Liquid Chromatography (LC/HPLC)

In this month's installment of "Directions in Discovery," the authors discuss how, with the arrival of combinatorial libraries and high-throughput screening, pharmaceutical firms can develop new models of drug discovery that not only lessen the initial capital outlay involved in drug discovery, but also refine the discovery process.

This month, John Dolan underlines the importance of building safety factors into laboratory methods so that costly and frustrating problems that can arise during routine operation can be avoided.

A simple HPLC procedure is described for the determination of bendroflumethiazide (BMFT) in pharmaceutical formulations and urine samples. No interferences from common additives or other drugs frequently administered with BMFT or from endogenous compounds in urine samples were found. The lack of an organic solvent in the mobile phase reduces the risk of environmental contamination and human toxicity.

The authors discuss the issue of meeting the demands of regulatory compliance whilst ensuring good scientific practice. A number of requirements from 21 CFR Part 11 are cited to demonstrate the importance of applying the principles of risk analysis.

When a column is replaced with a new or "equivalent" column, sometimes the chromatogram can change so much that it is no longer suitable for its intended use. In such cases, method adjustment is necessary to correct the change. How much can the chromatographic variables be changed before revalidation is required? What do the regulatory agencies have to say about method adjustment? The authors discuss these issues and propose a technique that can be used to speed selection of new operating conditions.

A new detection method based upon aerosol charging was examined for its applicability and performance with high performance liquid chromatography (HPLC). Our results demonstrate universal detection of nonvolatile analytes with response magnitude that is independent of analyte chemical properties, four orders of magnitude dynamic range, low nanogram, lower limits of detection, and < 2% relative standard deviation response variability. Broad applicability was demonstrated for a range of methods including those using gradient elution, reversed phase, hydrophilic interaction, and ion chromatography; normal and narrow bore column formats; and in combination with other detectors (for example, UV detectors, evaporative light-scattering detectors, and mass spectrometers).

Formic acid often is used for the analysis of peptides in proteomic studies by HPLC-MS, due to its volatility and reduced signal suppression. However, poorer chromatographic performance can be obtained in comparison with trifluoroacetic acid or nonvolatile phosphate buffers due to increased overloading, which can occur even for extremely small sample masses. Comparison of a highly inert silica-ODS and a wholly polymeric phase indicated that overloading effects on both are very similar and caused by the mutual repulsion of solute ions on the hydrophobic column surface.

When you look at the manufacturer's literature or examine the performance sheet included with a new column, you'll see a list of column specifications, including the column plate number N. For a 5-?m particle size, column N generally will be 80,000 plates/m or more, whereas a 3-?m column will exhibit 100,000 or more theoretical plates. Your first response might be, "Get real!" After all, when real samples are analyzed on typical liquid chromatographic (LC) systems, rarely do we observe plate numbers anywhere near the manufacturer's claim.

A new detection method based upon aerosol charging was examined for its applicability and performance with high performance liquid chromatography (HPLC). Our results demonstrate universal detection of nonvolatile analytes with response magnitude that is independent of analyte chemical properties, four orders of magnitude dynamic range, low nanogram, lower limits of detection, and < 2% relative standard deviation response variability. Broad applicability was demonstrated for a range of methods including those using gradient elution, reversed phase, hydrophilic interaction, and ion chromatography; normal and narrow bore column formats; and in combination with other detectors (for example, UV detectors, evaporative light-scattering detectors, and mass spectrometers).

In this article, the authors present a simple high performance liquid chromatographic procedure for the determination of bendroflumethiazide (BFMT) in pharmaceutical formulations and urine samples. They demonstrate how the lack of an organic solvent in the mobile phase reduces the risk of environmental contamination and human toxicity.

Reagent water is used in all aspects of liquid chromatography (LC) technology, from preparation of mobile phase to preparation of standards, blanks, and samples. Reagent water is the most widely used analytical solvent, yet it is the least characterized, especially in total organic carbon (TOC) content. TOC adversely effects performance of LC methods and hence, reagent water quality is a major issue. Organics initially present in tap water will be reduced efficiently to low parts-per-billion concentrations by combining several technologies embedded in a water purification system. Monitoring the TOC concentrations gives chromatographers added confidence in their results.

This month, John Dolan looks at how changes in retention time can help to identify the source of some LC problems and demonstrates what can be learned from observing how the retention varies.

This column focuses on some of the latest developments in preparative-scale columns, bulk packing materials and column hardware designs. Silica, polymeric and other packings are discussed together with the newest monolithic columns.

In this month's column, John Dolan reveals a number of simple and useful shortcuts or rules of thumb that he has acquired over the years that allow quick estimates to guide method development or help solve a problem.

In this month's column the authors present a case study to outline several gradient quality checks that can be performed periodically to ensure proper performance of LC instrumentation. The example shows how by taking the time to perform such tests, potential problems can be identified before they reach a critical point.

In this article, the authors present a column ranking system to facilitate column selection in reversed-phase liquid chromatographic analysis. The system ranks columns based on their similarity to a reference column of choice and its applicability to a real pharmaceutical analysis is demonstrated.

Column Editor Ron Majors turns his attention to preparative chromatography. He focuses on the columns used in preparative chromatography, including how to select the appropriate mode, mobile-phase system and operating conditions.

Are you guilty of strange troubleshooting and maintenance habits that have been passed on to you from predecessors? John Dolan looks at some such practices, which were once acceptable, but don’t make sense in today’s lab.

In this article the authors review the use of elevated temperatures in HPLC, and provide examples covering separations of both small molecules and biomolecules. Generic issues of temperature dependence of retention and plate height are discussed, and comparisons are made between temperature gradient and solvent gradient elution. They describe how the use of elevated temperatures allow good chromatographic efficiency to be obtained at flow-rates higher than those optimal at ambient temperature, thus increasing the speed of separation.

The guest authors describe the use of LC–MS for the analysis of peptides. They deal with peptide extraction, peptidomic analysis in organisms with and without genomic databases, identification of peptides with conserved N- and C- terminal sequences, and in situ MALDI-TOF peptide profiling.

Maltodextrin is an easily digested carbohydrate made from corn starch...

VENTURE A affinity columns utilize ICEtechTM surface passivation technology...

Mini application note summaries...

Polymeric monolithic stationary phases offer an alternative to the classical microparticulate sorbents...

A report on the meeting that took place in April this year in York.

Column author, John Dolan, presents a simple but powerful isolation technique for identifying problems with LC methods and equipment.

The authors use the stochastic model to estimate the fundamental characteristics of the separation process.

A C18 stationary phase based upon a titanized silica support was prepared through reaction with octadecyltrimethoxysilane.

A look at the latest developments in preparative-scale columns, bulk packing materials, and column hardware designs.

The author focuses on a simple but powerful tool for isolating problems in the laboratory.