LC Modes Research Articles

Online multimethod platform for comprehensive characterization of monoclonal antibodies in cell culture fluid from a single sample injection - Intact protein workflow

BackgroundTherapeutic monoclonal antibodies (mAbs) comprise a large structural variability with respect to charge, size and post-translational modifications. These critical quality attributes (CQAs) need to be assessed during and after the production of mAbs. This normally requires off-line purification and sample preparation as well as several chromatographic selectivities, which makes the whole process time-consuming and error-prone. To improve on this, we developed an integrated and automated multi-dimensional analytical platform for the simultaneous assessment of multiple CQAs of mAbs in cell culture fluid (CCF) from upstream processes. ResultsThe on-line system allows mAb characterization at the intact level, combining protein A affinity chromatography (ProtA) with size-exclusion, ion-exchange, and reversed-phase liquid chromatographic modes with UV and mass spectrometric detection. Multiple heart cuts of a single mAb elution band from ProtA are stored in 20-μL loops and successively sent to the multimethod options in the second dimension. ProtA loading and elution conditions and their compatibility with second-dimension LC modes were studied and optimized. Subsequently, heart-cutting and valve-switching schemes were investigated to achieve effective and reproducible analyses. The applicability of the developed workflow was demonstrated by the direct analysis (i.e. not requiring off-line sample preparation) of a therapeutic mAb in CCF, obtaining useful information on accurate molecular mass, glycosylation, and charge and size variants of the mAb product at the same time and in just over 1 h. SignificanceThe developed multidimensional platform is the first system that allows for multiple fractions from a single ProtA band to be characterized using different chromatographic selectivities in a single run allowing direct correlation between CQAs. The performance of the system is comparable to established off-line methods, fully compatible with upstream process samples, and provides a significant time-reduction of the characterization procedure.

Sub/supercritical fluid chromatography versus liquid chromatography for peptide analysis

The aim of this study was to evaluate the potential of ultra-high performance supercritical fluid chromatography (UHPSFC) for peptide analysis by comparing its analytical performance to several chromatographic approaches based on reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC) and mixed-mode liquid chromatography. First, the retention behavior of synthetic peptides with 3 to 30 amino acids and different isoelectric points (acid, neutral, and basic) was evaluated. For all the tested conditions (13 peptides in 8 conditions), only 4 results were not exploitable (not retained or not eluted), confirming that all the tested chromatographic conditions can be successfully applied when analyzing a wide range of diverse peptides. Average tailing factor were quite comparable across all chromatographic modes, while the best peak capacity values were obtained under mixed-mode LC conditions. Selectivity for each chromatographic mode was also evaluated for six closely related peptides having minor modifications on their structures. The LC-based chromatographic modes confirmed their superior selectivity over UHPSFC. By contrast, when analyzing short peptides (di- or tripetides), UHPSFC was the only technique allowing to simultaneously separate highly polar and less polar peptides within the same run confirming its unique versatility. In addition, the sensitivity of each chromatographic approach was accessed by for two representative peptides by both UV and MS detection. With UV detection, limit of detection (LOD) values were comparable among the different chromatographic modes, ranging from 0.5 to 2 µg mL−1. However, major differences were found when employing MS detection (LOD values ranged from 0.05 to 5 µg mL−1). The best results were obtained under HILIC conditions, followed by SFC, and finally mixed-mode LC and RPLC modes.

Miniaturized liquid chromatography applied to the analysis of residues and contaminants in food: A review.

The humankind is pretty dependent on food to control several biological processes into the organism. As the world population increases, the demand for foodstuffs follows the same trend claiming for a high food production situation. For this reason, a substantial amount of chemicals is used in agriculture and livestock husbandries every year, enhancing the likelihood of contaminated foodstuffs being commercialized. This outlook becomes a public health concern; thus, the governmental regulatory agencies impose laws to control the residues and contaminants in food matrices. Currently, one of the most important analytical techniques to perform it is LC. Despite its already recognized effectiveness, it is often time consuming and requires significant volumes of reagents, which are transformed into toxic waste. In this context, miniaturized LC modes emerge as a greener and more effective analytical technique. They have remarkable advantages, including higher sensitivity, lower sample amount, solvent and stationary phase requirements, and more natural coupling to MS. In this review, most of the critical characteristics of them are discussed, focusing on the benchtop instruments and their related analytical columns. Additionally, a discussion regarding the last 10 years of publications reporting miniaturized LC application for the analysis of natural and industrial food samples is categorized. The main chemical classes as applied in the crops are highlighted, including pesticides, veterinary drugs, and mycotoxins.

