Perfusion Chromatography Research Articles

Development of a fast LC-MS/MS protocol for combined measurement of six LSDs on dried blood spot in a newborn screening program

New treatment options and improved strategies for Lysosomal Storage Disorders (LSDs) diagnosis on dried blood spot (DBS) have led to the development of several pilot newborn screening programs.Building on a previously published protocol, we devised a new 6-plex assay based on a single DBS punch incubated into a buffer containing a combination of substrates (GAA, GLA, ASM, GALC, ABG and IDUA). This new protocol incorporates a new trapping and clean-up procedure using perfusion chromatography connected on-line with an analytical column for analyte separation, after enzymatic reaction. Results are available after 4.5 min.Several incubation times were tested in order to reduce sample preparation times and to improve accuracy and reproducibility, also regarding the quenching of the reaction within the time window of linear product accumulation. The collected data demonstrate that an incubation time of 4 h is enough to achieve good reaction efficiency without any impact on sensitivity.The method proved versatile and robust for various instrument configurations. The fast sample preparation and running times allow a high sample throughput; an advantage in newborn screening procedures. This method can also be used for diagnostic purposes, allowing a rapid diagnosis in a few hours.

Plasmid pVAX1-NH36 purification by membrane and bead perfusion chromatography.

The demand for plasmid DNA (pDNA) has increased in response to the rapid advances in vaccines applications to prevent and treat infectious diseases caused by virus, bacteria or parasites, such as Leishmania species. The immunization protocols require large amounts of supercoiled plasmid DNA (sc-pDNA) challenging the development of efficient and profitable processes for capturing and purified pDNA molecules from large volumes of lysates. A typical bioprocess involves four steps: fermentation, primary recovery, intermediate recovery and final purification. Ion-exchange chromatography is one of the key operations in the purification schemes of pDNA owing the chemical structure of these macromolecules. The goal of this research was to compare the performance of the final purification step of pDNA using ion-exchange chromatography on columns packed with Mustang Q membranes or perfusive beads POROS 50 HQ. The experimental results showed that both matrixes could separate the plasmid pVAX1-NH36 (3936bp) from impurities in clarified Escherichia coli lysates with an adequate resolution. In addition, a 24- and 21-fold global purification factor was obtained. An 88 and 63% plasmid recuperation was achieved with ion-exchange membranes and perfusion beads, respectively. A better understanding of perfusion-based matrices for the purification of pDNA was developed in this research.

Comparison of perfusion media and monoliths for protein and virus-like particle chromatography

Structural and performance characteristics of perfusion chromatography media (POROS HS 20 and 50) and those of a polymethacrylate monolith (CIM SO3-1 tube monolith column) are compared for protein and virus-like particle chromatography using 1mL columns. Axial flow columns are used for POROS while the monolith has a radial flow configuration, which provides comparable operating pressures. The POROS beads contain a bimodal distribution of pore sizes, some as large as 0.5μm, which allow a small fraction of the mobile phase to flow within the particles, while the monolith contains 1–2μm flow channels. For proteins (lysozyme and IgG), the dynamic binding capacity of the POROS columns is more than twice that of the monolith at longer residence times. While the DBC of the POROS HS 50 column decreases at shorter residence times, the DBC of the POROS HS 20 column for IgG remains nearly twice that of the monolith at residence times at least as low as 0.2min as a result of intraparticle convection. Protein recoveries are comparable for all three columns. For VLPs, however, the eluted peaks are broader and recovery is lower for the monolith than for the POROS columns and is dependent on the direction of flow in the monolith, which is attributed to denser layer observed by SEM at the inlet surface of the monolith that appears to trap VLPs when loading in the normal flow direction.

