Preparation and chromatographic performance evaluation of hydrophilic interaction chromatography stationary phase based on amino acids

To overcome current limitations in polar compound separation and better understand hydrophilic interaction liquid chromatography （HILIC） retention mechanisms， we designed and synthesized two novel amino acid-functionalized stationary phases using highly hydrophilic L-hydroxyproline and L-proline as modifiers through a continuous solid-liquid reaction method. The synthesized stationary phases were thoroughly characterized using Fourier-transform infrared spectroscopy （FT-IR）， thermogravimetric analysis （TGA）， and elemental analysis. Comparative elemental analysis revealed a substantial increase in carbon （C）， hydrogen （H）， and nitrogen （N） contents in both L-hydroxyproline-functionalized （L-OH-PSil） and L-proline-functionalized （L-PSil）stationary phases relative to the cyanuric chloride-bonded aminopropyl silica gel （TCT-Sil） intermediate， confirming successful functionalization. Quantitative analysis demonstrated distinct ligand densities for each phase， with L-OH-PSil exhibiting a higher loading （0.193 mmol/g） compared to L-PSil （0.178 mmol/g）. Thermal stability assessments indicated both materials maintained excellent structural integrity across a wide temperature range （20-600 ℃）， as evidenced by TGA results. To explore the chromatographic separation performance of the prepared L-OH-PSil and L-PSil stationary phases， sulfonamides were selected as solutes， and preliminary chromatographic separation investigations were conducted. The sulfonamide compounds exhibited excellent separation efficiency on both stationary phases， with retention behavior following consistent elution orders strongly correlated with analyte polarity. This observed retention pattern strongly suggested hydrophilic interactions constituted the predominant retention mechanism between the amino acid-functionalized stationary phases and sulfonamide analytes. Further supporting this conclusion， a systematic decrease in retention factors （lg <i>k</i> values） with increasing aqueous content in the mobile phase was observed as a characteristic feature of HILIC. Under optimized HILIC conditions， we further systematically evaluated the separation performance of both stationary phases using heterocyclic amines and nucleosides as model analytes， with direct comparison to a commercial Hypersil NH₂ column. Both custom phases exhibited exceptional column efficiency， with L-OH-PSil achieving 11 582.87 theoretical plates for 2-amino-3-methyl-9<i>H</i>-pyrido［2，3-b］indole （MeAαC） compared to 8 661.45 for L-PSil， while maintaining excellent performance across diverse analyte classes including plant growth hormones， flavonoids， and amines. The L-OH-PSil phase demonstrated superior chromatographic performance relative to both its L-PSil counterpart and the commercial NH₂ column. This superiority is attributable to its unique bifunctional design incorporating two hydroxyl groups， which combine the advantageous features of amino acid and diol-based stationary phases. This structural characteristic enables multiple synergistic interaction mechanisms， including <i>π-π</i> stacking， enhanced ion-exchange capacity， and additional hydrogen bonding sites， collectively yielding improved selectivity for polar small molecules. To further evaluate the chromatographic performance of the amino acid-based stationary phases， we investigated the effects of flow rate and column temperature using nucleosides and heterocyclic amines as model analytes. Remarkably， baseline separation was maintained even at elevated flow rates， demonstrating the robustness of both phases under high-throughput conditions. Temperature-dependent studies revealed that retention times exhibited only minor decreases or remained stable with increasing column temperature， suggesting minimal thermal effects on retention behavior. Van't Hoff analysis yielded excellent linear correlations （<i>r</i>²=0.992 9-0.999 7） for all tested analytes， confirming that the retention mechanism remains unchanged across the studied temperature range while indicating an exothermic separation process. Method validation confirmed the reliability of the developed system， with chromatographic peaks maintaining excellent shape and retention time stability across varying analyte concentrations. The relative standard deviations （RSDs） of retention times for six nucleosides ranged from 0.29% to 0.59%， underscoring the outstanding operational stability and analytical reproducibility of the L-OH-PSil stationary phase. These results collectively demonstrate the robustness of the amino acid-functionalized stationary phases under varying chromatographic conditions， further supporting their potential for practical applications in polar compound analysis. These results indicate that the L-OH-PSil stationary phase has excellent potential for broad applications in pharmaceutical analysis， environmental monitoring， and bioanalytical separations.