Band spreading, column chromatography

Overall, the most effective factor in Equation 5.20 is the particle size. The smaller the particle size, the higher the column efficiency. Equations 5.13, 5.15, and 5.18 are depicted in Figure 5.6 against flow velocity as A, B, and C, respectively. The band spreading is thus influenced by Equation 5.15 at a low flow rate. The band spreading is influenced by Equations 5.18 and 5.19 at a high flow rate. For gas chromatography curve D is obtained. 

Figure 23-19 Band spreading from multiple flow paths. The smaller the stationary phase particles, the less serious this problem is. This process is absent in an open tubular column. [Adapted from H m. McNair and E. J. Bonelli, Basic Gas Chromatography (Palo Alto. CA Varlan Instrument Division. 1968).]...

The volume of a chromatography system outside of the column from the point of injection to the point of detection is called the dead volume, or the extra-column volume. Excessive dead volume allows bands to broaden by diffusion or mixing. Use short, narrow tubing whenever possible, and be sure that connections are made with matched fittings to minimize dead volume and thereby minimize extra-column band spreading. 

Kauffman, A.D Kissinger, P.T. Exfra-column band spreading concerns in posf-column photolysis reactors for microbore liquid chromatography. Curr. Sep. 1998, 17, 9-16. 

Radial compression uses radial pressure applied to a flexible-wall column to lessen wall effects. The mobile phase has a tendency to flow slightly faster near the wall of the column because of decreased permeability. The solute molecules that happen to be near the wall are carried along faster than the average of the solute band, and, consequently, band spreading results. Preparative scale radial compression chromatography columns have been found to possess efficiencies close to those of analytical-scale columns when an adequate radial compression level is used. Radial compression technology also helps lower the cost by substituting reusable column holders in place of expensive steel columns. 

Glass columns used for low-pressure chromatography are nearly always equipped with an adjustable inlet plunger that can be moved up and down depending on the hei t of the bed. This way, any void space above the packed bed can be eliminated easily. The same principle can be applied to HPLC columns. When the bed collapses, the void space that is form above the bed contributes significantly to band spreading and peak distortion. However, the bed itself usually has become nonunifotm, and this distorts the peaks as well Thus an adjustable end fitting removes only part of the cause of p distortion and its effectiveness is limited. 

There is another packed column technique, referred to as porous HDC, or simply liquid exclusion chromatography or EEC, which has also been applied to particle size analysis. In this case the hardware nearly exactly duplicates that used for bulk polymers. Although the elution volume range is enhanced with these systems, the rather excessive band-spreading which results severely limits the resolution and therefore the practical use of the technique. 

Because the effect on resolution attributed to band spreading is innately important in chromatography, the loading capacity of a column directly influences the efficiency of separations performed on ion-exchange sorbents. As the concentration of protein injected overloads the column, the plate number decreases due to band broadening, reducing the resolution between separated species. Column efficiency can be increased by using microparticulate supports... 

The width of a band eluting from a column in linear chromatography may be calculated from the sum of the variances of the different processes which influence it. For the present purposes we assume that the load is low and the predominant band spreading mechanism is due to the injection. The band variance may be considered as the sum of the variances due to the column and the injection volume. (It should be remembered that the variance is the square of the standard deviation of the band the standard deviation can be measured as 1/4 of the baseline width, so variance is calculated as = w Vlb.) If the column efficiency is high, and the column variance is therefore small compared with that of the injection, the latter will dominate and the peak will become rectangular, with a width equal to that of the injection. Under such circumstances, two peaks will be just resolved if their centres are separated by an elution volume equal to that of the injection. 

One of the difficulties with any form of chromatography is that a band of solute is dispersed, becoming less concentrated as it travels through the system. The efficiency of the column is a measure of the amount of spreading that occurs. In the chromatogram in Fig. 2.3b, Vr = the retention volume of a solute and wg = the volume occupied by the solute. This is called the peak width, but remember it means a volume, not a length. 

A band of solute invariably spreads as it travels through a chromatography column (Figure 23-11) and emerges at the detector with a standard deviation o. Each individual mechanism... 

A similar result is accomplished by using as one phase a solid powder or fine "beads" packed in a vertical column or spread in a thin layer on a plate of glass. The methods are usually referred to as chromatography, a term proposed by Tswett to describe separation of materials by color. In 1903 Tswett passed solutions of plant leaf pigments (chlorophylls and carotenes) in nonpolar solvents such as hexane through columns of alumina and of various other adsorbents and observed separation of colored bands which moved down the column as more solvent was passed through. Individual... 

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