CE-ESI/MS system

Capillary electrophoresis (CE) is a powerful separation technique. It is especially useful for separation of ionic compounds and chiral mixtures. Mass spectrometry has been coupled with CE to provide a powerful platform for separation and detection of complex mixtures such as combinatorial libraries. However, the full potential of CE in the application of routine analysis of samples has yet to be realized. This is in part due to perceived difficulty in the use of the CE technique compared to the more mature techniques of HPLC and even SFC. Dunayevskiy et al. [136] analyzed a library of 171 theoretically disubstituted xanthene derivatives with a CE/ESI-MS system. The method allowed the purity and makeup of the library to be determined 160 of the expected compounds were found to be present, and 12 side products were also detected in the mixture. Due to the ability of CE to separate analytes on the basis of charge, most of the xanthene derivatives could be resolved by simple CE-MS procedures even though 124 of the 171 theoretical compounds were isobaric with at least one other molecule in the mixture. Any remaining unresolved peaks were resolved by MS/MS experiments. The method shows promise for the analysis of small combinatorial libraries with fewer than 1000 components. Boutin et al. [137] used CE-MS along with NMR and MS/MS to characterize combinatorial peptide libraries that contain 3 variable positions. The CE-MS method was used to provide a rapid and routine method for initial assessment of the construction of the library. Simms et al. [138] developed a micellar electrokinetic chromatography method for the analysis of combinatorial libraries with an open-tube capillary and UV detection. The quick analysis time of the method made it suitable for the analysis of combinatorial library samples. CE-MS was also used in the analysis... 

Phosphatase Treatment The specificity of phosphatases for the removal of the phosphate group has also been exploited to identify phosphopeptides and proteins selectively [38]. The molecular mass of phosphopeptides decreases by 80 Da for each phospho unit after the phosphatase treatment. The reaction is monitored by MALDI-MS [39]. The peptide maps can be compared before and after phosphatase treatment to identify phosphopeptides in the digest [40]. Sensitivity is a major issue in such experiments. One way to improve sensitivity is to perform dephosphorylation on the MALDI target [40]. Another way is to use immobilized phosphatase packed in a smaU-diameter colnmn in-line with an LC/ESI-MS or CE/ESI-MS system [39,41],... 

Because there is no ionizable groups of the coating in the neutral capillary, the interaction between charged molecules with ionic capillary surface is eliminated. Also, the electro-osmotic flow (EOF) of a neutral capillary is eliminated. However, a continuous and adequate flow of the buffer solution toward the CE capillary outlet is an important factor for routine and reproducible CE-ESI-MS analysis in order to maintain a stable ESI operation, some low pressure applied to the CE capillary inlet is usually needed, especially when the sheathless interface is employed. The disadvantage of the pressure-assisted CE-ESI-MS is the loss of some resolution because the flat flow profile of the EOF is partially replaced by the laminar flow profile of the pressure-driven system. A typical neutral capillary is a LPA (linear polyacrylamide)-treated capillary. Karger and co-workers [6] used mixtures of model proteins, a coaxial sheath flow ESI interface. 

Similarly to LC (section 4.3.4), also CE instruments can be coupled to a mass spectrometer, providing a powerful system for analysis of complex samples. The output of the electrophoresis capillary is connected to an electrospray ionisation (ESI) source, CE-ESI-MS. Usually a make up flow is necessary to increase the flow rate for a stable spray. To avoid contamination of the ion source, it is essential to utilise only volatile buffer components such as ammonium acetate and volatile additives such as methanol, acetonitrile and acetic acid. 

Microsphere-filled capillary microreactor TPCK-treated trypsin Covalent linking Magnetic beads with carboxylic acid functionalized surface immobilization of trypsin onto beads via EDC activation microreactor coupled to CE-ESI-MS for digestion of insulin chain b and P-casein, that is, a 2-dimensional CE system. Platform delivers tryptic digests to the MS within a short window [132]... 

The capillary reactor was coupled to CE-ESI-MS. The performance efficiency of the system was checked by digestion and separation of model proteins, insulin chain b oxidized, and P-casein. This platform delivers tryptic digests to the MS within a short window. The two-dimensional separation requires roughly 30 min to digest and separate 30 fractions. 

The evolution of the ESI source has been marked by the use of electrospray devices as interfaces between the separation systems such as HPLC or CE and MS detectors, the earliest instances of which were reported by Yamashita and Fenn [59] and Aleksandrov et al. [60] in the mid-1980s. Because ESI-MS is used in many areas of chemistry, a vast number of articles reporting specific modifications of the electrospray interface has been published so far. Also, instrument manufacturers have provided innovative solutions for more sensitive and reliable mass spectrometers. 

An ESI interface for the coupling of capillary electrophoresis (CE) and MS was developed by the group of Smith [15-17]. A schematic diagram of the system is shown in Figure 5.4. A hot 2 5-1/min nitrogen curtain gas is used to clean the... 

An incompatability that does need to be considered in CE-MS method development is the use of certain CE buffer systems and additives which are detrimental to the ESI process. For example, although sample concentration can be increased by the use of more conductive buffers, this approach is not advantageous for ESI-MS detection. These characteristics result in a significant demand upon ESI interface efficiency [11]. Ideally, the chosen CE buffer should be volatile, such as ammonium acetate or formate. The use of pure acids or bases rather than a true buffer has also been shown to be advantageous for certain molecules. Nonaqueous buffer systems are also being employed more widely. 

Capillary electrokinetic chromatography (CEKC) with ESI-MS requires either the use of additives that do not significantly impact the ESI process or a method for their removal prior to the electrospray. Although this problem has not yet been completely solved, recent reports have suggested that considered choices of surfactant type and reduction of electro-osmotic flow (EOF) and surfactant in the capillary can decrease problems. Because most analytes that benefit from the CEKC mode of operation can be effectively addressed by the interface of other separations methods with MS, more emphasis has until now been placed upon interfacing with other CE modes. For small-molecule CE analysis, in which micellar and inclusion complex systems are commonly used, atmospheric pressure chemical ionization (APCI) may provide a useful alternative to ESI, as it is not as greatly affected by involatile salts and additives. 

The further development of microscale preconcentration and cleanup techniques and the resulting improvements in CE-MS concentration detection limits are likely to expand the use of this analytical technique. The more common use of small-diameter capillaries and even tiny etched microplate devices [10], along with the improvements in ESI spray techniques are pushing research along. Further investigations into improving interface design, durability, reproducibility and sensitivity are still necessary. The availability of improved, less expensive, and smaller mass spectrometers will almost certainly lead to increased use of CE-MS. However, the sensitivity and selectivity already demonstrated by CE-MS systems, in combina-... 

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