The influence of the thin-layer flow cell design on the mass spectra when coupling electrochemistry to electrospray ionisation mass spectrometry

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Abstract

The influence of the flow cell configuration on the mass spectra obtained when coupling an electrochemical thin-layer flow cell to electrospray mass spectrometry (ESI-MS) has been investigated. It is shown that interferences due to the electrochemical reaction on the counter electrode and/or the absence of 100% conversion efficiency can alter the mass spectra when conventional thin-layer flow cells are used in conjunction with ESI-MS. The effects, which affect the intensities and distribution of the peaks in the mass spectra, can result in the inability to detect products formed at the working electrode. Comparisons of mass spectra, generated after the electrochemical oxidation of a dinuclear Mn complex [Mn2II,II(bpmp)(μ-OAc)2]+ (where bpmp = 2,6-bis[bis(2-pyridylmethyl) amino]methyl-4-methylphenol) using two different thin-layer flow cells clearly show that the potential dependence and appearance of the mass spectra depend on the flow cell configuration used. The use of a modified thin-layer flow cell, in which the counter electrode had been separated from the working electrode, gave rise to significantly increased intensities for the oxidised MnIII,IV state of the complex. With the conventional unmodified cell, the corresponding complex was only seen for considerably higher oxidation potentials. The different results can be explained by the reduced risk of redox cycling and interferences due to species generated at the counter electrode with the modified cell. As interferences due to the counter electrode reactions likewise may be expected with many coulometric flow cells, the electrochemical cell design clearly needs to be considered when using electrochemistry coupled to ESI-MS to study electrochemical reactions.

Introduction

Mass spectrometry (MS) is one of the most powerful analytical techniques available today with many possibilities and an increasing number of applications in numerous scientific fields [1]. There is currently an increasing interest in coupling electrochemical flow cells to mass spectrometry. The main reason for this has been to facilitate studies of electrochemical reactions by identification of the generated products [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14] but it has also been demonstrated that electrochemical reactions can be used for ionisation and derivatisation of analytes prior to their detection by mass spectrometry [15], [16], [17], [18], [19]. An important reason for the recent growing interest in the combination of electrochemistry (EC) and mass spectrometry is the development of the electrospray ionisation (ESI) technique. Electrospray is a mild ionisation method giving rise to few fragmentation products. The combination of flow cell electrochemistry and electrospray ionisation mass spectrometry, which has been described comprehensively by Van Berkel and co-workers [12], [20], [21], [22], [23], facilitates identifications of electrochemically generated products present in small sample volumes [20], [21], [22]. It is also possible to detect unstable products as transfer times between the flow cell and the mass spectrometer of down to a few seconds can be realised [5], [7], [23]. Another advantage of combining electrochemistry and ESI-MS is that electrochemical reactions can be used for tagging of analytes [24], [25] and for ionisation of neutral analytes [6], [26], [27], [28] that otherwise are difficult to analyse with ESI-MS. EC–ESI-MS has also been used by Bruins and co-workers to simulate metabolic oxidations [29] and to study electrochemically assisted Fenton reactions using LC–MS–MS [30]. Comparisons of the products of EC–MS and cytochrome P450 catalysed oxidation reactions as well as cleavages of peptides with EC–MS–MS were likewise described [31], [32]. Other important contributions to the development of EC–ESI-MS have been made by Lev and co-workers [33], [34], [35].

