Sample extracts, cleanup

Principles and Characteristics Because of the limited selectivity of extraction, a chromatographic analysis is almost always needed. Recently, a fair amount of progress has been made regarding the front end of the total analysis procedure, namely the integration of sample preparation (this being the analytical bottleneck) and separation. The idea behind such systems is to perform sample extraction, cleanup and concentration as an integral part of the analysis in a closed system. Scheme 7.2 shows the main procedures related to sample preparation for chromatographic analysis. 

Whereas the use of conventional fast atom bombardment (FAB) in the analysis of polymer/additive extracts has been reported (see Section 6.2.4), the need for a glycerol (or other polar) matrix might render FAB-MS analysis of a dissolved polymer/additive system rather unattractive (high chemical background, high level of matrix-, solvent- and polymer-related ions, complicated spectra). Yet, in selected cases the method has proved quite successful. Lay and Miller [53] have developed an alternative method to the use of sample extraction, cleanup, followed by GC in the quantitative analysis of PVC/DEHP with plasticiser levels as typically found in consumer products (ca. 30 %). The method relied on addition of the internal standard didecylphthalate (DDP) to a THF solution of the PVC sample with FAB-MS quantitation based on the relative signal levels of the [MH]+ ions of DEHP and DDP obtained from full-scan spectra, and on the use of a calibration curve (intensity ratio m/z 391/447 vs. mg DEHP/mg DDP). No FAB-matrix was added. No ions associated with the bulk of the PVC polymer were observed. It was... 

To eliminate the interference effect of other contaminants and for dirty sample extracts, cleanup may become necessary. The extract is either passed through a florisil column or an alumina column and the phthalate esters are eluted with ether-hexane mixture (20% ethyl ether in hexane, v/v). 

Tomlinson, A.J., Guzman, N.A. and Naylor, S. (1995) Enhancement of concentration limits of detection in CE and CEMS a review of on-line sample extraction, cleanup, analyte preconcentration, and microreactor technology. J. Capillary Electrophor., 2 (6), 247-66. 

An on-line concentration, isolation, and Hquid chromatographic separation method for the analysis of trace organics in natural waters has been described (63). Concentration and isolation are accompHshed with two precolumns connected in series the first acts as a filter for removal of interferences the second actually concentrates target solutes. The technique is appHcable even if no selective sorbent is available for the specific analyte of interest. Detection limits of less than 0.1 ppb were achieved for polar herbicides (qv) in the chlorotriazine and phenylurea classes. A novel method for deterrnination of tetracyclines in animal tissues and fluids was developed with sample extraction and cleanup based on tendency of tetracyclines to chelate with divalent metal ions (64). The metal chelate affinity precolumn was connected on-line to reversed-phase hplc column, and detection limits for several different tetracyclines in a variety of matrices were in the 10—50 ppb range. 

The predominant method of analyzing environmental samples for methyl parathion is by GC. The detection methods most used are FID, FPD, ECD, and mass spectroscopy (MS). HPLC coupled with ultraviolet spectroscopy (UV) or MS has also been used successfiilly. Sample extraction and cleanup varies widely depending on the sample matrix and method of detection. Several analytical methods used to analyze environmental samples for methyl parathion are summarized in Table 7-2. 

Analysis of methyl parathion in sediments, soils, foods, and plant and animal tissues poses problems with extraction from the sample matrix, cleanup of samples, and selective detection. Sediments and soils have been analyzed primarily by GC/ECD or GC/FPD. Food, plant, and animal tissues have been analyzed primarily by GC/thermionic detector or GC/FPD, the recommended methods of the Association of Official Analytical Chemists (AOAC). Various extraction and cleanup methods (AOAC 1984 Belisle and Swineford 1988 Capriel et al. 1986 Kadoum 1968) and separation and detection techniques (Alak and Vo-Dinh 1987 Betowski and Jones 1988 Clark et al. 1985 Gillespie and Walters 1986 Koen and Huber 1970 Stan 1989 Stan and Mrowetz 1983 Udaya and Nanda 1981) have been used in an attempt to simplify sample preparation and improve sensitivity, reliability, and selectivity. A detection limit in the low-ppb range and recoveries of 100% were achieved in soil and plant and animal tissue by Kadoum (1968). GC/ECD analysis following extraction, cleanup, and partitioning with a hexane-acetonitrile system was used. 

The natural matrix materials, which are similar to the actual environmental, clinical, food, or agricultural samples analyzed, are used to validate the complete analytical measurement process including extraction, cleanup and isolation procedures, and the final chromatographic separation, detection, and quantification. 

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. 

After sampling, homogenization, extraction, cleanup, concentration and possible derivatization, use a suitable determination method which provides sufficient selectivity or specificity and sufficient sensitivity. 

Until this point, the sample preparation techniques under discussion have relied upon differences in polarity to separate the analyte and the sample matrix in contrast, ultraflltration and on-line dialysis rely upon differences in molecular size between the analyte and matrix components to effect a separation. In ultrafiltration, a centrifugal force is applied across a membrane filter which has a molecular weight cut-off intended to isolate the analyte from larger matrix components. Furusawa incorporated an ultrafiltration step into his separation of sulfadimethoxine from chicken tissue extracts. Some cleanup of the sample extract may be necessary prior to ultrafiltration, or the ultrafiltration membranes can become clogged and ineffective. Also, one must ensure that the choice of membrane filter for ultrafiltration is appropriate in terms of both the molecular weight cut-off and compatibility with the extraction solvent used. 

