Pretreatment, samples

Sample preparation for MALDI is relatively straightforward. The sample could be a commercially obtained protein or peptide, or a band from a dried SDS gel (section 3.2.3) or a spot from a dried 2D gel (section 3.2.5). Solutions of the sample and the matrix are made up and mixed either in a tube prior to placing onto the target plate or on the target plate itself. To obtain good spectra, it is essential to keep the salt concentrations in buffers to a minimum. Two common methods are described below. 

The dried droplet method is the method originally introduced by Hillenkamp and Karas (Fig. 4.7). A saturated matrix solution, 5-10 g depending on the solubility of the matrix, is prepared in water, water-acetonitrile, or water-alcohol mixtures. In a second vessel, the sample is diluted to about 100 mg L Mn a solvent that is miscible with the matrix solution. The matrix and sample solutions are then mixed such that the final molar ratio is 10,000 1 with a final volume of a few tiL. 

A homogenous mixture is essential for obtaining good spectra. A droplet with a volume of 0.5 to 1 tiL is placed onto the stainless steel target plate and dried by ambient pressure evaporation, heating with a stream of warm air or under vacuum until crystallisation occurs. 

For the fast evaporation method (Fig. 4.8), a water-insoluble matrix is used. The matrix is dissolved in an organic solvent like acetone and a drop is applied to the target plate. The solvent evaporates within a few seconds leaving a dry thin film of the matrix on the target. A drop of analyte solution is then applied on top of the dried matrix. The analyte molecules are absorbed into the matrix crystal close to the matrix surface. It is possible to wash the crystal with water several times to remove impurities, especially alkaline metal ions from buffers. With the fast evaporation method, often spectra of high sensitivity and high resolution can be obtained. 

The extent to which environmental samples are treated prior to testing depends on the objectives of the study and should be the subject of discussions between interested parties (e.g. dischargers and regulators). There are two possible approaches  

Testing of samples unadjusted in order to gain information on the total biological effects, including the influence of potentially confounding physicochemical parameters such as pH, dissolved oxygen, suspended solids and turbidity, hardness or salinity and colour. This approach could mean that in certain instances it will not be possible to carry out certain methods because 

Adjusting either the sample or specific test solutions so that all potentially confounding physicochemical parameters specified for a particular method are met (see Table 2.2). Modification of the sample or test solutions will remove the influence of these parameters and reflect residual chemical toxicity. 

Described below are two wet-ashing techniques for water and wastewater samples prior to AAS. 

Samples that do not require preconcentration may contain substances that may adversely affect chromatographic performance. These substances may mask peaks of interest or be irreversibly retained, permanently damaging the column. In such cases sample pretreatment is necessary before an IC separation can be attempted. 

4 Potential Applications in the Fields of Process Development and Microreactor Technology 

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Anderson, R. Sample Pretreatment and Separation, Wiley Chichester, 1987. 

We have developed pre-eoneentration proeedures based on distillation of matrix elements after its ehemieal transfonnation in a volatile form. Residues of matrix oxides were used as the eolleetors for miero-elements. Main advantages of distillation are realization of proeess in the elose volume, whieh let us to exelude a eontamination minimal quantity of ehemieals used for sample pretreatment proeedure eonservation up to 40 mieroelements in the eoneentrate aehievement the faetor of eoneentration of about 10 easy solubility of the eoneentrates in nitrie and hydroehlorie aeids. 

In frames of the present work the problems of elemental analysis of human bio-substrates (blood semm, hair and bones) are diseussed. Sample pretreatment proeedures using ash and mineral aeids digestion were developed. The main sourees of systematie errors were studied and their elimination ways were suggested. 

In this presentation prineiples and applieations of liquid membrane extraetion teehniques for sampling and sample pretreatment in environmental analytieal ehemistry will be deseribed. 

