Capillary Electrophoresis Separation Research Articles

Improving separation optimization in capillary electrophoresis by using a general quality criterion

In this paper we extend the use of the quality criterion t’ to optimize separations in capillary electrophoresis (CE). The theoretical parameter t’ takes into account not only the relative separation between a given pair of compounds but also their separation from the neutral species migrating with the electroosmotic flow (EOF). Furthermore, it can be composed for complex mixtures as a global multicriterium optimization function T′, for a rapid, simple and reliable selection of optimized separation conditions by mathematical maximization. Here, we demonstrate the applicability of T′ using as a variable the electrophoretic mobility (me) for the optimization of pH in the separation of a mixture of amyloid beta (Aβ) peptide fragments. In addition, it is shown the versatility of T′ using other variables related to me, which do not require experimental measurements. This is the case with ionizable compounds as the Aβ peptide fragments, whose charge-to-mass ratios can be calculated if accurate pKa values are available in the literature. The excellent performance of T’ for Aβ peptide fragments is further validated optimizing the pH for the separation of mixtures of harmala alkaloids (HAlks) and quinolone antibiotics.

A novel diazoresin/polystyrene-poly(ethylene oxide) covalent capillary coating for the analysis of proteins by capillary electrophoresis

A novel method for the preparation of covalently linked capillary coatings of polystyrene-poly (ethylene oxide) (PS-PEO) was demonstrated by using photosensitive diazoresin (DR) as coupling agents. Layer by layer (LBL) self-assembly membrane of DR and PS-PEO based on hydrogen bonding firstly was fabricated on the inner wall of capillary, then with the treatment of UV light through the unique photochemistry reaction of DR, the hydrogen bonding was converted into covalent bonding. The covalently bonded coatings have the ability of inhibiting protein adsorption on the inner surface of capillary, and thus a baseline separation of Lysozyme (Lys), cytochrome c (Cyt-c), bovine serum albumin (BSA), ribonuclease A (RNase A) and myoglobin (Mb) was achieved by using capillary electrophoresis (CE). Compared with bare capillary or non-covalently bonded DR/PS-PEO coatings, the covalently linked DR/PS-PEO capillary coatings improved the CE separation performance for proteins; what’s more, it exhibited good stability and repeatability. This method can provide a green and easy way to make the covalently coated capillaries for CE, replacing of the old way which using highly toxic and moisture sensitive silane coupling agent by DR in the covalent coating preparation.

Online capillary zone electrophoresis negative electron transfer dissociation tandem mass spectrometry of glycosaminoglycan mixtures

Glycosaminoglycans (GAGs) are important biological molecules that are highly anionic and occur in nature as complex mixtures. A platform that combines capillary zone electrophoresis (CZE) separations with mass spectrometry (MS) and gas-phase sequencing by using negative electron transfer dissociation (NETD) is shown to be efficacious for the structural analysis of GAG mixtures. CZE is a separation method well suited to the highly negatively charged nature of GAGs. NETD is an electron-based ion activation method that enables the generation of informative fragments with retention of the labile sulfate half-ester modification that determine specific GAG function. Here we combine for the first time NETD and CZE for assigning the structures of GAG oligomers present in mixtures. The speed of ion activation by NETD is found to couple well with the narrow peaks resulting from CZE migration. The platform was optimized with mixtures of GAG tetrasaccharide standards. The potential of the platform is demonstrated by the analysis of enoxaparin, a complex mixture of low molecular weight heparins, which was separated by CZE within 30 min and characterized by NETD MS/MS in one online experiment. 37 unique molecular compositions have been identified in enoxaparin using CZE-MS and 9 structures have been assigned with CZE-NETD-MS/MS.

Online preconcentration and determination of chondroitin sulfate, dermatan sulfate and hyaluronic acid in biological and cosmetic samples using capillary electrophoresis.

Capillary electrophoresis with large-volume sample stacking using an electroosmotic flow pump was developed for the determination of chondroitin sulfate, dermatan sulfate, and hyaluronic acid. Central composite design was used to simultaneously optimize the parameters for capillary electrophoresis separation. The optimized capillary electrophoresis conditions were 200mM sodium dihydrogen phosphate, 200mM butylamine, and 0.5% w/v polyethylene glycol as a background electrolyte, pH 4 and -16kV. Exploiting large-volume sample stacking using an electroosmotic flow pump, the sensitivity of the proposed capillary electrophoresis system coupled with UV detection was significantly improved with limits of detection of 3, 5, 1mg/L for chondroitin sulfate, dermatan sulfate, and hyaluronic acid, respectively. The developed method was applied to the determination of chondroitin sulfate and hyaluronic acid in cell culture media, cerebrospinal fluid, cosmetic products, and supplementary samples with highly acceptable accuracy and precision. Therefore, the proposed capillary electrophoresis approach was found to be simple, rapid, and reliable for the determination of chondroitin sulfate, dermatan sulfate, and hyaluronic acid in cell culture media, cerebrospinal fluid, cosmetic, and supplementary samples without sample pretreatment.

