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J. Chem. Soc. Pak., Vol. 35, No.3, 2013 J.Chem.Soc.Pak.,Vol. 35, No.3, 2013 783
Development of Iodimetric Redox Method for Routine Estimation of Ascorbic Acid from Fresh Fruit and Vegetables 1
MUHAMMAD MUNIR, 1AHMAD KHAN BALOCH*, 2WAQAR AHMAD KHAN 3 FARZANA AHMAD AND 4MUHAMMAD JAMIL 1 Department of Food Science and Technology, Gomal University, Pakistan. 2 Department of Food, Agriculture and Environmental Chemistry, University of Glasgow G12 8QQ, UK. 3 Department of Chemistry, Konkuk University, Seoul 143-701, Korea. 4 Division of International Studies, Konkuk University, Seoul 143-701, Korea.
[email protected]* (Received on 8th November 2012, accepted in revised form 13th February 2013) Summary: The iodimetric method (Im) is developed for rapid estimation of ascorbic acid from fresh fruit and vegetables. The efficiency of Im was compared with standard dye method (Dm) utilizing a variety of model solutions and aqueous extracts from fresh fruit and vegetables of different colors. The Im presented consistently accurate and precise results from colorless to colored model solutions and from fruit/vegetable extracts with standard deviation (Stdev) in the range of ±0.013 - ±0.405 and ±0.019 - ±0.428 respectively with no significant difference between the replicates. The Dm worked also satisfactorily for colorless model solutions and extracts (Stdev range ±0.235 - ±0.309) while producing unsatisfactory results (±0.464 - ±3.281) for colored counterparts. Severe discrepancies/ overestimates continued to pileup (52% to 197%) estimating the nutrient from high (3.0 mg/10mL) to low (0.5 mg/10mL) concentration levels, respectively. On the basis of precision and reliability, the Im technique is suggested for adoption in general laboratories for routine estimation of ascorbic acid from fruit and vegetables possessing any shade.
Key Words: Ascorbic acid, iodimetric estimation, redox method, dyes method, colored extracts. Introduction Vitamin C is chemically L-ascorbic acid that participates in a wide variety of biological processes necessary to continue normal growth and maintenance of good health, and to provide resistance against infections to human body [1, 2]. Stimulation of certain enzymes, collagen biosynthesis, hormonal activation, antioxidant, detoxification of histamine, phagocytic capacity against leukocytes, and proline hydroxylation are some important mechanisms responsive to this nutrient. Vitamin C has been also associated with reduction in cancer incidence, blood pressure, immunity and tissue regeneration, and in the enhancement of iron absorption from foods. Its severe deficiency typically results in scurvy [2]. Vitamin C like many other vitamins cannot be synthesized in the human body, and hence must be provided in diets. Relatively small amounts of natural vitamin C are found in animal products including kidney, liver and milk; however fruit and vegetables are the main contributors of this vitamin in human diet [3, 4]. Measurement of the concentration of this nutrient commonly signifies food quality and product stability. Due to immense importance, numerous methods had been developed for accurate estimation of this vitamin [4 - 8] but most of them lack in application quality for routine use. The conventional AOAC titrimetric method [9] commonly employed as routine technique apparently causes problem on *
To whom all correspondence should be addressed.
evaluating from colored solutions with particular reference to nutrient in small quantities [10]. Owing to these and other limitations the quantification of this nutrient is under continuous investigation by analysts all over the world. Direct spectrophotometric methods [4, 11-13] have not received widespread application for routine analysis in foods as the technique requires sufficiently pure solution and other measures for highly colored solutions. The colorimetric, photoelectric and fluorometric methods employed by several workers [10, 14-17] have also been regarded to have limited applications in food analysis. High performance liquid chromatography (HPLC) has also been applied [15, 18-21], however presence of interfering substances necessitated sample purification. For this and other reasons, the technique is not recommended for general use as a routine method. Similarly the conventional spectroscopic, potentiometric and enzymatic methods [21-23] though possess their own worth yet not suitable for rapid estimation. The present work is aimed to develop a simple, pragmatic and accurate technique for routine use employing iodine as a redox reagent. The iodine oxidizes ascorbic acid quantitatively to dehydroascorbic acid and itself reduced to iodide ions and the excess of the reagent in trace amount complexes with starch forming a prominent blue color. The potential of the proposed
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mechanism for ascorbic acid estimation is evaluated employing model systems as well as extracts from raw/fresh fruit and vegetables. The interfering impact of various hues by certain fruit and vegetables is also examined, and the performance compared with the standard dye (2,6-dichlorophenolindophenol) reduction method [9].