The liquid was applied and dried on cellulose filter (diameter 25 mm). In the present work as an analytical signal we took the relative intensity of analytical lines. This approach reduces non-homogeneity and inequality of a probe. Influence of filter type and sample mass on features of the procedure was studied. The dependence of analytical lines intensity from probe mass was linear for most of above listed elements except Ca presented in most types of filter paper. The relative intensities (reduced to one of the analysis element) was constant or dependent from mass was weak in determined limits. This fact allows to exclude mass control in sample pretreatment. For Ca this dependence was non-linear, therefore, it is necessary to correct analytical signal. Analysis of thin layer is characterized by minimal influence of elements hence, the relative intensity explicitly determines the relative concentration. As reference sample we used solid synthetic samples with unlimited lifetime. 

Presently, the on-line coupling of NPLC and GC via heart-cutting is an established procedure which has been used successfully for several bioanalytical applications. Obviously, dfrect analysis of aqueous samples is not possible by NPLC, and therefore, a solvent switch by a sample pretreatment step (e.g. liquid-liquid extraction or SPE) is always requfred when biological samples are analysed by NPLC-GC. 

Figure 11.13 (a-c) Immunoaffinity exti action-SPE-GC-FID ti aces of (a) HPLC-grade water (b) urine (c) urine spiked with /3-19-noitestosti one (0.5 p.g/1) or norethindrone and norgestrel (both 4 p.g/1) (d) SPE-GC-FID ti ace of urine. Reprinted from Analytical Chemistry, 63, A. Faijam et al., Direct inti oduction of large-volume urine samples into an on-line immunoaffinity sample pretreatment-capillary gas cliromatography system, pp. 2481-2487,1991, with permission from the American Chemical Society. 

Figure 11.16 Chromatograms of plasma samples obtained by using SPE-SFC with super-aitical desorption of the SPE cartridge (a) blank plasma (20 p.1), UV detection at 215 nm (b) blank plasma (20 p.1), UV detection at 360 nm (c) plasma (1 ml) containing 20 ng mitomycin C (MMC), UV detection at 360 nm. Reprinted from Journal of Chromatography, 454, W. M. A. Niessen et al., Phase-system switching as an on-line sample pretreatment in the bioanalysis of mitomycin C using supercritical fluid cliromatography, pp. 243-251, copyright 1988, with permission from Elsevier Science.

W. Haasnoot, R. Scliilt, A. R. M. Hamers, F. A. Huf, A. Farjam, R. W. Frei and U. A. Th Brinkman, Determination of /3-19-nortestosterone and its metabolite a- 9-nortestosterone in biological samples at the sub parts per billion level by high-performance liquid cliromatography with on-line immunoaffinity sample pretreatment , J. Chromatogr. 489 157-171 (1989). 

A. Farjam, N. C. van de Merbel, A. A. Nieman, H. Lingeman and U. A. Th Brinkman, Determination of aflatoxin Ml using a dialysis-based immunoaffinity sample pretreat-ment system coupled on-line to liquid cliromatography , ]. Chromatogr. 589 141-149(1992). 

The usual means of identifying and quantifying the level of these additives in polymer samples is performed by dissolution of the polymer in a solvent, followed by precipitation of the material. The additives in turn remain in the Supernatant liquid. The different solubilites of the additives, high reactivity, low stability, low concentrations and possible co-precipitation with the polymer may pose problems and lead to inconclusive results. Another sample pretreatment method is the use of Soxhlet extraction and reconcentration before analysis, although this method is very time consuming, and is still limited by solubility dependence. Other approaches include the use of supercritical fluids to extract the additives from the polymer and Subsequent analysis of the extracts by microcolumn LC (2). 

A multidimensional system using capillary SEC-GC-MS was used for the rapid identification of various polymer additives, including antioxidants, plasticizers, lubricants, flame retardants, waxes and UV stabilizers (12). This technique could be used for additives having broad functionalities and wide volatility ranges. The determination of the additives in polymers was carried out without performing any extensive manual sample pretreatment. In the first step, microcolumn SEC excludes the polymer matrix from the smaller-molecular-size additives. There is a minimal introduction of the polymer into the capillary GC column. Optimization of the pore sizes of the SEC packings was used to enhance the resolution between the polymer and its additives, and smaller pore sizes could be used to exclude more of the polymer... 

