Extra column variances

There are four major sources of extra-column dispersion which can be theoretically examined and/or experimentally measured in terms of their variance contribution to the total extra-column variance. They are as follows ... 

Another critical instrument specification is the total extra-column dispersion. The subject of extra-column dispersion has already been discussed in chapter 9. It has been shown that the extra-column dispersion determines the minimum column radius and, thus, both the solvent consumption per analysis and the mass sensitivity of the overall chromatographic system. The overall extra-column variance, therefore, must be known and quantitatively specified. 

Dispersion in column frits was originally thought to be large and thus, made a significant contribution to the overall extra column variance. It was not until the introduction of low-dispersion unions that it was found that most of the dispersion that was thought to occur in the frits, actually occurred in the unions that contained the frits. Scott and Simpson (11) measured the dispersion that occurred in some commercially available column frits and demonstrated that their contribution to dispersion to be insignificant compared with other sources of extra column dispersion. 

One way to calculate the extra column band broadening [Pg.7]

The plate number, i.e., the chromatographic efficiency, is a parameter difficult to estimate correctly [15]. The use of methods assuming Gaussian peaks may produce grossly overestimated peak efficiency. The asymmetry-based method [9] (eq. 6.5) was designated as the manual method giving plate numbers closest to the exact plate numbers obtained with the moment method [15, 16]. The second central reduced moment of a peak corresponds to its variance [17]. It is known, but too often overlooked, that the overall peak variance, a, is a combination of the column variance plus all the other extra-column variances... 

It is possible and desirable to reduce all extra-column variances. However, external band broadening cannot be fully eliminated. The measured variance is always the sum of the actual column variance, and a... 

It follows that the total permitted extra column variance, i.e. 10% of the column variance (o2) as suggested by Khnkenberg (4), has to be shared between each source of dispersion. 

Figure 5.5 Plots of extra-column variance versus mobile phase flow rate. Instruments Waters Acquity UPLC, Agilent 1200, Agilent 1100, Shimadzu Nexera, Peridn Elmer Flexar, Merck Hitachi LaChrom. The possible maximum acquisition rate was set on each instrument (10 Hz on Merck Hitachi LaChrom system, 80 Hz on Agilent 1100 and 1200 systems, and 100 Hz on Shimadzu Nexera, Perkin-Elmer Flexar, and Waters Acquity systems). From Fekete, S., Fekete, J. J. Chromatogr. A. 2011, with permission.

According to a recent study, the commercially available LC systems can be classifled in three groups (1) optimized systems for fast separation with very low dispersion (crv,ext < 0 P-L ) (2) hybrid LC systems recommended for both fast and conventional separations ((Tv,ext = 10-30 p-L ) and (3) conventional LC systems with an extra-column variance over 50 pL (Figure 5.5). These major differences in extracolumn peak variance have a significant impact on measured column performance and achievable analysis time (63). Further improvements in instrument design (smaller dispersion) are necessary to take the full advantage of the most recent very efficient small columns. Today it is not always possible to utilize the potential of these small columns. The loss in efficiency can reach 30%-55% with commercially available optimized UHPLC systems (63). 

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