The rational design of layer-by-layer deposited biopolymer films is underpinned by measurements of their growth characteristics and functional properties. In this study we compare measurements of the hydrated mass, polymer mass and concentration, density and composition of the films measured using a quartz crystal microbalance with dissipation monitoring (QCMD), a dual polarization interferometer (DPI) and a Fourier-transform infrared spectrometer in attenuated total reflectance mode (FTIR-ATR). The biopolyanion used for the deposition was polygalacturonic acid (PGA) and the biopolycations were poly-L-lysine (PLL), chitosan (Chi) and lysozyme (Lys). A 10 step deposition of PGA–PLL and PGA–Chi from 10 mM pH 7.0 phosphate buffer with 30 mM NaCl led to 18.3 nm and 32 nm thick multilayer films, respectively, and the PGA–Lys deposition to a 3.2 nm thick mixed adsorbed film. The PGA–PLL multilayer assembled with a relatively small polycation showed exponential growth and QCMD showed its material properties remained constant during assembly. The PGA–Chi multilayer also showed exponential growth and, despite some stripping of Chi when PGA was deposited, grew more rapidly than the PGA–PLL multilayer. The QCMD showed that the multilayer was more viscous when the Chi was uppermost and more elastic when PGA was uppermost. The PGA–Lys deposition showed a cyclical behaviour corresponding to repeated deposition and stripping with little net growth after the first 3–4 deposition cycles. Further analysis of the PGA–Chi acoustic and optical hydrated mass measurements suggested that the refractive index of the film was relatively high in the region close to the adsorbing surface, and an extensive lower refractive index region stretched out from the surface. The polymer mass measured by FTIR-ATR was up to 1.5 times greater than that measured by DPI, a discrepancy which could be explained in terms of the known sensitivity of the FTIR-ATR amide I band to conformational changes of these polycations which change upon deposition. The strength of the FTIR-ATR method was that it yielded film compositions which indicated that the polymers in the multilayer films carried an excess of negative charge, evidence for extrinsic charge balance within the multilayer films.
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