Sample cleanup

Sample cleanup is particularly important for analytical separations such as GC, HPLC, and electrophoresis. Many solid matrices, such as soil, can contain hundreds of compounds. These produce complex chromatograms, where the identification of analytes of interest becomes difficult. This is especially true if the analyte is present at a much lower concentration than the interfering species. So a cleanup step is necessary prior to the analytical measurements. Another important issue is the removal of high-boiling materials that can cause a variety of problems. These include analyte adsorption in the injection port or in front of a GC-HPLC column, false positives from interferences that fall within the retention window of the analyte, and false negatives because of a shift in the retention time window. 

In extreme cases, instrument shut down may be necessary due to the accumulation of interfacing species. 

Complex matrices such as, soil, biological materials, and natural products often require some degree of cleanup. Highly contaminated extracts (e.g., soil containing oil residuals) may require multiple cleanup steps. On the other hand, drinking water samples are relatively cleaner (as many large molecules either precipitate out or do not dissolve in it) and may not require cleanup [5], 

The following techniques are used for cleanup and purification of extracts. 

The solvent extracts can be cleaned up by traditional column chromatography or by solid-phase extraction cartridges. This is a common cleanup method that is widely used in biological, clinical, and environmental sample preparation. More details are presented in Chapter 2. Some examples include the cleanup of pesticide residues and chlorinated hydrocarbons, the separation of nitrogen compounds from hydrocarbons, the separation of aromatic compounds from an aliphatic-aromatic mixture, and similar applications for use with fats, oils, and waxes. This approach provides efficient cleanup of steroids, esters, ketones, glycerides, alkaloids, and carbohydrates as well. Cations, anions, metals, and inorganic compounds are also candidates for this method [7], 

Proper preparation of the sample is the most important preliminary step in conducting any anal-y.sis (- Sample Preparation for Trace Analysis), and this is especially true in chromatography. Of particular concern is the amount of extraneous material present in the sample solution. The greater the amount of junk, the more sample preparation that will be required. 

The Sample. Before undertaking the preparation of any sample, an aliquot of material representative of that sample must be obtained. Otherwise the analysis itself may be meaningless. The best way to assay a solid sample is to collect random aliquots from the whole and then mix 

Sample Extraction. Several alternatives are available for extracting the sample from its solvent. These include  

1) Direct application of the solution, with extraction taking place on the TLC layer 

2) Application of a solution of the sample on a preadsorbent layer, followed by direct development 

Trace analysis of soil samples often requires post-extraction cleanup to remove coextracted matrix interferences. There are several difficulties that may arise during chromatographic analysis due to interferences present in sample extracts. To avoid these and other issues, one or more of the following cleanup techniques are often used. 

This technique is based on the same separation mechanisms as found in liquid chromatography (LC). In LC, the solubility and the functional group interaction of sample, sorbent, and solvent are optimized to effect separation. In SPE, these interactions are optimized to effect retention or elution. Polar stationary phases, such as silica gel, Florisil and alumina, retain compounds with polar functional group (e.g., phenols, humic acids, and amines). A nonpolar organic solvent (e.g. hexane, dichloromethane) is used to remove nonpolar inferences where the target analyte is a polar compound. Conversely, the same nonpolar solvent may be used to elute a nonpolar analyte, leaving polar inferences adsorbed on the column. 

The most common technique used for agrochemicals is reversed-phase SPE. Here, the bonded stationary phase is silica gel derivatized with a long-chain hydrocarbon (e.g. C4-C18) or styrene-divinylbenzene copolymer. This technique operates in the reverse of normal-phase chromatography since the mobile phase is polar in nature (e.g., water or aqueous buffers serve as one of the solvents), while the stationary phase has nonpolar properties. 

Ion-exchange solid-phase extractions are used for ionic compounds. The pH of the extracts is adjusted to ionize the target analytes so that they are preferentially retained by the stationary bonded phase. Selection of the bonded phase depends on the pK or pA b of the target analytes. Sample cleanup using ion exchange is highly selective and can separate polar ionic compounds that are difficult to extract by the liquid-liquid partition technique. 

A variety of solid-phase cartridges are available from a number of different manufacturers (e.g. J.T. Baker, Varian). Most cartridges, however, use a similar extraction procedure that consists of these basic steps  

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Sample Cleanup. The recoveries from a quick cleanup method for waste solvents based on sample filtration through a Elorisd and sodium sulfate column are given in Table 2 (40). This method offers an alternative for analysts who need to confirm the presence or absence of pesticides or PCBs. 

M. Tanaka and H. Yamazaki, Dkect detemination of pantoprazole enantiomers in human serum by reversed-phase liigh peifomance liquid chi omatography using a cellulose-based cliiral stationaiy phase and column-switching system as a sample cleanup procedure , Aim/. Chem. 68 1513-1516(1996). 

