This month's column continues with Part II of the three-part series, "A Mass Spectrometry Primer." To recap the basis for undertaking this work, technological changes that affect our knowledge, in terms of its depth and our speed of accessing it, spurs a couple of observations about print media versus electronic. Anything published must be scholarly because it often serves as a primary resource for certain facts, equations, and other things we can't, or won't, remember.
HPLC and GC Articles
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 variety of chromatographic sorbents are available commercially for reversed-phase liquid chromatography (LC), and while many of these columns are nominally similar, in practice, the columns can provide significantly different separations. An understanding of the nature of these differences is essential for efficient and appropriate method development. Column differences are evident particularly for the separation of geometric isomers.
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.
Mobile phase degassing is the single most effective way to avoid problems with an LC system. Liquid chromatographs and air just weren't meant to be together! LC pumps are very effective at pumping liquids, but introduce an air bubble and in the best circumstances you will observe a momentary reduction in the flow-rate and a drop in the system back pressure. If the bubble is large enough, the pump will not deliver any solvent and if the pressure drops below a preset low-pressure limit, the pump will stop. Some pump designs will pass bubbles fairly well, whereas other designs will fail to operate when a bubble is present.
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.
To routinely obtain reliable results and develop HPLC methods efficiently, modern laboratories require not only advanced instruments and tools, but also precisely defined procedures for verifying performance. The reproducibility and accuracy of any HPLC analysis is strongly dependent on the solvent composition, especially when working in isocratic mode. In gradient mode, however, the quality of the analysis will be influenced by the performance of the proportioning and mixing of the eluents at any time during a given chromatogram. Therefore, routine testing of the gradient performance of the delivery system within well-defined intervals is, at least, of the same importance as testing column efficiency1,2 and selectivity.3,4
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.
The term Good Laboratory Practice (GLP) is a well known acronym for quality in the laboratory but how much do you know about the term, its background and what does it really mean in practice?
OK, let's start from the beginning: GLP is a formal regulation that was formulated in 19781 by the US Food and Drug Administration (FDA) that impacted the pharmaceutical industry. However, the primary focus of the regulation was not the chromatography laboratory nor even the analytical laboratory. GLP was intended to regulate non-clinical laboratory safety studies (i.e., animal toxicology testing) to ensure that any new molecular entities developed by the pharmaceutical industry were safe before administration to human volunteers and patients in clinical trials. This original aim has not changed since the regulation was issued as the title of the regulation makes clear: Good Laboratory Practice for Nonclinical Laboratory Studies.
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.
It is strange how we automatically apply the scientific method to most of our work in the laboratory, but somehow we discard it when it comes time to troubleshoot an LC problem. For troubleshooting purposes, I summarize the recommended technique as the of One". This reminds us to change just one variable at a time when investigating a problem.
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.
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.
Countercurrent chromatography (CCC) is basically the liquid–liquid partition of a sample between two immiscible liquid phases. This is the same separation principle as separating two mixtures using a simple separating funnel experiment. The technique developed from countercurrent distribution, which was investigated in the 1940s to provide separations without a solid chromatographic support. The term "countercurrent chromatography", although generally accepted nowadays, is in fact a misnomer because most systems involve the passage of one phase (the mobile phase) of a biphasic solvent system through a second non-mobile (stationary phase) component of the solvent system.
This article will provide a review of CCC because the technique is at last becoming more widely accepted by the scientific community. Reliable instruments are now being produced and applications in university and industrial circles are increasing.1
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
Computerized pneumatics facilitate set-up and operation of both capillary and packed columns and offer chromatographers improved performance over manually operated pneumatics in terms of retention time stability and split or splitless quantification. The first part of this series introduced the basics of computerized pneumatics,1 showed how a gas chromatography (GC) system controls flow or pressure and discussed the fundamentals of operation as well as some pitfalls that can arise when changing the pneumatic configuration.
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.
Tetracycline antibiotics (TCAs) are used globally in aquaculture to control disease and promote growth. The tetracyclines used in veterinary medicine in Japan account for more than half the total of antibiotics used.1 The overuse of TCAs can result in their presence in seafood for human consumption.
This is a serious problem in Japan where seafood is consumed in copious amounts and prawns are the Japanese consumer's favourite choice. A report that 2.3 ppm of oxytetracycline (OTC) was detected in imported Japanese Tiger Prawn (Marsupenaeus japonicus) magnified these concerns. This level is greater than the minimal risk level (MRL) of 0.1 µg/g in fish — singly or in combination — the EU has set for OTC, chlortetracycline (CTC) and tetracycline (TC).2 Correspondingly, the Codex MRL for OTC is 0.2 µg/g for Black Tiger Prawn (Penaeus monodon),3 which is consistent with the value set for fish by the Japan Ministry of Health, Labor, and Welfare.4
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.
The Transport Modelling and Analytical Separation Science group is part of the Department of Chemical Engineering of the Faculty of Engineering Sciences. The group currently has eleven PhD students and one post-doc and is headed by Gert Desmet, who is also head of the department. The group focuses on the miniaturization of separation methods and on the investigation and the modelling of flow effects in commercial and innovative chromatographic systems. The activity of the group in the field of chromatography started in 1999 by practically demonstrating the giant leap in separation speed that can be achieved by using shear-driven flows in 100 nanometere-deep channels.
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.
For the last six months, "LC Troubleshooting" has been concentrating on the process for the development of isocratic methods (those for which the mobile phase composition is constant throughout the run). This worked through the sequence of goal setting,1 selecting the right starting conditions,2 adjusting retention,3 changing peak spacing4,5 and fine-tuning the column efficiency.6 Hopefully you have gained additional insight into the liquid chromatography (LC) process, as well, so your methods will be less likely to fail and will be easier to troubleshoot.