Journal of Chromatography A, 1360 (2014) 164–171 Contents lists available at ScienceDirect Journal of Chromatography A jo ur nal ho me pag e: www.elsevier.com/locate/chroma A generic approach to post-column refocusing in liquid chromatography Jelle De Vos, Gert Desmet, Sebastiaan Eeltink ∗ Vrije Universiteit Brussel, Department of Chemical Engineering, Pleinlaan 2, B-1050 Brussels, Belgium a r t i c l e i n f o Article history: Received 8 May 2014 Received in revised form 9 July 2014 Accepted 23 July 2014 Available online 1 August 2014 Keywords: Detection limits Solid-phase extraction Heart-cut LC Solvent switch Modulation a b s t r a c t To increase detection sensitivity in liquid chromatography, a generic post-column refocusing strategy has been developed to enrich (target) analytes prior to detection. In this strategy, after separation on the ana- lytical column, the analytes are led to a trap column preferably containing a stationary phase with strong retentive properties (e.g. silica C 30 ). They are then eluted using a strong solvent in a backward-elution mode. A first key element of the proposed strategy is that the trapping time should be at least equal to the time the front of the remobilization solvent needs to cover the entire length of the trap column, divided by the ratio of the flow rates used for trapping and remobilization. This condition is independent of the retention properties of the analytes in the trapping and remobilization solvent. Another essential element is the addition of a third solvent (isopropanol in the present case) to the remobilization solvent to overcome viscous-fingering effects caused by the viscosity difference between the trap and the remo- bilization solvents. The potential of the proposed post-column refocusing strategy is demonstrated for an isocratic separation of KI (t 0 marker), an antibiotic (sulfamethazine), and acetophenone as a case study. Using optimized remobilization conditions a maximum signal-enhancement factor of 8 was achieved. Higher enhancement factors using a remobilization solvent with slightly higher elution strength were prohibited by disturbances of the UV background signal. © 2014 Elsevier B.V. All rights reserved. 1. Introduction High-performance liquid chromatography (HPLC) has emerged as one of the most important techniques for the analysis of non- volatile (contaminant) samples and is successfully applied in many application areas. However, the fundamental nature of the separa- tion process is such that analytes in a sample mixture are diluted when components are distributed between mobile and station- ary phases. Dilution is a problem for impurity profiling, in which the aim is the detection, identification/structure elucidation, and quantification of sub-ppm components and residuals in a wide variety of matrices [1]. Advances in column technology and the development of UHPLC instrumentation have led to better separa- tion efficiency and hence increased detection sensitivity because analytes elute in narrower highly concentrated zones [2,3]. Fur- thermore optimization of the design of detector flow cells and advances in mass spectrometry interfacing via electrospray ioniza- tion (ESI), has resulted in considerable improvements in detection sensitivity [4–6]. However, even when HPLC is hyphenated to ∗ Corresponding author. Tel.: +32(0)26293324; fax: +32(0)26293248. E-mail address: seeltink@vub.ac.be (S. Eeltink). sensitive detectors such as mass spectrometers (MS), the detec- tion limits are not always low enough to detect at the required trace levels set by authorities. For example, the detection and accu- rate analysis of trace-levels of antibiotics and their metabolites in agricultural and food products is a major bottleneck and several classes are even undetectable using current LC–MS methods [7]. The presence of sub-therapeutic doses in food products increases the prevalence of antibiotic-resistant bacteria [8]. In addition, con- cerns have been raised regarding the carcinogenicity of the residues [9]. When considering small-molecule method development in two-dimensional liquid chromatography (LC × LC), dilution is an even larger bottleneck since the dilution factors in the two consec- utive chromatographic processes are multiplicative when analyte focusing between the two developments is not possible [10,11]. On-column preconcentration procedures in HPLC have been described by several authors as a means to enhance detection limits [12,13]. However, when the sample volume is increased, the injected volume will cover a more signifi- cant fraction of the column, decreasing column performance, which in turn severely compromises sensitivity of the ana- lytical method. Hence, a trade-off needs to be made between detection limits and chromatographic resolution. A solid- phase-extraction (SPE) column is often applied prior to the http://dx.doi.org/10.1016/j.chroma.2014.07.072 0021-9673/© 2014 Elsevier B.V. All rights reserved.