Cell retention by encapsulation for the cultivation of Jurkat cells in fixed and fluidized bed reactors.

Jurkat cells are accepted model cells for primary human T lymphocytes, for example, in medical research. Their growth to tissue-like cell densities (up to 100 × 10(6) cells/mLcapsule ) in semi-permeable (molecular weight cut off <10,000 Da) sodium cellulose sulfate/poly(diallyldimethylammonium chloride) polyelectrolyte capsules has previously been shown by us [Werner et al. (2013). Use of the mitochondria toxicity assay for quantifying the viable cell density of microencapsulated jurkat cells. Biotechnol Prog 29(4): 986-993]. Herein, we demonstrate that encapsulation can be used to retain the cells in continuously operated bioreactors, which opens new possibilities for research, for example, the use of Jurkat cells in pulse response experiments under steady state conditions. Two reactor concepts are presented, a fluidized and a fixed bed reactor. A direct comparison of the growth kinetics in batch and repeated batch spinner cultivations, that is, under conditions where both encapsulated and non-encapsulated cells can be cultivated under otherwise identical conditions, showed that maximum specific growth rates were higher for the encapsulated than for the non-encapsulated cells. In the subsequent batch and repeated batch bioreactor experiments (only encapsulated cells), growth rates were similar, with the exception of the fixed bed batch reactor, where growth kinetics were significantly slower. Concomitantly, a significant fraction of the cells towards the bottom of the bed were no longer metabolically active, though apparently not dead. In the repeated batch fluidized bed reactor cellular division could be maintained for more than two weeks, albeit with a specific growth rate below the maximum one, leading to final cell densities of approximately 180 × 10(6) cell/gcapsule . At the same time, the cell cycle distribution of the cells was shifted to the S and G2/M phases.

Supposedly identical microplastic particles substantially differ in their material properties influencing particle-cell interactions and cellular responses.

Microplastics and its putative adverse effects on environmental and human health increasingly gain scientific and public attention. Systematic studies on the effects of microplastics are currently hampered by using rather poorly characterised particles, leading to contradictory results for the same particle type. Here, surface properties and chemical composition of two commercially available nominally identical polystyrene microparticles, frequently used in effect studies, were characterised. We show distinct differences in monomer content, ζ-potentials and surface charge densities. Cells exposed to particles showing a lower ζ-potential and a higher monomer content displayed a higher number of particle-cell-interactions and consequently a decrease in cell metabolism and proliferation, especially at higher particle concentrations. Our study emphasises that no general statements can be made about the effects of microplastics, not even for the same polymer type in the same size class, unless the physicochemical properties are well characterised.

Isolation of primary human B lymphocytes from tonsils compared to blood as alternative source for ex vivo application.

B lymphocytes ('B cells') are components of the human immune system with obvious potential for medical and biotechnological applications. Here, we discuss the isolation of primary human B cells from both juvenile and adult tonsillar material using a two-step procedure based on gradient centrifugation followed by separation on a nylon wool column as alternative to the current gold standard, i.e., negative immunosorting from buffy coats by antibody-coated magnetic beads. We show that the nylon wool separation is a low-cost method well suited to the isolation of large amounts of primary B cells reaching purities ≥ 80%. More importantly, this method allows the preservation of all B cell subsets, while MACS sorting seems to be biased against a certain B cell subtype, namely the CD27+ B cells. Importantly, compared to blood, the excellent recovery yield during purification of tonsillar B cells provides high number of cells, hence increases the number of subsequent experiments feasible with identical cell material, consequently improving comparability of results. The cultivability of the isolated B cells was demonstrated using pokeweed mitogen (PWM) as a stimulatory substance. Our results showed for the first time that the proliferative response of tonsillar B cells to mitogens declines with the age of the donor. Furthermore, we observed that PWM treatment stimulates the proliferation of a dedicated subpopulation and induces some terminal differentiation with ASCs signatures. Taken together this indicates that the proposed isolation procedure preserves the proliferative capability as well as the differentiation capacity of the B cells.

Antihelminthic action of the Anethum graveolens essential oil on Haemonchus contortus eggs and larvae.