Theoretical Investigation on Microcavity Coupler for Terahertz Quantum-Well Infrared Photodetectors

Resonant behaviors and absorption enhancement of metallic grating-dielectric-metal (GDM) microcavity are theoretically investigated. The GDM structure is treated as periodic unit cells of two serially-connected metal-dielectric-metal (MDM) and air-dielectric-metal (ADM) waveguide resonators. The resonant modes can be divided into three types: the LC mode, the TEM modes supported by the MDM waveguide resonator, and the TEM-TM hybrid modes supported by the whole GDM structure. The resonant frequencies of LC and TEM modes are independent on a small incident oblique angle. However, for the TEM-TM hybrid modes, each mode splits into two branches with non-zero incident angles. Based on the temporal coupled-mode theory, the intersubband absorption efficiency of multi quantum wells inserted into the GDM microcavity is calculated and discussed qualitatively. We point out that the condition of critical coupling is not the optimal condition for intersubband absorption efficiency when the metallic loss cannot be neglected. An optimization procedure is given for the design of high performance GDM-microcavity-coupled terahertz quantum-well infrared photodetectors.

Exploiting the Dynamic Relationship between Peptide Separation Quality and Peptide Coisolation in a Multiple-Peptide Matches-per-Spectrum Approach Offers a Strategy To Optimize Bottom-Up Proteomics Throughput and Depth.

Peptide cofragmentation leads to chimeric MS/MS spectra that negatively impact traditional single-peptide match-per-spectrum (sPSM) search strategies in proteomics. The collection of chimeric spectra is influenced by peptide coelution and the width of precursor isolation windows. Although peptide cofragmentation can be reduced by advanced chromatography, such as UHPLC and 2D-HPLC separation schemes, and narrower isolation windows, chimeric spectra can still be as high as 30-50% of the total MS/MS spectra collected. Alternatively, cofragmented peptides in chimeric spectra and the use of wider isolation windows benefit multiple-peptide matches-per-spectrum (mPSM) algorithms, such as CharmeRT, which facilitate the identification of several cofragmented peptides. Considering recent advancements in LC and mPSM methodologies, we present a comprehensive examination of the levels of chimeric spectra collected in the analysis of a HeLa digest measured using different LC modes of separation and isolation windows and compare the depth of identifications obtained when these data are annotated using a sPSM or a mPSM approach. Our results demonstrate that MS/MS data derived from 1D-HPLC strategies under different gradient schemes and searched with CharmeRT yielded higher average numbers of PSMs (11%-49%), peptide analytes (10%-16%), and peptide sequences (3%-10%) compared to data derived from 1D-UHPLC runs but searched with a sPSM strategy. Interestingly, data from a 2D-HPLC separation strategy benefits more from the application of CharmeRT results when compared to a 50 cm 1D-UHPLC column employing a 500 min gradient. Overall, these results provide new insights into how to better configure LC-MS/MS measurements for improved throughput and peptide identification in complex proteomes.

Computer-aided gradient optimization of hydrophilic interaction liquid chromatographic separations of intact proteins and protein glycoforms

