Nowadays, wellness has become the most of import belongings of homo ‘s life. Normally, diets with high contents of fruits are protective against several human diseases such as cardiovascular diseases and even malignant neoplastic disease. Therefore, people are seting more and more attending on antioxidant substances such as vitamin C which is besides known as ascorbic acid or more specifically L-ascorbic acid. Vitamin C is likely one of the most extremely good known. Furthermore, people have become more cognizant to the importance of vitamin C. Hence, this causes the planetary market flooded with vitamin C fortified nutrients ( Arya, Mahajan and Jain, 2000 ) .
The term of vitamin C is used as generic term for all compounds exhibiting qualitatively the biological activity of ascorbic acid. The molecular construction of vitamin C is C6H8O6 and the molecular weight is 176.1 ( Ball, 2006 ) . Vitamin C is extremely polar and readily soluble in aqueous solution and indissoluble in less nonionic dissolvers ( Fennema, 1996 ) . It is an acidic compound due to the facile ionisation of hydroxyl group on C 3 ( pK1 = 4.17 ) while the hydroxyl group on C 2 is much more immune to ionisation ( pK2 = 11.79 ) . The construction of L-ascorbic acid is shown in Figure 1 ( Ball, 2006 ) .
Ball ( 2006 ) besides stated that ascorbic acid is easy and reversibly oxidized to dehydroascorbic acid, organizing the ascorbyl extremist anion which is besides known every bit semidehydoascorbate as an intermediate as shown in Figure 2. Dehydoascorbic acid possesses full vitamin C activity because it is readily reduced to ascorbic acid in the animate being organic structure. However, dehydoascorbic acid is non an acid in the chemical sense, as it does non hold the separable protons that ascorbic acid has at C 2 and carbon 3 places.
One of the most of import belongingss of vitamin C is that it is an antioxidant. However, it has a broad scope of antioxidant belongingss outside the organic structure and can slake most biologically active groups. It scavenges superoxide, nitroxide, hydrated oxide, H peroxide and will cut down vitamin E ( Hickey and Roberts, 2004 ) . It is besides found to be a strong antioxidant as it helps to neutralize harmful free groups ( Izuagie and Izuagie, 2007 ) .
Vitamin C is an about odourless white or pale xanthous crystalline pulverization with a pleasant crisp gustatory sensation and runing point of about 190 & A ; deg ; C. It is non a carboxylic acid but a lactone and easiness of oxidization to the presence of an enediol grouping ( Izuagie and Izuagie, 2007 ) . Vitamin C is extremely susceptible to oxidization, particularly when catalyzed by metal ions such as Cu ( II ) ion and Fe ( III ) ion. The maps and activities of vitamin C are based on its belongingss as a reversible biological reducing agent ( Hickey and Roberts, 2004 ) .
Vitamin C participates for the growing and fix of tissues in all parts of the organic structure ( Kleszczewski and Kleszczewska, 2002 ) . Vitamin C is a natural antioxidant that largely found in fruits and veggies. The chief beginnings of vitamin C are citrus fruits, strawberries, Piper nigrums, tomatoes, chou, and Spinacia oleracea. Vitamin C plays important functions in negatron conveyance, hydroxylation reactions and oxidative katabolism of aromatic compounds in carnal metamorphosis ( Gazdik et al, 2008 ) .
Vitamin C can assist to forestall and handle common cold, mental unwellness, sterility, scorbutus, malignant neoplastic disease and acquired immune lack syndrome ( AIDS ) ( Yusuf and Gurel, 2005 ) . It is reported to lower malignant neoplastic disease hazard and besides said to hold of import interactions with other vitamins. For illustration, inordinate consumption of vitamin A is less toxic to the organic structure when vitamin C is readily available ( Izuagie and Izuagie, 2007 ) . Due to the great importance of vitamin C in human existences, the quantitative analysis of vitamin C has gained increased significance in several countries of analytical chemical science such as pharmaceutical and nutrient applications ( Yusuf and Gurel, 2005 ) .
Vitamin C is besides used as an index of the alimentary quality for fruit and vegetable merchandises. This is because it is much more sensitive to assorted manners of debasement in nutrient processing and subsequent storage ( Ozkan, Kirca and Cemero, 2004 ) . It is good known that vitamin C is easy oxidized to dehydroascorbic acid in alkalic solutions, while it is comparatively stable in acidic solution. Vitamin C of fruit juices is readily oxidized and lost during remaining of the juices ( Kabasakalis, Siopidou and Moshatou, 2000 ) .
In the nutrient industry, vitamin C is used as nutrient additive ( Mai and Mohammed, 2004 ) . It preserves and protects nutrient from any coloring material alterations and act as an of import constituent of our nutrition every bit good. Vitamin C helps to forestall the debasement of soft drinks and juice which helps to retain their spirits. Hence, it increases the quality of nutrient and nutritionary value every bit good ( Burdurlu, Koca and Karadeniz, 2005 ) .
Degradation of vitamin C undergoes both anaerobiotic and aerophilic tracts. Qxidation of vitamin C in aerophilic tract occurs chiefly during the processing of nutrient whereas anaerobiotic debasement of vitamin C chiefly during storage. Hydroxymethylfurfural ( HMF ) is one of the decomposition merchandises of vitamin C and acts as precursor of brown pigments ( Burdurlu, Koca and Karadeniz, 2005 ) . Vitamin C debasement in packaged fruit juices depends chiefly on storage temperature, pH, dissolved oxygen degree, residuary H peroxide, H2O2 left after the sterilisation of boxing stuff and hint metal ions ( Ozkan, Kirca and Cameroglu, 2004 ) .