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from iodine and dye methods for the colorless solution with respective confident interval of 2.982 3.014mg and 2.699 - 3.301mg which overlapped each other (Fig. 1). Both of the techniques performed equally well when applied to solutions that have no color, and are clean and transparent to view through easily color changes close to the endpoint.
Results and discussion 12
A variety of model studies is designed to compare performance and efficiency of iodine developed technique with that of a standard dye method for ascorbic acid estimation. Model studies were selected for its convenience in taking known and variable amounts of ascorbic acid for estimation, and its ease to vary the contents and color intensity in order to evaluate their effect more precisely with more certainty. In this study, ascorbic acid is measured from 0.5-5.0 mg range with the consideration that such amount of ascorbic acid is normally present in a titrate for the analysis from the natural ascorbic acid sources, and similarly colorless to colored solutions of various hues prepared from known synthetic dyes were employed in order to match fruit/vegetable shades. Performance of each method is examined after estimating and comparing ascorbic acid recovery percentage, mean replicate values, standard deviation, standard error (SE), coefficient of variation (CV) and other appropriate estimates. The findings are also verified by plotting 95% confident intervals from interval plots within the perspective of interference from color intensity on ascorbic acid determinations from colored solutions. The results are tabulated or plotted in graphic forms, accordingly. The study was further extended to ascorbic acid estimation from fresh fruit and vegetables having diverse color shades. Colorless solutions The estimation of ascorbic acid from colorless solution was aimed to define workability of the methodology, and to establish baseline for the developed iodine technique. Results are given in Table-1. The amount of ascorbic acid as determined by iodine method recorded a mean value of 2.998 ±0.013 (SE 0.006) which is very close to the actual amount of 3.0 mg given 99.93% ascorbic acid recovery. The dye method had also given 100% recovery, however there was a wide dispersion between the determinants (Stdev 0.242, SE 0.11, CV 8.1%). Statistically there appeared no significance difference (p = 0.986) between the two estimates
Ascorbic acid recovery (mg)
Model System Studies
10 8 6 4 2 Im Dm l Im l Dm d Im Dm n Im Dm t Im t Dm e d e n ss e e e ss e e e e m ol e e R m e ol r a L R a L Vi G C ar Vi Gr C ar C C
*
P Values between iodine and dye method estimates are 0.896, 0.026,
0.016, 0.012 and 0.025 for colorless (CLess), caramel, red, green and violet colored, respectively.
Fig. 1: Interval plot of ascorbic acid estimates from colorless, caramel, red and violet colored model solutions using dye and iodine methods. Colored solutions The ascorbic acid recovery values obtained from the caramel colored solutions are given in Table-1. Using Im, a recovery very close to actual value (101.07% ±0.047) was recorded, whereas a significantly over estimate of 27.3% was observed by Dm and according to interval plots (3.192 - 4.448, Fig. 1) more than 13% variation appeared amongst the replicates. The Im emerged to work significantly much better (p≤0.026) as compared to Dm evaluating ascorbic acid from caramel colored media. Estimation efficiency of the two techniques is also compared using red, green and violet colors that have hue resemblance with color of the titrating dye or to that resulted at the endpoint. The Dm had given significantly higher overestimates, which went on amplifying from 49.3, 73.3 and to 178% on change in color of ascorbic acid solutions from red, green and to violet respectively. The 95% confidence intervals between replicates also increased
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significantly for red (3.529 - 5.431 mg, Stdev ±0.766, p≤0.016), green (4.049 - 6.351 mg, Stdev ±0.927, p≤0.012) and violet (4.476 - 12.204 mg, Stdev 3.111, p≤0.025) colored solutions presenting 17.0, 17.8 and 37.3% variances, respectively (Fig. 1). Whereas the levels of interval for Im were relatively very narrow within the acceptable ranges of 2.914 - 3.246 mg Stdev ±0.134 for red, 2.874 - 3.690 mg Stdev ±0.329 for green, and 2.945 - 3.950 mg Stdev ±0.405 for violet color, and as such the Im technique offered consistent results with marked performance in each color. The increased variations amongst the replicates and enlarged level of uncertainty in the estimates posed serious objections to the application of Dm to