4 results
5 - The importance of a quality assurance plan for method validation and minimizing uncertainties in the HPLC analysis of phytoplankton pigments
- Edited by Suzanne Roy, Carole A. Llewellyn, Plymouth Marine Laboratory, Einar Skarstad Egeland, Geir Johnsen, Norwegian University of Science and Technology, Trondheim
-
- Book:
- Phytoplankton Pigments
- Published online:
- 05 March 2012
- Print publication:
- 27 October 2011, pp 195-256
-
- Chapter
- Export citation
-
Summary
Introduction
A quality assurance plan (QAP) describes a process to ensure an analytical method fulfils the agreed upon accuracy objectives at all points during the analysis of samples. A QAP includes such things as standardized procedures, method validation, quality control (QC) measurements, and quality assessment (QA); the latter quantitatively describes how results of QC measurements are used to determine whether a method is performing within expectations. By analogy, a QAP describes what can be considered, in a more general sense, a ‘holistic’ approach to sample analysis, whereby the ‘whole is considered to be a result of the interdependence of all parts’. Here, the ‘whole’ represents the overall combined uncertainty of a final data product, and ‘the interdependence of all parts’ represents the uncertainties contributed by the many individual procedures required to produce that final data product.
An in depth discussion of uncertainty analysis in the chemical laboratory, given in EURACHEM (2000), is beyond the scope of this chapter, but the importance of a so-called holistic approach to sample analysis, whether this includes a formalized QAP or not, is necessary to provide knowledge of the uncertainties associated with measured values and, thus, facilitate confidence in the data products. Such knowledge is important, because pigment data are often compiled in increasingly large databases, and end users of the data are remote (in space and time) from the data providers. Knowing the accuracy of the data facilitates their wider utility, for both current and unanticipated future applications. This chapter describes components of a QAP in the context of knowledge gained during intercomparisons sponsored by the National Aeronautics and Space Administration (NASA). A primary objective of these activities was to determine if the myriad providers of HPLC analyses to NASA researchers were satisfying the accuracy requirements for field observations.
Appendix A - Update on filtration, storage and extraction solvents
- Edited by Suzanne Roy, Carole A. Llewellyn, Plymouth Marine Laboratory, Einar Skarstad Egeland, Geir Johnsen, Norwegian University of Science and Technology, Trondheim
-
- Book:
- Phytoplankton Pigments
- Published online:
- 05 March 2012
- Print publication:
- 27 October 2011, pp 627-635
-
- Chapter
- Export citation
-
Summary
Filtration
In Chapter 10 of Jeffrey et al. (1997), Whatman GF/F (or equivalent) filters (0.7 μm nominal pore size) were recommended for sample filtration. With the exception of targeted studies, GF/F filters remain the most commonly used media for routine filtration and accompanying in vitro analyses by HPLC, fluorometry and spectrophotometry. As indicated in the above-mentioned Chapter 10 (p. 284–287), many studies have compared the effectiveness of different filter types and highlighted their advantages/disadvantages. Of particular note are three relatively recent papers highlighting the limitations of GF/F filters. Knefelkamp et al. (2007) compared six different filter types and concluded that Whatman nylon membranes (0.2 μm pore size, 47 mm diameter) provided the most consistent results with respect to chlorophyll a analyses. Nucleopore filters (0.2 μm) have been reported to retain as much as four times the amount of chlorophyll a as GF/F filters in open ocean samples (Dickson and Wheeler, 1993). Furthermore, Lee et al. (1995) reported that GF/F filters retained only 13–51% of small bacterioplankton (< 0.8 μm diameter) in natural samples. In contrast, recent comparisons of filter types have reported no differences in pigment concentrations obtained using GF/F and membrane filters in a variety of aquatic habitats (Chavez et al., 1995; Morán et al., 1999). The choice of filter type, whether glass-fibre or membrane, should be determined by the individual investigator for their particular application. However, once a filter type is selected, it should be used uniformly for sample filtrations to insure consistency between samples.
In estuarine and coastal waters, particulate matter (seston) may result in rapid saturation and ‘clogging’ of filters. Continued vacuum filtration of ‘clogged’ filters may promote mechanical stress and induce cell lysis, potentially resulting in underestimation of the actual pigment concentrations in the sample (Goldman and Dennett, 1985; Taguchi and Laws, 1988; Richardson and Pinckney, 2004). The total time for sample filtration should not exceed 5–10 min to minimize filter saturation (Wasmund et al., 2006). Filters should be removed as soon as the passage of water through the filter is undetectable and the vacuum should never exceed 50 kPa. Although not commonly used, positive pressure filtration (7–14 kPa) reportedly allows the filtration of larger volumes of water with reduced filtration times (Gibb et al., 2001; Bidigare et al., 2002). Regardless of the filtration method used, multiple filters can be pooled to achieve the biomass necessary for HPLC analyses. After filtration, filters should be folded in half, blotted on absorbent paper to remove excess water, and immediately flash frozen and stored in liquid nitrogen or at −80 °C (Wright and Jeffrey, 2006).
Appendix B - HPLC instrument performance metrics and validation
- Edited by Suzanne Roy, Carole A. Llewellyn, Plymouth Marine Laboratory, Einar Skarstad Egeland, Geir Johnsen, Norwegian University of Science and Technology, Trondheim
-
- Book:
- Phytoplankton Pigments
- Published online:
- 05 March 2012
- Print publication:
- 27 October 2011, pp 636-649
-
- Chapter
- Export citation
-
Summary
Currently, there are over 90 companies that offer HPLC hardware and accessories, and more than 30 that offer complete systems. Given the myriad choices available in the marketplace, the discerning chromatographer needs to approach equipment purchases with a critical mindset and a clear understanding of what they require from an HPLC system or component. This appendix covers some of the features available in HPLC autosamplers, pumps, detectors and ovens. It is not meant to be a definitive catalog of available HPLC hardware components and design elements. Instead, it is designed to call attention to some of the features available in specific HPLC hardware that the authors of this appendix have researched in the context of how these decisions can affect one's ability to produce consistent, high quality pigment results. A thorough review of the basics and advancements in HPLC hardware is covered in the third edition of Introduction to Modern Liquid Chromatography (Snyder et al., 2010).
To make informed decisions regarding one's needs in HPLC hardware, one must understand the component design (and software control thereof) from the perspective of its contribution to combined uncertainty. Uncertainties in pigment results related to hardware characteristics are most often associated with injectors and detectors, and to a lesser extent, column oven design and pump capabilities. The uncertainties of the latter are often related to implementation of a method (e.g. baseline disturbance).
4 - New HPLC separation techniques
- Edited by Suzanne Roy, Carole A. Llewellyn, Plymouth Marine Laboratory, Einar Skarstad Egeland, Geir Johnsen, Norwegian University of Science and Technology, Trondheim
-
- Book:
- Phytoplankton Pigments
- Published online:
- 05 March 2012
- Print publication:
- 27 October 2011, pp 165-194
-
- Chapter
- Export citation