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Procedures for the determination of vitamin C in foods have continued to draw the attention of analysts. Recent reviews in this subject include those of Christie and Wiggins (1978), Cooke and Moxon (1981), Sauberlich et al. (1982) and Pachla et al.(1985). The complex biological relationship between the compound(s) possessing vitamin C activity, as well as the chemical similarity of these compounds to others that are inactive, has made the existence of a single, simple, and specific method clo se-to impossible (Sauberlich et al., 1982). Thus, there has been a proliferation of methods developed, resulting in hundreds and possibly thousands of papers reported. However, they mostly describe minor variations on a fairly limited range of possible pr ocedures, based essentially on the chemical reactions of ascorbic acid and DHAA(Cooke and Moxon,1981). The Association of Official Analytical Chemists (AOAC) has designated two official methods for the determination of vitamin C: the dye -titration method and the microfluorometric method (AOAC, 1984). The former method makes use of the reducing power of the vitamin, and employs 2, 6 dichlorophenolindophenol (DCIP) as the redox indicator for the determination of ascorbic acid. In the micro-fluorometric method, ascorbic acid is oxidized with active carbon to dehydroascorbic acid to form a fluorescent quinoxaline compound which is measured in a fluorometer.
DETERMINATION OF VITAMIN C IN FRESH FRUITS AND VEGETABLES USING THE DYE-TITRATION AND MICROFLUOROMETRIC METHOD
Determination of Vitamin C in Fresh Fruits and Vegetables Using the Dye-titration and Microfluorometric Method has been Studied 2. The vitamin C content of 19 types of fresh fruits and 24 vegetables was determined by the official AOAC methods of dye-titration and microfluorometry. As expected, values obtained by the latter method, which estimated ascorbic acid plus dehydroasorbic acid (DHAA), were clearly higher than those given by the titration method, which determined only ascorbic acid. There were considerable differences in the values obtained by the two methods, depending on the concentration of DHAA in the foods. Larger differences were obtained for the vegetables. The mean recovery value obtained by the dye -titration method was significantly higher than that given by the fluorometric method (p<0.01). Reproducibility studies showed that the two methods did not give significantly different variances (p<0.05). If only ascorbic acid values were required, the titration procedure would give good results, and it may be carried out rapidly using simple laboratory equipment. If a fluorometer was available, total vitamin C values, which would be more useful from the nutritional point of view, could be determined.
DETERMINATION OF ASCORBIC ACID (VITAMIN C) IN COMMERCIAL TABLET BY IODOMETRIC TRTRATION
Determination of Ascorbic acid (Vitamin C) in Commercial Tablet by Iodometric Trtration has been Studied 3. A 0.010 M solution of KIO3 was prepared as a primary standard and titrated by an iodometric procedure a gainst a solution of sodium thiosulfate. In this way, the concentration of the thiouslfate solution was determined to be 0.075 M. Using 50.00 mL of this standard iodate solution, a corresponding quantity of I 3- was allowed to react with ascorbic acid present in a known quantity of a crushed vitamin C tablet. By determining the left over amount of I3-, the amount of ascorbic acid present in vitamin C tables was determined to be 59.30% w/w.
EFFECT OF PRESSURE COOKING ON VITAMIN C CONTENT OF VEGETABLES
Effect of pressure cooking on vitamin c content of vegetables has been studied4. A comparison has been made of the effect upon the retention of vitamin C in 10 different vegetables when cooked in a modern pressure saucepan and in an ordinary saucepan accord ing to the recommended methods. On the average the retention was higher in vegetables cooked in the pressure cooker. The method of estimation used was that of Harris and Olliver (1942).