colored solutions downgrading credibility of the technique. Instead, the Im furnished accurate and precise results from colorless to colored model solutions of different hue establishing its excellent performance efficiency convincing to trust in the technique. In order to examine the interfering impact of color intensity on ascorbic acid estimation, a solution with caramel color was selected for the reason the Dm method gives relatively better performance from this color medium. A model was set up to find out as to whether evaluation of ascorbic acid is hampered by caramel color with variable intensity. For the purpose a gradually increased amount of caramel solution was added to a solution containing variable amount of ascorbic acid (0.5 - 5.0 mg) so as to give 0.01 to 0.10 optical densities, and ascorbic acid determined by the two methods (Table-2). The Im produced almost 100% recovery from all levels of color intensity recording least variation in ascorbic acid recoveries (Stdev range ±0.024 - ±0.035). However, in case of Dm the results were inconsistent with wide variations (Stdev range ±0.254 - ±1.042) given excess estimates, and beyond 0.047 OD overestimated amounts were significantly high (23.7 – 46.2% with p≤0.038 - 0.008). On plotting ratios of ascorbic acid recovery against optical color density Dm deliberated as high as 94% linear relationship (Fig. 2) showing a definitely high effect of color intensity on ascorbic acid measurements, and hence color intensity is regarded a significantly great interfering factor affecting ascorbic acid estimates on using Dm technique, whereas no such relationship appeared in case of Im. We agree with the findings of many other workers reporting severe discrepancies in ascorbic acid estimation from colored solutions/extracts using 2, 6dichlorophenolindophenol dye method, and endorse their views to give overestimates in ascorbic acid contents due to color interference from food and feed,
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and from pharmaceutical and natural products [8, 12, 21 - 23].
Fig. 2: Effect of color intensity on vitamin C estimation from variable amount of ascorbic acid. To compare performance of the two methods to low ascorbic acid concentrations in the presence of colorant, the solutions of caramel color with optical density varying from 0.01 to 0.10 OD were fortified with 0.5 or 3.0 mg ascorbic acid, and quantified by the two methods (Table-3). The Im gave consistent recovery from 98.03 to 100.8% with little variation between the replicates (Stdev ±0.027 ±0.035), whereas the recoveries from Dm varied from 100.2 to 297.4% with wide variation between the replicates (Stdev ±0.249 - ±0.923). Further, the overestimates given by Dm from 0.5 mg compared to samples with 3 mg ascorbic acid were exceptionally high and fluctuated extensively. To compare their impact further the estimate ratios were determined dividing estimated values of ascorbic acid by the actual amount of ascorbic acid for each ascorbic acid concentration and optical density. On plotting such ratios of ascorbic acid estimate versus optical density of the solution a significantly high relationship appeared by either concentration as a result of estimation by Dm (Fig. 3), and the slope of the line for 0.5 mg sample was very high compared to that for 3.0 mg ascorbic acid concentration, and as such recorded almost 4 times higher overestimates. Whereas a negative value for R2 was given out from each sample subjected to estimation by Im. Therefore Dm demonstrated to give highly unsatisfactory results for colored solutions in general and with low concentrations in particular. The endpoint color in the dye method is masked with the color of solutions under study leading to erroneous results, and the interfering effect becomes much higher at lower
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ascorbic acid values. The developed iodimetric titration technique instead renders the ascorbic acid estimation more accurate and reproducible even from colored samples containing wide range (0.5 - 3.0 mg) of ascorbic acid. The basis of adoption of the iodine redox technique lies in the fact that excess/available iodine in trace amount from complete ascorbic acid oxidation spontaneously reacts with starch indicator
causing a dramatically sharp endpoint color change from colorless to a dense purple color. This endpoint color change is highly distinctive producing pragmatic contrast from the caramel, red or green colors to view endpoint vividly. The solutions with blue/ purple hue had insignificant interference as iodine-starch interaction produces dominantly rich color overshadowing the initial solution color.
