Free-solution separations

The supporting electrolyte for all free-solution separations reported here was a borate buffer (pH 8.1 or 8.26, 0.10 M or 0.20 M), prepared from reagent-grade sodium borate decahydrate and boric acid (J.T Baker). 

The capillary gel electrophoresis separation of a three-component, 3 P-labeled, nucleotide mixture is illustrated in Figure 10. It is interesting to note that the migration order of ATP and CTP is reversed with respect to the free solution separations presented in Figures 8 and 9. This is caused by the absence of electroosmotic flow in the gel-filled capillary. 

Fig. 23 Superimposed electropherograms of p(4-VPy) a-e in free solution. Separation conditions were as for Fig. 22...

FIGURE 2 Free solution separation of model peptides and tryptic digest of cytochrome c. Capillary 72 cm (50 cm to detector), 50- m id injection 1-sec vacuum analysis 200 nm, 30°C sample 100 g/mL each peptide in water, 100 g/mL tryptic digest buffer sodium phosphate (pH 2.5, 7.0), sodium acetate (pH 4.0), sodium tetraborate (pH 9.0), 20 mM in all cases. 

Transfer the quinoline chlorozincate to a beaker, add a small quantity of water, and then add 10% sodium hydroxide solution until the initial precipitate of zinc hydroxide completely redissolves, and the free quinoline separates. Transfer the mixture to a separating-funnel, wash out the beaker with ether, adding the washings also to the solution in the funnel, and then extract the quinoline twice with ether. Dry the united ethereal extracts by adding an ample quantity of powdered potassium hydroxide and... 

Into a 750 ml. round-bottomed flask furnished with a reflux condenser place a solution of 34 g. (18-5 ml.) of concentrated sulphuric acid in 100 ml, of water add 33 g. of di-n-butyl cyanamide and a few fragments of porous porcelain. Reflux gently for 6 hours. Cool the resulting homogeneous solution and pour in a cold solution of 52 g. of sodium hydroxide in 95 ml. of water down the side of the flask so that most of it settles at the bottom without mixing with the solution in the flask. Connect the flask with a condenser for downward distillation and shake it to mix the two layers the free amine separates. Heat the flask when the amine with some water distils continue the distillation until no amine separates from a test portion of the distillate. Estimate the weight of water in the distillate anp add about half this amount of potassium hydroxide in the form of sticks, so that it dissolves slowly. 

Bromo-4-aminotoluene, Suspend the hydrochloride in 400 ml, of water in a 1-Utre beaker equipped with a mechanical stirrer. Add a solution of 70 g. of sodium hydroxide in 350 ml. of water. The free base separates as a dark heavy oil. After cooUng to 15-20°, transfer the mixture to a separatory funnel and run off the crude 3-bromo-4-amino-toluene. This weighs 125 g. and can be used directly in the next step (3). 

The sohd can be contacted with the solvent in a number of different ways but traditionally that part of the solvent retained by the sohd is referred to as the underflow or holdup, whereas the sohd-free solute-laden solvent separated from the sohd after extraction is called the overflow. The holdup of bound hquor plays a vital role in the estimation of separation performance. In practice both static and dynamic holdup are measured in a process study, other parameters of importance being the relationship of holdup to drainage time and percolation rate. The results of such studies permit conclusions to be drawn about the feasibihty of extraction by percolation, the holdup of different bed heights of material prepared for extraction, and the relationship between solute content of the hquor and holdup. If the percolation rate is very low (in the case of oilseeds a minimum percolation rate of 3 x 10 m/s is normally required), extraction by immersion may be more effective. Percolation rate measurements and the methods of utilizing the data have been reported (8,9) these indicate that the effect of solute concentration on holdup plays an important part in determining the solute concentration in the hquor leaving the extractor. 

Ultrafiltration utilizes membrane filters with small pore sizes ranging from O.OlS t to in order to collect small particles, to separate small particle sizes, or to obtain particle-free solutions for a variety of applications. Membrane filters are characterized by a smallness and uniformity of pore size difficult to achieve with cellulosic filters. They are further characterized by thinness, strength, flexibility, low absorption and adsorption, and a flat surface texture. These properties are useful for a variety of analytical procedures. In the analytical laboratory, ultrafiltration is especially useful for gravimetric analysis, optical microscopy, and X-ray fluorescence studies. 

P. D. Grossman, J. C. Colburn, H. H. Lauer, R. G. Nielsen, R. M. Riggin, G. S. Sittampalam and E. C. Rickard, Application of free-solution capillary electrophoresis to the analytical scale separation of proteins and peptides . Anal. Chem. 61 1186-1194 (1989). 

When an electric field is imposed perpendicular to the flow, differential interaction between the various solutes and the electric field produce a lateral displacement of individual analyte streams between the two electrodes (Fig. 11-5). Thus, the separations are accomplished in free solution. Individual fractions are collected through an array of closely spaced ports evenly placed across the other end of the chamber. 

