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.
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).
- 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.
or solid samples of metals deposit on glass or quartz
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.