Seamless Combination of High-Pressure Chip-HPLC and Droplet

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Seamless combination of high pressure chip-HPLC and droplet microfluidics on an integrated microfluidic glass chip Renata Gerhardt, Andrea J. Peretzki, Sebastian K. Piendl, and Detlev Belder Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04331 • Publication Date (Web): 02 Nov 2017 Downloaded from http://pubs.acs.org on November 5, 2017

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Analytical Chemistry

Seamless combination of high pressure chip-HPLC and droplet microfluidics on an integrated microfluidic glass chip Renata Gerhardt, Andrea J. Peretzki, Sebastian K. Piendl, Detlev Belder* Institute of Analytical Chemistry, University of Leipzig, Linnéstraße 3, 04103 Leipzig ABSTRACT: We introduce an approach for the integration of high performance liquid chromatography and droplet microfluidics on a single high pressure resistant microfluidic glass chip. By coupling these two functionalities, separated analyte bands eluting from the HPLC column are fractionated into numerous droplets in a continuous flowing oil phase. The compartmentalisation of the HPLC-eluate in a segmented flow was performed with droplet sizes of approximately 1 nL and with droplet frequencies reaching up to 45 Hz. This approach prevents peak dispersion and facilitates post column processing of chromatographic fractions on chip. A reliable generation of droplets is also possible in reversed phase gradient elution mode as demonstrated by applying a solvent gradient from 20% to 100% acetonitrile. A chip design with an incorporated dosing unit enabled the directed post-column addition of reagents to individual droplet fractions. The capability of this dosing function was successfully evidenced by post column addition of a reagent which quenches the fluorescence signal of the analytes. The chip-integration of gradient HPLC, fractionation, detection and post column addition of reagents opens up new avenues to perform multistep chemical processes on a single lab-on-a-chip device.

One of the most appealing features of lab-on-a-chip technology is the building block-like ability to integrate various operations such as synthesis, separation and detection on a single device1–3. This is especially appealing for chip chromatography4–6, as the microchip format allows monolithic and dead-volume free interconnections, eliminating extra column band broadening and void or swept volumes7,8. However, if such an HPLC chip is coupled to the outer world, this is usually performed using traditional HPLC tubings and fittings, and it is often extremely challenging to preserve the high separation performance. This problem can be avoided if as many processes as possible are carried out on the same device, thereby avoiding off-chip transfer altogether. Accordingly, detection of the separated analytes is usually performed onchip, either optically or, more prominently, by an integrated ESI-emitter for MS coupling9,10. While sample injection11 or preconcentration12 can also be integrated on the same device, fractionation for post collection processing is more challenging. Such a combination of preparative HPLC and fraction collection, including subsequent treatment of purified compounds, is a well-established workflow in synthetic chemistry and drug development13–15. An appealing microfluidic analogue to such traditional technology is the combination of chip-HPLC and droplet microfluidics. Herein, the column eluate is fractionated into small segments by introduction of an immiscible phase directly after the separation is complete. The eluate is thereby segmented into discrete droplets, separated from one another by the continuous carrier stream, which prevents peak dispersion. This approach connects chipseparations with the field of droplet microfluidics16–21 and provides access to the droplet microfluidic toolbox for controlling processes in segmented flow, such as droplet splitting, merging, sorting, extraction or addition22–25. Due to its powerful capabilities droplet microfluidics has found wide-spread

