A novel method for measuring concentrations of phase separated biopolymers: the use of confocal Raman spectroscopy with self-modelling curve resolution

Abstract

This paper examines the potential use of confocal Raman spectroscopy in measuring the concentration of phase separated biopolymers. It demonstrates that this can be done for both bulk phase separated systems and gelled microstructures. The developments in this work mean that the Raman method can now be applied to any mixtures of biopolymers, even when their spectra are highly overlapping. The key to this universality is the data analysis method. The method chosen was a chemometrics approach called multivariate curve resolution (MCR) which falls into the more general class of factor analysis methods called self-modelling curve resolution. This method allows separation or purification of each individual ‘spectral’ component of the mixed biopolymer system. The MCR method was further developed so that the resolved intensities corresponding to the separated ‘spectra’ could be calibrated for absolute concentration. The limitations of the experimental method have been examined in detail, especially the spatial resolution. It is shown that, with the right experimental set-up, i.e. use of an oil immersion objective, accurate concentrations can be measured in particles of less than 10 μm. Results are presented for a bulk phase separated system, gelatin/dextran and a gelled microstructure of two carbohydrate polymers, gellan/κ-carrageenan, which is phase separated on the micron scale. The carbohydrate system demonstrates it is applicable to very similar polymers. This work demonstrates that the methodology for confocal Raman spectroscopy is now in place and is able to accurately measure biopolymer concentrations in any phase separated biopolymer system. The methodology is equally applicable to any system that has a microstructure on a similar scale to that described here for mixed biopolymers, where different component locations and concentrations are needed.