Development of a miniaturized injection cell for online electrochemistry–capillary electrophoresis–mass spectrometry
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The elucidation of oxidation or reduction pathways is important for the electrochemical characterization of compounds of interest. In this context, hyphenation of electrochemistry and mass spectrometry is frequently applied to identify products of electrochemical reactions. In this contribution, the development of a novel miniaturized injection cell for online electrochemistry–capillary electrophoresis–mass spectrometry (EC–CE–MS) is presented. It is based on disposable thin-film electrodes, which allow for high flexibility and fast replacement of electrode materials. Thus, high costs and time-consuming maintenance procedures can be avoided, which makes this approach interesting for routine applications. The cell was designed to be suitable for investigations in aqueous and particularly non-aqueous solutions making it a universal tool for a broad range of analytical problems. EC–CE–MS measurements of different ferrocene derivatives in non-aqueous solutions were carried out to characterize the cell. Oxidation products of ferrocene and ferrocenemethanol were electrochemically generated and could be separated from the decamethylferricenium cation. The importance of fast CE–MS analysis of instable oxidation products was demonstrated by evaluating the signal of the ferriceniummethanol cation depending on the time gap between electrochemical generation and detection.
KeywordsCapillary zone electrophoresis Electrochemistry Mass spectrometry Reaction mechanisms
Electrochemical methods are of high relevance in many fields of research. They are essential tools for studies in the context of material sciences such as corrosion studies , the development of energy carriers , microbial fuel cells , or electrosynthetic processes . Electrochemistry is also widely applied in bioanalytical studies such as the electrochemical simulation of oxidative stress [5, 6, 7, 8] or metabolic processes [9, 10, 11, 12, 13, 14, 15, 16, 17].
Pure electrochemical investigations such as cyclic voltammetry are well suited for the characterization of redox activities and reversibility of redox processes , but lack of qualitative information regarding mechanistic details. Thus, hyphenation to powerful detection techniques is in demand to obtain additional information on processes taking place on the electrode surface. In this context, hyphenation of electrochemistry (EC) to electrospray ionization–mass spectrometry (ESI–MS) is a frequently applied method, as recent reviews point out [16, 19]. ESI–MS offers high sensitivity and the possibility of identifying products of electrochemical reactions by their molecular masses and isotopic patterns with low fragmentation in the ionization process . Thus, this technique can help in the elucidation of possible reaction mechanisms. However, EC and ESI have to be decoupled as electrochemical cells are operated at low voltages, while in ESI high-voltage conditions are applied . This can be achieved using setups with grounded ESI interfaces .
Typical approaches to EC–MS comprise the direct coupling of electrochemical flow cells such as coulometric flow-through cells with porous electrodes or thin-layer flow cells with planar electrodes to mass spectrometry [4, 19, 23, 24]. Furthermore, efforts towards miniaturized setups using microfluidic electrochemical cells and nanoscale electrochemical reactors were made . The advantage of such an approach is the simplicity of the experimental setup and the possibility of very fast detection within seconds , as the analytes are directly transported to MS via pumps. However, based on the cell type, the dependence of the conversion efficiency on the flow rate and the electrode surface area has to be kept in mind [23, 24]. An innovative approach to EC as online sample preparation technique for ESI–MS was developed by Dytrtová et al. , who coupled an electrochemical cell with switchable working electrodes to ESI–MS to ionize even non-polar organic compounds by adduct formation with electrochemically generated reactive metallic ions.
However, there are also some limitations of direct EC–MS. In complex samples, additional separation steps are necessary, as ion suppression effects in the ion source of MS can influence the detection, especially if mixtures of products are formed or if product and educt species show significant differences in their ionization properties. Thus, a quantification of oxidation or reduction products is difficult. Overlapping mass spectra can prevent the clear identification of individual species. Moreover, the separation behavior can give important additional information on the analytes, such as the presence of functional groups or polarity.
