Adsorption of Water to Collagen as Studied Using Infrared (IR) Microspectroscopy Combined with Relative Humidity Control System and Quartz Crystal Microbalance

Infrared (IR) microspectroscopy combined with a quartz crystal microbalance (QCM) together with an original relative humidity (RH) control system has been developed for studying water adsorption on a collagen film. The adsorbed water weights measured by QCM are almost similar for wetting and drying processes at 28 ℃, indicating that the collagen film is close to the water adsorption/desorption equilibria. A broad OH + NH stretching band area (3000–3700 cm⁻¹) in the IR spectra of the collagen film increased linearly with the adsorbed weight until about 1.2 μg/8.0 μg dry collagen film at relative humidity (RH) = 40%, while at higher RH (60%, 80%), the band area deviates from the linear trend to the lower side, due to viscoelasticity and others. The OH + NH band can be simulated by four Gaussian components at 3440, 3330, 3210, and 3070 cm⁻¹ with the relatively constant band areas of 3330 and 3070 cm⁻¹ components due to amide A and B (NH) for increasing and decreasing RH. Bound water (3210 cm⁻¹ component: short H bond) constitutes around 70% of total water (3440 + 3210 cm⁻¹ band areas) at RH = 4.9% but decreases to 23% at RH = 80.3%, where free water (3440 cm⁻¹ component: long H bond) becomes dominant over 70%. The peak shifts of C=O stretching (Amide I) and N–H bending (Amide II) can be understood by increasing hydrogen bonding of water molecules (bound water) bound to peptides at lower RH. The higher wavenumber shifts of CH stretching can be due to the loose binding of water molecules (free water) to aliphatic chains on the collagen surface, especially at higher RH. The present combined QCM-IR method is useful for studying amounts and natures of water adsorbing on biomolecules.