applications in areas such as biochemical analysis26–29, particle synthesis30,31, cell interrogation32,33, drug discovery34 microbiology35, as well as for high-throughput screening36–40, and for studying and optimising chemical transformations at the nanoscale41,42. The combination of HPLC and droplet microfluidics was first accomplished by coupling (commercially-) packed nanoLC capillaries with a chip interface, which included a droplet generator, enabling the separation and analysis of biological samples43–48. Interconnection of two conventional separation capillaries via an intermediate chip-based droplet generator is an interesting tool for interfacing capillary HLPC and capillary electrophoresis49,50. The first pioneering works, which integrated separation and droplet functionality on a single device, showed the combination of chip electrophoresis and droplet microfluidics51. Recently, a related approach was used to combine capillary gel electrophoresis and downstream droplet generation on-chip52. While chip electrophoresis is applied at ambient pressure, the system integration of pressure-driven liquid chromatography with droplet microfluidics is more challenging. In addition to the necessity of employing high pressure resistant chips and world-to-chip interfaces, the high pressure pumping of the HPLC-eluate must not negatively affect the delivery of the dispersing oil carrier phase, which is usually at low pressure. For regular fractionation frequencies, highly precise and stable flowrates have to be ensured for both pressure regimes. A first approach for interfacing chip LC and droplet microfluidics on a single device was accomplished by Kim et al. utilizing a thermoset polyester (TPE)/PDMS hybrid chip53. Later this was extended to the integration of a monolithic porous polymer as column material54. In these initial studies it could be elegantly shown that such an integrated approach avoids dispersion during compartmentalisation. The chroma-

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tographic performance of this chip was, however, rather limited as evidenced by plate numbers of 19 and 63 for the two separated fluorescent dyes AF 488 and FITC. As stated by the authors an improved chromatographic separation would need an enhanced column quality. However, as the proper use of such high quality HPLC-type columns on chip is accompanied with relatively high pressure55,56 this would require microfluidic chips with pressure resistance much higher as the 15 bar reported for the TPE polymer chip. This is the starting point of this work where we intend to integrate a high performance chip LC column with droplet microfluidics functionalities on a single high pressure resistant glass chip. Furthermore, this approach will be significantly extended by investigating the compatibility of droplet generation with gradient elution as well as the integration of additional functionalities for droplet processing.

Experimental Chemicals and materials Analytical standard grade 2-acrylamido-2-methylpropane sulfonic acid, anthracene, 7-amino-4-methylcoumarin, azobisisobutyronitrile, butyl acrylate (BA), 1,3-butanediol diacrylate (BDDA), 2,2-dimethoxy-2-phenylacetophenone (DMPA), fluoranthene, 3-(trimethoxysilyl)propyl methacrylate (z-6030), benzo[a]anthracene, benz[a]-pyrene, isooctane, perfluorodecalin and trichloro(1H,1H,2H,2H-perfluorooctyl)silane were purchased from Sigma–Aldrich GmbH (Taufkirchen, Germany). Anthracene-9-carbaldehyde and KI were purchased from Merck KGaA (Darmstadt, Germany). Krytox® 157FS was purchased from DuPont de Nemours GmbH (Neu-Isenburg, Germany), HPLC-grade acetonitrile was purchased from Carl Roth GmbH+Co.KG (Karlsruhe, Germany) and high-purity water was provided by a Smart2Pure purifying system (18.2 MΩ cm, TKA Wasseraufarbeitungssysteme GmbH, Niederelbert, Germany). Stock solutions of the polyaromatic hydrocarbons (PAHs) at 1 mg/mL and 5 mg/mL for anthracene-9carbaldehyde, were prepared with acetonitrile and diluted with a mixture of 60% methanol/40% water (v:v) for the isocratic experiments and 60% acetonitrile/40% water for the gradient experiments. Fabrication and design of microchips The HPLC microchips were fabricated in-house and consisted of two 1 mm thick soda-lime glass slides (76x26 mm). Common photolithography followed by a wet-etching procedure and high temperature bonding was employed. A detailed description can be found in the Supporting Information section (Figure S-1). The chip design was devised with the open-source vector graphics editor Inkscape. Foil masks were inkjet-printed and glued onto glass slides via 3M double-sided adhesive tape (3M Deutschland GmbH, Neuss, Germany). Photomasks were fabricated by spin-coating the positive photo resist AZ 1518 (MicroChemicals GmbH, Ulm, Germany) onto pre-cleaned, 150 nm thick, chrome-coated soda-lime glass slides. After a pre-bake (5 min for 110°C, 2 min 90°C), the slides were exposed to UV light (14 mJ/cm2, 30 s), applying the foil mask to pattern the photoresist. The development step with AZ 351B 1518 (MicroChemicals GmbH, Ulm, Germany) for 60 s was followed by an etching step with the chrome etchant TechniStrip Cr01 for 1 min and a wet etching step with BOE 7:1 (HF:NH4F = 12.5 : 87.5 %) for 60 min (MicroChemicals