AN ENZYMIC METHOD FOR THE ESTIMATION OF TRUE VITAMIN C*
An enzymic method for the estimation of true vitamin c * has been studied5. The quantitative determination of vitamin C by chemical methods has been the subject of a number of recent investigations. Biologic assay is usually more difficult, more costly, and necessarily much slower. It has been more accurate, however, up to the present time. The chief inaccuracy of the volumetric method of Tillmans and associates (1) is that certain other constituents of plant and animal tissues reduce the dye, since their reduction potential is lower that that of the indicator. The recent isolation (2 , 3) of a powerful and specific ascorbic acid oxidase afforded us an opportunity of devising a method for the determination of true vitamin C content. In brief, this consists of determining by a suitable procedure the reducing power of tissue extract before and after the action of the enzyme. The difference, of course, is a measure of the amount of vitamin C present. All tissues, juices, and body fluids cannot be treated alike. If it is suspected that some of the vitamin is present in the oxidized (non -reducing) form, it may be reduced. This is accomplished in the course of the preliminary mercuric acetate purification procedure (4), which is ofte n essential for accurate results. We are therefore reporting the technique as we have applied it to several typical foodstuffs. The reasons for the variations are usually self evident and their applications to other materials will be easily appreciated.
TABLE I Analysis for Vitamin C per Gm. Of Substance
Reduction not due to ascorbic acid
True ascorbic acid
mg. Orange juice «««««««««« Lemon juice ««««««««««. Tangerine juice ««««««««« Grapefruit juice ««««««««... Hubbard squash* «««««««« Tea* «««««««««««««. Beer* ««««««««««««« 0.48 (?) 0.52 0.61 0.50 0.62
mg. 0.05 0.00 Trace 0.00 0.00 0.26
mg. 0.47 0.61 0.50 0.62 0.015 0.22
No reduction after mercuric acetate
Mick (grade B, pasteurized) per 10 cc «««««««««««««...... 0.07 0.00
* Prolonged bleaching occurred until tannins, pigments, etc., were removed by treatment with mercuric acetate.
SPECTROPHOTOMETRIC DETERMINATION OF VITAMIN C IN CITRUS FRUITS USING PERI-NAPHTHINDAN-2, 3, 4-TRIONE
spectrophotometric determination of vitamin c in citrus fruits using peri naphthindan-2, 3, 4-trione has been studied6. A spectrophotometric method for the determination of vitamin c in lemon, orange, and grapefruit juices is described. It is based on the reaction with peri -naphthindan-2, 3, 4-trione hydrate at pH 3 to give dihydroxy-peri-naphthindenone which is dissolved in methanol and measured at 460 nm. The method shows good precision and its accuracy compares favourably with the standard titrimetric procedure of the U.S.P. No interferences are caused by dehydroascorbic acid as well as many other juice ingredients.
APPLICATION OF DIFFERENTIAL PULSE POLAROGRAPHY TO THE ASSAY OF ASCORBIC ACID
Application of differential pulse polarography to the assay of ascorbic acid has been studied7. A polarographic method for the determination of ascorbic acid in pharmaceutical preparat ions and fruit juices is described. The method is based on the oxidation of ascorbic acid by iodine, and the resulting iodide, after removal of excess iodine, is oxidized with bromine to iodate which is measured polarographically. The method enables analys is of solutions as dilute as 1.7 X 10 -6 M ascorbic acid.
THALLIC PERCHLORATE IN ACID MEDIUM AS A REAGENT FOR OXIDIMETRIC DETERMINATION OF ASCORBIC ACID IN DRUGS AND FRUIT
Thallic perchlorate in acid medium as a reagent for oxidimetric determination of ascorbic acid in drugs and fruit has been studied 8. Thallic perchlorate is used for determination of ascorbic acid either by direct titration in acid medium (0.5N) or indirectly by addition of excess of the reagent and iodometric determination of the excess. The method is applicable to fruit juices and vitamin C tablets.
