Miniaturized HPLC Systems

Science or Fiction?

  • Dr. Thorsten TeutenbergDr. Thorsten Teutenberg
  • Dr. Thorsten Teutenberg
  • Dr. Juri Leonhardt
  • Dr. Terence Hetzel
  • Fig. 1: Schematic comparison of HPLC systems Anno 1980 and Anno 2018.

In the academic environment, micro-LC played a major role in the early commercialization of HPLC systems in the 1960s. Although the technical requirements were significantly worse than today, many university research groups have succeeded in successfully demonstrating the much-cited advantages of miniaturized HPLC systems in the form of proof-of-concept studies.

The biggest obstacle to the widespread use of micro-LC in routine and research laboratories was the reduction of critical system volumes. Following the successful market introduction of UHPLC technology at the beginning of the new millennium, the conditions were ideal for exploiting the potential of micro-LC for routine applications. In fact, it is noted that the development of HPLC systems designed for low flow rates has recently gained considerable momentum. The manufacturers pursue different technical approaches. While some of them focus on retrofitting their existing systems, others design special low-flow systems, some of which take up less space.

To ensure high separation efficiency when using columns with an inner diameter (ID) of 300 µm, all volumes leading to band broadening must be significantly reduced. We have explained this in detail in the article „Micro LC Basics” [1].

In addition to reducing dead and system volumes, a micro-LC system must be capable of generating a flow rate between 10 µL min-1 and 50 µL min-1. This can be achieved using two different approaches.


In one variant, a conventional system designed for „high-flow“ applications is technically modified. This can be done e.g. via an electronic flow control. A micro- or nano-flow is generated by real-time adjustment of the flow rate in the form of a flow splitter in front of the separation column. Furthermore, the gradient dwell volume (GDV) must be reduced, e.g. by removing the mixer or mixing chamber. At flow rates of less than 50 µL min-1, a precise mixing of the eluent streams is already achieved by the axial diffusion in the capillaries if the diameter of the capillaries is less than 50 µm [2].

Hence, a simple mixing tee is sufficient.

A glance at the technical specifications of „classic“ UHPLC systems reveals that the pumps are often specified for a micro-flow of up to 10 µL min-1. If the GDV is 50 µL and the analysis is performed at 10 µL min-1, it would take five minutes for the gradient to reach the column. Short cycle times cannot be achieved in this way. In the second variant, for example, pneumatic syringe pumps are used that generate a real micro or nano flow. The advantage is that solvents are saved because the eluent flow does not have to be split up in front of the column.

The second point concerns the injection volume. As we explained in the article „The injection, the unknown and complex being” [3], technical solutions exist for reproducibly transferring volumes of less than 100 nL to the separation column using standard autosamplers. A disadvantage of conventional systems that can be technically modified for micro or nano HPLC applications, is the sometimes considerable distance of more than 50 cm between the injector and the separation column. This can lead to a strong band broadening in front of the column, as well as to long delay times until the injection plug actually reaches the column. All this should be taken into account, even if the technical specifications of the manufacturers specify a micro-LC flow.

The third point concerns the detector. As explained in the article „The Detector“ [4], a technical solution exists for each type of detection, which is suitable for coupling with micro-LC and only leads to a negligible band broadening. Coupling with mass spectrometry is particularly well suited because band broadening can be reduced by simply exchanging the emitter tip. Unfortunately, when using spectroscopic detectors, the reduction of the system or cell volume is always associated with a loss of signal intensity.

With regard to the general system design, many HPLC systems that can be used as micro LC systems are characterized by a classical structure. This is illustrated in Figure 1. Although current micro-LC systems incorporate many technical innovations, not much has changed in terms of space requirements.

Low Flow

Some companies have introduced numerous innovations to the market in recent years. These are mainly special pump systems. However, most of them are stand-alone solutions and therefore not integrated into an overall system. The problem for the user is then to combine different individual modules into a communicating overall system that can be controlled via a single software. It is precisely this problem that is currently delaying the large-scale use of miniaturized HPLC systems. No routine laboratory has the human and/or technical resources to perform development work. Manufacturers are still required to provide complete solutions for the customer. Large companies generally focus on more profitable market segments. Unfortunately, micro-LC is still a niche technology. Many SMEs are innovation drivers with highly specialized individual products. However, they have neither the appropriate marketing capacities nor sufficient research resources to solve the problem of holistic system integration. This is only possible in a joint network.


