Mobile phases salts

The negative charge of silanols can be minimized by utilizing competing ions, such as salts, amines, or zwitterions. The tailing peaks frequently observed with fused silica capillaries can be sharpened by increasing mobile phase salt concentration to 100 mM or more. However, the limitation of this approach is that joule heating increases in direct proportion to the ionic... 

In contrast to high molecular weight polyelectrolyte displacers, the efficacy of low molecular weight displacers is dependent on both mobile phase salt and displacer concentrations. This sensitivity to the operating conditions opens up the possibility of carrying out selective displacement where the products of interest can be selectively displaced, while the low affinity impurities can be desorbed in the induced salt gradient ahead of the displacement train and the high-affinity impurities either retained or desorbed in the displacer zone.43 A... 

The steric mass action (SMA) model has been shown to successfully predict nonlinear, multicomponent behavior in ion-exchange systems over a range of mobile phase salt concentrations.71-75 It has also been widely employed as a methods development tool for displacement separations.42,45,50 In this section, we will describe several graphical techniques derived this theory which can facilitate methods development in ion-exchange displacement systems. 

Isocratic elution under linear conditions at various mobile phase salt concentrations can be employed to determine v and K26 by the following expression ... 

The parameter cr can be determined by nonlinear frontal experiments).26 Once the SMA parameters have been obtained, they can be employed to predict the nonlinear behavior of solutes under any mobile phase salt concentration. 

Tugcu, N., Song, M., Breneman, C.M., Sukumar, N., Bennett, K.P. and Cramer, S.M. (2003) Prediction of the effect of mobile-phase salt type on protein retention and selectivity in anion exchange systems. Anal. Chem., 75, 3563-3572. 

Gradual desorption of the immobilised analytes is achieved either by a continuous increase in ionic strength or a change in pH of the mobile phase. Salt gradients,... 

Retention in ion exchange involves a competition between solute and mobile-phase ions for ion-exchange sites on the stationary phase. This leads to the following expresaon for Ac versus mobile-phase salt concentration c 20,21) ... 

It is established in ion-pair chromatography that salts always reduce the ion-pair retention factors by shielding ion-ion interactions. The plot of the retention factor, k, of a ionic solute versus the inverse of the mobile phase ionic strength, I , is linear with a positive slope [9]. Often, an increase in ionic strength decreases the retention factor and reduces the efficiency of the system. From a practical point of view, the mobile phase salt concentration (ion-pairing agent, solute and buffer) should be maintained below the 0.02 M limit [10]. 

The distinction between the elution of 22 ia+ and the apparent Na" " peak was further discussed by Ujimoto (18) in reply to de Ligny (19). Charged gels act as ion-exchange resins, binding their counterion. When Na is injected onto Sephadex for example, it displaces eluent cations previously bound to the gel. If the mobile phase salt also contains Na+, then "the ions of the sample cannot be differentiated from the corresponding ions of the eluent" (18). 

Composition of mobile phase Salt concentration (mol/dm ) (Ri X 100) values ... 

Reversed-phase chromatography is widely used as an analytical tool for protein chromatography, but it is not as commonly found on a process scale for protein purification because the solvents which make up the mobile phase, ie, acetonitrile, isopropanol, methanol, and ethanol, reversibly or irreversibly denature proteins. Hydrophobic interaction chromatography appears to be the least common process chromatography tool, possibly owing to the relatively high costs of the salts used to make up the mobile phases. 

When the mobile phase modulator is a ddiite solute in a solvent, as when the modulator is a salt,

[Pg.1536]

In ion-exchange chromatography (lEC), the mobile phase modulator is typically a salt in aqueous solution, and the stationary phase is an ion-exchanger. For ddnte conditions, the solute retention faclor is commonly found to be a power-law function of the salt uormahty [cf. Eq. (16-27) for ion-exchange equilibrium]. 

Peroxodisulphate salts in air Lab method using mobile phase ion chromatography 79... 

Organic acids yield lemon-yellow zones on a blue background [1]. Halide ions migrate as ammonium salts in ammoniacal mobile phases and are also colored yellow. The colors fade rapidly in the air. This can be delayed for some days by covering the chromatogram with a glass plate. 

Cationic samples can be adsorbed on the resin by electrostatic interaction. If the polymer is strongly cationic, a fairly high salt concentration is required to prevent ionic interactions. Figure 4.18 demonstrates the effect of increasing sodium nitrate concentration on peak shapes for a cationic polymer, DEAE-dextran. A mobile phase of 0.5 M acetic acid with 0.3 M Na2S04 can also be used. 

If the column is contaminated with basic compounds, clean it with a concentrated salt in the normal mobile phase, e.g., 0.5-1.0 M K2SO4. Avoid the use of halides, as they will corrode stainless steel over time. Buffered solutions at low pH (2-3) or high pH (11-12) can also be used. 

Controlling for these forces requires variation in the amount of salt, organic solvent, and the pFI of the mobile phase. It is impractical to perform such experiments with 50 mM formic acid an alternative additive must be used that maintains its chaotropic properties independent of salt content or pFI. Fortunately, mobile phases containing 50 mM hexafluoro-2-propanol (HFIP) afford a fractionation range comparable to that of the formic acid (Fig. 8.6), permitting the effects of these variables to be studied systematically. 

Electrostatic effects have long been recognized in commercial HPLC columns for SEC of proteins (15,21,22). The usual remedy is to add 100 mM salt to the mobile phase. This works here too the Lys and Asp peaks collapse into the Gly peak with 100 mM salt (Eig. 8.8). High concentrations of sodium sulfate were added to determine the role played in SEC by hydrophobic interactions (sodium sulfate, a structure-forming salt, strengthens such interactions). Sodium sulfate increased the retention only of the most hydrophobic amino acids to any extent, and then only when the concentration approached 1 M. Clearly, hydrophobic interaction cannot account for the elution order of amino acids on PolyHEA. 

FIGURE 8.8 Effect of salt on retention of amino acids. Column and flow rate Same as Fig. 8.1. Mobile phase 10 mM potassium phosphate + sodium sulfate (as noted), pH 3.0, with 50 m/VI HFIP. 

Steven Carr (SmithKline Beecham) has used microbore columns to desalt proteins prior to ES-MS (32). The pore diameter of PolyHEA used (usually 200 A) was selected so that all proteins of interest would elute at Vo with 50 mM formic acid. Only the Vo peak was allowed to flow into the ES-MS nebularizer the rest of the SEC effluent (including the salts) was diverted to waste by opening a microdumper valve between the column and the nebularizer. The properties of the mobile phase were quite compatible with ES-MS analysis. 

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