The mixture of alkaline copper tartarate and potassium ferrocyanide was found to be useful in the spectrophotometric determination of reducing sugar from papaya peel by hydrolyzing the polysaccharide by varying the concentration of acid, temperature and time of exposure. The absorbance formed at 670 nm was proportional to the concentration of copper; with reference to Cu-glucose equivalence table quantitative estimation of reducing sugar is arrived. The novelty in this method is utilization of Cu (II) oxidative state instead of reduced Cu (I) for analysis which makes it different from usual standard methods. This method is rapid, convenient with a minimal relative percentage error i.e. 0.8%.

Keywords: Spectrophotometric estimation, Reducing sugar, Cu (II), Potassium ferrocyanide, Minimum error.

INTRODUCTION

Carica papaya contains an enzyme known as papain, present in the fruit, stem and leaves (Oderinde et al., 2002). The milky juice is extracted, dried and used as chewing gum, medicine (digestion problems), and toothpaste and meat tenderizers (Lohiya et al., 2002). Meat can be tenderized by wrapping it in a bruised papaya leaf before it is cooked. Carica papaya contains many biologically active compounds. Two important compounds are chymopapain and papain, which are supposed to aid in digestion (Morton et al., 1987). Papain also is used to treat arthritis. The level of the compounds varies in the fruit, latex, leaves, and roots. Papaya has been used for digestive problems and intestinal worms. The softening and disintegrating qualities of papain (generally in alkaline combination, as with borax or potassium carbonate), have been taken advantage of in the treatment of warts, corns, sinuses, and chronic forms of scaly eczema, cutaneous tubercles, and other hardness of the skin, produced by irritation, etc. (Echeverri Torres., et al., 1997) and injected into indolent glandular tumors to promote their absorption.
Green fruits are used to treat high blood pressure and also used as an aphrodisiac. It is useful in round worm infestation, stomachalgia, dyspepsia, constipation, amenorrhoea, skin diseases and general debility (Titanji et al., 2008). In spite of all these advantages consumption of these fruits generates outer skin wastes that could bring about environmental pollution if not properly handled. Towards recycling of vegetative wastes avoiding littering and waste-related environmental degradation, this study was carried out to explore the sugar component of papaya peels with a view to establishing their raw material potentials.

MATERIALS AND METHODS

Reagents
All the used reagents were of analytical grade. These reagents include: Fehling A: 6.9280 g of Cupric sulphate in 100 ml of deionized water, Fehling B: diluted to 100 ml, 10% potassium ferrocyanide, Papaya peel, 1M sulphuric acid, barium hydroxide hepta-hydrate. All the solutions were stored at room temperature.
Bertrand’s method is based on the reducing action of sugar on the alkaline solution of tartarate complex with cupric ion; the cuprous oxide formed is dissolved in warm acid solution of ferric alum. The ferric alum is reduced to FeSO4 which is titrated against standardized KMnO4; Cu equivalence is correlated with the table to get the amount of glucose. While filtering the target, red cuprous oxide, there is every chance for it to get oxidized to cupric by aerial oxidation and secondly the end point is not stable as it immediately disappears because it is reversible. Due to these two logical points error is more feasible (Khanna et al., 1942).
In Lane-Eynon method (SausenSilmi et al., 1997) sugar solution is titrated by maintaining the temperature at 65-70 °C. Titration is continued till it acquires a very faint blue colour. At this stage three drops of methylene blue indicator is added. The dye is reduced to a colorless compound immediately and the end point is change of colour from blue to red (Davis, 1963) the result depends on the precise reaction time, temperature and concentration of reagent used. In this method it is susceptible for interference from other types of molecules that act as reducing agents.
Sample solution preparation
About 20 ml of Fehling A and 20 ml of Fehling B was taken in two 250 ml beakers each under identical conditions. The solution in the first beaker is transferred quantitatively into a 100 mL volumetric flask and diluted with deionized water followed by 5 times dilution (Stock solution). To the other beaker standard glucose solution (250 mg in 100 ml) was added and heated around 60- 65 °C over hot plate for about 10-15 minutes. Red cuprous oxide formed was cooled to room temperature, carefully filtered and the filtrate is collected for further use. The filtrate was transferred into a 100 ml volumetric flask quantitatively using deionized water followed by 5 times dilution as in the unknown solution.
Objectives
Benedict quantitative reagent over comes many drawbacks of the above methods. For instance end point is blue to white by using potassium thiocyanate which converts the red cuprous oxide to white crystals of cuprous thiocyanate, it helps in visual view but here also the condition plays the integral role which may lead to error. The conventional standard methods taken for our study is Bertrand’s method, Lane – Eynon method and Benedict quantitative method.
UV-Vis Spectroscopy
Aliquots, say, 2, 4, 6, 8, 10 ml etc. were pipette out from the stock and transferred into a 25 ml volumetric flask. To this 8 ml of 10% potassium ferrocyanide was added and made up to the mark using deionized water. Keeping 10% potassium ferrocyanide as the blank, the absorbance of the complex is measured spectrophotometrically at 670 nm (Table 1) and the λmax value is depicted (Fig. 1). The same procedure is repeated for the unknown by taking anyone of aliquot as in the case of stock (Table 2). The observed values are plotted in the graph by taking concentration in the X- axis and absorbance in the Y- axis which obeys Beer’s law the obtained absorbance value for unknown is interpolated to get the concentration.
Conversion of Papaya peel to Reducing Sugar
Papaya peel is composed of polysaccharides (starch and cellulose). It was powdered well using a blender, dried and about 1 g was weighed accurately hydrolyzed under the conditions as mentioned in i, ii & iii. It was neutralized using barium hydroxide, to give a precipitate of barium sulphate which was filtered off. The acidity is neutralized by monitoring pH, using pH paper. Hot air oven was used to set up the appropriate temperature to maintain uniform environment (Venkatesh et al., 2014). (i) By varying the concentration of sulphuric acid ranging 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, & 0.6 M keeping time 60 minutes and temperature 75 °C as constant (Chandraju et al., 2011). (ii) By varying the temperature 60 °C, 75 °C, 90 °C respectively keeping time 60 minutes and concentration 0.3 M as constant (Venkatesh et al., 2014). (iii) By varying the time 30, 60, 90 minutes respectively keeping temperature 75 °C and concentration 0.3M constant, the quantitative values are tabulated in Tables 4, 5 and 6 respectively (Chidankumar et al., 2013).