Consequently, surveies on vitamin C content in nutrients are of import in relation to the control of nutritionary labels, the update of nutrient databases and the constitution of dietetic mention consumptions. Orange juice is likely the most globally accepted fruit juice and it is recognized worldwide as a good beginning of ascorbic acid ( Sharma, Singh and Saxena, 2006 ) .
In add-on, there are many analytical methods used to find the concentration of vitamin C in the pharmaceutical samples which are colorimetric method, titration, enzymatic method, flow injection analysis ( FIA ) and high- public presentation liquid chromatography ( HPLC ) ( Arya and Mahajan, 1997 ) . Reflectometer is an instrument that can used to analyse many different types of trial which include ascorbic acerb trial that is concerned in this undertaking. It provides a simple and rapid finding of vitamin C content in many pharmaceuticals merchandise.
Assorted methods used in finding of Vitamin C
In recent old ages, the finding of vitamin C has become an of import topic in the field of biochemistry and commercial nutrients. This is because vitamin C plays an of import function in keeping human wellness ( Chen and Sato, 1995 ) . Due to the importance of vitamin C in human existences, the quantitative analysis of vitamin C has gained a important addition in several countries of analytical chemical science such as pharmaceutical and nutrient applications ( Yusuf and Gurel, 2005 ) .
There are legion methods for the finding of vitamin C in a assortment of natural samples, biological fluids and pharmaceutical preparations. The methods for the finding of vitamin C are spectrophotometric methods and non-spectrophotometric methods ( Arya and Mahajan, 1997 ) . For non-spectrophotometric methods are such as high-performance liquid chromatography ( HPLC ) , titration, enzymatic method and fluorometry ( Arya, Mahajan and Jain, 2000 ) . Direct spectrophotometry besides has been applied to find the vitamin C content in soft drinks, fruit juices, and liqueurs after rectification for background soaking up in the UV part.
Flow-injection analysis ( FIA )
In FIA, there is no air cleavage and it is non necessary for a province of chemical equilibrium to be reached. The sample is introduced into a bearer watercourse as a distinct stopper. The presence of a sample-carrier interface allows diffusion-controlled scattering of the sample as it is swept through narrow-bore tubing to make a concentration gradient. The flow-through sensor proctors the alteration in concentration of the reaction merchandise, which is displayed as a chiseled extremum ( Ball, 2006 ) .
Flow-injection analysis permits a simple, rapid and sensitive method for the finding of vitamin C where its systems allow faster trying rates and consumed fewer reagents compared with segmented-flow analysis ( Kleszczewki and Kleszczewska, 2002 ) . Memon, Dahot and Ansari had proposed a method by utilizing single-channel 1, 10-phenanthroline-iron ( III ) composite as oxidizer. This experiment was based on its cut downing reaction on glandular fever ( 1-10-Phenanthroline ) -iron ( III ) to tris ( 1,10-Phenanthroline ) -iron ( II ) ( ferroin ) and the optical density of ferroin was monitored at 510nm through spectrophotometer equipped with a flow through cell ( Memon et al, 2000 ) .
In this analysis individual channel manifold is used as shown in Figure 3. The reagent watercourse is pumped at the flow rate 1.1mL/min via a peristaltic pump equipped with PVC pump tube. The vitamin C sample is introduced into the reagent watercourse via a rotary Teflon valve. A standardization curve for vitamin C in the scope 0-50ppm was plotted from the consequences obtained by Memon, Memon, Dahot and Ansari which are shown in Figure 4. They besides studied about the consequence of reaction spiral and reagent concentration. From the graph ( Figure 5 ) , the maximal strength was observed at 50cm reaction spiral. While the consequences of the consequence of reagent concentration obtained is shown in Figure 6 indicating that the maximal signal could be obtained at 35 % reagent ( Memon, Memon, Dahot and Ansari, 2000 ) .
This method can be improved within certain bounds by increasing the volume of the injected sample in flow injection analysis. The sensitiveness is increased two crease with the addition of sample volume. As decision, since the clip required for sample readying is short and reagent ingestion is low, therefore the method is extremely economical and is suited to utilize on everyday footing for finding of ascorbic acid in pharmaceutical readyings ( Memon et al, 2000 ) .
Ultraviolet ( UV ) spectrophotometry
Direct UV spectrophotometry is a fast, simple and dependable method for the finding of vitamin C. This method can be done through alkaline intervention and the maximal soaking up of vitamin C falls at 243nm at pH2 ( Yanshan, 1997 ) . The soaking up of UV visible radiation by the sample matrix was the major job in this method. Therefore, alkalic intervention method was found to be used as background rectification in space. This is because more than 95 % of vitamin C will be destroyed in 10 proceedingss after alkaline intervention which is in the scope of pH 12 to 13 ( Salkic and Kubicek, 2008 ) . UV spectrophotometry method was found to be applicable for most fruits, fruit juices and soft drinks except those that are unstable to alkaline intervention, and were profoundly colored, or contained high concentration of caffeine, saccharin, caramel and tannic acid ( Yanshan, 1997 ) .
