Chromatography sample capacity

Three elements interact in chromatography resolution, analysis speed and capacity. Any one component can be improved at the expense of the others. In analytical chromatography, sample capacity is not a priority resolution and speed are required. However, when performing preparative chromatography, sample capacity and resolution are important to obtain large amounts of pure compounds, therefore speed must be sacrificed. 

The limited sample capacity and low carrier gas flow rates characteristic of open tubular column gas chromatography give rise to certain difl icultles in sample introduction. Direct sample... 

Totally porous particles of relatively large particle sizes were widely used in low pressure liquid chromatography for many years. These column packings had good sample capacity but only limited efficiency accompanied by long separation times, due to the large size and unfavorable size distribution of the particles and the presence of relatively deep pores within the particles through whick sample molecules diffused in and out of very slowly. 

The loading capacity of SEC columns is quite modest compared to interactive modes of chromatography. A rule of thumb dictates that the sample volume capacity is about 2% of the column volume. A typical analytical SEC column with dimensions of 8 x 300 mm has a VM of 10 to 11 ml, providing a sample volume limit of about 200 pi. The mass loading limit for such a column is about 1 to 2 mg. Above these volume and mass limits, resolution will be compromised. Sample capacity will scale in proportion to column volumes for different column lengths and diameters. 

Packed-column SFC also is suitable for preparative-scale enatioseparations. Compared with preparative LC, sub- or supercritical fluid chromatography results in easier product and solvent recovery, reduced solvent waste and cost, and higher output per unit time. Because of its reduced sample capacity, SFC usually allows the separation of 10-100 mg samples per run. Chromatographers can compensate for these sample amounts by using shorter analysis times and repetitive injections (Wolf and Pirkle, 1997). 

In gas chromatography, we have a choice of using open tubular columns or packed columns. For similar analysis times, open tubular columns provide higher resolution and increased sensitivity to small quantities of analyte. Open tubular columns have small sample capacity, so they are not useful for preparative separations. 

Theoretical performance in gas chromatography. As the inside radius of an open tubular gas chromatography column is decreased, the maximum possible column efficiency increases and sample capacity decreases. For a thin stationary phase that equilibrates rapidly with analyte, the minimum theoretical plate height is given by... 

Preparative-scale chromatography relies on a compromise between three variables (cf. Figure 1) (i) component resolution (determined by selectivity, efficiency and retention factor), (ii) speed of analysis and (iii) column sample capacity (Pescar, 1971). Any two of the desired goals may be realized only at the expense of the third. If a large amount of sample is required in a short time, resolution must be high. If resolution is insufficient, either the column load is limited or the time required for separation is long. 

Thin-layer chromatography (TLC) is another liquid-liquid partition technique applicable to polysaccharides, but in two dimensions. In TLC, the M cutoff boundaries between separated molecules are sharpened, because diffusion is minimized or eliminated in favor of capillary transport. The sample capacity of a TLC plate is in microliters. Resolution is enhanced further at high solvent pressure (Rombouts and Thibault, 1986). 

In addition to different column characteristics, one more factor must be taken into consideration in gas chromatography, namely the sample capacity. This is defined as the maximum permissible sample size that can be injected into a column without more than 10% loss of efficiency, and it is expressed as... 

For biological samples where the analytes often are non-volatile and/or occur in an aqueous matrix, the reversed-phase mode, using a hydrophobic stationary phase and an aqueous mobile phase is extremely useful. The usefulness and popularity of HPLC was further increased by the possibility to automate and computerize the systems providing unattended operations and high sample capacities. Many Nobel Prize awards have been based upon work in which chromatography played an important role [5], Most recently, the 2002 Nobel Prize in chemistry was awarded to the development of methods for identification and structure analyses of biological macromolecules" in which HPLC and Mass spectrometry (MS) were used [6],... 

Finely divided silica and alumina are the only stationary phases that find extensive use in adsorption chromatography. Silica is preferred for most (but not all) applications because of its higher sample capacity and its wider range of useful forms. The adsorption characteristics of the two substances parallel one another. For both, retention times become longer as the polarity of the analyte increases. 

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