Single-Shot Capillary Zone Electrophoresis–Tandem Mass Spectrometry Produces over 4400 Phosphopeptide Identifications from a 220 ng Sample

The dependence of capillary zone electrophoresis (CZE) separations on the charge state of the analyte is useful for the analysis of many post-translational modifications in proteins. In this work, we coupled CZE to an Orbitrap Fusion Lumos Tribrid platform with an advanced peak determination algorithm for phosphoproteomics analysis. A linear-polyacrylamide-coated capillary with very low electroosmotic flow was used for the separation. The optimal injection volume was between 100 and 150 nL of a solution of phosphopeptides in 30 mM ammonium bicarbonate (pH 8.2) buffer, which produces a dynamic pH junction sample injection. Larger injection volumes resulted in serious peak broadening and decreased numbers of phosphopeptide identifications. The optimized system identified 4405 phosphopeptides from 220 ng of enriched phosphopeptides from mouse brain, which represents the state-of-the-art result for single-shot CZE-ESI-MS/MS-based phosphoproteome analysis. We found that the migration time for phosphopeptides is much longer than that for non-phosphopeptides and increased along with the number of phosphorylation sites on the peptides, as expected for the additional negative charges associated with the phosphate groups. We also investigated the phosphorylation site motifs; a number of motifs appeared in the CZE-ESI-MS/MS data but not in LC-ESI-MS/MS data, which suggested the complementary performance of the techniques. The data are available via ProteomeXchange with identifier PXD012888.

PS1427 THE SIGNIFICANCE OF UNEXPECTED PHARMACOKINETICS OF DARATUMUMAB DURING LONG‐TERM MONTHLY TREATMENT FOR MYELOMA

Background:Daratumumab, a drug used in the treatment of multiple myeloma appears as a small IgGK band on serum protein electrophoresis that can be quantified alongside any monoclonal proteins that are present in a patient's serum.Aims:To assess the value and significance of the Daratumumab concentration over the course of treatment.Methods:Daratumumab concentrations have been measured in the serum in 33 patients being treated for B cell malignancies (the paraprotein types were IgG n = 15, IgA n = 11, BJP n = 5, no paraprotein n = 2). The mean duration of Daratumumab treatment was 15.8 months (range 1–31 months) and all patients were treated according to standard Daratumumab protocol. The quantifications were obtained from the densitometry of the Daratumumab peak from capillary zone electrophoresis separation peak converted into g/L using the samples serum total protein concentration.Results:Five of the 33 patients never shown a band of Daratumumab on the serum protein electrophoresis. Interestingly all these patients had IgG paraproteins (4 IgGK, 1IGgL) and it might be that the electrophoretic mobility of their paraprotein overlapped that of the Daratumumab. Their total IgG concentrations (measured immunochemically) were all above 12 g/L. All other patients with IgG paraproteins and comparable total IgG concentrations maintained detectable Daratumumab during their treatment.There were 23 patients where (to date) the duration of the Daratumumab treatment is more than 6 months and who showed a quantifiable Daratumumab band during their treatment. Eight of the 23 patients (2 IgAK, 1 IgAL, 1 IgGK, 2 IgGL, 1 BJP and 1 non‐secretory) had quantifiable Daratumumab throughout their Daratumumab treatment (range 10–31 months). In fifteen patients (6 IgGk, 3 IgAk, 2 IgAl, 3 K BJP and 1 L BJP) the Daratumumab band disappeared from the serum protein electrophoresis at some point during their treatment. The mean time to loss of the Daratumumab band was 12.4 months (range 6–26 months, median 10 months). Following the recommended dose of 16 mg/kg, the Cmax value is reported to be 0.915 g/L which is consistent with our findings of Daratumumab concentrations between 0.46 and 2.01 g/L (included data from all patients with measureable concentrations).Summary/Conclusion:The half‐life of Daratumumab is reported to be 18–23 days so while patients are on weekly or even two‐weekly infusions at the start of Daratumumab therapy, a progressive increase in the Daratumumab may be expected but this is not reflected in our data. The concentration and duration of the Daratumumab band, particularly during the monthly infusion phase is surprising; the concentration remains comparable to the more frequent infusion phases. However, the most surprising findings are i) the disappearance of the Daratumumab band in a significance number of patients during the monthly infusion period, and ii) the wide range in the “time to disappearance” in the context of the reported 18–23 half‐life of Daratumumab. It would be expected that daratumumab would detectable in all patients throughout all treatment phases.The turnover of IgG is complex and in addition to normal catabolic processes the neonatal Fc‐receptors recirculate IgG and prolong its half‐life when IgG concentrations fall; the interaction of Daratumumab in this process may offer some explanation for unexpected patters of Daratumumab concentrations that we have seen in a cohort of 33 patients. The presence, concentration or duration of the Daratumumab band had no correlation to patient outcome.

