Antec Scientific SenCell User manual

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SenCellTM
Electrochemical Flow cell
User manual
116.0010, Edition 1, 2012
Antec Scientific
Industrieweg 12
2382 NV Zoeterwoude
The Netherlands

Copyright ©2012, Antec, The Netherlands. Contents of this publication may not be reproduced in any form or by any means
(including electronic storage and retrieval or translation into a foreign language) without prior agreement and written consent from the
copyright of the owner. The information contained in this document is subject to change without notice.
ROXY, ALEXYS, DECADE, DECADE II, INTRO, Flexcell, ISAAC, HyREF, SenCell aretrademarks of Antec. Whatman™ (word and
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property of their respective owners.
Thesoftware and the information provided herein is believed to be reliable. Antec shall not be liablefor errors contained herein or for
incidental or consequential damages in connection with the furnishing,performance, or use of software orthis manual. All use of the
software shall be entirely at the user’s own risk.

INTRODUCTION Table of contents 3
Symbols
The following symbols are used in this guide:
The warning sign denotes a hazard. It calls attention to a
procedure or practice which, if not adhered to, could result
in severe injuryor damage or destruction of parts or all of
the equipment. Do not proceed beyond a warning sign until
the indicated conditions are fully understood and met.
The attention sign signals relevant information. Read this
information, as it might be helpful.
The note sign signals additional information. It provides
advice or a suggestion that may support you in using the
equipment.
Intended use
This hardware should be used by trained laboratory personnel only
with a completed degree as chemical laboratory technician or
comparible vocational training. The operator should have fundamental
knowledge of liquid chromatography.
Use proper eye and skin protection when working with solvents.
Additional safety requirements or protection may be necessary
depending on the chemicals used in combination with this equipment.
Make sure that you understand the hazards associated with the
chemicals used and take appropriate measures with regards to safety
and protection.

4SenCell flow cell user manual, ed. 1
(U) HPLC: (Ultra) High Performance Liquid Chromatography (HPLC) is a
method for separating substance mixtures, determining substances and
measuring their concentration. This device is suitable for high-performance
liquid chromatography. It is suitable for laboratoryuse, for analyzing
substance mixtures that can be dissolved in a solvent or solvent mixture.
Check intended use: Only use the device for applications that fall within the
scope of the specified intended use. Else the protective and safety
equipment of the device could fail.
Laboratory use:
•Biochemistry/bioanalytical analyses
•Chiral analyses
•Food analyses
•Pharmaceutical analyses
•Environmental analyses
•Clinical analyses (research purpose only)
With respect to clinical analyses the device is intended for research purposes
only. While clinical applications may be shown, this device is not tested by
the manufacturer to comply with the In Vitro Diagnostics Directive.
Laboratory regulations
Observe national and international regulations pertaining to laboratory work!
For example:
•Good LaboratoryPractice (GLP) of the American Food & Drug
Administration
•For development of methods and validation of devices:
•Protocol for the Adoption of Analytical Methods in the
•Clinical ChemistryLaboratory, American Journal of Medical
Technology, 44, 1, pages 30–37 (1978)
•Accident prevention regulations published bythe accident insurance
companies for laboratory work

INTRODUCTION Table of contents 5
Solvents
Organic solvents are highly flammable. Since capillaries can detach from
their screw fittings and allow solvent to escape, it is prohibited to have any
open flames near the analytical system!
Regularly check for leaks and clogged LC tubing and connections. Test back
pressure without column. Do not close or block drains or outlets. Do not allow
flammable and/or toxic solvents to accumulate. Follow a regulated, approved
waste disposal program. Never dispose of such products through the
municipal sewage system.
Toxicity: Organic solvents are toxic above a certain concentration.
Ensure that work areas are always well-ventilated! Wear protective
gloves, safety glasses and other relevant protective clothing when
working on the device!

