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use. Rapid Separations Employing Gradient Elution Rapid LC separations are relatively easy to accomplish with isocratic development assuming low dispersion instrumentation with a fast response is available. However, if a sample mixture contains components that extend over a wide polarity or molecular weight range then gradient elution development will be necessary and fast gradients are almost impossible to form with conventional LC solvent programmers. As a consequence, for high speed gradient separations, a unique procedure must be used in conjunction with specially designed apparatus. The solution to the problem of fast gradient generation is to employ a preformed gradient a concept that was first introduced by Snyder and Saunders (13) as long ago as 1969. A diagram of a gradient preformer is shown in figure 27. In the particular apparatus that was used to provide the fast analyses that are described below the required gradient was formed in a column 25 cm long, 4.6 mm

to provide a solvent concentration profile is formed over a period of time and pumped into the gradient storage column. During the process of loading the gradient into the storage column, the solvent content of the storage vessel is passed to waste. When the complete solvent program is contained in the storage column, the flow is arrested. The sample is then charged into the sample loop (an internal sample valve loop should be used). The loop is then placed in line with the column and the gradient is discharged at full flow rate through the sample loop and column. An example of the rapid separation of a thirteen component mixture in just over 20 seconds is shown in figure 28. J. Chromatogr.,253(1982)159 Column Length 2.5 cm, column I.D. 2.6 mm, packing, C18 reversed phase, particle size 3 mm, solvent program 25% v/v acetonitrile in water to 100 % acetonitrile, flow rate 5 ml/min. Figure 28. The fast Separation of a Wide-Polarity Range Mixture by Preformed Gradient

Although the actual elution took only 22 seconds, due to the time required to form the gradient and regenerate the column the total gradient cycle was 5-6 minutes. To fully utilize the speed of the system a number of gradient storage columns would be necessary, that could be operated in parallel, if the gradient analysis was to be repeated continuously. The quantitative repeatability of the system was tested with 8 replicate analyses of the mixture. The results obtained are shown in Table 6. It is seen that despite the complexity of the analytical procedure, and the need for

nbsp; Figure 44  The Separation of Blood Liquids Employing Incremental Gradient Elution and Monitored by the Modified Moving Wire Detector However, due to the limited number of compounds that were tested this relationship should be assumed only with caution. A chromatogram of blood lipids obtained by incremental gradient elution and monitored by the modified detector is shown in figure 44. As incremental gradient elution involves a program of 12 solvents ranging from hydrocarbons, chlorinated hydrocarbons, nitro-paraffins, esters, ketones and alcohols. This

of the unique characteristics of mobile phases consisting of methanol water mixtures when used in reversed phase LC. From figure 47 it is seen that when the original mixture contains 50%v/v of methanol there is little free methanol available in the mobile phase to elute the solutes as it is mostly associated with water. Subsequently, however, the amount of methanol unassociated with water increases rapidly in the solvent mixture and this rapid increase must be accommodated by the use of a convexgradient profile when employing gradient elution. The convex gradient will compensate for the strongly concave form of the unassociated methanol concentration profile shown in figure 47 which will be the strongest eluting component of the mobile phase. The strong association of methanol with water could also account for the fact that proteins can tolerate a significant amount of methanol in the mobile phase before they become denatured. It is clear that this is because there is virtually no

and the reference electrode (which compensates for any changes in the background conductivity of the mobile phase). The processes taking place at the electrode surface can be very complex; nevertheless, the dominant reaction can be broadly described as follows. At the actual electrode surface the reaction is extremely rapid and proceeds almost to completion. This results in the layer close to the electrode being virtually depleted of reactant. As a consequence, a concentration gradient is established between the electrode surface and the bulk of the solution. This concentration gradient causes solute to diffuse into the depleted zone at a rate proportional to the solute concentration in the bulk of the mobile phase. Thus, the current generated at the electrode surface will be determined by the rate at which the solute reaches the electrode and consequently, as the process is diffusion controlled, will depend on solute concentration and the magnitude of solute