Hyphenation to separation techniques is often achieved by coupling EC to HPLC via electrochemical flow cells installed prior to or after the separation column [7, 13, 24]. However, the instrumental setups for this purpose are quite complex as different pumps and valves are needed, if EC is carried out before HPLC . Another disadvantage that can arise is the compatibility between the conditions needed for electrochemical reactions and the separation conditions . Contrary to direct EC–MS, the time gap between formation and detection of products is longer if a separation step is carried out after oxidation or reduction. Typical analysis times are in the range of several minutes [7, 14, 23]. Working with reversed phase HPLC–MS it has to be considered that non-polar analytes are usually favored for HPLC, but polar analytes are better compatible to ESI–MS conditions .
Due to the aspects mentioned above, capillary electrophoresis (CE) is the method to be preferred in some cases. It offers fast separation and low solvent consumption [29, 30] and is suitable for separation of charged species. Thus, it is an ideal separation method for many biomolecules that contain functional groups which can be protonated or deprotonated depending on pH. In contrast to HPLC, separations are possible under nearly physiological conditions  and can be carried out in the same electrolyte as the oxidation or reduction, so that the migration behavior in CE is representative for the state of charge of the analytes in the electrolyte. Due to the charge-dependent migration behavior, CE allows for differentiation between ions, which are generated by electrochemical processes and ions generated in the ionization prior to MS detection. This information cannot be obtained in direct EC–MS or EC–HPLC–MS.
First online EC–CE approaches were established in 2003 [31, 32] using batch electrolysis cells and classical three-electrode setups. Thus, they had the disadvantage of time-consuming electrode maintenance procedures, which are necessary to avoid electrode fouling and require experienced users. Additionally, a comparably high sample volume is needed. In contrast to that, Palatzky et al.  developed a fully automated device for online EC–CE–MS based on disposable screen-printed electrodes (SPEs). Hence, compared to classical cells, significantly lower sample consumption (about 50 mm3 is sufficient), easy replacement of electrodes avoiding time-consuming cleaning and polishing procedures, and high flexibility concerning electrode materials could be achieved. The electrochemical cell consisted of a droplet of solution placed onto the three-electrode structure of the electrode and sample injection into the CE-system was achieved by placing the separation capillary into this droplet directly above the working electrode. However, this system was not compatible to non-aqueous solutions due to the screen-printed electrode materials, which is a major drawback when it comes to the investigation of analytes that are not readily soluble or stable  in water.
This contribution presents an instrumental approach to online EC–CE–MS with disposable electrodes, which is applicable under non-aqueous conditions. It is based on the existing fully automated EAI–CE–MS device described in . To allow for investigations in non-aqueous solutions, different problems had to be addressed. As already mentioned above, screen-printed electrodes are attacked by organic solvents, so that alternative electrode types had to be used. Commercially available thin-film electrodes consist of metal electrode materials fabricated on glass substrates and are ideal for this purpose as they are solvent-resistant . However, simply applying droplets of solution onto the electrode surface as it could be done with aqueous solutions [5, 33] was not possible, as organic solvents easily spread due to low surface tension. This can lead to electrical shortcuts and corrosion problems, when the liquid flows into electrical contacts. Therefore, the cell volume had to be delimited physically to prevent spreading of the liquid. To overcome these problems, a novel miniaturized injection cell for online EC–CE–MS with integrated thin-film electrodes was developed, capable of measurements in aqueous and especially non-aqueous media. A model mixture consisting of ferrocene (Fc), ferrocenemethanol (FcMeOH), and decamethylferrocene (dMFc) was used to characterize this injection cell. The importance of short separation times was demonstrated by evaluation of the dependency of the FcMeOH+ signal on the separation time.
Results and discussion
Design and fabrication of the injection cell
Polyether ether ketone (PEEK), a highly chemical resistant and mechanically stable material, was used for fabrication of the cell body. To prevent leakage, a sealing ring was integrated at the bottom of the open cell chamber. Thus, spreading of droplets could be avoided. As commercially available O-ring materials were attacked by organic solvents, a custom silicone sealing ring with appropriate dimensions (inner diameter 2 mm, outer diameter 4 mm, thickness 1 mm) was prepared. The electrical contact to the implemented thin-film electrode was achieved via spring contact probes. To facilitate a fast assembling and disassembling of the cell, magnets were integrated to keep the cell closed. Due to materials and modularity of the cell, it could be cleaned easily and was suitable for measurements in aqueous as well as non-aqueous solutions. Electrodes could easily be exchanged, which allowed for high flexibility regarding electrode materials. When installed in the EC–CE–MS device, a fully automated hydrodynamic injection of sample directly from the working electrode surface was possible by placing the tapered tip of the fused silica separation capillary onto the electrode surface. The overall experimental setup is illustrated in Fig. 5 in the experimental section.