The coupling of electrochemical cells to ESI-MS is complicated by the fact that electrochemical reactions take place in the electrospray itself [15], [36], [37], [38], [39] and the need to decouple the electrochemical flow cell from the ESI high voltage. The latter can be accomplished using sufficiently long transfer lines (∼30 cm) between the electrochemical cell and the mass spectrometer [6], [17], the insertion of a ground point prior to the ESI-MS system [20], [21], [22], [23], [40], [41], [42], [43], [44] or by allowing the whole EC–ESI-MS system to float on the potential induced by the ESI high voltage, as described by Cole and co-workers [7], [8], Nyholm and co-workers [27], [28], [45], [46], [47], and Van Berkel et al. [48]. The main disadvantage with the use of a long transfer line is the increased transfer time between the flow cell and the ion source which makes the detection of unstable electrochemical reaction products difficult. The insertion of a ground point (electrode) between the flow cell and the ESI-MS is undesirable since electrochemical reactions (which could involve the redox species of interest) take place on the ground electrode [27], [49]. Although, ground points are often employed in commercial ESI-MS systems for safety reasons, such ground points should generally not be used in EC–ESI-MS studies to avoid losses of the generated species as well as the appearance of additional unintended species in the mass spectra. For similar reasons, the conversion efficiency (with respect to the species under study) in the electrospray should also be made negligibly small [27]. This means that metal capillary electrospray emitters are less suitable in this kind of EC–ESI-MS systems. Although it has been demonstrated that the electrochemical reactions in the electrospray can be used to oxidise or reduce species in the solution or the electrospray capillary itself [7], [41], [42], [50], [51], it is generally difficult to utilise this approach for the present purposes as the electrochemical potential of the spray capillary is determined by the magnitude of the electrospray current, the capillary material and the concentrations of the available electroactive species in the solution [26]. The most appropriate way to decouple the electrochemical cell from the ESI high voltage is consequently to allow the EC system to float on the potential induced by the ESI high voltage either by using a battery-operated potentiostat [7], [8], [47] or an isolation transformer [27], [28], [46], [48]. The main draw back with this approach is the hazard associated with the use of a (high voltage) floating system, which is why particular care needs to be taken when employing such EC–ESI-MS systems. An important advantage with the floating potentiostatic approach is that the electrochemical cell is effectively decoupled from the electrochemical reactions associated with the electrospray. This means that oxidations and reductions straightforwardly can be performed in the electrochemical cell irrespective of whether positive or negative electrospray is used. It is clearly also possible to switch the electrochemical reactions on and off without having to alter the electrospray current. The latter facilitates the alternating recording of the mass spectra for the reactant and products, a feature which is particularly convenient when the conversion efficiency in the electrochemical cell is close to 100%.

As pointed out by several authors [3], [9], [14], [27], [28], [46], [47], [48], [49], [50], the flow cell configuration needs to be considered when coupling electrochemical flow cells to ESI-MS. This is particularly true when flow cells originally designed for electrochemical detection purposes are utilised in EC–ESI-MS. In such experiments, the composition of the electrosprayed solution will be affected by both the working and counter electrode reactions. Species generated on the working and counter electrodes may then undergo additional (undesirable) redox reactions in the solution on their way to the ESI-MS system. This problem was discussed by both Bökman et al. [27] and Van Berkel et al. [48] and it was concluded that the counter electrode should be positioned in such a way that it is not in direct contact with the flowing solution.

In thin-layer electrochemical detection cells, the working and counter electrodes are commonly separated only by a thin insulating spacer. In these cases, the products formed on the electrodes can readily diffuse between the electrodes, which may alter the concentrations of the reaction products expected to be formed on the working electrode. Although it is well-known that reversible electrochemical systems can exhibit significant redox cycling [52], [53], [54] under such experimental conditions, it is still not clear how this affects the mass spectra obtained with electrochemical cells of different design. Another important issue involves the conversion efficiency of the electrochemical cell. Even though the conversion efficiency can be increased by utilising thin spacer gaskets and low flow rates, it is generally difficult to reach 100% electrochemical conversion efficiency with common thin-layer flow cells. This means that the sprayed solution will contain the reactant compounds as well as the generated reaction products. The latter issue has so far received little attention despite the fact that this type of thin-layer flow cells often are coupled to ESI-MS to study the products of different electrochemical reactions. In such cases, unintended homogeneous follow-up reactions may take place in the solution prior to the electrospray. Although the latter problem can be minimised by using coulometric cells with 100% conversion efficiencies, the risk of interferences due to species generated at the counter electrode generally persist with many coulometric detection cells in which both the counter and working electrodes are in direct contact with the flowing solution. Recently, Van Berkel et al. [51] were able to obtain 100% conversion efficiency with a porous electrode emitter in an electrospray ion source combined with an upstream ground point generating an additional current loop, which increased the total current significantly. A similar approach to control the electrospray electrochemical reaction has also been employed [55] to enhance the stability of sheathless electrospray emitters when coupling capillary electrophoresis to ESI-MS.

