HPLC

Faster isn't always better

Modern gas chromatography (GC) capillary columns are rugged and forgiving but some care should be taken when handling and using these high-efficiency columns. In this instalment of "Column Watch", the authors discuss the issues surrounding avoiding column breakage, stationary phase damage and column contamination. Prolonging the life of a column by keeping an oxygen-free system, providing a cleaner sample and not exceeding the upper temperature limit of the stationary phase are highlighted in this practical discussion.

An evaporative light scattering (ELS) detector is a powerful detection tool if the solutes are less volatile than the eluent. Three main processes occur successively: nebulization, evaporation of the liquid chromatographic (LC) effluent and measurement of the light scattering by the residual particles. This leads to a non-linear calibration curve such as, A= a.m b where A is the peak area,m the sample mass and b the response coefficient measured as the slope of Log A = b>Log m + Log a.

Column temperature plays an important role in controlling peak spacing (selectivity) in reversed-phase liquid chromatography (LC) separations. Temperature has long been known to affect retention time, and more recently, its use in adjusting selectivity has gained popularity (see reference 1 for a review of temperature selectivity). In preparation of a paper for the most recent Pittsburgh Conference, I had an opportunity to reexamine some data that compare temperature selectivity with other variables used to control selectivity in LC separation. This month's instalment of "LC Troubleshooting" examines temperature selectivity and its relationship to pH selectivity.

This article describes the use of ultrahigh-pressure liquid chromatography (UHPLC) in pharmaceutical analysis of drug substances and drug products with UV absorption detection.

Getting started on the right foot is important for efficient method development.

In early 2007, a web-based HPLC columns survey was conducted. Current usage was compared to a 1997 survey.

High-performance liquid chromatography (HPLC) is a powerful tool for the enantioselective separation of chiral drugs. However, the selection of an appropriate chiral stationary phase (CSP) and suitable operating conditions is a bottleneck in method development and a time- and resource-consuming task. Multimodal screening of a small number of CSPs with broad enantiorecognition abilities has been recognized as the best strategy to achieve rapid and reliable separations of chiral compounds. This paper describes the generic screening strategy developed at Johnson & Johnson Pharmaceutical Research and Development (J&J PRD) to successfully develop enantioselective HPLC methods for chiral molecules of pharmaceutical interest.

lthough liquid chromatography (LC) is most commonly associated with analytical techniques that are found inside the laboratory, LC also has been used in industrial applications for separation of a wide range of products for decades. The technique recently has been applied to requirements in the pharmaceutical and biotechnology industries, both inside the laboratory and in industrial settings. With global sales of over $1.7 billion, the market for preparative LC has encountered a stellar growth over the past few years.

The topic of this month's instalment of "LC Troubleshooting" was prompted by a manuscript I recently reviewed and a question I received from a reader of this column. Both inputs related to the variability of retention times observed in liquid chromatography (LC) methods. Variable retention is a topic that has been touched on many times over the history of this column, sometimes just in passing and other times in depth. Yet, it seems to be a problem that keeps recurring, so I think it is worth considering again.

Different methods require different strategies.

Chiral technology has become a very important aspect for scientists involved in the pharmaceutical, chemical, and agricultural industries. The chiral enantiomers can have vitally different pharmacological effects in biological systems. In fact, the U.S. Food and Drug Administration mandates that only therapeutically active isomers must be introduced to the prescription drug market.

Peptide mapping is one of the preferred techniques for the comprehensive characterization of biopharmaceutical products and is often the analytical method of choice for studying a protein's primary structure.

The terms hydrophilic interaction liquid chromatography (HILIC) and aqueous normal phase (ANP) chromatography very often are used interchangeably.

Over the years, LC instrumentation has undergone continuous development in pursuit of greater performance. More recently, the focus of progress has been on shorter run times, as a direct response to greater user demand to perform faster chromatographic analyses, particularly for their LC–MS applications. This has led to separations on short (30–50 mm) columns with a small internal diameter (i.d. ~2.0 mm), packed with small particle size phases (1.5–3.0 μm). The trend for smaller column particle size has now reached a practical limit on current hardware and innovative technological solutions for further gains in performance are required. Several manufacturers offer fast LC instruments designed for greater productivity, while maintaining low carryover, high sample capacity, resolution and reliability. With ultra-fast run times of under 1 minute, these companies have achieved increased throughput using contrasting technological approaches. Here we examine the background to this current trend,..

Is it more than meets the eye?

...it is important to acknowledge that environmental concerns can be more important than the economics of the reduction of solvent consumption.

Detector selection for a method should be made on a case-by-case basis.

his article reveals the first liquid chromatography (LC) separations performed on a microfabricated pillar array column under pressure-driven conditions. The pillars were non-porous and produced using a Bosch-type deep reactive ion etch (DRIE) to pattern the surface of a silicon wafer and had a diameter of approximately 5 μm. Two different packing densities were compared: one similar to the packing density of a packed bed (external porosity of approximately 49%) and one similar to the packing density of monolithic columns (external porosity of approximately 70%).

Prompted by a recently reviewed manuscript and a question from a reader, John Dolan examines the variability of retention times observed in LC methods in this month's installment of "LC Troubleshooting."

This month's installment of "Column Watch" is the conclusion of a two-part series in which columnist Ron Majors examines trends in column and sample prep introductions at Pittcon 2007.

The major advantages of monolithic phases are their ability to withstand high mobile phase flow rates and the rapid separations at low back pressure that can be achieved with their use.

it has been more than 10 years since solvent recycling has been the main subject of an "LC troubleshooting" column, so this month, John Dolan revists this subject.

Fast, reliable measurements of lipophilicity has a great impact on the compounds' selection process at an early stage of dug discovery. The guest columnit examines HPLC methods for this purpose.

Separation instrument techniques are among the most widely used technologies in the analytical instrumentation market. They span the entire industrial and regional marketplace. The lab separations market includes chromatographic techniques such as analytical and preparative HPLC, GC, IC, TLC, flash, and low pressure LC.

An overview is presented of possible pathways to enhance peak capacity in liquid chromatography (LC). The peak capacity in a chromatographic separation is directly related to the plate number and thus to column length and particle size. Serial coupled columns can be used to obtain long effective column lengths, reaching over 100000 theoretical plates and peak capacities up to 900. Some theoretical considerations are made on column dimensions and particle size and examples are given of high resolution "GC-like" separations in LC using state-of-the-art LC hardware. Recent developments in LC hardware have also enhanced the applicability of two-dimensional LC–LC and comprehensive LCÃ-LC. Both techniques are extremely powerful to unravel complex samples.

This month's installment of "Column Watch" is the first of a two-part series in which Ron Majors examines the trends and highlights in columns and consumables at Pittcon 2007.

A reluctance to make any changes to a validated or compendial method is common, and often well founded.