The development of increasingly robust low-flow systems is currently being addressed by all manufacturers. The isolated solutions mentioned have their charm, but currently there is still a lack of concepts to combine different modules from different manufacturers into a holistic approach for the user.

On the other hand, many industries lack the courage to introduce a new technology that even pays for itself relatively quickly. Again and again, it is noticeable that academic problems are used to justify the disadvantages of miniaturized HPLC systems for industrial routine analysis. In many cases, this concerns the separation performance. With reference to scientific results published in renowned peer-reviewed journals, critics refer to the lower system or separation efficiency. A closer look and analysis of these statements reveals that these alleged disadvantages play no or at least a subordinate role in practice. Another point concerns the detection sensitivity of the overall procedure. Micro-LC-MS coupling has not established itself – at least at the present time –as the method of choice when the focus is on an extremely low detection limit. One reason for this is that the interface between the HPLC and the MS or the ion source have not been optimized for the micro-LC flow in the range between 10 µL min-1 and 50 µL min-1. If it is possible to eliminate this fundamental disadvantage, many environmental analytical laboratories would certainly consider micro-LC-MS coupling.

On the other hand, there are many fields of application in the pharmaceutical industry where high sensitivity is not a priority. There would be a great deal of leverage here, to use the new technology and take advantage of the reduced consumption of resources and space. Finally, the question arises as to how far chip technologies will replace capillary-based systems. A major advantage of chip-based systems is the integration of the connection points between the injection, separation and detection unit. With a design that is easy for the user to handle, this means that installation or integration of the separation unit into the overall system is very simple. With the IonKey from Waters, such a technology is already available today. The far greater advantage of chip-based systems, namely the integration of reaction and flow channels for carrying out chemical reactions and simultaneous separation of reaction and synthesis products, is not commercially available, although there are numerous examples in the scientific literature that clearly demonstrate the benefits of so-called „vest-pocket laboratories“. When implementing chip technology, not only the flexibility of chip design production but also the costs must be considered. A frequent change of a chip only makes sense if the manufacturing costs are correspondingly low.

Do you have problems with micro- or 2D-LC? Ask the experts at:

Thorsten Teutenberg1, Terence Hetzel2, Juri Leonhardt3


Dr. Thorsten Teutenberg studied Chemistry at Ruhr University Bochum. Here, he studied for a doctorate in Analytical Chemistry, submitting a thesis on “High-temperature HPLC”. In 2004, his career took him to the Institute of Energy and Environmental Technology (IUTA) in Duisburg as a research associate. Since 2012, he has been in charge of the Research Analysis Department, mainly working on the various aspects of high-temperature HPLC, miniaturized separation and detection techniques, and multi-dimensional chromatography processes.


Dr. Juri Leonhardt finished his studies with the focus on instrumental analysis and laboratory management at the University of Applied Sciences in Krefeld in 2011. Afterwards he began his doctorate at the faculty of chemistry at the chair of “Instrumental Analytical Chemistry” at the University Duisburg-Essen. His research was focused on the development of miniaturized multidimensional liquid chromatography systems on the basis of nano and micro liquid chromatography and their hyphenation to different detection techniques. Until 2017 he was research assistant at the department of Research Analysis at the Institut für Energie- und Umwelttechnik e. V. (Institute of Energy and Environmental Technology) in Duisburg. Since 2018 he has been head of laboratory at the department of production analytics at Currenta GmbH & Co. OHG, Dormagen, Germany.


Dr. Terence Hetzel studied “Instrumental analysis and laboratory management” at the University of Applied Sciences in Krefeld. From 2013 to 2017, he did his PhD thesis at the faculty of chemistry at the chair of “Instrumental Analytical Chemistry” at the University Duisburg-Essen. His research was primarily focused on the characterization and development of miniaturized separation techniques in combination with mass spectrometry. From 2012 to 2017, he worked as scientist in the department research analysis at the Institut für Energie- und Umwelttechnik e. V. (Institute for Energy and Environmental Technology) in Duisburg. In 2017, he joined the Research and Development division of the Bayer AG and acts since then as lab head in the field of bioanalysis in Wuppertal.

1Institut für Energie- und Umwelttechnik e. V., IUTA, Duisburg, Germany
2Bayer AG, Wuppertal, Germany

Dr. Thorsten Teutenberg

Institut für Energie- und Umwelttechnik e. V. (IUTA)
Duisburg, Germany

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Split or Spitless: Pumps

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The Detector


Institut für Energie- und Umwelttechnik e.V. (IUTA)
Bliersheimer Str. 60
47229 Duisburg
Phone: +49 2065 418 0
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