Column, capillary dispersion, extra

Conventional LC-NMR employs separation columns which are connected to NMR detection probes via open tubular capillaries with internal diameters substantially smaller than the column to avoid extra-column band broadening. Since the band broadening introduced by the connections and transfer lines causes significant peak dispersion in capillary separations, on-column detection for small-volume LC is desirable. Because of the need for deuterated solvents for NMR detection, the relatively large volumes and flow rates used with conventional analytical HPLC columns make coupling of LC and NMR an expensive experiment. 

Equations (2) and (4) allow the permissible extra-column dispersion to be calculated for a range of capillary and packed columns. To allow comparison, data was included for a GC column, in addition to LC columns. The results are shown in Table 1. 

The standard deviation of the extra-column dispersion is given as opposed to the variance because, as it represents one-quarter of the peak width, it is easier to visualize from a practical point of view. It is seen the values vary widely with the type of column that is used, (ag) values for GC capillary columns range from about 12 pi for a relatively short, wide, macrobore column to 1.1 pi for a long, narrow, high efficiency column. 

By replacing conventional 3.5 or 5 jtm columns with sub-2-micron columns, gradient time can be reduced dramatically. The flow rate must be increased for optimal conditions as well but solvent consumption will be less than the amount used by the original method. To use the full power of these columns, an LC instrument must be thoroughly optimized toward lowest extra-column dispersion. The smaller the column (small ID and short length), the more sensitive the performance is to dispersion. With smaller internal diameter columns, the injection volumes and internal diameters of the capillaries should be reduced. 

Clearly, the column dispersion in capillary chromatography is a very strong function of the diameter of the column. If the column diameter is decreased, then the column dispersion will decrease strongly and therefore, according to eqn.(7.33), the demands imposed on the maximum allowable extra-column dispersion will become increasingly severe. 

Although the above calculation is somewhat oversimplified because the effects of the compressibility of the gas have been neglected, it serves to illustrate that a reduction of the column diameter cannot be fully compensated by an increase in the column length to keep the column dispersion constant. Therefore, when narrow-bore capillary columns are to be used in GC, the extra-column contribution to band broadening will need to be reduced. 

In contrast, the use of wide-bore capillary columns allows a considerably larger extra-column dispersion. Consequently, these columns may readily be used in instruments that are compatible with conventional capillary columns. Moreover, they may be used instead of packed columns to yield greatly increased plate numbers without the requirement of major modifications to the instrument. One of the major reasons for the popularity of wide-bore capillary columns may therefore be their role as intermediates in the gradual replacement of packed columns by capillary columns in GC. 

It is clear from table 7.2 that in terms of extra-column dispersion a wide-bore capillary column requires instrumentation similar to that used for the packed column. However, the capillary column provides eight times as many plates (in a fifteen-fold analysis time). Conventional capillary columns require a reduction in the dispersion by about an order of magnitude, whereas narrow-bore columns require a further reduction by a factor of about 100. This, combined with the high pressures required, puts narrow-bore columns out of reach for current GC instruments. 

In GC the use of wide-bore" capillary columns allows the use of instruments designed to accommodate packed columns (in terms of extra-column dispersion). For capillary columns of conventional diameter a reduction of the extra-column dispersion by a factor of /0, and for narrow bore columns a reduction by a factor of 1000, are required. 

Instrument dispersion can be reduced by optimizing injector and detector systems and reducing diameter of connection capillaries. The individual sources of volumetric extra-column broadening specified in equation (17-26)... 

A significant reduction of the column ID put stringent demands on the instrumentation for micro-LC as all volumetric extra-column dispersion contributions must be down scaled accordingly. Initial developments in this area were achieved on modified standard LC instruments. For approximately 10 years, dedicated instrumentation for micro-, capillary-, and nano-LC has become commercially available and was recently discussed [33]. 

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