Column length selectivity

If the separation is still unsuitable after optimizing the experimental conditions and column length, selectivity must be optimized further by changing the stationary phase, the type of column, or the mobile phase by changing it or adding a modifier. These choices were described earlier in this chapter, because the changes needed depend so heavily on the nature of the analytes and/or sample matrix, it is difficult if not impossible to provide concise, general recommendations. 

Recovery factor Reduced column length Reduced plate height Reduced velocity Relative retention ratio Retardation factor d Retention time Retention volume Selectivity coefficient Separation factor... 

The diameter of the column is selected from the volume of sample that is to be processed. As a rule of thumb the maximum productivity is obtained at a sample volume of 2-6% of the bed volume in preparative gel filtration on a 50-/rm chromatographic medium (Hagel et al., 1989). Thus, the required column diameter is calculated from the bed volume needed to cope with the sample volume and the column length needed to give the resolution desired. 

Generally, optimizing the selectivity by choosing a gel medium of suitable pore size and pore size distribution is the single most important parameter. Examples of the effect of pore size on the separation of a protein mixture are given in Fig. 2.15. The gain in selectivity may then be traded for speed and/ or sample load. However, if the selectivity is limited, other parameters such as eluent velocity, column length, and sample load need to be optimized to yield the separation required. 

The important parameters to consider are the selectivity (dKJdlogR), the ratio of pore volume, Vp, over void volume, Vq, the plate height, H, and the column length, L. The distribution coefficient, Kq, has a slight effect on resolution (with an optimum at Kp 0.3-0.5). In addition to this, extra column effects, such as sample volume, may also contribute to the resolution. 

Below the dotted line in Table I we list less fundamental differences between the two methods. Column lengths tend to be somewhat shorter in HPLC using small particle PB as a consequence of the high efficiencies that can be generated with the smaller particle sizes. For analytical scale HPLC, tube diameters of 3-4 mm are selected however, for preparative scale, tube diameters of 1 cm or above are not uncommon. 

Once the selectivity is optimized, a system optimization can be performed to Improve resolution or to minimize the separation time. Unlike selectivity optimization, system cqptimization is usually highly predictable, since only kinetic parameters are generally considered (see section 1.7). Typical experimental variables include column length, particle size, flow rate, instrument configuration, sample injection size, etc. Hany of these parameters can be. Interrelated mathematically and, therefore, computer simulation and e]q>ert systems have been successful in providing a structured approach to this problem (480,482,491-493). 

Some kinds of chromatography require relatively little optimization. In gel permeation chromatography, for example, once the pore size of the support and number of columns is selected, it is only rarely necessary to examine in depth factors such as solvent composition, temperature, and flow rate. Optimization of affinity chromatography is similarly straightforward. In RPLC or IEC, however, retention is a complex and sensitive function of mobile phase composition column type, efficiency, and length flow rate gradient rate and temperature. 

Trying to determine which column is ideal for a specific analysis can be difficult with over 1000 different columns on the market [74]. A proper choice implies a definition of parameters such as column material, stationary phase (polarity), i.d., film thickness and column length. Guides to column selection are available [74,75]. The most important consideration is the stationary phase. When selecting an i.d., sample concentration and instrumentation must be considered. If the concentration of the sample exceeds the column s capacity, then loss of resolution, poor reproducibility and peak distortion will result. Film thickness has a direct effect on retention and the elution temperature for each sample compound. Longer columns provide more resolving probe, increase analysis times and cost. 

FIGURE 9.1 Comparison of resolution and selectivity with increased speed. L = column length. dp= particle size. (Courtesy of Waters Corp.)... 

A parameter defined as the relative retention, or selectivity, is often reported for a given instrumental chromatography system as a number that ought to be able to be reproduced from instrument to instrument and laboratory to laboratory regardless of slight differences that might exist in the systems (column lengths, temperature, etc.). This parameter compares the retention of one component (1) with another... 

As mentioned in Section 11.8.4, the parameters that are most important for a qualitative analysis using most GC detectors are retention time, tR adjusted retention time, t R and selectivity, a. Their definitions were graphically presented in Figures 11.16 and 11.17. Under a given set of conditions (the nature of the stationary phase, the column temperature, the carrier flow rate, the column length and diameter, and the instrument dead volume), the retention time is a particular value for each component. It changes... 

FIGURE 5.11 Variations in column shape selectivity (axBN/BaP) as afunctionof aUcyl-phase length for monomeric and polymeric phases. (Adapted from Sander, L.C. and Wise, S.A., Anal. Chem., 59, 2309, 1987. With permission.)... 

This chapter provides an overview of essential concepts in HPLC including retention, selectivity, efficiency, and resolution as well as their relationships with key column and mobile phase parameters such as particle size, column length and diameter, mobile phase strength, pH, and flow rate. The significance of several concepts important in pharmaceutical analysis such as peak capacity, gradient time, void volume, and limit of quantitation are discussed. 

Equation 6 Calculation of optimum ratio of particle size and column length, with selectivity factor, a capacity factor of second component of critical pair under analytical chromatography conditions, fe 02 diffusion coefficient, (cm /s) (typical value for MW 1000 10 cm /s) viscosity, p (cP) specific permeability (1.2 X 10 for spherical particles), feo third term of the Knox equation, C and maximum safe operating pressure, Ap, (bar). 

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