Vitamin C, also known as L-ascorbic acid, is an essential component of our diets. It is a good antioxidant, a key component that helps to prevent damage to proteins and deoxyribonucleic acids. In the food industry, vitamin C is added to increase the nutritional content of food products and also for preservative purposes. As the human body is unable to synthesise vitamin C, it would have to be consumed as part of our diet. Fresh fruits and vegetables for instance are common sources of vitamin C.
Vitamin C is a labile compound and is easily degraded by enzymes and atmospheric oxygen. Its oxidation can be accelerated by excessive heat, light, and heavy metal cations (Pisoschi, Danet, & Kalinowski, 2008). During the manufacturing process, some vitamin C would be lost. Thus, in fruit juice products, vitamin C level is commonly used as a gauge for quality. This prompts manufacturers to fortify their products with high levels of vitamin C to ensure that sufficient vitamin C is present in the product throughout the storage process. Due to the wide use of ascorbic acid in both food products and in the pharmaceutical industry, many analytical methods exist for the determination of ascorbic acid, including titrimetric, spectrophotometric and chromatographic methods, each with their advantages and disadvantages.
An example of a titrant used is 2,6-dichlorophenolindophenol (DCIP), which will oxidise the ascorbic acid that is present in the sample. While titrimetric methods are simple to use, they are also known to overestimate the amount of ascorbic acid present due to the presence of oxidisable species other than ascorbic acid (Hernandez, Lobo, & Gonzalez, 2006). In addition, many interferences often occur with coloured samples (Arya, Mahajan, & Jain, 2000). An example would be the masking of colour change at the end point of titration by highly coloured extracts from fruits and vegetables (Eitenmiller, Landen, & Ye, 2007).
Spectrophotometric methods work by determining the absorbance of vitamin C which is compared against standard concentrations. However, such methods are susceptible to possible interference due to absorbance exhibited by other components that is present in the sample matrix.
Lastly, chromatographic methods are commonly used because of their simplicity, short analysis time and sensitivity (de Quiros, Fernandez-Arias, & Lopez-Hernandez, 2009). The sample is separated into its components based on their relative affinity with the mobile and stationary phase. Reversed-phase high performance liquid chromatography (RP-HPLC) for instance is a very efficient method that is used in ascorbic acid analysis of fruits, vegetables and beverages. Ascorbic acid is relatively hydrophilic due to the presence of several hydroxyl groups. Thus, it has a higher affinity to the polar mobile phase than the non-polar stationary phase, allowing it to be separated from the other components in a sample. The retention time gives a qualitative analysis of the sample while the area under the peak allows for the quantitative determination of ascorbic acid content present. However, a major disadvantage of this method is its high cost compared to other conventional methods.
The objective of this experiment is to determine the ascorbic acid content in commercial guava juice by RP-HPLC.
Materials and Methods
The product analysed was commercial guava juice.
The experimental procedure was as stated in the laboratory manual, with the slight amendments as follows. The filtrate (2mL) from the centrifuged sample was diluted (1 part sample: 4 part acetic acid) with 2% acetic acid. Five sets of standard ascorbic acid solutions were prepared (40ppm, 80ppm, 120ppm, 160ppm and 200ppm for each set) using a stock solution (1000ppm ascorbic acid) and 2% acetic acid for dilution. The standard solutions were filtered using the 0.45 micron cellulose acetate syringe filter, beginning with the lowest concentration. The column used was a Phenomenex Ultrasphere 5u C18 column (150?4.6mm) and the wavelength used was 254nm.
Results and Discussion
A calibration graph was plotted (Figure 1) using average peak area (Table A1 in appendix) for standard solutions versus their respective concentrations.
For each standard solution, four duplicates were prepared. This was done to increase the accuracy of the calibration curve. The r2 value obtained (0.9984) was close to the ideal value 1, indicating a good linear correlation between the area under peak of interest and ascorbic acid concentration. This allows good estimates of ascorbic acid content to be made given the area under peak of interest for each sample.