Table-1: Recovery from colorless, and caramel, red, green and violet colored solutions containing 3.0mg ascorbic acid as determined by iodimetric and dye titration techniques. Replicate
Colorless
Im 1 2.980mg 2 3.000 3 3.010 4 2.990 5 3.010 Mean 2.998 Stdev 0.013 S.E 0.006 CV % 0.4 P Value* 0.986 * Significant at 95% level of confidence.
Dm 2.750 3.300 2.950 2.800 3.200 3.000 0.242 0.108 8.1
Caramel colored Red colored Green colored Estimated amount of ascorbic acid (mg) Im Dm Im Dm Im Dm 3.030 3.600 3.100 3.700 3.450 5.800 3.100 4.050 3.020 5.700 3.050 5.050 3.040 4.600 2.950 4.600 3.780 4.500 3.020 3.400 3.300 4.000 3.130 6.450 2.970 3.450 3.030 4.400 3.000 4.200 3.032 3.820 3.080 4.480 3.282 5.200 0.047 0.506 0.134 0.766 0.329 0.927 0.021 0.226 0.056 0.343 0.147 0.415 1.6 13.3 4.4 17.8 10.3 17.8 0.026 0.016 0.012
Violet colored Im 3.200 3.020 3.700 3.300 4.020 3.448 0.405 0.181 11.8
Dm 5.500 8.700 13.50 7.400 6.600 8.340 3.110 1.391 37.2 0.025
Fig. 3: Effect of optical density on vitamin C estimation from 0.5 mg or 3.0 mg of ascorbic acid solutions.
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Table-2: Ascorbic acid recovery from solutions having variable ascorbic acid contents and color intensity. Optical Density (540nm)
Solution composition Ascorbic acid (mL of 0.1%)
Caramel solution (mL)
Ascorbic acid estimated (mg) (Im)
MPA 4% (mL)
0.5
0.5
9.0
0.010
1.0
1.0
8.0
0.015
1.5
1.5
7.0
0.021
2.0
2.0
6.0
0.028
2.5
2.5
5.0
0.037
3.0
3.0
4.0
0.047
3.5
3.5
3.0
0.059
4.0
4.0
2.0
0.070
4.5
4.5
1.0
0.085
5.0
5.0
0.0
0.100
0.46 0.49 0.53 0.52 0.53 0.506± 0.031 0.99 1.01 0.97 1.06 1.03 1.012±0.035 1.46 1.49 1.53 1.51 1.47 1.492±0.029 2.02 1.97 1.99 2.03 2.05 2.012±0.032 2.48 2.47 2.51 2.53 2.46 2.490±0.029 2.99 2.97 2.96 3.01 3.02 2.990±0.026 3.48 3.49 3.51 3.54 3.53 3.510±0.026 3.96 3.98 4.01 4.03 4.04 4.004±0.034 4.49 4.46 4.51 4.52 4.48 4.492±0.024 5.09 5.01 5.05 5.03 5.05 5.046±0.030
(Dm)
0.33 0.72 0.26 0.86 0.53 0.540± 0.254 1.36 1.47 0.70 1.07 0.85 1.090±0.327 1.45 1.91 2.00 1.10 1.85 1.662±0.378 1.71 2.76 2.35 2.40 2.70 2.384±0.417 2.91 3.61 2.85 2.48 3.40 3.050±0.453 3.8 4.44 2.96 3.70 3.65 3.710±0.526 4.40 3.85 4.72 4.6 5.3 4.574±0.525 6.1 5.00 5.9 5.5 4.85 5.470±0.545 5.8 5.50 7.7 6.6 5.3 6.180 ±0.983 6.7 6.96 7.75 8.9 6.25 7.310±1.042
P value*
0.781
0.624
0.373
0.118
0.051
0.038
0.011
0.004
0.019
0.008
*
Significant at 95% confident level.
