Elution conditions

The design procedure described above will, in theory, be applicable only to samples that are separated by isocratic development. Under gradient elution conditions the (k ) value of each solute is continually changing, together with the viscosity of the... 

Dias, N. C., Nawas, M. I., Poole, C. F. Evaluation of a reversed-phase column (Supelcosil LC-ABZ) under isocratic and gradient elution conditions for estimating octanol-water partition coeffidents. 

Load and elution conditions for experiments in Table 2 were as follows ... 

Hallgren, E., Prediction of protein retention at gradient elution conditions in ion-exchange chromatography, /. Chromatogr. A, 852, 351, 1999. 

In an off-line configuration, a complex peptide mixture from a proteomic sample is loaded onto a SCX column and fractions collected (Fig. 11.1). After the collection of fractions, they are then loaded into an autosampler and analyzed via the traditional RP/ MS/MS approach. Using this system, a variety of buffers and elution conditions may be used (Table 11.1). For example, one may use a volatile salt such as ammonium formate (Adkins et al., 2002 Blonder et al., 2004 Fujii et al., 2004 Yu et al., 2004 Qian et al., 2005a and b) or ammonium acetate (Cutillas et al., 2003 Coldham and Woodward, 2004), collect SCX fractions, lyophilize, resuspend in low acetonitrile and acid, and then directly analyze via RP/MS/MS. In most of the cases, when ammonium acetate or ammonium formate are used, a 20-minute wash period is used to remove the ammonium acetate or ammonium formate prior to the reversed-phase gradient (Table 11.1). However, because fractions are collected and can be buffer exchanged,... 

TABLE 12.2 Theoretical Peak Capacity, Orthogonality, and Practical Peak Capacity of Investigated 2DLC Setups. Second Dimension was in All Cases Carried Ont Using Cig Column and Typical LCMS Compatible Elution Conditions... 

In studies for the U.K. Food Standards Agency, Sidwell and Zondervan [10] also examined the determination of benzophenone and benzotriazole UV stabilisers by LC-MS. Specific detection parameters using the elution conditions given in Section 3.4 are given in Table 9. 

The iminobiotin-avidin interaction also can be utilized in the opposite approach. Immobilized iminobiotin affinity columns can be used to purify avidin- or streptavidin-containing complexes under mild elution conditions (Hofmann et al., 1980). 

Reverse-phase chromatography may also be used to separate proteins on the basis of differential hydrophobicity. This technique involves applying the protein sample to a highly hydrophobic column to which most proteins will bind. Elution is promoted by decreasing the polarity of the mobile phase. This is normally achieved by the introduction of an organic solvent. Elution conditions are harsh and generally result in denaturation of many proteins. 

Gradient elution is a procedure whereby the conditions under which the sample is eluted are progressively varied throughout the separation so as to speed up the process. This can be achieved by altering the composition of the mobile phase or increasing the temperature or flow rate. The effect is to elute components more rapidly in the latter stages and sharpen their elution profiles. Stepwise elution is a similar procedure in which elution conditions are changed at predetermined times rather than continuously. 

Elution step Specific elution conditions (extreme pH. high salinity, organic solvents, etc.) lo dissociate the complex are usually required. 

Gradient Elution Conditions in Multicomponent Preparative Liquid Chromatography. 

Figure 1 shows a schematic drawing of a CAC apparatus. The apparatus consists of two concentric cylinders standing one inside the other, forming an annulus into which the stationary phase is packed. This annular bed is slowly rotating about its vertical axis. Under isocratic elution conditions the feed mixture to be separated is introduced continuously at the top of the bed at a space that remains fixed in space while the rest of the annulus is flooded with elution buffer. As time progresses, helical component bands develop from the feed point, with... 

Proteins contain a variety of functional groups and interact with the stationary phase at a number of simultaneous sites on the protein molecule, each more or less affected by change in eluent, or mobile-phase, pH or ionic strength. The equilibrium constant for the dissociation of the adsorption complex thus contains a product of many eluent-sensitive concentration terms, and the equilibrium position is very sensitive to elution conditions. Under the elution conditions, some proteins in a mixture may be tightly bound by the stationary phase (t), oo) while others are unretained (t R 0). Differential migration (Section 19.2.1) is replaced by extreme retention values. 

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