28 Buyers’ Guide 2021 plate height.
The results illustrate the importance of matching the column format to the instrument in order to obtain reasonable effi ciencies. The Nexera XS with a variable volume injector and SPD-M30A capillary fl ow cell has a bandwidth of 6 µL as standard, but further optimisation with loop injector is also available. However, when chasing an ever- lower dispersion volume, there are consequences which may prove detrimental to the robustness of the system. Fixed loop injectors have the potential for greater carryover which can impede on accurate and reproducible quantitative data. The use of narrower bore tubing also can have potential issues. Stainless steel tubing can be manufactured by different pathways, however, there is a tolerance for the tubing where the narrower the tubing, the greater the impact of errors, therefore a reproducible source of tubing is a necessity. In addition, extra narrow bore tubing will increase the inherent back pressure of the LC system, excluding the column. A change in pressure can cause pressure sensitive peaks from an established method to move which can change the resolution between analytes. Finally, narrow bore tubing is prone to blockages, particularly if the sample matrixes is dirty or even from the mobile phase preparation.
Although it is possible to reduce the extra column band broadening using fi xed loops and narrow bore tubing, from a practical point of view, does the dispersion need to be reduced to such extremes as
Concluding Remarks
A simple and quick test can easily provide information regarding both the extra column band broadening, referred to as dispersion, as well as the system volume. This information gives crucial information regarding suitable column formats for that system, which can save buying expensive columns. For example, a UHPLC column format can never achieve its full chromatographic potential on a HPLC confi guration, therefore, an appropriately optimised system should be employed.
The 2.1 mm column internal diameter is most suited to systems with dispersion of less than 12 µL, which can be achieved on UHPLC or UHPLC-Like systems which have been optimised. 3.0 mm column formats are most appropriate on UHPLC-Like instruments. The % effi ciency yield of a 4.6 mm ID is largely unaffected by dispersion of less than 40 µL, even with retention factors of 2, thus HPLC systems are most compatible.
Although the 2.1 mm column format does require minimal extra column band broadening, there are other factors which can impact on obtaining reasonable effi ciencies, such as operating at optimal fl ow rates. This has a bigger impact on the expected yields than the effect of reducing the dispersion. Therefore, although it is
Figure 3: Establishing the effect of dispersion on the theoretical % effi ciency yield expected for different totally porous particle column formats. (a) Effect of k, column 50 x 2.1 mm, 1.7 µm, k = 2, 5 or 10, and comparison of UHPLC vs HPLC, column 150 x 4.6 mm, 5 µm, k = 2, (b) Effect of column length, column 50 x 2.1 mm, 100 x 2.1 mm, 150 x 2.1 mm, 1.7 µm, k = 5, (c) Effect of column internal diameter, column 50 x 2.1 mm, 50 x 3.0 mm, 50 x 4.6 mm, 1.7 µm, k = 5 (d) Effect of particle size, column 50 x 2.1 mm, 1.7 µm, 3 µm or 5 µm, k = 5.
imperative to operate using minimal bandwidth, the practical implications intimate that there are other factors which may play a larger role, thus diminishing the impact of minimising the dispersion excessively.
Figure 4: Overlaid chromatograms of alkylphenones chromatographed on a 1.5 minute gradient using an 6 µL dispersion system (black trace) and a 15 µL dispersion LC system (pink trace).
~5 µL, with its added complications? A series of alkylphenones were chromatographed using a ballistic gradient on a UHPLC C18 column (50 x 2.1 mm, 1.7 µm, Figure 4). The seven peaks were assessed on an LC system with 6 µL extra column band broadening and volume added to increase dispersion to 15 µL. The results denote there is an improvement in effi ciency. The peak width at 10% height saw a 7.9% decrease between the UHPLC-Like and UHPLC conditions, whilst the peak capacity saw an increase of 8.4%. However, this is not as signifi cant as might be expected. Ballistic gradients are usually performed at fl ow rates greater than the optimal linear velocity. Although 1.7 µm particle sizes have a fl atter C-term in the van Deemter [4], which provides a wider optimal linear velocity range, the fl ow rate can still have a greater impact than the dispersive effects. Therefore, although in theory the dispersion can be reduced signifi cantly, other factors play a more crucial role in affecting effi ciency, therefore the bandwidth improvements are negligible.
Most laboratories aspire for robustness in combination with performance. Excellent dispersion can be achieved on the variable volume Nexera XS system with fantastic performance, however, if deemed necessary, it is possible to push it even further by introducing fi xed loop autosamplers and extra narrow tubing. Nevertheless, with these changes, it can negatively
impact on the ruggedness of any LC system. These considerations should be thought through thoroughly in combination with the purpose of the instrument, and column formats to truly attain the desired results.
References
[1.] J.W. Dolan, Extracolumn Effects, LCGC North America, 2008, 26, 1092-1098
[2.] M.W. Dong, UHPLC, Part 1: Perspectives and Instrumental Features, LCGC North America 2017, 35, 374-381
[3.]
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/ Supelco/General_Information/t408143.pdf (accessed 23/10/2020)
[4.] U.D. Neue, HPLC Columns: Theory, Technology and Practice, 1997, 1st ed. Wiley VCH
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