To find the entire content of vitamin C in nutrient samples, a well-established method was investigated by Khan, Rahman, Islam and Begum, 2006 by utilizing the 2,4-dinitrophenyl hydrazine methods ( DNPH ) . This is a simplified method for the coincident finding of entire vitamin C employed matching reaction of 2,4-dinitrophenyl hydrazine dye with vitamin C and followed by spectrophotometric finding. The spectrophotometric method involves the oxidization of ascorbic acid to dehydroascorbic acid by the action of bromine solution in the presence of acetic acid. Chemical reaction between dehydoascorbic acid and 2,4-dinitrophenyl hydrazine at 37 & A ; deg ; C temperature for three hours will organize an osazone. The solution is treated with 85 % H2SO4 to bring forth a ruddy colour composite. The optical density of all criterions was measured at 521 nanometers by utilizing a UV-spectrophotometer. The consequences obtained were taken to contruct a standardization curve ( Khan et al, 2006 ) .
The standardization curve was constructed by plotting the concentration versus the corresponding optical density as shown by Figure 7. The molar absorption factor, ? can be obtained utilizing Beer-Lambert secret plans. The reliabilty of this method was justified by the computations of the % of standard divergence and it was found to be varied within the scope from 0.20 to 2.45 % . The dependability of this method was besides confirmed from the consideration of the undermentioned expected interventions ( Khan et al, 2006 ) .
There are a few interventions that might impact the consequences. First, the intervention was due to the diketogulonic acid. At higher pH, destructive oxidization hydrolysis might happen. This consequences in the gap of the lactone ring of the ascorbic acid and free the vitamin activity. These procedures are of course occurred in fruits and some sums of diketogulonic acid are presence in the fruits. Besides that, diketogulonic acid has keto group that might organize osazone when react with DNPH. Hence, there is a opportunity of mistake in this method which may give false consequences ( Khan et al, 2006 ) .
Another intervention was due to the extracted glucose which contains similar construction like vitamin C. Therefore, some of the glucose may be extracted in the meta-phosphoric acid during the extraction of ascorbic acid from sample. Glucose may besides do the formation of coloured complex with DNPH and gives the false consequence in the finding of vitamin C. This was proven in Figure 8 where there is no soaking up extremum around the interested extremum at 521nm ( Khan et al, 2006 ) .
As decision, the method is simple and first-class for the finding of entire vitamin C in fruits and veggies ( Khan, Rahman, Islam and Begum, 2006 ) .
Fluorometric analysis has been used for ascorbic acid assay in pharmaceutical readyings, drinks, particular dietetic nutrients and even for human serum ( Arya, Mahajan and Jain, 2000 ) . This method had been reported to hold successful application to a broad scope of groceries, including liver, milk, fresh and transcribed fruit, natural and cooked veggies, and murphy pulverization ( Ball, 2006 ) . Previously, fluorometric findings of vitamin C have been developed based on condensation reactions of vitamin C with o-phenylenediamine and on the oxidization with quicksilver ( II ) of vitamin C to organize quinoxaline derived function. The reaction merchandises of these methods exhibit fluorescensce ( Yusuf and Gurel, 2005 ) .
Figure 9 shows the reaction of the dehydroascorbic acid with 1,2-phenylenediamine dihydrochloride to organize the fluorescent quinoxaline derivative 3 ( 1,2dihydroxyethyl ) furol [ 3,4-b ] quinoxaline-1-one. The space can be prepared by complexing the oxidised vitamin with boracic acid to forestall the formation of the quinoxaline derived function. It is used to uncover any fluorescence due to interfering substances ( Ball, 2006 ) .
Yusuf and Gurel have described a method by utilizing Methylene Blue ( MB ) for the finding of vitamin C. This experiment was run by utilizing a spectrofluorimeter to enter the spectra and transport out fluorescence measurings. This method was used to find the sum of vitamin C in the purified stuffs, specifically vitamin C tablets. MB is a member of thiazine dye group. It is widely used in many different countries. For illustration, a exposure sensitiser is used to bring forth vest O in photodynamic therapy for the intervention of malignant neoplastic disease. The extremely coloured oxidised signifier of MB can be reduced to be colourless leuco signifier, Leuco-Methylene Blue ( LMB ) which is shown in Figure 10. LMB is the decreased and colourless signifier of methylene blue ( Yusuf and Gurel, 2005 ) .
Harmonizing to Yusuf and Gurel, the fluorescence sets of MB were obtained at 664nm for excitement province and 682nm for emanation extremums. This was proven by the other research workers who besides examined the emanation bands at 682nm for MB and 452nm for LMB. In Figure 11, the emanation extremum of MB at 682nm increased due to the addition of its concentration. A additive relationship between MB concentration and strength was obtained over the concentration scope of mol L-1 MB ( y= 49.082x + 94.46, r2=0.9969 ) . The excitement extremum of MB at 664 nanometers besides linearly increased depending on the addition of its concentration ( Yusuf and Gurel, 2005 ) .
The surveies of the consequence of vitamin C on the fluorescence of MB is made to avoid any mistakes that might impact the truth of the consequences. In order to analyze the consequence of vitamin C on the fluorescence of MB at 664 nanometers, mol L-1 MB solutions, each solution was added with different concentration of vitamin C and were prepared under N ( N2 ) atmosphere. This was shown in Figure 12 where the spectra were recorded at 664nm ( Yusuf and Gurel, 2005 ) .