Particle size effects on protein and virus-like particle adsorption on perfusion chromatography media

The resin structure, chromatographic behavior, and adsorption kinetics of proteins and virus-like-particles (VLPs) are studied for POROS HS 20 and POROS HS 50 (23 and 52μm mean diameter, respectively) to determine the effects of particle size on perfusion chromatography and to determine the predictive ability of available models. Transmission electron microscopy (TEM) and inverse size-exclusion chromatography (iSEC) show similar structures for the two resins, both containing 200–1000nm pores that transect a network of much smaller pores. For non-binding conditions, trends of the height equivalent to a theoretical plate (HETP) as a function of reduced velocity are consistent with perfusion. The estimated intraparticle flow fractions for these conditions are 0.0018 and 0.00063 for POROS HS 20 and HS 50, respectively. For strong binding conditions, confocal laser scanning microscopy (CLSM) shows asymmetrical intraparticle concentrations profiles and enhanced rates of IgG adsorption on POROS HS 20 at 1000cm/h. The corresponding effective diffusivity under flow is 2–3 times larger than for non-flow conditions and much larger than observed for POROS HS 50, consistent with available models. For VLPs, however, adsorption is confined to a thin layer near the particle surface for both resins, suggesting that the bound VLPs block the pores.

Protein and virus-like particle adsorption on perfusion chromatography media

The structural and protein adsorption characteristics of the perfusion chromatography matrix POROS® HS 50 are determined. Transmission electron microscopy shows a broad distribution of pore sizes with 100–500nm through-pores transecting a network of much smaller pores formed by aggregates of microgranules about 100nm in size. Dextran standards, proteins, and virus-like particles (VLPs) show size-exclusion behavior consistent with such a bimodal distribution of pore sizes. For non-binding conditions, the trends in height equivalent to a theoretical plate (HETP) as a function of mobile phase velocity and molecular size are consistent with perfusion suggesting that a fraction of the mobile phase between 0.0005 and 0.0008 flows through the particles. This small fraction provides little or no enhancement of intraparticle mass transfer for relatively small proteins (lysozyme and IgG) even at 1000cm/h, but can contribute substantially to transport for large proteins (thyroglobulin) and VLPs. Intraparticle concentration profiles during transient adsorption are determined by confocal microscopy in batch and flow systems. The profiles are spherically symmetrical indicating a dominance of diffusion for smaller proteins in both batch and flow systems but become highly asymmetrical and skewed in the direction of flow for thyroglobulin at 1000cm/h. Estimates of the convective enhancement of intraparticle transport for these conditions based on the confocal measurements are consistent with estimates of the intraparticle Peclet number and previously published models. Adsorption of VLPs, however, was found to be confined to a thin layer on the outer surface of the particles indicting that bound VLPs block access to the underlying pore network and suggesting that pores larger than those present on the resin studies are needed to take advantage of the effects of perfusion for the adsorption of large VLPs.

Glandular kallikrein in the innate immune system of Atlantic salmon ( Salmo salar)

Glandular Kallikrein is a serine-protease with trypsin-like activity and is able to generate bioactive peptides from inactive precursors. We have evaluated the presence of this protease in the different organs of the Atlantic salmon ( Salmo salar). The results clearly indicate that GK and PRL are generated in the same pituitary cells based on a co-localization by confocal microscopy. Based on probed cross-reactivity between C. striata and C. carpio glandular anti-GK antibodies, we used a homologous antibody to detect the presence of GK in several salmon tissues. We have evaluated the GK expression in healthy and defied fish. P. salmonis and V. ordalii. The GK immunoreaction in organs such as leukocytes, gills and skin is considerably increased in defied fish compared to healthy fish. This increase was present in the cells of the excretory kidney and in the intercellular tissue, where the development of hematopoietic and lymphocytic lines in fish take place. One of the most interesting organs to study was the skin, bearing in mind that this is a primary barrier to all pathogens. The skin of the defied fish exhibited an increase in immunoreactivity for glandular kallikrein similar to the protease found in mucus. An immunoreactive tissue kallikrein-like protein was identified and partially separated by perfusion chromatography. Enzymatic activity of salmon muscle prokallikrein was determined before and after trypsin activation. Kallikrein activity was characterized with respect to their ability to cleave the chromogenic leaving group, p-nitroanilide, from the peptidyl kallikrein and trypsin substrate. These findings constitute a important contribution to reveal the role of kallikrein in the innate immune system of fish.

