R-Biopharm KOBRA CELL User manual

R-Biopharm Rhône Ltd
R-Biopharm – for the safety of your analysis.
KOBRA®CELL Art. No. K01
Instruction Manual

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Contents
1. Introduction to the KOBRA®CELL .................................................................................................... 4
1.1 Intended Use ................................................................................................................................. 4
1.2 Derivatisation Reaction .................................................................................................................. 5
2. KOBRA®CELL Contents..................................................................................................................... 6
2.1 Contents........................................................................................................................................ 6
2.2 Reagents Required......................................................................................................................... 6
2.3 Accessory Products ........................................................................................................................ 6
2.4 Hazards ......................................................................................................................................... 6
2.5 Decontamination........................................................................................................................... 6
3. Assembly of the KOBRA®CELL.......................................................................................................... 7
3.1 Components ................................................................................................................................. 7
3.2 Precautions When Assembling....................................................................................................... 8
3.3 Assembly ....................................................................................................................................... 8
4. KOBRA®CELL Installation.................................................................................................................. 9
4.1 Power Pack Notes .......................................................................................................................... 9
4.2 Power Pack Technical Specification ................................................................................................ 9
4.3 Installation Guide .........................................................................................................................10
4.4 Installation Instructions ................................................................................................................11
4.5 Precautions to Take Before Running the KOBRA® CELL...................................................................12
4.6 Operating the KOBRA® CELL .........................................................................................................12
4.7 Error LED ......................................................................................................................................12
5. HPLC Conditions ............................................................................................................................. 13
6. KOBRA®CELL Maintenance ............................................................................................................ 14
6.1 Storage.........................................................................................................................................14
6.2 Daily Cleaning ..............................................................................................................................15
6.3 Monitoring Performance...............................................................................................................16
6.4 Changing the Membrane..............................................................................................................16

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7. Troubleshooting.............................................................................................................................. 17
7.1 No Peaks Observed.......................................................................................................................17
7.2 Reduced / No Aflatoxin B1 and G1 Peaks Observed ......................................................................18
7.3 Abnormal Peaks Observed............................................................................................................19
7.4 Error Light Observed on Power Pack ............................................................................................ 20
7.5 Leakage from KOBRA® CELL ......................................................................................................... 20
7.6 KOBRA® CELL Causing High Pressure on the HPLC ....................................................................... 20
7.7 Storage Plug Broken Off or Lodged in KOBRA® CELL .................................................................... 20
8. General Information ....................................................................................................................... 21
8.1 Quality .........................................................................................................................................21
8.2 Technical Support.........................................................................................................................21
8.3 Warranty.......................................................................................................................................21

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1.1 Intended Use
Confirmation of the presence of aflatoxins in a
sample by HPLC requires derivatisation of aflatoxins
B1 and G1 in order to enhance their natural
fluorescence and make them more easily detected.
Previously, the only options available for derivatising
aflatoxins involved the use of trifluoroacetic acid
(TFA), pyridinium bromide perbromide (PBPB)
or iodine. All of these methods have significant
limitations which can be overcome by use of the
KOBRA® CELL.
Pre-column derivatisation with TFA requires the
solution containing aflatoxin to be blown to dryness
under a stream of nitrogen, potentially leading to a
loss of toxin. Further limitations are that the reaction
takes 30 minutes at 50 °C, and the TFA reagent is
itself corrosive and harmful to handle.
The post-column PBPB method involves addition of
the diluted reagent into the eluate from the HPLC
column. The limitations of this method are the use
of a second pump and the difficulty in dissolving the
PBPB as well as the hazardous nature of the reagent.
Post-column derivatisation with iodine also has
some limitations including the need for a second
pump, water bath or oven which can be expensive.
It is necessary to clean the equipment regularly in
order to avoid iodine crystals forming inside the
reaction coil. Finally, the iodine must be prepared
fresh each day due to its unstable nature.
The KOBRA® CELL overcomes the problems relating
to alternative derivatisation procedures. It is an
electrochemical cell connected to an HPLC system
downstream from the HPLC column and in line with
the column effluent and the fluorescence detector.
The KOBRA® CELL generates a reactive form of
bromine for derivatisation of aflatoxins B1 and G1,
resulting in enhanced fluorescence and thus more
sensitive detection.
The KOBRA®CELL is used by hundreds of labs
around the world and is mentioned in several EU
and other international standard methods.
1Introduction to KOBRA®CELL

