Cationic Covalent Organic Framework as an Ion Exchange Material for Efficient Adsorptive Separation of Biomolecules.

Although covalent organic frameworks (COFs) have earned significant interest in separation applications, the use of COFs in biomolecule separation remains unexplored. We examined the ionic COF Py-BPy2+-COF as an ion exchange material for biomolecule separation. After characterizing the properties of the synthesized COF with a variety of techniques, binding experiments with both large and small biomolecules were performed. High adsorption capacities of amino acids with different hydrophobicity and charge, as well as proteins of different isoelectric points and molecular weights, were determined in batch equilibrium experiments. Desorption experiments with mixtures of model proteins demonstrated an ability to successfully separate one protein from another with the selectivity hypothesized to be a combination of the isoelectric point, hydrophobicity, and ability to penetrate the crystalline material. Overall, the results demonstrated that Py-BPy2+-COF can be exploited as a robust crystalline anion exchange biomolecule separation material.

Fabrication of Raspberry-like Cytochrome C Surface-Imprinted Nanoparticles Based on MOF Composites for High-Performance Protein Separation.

The development of high-performance protein-imprinted materials is vital to meet the requirements of proteomics research but remains a challenge. Herein, a new type of raspberry-like cytochrome C-imprinted nanoparticle was first designed and fabricated via surface imprinting technology combined with a template immobilization strategy. In particular, the state-of-the-art metal-organic framework (MOF)/carbon nanoparticle (CN) composites were selected as protein immobilization carriers for two advantages: (1) the composites reflected the intrinsic characteristics of MOFs including flexible design, facile preparation, and extensive interactions with proteins and (2) the utilization of composites also overcame the issue associated with the severe agglomeration of individual MOFs during the post-use process. Therefore, the as-prepared composites exhibited a regular raspberry-like shape with good dispersion (polydispersity index (PDI) < 0.25), high specific surface area (551.4 m2 g-1), and outstanding cytochrome C immobilization capacity (900 mg g-1). Furthermore, a zwitterionic monomer was chosen to participate in the synthesis of an imprinting layer to reduce the nonspecific binding with proteins. As a result, the unique design presented here in both the protein immobilization carrier and the selected polymer composition endowed the imprinted material (noted as CN@UIO-66@MIPs) with the excellent ability for cytochrome C enrichment with extremely high recognition ability (imprinting factor (IF) = 6.1), rapid adsorption equilibrium time (40 min), and large adsorption capacity (815 mg g-1). Furthermore, encouraged by the experimental results, we successfully used CN@UIO-66@MIPs to specifically capture cytochrome C in mixed protein solutions and biological samples, which proved them to be a potential candidate for protein separation and purification.

Altered structure and dynamics of pathogenic cytochrome c variants correlate with increased apoptotic activity.

Mutation of cytochrome c in humans causes mild autosomal dominant thrombocytopenia. The role of cytochrome c in platelet formation, and the molecular mechanism underlying the association of cytochrome c mutations with thrombocytopenia remains unknown, although a gain-of-function is most likely. Cytochrome c contributes to several cellular processes, with an exchange between conformational states proposed to regulate changes in function. Here, we use experimental and computational approaches to determine whether pathogenic variants share changes in structure and function, and to understand how these changes might occur. Three pathogenic variants (G41S, Y48H, A51V) cause an increase in apoptosome activation and peroxidase activity. Molecular dynamics simulations of these variants, and two non-naturally occurring variants (G41A, G41T), indicate that increased apoptosome activation correlates with the increased overall flexibility of cytochrome c, particularly movement of the Ω loops. Crystal structures of Y48H and G41T complement these studies which overall suggest that the binding of cytochrome c to apoptotic protease activating factor-1 (Apaf-1) may involve an 'induced fit' mechanism which is enhanced in the more conformationally mobile variants. In contrast, peroxidase activity did not significantly correlate with protein dynamics. Thus, the mechanism by which the variants increase peroxidase activity is not related to the conformational dynamics of the native hexacoordinate state of cytochrome c. Recent molecular dynamics data proposing conformational mobility of specific cytochrome c regions underpins changes in reduction potential and alkaline transition pK was not fully supported. These data highlight that conformational dynamics of cytochrome c drive some but not all of its properties and activities.

Particle packed mixed-mode chromatographic stationary phase for the separation of peptide in liquid chromatography.

