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Surface coatings are extremely important for CE analysis to prevent analyte adsorption and control the EOF. An issue affecting separation efficiency of CE is adsorption of

biomolecules to the channel wall through electrostatic and hydrophobic interactions. A common approach to assuage this is to use a surface coating.52–55 _{For CE-MS analysis of biomolecules} these coatings are often neutral polymers or cationic in nature to prevent analyte adsorption through electrostatic repulsion. Polybrene, polyethyleneimine, polyacrylamide, cellulose, dextran, poly(vinyl alcohol), and silane based coatings have been reported in the literature for use with CE-MS.55–63 However, when compared with theoretical separation performance for CE, it can be determined that most separations presented in the literature could be improved several fold.2 For instance, a polyacrylamide based coating was recently reported.62 Δ values were calculated from the electropherograms and the average Δ was found to be 5.5. This indicates that there is significant band broadening in the CE system. If surface coatings are not applied

uniformly, electroosmosis will be inconsistent throughout the capillary. This creates pressure gradients during the CE separation which degrades the separation efficiency through Taylor dispersion. Therefore, the ideal surface coating for CE-MS prevents analyte adsorption, is easily and reproducibly applied to generate uniform electroosmosis, and will not introduce background during MS analysis.

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1.4.1 Coating technology for microfluidic CE-MS

Around 2010 the Ramsey group began experimenting with applying surface coatings for CE-MS in the gas phase. A method was developed based on previously published work64 to deposit cationic aminopropylsilanes (APS) on the microfluidic channel surfaces via chemical vapor deposition (CVD). This technique proved to produce highly uniform, dense coatings. APS coatings using trifunctional 3-aminopropyltriethoxysilane (APTES) and monofunctional 3- aminopropyldiisopropylethoxysilane (APDIPES) have generated near diffusion limited separations of fluorescent dyes with Δ values of 1.2 and 1.09 for APTES and APDIPES,

respectively.2 _{Analyses of peptide and protein standards using an APDIPES coating indicate that} the APS surface prevents adsorption of biomolecules and maintains separation efficiency. Figure 1.6 shows the separation of bradykinin, methionine-enkephalin, thymopentin, and angiotensin II, four peptides commonly used to characterize the performance of our devices. The average Δ value for the peptides is 1.4 with an average efficiency of 6.8x105 plates, and according to Equation 1.8 theoretically this could be further improved to 9.5x105 _{plates. In comparison, most} CE separations reported in the literature using coatings generate efficiencies below 200,000 plates for small molecules and in the tens of thousands for intact proteins.54,56 _{Thus, the gas} phase APS coating generates superior CE performance for biomolecules.

A characteristic of these coatings is that there is a high level of EOF. This reduces

analysis time, but the resulting resolution is often insufficient to separate similar species. For CE analysis the resolution between analyte species can be characterized by the following equation7:

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𝑅_𝑠 = 0.177(𝜇_𝐸𝑃1− 𝜇_𝐸𝑃2)√ 𝑉

𝐷̅(𝜇̅_𝐸𝑃+ 𝜇_𝐸𝑂𝐹) (1.9)

where µEP1,2are the electrophoretic mobilities of the analyte ions, 𝜇̅_𝐸𝑃is the average

electrophoretic mobility, µEOF is the EOF magnitude, 𝐷̅ is the average diffusion coefficient of the

analytes and V is the applied voltage for separation. Based on this equation, Rs is maximized

when the product µEP and µEOF is 0. However, this results in impractical migration times. An

alternative approach is to suppress the EOF. The EOF magnitude can be affected by several factors including buffer ionic strength and pH, or through the use of buffer additives, such as surfactants. However, with CE-MS many of these approaches cannot be utilized due to the need to maintain MS compatibility.

In the Ramsey lab, the approach to reduce the EOF in the separation channel is to use surface chemistry rather than modifying the separation buffer so that it can remain simple and MS compatible. A novel modification to the APS coating has been developed to achieve EOF reduction. The APS coatings are reacted with polyethyleneglycol (PEG) chains as illustrated in Figure 1.7. The PEG chains are available in various lengths and terminate with an N-

hydroxysuccinimide (NHS) ester that reacts spontaneously with the primary amine of the APS coating, forming an amide bond between the APS and PEG chain. Previous work determined that the degree of EOF suppression is dependent on the length of the PEG chain used; the longer the chain, the lower the EOF (unpublished data).65

As mentioned previously, a significant amount of separation resolution is necessary to successfully resolve proteins at the intact level since protein variants are often very similar in structure and net charge. Thus, as described by Equation 1.9, the EOF should be reduced to

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maximize the Rs between similar species. Using this strategy, the APS-PEG combination that

resulted in the highest degree of EOF reduction was chosen to be optimized for intact protein separations. The EOF reduction was characterized through microfluidic CE separations of fluorescent dyes using laser induced fluorescence (LIF) detection and chips without integrated ESI and 3 cm long separation channels. The APS-PEG450 coating was been found to reduce the EOF from approximately 9.5x10-4 cm2/Vs to 0.8x10-4 cm2/Vs. However, this coating scheme had not yet been successfully applied to microfluidic CE-ESI devices with longer separation

channels. A modified coating procedure was developed that has successfully been used to coat microfluidic CE-ESI devices with separation channel lengths of up to 46 cm. The optimized coating procedure can be found in Appendix 2.

Figure 1.8 compares the separation of a five protein mixture (carbonic anhydrase I, hemoglobin, human serum albumin, cytochrome c, and lysozyme) using the APS and the optimized APS-PEG450 coating for intact protein separations. It should be noted that due to the EOF suppression, the APS-PEG450 separations were performed in reverse polarity in order to direct cations into the separation channel. As such, the migration times of the proteins are

reversed compared to the separations performed with the APS coating. As theorized, suppressing the EOF increased the separation resolution by enhancing differences in apparent analyte

mobility. From the separations in Figure 1.8a and b, a significant improvement in resolution can be seen between neighboring peaks. The APS-PEG450 device generated a resolution of 2.9

between cyt-c (Peak 5) and HSA (Peak 4) while the APS coated device resulted in a resolution of 1.5. Unresolved Hb species can be detected in the mass spectra of the β-Hb peak from the APS separation. As seen in Figure 1.8c the mass spectra of peaks 2a-2c spectrally combine to give the spectrum obtained from peak 2 of the APDIPES-based separation, indicating that these are the

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unresolved charge variants detected in peak 2. Overall, the APS-PEG450 coating resulted in fast, highly efficient separations of intact proteins with improved resolution as compared to

APDIPES. Additionally, as will be discussed later in the text, this low EOF coating has proven useful for a variety of analyte molecules ranging in size from large intact proteins to small metabolites.