Band Broadening outside the Column

The most common HPLC column diameter is 4.6 mm. There is a trend toward narrower columns (2 mm, 1 mm, and capillary columns down to 25 pm) for several reasons. Narrow columns are more compatible with mass spectrometers, which require low solvent flow. Narrow columns require less sample and produce less waste. Heat generated by friction of solvent flow inside the column is more easily dissipated from a narrow column to maintain isothermal conditions. Instruments must be specially designed to accommodate column diameters <2 mm or else band broadening outside the column becomes significant. 

Band broadening outside the column was discussed in Section 23-5. 

Care must be taken to keep additional band-broadening outside the column to a minimum. Extra-column contributions to band broadening, such as mixing or poor sample-introduction technique, will increase the //-value. Because of the slow diffusion of samples in liquid phases, extra-column volumes are more detrimental to efficiency in LC than in GC hence, great effort should be exercised to keep those volumes between the point of injection and the top of the column to a minimum. Likewise, the volume between the column exit and the detector, and the volume of the detector itself, should be minimized. In TLC or PC, the applied spot should be kept as small as possible. 

Some factors outside the column contribute to broadening. For example, solute cannot be applied to the column in an infinitesimally thin zone, so the band has a finite width even before it enters the column. If the band is applied as a plug of width At (measured in units of time), the contribution to the variance of the final bandwidth is... 

Achieving the theoretically expected performance of high-efficiency columns requires proper instrument design to ensure that band broadening outside of the column is negligible. Sources of extra-column broadening can be classified into two categories volumetric effects and electronic effects. Those associated... 

Here, y is a tortuosity factor ( 0.64), jc is the fraction of mobile phase iside the column) that is outside the column-packing pores, Dp is the solute iranision coefficient in the mobile phase inside the pores (Dp Z7 ), and Z), is the solute diffusion coefficient for molecules in the stationary phase. Equation (36) recognizes two effects that are often overlooked in d ribing band broadening (1) difiusion of retained solute molecules (stationary-phase or Surfiioe dif on) and (2) reduced solute diffusion in pores versus the bulk iaol phase. 

Considering a chromatographic process controlled by a partition equilibrium and neglecting extracolumn effects (i.e., band broadening caused by factors outside the column, e.g., tubings, detector etc.), several factors can contribute to the overall solute band broadening eddy diffusion, longitudinal diffusion, and resistance to mass transfer in mobile and stationary phase. 

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. 

In near-isothermal systems, it is assumed that the heat transfer between mobile and stationary phases is slow. This causes an additional band broadening contribution to appear [53]. Such a contribution can be especially important on the front of a sharp concentration profile. On the other hand, the heat transfer between the chromatographic column and the outside is fast enough to prevent the formation of a temperature front and of an associated secondary mass transfer zone. 

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