A non-destructive test to assess the axial heterogeneity of in situ modified monoliths for HPLC

This paper describes a non-destructive "peak parking" protocol in order to assess the axial heterogeneity of an in situ modified monolithic column for high performance liquid chromatography; a "gradient stationary phase" was designed whereby the ligand density decreases along the length of the rod in the "forward flow" configuration. The results of multi-location peak parking demonstrated a consistent increase in peak variance from the 1 cm position of the column to the 9 cm location. This increase in band broadening supported the theory of a decreasing ligand density along the length of this gradient column. This is consistent with efficiency measurements performed in both the forward and reverse flow directions, with an improved efficiency (15% increase in N m^-1) in the reverse direction. These results are consistent with theoretical investigations into stationary phase gradients.

Multiplexed detection: fast comprehensive sample analysis of tobacco leaf extracts using HPLC with AFT columns

Detector-based comprehensive screening analysis of complex samples of natural origin using High Performance Liquid Chromatography (HPLC) can be a complicated and time-consuming task. There are a number of ways multidetection characterization can be achieved; however, there are limitations associated with each technique. Active Flow Technology (AFT) in Parallel Segmented Flow (PSF) mode allows for multiplexed detection HPLC analysis within a single injection, whereas maintaining chromatographic performance and allowing the use of multiple destructive detectors to achieve a comprehensive yet efficient screening of a complex sample. In this study, a comprehensive characterization analysis of tobacco leaf extract was carried out through multiplexed detection using a PSF column for the detection of biomolecules by UV-Vis detection, DPPH• for reactive-oxygen species (ROS) detection, and mass spectrometry, the latter two detection methods being sample destructive.

Outlining a multidimensional approach for the analysis of coffee using HPLC

This study investigated approaches for the profiling of coffee using two multidimensional approaches: (1) a multi-detection process and (2) a multi-separation process employing HPLC. The first approach compared multidetection techniques of conventional High Performance Liquid Chromatography (HPLC) hyphenated with a detector (DPPH•, UV-Vis and MS), and multiplexed mode via HPLC with an Active Flow Technology (AFT) column in Parallel Segmented Flow (PSF) format with DPPH• detection, UV-Vis and MS running simultaneously. Multiplexed HPLCPSF enabled the determination of key chemical entities by reducing the data complexity of the sample whilst obtaining a greater degree of molecule-specific information within a fraction of the time it takes using conventional multi-detection processes. DPPH•, UV-Vis and MS (TIC) were multiplexed for the analysis of espresso coffee and decaffeinated espresso coffee. Up to 20 DPPH• peaks were detected for each sample, and with direct retention time peak matching, 70% of DPPH• peaks gave a UV-Vis response for the espresso coffee and 95% for the decaffeinated espresso coffee. The second approach involved the use of a two-dimensional (2D) HPLC system to expand the separation space and separation power for the analysis of coffee, focusing on the resolution and detection of coeluting and overlapping peaks, which was beyond the limits of conventional HPLC in resolving complex samples. The 2DHPLC analysis resulted with the detection of 176 peaks and a closer observation showed the presence of an additional 17 peaks in a cut section where in 1D mode only one peak was observed.

Overcoming solvent mismatch limitations in 2D-HPLC with temperature programming of isocratic mobile phases

This work describes a method for two-dimensional high performance liquid chromatography (2D-HPLC) that uses an isocratic mobile phase with a temperature gradient in the first dimension. Temperature programming was used to manipulate solvent elution strength in place of a mobile phase concentration gradient. This ensured that all eluent fractions transferred into the second dimension were of an identical solvent composition, i.e. the second dimension injection solvent did not increase during the course of the analysis. When applied to a complex natural product extract of coffee, the separation was completed in 35 min and had an orthogonality of 35% (calculated using the bins method) and a spreading angle of 52° as determined via a geometric approach to factor analysis. This approach, incorporating a temperature gradient in the first dimension, compared favourably to previously reported 2D-HPLC separations of coffee, with similar or shorter analysis times.

Stationary phase characterisation and method development for fast two-dimensional HPLC

Fundamental improvements to the experimental protocol of two-dimensional high performance liquid chromatography were developed to accomplish truly quantitative analyses of complex natural products. This included developing novel measurement techniques to elucidate the retention behaviour of monolithic columns. Approaches to streamline chromatographic method development were also explored.