Tracer purification

TABLE IV. Tracer Purification and Oxidation State Selection... 

Isotope Dilution Another important quantitative radiochemical method is isotope dilution. In this method of analysis a sample of analyte, called a tracer, is prepared in a radioactive form with a known activity. Ax, for its radioactive decay. A measured mass of the tracer, Wf, is added to a sample containing an unknown mass, w, of a nonradioactive analyte, and the material is homogenized. The sample is then processed to isolate wa grams of purified analyte, containing both radioactive and nonradioactive materials. The activity of the isolated sample, A, is measured. If all the analyte, both radioactive and nonradioactive, is recovered, then A and Ax will be equal. Normally, some of the analyte is lost during isolation and purification. In this case A is less than Ax, and... 

The small synthetic scale used for production of many labeled compounds creates special challenges for product purification. Eirst, because of the need for use of micro or semimicro synthetic procedures, the yield of many labeled products such as high specific activity tritiated compounds is often low. In addition, under such conditions, side reactions can generate the buildup of impurities, many of which have chemical and physical properties similar to the product of interest. Also, losses are often encountered in simply handling the small amounts of materials in a synthetic mixture. As a consequence of these considerations, along with the variety of tracer chemicals of interest, numerous separation techniques are used in purifying labeled compounds. 

These reactions are useful because they run under mild conditions, use inexpensive or easily recoverable starting materials, and have short reaction times. The major problem in purification is the separation of the sodium pyridone sulfonate from excess sodium sulfite, sodium bromide, and sodium bromoalkyl sulfonate. However, these latter compounds usually would not interfere with the use of the pyridone sulfonate as a water tracer. From a practical point of view, the pyridone sulfonates need not be purified, but can be used directly. A modified synthetic procedure involves the treatment of the pyridone sodium salt with a tenfold excess of a,iu-dibromoalkane in acetonitrile, followed by removal of the excess dibromide by vacuum distillation. The resulting product is treated with an excess of sodium sulfite in aqueous ethanol. Evaporation of the solvent yields a useful tracer. Procedures given in the experimental section were... 

Purification of the radioactive tracer was modified to include a fractional sublimation before a single extraction—recrystallization cycle to conserve the tracer material. Microgram samples were prepared in melting point capillaries for assay by mass spectroscopic analysis (Table III), made by direct probe injection of the sample into the ion source (18). The probe was heated rapidly to 200°C, and mass spectra were obtained during vaporization of the sample. Tri-, tetra-, and pentachlorodibenzo-p-dioxins vaporized simultaneously with no observed fractionation. 

Novel biomarkers, i.e. tracer derivatives from unknown natural products, are sometimes encountered in geological or environmental samples, typically as hydrocarbons. The detection and determination of these compounds are usually based on the interpretation of mass spectra in GC-MS analyses. The proofs of chemical structures are based on the proposed interpretation of the MS data, separation and purification of the unknown compounds, exact structure determination by NMR methods or X-ray crystallography (if the compound is a solid that can be crystallized), and finally, comparison with a synthetic standard. The next question concerns the biological source of the biomarker precursor compound. Many biomarkers still have no proven natural product precursors nor known biological sources (e.g. perylene, tricyclic terpanes). " ... 

Elemental carbon has many important applications. The diamond is a precious gem, known to mankind for ages graphite is used as an electrode and has numerous other applications carbon-14 isotope is used in carbon dating and the isotope carbon-13 in tracer studies and NMR. Carbon black is used in paints, pigments and inks. Activated carbon is used as an adsorbent for purification of water and separation of gases. Coke is used for electrothermal reduction of metal oxides to their metals. These applications are discussed below in more detail. 

On the other hand, the use of the total count method (7) largely forfeits the advantages which are inherent in the tracer method. Because the samples are extracted rather than assayed by combustion techniques, incomplete material recovery becomes a contributing factor. Furthermore, since the extracts are counted without preceding purification, the results cannot discriminate between residual gibberellie acid and possible degradation products. In spite of potentially increased sensitivity, the total count method offers no advantages over the bioassay or fluorometric procedures. 

Several tracers have been used in experiments describing axial mixing in fluidized beds of porous particles, e.g. acetone [37,57], Tryptophane [47], NaCl [49,56], radioactive tracers [58] and dextrane blue [59], It should be noted at this point, that measurement of RTD is not only important for determining possible domination of the chromatographic result by liquid mixing, Bo may as well be taken as a measure for the existence of a stable classified fluidized bed which is ready for sample application. Measurement of RTD in this case will provide a rational basis for the decision to start a large scale protein purification using a fluidized bed or to take measures for improvement of bed stability before application of valuable material. 