Capillary zone electrophoresis-native mass spectrometry for the quality control of intact therapeutic monoclonal antibodies

Therapeutic monoclonal antibodies (mAbs) are complex glycoproteins and ensuring their safety, efficacy and quality is still challenging. Indeed, during their manufacturing process, they are exposed to several stresses that can lead to their denaturation, misfolding or dimerization. We report here a new method based on capillary electrophoresis coupled to native mass spectrometry (MS) with a sheath liquid interface to analyze an intact therapeutic mAb, Infliximab, under non-denaturing conditions that preserve its conformational heterogeneity as well as self-association without inducing further unfolding / denaturation. For capillary zone electrophoresis (CZE) separation, a triple layer coating using polybrene-dextran sulfate-polybrene was employed. A sheath liquid composed of isopropanol - water - acetic acid with a flow rate of 10 μL min−1 and mild MS conditions allowed optimal signal intensities. A specific mass spectrum was obtained for each Infliximab conformation in a “stressed” formulated preparation. This is the first time that within a single analysis different conformational states, i.e. native and unfolded monomers as well as dimers are simultaneously detected. The results and the lack of analytical bias arising from the CZE-MS conditions were confirmed by using atomic force microscopy (AFM) as an orthogonal technique. A middle-up approach combined to CZE-MS analysis of the stressed samples suggested that the dimer formation involved mostly Fab-Fab interactions.

Carbon Dot-Mediated Capillary Electrophoresis Separations of Metallated and Demetallated Forms of Transferrin Protein.

Carbon dots (CDs) are fluorescent nanomaterials used extensively in bioimaging, biosensing and biomedicine. This is due in large part to their biocompatibility, photostability, lower toxicity, and lower cost, compared to inorganic quantum dots or organic dyes. However, little is known about the utility of CDs as separation adjuvants in capillary electrophoresis (CE) separations. CDs were synthesized in-house according to a ‘bottom-up’ method from citric acid or other simple carbon precursors. To demonstrate the applicability of CDs as separation adjuvants, mixtures of holo- (metallated) and apo- (demetallated) forms of transferrin (Tf, an iron transport protein) were analyzed. In the absence of CDs, the proteins were not resolved by a simple CE method; however, upon addition of CDs to the separation buffer, multiple forms of Tf were resolved indicating that CDs are valuable tools to facilitate the separation of analytes by CE. CE parameters including sample preparation, buffer identity, ionic strength, pH, capillary inside diameter, and temperature were optimized. The results suggest that dots synthesized from citric acid provide the best resolution of various different forms of Tf and that CDs are versatile and promising tools to improve current electrophoretic separation methods, especially for metalloprotein analysis.

Synthesis and application of amino alcohol-derived chiral ionic liquids, as additives for enantioseparation in capillary electrophoresis

In this study, three functionalized chiral ionic liquids (CILs) derived from l-valinol, l-prolinol and l-phenylalaninol, namely N,N,N-trimethyl-l-valinol-bis(trifluoromethanesulfon)imide ([TMLV]+[Tf2N]−, CIL1), N,N-dimethyl-l-prolinol-bis(trifluoromethanesulfon)imide ([DMLP]+[Tf2N]−, CIL2) and N,N,N-trimethyl-l-phenylalaninol-bis(trifluoromethanesulfon)imide ([TMLP]+[Tf2N]−, CIL3), were synthesized and subsequently utilized for enantiomeric separation in capillary electrophoresis (CE) with 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral selector for the first time. Compared with traditional single HP-β-CD separation system, the synergistic system exhibited substantially improved separations of six tested drugs. Using the CIL1/HP-β-CD as a model system, the influence of crucial parameters including the type and proportion of organic modifier, CILs concentration, HP-β-CD concentration and buffer pH was investigated in detail. Additionally, molecular modeling with AutoDock was applied to elucidate the enhanced enantioselectivity in the presence of CILs, which has certain guiding value in predicting the migration order of the enantiomers and studying the interactions important for the chiral recognition.

