27
Table 1: Comparison of UHPLC, UHPLC-Like and HPLC systems. *Other vendor information obtained from Reference [2]
was compared for a 50 x 2.1 mm column format, with 1.7 µm particle size, with retention factors, k, of 2, 5 and 10 (the optimum region for retention factors as described by the Purnell Fundamental Resolution Equation). The dispersion regions for UHPLC, UHPLC-Like and HPLC systems were also denoted on the graph (Figure 3(a)). The 50 x 2.1 mm column format with the 1.7 µm particle is suitable for UHPLC analyses and is the harshest condition to assess, therefore, any other column will have better performance. The largest value of k assessed (k = 10) achieved >90% efficiency yield for dispersion <13 µL. A k of 5 could achieve above 90% of the efficiency yield at the smallest dispersion values (<7 µL). The minimum retention factor of 2 was below 80% thus indicating that greater retention factors should be applied to achieve adequate chromatographic performance. In comparison, a HPLC column format (150 x 4.6 mm, 5 µm particle), with a k of 2 was mostly unaffected by the extra column band broadening, where the % efficiency yield was greater than 90% up to maximum 40 µL of dispersion assessed.
Column dimensions
Figure 2: Comparison of the 1% acetone peak measured for dispersion on a UHPLC with variable volume injector. Extra column volume was added to simulate the effect of dispersion.
Impact of Dispersion
Dispersion can be measured by injecting a sample of acetone onto a ZDV union to quantify the extra column band broadening effect. The method conditions are in the experimental section, with an example of instruments with different system volumes and bandwidths in Figure 2.
To determine the instrument bandwidth, the retention time (tR ) and
efficiency (N) at half height should be recorded. These can be inserted into Eq. 1 to calculate sigma (σ), which in turn can calculate instrument bandwidth (4σ, Eq. 2). Consequently, the system volume can also be determined by this test by multiplying the average retention time of acetone by the flow rate (Eq. 3, µL/min). Nine repeat measurements should be recorded to generate a statistically significant result.
The effect of dispersion is magnified for column formats with smaller column volumes, as the ratio between the two parameters is changed (Figure 3(b) and
(c)). Thus, as expected, the longer the column length or wider the internal diameter, the less affected by dispersion. The retention factor was maintained at 5 and the particle size was standardised on 1.7 µm. The results indicate that the internal diameter has the greatest impact, where the 4.6 mm ID achieved greater than 90% efficiency yield below 30 µL dispersion. The 3.0 mm ID is a good compromise for reduced flow rate, solvent consumption and faster runs, for UHPLC and UHPLC-Like type systems. The 2.1 mm ID requires the LC system to be optimised by reducing the volume of tubing, the ID of tubing, optimising the flow cell, and potentially removing column switching valves.
Particle Size
What influences the impact of dispersion? Retention factor, k
The speed the peak band migrates through the system can be highly influential for the effect of dispersion. The % efficiency yield
The more efficient the column, the more the dispersion volume can have a negative impact (Figure 3(d)). The graph illustrates this, where the smallest 1.7 µm requires the most optimised UHPLC systems, and the 3 and 5 µm achieve a greater degree of the expected efficiency using systems up to UHPLC-Like. Again, the retention factor was standardised at 5, and the column format was set to 50 x 2.1 mm, therefore, if the internal diameter or column length was increased, this would also offer additional support for improving the efficiency yield. Totally porous particles were applied in this theoretical study, where superficially porous particles have a different particle structure which alters their diffusion pathway, thus decreasing the reduced
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