Extra column band broadening

The overall peak dispersion produced is a combination of column and extra column effects, is additive and can be expressed in terms of their variances (see Chapter 2)  

It can be shown that the contribution to the overall plate height from these extra column effects is a function of a number of instrumental parameters, i.e. 

With narrow bore columns the flow-cell volume must be 3 pi in order that the resolution achieved on the column is not lost due to analyte spreading in the detector cell. 

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 

The instrument band-spreading (ITec) can be measured by replacing the column with a zero dead-volume restrictor, injecting a very small volume of sample, and measuring the resulting peak width. Table 17-5 provides band- 

TABLE 17-5. Extra-column Volumes and Band-Spreading of Different Commercial 

System Detector Cell Volume, Path Length Fec [rL] (Extra-column Volume) Wec [pL] (Extra-column Band Spreading) 

Eigure 17-17. Theoretical plates versus retention factor for series of parabens separated by isocratic chromatography. Extra-column effects result in decreased efficiency for early-eluting components (low values of k). 

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The efficiency, or plate count of a column N is often calculated as 5.54 (tr/a)2, where tr is the retention time of a standard and a is the peak width in time units at half-height.1 2 5 This approach assumes that peaks are Gaussian a number of other methods of plate calculation are in common use. Values measured for column efficiency depend on the standard used for measurement, the method of calculation, and the sources of extra-column band broadening in the test instrument. Therefore, efficiency measurements are used principally to compare the performance of a column over time or to compare the performance of different columns mounted on the same HPLC system. 

Monoliths Low backpressure, suited for conventional HPLC Higher separation efficiency by column coupling Rugged against delay volume and extra-column band broadening fast column re-equilibration Reduced maintenance on pumps and injector seals Reduced need for sample pre-treatment... 

One way to calculate the extra column band broadening extra column variance is rather low or negligible for solutes of high capacity. In this way we can write... 

The detector flow-cell, the contribution of which to ctv is approximately equal to its volume [707], represents a considerable and recognizable contribution to the extra-column band broadening. Typical conventional flow-cells have a volume of 8 pi, which is quite substantial compared with the maximum allowable extra-column dispersion. 

Presently [707], we may be able to reduce the extra-column band broadening by modifying conventional H PLC equipment to a total of 1 or 2 pi. This implies that with these modifications short bulky 3 pm columns or narrow (2 mm) 5 pm columns may be used. 

The main disadvantage of size-exclusion chromatography is its low peak capacity, which arises from the small elution volumes of the peaks. In addition because peak dispersion is small, extra-column contributions to band broadening in size-exclusion chromatography have to be kept to a minimum. The relative contribution of extra-column band broadening can be minimized by the use of small particles = 3-10 pm) and long (L = 50 100 cm), wide-bore (d = 8-10 mm) columns. 

Even with all these chromatographic parameters optimised, sensitivity may not be enough. The simplest way to increase sensitivity further is to inject more sample. Valve injectors are usually supplied with 20-pl loops but there are few assays for which 100-200 pi of sample cannot be injected simply by changing the loop size. A further way is to use minibore (1 mm or 2mm i.d.) or even microbore (< 1 mm) columns. Such columns give increases in sensitivity due to the narrower peaks eluted but this may reduce column loading volumes and with some equipment the extra sensitivity gained may be lost due to extra column band broadening. 

Detectors for HPLC must have low dead volume to minimize extra-column band broadening. The detector should be small and compatible with liquid flow. No highly sensitive, universal detector system, such as those for gas chromatography, is available for high-performance liquid chromatography. Thus, the detector used will depend on the nature of the sample. Table 32-1 lists some of the common detectors and their properties. 

In order to obtain the characteristics of the column the values of and must be first corrected for the extra-column band broadening contributions that... 

With the trend toward smaller columns, extra care should be taken to minimize extra column band broadening by using reduced diameter tubing, smaller volume detector cells, and properly fitting connections (9). 

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