Protein glycosylation is one of the most common and critical post-translational modification, which results from covalent attachment of carbohydrates to protein backbones. Glycosylation affects the physicochemical properties of proteins and potentially their function. Therefore it is important to establish analytical methods which can resolve glycoforms of glycoproteins. Recently, hydrophilic-interaction liquid chromatography (HILIC)-mass spectrometry has demonstrated to be a useful tool for the efficient separation and characterization of intact protein glycoforms. In particular, amide-based stationary phases in combination with acetonitrile-water gradients containing ion-pairing agents, have been used for the characterization of glycoproteins. However, finding the optimum gradient conditions for glycoform resolution can be quite tedious as shallow gradients (small decrease of acetonitrile percentage in the elution solvent over a long time) are required. In the present study, the retention mechanism and peak capacity of HILIC for non-glycosylated and glycosylated proteins were investigated and compared to reversed-phase liquid chromatography (RPLC). For both LC modes, ln k vs. φ plots of a series of test proteins were calculated using linear solvent strength (LSS) analysis. For RPLC, the plots were spread over a wider φ range than for HILIC, suggesting that HILIC methods require shallower gradients to resolve intact proteins. Next, the usefulness of computer-aided method development for the optimization of the separation of intact glycoform by HILIC was examined. Five retention models including LSS, adsorption, and mixed-mode, were tested to describe and predict glycoprotein retention under gradient conditions. The adsorption model appeared most suited and was applied to the gradient prediction for the separation of the glycoforms of six glycoproteins (Ides-digested trastuzumab, alpha-acid glycoprotein, ovalbumin, fetuin and thyroglobulin) employing the program PIOTR. Based on the results of three scouting gradients, conditions for high-efficiency separations of protein glycoforms varying in the degree and complexity of glycosylation was achieved, thereby significantly reducing the time needed for method optimization.

Liquid-Crystal-on-Silicon for Augmented Reality Displays

In this paper, we review liquid-crystal-on-silicon (LCoS) technology and focus on its new application in emerging augmented reality (AR) displays. In the first part, the LCoS working principles of three commonly adopted LC modes—vertical alignment and twist nematic for amplitude modulation, and homogeneous alignment for phase modulation—are introduced and their pros and cons evaluated. In the second part, the fringing field effect is analyzed, and a novel pretilt angle patterning method for suppressing the effect is presented. Moreover, we illustrate how to integrate the LCoS panel in an AR display system. Both currently available intensity modulators and under-developing holographic displays are covered, with special emphases on achieving high image quality, such as a fast response time and high-resolution. The rapidly increasing application of LCoS in AR head-mounted displays and head-up displays is foreseeable.

Fast response time of liquid crystal display modes using band separation UV exposure method

ABSTRACTWe propose a double frequency ultraviolet (UV) exposure process that can improve the surface anchoring energy of photopolyimide (PI) embedded with reactive mesogens (RMs) for the high speed liquid crystal modes. In experiment, we polymerize the RM materials using long wavelength UV light over 340 nm, and then accomplish the polymerization of the UV alignment layer using short UV light between 254 nm and 340 nm. As results, we demonstrate the improved surface anchoring energy and the fast response time of an in-plane switching (IPS) and a twisted nematic (TN) LC modes in this paper.

Characterization of Recombinant Human Erythropoietin Isoforms by Capillary Isoelectric Focusing with Hydroxypropyl Cellulose-Coated Capillaries

Capillary isoelectric focusing (cIEF) has become a powerful method for protein analysis and has shown promising perspectives in biopharmaceutical research, because it allows fast and automated analysis of proteins with high resolution. Here, an improved cIEF system was set up for exploring better characterization of recombinant human erythropoietin (EPO) with hydroxypropyl cellulose (HPC)-coated capillaries and an offline fast and convenient desalting treatment for EPO real sample. With the cIEF system, several separation conditions for EPO isomers were optimized and high resolution results were obtained. The use of HPC-coated capillary columns allowed for no additive needed for the elimination of EOF. The cIEF system might be applied to further complementary characterization of EPO by comparison with LC and other modes. And the cIEF system was considered promising for online coupling with MS.

Gold and silver resonators and their optical properties

We present fabrication and optical properties of sub-wavelength metallic structures, including the sub-100 nm size gold and silver split ring resonators (SRRs). It is shown that introducing rotational symmetry into SRR array could enhance the resonance magnitude of magnetic and electric modes under unpolarised light illumination, thus enabling SRR optical characterisation using simple unpolarised light. Sub-100-nm SRRs will be shown to have magnetic resonance entering in the visible frequency range, where silver SRR as small as ~60 nm had been successfully fabricated with fundamental magnetic resonance at ~604 nm. The resonance wavelengths of the LC and plasmon modes of the sub-100-nm SRRs are found to linearly decrease with the decrease of SRR size, suggesting that kinetic inductance has not yet occurred at such frequency and also the possibility of having shorter magnetic resonance wavelength.

