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

is employed, the supply from each reservoir may pass either to a pump or to a valved blending device. Solvent reservoirs are not usually thermostatted but, when necessary, the solvent can be brought to the column temperature by the use of an appropriate heat exchanger. The solvent containers are often situated in an enclosure that protects the user from toxic solvent vapors such as chloroform or aromatic hydrocarbons. Such enclosures also isolate the solvents from atmospheric moisture. The Gradient Programmer The High Pressure Programmer There are two basic types of solvent programmer. In the first, the solvent mixing occurs at high pressure and in the second the solvents are premixed at low pressure and then passed to the pump. The high pressure programmer is the simplest but most expensive as each solvent requires its own pump. Theoretically, there can be any number of solvents involved in a mobile phase program, however, most LC analyses require only two solvents,

Low Dispersion Gradient Elution Apparatus Gradient elution with high efficiency low dispersion columns, particularly small bore columns that operate at very low flow rates, present another instrumental problem for the designer of the modern chromatograph. Not only must the gradients be formed accurately and precisely, they must be also formed at very low flow rates and sometimes the total volume employed for the analysis will be less than 1 ml.  The apparatus can be basic and accommodate only to solvents

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

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

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