Liquid Chromatography (LC/HPLC)

No column lasts forever, but in this month's installment of "LC Troubleshooting," John Dolan offers some tricks to help extend a column's life.

Once the visual problems have been eliminated as problem sources, you are left with problems related to system performance.

Because of progress in liquid chromatography/time-of-flight mass spectrometry (LC/TOF-MS) instrumentation, data processing and reporting, the measurement of compounds' accurate masses is becoming routine practice in screening analysis based on target databases. As such databases of monoisotopic masses can be easily updated with recent data from the literature; rapid characterization of new compounds and metabolites is possible without the need for primary reference standards. This approach has already been established in comprehensive toxicological urine screening and in analysis of drugs-of-abuse in seized street drug samples. Currently, a mass accuracy within 5 ppm can be routinely achieved, and confirmation via a numerical isotopic pattern match (SigmaFit) is provided by a new generation LC/TOF-MS instrument.

Analytical chemists are concerned with the quality of their methods and results. An important question in this context is whether the precision of a newly developed and validated method is up to standard. In other words: is the precision of the newly developed method comparable to what could be expected? This article looks at how the Horwitz equation can answer this. It also describes the results of an extensive study involving 10000 laboratories which indicates that the relative reproducibility approximately doubles for every 100-fold decrease in concentration and that, surprisingly, it does not depend on the type of material or method.

A problem that is encountered occasionally in liquid chromatography (LC) separations is the presence of unusually broad peaks in the chromatogram. This problem is seen most often in isocratic separations, but it can occur with gradients as well. This month's instalment of "LC Troubleshooting" will cover some techniques to help determine the reason these wide peaks are seen.

The authors describe a new laser-induced flourescence detector for application to both capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC).

Ron Majors discusses advances in multidimensional chromatography and comprehensive multidimensional chromatography.

This month's "LC Troubleshooting" is aimed at those who are just starting out in the liquid chromatography business, as John Dolan presents some helpful tips.

HPLC 2005 was held in Stockholm, Sweden, June 26-30. In this month's installment of "Column Watch," Ron Majors covers this year's hot topics including proteomics, column technology - especially monoliths - microfluidics, and multidimensional and high-throughput separations.

The use of a simple Excel-based "Golf Score Card" tool can facilitate assessments and instrument acquisistion decisions by collating and weighting the relative importance of the many criteria that impact the final purchase decision. The authors describe this process.

Some simple practices will help to make problems with gradient methods rare rather than frequent. this month's installment of "LC Troubleshooting" presents a baker's dozen of these tips.

Sometimes the mobile phase conditions selected for reversed-phase and ion-pairing methods seem either arbitrary or mysterious.

The implementation and validation of a new site-wide chromatography data system for a major active pharmaceutical ingredient custom manufacturer provided the opportunity to map and optimize the GMP and ISO business processes to use electronic signatures effectively. This article describes the process optimization and how it integrates with the validation activities of the system. This overall approach provides substantial business benefits from the use of electronic signatures as evidenced by the savings resulting from process improvement and by the fact that the non-GMP laboratories implemented a similar process to analyse and approve results electronically.

The authors explain why sample diluent is more than just a solubilizing agent and describe a systematic process for diluent selection when developing an HPLC assay of an active pharmaceeutical ingredient.

In this month's "LC Troubleshooting," we look at two questions submitted by readers that relate to mobile phase.

The established kinetic plots clearly show that monolithic columns are suited for large plate number separations whereas in the small plate number range the 3 ?m packed bed system is to be preferred.

Although there are other possible causes, the most common source of fronting peaks is a disturbance in the column packing bed. The fix is straightforward: replace the column.

A good pretreatment system combines reverse osmosis and electrodeionization technologies.

The authors look at three column performance problems encountered frequently in today's laboratory.

A detection method based upon aerosol charging was examined for its applicability and performance with high performance liquid chromatography.

The authors address the specific separation problems of proteins and assess whether 2D liquid chromatography could possibly be considered as an alternative to traditional 2D gel electrophoresis in the future.

In the last part of this series, the authors provide a technical review of the means for method adjustment and the pertinent regulations concerning the required validation of adjusted methods.

This month, John Dolan offers some suggestions for distinguishing between overload and the presence of a minor peak.

Guest columnist Matt Przybyciel reviews the structure, behavior, and applications of both alkyl- and phenyl-fluorinated phases in HPLC.

Some possible solutions to the ubiquitous paek-tailing problem are presented by the author.

This month, John Dolan turns his attention to the unexplained forces that seem to inhabit laboratories in spite of the most ardent troubleshooting efforts.

As LC-UV and LC-MS have become preferred instrumental methods for fast, sensitive analysis, many scientists are searching for ways to optimize performance.

This month’s column provides an overview of green chemistry issues relating to preparative chiral SFC chromatography in support of preclinical development in the pharmaceutical industry.

Bob McDowall stresses the importance of using accurate and precise time and date stamps to ensure the integrity of the data and results generated by computerized systems in any chromatography laboratory.

Columnist Leslie Ettre looks back at the life and contributions of Csaba Horvath, one of the giants in the field of chromatography.