3.2: Distinguished Student Paper: High Image Quality Wearable Displays with a Fast‐Response Liquid Crystal

We report two ultra‐low viscosity liquid crystals for high image quality wearable displays with a field sequential color LCOS. Our mixtures offer ~4X faster response time than a commercial material at 20°C and ~8X faster at −20°C. Such a fast response time offers vivid color, high ambient contrast ratio, low power consumption, and mitigated color breakup even at −20°C. Different LC modes and frame rates are also evaluated.

42.2: Stress Induced Substrate Mura in Curved LCD

Curving flat substrates creates retardance proportional to the square of the thickness, approximately three times the typical max value of flat substrates. These stress patterns have distinctive interactions with dark state VA and IPS LC modes. Curved substrate stress‐birefringence can create unique circular polarized edge mura through liquid crystal and substrate interactions.

Fringe-Field Switching (FFS) 액정 소자의 전기광학 특성, 응용 현황 및 향후 이슈

Recently, the fringe-field switching (FFS) mode in liquid crystal displays has been used mainly for high image quality and high-resolution liquid crystal displays (LCDs). In this review paper, the fundamental switching principle of the FFS mode, with its excellence over other LC modes in electro-optic performance, will be described. In addition, the development history, present technical issues, and future of the FFS LCD will be discussed.

Optically Modulated Multiband Terahertz Perfect Absorber

Development of tunable, dynamic, and broad bandwidth metamaterial designs is a keystone objective for metamaterials research, necessary for the future viability of metamaterial optics and devices across the electromagnetic spectrum. Yet, overcoming the inherently localized, narrow bandwidth, and static response of resonant metamaterials continues to be a challenging endeavor. Resonant perfect absorbers have flourished as one of the most promising metamaterial devices with applications ranging from power harvesting to terahertz imaging. Here, an optically modulated resonant perfect absorber is presented. Utilizing photo‐excited free carriers in silicon pads placed in the capacitive gaps of split ring resonators, a dynamically modulated perfect absorber is designed and fabricated to operate in reflection. Large modulation depth (38% and 91%) in two absorption bands (with 97% and 92% peak absorption) is demonstrated, which correspond to the LC (0.7 THz) and dipole (1.1 THz) modes of the split ring resonators.

Emerging Quantum-Dots-Enhanced LCDs

Quantum dots (QDs)-based backlight greatly enhances the color performance for liquid crystal displays (LCDs). In this review paper, we start with a brief introduction of QD backlight, and then present a systematic photometric approach to reveal the remarkable advantages of QD backlight over white LED, such as much wider color gamut, higher optical efficiency, enhanced ambient contrast ratio, and smaller color shift. Some popular LC modes are investigated, including twisted nematic, fringing field switching (FFS) for touch panels, multi-domain vertical alignment (MVA) for TVs, and blue phase liquid crystal (BPLC) for next-generation displays. Especially, QD-enhanced BPLC combines the major advantages of FFS and submillisecond response time. It has potential to become a unified display solution.

Optical design and roll‐to‐roll fabrication process of microstructure film for wide viewing LCDs

AbstractWe have developed a new microstructure film for wide viewing liquid crystal displays (LCDs). By attaching it to the surface of a conventional LCD, the viewing angle characteristics of LCD has drastically improved without causing a blur of the frontal image and a decrease in the contrast ratio under bright ambient light conditions. This film can be applied to various LC modes including twisted nematic and multidomain vertical alignment by changing its internal micrometer‐size 3D structure. Further, this film can be mass‐produced efficiently by self alignment roll‐to‐roll process.

