Chitra K. Ratnayake

High Capacity Capillary Electrophoresis-Electrospray Ionization Mass Spectrometry: Coupling a Porous Sheathless Interface with Transient-Isotachophoresis

A sheathless interface making use of a porous tip has been used for coupling capillary electrophoresis and electrospray ionization mass spectrometry. First, effective flow rates using the interface have been characterized. It was found that the interface is capable of generating a stable spray with flow rates ranging from below 10 nL/min to >340 nL/min, enabling its use in either the mass or concentration-sensitive region of the electrospray process. Subsequently, by analyzing peptide mixtures of increasing complexity, we have demonstrated that this platform provides exquisite sensitivity enabling the detection of very low amounts of materials with very high resolving power. Transient isotachophoresis (t-ITP) can also be integrated with this setup to increase the mass loading of the system while maintaining peak efficiency and resolution. Concentration limits of detection in the subnanomolar or nanomolar range can be achieved with or without t-ITP, respectively. The application of a vacuum at the inlet of the separation capillary further allowed the peak capacity of the system to be improved while also enhancing its efficiency. As a final step in this study, it was demonstrated that the intrinsic properties of the interface allows the use of coated noncharged surfaces so that very high peak capacities can be achieved.

Performance of a sheathless porous tip sprayer for capillary electrophoresis-electrospray ionization-mass spectrometry of intact proteins

The performance of a prototype porous tip sprayer for sheathless capillary electrophoresis-mass spectrometry (CE-MS) of intact proteins was studied. Capillaries with a porous tip were inserted in a stainless steel needle filled with static conductive liquid and installed in a conventional electrospray ionization (ESI) source. Using a BGE of 100 mM acetic acid (pH 3.1) and a positively charged capillary coating, a highly reproducible and efficient separation of four model proteins (insulin, carbonic anhydrase II, ribonuclease A and lysozyme) was obtained. The protein mass spectra were of good quality allowing reliable mass determination of the proteins and some of their impurities. Sheath-liquid CE-MS using the same porous tip capillary and an isopropanol-water-acetic acid sheath liquid showed slightly lower to similar analyte responses. However, as noise levels increased with sheath-liquid CE-MS, detection limits were improved by a factor 6.5-20 with sheathless CE-MS. The analyte response in sheathless CE-MS could be enhanced using a nanoESI source and adding 5% isopropanol to the BGE, leading to improved detection limits by 50-fold to 140-fold as compared to sheath liquid interfacing using the same capillary - equivalent to sub-nM detection limits for three out of four proteins. Clearly, the sheathless porous tip sprayer provides high sensitivity CE-MS of intact proteins.

A systematic study in CIEF: Defining and optimizing experimental parameters critical to method reproducibility and robustness

A systematic study of two‐step CIEF analysis was completed to identify key components that could be optimized to enhance the performance of mAb analysis by CIEF. Resolution between mAb isoforms was increased by selecting a narrow detector aperture, utilizing chemical rather than pressure mobilization, and improving protein solubility by incorporating urea into the carrier ampholyte (CA) solutions. Loss of the extreme pI CAs and sample components by the bidirectional ITP inherent to IEF was avoided by setting the concentration of the phosphoric acid anolyte to 200 mM and sodium hydroxide catholyte to 300 mM and by adding sufficient amounts of an acidic (pI<3) and basic (10

Particle Loaded Monolithic Sol-Gel Columns for Capillary Electrochromatography: A New Dimension for High Performance Liquid Chromatography

Particle-loaded (3 pm, octadecylsilica) monolithic sol-gel columns have been prepared and selected characteristics measured. Several electrical properties may be calculated from simple current measurements in the column as a whole. Resistivity in the packed segment is approximately three times that in open segments, resulting in a 60% increase in field strength in the packed regions compared to the capillary with no packing. The surprisingly high specific permeability of these sol-gel columns is characteristic of 8-pm particles, which allows their operation in the microLC mode at pressures as low as 69 kPa where their efficiency is about 50,000 plates per meter and in the CEC mode where efficiency is about 106,000 plates per meter at 5 kV. There is a relatively rapid loss of efficiency with increasing linear velocity beyond 0.2 mm/s in microLC mode, which may be due to additional diffusion processes in the inter-particulate voids. A rapid loss of efficiency above 0.5 mm/s is also observed in the CEC mode, for the same reasons. Chromatographic retention behavior in either separation mode is characteristic of conventional octadecylsilica particles, indicating that analytes have significant access to the surface within the pores of the immobilized bonded phase.

Flow-Through Microvial Facilitating Interface of Capillary Isoelectric Focusing and Electrospray Ionization Mass Spectrometry

A capillary electrophoresis mass spectrometry (CE-MS) interface utilizing a flow-through microvial is used to ensure the electric continuity and supply the catholyte and mobilizer solutions during the capillary isoelectric focusing (cIEF) and mobilization process. The flow-through microvial provides a stable chemical environment and helps to improve the ionization efficiency without significantly diluting the analyte. The CE-MS interface facilitates the transfer of the mobilized cIEF effluent to the site of electrospray ionization, and the gaseous ions can be detected directly by a mass spectrometer. It also allows for complete focusing and mobilization processes to be performed automatically in programmed sequences with commercial CE systems. Two different strategies, using either a part of the capillary or the flow-through microvial of the CE-MS interface as the catholyte reservoir for bare fused silica capillaries or neutral coated capillaries, respectively, were developed for automated cIEF-electrospray ionization (ESI)-MS. Reasonable separation efficiency was achieved using proper concentration of carrier ampholytes and suitable strategies of electroosmotic/electrophoretic mobilization.

