Ion desorption efficiency and internal energy transfer in polymeric electrospun nanofiber-based surface-assisted laser desorption/ionization mass spectrometry.

The understanding of the desorption mechanism in surface-assisted laser desorption/ionization (SALDI) remains incomplete because there are numerous types of SALDI materials with a broad range of physical and chemical properties, many of which impact the ultimate analytical performance in terms of signal generation. In this study, the chemical thermometer molecule, benzylpyridinium chloride, is applied to investigate the desorption process of SALDI using electrospun nanofibrous polymer and polymer composite substrates. The ion desorption efficiency was inversely related to the ion internal energy, which could not be fully explained by a thermal desorption mechanism. A competing non-thermal desorption (i.e., phase transition/explosion) was proposed to be involved in this SALDI process. The influence of the orientation and dimension of the nanofiber structure revealed that a cross-linked nanofiber network with a small diameter favored the nanofiber-assisted LDI to provide efficient ion desorption. Graphical abstract.

Separation and characterization of KRas proteins and tryptic peptides using enhanced-fluidity liquid chromatography tandem mass spectrometry.

Enhanced-fluidity, reversed-phase liquid chromatography was developed using custom instrumentation for separation and characterization of intact KRas proteins and tryptic peptides. The KRas, HRas and NRas function as GDP-GTP regulated binary switches in many signalling pathways, and mutations in Ras proteins are frequently found in human cancers and represent poor prognosis markers for patients. Mutations of the KRas isoform constitute some of the most common aberrations among all human cancers and intensive drug discovery efforts have been directed toward targeting the KRas protein. Separation and characterization of the KRas protein and tryptic peptides are helpful for exploring targeting, which has not been fully investigated using liquid chromatography-tandem mass spectrometry. EFLC-MS provided improved chromatographic performance compared to traditional HPLC-MS in terms of shorter analysis time, increased ion intensity and a shift to higher charge states for intact KRas proteins.

Enhanced-Fluidity Liquid Chromatography-Mass Spectrometry for Intact Protein Separation and Characterization.

Recent advances in the analysis of proteins have increased the demand for more efficient techniques to separate intact proteins. Enhanced-fluidity liquid chromatography (EFLC) involves the addition of liquefied CO2 to conventional liquid mobile phases. The addition of liquefied CO2 increases diffusivity and decreases viscosity, which inherently leads to a more efficient separation. Herein, EFLC is applied to hydrophobic interaction chromatography (HIC) stationary phases for the first time to study the impact of liquefied CO2 to the chromatographic behavior of proteins. The effects of liquefied CO2 on chromatographic properties, charge state distributions (CSDs), and ionization efficiencies were evaluated. EFLC offered improved chromatographic performance compared to conventional liquid chromatography (LC) methods including a shorter analysis time, better peak shapes, and higher plate numbers. The addition of liquefied CO2 to the mobile phase provided an electrospray ionization (ESI)-friendly and "supercharging" reagent without sacrificing chromatographic performance, which can be used to improve peptide and protein identification in large-scale application.

Separation of PEGylated Gold Nanoparticles by Micellar Enhanced Electrospun Fiber Based Ultrathin Layer Chromatography.

Gold nanoparticles (AuNPs) are of great interest in many fields, especially in biomedical applications. Thiol terminated polyethylene glycol (PEG) is the most widely used polymer to increase the biocompatibility of nanoparticle therapeutics. Herein, a rapid method for separation and characterization of PEGylated AuNPs on an ultrathin layer chromatographic (UTLC) plate using electrospun polyacrylonitrile (PAN) nanofibers as the stationary phase is described. AuNPs with sizes ranging from 10 to 80 and 30 nm AuNPs coated with various molecular weight of PEG (2, 5, 10, and 20 kDa) were all successfully separated by UTLC using optimized conditions. The fabrication of electrospun UTLC is simple, fast, and inexpensive. The UTLC, with much thinner sorbent layer (10× thinner than traditional TLC) and small fiber size (∼300 nm), requires minimal mobile phase solvent and provides faster separation and higher resolution compared to other separation methods for AuNPs. AuNPs with different sizes and different PEG molecular weights were well separated within 5 min with lowest plate height <2 µm and resolution value >1.5. As an example of this method, the size transformation of AuNPs in serum protein was determined quantitatively.

