Packed columns composition gradients

Column pressure usually has little effect on enantioselectivity in SFC. However, pressure affects the density of the mobile phase and thus retention factor [44]. Therefore, similar to a modifier gradient, pressure or density programming can be used in fast separation of complex samples [106]. Later et al. [51] used density/temperature programming in capillary SFC. Berger and Deye [107] demonstrated that, in packed column SFC, the effect of modifier on retention was more significant than that of pressure. They also showed that the enhanced solvent strength of polar solvent-modified fluid was nof due fo an increase in densify, caused by fhe addition of fhe liquid phase modifier, buf mainly due fo fhe change in composition. 

Figure 7.8. Separation of Triton X-114 by SFC using programmed elution on a 10 cm x 2 mm I.D. Nucleosil Cig column, 3 p,m packing, at 170°C with UV detection at 278 nm. Separation on the left isobaric conditions at 210 bar with a mobile phase of carbon dioxide and methanol (2 + 0.125) ml/min. Center separation composition gradient from 0.025 to 0.4 ml/min methanol over 8 min with other conditions as above. Separation on the right pressure program from 130 to 375 bar over 8 min with the same mobile phase used for the isobaric separation. (From ref. [137] Copyright Preston Publications, Inc.)...

In column fractionation (157,158) the polymer is precipitated onto an inert support, which is placed at the top of a packed column (159). A solvent mixture of increasing solvent power is pumped through the column a temperature gradient is often maintained. This is known as Baker-Williams fractionation (160). This technique is applicable to all amorphous homopolymers and crystalline ho-mopol5miers above the melting point. For copol5miers and more complex compositions, the same technique may be employed, but the analysis is considerably more difficult. 

The setnp for polymer HPLC is qnite similar to SEC systems with a few modifications. Liqnid Adsorption Chromatography (LAC) requires the adsorption and desorption on a stationary phase. Therefore, in most cases isocratic separation is not sufficient. Gradients with respect to pH valne, ionic strength, eluent composition, or temperature are applied. The most common approach is to use eluent composition gradients. In contrast to SEC where polymeric phases dominate, silica-based column packings are the most important stationary phase. Both normal phase and reversed phase separations have been described. A summary of different applications in copolymer separation is offered by Pasch [27]. Detectors used in gradient LAC are mainly UV/DAD detectors and ELSD. 

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. 

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