Mobile phase organic solvent gradients

Once separation conditions are selected, column, mobile phase organic solvent, and buffer are known. However, column dimension, flow rate, column temperature, mobile phase organic solvent gradient range and gradient time, and buffer pH need to be optimized for better resolution, robustness, and shorter run time. If necessary, buffer concentration needs to be optimized or triethylamine (TEA) needs to be added for better resolution or tailing factor. 

By appropriate choice of the type (or combination) of the organic solvent(s), selective polar dipole-dipole, proton-donor, or proton-acceptor interactions can be either enhanced or suppressed and the selectivity of separation adjusted [42]. Over a limited concentration range of methanol-water and acetonitrile-water mobile phases useful for gradient elution, semiempirical retention equation (Equation 5.7), originally introduced in thin-layer chromatography by Soczewinski and Wachtmeister [43], is used most frequently as the basis for calculations of gradient-elution data [4-11,29,30] ... 

RPC with mobile phases containing relatively high concentrations of salts (0.1-0.5M) is occasionally employed for separations of organic acids or bases [67-69], Eqnation 5.7 can often describe the effect of the concentration of organic solvents on the retention in these phase systems, so that Eqnations 5.8 and 5.9 can be principally used for calculation of the retention data in the LC with organic solvent gradients. 

The final approach to the reduction of eluent consumption is the optimal recycling of solvents. Indeed, preparative and industrial chromatography can be designed as a unit operation that includes solvent recycling dry feed mixture is injected while dry separated compounds are recovered. Many techniques can be applied depending on the situation in isocratic (that is with a constant mobile phase composition) or gradient conditions, and with organic and/or supercritical eluents. 

The concentration of the organic solvent is lower in the initial mobile phase (mobile phase A) than it is in the final mobile phase (mobile phase B). The gradient then, regardless of the absolute change in percent organic modifier, always proceeds from a condition of... 

Reverse phase chromatography is finding increasing use in modern LC. For example, steroids (42) and fat soluble vitamins (43) are appropriately separated by this mode. Reverse phase with a chemically bonded stationary phase is popular because mobile phase conditions can be quickly found which produce reasonable retention. (In reverse phase LC the mobile phase is typically a water-organic solvent mixture.) Rapid solvent changeover also allows easy operation in gradient elution. Many examples of reverse phase separations can be found in the literature of the various instrument companies. 

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