6SenCell flow cell user manual, ed. 1

INTRODUCTION Table of contents 7
Table of contents
I N T R O D U C T I O N
Symbols 3
Intended use 3
Laboratory regulations 4
Solvents 5
Table of contents 7
The electrochemical flow cell 9
Introduction 9
Three-electrode configuration 10
Working electrode 11
Detection limit 12
Cell working volume adjustment 13
Reference electrodes 18
ISAAC reference electrode 18
Salt bridge Ag/AgCl reference electrode 20
HyREF reference electrode 20
Installation 22
Introduction 22
Adjusting the SenCell working volume 23
Installation in LC system 24
Maintenance 28
Assembling/Disassembling the Cell 28
HyREF 30
ISAAC 30
Polishing 30
Coating with ISAAC solution 31
Ag/AgCl salt bridge 32
Saturation and air bubbles 32
Material 33
Procedure 33
Maintenance of the cotton wool frit 34
Working electrode 35
Polishing 35
Specifications 38
Part list 40
Index 42

8SenCell flow cell user manual, ed. 1

CHAPTER 1 The electrochemical flow cell 9
C H A P T E R 1
The electrochemical flow cell
Introduction
Congratulations on your purchase of the SenCellTM, a new electrochemical
flowcell for (U)HPLC with ECD. The SenCell has several unique features
(Patent Pending) like a stepless adjustable working volume (spacerless
concept) and toolless assembly.
The SenCell is available with a glassy carbon working electrode (WE). The
SenCell design eliminates the use of plastic/metal spacers. The working
volume of the electrochemical cell can be stepless adjusted without opening
the cell bymeans of a special key, allowing easy optimization of the detection
sensitivity for anyLC application. The working volume can be adjusted
between roughly 0 –300 nL (based on a 2 mm diameter WE). As a standard,
the salt bridge Ag/AgCl reference electrode is advised. For special
applications the HyREF™reference electrode is available. A third reference
electrode is the in situ Ag/AgCl (ISAAC™).
Fig. 1. Left side: assembled SenCell electrochemical flow cell with ISAAC
inlet block (green). The upper part, the inlet block, is separated from the
working electrode block. Right side: SenCell WE block. .
The SenCell has been developed for ultra-trace analysis in standard,
microbore and capillaryLC-EC. After extensive testing it was established that
the confined wall-jet configuration gave the very best results. In addition it
was found that the electrode materials qualityand the finishing of the
electrodes in the flow cell are decisive factors for the performance of an EC
detector.While competitive designs usually deteriorate when in use, this flow

10 SenCell flow cell user manual, ed. 1
cell, by design, improves in performance. The flow cell permit unusuallyshort
stabilisation times: trace analysis within a few hours after starting up may be
expected.
Three-electrode configuration
In the SenCell flow cell a three-electrode configuration is used (Fig. 2). The
working potential is set between the working electrode (WE) and the auxiliary
electrode (AUX). The AUX is kept at a preciselydefined reference electrode
(REF) potential by means of the so-called voltage clamp. This is an electronic
feedback circuit that compensates for polarisation effects at the electrodes.
At the WE, which is kept at virtual ground, the electrochemical reaction takes
place, i.e. electrons are transferred at theWE. This results in an electrical
current to the I/E converter, which is a special type of operational amplifier.
The output voltage can be measured by an integrator or recorder.
Fig. 2. Schematic representation of an electrochemical cell with a three-
electrode configuration.
Essentially, for the oxidation or reduction reaction it would be sufficient to use
only two electrodes. However, the three-electrode configuration has several
advantages over a two-electrode configuration. If the working potential would
be applied only over an AUX versus the WE (without REF), the working
potential would continuously change due to polarisation effects at the
electrodes, resulting in highly unstable working conditions.

