Investigating carotenoid loss after drying and storage of

58
2. Assessment of Methods
to amber vials before analysis on both spectrophotometric and HPLC methods. For
HPLC analysis samples were diluted in the solution ten times for fresh and twenty times
for dried samples. For UVIKON 933 UV/Visible double beam spectrophotometer
readings, samples were diluted in Petroleum Ether 100 "l/10 ml for fresh samples and 50
"l/10 ml for dried samples respectively.
Spectrophotometric reading
Absorbance at 450 nm was read in PMMA (Poly meta meta acrylate cells, with a range
between 300-700 nm). No difference of reading was noticed when compared with quartz
cells. Petroleum Ether is a volatile solvent and reading had to be carried out quickly in
order to prevent bias and tubes were also sealed with Parafilm. Two readings were taken
on the same sample tube. Total carotenoids levels were determined by comparison with
a standard curve using pure !-carotene (Purity > 97%; Extrasynthese, Genay, France).
Concentration (C in mg.l -1 ) was calculated by Lambert Beer law from the absorbance
(A). The concentration is given by Equation 2-1 (Britton et al. 1995):
3 MA
C = 10 (Equation 2-1)
Lξ
Where: the Molar extinction coefficient for !-carotene in Petroleum Ether (!) is 138900
L.mol. -1 .cm. -1 ; and !-carotene molecular weight (M) is 536.88g.mol -1 . L is the path
length (generally equal to 1 cm -1 ).
An example of standard curve on the spectrophotometer is given in Figure 2-2.
B-carotene content in solution
(mg/L)
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
y = 3.8720x
R 2 = 0.9945
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Absorbance
Figure 2-2: β-carotene standard curve at 450nm absorbance using a UVIKON 933
Spectrophotometer, France