4
$\begingroup$

I am working on a project in my lab, and we are analyzing capsaicin using HPLC. I am trying to find an appropriate wavelength for the HPLC using a UV-Vis spectrometer. When analyzing the pure capsaicin standard dissolved in methanol, I of course use methanol as a blank. However, I got two peaks, one around 230nm and one at 280nm. Since the system automatically subtracts the methanol, I can't understand why I am getting two peaks. Anyway, 230 yielded the maximum absorbance, so I used that in my HPLC. I didn't get great results and decided to read some literature on the project. I saw that most other people had a UV-Vis spectrum like mine, but they decided to use 280nm. Why is that?

$\endgroup$
  • $\begingroup$ Welcome to Chemistry.SE! To acquaint yourself with this page, take the tour and visit the help center. Furthermore this tutorial shows you how math and chemical formulae can be nicely formatted on this site. $\endgroup$ – Philipp Nov 19 '14 at 23:22
4
$\begingroup$

Different functional groups may show different absorptions and even a single functional group may exhibit two signals in your UV-VIS spectrum, e.g. corresponding to $\pi,\pi^*$ or $n,\pi^*$ transitions.

While HPLC grade methanol is typically much cheaper than acetonitrile, it might not be the best choice here. You might want to have a look at the cutoff wavelengths of different solvents.

I'd definitely give measurements at $\lambda = $ 280 nm a try.

$\endgroup$
2
$\begingroup$

Many organic compounds give more than one maximum peak when its UV-Vis spectra is analyzed. Each peak correspond to a electron transition from a ground state to an excited state, and more than one different transitions (with different energy, and therefore, different wavelenght) are allowed. To determine the concentration of a compound, in principle, any fairly strong peak would be valid, but depending on the experimental conditions, sometimes one gives a stronger signal, and other times that one gives a worst signal.

The peak employed on your determinations can also be selected based on your instrument conditions, i.e., if one of the peaks is close to your measurement limits, you may prefer to use another, or if one of them has a better shape, etc.

$\endgroup$

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.