The extent of cleanup needed depends on the target analyte, the quality of the sample extract, the method of detection and sensitivity. Liquid-liquid partition (LLP) and solid-phase extraction (SPE) columns such as the Cig cartridge and macroporous diatomaceous column are the cleanup method of choice. 

A sensitive method has been developed to determine the aged residues of diflufenican in soil by GC/ECD. A sample extraction using 100% methanol with extended shake was performed. The extract was concentrated and purifled using a Cig SPE column. Further cleanup was effected by using a silica SPE column. The LOD for diflufenican in soil was 0.001 mgkg The recovery of diflufenican at fortiflcation levels from 0.02 to 0.2 mg kg in soil by this method was between 94 and 121%. ... 

Sample extracts are cleaned up with a cartridge column before the acetylation of E-2 to E-16 and of E-1 to E-15. The final cleanup, plant material and soil samples are analyzed by gas chromatography (GC)/MSD. The GC/NPD method is applicable to water samples. 

The proper elution and wash solvent composition and the volume and flow rate through the cartridges must be determined. The SPE steps are critical to the separation and cleanup of the sample extract. Listed brands for Cg and silica gel cartridges should be used, if possible. 

The following methods serve as typical examples of immunoassay-based analytical methods applied to biomonitoring, environmental, and crop tissue analyses. Each method utilized a commercially available immunoassay kit that was combined with sample extraction and cleanup steps as part of an overall residue method. These methods can serve as models for resolution of similar problems. 

Most modern methods of analysis to determine pesticide residues in food commodities, whether a multi-residue method (MRM) or a single-residue method (SRM), can be broken down into three or four basic steps sample processing, sample extraction, extract cleanup (optional) and instrumental determination. 

It is often difficult to define where sample extraction ends and cleanup procedures begin. Sample extracts may be injected directly into a gas or liquid chromatograph in certain cases, but this will be dependent on the analyte, sample matrix, injection, separation and detection system, and the limit of determination (LOD) which is required. It is also more likely that matrix-matched calibration standards will be needed in order to obtain robust quantitative data if no cleanup steps are employed. 

The separation of analytes from undesirable matrix components, or cleanup , of sample extracts can be accomplished through a variety of techniques that take advantage of differences in the physicochemical properties of the analytes from co-extracted matrix components. 

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. 

Cleanup of sample extract. Pipet 2.5 mL of the solution derived from Module GPC into a long-necked round-bottom flask or a pear-shaped flask and add 10 mL of isooctane. By rotating the flask slowly, carefully evaporate the solution to ca 1 mL in a rotary evaporator (water-bath temperature set at 30 0 °C). If an odor of ethyl acetate is still present, add isooctane again and repeat the evaporation. Repeat, if necessary, until no odor of ethyl acetate is present the ethyl acetate must be completely removed. Allow the solution to drain to the upper surface of the column packing and then place a graduated test-tube under the column. 

HPLC/MS and HPLC/MS/MS analyses are susceptible to matrix effects, either signal enhancement or suppression, and are often encountered when the cleanup process is not sufficient. To assess whether matrix effects influence the recovery of analytes, a post-extraction fortified sample (fortified extract of control sample that is purified and prepared in the same manner as with the other samples) should be included in each analytical set. The response of the post-extraction fortified sample is assessed against that of standards and samples. Matrix effects can be reduced or corrected for by dilution of samples, additional cleanup, or using calibration standards in the sample matrix for quantitation. 

A cleanup procedure is usually carried out to remove co-extracted matrix components that may interfere in the chromatographic analysis or be detrimental to the analytical instrument. The cleanup procedure is dependent on the nature of the analyte, the type of sample to be analyzed, and the selectivity and sensitivity of the analytical instrument used in the analysis. Preliminary purification of the sample extracts prior to chromatographic separation involves liquid-liquid partitioning and/or solid-phase extraction (SPE) using charcoal/Celite, Elorisil, carbon black, silica, or aminopropyl-silica based adsorbents or gel permeation chromatography (GPC). 

For milk, transfer the entire sample extract into a separatory funnel (250-mL), add an equivalent volume of dichloromethane plus a half equivalent volume of sodium chloride solution (5%, w/v). Shake the separatory funnel for 2 min and allow the phases to separate. Partially fill a glass filter funnel with anhydrous sodium sulfate (approximately 10 g) and filter the lower dichloromethane layer through the sodium sulfate, collecting the filtrate in a round-bottom flask (250-mL). Wash the sodium sulfate with dichloromethane (5 mL) and collect the washings in the same round-bottom flask. Rotary evaporate the sample to dryness under reduced pressure with a water-bath temperature of 40 °C. Dissolve the residue in 4 mL of ethyl acetate-toluene (3 1, v/v) and transfer the solution to a suitable vial ready for GPC cleanup. 

Cleanup of highly colored samples (e.g., mustard greens) on silica columns may require that only half of the sample extract be passed through the silica columu. 

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