Application of resonance Raman spectrometry to the determination of vitamin B12.

The resonance Raman effect has been known for many years’. Its potential for trace analysis was pointed out by Brandmiiller2 in 1959. However, full exploitation of resonance Raman spectrometry did not begin until the development of high power, multiline gas lasers as Raman excitation sources. The recent application of tunable dye lasers as excitation sources3 promises even broader applications of resonance Raman spectrometry. The resonance Raman effect is extremely sensitive: Rimai et cd.” have observed resonance Raman spectra from /I-carotene solutions as dilute as IO-’ M. Moreover, Raman bands are typically .quite narrow. IO-20 cm-l half-width. This width corresponds to 0.3-0.6. nm at 500 nm. Thus, while resonance Raman spectrometry has a sensitivity comparable to that of spectrophotometry, it offers a selectivity which spectrophotometry cannot match. In the present work, resonance Raman spectrometry was investigated as an alternative to spectrophotometry for the determination of vitamin B, 2. The standard spectrophotometric determination at 360 nm offers good sensitivity, but suffers from many potential interference$. Microbiological methods have detection limits approaching log M, but require very careful control of experimental conditions in order to achieve reproducibility6. Recently, Hester st al. 7-g have examined the resonance Raman spectra of . . vttarnrn B12, cyanocobalamin. Similar. less extensive work has been reported by George and Mendelsohn”. Wozniak and Spiro l l have shown that various cobalt corrins have resonance Raman spectra similar to that of cyanocobalamin. Although several bands of the cyanocobalamin Raman spectrum are resonance-enhanced. the strongest is a ring stretching vibration at 1504 cml. This band can be strongly enhanced using either the 488,O nm or 514.5 nm line of an Ar+ laser. Both of these lines lie in the a-/I band system of the absorption spectrum of cyanocobalamin. Previous researchers have worked with solutions in the 10-4-10-3 M range. Such concentrations yield excellent spectra in which all the minor bands