Figure 12 above shows the excitement strength of mol L-1 without adding vitamin C was about 1000.0 and supra. The strength was decreased by the addition of vitamin C concentration in MB solutions ( Yusuf and Gurel, 2005 ) .
Figure 13 shows the emanation spectrum of mol L-1 MB as a map of clip. Each spectrum was recorded at 1 minute intervals. The consequences showed that the fluorescence was non changed with clip, reflecting that the fluorescence spectrum of MB was extremely stable with clip ( Yusuf and Gurel, 2005 ) .
In the redox reaction between ascorbic acid and MB, the ascorbic acid is oxidized to dehydroascorbic acid, while MB was reduced to colorless LMB as shown in the followers:
The standardization curve was made based on the concentration of MB ( mol L-1 ) . The consequences indicate that the fluorescence strength of the system is a additive map of vitamin C concentration in the scope of mol L-1 and the arrested development coefficient is 0.9941 as shown in Figure 15 ( Yusuf and Gurel, 2005 ) .
Table 1 below shows the tolerance towards different compounds that might do interventions in this method. These compounds are normally present in most vitamin C tablets. The experimental consequences showed that the presence of hundred-fold surplus of the all contaminant compounds and twenty-fold surplus of citric acid did non significantly act upon the finding of vitamin C utilizing this method. Therefore, it can be concluded that there is no major intervention caused by these compounds ( Yusuf and Gurel, 2005 ) . So it is possible to utilize this method for direct finding of vitamin C in pharmaceuticals without dividing the interfering stuffs.
Table 2 lists the consequences obtained by the proposed method with triiodide method. It can be clearly seen that the consequences are in good understanding with the triiodide method ( Yusuf and Gurel, 2005 ) .
Therefore, the proposed method provides a simple and sensitive fluorimetric process by utilizing MB for the finding of vitamin C. This experiment besides shows that MB could be used for fluorimetric finding of vitamin C in vitamin C tablets although it has merely somewhat fluorescence belongings compared to LMB. Therefore, as decision, it can be explained that the fluorescence strength of MB was more sensitive to find vitamin C concentration ( Yusuf and Gurel, 2005 ) .
Stability of Vitamin C in Orange Juice
Vitamin C is really susceptible to chemical and enzymatic oxidization during the processing, storage, and cookery of nutrient. The catalyzed oxidization tract of vitamin C debasement is the most of import reaction tract for the loss of vitamin C in nutrients. Therefore, vitamin C of orange juice is readily oxidized and lost during remaining of the juice ( Ball, 2006 ) . On the other manus, there are several factors that will besides impact the stableness of vitamin C in orange juice. The factors are such as the consequence of vitamin E, pH, and parametric quantities which include air, heat, H2O every bit good as prolonged storage and overcooking ( Kabasakalis, Siopidou, and Moshatou, 2000 ) .
Harmonizing to Ball, a meta-oxygen-ascorbate composite is formed in the presence of molecular O and hint sums of passage metal which peculiarly are Cu ( II ) and Fe ( III ) . This complex contains a resonance signifier of a diradical that quickly decompose to give the ascorbate extremist anion, the original metal ion, and H peroxide. This extremist anion will in bend reacts with the O to give dehydroascorbic acid ( DHAA ) . For anaerobiotic tract of vitamin C which occurs in the absence of free O, the debasement is caused by the formation of diketogulconic acid. As the rate of debasement is maximal at pH 3 to pH 4, hence this tract is largely responsible for anaerobiotic loss of vitamin C in transcribed Citrus paradisi and orange juices ( Ball, 2006 ) .
Consequence of vitamin E on the stableness of vitamin C in orange juice
Vitamin E is a fat soluble antioxidant that has four vitamin Es and four tocotrienols. In nature, these four vitamin Es and four corresponding tocotrienols are designated as alpha- ( ? ) , beta- ( ? ) , gamma- ( ? ) and delta- ( ? ) harmonizing to the figure and place of methyl substituent in chromonal ring ( Ball, 2006 ) .
The vitamin E maps as a biological antioxidant by protecting the critical phospholipids in cellular and subcellular membranes from peroxidative devolution. Vitamin E largely accumulates in organic structure which are liver and pancreas. But unlike vitamins A and D, vitamin E is basically atoxic ( Ball, 2006 ) .
Nagymate and Fodor ( 2008 ) have designed a method to analyze the consequence of vitamin E on the stableness of vitamin C. In this experiment, vitamin E stock solution was prepared by fade outing? -tocopherol in absolute ethyl alcohol. The orange juice which contained vitamin E and vitamin C was used as sample. The storage temperature of the phials was 4 & A ; deg ; C and they were covered with aluminium foil to forestall the consequence of sunshine. Besides, two different temperatures were used to analyze the consequence of vitamin E at that temperature which half of the samples were stored at 20 & A ; deg ; C. On the other manus, the linear consequence of these vitamins was besides examined but merely cool samples ( 4 & A ; deg ; C ) were used for this experiment. Two samples were prepared which one contained vitamin E stock solution and vitamin C stock solution while another contained merely vitamin C stock solution. The samples were analysed one time a hebdomad for five hebdomads ( Nagymate and Fodor, 2008 ) .
The consequences of the stableness of vitamin C show that the presence of vitamin E influenced the decay of vitamin C. Figure 17 shows that there were differences between samples with or without vitamin E. From figure 17, it can be clearly seen that the concentration of vitamin C without vitamin E fell down to 1.2mg/L on the 2nd twenty-four hours. However, in the presence of both vitamins, the decay was besides observed, but it was lesser. The concentration of vitamin C in the orange juice with vitamin E was 13mg/L in the 5th hebdomad.