The following description of the isolation, purification and structure proof of a naturally occurring phosphatidylserine will be treated in exactly the same manner as with the previous phosphoglycerides. The methodologies described here can be used with any cell type and can be adapted for nearly any size sample. The exception, of course, would be if interest centered on the possible role of phosphatidylserine in a specific cellular reaction. Usually this would involve a small cell sample, and hence radioactive tracers would be needed. However, the biochemical pattern can be followed easily using exactly the same techniques. 

Success in the synthesis of new catalytic antibodies (CAs) depends on the efficiency of each of the following steps 1) hapten design, 2) immunogen synthesis, 3) preparation of the enzymatic tracer 4) generation and purification of antibodies and 5) kinetic assays. 

Incorporations of [14C]isobutyrate, [I4C]isovalerate,398 and [14C]acetate399 into humulone (132) and derived compounds in Humulus lupulus have been reported. The first two are considered to be precursors of MVA, but the percentage incorporations recorded may not be meaningful, as adequate purification of the product was not carried out. As the location of tracer in the mixed terpenoid-polyketide was not determined, any biogenetic speculations must be tentative. 

Neutron Activation Analysis. Magnesium-26 has a small cross section of 0.03 b. The product of irradiation with thermal neutrons is Mg (1 9.5m). As shown in Table 1, several elements commonly present in biological materials give rise to radioactive nuclides with radiations at energy levels close to those characteristic of Mg. Neutron activation was used in the first trials of Mg as an in vivo tracer when measurements were made with a well-type Nal-Tl crystal detector (14,21). Under these conditions the presence of sodium, altuninum and manganese in the samples interfered in the accurate detection of Mg, but could be reduced or eliminated by sample purification. 

New methods for the isolation and purification of inorganic phosphate (Pj) from natural waters (Colman et al., 2000 Colman, 2002) have permitted phosphate-oxygen isotopic (5 0-Pj) analysis of dissolved seawater inorganic phosphate as a tracer of phosphate source, water mass mixing. 

Calcium-45 can also be used as a tracer in the study of glassy materials, detergents, and water purification systems. 

For gel purification, 20CM-00 pmol of 32P-labeled RNA crude mixture is mixed as a tracer with 40,000 pmol of unlabeled crude substrate. Measure the radioactivity of a small aliquot of the mixture in a scintillation counter to calculate the specific activity of the RNA. The dsRNA and ssRNA in the crude mix are separated by electrophoresis in a preparative 8% polyacrylamide gel. After visualization by autoradiography, the region of the acrylamide gel containing the dsRNA is excised with a razor blade and the RNA is extracted from the gel. After gel purification, the dsRNA substrate is suspended in buffer I, and the radioactivity of an aliquot is measured on the scintillation counter. From the specific activity of the dsRNA, the amount of gel-extracted substrate can be determined. This is called the cold substrate because the specific activity is lower than that of the hot substrate, which is freshly labeled with 33P for use in the assay. Since a small amount of the purified dsRNA is labeled with 32P, aliquots of gel-purified cold substrate should be stored in an acrylic (3-radiation storage container until they the radiation has decayed with time. 

Fortunately the following advances permitted the development of noncompetitive methods for intact PTH (1) the determination of the amino acid sequence of human (h)PTH (2) increased understanding of the secretion, metabolism, clearance, and circulating forms of PTH and (3) the synthesis of hPTH, fragments of hPTH, and analogues of fragments for use as immunogens, tracers, and cahbrators, for characterization of antiserum and antibody specificity, and for affinity purification of antibodies. 

A binding of 13.2 0.8% ( = 3) was observed with 5 x 10 MCF-7 cells for 1 gg of the Lu-p-NCS-benzyl-DOTA-estradiol complex. With SepPak purification, the cell uptake improved to 17.1 1.6% (n = 3). No further increase in cell uptake was observed with HPLC purification. It was observed that the tracer uptake in the cells decreased to 8.3 2.0% (n = 3) with the addition of 100 gg of cold estradiol. Similar results were observed when these experiments were carried out on 24 well plates where cells were plated 1 d prior to the experiments. The decrease in cell uptake with the addition of estradiol indicated the specificity of the radiolabelled conjugate for the MCF-7 cell lines. Blank experiments were carried out with Lu labelled BFCA under similar experimental conditions. No retention of the activity in the cell pellet was observed, ruling out the possibility of carrier mediated uptake. 

Which mode or production is utilized dep ds on several factors. The length of time for production, separation and purification processes, the time from production to shipping, and the shipping time determine the half-life with which it is practical to work. The type of experiment and equipment available for handling and measurement determines the preferable decay type and intensity. All these factors must be considered in determining which nuclide is to be used. For example, if in an experiment requiring sodium tracer these aspects indicate the necessity of a time of at least several weeks, the use of... 

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