3.4 Quantification by
Absolute Levels and/or
Determining
the Percent
Composition
1. Open data files in the HPLC instrument software (e.g., Agilent
ChemStation off-line software).
2. Identify carotenoid species according to their retention time,
spectral features, and ratios of maximum absorption peaks (λ)
(see Notes 11 and 12). A detailed DAD spectra analysis (Fig. 3)
and retention times of some of the identified carotenoids on
C30 and C18 columns have been provided in Tables 1 and 2.
3. Perform peak integration and calculate individual peak area
using HPLC instrument software (see Note 13).
4. If calibration curves and slope were predetermined, use the formula below to calculate carotenoid abundance in grams
per gram fresh weight (g/gfw). Otherwise, perform fraction
collection (step 4) and/or establish standard calibration
curves using commercially available carotenoids (step 15) (see
Note 14).
Formula:
Carotenoid weight g=gfw
ð
Þ¼ Peak area AU
ð
ÞÂ1=Slope
 Dilution factor DF
ð ÞÂg grams
ð
Þ
5. Program HPLC instrument software and fraction collector to
collect individual carotenoid fractions according to their retention times or spectral identification.
6. Collect each fraction in a new dark amber colored glass vial.
7. Validate the purity of carotenoid fractions using HPLC according to the instructions given at step 2.
8. Repeat steps 5 and 6 until pure carotenoid fractions have been
prepared.
9. Evaporate the mobile phase within the collected fractions
under a steam of nitrogen gas.
10. Redissolve carotenoid precipitate in 1 mL of organic solvent
(see Notes 15 and 16).
11. Prepare the spectrophotometer at RT and zero the spectrometer with the same solvent used to dissolve the precipitated
carotenoid (see Note 17).
12. Transfer 1 mL of redissolved carotenoid sample (from step 10)
into a glass or disposable plastic cuvette and measure absorbance at specific wavelengths (λ) (see Table 3) for each carotenoid using a UV-vis spectrophotometer (see Note 18).
13. Calculate the carotenoid concentration using this formula:
Formula:
Absorbance read AU
ð
ÞÂDilution factor DF
ð ÞÂConcentration mg=mL
ð
Þ
A
1%
1cm
Profiling Carotenoids in Plants Using HPLC
153
Absolute Levels and/or
Determining
the Percent
Composition
1. Open data files in the HPLC instrument software (e.g., Agilent
ChemStation off-line software).
2. Identify carotenoid species according to their retention time,
spectral features, and ratios of maximum absorption peaks (λ)
(see Notes 11 and 12). A detailed DAD spectra analysis (Fig. 3)
and retention times of some of the identified carotenoids on
C30 and C18 columns have been provided in Tables 1 and 2.
3. Perform peak integration and calculate individual peak area
using HPLC instrument software (see Note 13).
4. If calibration curves and slope were predetermined, use the formula below to calculate carotenoid abundance in grams
per gram fresh weight (g/gfw). Otherwise, perform fraction
collection (step 4) and/or establish standard calibration
curves using commercially available carotenoids (step 15) (see
Note 14).
Formula:
Carotenoid weight g=gfw
ð
Þ¼ Peak area AU
ð
ÞÂ1=Slope
 Dilution factor DF
ð ÞÂg grams
ð
Þ
5. Program HPLC instrument software and fraction collector to
collect individual carotenoid fractions according to their retention times or spectral identification.
6. Collect each fraction in a new dark amber colored glass vial.
7. Validate the purity of carotenoid fractions using HPLC according to the instructions given at step 2.
8. Repeat steps 5 and 6 until pure carotenoid fractions have been
prepared.
9. Evaporate the mobile phase within the collected fractions
under a steam of nitrogen gas.
10. Redissolve carotenoid precipitate in 1 mL of organic solvent
(see Notes 15 and 16).
11. Prepare the spectrophotometer at RT and zero the spectrometer with the same solvent used to dissolve the precipitated
carotenoid (see Note 17).
12. Transfer 1 mL of redissolved carotenoid sample (from step 10)
into a glass or disposable plastic cuvette and measure absorbance at specific wavelengths (λ) (see Table 3) for each carotenoid using a UV-vis spectrophotometer (see Note 18).
13. Calculate the carotenoid concentration using this formula:
Formula:
Absorbance read AU
ð
ÞÂDilution factor DF
ð ÞÂConcentration mg=mL
ð
Þ
A
1%
1cm
Profiling Carotenoids in Plants Using HPLC
153