Gastrointestinal nematodes are responsible for great economic losses in sheep raising, and their control has long been carried out almost exclusively by the administration of anthelmintics, which have led to serious resistance problems. In the search for alternative control measures, phytotherapic research is highlighted. The aim of this study was to evaluate the action of Anethum graveolens (dill) essential oil on different stages of Haemonchus contortus life cycle, as well its cytotoxicity MDBK (Madin-Darby bovine kidney) cells. H. contortus larvae and eggs were obtained from infected sheep feces, and essential oil extracted from plant seeds through the Clevenger apparatus. 9.4, 4.7, 2.35, 1.17. 0.58 and 0.29 mg/mL concentrations were evaluated. The Egg Hatch Inhibition (HI), Larval Development Inhibition (LDI) and Larval Migration Inhibition (LMI) techniques were used. Thybendazole 0.025 mg/mL in HI and Levamisole 0.02 mg/mL in the LDI and LMI tests were used as positive controls, while distilled water and a Tween 80 solution were used as positive negative controls. The inhibition results obtained for the highest oil concentration were: HI 100%, LDI 98.58% and LMI 63.7%, differing (𝑝 <0.05) from negative controls. Main A. graveolens oil components present in 95.93% of the total oil were Dihydrocarvone (39.1%), Carvone (22.24%), D-Limonene (16.84%), Apiol (10.49%) and Trans-dihydrocarvone (7.26%). Minimum A. graveolens essential oil concentrations required to inhibit 50% (IC50) of egg hatching, larval development and larval migration were 0.006 mg/mL, 2.536 mg/mL and 3.963 mg/mL, respectively. Cell viability in MDBK (Madin-Darby bovine kidney) cells, when incubated with A. graveolens essential oil, was 86% for the highest (9.4 mg/mL) and 99% for the lowest concentration (0.29 mg/mL). A. graveolens essential oil, according to the results obtained in this study, is a promising alternative in sheep gastrointestinal nematode control.

Can the essential oil of rosemary (Rosmarinus officinalis Linn.) protect rats infected with itraconazole-resistant Sporothrix brasiliensis from fungal spread?

INTRODUCTION: Itraconazole is the first-choice option to treat human and animal sporotrichosis. However, the emergence of itraconazole-resistant strains has encouraged research on new active antifungals. Among them, the essential oil of rosemary (Rosmarinus officinalis Linn., Lamiaceae) has shown antifungal activity in vitro. OBJECTIVE: Assessing, for the first time, the effectiveness of rosemary essential oil in vivo in experimental cutaneous sporotrichosis, as well as its chemical composition and action mode. METHODS: Itraconazole-resistant Sporothrix brasiliensis was inoculated in the left foot pad of 30 Wistar rats, which were randomized (n=10) for treatment with saline solution (control, CONT), itraconazole (ITRA, 10 mg/kg) and rosemary oil (ROSM, 250 mg/kg) for 30 days at an oral dose of 1 mL, daily. Clinical evolution, histopathology and fungal burden were investigated. GC-MS was used for chemical analysis; sorbitol protection and ergosterol effect were used to evaluate the action mechanism of rosemary oil. RESULTS: ROSM was the only group evolving to skin lesion remission, lack of edema and exudate, and mild-to-absent yeast cells. Rosemary oil delayed fungal spreading and protected systemic organs, mainly liver and spleen. The ROSM group presented lower fungal load than that observed for the CONT and ITRA groups (p<0.05). Antifungal action took place at complexation level after ergosterol application. Most compounds were 1,8-cineole/eucalyptol (47.91%), camphor (17.92%), and α-pinene (11.52%). CONCLUSIONS: These findings have evidenced that rosemary oil is a promising antifungal to treat sporotrichosis, since it protects systemic organs from fungal spread.

Megabenthic assemblages at the southern Central Indian Ridge - Spatial segregation of inactive hydrothermal vents from active-, periphery- and non-vent sites.

Active hydrothermal vents are small-scale habitats hosting endemic fauna in a well-defined zonation around fluid effluents. The fauna of inactive hydrothermal vents and its relation to active vents and non-vent area is poorly known. Characterizing inactive areas is prerequisite to establish protected areas, especially in the context of potential seafloor massive sulfide mining, which targets inactive sites. Hierarchical clustering and Distance-based Redundancy Analysis revealed five assemblages, with significantly associated substrate types: I) active hydrothermal vent, II) periphery, III) inactive hydrothermal vent and IV) soft- and V) hard-substrate within the non-vent area. For the first time, a unique inactive faunal assemblage could be identified within the hydrothermally extinct inactive Gauss field and on adjacent hard substrates. The spatial separation from the active Edmond field and periphery and the non-vent area indicates the existence of an inactive assemblage.

