One view of Ultra High Performance Liquid Chromatography is that it is the culmination of many small improvements. As applications are transitioned to UHPLC methods, one might ask, is it the column, the extra-column flow path, the instrument, optimized flowrate, or some other characteristic of the system which is responsible for this ‘Ultra’ level of performance? Some scientists look to the instrument, as this is the source of high operating pressures, fast digitization rates, and many extra-column factors required to achieve low dispersion, and fast, high resolution separation. But this hardware based approach does not fully take into account the contributions of advanced stationary phase designs such as the fused core particle, which also attains high, ‘Ultra’ levels of resolution. And yet, these particle geometries do not rely on extremely high operating pressures, and the hardware to produce such pressures. So, UHPLC performance is not solely a function of an instrument capable of attaining high pressures. Keep in mind that it is also quite possible to utilize a state-of-the-art UHPLC instrument, and to fail to achieve ‘Ultra’ levels of performance. Method optimization, stationary phase selection, solvent selection and other optimizations which apply to HPLC certainly apply to UHPLC? Not least among these simple optimizations is the extra-column flow path, that is, the fittings and tubing used to connect the column to the instrument.
The effects of sample dispersion, both internal to, and external to the column, have limited traditional HPLC resolution and speed. Advanced columns and stationary phases, coupled with a low dispersion instrument, can provide the new analytical insights and productivity offered by UHPLC. There are just a few pitfalls to achieving these performances and they are easily avoided. As has been mentioned, an absolutely critical component of extra-column dispersion is the selected tubing and fittings.
It should be noted that UHPLC fluidic hardware (tubings and fittings) have undergone a second generation of evolution to keep pace with advances in stationary phase. In the 2005-2012 era, fittings evolved to provide minimized dead volumes. Advertized as ‘zero dead volume’, in actuality, they were ‘nearer zero’, but as in many fields, a value of zero for a given parasitic is an asymptote over time. The fittings of the 2005-2012 era fittings were in the range of 10% of the dead volume of the traditional one-piece PEEK fittings which were commonplace prior to 2005. That is, the potential improvement in dead volume, (on a per fitting basis) went from approximately 1.8uL (worst case) for the PEEK one piece fittings of the era, down to about 0.25uL for the first generation of ‘UHPLC’ fittings. From 2005 to 2012, of course ‘state of the art’ has evolved and the most advanced fittings of today provide approximately another 10:1 reduction in dead volume.
Instrument bandwidths or IBW, is a direct measure of an instruments fidelity and appropriateness to attain the performance which the latest UHPLC columns offer. Sub-2 micron stationary phases, and fused core particles offer such low variance in interstitial pathways that dispersion due to the instrument, which includes the fluidic conduits to and from the column, can be seen to influence resolution. The general conclusion should be to be aware of all sources of dispersion so maximum potential of UHPLC columns can be attained. This is easily tested with an instrument IBW test.
UHPLC columns attain high resolution and therefore higher practical speeds in large part by tightening the distribution of mean free paths encountered by the mobile phase. Smaller stationary phase media is therefore desirable in UHPLC columns, and with few stationary phase exceptions (such as those columns using fused core media) higher operating pressures are encountered. Sources of internal dispersion are minimized, such as those dispersions arising from internal gradients. Extra-column sources of dispersion must be considered by the user to achieve the total benefit offered by the latest advances in columns.
The latest generation of HPLC fittings, called TSipTM (Tip Sealing Intellectual Property) by Best Instrument, Inc. offers the consistently lowest possible extra column dispersion of any fitting currently on the market. In this the study which resulted in this publication, http://www.sigmaaldrich.com/content/dam/sigma-aldrich/countries/european-images/events/hplc2013/HPLC2013-dorina-kotoni.pdf TSipTM fittings were provided to acheive the low extra-column dispersion required to acheive maximum resolution of this new stationary phase. And the result is yet another advance in resolution. The fittings used in Dr. Kotoni's study can be obtained here: http://www.best-instrument.com/
But it must be noted resolution and selectivity are two different things. Certainly when careful attention to phase selection is made to acheive maximum selectivity, the work always produces better results when the gains are not being lost by extra-column dispersive effects. A very good overview of total optimization can be found here: http://www.sigmaaldrich.com/video/analytical/hplc-optimization.html In this video, Dr. Henry discusses the variety of factors which must be taken into account to optimize HPLC and certainly UHPLC systems.