Sample Clean-up

The nature of environmental samples often requires a clean-up step between sampling and the application of the analytical method. There are a variety of clean-up procedures available, most of which can be implemented in an automated fashion (see Chapters 3 and 4), in both segmented and unsegmented flow-methods, robotic methods and HPLC. Liquid-liquid and solid-liquid extraction [12-14], filtration [14,15], dialysis [12,15], evaporation [12,13], low-temperature precipitation of lipids and column-switching methods [16] are all cleanup methodologies of proven efficiency with environmental samples. 

The automatic sampling of liquids can be carried out with off-line and online samplers. Off-line assemblies consist essentially of an suction pump, a series of flasks and microprocessor allowing programming of the Intervals between successive samplings. An example of this type of set-up is the portable automatic sampler S-4000 of the Philips Environmental Protection Series, a scheme of which Is depicted In Fig. 15.1a. The operational sequence Involved the following events  

The instruments with on-line sampling units used in surveillance stations can also include a programmed system for emergency samplings, allowing samples to be collected as soon as any of the controlled parameters exceeds a preset limit. This allows samples to be obtained in cases of transient or unexpected pollution. 

Mass loadability of SPE and RAM columns play a key role in executing the sample clean-up. It is advisable to work below the overload regime of the column. Otherwise, displacement effects and other phenomena such as secondary interaction by adsorbed species might take place, which will lead to nonrepro-ducible results. This last statement is particularly important when the task is to monitor medium-to-low-abundance proteins large sample volumes in the milliliter range are therefore usually applied. 

Optimum chiral resolution without any racemization and reliable interpretation of chromatographic behaviour of enantiomers have to be considered as the first targets in enantioselective analysis (section 6.2.3.1.). Even if a universal recommendation on sample clean-up cannot be given, depending on the complexity of samples to be analyzed, preseparation procedures of highest efficiency are needed if a reliable stereodifferentiation should be achieved. 

In particular, enantioselective multidimensional gas chromatography (enantio-MDGC) with the combination of a non-chiral precolumn and a chiral main column 

A schematic diagram of enantio-MDGC, well proved in quality assurance and origin control of flavourings and fragrances is outlined in Fig. 6.24. 

A double-oven system with two independent temperature controls, two detectors (DM1, DM2) and a live switching coupling piece, is used. At optimum pneumatic adjustment of the MDGC-system definite fractions, eluted from the precolumn, are selectively transferred onto the chiral main column (heart-cutting technique). 

At first the chromatographic conditions of the chiral main column must be optimized carefully. Capillary columns coated with chiral stationary phases of suitable enantio-selectivities are used as main columns. Chiral resolutions are commonly achieved isothermally or by low temperature programming rates, starting at least 20°C below precolumn temperature. Precolumns are chosen with respect to (i) the versatility of application, to (ii) the direct injection of high sample volumes and (iii) with respect to the requisite time of analysis. 

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Tandem mass spectrometry or ms/ms was first introduced in the 1970s and gained rapid acceptance in the analytical community. The technique has been used for stmcture elucidation of unknowns (26) and has the abiUty to provide sensitive and selective analysis of complex mixtures with minimal sample clean-up (27). Developments in the mid-1980s advancing the popularity of ms/ms included the availabiUty of powerhil data systems capable of controlling the ms/ms experiment and the viabiUty of soft ionisation techniques which essentially yield only molecular ion species. 

The main advantages of the ms/ms systems are related to the sensitivity and selectivity they provide. Two mass analyzers in tandem significantly enhance selectivity. Thus samples in very complex matrices can be characterized quickly with Htde or no sample clean-up. Direct introduction of samples such as coca leaves or urine into an ms or even a gc/lc/ms system requires a clean-up step that is not needed in tandem mass spectrometry (28,29). Adding the sensitivity of the electron multiplier to this type of selectivity makes ms/ms a powerhil analytical tool, indeed. It should be noted that introduction of very complex materials increases the frequency of ion source cleaning compared to single-stage instmments where sample clean-up is done first. 

