Column metal contamination

Heavy metal contamination of pH buffers can be removed by passage of the solutions through a Chelex X-100 column. For example when a solution of 0.02M HEPES [4-(2-HydroxyEthyl)Piperazine-l-Ethanesulfonic acid] containing 0.2M KCl (IL, pH 7.5) alone or with calmodulin, is passed through a column of Chelex X-100 (60g) in the K" " form, the level of Ca ions falls to less than 2 x 10" M as shown by atomic absorption spectroscopy. Such solutions should be stored in polyethylene containers that have been washed with boiling deionised water (5min) and rinsed several times with deionised water. TES [, N,N, -Tetraethylsulfamide] and TRIS [Tris-(hydroxymethyl)aminomethane] have been similarly decontaminated from metal ions. 

Because of the complex nature of most biological samples, a single fractionation technique may not be adequate for the separation of the wide range of molecules present. Better resolution of some molecules is obtainal when properties other than differences in size are exploited. These include differences in ionic characteristics, affinity for other molecules and hydrophobicity. In separations that involve any one or more of these properties, the sample constituents interact with the column material and are then eluted with a suitable eluant. As a consequence of this interaction, and the use of eluants, whose properties may not closely resemble those of the medium found in vivo, the metal may dissociate from the ligand. In addition, as the complexity of the sample increases it is difficult to predict the behaviour of the various constituents. Undesirable effects leading to irreversible interaction between some molecules in the sample and the column packing material, degradation and decomposition of some constituents may result. Furthermore, it may be difficult to rid the column of certain trace metal contamination. 

The reactor effluent contains about 25% formaldehyde, which is absorbed with the excess methanol and piped to the make tank. The latter feeds the methanol column for separation of recycle methanol overhead, the bottom stream containing the formaldehyde and a few percent methanol. The water intake adjusts the formaldehyde to 37% strength (marketed as formalin). The catalyst is easily poisoned so stainless-steel equipment must be used to protect the catalyst from metal contamination. 

A small flow of 2M NaN03 was routinely introduced to the bottom of the WE-1 Column to scrub aluminum, iron, and other metallic contaminants from the DBBP phase. Even so, americium in the SIP stream is extensively contaminated with plutonium, aluminum, iron and other metallic impurities. Ion exchange procedures, both simple load-elute and chromatographic, are used to yield highly purified 2lflAm. 

Bisdom E. B. A., Boekestein A., Curmi P., Legas P., Letsch A. C., Loch J. P. G., Nauta R., and Wells C. B. (1983) Submicroscopy and chemistry of heavy metal contaminated precipitates from column experiments simulating conditions in a soil beneath a landfill. Geoderma 30, 1-20. 

The impurities from accompanying trace levels of metal contaminants in the radionuclide solution, mainly iron and zinc, are critical for the labelling yields. If the level of either metal contaminant exceeds 1 pg/Ci, the labelling yields will be adversely affected. With reputed commercial suppliers, specific activities of 1 mCi/pg can be routinely obtained. A value lower than this reference specific activity can be indicative of the presence of the above metal impurities. The level of impurities can be reduced by passing the LuCl, or °YCl3 solutions through a cation exchange column (such as Chelex) that retains the other metal contaminants and effects purification. 

If ferrous ions are present during assays of DNA and RNA polymerases in which radioactive newly synthesized polynucleotides are precipitated on to filter paper discs in trichloroacetic acid, spuriously high results are obtained, apparently due to acid-insoluble complexes of Fe with substrate nucleotides precipitating on the paper. Clearly ferrous ions should be avoided in assays of this type. The complex Al. ATP is a potent inhibitor of hexokinase, and neutron activation analysis of many commercial samples of ATP has shown that Al is ubiquitous, and the most common metal contaminant It is best removed by passing the ATP preparation over a cellulose polyphosphate column at pH 5. Chelates of ATP with divalent metal ions have been separated from non-chelated ATP using reverse-phase h.p.l.c. ... 

First, any residual metal contamination from the buffer and from other sources is concentrated on the column. As a result ligands in the sample that form thermodynamically stable complexes with the metals will bind to them. It is also possible that non-specific effects such as local changes in ionic strength may lead to the elution of some of the metals with the sample. In either case new species are introduced into the fractions. Furthermore, the amount of metal associated with specific ligands may be over-estimated. Experiments by Woittiez (1984) have shown that zinc, copper, manganese and vanadium contamination on a column eluate with the protein fractions. This will doubtless lead to the over-estimation of the protein-bound fraction. 

However, one has to be careful with tests for metal cootamisatioa of the silica surface. HPLC systems are usually made of steel, as is the column hardware, including the frits. All steel will slowly bleeo metals, especially iron, which accumulates on the surface of the packing. Thus the test for metal contamination is a test of the status of the contamination of a column rather than a test of a permanent property of the packing. The test is meaningful only, when carried out using an HPLC system with a metal-free fluid path and a nonmetallic column. 

Transition-metal contamination of ion exchangers can be removed by washing the column with an EDTA-containing eluent. 

DNA and RNA chromatography require that the instrument, column and eluent be completely free of metal contamination. The effect, source, and control of metal contamination wiU be discussed. A high-quality oven must be used. The instrument normally includes the option of collecting the nucleic acid in a fragment collector for further research and processing. 

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