Measurements of multiply scattered light for on-line monitoring of changes in size distribution of cell debris suspension.

Dual wavelength frequency-domain measurements of photon migration (FDPM) are conducted on filtrate samples obtained from an industrial centrifugation process designed to separate Escherichia coli cell debris from the inclusion bodies. FDPM measurements consist of detecting phase delay of intensity-modulated light at 670 and 820 (or 830) nm. Optical properties of isotropic scattering and absorption are obtained from the regression of phase delay data to the optical diffusion equation. We show that the corresponding intensity-based measurements alone cannot provide accurate and independent estimates for these optical properties. However, FDPM-derived scattering coefficients of filtrate solutions (primarily consisting of 0.1-0.2 micrometer E. coli cell debris) are sensitive to approximately 1 vol % of added inclusion bodies (of 1-2 micrometer size). The technique, theory, and future adaptation of FDPM as an on-line monitor to detect the loss of inclusion bodies in centrifugation following homogenization are presented and contrasted to conventional, intensity-based measurements.

Frequency-domain photon migration measurements for quantitative assessment of powder absorbance: A novel sensor of blend homogeneity.

The measurement and analysis of frequency-domain photon migration (FDPM) measurements of powder absorbance in pharmaceutical powders is described in the context of other optical techniques. FDPM consists of launching intensity-modulated light into a powder and detecting the phase delay and amplitude modulation of the re-emitted light as a function of the modulation frequency. From analysis of the data using the diffusion approximation to the radiative transport equation, the absorption coefficient can be obtained. Absorption coefficient measurements of riboflavin in lactose mixtures are presented at concentrations of 0.1 to 1% (w/w) at near-infrared wavelengths where solution absorption cross sections are difficult to accurately measure using traditional transmission measurements in nonscattering solutions. FDPM measurements in powders enabled determinations of absorption coefficients that increase linearly with concentration (w/w) according to Beer-Lambert relationship. The extension of FDPM for monitoring absorbance of low-dose and ultralow-dose powder blending operations is presented.

Investigation of static structure factor in dense suspensions by use of multiply scattered light.

Near-infrared, frequency-domain photon migration measurements of phase shift are used to derive accurate values of isotropic scattering coefficients in concentrated, interacting suspensions of aqueous polystyrene microspheres with volume concentrations ranging from 1% to 45% by solids and mean diameters ranging from 135 to 500 nm. Under conditions of high ionic strength, the isotropic scattering coefficient can be quantitatively predicted by the Percus-Yevick model for hard-sphere interactions and Mie theory. In addition, the attractive interactions between scatterers arising from the addition of soluble poly(ethylene glycol) with molecular weights of 100 and 600 K cause hindered scattering. The increases in static structure and decreases in isotropic scattering coefficient agree with that predicted by Mie theory and the depletion interaction model developed by Asakura and Oosawa [J. Chem. Phys. 22, 1255 (1954)]. These results demonstrate the success of monitoring interaction between particles by use of multiple-scattered light and the necessity of incorporating models for these interactions when predicting scattering of dense, concentrated suspensions.

Transport effects on the kinetics of protein-surface binding.

A detailed model is presented for protein binding to active surfaces, with application to the binding of avidin molecules to a biotin-functionalized fiber optic sensor in experiments reported by S. Zhao and W. M. Reichert (American Chemical Society Symposium Series 493, 1992). Kinetic data for binding in solution are used to assign an intrinsic catalytic rate coefficient k to the biotin-avidin pair, deconvoluted from transport and electrostatic factors via application of coagulation theory. This intrinsic chemical constant is built into a reaction-diffusion analysis of surface binding where activity is restricted to localized sites (representing immobilized biotin molecules). The analysis leads to an effective catalytic rate coefficient keff characterizing the active surface. Thereafter, solution of the transport problem describing absorption of avidin molecules by the macroscopic sensor surface leads to predictions of the avidin flux, which are found to be in good agreement with the experimental data. The analysis suggests the following conclusions. 1) Translational diffusion limitations are negligible for avidin-biotin binding in solution owing to the small (kinetically limiting) value k = 0.00045 m/s. 2) The sparse distribution of biotin molecules and the presence of a repulsive hydration force produce an effective surface-average catalytic rate coefficient keff of order 10(-7) m/s, much smaller than k. 3) Avidin binding to the fiber optic sensor occurs in an intermediate regime where the rate is influenced by both kinetics and diffusion.