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Capillary Flow LC-MS Unites Sensitivity and Throughput
by Stephan Meding, Alexander Boychenko Thermo Fisher Scientific, Dornierstr. 4, 82110 Germering, Germany
There is a continuous demand for improved analytical depth and higher throughput capabilities for liquid chromatography mass spectrometry (LC-MS) based analyses in both research and applied markets. The need for greater sensitivity, selectivity and faster cycle times has led to impressive improvements in instrumentation and column technology.
Liquid chromatography has seen the adoption of high performance systems (HPLC) and then a transition to ultra-high performance systems (UHPLC, >400 bar) [1]. Nowadays, pressure capabilities beyond 1000 bar are widely available for analytical flow LC-MS systems. Together with ultra- high performance columns, packed with small particle size stationary phases (2 µm and below), fast separations with high resolution are now possible. Additionally, instrument development has led to commercially available LC systems capable of nano flow rates and UHPLC performance levels [2].
The combination of LC systems with mass spectrometers, for analyte identification and quantification, has further improved the analytical capabilities. The development of mass spectrometers has led to detection limits in the attomole range, with the latest generation high-resolution accurate-mass (HRAM) mass spectrometers being able to achieve sub-part-per-million (ppm) mass accuracy and mass resolution approaching 250,000 [3]. At the same time commercial LC-MS manufacturers have worked hard on increasing the system robustness and usability.
Unfortunately, obtaining the best sensitivity and the highest throughput is not always possible, as these two demands often counteract each other. Reducing flow rates increases the efficiency of the electrospray ionisation (ESI) process but at the same time increases the run time, and will also have an effect on the chromatographic performance. This results in the use of different instrumentation and the development of different methods which provide either ultimate sensitivity with limited throughput
Table 1. Flow rate regimes in HPLC
Flow range Analytical flow Micro Flow
Capillary Flow Nano Flow
or highest throughput with limited sensitivity. As a result, chromatographers need to consider carefully which requirement is more important to them.
Capillary LC-MS can provide the solution to the problem of delivering high sensitivity and throughput at the same time [4,5]. It is usually defined by the flow rate (1-10 µL/min) used, which is a consequence of using column diameters in the range 100-500 µm i.d. (see Table 1) [6]. As a consequence, the ESI source, which interfaces the LC with the MS system, has to be modified from the sources used for analytical flow rates (>100 µL/min). The inner diameter of the flow emitter needles used in analytical flow systems is too wide resulting in sample dispersion and reduced sensitivity when used in capillary systems. Efficient electrospray ionisation at analytical flow rates requires a supporting gas flow around the emitter, to aid the desolvation process. At lower flow rates, such as nano or capillary flow rates, this is no longer needed. Therefore, existing LC-MS sources designed for nano flow rates can often be adapted to capillary flow rates. This is done by using emitters with an appropriate inner diameter for capillary flow rates (20-50 µm). To facilitate operation these emitters are usually
>100 µL/min 10-100 µL/min 1-10 µL/min <1 µL/min
incorporated into easy-to-install transfer lines or chip based devices.
As mentioned previously, reducing flow rate results in increased sensitivity due to better desolvation of the analytes, a more efficient electrospray ionisation process and a higher sampling of analytes into the mass spectrometer [7]. Wilm and Mann observed an improvement in sensitivity of approximately two orders of magnitude for nano compared to analytical flow LC-MS systems [7]. The effect of the sample matrix on ionisation efficiency can also contribute considerably to sensitivity improvements, when using reduced flows. Unfortunately, the combined effects are hard to predict and would require experimental data for each analyte / sample combination. As a rule of thumb, however, capillary LC-MS has approximately a 10-20 times higher sensitivity than analytical flow LC-MS (with 2.1 mm columns), when optimised LC conditions and appropriate emitter needles are used (Figure 1). We have verified these findings with our own set-up and observed significant impact of the flow rate and column diameter on the sensitivity. At a given internal diameter sensitivity increases with lower flow rates.
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