CHAPTER 1 The electrochemical flow cell 11
If the working potential would be applied only over the REF versus the WE
(without AUX), the working potential would be very well defined. However,
the potential of a REF is only well defined if the current drawn is extremely
low (pico-amperes) resulting in a very limited dynamic range.
A three-electrode configuration, combines the best of both electrodes. The
REF stabilises the working potential and the AUX can supply high currents.
This results in the tremendous dynamic range of a three-electrode system.
Working electrode
Electrochemical detection puts high demands on the WE material. The WE
should be made of a (electro-)chemically inert material. Furthermore, to avoid
an irregular flow profile over the electrode, it should have a very well defined
surface. Finally, it is important that the analyte of interest can be oxidised (or
reduced) with favourable I/E characteristics. This in fact means that a high
signalmust be obtained at a low working potential. For most applications
glassy carbon will be the WE material of choice. The SenCell is currently
available with 2 mm diameter Glassy Carbon electrode only. Under certain
circumstances other materials are favourable.
For example, for the analysis of iodide a silver WE can be used. At the silver
WE the following oxidation reaction occurs for iodide:
Ag + I-→AgI + e-
This reaction alreadytakes place at a very low working potential (1 mV !),
which results in an extremelyhigh selectivity. This allows the determination of
iodide in urine samples to take place almost without any sample pre-
treatment.
Table I. Working potential limits and application area for different WE
materials.
WE material
potential limits (V)
major application
alkaline
acidic
Glassy carbon
-1.50
+0.60
-0.80
+1.30
catecholamines
Gold
-1.25
+0.75
-0.35
+1.10
carbohydrates
Platinum
-0.90
+0.65
-0.20
+1.30
alcohols, glycols
Silver
-1.20
+0.10
-0.55
+0.40
halides, cyanide
Copper
-
+0.60
-
-
amino acids,
carbohydrates

12 SenCell flow cell user manual, ed. 1
Another consideration in choosing a WE is the oxidation or reduction of
mobile phase constituents or WE material, that occurs when the potential
exceeds the limits as given in Table I. At high positive working potentials the
water in the mobile phase electrolyses and results in an strong increase of
the background current and noise. Formation of metal oxides, resulting in an
increase in background current is a limiting factor for metal electrodes.
Glassy carbon and platinum have the highest positive potential limits and are
therefore often used in oxidative ECD. For negative potentials the use of
platinum electrodes is limited by the ease of reducing hydrogen ions to
hydrogen gas.
Detection limit
One of the most important parameters used to characterise the performance
of a detection system is the signal-to-noise ratio (S/N ratio) from which the
concentration detection limit is derived. It enables objective comparison not
only between different electrochemical detectors but also between complete
analytical methods irrespective what detection system is used.
Table II. LC-EC conditions for analysis of norepinephrine.
column
ODS-2, 3 µm, 100 x 4.6 mm
flow rate
1.0 ml/min
mobile phase
H3PO450 mM, citric acid 50 mM, 20 mg/l EDTA, 100 mg/l
octane sulphonic acid (OSA), pH=3.1 with KOH, 5%
methanol
sample
1.0 µmol/l norepinephrine, 20 µl injection
temperature
30 oC
Cell
Flow cell with 3 mm GC WE, SB REF with 50 µm spacer
E cell
800 mV (vs. Ag/AgCl, filled with saturated KCl)
Icell
ca. 3 nA
In literature several ways are described to determined the detection limit. In
principle, it does not matter which definition of detection limit is used, as long
as the definition is precisely described.
In this manual the concentration detection limit (cLOD) for a certain compound
is defined as the analyte concentration that results in a signal that is 3 times
the standard deviation of the noise:
c = 3
signal c
LOD noise A
 
where sigma-noise is 0.2 x peak-to-peak noise and cAis the concentration of
analyte injected.
In Fig. 3 a typical S/N ratio for a flow cell with 2.74 mm WE is shown. In this
example the concentration detection limit for norepinephrine based on three

CHAPTER 1 The electrochemical flow cell 13
times the sigma-noise is 11 pmol/L (see Table II for conditions). Expressing
the performance of a detection system byonly the peak height makes no
sense.
A system can easilybe changed in a way that a larger peak height is
obtained. However, if the noise increases similarly, it has the same effect as
switching a recorder to a higher sensitivity: peaks appear higher but the S/N
ratio is the same. Expressing the limit of detection in an absolute amount (i.e.
in picomoles) without mentioning the injection volume, makes a good
comparison between different systems difficult.
Fig. 3. Example S/N ratio for norepinephrine (peak height: 80 nA, peak-to-
peak noise: 1.5 pA). The amount injected is 20 pmol (1.0 µmol/l). The
concentration detection limit based on three times sigma-noise in this case is
11 pmol/l.
Cell working volume adjustment
In a traditional electrochemical flow cell which uses metal/plastic gaskets
(spacers) the thickness of the gasket affects the linear flow velocity in the cell.
With a thinner gasket the cell working volume is decreased, resulting in a
higher linear flow velocity. For example the working volume of a cell with a 2
mm diameter electrode with 25 and 50 µm spacer is 80 nL and 160 nL,
respectively.

