Antec Scientific VT-03 User manual

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Antec Scientific
Industrieweg 12
2382 NV Zoeterwoude
The Netherlands
VT-03
Electrochemical Flow cell
User manual
110.0010, Edition 9, 2012

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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)

CHAPTER 1 The electrochemical flow cell 11
Table IV. Flow cell volume
WE diameter (mm)
3.00
2.74
2.54
2.00
1.90
1.00
0.75
0.50
spacer (µm)
cell volume (µl)
25
0.18
0.15
0.13
0.08
0.07
0.020
0.011
0.005
50
0.35
0.29
0.25
0.16
0.14
0.039
0.022
0.010
120
0.85
0.71
0.61
0.38
0.34
0.094
0.053
0.024

12 VT03 flow cell user manual, ed. 8
C H A P T E R 2
Reference electrodes
The VT-03 flow cell 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 Faradayconstant (96485 Cmol-1).
The potential of the ISAAC at 2 mmol/l KCl is 379 mV (Table V). 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 13
Fig. 6. Dependence of the Ag/AgCl REF potential on the chloride
concentration.
Table V. 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.

14 VT03 flow cell user manual, ed. 8
Fig. 7. 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 VT-03 flow cell 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 fullycomparable 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,
especiallyin case of high modifier concentrations (i.e. analysis of fat-soluble
vitamins) or high pH (analysis of carbohydrates, PAD).
Depending on the pH of the mobile phase, the potential setting of the working
electrode vs. the HyREF maydiffer significantlycompared to Ag/AgCl.
I/E curves show a shift of more than 200 mV at pH 3.1 (e.g. catecholamines),
no shift appears at pH 12 (e.g. PAD of carbohydrates). Therefore, it is

CHAPTER 2 Reference electrodes 15
advisable first to construct a hydrodynamic (or scanning) voltammogram
when using the HyREF. In Table VI the potential of the HyREF is measured
against the Ag/AgCl (in sat'd KCl) electrode at different pH values.
Table VI. Measured cell potential (HyREF - Ag/AgCl) versus pH.
PH
EHyREF - Ag/AgCl (mV)
3.3
232
6.2
130
7.5
90
11.8
0
So, if an Ag/AgCl REF is replaced bya HyREF, the pH effect must be taken
into account (Table VI). The pH vs. voltage relation is described by:
EHyREF = EAg/AgCl - 328 + 29.9 pH (1)
For example: a working potential of 800 mV (vs. Ag/AgCl with sat’d KCl) at
pH 3, has to be changed to: EHyREF = 800 - 328 + 29.9*3 = 561.7 mV (vs.
HyREF)

16 VT03 flow cell user manual, ed. 8
C H A P T E R 3
Installation
VT-03 flow cell with HyREF or ISAAC
The flow cell is assembled properly when it arrives. The force on the bolts is
pre-set to 13 Ncm (“a little bit beyond fingertight”). Familiarise yourself with
this force, since over-tightening of the bolts strongly deteriorates the S/N ratio
and eventually the cell itself. Also, be aware that the black marks on both
blocks should be in line. This ensures the best performance. For instructions
about assembling the cell refer to page 20.
Fig. 8. Installation of flow cell (Intro™or DECADE™). WORK, AUX and REF
are connected using the red, blue and black cell cable. LC out should be on
top to prevent entrapment of bubbles.
Fig. 9. Installation of flow cell (DECADE II™). WORK, AUX and REF are
connected using the red, blue and black cell cable. LC out should be on top
to prevent entrapment of bubbles.

CHAPTER 3 Installation 17
The ISAAC reference electrode requires 2 mmole/l chloride ions (KCl or
NaCL) in the mobile phase. Add and equilibrate before installation of
the ISAAC. See manual electrochemical detector for optimisation of
working potential
1. Connect the column outlet to the flow cell inlet, using small-bore PEEK
tubing (0.3 mm ID) and one of the fingertights supplied. Use only our
factory supplied fingertights in the flow cell, others may cause
serious damage! Let the tubing protrude for ca. 1.5 cm from the
fingertight fitting and tighten it such that the tubing is not or slightly
indented by the fitting.
2. Do not over-tighten the fingertight. Over-tightening affects the flow
pattern through the tubing (turbulence) and may strongly decrease the
flow cell performance.
3. Connect 0.5 mm ID PEEK tubing to the outlet of the flow cell. Use only
our factory supplied fingertights in the flow cell, others maycause serious
damage! Again (see above), do not over-tighten the fingertight.
4. Turn on the HPLC pump. Keep some tissues at hand as you probably
will spill some mobile phase during this mounting procedure.
5. Fill the flow cell, by keeping it in an angle of about 45° with the outlet (LC
out) on top to force the air through the outlet.
6. Position the flow cell in its clamp in the controller with the REF at the
lower side and the outlet at the upper side. This excludes trapping of air
bubbles.
7. Connect the cell cable as illustrated in Fig. 8.
Never switch ON the flow cell when:
- the cell cable is not correctly connected
- the cell is only partly (or not at all) filled with buffer/electrolyte
- the outside of the flow cell is wet, particularly the part between the
auxiliary and working electrode connection
because substantial damage to the working electrode or electronics
may occur.
The maximum detection stability is attained when not only the flow cell, but
also the HPLC column is incorporated in the controller. The controller has an
integrated Faradaycage and an accurately thermostatted oven compartment

