HPLC

Guest authors show how mixed modes can be used successfully in the optimization of protein purification, and discuss how various experimental parameters can be used to regulate the binding of proteins to mixed-mode sorbents.

Cytosine (chemical name 4-amino-2-hydroxypyrimidine) is a pyrimidine derivative with a hetereocyclic aromatic ring and two substituents (amine and keto groups) attached and is a polar compound of significant biological and pharmaceutical interest. In response to the intended use of bulk cytosine as a raw material in pharmaceutical manufacturing, a method for the determination of the purity of cytosine was developed.

This month's "LC Troubleshooting" installment will take a look at the design of these durable pumps and also examine some of the potential weaknesses and how to overcome them.

An alphabetical listing of all 2008 LCGC articles by author and subject.

This installation of "Validation Viewpoint" describes how statistically rigorous quality-by-design (QbD) principles can be put into practice to accelerate each phase of liquid chromatography (LC) instrument method development.

All the buzz lately about LC columns packed with particles smaller than 3 mm often comes with warnings about extracolumn effects. This month's installment will take a look at what these effects are and more.

The combination of reversed-phase high performance liquid chromatography (RP-HPLC), atmospheric pressure ionization (API), mass spectrometry (MS) and tandem mass spectrometry (MS/MS) is ideal for determining and characterizing analytes in complex biological matrices. This review looks at the importance of parameters such as hydrophobicity, ionization properties, molecular mass and, partially, the molecular structure resulting from applied LC–MS–MS systems in analytical laboratories. The use of these parameters to investigate biomolecules and their unambiguous identification is also described.

HPLC with ELSD continues to grow in popularity as a "quasi- universal" detector. Improvements in ELSD instrument design, including low temperature evaporation, have recently been commercialized...

This "LC Troubleshooting" installment marks the beginning of the 26th year that John Dolan has written this column.

A variety of chromatographic sorbents are commercially available for reversed-phase liquid chromatography (RPLC) and while many of these columns are nominally similar, in practice the columns may provide significantly different separations.

Applied Biosystems Inc., (Norwalk, CT) announced that the U.S. Food and Drug Administration has purchased seven of its 4000 QTRAP Systems for the analysis of potential harmful pesticides in the U.S. food supply. These systems will be deployed at FDA field offices in Jefferson, AR.; Irvine, CA.; Lenexa, KA.; Jamaica, NY; Bothell, WA.; Atlanta, GA; and College Park, MD.

Proxeon (Denmark) has announced it has integrated the software for its liquid chromatography platform with Thermo Fisher Scientific?s (San Jose, CA) mass spectrometry data-management system.

Very short columns filled with 1.9 µm particles were evaluated for the ultra-fast analysis of pharmaceutical formulations. Local anæsthetic, mydriatic and anti-hypertensive agents were chosen as analytes and a method was developed and validated for each of these substances, according to ICH guidelines. Excellent quantitative performance was obtained using an optimized chromatographic system that reduces the importance of extra-column effects and cuts the analysis time to less than 15 s.

Microemulsion liquid chromatography (MELC) is a recent development offering reduced sample preparation times for complex samples and generic separation conditions applicable to a wide range of solutes. This article introduces the concepts of MELC and discusses the possible benefits and future applications.

A number of commercial in-solution, and in-gel IEF fractionation systems are described in this month's column.

Ron Majors continues his summary of the technical highlights of HPLC 2008, discussing more hot topics from the show.

John Dolan regularly receive e-mails from readers with a wide variety of questions. As he cleans out his inbox, he chooses a few of these to share.

Micellar liquid chromatography (MLC) is a reversed-phase liquid chromatographic mode with a solution of surfactant forming micelles as the mobile phase. The interaction of solutes with the stationary phase coated with surfactant monomers, combined with the increased solubilization capability of micelles, have profound implications with regard to retention, selectivity and efficiency. Practical steps that a chromatographer involved in MLC should consider when developing an analytical procedure are described, including mobile phase preparation, column conditioning and cleaning.

The move from conventional particle sizes (5 μm or higher) to smaller diameter packing materials is one of the most attractive approaches to achieve higher separating efficiency. Recently developed 3 μm polysaccharide-derived chiral stationary phases demonstrate characteristics of favourable mass transfer kinetics, high column efficiency and good column permeability. This allows the fast analysis of enantiomers using conventional HPLC instruments.

HPLC 2008 was held in Baltimore in May. Columnist Ron Majors covers the highlights of this major symposium, held in the U.S. every two years.

After returning from Israel, John Dolan dedicates this month's column discussion to three problems that know no international boundaries: how to condition a new column, the effect of washing a column with water, and ...

Nanostream, Inc. (Pasadena, California), a developer of microfluidics-based parallel HPLC technology and a provider of assay development has closed all business operations.

The authors meet the need for a method for the determination of cytosine purity by developing a hydrophilic interaction chromatography (HILIC) method with demonstrated advantages in comparison to other separation techniques.

This month's column will consider two problem areas; peaks that have severely tailing peaks, or split or doubled peaks, and the peaks that front badly.

Guest Editor Peter Schoenmakers provides an introduction to LCxLC, and then goes on to talk about peak capacity, sample dimensionality, phase orthogonality, and some of the successes of the technique and the obstacles yet to be overcome.

In gradient mode, the quality of the analysis will be influenced by the performance of the proportioning and mixing of the eluents at any time during a given chromatogram.

I remember reading a study on learning in the all-day short-course format. Because teaching liquid chromatography (LC) classes is a significant part of my work, my attention was captured. The writers claimed that in a 6–8 h class, only three points would be remembered. One of my LC troubleshooting classes has approximately 200 slides - what does this say about how effective a short course is at conveying critical knowledge? As the saying goes, I have tried to make lemonade out of these lemons, and use the "only three things" concept to help reinforce what I think are the key points. So, this month's "LC Troubleshooting" instalment will use these points to form the core of a preventive maintenance programme for your LC system.

Only a generation ago, HPLC was considered an exclusive, futuristic technique that only a select few laboratories could afford to use. Today, HPLC and its various related techniques continue to dominate the world of chromatography. From HILIC to Chiral technology to hyphenated techniques such as HPLC-MS, it is without a doubt the most widespread technique currently used in the field of analytical chemistry. This month E-Separations Solutions' Technology Forum looks at the topic of HPLC-Chiral and the trends and issues surrounding it. Joining us for this discussion is Elena Eksteen, Ph.D., of Chiral Technologies, Inc., Todd Palcic of Thar Instruments, Inc., and Bill Ciccone of Microsolv.

This article investigates the different methods that can be used to compare the performance of liquid chromatography (LC) columns to assess the advantage of using them at high pressures and/or high temperatures. The main focus is on the kinetic plot method. This method, which is based on two simple equations, allows the user to transform the more common Van Deemter curve into a curve describing the ultimate separation speed as a function of the required plate number, or the required peak capacity or the required resolution.

What do you do when the method can't be reproduced?