Stepwise elution fractionation procedure

An affinity sorbent based on WPA-PG carrying immobilized human IgG was applied to the isolation of the first component of the complement (Cl) from human serum and for its separation into subcomponents Clr, Cls and Clq by the one-step procedure [126,127]. Cl was quantitatively bound to the sorbent at 0 °C. The activities of subcomponents Clq and Clr2r2 in the unbound part of the serum were found to be 0.8% and 3.3% of the initial activities in serum. This fraction, therefore, could be used as a R1 reagent for determining the hemolytic activity of Cl. Apparently, the neighboring macromolecules of immobilized IgG resemble to some extent an immune complex, whereas Cl formation is facilitated due to the mobility of polymer chains with the attached IgG macromolecules (Cl is usually dissociated in serum by 30%). After activation of bound Cl by heating (30 °C, 40 min) the activated subcomponent Clr is eluted from the sorbent. Stepwise elution with 0.05 mol/1 EDTA at pH 7.4 or with 0.05 mol/1 EDTA + 1 mol/1 NaCl at pH 8.5 results in a selective and quantitative elution of the activated subcomponent Cls and subcomponent Clq. 

Samples of branched polyethylene were investigated by the combination of crystal-lizability fractionation and fractionation by molar mass. The first step was the precipitation of the polymer sample onto the glass-beads in a column (150 x 8 mm) used for the fractionation by crystallinity. The precipitation was performed by cooling a solution of 10 g/1 polyethylene in o-dichlorobenzene from 140 to 40 °C within 60 min. Subsequently, the polymer was extracted from the column by the same solvent at stepwise increased temperature. In an example given, the first fraction was eluted at 40 °C and additional 17 fractions at temperatures each raised by 2-10 K. The finer steps were employed in the middle of the fractionation procedure the last one reached even from 110 to 140 °C. About 10 min equilibration time proved to be adequate. The fractions were analyzed subsequently by SEC on a polystyrene-gel column. The whole process was automated 128). 

The functionality distribution of HTPB 134> can be obtained using the double detection procedure after derivatization of the OH groups with phenyl isocyanate. However, a true fractionation can been obtained by stepwise elution from silica gel n8,120) and, coupled with GPC, it enables establishment of the relationship between functionality and molecular weight distributions. Precipitation fractionation can also be used... 

There are three principal elution procedures isocratic (from the Greek isochros, meaning equal strength) stepwise (or fractional) and gradient. 

Once the sample has been loaded onto the column, there are several different techniques that may be used to elute its components. In a simple elution experiment, a single solvent is passed through the column during the entire course of the separation. This procedure works well for the separation of mixtures containing only two or three compounds having similar polarities. However, the more common chromatographic procedure is stepwise or fractional elution. In this technique, a series of increasingly more polar solvents is used to elute the mixture from... 

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