Fresh Fruit and Vegetables A total of 51 fruit and vegetables comprising from light to dense color of various shades were included for this study. Vitamin C content of 29 types of fresh fruit is tabulated in Table-4, whilst those for
the 22 fresh vegetables are given in Table-5. There appeared a definite difference between vitamin C estimates as determined by the two techniques. The ascorbic acid values of the fruit and vegetables as obtained by Dm were found much higher and highly scattered (Stdev range ±0.235 - ±3.281) than those
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determined by Im (Stdev range ±0.019 - ± 0.428). The comparative performance of Im and Dm techniques for determining ascorbic acid from fresh fruit/vegetables is discussed below. Light Colored Fruit and Vegetables Despite the fact that the mean recovery values obtained by the dye-titration method is slightly higher compared to that from the Im, both of the methods demonstrated satisfactory performance on ascorbic acid evaluation from colorless to light colored fruit and vegetables (Tables 4 and 5). The reproducibility studies showed that there is no significant difference (p < 0.05) between respective determinants by the two methods. However, the results given out by iodimetric developed technique appeared more precise Stdev range (±0.019 - ±0.032) as compared to dye method given scattered effects (Stdev range ± 0.235 - ±0.309). Dark Colored Fruit and Vegetables The ascorbic acid estimates from fresh fruit and vegetables as well as the extent of replicate variability in terms of standard deviation are given in Tables 4 and 5. The ascorbic acid contents determined by Im are close to the literature values [3] and are invariably lower than to those measured by Dm. The difference in ascorbic acid contents determined by Im and Dm may have three major ranges of 10 – 29 %, 30 – 49% and ≥50%. Fruit and vegetables with 10-29% difference include oranges, dates, mango, peach, papaya, sweet potato, cucumber, cauliflower and green chilies. The replicate variability for these commodities as determined by Im is much lower in size (±0.046 - ± 0.072) as compared to that from Dm (±0.464 – ±0.564), and hence the determinations are more precise. Those fruit having estimation difference ranging from 30 to 49% are watermelon, mandarin
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‘kino’, mandarin ‘narangi’, strawberry and pomegranate. The vegetables of this range are bitter gourd, mung bean, pepper red, mint leaves, cabbage, spinach and pumpkin. The standard deviation between replicates range for Dm (±0.748 – ±1.422) compared to Im (±0.102 – ±0.242), appeared quite wide and thus casts doubts on the validity of the results from Dm. Several other highly colored fruit/vegetables caused much higher estimate differences (≥50%) resulting into exceedingly broader standard deviation ranges (Im ±0.299 – ±0.428; Dm ±2.305 – ±3.281). The fruit/vegetables under this class are ‘jaaman’, plum, cherry, mulberry, raspberry, persimmon, ‘faalsa’, mustered leaves and lettuce. From application to such highly colored blue/black fruit/vegetables, it becomes evident that the Dm is simply flap, and is least reliable on account of giving exceedingly high overestimates. Such results are likely to occur due to color interference near to end point titration overshadowing completely the resulting color between the dye and ascorbic acid. Similar impression emerged from the recovery study conducted previously on model colored solutions wherein the Dm technique appeared less precise and inaccurate to give overestimates to the extent of 197.4%. There are also reports raising serious objections about conventional dye method to colored extracts containing vitamin C in small amounts [13, 22, 24 - 25]. The Im technique has also given slightly wider variation, nevertheless of insignificant level. The technique worked flawlessly on account of distinct end point detection from exceedingly rich purple color produced by the residual iodine with starch. Owing to simplicity in manipulation, accuracy and reliability it becomes highly convincing to apply the technique for the measurement of ascorbic acid from colorless to colored extracts from fresh fruit/vegetables and other similar products. The iodimetric technique is considered most suitable for routine analysis and is suggested for adoption by diagnostic institutes in general and those undergoing to establish new analytical setups in particular.