Torquetenovirus viremia for early prediction of graft rejection after kidney transplantation.

OBJECTIVES: New biomarkers reflecting the degree of immunosuppression in transplant recipients are needed to provide an optimal personalized balance between rejection and infection risks. METHODS: For this purpose, we investigated TTV viremia dynamics in 66 kidney transplant recipients followed up for two years after transplantation, in relation to BK virus infection and graft rejection. RESULTS: After transplantation, TTV viremia rose by ≥2 log10 copies/mL from baseline to month 3, then declined by ≥1 log10 copies/mL thereafter. Higher TTV viremia was associated with recipients of a deceased donor, a lower count of CD8+ T cells and a higher BKV viremia. Importantly, TTV loads were significantly lower in KTR who would later display graft rejection; indeed, patients with TTV viremia lower than 3.4 log10 copies/mL at transplantation or lower than 4.2 log10 copies/mL at month 1 had a higher risk of developing graft rejection in the two following years (hazard ratio (HR) at D0 = 7.30, p = 0.0007 and HR at M1 = 6.16, p = 0.001). CONCLUSIONS: TTV viremia measurement at early times post transplantation predicts graft rejection and would represent a useful tool to improve kidney transplant monitoring.

Chemical composition of essential oils from the apiaceae family, cytotoxicity, and their antifungal activity in vitro against candida species from oral cavity.

The aims of this research were: evaluate the chemical composition and the cytotoxicity of the Cuminum cyminum (cumin), Anethum graveolens (dill), Pimpinella anisum (anise) and Foeniculum vulgare (fennel) essential oils, as well as their antifungal activity in vitro against ten Candida spp. isolates. The chemical composition of the oils was analyzed by means of gas chromatography coupled with mass spectrometry (GC/MS). The cytotoxicity assays were performed, using the cell proliferation reagent WST-1 in L929 mouse fibroblasts (20x103 well-1). The determinate the Minimum Inhibitory Concentration (MIC), was performed through the Broth Microdilution technique (CLSI). The chemical main components were the cuminaldehyde (32.66%) for cumin, carvone (34.89%) for the dill, trans-anethole (94.01%) for the anise and anethole (79.62%) for the fennel. Anise and fennel did not were cytotoxic in all the tested concentrations, however the cumin oil was cytotoxic in the concentration of 20 mg.mL-1 and the dill in the concentrations of 20 and 8 mg.mL-1. All yeasts were susceptible against the evaluated essential oils. Cumin presented the lowest MIC against yeasts. We concluded that all the essential oils presented inhibitory action against Candida spp., and C . cyminum, P. anisum and F. vulgare were not cytotoxic in the same minimum inhibitory concentrations for the fungi.

Evaluation of anti-enzyme properties of Origanum vulgare essential oil against oral Candida albicans.

This study aimed to evaluate the anti-enzymatic activity of Origanum vulgare (oregano) essential oil against 15 strains of Candida albicans. Candida albicans samples were isolated from the oral mucosa of patients with denture stomatitis treated in a Dentistry school on a public university. Preparation of the inoculum was performed with a suspension of C. albicans reactivated 24h earlier in 5mL of sterile phosphate buffer saline (PBS) adjusted to a 0.5-turbidity on the MacFarland scale (1,5×108UFC/mL). The essential oil was obtained by hydrodistillation in a Clevenger-type machine and analyzed by gas chromatography. Enzymatic assay was performed to test phospholipase anti-enzymatic properties. Chromatography analysis revealed that the main compounds present in the essential oil were 4-terpineol (41.17%), thymol (21.95%), γ-terpinene (5.91%) and carvacrol (4.71%). For the anti-enzymatic test, the statistical analysis showed that there was found statistically significant interactions between the factors time and concentration (P≤0,001). Thus, essential oil of oregano at 1%, 5% and 10% presented significant reductions in the production of the phospholipase enzyme produced by Candida albicans strains. However, the longer the incubation time of the essential oil, there is a relatively moderate reduction in its anti-enzymatic activity.

Atypical symptoms in patients with herpesvirus DNA detected by PCR in cerebrospinal fluid.

