Antec Scientific VT-03 User manual

T [phone] | [e-mail] | https://AntecScientific.com
Antec Scientific
Industrieweg 12
2382 NV Zoeterwoude
The Netherlands
VT-03
Electrochemical Flow cell
User manual
110.0010, Edition 9, 2012

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 writtenconsent 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 are trademarks of Antec.Whatman™ (word and device)
and Whatrnan™ (word only) are trademarks of Whatman lnternational Ltd. SOLVENT IFD™ and AQUEOUS IFD™ are trademarks
of Arbor Technologies, Inc. Clarity®, DataApex® are trademarks of DataApex Ltd. Microsoft® and Windows™ are trademarks of
Microsoft Corporation. Excel is a registered trademark of the Microsoft Corporation.
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 or this manual. All use of the
software shall be entirely at the user’s own risk.

INTRODUCTION Table of contents 3
Table of contents
I N T R O D U C T I O N
Table of contents 3
The electrochemical flow cell 4
Introduction 4
Three-electrode configuration 5
Working electrode 6
Detection limit 7
Working electrode diameter 9
Spacer thickness 9
Reference electrodes 12
ISAAC reference electrode 12
Salt bridge Ag/AgCl reference electrode 14
HyREF reference electrode 14
Installation 16
VT-03 flow cell with HyREF or ISAAC 16
VT-03 flow cell with salt bridge REF 18
VT-03 micro flow cell 21
Assembling the micro flow cell 21
Capillary connections 24
Maintenance 28
HyREF 28
ISAAC 28
Polishing 28
Ag/AgCl salt bridge 29
Saturation and air bubbles 29
Material 30
Procedure 30
Maintenance of the cotton wool frit 31
Working electrode 32
Decreased flow cell performance 32
Polishing 33
Index 34

4VT03 flow cell user manual, ed. 8
C H A P T E R 1
The electrochemical flow cell
Introduction
The VT-03 flow cell is available with a glassy carbon, platinum, gold, silver
and copper working electrode. In combination with the spacer set (25, 50 and
120 µm) a variety of detection volumes (down to 5 nl) can be attained. 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. The VT-03 electrochemical flow cell. The upper part, the inlet block, is
separated from the working electrode block by means of a gasket (spacer,
not shown).
The VT-03 electrochemical flow cell has been developed for ultra-trace
analysis in standard, microbore and capillary LC-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 quality and 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 cell, by design, improves in performance. The flow cell
permits unusually short stabilisation times: trace analysis within half an hour

CHAPTER 1 The electrochemical flow cell 5
after starting up maybe expected.We have so much confidence in our flow
cell that we warrant the glassy carbon flow cell for a period of 5 years.
Three-electrode configuration
In the VT-03 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
feed back 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 the WE. 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 theWE
(without REF), the working potential would continuously change due to
polarisation effects at the electrodes, resulting in highly unstable working
conditions.

6VT03 flow cell user manual, ed. 8
If the working potential would be applied only over the REF versus theWE
(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 factmeans that a high
signalmust be obtained at a low working potential. For most applications
glassy carbon will be the WE material of choice. 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 already takes 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.

CHAPTER 1 The electrochemical flow cell 7
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
Another consideration in choosing aWE 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
flow cell
VT-03 flow cell with 3 mm GCWE mounted with 50 µm
spacer
E cell
800 mV (vs. Ag/AgCl, filled with saturated KCl)
Icell
ca. 3 nA

8VT03 flow cell user manual, ed. 8
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 of a VT-03 glassy carbon flow cell with 2.74 mm
WE is shown. In this example the concentration detection limit for
norepinephrine based on three times the sigma-noise is 11 pmol/l (see Table
II for conditions).
Expressing the performance of a detection system by only 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. Typical S/N ratio for norepinephrine measured with a VT-03 glassy
carbon flow cell (peak height: 80 nA, peak-to-peak noise: 1.5 pA). The

CHAPTER 1 The electrochemical flow cell 9
amount injected is 20 pmol (1.0 µmol/l). The concentration detection limit
based on three times the sigma-noise is 11 pmol/l.
Working electrode diameter
The size of theWE is an important factor in LC-EC, it affects both the signal
and the noise. For the VT-03 flow cell several glassy carbon WE diameters
are available (0.7, 2 and 3 mm). In a standard LC system the signal and the
noise increases linearly with the WE diameter. This means that he S/N ratio
remains more or less the same. In case of micro-LC an increase of theWE
diameter will increase the noise more than the signal. Therefore, in micro-LC
a decrease of the WE diameter will result in a better S/N ratio.
Table III. Flow cell recommendations.
Column diameter (mm)
Recommended flow cell
3 and higher
3 mm GC
3 - 1
2 mm GC
1 and below
0.7 mm µGC
The choice for a flow cell is primarilybased on the HPLC column diameter
(Table III). This waythe best possible detection limit for a standard,
microbore or capillary column is warranted.
The recommended combinations are giving the best S/N ratios. It should be
kept in mind that other combinations are possible that still result in acceptable
sensitivities for many applications. All VT-03 flow cells are individually tested
and meet our high standards of qualityand detection sensitivity.
Spacer thickness
The thickness of the gasket affects the linear flow velocity in the cell.With a
thinner spacer the cell volume is decreased (Table IV), resulting in a higher
linear flow velocity. The signal increases with thinner spacers while the noise
remains more or less constant (Fig. 4).
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. Also for the VT-03
flow cell the relation between S and b-2/3 results in a straight line (Fig. 5).

10 VT03 flow cell user manual, ed. 8
Fig. 4. The signal and noise for 1.0 µmol/l nor-epinephrine measured at
variable spacer thickness (given in µm). See Table II for other conditions.
Fig. 5. Peak height versus spacer thickness to the power -2/3.
Decreasing the spacer thickness is limited by an increased pressure drop
over the flow cell which eventually will lead to an obstruction of the flow. The
minimum spacer thickness available is 25 µm. Applying these small spacers
should be done with care. Over-tightening of the bolts maycause an
excessive pressure built up over the flow cell and increase the noise
considerably.
0.120.100.080.060.040.02
0
100
200
b^(-2/3)
S (nA)