RESULTS

The result obtained from spectrum at absorbance of 670 nm alkaline copper tartarate ferrocyanide complex was shown in figure 1 and the concentration of copper and corresponding absorbance measurements was indicated in table 1. The extrapolated unknown concentration shown in the table 2, were compared to table 1 to get the unknown sample concentration. The table 3 shows the estimated glucose (230, 234, 238, and 242 mg) and percentage of error (6.5, 5.1, 3.2, and 0.8) in Benedict’s, Bertrand’s, Lane-Eynon and spectrophotometric methods respectively. And the figure 2 shows the comparative error percentage from the above methods. Comparative report by varying the concentration of acid, temperature and time (75 °C & 60 min) constant were shown in the figure 3 and table 4 respectively. By varying the temperature (t), C=0.3M, t=60 min constant shown in the figure 4 and table 5. In the same way by varying time (t), C=0.3M, T=75°C constant figure 5 and table 6, respectively.

Figure 1. Strong absorbance at 670 nm of alkaline copper tartarate ferrocyanide complex. Click here to view full image

Table1. Concentration of copper and corresponding absorbance measurements. Click here to view full image

Table 2. Extrapolated unknown concentration from Beer’s plot. Click here to view full image

Table 3. Estimated glucose amounts and percentage error of the currently taken methods.. Click here to view full image

Table 4. Varying concentration of acid (C), T = 75 °C, t = 60 minutes constant. Click here to view full image

Table 5. Varying temperature (T), C = 0.3 M, t = 60 minutes constant. Click here to view full image

Table 6. Varying Time (t), C = 0.3M, T = 75 °C constant. Click here to view full image

Figure 2. Comparative error percentages of various methods. Click here to view full image

Figure 3. Comparative report varying concentration of acid (C), T = 75°C, t = 60 minutes constant. Click here to view full image

Figure 4. Comparative report varying temperature (T), C = 0.3M, t = 60 minutes constant. Click here to view full image

Figure 5. Comparative report varying time (t), C = 0.3M, T = 75 °C constant. Click here to view full image

DISCUSSION

Copper (Cu2+) in aqueous solution exhibits a strong absorbance in the spectral range of 460–800 nm, which corresponds to the yellow-orange-red region of the Electro-Magnetic spectrum (Scott and Lukehart, 2007). The absorbance around 300–450 nm is almost negligible. Hence it appears blue in colour. Copper tartarate, deep blue complex appears at λmax 620 nm (Agrawal et al., 1976; ChidanKumar et al., 2012). The spectrum of complex read using Perkin Elmer UV/VIS Spectrometer. Copper forms an intense green colored complex with sodium potassium tartarate and potassium ferrocyanide which shows a strong absorbance at 670 nm. The absorbance from 400–800 nm is intense when compared to that of free Cu2+ and tartarate complexed ion in aqueous solution.
The color of the complex is attributed to the Metal-Ligand charge transfer or vice-versa. This may be due that copper is complexed with tartarate and cyano ligands.

CONCLUSION

In the present study, a novel spectrophotometric determination of reducing sugar using potassium ferrocyanide was successively developed. These results obtained are well compared with the established quantitative methods. This study revealed the error factor of standard methods and suggested a remedy to minimized it by following this rapid, ease and time consuming procedure. Since promising results are arising for the structural elucidation of the synthesized complex is under progress and application of this technique to various sugar residues are also undergoing.

ACKNOWLEDGEMENTS

The authors thank the financial support from University Grants Commission (UGC) Delhi, and Department of Sugar Technology, Sir MV Post Graduate Centre, University of Mysore, Mandya (Where the research work carried out).

CONFLICT OF INTEREST
None declared.

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