Confocal Laser Scanning Microscopy Method for Quantitative Characterization of Silica Monolith Morphology

Abstract

We present a fast, nondestructive, and quantitative approach to characterize the morphology of capillary silica-based monolithic columns by reconstruction from confocal laser scanning microscopy images. The method comprises column pretreatment, image acquisition, image processing, and statistical analysis of the image data. The received morphological data are chord length distributions for the bulk macropore space and skeleton of the silica monolith. The morphological information is shown to be comparable to that derived from transmission electron microscopy, but far easier to access. The approach is generally applicable to silica-based capillary columns, monolithic or particulate. It allows the rapid acquisition of hundreds of longitudinal and cross-sectional images in a single session, resolving a multitude of morphological details in the column.

Bed morphological features associated with an optimal slurry concentration for reproducible preparation of efficient capillary ultrahigh pressure liquid chromatography columns

“…The three selected columns were prepared for CLSM imaging following established procedures [2, 12, 47, 48]. Detection window son the packed columns were created at about 2/3 of the packed bed length from the inlet (i.e., after ~30 cm) using a drop of warm sulphuric acid.…”

“…Image quality of the raw images (Fig. 1A and C) from the microscope was enhanced in a multiple-step process following established procedures [2, 12, 47, 48]. If not stated other-wise, custom software written in C# using Microsoft Visual Studio 2008, 2012, and 2013 (Microsoft Corporation, Redmond, WA) was used.…”

Low-flow-resistance Methacrylate-based Polymer Monolithic Column Prepared by Low-conversion Ultraviolet Photopolymerization at Low Temperature

“…[29][30] A fluorescent dye was adsorbed onto the monolith according to the following procedure: first, the column was filled with 0.01% BODIPY FL C16 in ACN to stain the monolith, followed by flushing with 50% ACN aqueous solution (v/v), which is the same composition used for the mobile phase in the isocratic separation mode. The teflon outer coating of the capillary was removed, and the column was cut into pieces about 5 mm in length.…”

Focussed ion beam serial sectioning and imaging of monolithic materials for 3D reconstruction and morphological parameter evaluation

“…Alternatively, confocal laser scanning microscopy (CLSM) has been used for analysis of silica monoliths in capillary format. This optical "slicing" of specimens by CLSM allowed to reconstruct 19 and compare 20 the macropore structure of silica-based monoliths, and in turn to conduct benchmark simulations of flow 21 and mass transport 22 using these 3D reconstructions for derivation of quantitative morphology-transport relationships.…”

“…On the other hand, SBF-SEM was still found unsuitable for analysis of silica monoliths since the diamond knife of the ultramicrotome would not withstand the cutting process. 24 Refractive index matching between sample and objective can also pose a significant challenge in CLSM, 19 as pointed out by Tallarek's group, who found CLMS unsuitable for imaging polymer monoliths as a result of their high refractive index. 18 In addition, one of the main drawbacks of CLSM compared to FIB-SEM and SBF-SEM is its lower resolution.…”