets to read out the (ΔT) obtained. The calibration curve (figure -3-) obtained by drawing the (ΔT) obtained against the amount of ascorbic acid in(0.5-5 mg), as shown in (figure -3-). The amount or percentage of the ascorbic acid in the different vitamin C tablets from different pharmaceutical preparations were obtained from the calibration curve. The results were described in (table -1- ) C- Juices of different fruits: Some fresh ripe orange, lemon and grapefruit were weighed accurately and the fruit were peeled and cut into two transversely and then squeezed to discharge their juice into a pre-washed beaker, sufficient juice was obtained for each kind. A 25ml of a liquate of each juice after acidification with H2SO4 solution was put in the plastic beaker and the ascorbic acid content was determined as performed for vitamin C tablets. Results and Discussion The sample matrix in pharmaceutical formulation is complex due to presence of a variety of complexs such as active substances and a large number of additives which act as supporting material, binders or stabilizers. Also the fruit juice samples are complex 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 spectroscopic methods or ionic strength of the ة و التطبیقیةمجلة إبن الھیثم للعلوم الصرف 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 sample mixture like 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 process of mixing of sulfuric acid with aqueous solution of the pharmaceutical samples and juice is an exothermic, but during the preparation of sample solution for injection, the reagent was not injected until the thermal equilibrium was obtained as described in the recommended procedure. K2Cr2O7 Concentration: In DIE technique the main condition is that the amount of the reagent injected in each injection must be in excess, and the addition must be rapid as a pulse 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 purpose specially for the range of ascorbic acid amount which we work on it. The main advantage of DIE technique as described before is that the standardization of reagents not required, this p rovides a fast, simple procedure for application. Determination of Ascorbic Acid % in different fruits and pharmaceutical preparations: The ascorbic acid content in juices of three different fruits (orange, lemon and grapefruit) were successfully determined by the proposed DIE method, results are shown in table-1- which is the average value 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 obtained for these fruits are agreed with the average contents of ascorbic acid in these fruits. In order to establish the validity of the new DIE method for determination of ascorbic acid, different types of vitamin C were analyzed, the same type of these tablets were also analyzed by the reference titrimetric method of “ British pharmacopeias” [15], results of the two method as shown in table-2- show a good agreement. Also the ascorbic acid contents of the juices which found are with the acceptable range of these types of the fruits. The accuracy of the proposed DIE method was also obtained by performing a standard addition method by addition of different amounts (25, 50, 75 and 100 mg) of pure solid ascorbic acid to the solution of vitamin C tablet (250 mg from Alta pharma). The recoveries which obtained as shown in (table-3- ) are with acceptable range: The results obtained from each tablet are the average of four measurements for each tablets, the standard deviations of the four repeated measurements for each tablet was between (0.3 – 0.5), this also indicates the good precision of the results obtained. Conclusion ة و التطبیقیةمجلة إبن الھیثم للعلوم الصرف 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 The proposed DIE method is very simple, speed, precise and accurate. The main advantage of this method is that it does not suffer from the effect of interferences which present in the sample matrix (juice or pharmaceutical preparations), the method also can be successfully applied for different samples containing ascorbic acid with acceptable accuracy and precision. The sensitivity of the method can be greatly improved by using a more sensitive heat sensor than thermocouple which used, like a thermistor or any other sensitive heat sensor. References 1. Ohio state 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 dietary allowance for 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 you need? The Journal of American Medical Association., 281 (15): 1640. 