Lets say I prepare 50 ml sample and took 10 ml for UV analysis at 650,660,670 nm (Abs. recorded i.e 0.5,1.5,1.3 respectively).
after short interval similarly when another 10 ml from sample was taken for analysis at same wavelength the absorbance values recorded were of different range. why?
How different are the absorbance? Are they decreasing with time? Or the spectral profile is changing? Perform a time dependent absorbance study of the same sample. Ideally the absorbance should not change. If the absorbance changes (decrease) with time then sample degradation/ photobleaching is suspected.
At some wavelength its increasing and at some in decreasing manner. However sample kept at dark place and analysis carried out in absence of light. so photo bleaching chances are very low in this case. Its really confusing.
Thanks Dear Tuhin for your valuable thoughts.
Could you mention if this is a scanning UV-Vis instrument? Is it a double or a single beam? Depending on the instrument, an absorbance of 1.5 is slightly towards the end limit. Secondly, the long wavelengths are also towards the end of typical UV-Vis performance limits. Although manufacturers claim absorbance range of 0 to 3.0, the error is minimum when absorbance is under 1. Do you autozero every time before collecting the data? Older spectrometers can also drift in values with time.You might have to do a little bit of statistical analysis as function of time to judge the stability of both samples as well as the instrument. A simple test is to use pure deionized water (in both cells, if it is a double beam) and see the variation in absorbance. Is there is a drift (i.e. trending absorbance) or just random variation. If everything appears to be okay from the instrument's side, it is just the sample nature.
ideally it should not happen. I think before taking absorbance you should centrifuge your sample and properly mix it each time.
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