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1.2 Derivatisation Reaction
The aflatoxins and the mobile phase enter the
KOBRA® CELL and the electrochemical reaction
occurs generating the reactive form of bromine.
The reaction between the reactive bromine and the
aflatoxins must take place before the derivatised
aflatoxins enter the fluorescence detector. Hence
the length of the reaction coil is critical. A minimum
reaction time of 4 seconds is required. Aflatoxin G1
Aflatoxin B1
+ Br2
+ Br2
H
H
H
H H
H
H
H

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2.1 Contents
•1 x KOBRA® CELL
•1 x Power Pack (including 1 red and 1 black
connection lead)
•1 x Electrical adapter (with various adapters)
•1 x 1 m length of 0.5 mm ID PEEKTM tubing
•1 x Spare membrane
2.2 Reagents Required
•Distilled Water
•Methanol
•Potassium Bromide
•Nitric Acid
2.3 Accessory Products
•1 x KOBRA® CELL Installation Pack
(Product Code: K03)*
•Guard column: Inertsil ODS-3, 5 µm,
4 mm x 10 mm or equivalent
*Available from R-Biopharm. Please
contact your local distributor for further
information.
2.4 Hazards
Mycotoxins are very hazardous substances. Only
laboratories equipped to handle toxic materials
and solvents should perform analyses. Suitable
protective clothing, including gloves, safety glasses
and lab coats should be worn throughout the
analysis.
2.5 Decontamination
Prior to disposal, excess standard solutions should
be treated with at least one-tenth their volume of
5 % sodium hypochlorite. Labware and
contaminated waste should be immersed in
5 % sodium hypochlorite solution for 30 minutes
followed by the addition of 5 % acetone for
30 minutes. Flush with copious amounts of water
before disposal. After decontamination labware
should be thoroughly washed. Incinerate waste if
regulations permit.
2KOBRA®CELL Contents

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3.1 Components
3Assembly of the KOBRA®CELL
1. 6 locating screws
2. White plastic housing with red
terminal (top)
3. PTFE spacer with reaction
area: 0.1 mm
(K09)
4. Platinum electrode
(K05)
5. PTFE spacer: 0.25 mm
(K07)
6. Ion exchange membrane
(K02)
7. PTFE spacer: 0.25 mm
(K07)
8. Spacer grid
(K10)
9. PTFE spacer: 0.25 mm
(K07)
10. Stainless steel electrode
(K04)
11. PTFE spacer: 0.1 mm
(K08)
12. White plastic housing with
black terminal (bottom)
Fig 1. KOBRA®CELL Components
11
2
3
4
5
6
7
8
9
10
11
12
Contact
Area for
Electrodes
Contact
Area for
Electrodes

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3.2 Precautions When
Assembling
•To reassemble the KOBRA® CELL it is important to
tighten the locating screws at opposite corners
before tightening the middle screws. This will
ensure that the KOBRA® CELL is correctly
assembled and will not leak.
•Do not over tighten locating screws as this will
damage the threads.
•Never use metal nuts or ferrules directly with the
KOBRA® CELL.
•Use a guillotine tubing cutter to prevent distortion
of the diameter of the PEEKTM tubing used when
assembling the KOBRA® CELL.
Topside view of all
KOBRA® CELL electrodes,
membranes and spacers.
Smallest fitting hole for location peg on left
side.
Largest fitting hole for location peg on right
side.
Corner‚ cut out‘ on bottom right.
3.3 Assembly
Fig 2. Assembly of KOBRA®CELL

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4.1 Power Pack Notes
It should be noted that the KOBRA® CELL power
pack has been improved and as such is of a slightly
different design. The new power pack is still 9 V and
is supplied with an adapter for use in the UK, USA,
Europe and Australia. The plug can be converted
by depressing the button located at the top of
the adapter using a screwdriver. This releases the
adapter that is in position. Slide to remove. To attach
the required adapter place on the two pins and slide
to connect. The power pack can now only be used
at 100 µA and there is a light to show it has been
switched on (top right). There is also an error light
to indicate if there is a problem (bottom left).
4.2 Power Pack Technical
Specification
Power Supply Primary 110 - 240 V
Secondary 9 V
Exit Power
Current Source
100 µA
Maximum
Output Power
9 V
Loading Time
NICAD 9 V
Approximately 1 hour
15 minutes.
Accuracy 0.5 % (This is an improvement
on the previous model which
was 2 %.)
Working Time
with Battery
20 hours
Approval CE approval conforms to
European Directives 73/23/EEC
and 89/336/EEC.
4KOBRA®CELL Installation