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GmbH, Ulm, Germany). The photoresist was removed with acetone and the remaining chrome was stripped with chrome etchant. The structured glass slide was then bonded to the cover slide, containing powder-blasted inlet holes via a high temperature glass-glass bonding process (muffle furnaces P330, Nabertherm GmbH, Lilienthal, Germany). The chip design can be subdivided in a high pressure region, where the chromatographic separation takes place, and a low pressure region for segmentation and further downstream processing. The main channels were 200 µm wide and 40 µm deep. The microfluidic channels were modified to render the channels hydrophobic. Before silanisation, the channels were subsequently rinsed with a mixture of methanol and concentrated HCl (1:1, v:v), followed by water, then H2SO4, and finally water. After drying, water-free isooctane was flushed through the channels, followed by a 1 vol-% solution of trichloro(1H,1H,2H,2H-perfluorooctyl)silane in isooctane for 10 minutes (10 µL silane + 990 µL isooctane). Integration of the column is based on our previous work57. Briefly, the designated chromatographic column channel was slurry packed with 3 µm ProntoSil C18 SH stationary phase material (Bischoff Analysentechnik und -geräte GmbH, Leonberg, Germany) via a packing channel. The back-pressure was adjusted to approximately 200 bar during the packing and densification procedure. As can be seen at the zoomed-in areas in Figure 1, the separation channel had 80 µm bottleneck-like structures at both ends. Before each narrowing, a porous frit was integrated via laser-assisted photopolymerisation to retain the particles. The packing channel was subsequently sealed with a photopolymerised, nonporous polymeric structure via laser-induced photopolymerisation. A solution of 3.75 µL 3(trimethoxysilyl)propyl methacrylate, 746.25 µL 1,3butanediol diacrylate, 100 µL methanol, and 5 mg 2,2dimethoxy-2-phenylacetophenone was deoxygenated under nitrogen flow. As in the slurry packing procedure, the monomer mixture was filled in the reservoir and pumped into the packing channel. A 355 nm, frequency-multiplied Nd:YVO4 laser was focused onto the T-junction between the column compartment and the packing channel. Afterwards, the residual monomers were removed by flushing the system extensively with methanol.

Figure 1. Schematic representation of the chip design. Channel width equates to 200 µm and 80 µm, respectively. Arrows represent the direction of flow.


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The outlet of the column led to a T-junction in the low pressure area of the chip, where the column eluate was segmented by the carrier oil stream. Further downstream, a dosing unit was placed to dispense reagents into the droplets. The dosing channel had a width of 130 µm. A schematic drawing of the chip design is shown in Figure 1.

the dosing unit and flow rate was controlled via the syringe pump. By adjusting the flow rate it was possible to inject each droplet with the fluorescence quenching solution and its quenching effect was monitored shortly after the dosing unit. The fluorescence intensities before and after the addition of KI were compared.

