Chromatography Forum

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

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

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 ?

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.

Tankyou...