Every ferrule type needs an appropriate length of tubing for connecting it to the connection port,
depending on the depth of the connection port. Refer to the information provided by the
manufacturer.
2.7.2 Mobile phase
Electrochemical detection is a sensitive detection technique characterized by extremely low
detection limits. A typical detection limit of 100 pmol/L or lower for catecholamines is no
exception. Improving detection limits is always limited by the weakest link in an LC-EC system. In
daily practice, a few rules must be followed to fully exploit the linear dynamic range and low
detection limits of an EC detector. These rules are related not only to hardware but also to mobile
phase composition, degassing, temperature and pH stability, and several other factors.
Mobile phase requirements:
• Electrochemically clean, HPLC-grade or better
• Ion strength 20 - 200 mmol/L
• Buffer pH near pKa
• In-line 0.2-µm filter and degassing of mobile phase
• EDTA for trapping of metal ions
Mobile phase must be clean because it is in direct contact with the working electrode in EC
detection. High-purity chemicals, including water, are prerequisites. In some applications, EDTA
is added to the mobile phase to electrochemically trap metals such as Fe
2+
by forming an inactive
complex. However, at higher working potentials (typically > 1.2 V vs. salt bridge AgCl REF),
EDTA can become electrochemically active and is not recommended. In that case only a
passivation step with 30% H
3
PO
4
is recommended.
Electrolytes ensure contact between the three electrodes in an electrochemical flow cell. Low ion
strength destabilizes an EC system and noise increases. Extremely high buffer concentrations
cause problems of salt formation. Therefore, concentrations between 20 and 200 mmol/L are
recommended.
Also, constant pH is important for baseline stability and reproducible results. Stability of pH is
best when close to pK
a
of a buffer ion. Frequently used buffers include phosphate, carbonate,
acetate, and citrate. Modifiers such as methanol, propanol, and acetonitrile can be used without
problems in DC amperometry, but not in pulsed amperometric detection because peaks are
strongly attenuated. In our experience, the quality and useful life of organic modifiers can be
problems that result in increased noise levels. Metal inlet frits in mobile phase bottles may
increase baseline noise. If so, alternative filters are available.
Vacuum degassing: Considerable amounts of the gases N
2
, O
2
, and CO
2
may be dissolved in
HPLC mobile phases. Whenever the temperature changes, solvents mix, or a pressure reduction
occurs, these gases may show up as very small air bubbles. To avoid noise baselines, use the
inline degasser incorporated in the Waters instruments.
Helium degassing: Degassing using helium is an effective and universally applicable method, but
it is recommended only when working in reductive electrochemical detection and pulsed
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