DETERMINATION OF TOTAL VITAMIN C IN FRUITS BY CAPILLARY ZONE ELECTROPHORESIS MARCELLA CHIARIa, b, MARINA NESIa, b, GIACOMO CARREAC AND PIER GIORGIO RIGHETTIa, b
Determination of total vitamin c in fruits by capillary zone electrophoresis marcella chiari a, b, marina nesi a, b, giacomo carreac and pier giorgio righetti a,
has been studied 9. A simpje capillary zone electrophoretic
(CZE) method is described for the rapid determination of ascorbic acid dehydroascorbic acid, the physiologically active forms of vitamin C, in fruits. The electropheretic run was accomplished in 9 min on a coated capillary column using 20mM phosphate buffer (pH 7.0). total ascorbic acid was determined by first reducing the deydroascorbic acid to ascorbic acid by
treatement with DL-homocysteine. This reaction was complete in 15 min and total ascorbic acid determination was performed immediately. The data obtained by CZE were in good agreement with HPLC data.
QUANTITATIVE ESTIMATION OF VITAMIN C IN LOCAL FRUITS
Quantitative estimation of vitamin c in local fruits has been studied 10. Four locally available and non -seasonal fruits namely: Adansonia digitata L. (A). Ziziphus jujube L. (B). Dialium guineense W. (C). and the calyx Hibiscus sabdariffa L. (D) were evaluated for their vitamin C (ascorbic acid) content using an official methods (AOAC methods. 1980). The results obtained ranged form 40-50mg vitamin C in 100g powdered dried fruits; the results showed the vitamin C content sample C > B > D > A at 49.50, 44.60, 43.20 and 39.50mg/100g dried powdered samples respectively. Therefore, these fruits when consumed will contribute to the supply the daily requirement the vitamin by man.
MODIFIED METHOD FOR THE ESTIMATION OF ASCORBIC ACID FROM PRESERVED JUICES AND SQUASHES
Modified method for the estimation of ascorbic acid from preserved juices and squashes has been studied11. A displacement titration method for the estimation of vitamin C from juices and squashes has been developed. Acetone is used as complexing agent for bisulfate because formaldehyde is not suitable
as it reacts with N-bromosuccinimide and also combines with ascorbic acid. N Bromosuccinimide is used as the titrant, which pre ferentially oxidizes iodide to iodine which in turn reacts with ascorbic acid. End -point is reached when bluish colour persists for 30 seconds. The method is rapid and precise. The maximum relative standard deviation is 2.4% in case of orange squash .
ASCORBIC ACID DETERMINATION IN COMMERCIAL FRUIT JUICE SAMPLES BY CYCLIC VOLTAMMETRY
Ascorbic acid determination in commercial fruit juice samples by cyclic voltammetry has been studied12. Ascorbic acid (vitamin C) is a water-soluble vitamin which can be found in many biological systems and foodstuffs (fresh vegetables and fruits, namely, citrus). Ascorbic acid plays an important role in collagen biosynthesis, iron absorption, and immune response activation and is involved in wound healing and osteogenesis. It also acts as a powerful antioxidant which fights against free-radical induced diseases [1-5].
Nevertheless, an ascorbic acid excess can lead to gastric irritation, and the metabolic product of vitamin C (oxalic acid) can cause renal problems . In some cases, excessive quantities of ascorbic acid may result in the inhibition of natural processes occurring in food and can contribute to taste deterioration; added to apple pulp (250 mg/kg), vitamin C inhibits oxidation processes responsible for apple juice aroma . Ascorbic acid is a labile substance, as it easily degraded by enzymes and atmospheric oxygen. Its oxidation can be
accelerated by excessive heat, light, and heavy metal cations . That is why ascorbic acid content of foodstuffs and beverage s represents a relevant indicator of quality which has to be carefully monitored, regarding its variation during manufacturing and storage.