As a consequence, it seems that vitamin E stabilized vitamin C in orange juice at a determined concentration. This is because vitamin E delay the oxidization of vitamin C therefore, enhances the stableness of vitamin C in orange juice. The combination of vitamin C with vitamin E makes the orange juice more stable and slower the debasement of orange juice. This concluded that orange juice with vitamin E add-on is a good manner to continue the vitamin C content during storage ( Nagymate and Fodor, 2008 ) .
Consequence of temperature on the stableness of vitamin C in orange juice
Vitamin C of fruit juice is readily oxidized and lost depends on the conditions of storage. There are surveies about the finding of the sums of vitamin C content in fruit juices under different storage conditions. Kabasakalis, Sipadou and Moshatou had done an experiment to find the rate loss of vitamin C with regard to clip and temperature of storage. A long-life and short-life commercial orange juice 100 % without preservatives and fresh orange juice were used for analysis. In this experiment, the yearss before the termination day of the month were recorded in Table 3 and Table 4 to detect the loss of vitamin C in short-life and long-life orange juice 100 % as the termination day of the month was approached ( Kabasakalis, Siopidou and Moshatou, 2000 ) .
Table 5 shows the loss of vitamin C from fresh and long-life commercial orange juice 100 % during a 31 yearss period, with measurings made every 1 to 3 yearss. The samples were refrigerated into containers which after the initial measuring remained either unfastened or with closed cap until the following measuring. Based on the consequences shown in table 5, the magnitude of vitamin C did non differ significantly between unfastened and closed cap for both juices. The commercial orange juice lost higher sums of vitamin C compared with fresh orange juice. As reported, lessenings of vitamin C upon storage did non match to additions of dehydroascorbic acid degrees. In fact, there was an addition of dehydoascorbic acid degrees in aseptically packaged orange juices. This means that the overall nutritionary quality of orange juices is affected upon storage ( Kabasakalis, Siopidou and Moshatou, 2000 ) .
The loss of the vitamin C in a commercial long-life orange juice 100 % stored in icebox and non-refrigerated for a period of 10 yearss in unfastened containers were shown in Figure 18 ( Kabasakalis, Siopidou and Moshatou, 2000 ) .
Harmonizing to Figure 18, non-refrigerated samples show higher per centum loss of vitamin C as compared to refrigerated samples. This is because the dehydoascorbic acid, the oxidised signifier of ascorbic acid was more stable at lower temperatures. Therefore, the vitamin C, in the signifier of dehydroascorbic acid for refrigerated orange juice was good retained than non-refrigerated orange juice ( Kabasakalis, Siopidou and Moshatou, 2000 ) .
Consequence of H peroxide on the stableness of orange juice
Hydrogen peroxide, H2O2 is the primary chemical for sterilisation of fictile packaging stuff used in sterile system. Aseptic packaging engineering is widely used by fruit juice industry for the production of shelf-life stable fruit juices. A Food and Drug Administration ( FDA ) ordinance presently limits the remainder of H2O2 to 0.5ppm, leached into distilled H2O, in finished nutrient bundles which stated in Code of Federal Regulations, 2000. However, during the sterilisation of sterile Chamberss or boxing stuff with H2O2, some residues will still be left on the packaging stuff or bluess generated during drying may acquire trapped inside the bundle upon sealing. These residues will so do the debasement of vitamin C ( Ozkan, Kirca and Cemeroglu, 2004 ) .
An experiment was proposed by Ozkan, Kirca and Cemeroglu to find the rates of vitamin C debasement in orange juice with or without add-on of H2O2 at assorted storage temperatures. In this experiment, the orange juice sample was thawed at room temperature and Na benzoate was added to forestall spoilage. The debasement surveies were done at H2O2 with 0.5ppm concentration at 20 & A ; deg ; C, 30 & A ; deg ; C and 40 & A ; deg ; C severally. At regular clip intervals, samples were removed from the H2O bath or brooder ( Ozkan, Kirca and Cemeroglu, 2004 ) . Then, the preset sums of diluted Na hydroxide solution were added quickly to the samples to hold the reaction between H2O2 and vitamin C. The samples were so quickly cooled by stop uping into an ice H2O bath and held at -30 & A ; deg ; C until analyzed for vitamin C content. Vitamin C concentration was measured by utilizing HPLC method. Qzkan, Kirca and Cemeroglu had modified the method by intermixing the orange juice sample with metaphosphoric acid. The sample was filtered through a membrane filter and was analyzed utilizing HPLC ( Shimadzu trade name ) ( Ozkan, Kirca and Cemeroglu, 2004 ) .Vitamin C contents of orange juice were plotted for assorted temperatures at 0.5ppm H2O2 concentration which is shown in Figure 19 below.
From Figure 19, the consequences show that at higher temperature, the rate of vitamin C debasement besides increased. The add-on of 0.5ppm H2O2 did non greatly increase the debasement of vitamin C. However, raising H2O2 concentration from 0.5ppm to 5ppm resulted in a enormous addition in debasement rates which was recorded in Table 6. At 0.5ppm H2O2, the antioxidant substances in orange juice which was flavonols reacted with H2O2, thereby forestalling the autoxidation of vitamin C. The protective mechanism of flavanols was chiefly due to chelation of metal ions and action of antioxidant. Flavanols map as antioxidants by donating the H ions to reactive free groups which may otherwise do the autoxidation of vitamin C ( Ozkan, Kirca and Cemeroglu, 2004 ) .
