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G.
Chromatography
(a)
Conditions
Analytical column
YMC C30 3 μm, 250 x 2.0 mm
Guard column
YMC C30 3 μm, 10 x 2.0 mm
Column temperature 30 °C
Mobile phases
A: 20 mM ammonium acetate in MeOH
B: MTBE
Gradient
Time (min)
%B
0
5
1
10
10
10
25
100
25.1
5
30
5
Flow rate
0.25 mL/min
Backpressure
ca. 185 bar
Injection volume
5 μL
UV/Visible detection 450 nm
(b)
Resolution between isomers
(1)
Resolution between lutein
cis
and
trans
isomers. – Inject the qualitative
cis
/
trans
lutein
solution and determine the resolution between the two major
cis
isomers and all-
trans
-
lutein.
(2)
Resolution between β-carotene
cis
and
trans
isomers and α-carotene. – Inject the β-
carotene system suitability solution and determine the resolution between the two major
cis
isomers, all-
trans
-β-carotene, and α-carotene.
(3)
Resolution between all-
trans
-lutein, zeaxanthin, and apocarotenal. – Inject the mixed
carotenoid working solution and determine the resolution between all-
trans
-lutein,
zeaxanthin, and apocarotenal.
(c)
Calibration. – Inject the mixed carotenoid WS three times before and after each set of sample
injections. Calculate the response factor based on average peak areas of analytes and the
internal standard.
H.
Calculations
(a)
Determine the purity of lutein and β-carotene standards by first determining the
spectrophotometric purity and then the chromatographic purity of each. The overall purity is
calculated as the product of the two measured purities
(1)
Spectrophotometric Purity (SP)
Measure each standard measuring solution against an MTBE blank at its absorbance maximum
(444 nm for lutein and 450 nm for β-carotene). Calculate the spectrophotometric purity of each
reference standard as the observed absorbance over the expected absorbance:
SP = (Abs
MS
x 100 x 1000)/(E
1%,1cm
x W)
Carot-02 (February 2016)
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