Coupled liquid chromatography-gas chromatography is an excellent on-line method for sample enrichment and sample clean-up. Recently, many authors have reviewed in some detail the various LC-GC transfer methods that are now available (1, 43-52). For the analysis of normal phase eluents, the main transfer technique used is, without doubt, concurrent eluent evaporation employing a loop-type interface. The main disadvantage of this technique is co-evaporation of the solute with the solvent. 

Trace enrichment and sample clean-up are probably the most important applications of LC-LC separation methods. The interest in these LC-LC techniques has increased rapidly in recent years, particularly in environmental analysis and clean-up and/or trace analysis in biological matrices which demands accurate determinations of compounds at very low concentration levels present in complex matrices (12-24). Both sample clean-up and trace enrichment are frequently employed in the same LC-LC scheme of course, if the concentration of the analytes of interest are Sufficient for detection then only the removal of interfering substances by sample clean-up is necessary for analysis. 

Chlorophenoxy acids are relatively polar pesticides which are usually determined by LC because volatile derivatives have to be prepared for GC analysis. This group of herbicides can be detected by multiresidue methods combined with automated procedures for sample clean-up, although selectivity and sensitivity can be enhanced by coupled-column chromatographic techniques (52). The experimental conditions for Such analyses are shown in Table 13.1. 

E. A. Hogendoom, G. R. van der Hoff and P. van Zoonen, Automated sample clean-up and fractionation of organochlorine pesticides and polychlorinated biphenyls in human milk using NP-HPEC with column-switching , J. High Resolut. Chromatogr. 12 784-789 (1989). 

One approach to the problem of matrix effects is to prevent the matrix materials reaching the electrospray source by carrying out some form of clean-up prior to analysis and/or to employ chromatographic separation. It is not always possible, however, to develop a simple procedure for sample clean-up and since this approach involves further work-up with the associated increase in analysis time and potential for sample loss it is therefore not ideal. 

GC/ECD or a halogen-specific detector (HSD) (Method 8080) is the technique recommended by EPA s Office of Solid Waste and Emergency Response for determining a- and [3-endosulfan and endosulfan sulfate in water and waste water at low-ppb levels (EPA 1986a). At these low concentrations, identification of endosulfan residues can be hampered by the presence of a variety of other pesticides. Consequently, sample clean-up on a Florisil column is usually required prior to analysis (EPA 1986a). 

Bartok, T., Borsok, G., and Sagi, F., RP-HPLC separation of polyamines after automatic FMOC-C1 derivatization and precolumn sample clean-up using column switching, /. Liq. Chromatogr., 15, 777, 1992. 

Urine Sample cleaned-up by coprecipitation, treated with HN03 and H202, wet ashed Biphasic liquid scintillation 1 pCi/200 cm3 84% Bomben et al. 1994... 

Urine Spiked sample clean-up by co-precipitation, purified by TRU-spec column and electrodeposition a -Spectrometry 0.016 pCi/800 cm3 95% at 0.1-100 pCi/sample Goldstein et al. 1991... 

Post-extraction manipulation (minimisation of sample clean-up procedures)... 

A single SFE/ESE instrument may perform (i) pressurised C02 (SFE), (ii) pressurised C02/modifier and (iii) pressurised modifier (i.e. ASE /ESE , organic solvent) extractions. The division between SFE and ASE /ESE blurs when high percentages of modifier are used. Each method has its own unique advantages and applications. ESE is a viable method to conduct matrix/analyte extraction provided a solvent with good solvating power for the analyte is selected. Sample clean-up is necessary for certain matrix/analyte combinations. In some circumstances studied [498], SFE may offer a better choice since recoveries are comparable but the clean-up step is not necessary. 

