Eluent salt concentrations

In HIC, the hydrophobic interactions are relatively weak, often driven by salts in moderate concentration (I to 2 M), and depend primarily on the exposed residues on or near the protein surface preservation of the native, biologically active state of the protein is an important feature of HIC. Elution can be achieved differentially by decreasing salt concentration or increasing the concentration of polarity perturbants (e.g., ethylene glycol) in the eluent. 

For some nonionic, nonpolar polymers, such as polyethylene glycols, normal chromatograms can be obtained by using distilled water. Some more polar nonionic polymers exhibit abnormal peak shapes or minor peaks near the void volume when eluted with distilled water due to ionic interactions between the sample and the charged groups on the resin surface. To eliminate ionic interactions, a neutral salt, such as sodium nitrate or sodium sulfate, is added to the aqueous eluent. Generally, a salt concentration of 0.1-0.5 M is sufficient to overcome undesired ionic interactions. 

Mass spectrometers that use electrospray ionization (ESI) do not function well if the eluent contains low volatility salts. This is a major concern when an ion-exchange column is used as a first-dimension column and the salt concentration is used to modulate the retention in this column. In this case, another valve can be connected between the second-dimension column and the detector so that any salt from the second-dimension elution process that is either unretained or weakly retained can be diverted prior to feeding zones to the mass spectrometer. 

System B is the most widely used for CL detection after an HPLC separation. In this system, two pumps are required for delivering the reagent solutions in the following cases (1) the solutions for CL reaction are first combined and then mixed with an eluent (2) CL reaction conditions (e.g., pH, water and organic solvent contents, and salt concentration) need to be optimized before mixing with the CL reagent. 

The ESI-MS is nowadays the most commonly used LC-MS coupling device for the analysis of LAS and SPC. But also for the analysis of these anionic analytes the most serious drawbacks of this ionisation technique are matrix effects. A crucial role is played by mobile phases containing relatively high salt concentrations or ion-pair reagents that lead to signal instability or even, in some cases, to plugging of the orifice plate. For successful removal of alkaline salts, a suppressor was incorporated between the LC column and the mass spectrometer [24]. With this set-up it was possible to reduce a spiked sodium concentration of 15 mg L-1 in the HPLC eluent before the suppressor by more than 99.8% at the entry into the MS. 

The mixture could be eluted by decreasing the pH of the eluent. However, it is usually easier to elute by increased salt concentration since this is simpler to control. 

Because of the cobalt(II) contamination, the perchlorate salt is synthesized and then converted to other salts. Cobalt(II) chloride 6-hydrate (1.0 g, 4 mmole) and free [15]aneN4 (05 g, 4.2 mmole) are warmed in 100 mL of methanol for 15 minutes. Concentrated hydrochloric add is added dropwise (1 mL) and air is bubbled through the solution for at least 2 hours. Several drops of perchloric acid are added to precipitate the products as the perchlorates. The yield is 72%. The product is brown and is about 90% pure isomer I. The brown perchlorate salt is dissolved in acetonitrile and passed through a Dowex 1X8,200-400 mesh anion exchange column (Cl form) at a rate of 10 sec/drop. The eluent is concentrated by rotary evaporation. Some green solid (isomer II) precipitates. This is filtered and the volume of the filtrate is further reduced to precipitate the tan isomer (I) as the chloride salt. Yield 60% Anal. Calcd. for CoC2r H26N4a3 C, 34.80 H, 650 N, 14.76. Found C, 34.35 H, 7.09 N, 14.50. 

An increase in salt concentration in the bulk mobile phase provides those counterions which are able to reduce, according to the Gouy-Chapman electrostatic theory, the electrostatic stationary-phase surface potential. Hence, the adsorption of the HR may increase, even if its concentration in the eluent is the same, because of lower electrostatic self -repulsion. However,... 

---