1: Experimental background

The active ingredient in Nongda herbicide is a chemical substance called glyphosate, in which glyphosate, N - (phosphonomethyl), is a form of glycine isopropylammonium salt. This form of glyphosate is easy to handle and has strong efficacy. The content of glyphosate in general agricultural herbicides is about 2%, and in actual use, it also needs to be diluted with water.

In this experiment, we used variable optical path immersion probes with optical path lengths of 10mm and 5mm to test the absorbance of these low concentrations of glyphosate and verify their detection limits. In absorbance measurement, the optical path length is negatively correlated with the detection limit, so we predict that a 10mm optical path length will provide more accurate and reliable results. We used a probe with a 5mm optical path length to verify whether a shorter optical path length can also detect glyphosate, and whether it will obtain similar results as a 10mm optical path length probe.

2: Spectral system construction

In the experiment, we used Avantes' research grade ultraviolet/visible spectrometer AvaSpec HERO (Figure 1). The AvaSpec HERO spectrometer is based on a high-sensitivity compact optical platform (focal length=100mm, numerical aperture=0.13) and a 1024 × 58 pixel backlit CCD detector, which has high sensitivity and resolution. AvaSpec HERO uses an advanced AS7010 circuit board, which includes a high-performance AD converter with high signal-to-noise ratio and dynamic range. USB 3.0 and Ethernet interfaces enable high-speed communication. Due to the high sensitivity required in the ultraviolet range for this experiment, we used this spectrometer.

Figure 1 Experimental System: AvaSpec HERO Spectrometer, AvaLight KE High Power Pulse Xenon Lamp, and Variable Path Immersion Probe

The light source adopts a pulsed xenon lamp AvaLight XE-HP, which has excellent performance in the extreme ultraviolet region and is connected to the spectrometer through a Y-shaped trigger line. It starts working after receiving the TTL signal from the spectrometer to ensure that the spectrum is only measured when the light source is working. The number of flashes per measurement can be set in the AvaSoft software to avoid spectral saturation (which occurs when excessive light enters the spectrometer). We set the number of flashes to 3 in the 10mm optical path length experiment and 2 in the 5mm optical path length experiment.

Figure 2: Variable optical path immersion probe with an optical path length of 10mm

The last component in the experimental system is Avantes' variable path immersion probe. Avantes provides fixed or variable optical path length immersion probes (Figure 2) for scientific research and industrial customers. The change in optical path length can be achieved by loosening and tightening the copper tube fittings. A longer optical path length is suitable for measuring low concentration samples, while a shorter optical path length is suitable for measuring high concentration samples. This is because longer distances allow more light to enter the sample, and a probe with an optical path length of 10mm is more suitable for samples with lower concentrations than a 5mm length.

3: Experimental process

As shown in Figure 3, the recommended dilution ratio for Nongda herbicide is 1.5 ounces (3 tablespoons) of Nongda herbicide mixed with 1 gallon of water, resulting in a concentration of approximately 1.2% for the Nongda herbicide sample. While keeping the solute weight constant, the other three samples were diluted 25%, 50%, and 75% more than the recommended concentration, respectively. (This concentration is determined based on three other concentrations, which have dilution rates 25%, 50%, and 75% higher than the recommended concentration, respectively.)

Figure 3 Concentration and percentage of diluted Nongda herbicide samples

The solvent is deionized water, used to dilute a 2% glyphosate solution of Nongda. After dilution, four sample concentrations were obtained, which were 1.2%, 0.88%, 0.59%, and 0.29%, respectively (Figure 3). The percentages of glyphosate in these four samples were 0.024%, 0.018%, 0.012%, and 0.0058%, respectively (Figure 3). The four diluted samples are shown in Figure 4.

Figure 4: Diluted solutions of four different concentrations of Nongda herbicide

4: Experimental data and results

Before testing the sample, we set the integration time of the spectrometer to 30ms and the flash frequency of the AvaLight XE-HP light source to 3 times. We first tested four samples using a 10mm optical path length. We chose the absorbance mode and stored the dark noise to eliminate the instrument's inherent noise, using the solvent (deionized water) as a reference. The absorbance measurement results using a 10mm optical path length probe are shown in Figure 5.

Figure 5: Measurement results of absorbance using a 10mm optical path length probe

The absorbance peak of pure glyphosate is around 265 nm, while the absorbance peak of diluted glyphosate can be located at any wavelength between 200 and 300 nm. The peak we measured in the experiment was around 200nm, and the spectral shape was consistent with the spectral data of other researchers. Therefore, it can be proven that using a probe with a 10mm optical path length can effectively measure glyphosate in diluted solutions of four types of herbicides.

The second experiment used a 5mm optical path length probe. We set the integration time of the spectrometer to 20ms and the AvaLight XE-HP light source to flash twice to avoid saturation. The measurement results are shown in Figure 6

Figure 6: Measurement results of absorbance using a 5mm optical path length probe

5: Discussion on Results

The consistency of spectra measured by a 5mm optical path probe is not as good as that measured by a 10mm probe. The spectral shapes of Sample 2 (purple, diluted by 25%) and Sample 4 (black, diluted by 75%) are different from the other two samples. And the absorbance spectra of samples 2 and 4 did not rapidly decrease after reaching their peak, but did not truly decrease. These results demonstrate that the measurement results of the 5mm optical path probe are not as reliable as those of the 10mm optical path probe. This is consistent with our initial assumption: because all four samples were fully diluted, a 10mm optical path probe can obtain better measurement results. The following figures (7, 8, 9, and 10) compare the test results of the 10mm optical path probe and the 5mm optical path probe for each sample. It can be seen that the measurement results using a 5mm optical path probe with a 10mm optical path probe are more accurate. Figure (11) is a summary of all spectral results from this experiment.

Figure 7: Measurement of suitable concentration solution using 5mm/10mm optical path probe

Figure 8: Measurement of a solution with a 25% dilution increase using a 5mm/10mm optical path probe

Figure 9: Measurement of a solution with a 50% dilution increase using a 5mm/10mm optical path probe

Figure 10: Measurement of a solution with a 75% dilution increase using a 5mm/10mm optical path probe

Figure 11: All results measured using a 5mm/10mm optical path probe

6: Conclusion

As predicted, a 10mm optical path variable immersion probe can better identify the absorbance peak of glyphosate. The 5mm optical path probe can also detect the signal of glyphosate, but the spectral shape is not as uniform as that of the 10mm optical path probe. These four highly diluted samples have all demonstrated better experimental results at longer optical path lengths. If our hypothesis is correct, then if the sample concentration is significantly increased in another experiment, the 5mm optical path probe can provide better experimental results.

This experiment proves that the variable optical path immersion probe can be used for absorbance measurement of low concentration samples and can cover a wide range of concentrations. This experiment has demonstrated that when the concentration of the tested sample is different, different optical paths provided by the variable optical path immersion probe can achieve good test results.