Enhanced fluidity liquid chromatography of inulin fructans using ternary solvent strength and selectivity gradients.

The value of exploring selectivity and solvent strength ternary gradients in enhanced fluidity liquid chromatography (EFLC) is demonstrated for the separation of inulin-type fructans from chicory. Commercial binary pump systems for supercritical fluid chromatography only allow for the implementation of ternary solvent strength gradients which can be restrictive for the separation of polar polymeric analytes. In this work, a custom system was designed to extend the capability of EFLC to allow tuning of selectivity or solvent strength in ternary gradients. Gradient profiles were evaluated using the Berridge function (RF1), normalized resolution product (NRP), and gradient peak capacity (Pc). Selectivity gradients provided the separation of more analytes over time. The RF1 function showed favor to selectivity gradients with comparable Pc to that of solvent strength gradients. NRP did not strongly correlate with Pc or RF1 score. EFLC with the hydrophilic interaction chromatography, HILIC, separation mode was successfully employed to separate up to 47 fructan analytes in less than 25 min using a selectivity gradient.

Protein separations using enhanced-fluidity liquid chromatography.

Enhanced-fluidity liquid chromatography (EFLC) methods using methanol/H2O/CO2 and hydrophilic interaction liquid chromatography (HILIC) were explored for the separation of proteins and peptides. EFLC is a separation mode that uses a mobile phase made of conventional solvents combined with liquid carbon dioxide (CO2) in subcritical conditions. The addition of liquid CO2 enhances diffusivity and decreases viscosity while maintaining mixture polarity, which typically results in reduced time of analysis. TFA additive and elevated temperature were leveraged as key factors in the separation of a 13-analyte intact protein mixture in under 5min. Under these conditions EFLC showed modest improvement in terms of peak asymmetry and analysis time over the competing ACN/H2O separation. Protein analytes detected by electrospray ionization - quadrupole time of flight, were shown to be unaffected by the addition of CO2 in the mobile phase. Herein, the feasibility of separating hydrophilic proteins up to 80kDa (with transferrin) is demonstrated for CO2-containing mobile phases.

Electrospun Nafion-Polyacrylonitrile nanofibers as an ion exchange ultrathin layer chromatographic stationary phase.

An ion-exchange method to separate charged biomolecules on ultrathin layer chromatographic (UTLC) plates using electrospun Nafion-Polyacrylonitrile (PAN) nanofibers as the stationary phase is described. Sulfonate groups on Nafion provide the ion-exchange sites. The addition of PAN (a higher molecular weight polymer than Nafion) was used to facilitate the nanofiber formation process using electrospinning. Electrospinning parameters and separation conditions were optimized using fractional factorial design and response surface methodology. Nafion-PAN nanofibers containing 45% (w/w) Nafion with 0.407 mmol/g of SO3H group and 16.0 mmol/g of fluorine as an ion exchange stationary phase for UTLC were evaluated using the separations of amino acids and proteins, followed by visualizations using ninhydrin and fluorescamine, respectively. The electrospun Nafion-PAN plates showed high chemical stability under various mobile phase conditions. Mobile phase velocity decreased with the addition of Nafion into the electrospinning solutions. The sources of band broadening of analyte spots were investigated. The separation of amino acids showed high selectivity and separation efficiency. The separation of four proteins demonstrated the feasibility of Nafion-PAN UTLC for separating large biomolecules.

Surface-assisted laser desorption/ionization time-of-flight mass spectrometry of small drug molecules and high molecular weight synthetic/biological polymers using electrospun composite nanofibers.