Ozkan, Kirca and Cemeroglu besides studied the debasement of vitamin C in the absence of H2O2. In this instance, the activation energy, Ea was taken into history to find the stableness of vitamin C in orange juice. The temperature dependance of the debasement of vitamin C in orange juice was compared by ciphering Ea and temperature quotients ( Q10 ) at 20 & A ; deg ; to 40 & A ; deg ; C from the undermentioned equation:
These consequences clearly indicate that the rate of vitamin C debasement in the presence of H2O2 was slower at 30 & A ; deg ; C to 40 & A ; deg ; C than 20 & A ; deg ; C to 30 & A ; deg ; C. This indicates that at 30 & A ; deg ; C to 40 & A ; deg ; C, the least consequence of temperature rise on vitamin C debasement. The consequences obtained for Ea shows that higher Ea in the presence of H2O2. This means that higher energy needed for the debasement of vitamin C. Therefore, the reaction clip is slower and the debasement of vitamin C besides slower. As decision, the consequence of temperature on the debasement rates of vitamin C in orange juice was more marked at higher H2O2 concentrations. Therefore, greater vitamin C losingss should be expected as residuary H2O2 concentration and storage temperature addition in aseptically packaged fruit juices ( Ozkan, Kirca and Cemeroglu, 2004 ) .
Consequence of pH on the stableness of vitamin C
pH is a step of sourness or basicity of a solution. pH is one of the primary factor that would impact the stableness of vitamin C in orange juice. Hence, the pH value of the matrix has an influence on the stableness of vitamin C. Harmonizing to FAO/WHO Expert Consultation on Human Vitamin and Mineral Requirements, Bangkok, Thailand, 1998, the vitamin C will disintegrate if the pH higher than 4 ( Nagymate and Fodor, 2008 ) .
Vitamin C is unstable in impersonal and alkalic environments, hence the higher the pH value and the longer the exposure, the greater the loss of vitamin C. This is because the higher the pH value, the faster the oxidization reaction of vitamin C and causes the debasement of vitamin C. Besides that, the addition in pH besides related to impairment of fruit feature which in this literature reappraisal, orange juice is more concerned. Table 8 below shows the pH value of the fruit juice with storage clip ( Ajibola, Babatunde and Suleiman, 2009 ) .
In this Table 8, the pH values of the orange juice were higher at room temperature and maintain increasing from hebdomad to hebdomad. This survey concluded that, though pH was important for the stableness of vitamin C, it was non the exclusive factor in commanding the impairment of vitamin C in orange juice with storage life ( Ajibola, Babatunde and Suleiman, 2009 ) . On the other manus, the loss of vitamin C activity during oxidative debasement of vitamin C occurs with the hydrolysis of the dehydroascorbic acid lactone to give 2,3-diketogulonic acid. This hydrolysis is favored by alkalic solution. Dehydroascorbic acid is most stable at pH 5.5 but lessening in stableness as pH additions which is more than pH 5.5 ( Fennmena, 1996 ) . For illustration, half-time values of dehydroasorbic acerb hydrolysis at 23 & A ; deg ; C were 100 and 230 proceedingss at pH 7.2 and pH 6.6 severally as shown in Figure 20. At pH 5.0 or below, dehydroascorbic acid was rather stable which decayed by less than 3 % over 4 hours. This experiment evaluated the consequence of H ion concentration on delactonization of dehyroascorbic acid over the scope of pH 3.0 to pH 8.0. The possible influence of the presence of O was done by equilibrating the reaction mixture before and during the incubation with 100 % O or with 100 % N. The consequences indicated no alteration in the decay rate of dehydoascorbic acid was obvious with these changes of atmospheric conditions. The rate of dehydroascorbic acerb hydrolysis markedly increases with increasing temperature but was unaffected by the presence of O ( Bode, Cunningham and Rose, 1990 ) .
Other research workers had proposed a method to find the consequence of pH on the debasement of vitamin C in orange juice. The purpose of their experiment was by comparing the stableness of vitamin C at different concentrations at lower pH value. An acidic sample was prepared from orange juice with medium intoxicant content. The original pH value of sample was subsequently modified by add-on of concentrated phosphorous acid. After that, different concentrations of vitamin C stock solutions were added and analysed for five hebdomads ( Nagymate and Fodor, 2008 ) .
The consequences showed that, for the experiment done in original pH value of the orange juice which was pH 4.0, the decrease of the sum of vitamin C content decreased with the increasing ascorbic acid concentration ( p & gt ; 0.05 ) , so the velocity of decay was higher at lower concentrations. In the instance of orange juice, the highest standard divergence of the repeated information was 3.14 % and the lowest standard divergence was 1.48 % . This indicates that the consequences are accurate and consistent. ( Nagymate and Fodor, 2008 ) .
When lower pH ( pH 3.0 ) was used, the velocity of decay for orange juice grew with the turning vitamin C concentration, and the highest value was at 50mg/L vitamin C concentration ( p & gt ; 0.05 ) . These consequences were tabulated in Table 10. The highest standard divergence of the repeated information was 3.42 % and the lowest standard divergence was 1.53 % . By comparing the statistical information in both Table 9 and Table 10, it shows that the lower pH values increased the vitamin C content measured at the terminal of 5th hebdomad ( Nagymate and Fodor, 2008 ) .