Characteristics of particle-loaded monolithic sol–gel columns for capillary electrochromatography: I. Structural, electrical and band-broadening properties

Particle-loaded (3 microm, C18) monolithic sol-gel columns have been prepared and selected characteristics measured. They have a surprisingly high permeability, allowing their operation in the microLC mode at pressures as low as 69 kPa where their efficiency is about 50000 plates per meter and the CEC mode where efficiency is at least 106000 plates per meter. These columns can withstand over 13.8 MPa pressure without compression or movement within the 75 microm capillary. Field strengths in the packed segments are approximately 50% greater than those in the open segments, due to the higher resistivity of the particle-laden regions. There is a relatively rapid loss of efficiency with increasing linear velocity in both the CEC and microLC modes, which may be due to a tortuosity effect in the inter- and intra-particulate voids. Chromatographic behavior is characteristic of conventional C18 particles, indicating that analytes have significant access to the surface within the pores of the immobilized bonded phase.

A promising capillary electrophoresis-electrospray ionization-mass spectrometry method for carbohydrate analysis.

A method for adapting widely used CE conditions for the separation of fluorescently labeled carbohydrates to permit online ESI‐MS detection is presented. Reverse polarity separations were performed in bare fused‐silica capillaries with an acidic BGE. Under these conditions, negatively charged 8‐aminopyrene 1,3,6‐trisulfonate‐labeled carbohydrates migrate forward against the EOF, which is towards the capillary inlet. Therefore, the CE‐MS interface must simultaneously back‐fill the capillary, in order to maintain the CE circuit, and provide a stable forward flow at the sprayer tip to support the electrospray process. This was achieved using a junction‐at‐the‐tip interface, which provides a flow of solution to the junction formed by the capillary terminus and the inner wall of the emitter needle tip. Because the flow rate required for this arrangement is much less than in conventional sheath flow interfaces, dilution of the analytes is minimized. Optimized separation conditions permit baseline resolution of glucose oligomers containing up to 15 glucose units, while longer oligomers, up to 33 glucose units, were observed as resolved peaks in the negative ion mode mass spectrum.

Capillary electrophoresis-mass spectrometry using an in-line sol-gel concentrator for the determination of methionine enkephalin in cerebrospinal fluid

In this study, a CE-MS method using a monolithic sol-gel concentrator for in-line solid-phase extraction (SPE) is evaluated for the analysis of methionine enkephalin in biological samples. Operational SPE parameters such as sample pH, loading volume, elution volume and composition have been studied. After optimization of the in-line preconcentration methodology, a 40-fold preconcentration was demonstrated for a methionine enkephalin test solution using a loading volume of 3200 nL. The method was linear in the range from 62.5 to 1000 ng/mL (R(2)>0.99). R.S.D. values for migration times and peak areas were 1.2% and 8.4%, respectively. Finally, the analysis of cerebrospinal fluid samples spiked with methionine enkephalin and deproteinized with perchloric acid (1:1, v/v) showed a detection limit (S/N=3) of approximately 1 ng/mL (ca. 5 nM). The recoveries of methionine enkephalin for three concentration levels (100, 10 and 1 ng/mL) were in the range of 74-91%, demonstrating the promising potential of the methodology for the analysis of biological samples.

Lateral interaction between electrostatically adsorbed and covalently immobilized proteins on the surface of cation-exchange sorbents

This paper examines the nature of chromatographic separations on a weak cation-exchange material in which immobilized proteins coats 50% or less of the sorbent surface. It was found that although these sorbents still function as cation exchangers, covalently immobilized proteins frequently contribute to the ion-exchange behavior of some protein analytes. Chromatographic retention of analytes was equal to or greater on immobilized protein derivatized columns than underivatized sorbents. Anionic proteins, in contrast, were not adsorbed, indicating that immobilized proteins were acting synergistically with ionic stationary phase groups to enhance retention. It was concluded that electrostatic adsorption is a prerequisite for analyte protein/immobilized protein interactions of sufficient magnitude to impact ion-exchange separations. Large differences in protein resolution were observed on columns that were identical in all respects except for the immobilized protein, further confirming that analyte/immobilized protein interactions were unique to the interacting pair. The extent of interaction was also influenced by concentration of the immobilized protein in the case of lysozyme. Interactions between the analyte and immobilized protein were found to occur between both the same two proteins and dissimilar species. It was concluded that these phenomena are due to lateral interactions between immobilized proteins and analyte proteins subsequent to electrostatic adsorption of the analyte on the underivatized surface of ion-exchange sorbents.

Study of protein binding to a silica support with a polymeric cation-exchange coating.

A silica-based, polyacrylate ion-exchange stationary phase has been prepared using Ce(IV) as the initiator. Analysis of the physical properties of the polymeric layer separated from the silica surface indicates that the polymeric coating is cross linked to some extent. The polymerization carried out at different concentrations of Ce(IV) demonstrated that the effective surface area can be increased by lowering the Ce(IV) concentration at higher monomer concentrations of the reaction mixture. These materials are quite reproducible and of high electrostatic binding capacity; 1.485 mumol/m2. The electrostatic binding capacity of a non-polymeric stationary phase reached the theoretical limit for a monolayer (0.16 mumol/m2). However, the covalent binding capacity of the same stationary phase was only 50% of the electrostatic binding capacity. The same trend was observed in all the polymeric stationary phases tested. This shows that the mechanism of protein binding in polymeric and conventional stationary phases is similar, and multilayer electrostatic binding is highly unlikely in these sorbents examined. Z numbers revealed that the contact area of the protein is independent of the polymeric character of the stationary phase and therefore, the increased loading of these polymeric stationary phases is due to the increased surface area.