I forgot to autozero during the run (Size exclusion chromatography.) and later i realised i forgot to do that and the baseline was not zero but below zero (and in some cases it above zero). I would like to present the data in a report, however, was wondering if it possible to correct or refine this absorbance values by any means.
what I have tried so far is try to subtract the absorbance value obtained at 0 ml. Any help would be appreciable!
Zeroing the baseline is just a simple vertical translation. You can pick any time point to be zero by adding or subtracting the value at that time point to the absorbance at every other time point. The only reason to have to do this is to calculate the area under the curve when your software can't otherwise draw a baseline from the start to the end of the peak. You have to make the assumption that the baseline is flat.
Adam B Shapiro DO you mean that i subtract 1,30 (an example below) from all the other absorbance values at a given volume?
ml mAU
0,00 1,30
0,03 0,93
0,06 0,59
0,09 0,31
0,12 0,01
assuming the baseline is zero, untill the peak appears...can you do that for scientific reports or papers?
That is what I meant, but in my opinion you don't have to zero the axis at all. You can just show the chromatogram with no y-axis, just a vertical scale bar to show how much distance corresponds to a certain absorbance change. The absolute baseline value isn't important.
Adam B Shapiro what i need to show is an elution profile of a protein. Y-axis is absorbance at 280 nm and X-axis is elution volume. i have to show both axis. The baseline has to zero because at that elution volume, nothing is supossed to elute, therefore absorbance is zero. however, in some cases even after autozero within the system, the baseline was falling zero. i believe this really will be a concern from the reviewers of the report.
It's not unusual for the baseline of a chromatogram to depart from the starting point at zero due to changes in the elution conditions or drift in the detector. I don't think it will be a problem for a journal publication. However, excessive drift in the baseline could indicate that your column or the flow cell in the detector needs to be cleaned.
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