Purification of the Sample

For purposes of structure elucidation, it is generally not necessary to eliminate paramagnetic impurities since most spectra are obtained in the proton decoupled mode which is usually the source of signal broadening (M 31n). However, filtration of the sample solution may be advisable to avoid the field inhomogeneities arising from the fluctuation of small solid particles in the liquid. 

When relaxation times, NOE enhancement, ri/ or exchange mechanisms are to be investigated, it is then strongly recommended (see Sect. 2.2) to eliminate paramagnetic impurities, namely paramagnetic metal ions. 

Finally degassing under a vacuum may be useful to eliminate the small quantities of paramagnetic oxygen dissolved in the sample the freeze-pump- thaw cycle technique appears to give the best results for eliminating oxygen in purified organic solutions. 

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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). 

A two-dimensional technique involving initial separation by high voltage electrophoresis at pH 2.0 followed by chromatography is a useful means of separating similar amino acids and short peptides and does not require desalting or excessive purification of the sample (Figure 10.17). 

Phenolic compounds in Sicilian wines were directly detected by La Torre et al. [373] using an HPLC with a DAD coupled on-line with a MS system equipped with ESI source operating in the negative-ion mode and a quadruple mass analyzer. The structure was elucidated by recording MS spectra at different voltages, in addition to the molecular mass information. The method allowed both the identification and determination of 24 phenolic compounds in 22 different commercial Sicilian red wines by direct injection without any prior purification of the sample. Figure 19.10 reproduced an HPLC trace obtained in this work. 

Should the temperature rise steadily, instead of remaining virtually constant, it is then clear that this simple distillation procedure is unsuitable for the purification of the sample and some form of fractional distillation (Section 2.26) will have to be used. 

A significant source of false-negative scores stems from DNA samples containing impurities that inhibit PCR amplification. Such samples are readily detected by noting the relative intensities of PCR products in an RAPD pattern other than the particular polymorphism being scored (Fig. 4, lane 9). If bands that are monomorphic throughout a population are weak or absent in an individual sample, it is likely that this sample contains some inhibitory contaminant. Further purification of the sample by any of a number of standard protocols normally corrects this problem. 

The peak is not symmetric. A first reason can be a defect of the spectrometer. In this case all the peaks in the same mass region should have a similar distortion. This can in some cases be corrected through the calibration. If not, a mathematical treatment of the peak can be performed if available in the software, or, better, another mass spectrometer can be used Another reason may be that the peak actually contains more than one component with similar masses. This should only be observed on this peak. Purification of the sample or better direct chromatographic separation can be tried if it is believed that this is due to an impurity. Otherwise a higher resolving power should be used to separate the two masses. 

Low molecular weight compounds may be removed, and a variety of methods are available, including dialysis and gel filtration chromatography. The removal of excess protein may be more complicated. It can be dealt with before the assay by further purification of the sample. Alternatively, the excess can remain during the incubation and be removed after the assay by introducing a termination step to precipitate all proteins, which are then removed by filtration. And finally, proteolytic activities can be eliminated by the addition of the inhibitory cocktail mentioned below. 

Extraction and purification of the sample containing is not required as y-rays penetrate coloured solutions and soft tissue with negligible loss of energy. In the case of p-emitters, the scintillation solution must be colourless or quenching corrections must be applied. 

Cai et al. digested about 10 g of crab tissue with a 30% KOH solution in a mixture of water and ethanol (3 1) at room temperature. Sulfuric acid washing and column chromatography with nonactivated silica, neutral alumina, and activated carbon/silica were used in the purification of the samples [48]. 

Due to the high specificity and sensitivity of immunoassays, there are bioanalytical methods for the measurement of an analyte of interest, with little or without preconcentration or purification of the samples. The principle behind immunoassays is based on an interaction between an antibody and a corresponding antigen, and the detection of the specific interaction using radiolabels (247), enzyme, fluorescent and luminescent compounds (178, 179,181,183), electroactive markers (177,180, 228, 248), or nanomaterials (249-251). 

Aqueous samples are treated similarly beginning with the acidification step. The entire sample is then put through the hydrophobic resin, and the fulvic acids are eluted at pH 7. The humic acids are removed with 0.1 M NaOH (2). After extraction, purification of the samples can be accomplished by freeze-drying and dialysis. The use of strong acids and bases has been criticized for several reasons. They can promote degradation, decarboxylation, oxidation, and condensation reactions. Strong acids and bases can also dissolve siliceous materials and lyse cells, resulting in contamination of the sample. Other extractants have been proposed, such as sodium pyrophosphate or sodium fluoride however, the classical procedure offers the most complete dissolution of humic material from solid samples and is still most often used (72). 

Methods of analysis by direct injection in the column of sample were proposed (Lea et al., 1979 Nagel et al., 1979 Ong and Nagel, 1978 Wulf and Nagel, 1976 Roggero et al., 1989 Lamuela-Raventos and Waterhouse, 1994), but usually, prior to analysis, the different classes of compounds are fractionated on absorbent polymers such as polyamide, Sephadex LH20 or C18. The stationary phase C18 is also used for concentration and purification of the sample by solid phase extraction (SPE) prior to analysis. 