Instrumentation, method and data processing Setup The pump setup consisted of the NEM-B003-02 D starter module, the low pressure B002-02 D module and the high pressure pulsation-free syringe pump B207-01 A module (cetoni GmbH, Korbussen, Germany). The high pressure syringe pump delivered the mobile phase. Samples were injected via a 4-port nano valve equipped with a 20 nL sample loop (VICI AG, Schenkon, Switzerland). Polyetheretherketone (PEEK) tubings (360 µm OD, 75 µm ID, VICI AG, Schenkon, Switzerland) were used for interconnection. The microchip inlet for the mobile phase was connected to the 4-port nano valve via steel connection clamps, which utilize perfluoro-elastomeric (FFKM) ferrules and headless 632 PEEK screws (N-123-04 and N-123H, IDEX Cop., Lake Forest, USA)56 (Figure S-2). Low pressure syringe pumps provided the continuous oil phase perfluorodecalin mixed with 1% Krytox (v:v), as well as the KI solution. Oil and dosed reagent were filled into 100 µL glass syringes (ILS, Stützerbach, Germany), positioned in the pump module and connected via PTFE tubing (0.3 mm ID) (SUPELCO, Bellfonte, USA) with a LuerTight fitting (P-835 IDEX Cop., Lake Forest, USA) to approximately 1 cm cut PTFE tubing pieces (1/16’’ ID), which were glued onto the remaining inlet holes of the chip. To perform the gradient elution experiments the high pressure pump was interchanged with the Agilent 1260 Infinity Binary Pump (Agilent Technologies Inc., Santa Clara, USA) which was used in combination with a flow splitter consisting of a T-piece (JR-C360QTPK4, VICI AG, Schenkon, Switzerland) with a 15 mm long restriction capillary (fused silica, 360 µm OD, 20 µm ID, CS GmbH, Langerwehe, Germany). The rest of the setup remained unchanged. Separation and droplet generation The isocratic separation was performed employing a mobile phase with 70% acetonitrile and 30% water (v:v). After injection of 20 nL of the sample the components were chromatographically separated on the 5 cm long particulate column. Back-pressure depended on the flow rate and varied between 20 bar at 0.5 µL/min and 120 bar at 3.5 µL/min. The column eluate was fractionated at the T-junction by the oil phase, employing a passive droplet generation scheme58. By varying the flow rate of the oil and the mobile phase, droplet size and frequency could be adjusted. For gradient elution the primary flow rate was adjusted to 150 µL/min, the oil flow rate was 1 µL/min and 5 nL of the sample was injected 5.5 min after starting the gradient program at the pump. Reagent addition via dosing unit As an example of downstream processing, each droplet was injected with a fluorescence quenching solution. For this purpose a stock solution of KI in water was diluted with mobile phase to give a concentration of 0.5 mol/L. The syringe containing the quenching solution was connected to the inlet of

Fluorescence detection The separated bands and the effect of the fluorescence quenching agent were detected by epi-fluorescence, employing an IX71 microscope from Olympus (Hamburg, Germany) equipped with a HBO103W/2 mercury lamp (OSRAM, Berlin, Germany), a 40x LUCPlanFLN objective (NA = 0.6, Olympus), a filter cube U-MWU from Olympus (λex = 330–385 nm, 400 dichroic, λem > 420 nm, Hamburg, Germany) and a photomultiplier tube (PMT, H 7711-03, Hamamatsu Photonics Deutschland GmbH, Herrsching, Germany). Chromatograms were recorded and analysed at a data rate of 400 Hz by the Clarity software (DataApex, Prague, Czech Republic). Flow rates were controlled by neMESYS UserInterface (cetoni GmbH, Korbussen, Germany) and the back-pressure monitored. Data analysis/Chromatographic parameters Analytical parameters were calculated based on the fullwidth-at-half-maximum of chromatographic signals. To enable analysis of the fractionated chromatograms, an upper envelope function was calculated using OriginPro 8G (Origin Lab Corporation, Northampton, USA). This function was smoothed and again analysed using Clarity software (DataApex, Prague, Czech Republic).

Results and discussion In order to combine high performance liquid chromatography and downstream droplet microfluidics, we developed a device which seamlessly integrates a high pressure packed bead HPLC-column, a downstream T-junction for droplet generation, and a dosing unit. A schematic drawing of the layout is shown in Figure 1.

Figure 2. Photograph of an in-house manufactured glass chip with a packed column. The upper microscopic image (40 fold magnification) shows the bottleneck-like structure at the



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beginning of the column with the integrated porous frit and retained 3 µm C18 particles. The lower microscopic image shows the droplet generation at the T-junction subsequent to the column outlet.

