Quantifying the microstructures of soft solid materials by confocal Raman spectroscopy Paul. D.A. Pudney a,* , Tom. M. Hancewicz b , Dale. G. Cunningham a , Michael. C. Brown a a Unilever Bestfoods R&D Colworth Laboratory, Sharnbrook, Bedford MK44 1LQ, UK b Unilever R&D US, Edgewater, NJ, USA Received 2 February 2003; received in revised form 11 August 2003; accepted 12 August 2003 Abstract Quantifying the microstructures of complex composite soft solid materials presents a great measurement challenge. Confocal Raman spectroscopy has all the attributes to potentially measure these structures quantitatively. However, for this potential to realised a number of measurement and data analysis obstacles need to be examined and overcome. This paper addresses these issues and shows how they can be tackled successfully. Experimentally it is shown that with the use of an external intensity standard and knowledge of instrumental stability, long-term concentration calibrations can be produced. In mapping, spatial resolution is limited in the Z-direction, but it is demonstrated that this limit can be minimised with the correct experimental set-up and the use of index matching objectives. Once the spectral image data set has been collected highly overlapping spectra can cause great problems in extracting the pertinent information inherent in the data. It has been shown previously that this can be overcome by statistical methods like self-modelling curve resolution (SMCR) to produce individual component images. Here, it is shown that this method can be extended to produce calibrated concentration images of the components present. These methods have been successfully applied to determine the microstructure of a gelled soft solid; a phase-separated system made from two carbohydrate biopolymers, gellan and k-carrageenan, in water. Raman maps with additional calibration spectra have been used to produce concentration images of both carbohydrates within the microstructure. It is also shown how components of a very complex ‘real life’ system, a dairy spread, can be separated to produce component images. Thus, it is demonstrated that confocal Raman spectroscopy can image complex soft solid microstructures quantitatively, and can play a large role in future research in this and other related areas. # 2003 Elsevier B.V. All rights reserved. Keywords: Raman spectroscopy; Gellan; k-Carrageenan 1. Introduction Soft solid materials are technologically important in many areas, e.g. foods and pharmaceuticals [1], and also present a great academic challenge. These materials are most com- monly complex composites of mixtures of bioploymers, usually in water. Their macroscopic and rheological proper- ties depend crucially on their morphology, especially at the micron length scale. These microstructures are often formed by phase separation of the biopolymers, however bulk phase separation at equilibrium conditions is not always reached. The kinetic trapping of structures is produced by a number of effects and processes, common ones including temperature quenching, which often induces physical gelation of one or more components present, and perturbation by shear [2]. To gain an understanding of these processes, and the proper- ties of the materials produced, a detailed knowledge is required of their structures and compositions. Due to the structures varying on the micron length scale many forms of microscopy have been used to observe them. These methods have, and do, provide much valuable information [3]. How- ever with many systems, labelling for specific components is necessary, which is never ideal. In many cases labels are not readily available, especially given the wide variety of bio- polymers that are used, or are not fully discriminating. These techniques can give locations of some components but are never quantitative. Spectroscopic microscopy would appear to have the appropriate properties to overcome some of these limitations and provide more detailed information about composition. FT-IR, for example, has been applied to such systems [4], it having been used successfully to study phase-separated systems in a number of ways, including measurement the phase diagram of bulk phase-separated amylopectin/gelatin Vibrational Spectroscopy 34 (2004) 123–135 * Corresponding author. Tel.: þ44-1234-222486; fax: þ44-1234-222757. E-mail address: paul.pudney@unilever.com (P.D.A. Pudney). 0924-2031/$ – see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.vibspec.2003.08.006