Table-3: Vitamin ‘C’ recovery* from solutions containing 0.5 mg or 3.0 mg of ascorbic acid at variable color intensity. Color density (540nm) 0.5 mg (Im) (Dm) 0.010 0.499±0.03 0.501±0.249 0.015 0.502±0.029 0.503±0.335 0.021 0.498±0.027 0.517±0.395 0.028 0.497±0.03 0.524±0.426 0.037 0.499±0.033 0.961±0.471 0.047 0.497±0.035 1.064±0.581 0.059 0.503±0.031 1.231±0.751 0.070 0.501±0.029 1.322±0.811 0.085 0.501±0.034 1.351±0.850 0.100 0.499±0.028 1.487±0.923 *Mean of five replicates; +Significant/Non-significant at 95% confident level
Ascorbic acid recovery (mg) S/NS+ NS NS NS NS S S S S S S
(Im) 2.991±0.029 3.011±0.031 2.973±0.035 3.001±0.027 3.012±0.029 3.023±0.03 2.941±0.033 3.001±0.028 2.981±0.032 3.022±0.029
3.0 mg (Dm) 3.012±0.306 3.022±0.315 3.153±0.395 3.211±0.408 3.392±0.426 3.645±0.529 3.822±0.540 3.896±0.567 4.169±0.606 4.555±0.628
S/NS+ NS NS NS NS NS S S S S S
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Table-4: Ascorbic acid contents*of the selective fruits as estimated by iodimetric (Im) and dye (Dm) methods. Fruit Common name (Botanical name)
(Im)
Orange (Citrus sinensis) Water melon (Citrullus vulgaris) Guava (Psidium guajava) Lemon (Citrus limon) Dates (Phoenix dactylifera) Mango (Mangifera indica) Apple (Pyrus malus) Muskmelon (Cucumis melo) Peach (Prunus persica) Banana (Musa paradisiacal) Loqat (Eriobotrya japonica) Jaman (Syzygium cuminii) Grapes (Vitis vinifera) Grapefruit (Citrus grandis) Papaya (Carica papaya) Plum (Pronus domestica) Lichi (Litchi chinensis) Mandarin ‘Kino’ (Citrus reticulate) Mandarin ‘Narangi’ (Citrus grandis) Cherry (Prunus cerasus) Apricot (Prunus armeniaca) Strawberry (Fragaria vesca) Mulberry Black (Morus niger) Raspberry (Pubus idacus) Persimmon (Diospyros kaki) Lime (Citrus aurantifolia) Falsa (Grewia asiatica) Pomegranate (Punica granatum)
59.98±0.065 9.98±0.134 179.99±0.030 54.97±0.029 12.0±0.068 46.0±0.058 14.93±0.026 29.99±0.032 18.01±0.151 9.8±0.022 6.25±0.026 18.36±0.428 6.44±0.027 39.99±0.024 44.5±0.046 10.05±0.214 44.97±0.024 20.01±0.106 29.51±0.102 10.01±0.406 11.18±0.024 52.34±0.156 30.6±0.421 33.01±0.137 30.04±0.177 44.99±0.026 22.14±0.401 12.2±0.148
Ber (Zizyphus Jujuba)
75.91±0.027
Ascorbic acid contents (mg/100g) (Dm)
Difference (Dm)–(Im)
S/NS+
67.7±0.462 13.86±0.757 180.03±0.247 55.0±0.262 13.48±0.504 52.39±0.524 15.2±0.267 30.1±0.244 24.94±0.764 10.02±0.251 6.5±0.301 30.3±3.182 6.67±0.303 40.04±0.264 50.8±0.461 13.68±0.807 45.45±0.255 25.31±0.672 35.86±0.632 14.64±3.146 11.43±0.272 71.68±0.799 43.76±3.172 45.63±0.778 42.02±0.804 45.08±0.255 31.62±2.744 16.36±0.856
7.72 3.88 0.04 0.03 1.48 6.39 0.27 0.11 6.93 0.22 0.25 11.94 0.23 0.05 6.3 3.63 0.48 5.3 6.35 4.63 0.25 19.34 13.16 12.62 11.98 0.89 9.48 4.16
S S NS NS S S NS NS S NS NS S NS NS S S NS S S S NS S S S S NS S S
76.07±0.247
0.16
NS
*Mean of five replicates, +Significant/Non significant at 95% confident level
Table-5: Ascorbic acid contents*of the selective vegetables as estimated by iodimetric (Im) and dye (Dm) methods. Vegetables Common name (Botanical name)
Potato (Solanum tuberosum) Tomato (Lycopersicum esculentum) Mustard leaves (Brassica juncea) Peas (Pisum sativum) Bitter gourd (Momordica charantia) Radish (Raphanus sativus) Mung bean (Vigna radiate) Okra (Hibiscus esculentus) Pepper red (Capsicum annum) Sweet potato (Ipomoea batatas) Cucumber (Cucumis sativus) Cauliflower (Brassica oleracea) Lettuce (Letuca sativa) Turnip (Brassica napus) Onion (Allium fistulosum) Green chilli (Capsicum annum) Mint leaves (Mentha cordifolia) Cabbage (Brassica oleracea) Garlic (Allium sativum) Carrot (Daucas carota) Spinach (Spinacia obracea) Pumpkin (Cucurbita moschata) *Mean of five replicates, +Significant/Non significant at 95% confident level
