Liquid Phase Separations with MS Detection

Coupling HPLC Separations with MS Detection

Since HPLC grew into maturity in the early eighties, coupling with mass spectrometry has been a challenge. Bringing the analytes out of the liquid phase, ionizing them, and transferring the molecular ions into the ultra-high vacuum, is far more difficult in HPLC than in the case of gas phase separations (GC-MS). After multiple iterations in interfacing methodology, e.g., by transport of the column eluate via a moving belt or moving wire into the vacuum, by direct liquid inlet introduction ¹, by thermo-spray ionization², by particle beam ionization approaches³ or by offline coupling via MALDI plates, electrospray ionization (ESI) at atmospheric pressure (AP) has become established as the de facto standard interfacing method since >20 years.

Fenn first reported atmospheric pressure electrospray ionization et al.⁴. The method allows the formation of a spray of (sub) micrometer-sized charged droplets under the influence of an electrical field from which eventually charged molecules (ions) evaporate and enter the mass spectrometer via an inlet orifice. Despite great promises, AP electrospray ionization was initially confined to flow rates of maximally 10 µL/min and to mobile phases with low aqueous content in order to allow for a stable electrospray.

Given the low flow rate demanded by ESI (< 10 µL/min), in the late nineties and following years, ESI was an exceptionally versatile and sensitive LC-MS interfacing method in proteomics, biomarker discovery, and in metabolomics. In these fields, very small sample amounts (low μL) are available, which mandate the use of low i.d. HPLC separation columns (< 150 μm). These are typically operated at extremely low flow rates (<< 1 μL/min) and therefore a good match with the flow requirements for electrospray ionization. In addition, low flow rates enhance sensitivity significantly while at the same time the effect of ion suppression and the matrix effect become less prominent. The technique is now generally called nano-electrospray ionization.

On the other hand, in HPLC separations many columns used have i.d.'s (1)2.1 - 4.6 mm and are typically operated at flow rates 0.1 - 1.0 mL/min. To establish a stable spray at higher flow rates, Henion et al. came up with an interface concept which they called "ion spray" to emphasize the difference between electrospray ionisation^5,^6,⁷ and US Patent 4861988. In this work, spray formation was assisted with a co-axially delivered flow of nitrogen gas. This approach was readily made commercially available by Sciex and other companies with further improvements allowing nowadays flow rates up to 1 mL/min.

While the inventors of "ion spray "clearly realized that they were operating in a different mode from what was designated as electrospray ionization, meanwhile the term has ESI been adopted as a general acronym for LC-MS coupling.