are usually only visible after considerable magnification of the chromatogram. If an AD converter
is used to record the analog signal, it is recommended that you keep the sampling rate of the AD
converter in pace with the data rate of the detector. For example: for a waveform with a pulse
duration of 0.5 seconds (2 Hz data rate), use a sampling rate of 2 Hz on your AD converter. Or, if
this is not selectable, use the closest possible higher sampling rate.
In FW1.09, an extended pulse mode, “pulse mode 2”, was introduced, in addition to the existing
pulse mode. In pulse mode 2 it is possible to program a multi-step waveform with up to 30 time-
potential (t,E) coordinates and a maximum pulse duration of 4 seconds. The time interval in
which the current is measured (marked by “Begin” and “End” markers) is freely programmable in
a pulse table. This new pulse mode extends the application areas of the 3465 Detector to
analysis using PAD detection with more sophisticated potential waveforms, such as that used in
amino acid analysis. See
8.1.2 High pH of mobile phase
In carbohydrate analysis, another special consideration must be taken into account. Pulse mode
detection uses a flow cell with a gold electrode. Separation employs an anion exchange column
and a mobile phase of 20 to 100 mmol/L sodium hydroxide. The pH is between 12 and 14. This
puts some demands on the HPLC system and flow cells. After prolonged use of a flow cell with a
gold working electrode (WE) in pulse mode, the gold oxide that is generated at the WE
precipitates on the auxiliary electrode (AUX). This gold oxide coating may electrically isolate the
AUX and result in an increase in noise. Cleaning the AUX electrode with metal wool can remove
this coating.
Reference electrodes of the Ag/AgCl type are not suitable for carbohydrate analysis. Due to silver
oxide formation, they require regular (monthly) maintenance. HyREF reference electrodes are
maintenance-free under these conditions and are therefore well-suited.
If a mobile phase with a high pH (pH >10 in carbohydrate analysis) is used, the standard Vespel
rotors from the injection valve should be replaced by Tefzel rotors, which are pH-resistant. For
carbohydrate analysis, only CO2-free sodium hydroxide should be used because carbonate
anions may disturb the ion exchange chromatography. The CO2-free sodium hydroxide is
available from several suppliers as a 50% solution (19.2 mol/L). NaOH pellets are not
recommended because of their high CO2 content. Organic modifiers (including acetonitrile)
strongly attenuate the signal of most carbohydrates in PAD and are therefore not recommended.
8.1.3 Pulse settings
In PAD of carbohydrates, a series of potentials is applied in a continuous, cyclic manner. The
detection potential is applied during time interval t1. Data collection occurs within t1, during time
interval ts (sampling time). The time difference t1-ts is the stabilization time.
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