(Im)
19.99±0.024 35.45±0.024 33.08±0.271 56.9±0.022 119.9±0.296 30.99±0.026 14.93±0.116 15.99±0.030 189.98±0.132 22.99±0.057 24.12±0.054 44.99±0.069 13.89±0.281 16.1±0.026 7.99±0.019 149.97±0.072 15.03±0.374 50.02±0.242 12.39±0.027 9.99±0.024 45.34±0.268 14.98±0.027
Experimental Sample Selection Fresh fruit and vegetables were purchased from the local market and the wholesome ones were
Ascorbic acid contents (mg/100g) (Dm)
20.0±0.278 35.57±0.298 45.08±1.332 57.1±0.248 148.76±1.447 31.1±0.244 17.71±0.494 16.79±0.257 243.06±0.832 26.31±0.507 28.70±0.581 50.92±0.526 18.74±1.305 16.24±0.235 8.11±0.244 169.45±0.609 20.31±1.317 68.86±1.321 12.64±0.298 10.27±0.309 62.68±1.422 17.73±0.482
Difference (Dm)–(Im)
S/NS+
0.01 0.12 12.00 0.20 28.90 0.11 2.78 0.80 53.08 3.32 4.58 5.93 4.85 0.14 0.12 19.48 5.28 18.84 0.25 0.28 17.34 2.75
NS NS S NS S NS S NS S S S S S NS NS S S S NS NS S S
selected for the study. The sorted fruit and vegetables were rinsed in tap water to remove the adhering contaminants. The samples were prepared by cutting the material into small pieces and mixing thoroughly for subsequent ascorbic acid extraction.
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Fresh fruit/vegetable samples (100 g) were immediately crushed in freshly prepared 4% aqueous m-phosphoric acid (MPA) solution (150 mL) using electric grinder and filtered through cotton plug. After repeated washing of the cake total volume of 200 mL was made in a volumetric flask using 4% MPA solution. Ascorbic acid was estimated on the same day by means of iodine and dye methods.
help of microburette in a titration flask was added 1 mL of freshly prepared soluble starch solution (1%). The contents mixed and titrated with iodine solution (0.001 M) with the help of microburette. Titration continued till appearance of bluish color persisting for 10-15 seconds. Titration replicated 5 times and the amount of ascorbic acid equivalent to iodine solution calculated from mean volume of iodine solution used. Iodine solution was standardized daily twice whenever used.
Chemicals and Apparatus
Model Solutions
The chemicals and the dyes used were of analytical-reagent grade, and distilled water was boiled and cooled before use. Color intensity was measured with the help of U.V double beam spectrophotometer (U-2000, Hitachi).
Colorless as well as colored solutions of caramel, green, red and violet hue were employed for ascorbic acid recovery studies. These dyes were selected to give resemblance with the widely occurring natural colors in fruit and vegetables. The solution of caramel color was prepared on heating 50 gm sucrose crystals dissolved in 100 mL distilled water containing 5 mL of 1.0 N NaOH solution. The contents were kept in an oven working at 40ºC for five days till the development of dense brown color. The brown material was dissolved to make 100 mL with aqueous 4% MPA solution. The solutions representing green, red and violet color were made dissolving 0.1g of malachite green, methyl red and crystal violet dyes respectively in 20 mL of methylated spirit and made to 100 mL with the same reagent.
