Special Issues-04-01-2008

It has often been stated (or maybe overstated) that the column is the heart of the chromatograph. Without the proper choice of column and appropriate operating conditions, method development and optimization of the high performance liquid chromatographic (HPLC) separation can be frustrating and unrewarding experiences. Since the beginning of modern liquid chromatography, column technology has been a driving force in moving separations forward. Today, the driving forces for new column configurations and phases are the increased need for high throughput applications, for high sensitivity assays and to characterize complex samples such as peptide digests and natural products.

This article details the principles of hydrophilic interaction chromatography (HILIC) and its complementary selectivity to reversed-phase high performance liquid chromatography (HPLC). Advantages of the technique that result from the use of low-viscosity, high-organic concentration mobile phases will be demonstrated. For example, LC–mass spectrometry (MS) sensitivity is enhanced and higher flow rates and longer columns can be used effectively with such mobile phases in HILIC. Common stationary phases employed in HILIC are reviewed.

The never-ending quest for separation media that enable efficient high speed/high throughput chromatography has led to the design of stationary phases in monolithic formats with both vastly improved mass transfer properties and reduced discontinuity. Historically, porous polymer monoliths have first emerged in the late 1980s/early 1990s followed by their silica-based counterparts in the mid 1990s. The common denominator for both organic and inorganic monoliths was originally their use in HPLC columns. However, the range of applications of monolithic materials grew significantly since their early times. This short review summarizes information about monoliths produced in different shapes such as discs, tubes, columns, polymer layer open tubes (PLOT capillaries), and microfluidic devices, and presents selected applications including chromatographic separations, sample preparation, and enzyme immobilization.

This article presents an overview of high performance liquid chromatography stationary phases with enhanced stability at high pH, focusing on the methods by which they were prepared. Among the many alternatives, the authors introduce reversed phases based upon metallized silica supports that show superior performance during stability testing at high pH, when compared with conventional C18 phases based upon bare silica.

The use of high temperature is playing an increasingly important role in high performance liquid chromatography (HPLC) method development and optimization. Major advantages of high-temperature LC (HTLC) include shortened separation time, increased efficiency, and reduction in the use of organic solvent, but the accompanying decrease in mobile phase viscosity provides a lowering of column back pressure, allowing even faster separations, use of longer columns, and use of smaller particles. Here the author summarizes some of the latest findings in HTLC and addresses issues raised when columns and analytes are heated beyond the "normal" operating conditions.

An ever-increasing need for chiral separations has led to a more generic approach for screening a variety of chiral stationary phases. These new screening methodologies have been supported by new instrument development, new chiral product performance, and a new level of user knowledge. Supercritical fluid chromatography has continued to grow, supported by published applications from the separations industry. An expanded field of polysaccharide phases has been made available from a variety of sources with some unique variants of the most common cellulose and amylose derivatives.

In the leadoff article, columnist Ron Majors provides an overview of column developments. He looks at various alternatives to high-throughput separations including small porous particles, monoliths and superficially-porous particles. Microfluidics and parallel column systems provide further alternatives. An alternative approach to isocratic method development uses optimized stationary phase combinations. Brief coverage of new phases for hydrophilic interaction chromatography, high temperature operation, chiral and mixed mode columns and finally supercritical fluid chromatography columns round out the overview. At the conclusion, Majors speculates on future directions in column technology.

In this article, silica-based monolithic columns are compared and contrasted to packed microparticulate columns. Some of the challenges developing commercial silica rods and encapsulated monolith columns are described, including the development of a 2-mm i.d. column. A study of wall effects in these monolith columns was performed. Future trends and challenges in improving the performance of silica-based monolith columns are described.

Based upon early theoretical predictions of thought leaders in the beginnings of high performance liquid chromatography (HPLC), the continuous evolution of a reduction of particle sizes in HPLC column technology along with improvements in instrumentation has led to the increased use of particles in the sub-2-mm range, which places certain constraints on operating conditions. In this article, Gerard Rozing puts theory and practice into perspective when using small particles at increased operating pressure and, in particular, looks at thermal effects that can affect overall performance.