A particle-based stationary phase has been prepared for the separation of five synthetic peptides and a mixture containing tryptic digest of cytochrome C in liquid chromatography. Particles originating from silica monolith were differentially sedimented to obtain 1-2 µm particles. A stationary phase was achieved by the coating of poly(styrene-methacrylic acid-N-phenylacrylamide) copolymer onto the particles via reversible addition-fragmentation chain transfer polymerization reaction. Stainless steel column (30 cm long and 1 mm internal diameter) was packed with stationary phase. Very high separation efficiency (ca. 351 000 plates/m) was achieved for five commercial peptides with a percent relative standard deviation of less than 1%. Protocol for the synthesis and modification of silica monolith particles has been well optimized with a good reproducibility both in particle and pore size. The column resolved about 21 peptide components from a mixture containing tryptic digest of cytochrome C, under the elution conditions of acetonitrile/15 mM ammonium format (65/35 v/v%) with a flow rate of 28 µL/min.

Partition Efficiency of Eccentric Coil for Countercurrent Chromatographic Separation of Proteins Using Small-scale Cross-axis Coil Planet Centrifuge with Circular and Elliptic Cylindrical Columns.

The partition efficiency of the double-spaced coil for eccentric and toroidal coils on countercurrent chromatographic separation of proteins was evaluated using the small-scale cross-axis coil planet centrifuge (CPC) equipped with circular and elliptic cylindrical columns. Standard cytochrome c, myoglobin and lysozyme samples were used for separation with the 12.5% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate system. In the circular column, the double-spaced eccentric coil yielded better peak resolution than the double-spaced toroidal coil, and the double-spaced eccentric coil yielded better peak resolution than the single-spaced eccentric coil. In the elliptic column, the double-spaced eccentric coil also produced better peak resolution than the double-spaced toroidal coil, but the single-spaced eccentric coil yielded better peak resolution than the double-spaced eccentric coil. The overall results indicated that the double-spaced eccentric coil for the circular column and the single-spaced eccentric coil for the elliptic column yielded better protein separation using the small-scale cross-axis CPC with aqueous two-phase solvent systems.

Use of Ultra-short Columns for Therapeutic Protein Separations, Part 2: Designing the Optimal Column Dimension for Reversed-Phase Liquid Chromatography.

In the first part of the series, it was demonstrated that very fast (<30 s) separations of therapeutic protein species are feasible using ultra-short (5 × 2.1 mm) columns. In the second part, our purpose was to find the appropriate column length; therefore, a systematic study was performed using various custom-made prototype reversed-phase liquid chromatography (RPLC) columns ranging from 2 to 50 mm lengths. It was found that on a low dispersion ultrahigh-pressure liquid chromatography instrument, columns between 10 and 20 mm were most effective when made with 2.1 mm i.d. tubing. However, with the same LC instrument, 3 mm i.d. columns as short as ∼5 to 10 mm could be effectively used. In both cases, it has been found to be best to keep injection volumes below 0.6 µL, which presents a potential limit to further decreasing column length, given the current capabilities of autosampler instrumentation. The additional volume of the column hardware outside of the packed bed (extra-bed volume) of very small columns is also a limiting factor to decrease the column length. For columns shorter than 10 mm, columns' extra-bed volume was seen to make considerable contributions to band broadening. However, the use of ultra-short columns seemed to be a very useful approach for RPLC of large proteins (>25 kDa) and could also work well for ∼12 kDa as the lowest limit of molecular mass. In summary, a renewed interest in the use of ultra-short columns is warranted, and additional method development will be to the benefit of the biopharmaceutical industry as there is an ever-increasing demand for faster, yet accurate assays (e.g., high-throughput screening) of proteins.

Electrochemical sensing of cytochrome c using Graphene Oxide nanoparticles as platform.

An improved cytochrome c (Cyt c) biosensor based on immobilization of cytochrome c oxidase (COx) on the surface of graphene oxide nanoparticles (GONPs) electrodeposited onto pencil graphite (PG) electrode. Characterization of graphene oxide nanoparticle was done by Transmission electron microscopy (TEM), Fourier transform infra-red spectroscopy (FTIR) and X-ray diffraction study (XRD). The working electrode (COx/GONPs/PG) was characterized at its different stages of fabrication by scanning electron microscopy (SEM) and FTIR. Fabrication of Cyt c biosensor was done by connecting COx/GONPs/PG as working electrode, Ag/AgCl as reference electrode and Pt as auxiliary electrode to potentiostat. The mechanism of detection of present biosensor was based on oxidation of Cyt c (reduced) to Cyt c (oxidized) by COx resulting in flow of electrons through GONPs to the PG electrode, hence current generated is proportional to the concentration of Cyt c. Present biosensor exhibited optimum potential at 0.49 V with optimum pH 7.5 and optimum temperature 35°C. Biosensor showed linearity within 40-180 ng/ml having 40 ng/ml limit of detection. The precision i.e. within and between-batch coefficients of variation (CVs) were found <0.04% and <0.21% respectively. The enzyme electrode lost 50% of its initial activity when operated for more than 6 months on weekly basis. It was applied for detection of Cyt c level in in apparently healthy and diseased human sera. The present biosensing method was co-related with standard colorimetric method and co-relation coefficient was found 0.99.