Sample Extraction
Preparation of Standard Solutions Ascorbic acid A 0.1% ascorbic acid solution (1.0 mg/mL) was prepared dissolving 0.2500 g of pure ascorbic acid in 250 mL of 4.0 % MPA cold solution. The solution was kept in cool place until used within a week period. Indophenol dye solution Weighed 0.05 g sodium 2,6dichlorophenolindophenol dye and dissolved in 100 mL of water. The solution after being filtered through cotton plug was stored in a refrigerator until used within a week period. Ten mL of the standard solution of ascorbic acid (1mg/mL) was taken into a titration flask, and using microburette it was titrated with indophenol dye solution until development of faint pink color persisting for 10-15 seconds. Five replicates were conducted at a time and the mean value was expressed as the concentration (mg) ascorbic acid equivalent to 1 mL of the dye solution. The dye solution was standardized twice daily before use. Iodine solution Dissolved 3.175 g pure resublimed iodine in 100 mL of 2% aqueous potassium iodide solution and volume made with distilled water to 250 mL for 0.1 M solution. The solution was kept in dark at cool place until required within 2 months. A working solution (0.001 M) was prepared by diluting 10 mL of the stock solution to a liter volume. To ten mL of pure ascorbic acid solution (1mg/mL) taken with the
Analytical Procedure for Iodine and Dye Methods The indophenol dye reduction method of AOAC [9] was followed for ascorbic acid estimation. Sample extract (10-15mL) from fresh fruit/vegetables or appropriate aliquot of ascorbic acid solution of known concentration prepared for model studies was titrated against standardized indophenol dye solution from microburette using magnetic stirrer until a faint pink color persisted for 15 seconds. In another experimental set appropriate volume of sample extracts or sample containing known amount of ascorbic acid was taken in titration flask, and 1 mL of 1% freshly prepared starch solution was added to it. The contents mixed and rapidly titrated under constant stirring against standardized iodine solution (0.001 M) from micro burette until the solution changed color to bluish tint persisting for 10-15 seconds. Ascorbic Acid Recovery Studies For recovery measurement from colorless solution a 3 mL of ascorbic acid solution (1.0 mg/
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mL) was added to 7 mL of 4% aqueous MPA taken in a titration flask. For recovery from colored solutions same amount of ascorbic acid was added to7 mL of 4% aqueous MPA in addition to two drops of respective dye solution prepared from malachite green, methyl red or crystal violet as a coloring material. Similarly to 3 mL of ascorbic acid (1.0 mg/ mL) solution a 3 mL of caramel and 4mL of MPA solution were added. Ascorbic acid determined using iodine and dye methods (Table-1). In order to evaluate the impact of color intensity on ascorbic acid estimation a variable volume of caramel solution (0.5-5.0 mL) was added to an increasing amount of ascorbic acid (0.5-5.0 mg). Normally the fruit /vegetable extracts required for singe estimation of the nutrient vary in the range of 0.5 –5.0 mg ascorbic acid, therefore such amount of ascorbic acid is taken to conduct model studies and ascorbic acid measured by the two methods. Optical density of the prepared mixture was also measured (Table-2).
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and decrease in ascorbic acid levels. Alternately, the developed iodimetric method offered accurate and precise results for low to high color intensity and hue of fresh fruit and vegetables with ascorbic acid in wide range. The validity of both the techniques is authenticated by conducting recovery studies on model solutions and fresh fruit/vegetables. The choice of Im technique is rooted in the production of highly distinctive and rich color resulting from interaction of residual traces of iodine with soluble starch rendering the technique versatile and innovative. References 1. 2. 3.
In other experiments a constant quantity of ascorbic acid (0.5 or 3.0 mg) was added to a solution having a variable density of caramel color, and ascorbic acid determined by iodine and dye methods (Table-3). For the measurement of optical density the sample was taken in a thoroughly cleaned and washed cuvette and absorption measured at 540 nm against 4% MPA as a blank solution using U.V double beam spectrophotometer. Statistical Analysis The performance of the two methods was compared taking mean values, standard deviations (Stdev), standard error (SE) and coefficient of variation (CV) on five times replicated determinants. Comparison is also made by taking interval plots and 2-sample T test at 95% level of significance using Minitab 16 package. Figures were plotted using MSExcel Office, 2007. Linear equations of trend lines and R2 were recorded to make the study more conclusive.
4. 5.
6. 7.
8. 9.
Conclusion
10.
The conventional dye method is good enough for the ascorbic acid analysis from colorless solutions/extracts. However, it did not give promising results for colored solutions whether taken from model studies or actually extracted from fresh fruit and vegetables of any shade. The technique extended approximates with increased replicate variations with error levels mounting with rise in intensity of color
11. 12. 13.
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