I have made significant progress in my research since the posting of my first blog. My research time has been split between literary research and research in the laboratory. In regards to literary research, I have been trying to find more assays than the two I have chosen that would produce useful results. Aside from the DPPH and Folin-Ciocalteu assays, I have been studying the 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid or ABTS assay. The ABTS assay allows for antioxidant activity to be measured and so it will be an additional assay to test antioxidant activity of the tea samples. Dr. Mojica and I are in the process of finalizing a protocol for this assay as well as ordering the necessary chemical reagents needed.
In the laboratory, bags from the tea samples were dipped into 200 mL of freshly boiled water for ten minutes. Once the tea was made, the bags were removed and the tea infusions were allowed to completely cool. The infusions need to be cool or room temperature in order to ensure that the results produced by the UV-Vis are as accurate as possible. In the Folin – Ciocalteu assay, 0.1 mL of the tea infusion in gallic acid solution was mixed with 0.1 mL of the Folin – Ciocalteu reagent and 0.9 mL of water. The mixture stood for five minutes and then was added to 1.0 mL of sodium carbonate and 0.4 mL of water. The absorbance reading at 765 nm was obtained after initial mixture, thirty minutes, and then sixty minutes. In the DPPH assay, DPPH (2,2-diphenyl-1-picrylhydrazyl) solution in 2.4 mL of methanol was mixed with 0.1 mL of the tea infusion. The absorbance reading at 539 nm was obtained after initial mixture, thirty minutes, and then sixty minutes.
My next step is to enter the results obtained from the UV-Vis into an Excel spreadsheet and create graphs using Excel and the program IGOR. Mixing and reading all twelve of my samples has been very time-consuming. Since half of the samples are green tea and the other half are black tea, I have been trying to mix and read all green teas at once and all black teas at once. Labeling the samples has helped tremendously because samples are the same color when made and it avoids contamination or incorrect readings. So far the readings are making sense and there is a direct relationship between antioxidant activity and phenolic content. My goal when I enter the data into a processing program is that the relationship will become much clearer. I will mix and read the samples again so that I can confirm my original research findings. Since the semester is winding down and finals are approaching, it has become difficult to dedicate as much time in the laboratory as I would like but Dr. Mojica and I will meet many times over the winter break to continue testing and reading my samples. I have placed my samples in the fridge and the next time I read them, I would like to see if there is any change in the results since they have been chilled as opposed to just being made and at room temperature. This will allow me to look at the data in a new light and see if temperature affects antioxidant activity and phenolic content.