Sample concentration and clean-up solid phase extraction

Representative Samples and Sample Storage. Sample Concentration and Clean-up Solid Phase Extraction. 

SAMPLE CONCENTRATION AND CLEAN-UP SOLID PHASE EXTRACTION... 

Extracellular toxins are typically present in very low concentrations and thus are difficult to analyze directly. Therefore the use of solid-phase extraction (SPE) or liquid-liquid extraction (LEE) for sample concentration and clean up are required [114,127,133,134]. 

Principles and Characteristics Solid-phase extraction (SPE) is a very popular sample preparation and clean-up technique. In SPE solutes are extracted from a liquid (or gaseous) phase into a solid phase. Substances that have been extracted by the solid particles can be removed by washing with an appropriate liquid eluent. Usually, the volume of solvent needed for complete elution of the analytes is much smaller (typically < 1 mL) than the original sample volume. A concentration of the analytes is thus achieved. 

Supercritical fluid extraction (SFE) and Solid Phase Extraction (SPE) are excellent alternatives to traditional extraction methods, with both being used independently for clean-up and/or analyte concentration prior to chromatographic analysis. While SFE has been demonstrated to be an excellent method for extracting organic compounds from solid matrices such as soil and food (36, 37), SPE has been mainly used for diluted liquid samples such as water, biological fluids and samples obtained after-liquid-liquid extraction on solid matrices (38, 39). The coupling of these two techniques (SPE-SFE) turns out to be an interesting method for the quantitative transfer... 

The Gilson Aspec automatic sample preparation system is a fully automated system for solid-phase extraction on disposable columns and online HPLC analysis. The Aspec system offers total automation and total control of the entire sample preparation process including clean-up and concentration. In addition, Aspec can automatically inject prepared samples into on-line HPLC systems. 

Dieldrin analysis. Dieldrin is an organochlorine pesticide. It is recommended that 500 ml of the water sample is introduced into a pre-cleaned and rinsed glass bottle. For preservation of the sample, two options are possible, i.e. either add 1 ml of a 10 mg ml-1 HgCh solution, or add the appropriate extraction solvent (or pre-concentrate the sample on a solid-phase extraction cartridge (see Chapter 8)). Ensure that the container is completely full of the sample. In this situation, the sample can be held for up to seven days if using HgCl2 solution (or 40 days, if extraction solvent is added) for the analysis of dieldrin. 

Solid phase extraction has several advantages over conventional liquid-liquid extraction when trace components are of interest. It is faster, requires less solvent, reduces the need for large concentration steps, and is easily automatable. Several liters, if necessary, can be poured through the column, and the trace components collected. These can be eluted with only a few mL of solvent, so the solvent removal for further concentration is nearly or completely eliminated. However, an HPLC column of 30 cm can provide 15,000 plates, whereas a solid phase disc can provide only 10 to 50 plates. What this means is that a solid phase disc can separate classes of compounds, but HPLC can then separate the compounds within that class. In fact, one of the major uses for SPE is as a clean-up for HPLC. The sequence is (1) select the proper size SPE tube, (2) condition the tube, (3) add the sample, (4) wash the packing, and (5) elute the compounds of interest. 

The first publication that reported the use of LC—MS for quantification of IsoPs in urine used reversed-phase LC coupled with ESI/MS. The method used only 1 mL urine and the clean-up procedure using solid phase extraction (SPE) columns gave quantitative recovery of the IsoP. The chromatographic runs and SPE purification methods are short, and this results in a very easy and user-friendly procedure (Li et al., 1999). A comprehensive review by Tsikas et al. describes sample preparation techniques and compares GC—MS methods with the most recent LC—MS/MS methods (Tsikas et al., 2003). The focus is primarily on 8-f5o-PGF2ci and highlights the difficulty to detect only one IsoP isomer without immunoaffinity chromatography preparation. The large concentration differences for the various IsoP classes in urine are also addressed. 

High-performance affinity chromatography has recently been reported with trypsin-modified avidin supported on 5 pm silica. While the separations were successful and a wide range of foods were studied, elution times were 80 minutes and ADAM post-column reactions were still required (Hayakawa et al. 2009). However, such affinity columns within a solid-phase extraction (SPE) platform make realistic choices for sample preparation, whereby the biotin can be purified and concentrated prior to reversed-phase HPLC. R-Biopharm has recently developed a commercially available antibody-based immunoaffinity column to bind biotin from aqueous extracts, providing an excellent technique to clean up complex samples. 

Solid-phase extraction (SPE) can be used to separate an organic analyte from the aqueous phase and to concentrate the analyte in a few milliliters of solvent. SPE also can be used to clean up a sample matrix and remove concomitant contaminants from the analyte. In some cleanups the analyte is absorbed onto the solid phase and the interferences pass through unretained. In the opposite cleanup strategy the solid phase retains the interferences and allows the analyte to pass through the cartridge with the mobile phase. In some environmental application, SPE performs all three roles—extraction, concentration, and cleanup. 

Several sample pre-treatment methods have been proposed, aiming both at sample clean-up to reduce interferences and pre-concentration of which some have been duly validated and integrated in standardised analysis procedures and automated devices (Pinheiro et al. 2004). Those pre-treatments will increase the accuracy and sensitivity of the detection and lead to faster analyses and better reproducibility. Besides, these procedures may attain minimal sample volume requirements and avoid of analyte losses through evaporation. Liquid-phase extraction, LPE, (Alaejos et al. 2008 Sun et al. 2012b) solid-phase extraction, SPE, (Alaejos et al. 2008 Martinez et al. 2000), solid-phase microextraction, SPME, (Alaejos et al. 2008 DeBruin et al. 1999 Sharma et al. 2011), liquid-liquid-liquid microextraction. 

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