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

doi:10.3724/SP.J.1123.2025.04015

Abstract

Abstract:

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 k 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-9H-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 π-π 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 （r²=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.

Key words: L-hydroxyproline, L-proline, amino acid-functionalized stationary phase, hydrophilic interaction liquid chromatography （HILIC）

CLC Number:

O658

XU Gaigai, YI Yang, LIU Pingping, ZHANG Wenfen. Preparation and chromatographic performance evaluation of hydrophilic interaction chromatography stationary phase based on amino acids[J]. Chinese Journal of Chromatography, 2025, 43(7): 734-743.

Figures/Tables 13

Fig. 1 Synthetic route of amino acid-functionalized HILIC stationary phasesDIPEA： N，N-diisopropylethylamine； HILIC： hydrophilic interaction liquid chromatography.

Table 1 Elemental analysis results

Analyte	Elemental contents/%			Surface coverage/（mmol/g）
Analyte	N	C	H	Surface coverage/（mmol/g）
APS-Sil	1.558	5.570	1.282	0.928
TCT-Sil	2.831	6.482	1.227	0.253
L-OH-PSil	2.885	8.801	1.520	0.193
L-PSil	2.888	8.614	1.320	0.178

Fig. 2 Characterization of L-OH-PSil and L-PSila. FT-IR spectra of SiO2， APS-Sil， TCT-Sil， L-OH-PSil and L-PSil； b. TGA of L-OH-PSil and L-PSil.

Fig. 3 Separation performance and retention behavior of sulfonamides on L-OH-PSil and L-PSilMobile phases：（a， b） L-OH-PSil， ACN-H2O （93∶7， v/v）；（c， d） L-PSil， ACN-H2O （91∶9， v/v）.

Fig. 4 Chemical structures of （a） eight heterocyclic amines and （b） seven nucleosidesGlu-P-1： 2-amino-6-methyldipyrido［1，2-a：3′，2′-d］imidazole； MeAαC： 2-amino-3-methyl-9H-pyrido［2，3-b］indole； Glu-P-2： 2-aminodipyrido［1，2-a：3′，2′-d］imidazole； PhIP： 2-amino-1-methyl-6-phenylimidazo［4，5-b］pyridine； Norharman： 9H-pyrido［3，4-b］indole； DMIP： 2-amino-1，6-dimethylimidazo［4，5-b］pyridine； Harman： 1-methyl-9H-pyrido［3，4-b］indole； IQ： 2-amino-3-methyl-3H-imidazo［4，5-f］quinoline.

Fig. 5 Chromatograms of （a） the eight heterocyclic amines and （b） the seven nucleosides on L-OH-PSil， L-PSil， and Hypersil NH2 columnsMobile phase： ACN-H2O （87∶13， v/v）.

Fig. 6 Separation of hydrophilic compounds on L-OH-PSil and L-PSil columnsMobile phases：a. ACN-H2O （92∶8， v/v）； b. ACN-H2O （87∶13， v/v）； c. ACN-H2O （87∶13， v/v）； d. ACN-H2O （89∶11， v/v）； e. ACN-H2O （90∶10， v/v）； f. ACN-H2O （89∶11， v/v）.

Fig. 7 Separation characteristics of nucleosides on L-OH-PSil and heterocyclic amines on L-PSil columnsSeparation efficiency of nucleosides on the L-OH-PSil column at （a） different flow rates and （b） different column temperatures；（c） Van’t Hoff plots of nucleosides on the L-OH-PSil column； separation efficiency of heterocyclic amines on the L-PSil column at （d） different flow rates and （e） different column temperatures；（f） Van’t Hoff plots of heterocyclic amines on the L-PSil column.Mobile phase： heterocyclic amines， ACN-H2O （87∶13， v/v）； nucleosides， ACN-H2O （89∶11， v/v）.

Table 2 Theoretical plate numbers of the eight heterocyclic amines on two amino acid-functionalized stationary phases

Column	Glu-P-1	MeAαC	Glu-P-2	PhIP	Norharman	DMIP	Harman	IQ
L-OH-PSil	3696.98	11582.87	3414.64	3889.27	4174.57	3701.19	3666.03	3299.56
L-PSil	2292.08	8661.45	3628.90	4376.20	4696.48	3377.73	3821.84	2950.15

Table 3 Selectivity factors of the six nucleosides on L-OH-PSil at different temperatures

T/℃	4，6-Dichloropyrimidine	2-Fluoro-6-chloro-purine	Uracil	Adenosine	Adenine	Acyclovir
15	-	10.88	1.61	1.84	1.51	1.44
20	-	10.28	1.72	1.78	1.49	1.46
25	-	10.20	1.79	1.82	1.37	1.50
30	-	11.35	1.76	1.80	1.43	1.52
35	-	9.96	1.87	1.77	1.45	1.50
40	-	9.48	1.89	1.76	1.43	1.52

Table 4 Selectivity factors of the six heterocyclic amines on L-PSil at different temperatures

T/℃	Glu-P-1	AαC	Norharman	Harman	DMIP	IQ
15	-	5.19	2.77	1.41	1.43	2.01
20	-	5.12	2.64	1.36	1.51	1.83
25	-	4.94	2.54	1.33	1.59	1.73
30	-	4.86	2.46	1.30	1.62	1.64
35	-	4.96	2.37	1.26	1.74	1.51
40	-	4.94	2.29	1.22	1.86	1.41

Table 5 Thermodynamic parameters （ΔH， ΔS） and fitting quality （r2） of nucleosides on L-OH-PSil column and heterocyclic amines on L-PSil column

Column	Analyte	ΔH/（kJ/mol）	ΔS/（J/（mol·K））	r²
L-OH-PSil	4，6-dichloropyrimidine	-10.54	-60.81	0.9959
	2-fluoro-6-chloro-purine	-13.31	-50.95	0.9961
	uracil	-8.91	-31.21	0.9953
	adenosine	-9.94	-29.76	0.9968
	adenine	-11.29	-31.22	0.9942
	acyclovir	-9.71	-22.63	0.9949
L-PSil	Glu-P-1	-1.85	-27.06	0.9961
	AαC	-3.33	-18.93	0.9978
	Norharman	-9.07	-30.08	0.9931
	Harman	-13.31	-42.29	0.9962
	DMIP	-5.93	-13.38	0.9997
	IQ	-16.08	-42.87	0.9928

Fig. 8 Separation stability of the six nucleosides on L-OH-PSil stationary phase under varying injection volumesMobile phase： ACN-H2O （87∶13， v/v）.

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