how i can increase the sensitivity of my method

[shantaram]

I want to increase the response of the compound in my method. the chromatographic conditions of my method are as follows:
Mobile phase: MeOH:25mM potassium dihydrogen phosphate pH5.5(32:68 %v/v)
Column: Lichrosphere RP 18e (5µ)
250*4.6i.d.
Oven temp.: 40ºc
Flow rate: 0.8mL/min
Detector :UV
Inj. vol :40µL
Physicochemical properties of analyte:
Solubility: soluble in water, slightly soluble in methanol
pKa : 5.01
Mol. Wt.: 670.76

[tom jupille]

Detectability (LOD or LLOQ) is all about signal/noise ratio. There are only two ways to improve it:
- more signal
- less noise

More signal:
- smaller, narrower column
- earlier elution (lower k' = narrower peak)
- smaller particle size (higher N = narrower peak)
- better wavelength (check spectrum, make sure you are working at an absorbance maximum)
- if you have very weak UV, consider a different detector (ELSD, CAD, or MS)
- make a derivative (fluorescence ?)

Less noise
- check your detector performance vs. specification
- get a better, newer detector
- new lamp
- better temperature control
- detector/data system settings for things like time constant

[HPLCCONSULT]

If your detector has the option of different flow cells, then you might be able to opimize the flow cell dimensions for your conditions. For example: If you have available a longer path length cell for your detector, then you could increase the signal response obtained by your detector by using it (simplistic explaination as their are many parameters to consider). *An optimal flow cell size for the instrument conditions can be easily calculated for any conditions.

Do you have control of the signal bandwidth ? If so, increasing it can 'add' signal to your response as well. However; (1) no other peaks must be nearby whose spectra may overlap ; (2) the spectra range you cover must not contribute more noise (such as very low UV ranges) than you would have without adding the extra bandwidth ; (3) adding extra bandwidth will add extra sensitivity, but decrease selectivity.

Lastly, optimize your method. Ask yourself if the method you have is really the best one ?

[Uwe Neue]

An additional option not mentioned above is an increase in the injected amount, if enough sample is available. Issues with overload from a large-volume injection can be solved by dissolving the sample in a solvent composition that is more polar than the mobile phase.

[shantaram]

Tankyou...