Session 3

Dušan Berek
Polymer Institute, Slovak Academy of Sciences, Dubravska cesta 9, 84541 Bratislava, Slovakia

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Block copolymers, BLC belong to the group of synthetic nonmetallic materials called complex polymers. They have valuable properties and found applications for example as flocculants, carriers of drugs and compatibilizers of polymer blends, etc. BLC are formed with two or several (only rarely with more than three) polymeric chains called blocks. Blocks have different chemical composition or physical architecture and they are mutually connected with a chemical bond. The structure of common diblock and triblock copolymers is schematically displayed as A-B, A-B-A or A-B-C. Most BLC contain their parent homopolymers, the “free blocks”, which may spoil their utility properties. Therefore, it is important to determine their amount and to adjust the synthesis of BLC to reduce their formation. The molecular characterization of BLC and determination of parent homopolymers is an analytical challenge. The limited separation selectivity of the most commonly polymer characterization method, size exclusion (gel permeation) chromatography, SEC/GPC is usually not sufficient to fulfill the task. Other, advanced methods of polymer liquid chromatography must be applied. Three of them will be presented in the contribution.

1. Liquid chromatography under critical conditions, based on unique mutual compensation of entropic partition (exclusion) and enthalpic interactions of macromolecules within column
2. Full adsorption of samples containing distinct macromolecules on a nonporous microparticulate sorbent, followed with their sectional, selective elution into an SEC/GPC column
3. Barrier methods, the emerging techniques, which are based on the action of slowly eluting, narrow liquid zones, barriers, which selectively decelerate fast elution of macromolecules of distinct nature.

Sunkyung Jeong, Doo Soo Chung
Department of Chemistry, Seoul National University, Seoul 08826, Korea

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Liquid extraction surface analysis (LESA) has the advantage of directly sampling analytes on a surface, thus avoiding unnecessary dilution by homogenization of the bulk sample commonly practiced in solid sample analysis. By combining LESA with capillary electrophoresis (CE), the additional advantage of separating analytes before detection can be accomplished. As a representative application of LESA-CE to a solid surface sample, non-infiltrative pesticides on the apple skin were analyzed. When non-infiltrative pesticides are applied to fruits such as apples, they remain on the fruit skin without being translocated to the fruit pulp. First, glufosinate-ammonium and glyphosate on the apple skin were directly extracted and injected into the separation capillary. After in-capillary derivatization with a fluorophore NBD-F, the pesticides were analyzed by CE-LIF. Secondly, three pesticides, chlorantraniliprole, kresoxim-methyl, and pyraclostrobin on the apple skin were analyzed by LESA-CE. For these water-insoluble neutral analytes, micellar electrokinetic chromatography combined with an on-line sample stacking method, analyte focusing by micelle collapse was employed. In addition, using a commercial CE instrument, the LESA process was performed much faster and more reliably compared to LESA-CE using a homemade CE setup. Thirdly, to analyze both cations and anions on a solid surface with a single run of a reasonable detection time, two contactless capacitively coupled conductivity detectors (C4D) were placed on a single capillary. After LESA using the inlet of a coated fused silica capillary having a weak but reversed EOF, the extracts were placed between the two C4Ds. When a reverse electric field is applied, the cations and anions migrated in the opposite directions, and were quickly detected with the C4D located in each direction. The ionic composition of a lithium battery anode surface was investigated by this LESA-CE/2C4D. The developed LESA-CE is promising for the analysis of biological surfaces of cells and tissue as well as technical surfaces.

Acknowledgements
The authors acknowledge the supports from the National Research Foundation of Korea (2021R1F1A1045947).

