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Capillary Electrophoresis coupling to Mass Spectrometry (CE-MS), an advanced technique orthogonal to LC-MS for high resolution separation and accurate molecule identification


Martin Greiner, Agilent Technologies R&D and Marketing GmbH & Co. KG, Waldbronn, Germany Capillary Electrophoresis (CE) coupled with Mass Spectrometry (MS) has been under investigation for the past couple of decades1 . Combining


the high separation power of CE with MS promises to yield more sensitive detection and higher information content on the analytes than current methods such as standard UV detection. Since those early days, there have been many improvements and technology advancements on CE and MS instruments, as well as the interfacing devices. Modern Time of Flight (TOF) MS instruments and robust commercial interfaces provide increasingly sensitive routine analysis with accurate mass data.


The basic approach to CE-MS is to use Electrospray Ionization (ESI) techniques. The advantage to this system is that typically ionized or polar compounds separated by the CE under low flow conditions (low nl/min range) can then be efficiently transferred from the liquid phase into gas phase ions required by MS instruments. In addition, ESI does not decompose fragile molecules, like proteins.


Routine CE-MS requires the use of volatile buffers or low concentration salts. This avoids contamination of MS instruments and the suppression of analyte ions by salts competing for entrance to the MS. Separation by CE and ionization of molecules in the ESI source are both based on analyte chemistry and require optimization for best results. It is critical to choose the correct solvents and pH ranges for solubility, to allow for optimal CE mobility and for high ionization efficiency during desolvation. This may call for different chemistries for the separation buffers and for a sheath liquid controlling the ESI processes.


High resolution in CE provides narrow peak widths, often just a few seconds wide, that require rapid scanning by the MS if full spectra are needed. For this reason TOF-MS is often the chosen tool providing sufficient data points over a peak, yet still offering full mass range. Since TOF or Quadrupole-TOF (allowing specific preselection of ions and


Figure 1. Sheath Liquid Interface for CE-MS as triple tube setup. The inner tube is the fused silica CE-capillary followed by grounded stainless steel (or platinum) tube guiding sheath liquid from an LC pump or other device. The nebulizing gas used for enhanced droplet formation is guided through the outer tube.


MS/MS fragmentation capabilities) can have very high resolution (e.g. 40.000 resolving power), exact masses and isotopic relations can be determined leading to molecule identification.


Why use CE or CE-MS? CE is an orthogonal method to HPLC where separation is based on mobility of ions in an electrical field, instead of chromatography. Intrinsically, CE can handle ionized or very polar compounds best, which are often difficult to separate on a LC. CE-capillaries are usually fused silica based open tubes with a 25-100 µm inner diameter. Adsorbance of compounds such as proteins to the inner wall is less critical compared to HPLC columns. This is due to the much larger surface area of HPLC packing


materials and the risk of getting physically stuck. The adsorbance effects to fused silica in CE can be counteracted by using specifically coated capillaries. This could be permanently bound coatings (e.g. polyvinyl alcohol) or various dynamic coatings with cationic or anionic modifiers added to the liquid phase. Hence, CE may also be used as a separation tool for large polymers or biological compounds, like proteins or nucleic acids.


Interfaces for CE-MS need to accomplish various tasks: position the CE capillary physically close to the MS entrance, provide electrical contact to the free end of the CE capillary and support the right droplet formation needed for desolvation and creation of free gas phase ions.


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