4. G.K.Mc Evoy; AHFS Drug (2005). information “American Society of health System pharmacists, USA, P. 3536 5- Izuagic ,A.A. and Izuagic, F.O. (2007). Journal of Agriculture and biological Science, 3(5), 367 – 369). Cited from A. M. Pisoschi, A. F. Danet, and S. Kalinowski, (2008) Ascorbic Acid Determination in Commercial Fruit Juice Samples by Cyclic Voltammetry. Journal of Automated Methods and Management in Chemistry, Hindawi Volume 2008, Article ID 937651, 8 pages. doi:10:1155/2008/937651. 6- Rrdy, L. and Svehla, G. (1963). chemist – Analyst, 52, 24. Cited from Ana-M. Hossu, and V. Magearu, (2004) Determination of vitamin C in pharmaceutical products with physico-chemical and bioanalytical technics. Roum. Biotechnol. Lett., Vol. 9(1): 1497-1504. 7- Hughes ,D.E. (1983). Titrimetric determination of ascorbic acid with 2,6- dichlorophenol indophenol in commercial liquid diets. Journal of pharmaceutical Sciences, 72(2), 126. 8- Sicki,B.I. ; Mimnough, E.G. and Gram, T.E. (1977). Effects of dietary ascorbic acid supplementation on hepatic drug-metabolizing enzymes in the guinea pig. Biochem. Pharmacol., 26: 2037. 9- Arya, S.P. and Mahajan, M. (1977). proc, Nat. Acad. Sci. India, 67, 39. Cited from Ana-M. Hossu, and V. Magearu, (2004) Determination of vitamin C in pharmaceutical products with physico-chemical and bioanalytical technics. Roum. Biotechnol. Lett., Vol. 9(1):1497-1504. 10- Okamura, M. (1981) A specific method for determination of total ascorbic acids in urine by the α,α-dipyridyl 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 plasma. Clin. Chim, Acta, 103, 259. ة و التطبیقیةمجلة إبن الھیثم للعلوم الصرف 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 12- Ursic, S.; Luteratti, S. and Lijubas, D.(2001). Partial multifactorial design in modelling of UV-spectrometric assays 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-performance liquid chromatography. Analytical Biochemistry., 114, 140. 14- Hu., X. and Leng, Z. (1995). Anal. Lett. 28: 2263. Cited from A.H. Saheed and K.M. Mahmood (2008). M.Sc. Thesis, chem. Dept., Salahaddin Univ., Iraq. 15- Greenway, G. M. and Onrgomo, P. (1990). Determination of L-ascorbic acid in fruit and vegetable juices by flow injection with immobilised ascorbate oxidase. Analyst. , 115, 1297. 16- Kozar, S.; beijak, A.; Trifunovic, J.E.; Kniewald, 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-phenylenediamine, Vol. 75, Issue 7, pp. 731–741. Collect. Czech. Chem. Commun., 75(7), 731-741. doi:10.1135/cccc2010026. 17- E.Lean ,L. (1996). Amperometric flow-injection method for the assay of l-ascorbic acid based on the photochemical reduction of Methylene Blue. Talanta, 43:1275. 18- Perez-Ruiz, T. ; Martinez-Lozano, C. ; Tomas V. and Fenol J. (2001). Fluorimetric determination of total ascorbic acid by a stopped-flow mixing technique. Analyst., 126:1436. 19- Perez-Ruiz, T. ; Martinez-Lozano, C. ; Tomas, V. and Sidrach, C. (1997). Flow injection Fluorimetric Determination of Ascorbic Acid Based on its Photooxidation by Thionine Blue. Analyst., 122:239. 20- Saheed, A.H. and Mahmood, K.M . (2008). M.Sc. Thesis, chem. Dept., Salahaddin Univ., Iraq. 21- Boric, L.S. and Bark, S.M. (1969) (Thermometric Titrimetry), 1st Edition, Pergamon Press Ltd. Headington Hill Hall, Oxford. London W. 1. Table(1):Determination of ascorbic acid in different juices Juice Sample ppm Ascorbic Acid Orange 580 Lemon 300 Grupe fruit 450 Table(2): Comparison of the results obtained for determination of ascorbic acid content in different pharmaceutical preparations by DIE method and reference 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 Al-Haitham Journal for 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-pol (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. “Syria” 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 pure A.A. added mg pure A.A. present mg pure 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 Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 Fig.(1):DIE Thermogram 1. A Styrofoam block 2. Plastic beaker 3. A Styrofoam cover 4. Small syringe for injection 5. Thermocouple sensor 6. Temperature reading Fig.( 2): The arrangement for the Injection Process ة و التطبیقیةمجلة إبن الھیثم للعلوم الصرف 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for 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. (mg) Ascorbic 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 Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 بوساطة تقنیة تقدیر المحتوى الحراري عند الحقن المباشر ) C )فیتامین ك تقدیرحامض األسكوربی DIRECT INJECTION ENTHALPIMETRY (DIE) احمد عبد اهللا العلوم جامعة السلیمانیةقسم الكیمیاء كلیة الخالصة امین (في عصائر مختلف الفواكه والمستحـضرات الـصیدالنیة ) cفیتامین (قدرت نسبة حامض االسكوربیك حبـوب فیتـ 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