Investigating carotenoid loss after drying and storage of

59
2. Assessment of Methods
!-carotene standard curve was determined by plotting five concentration levels (0.8579,
1.7158, 2.1447, 2.5737, 3.4315 mg.l -1 ). A coefficient of correlation of 0.9945 indicating
excellent linearity (y=3.8720x where y is the concentration in mg.l -1 and x the
absorbance). l -1
HPLC analysis
Carotenoids were analysed by reverse-phase high performance liquid chromatography
using an Agilent 1100 system (Massy, France) equipped with autosampler injector,
degasser, pump and photodiode array detector set at 450 nm (Dhuique-Mayer et al.
2005). The mobile phases were distilled water as eluent A, methanol as eluent B, and
MTBE as eluent C. A solvent gradient was programmed in order to enhance compound
separation: 0 min.: 40% A /60% B; 0-5 min.: 20% A/ 80% B; 5-10 min.: 4% A /81% B
/15% C; 10-60 min.: 4% A /11% B /85% C; 60-71 min.: 100% B; 71-72 min. back to
the initial condition for re-equilibration. Carotenoids were separated through a polymeric
C30 reverse phase column (250 x 4.6 mm inner diameter; 5 µm particle size YMC
EUROP GmbH) with a flow rate of 1 ml.min -1 , a column temperature at 25ºC and an
injection volume of 20 µl. The data were stored and processed by Agilent Chemstation
Plus software. Concentrations were determined by comparison to a standard curve using
pure !-carotene (Extrasynthese, Genay, France). An example of a standard curve is
given in Figure 2-3.
!-carotene content (mg/L)
70
60
50
40
30
20
10
0
y = 4.9565E-03x
R 2 = 0.9986
0 2500 5000 7500 10000 12500 15000
Peak area (units)
Figure 2-3: β-carotene standard curve at 450nm absorbance. Agilent 1100 system
HPLC, France