Equilibration behavior of superficially and fully porous ZIC®-cHILIC columns: A comparative study of Ascentis® Express (SPP) and SeQuant® (FPP) architectures
Abstract
The equilibration performance of hydrophilic interaction liquid chromatography (HILIC) columns packed with superficially porous particles (SPPs) and fully porous particles (FPPs) was evaluated using a gradient method. Stable retention factors for representative marker compounds were assessed following gradient elution. The SPP column reached stable retention factors more rapidly than the FPP column, requiring fewer equilibration cycles. These findings suggest that SPP-based HILIC columns can improve analytical efficiency and reduce solvent consumption in gradient applications.
Section overview
Introduction
Equilibration of hydrophilic interaction liquid chromatography (HILIC) columns is a critical step, particularly in gradient applications, to ensure consistent and reproducible chromatographic performance. The re-equilibration time depends on factors, including the type of column modification, column length and inner diameter of the column, as well as the particle architecture.
In this study, the required (re-)equilibration time for a HILIC gradient method was investigated using two columns with polyphosphorylcholine (PC) grafted surface, a superficially porous particle (SPP) Ascentis® Express ZIC®-cHILIC column and a fully porous particle (FPP) SeQuant® ZIC®-cHILIC column. The equilibration procedure followed the steps described by McCalley (2018).1 Uridine, 4-hydroxybenzoic acid, and nortriptyline (Figure 1) were employed as indicator/marker compounds.
Figure 1.Structures of marker compounds used in the study.
Experimental
The equilibration study was performed by injecting a standard mixture containing uridine, 4-hydroxybenzoic acid, and nortriptyline after different equilibration times on an Ascentis® Express ZIC®-cHILIC, 2.7 µm, 150 x 2.1 mm column, and a SeQuant® ZIC®-cHILIC, 3 µm, 150 x 2.1 mm column, using UV detection. The applied HILIC method employed a solvent and ionic strength gradient (Table 1). Flow rates and gradients were adjusted according to particle size.
The columns were equilibrated with varying column volumes (CV; empty tube volume: 519.5 µL) prior to injection of the test mixture containing three indicator compounds. The test procedure was as follows:
- Flush the columns with mobile phase A/B (40/60, v/v), representing the end-of-gradient conditions.
- Perform three equilibration cycles (8 CV) to A/B (5/95, v/v), each followed by a gradient run with sample injection.
- Perform three equilibration cycles (10 CV) to A/B (5/95, v/v), each followed by a gradient run with sample injection.
- Perform three equilibration cycles (15 CV) to A/B (5/95, v/v), each followed by a gradient run with sample injection.
- Continue the procedure with higher equilibration volumes as indicated.
The results of every third sample run from each equilibration cycle set (CV setting) were recorded.
Results and discussion
For the equilibration evaluation study, the SSP (Ascentis® Express ZIC®-cHILIC) and FPP (SeQuant® ZIC®-cHILIC) columns were initially flushed to the end-of-gradient conditions (A/B:40/60, v/v). Subsequently, for each CV setting, three cycles of equilibration to the initial gradient condition (A/B:5/95, v/v) followed each by a sample run were performed, before progressing to the next higher CV equilibration setting. The chromatograms of every third run from each equilibration cycle (CV applied) are shown in Figures 2 & 4 and the corresponding chromatographic data are summarized in Tables 2 & 3.
The development of retention factors (k’) for the three marker compounds as a function of the column volume (CV) is depicted in Figures 3 & 5.
Superficially porous particle (SPP) column
Chromatograms and chromatographic data obtained using the Ascentis® Express ZIC®-cHILIC column are shown in Figure 2 and Table 2.
Figure 2.Chromatograms of third injections of the standard mixture after equilibration with the indicated CVs on an Ascentis® Express ZIC®-cHILIC 150 x 2.1 mm column. Peak IDs: (1) void, (2) 4-hydroxybenzoic acid, (3) nortrypyline, (4) uridine.
Figure 3.Retention factor (k’) of marker compounds vs number of column volumes (CV) used during equilibration on the Ascentis® Express ZIC®-cHILIC column.
Fully porous particles (FPP)
Chromatograms and chromatographic data obtained using the SeQuant® ZIC®-cHILIC column are presented in Figure 4 and Table 3.
Figure 4.Chromatograms of third injection of the standard mixture obtained after equilibration with the indicated CVs on a SeQuant® ZIC®-cHILIC 150 x 2.1mm. Peak IDs: (1) void, (2) 4-hydroxybenzoic acid, (3) nortriptyline, (4) uridine.
Figure 5.Retention factor (k’) of marker compounds vs number of column volumes (CV) used during equilibration on the SeQuant® ZIC®-cHILIC column.
Comparison of SPP and FPP columns
Across all runs, uridine exhibited stable retention behavior irrespective of the equilibration process. In contrast, nortriptyline and 4-hydroxybenzoic acid showed changing retention times with a reversal in the elution order taking place between 15 and 25 column volumes (CV), which is attributable to incomplete equilibration. On the superficially porous particle (SPP) column, stable retention factors were obtained after 25 CV equilibration for all the three analytes (Figure 3), whereas 40 CV were required on the fully porous particle (FFP) column (Figure 5). These findings indicate that particle architecture exerts a significant impact on the required equilibrium volume, which in turn influences overall runtime and solvent consumption. In this study, the SPP column reached equilibration significantly faster (25 CV) compared to the FPP column (45 CV), resulting in 37% reduced solvent usage and 43% faster analysis time.
Conclusion
The results demonstrate that sufficient and reliable (re-)equilibration is a crucial requirement in HILIC chromatography, particularly under gradient conditions. For two of the three analytes, substantial shifts in retention time were observed when equilibration was inconsistent or inadequate. The superficially porous particle (SPP) Ascentis® Express ZIC®-cHILIC column achieved faster equilibration, enabling shorter run times and higher sample throughput compared to the FPP alternative.