Gradient elution in LC

The program starts and ends at the purge segment (P). The reason for this is related to the typical baseline observed in a gradient elution LC experiment (figure 6.6b). Unlike the situation in GC, the main cause of the blank signal in programmed solvent LC is formed 

A second factor that contributes to the baseline variation is the difference in the background signal (absorption fluorescence) between the two solvents. This effect causes the difference in the baseline level between the left and the centre in figure 6.6b. A more extensive discussion on baseline variations in programmed solvent LC can be found in ref. [607]. 

In RPLC retention varies exponentially with the composition of the mobile phase, i.e. approximately straight lines are obtained in a plot of In k vs. p (see section 3.2.2). If we look at the retention behaviour of each individual solute, then the optimum conditions (LSS gradient, see section 5.4) correspond to a linear gradient (figure 6.2b). Linear gradients will indeed be optimal when acetonitrile-water mixtures are used as the mobile 

In eqns.(6.1) and (6.1a) a, b9 c and d are constants. Because p should increase with time, a is a positive constant in both equations and hence a concave gradient (figure 6.2d) is optimal for LSC. 

It will be clear from figure 6.7 that the nature of the mobile phase (compare figures 6.7a and 6.7b) and the stationary phase (compare figure 6.7c with figures 6.7a and 6.7b) have a great effect on the character of the retention vs. composition plots and hence on the shape of the required (optimum) gradient. It will also be clear that, unlike the situation in GC, the selectivity may be greatly influenced by variations in the mobile phase. 

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Peak capacity can be very effectively improved by using temperature programming in GC or gradient elution in LC. However, if the mixture is very complex with a large number of individual solutes, then the same problem will often arise even under programming conditions. These difficulties arise as a direct result of the limited peak capacity of the column. It follows that it would be useful to derive an equation that... 

Bulk property detectors function by measuring some bulk physical property of the mobile phase, e.g., thermal conductivity or refractive index. As a bulk property is being measured, the detector responses are very susceptible to changes in the mobile phase composition or temperature these devices cannot be used for gradient elution in LC. They are also very sensitive to the operating conditions of the chromatograph (pressure, flow-rate) [31]. Detectors such as TCD, while approaching universality in detection, suffer from limited sensitivity and inability to characterise eluate species. 

Figure 6-16 Examples of isocratic and gradient elution in LC. LC, Liquid chromatography.

For complex mixtures, especially those analyzed with temperature programming (in GC) or gradient elution (in LC), it is impossible to select a single reference compound. The Kovats retention-index system [39], based on homologous series of reference compounds, provides an elegant solution in GC, one which has been widely accepted. In LC, the absence of suitable homologous series, and the fact that retention depends more than in the case of GC on the polarities of compounds and less on molecular weight makes the use of an index system impractical. 

GRADIENT ELUTION IN VARIOUS LC MODES 5.4.1 Reversed-Phase Chromatographv... 

A solvent system consisting of chloroform and methanol or hexane and isopropanol with or without addition of a small volume of water is commonly used for this mode to separate lipid classes. These solvent systems can be readily compiled with an ESI ion source. Unfortunately, due to the toxicity of chloroform, inclusion of this solvent in LC-MS is seldom. The use of inorganic salts is incompatible with MS, a small amount of organic salt or acid may be used as a modifier. Isocratic elution by using a mobile phase with a constant composition may be used for a certain lipid class, but gradient elution in which the polarity of the mobile phase is increased at a controlled rate affords greater versatility. 

For LC, temperature is not as important as in GC because volatility is not important. The columns are usually metal, and they are operated at or near ambient temperatures, so the temperature-controlled oven used for GC is unnecessary. An LC mobile phase is a solvent such as water, methanol, or acetonitrile, and, if only a single solvent is used for analysis, the chromatography is said to be isocratic. Alternatively, mixtures of solvents can be employed. In fact, chromatography may start with one single solvent or mixture of solvents and gradually change to a different mix of solvents as analysis proceeds (gradient elution). 

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. 

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