Polyacrylonitrile/Nafion®/carbon nanotube (PAN/Nafion®/CNT) composite nanofibers were prepared using electrospinning. These electrospun nanofibers were studied as possible substrates for surface-assisted laser desorption/ionization (SALDI) and matrix-enhanced surface-assisted laser desorption/ionization time-of-flight mass spectrometry (ME-SALDI/TOF-MS) for the first time in this paper. Electrospinning provides this novel substrate with a uniform morphology and a narrow size distribution, where CNTs were evenly and firmly immobilized on polymeric nanofibers. The results show that PAN/Nafion®/CNT nanofibrous mats are good substrates for the analysis of both small drug molecules and high molecular weight polymers with high sensitivity. Markedly improved reproducibility was observed relative to MALDI. Due to the composite formation between the polymers and the CNTs, no contamination of the carbon nanotubes to the mass spectrometer was observed. Furthermore, electrospun nanofibers used as SALDI substrates greatly extended the duration of ion signals of target analytes compared to the MALDI matrix. The proposed SALDI approach was successfully used to quantify small drug molecules with no interference in the low mass range. The results show that verapamil could be detected with a surface concentration of 220 femtomoles, indicating the high detection sensitivity of this method. Analysis of peptides and proteins with the electrospun composite substrate using matrix assisted-SALDI was improved and a low limit of detection of approximately 6 femtomoles was obtained for IgG. Both SALDI and ME-SALDI analyses displayed high reproducibility with %RSD ≤ 9% for small drug molecules and %RSD ≤ 14% for synthetic polymers and proteins.

Gradient separation of oligosaccharides and suppressing anomeric mutarotation with enhanced-fluidity liquid hydrophilic interaction chromatography.

Enhanced fluidity liquid chromatography using the hydrophilic interaction retention mechanism (EFLC-HILIC) is studied as an alternative separation mode for analyzing oligosaccharides and other sugars. These carbohydrates, which are important for the study of foods and biological systems, are difficult to comprehensively profile and either require a non-green, expensive solvent (i.e. acetonitrile) or derivatization of the analytes at the expense of time, sample loss, and loss of quantitative information. These difficulties arise from the diverse isomerism, mutarotation, and lack of a useable chromophore/fluorophore for spectroscopic detection. Enhanced fluidity liquid chromatography is an alternative separation method that involves the use of conventional polar solvents, such as methanol/water mixtures, as the primary mobile phase component and liquid carbon dioxide (CO2) as the modifier in subcritical conditions. The addition of liquid CO2 enhances diffusivity and decreases viscosity while maintaining mixture polarity, which typically results in reduced time of analysis and higher efficiency. This work illustrates an optimized EFLC-HILIC separation of a test mixture of oligosaccharides and simple sugars with a resolution greater than 1.3 and an analysis time decrease of over 35% compared to a conventional HPLC HILIC-mode analysis using acetonitrile/water mobile phases.

Gradient enhanced-fluidity liquid hydrophilic interaction chromatography of ribonucleic acid nucleosides and nucleotides: A "green" technique.

A "green" hydrophilic interaction liquid chromatography (HILIC) technique for separating the components of mixtures with a broad range of polarities is illustrated using enhanced-fluidity liquid mobile phases. Enhanced-fluidity liquid chromatography (EFLC) involves the addition of liquid CO2 to conventional liquid mobile phases. Decreased mobile phase viscosity and increased analyte diffusivity results when a liquefied gas is dissolved in common liquid mobile phases. The impact of CO2 addition to a methanol:water (MeOH:H2O) mobile phase was studied to optimize HILIC gradient conditions. For the first time a fast separation of 16 ribonucleic acid (RNA) nucleosides/nucleotides was achieved (16min) with greater than 1.3 resolution for all analyte pairs. By using a gradient, the analysis time was reduced by over 100% compared to similar separations conducted under isocratic conditions. The optimal separation using MeOH:H2O:CO2 mobile phases was compared to MeOH:H2O and acetonitrile:water (ACN:H2O) mobile phases. Based on chromatographic performance parameters (efficiency, resolution and speed of analysis) and an assessment of the environmental impact of the mobile phase mixtures, MeOH:H2O:CO2 mixtures are preferred over ACN:H2O or MeOH:H2O mobile phases for the separation of mixtures of RNA nucleosides and nucleotides.

