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Coring Cell

An Electrochemical Cell for the


Derivatisation of Aflatoxins
Code 3200130
Release Julyr 2005

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

TFA method as pre-column derivatising technique requires a very


accurate reaction performance and results in Aflatoxin derivatives
which do elute from HPLC very early and therefore can be easily
covered by matrix peaks usually eluting at retention times similar to
Aflatoxin B1 and G1.
Iodine derivatisation as a post column technique does have other
disadvantages like longer reaction time at higher temperatures (peak
broadening), additional HPLC pump necessary and daily preparation
of the corrosive iodine solution.

AF B1

AF G 1

400

450

500

nm

Emmissionswellenlnge

Fluorescense spectrum of underivatised Aflatoxins

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:

no daily preparation of derivatising reagent necessary


no stability problems of mobile phase even after addition of potassium bromide and nitric acid
no handling with halogeneous solutions (f.e. iodine)
no additional pump for addition of derivatising reagent
no corrosion of pumps
low maintenance costs
high increase of Aflatoxins fluorescence activity
derivatisation in 4 seconds at ambient temperature, no heating block necessary
simple to install / uninstall
high reproducibility of results

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

Working pressure: max. 2 bar

Membrane
Fluorescense
detector

Spacer
Stainless steel electrode

2. Coring Cell Components


1 x Coring Cell
1 x Current Source
1 x Electrode Connection Lead
1 x Spare Membrane

Black contact

Waste

Figure 1: Diagram of the CoBrA Cell Connections

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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.

4. Installation of the Coring Cell


a) Prepare one litre of mobile phase, methanol: acetonitrile: water (20:20:60 v/v), containing 119mg of potassium
bromide and 100l of 65% nitric acid. Connect the reservoir of freshly prepared mobile phase to the HPLC, by
immersing the tubing of the HPLC pump feed into the reservoir.
b) Disconnect all capillary tubing from the Coring Cell and connect the HPLC column with a plastic tubing to the
Coring Cell inlet (marked RP18). Connect Coring Cell outlet (marked FD-in) with a plastic tubing to the
detector inlet - this length of tubing is critical, and the length is dependent upon flow rate and internal diameter (i.d)
of the tubing. Length calculation: see equation and table 1.
c) Connect the detector outlet with a plastic tubing to the Coring Cell inlet (marked FD-out) and the Coring Cell
outlet (marked waste) with a plastic tubing to waste. All connections are made to hand tightness (CAUTION: do
not over tighten!) and should be plastic not metal or stainless steel.
d) Switch on HPLC pump.
e) Connect the Coring Cell current source to the cell, red lead to red terminal and black lead to black terminal. Plug in
current source and switch it on . One green and 2 red LED lights on the current source indicate the functions as
listed in the table:
LED
OK

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.

Equation: L[cm] = 8.5 x Flowrate [ml/min] / ID [mm]

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

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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

7. Daily Cleaning Routine for the Coring Cell


The HPLC system can be left running overnight at a reduced flow rate (eg 0.1ml/min) and with the fluorescence
detector and Coring Cell current source unplugged. Alternatively, a better practice to prolong the life of the system is
to clean each day, as follows:
1.
2.
3.
4.
5.
6.

Switch off Coring Cell current source and fluorescent detector.


Switch off HPLC pump.
Disconnect Coring Cell from HPLC system, and reconnect HPLC column directly to fluorescence detector.
For washing the HPLC column change the mobile phase to acetonitrile (100% v/v).
Switch on the HPLC pump and flush system for at least 30 minutes before switching "off" system.
Meanwhile, manually flush Coring Cell through with 5-10 mls distilled water using a syringe. Then store Coring
Cell with water inside the cell by closing off both ports on each housing.

8. Monitoring the Sensitivity of the Coring Cell


It is necessary to regularly monitor the performance of the Coring Cell in order to detect any deterioration in the
membrane contained within. The sensitivity should be checked at the time of installation and then weekly by comparing
the peak areas of a known aflatoxin standard solution from one week to another.
Deterioration will occur over a period depending upon the frequency of use and the type of samples analysed. When the
level of sensitivity becomes unacceptable the membrane should be replaced.
Normally, it is found that even under extreme workloads the membrane need not be replaced for at least 6 months.

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9. Maintenance: Changing the Membrane


Change the membrane when a decrease in sensitivity
is observed. Additional membranes are available
separately.
Using the integrated hexagon key remove the six
locating screws in the top housing over cross.
Carefully separate the top housing, and remove in turn
the internal layers of the cell using forceps. Make a
note of the position and orientation of each layer as it
is removed. Continue removing the internal layers
until the brown membrane is exposed.
Remove the old membrane and replace it by a new
membrane just having taken it out of the storage
bottle. Take care not to touch the middle of the
membrane with hands or fingers. DO NOT ALLOW
THE MEMBRANE TO DRY-OUT. ADD
DISTILLED WATER IF NECESSARY.
Carefully replace the layers to be positioned on top of
the membrane remembering their correct order and
orientation. Secure the layers in by re-fitting the six
locating screws in the housing over cross. Tighten the
screws just by hand, do not over tighten the screws.
Regularly monitor the sensitivity of the Coring Cell
in order to detect any significant deterioration of the
membrane contained within.

10. Chromatogram of Total Aflatoxin Standard

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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

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