Two-dimensional liquid chromatography (2D-LC), and in particular comprehensive two-dimensional liquid chromatography (LC×LC), offers increased peak capacity, resolution and selectivity compared to one-dimensional liquid chromatography. It is commonly accepted that the technique produces the best results when the separation mechanisms in the two dimensions are completely orthogonal; however, the use of similar separation mechanisms in both dimensions has been gaining popularity as it helps avoid difficulties related to mobile phase incompatibility and poor column efficiency. The remarkable advantages of using reversed phase in both dimensions (RPLC×RPLC) over other separation mechanisms made it a promising technique in the separation of complex samples. This review discusses some physical and practical considerations in method development for 2D-LC involving the use of RP in both dimensions. In addition, an extensive overview is presented of different applications that relied on RPLC×RPLC and 2D-LC with reversed phase column combinations to separate components of complex samples in different fields including food analysis, natural product analysis, environmental analysis, proteomics, lipidomics and metabolomics.

The understanding of principles that drive the separation in reversed-phase chromatography plays an important role in the prediction of the elution of solutes in RP-HPLC. The separation in RP-HPLC is based on the principle of adsorption and partition. In addition, the logP value, the pKa value of the drug and chromatographic parameters like mobile phase pH, buffer concentration, organic modifier and mobile phase additives also influence the retention and selectivity of the analyte. It was found that hydrophobic, electrostatic, hydrogen bonding and other specific interactions between the stationary phase and the solutes, along with the hydrophobicity of an analyte molecule (logP), modify the retention behaviour of the analytes. This article gives special attention to the influence of ionization and ion interaction on the separation of analytes. The drug molecules with different logP values containing protonated and deprotonated acids, bases and zwitterions are selected as examples and this article addresses various issues related to the method development, relationships between analyte retention and mobile phase pH and the pKa value of the analyte. The advances in this regard, with highlights on topics such as mechanisms of retention and various factors that influence the retention behaviour of analytes, are also updated with suitable examples.

Hydrophilic interaction liquid chromatography on polar columns in aqueous-organic mobile phases has become increasingly popular for the separation of many biologically important compounds in chemical, environmental, food, toxicological, and other samples. In spite of many new applications appearing in literature, the retention mechanism is still controversial. This review addresses recent progress in understanding of the retention models in hydrophilic interaction liquid chromatography. The main attention is focused on the role of water, both adsorbed by the column and contained in the bulk mobile phase. Further, the theoretical retention models in the isocratic and gradient elution modes are discussed. The dual hydrophilic interaction liquid chromatography reversed-phase retention mechanism on polar columns is treated in detail, especially with respect to the practical use in one- and two-dimensional liquid chromatography separations.

Residues of antibiotics (ABs) in the aquatic environment and in food of animal origin represent a major concern, as prolonged exposure to ABs is a serious health hazard, related to both the side effects of prolonged use and the risk of developing bacterial resistance to various ABs. Given the low levels of the AB residues in complex matrices, the development of sensitive analytical methods represents a major challenge. This is certainly true for the aminoglycoside ABs (AGs) which lack a chromophore and show poor chromatographic properties in reversed-phase liquid chromatography. This paper reviews the current state of the art in the determination of AGs. Attention is paid to extraction, sample clean-up, chromatographic separation, and detection of AGs in both environmental and food samples and in plasma and serum. A general workflow for the analysis of AGs is presented which takes into account the matrix and required level of information.

This review aims to summarize the available analytical methods in the open literature for the determination of some aliphatic and cyclic nitramines. Nitramines covered in this review are the ones that can be formed from the use of amines in post-combustion CO2 capture (PCC) plants and end up in the environment. Since the literature is quite scarce regarding the determination of nitramines in aqueous and soil samples, methods for determination of nitramines in other matrices have also been included. Since the nitramines are found in complex matrices and/or in very low concentration, an extraction step is often necessary before their determination. Liquid-liquid extraction (LLE) using dichloromethane and solid phase extraction (SPE) with an activated carbon based material have been the two most common extraction methods. Gas chromatography (GC) or reversed phase liquid chromatography (RPLC) has been used often combined with mass spectrometry (MS) in the final determination step. Presently there is no comprehensive method available that can be used for determination of all nitramines included in this review. The lowest concentration limit of quantification (cLOQ) is in the ng L(-1) range, however, most methods appear to have a cLOQ in the μg L(-1) range, if the cLOQ has been given.

Plasmid DNA (pDNA)-based vaccines offer more rapid avenues for development and production if compared to those of conventional virus-based vaccines. They do not rely on time- or labour-intensive cell culture processes and allow greater flexibility in shipping and storage. Stimulating antibodies and cell-mediated components of the immune system are considered as some of the major advantages associated with the use of pDNA vaccines. This review summarizes the current trends in the purification of pDNA vaccines for practical and analytical applications. Special attention is paid to chromatographic techniques aimed at reducing the steps of final purification, post primary isolation and intermediate recovery, in order to reduce the number of steps necessary to reach a purified end product from the crude plasmid.

In recent years the use of monolithic polymers in separation science has greatly increased due to the advantages these materials present over particle-based stationary phases, such as their relative ease of preparation and good permeability. For these reasons, these materials present high potential as stationary phases for the separation and purification of large molecules such as proteins, peptides, nucleic acids and cells. An example of this is the wide range of commercial available polymer-based monolithic columns now present in the market. This review summarizes recent developments in the synthesis of monolithic polymers for separation science, such as the incorporation of nanostructures in the polymeric scaffold as well as the preparation of hybrid structures. The different methods used in the surface functionalization of monolithic columns are also reviewed. Finally, we critically discuss the recent applications of this column technology in the separation of large molecules under different chromatographic mode.

Proteomics is the large-scale study of proteins, particularly their expression, structures and functions. This still-emerging combination of technologies aims to describe and characterize all expressed proteins in a biological system. Because of upper limits on mass detection of mass spectrometers, proteins are usually digested into peptides and the peptides are then separated, identified and quantified from this complex enzymatic digest. The problem in digesting proteins first and then analyzing the peptide cleavage fragments by mass spectrometry is that huge numbers of peptides are generated that overwhelm direct mass spectral analyses. The objective in the liquid chromatography approach to proteomics is to fractionate peptide mixtures to enable and maximize identification and quantification of the component peptides by mass spectrometry. This review will focus on existing multidimensional liquid chromatographic (MDLC) platforms developed for proteomics and their application in combination with other techniques such as stable isotope labeling. We also provide some perspectives on likely future developments.

Arsenic-containing carbohydrates, generally termed arsenosugars, have been the subject of increasing analytical interest in arsenic speciation analysis. The present review gives an overview concerning achievements and trends in the field of instrumental analysis of arsenosugars. The typical experimental approaches for sample pre-treatment, extraction, separation and detection are discussed. Current possibilities and limitations of modern instrumental techniques are pointed out.