Construction of polymer monolithic columns in polypropylene ink-pen tubes for separation of proteins by cation-exchange chromatography.

We describe the synthesis of polymer monoliths inside polypropylene tubes from ink pens. These tubes are cheap, chemically stable, and resistant to pressure. UV-initiated grafting with 5 wt% benzophenone in methanol for 20 min activated the internal surface, thus enabling the covalent binding of ethylene glycol dimethacrylate, also via photografting. The pendant vinyl groups attached a poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) monolith prepared via photopolymerization. These tubes measured 100-110 mm long, with 2 mm of internal diameter. The parent monoliths were functionalized with Na2 SO3 or iminodiacetate to produce strong and weak cation exchangers, respectively. The columns exhibited permeabilities varying from 2.7 to 3.3 × 10-13  m2 , which enabled the separation of proteins at 500 µL/min and back pressures <2.8 MPa. Neither structure collapse nor monolith detachment occurred at flow rates as high as 2.0 mL/min, which produced back pressures between 6.9 and 9.0 MPa. The retention times of ovalbumin, ribonuclease A, cytochrome C, and lysozyme in salt gradient at pH 7.0 followed the order of increasing isoelectric points, confirming the cation exchange mechanism. Separation and determination of lysozyme in egg white proved the applicability of the columns to the analysis of complex samples.

The Human Cytochrome c Domain-Swapped Dimer Facilitates Tight Regulation of Intrinsic Apoptosis.

Oxidation of cardiolipin (CL) by cytochrome c (cytc) has been proposed to initiate the intrinsic pathway of apoptosis. Domain-swapped dimer (DSD) conformations of cytc have been reported both by our laboratory and by others. The DSD is an alternate conformer of cytc that could oxygenate CL early in apoptosis. We demonstrate here that the cytc DSD has a set of properties that would provide tighter regulation of the intrinsic pathway. We show that the human DSD is kinetically more stable than horse and yeast DSDs. Circular dichroism data indicate that the DSD has a less asymmetric heme environment, similar to that seen when the monomeric protein binds to CL vesicles at high lipid:protein ratios. The dimer undergoes the alkaline conformational transition near pH 7.0, 2.5 pH units lower than that of the monomer. Data from fluorescence correlation spectroscopy and fluorescence anisotropy suggest that the alkaline transition of the DSD may act as a switch from a high affinity for CL nanodiscs at pH 7.4 to a much lower affinity at pH 8.0. Additionally, the peroxidase activity of the human DSD increases 7-fold compared to that of the monomer at pH 7 and 8, but by 14-fold at pH 6 when mixed Met80/H2O ligation replaces the lysine ligation of the alkaline state. We also present data that indicate that cytc binding shows a cooperative effect as the concentration of cytc is increased. The DSD appears to have evolved into a pH-inducible switch that provides a means to control activation of apoptosis near pH 7.0.

Efficiency of Ionic Liquids-Based Aqueous Two-phase Electrophoresis for Partition of Cytochrome c.

Cytochrome c is a small water-soluble protein that is abundantly found in the mitochondrial intermembrane space of microorganism, plants and mammalians. Ionic liquids (ILs)-based aqueous two-phase electrophoresis system (ATPES) was introduced in this study to investigate the partition efficiency of cytochrome c to facilitate subsequent development of two-phase electrophoresis for the separation of cytochrome c from microbial fermentation. The 1-Hexyl-3-methylimidazolium bromide, (C6mim)Br and potassium citrate salt were selected as the phase-forming components. Effects of phase composition; position of electrodes; pH and addition of neutral salt on the partition efficiency of cytochrome c in the ATPES were evaluated. Highest partition coefficient (K = 179.12 ± 0.82) and yield of cytochrome c in top phase (YT = 99.63% ± 0.00) were recorded with IL/salt ATPES composed of 30% (w/w) (C6mim)Br and 20% (w/w) potassium citrate salt of pH 7 and 3.0% (w/w) NaCl addition with anode at the bottom phase and cathode at the top phase. The SDS-PAGE profile revealed that cytochrome c with a molecular weight of 12 kDa was preferably partitioned to the IL-rich top phase. Present findings suggested that the single-step ATPES is a potential separation approach for the recovery of cytochrome c from microbial fermentation. Graphical Abstract.

