Solvent Extraction and Precipitation

Phenolic extraction of cell lysates is one of the oldest techniques in DNA preparation. Examples of these have been presented in Chapters 6 and 7. Single cells in suspension are lysed with a detergent, and a proteinase enzyme is used to break down the protein molecules. Non-nucleic acid components are then extracted into an organic (phenol-chloroform) solvent, leaving nucleic acids in the aqueous layer. Two volumes of isopropanol are added to the isolated aqueous phase to precipitate the high-molecular-weight nucleic acids as a white mass. These are then treated with DNase-free ribonuclease (RNase) to remove the RNA. This is followed by a second treatment with proteinase, phenol extraction, and isopropanol precipitation. After precipitation, the DNA is separated from the isopropanol by 

A recent development is to combine filtration with solid-phase extraction separation. These filter modules contain a unique silica gel membrane that binds up to 20 pg of DNA in the presence of a high concentration of chao-tropic salt and allow eventual elution in a small volume of low-salt buffer. They also contain an asymmetric laminar membrane with a gradation of pore sizes for efficient removal of material precipitated in the lysate. Such membrane filters eliminate time-consuming phenol-chloroform extraction and alcohol precipitation. The impregnation of silica in the membrane matrix also prevents the problems associated with loose resins and slurries. High-purity plasmid DNA eluted from such modules is ready to use and often needs no further precipitation, concentration, or desalting. 

It is often desirable to go through a postlysis separation/concentration step prior to chromatography. Concentration methods involve the use of ammonium acetate and polyethylene glycol precipitation to further remove host proteins and small nucleic acids. These methods also reduce the volume of the sample (or the process streams) prior to chromatographic purification. The separation may also involve centrifugation and filtration to remove cell debris. 

Chromatography is relatively easy to optimize and scale up, and several plasmid properties, such as charge and size, can be exploited in the design of these separations. Typically, plasmid DNA of 3000-base pair (bp) size has an average length of 10,050 A (based on 3.35 A/bp). However, upon super-coiling, plasmids adopt a branched interwined shape and become more 

---

An overview is presented of plutonium process chemistry at Rocky Flats and of research in progress to improve plutonium processing operations or to develop new processes. Both pyrochemical and aqueous methods are used to process plutonium metal scrap, oxide, and other residues. The pyrochemical processes currently in production include electrorefining, fluorination, hydriding, molten salt extraction, calcination, and reduction operations. Aqueous processing and waste treatment methods involve nitric acid dissolution, ion exchange, solvent extraction, and precipitation techniques. 

Radiotracers are uniquely well suited to such studies. The sensitivity of detection means that only very small amounts of tracer need be added to follow the chemical pathway of the relevant species. Furthermore, it matters little what the physical or chemical state of the tracer is, for measurements may be made on liquids, solids or gases. Chromatography, solvent extraction and precipitation are amongst separation methods widely studied by means of radiotracers. In the individual separation steps the distribution of the species may be studied by simple radioactivity measurements, and subsequently the tracer will serve as a yield indicator for the overall procedure. 

Indium may be recovered from zinc ores by several patented processes. Usually it is recovered from residues obtained from zinc extraction. The residues, slags, fume, or dusts from zinc smelting or lead-zinc smelting are treated with a mineral acid. Other steps involved in recovery often vary, but mostly use solvent extraction and precipitation steps. In some processes, treatment with caustic soda yields indium hydroxide. The hydroxide is calcined to obtain oxide, which then is reduced with hydrogen at elevated temperatures to obtain the metal. Distillation or electrolysis are the final steps to... 

DNA purification Solvent extraction and precipitation, gel electrophoresis, chromatography size exclusion, ion exchange, solid-phase extraction, SPRI, affinity purification... 

In Part VI, Chapter 30 is now a general introduction to separations. It includes solvent extraction and precipitation methods, an introduction to chromatography, and a new section on solid-phase extraction. Chapter 31 contains new material on molecular mass spectrometry and gas chromatography/mass spectrometry. Chapter 32 includes new sections on affinity chromatography and chiral chromatography. A section on LC/MS has been added. A new Chapter 33, Miscellaneous Separation Methods, has been included. It introduces capillary electrophoresis and field-flow fractionation. 

Separation of a new element is a key problem. Separations involve methods such as volatilization, electrodeposition, ion-exchange, solvent extraction and precipitation/ adsorption. Separation relies on the unique chemistry of each element although not heavy elements, but useful as an illustration, 643oZn/6429Cu are separated by dissolution in dilute HN03 followed by selective electrodeposition of Cu (a very simple task, as the CuII/0 and ZnII/0 redox potentials differ by 1 V). 

Isolation of crude saponins. Although the saponins are the dominating constituents of the leaves of G. sylvestre they are present as a very complex mixture. As already mentioned several workers have tried to isolate the saponins by the usual solvent extraction and precipitation methods. A procedure for isolation of the gymnemic acids reported by... 

Numerous techniques have been reported (Kusaka and Meinke 1961). Fundamentally, all processes for sample preparation, pnrilication, and measnrement must be brief and highly effective. Sample pnrilication before production is helpful. Miniature ion exchange, solvent extraction, and precipitation systems have been developed for processing small volumes. Mechanical transfer from pnrilication to the counting system enhances prompt measnrement. Measnrement results must take into account the radioactive decay of the radionuclide during measnrement shown in Eq. (10.2). 

Separation of products from other components Ion exchange, solvent extraction, and precipitation... 

Hg was often separated by wet chemical procedures containing solvent extraction and precipitation steps in the form of Hgl2, Hg(SCN)2, HgS, or elementary Hg (Grimanis and Kanias 1982 Biso et al. 1983 Pietra et al. 1986). 

Traditionally, the recovery and purification of americium has utilized primarily aqueous ion-exchange, solvent extraction, and precipitation processes. Departures are represented by the pyrochemical process and distillation of americium metal in high vacuum used at the Rocky Flats plant (Colorado) [333]. 

---