Interaction Domain on Thioredoxin for Pseudomonas aeruginosa 5′-Adenylylsulfate Reductase

NMR spectroscopy has been used to map the interaction domain on Escherichia coli thioredoxin for the thioredoxin- dependent 5'-adenylylsulfate reductase from Pseudomonas aeruginosa (PaAPR). Seventeen thioredoxin amino acids, all clustered around Cys-32 (the more surface-exposed of the two active-site cysteines), have been located at the PaAPR binding site. The center of the binding domain is dominated by nonpolar amino acids, with a smaller number of charged and polar amino acids located on the periphery of the site. Twelve of the amino acids detected by NMR have non-polar, hydrophobic side chains, including one aromatic amino acid (Trp-31). Four of the thioredoxin amino acids at the PaAPR binding site have polar side chains (Lys-36, Asp-61, Gln-62 and Arg-73), with three of the four having charged side chains. Site-directed mutagenesis experiments have shown that replacement of Lys-36, Asp-61, and Arg-73 and of the absolutely conserved Trp-31 significantly decreases the V(max) for the PaAPR-catalyzed reduction of 5'-adenylylsulfate, with E. coli thioredoxin serving as the electron donor. The most dramatic effect was observed with the W31A variant, which showed no activity as a donor to PaAPR. Although the thiol of the active-site Cys-256 of PaAPR is the point of the initial nucleophilic attack by reduced thioredoxin, mutagenic replacement of Cys-256 by serine has no effect on thioredoxin binding to PaAPR.

Protein Purification

The science, art, and practice of protein purification have been with us for more than a century, yet, in many respects, the field is only now evolving past its adolescent roots. New methods are replacing old methods at such a dizzying pace, that even life-long experts in protein purification cannot keep up. In this article, we present many state-of-the-art protein purification techniques without totally ignoring the past. Our goal is to enable those relatively new to the field of protein purification to choose the best methods to solve their own purification problems. Each method we describe has been used and validated in our own research. We describe these methods, pointing out advantages, disadvantages, and limitations with practical examples rather than with complex, theoretical equations. This paper covers methods of extraction, clarification, batch purification, low pressure column chromatography, HPLC, and electrophoresis as applied to both genetically engineered, recombinant proteins and proteins isolated from natural sources. The relatively new methods of three-phase partitioning, hydrophobic charge induction chromatography, immobilized metal affinity chromatography, and perfusion chromatography are featured. Keywords: Protein purification, Extraction, Clarification, Chromatography, HPLC, Electrophoresis, Filtration

Ultrarapid Quantitation of Maize Proteins by Perfusion and Monolithic Reversed-Phase High-Performance Liquid Chromatography

The main objective of this study was to develop a new methodology alternative to the classical Kjeldahl analysis for determining maize proteins in maize products and seeds. For that purpose, two different chromatographic methodologies using perfusion and monolithic stationary phases, both enabling rapid separations of maize proteins, were investigated. Due to the difficulty to find suitable standards for this type of analysis, three different maize products were initially tested as proteins standards: zein F4000, corn gluten meal, and maize flour. Different figures of merit (i.e., linearity, correlation coefficient, precision, limits of detection and quantitation), as well as the presence of matrix inferences, were investigated. The results obtained for the different chromatographic stationary phases and protein standards were compared in order to select the most suitable analytical conditions. Despite both perfusion and monolithic methodologies resulting, in general, as appropriate for the quantitation of maize proteins, the highest reduction of analysis time and lowest detection and determination limits provided by perfusion methodology enabled to select this one as the method of choice for the quantitation of maize proteins. Regarding the different protein standards studied in this work, in general the best results were obtained using the zein standard. Compared to Kjeldahl methodology, perfusion chromatography yields total protein contents in shorter analysis time while enabling the separation of the different kinds of proteins. Due to the high diversity and complexity of industrial maize products, the proposed chromatographic method could be a very useful tool for their routine analysis.

