{"doi":"10.1002/jssc.70288","title":"Quantifying Biomolecular Interactions in High‐Conductivity Samples With Capillary Electrophoresis","abstract":"ABSTRACT Capillary electrophoresis (CE) is a powerful tool for studying biomolecular interactions due to its high speed, low sample consumption, and adaptability. However, challenges arise when sample buffers possess higher conductivity than the background electrolyte (BGE), leading to peak distortions and reduced measurement accuracy in binding assays such as affinity probe CE and nonequilibrium CE of equilibrium mixtures. This study investigates these effects using a combination of simulation and experiment, focusing on aptamer–protein interactions. Moderate conductivity mismatches (e.g., sample buffer = 2 × tris glycine, BGE = 30 mM tricine) led to peak splitting artifacts, whereas large mismatches (e.g., sample buffer = phosphate‐buffered saline, BGE = 30 mM tricine) produced broad, indistinct peaks, obscuring free and bound species. Simulations revealed that these artifacts arise from analyte ions trapped in high‐conductivity sample plugs and are exacerbated by longer injection times. Experimental results confirmed that reducing plug length and selectively excluding artifact peaks during analysis improves quantification accuracy. When traditional separation fails under high‐conductivity conditions, we propose an alternative method based on quantifying the “de‐stacked” fraction of aptamers escaping the sample zone. This approach yielded values for the dissociation constant ( K d ) and Hill coefficient ( n ) comparable to those obtained using fluorescence anisotropy, demonstrating its viability. The method was further validated by measuring the binding of an integrin‐targeting aptamer (S10yh2) to human serum albumin. Overall, this work provides practical guidelines and analytical strategies for accurate quantification of binding interactions in CE under nonideal conductivity conditions, broadening the applicability of CE for bioanalytical research.","journal":"Journal of Separation Science","year":2025,"id":547496,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9555,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1440363,"name":"Samson Aruna","orcid":null,"position":1,"is_corresponding":false},{"id":1439873,"name":"Bhagya Roshani Samarakoon","orcid":"0009-0007-9079-0307","position":2,"is_corresponding":false},{"id":1010702,"name":"Rebecca J. Whelan","orcid":"0000-0002-9293-1528","position":3,"is_corresponding":false},{"id":1252797,"name":"Miyuru De Silva","orcid":"0009-0002-5055-5103","position":0,"is_corresponding":true}],"reference_count":60,"raw_metadata":null,"created_at":"2026-07-19T02:53:41.314800Z","pmid":"41057289","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}