The technology for the integration of an HPLC column in microfluidic glass chips builds upon previous publications56,57. While the HPLC-glass chips in our earlier work were custom manufactured by a MEMS foundry (IX-factory), using high quality borofloat glass and chrome masks, the substrates in the present study were economically prototyped in-house using soda-lime glass slides and inkjet-printed foil masks. This allowed a cost-effective optimization and evolution of the functional design over various chip generations. A photograph of such a chip is shown in Figure 2. As evident from the photograph, as well as from microscopic images of the channels in Figure 2, the chips were visually imperfect with uneven and rather rough channel walls. This obviously also affected the column packing procedure as evidenced by rather high values for reduced plate heights of about 12. Although not optimally packed it was possible to achieve well resolved liquid chromatographic separations with such a chip applying pressures up to 120 bar.

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obtained theoretical plates and resolution for this separation are listed in Table 1 After successful integration of the HPLC-functionality, we continued with the development of the chip layout to integrate a functional droplet generator in an iterative process. This involved the optimization of channel dimensions, evaluation of different droplet generator geometries, and evaluation of different dosing units. An important aspect in the device and method development was also to adopt the flow rate of the carrier phase to ensure a reliable droplet generation at optimal HPLC column flow velocities. The aim was a functional fullbody glass chip to perform HPLC separations under high pressure conditions and a robust droplet generator to compartmentalise the separated bands in combination with a downstream dosing unit. For reliable droplet generation it was necessary to render the droplet-channels hydrophobic with a perfluorosilane (see SI). The performance and joint functionality of the HPLC column with the downstream droplet generator was evaluated by monitoring the eluting compounds directly after fractionation. A representative result, detected 0.1 cm behind the droplet generator, is shown in Figure 4.

Figure 3. Non-fractionated chromatogram of a separation of (1) Coumarin 120 (0.057 mmol/L), (2) Anthracen-9-carbaldehyd (7.3 mmol/L), (3) Fluoranthene (0.37 mmol/L), (4) Benz[a]anthracene (1.1 mmol/L) on a ProntoSilC18-SH column 3 µm, column length: 5 cm; mobile phase: 70% acetonitrile/30% water (v/v). Eluent pump: 2.5 µL/min.

Table 1. Chromatographic parameters for the separation before compartmentalisation

N R

(1) C120

(2) A9C

(3) FA

(4) B[a]a

1200

1400

1300

1200

2.4

2.5

2.0

To assess the chromatographic performance of the column, a mixture containing Coumarin 120 and polycyclic aromatic hydrocarbons (PAHs) was eluted under reversed-phase conditions and detected via fluorescence spectroscopy immediately after the column. The corresponding chromatogram, documenting the high separation performance (25000 theoretical plates/m for compound 1), is shown in Figure 3. Mobile phase velocity was determined to be approximately 3 mm/s. The

Figure 4. (above) Fractionated chromatogram after a separation of (1) Coumarin 120 (0.057 mmol/L), (2) Anthracen-9-carbaldehyd (7.3 mmol/L), (3) Fluoranthene (0.37 mmol/L), (4) Benz[a]anthracene (1.1 mmol/L) on a ProntoSilC18-SH column 3 µm, column length: 5 cm; mobile phase: 70% acetonitrile/30% water (v/v). Eluent pump: 2.5 µL/min, Oil flow 2 µL/min. (below) Section of the first peak illustrating the segmentation into droplets.


