14. If absolute quantification of carotenoids is necessary, perform
calibration for each specific carotenoid that you need to quantify with available standards according to steps between 15 and
18. Alternatively, collect carotenoid fractions by performing
the steps between 4 and 14, then perform calibration analysis
again. It is recommended to perform a calibration analysis
every time when you start using a new column and/or instrument or any other HPLC protocol as coefficients for the absolute quantification might be slightly different depending on
these variables.
15. It might be necessary to use higher volume of solvents as
necessary to completely dissolve carotenoids before spectral
measurements.
16. Once prepared, stock solutions can be stored at À20
C freezer
for a short period of time. Bring standards to RT before taking
UV-Vis measurements as temperature impacts absorption
measurements.
17. Make sure that there is no substance (dust from gloves, finger
prints, etc.) to block the passage of light through the cuvette
surfaces during spectrophotometric measurements. Avoid
touching the sides of the cuvettes. Lint-free wipes are recommended to clean the cuvette surface.
λmaxIII
λmaxII
λbaseline
Absorbance (λ)
Wavelength (nm)
Peak II
Peak III
Fig. 5 Carotenoid peak ratios help determine isomers. A basic demonstration of
how to calculate %III/II ratios
160
Yagiz Alagoz et al.
calibration for each specific carotenoid that you need to quantify with available standards according to steps between 15 and
18. Alternatively, collect carotenoid fractions by performing
the steps between 4 and 14, then perform calibration analysis
again. It is recommended to perform a calibration analysis
every time when you start using a new column and/or instrument or any other HPLC protocol as coefficients for the absolute quantification might be slightly different depending on
these variables.
15. It might be necessary to use higher volume of solvents as
necessary to completely dissolve carotenoids before spectral
measurements.
16. Once prepared, stock solutions can be stored at À20
C freezer
for a short period of time. Bring standards to RT before taking
UV-Vis measurements as temperature impacts absorption
measurements.
17. Make sure that there is no substance (dust from gloves, finger
prints, etc.) to block the passage of light through the cuvette
surfaces during spectrophotometric measurements. Avoid
touching the sides of the cuvettes. Lint-free wipes are recommended to clean the cuvette surface.
λmaxIII
λmaxII
λbaseline
Absorbance (λ)
Wavelength (nm)
Peak II
Peak III
Fig. 5 Carotenoid peak ratios help determine isomers. A basic demonstration of
how to calculate %III/II ratios
160
Yagiz Alagoz et al.