29] Principles of differential spectrophotometry with special reference to the dual wavelength method

Abstract

This chapter discusses the principles of differential spectrophotometry with special reference to the dual wavelength method. The spectroscopic technique supplemented with an appropriate array of inhibitors, suitable and adequately controlled redox poises, and variation of temperature over the widest possible range provides detailed and versatile information on the system investigated. The dual wavelength technique supplements the information obtainable from the single wavelength technique with important signals from a neutral reference wavelength, which is either continuously or intermittently subtracted from that of the measuring wavelength. The information from the reference wavelength may be of low-frequency character such as light source fluctuations or scattering changes in the sample, or it may be of high-frequency character such as artifacts from actinic illumination or a portion of the signal itself, which is either redundant or undesirable. The proximity of the detector and the sample is essential for operation with highly scattering material, such as chloroplasts and mitochondria.