A snake venom phospholipase A 2 with high affinity for muscarinic acetylcholine receptors acts on guinea pig ileum

A group of small proteins, designated as muscarinic toxins (MTs), have been isolated from the venom of African green mamba ( Dendroaspis angusticeps) and documented to bind selectively to individual muscarinic acetylcholine receptor (mAChR) subtypes. These components have less been reported to be isolated from other snake venoms. In this study, we have isolated a snake factor with high affinity for mAChR from the venom of Naja atra (Chinese cobra) by column chromatography on Sephadex G-50, Sephadex G-150, CM-Sepharose Fast Flow and Poros ®CM 4.6/100 Perfusion Chromatography Column. The final preparation was homogeneous as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and HPLC. The isolated active component, which was designated muscarinic protein (MP), was found to displace [ 3H]quinuclidinyl benzilate binding to rat cortex synaptosomes in a dose-dependent manner, and the K i value estimated was 10.1 nM. The isolated MP was determined to have a molecular weight of 13.3 kDa and an N-terminal amino acid sequence of NLYQFKNMIQCTVPSR, which is highly homologous with phospholipase A 2 from the venoms of genus Naja. The N. atra MP could hydrolyze phosphatidylcholine in a dose-dependent manner. In guinea-pig ileum, MP produced an onset and dose-dependent contraction, which could be reversed by atropine indicating the involvement of mAChR. The EC 50 value of MP for guinea-pig ileum contraction was estimated as 30 nM, and the maximum contraction caused by MP was approximately 43% of that obtained from carbachol. These results seem to suggest that the snake venom phospholipase A 2 may not only have high affinity for mAChRs but also have the ability to activate mAChRs. However, it is possible that the toxin caused the contraction in the guinea-pig ileum by inducing acetylcholine release via another mechanism.

Tumor Cell Targeting of Transferrin−PEG−TNF-α Conjugate via a Receptor-Mediated Delivery System: Design, Synthesis, and Biological Evaluation

PEGylation is a procedure of growing interest for enhancing the therapeutic and biotechnological potential of peptides and proteins. Transferrin (Tf) has been proposed to be useful for targeting cancer cells. The aim of this study was to modify PEGylated recombinant human tumor necrosis factor alpha (PEG-TNF-alpha) with Tf to form Tf-PEG-TNF-alpha conjugates, which would maintain the advantages of PEGylation and also achieve the function of active targeting to tumor cells. In PEGylation reactions with 5-, 20-, 40-, and 60-fold molar excess of 3.4 kDa N-hydroxysuccinimide-PEG-maleimide (PT1, PT2, PT3, and PT4, respectively), PEG-TNF-alpha conjugates with different PEG chains were synthesized. A perfusion chromatography technique using a cation-exchange column was introduced to purify PEG-TNF-alpha conjugates. PT4 with about five PEG chains was selected as a lead candidate due to highest extent of PEGylation and maximum reaction yield. Thiolated Tf was conjugated to the maleimide group at the distal end of the PEG chains on the PEG-TNF-alpha conjugates, with the resulting Tf-PEG-TNF-alpha conjugates after purification containing approximately one Tf ligand on one TNF-alpha molecule. The conjugate of Tf and PT4 (TPT4) was selected to assess the specificity and affinity to transferrin receptor (TfR) on two kinds of tumor cells, K562 and KB. Both the receptor binding assays and the competition experiments were performed using radioligand binding analysis. The results demonstrated that TPT4 as well as Tf bound specifically to the TfR on the tumor cell surface and the affinity of the conjugate to TfR was similar to that of native Tf. In contrast, PEG-TNF-alpha demonstrated no specificity. The biodistribution and antitumor effects were investigated in S-180 tumor-bearing mice. It was found that TPT4 could markedly alter in vivo behavioral characteristics of TNF-alpha. Compared with TNF-alpha and PT4, extravasated TPT4 in tumor tissues exhibited a significantly delayed blood clearance and the highest intratumoral TNF-alpha levels. Furthermore, the inhibitory rate of tumor of TPT4 enhanced 5.3- and 1.8-fold over that of TNF-alpha and PT4, indicating that TPT4 exhibited the highest antitumor activity. These results suggested that Tf-PEG-TNF-alpha was a useful long circulating conjugate with the capabilities of specific receptor binding resulting in enhanced antitumor activity of TNF-alpha.