14 SenCell flow cell user manual, ed. 1
The signal increases with thinner spacers while the noise remains more or
less constant, which can lead to improvement of the detection sensitivity
(signal-to-noise ratio). Several authors have described the relation between
the layer thickness (i.e. spacer thickness) in a thin layer flow cell and the
measured current (S) as S = k b-2/3 where b is the spacer thickness and k a
constant.
The SenCell design eliminates the use of polymeric or metal gaskets. The
working volume of the electrochemical cell can be stepless adjusted without
opening the cell using the supplied adjustment key (p/n 116.1400). This
allows easy optimization of the cell working volume and thus detection
sensitivity (signal-to-noise ratio) for any LC application.
Fig. 4. Example chromatograms of 100 nM standard of catecholamines in 10
mM HAc recorded with the Sencell spacing adjustment set to position 3 and
0.5, corresponding with an approximate spacing setting of 100 µm and 12 µm
respectively.
In figure 4 an example is shown to demonstrate the effect of cell working
volume on signal. In figure 5 the peak height (normalized) as a function of
spacing is shown for Dopamine based on the data from the example shown
in figure 4.

CHAPTER 1 The electrochemical flow cell 15
Fig. 5. Normalized Peak height of Dopamine as a function of spacing setting
(red curve) based on chromatograms recorded with a 100 nM standard of
catecholamines in 10 mM HAc with a SenCell. The dotted curve is a
simulated curve based on the Cotrell equation (Lit ref F.G. Cottrell, Z. Phys.
Chem 42 (1903) 385).
Decreasing the spacing/working volume is limited by an increased pressure
drop over the flow cell which eventually will lead to an obstruction of the flow.
The onset is typically characterized by an increased noise level and the rise
of the system back pressure.
Applying small working volume settings should be done with great
care, it may cause excessive pressure built-up over the flow cell,
excessive baseline noise and may damage the cell. In any case DO
NOT OPERATE THE CELL AT POSITION 0.

16 SenCell flow cell user manual, ed. 1
This is illustrated in figure 6. It is evident that the noise remains relatively
constant as a function of spacing, but at a spacing of approximately6 µm a
significant increase in noise is observed accompanied with a rise in system
pressure due to a restriction build over the cell. So in this example setting the
cell spacing less than approximately 12 µm is not advisable.
Fig. 6. Left side: ASTM noise values as a function of cell spacing. Right side:
Noise traces as a function of cell spacing. SenCell spacing position 3
corresponds with approximately 100 ± 10 µm. The spacings used with this
particular SenCell under test in this experiment were determined using a
stylus profilometer.
So optimization of the cell working volume is focused on finding the right
balance between signal height and noise level for your SenCell, under your
specific LC-EC condition. Optimization can be achieved by decreasing the
cell spacing in small steps in a systematic wayand evaluating the baseline
noise and peak height of the analytes of interest till you find the optimal
Signal-to-Noise ratio. Note that with LC applications using larger ID columns
in combination with higher flow rates the minimum spacing which can be
used will be larger.
Inexperienced users are advised to use the factory pre-set cell working
volumes (position 1 or 2) with their SenCell. See chapter 3 installation.
0.0
0.5
1.0
1.5
2.0
2.5
3.0
020 40 60 80 100
Space setting SenCell (µm)
Unfiltered noise (pA)
[min.]
Time
910 11 12 13 14 15 16
[pA]
Current
-10
-5
0
56 um
12 um
25 um
50 um
100 um
Pump pulsations
& pressure rise