Matthias Guggenberger¹, Josua T. Oberlerchner¹, Heinrich Grausgruber², Thomas Rosenau¹, Stefan Böhmdorfer^1
1University of Natural Resources and Life Sciences, Vienna (BOKU), Institute of Chemistry of Renewable Resources, Konrad-Lorenz-Straße 24, 3430 Tulln, Austria
²University of Natural Resources and Life Sciences, Vienna (BOKU), Institute of Plant Breeding, Konrad-Lorenz-Straße 24, 3430 Tulln, Austria

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Chromatograms obtained by HPTLC are highly suited for visual comparison and classification of samples, but manual evaluation might suffer from operator bias or fatigue. Common chemometric methods for classification struggle with the non-uniform background noise of HPTLC plates and limited plate-to-plate reproducibility.
We employed the Self Organizing Map (SOM) algorithm to overcome these limitations.[1] It uses iterative and distance-dependent learning to prepare a map-like projection, on which chromatograms of high similarity are located close together, while less similar ones are placed further apart. The maps were used for direct visualization of similarity relations of the samples, implementation of multi-class and one-class classifications, determinations of signals that contributed to misclassification (which was also useful for validation of method robustness), and detection unusual samples for a detailed evaluation or quality control.
We analyzed a set of 495 HPTLC chromatograms of essential oils with conventional multiclass SOMs as well as one-class SOM Quality Control Index (SOMQC, [2]). Both approaches give convincing results (95% correct classification). SOMQC offers the exceptional advantage, that it assigns indistinct samples to several classes. This is a clear indicator that the sample is faulty or demands manual verification.
The classification is tolerant towards noise and inevitable variations among replicates. Only minimal data preprocessing is required. Grouping by plates was never observed, even though simple RGB data were used as input.

Acknowledgments
We are grateful for the essential oils provided as a gift by Sonnentor Kräuterhandelsgesellschaft mbH and for the support of the Austrian Biorefinery Center Tulln (ABCT).

References
[1]  M. Guggenberger, J. T. Oberlerchner, H. Grausgruber, T. Rosenau, S. Böhmdorfer, Talanta 233 (2021) 122460.
[2]  S. Kittiwachana, D.L.S. Ferreira, L.A. Fido, D.R. Thompson, R.E.A. Escott, R. G. Brereton, Anal. Chem. 82 (2010) 5972–5982.

Urh Černigoj
BIA Separations, a Sartorius company, Mirce 21, 5270 Ajdovščina, Slovenia

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Plasmid DNA (pDNA) was key to the development of biologic drug manufacturing. Today, it plays a crucial role in the production of cell and gene therapies and vaccines. HPLC based analytical assay is a powerful technique that can be used to efficiently monitor pDNA purity, quantity and isoform composition throughout the entire purification process. Elution of weak anion exchangers, for example commercially available CIMac^TM pDNA analytical column, with sodium chloride gradients is commonly employed for analysis of sample mixtures containing different isomers of plasmid DNA [1]. We have found that gradient elution of a CIMac pDNA in the presence of guanidine hydrochloride (Gdn) roughly doubles resolution between open-circular (oc) and supercoiled (sc) isomers [2]. It also improves resolution among sc, linear, and multimeric/aggregated forms. Selectivity of the improved method is orthogonal to electrophoresis, but with better quantification than agarose electrophoresis, better quantitative accuracy than capillary electrophoresis, and resolution approaching capillary electrophoresis. Unfortunately, Gdn elution does not decrease a reversible entrapment of the oc isoform in the restrictions within the pores of the monolithic material for ≥7 kbp pDNA on a CIMac pDNA columns with 1.4 um channel diameter. Therefore, a monolithic polymer with 6 um channels was developed to enable robust and reliable analytical separation of plasmid isoforms larger than 10 kbp. The novel material in combination with Gdn gradient enables a baseline separation between oc and sc pDNA as well as ≥85% recovery for oc, sc and linear isoforms of 15 kbp large pDNA even at 10 CV/min flow rate. Presented improvements were successfully implemented in PATfixTM pDNA analytical platform package, designed to give »at-line« reliable insight during process development and production of every possible pDNA.

References
[1]  B. Gabor, U. Černigoj, M. Barut, A. Štrancar, J. Chromatogr. A, 1311 (2013) 106-114.
[2]  U. Černigoj, J. Vidič, A. Ferjančič, U. Sinur, K. Božič, N. Mencin, A. Martinčič Celjar, P. Gagnon, A. Štrancar, Electrophoresis 42 (2021) 2619-2625.