BACKGROUND: Polymerase chain reaction (PCR) detection of herpesvirus DNA in cerebrospinal fluid (CSF) is an important tool in the diagnosis of central nervous system (CNS) syndromes. The corresponding viral infections present with diverse clinical signs, which are often classical although no sign can be considered as specific. This retrospective study aims to describe atypical symptoms in patients with herpesvirus DNA detected in CSF by PCR. A total of 3452 cerebrospinal fluid samples from patients with suspected herpesvirus infection of the CNS were investigated between 1998 and 2003 in our clinical virology laboratory. "In-house" PCRs for each herpesvirus [herpes simplex virus (HSV), varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein Barr virus (EBV), or human herpes virus 6 (HHV6)] were used until 2001 and a commercially available "Herpes Consensus PCR" was used thereafter. One of the five herpesviruses investigated in this study was found in 71 (2.1%) of CSF samples (37 HSV, 14 VZV, 1 CMV, 9 EBV and 10 HHV6). These samples were obtained from 62 patients whose clinical findings were generally consistent with the PCR data. However, some little known features of herpesvirus-related symptoms, such as partial seizure associated with HSV infection, and unusual VZV or HHV6-related myelitis were also observed.

Antihelminthic action of the Anethum graveolens essential oil on Haemonchus contortus eggs and larvae / Ação anti-helmítica do óleo essencial de Anethum graveolens sobre ovos e larvas de Haemonchus contortus

Abstract Gastrointestinal nematodes are responsible for great economic losses in sheep raising, and their control has long been carried out almost exclusively by the administration of anthelmintics, which have led to serious resistance problems. In the search for alternative control measures, phytotherapic research is highlighted. The aim of this study was to evaluate the action of Anethum graveolens (dill) essential oil on different stages of Haemonchus contortus life cycle, as well its cytotoxicity MDBK (Madin-Darby bovine kidney) cells. H. contortus larvae and eggs were obtained from infected sheep feces, and essential oil extracted from plant seeds through the Clevenger apparatus. 9.4, 4.7, 2.35, 1.17. 0.58 and 0.29 mg/mL concentrations were evaluated. The Egg Hatch Inhibition (HI), Larval Development Inhibition (LDI) and Larval Migration Inhibition (LMI) techniques were used. Thybendazole 0.025 mg/mL in HI and Levamisole 0.02 mg/mL in the LDI and LMI tests were used as positive controls, while distilled water and a Tween 80 solution were used as positive negative controls. The inhibition results obtained for the highest oil concentration were: HI 100%, LDI 98.58% and LMI 63.7%, differing (�� <0.05) from negative controls. Main A. graveolens oil components present in 95.93% of the total oil were Dihydrocarvone (39.1%), Carvone (22.24%), D-Limonene (16.84%), Apiol (10.49%) and Trans-dihydrocarvone (7.26%). Minimum A. graveolens essential oil concentrations required to inhibit 50% (IC50) of egg hatching, larval development and larval migration were 0.006 mg/mL, 2.536 mg/mL and 3.963 mg/mL, respectively. Cell viability in MDBK (Madin-Darby bovine kidney) cells, when incubated with A. graveolens essential oil, was 86% for the highest (9.4 mg/mL) and 99% for the lowest concentration (0.29 mg/mL). A. graveolens essential oil, according to the results obtained in this study, is a promising alternative in sheep gastrointestinal nematode control.