In our previous work [27], [28] we have described a thin-layer set-up for EC–ESI-MS in which the counter electrode is separated from the working electrode to minimise the risk of interferences due to the counter electrode reactions. Using an isolation transformer to decouple the electrochemical cell from the ESI high voltage, it was shown that differences in the mass spectra were obtained with the modified cell set-up, when compared with the conventional thin-layer set-up, for the oxidation of an azo compound. However, due to the complexity of the latter oxidation reaction it was not possible to fully interpret the mass spectra and to reach a satisfying understanding of the reasons for the differences between the mass spectra.

The present study contains a systematic comparison of the mass spectrometric results obtained with the conventional and modified thin-layer cell, respectively, for the oxidation of a binuclear manganese complex ([Mn2II,II(bpmp)(μ-OAc)2]+) at different potentials. As this compound has a known electrochemical and mass spectrometric behaviour (studied with the modified cell set-up) [45], [56], [57], [58] and as the oxidation of the complex gives rise to a known set of different manganese oxidation states ranging from MnII,II to MnIII,IV, this compound should be particularly well-suited for investigations of the influence of redox cycling, homogeneous redox reactions and interferences due to counter electrode reactions on the EC–ESI-MS results.

Section snippets

Chemicals and solutions

The [Mn2II,II(bpmp)(μ-OAc)2][ClO4] compound (where bpmp = 2,6-bis[bis(2-pyridylmethyl)amino]methyl-4-methylphenol anion), which was kindly provided by Magnus Anderlund and Professor Licheng Sun (Stockholm University, Sweden), had been prepared according to reported methods [58]. All measurements were performed with a solution of 1.0 mM [Mn2II,II(bpmp)(μ-OAc)2][ClO4] in a solution of 90% acetonitrile and 10% (v/v) purified (osmotically cleaned) water. The acetonitrile was of LiChrosolv® isocratic

Open circuit conditions

In Fig. 2a and b, mass spectra are shown for the conventional and modified cell set-ups for open circuit potential (OCP) conditions. In agreement with previous results [45], obtained with the modified cell, the singly charged ion [Mn2II,II(bpmp)(μ-OAc)2]+ (labelled 1a) gives rise to a peak at m/z 757.0 under open circuit conditions for both cell set-ups. A peak at m/z 349.1 is also seen due to the presence of a doubly charged MnII,II ion (labelled 2) formed from 1a by the loss of acetate, as

Conclusions

The results of the present study demonstrate that different mass spectra can be obtained when coupling thin-layer cells of different design to ESI-MS. When conventional thin-layer cells (originally designed for electrochemical detection in liquid chromatography) are used care should be taken to position the counter electrode in a separate compartment. If this is not done, interferences due to the counter electrode reaction may be introduced. These interferences can affect both the potential

Acknowledgements

Financial support from The Swedish Research Council (grant no. 621-2003-3626 and 621-2001-3139), The Swedish Foundation for Strategic Research (SSF), MedChip, The Swedish Energy Agency and The Royal Swedish Academy of Sciences is gratefully acknowledged. Magnus Anderlund and Professor Licheng Sun (Stockholm University, Sweden) are acknowledged for kindly providing samples of the [Mn2II,II(bpmp)(μ-OAc)2][ClO4] compound. The authors also want to thank Andreas Dahlin for help with the preparation

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