Liquid Chromatography (LC/HPLC)

Mini application notes summaries

In this article, the authors look at the contemporary features of a UV detector and the design improvements that have been made over the last 30 years. Recommendations concerning technical details are also given that may influence the choice in purchase.

John Dolan concentrates on the impact plumbing can have on column performance and offers his advice on tubing selection.

Case studies are good ways to look at specific examples of common liquid chromatography (LC) problems and to draw general conclusions that can be applied to prevent similar problems from happening for other workers. The example in this month's installment of "LC Troubleshooting" comes from a reader who works in the pharmaceutical industry. The sample is a cold-cough syrup analyzed with an ion-pairing LC method. I have disguised the details somewhat to protect the proprietary nature of the method, but there should be sufficient information to help us gain some knowledge of the peak-splitting problem experienced by the user.

Pittcon 2005 - the 56th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy - returned to the Orange County Convention Center, Orlando, Florida, 27 February-4 March 2005. This year's event hosted more than 900 instrument manufacturers and 1aboratory suppliers in more than 2300 booths. In addition to attending the exposition, the conferees were able to listen to numerous oral presentations, view more than 900 posters, check out 38 seminar rooms, or attend one of 150 short courses.

It is hypothesized that in particular cases, conventional planar chromatography provides a more effective and robust system than column chromatography with regard to separation efficiency and peak distribution of mixtures composed of low-retarded analytes. Under similar reversed-phase experimental conditions, a regular distribution of thin-layer chromatography (TLC) spots of four natural estrogens (estetrol, estriol, 17?-estradiol, and estrone) corresponds to strong irregular dispersion of peaks in chromatograms generated by high performance liquid chromatography. In both cases, the efficiency of separation was assessed using simple optimization criteria such as selectivity (?min) and resolution (Rs min). The distribution of chromatographic spots was evaluated using the relative resolution product (r). The results revealed that an excellent separation of the components of interest could be achieved easily using simple nonforced and isocratic TLC. Such an interesting property of planar chromatography is mainly driven by the nonlinear relationship between k and Rf retention factors. This article also reports the practical advantages of TLC for the separation of estrogenic steroid mixtures at different temperatures.

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.