What detection limits are feasible in HPLC/ECD?
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:
- Sample pre-treatment (e.g. concentration)
- LC parameters (mobile phase, column, injection volume, selectivity etc)
- Detector settings (range, potential etc.)
- Flow cell parameters (spacer, working electrode)
- 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.