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4.3 Installation Guide
Column
Detector Inlet Detector Outlet
Detector
Reaction Coil
Outlet
Inlet
Red Terminal
Black Terminal
Inlet Outlet
Waste
Working Electrode
(platinum)
Spacer
Membrane
Spacer
Counter Electrode
(Stainless Steel)
Membrane: Reichelt Thomapor Anion.
Capillary connections: Valco 1/16 inch (do not use metal nuts or ferrules because these can
damage the electrochemical properties of the cell).
Working pressure: Max 2 bar.
Fig 3. KOBRA®CELL Installation

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4.4 Installation Instructions
Caution: Do not over tighten. Cell connections
should be made with plastic ferrules, not metal.
1. Unpack the KOBRA® CELL and check that all of
the components are present.
2. Add 119 mg of potassium bromide and 350 µl of
4 M nitric acid to 1 l of mobile phase.
3. Immerse the tubing from the HPLC pump into
the mobile phase.
4. Disconnect the storage plugs and capillary
tubing from the KOBRA® CELL. Connect the
HPLC column discharge to the KOBRA® CELL
inlet using plastic ferrules (hand tightened) and
a length of PEEKTM tubing supplied with the kit.
5. Connect the KOBRA® CELL outlet using ferrules
(hand tightened) and a length of PEEKTM tubing
to the detector inlet according to the table
below.
Caution: Use a guillotine tubing cutter to
prevent distortion of the diameter of the tubing.
6. Connect the detector outlet to the KOBRA® CELL
inlet using plastic ferrules (hand tightened) and
PEEKTM tubing.
7. Connect the KOBRA® CELL outlet to waste using
plastic ferrules (hand tightened) and PEEKTM
tubing.
8. Switch on the HPLC pump and flush mobile
phase through the system for approximately
5 minutes.
9. Connect the KOBRA® CELL power pack to the
cell, red lead to red terminal and black lead to
black terminal.
Caution: Do not switch on without mobile
phase flowing through the KOBRA® CELL or the
membrane will be damaged.
10. After the HPLC has been allowed to stabilise
(approximately 30 minutes) the KOBRA® CELL is
ready to use.
Determination of Reaction Coil / PEEKTM Tubing Length
Flow Rate 0.5 ml/min 0.6 ml/min 0.7 ml/min 0.8 ml/min 0.9 ml/min 1.0 ml/min
Internal
diameter
of reaction
coil /
PEEKTM
tubing (id)
0.2 mm 106.1 cm 127.3 cm 148.5 cm 169.8 cm 191.0 cm 212.2 cm
0.25 mm 67.9 cm 81.5 cm 95.1 cm 108.6 cm 122.2 cm 135.8 cm
0.4 mm 26.5 cm 31.8 cm 37.1 cm 42.4 cm 47.7 cm 53.1 cm
0.5 mm 17.0 cm 20.4 cm 23.8 cm 27.2 cm 30.6 cm 34.0 cm
0.6 mm 11.8 cm 14.1 cm 16.5 cm 18.9 cm 21.2 cm 23.6 cm
0.8 mm 6.6 cm 8.0 cm 9.3 cm 10.6 cm 11.9 cm 13.3 cm
1.0 mm 4.2 cm 5.1 cm 5.9 cm 6.8 cm 7.6 cm 8.5 cm

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4.5 Precautions to Take Before
Running the KOBRA®CELL
•This device has only to be used with the supplied
power pack.
•Always ensure there is a mobile phase flowing
through the KOBRA® CELL before switching on
the current source.
•Do not switch on without mobile phase flowing
through the KOBRA® CELL or the membrane will
be damaged.
•It is advised to run the mobile phase through the
KOBRA® CELL for 30 minutes prior to analysis.
•Always turn off the KOBRA® CELL power pack
before turning off the HPLC pump.
•Always use HPLC grade solvents (with minimal
benzene content) from a quality supplier and
maintain fresh supplies of potassium bromide.
•Never flush 100 % solvent through the
KOBRA® CELL as this can damage the membrane.
4.6 Operating the KOBRA®CELL
The KOBRA® CELL power pack is turned on by
moving the switch to the ‘On’ position. To shut
down the device move the switch to the ‘Off’
position.
4.7 Error LED
•The LED will light up if there is no KOBRA® CELL
attached to the current source.
•The LED will also light up when the KOBRA® CELL
is dry and the flow of the current source is
inhibited.
Power Light
Error Light

