3.1 Construction of the stand-alone nebulizer 20 cm × 10 cm

The Nebulizer for 10 cm × 10 cm HPTLC plates operated by the 2LabsToGo-Eco [11] was enlarged for use of up to 20 cm × 10 cm HPTLC plates and newly designed as an open-source stand-alone device with an integrated exhaust unit. The new Nebulizer 20 cm × 10 cm consisted of three parts, i.e., the 3D-printed casing, exhaust unit and control unit for stand-alone use (Fig. 1). A list of 20 components was compiled to be bought from nine different suppliers in advance (bill of materials, Table S1). The total material costs of 324 € consisted of 226 € for the Nebulizer 20 cm × 10 cm casing (including 2 × 50 atomizer discs), 64 € for the control unit, and 35 € for the exhaust unit. A detailed instruction for self-assembly, including 3D-printed parts, was written (Instruction S1). The CAD, STL, and KiCad files including the electronic parts list, and the software for the control board are freely available at https://github.com/OfficeChromatography/Nebulizer20x10.

Fig. 1

Newly developed Nebulizer 20 cm × 10 cm consisting of casing, control unit, and exhaust unit

3.1.1 3D-printed casing

The casing (Fig. 1) was designed with Autodesk Fusion 360 (www.autodesk.com), 3D-printed with polylactic acid (PLA, filamentworld.de) on a Prusa MK4+ 3D printer (www.prusa3d.com), and constructed from the 3D-printed parts, i.e., base with a 20 cm × 20 cm glass window, extension, and lid. The lid was designed to take up two atomizer holders (three-parted and 3D-printed with polyamide, Handtmann Lauramid PA12 natural, filamentworld.de) to guarantee the homogeneous reagent transfer on the HPTLC plate up to 20 cm × 10 cm (filling the two atomizer holders was needed also for smaller HPTLC plate pieces). The selected ultrasonic piezo atomizers of 5 µm or 11 µm pore size (Ø 16 mm, 108 kHz, PZT Electronic Ceramic, piezoelements.com) worked well for the exemplarily tested solutions and suspensions (Table 1).

Table 1 Nebulizer 20 cm × 10 cm application examples of tested solutions and suspensions successfully nebulized on HPTLC silica gel plates as well as parameters used (volume per atomizer holder, atomizer, and intensity of the control board)3.1.2 Exhaust unit

Although the Nebulizer 20 cm × 10 cm can safely be used inside a fume hood or clean bench, an exhaust unit widens the flexibility to use the Nebulizer 20 cm × 10 cm outside. The exhaust unit (Fig. 1) was constructed from 3D-printed parts (PLA), a housing containing a 12-V micro vacuum pump and a 250-mL washing bottle with a GL 45 screw cap and filled with water. Silicone tubings (6 mm × 4 mm) connected the vacuum pump to the Nebulizer 20 cm × 10 cm extension (by a hose coupling) and the washing bottle (Instruction S1). The 12-V power was supplied by the control unit that also allowed the vacuum pump to be switched on and off. Note that 2LabsToGo-Eco owners do not need this separate 12-V power supply as the exhaust unit also can be powered by the 2LabsToGo-Eco mainboard (external port) and controlled by the 2LabsToGo-Software (fan option in the finecontrol menu).

3.1.3 Control unit for stand-alone use

The control unit for stand-alone use (Fig. 2) consisted of a printed circuit board (PCB), designed in KiCad EDA (www.kicad.org) and equipped with the essential electronic parts to drive the piezo atomizers at 108 kHz, a potentiometer to manually adjust the energy (amplitude) and thus nebulizing speed, and a display showing the energy setting. Instead of using an oscilloscope to set the power inductor to the required frequency, an Arduino nano was attached to the board. With the functions of the avr timer register, an Arduino sketch (firmware) was written to control both the frequency and the amplitude of the atomizers. The value to control the frequency can easily be changed in the firmware, if an atomizer of a different frequency should be tested in the future (Instruction S2). The control unit board provides terminal blocks to connect the atomizers and the exhaust unit (see below) and is powered by a 12-V (1.5 A) power supply. The PCB board designed as small as possible (manual soldering the electronic parts should not need an electronic specialist) was mounted in a 3D-printed casing (Fig. 2). This way, the Nebulizer 20 cm × 10 cm can be used as a stand-alone device. Note that 2LabsToGo-Eco owners do not need this separate control unit because the two atomizer cables of the Nebulizer 20 cm × 10 cm can simply be connected to the 2LabsToGo-Eco mainboard to be controlled by the 2LabsToGo-Eco software.