Harmonizing to Nagymate and Fodor, vitamin C had a important decay independently from the storage temperature when the pH value was more than 4.0. However, under this pH bound, low storage temperature will assist in stabilising this vitamin. Hence, lower pH value was preferred to protract the shelf life of orange juice.
MATERIALS AND METHODS
Analysis of Vitamin C Content in Vitamin C Tablets
Instrument, Materials and Chemicals
The instrument that used in this research is Reflectometer.
The chemicals that used in this research are as below:
- 1 gdm-3 of Ascorbic Acid ( Sigma )
- 0.1M Sodium Hydroxide ( R & A ; M Chemicals )
- Phenolphthalein index
- Potassium acid phthalate ( Hamburg Chemical GMBH )
The stuffs that used in this research are as below ( refer to appendix 1 ) :
- Flavettes Vitamin C-Sugar-Free C
- Redoxon Orange
- Cebion Vitamin C
- Bio C Plus ( Anway )
Merchandise Sample Preparation
The vitamin C tablets were crushed into pulverization by utilizing howitzer and stamp. 1 g dm-3 of each sample was prepared by fade outing 0.1±0.05g of the samples with distilled H2O and exceed up to 100 milliliter. The solutions were exhaustively assorted. The solutions were kept off from direct sunshine and stopper was used to minimise the oxidization of ascorbic acid.
Reagent Solution Preparation
Preparation of Vitamin C Standard Solution
A newly prepared ascorbic acerb solution was prepared by fade outing 0.02g of ascorbic acid and exceed up to 100mL with distilled H2O in a volumetric flask. The solution was exhaustively assorted. The solution was kept off from direct sunshine and a stopper was used to minimise the oxidization of ascorbic acid.
Preparation of Sodium Hydroxide Solution
0.1M of Na hydrated oxide solution was prepared by adding 2.0g of Na hydrated oxide ( NaOH ) pellets and was topped up to 500mL into a volumetric flask. This solution was assorted and was standardized utilizing K acid phthalate ( KHP ) solution.
Determination of Vitamin C by utilizing Reflectometer ( MERCK )
As control method for ascorbic acerb trial, 0.02g of ascorbic acid was diluted with 100mL of distilled H2O. The ascorbic acerb solution should be newly prepared. Beside that, two extra trials were done by utilizing the same instrument but different trial kits which used to prove the glucose and hydroxymethylfurfural ( HMF ) trial. The several strip was immersed into the sample for few seconds. The trial strip was inserted into the strip arranger. At the terminal of reaction clip, the consequence was read from the show.
Determination of Vitamin C by utilizing titration method
Standardization of 0.1M Sodium Hydroxide ( NaOH )
Standard K acid phthalate ( KHP ) , KC8H4O4H solution was prepared by weighing 0.5g dried KHP into an Erlenmeyer flasks and was dissolved by adding 75mL of distilled H2O. The molecular weight of KHP is 204.23. Three beads of phenolphthalein index were added into each Erlenmeyer flask. For 0.1M NaOH standardisation, KHP solution was used and titrated with 0.1M NaOH until the faintest pink persists for 30 seconds. The concluding volume was recorded. The standardisation of NaOH was repeated for another two times.
Vitamin C Analysis: Quantitative method
0.2g of vitamin C was weighed and so added into an Erlenmeyer flask. 50mL of distilled H2O was added to fade out the vitamin C tablet. Three beads of phenolphthalein index were added into the Erlenmeyer flask. A burette which contains 0.1M of NaOH solution was set up every bit which as shown in Figure 22. The vitamin C solution was titrated with NaOH solution was until a pink colour that persists for 30 seconds which was the terminal point. The concluding volume was recorded. These processs were repeated for another two sets of sample.
RESULTS AND DISCUSSION
Determination of Vitamin C by utilizing Reflectometer ( MERCK )
In this undertaking, reflectometer ( Figure 21 ) and titration method ( Figure 22 ) were used to find the vitamin C content in four pharmaceutical merchandises which are Flavette, Cebion, Bio C Plus and Redoxon Orange. In reflectoquant analysis, Reflectoquant Test Strips are inserted into the instrument ( RQflex 10 ) which is a extremely sensitive reflectrometry instrument. The combination of these two trials which are the trial strips and the reflectometer aid to analyse the vitamin C content for a broad assortment of samples in merely few seconds. Reflectometer uses a dual ocular system which works in concurrence with a double reaction zone on Reflectoquant Test Strips to let for coincident dual measurings in one measure. It is a portable trial system that is little, compact, and battery-operated for rapid, quantitative analysis of assorted samples by rating of particular trial strips.
Based on the consequences in Table 14, the vitamin C contents in each sample which is determined utilizing reflectometer do non hold much different compared to the label value. Tablets are labeled harmonizing to their vitamin C content and non harmonizing to their weight. The per centum of divergence of each sample was calculated and the per centums of divergence obtained for all the samples are less than 5.0 % . This indicates that reflectometer can supply accurate and consistent consequences. The difference between label value and analysis consequences could be caused by the intervention substances such as the presence of binder. Binders are normally used when doing conventional tablets. Most binders are polymers which can increase the fictile distortion of the preparation. Binder can be used to forestall a rapid disintegration of the sparkling tablet such as Redoxon. Examples of binders are such as methyl cellulose and gelatin which map to keep the ingredients together to organize a tablet ( Swarbrick and Boylan, 1992 ) . For Redoxon, the analysis consequence is lower than the label value. This is due to the presence of foreign substances such as Zn citrate which may act upon the concentration of vitamin C.
