Comparison of the steric selectivity on hydrophilic interaction chromatography columns modified with poly(acrylamide) possessing different morphology.

Poly(acrylamide) (PAAm)-modified hydrophilic interaction chromatography (HILIC) columns were prepared via surface-initiated atom transfer radical polymerization (SI-ATRP) and free radical polymerization (FRP) to generate brush-like and mushroom-like polymer chains on silica particles, respectively. The maltose homologues (MHs) and cyclodextrins (CDs) were chosen as analytes to evaluate steric selectivity by the different polymer morphologies in the ATRP-PAAm and the FRP-PAAm columns. The ATRP-PAAm exhibited superior retention than the FRP-PAAm and three commercial HILIC columns. The house-made PAAm columns provided significant hydrophilicity that enabled to analysis the oligosaccharides even in 60:40 mixture of acetonitrile-aqueous buffer. In the case of three ATRP-PAAm columns characterized by different polymer lengths and the density on the silica particles, those are different thickness of the water-enriched layer, and phase ratio φ, based on hydrophilicity of them columns. The logarithm of the retention factor (ln k) displayed a non-linear dependence on the inverse of the temperature (1/T, T = 278-333 K). Notably, a similar correlation was observed to exist between the logarithm of the phase ratio (ln φ), and 1/T. A van't Hoff plot was used to determine the thermodynamic parameters of the partition process for each MH. The values of the Gibbs free energy (ΔG°) for the analytes partition on the ATRP-PAAm columns were smaller than their counterparts measured for the FRP-PAAm columns; by contrast, the opposite trend was observed for the ΔG° values measured for CDs. The standard entropy ΔS° for MHs and CDs were comparable for the two types PAAm columns, while, the standard enthalpy, ΔH° displays significant difference between the ATRP and the FRP PAAm columns. These findings indicate that the differences between PAAm morphology and polymer densities on the stationary phase surface affect analyte differentiation on the basis of molecular steric factors. The higher selectivity for MHs and CDs displayed by ATRP-PAAm columns with respect to their FRP-PAAm and commercial amide columns will be useful for the fine separation of oligosaccharides.

Functionalization using polymer or silane? A practical test method to characterize hydrophilic interaction chromatography phases in terms of their functionalization method.

Herein, commercially available columns employed in hydrophilic interaction chromatography (HILIC) were characterized by determining their ability to selectively distinguish the minute structural differences between small molecules such as nucleosides and xanthines in complex sample matrices. Principal component analysis (PCA) was applied to the data obtained from structurally similar analytes, and the results showed that HILIC columns could generally be classified into two groups: (i) silane-modified columns that were prepared from either native silica particles or silica particles modified with low-molecular-weight silanes and (ii) polymer-modified columns obtained from silica particles functionalized with organic polymers. These two groups could be further subdivided based on the functionalities attached to the respective stationary phases. These results were confirmed via cluster analysis by preparing a dendrogram using the morphology-based selectivity parameters associated with the respective columns. We were able to determine the selectivity of columns for the OH groups, i.e., α(OH) and the prevailing pH conditions (cation- and anion-exchanging natures) on the surface of the respective stationary phases; α(theobromine/theophylline) was employed to obtain a similar two-dimensional plot. This test scheme, in which five compounds were analyze for each column, was helpful for understanding the impact of factors such as the hydrophilicity, degree of hydration, acidity/basicity, or the weak ion-exchange nature of the respective stationary phases on the separation characteristics of new HILIC stationary phases. The selectivity of columns for the CH2 group was also examined. The cation-exchange nature of the HILIC columns significantly influenced native silica columns and some polymer-modified columns. Herein, 45 commercially available HILIC columns were classified according to this method, and the results proved useful for understanding distinct separation characteristics of each HILIC column, enabling improved column selection.

The relationship between polymer structures on silica particles and the separation characteristics of the corresponding columns for hydrophilic interaction chromatography.

Silica particles with various pore sizes were modified with poly(acrylamide) via surface-initiated atom-transfer radical polymerization (SI-ATRP) under different reaction conditions. Twenty different columns were prepared and characterized according to a test method for hydrophilic interaction chromatography (HILIC) columns. Hydrophilic retention by the SI-ATRP columns was much higher than that of poly(acrylamide) columns prepared via free-radical polymerization and many commercially available HILIC columns. The SI-ATRP columns displayed greater selectivity for -OH groups than any of the HILIC columns based on their α(U/2dU) values. SI-ATRP functionalization was used to increase the polymer chain density on the silica particles, which suggested a brush-type morphology, and improved hydrophilic selectivity. This indicated that hydrophilic retention and selectivity could be controlled by adjusting the morphology of the organic stationary phase. This stationary phase design strategy was validated experimentally by the effective separation of highly hydrophilic analytes. The findings of this study will greatly contribute to the creation of better separation media.