Enhanced-fluidity liquid chromatography using mixed-mode hydrophilic interaction liquid chromatography/strong cation-exchange retention mechanisms.

The potential of enhanced-fluidity liquid chromatography, a subcritical chromatography technique, in mixed-mode hydrophilic interaction/strong cation-exchange separations is explored, using amino acids as analytes. The enhanced-fluidity liquid mobile phases were prepared by adding liquefied CO2 to methanol/water mixtures, which increases the diffusivity and decreases the viscosity of the mixture. The addition of CO2 to methanol/water mixtures resulted in increased retention of the more polar amino acids. The "optimized" chromatographic performance (achieving baseline resolution of all amino acids in the shortest amount of time) of these methanol/water/CO2 mixtures was compared to traditional acetonitrile/water and methanol/water liquid chromatography mobile phases. Methanol/water/CO2 mixtures offered higher efficiencies and resolution of the ten amino acids relative to the methanol/water mobile phase, and decreased the required isocratic separation time by a factor of two relative to the acetonitrile/water mobile phase. Large differences in selectivity were also observed between the enhanced-fluidity and traditional liquid mobile phases. A retention mechanism study was completed, that revealed the enhanced-fluidity mobile phase separation was governed by a mixed-mode retention mechanism of hydrophilic interaction/strong cation-exchange. On the other hand, separations with acetonitrile/water and methanol/water mobile phases were strongly governed by only one retention mechanism, either hydrophilic interaction or strong cation exchange, respectively.

Homogeneous edge-plane carbon as stationary phase for reversed-phase liquid chromatography.

Carbon stationary phases have been widely used in HPLC due to their unique selectivity and high stability. Amorphous carbon as a stationary phase has at least two sites of interaction with analytes: basal-plane and edge-plane carbon sites. The polarity and adsorptivity of the two sites are different. In this work, the edge-plane carbon stationary phase is prepared by surface-directed liquid crystal assembly. Specific precursor polymers form discotic liquid crystal phases during the pyrolysis process. By using silica as the substrate to align the discotic liquid crystal, edge-plane carbon surfaces were formed. Similar efficiencies as observed for Hypercarb were observed in chromatograms. The column efficiency was studied as a function of linear flow rate. A minimum reduced plate height of 6 was observed in these studies. To evaluate the performance of the homogeneous edge-plane carbon stationary phase, linear solvation energy relationships were used to compare these ordered carbon surfaces to commercially available carbon stationary phases, including Hypercarb. Reversed-phase separations of nucleosides, nucleotides, and amino acids and derivatives were demonstrated using the ordered carbon surfaces, respectively. The column batch-to-batch reproducibility was also evaluated. The retention times for the analytes were reproducible within 1-6% depending on the analyte.

Development of a new separation media using ultra-thin glassy carbon film modified silica.

A self-polymerizable octatetrayne, 1,8-dialdehydebenzyl-1,3,5,7-octatetrayne, is synthesized and covalently attached to an amino-functionalized surface of silica particles. The silica particles with a monolayer coverage of octatetrayne were then thermally processed to various final temperatures of 200, 400 and 700°C. The amino-functionalization, covalent attachment of octatetrayne and thermal process of silica particles were monitored by scanning electron microscopy (SEM), infrared (IR) spectroscopy and thermogravimetric analysis (TGA). The thermally processed particles were then packed into a capillary column and evaluated as a stationary phase for HPLC. After chromatographic evaluation, the optimized temperature for thermal processing was determined to be 400°C, which provides the best modified silica particles SiO2-OCT-T400 with an ultra-thin glassy carbon film coating. The linear solvation energy relationship model indicated that the primary contributors in retention are dispersion and H-bond basicity. The application of SiO2-OCT-T400 as a stationary phase was further demonstrated by successful separation of nonpolar hydrocarbons mixture and a nucleosides mixture.