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As evident from Figure 4, all peaks are baseline resolved and zooming in on the chromatogram reveals that each peak consists of numerous sharp signals, illustrating the compartmentalisation of the chromatographic run into droplets. The droplet generation at a 10 Hz frequency was very regular and the fluorescence intensity distribution along the droplets exhibits the typical Gaussian form of chromatographic peaks. The back-pressure during the separation amounted to 87 bar. Droplet size and frequency could be varied by adjusting the oil flow rate and the geometry of the droplet generator (see Figures S-3 to S-7). Operating with a T-junction at 10 Hz yielded 1 nL droplets with an oil flow rate of 2 µL/min. Generating the same frequency at a T-junction with a channel narrowing resulted in a droplet volume of 0.2 nL with a lower oil flow rate of 1.5 µL/min. We successfully applied the design for fractionations at frequencies of up to 45 Hz. After coupling the droplet functionality with the on-chip separation process, we studied the effect of compartmentalisation on the chromatographic performance in more detail. Unless stated otherwise, the following experiments were conducted on the same chip system under separation conditions listed in Figure 4. We compared the chromatogram recorded before compartmentalisation with the chromatogram obtained after fractionation by detecting at the column end just before (P1 in Figure 5) as well as just behind (P2 in Figure 5) the droplet generator. The chromatograms are shown in Figure 5.

Figure 5. (A) Schematic drawing of the chip design with marking of the three different detection points. P1: at column end i.e. before compartmentalisation, P2: shortly downstream after droplet generation, P3: after the post-column addition of the quenching agent (B) Comparison of the chromatograms of the PAHs separation before compartmentalisation at P1 and after fractionation at P2.

A qualitative comparison of the respective peak widths and shape reveals that the compartmentalisation does not negatively affect the chromatographic performance. For a more quantitative analysis of the peak shapes, an upper envelope function was calculated, spanning alongside the droplet maxima, and

the resulting signals were compared with those from a noncompartmentalised chromatogram. This analysis revealed that all peaks are baseline resolved with the lowest value being R = 2.4 for the first two peaks (Coumarin 120 and Anthracen9-carbaldehyd). An interesting application of the successfully developed chip-HPLC droplet device is the post-column addition of reagents to the individual fractions. Such a device can be regarded as an analogue to the laboratory workhorse preparative HPLC followed by chemical processing and derivatisation of the collected fractions. To illustrate this approach, the chip design contained a functionality for a downstream addition of reagent. The flow rate of the post-column reagent was adjusted so that each droplet was injected with reagent and no additional droplets were produced. If the dosing flow rate is too low, droplets may pass without reagent injection and if the dosing flow rate is too high additional droplet containing only the reagent are produced which may lead to irregular droplet frequencies and droplet coalescence. As a proof of concept, we chose a simple model system to demonstrate the functionality of the dosing unit. Each droplet was dosed post-column with a solution containing potassium iodide which acts as a fluorescence quencher. The dosing and mixing function can than simply be evidenced by a decrease in fluorescence signal of the analytes utilizing the same detection setup as before. The dosing process was followed by video microscopy. Respective consecutive light microscopic images are shown in Figure 6. In the depicted example, the addition of reagent leads to a droplet volume increased by 33% from 0.9 nL to 1.2 nL.

Figure 6. Consecutive microscopic images visualising the process of reagent addition to individual droplets at the dosing unit.. The arrows symbolise the direction of droplet and dosing flow. Droplets arrive with intact droplet boundary at the orifice and are squeezed onto the flow arriving from the dosing channel (1). The droplet boundary opens up and reagent is injected into the droplet (2). The droplets breaks away from the dosing channel (3) and is carried downstream as the next droplet approaches the dosing structure (4).

Figure 7 shows a comparison of the chromatograms before and after the dosing process. Addition of iodide ions, which are well-known chemical fluorescence quenchers59,60, led to a decrease in signal intensity or to signal extinction. The degree of quenching is dependent on the ratio of analyte to quencher and the fluorescence quantum yield. The altered fluorescence intensity could again be readily monitored via fluorescence