Determination of Urinary Pterins by Capillary Electrophoresis Coupled with LED-Induced Fluorescence Detector.

Urinary pterins have been found as potential biomarkers in many pathophysiological conditions including inflammation, viral infections, and cancer. However, pterins determination in biological samples is difficult due to their degradation under exposure to air, light, and heat. Besides, they occur at shallow concentration levels, and thus, standard UV detectors cannot be used without additional sample preconcentration. On the other hand, ultra-sensitive laser-induced fluorescence (LIF) detection can be used since pterins exhibit native fluorescence. The main factor that limits an everyday use of LIF detectors is its high price. Here, an alternative detector, i.e., light-emitted diode induced fluorescence (LEDIF) detector, was evaluated for the determination of pterins in urine samples after capillary electrophoresis (CE) separation. An optimized method was validated in terms of linearity range, limit of detection (LOD), limit of quantification (LOQ), intra- and interday precision and accuracy, sample stability in the autosampler, and sample stability during the freezing/thawing cycle. The obtained LOD (0.1 µM) and LOQ (0.3 µM) values were three-order of magnitude lower compared to UV detector, and two orders of magnitude higher compared to previously reported house-built LIF detector. The applicability of the validated method was demonstrated in the analysis of urine samples from healthy individuals and cancer patients.

On-line preconcentration of sodium dodecyl sulfate-protein complexes using electrokinetic supercharging method with a prefilled water plug in capillary sieving electrophoresis.

An electrokinetic supercharging (EKS) method with a prefilled water plug at the head column of capillary was developed for on-line preconcentration of sodium dodecyl sulfate (SDS)-protein complexes in capillary sieving electrophoresis (CSE). Conventional EKS is a combination of electrokinetic injection with transient isotachophoresis (tr-ITP). The capillary is first filled with background electrolyte, then an appropriate amount of a leading electrolyte is filled and electro-injection is carried out for certain duration. After that, terminating electrolyte is filled, and tr-ITP is subsequently initiated, followed by capillary electrophoresis (CE) separation. In this work, the performance of EKS was evaluated by integrating multiple sub-methods step by step, and a water plug containing polymer was introduced before electrokinetic injection in order to further improve the concentration effect. The positive effects of the sub-methods were verified, including molecular sieving effect of polymer, field enhanced sample injection (FESI) with and without a water plug, and transient isotachophoretic electrophoresis-based FESI. It was observed that analyte discrimination usually encountered in conventional electrokinetic injection was eliminated due to the similar charge to mass ratios of SDS-protein complexes. Based on these results, a hybrid on-line preconcentration method, EKS with injecting a water plug containing polymer before sample electrokinetic injection, was proposed and used to indiscriminately preconcentrate SDS-protein complexes, which provided a sensitivity enhancement factor of more than 1000. It was very suitable for the analysis of low-abundance proteins, providing the information of their molecular mass.

The effect of membrane thickness on supported liquid membrane extractions in-line coupled to capillary electrophoresis for analyses of complex samples

The effect of membrane thickness on extraction performance was systematically examined in extractions through supported liquid membranes (SLM), which were in-line coupled to capillary electrophoresis (CE). Three porous polypropylene membranes with different thickness (25, 100 and 170 μm) were used as supports for SLM extractions of model basic drugs (nortriptyline, papaverine, haloperidol and loperamide) from complex samples. The analytes were transferred through the SLMs by a pH gradient and were in-line injected, separated and quantified using a commercial CE instrument with ultraviolet (UV) detection. Transfers of the model drugs through SLM decreased with the increased membrane thickness (in the order: 25 > 100 > 170 μm) and highest transfers were achieved for the thinnest membrane. Interferences from complex sample matrices were efficiently eliminated, moreover, impregnation of the 25 μm membrane required significantly reduced volume of organic solvent. Mechanical stability of the impregnated 25 μm membrane was excellent during in-line injections, which necessitated direct contact of CE separation capillary with the SLM. Repeatability of the hyphenated SLM-CE-UV method (using the 25 μm membrane) was lower than 11% (RSD values of peak areas) and calibration curves were strictly linear in 0.5–30 μg/mL concentration range (coefficients of determination ≥ 0.997). Transfers of the basic drugs from donor solutions (standard and undiluted human urine/plasma) through the SLMs ranged from 45 to 231% and limits of detection were between 0.02 and 0.15 μg/mL.