Planar electrochromatography using an electrospun polymer nanofiber layer.

Electrospun polymer nanofiber stationary phases were examined for their application to planar electrochromatography (PEC). Separations were performed on polyacrylonitrile nanofiber ultra-thin-layer chromatography (UTLC) plates in 1-2 min using a ternary mobile phase. The influences of buffer concentration and pH, ratio of organic modifier, and development time on analyte migration distances were studied. Band broadening in this system was studied as a function of distance. The plate height initially decreased and then plateaued with a minimum plate height value as low as 11 µm. Nanofiber alignment considerably increased analyte migration rate, but larger spot sizes were noted when nearly complete fiber alignment was used. The easily tunable stationary phase thickness can be tailored to a given separation, where thinner layers promote faster separations and thicker layers are ideal for more complex mixtures. Compared to UTLC, PEC offers unique selectivity and decreased analysis time (>4 times faster over 15 mm). Results for a two-dimensional separation using UTLC and PEC are also reported. These rapid separations required 11 min using a 40 × 40 mm plate and exhibited a significant increase in separation number (70-77).

Silica-based nanofibers for electrospun ultra-thin layer chromatography.

Nanofibrous silica-based stationary phases for electrospun ultra-thin layer chromatography (E-UTLC) are described. Nanofibers were produced by electrospinning a solution of silica nanoparticles dispersed in polyvinylpyrrolidone solutions to create composite silica/polymer nanofibers. Stationary phases were created from as-spun nanofibers, or the nanofibers were heated either to crosslink the polyvinylpyrrolidone or to calcine and selectively remove the polymer. As-spun, crosslinked, and calcined nanofibers with similar mat thicknesses (23-25 µm) were evaluated as stationary phases for E-UTLC separations of laser dyes and amino acids and compared to commercial silica TLC plates. As-spun nanofiber plates offered fast mobile phase velocities, but like other polymer-based nanofibers, separations were only compatible with techniques using nonsolvents of the polymer. Crosslinked nanofibers were not as limited in terms of chemical stability, but separations produced tailed spot shapes. No limitations in terms of mobile phases, analyte solvents, and visualization techniques were observed for calcined nanofibers. Highly efficient separations of amino acids were performed in 15 mm on calcined nanofiber plates, with observed plate heights as low as 8.6 µm, and plate numbers as large as 1400. Additional alignment of the nanofibers provided shorter analysis times but also larger spot widths. The extension of stationary phases to silica-based nanofibers vastly expands the range of mobile phases, analyte solvents, and visualization techniques which can be used for E-UTLC separations.

Carbon nanotube and carbon nanorod-filled polyacrylonitrile electrospun stationary phase for ultrathin layer chromatography.

The application of carbon nanotube or nanorod/polyacrylonitrile (PAN) composite electrospun nanofibrous stationary phase for ultrathin layer chromatography (UTLC) is described herein. Multi-walled carbon nanotubes (MWCNTs) and edge-plane carbon (EPC) nanorods were prepared and electrospun with the PAN polymer solution to form composite nanofibers for use as a UTLC stationary phase. The analysis of laser dyes demonstrated the feasibility of utilizing carbon nanoparticle-filled electrospun nanofibers as a UTLC stationary phase. The contribution of MWCNT or EPC in changing selectivity of the stationary phase was studied by comparing the chromatographic behavior among MWCNT-PAN plates, EPC-PAN plates and pure PAN plates. Carbon nanoparticles in the stationary phase were able to establish strong π-π interactions with aromatic analytes. The separation of five polycyclic aromatic hydrocarbons (PAHs) demonstrated enhanced chromatographic performance of MWCNT-filled stationary phase by displaying substantially improved resolution and separation efficiency. Band broadening of the spots for MWCNT or EPC-filled UTLC stationary phases was also investigated and compared with that for pure PAN stationary phases. A 50% improvement in band dispersion was noted using the MWCNT based composite nanofibrous UTLC plates.