Fig. 2

Nebulizer 20 cm × 10 cm control unit; view of the board with the electronic parts (A) and view of the 3D-printed housing (B)

3.2 Quantitative evaluation of the nebulization using the Nebulizer 20 cm × 10 cm

For operational qualification and quantification of the homogeneity of the nebulization process, a methanolic solution of the water-soluble blue food dye E 131 was nebulized onto a 20 cm × 10 cm HPTLC plate. After plate drying, an image was captured with the Visualizer 2 and evaluated with Gimp Image Manipulation software (Fig. 3). Already observed with the naked eye, the blue coloring was applied homogeneously. For the blue food dye E 131, the quantitative evaluation of the 8,044,784 pixels in both the blue channel and Value (grey) setting with Gimp revealed a mean intensity value of 0.86 (= B219) with a standard deviation of 0.011 (= 1.3%), giving proof of the very homogeneous nebulization of the E 131 solution onto the whole plate. Considering the subtraction of a blank plate image would improve the precision result (no performed here).

Fig. 3

Quantitative evaluation for the proof of the nebulization homogeneity: plate image after the application of E 131 solution with the Nebulizer 20 cm × 10 cm, and the evaluation results of Gimp Image Manipulation software. The somewhat bulging outer edges of the layer (10 mm marked by the frame) have been excluded from the evaluation

3.3 Nebulizer 20 cm × 10 cm application examples

Atomizers of two different pore sizes (5 µm and 11 µm) were studied. Aqueous or organic derivatization reagents and bacteria cell suspensions were preferably applied with the 5-µm atomizer, while yeast cell suspensions (including the comparatively large human androgen receptor yeast cells [13]) were preferably nebulized with the 11-µm atomizer. In some examples, it did not make a difference which atomizer pore size was used (Table 1). Exemplarily, two different fluorescence enhancer solutions, seven derivatization reagent solutions, three bioassay cell suspensions, and three substrate solutions were tested. As precautionary advice, corrosive reagents, such as hydrochloric acid, should be substituted by other non-corrosive acids. Advantageously, the substitution of a damaged atomizer disc costs only about 2 Euros.

In particular, biological and biochemical assay detections are able to detect known and unknown active compounds in complex samples [12,13,14], which is an important and unique feature in the whole field of chromatography. The operation including the cleaning process (< 5 min) of the Nebulizer 20 cm × 10 cm is described in detail (Instruction S3). The nebulization of the different solutions and suspensions tested [5, 6, 11, 14, 15] filled in each atomizer holder worked well using the indicated parameters (Table 1), whereby the listed volume was pipetted in each atomizer holder and the nebulization was immediately started to avoid potential droplet formation. Several plates using the same solution or suspension can be sprayed in sequence without intermediate cleaning. After plate drying, the plate images were recorded as indicated and showed very homogeneous plate backgrounds (Fig. 4). The zone patterns of the samples were detectable as usual (in reference to different parallel projects to be published separately). Thus, the resulting chromatograms and bioautograms successfully proved the excellent performance of the newly developed stand-alone Nebulizer 20 cm × 10 cm with integrated exhaust unit. Limitations of the Nebulizer 20 cm × 10 cm were not faced, but not all possible solutions and suspensions have been tested so far.

Fig. 4

Nebulizer 20 cm × 10 cm application examples of solutions and suspensions (Table 1) nebulized on HPTLC silica gel plates to detect the respective substances in the indicated samples for the given detection modes