Determination of glucose content and hydroxymethylfurfural ccontent in vitamin C tablet
The consequences in Table 15 besides showed that the glucose content in each tablet of different trade names is less than 1g/mL except for Flavette Vitamin C. Flavette Vitamin C contains 22mg/L of glucose content although this produc is labeled sugar free. Many industries use glucose, fructose or dextrose to dulcify a tablet for commercial intent even though the tablet labeled ‘no sugar ‘ . For hydroxymethylfurfural ( HMF ) trial, 5- ( Hydroxymethyl ) furfural ( 5-hydroxymethyl-2-furancarbaldehyde, HMF ) reacts with a barbituric derived function and an aminophenazone derived function to organize a red-violet compound that is determined reflectrometrically. HMF trial was done to prove the sum of unwanted merchandise such as 5-HMF in vitamin C tablet. From the consequences, all the merchandise samples contain about 1.3 to 1.5 mg/L of HMF. Based on the Commission Regulation, the mean use of HMF is 2.0mg/L and the maximal use is 10.0mg/L for non-alcoholic merchandises ( Berger, 2007 ) . HMF is a crystalline merchandise with a pleasant olfactory property. HMF is formed in nutrients by thermic intervention during storage ( Nollet, 2004 ) . High sum of HMF can do vitamin C loss, therefore impacting the quality of the merchandise ( Damasceno et al, 2008 ) .
Determination of Vitamin C by utilizing titration method
Standardization of Sodium Hydroxide ( NaOH ) solution utilizing K acid phthalate solution ( KHP )
In a titration, it is critical to cognize the exact concentration of NaOH in order to find the concentration of the solution being tested. KHP is a weak acid and reacts with base in the undermentioned manner:
In the titration method, phenolphthalein was used as an index which will be used to find when the reaction reaches its end point. End point is the point which the sum of NaOH added peers the sum of vitamin C. By cognizing the strength and the volume of NaOH required to wholly respond with vitamin C, the existent sum of vitamin C nowadays can be calculated. pH metre besides helps to find the terminal point which is about pH8.50. As vitamin C is a weak acid, the pH of the terminal point is detected by utilizing phenolphthalein index with the passage scope between pH8.0-9.8. Phenolphthalein will alter from colorless to tap when all of the acid has been neutralized.
The samples were analyzed by the reflectometer and the titration methods are summarized in Table 19. The consequences show that both methods are in understanding with the measures specified on the label. This indicates that the proposed method was applied successfully for the finding of vitamin C in commercial pharmaceutical merchandises.
Vitamin C is required for the optimum activity of several of import biosynthetic enzymes and it is hence indispensable for assorted metabolic tracts in the organic structure. However, harmonizing to RDA for vitamin C, 75mg/day and 90mg/day are required for normal adult females and work forces severally. This degree is believed sufficient plenty to forestall lack disease but non chronic disease. Owing to this, vitamin C should be taken each twenty-four hours to forestall chronic disease and the effectual doses are still remained ill-defined today. On the other manus, the Tolerable Upper Intake Level ( UL ) is 2000mg/day where excessively much of vitamin C may be unsafe due to the inauspicious effects such as kidney rock formation, addition of uric acerb elimination and overload Fe.
There are few factors that will impact the stableness of vitamin C which are vitamin E, pH, sum of H peroxide and temperature. Additionally, newly prepared orange juice should be taken in every bit shortly as possible. This because vitamin C may be oxidized during storage even we store it in icebox. It was found that vitamin C loss is the most when the orange juice was stored at room temperature. Well-pasteurized bundle juice from market can lose its vitamin C every bit good due to long storage clip even if it is non opened. The highest loss of vitamin C occurred with conventional boiling. The research besides shows that orange juice with vitamin Tocopherol can detain the debasement rate of vitamin C. Besides that, high concentration of H peroxide will do greater loss of vitamin C. Finally, lower pH value was preferred to protract the shelf life of orange juice.
Three different methods are studied in the reappraisal portion which is flow-injection analysis, ultraviolet spectrophotometry and fluorometric method. All these methods are utilizing spectrophotometer as the sensor to find the content of vitamin C in a sample. Nevertheless, all these methods required extremely cost equipment and reagent in order to execute an analysis than reflectometric method.
In my undertaking, the proposed method utilizing reflectometer can supply a simple analysis of vitamin C. The simpleness of the process permits rapid analysis for vitamin C content in pharmaceutical merchandises. This method is found to be more sensitive and dependable. Besides that, the clip required for sample readying is short and reagent ingestion is besides low, therefore this method is extremely economical. In add-on, by utilizing reflectometer, it is a good option method compared to some of the extremely cost instrument method. Therefore, it is suited to utilize on everyday footing for the finding of vitamin C in pharmaceutical readyings.
It is desirable that nutrition instruction should be brought into the public apprehension of scientific discipline. Furthermore, more researches should be done on the field of the relation between diseases and vitamin C consumption. Food or harvest technology is another critical field in the survey of vitamin C where this can better the concentration of ascorbic acid in natural nutrient.
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