I got positive OD values for pure sample. But I got negative OD values at certain regions of the spectra upon addition of nanoparticles successively in both the reference and sample cuvettes. what is the reason behind it?
If you need to add particles to your reference cuvette, then first have a look what happens in the reference cuvette. The reference cuvette is expected to stay stable over the whole course of measurement, so having a reference with varying concentration of nanpoarticles makes no sense. As there is an interaction between sample and nanoparticle, you want to measure two series:
1) adding nanoparticles to solvent
2) adding nanoparticles to sample solution
This will show you what truly happens
Have you measured fluorecence spectra? I mean, in order to appear that strongly in a UV/Vis-Spectrum they would have to be fluorescent as hell with a giant quantum yield, but that would be my first idea.
Negative absorbance has no physical meaning. It means that your blank absorbs more light than your sample. What are the compositions of your blank and sample at negative absorbances?
My guess is that the absorption of your nanoparticles (plasmonic nanoparticles?) redshift due to an increase of the index of refraction in their surroundings. I would assume that the sample preferentially concentrates around the nanoparticles, which causes this index of refraction change and thereby the shift of the band of the nanoparticles...
I think it is a "auto zero"/ "set zero" issue. Record the spectra without setting the absorbance value to zero at 800 nm (i.e., do not press the auto zero option). You have basically making a non zero value (say 0.5 absorbance) zero thereby leading to lesser absorbance value (say 0.2 absorbance) to negative. I can see the trend in the spectra. Stronger absorbance has larger negative value. Because you are setting larger non-zero value to zero leading to it going progressively negative. You can confirm this by recording spectra from 700 nm (and choosing auto zero). In this case the signal will go further negative. (In other word you are not recording the complete spectra. Try recording from 950 nm or further red.)
There are two things need to be understood from the graph.
As per the figure, with successive addition of nanoparticles to sample, a negative band is arising ~ 525 nm and another band arises in the region of ~ 650 nm with red shift. As you mentioned that nanoparticles were added to reference cuvette as well, the negative band may be due to addition of more nanoparticles in reference cuvette. This is visible from 250-500 nm range.
The new band ~650 nm may be due aggregation of nanoparticles.
The negative signal is cannot be entirely due to excess nanoparticle in reference cuvette as Bikash Chandra Swain suggested. If it was the case then the signal would progressively go negative (due to 1/wavelength^4 dependence). But the signal is practically flat in the region of 300-450 nm. (Unless you have something equally positive which nullifies it.) Again with increase in concentration the scattering would have been stronger but I see flat signal. I also do not expect strong scattering in the visible region due to its wavelength dependence. Though it depends on the size of the nanoparticle.
If you need to add particles to your reference cuvette, then first have a look what happens in the reference cuvette. The reference cuvette is expected to stay stable over the whole course of measurement, so having a reference with varying concentration of nanpoarticles makes no sense. As there is an interaction between sample and nanoparticle, you want to measure two series:
1) adding nanoparticles to solvent
2) adding nanoparticles to sample solution
This will show you what truly happens
Today I had a chat with Janmejaya Rout over mail and after looking at some additional data now I am almost sure why the signal goes negative. "Auto zero" does not have a major role to play here. It also seems that there is not much scattering from the nanoparticle. I should have noticed that the negative signal is exactly at the nanoparticle absorption area.
The reason foe negative signal is, after the addition of NP to the sample the NP has a smaller absorbance value (say it decreases from 0.5 to 0.4 after interaction with sample). But in the reference cuvette, since only NP is present, it has standard absorption (say 0.5). This results in a negative absorbance value. Instead of calling it an absorbance maybe it should be called difference absorbance. In one way Bikash Chandra Swain is right on the reason of negative signal. Though he assumed an excess nanoparticle in reference, in reality, there is lower effective NP concentration (smaller absorbance) in sample cuvette.
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