HPLC 2025 Preview: Functionalized Monoliths as Selective Sample Preparation Materials

This article highlights advancements in instrumentation for separation and detection, improving sensitivity and reducing analysis time. Analyzing trace compounds from complex samples often requires purification and pre-concentration, and online coupling of solid-phase extraction (SPE) with liquid chromatography (LC) helps minimize analysis time and solvent/sample use. Monoliths with large macropores are ideal for this coupling due to their low back pressure and versatility in various formats. Functionalizing monoliths with biomolecules or nanoparticles enhances their selectivity and sensitivity. Imprinted monoliths aid in selective extraction, eliminating matrix effects. Miniaturizing these monoliths for nanoLC reduces solvent consumption and lowers analytical costs.

Nathalie Delaunay, Audrey Combès, and Valérie Pichon © Images courtesy of authors.

Analytical chemists face challenges in many applicationn areas, including the environment, biomedicine, toxicology, and food, because of the huge number of compounds that need to be analyzed. In this context, the evolution of instrumentation in terms of separation and detection has led to a real improvement in sensitivity and a reduction in analysis time. The use of multidimensional separative systems combined with very high-resolution mass spectrometers now makes it possible to characterize very complex mixtures. However, the analysis of compounds at trace- or ultra-trace level in complex samples often requires a purification and pre-concentration step prior to chromatographic separations. This can be achieved by solid-phase extraction (SPE), which delivers high enrichment factors. This technique benefits greatly from the wide variety of sorbents available in disposable cartridges, includingC18-bonded silica, polymers and carbonaceous sorbents, and has the advantage of reducing the amount of organic solvent used during this sample preparation stage compared with the more conventional liquid–liquid extraction. The direct coupling of SPE with liquid chromatography (LC) also facilitates the automation of the entire analytical procedure while reducing analysis time, solvent, and sample consumption. Thanks to their porous structure with large macropores acting as flow-through channels, thus favoring percolation of the sample at high flow rate without generating high back pressure, and mesopores ensuring a high specific surface area, monoliths are attractive sorbents to develop such a type of coupling. The fact that they can be synthesized in a single step in a wide variety of formats, ncluding stainless steel columns and, silica capillaries, also means that they can be coupled with a wide variety of LC column formats. Moreover, because their properties can be controlled by polymerization conditions (nature and ratio of reagents, temperature, reaction time), their nature can be adapted to the targeted application (1,2).

The miniaturization of the separation column’s format also contributes to an increased sensitivity of the entire analytical method and to the reduction of solvent consumption and sample volume, which is essential for the analysis of samples available in small quantities. However, to be optimal, miniaturized separation devices need to be combined with equally miniaturized extraction devices. The development of monoliths in miniaturized format appears in this context as a powerful approach, and the introduction of porous crystals (metal–organic frameworks [MOFs], covalent organic frameworks [COFs]) or nanoparticles during their synthesis strongly contributes to the enhancement of their specific surface area to compensate for their reduced size (3,4). However, miniaturizing the separation device often reduces the separation length, leading to a loss in chromatographic resolution. This can be offset during sample pre-treatment by using sorbents that provide a selective extraction procedure to retain the target analytes while eliminating matrix components. A selective extraction procedure can be developed using monoliths functionalized by biomolecules such as antibodies or aptamers that present a high affinity for targeted analytes This enhancement in selectivity can also be achieved by developing monolithic molecularly imprinted polymers (MIPs). These tools enable matrix components to be eliminated during sample preparation, thus avoiding the risk of matrix effects widely encountered in LC–MS. These monoliths can be developed in a conventional format such as cartridges or columns for their coupling with LC. They can also be miniaturized in capillaries for coupling with nanoLC or on chip, reducing both solvent consumption and analytical costs.