PCR is a resilient process and does not require highly purified nucleic acid. In practice, however, clinical samples may contain unpredictable amounts of impurities that can inhibit polymerase activity. To ensure reliable amplification, some form of nucleic acid purification is often used. The idiosyncratic nature of PCR inhibitors within clinical specimens requires demonstration that the sample (or preparation of nucleic acid purified from it) will allow amplification. A control nucleic acid sequence, usually different from the target, can be added to the sample (or extract from the sample). Failure to amplify this control indicates that further purification of the sample is required to remove inhibitors of the reaction. 

An important result of the experience of Baaen c< al. (1964) with the differential method should be mentioned here, namely that isotopio analyses of the early (0 6%) fractions are often the least reliable indicators of the isotope effect the points calculated from these early fractions frequently do not lie on the curve derived from the remaining fractions, and repeated purification of the samples does not usually alter the analyses. This situation could result fromthenearly quantitative removal, with the first fractions, of materials present in trace quantities in the reaction mixture. Such observations indicate that caution must be exercised in the evaluation of isotope effects by the cumulative method. 

Remove a portion of each collected fraction evaporate the solvent and assay to determine which fraction(s) contain peptide(s) of interest prior to further purification of the sample(s) by HPLC see Note 15)... 

In the samples which were not purified, some high molecular weight compoimds could be found by UV detection. In most cases, no relation between the UV peaks and the polymer peaks detected by RI detection could be found. Purification of the samples seemed to remove these compounds and it is assumed that there were some proteins adsorbed to the polymer causing UV absorption by the aromatic groups of the amino acids. 

NMR is the most powerful technique available to identify the type of carrageenan, but it requires the purification of the samples, preferably under the sodium salt form, to be sure that the polymer is in the coiled conformation in dilute solution, even at ambient temperature. This technique also gives rapid access to the quantitative determination of the different substituents, if any, and to get information on the purity of the sample tested. This point is important because the chemical structure directly controls the physical propariies of the polysaccharide. 

Most clinical immunoassays require neither prior extraction nor purification of the sample containing the analyte before they are added to the reaction containing the appropriate amoimts of high-affinity specific antibody and label. This is not the case, however, for analytes such as thyroxin or cortisol, which are tightly boimd to highly avid binding proteins that compete with the antibody for both the analyte and the label. Some of the more important protein-bound analytes are shown in Table 3. 

Mainly collect particulate matter in gas or air sample. Ionic component could be determined by 1C, PAHs by LC after purification of the sample, and heavy metals by PIXE or INAA Classify and collect particulate matter in different sizes... 

The use of solid-phase extraction cartridges is now well established in the analysis of clinical specimens. However, although this method provides efficient purification of the sample, it may lead to a loss of protein-bound vitamins. Direct injection of plasma samples into liquid chromatography (LC) columns is possible in some applications. Dilute filtered or centrifuged urine can be injected in certain LC applications, as is the case in urinary riboflavin assay. 

I am absolutely sure that this is the case, and that very extensive further purification of the samples is needed. 

This method has been extensively utilized for PGs measurements and in fact the whole PGs field has been opened up with the help of Bioassay (4). The major advantage of it is that compounds with a very short half life can be measured if the preparation has been done properly. This is not possible with RIA and MF, since both these methods entail previous purification of the sample. 

The same sample workup procedure described in Table 2 was used. The data reported in Tables 6 and 7 are raw data obtained in cortex samples of different weight. As it can be seen, MF, Bioassay and RIA gave results of the same order of magnitude. It must be noted, however, that the data for MF are in many cases about twice those obtained by the other methods. It is difficult to attribute a statistical significance to this discrepancy it must be reminded, on the other hand, that only mass fragmentography is able to correct, for each sample, losses due to sample manipulation, since deuterated internal standards are added prior to purification of the sample. 

In chronometry, the age of the sample is defined not in terms of the decay of a parent nuclide, but rather as the in-growth of a daughter activity. Radionuclides that are linked to one another by the processes of radioactive decay have relative concentrations that can be calculated with the Bateman equations, which express the simple laws of radioactive decay and ingrowth. If there exists a time at which all the descendant radionuclides have been removed from the mother material, that time can be determined through the measurement of the relative concentrations of the mother and daughter nuclides at a later time. The time interval between the purification of the sample and the subsequent analysis of the sample is defined as the age of the material at the analysis time. The technique does not apply when the half-life of the daughter nuclide involved in the determination is significantly shorter than the elapsed time. 

Chemical Protocols There are two aspects— the chemical purification of the sample, which is often called pretreatment, and the conversion of this material, first to CO2, and then to graphite in a form suitable for loading into the cesium sputter source (gas sources, e.g., using CO2, have also been developed, but have so... 

Among the different experimental procedures which are routinely used in NMR spectroscopy, attention will be focussed on some specific topics concerning the NMR cell, the purification of the sample, the chemical... 

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