Sub‐100‐nm Sized Silver Split Ring Resonator Metamaterials with Fundamental Magnetic Resonance in the Middle Visible Spectrum

Split ring resonator (SRR) metamaterials working in the visible spectrum have great potential applications, but their fabrication is challenging due to the stringent requirements in the feature size and critical dimension. This paper reports fabrication and systematic characterization of silver SRRs with sub‐100‐nm sizes that can have magnetic resonance entering into the visible frequency spectrum. SRR size as small as ∼60 nm and fundamental magnetic resonance (LC‐resonance) as short as ∼604 nm have been successfully demonstrated, which to the best of our knowledge represent the smallest fabricated SRR and the shortest LC‐resonance based on SRR geometry. The resonance wavelengths of the LC and plasmon modes of the sub‐100‐nm SRRs are found to linearly decrease with SRR size. Excellent agreement between LC‐model and experimental data is obtained when the capacitance of the sub‐100‐nm SRRs is interpreted as capacitance between two spheres instead of between two parallel plates.

Enantiomeric separation of proton pump inhibitors on new generation chiral columns using LC and supercritical fluid chromatography

The enantioselectivity of proton pump inhibitors, namely, omeprazole, lansoprazole, rabeprazole, pantoprazole, tenatoprazole, and ilaprazole were studied using new generation chiral packing materials: CHIRALPAK IA, CHIRALPAK IB, and CHIRALPAK IC. Two versatile techniques, HPLC and supercritical fluid chromatography (SFC) were used in this study. CHIRALPAK IC has shown superior selectivity under both LC and SFC conditions, whereas CHIRALPAK IA has shown good selectivity in SFC when compared to LC under primary screening conditions. The chiral recognition ability in LC and SFC modes were found to be in the order CHIRALPAK IC > CHIRALPAK IA > CHIRALPAK IB. In addition to diode array detection, chiral detection was carried out using a laser polarimeter and the elution orders were found to be the same in both LC and SFC elution modes. Mobile phase modifiers and column temperature effects were also studied. In SFC, modifiers (cosolvent) elution strength was found to be in the order ethanol > methanol > 2-propanol > acetonitrile. In both LC and SFC, a decrease in retention and increase in resolution with an increase in temperature was noticed for all the proton pump inhibitors.

Systematic optimisation and evaluation of on-line, off-line and stop-flow comprehensive hydrophilic interaction chromatography × reversed phase liquid chromatographic analysis of procyanidins, Part I: Theoretical considerations

Comprehensive two-dimensional liquid chromatography (LC×LC) provides significantly improved separation for complex real-life samples. LC×LC can be performed in one of three different ways, using on-line, off-line, or stop-flow configurations. We have previously shown how off-line comprehensive hydrophilic interaction chromatography (HILIC)×reversed-phase liquid chromatography (RP-LC) provides a powerful separation system for procyanidins (PCs), one of the most complex fractions of natural phenolics. In the current contribution, a systematic approach for the optimisation and evaluation of each of the LC×LC methodologies is presented using HILIC×RP-LC analysis of PCs as application. Optimisation was performed using the peak capacities of individual one-dimensional separations measured for different gradient times and flow rates and their combination in each of the three LC×LC modes by taking into account the effects of first dimension under-sampling, the degree of orthogonality between the two dimensions and additional band broadening associated with stop-flow analysis. The performance of all three methods is compared in terms of practical peak capacities, analysis times and peak production rates. One-dimensional LC provided the best performance for separations requiring relatively low peak capacities, whereas the on-line LC×LC system was advantageous for required practical peak capacities up to ~600. For higher resolution, the off-line or stop-flow systems should be used. Especially noteworthy is the fact that, due to slow diffusion of PCs, the contribution of stop-flow to first dimension band broadening was negligible for stop-flow times of up to 15min. In a separate contribution, the experimental verification of the findings of this study will be reported.

Preparing arbitrary mode superconducting LC entangled coherent state via a superconducting charge qubit

We design a pure solid-device comprised by LC circuits, which are coupled to superconducting charge qubit to entangle superconducting LC coherent modes. The operation time of the state of the system does not increase with the increase of the number of LC modes. Based on the measurement of charge states, an arbitrary mode of the state can be easily generated. A brief discussion about the experimental feasibility of the scheme is also shown.