Fourier-transform infrared spectroscopy (FTIR) is a powerful analytical tool for studying molecular vibrations and identifying chemical compositions in various samples. However, its application in water-rich environments, such as biological fluids or environmental aqueous samples, presents unique challenges due to the strong infrared absorption of water. This article discusses the key obstacles and solutions for implementing FTIR in aqueous systems, with a focus on attenuated total reflectance (ATR)-FTIR and solvent suppression techniques, particularly in biological and environmental contexts.

One of the primary challenges in water-rich environments is the intense absorption bands of water in the mid-infrared region. Water exhibits strong peaks around 3400 cm⁻¹ (O-H stretching) and 1640 cm⁻¹ (H-O-H bending), which can overshadow the signals of analytes of interest. This interference complicates spectral interpretation, especially for biological molecules like proteins, lipids, and carbohydrates, which have overlapping absorption bands. Additionally, water’s high infrared absorption necessitates very short path lengths in transmission mode, often requiring specialized cells with spacers in the micrometer range, which can be impractical for many samples.

ATR-FTIR offers a practical solution to these challenges. In ATR-FTIR, the infrared beam penetrates only a few micrometers into the sample at the interface with an internal reflection element (IRE), such as diamond, zinc selenide, or germanium. This evanescent wave probes the sample surface, minimizing bulk water interference while still capturing molecular information. Diamond ATR is particularly advantageous for aqueous systems due to its chemical inertness, durability, and broad spectral range. ATR-FTIR also eliminates the need for thin sample preparation, making it suitable for heterogeneous or viscous biological samples like cell cultures, biofilms, or tissues.

Despite its advantages, ATR-FTIR still faces limitations in highly dilute aqueous systems where the analyte signal is weak compared to water. To address this, advanced signal processing techniques are employed. Water subtraction algorithms are commonly used, where a reference water spectrum is subtracted from the sample spectrum to isolate the analyte peaks. However, this requires precise matching of water bands, as slight variations in temperature or ionic strength can shift water absorption frequencies. Temperature control during measurements is critical, as water’s infrared spectrum is highly temperature-dependent.

Another challenge in aqueous FTIR is the potential for water to interact with analytes, altering their spectral signatures. For example, proteins may undergo conformational changes in solution, leading to shifts in amide I and II bands. To mitigate this, some studies use deuterated water (D₂O) as a solvent, which shifts the O-D stretching band to around 2500 cm⁻¹, reducing overlap with analyte peaks. However, D₂O can still interfere with certain regions of the spectrum and may not be suitable for all applications, particularly environmental studies where natural conditions must be preserved.

Solvent suppression techniques are essential for enhancing analyte detection in water-rich environments. One approach is the use of advanced background correction methods, such as dynamic subtraction, where the reference spectrum is continuously adjusted to account for solvent variations. Another technique involves optimizing the ATR crystal material and geometry to enhance the evanescent wave’s sensitivity. For instance, germanium crystals provide deeper penetration than diamond, which can improve signal strength for certain analytes but may also increase water interference. The choice of crystal depends on the specific application and trade-offs between sensitivity and water suppression.

In biological applications, ATR-FTIR has been successfully used to study protein secondary structures in aqueous solutions. The amide I band (1600–1700 cm⁻¹) is particularly informative for analyzing α-helix, β-sheet, and random coil conformations. By carefully controlling hydration levels and using buffer-matched reference spectra, researchers can minimize water contributions and focus on protein-specific signals. Similarly, lipid bilayers and cell membranes can be studied at the ATR interface, providing insights into molecular interactions in near-native environments.

Environmental applications of ATR-FTIR include monitoring pollutants in water, such as microplastics, organic contaminants, or heavy metal complexes. Here, the challenge lies in detecting trace analytes amidst a dominant water matrix. Pre-concentration techniques, such as solid-phase extraction or filtration, can enhance analyte signals before ATR-FTIR analysis. For in situ measurements, flow-through ATR cells enable real-time monitoring of dynamic processes like adsorption or degradation of contaminants in water.

Time-resolved ATR-FTIR is another powerful tool for studying kinetic processes in aqueous systems. By coupling ATR with rapid-scanning FTIR, researchers can track chemical reactions, phase transitions, or biological interactions with millisecond resolution. This is particularly useful for investigating enzyme catalysis, polymer dissolution, or environmental redox reactions. The key is to ensure that water’s strong absorption does not saturate the detector, which can be managed by adjusting the scan speed and spectral resolution.

Despite these advancements, some limitations persist. For example, highly ionic or turbid samples can scatter or absorb infrared radiation, reducing signal quality. In such cases, combining ATR-FTIR with other techniques like Raman spectroscopy or electrochemical methods can provide complementary data. Additionally, the cost and complexity of advanced FTIR systems may limit their accessibility for routine environmental monitoring.

In summary, FTIR spectroscopy in water-rich environments requires careful consideration of water’s spectral interference and its impact on analyte detection. ATR-FTIR and solvent suppression techniques offer viable solutions, enabling studies of biological molecules and environmental contaminants in aqueous media. Continued advancements in signal processing, crystal materials, and integration with other analytical methods will further expand the capabilities of FTIR for water-based applications. The key to success lies in optimizing experimental conditions to balance sensitivity, specificity, and practicality for real-world samples.