Few technologies are as widely used in the analytical lab as liquid chromatography (LC). The concept of LC arose from simple beginnings as partitioning of compounds or molecules based on migration of a liquid sample through a paper substrate. Though the concept behind thin layer chromatography is still relevant, LC has grown by leaps and bounds – merging advanced methods with state of the art instrumentation and applications.
Here we detail some of the fundamentals of LC and examine emerging technologies and new products.
Mobile and Stationary Phases in Liquid Chromatography
A fundamental principle of chromatography involves the interaction of two phases of material, the mobile phase and the stationary phase. The mobile phase may be an aqueous buffer, solvent, or gas, while the stationary phase is typically a solid support such as a column packed with resin or matrix material.
In LC, analytes suspended in the mobile phase interact to a greater or lesser degree with the stationary phase (in accordance with thermodynamics), which can be observed as retention and elution time and measured by absorbance or another physical detection method. The stronger the interaction, the longer then retention time and the stronger or more concentrated the solution required for elution. Results are displayed in the form of a chromatogram or spectrum.
Reverse Phase Liquid Chromatography
Reverse Phase Chromatography (RPC) involves a hydrophobic solid support such as alkyl chain polymers covalently linked to resin particles -- although any inert non-polar substance that can be uniformly packed can be used. The mobile phase includes a polar aqueous buffer or a water miscible solvent such as acetonitrile – a main requirement being delivery of analyte in solution and selective partitioning of said analyte to the stationary phase. Elution involves organic solvents such as methanol – disruption of hydrophobic interactions being key at this stage.
Popular RPC columns include C18 (octadecyl carbon chain-bonded silica), in which hundreds of products are available with different column dimensions, beads sizes, and preferred applications. C8-bonded, silica-bonded, phenyl- and cyano-bonded, and others are also available according to preferred chemistries and use. Subtle modifications in the surface chemistries, such as monomeric or polymeric end-capping, can lead to changes in the selectivity of solid phase media.
What are the Differences Between Reverse Phase and Normal Phase Chromatography?
The main difference between RPC and normal phase (NP) is the solid and mobile phases are reversed such that hydrophilic analytes in solution have affinity and will adsorb to a hydrophilic solid phase. Increasing concentrations of polar solvents are then used for column elution. The popularity of this technique has been overcome by RPC because of intrinsic properties of the hydrophilic solid silica or alumina support, and the residual presence of water or moisture in the solvent, thus resulting in drifting retention times and irreproducibility of results.
Two-Dimensional Liquid Chromatography
A maturing technology, 2D-LC is typically applied to the separation of complex samples where resolution of 1D-LC is fundamentally challenged. The use of RP columns for both dimensions (RPLC x RPLC) is common due to the compatibility of mobiles phases and the wide availability of column selectivities. Hydrophibic interaction (HILIC), normal phase and others are also used in 2D-LC, although method development and optimization can be more challenging.
Two main types of separation modes include Comprehensive 2D-LC, where the entire eluent of the first separation is analyzed in the second dimension, and Heart-cut 2D-LC, where co-eluting species in the first separation can be targeted for analysis in the second. The latter approach has been used for metabolite identification and clonal variation and separation of antibodies, among other applications.
The 1290 Infinity II 2D-LC System from Agilent is a state of the art platform that allows easy switching from 1D-LC to 2D applications, thus increasing the versatility and workflow of the instrument. Separations performance is boosted through ultrahigh peak capacity in excess of 1000, to meet the demands of highly complex samples. Powerful and versatile software enables a range of operations including heart-cut, multiple heart-cut, and comprehensive analysis. This system has shown proven performance in pharmaceutical impurity analysis and a wide range of challenging applications.
Microfluidic and Chip-based Liquid Chromatography
Microfluidics involves the manipulation of minute volumes of liquid in a miniaturized system. Chip-based LC is a nanoflow-scale HPLC concept which uses microfluidic polymer chips and nanoflow LC columns to maximize sensitivity of detection with minimal sample consumption. Agilent pioneered the HPLC-Chip to integrate sample preparation, separation, and electrospray tip on the single chip, to enable high-speed separations and streamlined processing for MS detection and analysis.
A problem that continues to challenge Chip-based HPLC, is the issue of high-pressure and columns of insufficient uniformity and capacity. New technologies which focus on micromachining of separation supports and media aim to increase the uniformity and performance of separations while lessoning requirements for ultrahigh pressures, longer columns lengths, and thus more complex methods.
Micro-chip Capillary Liquid Chromatography
Applications sometimes include analytes with a wide range of isomers, with similar yet unique chemistries and molecular weights. Areas such as lipidomics can involve a large number of these analytes over a wide dynamic range, such as those conditions found in human blood plasma.
Micro-chip-based pillar array chromatography columns can contain separation beds of perfectly ordered and freestanding pillars, formed by microfine etching of silicon wafers. PharmaFluidics has pioneered the technology to enhance the overall separation, resolution, and peak sensitivity of complex biological applications. These micro pillar array columns or microPAC units have proven useful in complex bottom-up proteomics, antibody characterization, and other areas where similar yet chemically diverse analytes are present in complex biological backgrounds.
The LC field is evolving inline with other fields which have borrowed from microchip and micro fabrication technologies. The result has been a wider and more diverse portfolio of LC technologies and products -- those designed to cover chemical isomer identification in complex biological matrices and high-throughput analysis of drug metabolites in biological fluids. Many more micro-products and applications are sure to follow.