Gradient packing material

The narrow range of organic modifiers required to elute and desorb polypeptides from the reversed-phase column packing material accounts for the separation of polypeptides from a short C18 capillary column. Because polypeptide elution and separation depend on the accuracy of solvent composition in gradient nano LC, it is very important to use a system that can precisely control the LC modifier concentration even at low percents of organic modifiers in chromatographic elution compositions. 

Fast gradient separations can be achieved, at a cost of decreased peak capacity, by using short packed columns and simultaneously decreasing the particle size, dp, of the packing material to keep a constant ratio of the column length to the mean particle diameter, lidp, e.g., with a 3 cm column packed with a 3 [im material, or even a 1 cm, 1 pm column for very fast separation instead of a conventional 5 cm, 5 pm or a 10 cm, 10 pm column. At constant Vq/V , and Hdp ratios, the... 

Gradient elution requires packing materials that can withstand the accompanied changes in solvent composition. 

Fig. 29. Influence of column length on gradient elution of SAN copolymers through iso-octane/THF. Column packing material Polygosil 60-5 silica in both cases, do = 6 nm dP = 5 pm. Upper chromatogram column 55x4.6 mm, lower one column 150x4.6 mm. (The lower chromatogram is from the same run as the dashed line in Fig. 28.) Injection 60 pg in 30 pi THF [124]...

In LC there is a relatively small number of stationary phase packing materials available. Therefore, it is necessary to vary solvent polarities to separate materials with vastly different k values. Often this guidance comes from the tabulation of the physical properties of the solvents (Table 6-3). In addition to polarity of the final mobile phase, there are two additional major considerations when selecting solvent pairs for either isocratic or gradient... 

Separations were performed on a Shimadzu HPLC instrument. A 50 pL aliquot was injected (in water acetonitrile 98 2 v/v) via a Rheodyne injector (Cotati, CA) onto a Vydac analytical column (4.6 cm x 250 mm) containing C-18 packing material (300A, 5 pm). Separations were achieved using a mobile phase of A) 0.06% TEA and B) 0.052% TEA in CH3CN. A solvent gradient of 2% —> 37.5% B (0-60 minutes) 37.5% 75% B (60-90... 

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