Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - STAMP (Separation Technology for A Million Peaks)

Teaser

The field of liquid chromatography (LC, i.e. liquid phase separations of mixtures) is challenged with increasingly complex mixtures. The current set of LC methods present a trade-off between separation time and the ability to separate complex samples: fast LC methods, with...

Summary

The field of liquid chromatography (LC, i.e. liquid phase separations of mixtures) is challenged with increasingly complex mixtures. The current set of LC methods present a trade-off between separation time and the ability to separate complex samples: fast LC methods, with typical separation times in the order of minutes, fall short in their ability to handle complex mixtures by several orders of magnitude. On the other hand, the current state-of-the-art in separating complex mixtures, known as 2D-LC, requires separation times in the order of hours.

The STAMP project aims to develop a new method of ultra-fast separations of complex mixtures such as those found in proteomics. The principle of the process differs from merely adding a dimension to existing 2D-LC setups. The method involves spatially fractioning the mixtures for two steps (i.e. in two dimensions) and eluting the fractions in the third step (or dimension). Crucially, the entire process should be performed within a chip as opposed to a typical LC column and system. The aims of the project therefore center around the production and functioning of a device for three-dimensional separations (as illustrated in Figure 1), referred to as the STAMP device.

The aims of the project can therefore be listed as follows:
1. Computationally studying the novel design aspects necessary for the STAMP device.
2. Developing manufacturing methods to create the STAMP device (e.g. various 3D-printing methods)
3. Studying the principles of retention necessary for the fractioning and separation within the device
4. Developing methods to implement stationary phase materials in-chip,
5. Devising new processes to detect and identify the separated analytes eluting out of the STAMP device

Work performed

The research activities thus have focused on developing and optimizing key enabling methods to address aims 1-5. A representative but non-exhaustive description of the results is provided here. Several aspects of the STAMP device have been explored computationally (example shown in Fig. 2). These include flow distributor designs, permeabilites of the first-, second- and third-dimension regions and structures of the second- and third-dimension regions.

An issue highlighted by the CFD simulations was the first-dimension fluid (or mobile phase) remaining in the first-dimension channel during the second-dimension step (Type II geometry in Fig 2). This could be fixed by the decreasing the permeability of the first-dimension region (Type V in Fig 2) but only at the expense of efficiency in the first-dimension step. To overcome this trade-off, novel flow-confinement methods have been developed by the STAMP project. Figure 2 shows the TWIST (Two-dimensional Insertable Separation Tool) valve concept where the first-and second-dimension regions are two separate pieces where their channels can be aligned (to open the valve) or one piece can be rotated to misalign (for a closed state).
Another flow-control method developed for the STAMP device is the freeze-thaw valve. Here, channels are enveloped by two sets of jackets. One jacket operates below the solvents’ freezing point and creates a frozen barrier (as shown in Fig 3), with the other jacket maintaining an ideal temperature for chromatographic separations.

Both these flow-control methods have been developed for the STAMP device and help address the trade-off between efficiencies in each stage of the three-dimensional separation. In addition to flow-control, the freeze-thaw concept provides thermal control over the entire device. A class of porous stationary-phases, necessary for chromatographic retention, are typically synthesized in ovens or water-baths at 70˚C. Here, the freeze-thaw setup is used to selectively create stationary-phases into targeted zones (e.g. the first-dimension region) and freeze the non-targeted zones. Figure 3 shows a demonstration of this process in a 3D-printed titanium device. A robust, reliable method for this synthesis in-chip¬ has been developed under the STAMP project.
In addition to demonstrating chromatographic separations on printed titanium devices, the STAMP project has developed methods to perform an electro-driven separation method (called isoelectric focusing) and RPLC in parts 3D-printed using polypropylene (PP). PP offers a low-cost in-house production option, with resolutions sufficient for the STAMP device. Figure 4 shows a stationary phase synthesized within a printed PP device.

The yeast proteome was digested and separated using comprehensive 2D-LC (shown in Fig 4). The combination of HILIC and RPLC (Hydrophilic interaction chromatography and Reverse phase liquid chromatography, respectively) has been found to provide complementary, or orthogonal separation methods for use in a STAMP device. Hyphenation to a high-resolution MS will provide improved identification to more accurately describe the two retention mechanisms.

Two patents were applied for, i.e. EP18184801.1, Device for Multi-Dimensional Liquid Analysis, by T. Adamopoulou*, S. Deridder, G. Desmet, P.J. Schoenmakers* (* denoting a STAMP-team member) and EP19170376.8, Stereo-lithographic 3D-printing assembly and stereo-lithographic 3D-printing method, by S. Nawada*.

Final results

Several enabling methods and techniques have been developed to address Aims 1-5 thus far. Further progress beyond state-of-the-art in the field involve implementing these methods into devices for two- and three-dimensional spatial separations. These include:
• Using CFD to obtain further insight obtained from simulations for the operation of the spatial 2D and 3DLC devices and design optimization of the spatial 3DLC device. More specifically, the effects of analyte spillover into the third-dimension zone will be investigated
• Implementing the developed flow control methods (TWIST and Freeze-thaw) into the STAMP device.
• Combining electro-focusing and reverse phase LC separations on a single polypropylene chip. Currently, both these methods have been established in standalone 3D-printed polypropylene devices.
• Using targeted synthesis of stationary phases to create a two- and three-dimensional separation device with two or three separate stationary phases in 3D-printed titanium devices
• Develop a performance metric to determine the complementary nature (known as orthogonality) of the three separation mechanisms in the STAMP device.
• Using the current detection stage built for the STAMP platform to perform matrix-assisted laser desorption/ionization (MALDI) and Surface enhanced Raman spectroscopy (SERS)

Because of the significant progress made regarding all sub-targets, a working prototype of a device for 3D spatial separations is still anticipated as the final deliverable of the STAMP project.