Table 3.46 compares SPME and SPE. Although SPME has in common with SPE that the analytes are concentrated by adsorption into a solid phase, SPE involves absorbing the analyte from the sample onto a modified solid support. In practice, the two techniques are quite different. SPME differs from conventional SPE in that SPE isolates the majority of the analyte from a sample (>90%) but injects only about 1 to 2% of the sample onto the GC. SPME isolates a much smaller quantity of analyte (2-20%), but that entire sample is injected into the GC. SPME is easy-to-perform and often significantly more rapid and simpler than SPE, but its quantitative aspect is exacting. Both conventional SPE and SPME minimise the use of solvents for sample preparation and free analysts from tedious sample clean-up. Where SPE can replace LLE... 

Function/objective Sample clean-up Sampling method... 

Hinman et al. [492] have compared SFE and ASE in the extraction of antioxidants from LDPE. Comparable extraction yields were obtained with both techniques. However, sample clean-up was necessary after ASE , while with SFE the extract could be analysed directly without any post-extraction clean-up. Supercritical fluid extraction of 15 polymer additives (AOs, UVAs, process lubricants, flame retardants and antistatic agents) from eight PS formulations was compared to dissolu-tion/precipitation extractions [557], Additive recoveries were comparable. Numerous additional comparisons can be found under the specific headings of the extraction techniques (Sections 3.3 and 3.4). 

Table 4.45 shows the main features of SEC. This technique has become an indispensable tool for polymer characterisation. SEC has some advantages over other LC methods, such as the predictability of the end of a chromatographic run and of the retention times in a calibrated chromatographic system. SEC is an attractive technique for prefractionation or sample clean-up prior to a more sensitive RPLC technique. This intermediate step is especially interesting for experimental purposes whenever polymer matrix interference cannot be separated from the peak of interest [647]. Disadvantages are that the whole separation must be eluted within the... 

Determination of caffeine in soft drinks was undertaken using the aerosol alkali flame ionization detector.24 Soft drinks studied were Coke, Diet Coke, Pepsi, Diet Pepsi, Dr. Pepper, and Mountain Dew. A sample clean-up and concentration procedure is employed followed by GC separation with alkali flame ionization detection. Results showed that Coke, Diet Coke, Pepsi, Diet Pepsi, Dr. Pepper, and Mountain Dew contained 41 2, 52 2, 43 4, 35 9, 46 6, and 60 15 mg caffeine per 355-ml serving. These values compared favorably with levels reported in the literature. 

Many of the adverse consequences of injecting dirty samples can be prevented or minimised by the use of guard columns, as discussed earlier, but often some form of sample clean-up is needed as well. The goal of sample preparation is to obtain, from the sample, the components of interest in solution in a suitable solvent, free from interfering constituents of the matrix, at a suitable concentration for detection and measurement. Naturally we want to do this with the minimum time and expense. 

With on-line techniques, the column switching operations are done using valves. Fig. 5.3d shows a simple arrangement for zone cutting that could be used for sample clean-up. The zone marked Y is to be determined and all other zones are to go to waste (this type of cut is called a heart cut). Initial separation takes place on column Cl so that early zones (X) are routed to waste. When zone Y is eluted from Cl, valve V2 is switched to elute this zone onto column C2. After complete transfer of Y onto C2, valve VI is switched to prevent further elution of unwanted zones (Z, for instance). Zone Y is eluted to the detector and Cl can be cleaned and re-equilibrated with mobile phase. 

Mangani, F., Luck, G., Fraudeau, C., Verette, E. (1997). Online column-switching high-performance liquid chromatography analysis of cardiovascular drugs in serum with automated sample clean-up and zone-cutting technique to perform chiral separation. J. Chromatogr. A 762, 235-241. 

If the extraction and/or sample clean-up procedures are particularly complicated, a separate document for these procedures may be justified. 

Increasingly used for sample clean-up prior to chromatographic analysis and pre-concentration of trace and ultra-trace levels of analytes. Largely replacing solvent extraction. 

Sample Clean-Up through Affinity Purification Employing Molecularly... 

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