Furthermore, about 1920 the idea had become prevalent that many common crystals, such as rock salt, consisted of positive and negative ions in contact. It then became natural to suppose that, when this crystal dissolves in a liquid, the positive and negative ions go into solution separately. Previously it had been thought that, in each case when the crystal of an electrolyte dissolves in a solvent, neutral molecules first go into solution, and then a certain large fraction of the molecules are dissociated into ions. This equilibrium was expressed by means of a dissociation constant. Nowadays it is taken for granted that nearly all the common salts in aqueous solution are completely dissociated into ions. In those rare cases where a solute is not completely dissociated into ions, an equilibrium is sometimes expressed by means of an association constant that is to say, one may take as the starting point a completely dissociated electrolyte, and use this association constant to express the fact that a certain fraction of the ions are not free. This point of view leads directly to an emphasis on the existence of molecular ions in solution. When, for example, a solution contains Pb++ ions and Cl- ions, association would lead directly to the formation of molecular ions, with the equilibrium... 

The precipitated precursor can be dissolved and re-crystallized from fluorine-free solutions. This provides excellent conditions for deep purification of the material and reduction of problematic impurities such as titanium, fluorine, etc. Peroxometalates decompose at relatively low temperatures forming tantalum or niobium oxides containing small amount of absorbed water. The absorbed water separation is achieved by further thermal treatment - drying and calcination - of the product ... 

Tran, A. D., Park, S., Lisi, P. J., Huynh, O. T., Ryall, R. R., and Lane, P. A., Separation of carbohydrate-mediated microheterogeneity of recombinant human erythropoietin by free solution capillary electrophoresis. Effects of pH, buffer type and organic additives,. Ckromatogr., 542, 459, 1991. 

The feedstreams can consist of either neat reactants or their solutions. When the feedstreams consist of solutions, the reaction mixture is pumped into a polymerization vessel where the reaction that started in the mixing head proceeds to its conclusion. The polymer is subsequently precipitated from solution, separated, dried, and pelletized. Solvent-free mixtures of reactants are pumped directly to a mold where polymerization proceeds. In this case, other additives, such as, fillers or fire retardants, are co-mixed with the reactants in the mixing head. These additives are permanently incorporated into the finished molding. 

Jorgenson reported the use of glass capillaries for free solution electrophoresis 25 years ago (Jorgenson andLukacs, 1981,1983). Aplug of analyte was introduced into a buffer-filled capillary and separated at high electric fields. Capillaries of 75 im inner diameter were employed, and detection of labeled amino acids and peptides was based on fluorescence. 

Recently the wall-PRISM theory has been used to investigate the forces between hydrophobic surfaces immersed in polyelectrolyte solutions [98], Polyelectrolyte solutions display strong peaks at low wavevectors in the static structure factor, which is a manifestation of liquid-like order on long lengths-cales. Consequently, the force between surfaces confining polyelectrolyte solutions is an oscillatory function of their separation. The wall-PRISM theory predicts oscillatory forces in salt-free solutions with a period of oscillation that scales with concentration as p 1/3 and p 1/2 in dilute and semidilute solutions, respectively. This behavior is explained in terms of liquid-like ordering in the bulk solution which results in liquid-like layering when the solution is confined between surfaces. In the presence of added salt the theory predicts the possibility of a predominantly attractive force under some conditions. These predictions are in accord with available experiments [99,100]. 

In a radioimmunoassay procedure, 1 pmol of labelled analyte was added to the sample. Antiserum equivalent to 75 pmol of analyte was then added, and after incubation the bound and free fractions separated by precipitation with ammonium sulphate solution. The activity ratio, bound free, was measured to be 2.88. Calculate the number of moles of analyte in the original sample. 

From a plot of the internalisation flux against the metal concentration in the bulk solution, it is possible to obtain a value of the Michaelis-Menten constant, Am and a maximum value of the internalisation flux, /max (equation (35)). Under the assumption that kd kml for a nonlimiting diffusive flux, the apparent stability constant for the adsorption at sensitive sites, As, can be calculated from the inverse of the Michaelis-Menten constant (i.e. A 1 = As = kf /kd). The use of thermodynamic constants from flux measurements can be problematic due to both practical and theoretical (see Chapter 4) limitations, including a bias in the values due to nonequilibrium conditions, difficulties in separating bound from free solute or the use of incorrect model assumptions [187,188],... 

Though electrophoretic separations were historically first studied in free solutions, more recent developments have extended its application to solid supports, including polyacrylamide, agarose, and starch gels. The purpose of a solid support is to suppress convection current and diffusion so that sharp separations may be retained. In addition, support gels of controlled pore sizes can serve as size-selective molecular sieves to enhance separation - smaller molecules experience less frictional resistance and move faster, while larger molecules move slower. Therefore, separation can be achieved based on molecular size. 

Gel electrophoresis provides a simple method for separating complex protein mixtures. Because proteins are visualized using stains that may not be linearly incorporated in the gel, the intensity of the stained bands may be poorly correlated with the amount of protein. For this reason, gel electrophoresis is at best a semiquantitative technique capable of generating relative purity results. In CE, separations are commonly performed in free solution, i.e., in the absence of any support such as gel matrices. This allows the replacement of the capillary s content in between analyses and therefore the automation of the process. The use of UV-transparent fused-silica capillaries enables direct on-line optical detection of focused protein zones, eliminating the requirement for sample staining. The detection systems available to CE provide true quantitative capabilities. 

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