Column length effect

Cobalt oxide, as adsorbent additive, 177 Column chromatography, see Elution Column length, effect on N, 115-117 Column packing, see HETP values Column pressure, effect on TV, 115-117 Coluisns, for liquid chromatography, 347-348... 

Figure 4 Column length effects in gradient elution. Separation of ribonuclease A, cytochrome c, ovalbumin. Cb columns of varying length, 0-100% propanol-water gradient 0.5mL/min. (a) 4.5 cm column (b) 0.63cm (o) 0.16cm. (From Ref. 7.)...

The analytical capability of a SEC column is sometimes judged by the peak capacity, which is the number of unique species that can be resolved on any given SEC column. This number will increase with decreased particle size, increased column length, and increased pore volume. Because small particlesized medium generally has a lower pore volume and a shorter column length, peak capacities of ca. 13 for fully resolved peaks can be expected for high-resolution modern media as well as traditional media, (see Eig. 2.5). It was found that SEC columns differ widely in pore volume, which affects the effective peak capacity (Hagel, 1992). 

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. 

Column length has an effect on resolution and analysis time. Longer columns provide higher resolution at the expense of longer analysis time, as shown in Fig. 4.41. The 60-cm-long GlOOOHg column has nearly identical resolution... 

The effect of flow rate on resolution by Toyopearl HW-55F and Toyopearl HW-55S columns has been studied using a bovine serum albumin sample. Eor both columns, resolution decreased with increasing flow rate (46). Resolution is increased, however, with decreasing particle size (47). Resolution is proportional to the square root of the column length, as theoretically expected, and indicates that longer columns can be packed as well as shorter columns. Therefore, for samples difficult to resolve, the solution may be to increase the column length. 

FIGURE 7.6 Effect of column length on the separation efficiency. Two different Fractogel EMD BioSEC columns (A 600 X 16 mm, B 1000 X 16 mm) were tested using BSA, ovalbumin, and cytochrome c (S/S/3 mg/ml) as sample (20 m/VI sodium dihydrogen phosphate, 300 m/VI NaCI, pH 7.2 0.5 ml/min). Better resolution can be achieved using longer columns. 

As we continue lowering the pressure, GC is the final limiting case when the mobile phase has zero solvent strength over the entire column length and where temperature is the only effective control parameter. Gas chromatography is shown in Figure 7.3. 

The plate height, and thus the total number of theoretical or effective plates, depends on the average linear carrier gas velocity (van Deemter relationship) and, for a particular carrier gas, the efficiency will maximize at a particular flow rate. Only at the optimum carrier gas flow rate are n, N, and HETP Independent of the column length. The efficiency will also depend on the column diameter (see section 1.7.1) where typical values for n, N, and HETP for different column types can also be found. Values for n, N, and HETP are reasonably independent of temperature but may vary with the substance used for their determination, particularly if the test substance and statioKary phase are not compatible. 

Koyama, J., Nomura, J., Shiojima, Y., Ohtsu, Y., and Horii, I., Effect of column length and elution mechanism on the separation of proteins by reversed-phase high-performance liquid chromatography, /. Chromatogr., 625, 217, 1992. 

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. 

Fig. 17.11. Bottom CGE separation of components of poly U (sigma) in 25% pluronic F127. Top Note the resolution of two contaminants between each of the oligonucleotides from about 15 to 27 nucleotides long in this expanded section of the bottom electropherogram. Electrophoresis was performed in 25% pluronic F127 in tris-borate-EDTA buffer (90 mM tris, 90 mM boric acid, 2 mM Na EDTA, pH 8.3.) (25°C, 500 V cm-1, effective column length 30 cm). Reprinted with permission from Ref. [82],...

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