Highly sensitive IRS based biosensor for the determination of cytochrome c as a cancer marker by using nanoporous anodic alumina modified with trypsin.

The determination of cytochrome c in the human serum sample is a regular medical investigation performed to assess cancer diseases. Herein, we used interferometric reflectance spectroscopy (IRS) based biosensor for the determination of cytochrome c. For this purpose first, the nanoporous anodic alumina (NAA) was fabricated. Then, the NAA pore walls were functionalized with 3-aminopropyl trimethoxy silane (NAA-NH2). Subsequently, the trypsin enzyme was immobilized on the NAA pore walls. The sensing principle of proposed IRS sensor to cytochrome c is based on a change in the intensity of the reflected light to a charge-coupled device (CCD) detector after digesting of cytochrome c by immobilized trypsin enzymes on NAA-NH2 into the heme-peptide fragment. The heme-peptide fragment then oxidized 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) to green color ABTS·- anion radical in the presence of hydrogen peroxide. The generated green color ABTS·- anion radical solution adsorbed the white light and therefore the intensity of the reflected light from NAA to the CCD decreased. The decrease in the intensity of the white light had a logarithmic relationship with the concentration of the cytochrome c in the range of 1-100 nM. The limit of detections (LOD) for cytochrome c was 0.5 nM. The proposed biosensor exhibited high selectivity, sensitivity, and good stability.

D-2-allylglycine embedded imidazolium-bridged polyhedral oligomeric silsesquioxane hybrid monolithic column for efficient separation of both small molecules and macromolecules.

The development of multifarious stationary phases is still a growing demand so as to solve the tasks of ever evolving actual applications. Herein, with D-2-allylglycine hydrochloride (AG·HCl) as the hydrophilic monomer, diene ionic liquid 1-allyl-3-vinylimidazolium bromide (AVI·Br) and polyhedral oligomeric silsesquioxane methacryl substituted (POSS-MA) as the dual crosslinkers, the highly cross-linked imidazolium-bridged POSS-AVI-AG hybrid monolithic column was fabricated via the "one-pot" free radical copolymerization. The AG·HCl embedded POSS-AVI-AG column displays typical reversed-phase liquid chromatography/hydrophilic interaction liquid chromatography mixed-mode retention mechanisms. Both hydrophobic phenols, alkylbenzenes, aromatic amines and hydrophilic nucleosides/nucleic acid bases, amides and thioureas were successfully separated with high column efficiencies (up to 571,000 plates/m for amides), outperforming our previously reported AVI·Br modified POSS-AVI column. Moreover, the column was also explored for the separation of cytochrome c tryptic digests and egg white protein extraction. All these results demonstrate that the POSS-AVI-AG column has a good potential in separation of both small molecules and complex biological samples with multiple mechanisms.

Aptamer-Based Fluorescent Biosensing of Adenosine Triphosphate and Cytochrome c via Aggregation-Induced Emission Enhancement on Novel Label-Free DNA-Capped Silver Nanoclusters/Graphene Oxide Nanohybrids.

Four fluorescent DNA-stabilized fluorescent silver nanoclusters (DNA-AgNCs) were designed and synthesized with differences in lengths of cytosine-rich DNA strand (as the stabilizing agent) and target-specific strand DNA aptamers for adenosine triphosphate (ATP) and cytochrome c (Cyt c). After their nanohybrid formation with graphene oxide (GO), it was unexpectedly found that, depending on the composition of the base and length of the strand DNA aptamer, the fluorescence intensity of three of the nanohybrids significantly enhanced. Our experimental observations and quantum mechanical calculations provided an insight into the mechanisms underlying the behavior of DNA-AgNCs/GO nanohybrids. The enhanced fluorescence was found to be attributed to the aggregation-induced emission enhancement (AIE) characteristic of the DNA-AgNCs adsorbed on the GO surface, as confirmed evidently by both fluorescence and transmission electron microscopies. The AIE is a result of hardness and oxidation properties of GO, which lead to enhanced argenophilic interaction and thus to increased Ag(I)-DNA complex shell aggregation. Consequently, two of the DNA-AgNCs/GO nanohybrids were successfully extended to construct highly selective, sensitive, label-free, and simple aptasensors for biosensing of ATP (LOD = 0.42 nM) and Cyt c (LOD = 2.3 nM) in lysed Escherichia coli DH5 α cells and mouse embryonic stem cells, respectively. These fundamental findings are expected to significantly influence the designing and engineering of new AgNCs/GO-based AIE biosensors.