Electrospun nanofibers as substrates for surface-assisted laser desorption/ionization and matrix-enhanced surface-assisted laser desorption/ionization mass spectrometry.

Electrospun polymeric nanofibers (polyacrylonitrile, poly(vinyl alcohol), and SU-8 photoresist) and carbon nanofibers pyrolyzed to final temperatures of 600, 800, and 900 °C were used as substrates for surface-assisted laser desorption/ionization (SALDI) and matrix-enhanced surface-assisted laser desorption/ionization (ME-SALDI) analyses. Sample preparation of polymeric analytes using the electrospun target plate for SALDI analysis is simple and fast. Signal enhancements for poly(ethylene glycol) were noted with nanofibrous carbon substrates compared to those obtained with commercially available stainless steel plates when no organic matrix is used. Minimal fragmentation was observed. Poly(ethylene glycol) with a molecular weight as high as 900 000 Da was successfully detected using the carbon nanofibrous substrate processed to 800 °C, which is the highest molecular weight that has been studied by SALDI. Small molecules were detected using nanofibrous carbon substrate processed to 800 °C. For example, spectra of glucose, arginine, and crystal violet were obtained with no observed interferences in the low molecular weight range. The SALDI results show enhanced shot-to-shot reproducibility compared to matrix-assisted laser desorption/ionization (MALDI). High-quality polystyrene spectra were obtained for the first time using SALDI nanofibrous polyacrylonitrile substrates. Significantly enhanced signal-to-noise ratios were obtained using ME-SALDI compared to conventional MALDI or SALDI for the studied analytes. A detection limit of 400 amol was achieved for angiotensin I using the nanofibrous carbon ME-SALDI substrate.

Aligned electrospun nanofibers for ultra-thin layer chromatography.

The fabrication and implementation of aligned electrospun polyacrylonitrile (PAN) nanofibers as a stationary phase for ultra-thin layer chromatography (UTLC) is described. The aligned electrospun UTLC plates (AE-UTLC) were characterized to give an optimized electrospun mat consisting of high nanofiber alignment and a mat thickness of ~25 µm. The AE-UTLC devices were used to separate a mixture of ß-blockers and steroidal compounds to illustrate the properties of AE-UTLC. The AE-UTLC plates provided shorter analysis time (~2-2.5 times faster) with improved reproducibility (as high as 2 times) as well as an improvement in efficiency (up to100 times greater) relative to non-aligned electrospun-UTLC (E-UTLC) devices.

Electrospun polyvinyl alcohol ultra-thin layer chromatography of amino acids.

Electrospun polyvinyl alcohol (PVA) ultrathin layer chromatographic (UTLC) plates were fabricated using in situ crosslinking electrospinning technique. The value of these ULTC plates were characterized using the separation of fluorescein isothiocyanate (FITC) labeled amino acids and the separation of amino acids followed visualization using ninhydrin. The in situ crosslinked electrospun PVA plates showed enhanced stability in water and were stable when used for the UTLC study. The selectivity of FITC labeled amino acids on PVA plate was compared with that on commercial Si-Gel plate. The efficiency of the separation varied with analyte concentration, size of capillary analyte applicator, analyte volume, and mat thickness. The concentration of 7mM or less, 50µm i.d. capillary applicator, minimum volume of analyte solution and three-layered mat provides the best efficiency of FITC-labeled amino acids on PVA UTLC plate. The efficiency on PVA plate was greatly improved compared to the efficiency on Si-Gel HPTLC plate. The hydrolysis products of aspartame in diet coke, aspartic acid and phenylalanine, were also successfully analyzed using PVA-UTLC plate.