18 VT03 flow cell user manual, ed. 8
which ensures stable working conditions. Installing the flow cell and column
within such a controlled environment is the minimum requirement for high-
quality LC-EC trace analyses.
VT-03 flow cell with salt bridge REF
The flow cell is assembled properly when it arrives. The force on the bolts is
pre-set to 13 Ncm (“a little bit beyond fingertight”). Familiarise yourself with
this force, since over-tightening of the bolts strongly deteriorates the S/N ratio
and eventually the cell itself. Also, be aware that the black marks on both
blocks should be in line. This ensures the best performance. For instructions
about assembling the cell refer to page 20.
Fig. 10. Installation of flow cell (in Intro or DECADE). WORK, AUX and REF
are connected using the red, blue and black cell cable. The LC outlet is
placed on top to prevent entrapment of bubbles.
Fig. 11. Installation of flow cell (DECADE II). WORK, AUX and REF are
connected using the red, blue and black cell cable. The LC outlet is placed on
top to prevent entrapment of bubbles.

CHAPTER 3 Installation 19
Use proper eye and skin protection when working with solvents.
1. Check the REF visuallyfor air bubbles and saturation with KCl. Some
KCl crystals should be visible. When no crystals are visible or air bubbles
are trapped the REF needs maintenance (see page 29). To prevent
drying-out the REF is sealed with a cap on arrival. Remove the cap.
2. Tighten the black swivel of the REF, a small droplet should appear at the
cotton-wool frit. Do not remove this droplet because it ensures proper
contact of the REF with the mobile phase.
3. Turn on the HPLC pump. Place some tissues as some mobile phase
may be spilled during this mounting procedure. Connect the column
outlet to the flow cell inlet, using small-bore PEEK tubing (0.3 mm ID)
and one of the fingertights supplied. Use only our factory supplied
fingertights in the flow cell, others maycause serious damage! Let the
tubing protrude for ca. 1.5 cm from the fingertight and tighten it carefully.
Over-tightening affects the flow through the tubing (turbulence) and
decreases the flow cell performance.
4. Connect 0.5 mm ID PEEK tubing to the outlet of the flow cell. Use only
our factory supplied fingertights in the flow cell, others maycause serious
damage! Again (see above), do not over-tighten the fingertight.
5. Fill the flow cell, by keeping it in an angle of 45° with the REF fitting on
top, is best done by blocking the outlet with a finger and letting the air
escape via the REF fitting. Carefully check the thread of the fitting for
trapped air bubbles.
6. When the REF fitting is completely filled with mobile phase, mount the
REF while slowly releasing the outlet. Make sure not to include an air
bubble!
7. Position the flow cell in its clamp in the controller with the REF at the
lower side and the outlet at the upper side. This excludes trapping of air
bubbles by the REF.
8. Connect the cell cable as illustrated in Fig. 10.
The maximum detection stability is attained when not only the flow cell, but
also the HPLC column is incorporated in the controller. The controller has an
integrated Faradaycage and an accurately thermostatted oven compartment
which ensures stable working conditions. Installing the flow cell and column

20 VT03 flow cell user manual, ed. 8
within such a controlled environment is the minimum requirement for high-
quality LC-EC trace analyses.
Never switch ON the flow cell when:
- the cell cable is not correctly connected
- the cell is only partly (or not at all) filled with buffer
- the outside of the flow cell is wet, particularly the part between the
auxiliary and working electrode connection
because substantial damage to the working electrode or electronics
may occur.
When not in use, please store the REF with the cotton wool frit immersed in a
saturated KCl solution to prevent drying out.
Fig. 12. Always install flow cell with outlet on top.