Resumo Os nematoides gastrintestinais são responsáveis ​​por grandes perdas econômicas na ovinocultura, e seu controle tem sido realizado quase exclusivamente pela administração de anti-helmínticos, que levaram a sérios problemas de resistência. Na busca de medidas alternativas de controle, destaca-se a pesquisa fitoterápica. O objetivo deste trabalho foi avaliar a ação do óleo essencial de Anethum graveolens (endro) em diferentes estágios de Haemonchus contortus, bem como testar a viabilidade celular para o óleo. Larvas e ovos de H. contortus foram obtidos de fezes de ovinos infectados e óleo essencial extraído de sementes de plantas através do aparelho de Clevenger. As concentrações avaliadas foram 9,4, 4,7, 2,35, 1,17, 0,58 e 0,29 mg/mL. Verificou-se a Inibição de eclosão dos ovos (IE), Inibição de Desenvolvimento Larval (IDL) e Inibição de Migração Larval (IML). Tiabendazol 0,025 mg/mL em IE e Levamisole 0.02 mg/mL nos testes IDL e IML foram usados ​​como controles positivos, enquanto água destilada e uma solução Tween 80 foram usados como controles negativos. Os resultados de inibição obtidos para a maior concentração de óleo foram: IE 100%, IDL 98,58% e IML 63,7%, diferindo (�� <0,05) dos controles negativos. Os principais componentes presentes em 95,93% do óleo total de A. graveolens foram Di-hidrocarvona (39,1%), Carvona (22,24%), D-Limoneno (16,84%), Apiol (10,49%) e Trans-di-hidrocarvona (7,26%). As concentrações mínimas de óleo essencial de A. graveolens necessárias para inibir 50% (IC50) de eclosão dos ovos, desenvolvimento larval e migração larval foram de 0,006 mg/mL, 2,536 mg/mL e 3,963 mg/mL, respectivamente. A viabilidade celular nas células MDBK (rim bovino Madin-Darby), quando incubadas com o óleo essencial de A. graveolens, foi de 86% para a maior (9,4 mg/mL) e 99% para a menor concentração (0,29 mg/mL). O óleo essencial de A. graveolens mostrou ser uma alternativa promissora no controle de nematoides gastrintestinais de ovinos.

Lipase of Pseudomonas cepacia for biotechnological purposes: purification, crystallization and characterization.

Commercial lipase (triacylglycerol lipase, EC 3.1.1.3) of Pseudomonas cepacia (Amano) has been purified to homogeneity by a single chromatography on phenyl Sepharose. The eluted lipase crystallized spontaneously at 4 degrees C in the eluent, containing 58-69% 2-propanol. The yield of the lipase was 87-100% and the specific activity during the hydrolysis of triolein 5800 U/mg protein. This protein has a molecular weight of 34.1 kDa as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Its purity was determined by SDS-PAGE and capillary zone electrophoresis to be > or = 99%. Immobilization on Sepharose increased its stability in organic solvents. This lipase of P. cepacia differs from that of other Pseudomonas strains in respect to substrate specificity and during crystallization. It exhibits a high stability in organic solvents and supercritical carbon dioxide.

Applied biosensors.

Biosensors are important analytical tools in clinical and environmental monitoring, biotechnological process control, medicine, and in the food and drink industry. This review devotes attention to the most common biosensor in biotechnology, the glucose biosensor, and to recent contributions to the rapidly growing field of optical biosensors. Trends and developments in these areas are discussed.

Comparison of particulate and continuous-bed columns for protein displacement chromatography.

A conventional anion exchange column packed with porous particles (BioScale Q2), and a novel continuous-bed column (UNO Q1) were compared for displacement separation of dairy whey proteins with polyacrylic acid as displacer. The steric mass action model was investigated as a means to aid and accelerate this development. Characteristic charges and steric factors were measured for the proteins and the displacer according to the model, and used together with the affinity constant derived from the adsorption isotherms for simulations, as well as for the construction of the affinity and operating regime plots. If possible, the latter two were used to select conditions for the actual experiments. In the case of the particle-based column, experimental results and simulations did not agree. In addition, the operating regime plot could not be constructed. The affinity plot did predict the order in the displacement train correctly, but gave misleading information concerning the possible effect of a change in displacer concentration. This is taken to be a result of the porous nature of the particles, which handicaps, to some extent, the interaction of the proteins and the displacer molecules with the adsorptive surface. Results were considerably better in case of the continuous-bed column, where there is no intraparticulate surface.

High performance flow injection analysis of recombinant protein G.

Chromatographic discs were investigated for their potential to substitute for the hitherto used cartridges in heterogeneous flow injection analysis. Originally designed for fast high performance liquid chromatography (HPLC) of biopolymers, the discs combine reliability with speed and resolution. This together with their price and their long-standing time made them attractive for use in flow injection analysis. The base material of the discs is a glycidyl methacrylate-co-ethylene dimethacrylate (GMA-EDMA) co-polymer. The epoxy groups inherent to this base structure can be used for immobilization purposes. In this first demonstration, antibodies were immobilized and the resulting affinity discs used for the fast analysis (< 5 min) of protein G from cell lysate of recombinant Escherichia coli. A linear calibration curve over several orders of magnitude as well as excellent reproducibility and correlation with data produced by conventional protein assay were obtained.

