IRsweep AG

Spectroscopic reaction monitoring is a proven tool in R&D and production environments, where it is used to gain insight into chemical reaction kinetics. In this application note, we showcase how dual-comb spectroscopy (DCS) can be used to monitor protein dynamics on microsecond to second timescales. Kinetics of irreversible protein reactions require an analytical technique that provides time-dependent infrared spectra in a single shot. First results have been validated with the recording of transients of the photoactivated proton pump bacteriorhodopsin with DCS and StepScan FT-IR (1).

Non-repetitive reactions with microsecond time resolution

Fourier transform infrared spectroscopy (FT-IR) with time resolutions faster than 10 ms is only possible with proteins, where the reactions can be repeated thousands of times. Many relevant protein reactions are nonrepetitive. In many cases reactions can be induced by caged compounds (2). As an example, we investigate the inhibiting Gi protein and the larger protein–protein complex of Gα

 with its cognate regulator of G-protein signaling (RGS). With DCS, we observe good data quality with 4 μs time resolution, three orders of magnitude faster than any FT-IR measurements.

Figure 1: (A) IR cuvette with photo-sensitive paper, exposed to the QC laser beam (burned point in the middle). (B) Contour-plot of the changes obtained from dual-comb spectroscopy.

Stopped-flow applications coupled with mid-IR spectroscopy—more than the sum of its parts! 

Additionally, DCS can be used to monitor fast kinetic reactions in combination with the stopped-flow technique. Mid-IR stopped flow was so far limited to timescales of RapidScan FT-IR on the order of 100 ms. The combination with DCS paves the way to new research possibilities for kinetic studies in the microsecond time regime. Simply speaking, it works by rapidly mixing reagents before a spectroscopic cell, as depicted in Figure 2.

Figure 2: Scheme of stopped-flow application with in situ spectroscopy.

For these applications, we partnered with TgK Scientific and BioLogic SA. It connects via an umbilical to a spectroscopic cell with CaF

The hydrolysis of methyl chloroacetate (MCA) is often carried out as a test for stopped flow systems and typically hydroxide concentrations of 0.1 M or less are used.

The results of a single shot of the hydrolysis reaction are shown in the Figure 3. Excellent signal to noise ratios are seen throughout the spectra and despite the low intensity of the MCA band, it can be fitted well with a monoexponential decay. A lifetime of 53 ms is found for this process at room temperature.

Figure 3: (A): Spectra of the hydrolysis reaction of MCA. 4.5 ms per spectrum. (B): resulted time traces taken at two selective wavelengths with 1000 spectra per s in situ.

High resolution dual-comb spectroscopy with QCLs

Optical frequency comb spectroscopy has further proven a very useful tool for high resolution molecular spectroscopy of gaseous samples. Frequency combs based on quantum cascade lasers (QCL) offer the possibility to easily explore the mid-infrared spectral range (4–12 μm) but suffer from very large repetition frequencies (~10 GHz) which make them seemingly unsuitable for high resolution spectroscopy.

We have developed techniques to overcome this limitation, which we call the rapid-sweep (3) and step-sweep (4) technologies. In rapid-sweep, full spectra can be measured in only 6 ms by simultaneously sweeping both frequency combs. The step-sweep technique guarantees a very accurate knowledge of the frequency axis in a record time of several min.

Both techniques have been applied to the measurement of absorption spectra of gases with narrow absorption lines at high resolution and sensitivity. The measured spectra cover a range of more than 60 cm

 and the narrowest observed lines have a full width at half maximum of 15 MHz (0.0005 cm

The broadband coverage is useful for the analysis of complex gas mixtures. The low measurement noise makes the technique the ideal tool for studying gas concentration in environmental or industrial applications (Figure 4).

Figure 4: Spectrum of methane with a spectral resolution of below 0.001 cm

(1) https://doi.org/10.1021/acs.analchem.8b02531.

(2) https://doi.org/10.1021/acs.analchem.1c00666.

(4) Quantum cascade laser dual-comb spectroscopy (DCS) for methane gas mixture studies (spiedigitallibrary.org)

Laubisruetistrasse 44, 8712 Staefa, Switzerland 

Articles

Mid-Infrared QCL Dual-Comb Spectroscopy: From Non-Repetitive Protein Dynamics to Stopped-Flow Applications with Microsecond Time Resolutions

This application note shows how the IRis-F1 dual-comb spectrometer can monitor curing reactions to provide unique insight on the performance of coatings and adhesives.

Mid-Infrared Reaction Monitoring of Curing Processes

	Fast spectroscopic monitoring is a proven tool in R&D and production environments to gain insight into chemical reactions. This application note showcases how the IRis-F1 dual-comb spectrometer can be used to monitor curing reactions, where it can provide invaluable insight into the performance of coatings and adhesives.

	Infrared spectroscopy is in many ways an ideal measurement technique for this application as it is capable of performing non-invasive in-situ measurements that provide direct information about the structure and bonding of the sample. However, many high-performance adhesives possess fast curing rates, and the speed of off-the-shelf spectroscopic techniques is the limiting factor in their analysis.

	The IRis-F1 is based on quantum cascade laser (QCL) frequency comb technology, which allows for measurements on microsecond to minute timescales to be carried out.

	Measurements were conducted with a single-reflection diamond Golden Gate ATR accessory (Specac). The sample is a UV-activated adhesive, which was kindly provided by DELO Industrial Adhesives (Germany). When uncured, it has an infrared band at 1613 cm

	The spectrometer was set to a resolution of 4 cm

 and the acquisition time was 1 ms, with a repetition rate of 40 Hz. Curing was triggered using a mercury lamp (LC8, Hamamatsu), via an optical fiber. After starting the measurement, the lamp is turned on to initiate the curing reaction. For each measurement, a rapid series of measurements spanning before, during, and after curing was taken.


	Figure 1: Set-up: IRis-F1 dual-comb spectrometer with ATR GoldenGate for µs/ms reaction monitoring; 40 Hz for curing was applied.

	Different spectra can be generated by subtracting a spectrum of the uncured glue (defined as a negative-time spectrum) from each of the subsequent positive-time spectra. Figure 2 shows the resulting difference spectra, which highlight the changes produced by the curing reaction in 25 ms increments. Negative bands show that a feature has disappeared, while positive bands show that a new feature has grown in. Here it is evident that not one, but two new bands grow in, at ca. 1638 and 1624 cm


	Figure 2: Time evolution of the curing reaction acquired in 25 ms time increments.

	The fringing, expected when using a laser-based instrument with an ATR prism, is caused by a change in the refractive index of the sample (for example, glue vs. air). Fringes could be removed with a simple Fourier transform (FT) filter.

	The kinetic traces at two points in the spectra (inset) clearly show the time-point where the lamp was turned on. By fitting the growth and decay of these points, the kinetic properties of the curing process can be determined.

	Rapid measurements with high signal-to-noise ratios were performed on a sample of glue during curing, giving insight into processes that happen on a millisecond timescale. This bridges the previously demonstrated capabilities of the IRis-F1 to measure on the microsecond timescale, with the second-to-minute timescale. It enables processes over many orders of magnitude in time to be studied.


	Laubisruetistrasse 44, 8712 Staefa, Switzerland
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IRsweep AG

Articles

Mid-Infrared QCL Dual-Comb Spectroscopy: From Non-Repetitive Protein Dynamics to Stopped-Flow Applications with Microsecond Time Resolutions

Mid-Infrared Reaction Monitoring of Curing Processes

Fast Curing Reaction Monitoring with Dual-Comb Spectroscopy