Online Comprehensive High pH Reversed Phase × Low pH Reversed Phase Approach for Two-Dimensional Separations of Intact Proteins in Top-Down Proteomics.

A proof-of-concept study is presented on the use of comprehensive two-dimensional liquid chromatography mass spectrometry (LC × LC-MS) for the separation of intact protein mixtures using a different mobile phase pH in each dimension. This system utilizes mass spectrometry (MS) friendly pH modifiers for the online coupling of high pH reversed phase liquid chromatography (HPH-RPLC) in the first dimension (1D) followed by low pH reversed phase liquid chromatography (LPH-RPLC) in the second dimension (2D). Owing to the ionic nature of proteins, the use of a different mobile phase pH was successful to provide altered selectivity between the two dimensions, even for closely related protein variants, such as bovine cytochrome c and equine cytochrome c, which differ by only three amino acids. Subminute gradient separation of proteins in the second dimension was successful to minimize analysis time, while maintaining high peak capacity. Unlike peptides, the elution order of studied proteins did not follow their isoelectric points, where acidic proteins would be expected to be more retained at low pH (and basic proteins at high pH). The steep elution isotherms (on-off retention mechanism) of proteins and the very steep gradients utilized in the second-dimension column succeeded in overcoming pH and organic solvent content mismatch. The utility of the system was demonstrated with a mixture of protein standards and an Escherichia coli protein mixture.

Isoelectric focusing on microfluidic paper-based chips.

Isoelectric focusing (IEF), a powerful technique for protein separation and enrichment, was successfully integrated into microfluidic paper-based analytical devices (µPADs) in this work. The µPADs for isoelectric focusing were fabricated by octadecyltrichlorosilane (OTS) silanization and subsequent region-selective plasma treatment. The system of IEF on µPADs could be easily assembled. And a series of conditions of the system were investigated, including the suitable concentration of ampholyte to create good pH gradient, the effect of polyvinylpyrrolidone (PVP) on electroosmotic flow (EOF) suppression, and focusing voltage applied on the paper channel. After optimization, simultaneous separation and enrichment of protein sample containing myoglobin and cytochrome C was successfully demonstrated. Besides, parallel IEF on multichannels were also achieved for the separation of multiple protein samples on one single chip, and their performance was compared with that of the conventional gel-IEF system. The developed IEF on µPADs exhibits appealing features such as low cost, simplicity, and disposability and are believed to have great application potentials.

Fast reversed-phase liquid chromatographic separation of proteins by flow-through poly(styrene-co-divinylbenzene) microspheres.

With the explosive growth of the bioscience and biopharmaceuticals, the demand for high efficient analysis and separation of proteins is urgent. High-performance liquid chromatography is an appropriate technology for this purpose, and the stationary phase is the kernel to the separation efficiency. In this study, flow-through poly(styrene-co-divinylbenzene) microspheres characteristic of the binary pores, i.e. flow-through pores and mesopores, were synthesized; this special porous structure would benefit the convective mass transfer while guarantee the high specific surface area. Owing to the hydrophobic nature, poly(styrene-co-divinylbenzene) microspheres were suitable as the reversed-phase stationary phase for separation of proteins. For the high permeability of the poly(styrene-co-divinylbenzene) microspheres packed column, fast separation of the studied six proteins in ∼2 min was achieved. The recoveries of studied proteins were acceptable in the range of 79.0-99.4%. The proposed column had good pH stability of 1-13 and repeatability. Moreover, the column was applied for egg white fast separation, further demonstrating its applicability for complex bio-sample separation. The flow-through poly(styrene-co-divinylbenzene) microspheres were promising for fast separation of large molecules.

QCQuan: A Web Tool for the Automated Assessment of Protein Expression and Data Quality of Labeled Mass Spectrometry Experiments.