With regard to monoliths functionalized with antibodies and aptamers, while the Sol-gel approach has been proposed for encapsulating antibodies during material synthesis, the most common approach is to first synthesize a porous monolith in the extraction device suited to the intended application (columns, capillaries, pipette tips, channels) and then immobilize the biomolecules by covalent grafting onto the monolithic surface (5,6). Organic, silica, and organic-inorganic hybrid monoliths have been largely reported to develop affinity-based extraction devices (2,5–8). Pore size and specific surface area are two important parameters to control to facilitate grafting. In addition, surface chemistry must be adapted to the grafting of selected biomolecules for compound trapping, while limiting the risk of non-specific interactions between targets and the monolithic surface. As antibodies and aptamers require percolation with aqueous media, hydrophilic monomers are generally preferred. Similar approaches have been selected for the grafting of other biomolecules such as enzymes, lectins, proteins A and G (3,5), and peptides (9).

Regarding MIPs, their synthesis is based on the complexation in solution of a template molecule, often via non-covalent interactions with functional monomers, and on the polymerization of these monomers around the template following the addition of a cross-linking agent and an initiator. After polymerization, the template molecule is removed from the polymer by washing steps with suitable solvent(s) to break the non-covalent interactions established within the cavities formed. The result is a polymer with cavities complementary to the template molecule in terms of size, shape, and position of functional groups. The choice of reagents involved in MIP synthesis must be carefully considered to create cavities that are highly specific to the target molecule (10). They can be synthesized in situ in the form of monoliths directly within a capillary or channel of a microchip, by anchoring it to the inner surface through prior activation. It is then necessary to determine the polymerization conditions to obtain a monolith that is sufficiently permeable to allow percolation of solutions without generating high back pressure and that possesses specific cavities (11)—two properties that result from compromises on the choice of synthesis solvent. It is thus possible to track a target molecule; for example, when analyzing cocaine in human plasma, injecting only 100 nL of diluted plasma can lead to an overall solvent consumption in the order of a microliter for each sample, while still achieving the necessary detection limits using a simple UV detector coupled with nanoLC (11). When only the target compound is retained by the MIP, it can be eluted for detection and quantification without the need for an analytical nanoLC column because no separation step is required (12).

Combining monolith functionalization with biomolecules or MIPs helps simplify extract composition by selectively targeting the analytes of interest. What’s more, their high permeability makes them very powerful high-throughput tools applied to real samples, as already demonstrated in the field of proteomics (13). The simplified extract composition may also open up the possibility of combining these functionalized monoliths with analytical devices that are very simple to use on site for monitoring purposes. Obtaining purified extracts is also an important consideration for the development of miniaturized analytical devices because this is usually accompanied by a loss of resolution due to reduced separation lengths—as is the case when developing on-chip devices. The miniaturization of these functionalized monoliths also enables the quantity of biomolecules (or template molecules for MIP synthesis) to be reduced, which lowers the cost of the final
analytical method.

Functionalized Monoliths for Sample Preparation (KN37)

WE-02 – Sample Preparation

Wednesday, June 18, 2025, 8:30–10:15 AM

References

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Audrey Combès has been an associate professor since 2013 at ESPCI Paris, PSL University in the Department of Analytical, Bioanalytical Sciences, and Miniaturization, headed by Valerie Pichon. Her main research interests include trace analysis, sample preparation, synthesis of selective phases using biological or biomimetic tools, and untargeted metabolomics for biomarker identification.

Nathalie Delaunay is research director at CNRS and works in the UMR CBI. Her main research interests include the development of new methodologies or stationary phases for separative sciences and the analysis of trace compounds in complex samples to make analytical methods more sensitive, more selective, and/or more miniaturized. She has been the president of AFSEP (Association francophone des sciences séparatives, www.afsep.com) since 2024.

Valérie Pichon is a professor at Sorbonne University and Director of UMR CBI. Her main research interests include the development of antibody- or aptamer-based stationary phases and molecularly- and ionically-imprinted polymers for the selective extraction of organic molecules, and more recently metals, from complex samples, always trying to miniaturize these tools.