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Scientist with beakers

Tech Tip: Understanding Negative Absorbance

How to Improve Experiment Parameters

by Derek Guenther, Senior Application Scientist


Have you ever looked at an absorbance plot with some spectral activity dipping into the negative region that left you scratching your head? Why would the sample have a negative absorbance? Is something spontaneously emitting light at those wavelengths? The answer could be a range of items, but nine times out of ten the answer is: It’s your reference condition.


Broadband spectroscopy can get tricky because you are dealing with activities across many energy levels (i.e., the broadband spectrum). For example, different compounds will interact with light in different regions. Also, depending on the concentration level or conditions of your absorbance reference, sample measurements will move as they change from that reference condition.


Negative Absorbance Scenarios

One scenario may be activity from your solvent, which I am very guilty of forgetting about when taking measurements. For example, let’s consider a fungicide dissolved in acetonitrile, specifically in the NIR range, that we measure with the Flame-NIR spectrometer.


The Flame-NIR spectrometer is responsive from 950-1650 nm.

Figure 1 shows the acetonitrile solvent’s absorbance in dotted orange, and the subsequent fungicide sample’s absorbance in solid blue after taking pure acetonitrile as the light reference. Note the negative absorbance in the fungicide sample spectrum correlates exactly where acetonitrile has strong activity. This is no mistake; as we are increasing the concentration of analyte in the solution, we are decreasing the concentration of our solvent. So anywhere that the solvent was optically active will now show a dip in absorbance as some of that optical real estate in the cuvette, dip probe and so on is now taken up by something else.


Figure 1. The dotted orange line shows acetrontrile absorbance and the solid blue line shows the fungicide sample absorbance.


Another scenario may be the analyte species itself changing forms, with each form having a unique optical activity. Perhaps the most common example of this are pH dyes such as in litmus paper that flip forms based on acidity or basicity. Figure 2 shows the absorbance response of the common pH dye bromocresol green with the light reference taken in the acidic condition. This means all the dye molecules were in the acidic form when the absorbance was set to zero everywhere. Most pH dyes typically have an isosbestic point, which is a wavelength that remains pH independent during migration between the forms. For this dye, the activity below the isosbestic point (lower wavelength) is representative of the acidic form of the dye, while activity above this point shows the basic form. As we push the pH level higher and move some percentage of dye molecules away from their acidic form and into the basic form, we see a negative dip in the acid peak region and a growing hump in the base peak region.


Figure 2. Absorbance response of a common pH dye reveals the influence of the reference being taken in the acidic condition.


So, do not be discouraged when you see negative absorbance in your spectra. Chances are the result is real, and may be a good catalyst for thinking more deeply about your experiment and how some parameters could be improved.


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