Advanced Liquid Chromatography: Sample Preparation and Automation

  • Fig. 1: Overview of an online-SPE-LC-MS/MS system: 1) HPLC pump, 2) PAL RTC autosampler, 3) automated cartridge exchanger, 4) high pressure dispenser (HPD), 5) HPLC oven, 6) tandem mass spectrometer.Fig. 1: Overview of an online-SPE-LC-MS/MS system: 1) HPLC pump, 2) PAL RTC autosampler, 3) automated cartridge exchanger, 4) high pressure dispenser (HPD), 5) HPLC oven, 6) tandem mass spectrometer.
  • Fig. 1: Overview of an online-SPE-LC-MS/MS system: 1) HPLC pump, 2) PAL RTC autosampler, 3) automated cartridge exchanger, 4) high pressure dispenser (HPD), 5) HPLC oven, 6) tandem mass spectrometer.
  • Fig. 2: Flow paths for the online-SPE system: a) enrichment step and b) elution step.
  • Fig. 3: Overview of a compact two-dimensional HPLC system including the injection unit, switching valves and pumps.
  • Fig. 4: Schematic presentation of the modular microfluidic chromatography chip (SlipChip).
The measurement of samples by liquid chromatography is one of the last steps within the entire analysis cycle. Depending on the matrix, a more or less time consuming sample preparation is mandatory. This often involves clean-up and / or enrichment steps.
In times of rising personnel costs and the general trend of increasing efficiency in the analytical laboratory, methods for automated sample preparation like e.g. online solid phase extraction (online-SPE) are an attractive alternative to increase throughput and reduce the costs per analysis. Again, miniaturization shows a clear advantage over classic procedures, which will be discussed in the following.
Sample Throughput
The increase of sample throughput in all areas of analytical chemistry and life sciences is a criterion of high priority. In many cases “only” the step of measurement, for example HPLC-UV or HPLC-MS, will be considered and the sample throughput is just correlated with the time of the chromatographic run. Besides that, the remaining work steps such as sample preparation, evaluation and plausibility control of results as well as report generation take significantly more time. Therefore, it is useful to consider the overall process. The automation yields a valuable contribution to increase the sample throughput, especially if the system can shift personal intensive working steps in night or weekend hours where the lab business in many cases rests or is at least strongly restricted.
A fully automated sample preparation system (online-SPE-HPLC-MS/MS) including clean-up and enrichment steps is shown in figure 1. This system consists of a “prep and load robotic tool change” (PAL, RTC) autosampler with syringes of a volume of 100 µL, 1 mL and 10 mL, two injection valves equipped with different loops and an enrichment unit with a cartridge exchanger for the automated online-SPE. The first sample loop has a volume of 50 µL.

Therefore, the system can be used as a conventional HPLC system with the possibility of injecting smaller volumes. The second sample loop has a volume of 10 mL. Herewith the enrichment of the sample takes place on a disposable SPE cartridge. Optionally, washing steps to remove salts or polar matrix constituents can be performed. As it is apparent from the instrumental setup depicted in figure 1, a large space must be provided to properly arrange all system components. Furthermore, it can be clearly seen that relatively complex valve circuits and long transfer capillaries are necessary to connect all tools and modules. 

Theoretically, the system configuration shown in figure 1 can also be understood as a two-dimensional HPLC system by using the SPE cartridge not only for enrichment or clean-up but also for a pre-separation, if a cartridge with orthogonal selectivity compared to the HPLC column is used. Furthermore, two SPE cartridges can be coupled serially. By means of frontal elution with an organic solvent the analytes trapped on an SPE cartridge can be eluted in a small plug which is then transferred directly to the HPLC column. Via an auxiliary pump or, as shown in figure 1, via the high pressure dispenser (HPD), the organic plug can be diluted with water to ensure a sample focusing on the analytical column. Figure 2 exemplarily shows two flow paths for sample enrichment as well as elution.
In the field of microscale two-dimensional HPLC, there are first approaches to develop compact systems including gradient pumps and switching valves. Such a system is shown in figure 3. A critical point is the connection between the column and the emitter tip of the mass spectrometer ion source. As shown in figure 3, an approximately 25 cm long capillary is needed for this connection. Especially those volumes behind the column have a great influence on the extra-column band broadening because the chromatographic bands cannot be refocused. A big advantage of this system is that the required laboratory space can be reduced significantly compared to the set up shown in figure 1. The optimal solution in terms of space requirements would be a separation unit on a chip, which can be placed directly in front of the ion source inlet of the mass spectrometer. Such a design circumvents the problem of extra-column band broadening and thus represents the best possible hyphenation between chromatography and detection.
Even Smaller
Current approaches of research are aimed at not only miniaturizing the two dimensional unit on chip size, but also to integrate sample preparation steps. Such a design is shown in figure 4. Establishing this approach would lead to an enormous reduction of needed laboratory space. 
In principle, the microfluidic chip is built of two microstructured layers with cavities and channels, which can be used as reservoirs and flow channels. By sliding these layers, micro cavities of upper- and lower level can be united and closed again. Moreover, flow channels can be generated by adding complements of structures. This approach is extremely innovative because it does not need chip integrated valves and thus promises an increased degree of robustness. Beyond that, extra-column volumes are extremely low, because this chip architecture includes the injection unit and a low dead volume connection between the chip and the MS is possible if the emitter is also integrated on the chip. It is to be noted that the miniaturization of the separation unit is only one important step for designing portable systems. To achieve the overall goal of miniaturization, all peripheral components such as pumps and detectors have to be miniaturized.
Despite the enormous advantages which are provided by chip technology, it is still a long way from research to routine. It can be clearly seen that the strength of integrated miniaturized systems is that all components such as sample clean-up and enrichment, one- or two-dimensional chromatography and possibly further functionalities like for example the integration of mixing or auxiliary channels for feeding ionization additives for sensitive MS detection can be combined on a single chip.
The authors would like to thank for financial support from the German Federal Ministry of Economic Affairs and Energy within the agenda for the Promotion of industrial cooperative research and development (IGF) based on a decision of the German Bundestag (IGF – Project No. 18199 BG).
T. Teutenberg1, T. Hetzel1, D. Loeker1, J. Leonhardt1
1Institut für Energie- und Umwelttechnik e. V., IUTA, Duisburg, Germany
Institute for Energy and Environmental Technology e. V. (IUTA)
Department Head research Analysis
Duisburg, Germany


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