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What detection limits are feasible in HPLC/ECD?

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Applies to

Electrochemical detection

Answer

Detection limit is usually defined as the peak height that gives a significantly (at least 3 times) higher signal than the baseline noise. Detection limits in LC-EC highly depend on the system configuration (injection volume, column efficiency and dimensions, retention time etc) and on the method itself (sample treatment or pre-concentration, use of enzyme reactor or derivatisation etc.). In Table 1 a few typical detection limits are given to give in idea of what is feasible. It is not always the best possible detection limit as that is largely depending on the many method optimization details.

Table 1. Typical detection limits, standards, without pre-concentration.

Example Column (mm) Inject. (µL) LOD (nmol/L) MDA (fmole)
catecholamines 4.6 x 100 20 0.05 1
catecholamines 1 x 100 4 0.05 0.2
PAD of carbohydrates 4 x 250 20 10 200
DNA adducts with 2D LC 3 x 100 5 10 50
ACh with IMER 2 x 100 20 0.5 10
3NT with reactor cell 1 x 50 10 0.5 5
VitK with reactor cell 2.1 x 50 20 0.3 6
Phenols with gradient LC 4.6 x 300 20 5 100

Abbreviations: PAD is pulsed amperometric detection, IMER is immobilized enzyme reactor, 3NT is 3-nitrotyrosine, ACh is acetylcholine, VitK is vitamin K. Improvements may include optimization of:

  1. Sample pre-treatment (e.g. concentration)
  2. LC parameters (mobile phase, column, injection volume, selectivity etc)
  3. Detector settings (range, potential etc.)
  4. Flow cell parameters (spacer, working electrode)
  5. Noise suppression

Keep in mind: no sensitivity without selectivity. It is difficult to improve detection sensitivity if detection selectivity is not optimized. In most LC-EC analyses in biological matrices (blood, urine, CSF) interfering peaks in the chromatogram are the limiting factor in getting better detection limits. Therefore, both sensitivity and selectivity must be optimized.  

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