Universal or Selective
- Fig. 1: Optimized system design when coupling a micro-LC system to the mass spectrometer.
- Fig. 2: Schematic representation of the coupling of a flexibly positionable UV cell with a mass spectrometer.
- Fig. 3: Comparison of peak capacity (np) when using a system-integrated micro-LC UV detector (black curve) and the UV cell (Knauer, red curve). The data were determined for gradient times (tG) of 0.15 to 0.5 minutes using a column packed with sub-2 μm particles.
- Fig. 4: Separation of steroids by evaporative light scattering detection. Red trace: conventional ELSD; black trace: Sedere 90 LT. Chromatographic conditions: Flow rate: 45 µL min-1; mobile phase: (A) water, (B) methanol; solvent gradient: see illustration; stationary phase: Phenomenex Kinetex C18 300 µm x 50 mm, 2.6 µm; injection volume: 0.15 µL; temperature: 60°C; analytes: 1: estriol; 2: 1,4-androstadiene-3,17-dione; 3: boldenone; 4: β-estradiol; 5: testosterone; 6: trans-dehydroandrosterone; 7: epitestosterone; 8: etiocholane-3α -ol-17-one.
In recent years, mass spectrometric detectors have increasingly found their way into analytical laboratories. Although the UV detector is still “the” standard HPLC detector, it is likely to be replaced by simple mass spectrometers (MS) in the near future. Why the coupling between micro-LC and MS is a true “marriage in heaven”, we would like to explain below.
Electrospray ionization (ESI) is the most commonly used ionization technique for coupling HPLC with MS. The interface between the HPLC and the mass spectrometer has the task of vaporizing the liquid mobile phase and generating analyte ions in the gas phase.
The evaporation of a relatively small liquid volume leads to a large volume of gas, which must be removed before the inlet into the high vacuum of the mass spectrometer. It is therefore obvious that the miniaturization of the HPLC and the associated reduction of the absolute flow rate to a few microliters per minute represent a clear advantage in terms of the HPLC-MS coupling. In order to maintain the quality of the separation, it is necessary to minimize all system volumes that contribute to a significant loss of separation efficiency. As we have already explained in the article “Micro-LC Basics”, all connecting capillaries between the injector and the column as well as between the column and the detector should have the smallest possible volume. This means adapting both the length and the inner diameter (ID) of the capillaries. For micro-LC columns with an ID of 300 µm, the ID of the connecting capillaries should be 50 µm, as we illustrated in the article G.I.T’N’T (capillaries). The positioning of the HPLC system and the mass spectrometer should be carried out in such a way that distances are as short as possible. Fig. 1 shows a configuration in which the micro-LC system is placed on top of the MS. Here, a column oven which can be freely positioned in space, can significantly reduce the distance between the injector and the column as well as between the outlet of the column and the inlet to the ion source.
It is also necessary to adjust the inner diameter of the emitter tip. If the ID of the emitter tip is significantly larger than 50 µm, this results in a loss of separation efficiency. Thus, the bands separated in the column will partially or even completely coelute. In many cases, the user cannot replace the emitter tip. However, some manufacturers offer the possibility of using emitter tips with different IDs, so that there is virtually no loss of the column’s intrinsic separation performance when using micro-LC-MS. Changing the emitter tip can then be done within a few minutes and means almost no effort for the user. Further modifications to the mass spectrometer are not necessary in this particular case, i. e. the standard ESI source can be used for both high-flow and low-flow applications. This is a decisive advantage over special nano-ESI sources. In some cases, considerable effort is required in the exact alignment and positioning of the emitter tip in order to achieve good spray stability and correspondingly high ionization efficiency.
In addition to mass spectrometry, other detection techniques such as UV, fluorescence or light scattering detection still play an important role. The availability of classic HPLC detectors in miniaturized format is therefore a further necessary criterion to use the micro-LC as comprehensively as possible in all relevant fields of application. Although there are a variety of different UV detectors suitable for micro or even nano-LC, relatively long transfer capillaries are often required to connect the column to the detector. This is due to the modular design of HPLC systems, which are constructed like a traditional “Hi-Fi” system. An elegant way to reduce the extra-column volume is the positioning of the detection cell directly behind the separation column and its optical coupling via fiber optic cables. This is particularly advantageous because the UV-cell can be integrated directly into the flow path between the column and the ion source of the mass spectrometer, as is shown schematically in figure 2.
Commercial UV detectors with a decoupled detection cell for micro- and nano-LC based on fiber optic technology are available. In a recent study, Hetzel et al. have demonstrated that a significantly higher peak capacity can be obtained with a new detection cell (Knauer) compared to a “standard micro-LC-UV detector”. The black curve in figure 3 represents the peak capacity as a function of gradient time when the standard UV detector of the micro-LC system is used, while the red curve is obtained when the standard UV detector is replaced by the new detection cell. For a gradient time of 0.5 minutes, the difference in peak capacity is already greater than 30% if a column filled with sub 2 µm particles is used. This example impressively proves that even small extra-column volumes have a large influence on the separation performance of the overall system.
The difference becomes even more impressive when a conventional ELSD (Evaporative Light Scattering Detector) and an ELSD developed for micro-LC are compared. Such detectors are used whenever the analytes have no chromophore and cannot be determined with UV detection. figure 4 shows two chromatograms. The red trace results if an ELSD suitable for conventional HPLC is used. The black trace is obtained if a detector modified especially for micro-LC (Sedere) is used. It can be clearly seen that the system volume of the conventional ELSD is far too large, so that no signal is obtained.
Although the availability of miniaturized detectors that are compatible with micro-LC is still limited, there is an appropriate system solution for micro-LC for almost every detection method. The micro HPLC-MS coupling is very easy to implement if the emitter tip of the mass spectrometer is interchangeable and its ID can be freely selected. Recent developments in the field of UHPLC have made it necessary to design ESI sources for higher flow rates up to 1 mL min-1. This, in turn, is a trend that opposes micro-LC coupling. It remains to be seen whether the MS manufacturers continue to pursue the goal of designing the conventional ESI sources for higher flow rates or whether a rethinking is taking place at this point.
Thorsten Teutenberg1, Terence Hetzel2, Juri Leonhardt1
1Institute for Energy and Environmental Technology e. V. (IUTA), Duisburg, Germany
2Bayer AG, Wuppertal, Germany