THE COMPARISON OF VITAMIN C CONTENT IN COMERCIALLY AVAILABLE SOURCES OF ORANGE JUICE USING HPLC
The comparison of vitamin c content in commercially available sources of orange juice using HPLC has been studied13.The present work utilized high performance liquid chromatography (HPLC) method for determining vitamin C content of commercially available orange juices by co mparing juices in carton, plastic jug, and frozen containers, as well as fresh orange. HPLC allows an efficient and quick way to relatively quantify vitamin C with a low cost of equipment and reagents. The results indicated higher daily percents of vitamin C than those provided on the container¶s label. The fresh sample yielded the largest amount of vitamin C per serving, followed by the frozen sample, the carton sample, and then the plastic jug sample. In conclusion, it was shown that HPLC method can be used to determine relative quantities of Vitamin C, but it is not an accurate method for determining the absolute quantities in samples containing other organic acids. In this study, the amount of ascorbic acid in orange juice is determined using HPLC witho ut separating ascorbic acid from the other organic acids present. The experiment
was able to rank containers with respect to vitamin C, the fresh sample have the largest concentration, followed by the frozen sample, the carton sample, and the plastic sample. The hypothesis was not correct. One cannot measure how much ascorbic acid oxidizes in different containers because HPLC measures both ascorbic and dehydroascorbic acids. For one to quantify only ascorbic acid content within a sample, voltammetry and titration are two good methods to use. Furthermore, voltammetry could be used because that method allows the determination of one chemical compound within a mixture. Therefore, it cannot be used to concruuently evaluate ascorbic and dehydroascorbic acids.
One way to determine the amount of vitamin C in food is to use a redox titration. The redox reaction is better than an acid-base titration since there are additional acids in a juice, but few of them interfere with the oxidation of ascorbic acid by iodine.
Iodine is relatively insoluble, but this can be improved by complexing the iodine with iodide to form triiodide:
I2 + I- <--> I3-
Triiodide oxidizes vitamin C to form dehydroascorbic acid: C6H8O6 + I3- + H2O --> C6H6O6 + 3I- + 2H+
As long as vitamin C is present in the solution, the triiodide is converted to the iodide ion very quickly. However, when the all the vitamin C is oxidized, iodine and triiodide will be present, which react with starch to form a blue -black complex. The blue-black color is the endpoint of the titration.
This titration procedure is appropriate for testing the amount of vitamin C in vitamin C tablets, juices, and fresh, frozen, or packaged fruits and vegetables.
The titration can be performed using just iodine solution and not iodate, but the iodate solution is more stable and gives a more accurate result.
1% Starch Indicator Solution
1. Add 0.50g soluble starch to 50 near -boiling distilled water.
2. Mix well and allow to cool before use. (doesn¶t have to be 1%; 0.5% is fine)
1. Dissolve 5.00g potassium iodide (KI) and 0.268g potassium iodate (KIO3) in 200 ml of distilled water. 2. Add 30 ml of 3 M sulfuric acid.
3. Pour this solution into a 500 ml graduted cylinder and dilute it to a final volume of 500 ml of distilled water.
4. Mix the solution.
5. Transfer the solution to a 600 ml beaker. Label the beaker as your iodine solution.
Vitamin C Standard Solution
1. Dissolve 0.250 g vitamin C ( ascorbic acid ) in 100 ml distilled water.
2. Dilute to 250 ml with distilled water in a volumetric flask. Label the flask as your vitamin C standard solution.
1. Add 25.00 ml of vitamin C standard solution to a 125 ml Erlenmeyer flask.
2. Add 10 drops of 1% starch solution.
3. Rinse your buret with a small volume of the iodine solution and then fill it. Record the initial volume.
4. Titrate the solution until the endpoint is reached. This will be when you see the first sign of blue color that persists after 20 seconds of swirling the solution.
5. Record the final volume of iodine solution. The volume that was required is the starting volume minus the final volume.
6. Repeat the titration at least twice more. The results should agree within 0.1 ml.
You titrate samples exactly the same as you did your standard. Record the initial and final volume of iodine solution required to produce the color change at the endpoint.
Titrating Juice Samples
1. Add 25.00 ml of juice sample to a 125 ml Erlenmeyer flask.
2. Titrate until the endpoint is reached. (Add iodine solution until you get a color that persists longer than 20 seconds.) 3. Repeat the titration until you have at least three measurement that agree to within 0.1 ml.
Titrating Real Lemon
Real Lemon in nice to use because the maker lists vitamin C, so you can compare your value with the packag ed value.
