2
$\begingroup$

I was collecting data to plot a calibration curve using pure beta-carotene sample. Prepared 5 different concentrations of beta-carotene dissolved in methanol. Pure methanol was used as blank. However, data collected for all 5 concentrations observed was negative (between -0.005 and -0.010) at 610nm ( orange region along the spectrum). Please advice.

$\endgroup$
6
  • 1
    $\begingroup$ Beta carotene does not absorb at 610 nm. Are you speaking of a slightly displaced baseline? $\endgroup$
    – Alchimista
    Feb 23 '20 at 7:54
  • $\begingroup$ I am aware that beta carotene does not absorb at 610nm, which gives rise to my confusion. however, the only peak in absorbtion was found at 610nm. it is also negative. hence i am checking what could have gone wrong. $\endgroup$
    – Markus
    Feb 23 '20 at 8:03
  • 3
    $\begingroup$ Even not absorption features? Add this info to the question. But I guess no one can speculate of what went wrong. It can be everithing including peaking up the wrong files, for what we know. One can only point to a typical error of novices, ie absorption and scattering exceedingly high, so the instruments handle a kind of 0 as numerator... $\endgroup$
    – Alchimista
    Feb 23 '20 at 8:40
  • 1
    $\begingroup$ Do the samples look orange? Are the solutions transparent or turbid? $\endgroup$ Feb 23 '20 at 11:44
  • 1
    $\begingroup$ What kind of spectrophotometer? What size cuvette/smple volume? As much detail as you can provide will be helpful. ALso, do you have a sample of B-carotene that is concentrated enough that you are able to see the peaks you are expecting? $\endgroup$
    – Andrew
    Feb 23 '20 at 14:57
6
$\begingroup$

Possible causes include:

  • If it is a dispersive instrument (simultaneous recording of blank and sample in two cells): is the logic about which one is sample and blank set correctly? At least some spectrometers by PerkinElmer and Shimadzu allow to swap how front and rear path are managed by the software.
  • How long did you run the spectrometer to warm up? Maybe the instrument wasn't yet stable, elder spectrometers need 10mn+ to warm up all their internals.

  • Check the lamp for its run time and compare with the specifications by the manufacture. Depending on the setup, the instrument's software may tell you this, or / and the lamps may have a counter (the little box between the plug and the mount of the lamp shown in the picture below). If the lamp's intensity over time is not stable any more while recording sequentially blanks and samples, you will have a hard time to record faithfully spectra. This cause typically remains unnoticed with mono-beam instruments less well maintained and run until the lamp «breaks» (multiple times older than the recommended run time).

enter image description here

(image credit)

  • Check your instrument with a known standard. This either may be a liquid solution of a known concentration (e.g., by SigmaAldrich) to purchase, or already sealed into a cell, e.g.

enter image description here

(image credit)

or solid samples of metals deposit on glass or quartz

enter image description here

(image credit)

Don't forget that groups active in materials science / spectroscpy / optics may have such references to calibrate their instruments regularly and may be happy to share with you how to use these accurately, too.

$\endgroup$

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.