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detection. In case of the two last eluting compounds peaks the amount of added quenching reagent was sufficient to completely suppress the fluorescence in each of the individual droplets. Accordingly, the respective fluorescent signals disappear after reagent dosing (see figure 7). For the first two eluting compounds the dosing was effective as well, as evidenced by the significant decrease in fluorescence signals. The lower degree of signal suppression, 90% for the first compound and 50% for the second, can be explained by the compound specific quenching process and was in accordance with off-chip experiments conducted in cuvettes with a bench-top spectrometer (see Figure S-10). A series of additional experiments was conducted with decreasing KI concentration as dosing reagent. The fluorescence intensity decreased with increasing KI concentration, confirming fast and effective mixing within the droplets (Figure S-9). This additional dosing functionality did not negatively affect the microfluidic functionality; the droplet frequency remained regular during and after reagent addition, and droplet coalescence was not observed. Moreover, connection of the dosing channel had no influence on the separation efficiency or droplet generation.

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After we successfully evaluated the device, applying isocratic reversed phase elution, we evaluated the feasibility of gradient elution. The extension to gradient elution would significantly broaden the applicability of a chip-HPLC droplet device. A challenge with gradient elution combined with droplet microfluidics is that the solvent composition can affect the process of droplet generation. Initially we performed a set of experiments with the device testing isocratic elution with an increasing acetonitrile content starting from 20% up to 100%. As shown in the data in the SI a reliable droplet generation was successful under all solvent conditions (see Figure S-12). Thereafter, we performed reversed phase separations with an acetonitrile water solvent gradient elution. To better illustrate the benefits of the gradient the late eluting compound benz[a]pyrene was added to the test mixture. The separation was performed, applying a gradient from 70% to 90% within 2 minutes. With this solvent gradient the total analysis time was reduced by 20% (see Figure S-14). We also applied a more wide-ranging gradient subjecting our test mixture to a gradient starting from 20% to 100% within 2 minutes (see Figure 8). Arguably, this gradient is not suitable to improve the chromatographic performance and prolongs the total analysis time. However, it demonstrates the robust droplet generation over a broad solvent composition range.

Figure 8. Chromatogram of a separation of (1) Coumarin 120 (0.029 mmol/L), (2) Anthracen-9-carbaldehyd (2.4 mmol/L), (3) Fluoranthene (0.15 mmol/L), (4) Benz[a]-anthracene (1.5 mmol/L) and (5) Benz[a]-pyrene (0.23 mmol/L) on a ProntoSilC18-SH column 3 µm, column length: 5 cm; gradient elution from 20% to 100% acetonitrile within 2 minutes. 2.1 min – 15 min: 100%, 15.1 min: 20 %, Primary flow 150 µL/min, Oil flow: 1 µL/min.

Conclusion

Figure 7. (above) Comparison of the chromatograms of the PAHs separation before the dosing unit at P2 and after addition of KI at P3. (below) Enlargement of the third peak reveals the stable droplet signal before dosing and the complete quenching of the fluorescence signal after the injection of KI. Eluent pump: 2 µL/min, Oil flow 1.5 µL/min, Dosing pump: 1.00 µL/min.

Herein, we presented the seamless integration of on-chip HPLC with droplet microfluidics and post-column reagent addition. High-pressure conditions and the possibility to perform gradient elution enabled high performance separations to be performed on chip with dead-volume free coupling to direct fractionation of the eluting analytes. The generated smallvolume segments are isolated by a continuously flowing oil phase, preventing peak dispersion, and thus maintaining the analytical information during transport and further operations. Integration eliminates the need for interconnection technologies, which are leakage prone and challenging to implement. This approach bridges high performance chip chromatography with the extremely versatile droplet microfluidic technology. It


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has high potential to significantly expand the chemist’s toolbox. The presented technique can be regarded as a highlyintegrated miniaturized analogue of micro-preparative HPLC with post-collection sample processing.

ASSOCIATED CONTENT Supporting Information

AUTHOR INFORMATION Corresponding Author * Tel.: +49 341 97 36091. E.mail: [email protected]

Author Contributions All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT We would like to thank Dr. Stefan Ohla for his interest and stimulating discussions of this work.

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