What is the concentration of analyte in SERS(C(sers))? Is it total concentration of analyte or concentration of capping molecules around nanoparticles?
enhancement factor (EF) is defined as {I(sers)/I(raman)}×{C(raman)/C(sers)}. I= peak intensity and C=concentration of analyte.
That depends on the type of measurement you are carrying out. I guess you are doing the SERS in solution, if you are going to use that formula: you should then use the total analyte concentration, and bear in mind that you may be doing an underestimation of the EF (as it is possible that not every molecule present in the medium is in the surroundings of the NPs, that will depend on the affinity of the analyte towards the NP surface). And of course it is necessary that the corresponding Raman measurements are taken in the same conditions (acquisition time, laser power, etc.) and changing only the concentration, otherwise the calculation will be inaccurate.
However, if you are trying to do Single Molecule SERS, you should calculate the number of molecules capped around the NPs, the focal volume of the laser, etc., and include such parameters in your calculation. Check the paper by Eric Le Ru on enhancement factor calculations, it is very clear and a general reference in this matter.
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