Factors Affecting Column Efficiency

As briefly mentioned earlier, multistage separation processes involve not only equilibrium considerations, but kinetic factors as well. The efficiency of a chromatographic process is subject to a number of these factors, such as longitudinal diffusion (B), eddy diffusion (A), mass transfer in the immobile (Cj), and mobile (Cj ) phases. A useful relation, proposed by van Deemter and subsequently elaborated by many others, sums all of these factors 

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First, we will explore the three fundamental factors in HPLC retention, selectivity, and efficiency. These three factors ultimately control the separation (resolution) of the analyte(s). We will then discuss the van Deemter equation and demonstrate how the particle diameter of the packing material and flow rate affect column efficiencies. 

Lack of the proper amount of packing in a gas chromatographic column often is the source of poor separation. How can one tell when a column is properly packed The answer is twofold by column performance (efficiency, i.e., number of theoretical plates) and by the peak symmetry (has a Gaussian or normal distribution shape). Many factors affect column performance loosely packed columns generally are inefficient and are easily noticeable in glass columns. A column that is too... 

Among the factors that affect column efficiency and selectivity are the structure of the mesogen (in relation to the solutes of interest), the phase type(s) and degree of ordering, temperature, column loading and coverage, concentration (solubility) effects, and the surface structure and specialized treatment of the support. The column surface can dramatically affect the orientation of the liquid crystalline molecules (especially for monomeric mesogens) and thus alter separation efficiency. For polymeric liquid... 

Three separate factors affect resolution (1) a column selectivity factor that varies with a, (2) a capacity factor that varies with k (taken usually as fej). and (3) an efficiency factor that depends on the theoretical plate number. 

Accordingly, two major parameters affect the band profiles in nonlinear chromatography the column efficiency, and the amount of sample injected or loading factor. Parameter F (phase ratio) depends on the total porosity of the packing and cannot be changed in practice. 

Separation speed and ease of use seem to be the primary factors driving changes in HPLC instrumentation. Resolution efficiency and stationary phase stability, especially at high pH, are the primary factors affecting current changes in column technology. 

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. 

As with the volatility range, recovery generally is most effectively increased by raising the sample temperature. Additional factors affecting sensitivity include trapping and desorption efficiencies, column resolution, interferences, and detector sensitivity. For oils the lower limit of detection for the majority of the compounds listed in Tables I and II is on the order of 1 to 100 ppb. For oil samples, nonane, which is often added as an internal standard, is detectable to less than 5ppb. 

Calculate the IBW (4 a) using the tangent method as shown in Figure 4.19b. An IBW of -60 pL was found for this HPLC. Figure 4.20 shows a chart plotting column efficiencies of 150-mm long 3-pm columns of various inner diameters versus retention factors (k) on an HPLC system with a 60-pL IBW. Note that efficiency loss from instrumental dispersion can be severe for small inner diameter columns where peak volumes are smaller. Note that peaks of low k are affected more by system dispersion since peak volumes are proportional to k. 

Improving the column efficiency, i.e., increasing N, will make the peaks sharper and narrower and hence will reduce the overlap between peaks, but it will not affect the flmdamental basis of the separation. The relative retention tunes of a mixture of components will remain the same. This factor in the above expression involves the square root of which means that in reality, an increase iniVhas to be fairly large to make any significant difference. This sort of improvement in separation is seen most dramatically m the difference between classical gravity-fed, large particle columns with plate values of hun-... 

While A is a useful concept to compare the efficiency of separation among columns, or between laboratories, it is difficult to use to assess the factors that affect that efficiency. This is due to the fact that it refers to the behavior of a single component during the separation process, and is unsuited to describing the separation of two components or the resolving power of a capillary. A more useful parameter is the height equivalent of a theoretical plate (HETP). ... 

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