Table 1. Ascorbic acid concentration in samples prepared by Groups 10 to 18
Area under peak of interest for sample
Ascorbic acid concentration in diluted sample (ppm)
Original Ascorbic acid concentration (ppm)
Original Ascorbic acid concentration (mg AA/ 100mL)
Average Original Ascorbic acid concentration (mg AA/ 100mL)
Coefficient of Variation (%)
Sample calculation for Group 11:
Ascorbic acid concentration in diluted sample = = 116ppm
Ascorbic acid concentration in original sample = 116ppm 5 = 581ppm = 58.1 mg / 100mL of juice
Average ascorbic acid concentration in original sample
=58.0 mg / 100mL of juice
The average retention time of the samples prepared by different groups was 2.557min (Table A3 in Appendix), which is highly similar to that of the ascorbic acid standard solutions was 2.559min (Table A2 in Appendix). This verifies that ascorbic acid was the component analysed.
The average ascorbic acid concentration in the guava juice product determined experimentally was 58.0mg/100mL of juice. This was approximately 3.9 times higher than the amount indicated on the packaging (15mg/100mL). As mentioned earlier, As the expiration date is approached, ascorbic acid would be lost to different extents depending on the storage conditions (Kabasakalis, Siopidou, & Moshatou, 2000). Manufacturers are known to add ascorbic acid to their products to improve their nutritional value and also to account for the ascorbic acid lost during the manufacturing and storage process (Ottaway, 2008). Since the experiment was conducted before the expiration date of the product (March 14, 2014), a higher ascorbic acid content would be expected.
The original ascorbic acid concentration for group 14 was excluded from the calculation as it was almost double of other results and thus likely to be an outlier. A possible reason might be an error in dilution during the preparation of the sample. The other results were found to be precise with a low standard deviation (1.4) and a low coefficient of variation (2.5%).
The ascorbic acid content of commercial guava juice determined using RP-HPLC was 58.0mg/100mL of juice.
Arya, S. P., Mahajan, M., & Jain, P. (2000). Non-spectrophotometric methods for the determination of Vitamin C. Analytica Chimica Acta, 417(1), 1-14. doi: http://dx.doi.org/10.1016/S0003-2670(00)00909-0
de Quiros, A. R.-B., Fernandez-Arias, M., & Lopez-Hernandez, J. (2009). A screening method for the determination of ascorbic acid in fruit juices and soft drinks. Food Chemistry, 116(2), 509-512. doi: http://dx.doi.org/10.1016/j.foodchem.2009.03.013
Eitenmiller, R. R., Landen, W. O., & Ye, L. (2007). Vitamin Analysis for the Health and Food Sciences, Second Edition: Taylor & Francis.
Hernandez, Y., Lobo, M. G., & Gonzalez, M. (2006). Determination of vitamin C in tropical fruits: A comparative evaluation of methods. Food Chemistry, 96(4), 654-664. doi: http://dx.doi.org/10.1016/j.foodchem.2005.04.012
Kabasakalis, V., Siopidou, D., & Moshatou, E. (2000). Ascorbic acid content of commercial fruit juices and its rate of loss upon storage. Food Chemistry, 70(3), 325-328. doi: http://dx.doi.org/10.1016/S0308-8146(00)00093-5
Ottaway, P. B. (2008). Food Fortification and Supplementation: Technological, Safety and Regulatory Aspects: Elsevier Science.
Pisoschi, A. M., Danet, A. F., & Kalinowski, S. (2008). Ascorbic Acid Determination in Commercial Fruit Juice Samples by Cyclic Voltammetry. Journal of Automated Methods and Management in Chemistry, 2008. doi: 10.1155/2008/937651
Table A1. Area under peak of interest for standard solutions
Area under peak of interest
Average area under peak for 40ppm
Table A2. Retention Times for standard solutions
Retention Time (min)
Table A3. Retention Times for samples prepared by Groups 10 to 18
Retention Time (min)