Rapid detection of the addition of soybean proteins to cheese and other dairy products by reversed-phase perfusion chromatography

The undeclared addition of soybean proteins to milk products is forbidden and a method is needed for food control and enforcement. This paper reports the development of a chromatographic method for routine analysis enabling the detection of the addition of soybean proteins to dairy products. A perfusion chromatography column and a linear binary gradient of acetonitrile-water-0.1% (v/v) trifluoroacetic acid at a temperature of 60°C were used. A very simple sample treatment consisting of mixing the sample with a suitable solvent (Milli-Q water or bicarbonate buffer (pH = 11)) and centrifuging was used. The method enabled the separation of soybean proteins from milk proteins in less than 4 min (at a flow-rate of 3 ml/min). The method has been successfully applied to the detection of soybean proteins in milk, cheese, yogurt, and enteral formula. The correct quantitation of these vegetable proteins has also been possible in milk adulterated at origin with known sources of soybean proteins. The application of the method to samples adulterated at origin also leads to interesting conclusions as to the effect of the processing conditions used for the preparation of each dairy product on the determination of soybean proteins.

Systematic Analysis of the Epidermal Growth Factor Receptor by Mass Spectrometry Reveals Stimulation-dependent Multisite Phosphorylation

Multisite phosphorylation of proteins is a general mechanism for modulation of protein function and molecular interactions. Definition of phosphorylation sites and elucidation of the functional interplay between multiple phosphorylated residues in proteins are, however, a major analytical challenge in current molecular cell biology and proteomic research. In the present study, we used mass spectrometry to determine the major phosphorylated residues of the human epidermal growth factor (EGF) receptor at various well defined cellular conditions. Activation of EGF receptor was achieved by several types of stimulation, i.e. by sodium pervanadate, EGF, and integrin-dependent adhesion. The contribution of cell-matrix adhesion was also determined by activating the EGF receptor by EGF in cells kept in suspension. We developed an analytical strategy that combined miniaturized sample preparation techniques and MALDI tandem mass spectrometry and determined a total of nine phosphorylation sites in the EGF receptor. We discovered one novel phosphorylation site (Ser967) and revealed constitutive phosphorylation of Thr669, Ser967, Ser1002, and Tyr1045 and stimulation-dependent differential phosphorylation of Tyr1068, Tyr1086, Ser1142, Tyr1148, and Tyr1173. The EGF receptor was purified from HeLa cells or ECV304 cells by immunoprecipitation and SDS-PAGE and then digested with trypsin. Phosphopeptides in the range of 0.8-3.7 kDa were recovered by combinations of IMAC, perfusion chromatography, and graphite powder chromatography and subsequently detected and sequenced by MALDI quadrupole time-of-flight tandem mass spectrometry. Two phosphorylation sites were detected in the peptide 1137GSHQISLDNPDYQQDFFPK1155; however, only Tyr1148 was phosphorylated upon EGF treatment; in contrast Ser1142 was only phosphorylated by integrin-dependent adhesion in the absence of EGF treatment, suggesting differential phosphorylation of this region by distinct stimuli. This MALDI MS/MS-based analytical approach demonstrates the feasibility of systematic analysis of signaling molecules by mass spectrometry and provides new insights into the dynamics of receptor signaling processes.

Quantification by affinity perfusion chromatography of phosphorylated BRCAl and BRCA2 proteins from tumor cells after lycopene treatment

A new procedure for the quantification of phosphorylated BRCA1 (P-BRCA1) and BRCA2 (P-BRCA2) proteins in breast cell lines after different treatments was carried out. Cells were cultivated with [ 35S]-methionine and extracts subjected to three perfusion chromatographies. First heparin affinity chromatography purified cellular DNA-binding proteins. Subsequent specific immunoprecipitation of BRCA1 and BRCA2 proteins was performed with antibodies raised against BRCA1 or BRCA2. The immune complexes were isolated by protein A affinity chromatography. Phosphorylated BRCA1 or BRCA2 proteins were then purified with a Poros 20 AL column where anti-phosphothreonine and anti-phosphoserine antibodies were previously bound. The percentage of phosphorylated BRCA1 or BRCA2 proteins was calculated as follows: 100 × dpm of P-BRCA1 or P-BRCA2 eluted from the POROS ® 20AL column/total dpm eluted from POROS ® 20AL column. Treatment with 10 μM lycopene increased P-BRCA1 and P-BRCA2 in the breast tumor cell line MCF7 but not in MDA-MB-231 or MCF-10a, breast tumor or fibrocystic cell lines, respectively.

