HPLC

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.

Just how good are your gradients?

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.

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.

The separation of individual congeners of commonly used fluorescent whitening agents is described.

The authors study the solubility of five buffers commonly used in reversed-phase liquid chromatography.