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5HPLC Conditions
HPLC Conditions
Derivatisation KOBRA® CELL at 100 µA setting
Guard Cartridge Inertsil ODS-3
5 µm, 4 mm x 10 mm or (Hichrom) equivalent
Analytical Column Inertsil ODS-3V
5 µm, 4.6 mm x 150 mm (Hichrom) or equivalent
Mobile Phase Water : Methanol (60 : 40 v/v)
Add 119 mg of potassium bromide and 350 µl 4 M Nitric Acid to 1 litre of mobile
phase
HPLC Pump To deliver mobile phase
Flow Rate 1.0 ml/minute
Fluorescence
Detector
Excitation: 362 nm
Emission: 425 nm (B1 and B2)
455 nm (G1 and G2)
Column Heater Maintain guard and analytical columns at 40 °C
Integrator / Data
Control System
From preferred supplier
Injector Autosampler / Reodyne valve
Injection Volume 100 µl
Elution Order G2, G1, B2, B1.

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6.1 Storage
•The KOBRA® CELL should be switched off and
disconnected for washing and storage (Fig. 5).
•Always store the KOBRA® CELL filled with water in
order to keep the membrane wet.
Fig 4. Storage of KOBRA®CELL
To fill the KOBRA® CELL with water:
1. Fill a syringe with 5 - 10 ml of water and attach
the tip of the syringe to a pipette tip.
2. Unscrew one of the storage plugs / ferrules from
one inlet / outlet position of the KOBRA® CELL,
leaving the other inlet / outlet positions sealed
with storage plugs or ferrules.
3. Using the syringe with the pipette tip, insert this
into the empty inlet / outlet position on the
KOBRA® CELL and fill the cell with water until
water overflows from the syringe connection.
4. When finished, reinsert the storage plug / ferrule
to contain the water within the cell and keep the
membrane wet.
6KOBRA®CELL Maintenance
Detector
HPLC Column
Out
Solvent waste
KC # 123
KC # 123
Connect
directly to the
detector
Dead end
ferrule to seal
port
KOBRA
®
CELL
waste
Inlet
Fig 5. KOBRA®CELL Switched Off for Washing and Storage

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6.2 Daily Cleaning
The HPLC system can be left running overnight at
a reduced flow rate (e.g. 0.1 ml/min) and with the
fluorescence detector and KOBRA® CELL power
source turned off, if necessary. Alternatively, a better
practice to prolong the life of the system is to clean
each day, as follows:
1. Turn off KOBRA® CELL power pack and
fluorescence detector.
2. Switch off HPLC pump.
3. Disconnect the KOBRA® CELL from the HPLC
system and reconnect the HPLC column directly
to the fluorescence detector.
4. Change the mobile phase to 5 % solvent and put
the HPLC column discharge to waste.
5. Switch on the HPLC pump and flush the system
for at least 30 minutes before switching off the
system.
6. Manually flush the KOBRA® CELL through with
distilled water several times using a syringe.
7. Store the KOBRA® CELL overnight with water
inside the cell by closing off both ports on each
housing using the storage plugs and capillary
tubing supplied with the cell.
8. The next day, change the mobile phase from
5 % solvent back to the normal mobile phase of
potassium bromide and nitric acid, and flush the
HPLC system through for at least 30 minutes
before re-connecting the KOBRA® CELL.
Caution: 100 % acetonitrile will damage the
KOBRA® CELL membrane.
9. Reconnect the KOBRA® CELL (Fig. 6).
Detector
HPLC Column
Out
Solvent waste
KC # 123
KC # 123
‘Fingertight’
Fittings
‘Union’Fittings
Outlet
Inlet
Fig 6. KOBRA®CELL Switched On for Analysis

