26 Buyers’ Guide 2021
Practical Impact of Dispersion on Fast Chromatographic Separations
Dispersion is often a criterion which is important to consider when investing in analytical equipment. Column formats should be appropriate for the volume of the system in order to obtain reasonable chromatographic performance from the stationary phase. However, in practical terms, do we need to be concerned about attaining such minimal dispersion volumes to obtain the best chromatography, particularly for fast, ballistic methods?
Introduction
As a peak band migrates through a chromatographic system, the analyte possesses a parabolic fl ow, where the band is distorted by friction against the tubing walls, and other forces. These components can be divided into two sections; extra column, and intra column broadening. The intra column forces occur in the porous particles, whilst extra column broadening is apportioned to the liquid chromatography (LC) system. The dispersive effects attributed to the components of the LC system can have a detrimental impact on the chromatographic performance. These elements include the injection port, loop, tubing, valves, fi ttings, detector and data collection rate (Figure 1), which applies to both isocratic and gradient systems. The dispersion is observed to greater detriment in isocratic separations with a constant mobile phase on later eluting peaks compared to the early eluting compound.
Figure 1: Schematic fl ow path of an LC system, with the components measured in dispersion encircled (blue dashed line).
could prove detrimental to critical pairs in fast separations. However, to what level does the dispersion need to be optimised to? This article will provide a means of estimating the extra column band broadening and evaluate the impact of different parameters on the theoretical % effi ciency yield.
The conventional HPLC with totally porous particle, 5 µm, 150 or 250 x 4.6 mm column formats are typically unaffected by dispersive effects as the peak variance caused by the column is greater than the variance caused by the LC extra-column components [1]. However, with the advancement in technology, including narrower bore columns, smaller particle sizes, and higher-pressure LC systems, the discussion regarding dispersion needs to be readdressed. Characteristically, conventional HPLC systems are not optimised where wider bore tubing is used, thus dispersion is measured at greater than 20 µL (Table 1). Nowadays, there are more LC system options available, where there are both UHPLC and UHPLC-Like systems available (please refer to Table 1 for differences). UHPLC systems are designed for greater throughput, performance and speed, and as such, requires the minimum possible extra column band broadening (<10 µL). The UHPLC-Like system offers greater capacity and performance than conventional HPLC, but without the added challenges associated with UHPLC analyses, thus the system can have a dispersion value of <15 µL.
The impact of dispersion can cause a loss of chromatographic performance, increased peak width, thus loss of resolution, which
Experimental
In order to assess dispersion and system volume, the following experimental parameters were applied: Water and methanol were proportionated using the pump to dispense 51:49 (v/v) respectively. The fl ow rate was set to 0.1 mL/min with a Zero Dead Volume (ZDV) union installed in place of a column. The sampling frequency was set to 40 Hz to adequately describe the peak and data collected at 254 nm (8 nm) reference 360 (100). A sample of 1% acetone in water / methanol (51:49 v/v) was prepared and chromatographed nine times after suffi cient equilibration of the system. The injection volume was 0.5 µL and the column oven controlled at 40 °C [3].
LC separations were performed on a Shimadzu Nexera XS UHPLC system equipped with binary pumps (LC-40D XS) and proportionating valves, degassers (DGU-405), autosampler with cooling capabilities (SIL-40C XS), column oven (CTO-40C), and photodiode array detector (UHPLC PDA or HPLC PDA). The system controller was integrated into the binary pump. A 20 µL 3-dimensional micro-reactor mixer was installed.
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