The 1.1-Å Structure of the Spindle Checkpoint Protein Bub3p Reveals Functional Regions

Bub3p is a protein that mediates the spindle checkpoint, a signaling pathway that ensures correct chromosome segregation in organisms ranging from yeast to mammals. It is known to function by co-localizing at least two other proteins, Mad3p and the protein kinase Bub1p, to the kinetochore of chromosomes that are not properly attached to mitotic spindles, ultimately resulting in cell cycle arrest. Prior sequence analysis suggested that Bub3p was composed of three or four WD repeats (also known as WD40 and beta-transducin repeats), short sequence motifs appearing in clusters of 4-16 found in many hundreds of eukaryotic proteins that fold into four-stranded blade-like sheets. We have determined the crystal structure of Bub3p from Saccharomyces cerevisiae at 1.1 angstrom and a crystallographic R-factor of 15.3%, revealing seven authentic repeats. In light of this, it appears that many of these repeats therefore remain hidden in sequences of other proteins. Analysis of random and site-directed mutants identifies the surface of Bub3p involved in checkpoint function through binding of Bub1p and Mad3p. Sequence alignments indicate that these surfaces are mostly conserved across Bub3 proteins from diverse species. A structural comparison with other proteins containing WD repeats suggests that these folds may bind partner proteins using similar surface areas on the top and sides of the propeller. The sequences composing these regions are the most divergent within the repeat across all WD repeat proteins and could potentially be modulated to provide specificity in partner protein binding without perturbation of the core structure.

Modelling and simulation in chemical engineering: Tools for process innovation

The Chemical Engineering Science movement has served well in solving problems from micro to macro scales. Ultimately, as Professor R. Sargent pointed out, it would be better if translated in “Scientific Engineering”.Examples of innovation as a combination of science (concept), technology and process/product will be presented.The first example is perfusion chromatography based on the concept of “diffusivity augmented by convection” and on the technology of fabrication of large-pore packings. The second example, Simulated Moving Bed (SMB), is based on a concept developed to overcome the difficulties in implementing True Moving Bed processes. Technological contributions come from adsorbent materials and rotary valve to simulate the solid movement. SMB is now a key technology for chiral separations. Modelling/Simulation tools provide sound basis for design/operation by using the concept of “separation volume”.The third example is from the area of multifunctional reactors. The SMB technology is extended to the simultaneous reaction/separation.

Isolation and quantification of dinucleoside polyphosphates by using monolithic reversed phase chromatography columns

In former studies, dinucleoside polyphosphates were quantified using ion-pair reversed-phase perfusion chromatography columns, which allows a detection limit in the μmolar range. The aim of this study was both to describe a chromatographic assay with an increased efficiency of the dinucleoside separation, which enables the reduction of analytical run times, and to establish a chromatographic assay using conditions, which allow MALDI-mass spectrometric analysis of the resulting fractions. We compared the performance of conventional silica reversed phase chromatography columns, a perfusion chromatography column and a monolithic reversed-phase C18 chromatography column. The effects of different ion-pair reagents, flow-rates and gradients on the separation of synthetic diadenosine polyphosphates as well as of diadenosine polyphosphates isolated from human platelets were analysed. Sensitivity and resolution of the monolithic reversed-phase chromatography column were both higher than that of the perfusion chromatography and the conventional reversed phase chromatography columns. Using a monolithic reversed-phase C18 chromatography column, diadenosine polyphosphates were separable baseline not only in the presence of tetrabutylammonium hydrogensulfate (TBA) but also in the presence of triethylammonium acetate (TEAA) as ion-pair reagent. The later reagent is useful because, in contrast to TBA, it is compatible with MALDI mass-spectrometric methods. This makes TEAA particularly suitable for identification of unknown nucleoside polyphosphates. Furthermore, because of the lower backpressure of monolithic reversed-phase chromatography columns, we were able to significantly increase the flow rate, decreasing the amount of time for the analysis close to 50%, especially using TBA as ion-pair reagent. In summary, monolithic reversed phase C18 columns markedly increase the sensitivity and resolution of dinucleoside polyphosphate analysis in a time-efficient manner compared to reversed-phase perfusion chromatography columns or conventional reversed-phase columns. Therefore, further dinucleoside polyphosphate analytic assays should be based on monolithic silica C18 columns instead of perfusion chromatography or conventional silica reversed phase chromatography columns. In conclusion, the use of monolithic silica C18 columns will lead to isolation and quantification of up to now unknown dinucleoside polyphosphates. These chromatography columns may facilitate further research on the biological roles of dinucleoside polyphosphates.