In the context of omics disciplines and especially proteomics and biomarker discovery, the analysis of a clinical sample using label-based tandem mass spectrometry (MS) can be affected by sample preparation effects or by the measurement process itself, resulting in an incorrect outcome. Detection and correction of these mistakes using state-of-the-art methods based on mixed models can use large amounts of (computing) time. MS-based proteomics laboratories are high-throughput and need to avoid a bottleneck in their quantitative pipeline by quickly discriminating between high- and low-quality data. To this end we developed an easy-to-use web-tool called QCQuan (available at qcquan.net ) which is built around the CONSTANd normalization algorithm. It automatically provides the user with exploratory and quality control information as well as a differential expression analysis based on conservative, simple statistics. In this document we describe in detail the scientifically relevant steps that constitute the workflow and assess its qualitative and quantitative performance on three reference data sets. We find that QCQuan provides clear and accurate indications about the scientific value of both a high- and a low-quality data set. Moreover, it performed quantitatively better on a third data set than a comparable workflow assembled using established, reliable software.

Retention Mechanism of Proteins in Hydroxyapatite Chromatography - Multimodal Interaction Based Protein Separations: A Model Study.

BACKGROUND: Retention mechanism of proteins in hydroxyapatite chromatography (HAC) was investigated by linear gradient elution experiments (LGE). MATERIALS AND METHODS: Several mobile phase (buffer) solution strategies and solutes were evaluated in order to probe the relative contributions of two adsorption sites of hydroxyapatite (HA) particles, C-site due to Ca (metal affinity) and P-site due to PO4 (cation-exchange). When P-site was blocked, two basic proteins, lysozyme (Lys) and ribonuclease A(RNase), were not retained whereas cytochrome C(Cyt C) and lactoferrin (LF) were retained and also retention of acidic proteins became stronger as the repulsion due to P-site was eliminated. The number of the binding site B values determined from LGE also increased, which also showed reduction of repulsion forces. CONCLUSION: The selectivity (retention) of four basic proteins (RNase, Lys, Cyt C, LF) in HAC was different from that in ion-exchange chromatography. Moreover, it was possible to tune the selectivity by using NaCl gradient.

Synthesis of penetrable poly(methacrylic acid-co-ethylene glycol dimethacrylate) microsphere and its HPLC application in protein separation.

In the present study, the narrow-dispersed penetrable poly(methacrylic acid-co-ethylene glycol dimethacrylate) (poly(MAA-co-EDMA)) microspheres were successfully synthesized based on the sacrificial support method. The poly(MAA-co-EDMA) microspheres mirrored the porous structure of the sacrificial support, i.e. penetrable silica, characteristic of copious mesopores and throughpores. In addition, they possessed large surface area, adjustable hydrophobicity and the cation-exchange ability. Owing to their multi functionalities, they were applied as chromatographic stationary phase to separate proteins in different separation modes, including reversed phase, hydrophobic interaction and weak cation exchange. Moreover, thanks to their throughpores, fast separation at low column backpressure could be achieved in these three modes. Both protein recovery and column stability were satisfactory. The penetrable poly(MAA-co-EDMA) microspheres were potential stationary phase matrix for fast protein separation.

Feasibility study for high-resolution multi-component separation of protein mixture using a cation-exchange cuboid packed-bed device.

In recent papers we have discussed the optimization of design and operating conditions for cuboid packed-bed device for chromatographic separations. The efficiency metrics used in these studies included the number of theoretical plates per unit bed height as well as attributes of flow-through and eluted peaks. These studies were carried out using equivalent columns as benchmarks. The cuboid packed-bed devices consistently outperformed the columns in terms of the above metrics. The current study examines how well, or indeed if at all these superior efficiency metrics translate to superiority in multi-component protein separation. Cation exchange resin was examined in the current study using appropriate multi-component model protein system which was chosen with close isoelectric points to make the separation challenging. Effects of operating and experimental parameters such as flow rate, loop size and linear gradient length on separation performance were systematically investigated. Separation metrics examined included peak width, tailing factor, asymmetry factor and resolution of separated protein peaks. The results obtained showed that the cation exchange cuboid packed-bed device significantly outperformed its equivalent commercial column (e.g., the number of theoretical plates per unit bed height was 8636/m for the cuboid packed-bed device as opposed to 1480/m for the column at a flow rate of 0.5 mL/min). The difference in efficiency was particularly high at lower flow rate and when shorter gradients were employed. The results suggest that the cuboid packed-bed devices could potentially have promising application in preparative separations such as biopharmaceutical purifications.