CHAPTER 1 The electrochemical flow cell 17

18 SenCell flow cell user manual, ed. 1
C H A P T E R 2
Reference electrodes
The SenCell is available with an ISAAC (in situ Ag/AgCl) reference electrode,
a salt bridge Ag/AgCl reference electrode and a HyREF reference electrode.
ISAAC reference electrode
The ISAAC reference electrode is in direct contact with the mobile phase
which contains chloride ions. The chloride concentration determines the
potential, therefore each time a fresh mobile phase is prepared it should
contain exactly the same concentration of chloride ions.
The standard electrode potential of the Ag/AgCl electrode (in 1.0 mol/l Cl-
solution) for the following half-reaction is defined as E0:
AgCl(s) + e-<=> Ag(s) + Cl-E0= 0.222 V
The potential of the REF is dependent from the chloride concentration as
described bythe following equation:
E = E - RT
Fln [Cl ]
cell AgCl
0 -
where R is the gas constant (8.314 Jmol-1K-1), T is the absolute temperature
(293 K) and F is the Faraday constant (96485 Cmol-1).
The potential of the ISAAC at 2 mmol/l KCl is 379 mV (
Table III). The potential difference (dE) between the saturated KCl Ag/AgCl
reference electrode and the ISAAC is 189 mV. If an application is running at
800 mV (vs. Ag/AgCl with sat’d KCl), the potential setting using the ISAAC
should be 611 mV (vs. Ag/AgCl in 2mmol/l KCl).

CHAPTER 2 Reference electrodes 19
Fig. 7. Dependence of the Ag/AgCl REF potential on the chloride
concentration.
Table III. Potential of the Ag/AgCl reference electrode, dE is the potential
difference with EAg/AgCl in saturated KCl.
Cl-(mmol/l)
E Ag/AgCl (mV)
dE (mV)
3500
190
0
2500
199
8
1500
212
21
500
240
49
100
280
90
20
321
130
10
338
148
8.0
344
154
6.0
351
161
4.0
361
171
2.0
379
189
1.0
396
206
0.5
414
224
The addition of chloride to the mobile phase has a few restrictions. For
example, the ISAAC is not recommended at a high working potential (> 1.2 V
vs. Ag/AgCl in 2 mmol/l KCl) because Cl-is oxidised and contributes to the
background current. In ion chromatography the addition of Cl-may lead to
undesired chromatographic changes. In case of a silver working electrode,
the addition of Cl-to the mobile phase will cause formation of an AgCl coating
on the working electrode leading to inactivation. At high pH or high modifier
concentrations the ISAAC is less suitable and a HyREF is recommended.

20 SenCell flow cell user manual, ed. 1
Fig. 8. Schematic representation of the Ag/AgCl reference electrode.
Salt bridge Ag/AgCl reference electrode
The reference electrode of the Ag/AgCl type with salt bridge consists of a
silver rod, coated with solid AgCl, immersed in a solution of saturated KCl,
containing KCl crystals. Electrical contact with the other electrodes in the flow
cell is made through a salt bridge consisting of a wetted cotton wool frit,
which is electrically conducting and slows down leakage of KCl. This REF for
the SenCell is factory filled with KCl. For certain applications another chloride
salt is to be preferred. In case of perchlorate containing mobile phases,
sodium chloride is mandatory, because potassium perchlorate precipitates
and will clog the cotton wool frit. At high modifier percentages, the REF must
be filled with lithium chloride for similar reasons.
HyREF reference electrode
The HyREF is a hydrogen reference electrode, its potential depends on the
pH of the mobile phase. The HyREF is fully comparable with the standard
Ag/AgCl REF as to baseline stability and S/N ratio. The HyREF is more user-
friendly and in principle this REF is completelyfree of maintenance. Trapping
of air bubbles like in the salt bridge Ag/AgCl type is impossible because of
the absence of a salt bridge. Consequently, refilling the REF with saturated
KCl is not longer required. Due to the absence of a salt bridge and its
inertness, the HyREF is an excellent alternative for the Ag/AgCl REF,