32 May / June 2016
[5]. It remains to be seen however, if this procedure will be universally adopted. Figure 5 shows measurements of the extra column bandspreading of an Acquity classic high pressure mixing binary UHPLC system, using the classical ZDV method and the 5-sigma peak width. The upper plot, (using a suffi ciently high data gathering rate of 80 Hz), shows that the extra-column variance increases with increase of fl ow rate until a broad maximum value is achieved. At low fl ow rate, radial inhomogeneity of the fl ow profi le can be counteracted by solute diffusion between the various fl ow streams. Using 90% ACN in the mobile phase, the bandspreading amounts to ~ 3.5 µl2
at a fl ow
Figure 4. Comparison of loading with nortriptyline on totally porous (Zorbax) and shell ODS columns (Poroshell) from same manufacturer. Mobile phase: acetonitrile-ammonium formate buffer pH 3. Table shows small mass peak asymmetry (As
) and efficiency (No reduce No to half its value (C0.5). Sample volume 1 µL. , plates) together with concentration necessary to
rate of 0.5 mL/min, a fl ow typically used with very small particle columns of i.d. 2.1 mm. A somewhat greater bandspreading value is obtained by use of 10% ACN as the mobile phase, a solvent of greater viscosity which reduces solute diffusion in the mobile phase. The lower plot of Figure 5 shows additionally the importance of using a suitably fast data gathering rate / small time constant in these experiments. A value of 20 HZ is clearly insuffi cient for such experiments. A similar experiment indicated instrumental bandspreading of 25 µl2
for a conventional
Figure 5. (Upper) Measurement of instrumental band broadening of Acquity Classic UHPLC system using different probes/mobile phases. (Lower) Measurement of band broadening for naphthopyrene in 90% acetonitrile at fast and low data gathering rates.
where σ2experimental variance, σ2column
is the measured peak is the variance caused by
the column alone, and σ2extracolumn is the extra-
column variance (all values usually measured in µl2
). The inherent column variance (and
from this its effi ciency) can be calculated from the measured experimental variance by measuring the extra-column bandspreading by substitution of a zero dead volume (ZDV) connector in place of the column. However, this procedure has become the subject of some controversy. For example, extra-column effects are measured at low (atmospheric) pressure, whereas the experimental value is measured at high
pressure (with the column in place [1]). Under the latter condition, the viscosity of the mobile phase may be increased somewhat leading to reduced solute diffusion in the mobile phase, and an underestimation of the extra-column contribution. A further problem is that the peak variance is usually measured by considering the peak width at a single position at some proportion of the peak height (such as the 5 sigma width at 4.4% of peak height). Desmet has proposed an improved deconvolution method for subtracting the shape of the extra column peak in its entirety from the experimentally measured peak shape
HPLC system (Agilent 1100) adapted with the use of small diameter connecting tubing (0.12 mm i.d.) and a micro fl ow cell (1 µL). Using these values for 10 cm columns with i.d. 2.1 mm and 4.6 mm, of true effi ciency 25000 plates operated on the two systems, the % loss in column effi ciency (N) caused by the instrumental bandspreading can be estimated (Figure 6). A 2.1 mm i.d. column operated on the Acquity system would show a loss of 40% in effi ciency for a peak of k =1, about double the value for the 4.6 mm column operated on the (much older) Agilent system! This result indicates clearly the problems of operation of high effi ciency, narrow bore columns even on modern UHPLC systems with apparently small extra- column band broadening contribution. Operation of the 4.6 mm column on the Acquity system shows understandably few problems; even with an unretained peak, the loss in N is ~ 10%. Furthermore, losses in effi ciency decrease as the retention of the solute increases, as the inherent band broadening caused by the column becomes large compared with the instrumental effects. Nevertheless, problems are increased if a shorter 5 cm column with the same reduced plate height is used (lower plot Figure 6) as the variance due to the column is reduced. Note that it is possible to use the wider 4.6 mm columns packed with shell particles of ~ 2.5 µm diameter as there are less problems with frictional heating with this type of phase.
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