I am trying to find the mass percentage of a particular analyte in my sample.
I carried out both external and standard addition calibration methods.
The external calibration method gave a result well within the mass of the original sample.
However, the results from standard addition gave consistent results of more than 200% that of the original mass. What are some possible reasons for this to occur?
Many thanks!
Dear Glenda,
Interesting question. What type of detection do you use? And the reference (standard), are they the same substance or different types of chemicals?
Regards,
Harrie
Dear Harrie,
The detector I am using is a photodiode array and it is set to display wavelengths of 265nm and 425nm since I am trying to measure the concentrations of ascorbic acid and tartrazine in a sample of vitamin c tablet.
The standards used for the standard addition are made up of differing amounts of the vitamin c tablet sample, ascorbic acid and tartrazine.
Many thanks for helping out.
Regards,
Glenda
Dear Glenda,
It could be that when you add ascorbic acid to the samples, that the azo bonds in the tartrazine molecule break down, resulting in a decrease of the absorption at 425 nm. If you take the tartrazine as reference, the observed ratio of ascorbic acid at 265 nm to the tratrazine at 425 nm will incrase beyond what you expect to see from adding extra ascorbic acid. The interaction of ascorbic acid and tartrazine has been described, but I do not have the refs at hand. What gradient are you using, column?
Regards,
Harrie
1. Wouldn't it be easier to determine ascorbic acid by a redox titration? I haven't got any useful references here at home (Sunday afternoon!), but a quick internet search suggests that an iodine titration should work OK. (Google "ascorbic acid" titration)
2. If I were trying to determine ascorbic acid by HPLC, I'd expect to have major problems with it oxidising in the sample vials. Obviously you can get rid of dissolved oxygen from the samples before you start, and maybe clear the headspace of oxygen as well; but it's rather more difficult to stop air getting into the sample vials as you withdraw solution from them for injection... and thus being able to oxidise the ascorbic acid. I've never found a way round this with air-sensitive samples - except to analyse them by some other technique!
Dear Glenda,
What do you think about absorbtion of your sample on the sides of the vials?
Does the performed HPLC experiments were carried out in different range of concentrations and with different tipes of vials to exclude the vial glass absorbtion effect?
Dear Glenda:
Maybe your sample (tablet of vitamin C) is too concentrate. In this case, the estimation of concentrations based on standard addition might be too high. Try to do the calibration by diluting the tablet of vitamin C (reduce its concentration) and adding smaller amounts of the target analytes.
Best,
Hi,
As vitamin C is sensitive to oxidation, you should priorly perform a stability assesment of your solution by injecting the same diluted stock solution at different times and monitor peak area.
Moreover you should perform a forced degradation on drug substance (vitamin C, tartrazine) to view if your method are stability indicating or not.
Regards,
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