Liquid Core Waveguide Cell with In Situ Absorbance Spectroscopy and Coupled to Liquid Chromatography for Studying Light-Induced Degradation

In many areas, studying photostability or the mechanism of photodegradation is of high importance. Conventional methods to do so can be rather time-consuming, laborious, and prone to experimental errors. In this paper we evaluate an integrated and fully automated system for the study of light-induced degradation, comprising a liquid handler, an irradiation source and exposure cell with dedicated optics and spectrograph, and a liquid chromatography (LC) system. A liquid core waveguide (LCW) was used as an exposure cell, allowing efficient illumination of the sample over a 12 cm path length. This cell was coupled to a spectrograph, allowing in situ absorbance monitoring of the exposed sample during irradiation. The LCW is gas-permeable, permitting diffusion of air into the cell during light exposure. This unit was coupled online to LC with diode array detection for immediate and automated analysis of the composition of the light-exposed samples. The analytical performance of the new system was established by assessing linearity, limit of detection, and repeatability of the in-cell detection, sample recovery and carryover, and overall repeatability of light-induced degradation monitoring, using riboflavin as the test compound. The applicability of the system was demonstrated by recording a photodegradation time profile of riboflavin.


Table of contents
The TooCOLD-LC-DAD set-up p. S3 A typical method programmed in Maestro for a degradation experiment p. S5 Schematic overview of transfer volumes tested p. S6 Effect of riboflavin concentration on degradation rate p. S7 Overview of significance testing by F-and t-tests p. S8 Calculation of LOD for riboflavin from in situ absorbance measurements p. S10 Data obtained from spectral repeatability experiments p. S11 Data for recovery experiments p. S12 Additional data for repeatability of analysis and degradation p. S13 Additional data from LC analysis of riboflavin time profile p. S14 (1) (3) Figure S2. A cross section of the filter wheel holding the optical elements on the left and the LID cell on the right. From left to right: a collimated beam (yellow) leaves the collimator (1), that is connected to a lens (3) via a short lens tube (2). The collimated beam is then focused by the lens, which has a focal length of 35 mm so that the focal point is positioned closely to the entrance of the LCW.

S5
A typical method programmed in Maestro for a degradation experiment Figure S3. An overview of a typical method programmed in Maestro to perform all steps during a 30 min degradation experiment. The first steps indicate that the lamp and detector are switched on. The 6-port valve is switched to position B (inject position). A sample of Milli-Q is injected to measure the baseline (I 0 ). 'Pulse Det ON' sends a trigger to the spectrometer to measure an absorbance spectrum. The command 'Sample 70 µL' injects a sample from the sample tray into the LID cell. The needle is then washed and real-time absorbance spectra are taken every 10 min. Then, the valve is switched to A (load) to transfer the sample to the sample loop. It is then injected and measured by LC. Afterwards, the LID cell and sample loop are flushed with wash solvent 2, followed by wash solvent 1.

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Schematic overview of transfer volumes tested Figure S4. A schematic overview of the transfer volumes tested to optimize the recovery of RF measured by LC; each rectangle represents 10 µL. The total volume of the LCW is 60 µL, the sample loop is 20 µL and there is a total of 15 µL of tubing volume that connects the injection port with the LID cell and the LID cell with the 6-port valve. The sample loop is depicted in green, the transfer volume in blue, the volume of the LCW in yellow. The middle of the cell is indicated with two X's. In the most ideal scenario the middle of the cell is transferred to the sample loop and the rest is sent to the waste channel. Figure S5. The remaining peak area of riboflavin measured by LC-DAD after 30 min of irradiation for different concentrations (n=3) compared to the peak area before irradiation. The degradation rate is dependent on the concentration, with a decreasing rate at higher RF concentrations. This is due to high absorbance at the front of the LCW, resulting in low light intensities reaching the middle section of the LCW (from where the solution will later be transferred for LC analysis). This effect becomes negligible at concentrations below 6.5*10 -6 M RF.

Calculation of LOD for riboflavin from in situ absorbance measurements
The LOD of riboflavin (on-line absorbance measurements in the 12-cm LCW) was calculated by means of the linear trend line, shown in Section 3.1.1. The LOD is determined as the concentration where the signal is three times the SD of the noise (σ noise ). The value for σ noise was obtained from the baseline of a spectrum of a blank measurement.
Formula of the linear trendline in Figure 2: G calculated is larger than G critical , and the value is therefore identified as an outlier and was excluded from the dataset. An overview of the new averages and RSDs are shown in Table   S3.

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Additional data for repeatability of analysis and degradation  Figure S7. LC-DAD spectra of the reaction products obtained after 3-hr degradation of RF, see   Figure S8. LC chromatogram of the same sample as in Figure 5 but DAD absorbance extracted at 450 nm, where a 7 th peak (indicated with ?) was observed.