Gradient Elution or Solvent Programming

Marcel Dekker, Inc. 270 Madison Avenue, New York, New York 10016 

The effect of solvent programming can be simulated by means of a computer in much the same way as temperature programming can be simulated. Reiterating equation (13) from chapter 4 

If the corrected retention volume in the pure strongly eluting solute is very small compared with the retention volume of the solute in the other pure solvent, i.e., V a V g which is very often the case in practical LC, then equation (10) 

In addition, under these conditions, the inverse will also apply, Le., 

---

Horizontal and descending TLC are other forms of chromatography which require special equipment. The advantages of these systems are not clear. They are usually more expensive than ascending equipment, but often have other features for stepwise gradient elution or continuous solvent programming. 

This equation, although originating from the plate theory, must again be considered as largely empirical when employed for TLC. This is because, in its derivation, the distribution coefficient of the solute between the two phases is considered constant throughout the development process. In practice, due to the nature of the development as already discussed for TLC, the distribution coefficient does not remain constant and, thus, the expression for column efficiency must be considered, at best, only approximate. The same errors would be involved if the equation was used to calculate the efficiency of a GC column when the solute was eluted by temperature programming or in LC where the solute was eluted by gradient elution. If the solute could be eluted by a pure solvent such as n-heptane on a plate that had been presaturated with the solvent vapor, then the distribution coefficient would remain sensibly constant over the development process. Under such circumstances the efficiency value would be more accurate and more likely to represent a true plate efficiency. 

Solute property detectors, such as spectroscopic andj electrochemical detectors, respond to a physical or chemical] property characteristic of the solute which, ideally, is] independent of the mobile phase. Althou this criterion is rarely met in practice, the signal discrimination is usually sufficient to permit operation with solvent changes (e.g., flow programming, gradient elution, etc.) and to provide high sensitivity with aj wide linear response range. Table 5.4. Solute-specific detectors complement ulk property detectors as they provide high ... 

The choice of solvents for gradient elution is still somewhat empirical however, using the data from Table 3.5 narrows the choices. Modern HPLC instruments are equipped with solvent programming units that control gradient elution in a stepwise or continuous manner. 

Isocratie elution uses a solvent of conslanl composition throughout the analysis. Gradient elution is a modification of the technique, in which the solvent is a mixture of two solvents that differ in solvent strength. The composition or raliu of the iwo is changed during the analysis in accordance with a predetermined program. The change may be continuous, linear or nonlinear, stepwise, or a combination. 

The solvent is moved through the system by constant-flow or constant-pressure pumps which arc driven mechanically (screw-driven syringe or reciprocating) or by gas pressure with pneumatic amplifiers. For gradient elution Iwo pumps may be synchronised and programmed to provide a controlled, reproducible composition change. 

The adaptation of the ABTS" method as an on-hne test required that the chromogen radical should be stable for sufficient time in different solvents to permit the utilization of isocratic or gradient elution programs. The on-line reaction time between ABTS" and potential antioxidants was an additional potential limiting factor. 

Another significant aspect was the stability of the radical chromogen ABTS in different solvents, in isocratic or gradient elution programs. We found that in the mobile phases used in our determinations (saline solutions and mixtures of organic solvents in different proportions), the observed fall was less than 0.01 expressed as — AAbs730nm/min. This stability is high... 

---