The results showed that electrochemical sample pretreatment and online analysis of oxidation products could be achieved within a short time scale. Very short oxidation times of only 10 s (injection during last 2 s of oxidation) were enough to generate a sufficient amount of product species for detection. The electrochemical generation and detection of Fc+ and FcMeOH+ were feasible within 90 s and both were separated from dMFc+.
Fast online analysis of oxidation products
A miniaturized injection cell for online EC–CE–MS was developed and characterized. It was capable of handling very small sample volumes of 10 mm3 or lower. Electrodes and injection cell were solvent-resistant, so that online investigations of electrochemical reactions in aqueous and particularly non-aqueous media were possible. The integration of disposable thin-film electrodes leads to a high flexibility in electrode materials and to an easy exchange of electrodes, which is minimizing artifacts due to adsorption or electrode fouling. Time-consuming electrode maintenance procedures that usually need experienced users can be avoided. In online EC–CE–MS using ferrocene derivatives, short analysis times within few minutes from generation to detection of oxidized species were possible. Fc+ and FcMeOH+ could be generated and separated from dMFc+ within less than 90 s. Due to that, the method is suitable for the investigation of instable products, which was demonstrated by evaluating the signal of electrochemically generated FcMeOH+ depending on separation speed. Fully automated oxidation and injection procedures allowed for reproducible measurements and a reliable control of the time gap between formation and detection of oxidized species.
In conclusion, this novel setup extends the applicability of online EC–CE–MS based on disposable electrodes to analytes that are only soluble in organic solvents, which was not possible using screen-printed electrodes. The high flexibility and possibility of fast online analysis make this setup attractive for further applications, such as kinetic studies or electrochemical simulation of metabolic processes with particular focus on reactive species.
Reagents and chemicals
The following chemicals were used, all of analytical grade or higher if not stated otherwise: acetic acid (Sigma Aldrich, MO, USA), acetonitrile, ammonium acetate (both Merck, Darmstadt, Germany), decamethylferrocene (purity 99%, ABCR, Karlsruhe, Germany), formic acid (Merck, Darmstadt, Germany), ferrocene (purity 98%, Riedel-de-Haën, Seelze, Germany), ferrocenemethanol (purity 99%, ABCR, Karlsruhe, Germany), isopropanol (Roth, Karlsruhe, Germany).
For evaluation of the cell performance, a solution of 1.5 mmol/dm3 Fc, 1 mmol/dm3 FcMeOH, and 40 µmol/dm3 dMFc in BGE was used. Fast detection studies were carried out with a solution of 1 mmol/dm3 FcMeOH in BGE. For the CE protocol, 8 mm3 of sample solution were filled into the cell. The sample was hydrodynamically injected into the CE system by placing the tapered end of the separation capillary onto the working electrode surface for 2 s at a difference in height of 18 cm between the injection end of the capillary and the detection end of the capillary (hydrostatic pressure). After the injection, the capillary was automatically placed into the 2-cm3 BGE reservoir and the separation voltage denoted in detail in the respective measurements in the results section was applied. Measurements were carried out without previous oxidation and after oxidation at 0.5 V for 10 s (injection during the last 2 s of oxidation). For data evaluation the extracted ion signals of Fc (m/z = 186.01), FcMeOH (m/z = 199.02; m/z = 216.02), and dMFc (m/z = 326.20) were used.
The parameters for the MS detection were as follows: acquisition: ion polarity: positive; mass range: m/z = 100–350; spectra rate 5 Hz; source: end plate offset: − 500 V; capillary: − 4000 V; nebulizer: 1.0 bar; dry gas: 4.0 dm3/min; dry temperature: 190 °C; transfer: capillary exit: 75.0 V; skimmer 1: 25.3 V; hexapole 1: 23.0 V; hexapole RF: 65.0 Vpp; skimmer 2: 23.0 V; lens 1 transfer: 38.0 µs; lens 1 pre pulse storage: 6.0 µs.
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