Hi. Last week I ran the analysis of ascorbic acid samples with concentrations of 2, 4, 6, 8, and 10 mg/ml that are dissolved in methanol. Every 3 mL of this sample was mixed with 1 mL of DPPH (0.1 mM). But when I ran the analysis using UV-VIS, I got a -ve reading at 517nm. The next day, I ran another analysis of ascorbic acid with the same method, but this time, no reading was recorded at 517nm for all the samples. So, I want to ask, is the procedure I used wrong that caused no reading at 517 nm, and why did it happen? Is there any idea you can give me about whether I should change the concentration of my sample or anything else?
Hello,
If I understood correctly, on the first day the reading was good, however on the 2nd day you had no reading. Did you use the same DPPH solution that you prepared the day before?
If so, potentially the DPPH has reduced when stored (try to see if its color has changed to yellow before using it for the test, which is a sign of its reduction) . Normally DPPH can be kept for a few days, but if it has reduced use a freshly made solution.
Sofiane Benyamina On the 2nd day, I made a new DPPH solution and not used the same DPPH solution that I prepared the day before.
Ankit Negi Is the concentration I used before too high and becomes the reason there is no reading recorded by uv-vis??
Yes, if your sample completely turns yellow after incubation, then you should start with the lowest concentration of sample/ascorbic acid. Alternatively, if it remains violet in color, you should begin with a higher concentration.
Better you repeat it with the lowest concentration and make sure you are the analyst. You knew the UV-Vis instrument functioning, please.
There is no way to get no results ???
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