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From the list below, circle the wavelength that would be the best setting for the UV-visible spectrophotometer used to determine the FITC concentration in the sample.
\begin{array} {} \mathrm{wavelengths:} & \pu{400 nm}& \pu{450 nm}& \pu{500 nm}&\pu{550 nm} &\pu{600 nm} \end{array}

The exam paper answer is $\pu{500 nm}$, because we need the wavelength of "maximum absorbance." But this graph shows two curves, and the absorbance curve is omitted.

But what is the difference between an emission, absorbance, and an excitation spectrum? What are they measuring? How do you interpret/read these graphs - those with multiple curves.


When you have an excitation spectrum and an emission spectrum, it is for fluorescence spectroscopy. To obtain these spectra, you hold either the emission wavelength or the excitation wavelength and scan the other to gather the information. The excitation spectrum shows at what wavelengths the solution uses to produce its fluorescence. The emission spectrum shows what wavelengths are given off from the solution.

For absorbency spectrum, the solution has all the wavelengths shining on the solution, and you monitor the wavelengths of interest. The absorbency shows which wavelengths are absorbed by the solution.

The excitation spectrum has the same peaks as the absorbency spectrum; although the absorbency spectrum may have more peaks.

  • $\begingroup$ i see... so in this case id be reading the excitation spectrum to find maximum absorbance? $\endgroup$
    – confused
    Apr 13 '14 at 2:31
  • $\begingroup$ It would be a good indication. There might be another peak in the absorbency spectrum that is greater, but you would have to have the spectrum to tell. $\endgroup$
    – LDC3
    Apr 13 '14 at 3:20

The energy gap of absorption is greater than that of emission. The gradient in between is termed stoke's shift. This because when the excited electron looses energy, it first gets to zero point level of excited state, emits and hits the potential well of the ground state after which it looses energy again to retire to the zero point level of the ground state. Emission takes longer time than absorption


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