What DAW software are you using? How many audio tracks are you running? What are the specs on your computer?
My philosophy is, one drive for audio, one for samples, and the other for everything else. Here's my setup. I'm using Logic Pro 6 on a G4 733 with 3 HDDs
1 60Gig 7200 RPM 2MB
2 120Gig 7200 RPM 2MB
the 60 houses my OS and all applications, including software instruments (in my case AUs). You won't get a speed increase by putting applications or VSTi's on a separate HDD because upon startup, the app gets loaded into RAM.
One of the 120s has vocals and other tracks.
The other 120 has samples.
Since the EXS does disk streaming, i thought it best to put the samples on a separate drive. If your sampler of choice does disk streaming, then I would suggest you put them on a separate drive. Even putting them on the 5400 RPM drive would be better. Or better yet, move the OS and apps to the 5400 RPM drive, and use the 40GB 7200 RPM drive for your samples.
Consider partitioning your OS drive, to provide storage for miscellaneous stuff.