Application of special FTIR ATR techniques for quantitative structural analysis of thin surface layers

FTIR ATR spectroscopy is increasingly used for in situ investigations of processes at or near a surface. Particularly when thin layers (biomembranes, monolayers, thin films) are investigated with respect to surface concentration and molecular structure, very sensitive techniques have to. be applied in order to achieve an adequate signal-to-noise ratio. This may lead to long measuring times due to extended data accumulation and averaging. However, this can cause new problems with respect to the stability of relevant experimental parameters, such as the sample itself, the spectrometer, and the atmosphere in the spectrometer. In this article we report on two techniques which were developed or improved in our laboratory and successfully applied over past years. Both methods, the so-called single-beam sample reference (SBSR) spectroscopy and the modulation or modulated excitation (ME) spectroscopy, are well suited to compensate instabilities that occur in the course of an experimental series. The SBSR technique converts a single-beam FTIR spectrometer into a pseudo double-beam instrument. By this technique there is always a reference with the same age as the sample available. Moreover, by alternating sample and reference measurements within short time periods, varying environmental conditions such as water vapor concentration in the spectrometer are easily compensated. Moreover SBSR technique enables data evaluation in the conventional single-beam mode (SB) in both the sample (S) and reference (R) channel. This kind of evaluation is important to gain information on the history of S and R. As examples for SBSR and SB applications we report on studies of the interaction of an endotoxin with an immobilized lipid bilayer membrane, as well as on the interaction of TNF alpha with a TNF alpha antibody. ME spectroscopy can be applied to systems that show a (pseudo-) reversible response to a periodic excitation. The response of the system measured with time-resolved FTIR spectroscopy is then processed by phase-sensitive detection (PSID). ME spectroscopy is able to determine kinetic constants of a system, allows a hardware separation of overlapping absorption bands, and eliminates all disturbing signal components which do not have the same frequency as the excitation itself. This improves the signal-to-noise ratio dramatically and leads in principal to a stable baseline. The binding of sodium cholate to an adsorbed protein layer of human serum albumin (HSA) is shown as an example that the required sensitivity to study specific molecular interaction is in the mu AU range and can be reached by FTIR ME spectroscopy. In a second example, the measurement of structural changes of PLL induced by temperature modulation shows the feasibility of band separation and indicates the possible determination of kinetic properties of a system.