In this video, we will talk about the analysis of ascorbic acid, also known as vitamin C using HPLC.
Banana has long known to be a superfood
It contains a lot essential nutrients that are beneficial to the human body, such as vitamins and minerals
Ascorbic acid, also known as vitamin C, is abundant in banana containing about 10% of daily amount required
Health benefits of ascorbic acid include prevention of infections, synthesis of collagen and production of iron
Quantitative analysis of ascorbic acid in various food is important in health and nutrition sciences, food processing and quality labelling
Some of the methods of analysis are AOAC 967.21 which is based on titrimetric method for vitamin C in preparations and juices, AOAC 2012.22 which is based on the measurement of vitamin C in infant and adult formula
Let’s start off with the sample preparation
Peel off the banana skin and cut the banana
Weigh the cut banana into a centrifuge tube
Add in a mixture of acetic acid, metaphosphoric acid and TCEP into the sample
TCEP is important in the extraction process. Ascorbic acid is easily oxidised into dehydroascorbic acid. TCEP helps to reduce dehydroascorbic acid into ascorbic acid.
Vortex the sample mixture
Centrifuge the sample mixture
Filter the supernatant with nylon filter
Dilute the filtered solution
Ascorbic acid stock solution at concentration of 1000 ppm was prepared using a diluent mixture of metaphosphoric acid, acetic acid and TCEP. The stock solution was diluted to 7 different concentrations as working standards using the same diluent as stock solution.
Let’s move on to the HPLC analysis
Load the samples into the HPLC system
Load analysis and inject the samples
A calibration curve is built with an ascorbic acid concentration ranging from 0.1 ppm to 10 ppm
Finally, this calibration curve is then used to quantitate ascorbic acid in banana samples
It is important for the ascorbic acid in sample to be stable and not oxidized during the analysis
In order to evaluate the stability of ascorbic acid in sample, an extracted banana sample was injected consecutively for 10 hours
The peak areas of the injections were compared
The peak area of ascorbic acid remains consistent throughout the period, showing that it is stable in the sample
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Analytical instrument
Liquid Chromatography