Behavior of Bacteria in the Inductively Coupled Plasma: Atomization and Production of Atomic Ions for Mass Spectrometry

The combination of perfusion chromatography (PC) and inductively coupled plasma mass spectrometry (ICPMS) is a fast, general way to monitor metal incorporation into bacteria. U+ signals from U incorporated intrinsically in Bacillus subtilis (B. subtilis) are measured with 4 ms time resolution to investigate the behavior of individual cells in the ICP. When intact B. subtilis cells are introduced directly into the ICP, occasional U+ spikes are observed. The positive U+ spikes suggest that bacteria behave more like solid particles than wet droplets in the ICP, compared to previous studies of such transient effects in the ICP. Drying the bacterial aerosol does not eliminate the spikes. Lysing the bacteria by sonication increases the U+ response by 30% compared to that from the untreated sample. PC results from a 10 ppb U standard, partially lysed and fully lysed bacteria samples show that the intracellular U-bound species are released by sonication and are small in size. The atomization-ionization efficiencies for different elements (U, Ca, and Mg) from cells differ somewhat. Reducing the aerosol gas flow rate by 0.1 L min-1 improves the relative U+ response for unlysed bacteria to 85% of that for lysed cells, although the absolute U+ signal is attenuated greatly.

Effects of adsorption kinetics and surface heterogeneity on band spreading in perfusion chromatography—a network model analysis

A network model was used to simulate the band-spreading of an eluting solute from a perfusive column under retained conditions. The computer experiments were carried out over a wide range of Peclet numbers. The effects of pore size distribution (PSD), pore inter-connectivity and linear adsorption/desorption kinetics were taken into account in the simulations. Surface heterogeneity effects were also discussed. Among these parameters investigated, adsorption/desorption kinetics was the most important parameter, which influenced the transit-time moments and, in turn, the band-spreading as well as band-tailing results significantly over the whole range of Peclet numbers investigated. In addition, pore inter-connectivity and PSD also play an important role in determining the band-spreading results. However, their effects are only important in the molecular diffusion- and convection-controlled regimes. Under strongly retained conditions, contribution of hydrodynamics can be neglected. Furthermore, surface energetic heterogeneity resulting from the wide pore size distributions could exacerbate both band spreading and band tailing; the effects on band tailing being more significant than those on band spreading.

Fast protein liquid chromatography.

High-resolution protein separation techniques critically depend on the availability of column packings of small average particle size. This gives a minimum of peak broadening on the column owing to the direct relationship between the theoretical plate height parameter, H, and particle size (lowest values of H give the highest resolution). High-performance liquid chromatography (HPLC) procedures exploit column packings with average diameters of as small as 5–40 μm. However, these are used in high-pressure systems (up to 400 bar) often with organic solvents and are generally limited to rather low sample loadings (). To provide a more biocompatible high-resolution separation of biopolymers, including (although not exclusive to) proteins, Pharmacia (Uppsala, Sweden; now known as, Amersham Biosciences) developed fast protein liquid chromatography (FPLC) in 1982 (). More recently, Regnier and his colleagues developed “perfusive” packings that contain large through-pores (). These allow high-resolution chromatography with ion-exchange groups similar to those of Amersham Biosciences columns but at much faster flow rates, giving remarkably short separation times (e.g., 5–7 min). Perfusion chromatography columns are also compatible with the FPLC system.