Application Prospect of Massively Parallel Sequencing in Mixed Stain Detection.

Mixed stains is the common biological sample in sexual crime cases. Its analysis and DNA profiles interpretation are one of the difficulties in forensic examination. The current genetic marking of mixed stain detection mainly rely on capillary electrophoresis （CE） separation technology, and the analysis methods of the results are mainly inclusion rate and likelihood methods. Because CE has limited resolution and is not able to exploit the efficacy of each genetic marker, its ability to split mixed stain is limited. In recent years, the emerging massively parallel sequencing （MPS） technique not only can obtain the base sequence information of genetic markers, but also is capable of detecting multiple genetic markers simultaneously, and thus derives new analytical methods, bringing new opportunities for forensic detection and analysis of mixed stain. This paper intends to review the application prospects of conventional mixed stain analyses and MPS technique, therefore to provide references for later research and practice.

Preparation of photosensitive diazotized poly (vinyl alcohol-b-styrene) covalent capillary coatings for capillary electrophoresis separation of proteins

In this paper, we have developed a novel method for the preparation of covalently connected capillary coatings in which diazotized poly (vinyl alcohol-b-styrene) (diazo-P(VA-b-St)) was used as a photosensitive coating agent. Firstly, the diazo-P(VA-b-St) coating was self-assembled on the inner surface of the capillary, and then irradiated by ultraviolet (UV) light to convert the ionic bonding into covalent bonding through the unique photochemical reaction of diazo groups. The covalently connected coatings inhibited the protein adsorption on the inner surface of the capillary, as a result, the baseline protein separation of ribonuclease A (RNase A), lysozyme (Lyz) and bovine serum albumin (BSA) were attained by utilizing the capillary electrophoresis (CE). The covalently connected diazo-P(VA-b-St) capillary coatings have greater CE separation performance with magnificent repeatability and enhanced stability, when compared with non-covalently coated or bare capillaries. This strategy to synthesize photosensitive diazo-P(VA-b-St) capillary coatings for their use in capillary electrophoresis separation of proteins is highly environment-friendly as it does not involve the use of extremely noxious and moisture penetrating coatings of silane.

A novel thread-based microfluidic device for capillary electrophoresis with capacitively coupled contactless conductivity detection

This work describes a novel thread-based microfluidic device to perform capillary electrophoresis separation and capacitively coupled contactless conductivity detection. Polyester threads with a diameter of about 315 μm were assembled (stretched) in a 3D-printed platform containing solution reservoirs and platinum electrodes. Separation of potassium, sodium, and lithium ions was achieved within 1 min with good peak resolutions and limits of detection close to 1 μmol L−1. Inversion of the electroosmotic flow direction by adding a cationic surfactant in the running electrolyte was demonstrated. The applicability of the thread-based microfluidic devices was successfully evaluated by the determination of sodium and potassium ions in commercial samples of diet soft drinks.

Capillary electrophoresis-mass spectrometry using robust poly(ether ether ketone) capillary for tolerance to high content of organic solvents

Nonaqueous capillary electrophoresis (CE) and organic-aqueous CE using high content of organic solvents are common techniques in capillary electrophoresis-mass spectrometry (CE-MS) due to various advantages. However, the long-term stability of commonly used polyimide coated fused silica capillary remains a major problem that polyimide coating is prone to swelling in organic solvents and aminolysis in alkaline buffer, which cause serious problems like irregular electrospray, current drop or clogging event. In this work, robust PEEK capillary was used as separation capillary for tolerance to high content of organic solvents in CE-MS system. It is interesting that the high mechanical and chemical stability of PEEK capillary ensured stable CE separation and electrospray when used in CE-MS. All quantitative experiments were accomplished by single PEEK capillary. In addition, the using of organic solvents as running buffer reduced adsorption of hydrophobic compounds on PEEK capillary wall and improved the symmetry of peak shape. The PEEK-based CE-MS method showed good separation performance in analysis of several groups of active alkaloids in traditional Chinese medicines and was applied for quantitative analysis of peimine and peiminine in Bulbus Fritillariae thunbergii herb with high sensitivity. Based on the advantages like simple pretreatment, easy to cut, stable and good separation performance of PEEK column in CE-MS, this study provides an effective way for improving the stability and employment of high content of organic solvents in CE-MS analysis.

