Parameters affecting chromatographic retention

As shown by Eq. 1 the resolution of components in a Uquid chromatographic separation is dependent on (1) their relative retention on a particular chromatographic system, and (2) their peak widths. To optimize these parameters for maximum resolution, a clear understanding of their nature and the factors that affect them is necessary. Although the retention time of a component adequately describes the amount of time a particular solute takes to elute from a chromatographic system, a more usefid parameter describing chromatographic retention is the capacity factor k. This parameter is defined as the ratio of time spent by a solute in the stationary phase to the time it spends in the mobile phase. It can be calculated by Eq. 2, where Tr is... 

In GC there are four operational parameters for a given stationary phase L, length of the column, u, velocity of the mobile phase (which affects the theoretical efficiency N, see Section 1.6.1), T, temperature of the column and /3, phase ratio (see Section 2.6.4), which affects the retention factor k (Section 1.7.4). The operating conditions of the chromatograph allows modifications in terms of Tand u and therefore affects both the efficiency of the column and the retention factors. 

The solution could be a quantitative estimation of retention parameters, carried out by using objective procedures based on predictive models. The establishment in GCxGC of quantitative relationships between chromatographic retention and molecular structure, and the study of how changes in chromatographic conditions affect the retention parameters can be useful for several purposes ... 

Models that describe the relationship between GCxGC retention and structural properties should not be restricted to a fixed set of columns and chromatographic conditions. In GCxGC, any alteration in the operation parameters of the first column will cause a change in the elution conditions (flow, temperature) in the second affecting the retention in both and columns. For these reasons, practical objectives require that retention GCxGC behaviour can be predicted for different chromatographic conditions, as described in the next section. 

Moreover, the chromatographic retention and selectivity is always affected to a certain extent by the interplay of the molecular configuration of analyte and stationary phase. This so-called molecular or steric recognition is one of the main reasons for the excellent selectivities that can be achieved in liquid chromatography. These parameters form the basis of the following equation (Eq. 6) to describe the retention relative to that of a reference solute. Therefore, Eq. (6) does not include an intercept value, which usually reflects, among other things, the phase ratio of the column. 

The second aspect of optimization in programmed analysis involves adapting the selectivity by variation of secondary parameters. The various secondary parameters listed in table 3.10 may be used to vary the selectivity of a chromatographic system without affecting retention to a great extent (see the discussion in section 3.6.1). 

The objective of all chromatographic separation is resolution. This experiment illustrates resolution and the factors that affect it. As discussed in Chapters 1 and 3 resolution cannot occur if the components are not partially retained or slowed down (retarded) by the column. Therefore, before calculating resolution, it is important to use the results of the experiment to calculate the fundamental chromatographic parameters of retention, capacity factor, selectivity, and efficiency. 

Therefore, it is possible to increase throughput, and thus the speed of analysis without affecting the chromatographic performance. The advent of UPLC has demanded the development of a new instrumental system for LC, which can take advantage of the separation performance (by reducing dead volumes) and consistent with the pressures (8000-15,000 psi, compared with 2500 to 5000 psi in HPLC). Efficiency is proportional to column length and inversely proportional to the particle size [41], Smaller particles provide increased efficiency as well as the ability to work at increased linear velocity without a loss of efficiency, providing both resolution and speed. Efficiency is the primary separation parameter behind UPLC since it relies on the same selectivity and retentivity as HPLC. In the fundamental resolution (Rs) equation [38], resolution is proportional to the square root of N. 

---