A new method of phase sensitive detection in modulation spectroscopy applied to temperature induced folding and unfolding of RNase A

Modulation spectroscopy can be an adequate tool to separate a weak system response from a huge background absorption, provided the process under consideration enables a periodic external stimulation. Phase sensitive detection (PSD) is then used to demodulate the periodic system response. We introduce a new method of PSD that can be used in modulated excitation (ME)-FTIR spectroscopy without the need of a lock-in amplifier of spectrometer build-in hardware. An advantage of this method is that only standard procedures included in all FTIR instruments, such as the measurement of time-resolved spectra, are required. The principal mathematical formalism of the used PSD is described. As an example we show phase-resolved spectra of the amid I' region obtained from temperature modulated FTIR spectroscopic experiments of RNase A. The advantage of the ME compared to a relaxation process is shown by the power of separation of overlapping absorption bands. (C) 2002 Elsevier Science B.V. All rights reserved.