Rapid and sensitive capillary electrophoresis method for the analysis of Ecstasy in an oral fluid

In the present study, a capillary electrophoresis method, with a native fluorescence detection for the quantification of three amphetamine derivatives, methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-methamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA) in an oral fluid is described. The reported CE method has made it possible to assess Ecstasy abuse in approximately 15 min, including a saliva sample collection, pretreatment procedures and capillary electrophoresis (CE) analysis. The proof of the principle that was demonstrated on a home-made lab scale instrument has had the potential to be easily translated onto a truly portable instrument for on-site measurements. The baseline CE separation of the three illegal drugs was achieved in 10 min, by applying an aqueous background electrolyte (BGE) that was composed of 40 mM phosphoric acid and 10 mM triethanolamine. The amphetamine derivatives were detected at their λex/λem maximum (280/326 nm) with LOD values of about 3 ng/mL for each amphetamine. The recovery of the compounds from the collection pad was about 40% of the LOQ concentrations and the inter-day precision was less than 6% for all of the analytes. The procedure was applied to a quantitation of oral fluid (OF) samples that were collected during the Baltic Weekend Music Festival that was held in Estonia.

Characterization of Amino Acid Based Molecular Micelles with Molecular Modeling.

The enantiomers of chiral drugs often have different potencies, toxicities, and biochemical properties. Therefore, the FDA and other worldwide regulatory agencies require manufactures to test and prove the enantiomeric purity of chiral drugs. Amino acid based molecular micelles (AABMM) have been used in chiral CE separations since the 1990's because of their low environmental impact and because their properties can easily be tuned by changing the amino acids in the chiral surfactant headgroups. Using molecular dynamics simulations to investigate the structures and properties of AABMM is part of an ongoing study focusing on investigating and elucidating the factors responsible for chiral recognition with AABMM. The results will be useful for the proper design and selection of more efficient chiral selectors. The micelles investigated contained approximately twenty covalently linked surfactant monomers. Each monomer was in turn composed of an undecyl hydrocarbon chain bound to a dipeptide headgroup containing of all combinations of L-Alanine, L-Valine, and L-Leucine. These materials are of interest because they are effective chiral selectors in capillary electrophoresis separations. Molecular dynamics simulation analyses were used to investigate how the sizes and positions of the headgroup amino acid R-groups affected the solvent accessible surface areas of each AABMM chiral center. In addition, headgroup dihedral angle analyses were used to investigate how amino acid R-group size and position affected the overall headgroup conformations. Finally, distance measurements were used to study the structural and conformational flexibilities of each AABMM headgroup. All analyses were performed in the context of a broader study focused on developing structure-based predictive tools to identify the factors responsible for (a) self-assembly, (b) function, (c) higher ordered structure and (d) molecular recognition of these amino acid based molecular micelles.

Achieving Stable Electrospray Ionization Mass Spectrometry Detection from Microfluidic Chips.

The past two decades have witnessed remarkable advances in the development of microfluidic devices as bioanalytical platforms for the analysis of biological molecules. The implementation of mass spectrometry (MS) detection systems on these devices has become inevitable, and various chip-MS ionization interfaces have been developed. As electrospray ionization (ESI) is particularly relevant for the analysis of large biological molecules such as proteins or peptides, efforts have focused on advancing interfaces that meet the demands of nano-separation techniques that are typically used prior to MS detection. Achieving stable ESI conditions that enable sensitive MS detection is, however, not trivial, especially when the spray is generated from a microfabricated platform. This chapter is aimed at providing a step-by-step protocol for producing stable and efficient electrospray sample ionization from microfluidic chips that are used for capillary electrophoresis (CE) separations.

Sample Injection Techniques.

Sample introduction is an important consideration in any microchip electrophoresis (ME) separation. The number of applications and level of complexity of ME devices continue to increase, but the introduction of sample into the separation channel has remained relatively constant. This work describes the most common electrophoretic methods of performing sample injection for ME applications using electroosmotic flow (EOF), providing both a transition from capillary electrophoresis (CE) separations and a straightforward entry point into ME.