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6.3 Monitoring Performance
It is necessary to regularly monitor the performance
of the KOBRA® CELL in order to detect any
deterioration in the membrane contained within.
The performance should be checked at the time of
installation and then weekly by comparing the peak
areas of a known aflatoxin standard. The same should
also be done in order to monitor the deterioration of
the lamp in the detector.
Deterioration of the membrane will occur over a
period of time depending upon the frequency of use
and the type of samples analysed (Fig. 7). When the
performance becomes unacceptable the membrane
should be replaced (Product Code: K02).
Normally, it is found that even under extreme
workloads the membrane does not need to be
replaced for at least 6 months or 1,000 injections.
Fig 7. Deterioration of KOBRA®CELL Membrane
6.4 Changing the Membrane
Change the membrane when a decrease in
performance is observed which cannot be attributed
to the detector lamp. Additional membranes are
available separately (K02).
1. Using a screw driver take off the white plastic
housing of the KOBRA® CELL by removing the
6 locating screws.
2. Carefully separate the top white plastic housing,
and remove in turn the internal layers of the cell
using tweezers. Make a note of the position and
orientation of each layer as it is removed.
3. Continue removing the internal layers until the
membrane is exposed. Before removal inspect
the membrane for damage, discolouration and
orientation. During the life of the KOBRA® CELL
the bromination reaction causes the centre of
the membrane to lose it’s colour. No holes
should be visible in the membrane other than
the pre-cut holes for the locating pins at either
end of the membrane. Note the orientation of
the membrane.
4. Remove the membrane from the KOBRA® CELL
ready for replacement using tweezers.
5. Carefully remove the spare membrane, holding
it by one end with the tweezers rather than the
middle.
6. Position the replacement membrane over the
different sized locating pegs, according to the
size of pre-cut holes in the membrane.
Caution: Do not allow the membrane to dry
out. Add distilled water if necessary.
7. Carefully replace the layers to be positioned on
top of the membrane, remembering their correct
order and orientation.
8. Secure the layers using the 6 locating screws.

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7Troubleshooting
7.1 No Peaks Observed
•If no peaks are observed on the HPLC
chromatogram it is probably not the KOBRA® CELL
that is at fault (Fig. 8). There may be a problem
with a component of the HPLC system or with the
standards used.
•Check that the bulb in the lamp does not need
replaced. It should be replaced every 1,000 - 2,000
hours depending on the detector.
•Check there is not a problem with the detector.
•Check the standard has been prepared correctly,
or the standard has not deteriorated. Standards
should be prepared fresh every day.
•Check the PEEKTM tubing leading to and from the
KOBRA® CELL is not blocked or distorted. Replace if
required.
•Check the internal components of the
KOBRA® CELL have been inserted in the correct
order, particularly if no peaks are observed
following the replacement of one of the
components (Fig. 1).
Fig 8. No Peaks Obtained in Chromatogram

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7.2 Reduced / No Aflatoxin B1
and G1 Peaks Observed
•Check the KOBRA® CELL is connected correctly to
the HPLC (Fig 6).
•Check the connections to the KOBRA® CELL from
the power pack.
•Check the KOBRA® CELL membrane. The
membrane is the site of the actual derivatisation
reaction (ion-exchange occurs on the membrane).
The membrane starts a deep amber colour and,
through time and use, the centre of the membrane
becomes discoloured, eventually turning a pale
colour. The pale colour indicates that the
membrane will need replaced.
•Check there is power in the power pack. Check
that the power pack has been turned on.
•Check the internal components of the
KOBRA® CELL are in the correct order (Fig. 1), or
that there is not a blockage in the system.
•No potassium bromide (or not enough potassium
bromide) was added to the mobile phase. Prepare
again.
•No nitric acid was added to the mobile phase.
Prepare again.
Fig 9. Reduced / No Aflatoxin B1 and G1 Peaks Observed

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7.3 Abnormal Peaks Observed
•If abnormal peaks are observed on the HPLC
chromatogram it is not normally the KOBRA® CELL
that is at fault (Fig. 10).
•Check the correct mobile phase has been used.
•Check the correct HPLC column has been used.
•Check the internal components of the
KOBRA® CELL have been inserted in the correct
order (Fig. 1).
•Analytical column could need changed and may
result in poor peak resolution or peak tailing.
Fig 10. Abnormal Peaks Obtained in Chromatogram