Gradient mixing chamber

In high pressure mixing devices each solvent is piunped separately in the proportions required by the gradient into a mixing chamber before being delivered to the column. Solvent compressibility and thermodynamic volume changes on mixing may Influence the accuracy of the composition delivered to the column. 

Where binary, ternary or quaternary gradient elution (p. 91) is required, a microprocessor controlled low-pressure gradient former is the most suitable (Figure 4.31(c)). The solvents from separate reservoirs are fed to a mixing chamber via a multiport valve, the operation of which is preprogrammed via the microprocessor, and the mixed solvent is then pumped to the column. For the best reproducibility of solvent gradients small volume pumps (< 100 gl) are essential. 

Fig. 7. Semi-Preparative Anion Exchange Purification of a 16-mer Oligodeoxynucleotide on a CIM DEAE Disk Monolithic Column. Conditions Column 0.34 ml CIM DEAE Disk (3X12 mm ID) Instrumentation Gradient HPLC system with extra low dead volume mixing chamber Sample 16mer oligodeoxynucleotide from the reaction mixture - bold line, standards of 1,2,3,4,5,6,7,9,10,11,12,14,15,16mer- thin line Injection Volume 20 pL Mobile Phase Buffer A 20 mM Tris-HCl, pH 8.5 Buffer B Buffer A+ 1 M NaCl Gradient as shown in the Figure Flow Rate 4 ml/min Detection UV at 260 nm...

Pour heavy gradient solution into the chamber closest to the outlet (mixing chamber) of the gradient maker and add a small stir-bar. Use 9.3 ml per chamber for 1-mm-thick standard (14 x 16-cm) gels. Use 2.3 ml per chamber for 1-mm-thick mini-format (8... 

The aspect of minimization of all void volumes is particularly important on a micrometer length scale since diffusion then becomes a very fast and dominating mixing process. Any small void volumes will act as efficient mixing chambers . For a typical diffusion coefficient of a small ion in solution (D= 10 5 cm2/s), diffusion over a distance of 10 pm will take place in approximately 100 ms over a distance of 1 pm only 1 ms is required. Any concentration gradients present on this length scale will be very readily leveled out, i.e. void volumes will be filled. The virtues of microfabrication for the production of virtually dead volume free interconnections can hardly be overestimated. It... 

A solution of 30.0 g 1-methoxyadenosine 3, 5 -cyclic phosphate (81.5 mmole), 20.0 g NaHC03 (238 mmole), and 300 ml H20 was refluxed 45 mm. The pH of the solution was adjusted to 2.5 with Dowex 50x8 (H)+ while warm, and a water pump vacuum was applied to mixture to remove C02. The pH was readjusted to 9-10 with NaOH, and the resin was removed by filtration. The solution was passed onto a column containing 400 ml Dowex 1x2 (formate, 100-200 mesh), and the column was washed well with water. The column was eluted with a gradient of 4 L water in the mixing chamber and 4 L 4 N formic acid in the reservoir. The first major product, coming after about 2 L eluate, was 5-amino-N-methoxy-l-p-D-ribofuranosylimidazole-4-carboxamidine 3, 5 -cyclic phosphate, giving 5.4 g (19%) after evaporation of the solvent and trituration of the residue with ethanol (recrystallization from water). 

Nearly any type of continuous eluent gradient can be generated by connecting two or more chambers containing solutions of different properties to a common mixing chamber (Fig. 4). (See also the description of UV- and carbohydrate analyzers.)... 

The outlet from the mixing chamber of the gradient generator may be led into a device such as that of Caton and Goldstein (1971), ( 8.3.4.1), or by way of a manifold into the tubes individually. Mirault and Scherrer, on the assumption that the polymerisation velocity is proportional to the acrylamide concentration on the one hand, and that of TEMED and persulfate on the other, arranged that both the latter components were added at a concentration inversely proportional to that of the acrylamide (e.g. for 10 ml of 2% acrylamide 50 pi each of 10% TEMED and 10% persulfate were added, but for 10 ml of 6% acrylamide 16.7 p only of each). The gels were prepared at room temperature but the acrylamide... 

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