This "LC Troubleshooting" installment marks the beginning of the 26th year that I have been writing this column. Over that time, changes in instrument design, and especially column technology, have occurred that have made liquid chromatography (LC) an easy-to-use and reliable process. Some of the major problems with routine operation now are minimal. For example, bubbles resulting from poorly degassed mobile phase no longer hold first place on the top 10 LC problems list — automatic in-line degassers are largely responsible for this change.

A decade or two ago, the primary users of mass spectrometers were probably graduates of doctoral programmes specializing in mass spectrometry (MS). But as the science evolved and merged with the analytical mainstream, manufacturers redoubled their efforts to make their instruments and operating software user-friendly. These days, specialists in various disciplines use MS as an analytical tool, a development that demands that instrument and software engineers pay close attention to ease-of-use issues and better understand how — and for what purposes — their instruments are used.

Research Focus: The group was set up in 1992 in the field of micellar liquid chromatography and focused on the analysis of drugs in physiological fluids, screening studies and the suppression of peak tailing for basic drugs. Subsequently, the group concentrated on more fundamental studies and chemometrics, with the aim of extracting the potential information contained in chromatographic signals and improving the separations, assisted by numerical methods. The group has a particular interest in the development of new optimization strategies, peak models, purity assays, deconvolution methods, and quantitative structure-retention relationships. Currently, it is involved in column characterization, development of clean analytical methods, bidimensional separations and fast chromatography.

Planar chromatography celebrates its 70th birthday. The technique dates back to 1938 when, at the Pharmaceutical Institute in Kharkov, Ukraine, N.A. Izmailov and M.S. Shraiber first devized a circular thin-layer chromatogram,1 which was called spread layer or spot chromatography at the time.

It is often overlooked that TLC has developed into a high-performance method (HPTLC) that can compete in terms of speed with ultra-rapid high performance liquid chromatography (HPLC) methods. Planar chromatography saves money by solving difficult problems in a simple way. The application of effect-oriented detection combined with high resolution mass spectrometry (MS) can identify bioactive substances with harmful effects that are not observed by common target analysis techniques, such as HPLC–MS–MS.

Instrument manufacturers are the best source of information about installation requirements. Following such guides is a very good habit to acquire. Detailed lists of the correct supplies and services that an instrument will require are readily available. For example, the electrical supply must be of the correct voltage and frequency, it must be properly grounded and it might require an individual connection and circuit breaker. Additional considerations. such as power filtering, surge suppressors and uninterruptible power supplies (UPSs), are important for the data handling system associated with an instrument. Also, the laboratory temperature and humidity should fall within specified guidelines. An approximate British thermal unit (Btu) output rating for an instrument helps estimate the load on laboratory air-conditioning systems. Sometimes adding a number of new gas chromatographs requires an upgrade to existing heating, ventilating and air conditioning (HVAC) systems.

For a successful method transfer column dimensions and flow-rates need to be considered. It may be necessary to re-calculate them. The mathematics are simple and should not present an obstacle, although some transfers are more complicated than others. Let us begin with the simple ones. The starting point is a method that has been successfully developed, but has perhaps been "over-solved" because the resolution is higher than is required.

Shorter Columns

Keynotes
For quantitative analysis it is best to look for baseline resolution which means that R should be 1.3 or higher. For peaks that differ greatly in size a high resolution of 2.0 or more is needed.1,2 However, if the resolution of the peaks to be quantified is much higher than required it is possible to use a shorter column (with an identical stationary phase and packing quality).

1Department of Analytical and Organic Chemistry, Rovira i Virgili University, Tarragona, Spain,
2Analytical Chemistry and Pharmaceutical Technology, Pharmaceutical Institute, Vrije Universiteit Brussel — VUB, Brussels, Belgium.

Laboratory managers and scientists who are responsible for the quality of analytical results need to be able to produce accurate results. Accuracy is extremely important because it assures the comparability of results produced by different laboratories, which gives decision makers and end-users confidence in reported results.

The ISO Guide 3534-1 defines accuracy as "the closeness of agreement between a test result and the accepted reference value," with a note stating that "the term accuracy, when applied to a set of test results, involves a combination of random components and a common systematic error or bias component."1

Institute of Chemical Technology, Faculty of Food and Biochemical Technology,
Department of Food Chemistry and Analysis, Prague, Czech Republic.

Techniques involving mass spectrometry (MS) as a detection tool in food analysis have evolved substantially. Gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS) are commonly used to detect, identify, quantify and confirm both natural and xenobiotic substances in the food production chain. One of the most distinctive trends in MS-based analysis is the use of time-of-flight mass spectrometry (TOF-MS) for both target and non-target analysis of a wide range of organic compounds that occur in biotic matrices.

In CE the typical length of an injection inside the capillary is a few mm. This is a significant length given that the total capillary length may be 30 cm and the detection window may be 0.1 mm. The peak width is directly related to the injection zone length. Ideally the injection zone length would be very small but this would result in sensitivity issues.

There are many different in-capillary concentration approaches in CE that can be used to improve method sensitivity and separation efficiency by reducing peak width after injection. These have been summarized and interested readers should read these more in-depth publications.1,2

I recently received a question from a reader regarding the impact of a flow-rate change on a validated method. The method was for the analysis of a multivitamin product using gradient elution with a reversed-phase liquid chromatography (LC) separation. The method ran over a 35 min period at 0.7 mL/min. The reader had observed small column-to-column changes over the several years that the method had been in use, but the main concern was that the retention times had all increased recently. It was found that an increase in the flow-rate to 0.8–0.85 mL/min adjusted the retention times sufficiently that the retention times specified in the system suitability parameters could be attained. The concern was how much variation in flow-rate was allowed (changes in flow-rate had not been investigated during method validation) and what could have caused the problem in the first place.

High-speed liquid chromatography is becoming important in many pharmaceutical and biochemical laboratories because of the demand to analyse complex samples quickly.1 The availability of small-particle columns (less than 2 µm particle size) has paved the way for LC separations with short analysis times, while maintaining — or even increasing — high separation efficiencies.

US Good Manufacturing Practice (GMP) regulations (21 CFR 211) have existed unchanged since 1978,1 although in 1996 a draft amendment was issued but was never implemented.2 However, on 4 December 2007, the FDA issued a Direct Final Rule for 21 CFR 211 that will make changes to GMP for finished pharmaceuticals,3 as well as withdrawing the draft 1996 amendment.4 This is the first phase in changes that will be made by the FDA to update and harmonize the GMP regulations over the next few years.

What is a Direct Final Rule?

The usual way for rulemaking by the FDA is a consultative process that involves requesting feedback from industry and other interested parties. For example, 21 CFR 11 had the following stages before the final regulation became law:

* Advance Notice of Proposed Rulemaking (1992).
* Draft rule 21 CFR 11 (1994).
* Final rule 21 CFR 11 and preamble (1997).

During the first two stages there was consultation and discussion with the industry.