I'm conjugatinig a peptide to AuNPs. Before washing the NPs by centrifugation, the spectrum is on the positive side of the y-axis but once I wash them, the spectrum moves to the negative side. do you know what may cause this? I would expect the spectrum to flat out if my NPs had aggregated.
NB: I've attached a picture a picture for reference.
Hi Keletso Modise , I've seen that happen in another situation, and it is normally caused by a foul reference measurement.
A negative absorbance basically means there is more light passing through your sample (system + environment) than through your reference (environment only), which doesn't make much sense, since your system should absorb a portion of light. A negative absorbance creates the impression that your system is creating light instead of absorbing. Nonsense. How could a more-stuffed medium be more transparent than less-stuffed medium?
Well, your sample is probably "less-stuffed" than your reference. That can happen if your reference contains more than just environment = cuvette + solvent.
If you make your reference to be environment = cuvette + solvent + AuNP(e.g.), then by washing, you are making your system be in a different environment than your reference measurement.
Remember that your reference measurement determines the environment, and your system of interest is assumed to remain in this environment at all times.
In simple terms, if you have the following situation:
* Reference = R = (cuvette + solvent + AuNP)
* Sample_Before-Washing = S1 = peptide + (cuvette + solvent + AuNP)
* Sample_After-Washing = S2 = peptide + (cuvette + solvent)
Then your S2 has a different environment than your R, which yields a negative absorbance spectrum.
I hope that helps.
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