ZANEDBÁVANÉ APLIKACE MONOLITICKÝCH STRUKTUR: POKROČILÉ STUDIE TENKOVRSTVÉ CHROMATOGRAFIE-HMOTNOSTNÍ SPEKTROMETRIE

Significance of the Topic

Thin‐layer chromatography (TLC) remains a versatile separation technique valued for its simplicity, low instrument cost and compatibility with diverse detection methods. Despite slower development compared to liquid and gas chromatography, recent advances in monolithic porous polymer layers have revitalized TLC by enabling direct mass spectrometric (MS) detection, multi‐dimensional separations and microfluidic formats.

Objectives and Study Overview

This work reviews cutting‐edge applications of monolith‐based TLC layers, focusing on:

• Direct MALDI‐MS detection from polymer monoliths without low‐molecular‐weight matrix
• Integration of polymerizable MALDI matrices for peptide and protein analysis
• Design of layers tailored for two‐dimensional (2-D) separations
• Emerging microfluidic and 3-D printed TLC formats

Methodology and Instrumentation

Monolithic layers were prepared by in situ free‐radical polymerization of monomers such as butyl methacrylate, ethylene dimethacrylate and glycidyl methacrylate in glass or polymer molds. Pore structure was tuned via porogen composition. For direct MS detection, monoliths served as matrix‐free MALDI targets under UV laser desorption/ionization. Instrumentation included:

• UV laser desorption/ionization time‐of‐flight mass spectrometers (MALDI‐TOF MS)
• Desorption electrospray ionization (DESI) interfaces for ambient MS imaging
• UV lamps (360 nm) for photopolymerization and surface functionalization
• Microfluidic devices fabricated by soft lithography (PDMS) or 3-D printing

Main Results and Discussion

Direct MALDI detection from porous polymer monoliths generated clean spectra for small molecules (e.g., caffeine) with minimal background interference and exceptional stability over weeks. Monoliths resisted oxidation and acid hydrolysis issues typical of conventional matrices. Polymerizable MALDI matrices (e.g., methacrylated cinnamic acid) were grafted into monolith pores via thiol–ene or photografting “click” reactions, enabling sensitive peptide analysis without external matrix deposition.

For 2-D TLC, superhydrophobic monoliths were photopatterned to embed hydrophilic ion‐exchange channels. Orthogonal separations combined ion‐exchange in one dimension and reverse‐phase in the second, achieving baseline resolution of peptides and revealing unexpected oligomeric forms. Gradient wettability patterns were also created by diagonal photografting of hydrophobic polymers, fine‐tuning retention in both dimensions.

Microfluidic pTLC chips incorporated sub-100 µm monolithic strips in etched glass with volumes as low as picoliters. These devices achieved high‐efficiency separations of fluorescent dyes and lipids extracted from single cells, demonstrating potential for single‐cell metabolomics. 3-D printed TLC substrates with interlayer porosity were rapidly fabricated to separate charged and neutral fluorescent analytes in alkaline mobile phases.

Benefits and Practical Applications

Monolithic TLC layers offer:

• Matrix‐free or built‐in matrix MS detection for small molecules, peptides and proteins
• Enhanced chemical stability and reduced spectral background
• Customizable pore size and surface chemistry for targeted selectivity
• Feasible 2-D separations on a single planar platform
• Microfluidic and miniaturized formats for limited‐volume samples, including single‐cell analysis
• Low capital investment compared to high‐end LC–MS systems

Future Trends and Opportunities

Emerging directions include:

• Integration with high‐throughput imaging MS for spatial metabolomics
• Advanced photopatterning to create multi‐zone orthogonal chemistries
• 3-D printing of hybrid inorganic–organic monoliths for tailored selectivity
• Automated microfluidic sampling and separation for point‐of‐care diagnostics
• Coupling with ambient ionization techniques beyond DESI, such as LAESI and DART

Conclusion

Monolithic porous polymer layers have expanded TLC capabilities by enabling direct and sensitive MS detection, complex multi-dimensional separations and micro- to nano-scale formats. Ongoing innovations in polymer chemistry, photolithography and additive manufacturing promise to further enhance the speed, resolution and applicability of TLC in analytical chemistry, bridging gaps between planar separations and modern high-throughput analyses.

References

Key literature includes studies on monolith‐based MALDI‐TLC, polymerizable matrices, 2-D photopatterned monoliths, microfluidic TLC chips and 3-D printed planar devices.