Professional Documents
Culture Documents
2132 Gd-Felsgd, Pf. 7 y Tel: (27) 330 149 y Fax: (27) 336 375
1. Introduction
Confirmation of the presence of aflatoxins in a sample by HPLC
requires derivatisation of the aflatoxins B1 and G1 in order to
enhance their natural fluorescence under UV light and make them
more easily detected. Previously, the only options available for
derivatising aflatoxins involved the use of either trifluoroacetic acid
(TFA) or iodine. Both of these methods are reliable, however they
do have some significant limitations which can be overcome by use
of the Coring Cell.
AF B2
AF G 2
AF B1
AF G 1
400
450
500
nm
Emmissionswellenlnge
The Coring (formerly: CoBrA) Cell is an electrochemical cell which generates the derivatising agent, bromine, from
potassium bromide present in the mobile phase. The derivatisation of aflatoxins occurs rapidly (reaction time is
approximately 4 seconds) at ambient temperature. Further advantages are:
When such a system is combined with the VICAM immunoaffinity columns, a significant amount of time can be saved
since the set up of the HPLC system and the maintenance of equipment is simpler. Optimising a system with such a
combined approach maximises the advantage of investing in an HPLC.
Membrane: Anion
Capillary connections: Valco 1/16"
(do not use metal nuts or tubes
because these can damage the cell!)
HPLC column
Reaction coil
Red contact
Platin electrode
Spacer
Membrane
Fluorescense
detector
Spacer
Stainless steel electrode
Black contact
Waste
2/5
3. Important Notes
Hazard: Aflatoxins are very hazardous substances. Suitable protective clothing, including rubber gloves, safety glasses
and laboratory coats should be worn throughout the assay.
When not using the Coring cell always store it filled with water in order to keep the membrane wet.
Never flush 100% v/v organic solvents through the Coring Cell as this can damage the membrane.
Always ensure there is flow through the Coring Cell before plugging in the current source.
Never turn off the HPLC pump before having unplugged the Coring Cell current source first.
Always use HPLC grade solvents from a quality supplier, and maintain fresh supplies of potassium
bromide.
Never connect metal nuts or tubes directly with the Coring Cell.
Always use plastic tubings (Teflon, PEEK...) for connections. Always keep tube i.ds the same.
Meaning
Error 1 Error 2
on
on
off
on
off
off
correct function
Coring not connected
on
off
on
Short circuit
Repair
-Connect red lead to red terminal and black lead to black
terminal
Connect leads the right way.
f) After the HPLC has been allowed to stabilise (no more baseline drift, 20-30 minutes) the Coring Cell is ready to be
used.
Table 1
0.20mm
0.25mm
0.40mm
0.50mm
0.80mm
0.4ml/min
84.9 cm
54.3 cm
21.2 cm
13.6 cm
5.3 cm
0.5ml/min
106.1 cm
67.9 cm
26.5 cm
17.0 cm
6.6 cm
3/5
0.6ml/min
127.3 cm
81.5 cm
31.8 cm
20.4 cm
8.0 cm
0.8ml/min
169.8 cm
108.6 cm
42.4 cm
27.2 cm
10.6 cm
1.0ml/min
212.3 cm
135.8 cm
53.1 cm
34.0 cm
13.3 cm
5.
HPLC Conditions
Inertsil ODS-2, 15 cm x 4.6 mm x 5m (INODS2-150A)
Inertsil ODS-2, 1 cm x 3.2 mm x 5m; 5/pk. (INODS2-10C)
Guard holder (HI-161) Column coupler (HI-081)
Fluorescense detector 362 nm excitation
440 nm emission
Mobile Phase
To one litre of mobile phase add 119mg of potassium bromide and 100l of
65% nitric acid
Flow rate
1 ml/min
Injection volume
100 l
Elution sequence
Aflatoxin G2, G1, B2, B1
Analytical column
Guard column
6. Derivatisation reaction
O
O
O
O
H
H
+
Br 2
Br
Br
A FLA T O X IN B 1
O
O
+
Br
H
2
Br
Br
A FLA T O X IN G1
4/5
5/5
RP-18
FD-in
Housing
Spacer 1
Platinum electrode
Spacer 2
Membrane
Spacer 2
Grid
Spacer 2
Stainless Steel Electrode
Spacer 1
Housing
FD-out
Waste