Spectroscopic or microscopic techniques have to be used to probe structural changes in molecular assemblies adsorbed on solid surfaces. Spectroscopic techniques operate by irradiation of the surface with electromagnetic radiation and collecting the reflected or transmitted radiation. Vibrational spectroscopy probes gas, liquid and solid phases with in the infrared wavelength range. Infrared spectroscopy (IRS) is particularly attractive for the structural analysis of molecular assemblies at interfaces because:

1. It allows identification of the molecular structure and conformation of complex molecules (e.g., biopolymers, macromolecules);
2. Different functional groups absorb the IR light at different wavelength allowing for simultaneous analysis of each kind of species present at the interface;
3. A complex sample composition does not limit the spectral resolution and sensitivity of IRS;
4. No labeling is required;
5. Reflection-based techniques are applicable for studies of material adsorbed at solid interfaces.

To probe in situ structural and conformation changes in molecular films at solid surfaces (e.g., electrodes), we use polarization modulation infrared reflection absorption spectroscopy (PM IRRAS).

Vertex-70 (Bruker) with external reflection unit (Bruler), polarisation modulator and electrochemical cells for this setup.

Greenler provided the theoretical bases for the development of infrared reflection-absorption spectroscopy (IRRAS). However, the absorption of thin molecular films present at interfaces is very low (in the range of 10^-3 – 10^-5 absorption units). In addition, the environmental background (H₂O vapor and CO₂) are not fully compensated by measuring the reference spectrum.

In 1979 Hipps and Crosby showed that a fast modulation of the polarization state of the incoming linearly polarized electromagnetic radiation, during the recording of IRS measurements gives a double modulated differential reflectance spectrum. It has a huge advantage of the cancellation of a strong background signal relative to the absorption of the molecules at the interface. A few years later, PM IRRAS was introduced to study the composition, structure and orientation of organic molecules adsorbed adsorbed on metallic surfaces. Thanks to an excellent signal-to-noise ratio, this technique was quickly adopted to study monolayers at water|air and solid|water interfaces. The mirrors for IR radiation are metallic surfaces (Au, Pt, Ag or Cu). These materials conduct electric current and are commonly used in electrochemical studies.

The interface between the electrode material and the electrolyte solution is in focus of electrochemical studies. PM IRRAS provides excellent possibilities to analyze potential-driven changes in the structure of molecular adsorbates in situ. This applies to changes in conformation, orientation and solvation of molecular films at electrode surfaces. Over the last years, we gained experience in studies of redox-inactive and redox-active mono- and multilayers of small organic and metalloorganic molecules, ionic liquids as well as films of macromolecules such as polymers, protein or DNA strands.

Models of biological cell membranes are an important research object for us. Lipids and membrane-associated proteins have anisotropic orientation with a clearly distinguishable stratified structure shown in Figure 4a. Due to the fact that lipids and proteins absorb IR light at different frequencies (e.g., CH stretching modes in acyl chains or C=O stretching modes in lipids and proteins) simultaneous analysis is possible for each kind of species present in the membrane on the gold surface. Thus, a supramolecular assembly of a model membrane shown in Figure 4a can be studied with a sub-molecular resolution as illustrated in Figure 4b.

Interestingly, the relative intensities of IR absorption modes in an anisotropic film may significantly differ from the spectra measured in a bulk phase. Figure 5a shows IR transmission spectrum of lipid vesicles in D₂O and of a lipid bilayer adsorbed on the gold surface.

DMPC is a phospholipoid molecule containing two saturated myristoyl (C₁₄) chains. One DMPC molecule contains 24 CH₂ and two CH₃ groups. Both methylene and methyl groups have comparable extinction coefficients. As expected, the CH₂ stretching modes in DMPC vesicles have significantly higher intensities than the CH₃ stretching modes. In the bilayer however, the methyl and methylene stretching modes have comparable intensities.

These spectral changes are due to the surface selection rule of IRRAS and are used to determine average orientation of different molecular fragments (e.g., hydrocarbon chains) in anisotropic films. The integral intensity of a given IR absorption mode is given by equation 1:

Thus, to calculate the theta angle, the following steps have to be done:

1. Determination of the integral intensity of a given IR absorption mode. In the case of overlapped modes: deconvolution of the IR absorption modes (determination of the second derivative: number of IR absorption modes, deconvolution using Gaussian and Lorenz functions)
2. Knowledge of the orientation of the transition dipole vector in of a given IR vibration
3. The electric field vector is normal to the surface in PM IRRAS.

Thus, attenuation of the intensities of the methylene stretching modes in the DMPC bilayer is connected to the well-defined orientation of the myristoyl chains in the membrane. Figure 6 illustrates this situation and shows exemplary orientations of a hydrocarbon chain fragment of an amphiphilic molecule adsorbed on a solid surface.

Note that the theta angle cannot be determined directly from the PM IRRA spectrum. A reference system is required. This reference system represents a PM IRRA spectrum of randomly oriented molecules in the film of a given thickness. This spectrum can be calculated from the transmission solution spectrum of the studied molecule, e.g., from lipid vesicles. In an isotropic (randomly oriented) film, the average theta angle between the two vectors equals 53.54 ° (magic angle). Knowing the integral intensities of the IR absorption mode form PM IRRAS experiments and for the random film the theta angle can be calculated from equation 2,