HPLC

On-line comprehensive two-dimensional liquid chromatography (on-line LC×LC) provides much higher separation power (higher peak capacity) than one-dimensional liquid chromatography (1D-LC). However, it is also often thought that a larger peak capacity should be obtained at the expense of a higher dilution (lower peak intensity). From a theoretical approach, it is demonstrated that both demands can go hand-in-hand in on-line reversed-phase LC×reversed-phase LC (higher peak capacity and higher peak intensity). Examples involving “sub-hour” separations of a tryptic digest show how this approach can be applied in practice.

This article reports on a recently developed zwitterionic-teicoplanin chiral stationary phase (CSP). As a result of the innovative chemistry used to immobilize the chiral selector, the CSP is characterized at neutral pH by the simultaneous presence of both a negative (carboxylate) and a positive (quaternary ammonium) charge. This feature has proved to be pivotal in efficiently and productively resolving very challenging problems. It is worth mentioning the simultaneous separation of chiral active pharmaceutical ingredients (APIs) from their counterions and the separation of neutral-nonpolar, neutral-polar, acidic, and basic N-Fmoc chiral amino-acids. Examples of highly-efficient ultrafast chiral separations achieved on this CSP, as a result of use of effective superficially- and porous-particles, are provided.

The hydrophobic subtraction model has been very successful. Nevertheless, the accompanying public database, which has parameters for 750 commercially available columns, is an underutilized column characterization tool. Here is some guidance on how to use both the model and the free database.

We explore what’s new in the liquid chromatography columns and accessories commercially released over the past year.

Our annual review of new high performance liquid chromatography and mass spectrometry instruments, chromatography data systems (CDS), and related products, including a summary of their significant features and user benefits.

How do I know when bioinert liquid chromatography columns, systems, or components are needed for my separations of biomolecules?

To avoid producing data not fit for purpose, chromatographers need to know how to identify worrying symptoms from HLPC instrument output.

Kevin Schug discusses the advantages of using mobile phases with different pH values in the first and second dimension when using comprehensive two‑dimensional liquid chromatography (2D‑LC) methods for top‑down protein analysis.

Aogu Furusho from the Graduate School of Pharmaceutical Sciences in Kyushu University, Fukuoka, Japan, spoke to us about his development of a highly selective three-dimensional chiral high performance liquid chromatography (HPLC) system for the determination of extraterrestrial amino acids in carbonaceous chondrites.

Partial coelution of chromatographic peaks is an often-encountered issue in high performance liquid chromatography (HPLC) analysis, despite best efforts in method development and optimization. Even though there are several ways of integration of overlapping signals, accurate quantification of single compounds using conventional photodiode array (PDA) detection is almost impossible without baseline separation. While extensive signal processing is well established in spectroscopic analyses such as infrared (IR) or nuclear magnetic resonance (NMR), it has not yet been commonly adapted to improve chromatographic data evaluation. This article introduces the theory and application of a novel data analysis technique for PDA detection to accurately determine and quantify single compounds, even from overlapping peaks, without the need for mass spectrometry (MS) detection.

If I increase the flow rate of my separation when using UV absorbance detection, should I expect peak area to change?

A deeper theoretical understanding the relationship between peak area and flow rate will help analysts diagnose problems when using UV absorbance detection.

Many users have questions about how long LC columns should be re-equilibrated following solvent gradient elution separations. Here we show satisfactory results with short re-equilibration periods, for both reversed-phase and HILIC separations.

The authors provide a technical overview of the design and operating principles of variable wavelength and photodiode array detectors, and include historical perspectives and common practices in operation and maintenance.

In this installment, tips, tricks, and suggestions are provided for best practices in reversed-phase separations, in both 1D and 2D LC, with gradient elution for minimum variations in retention time.

This note addresses the optimum solution for the true productivity in all nanoLC-MS proteomics workflows.

Carefully diluting a sample with weak solvent can mitigate the impact of sample solvent on peak shape in both reversed-phase and HILIC separations, but we need to understand how the choice of sample diluent can affect analyte recovery.

The high levels of non-volatile salts typically used in ion-exchange separations are incompatible with mass spectrometry. But the desire to directly couple these two powerful techniques has prompted researchers find solutions to this problem. Here are three approaches.

We explain recent trends in HPLC autosampler design, provide recommendations for selecting one, and offer guidelines for operation and troubleshooting.

In this instalment of the LCGC Blog, Tony Taylor discusses noisy baselines in high performance liquid chromatography (HPLC).

Many analysts have strongly held beliefs about buffer preparation methods, but these positions are not always supported by experimental evidence.

The effect of dwell volume on chromatographic selectivity can be successfully modelled using retention prediction software. Hence, the robustness of reversed-phase LC gradient methodologies, with respect to dwell volume, can be conveniently assessed.

Ultrahigh efficiency separations based on the presence of one deuterium in benzene, toluene, and naphthalene were achieved by recycle chromatography using C18 silica columns. The discrimination mechanism of H/D isotopic species is discussed based on the dispersion interactions of a CH/CD group of the solute with the stationary phase as well as the mobile phase.

The impact of ionic strength, buffer capacity, and pH-response on the retention behavior and peak shape of mAb species characterization is evaluated for IEX-MS. The aim of the present study was to understand the impact of ionic strength, buffer capacity, and pH-response on the retention behavior and peak shape of mAb species.

An in-line mixer between the sample injector and column may resolve problems with peak shape caused during sample dilution.

Legacy LC methods seem like they come from a different planet. This month, we look at which conditions to keep, and which ones to let go.

Ultrahigh-efficiency separations based on the presence of one deuterium in benzene, toluene, and naphthalene were achieved by recycle chromatography using C18 silica columns. Larger isotopic separation factors, α(H/D), were observed in methanol–water than in acetonitrile–water, when the mobile phases provided similar retention factors (k), or similar methylene selectivity, α(CH2). Isotopic resolutions between nondeuterated and perdeuterated aromatic hydrocarbons at long separation times were estimated by using the plate counts obtainable by recycle operation as a function of a cycle time, along with the retention factors and the separation factors experimentally observed.

This article gives a brief overview of the advantages and limitations of recently introduced mathematical procedures such as the Fourier deconvolution of extracolumn effects, iterative curve fitting, multivariate curve resolution, modified power law, and use of first and second derivatives in enhancing resolution. High-throughput analyses in gas chromatography (GC), LC, and supercritical fluid chromatography (SFC) could benefit from these simple and effective approaches in many challenging separations applications.

The design of a user-friendly vacuum-jacketed column (VJC) is described for improved LC–MS performance, which does not require a large vacuum chamber with multiple vacuum pumps. Using this configuration, the experimental peak capacities measured for a 2.1 mm × 100 mm column packed with sub-2-μm particles and placed in the VJC-MS probe are doubled with respect to standard LC–MS systems.

Re-equilibrating a reversed-phase stationary phase following aqueous gradient elution can be achieved much faster than you think.