LC-GC is a very powerful analytical technique because of its selectivity and sensitivity in analysing complex mixtures and therefore it has been used extensively to determine trace components in environmental samples (2, 5,77). LC allows preseparation and concentration of the components into compound types, with GC being used to analyse the fractions. The advantages of on-line LC-GC over the off-line System are, first, the less sample which is required and, secondly, that there is less need for laborious sample pretreatment because the method is automated (78). 

It seems justified to supplement the authors conclusions by adding that in the case of samples pretreated with hydrogen their higher energy of activation (12.3 kcal/mole) may result from the presence of a certain content of the /8-hydride phase in the a-solution phase. 

The procedure for the determination of total secondary alkanesulfonates with TLC and of total monosulfonates specified as homologs and isomers by derivatization GC-MS is shown in Fig. 18. The specific clean-up for sewage sludges prior to total secondary alkanesulfonate determination is outlined in Fig. 19. TLC conditions are given in Table 9. The limits of the quantification of secondary alkanesulfonates are summarized in Table 10. For eight samples and one operator the TLC time schedule is 4 days sample pretreatment and sublation, clean-up, TLC performance, and quantitative evaluation of TLC [24]. 

This separation technique produces very good results for acidic or anionic dye molecules containing carboxylic, sulfonic, and hydroxy groups that can be separated within short run times in an aLkaline medium in a single analysis step. - Natural colorants usually do not contain these functional groups they are usually more voluminous and strongly hydrophobic, properties that complicate their determination by CE. The sample pretreatment is more difQcult when CE (compared to HPLC) is used. 

ESCA Sample Pretreatment. Samples were pelleted and cut to fit into a rectangular depression in an ESCA sample probe similar in design to one used by Hercules (16). The portion of the probe holding the catalyst sample could be withdrawn into an outer cylinder and sealed under an atmosphere of the pretreatment gas. For pretreatment the calcined samples were exposed to a hydrogen flow at one atmosphere and heated to AOO C. After this pretreatment the sanqile was withdrawn into the insertion tube, sealed in the pretreatment gas, inserted into the ESCA, evacuated, and then the ESCA spectra were recorded. A similar procedure was followed for the uncalcined catalysts except that the temperature was first increased in hydrogen flow to SOO C and held at this temperature for 3 to 4 hours the sample was then heated to 400 C and held at this temperature for 18 hours. 

For this purpose we studied a temperature-programmed interaction of CH with a-oxygen. Experiments were carried out in a static setup with FeZSM-5 zeolite catalyst containing 0.80 wt % Fe203. The setup was equipped with an on-line mass-spectrometer and a microreactor which can be easily isolated from the rest part of the reaction volume. The sample pretreatment procedure was as follows. After heating in dioxygen at 823 K FeZSM-5 cooled down to 523 K. At this temperature, N2O decomposition was performed at 108 Pa to provide the a-oxygen deposition on the surface. After evacuation, the reactor was cooled down to the room temperature, and CH4 was fed into the reaction volume at 108 Pa. 

The pretreatment conditions were the same as those for IR study of adsorbed pyridine. The sample pretreated in a vacuum was exposed to ca. 300Torr D2 at different temperatimes for 1 h. After cooling to room temperature, the sample was outgassed for 10 min prior to TPD run. TPD was run at a heating rate of lOK min 1, and the desorbed gases were analyzed by mass spectrometry. 

Besides CO, hydrogen, oxygen, and other gases can also be used in chemisorption, provided suitable conditions are applied for sample pretreatment and chemisorption measurements (Overbury et ai, 1975 Bartley et al., 1988 Scholten et al., 1985 and Van Delft et ai, 1985). 

Ion-selective electrodes allow the measurement of ionic activity in diluted or undiluted whole blood, plasma or rum. The direct (undiluted) measurement may be preferred, since no sample pretreatment is necessary and the assay values are independent of hematocrit and amount of solids present. However, direct potentiometry by its very nature does not provide total concentration values similar to those obtained by flame photometry and indirect (diluted) potentiometry... 

Convenient and optimized method for sample pretreatment for the analysis of 70... 

Papadoyannis, N. and Samanidou, V.A., Sample pretreatment in clinical chemistry, in Separation Techniques in Clinical Chemistry, Aboul-Enein, H.Y., Ed., Marcel Dekker, New York, 2001, chap. 1. 

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