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In an extended introduction, key aspects of resonance Raman spectroscopy (RRS) such as enhanced sensitivity and selectivity are briefly discussed in comparison with normal RS. The analytical potential is outlined. Then achievements in different fields of research are highlighted in four sections, with emphasis on recent breakthroughs: (1) The use of visible RRS for analyzing carotenoids in biological matrices, for pigments and dyes as dealt with in art and forensics, and for characterizing carbon nanotubes. (2) The use of RRS in the deep UV (excitation below 260nm) in the bioanalytical and life sciences fields, including nucleic acids, proteins and protein-drug interactions. Metalloproteins can be studied by visible RRS in resonance with their chromophoric absorption. (3) Progress in theoretical calculations of RRS excitation profiles and enhancement factors, which ultimately might facilitate analytical RRS. (4) Instrumental and methodological achievements including fiber-optic UV-RRS, coupling of RRS to liquid chromatography and capillary electrophoresis. Sensitivities can approach the single-molecule level with surface-enhanced RRS or tip-enhanced RRS. Last but not least, promising fluorescence background rejection techniques based on time-gated detection will be presented. This review ends with a concluding section on future expectations for RRS, in particular its potential as an analytical technique.

A Raman instrument was assembled and tested that rejects typically 98-99% of background fluorescence. Use is made of short (picosecond) laser pulses and time-gated detection in order to record the Raman signals during the pulse while blocking most of the fluorescence. Our approach uses an ultrafast-gated intensified charge-coupled device (ICCD) camera as a simple and straightforward alternative to ps Kerr gating. The fluorescence rejection efficiency depends mainly on the fluorescence lifetime and on the closing speed of the gate (which is about 80 ps in our setup). A formula to calculate this rejection factor is presented. The gated intensifier can be operated at 80 MHz, so high repetition rates and low pulse energies can be used, thus minimizing photodegradation. For excitation we use a frequency-tripled or -doubled Ti : sapphire laser with a pulse width of 3 ps; it should not be shorter in view of the required spectral resolution. Other critical aspects tested include intensifier efficiency as a function of gate width, uniformity of the gate pulse across the spectrum, and spectral resolution in comparison with ungated detection. The total instrumental resolution is 7 cm(-1) in the blue and 15 cm(-1) in the ultraviolet (UV) region. The setup allows one to use resonance Raman spectroscopy (RRS) for extra sensitivity and selectivity, even in the case of strong background fluorescence. Excitation wavelengths in the visible or UV range no longer have to be avoided. The effectiveness of this setup is demonstrated on a test system: pyrene in the presence of toluene fluorescence (lambda(exc) = 257 nm). Furthermore, good time-gated RRS spectra are shown for a strongly fluorescent flavoprotein (lambda(exc) = 405 nm). Advantages and disadvantages of this approach for RRS are discussed.

Ultraviolet resonance Raman spectroscopy is carried out using a continuous wave frequency-doubled argon ion laser operated at 229, 244, and 257 nm in order to characterize the overtones and combination bands for several classes of organic compounds in liquid solutions. Contrary to what is generally anticipated, for molecules such as pyrene and anthracene, strong overtones and combination bands can show up; it is demonstrated that their intensity depends critically on the applied laser wavelength. If the excitation wavelength corresponds with a purely electronic transition--this applies to a good approximation for 244-nm excitation in the case of pyrene and for 257-nm excitation in the case of anthracene--mostly fundamental vibrations (up to 1700 cm(-1)) are observed. Overtones and combination bands are detected but are rather weak. However, if the laser overlaps with the vibronic region--as holds for 229- and 257-nm excitation for pyrene and 244-nm excitation for anthracene--very strong bands are found in the region 1700-3400 cm(-1). As illustrated for pyrene at 257 nm, all these bands can be assigned to first overtones or binary combinations of fundamental vibrations. Their intensity distribution can roughly be simulated by multiplying the relative intensities of the fundamental bands. Significant bands can also be found in the region 3400-5000 cm(-1), corresponding with second overtones and ternary combinations. It is shown that these findings are not restricted to planar and rigid molecules with high symmetry. Substituted pyrenes exhibit similar effects, and relatively strong overtones are also observed for adenosine monophosphate and for abietic acid. The reasons for these observations are discussed, as well as the potential applicability for analytical purposes.

The results of time-dependent density functional theory (TDDFT) calculations of resonance Raman intensities are combined with experimental deep-ultraviolet resonance Raman measurements at a single wavelength, i.e., 244 nm, in order to test the possibility to distinguish several very similar compounds. Pyrene and three of its substituted derivatives, in which a single hydrogen atom has been replaced by a halogen atom, are compared. The fixed 244 nm excitation wavelength overlapped with the same electronic transition of the four pyrenes. Ground-state calculations using the BP86 exchange-correlation functional were used to predict the Raman frequencies, whereas excited-state calculations have been carried out employing the "statistical averaging of (model) orbital potentials" (SAOP) potential within a linear-response TDDFT framework in combination with the short-time approximation of resonance Raman intensities. In view of the simplistic theoretical approach, we find a surprisingly good agreement between the simulated and measured resonance Raman spectra of pyrene and its substituted analogues in terms of frequencies and intensities, which shows that the calculations can be used reliably to interpret the experimental spectra. With this combined information, it is possible to find criteria to distinguish the compounds under investigation, although many features of their vibrational spectra are similar.

Capillary electrophoresis (CE) and resonance Raman spectroscopy (RRS) with excitation in the deep ultraviolet (UV) region (lambda(ex): 244 or 257 nm) were coupled on-line. The potential of this hyphenated technique, denoted as CE-UV-RRS, for analyte confirmation/identification purposes was explored with aromatic sulfonic acids and nucleotides as test compounds. Good-quality UV-RRS spectra could be recorded on-the-fly. Identification limits for the nucleotides were in the 10-125 microg/mL range. The RRS spectra showed sufficient characteristic features to enable analyte confirmation. In addition, the identification power of UV-RRS was studied with substituted pyrenes as model compounds. The compounds were distinguishable on the basis of their RRS spectra at 244 nm.