Are the Lights on Green?
Micro-LC’s Road to Routine Analysis
- Fig. 1: Van-Deemter curve for Naphthaline in dependence of the particle diameter of the stationary phase (YMC Triart C18, 50 x 0.3 mm, ❍ 1,9 μm, ■ 3.0 μm) at a temperature of 30 °C.
- Table 1: Information on the important parameters of the separation efficiency of commercially available Micro-LC columns. Stationary phases: see figure 1.
- Fig. 2: Schematic presentation of a multi detection process consisting of a flexible UV-cell and a mass spectrometer.
- Fig. 3: Separation of 15 polycyclic aromatic hydrocarbons. Stationary phase: YMC-PAH, 50 x 0.3 mm, 3.0 μm, Flow rate: 20 μL min-1, Temperature: 26 °C, Injection volume: 10 μL, Detector cell: Single Channel Fluorescence Cell Prototype.
It seems the time is ripe for Micro-LC’s comeback. At least that was the general tenor at the “Hyphenated Techniques in Chromatography” conference, held in Ghent at the beginning of this year.
Compared to the “classic” (U)HPLC-systems, Micro-LC offers a whole series of advantages. Its drastic reduction of toxic and expensive solvents is still a powerful argument for using miniaturised separation techniques. In addition, separation columns with a smaller internal diameter used with a constant injection volume, achieve a lower dilution of the elution band. This is a deciding factor for many applications in bio-analysis if the sample material is limited.
In spite of the above mentioned advantages there are also prejudices with regard to miniaturised analysis systems. A primary aspect is the commercial availability of the HPLC-systems that can be used with Micro-LC. Using a flow splitter makes it possible to use Micro-LC columns with classic systems. However, using such a system does not make sense if one wants to fully exploit the separation performance of the column, as the smaller the internal diameter of the column, the more important the minimisation of all system- and dead volumes. Contrary to common belief, dedicated Nano- or Micro-LC systems have been commercially available for the last 10 years. The company Eksigent (now part of Sciex) developed one of these devices based on the concept of microfluidic flow control. Pneumatic piston pumps facilitate a constant and steady transfer of the eluent, drastically reducing the consumption of solvent compared to the classic systems. The solvent gradient is very simply generated: the liquid flows of both mobile phase channels converge via a T-piece and mix passively due to the radial diffusion in the capillary tube. As an additional mixer is not necessary the dwell volume of the gradient can be reduced to a microliter. E.g. if an analysis is to be carried out using a flow rate of 50 µL min-1, the delay time for the gradient would only be 1.2 seconds. This means that extremely fast cycle times and therefore a higher sample throughput can be achieved.
A secondary aspect refers to the widely held belief that columns with an internal diameter of < 2.1 mm are more difficult to fill and would therefore exhibit much worse separation efficiency than classic separation columns with internal diameters of between 2.1 mm and 4.6 mm.
Therefore, in a recent study, the question of what separation efficiency could actually be achieved if Micro-LC columns with an internal diameter of 300 µm were used on a Micro-LC system, was addressed. A van-Deemter analysis for 2 stationary phases with particle diameters of 1.9 µm and 3.0 µm was carried out. In figure 1 it can be clearly seen that the column with the bigger particles achieves a higher plate height (H) and therefore provides poorer separation efficiency. This is because the eddy diffusion for bigger particles leads to a higher A-term in the van-Deemter equation. To compare the packing quality of the two columns, the minimum reduced plate height (hmin) was then calculated. For well-packed separation columns this should be in the range of 2.0 to 2.5. From the data in table 1 it can be seen that the column filled with sub 2 µm particles almost fulfils this condition, whereas the column filled with the 3.0 µm particles has the much higher value of 4.03. A possible explanation for this result is that the ratio between the internal diameter of the separation column and the particle diameter of the stationary phase is more favourable for the separation column filled with the sub 2 µm particles. It can therefore be filled more homogenously.
At this point it is important to note that the database available in scientific literature at present, is not sufficient for making generalized conclusions. In the main, it can be said that to date only a few manufacturers of columns have miniaturised separation columns with an internal diameter of < 1 mm in the product portfolio. This is because Micro-LC is currently not considered a standard method in routine or research laboratories and the demand for the necessary separation columns is therefore low.
Coupling Micro-LC with mass spectrometry (MS) is relatively easily accomplished without having to make serious changes to the MS-detector. Several manufacturers offer the possibility of exchanging the standard emitter tip on the available ESI source of the mass spectrometer. In contrast, when using Nano-LC, a special Nano-electrospray source has to be used for the mass spectrometer and this is generally difficult to adjust. Although mass spectrometry is well established in many areas of the life sciences, there are several applications where a mass spectrometric detector is not necessary. A good example is the field of quality control in the pharmaceutical industry. UV detection is the “Gold standard” here. The application of other detection techniques such as fluorescence or light scattering detection is of importance in many fields, which means that the availability of miniaturised HPLC detectors is a further necessary criterion for the possible comprehensive utilization of Micro-LC. Even though there are many different UV detectors that are appropriate for Micro- or even Nano-LC, relatively long transfer capillary tubes are often needed to connect the column to the detector. This is due to the widely used modular design of HPLC systems, which are built up like traditional “Hi-Fi” systems. An elegant way to reduce the extra column volume is to position the detector cell immediately behind the separation column and to set up the optical connection with an optical fiber. This is particularly advantageous when an additional MS-detector is being used, as the UV cell can be integrated directly between the separation columns and the ion source – as shown in figure 2.
Commercial UV detectors for Micro- and Nano-LC, based on optical fiber technology are available from the company Knauer. In addition, during a research project, we developed a Micro-LC compatible fluorescence detector whose cell is also driven by fibre optic cables. The detector volume (0.05 - 5 µL) depends on the capillary diameter. Figure 3 is an exemplary presentation of the separation of 15 polycyclic aromatic hydrocarbons. The detection limit with an injection volume of 10 µL is between 1 ng mL-1 und 10 ng mL-1 depending on the analyte being used.
Preparation of Samples
In addition to the aspects of chromatography and detection, the preparation of samples also plays a deciding role. Connecting sample robots to miniaturised HPLC systems is quite easily manageable in many cases, as system components are already commercially available. This guarantees that the whole analytic workflow can be mapped. A more critical point becomes apparent in the connecting and operating of the components as a whole. Many products in the field of miniaturised separation techniques are currently stand-alone solutions. Problems occur for example in the connecting and operating of a complete system that consists of two or more single components from different providers. So called “Master Software” such as Chronos (Axel Semrau) or Chromeleon (Thermo Fisher Scientific), needs to be developed further so that a simple and complete integration of different system components is possible.
Although the technical requirements for the implementation of Micro-LC in routine laboratories are fulfilled, the lights are still on amber. Micro-LC will only experience its “comeback” when all obstacles have been overcome and when automated sample preparation can be integrated in the miniaturised workflow. With this in mind, now is the right time to have your foot on the accelerator if you don’t want to miss the start.
The KF2025430AK3 research project was sponsored by the Federal Ministry of Economics and Technology (BMWi) as part of the ministry’s “Central Innovation Program for Medium Sized Companies” which was introduced on the basis of a decision in the German Bundestag.
Thorsten Teutenberg1, Terence Hetzel1, Denise Loeker1, Juri Leonhardt1, Philipp Schulze2
1Institut für Energie- und Umwelttechnik e. V., IUTA, Duisburg, Deutschland
2Max-Planck-Institut für Kohlenforschung, Mülheim (Ruhr), Deutschland
Dr. Thorsten Teutenberg
Institut für Energie- und
Umwelttechnik e. V. (IUTA)