1. Add 10.00 ml of Real Lemon into a 125 ml Erlenmeyer flask.
2. Titrate until you have at least three measurements that agree within 0.1 ml of iodine solution.
y Fresh Fruit Juice ± Strain the juice through a coffee filter or cheese cloth
to remove pulp and seeds, since they could get stuck in the glassware.
y Fruits & Vegetables ± Blend a 100g sample with ~ 50 ml of distilled
water. Strain the mixture. Wash the filter with a few milliliters of distilled water. Add distilled water to make a final solution of 100 ml in a volumetric flask. Titrate these samples in the same way as the juice sample described above.
2. E. S. TEE, S. I YOUNG, S. K. HO* and S. SITI MIZURA Division of Human Nutrition, Institute for Medical Research, Kuala Lumpur, Malaysia. 3. William S. Smith, * Carlton Green, and Mungo Jerry Department of Chemistry, stevens Institute of Technology, Castly Point on Hudson, Hoboken, NJ 07030. Performed September 19, 2003; submitted September 27, 2003. Harris, D.C. Quantitative Chemical Analysis. 5th ed.: W. H. Freeman & Company, NewYork., 2000. Chapter 7. 4. Gweneth M. Chappell, M.Sc., And Audrey M. Hamilton, B.Sc., 5. HENRY TAUBER and ISRAEL S. KLEINER 6. Chemistry and Materials Science Fresenius¶ Journal of Analytical Chemistry Volume 277, Number 5, 369-371. 7. Microchemical Journal Volume 28, Issue 2, June 1983, Pages 174 -179. 8. Dinesh Guptaa, P.D. Sharmaa and Y.K. Guptaa aChemical Laboratories, University of Rajasthan, Jaipur, India. Received 13 December 1974; accepted 26 March 1975. Available online 20 December 2001. 9. aIstituto di Chimica degli Ormoni, CNR, Via Mario Bianco 9, Milano 20133 Italy
Faculty of Pharmacy and Department of Biomedical
Sciences and Technologies, University of Milan, Via Celoria 2, Milan
20133 Italy cIstituto di Chimica degli Ormoni, CNR, Via Mario Bianco 9, Milan 20133 Italy Received 25 February 1993; 10. Science World Journal, Vol 3, No2 (2008) Hassan A.S., Hassan H. S. 11. Microchimica Acta Volume 64, Number 6, 699 -704, DOI:
10.1007/BF01225632. M. Sarwa r, Zafar Iqbal and Shaheen Zaidi. 12. J Autom Methods Manag Chem. 2008; 2008: 937651. Published online 2009 March 29. Doi:10.1155/2008/937651. Aurelia Magdalena Pisoschi, 1 Andrei Florin Danet, 2* and Slawomir Kalinowski 3. 13. Boostani, M.F.; Hamidi, Z. E.; Sahari, M. A. Effect of low temperature on the ascorbic acid content and quality characteristics of frozen strawberry Food Chem., 2004, 86, 357-363.
Collins, C.H.; Silva, C. R.; Simoni, J. A.; Volpe, P. L. O. Ascorbic acid as a Standard for Iodometric Titrations J. Chem. Educ., 1999, 76, 14211422.
Gritsanapun, W.; Nilkamhank, S.; Paochom, A.; Suntornsuk, L. Quantitation of vitamin C content in herbal juice using direct titration J. Pharm. Biomed. Anal., 2002, 28, 849-855.
Kabasakalis, V; Moshatou, E.; Siopidou, D. Ascorbic acid content of commercial fruit juices and its rate of loss upon storage Food Chem., 2000, 70, 325-328.
Lagier, C.M.; Verdini, R.A. Voltammetric Iodometric Titration of Ascorbic Acid with Dead-stop End-Point Detection in Fresh Vegetables and Fruit Samples J. Agric. Food Chem., 2000, 48, 2812 -2817.
Paim, A.P.; Reis, B. F. An Automatic Spectrophotometric Titration Procedure for Ascorbic Acid Determination in Fruit Juices and Soft Drinks Based on Volumetric Fraction Variation Anal. Sci ., 2000, 16, 487491.
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