# Natural Clays as Potential Amino Acids Carriers for Animal Nutrition Application
## 3 Results
### Effect of Multiple Intercalation Steps  
On ADS, i.e., the carrier selected for the target application, multiple contacting experiments were also performed.  
Each step was carried out with a fixed methionine concentration of 0.2 M, at room temperature and stirring at 600 rpm for 30 min. Upon liquid-solid separation, the liquid was analyzed by COD, while the solid by FTIR spectroscopy. In Figure 5, two sets of experiments, one considering three repeated intercalation steps, and another considering six intercalation steps, are plotted as a function of the steps number.  
A constant amount of methionine is captured in each step (on average, 0.17-0.18 g of methionine for g of carrier). Considering experimental evidence (always the same amount of methionine is captured), in the case of the MET-ADS system, the adsorption process, apparently, does not depend on site availability, but rather on the methionine concentration in solution. Indeed, it appears that a threshold methionine value must be present in solution to act as a driving force of the adsorption reaction. A total methionine content of 0.51 gMET/gADS and 1.02 gMET/gADS was supported, respectively, upon three and six steps. The total captured methionine linearly increased with number of steps only, the MET/ADS ratio upon three steps being almost the half of the one upon six steps.  
Similar results were also found for BIO, the reference zeolite material (not reported in the figure), where the same capture behavior was manifest, and a methionine capture of 0.50 gMET/gBIO was measured after three steps. Accordingly, this behavior can be related to the surface area of these carriers, i.e., 93 m\({}^{2}\)/g for ADS and 103 m\({}^{2}\)/g for BIO, which in turn depends on the presence of a large zeolitic component (see Table 1).  
Moreover, by comparing the three-step process (Figure 5, orange columns) with the first three steps of the six-step one (Figure 5, green columns), it can be concluded that the process is fully reproducible; once operating conditions and number of steps were fixed, the same amount of methionine was captured in two different experiments.  
Figure 5: Methionine capture as a function of repeated three or six intercalation steps for the system MET–ADS.  
Effect of the multiple intercalation steps was also studied by FTIR spectroscopy (Figure 6a,b).  
Over ADS, IR analysis evidenced, indeed, the presence of methionine, and multiple contacting resulted in an increased methionine-to-clay band intensity ratio (not-subtracted skeletal spectra are reported in Supporting S5), in agreement with data reported in Figure 5. As discussed previously, methionine is adsorbed in its zwitterionic form, as indicated by bands near 1655, 1620, 1580, and 1415-10 cm\({}^{-1}\), and by the weak absorptions above 2000 cm\({}^{-1}\). Moreover, in the low-frequency region, some bands due to side chain of the amino acid are detected almost unchanged. The comparison with spectra previously reported seems to indicate that the molecule is even less affected by interaction with matrix. Possibly, more than one layers of methionine are deposited, loosening the interaction with clay surface and therefore leading to features close to the methionine solid compounds. Methionine adsorbed in the same conditions over BIO matrix showed IR bands that were slightly broadened and shifted (see, for instance, CH stretching bands in the range 3000-2800 cm\({}^{-1}\), Figure 6b), evidencing a closer interaction with the host surface, in spite of multiple contacting runs.  
Figure 6: FTIR subtraction spectra (Adsorbene/BioKi + methionine) – (Adsorbene/BioKi) following multiple contacting tests: three steps and six steps. Pure methionine spectrum is also reported as reference: (**a**) low frequency region; (**b**) high-frequency region.