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112-R3
1. Scope
This
analysis
preparations
method
is
applied
to
food
require
the
cocoa.
Determination of the theobromine and caffeine
using
high
performance
liquid
chromatograph.
(3)
water:
4. Reagents
All chemicals must be JIS special reagent grade or
contents
phase,
procedure is as follows.
(2)
mobile
(1)
settings:
Theobromine
(b)
Caffeine
(c)
-Hydroxyethyltheophylline
(1,3-Dimethyl-7-2[2-hydroxyethyl]xanthine)
(d)
Petroleum ether
(e)
Deproteining agent
A: Dissolve 2 g of zinc sulfate heptahydrate
(ZnSO47H2O) in water and bring to 100 ml
3. Apparatus
follows:
(a) Separation column, Zorbax ODS, 4.6 mm I.D. 250
mm, or equivalent column
(b) Column temperature, 25C
(c) Mobile phase, water : acetonitrile (85 : 15) (Both
HPLC grade)
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JCAM No.112-R3
a 1,000-ml brown volumetric flask and dissolve.
(approximately
6,300g)
for
15
min.
After
stirring occasionally.
paper (e.g., JIS P 3801 Class 5, No. 5C) while still warm.
Note 2)
Kinds of substances
Cocoa powder
0.1
Cacao mass
0.2
Chocolate
0.3
Milk chocolate
0.3
Chocolate sandwich
0.5
Chocolate milk
1.0
1.0
Note 3)
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JCAM No.112-R3
7. Procedure
7.1. Preparation of calibration curve
Inject 20 l of each standard solution prepared in 5
(Wx/Ws) Ms
S 1000
100
Where -
Wx/Ws:
Ms:
S:
Are a ratio
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
formula:
curve
Weight ratio
(Wx/Ws)
0.500999
0.751499
1.001998
1.502997
2.504995
Where-
Weight ratio
A re a ratio
curve
Weight ratio
(Wx/Ws)
0.1001
0.2002
0.3003
0.4004
0.5005
(%)
in
test
sample
content
0.2
Caffeine
9. References
y = 1.1637x - 0.0032
R 2 = 1.0000
0.1
C:
determined in 7.2.
0.0
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
T:
0.5
0.6
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