T. Hyotylainen, T. Andersson and M. E. Riekkola, Eiquid cliromatographic sample cleanup coupled on-line with gas chromatography in the analysis of beta-blockers in human serum and urine , 7. Chromatogr. Sci. 35 280-286 (1997). 

To satisfy the requirement of having a total residue of only a few micrograms, the sample cleanup must be very thorough (10). The procedure which we have used to accomplish this is the following. A 10-... 

To obtain reliable chromatograms in the final step of the determination of the analytes by LC or GC, it is important to remove interfering signals resulting from coelution of other compounds. To this end, a variety of techniques are applied for cleanup of the sample extract. The most effective procedures for sample cleanup for PAH measurements are partitioning between M, N-dimethylformamide/water/cyclo-hexane and LC on silica and on Sephadex LH 20. Other cleanup procedures include LC on alumina or XAD-2 and preparative thin-layer chromatography. 

The other column chromatography cleanup procedure uses a macroporous diatoma-ceous column (e.g., Chem Elut column) and an SPE column Cig cartridges are effective columns for sample cleanup. 

Note each lot of Florisil must be checked for acceptable recovery of flumioxazin prior to initiating the column cleanup procedure. Adjust elution volumes and/or the solvent mixture as necessary to achieve >90% recovery for this step. Sample cleanup with Florisil may not be required for some water samples. 

For soil samples, sufficient sample cleanup could be conducted even if the alumina column was changed to a Sep-Pak Alumina N cartridge (Waters) by the following process. The entire sample of the dried n-hexane extract (Section 6.2) is introduced into a Sep-Pak Alumina N cartridge, and the column is washed with 50 mL of n-hexane. Subsequently, pyriminobac-methyl is eluted with 3 mL of ethyl acetate, the solvent is evaporate to dryness under reduced pressure and the residue is dissolved in an appropriate volume of acetone for GC analysis. 

Consistent with other analytical methods, immunoassays must be validated to ensure that assay results are accurate. Initial validation involves an evaluation of the sensitivity and specificity of the immunoassay, while later validation includes comparison with a reference method. Because a goal of immunoassays is to minimize sample preparation, validation also includes testing the effects of sample matrices and(or) sample cleanup methods on results. The final steps in validation involve testing a limited number of samples containing incurred residues to determine if the method provides reliable data. 

The development of a robust analytical method is a complex issue. The residue analyst has available a vast array of techniques to assist in this task, but there are a number of basic rules that should be followed to produce a reliable method. The intention of this article is to provide the analyst with ideas from which a method can be constructed by considering each major component of the analytical method (sample preparation, extraction, sample cleanup, and the determinative step), and to suggest modern techniques that can be used to develop an effective and efficient overall approach. The latter portion emphasizes mass spectrometry (MS) since the current trend for pesticide residue methods is leading to MS becoming the method of choice for simultaneous quantitation and confirmation. This article also serves to update previous publications on similar topics by the authors. ... 

Other ways to minimize matrix effects include improving the sample cleanup, diluting the sample, using labeled internal standards, using standard addition, or using matrix-matched standards. The last approach, however, is not permitted for enforcement methods at present by the US EPA or the US Pood and Drug Administration... 

Packing materials for column chromatography. Adequate cleanup can be achieved with alumina instead of silica gel. Activated carbon is not suitable for sample cleanup of ripe orange and leaf. 

The most common method of isolation and sample cleanup involves contacting a filtered aqueous solution with an appropriate immiscible organic solvent in a. aboratory separatory funnel of appropriate size. Some specific examples are discussed later. With multicomponent samples a single solvent or solvent mixture is unlikely to extract all components equally causing discrimination. Ihis discrimination may be useful if the solvent discriminates against the extraction of solutes that are not of interest in the analysis. 

Prepared from sodium silicate using the sol-gel procedure. Silica gel is the most widely used general purpose adsorbent for sample cleanup although it may irreversibly bind some strongly basic substances. Generally considered to be slightly acidic in character but this may vary with the method of prq ration. Activated by heating at about 180 C for 8-12 hours. 

Prechromatographic derivatization reactions re usually favored when it is desired to modify the properties of the sample to enhance stability during measurement (i.e., minimize oxidative and catalytic degradation, etc.), to improve the extraction efficiency of the substance during sample cleanup, to improve the chromatographic resolution, or to simplify the optimization of the reaction conditions [698-702]. As both pre- and postchromatographic methods enhance the sensitivity and selectivity of the detection process a choice between the two methods will usually depend on the chemistry involved, ease of optimization, and which method best overcomes matrix and reagent interferences. 

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