On-line immunoanalysis for bioprocess control.

Immunoanalytical techniques such as ELISA are often used for the detection of proteins produced in cultivation processes. Owing to the difficulty of automating of the time-consuming traditional ELISA, there is an intense demand for a suitable on-line monitoring method. Combining well-known immunoassays with the FIA technique, we present the heterogeneous and the turbidimetric immuno-FIA methods. The following proteins were investigated with these FIA methods: thermostable pullulanase, IgG, antithrombin III, and recombinant tissue-type plasminogen activator. In the cases of pullulanase and monoclonal mouse IgG, the turbidimetric immuno-FIA was used for on-line analysis of the cultivation process. Results are presented here to demonstrate the effectiveness and application of these immunoanalysis.

Immuno- and flow cytometric analytical methods for biotechnological research and process monitoring.

In this article, the applications of immunoanalysis and flow cytometry for research and process monitoring in biotechnology are discussed. Brief reviews of the two analytical methods are followed by descriptions of actual applications in various areas of biotechnology. In the case of immunoanalysis, emphasis is placed on systems for on-line bioprocess monitoring, and examples are given for a thermostable pullulanase, a mouse IgG, and antithrombin III. Although flow cytometry is not currently an on-line analytical technique, its value as an off-line method is illustrated by examples of the measurement of shear stress effects, lipid content, and sterol content.

Preparation of stationary phases for open-tubular capillary electrochromatography using the sol-gel method.

Capillary electrochromatography requires the deposition of a stationary phase inside the capillary. In this paper the sol-gel method is proposed for this purpose. The gels were prepared externally and injected into a fused-silica capillary, where anchorage to the capillary wall was possible through condensation reactions between the silanol groups of the capillary wall and the residual silanol groups the gel. Contrary to a commonly used practice, alkaline pretreatment of the inner capillary wall prior to the introduction and anchoring of the gel was found to be only marginally effective in improving the mechanical stability of the column. The influence of various parameters, such as the pH, the water content, the presence of alcohol (ethanol) on the formation of tetraethoxysilane (TEOS)-n-octyltriethoxysilane (C8-TEOS) hybrid gels of varied composition is discussed. The pH and the amount of water present were found to be the determining factors in the preparation of a stable gel with the desired mechanical and chromatographic properties. By carrying out the gel formation at 80 degrees C, capillary columns could be produced in 2.5 h. While an acidic pH was required during (external) gel formation, subsequent treatment of the gel inside the capillary with an alkaline solution ('aging') was found to improve separation and stationary phase capacity significantly. The capillary columns were subsequently used to separate a mixture of polycyclic aromatic hydrocarbons in less than 3 min.

Acrylamide-based monoliths as robust stationary phases for capillary electrochromatography.

A method is described for the synthesis of rigid, macroporous polymers (monoliths) to be used as stationary phases in capillary electrochromatography (CEC). The procedure reproducibly results in columns with good mechanical and chemical stability. Once the procedure was optimized, it yielded the desired CEC columns in nearly 100% of the cases. The batch-to-batch standard deviation of the migration of the electroosmotic flow (EOF) marker for nine randomly chosen columns was 5%. The polymerization is carried out inside the capillary, an aqueous phase is used as solvent. Monomers based on acrylamides with varying hydrophilicity were used to introduce the interactive moieties together with piperazine diacrylamide as cross-linker and vinylsulfonic acid as provider of the charged, EOF-producing moieties. The pore size of the monoliths was adjusted by adding varying amounts of ammonium sulfate to the reaction mixture. In this manner, the average pore size of a given monolith could be reproducibly adjusted to values ranging from 50 nm to 1.3 microm. The procedure was optimized for four particular types of monoliths, which differed in hydrophobicity. The latter was adjusted by introducing suitable co-monomers, such as alkyl chain-bearing molecules, into the monolithic structure. Attempts to systematically investigate the chromatographic behavior of the monolithic stationary phases were made, using a model mixture of aromatic compounds as sample. The standard deviations for the run-to-run reproducibility of the retention times for unretained and retained analytes were <1.5%. Flat Van Deemter curves were measured even at elevated flow-rates (2 mm/s). Plate heights between 10 and 15 microm were measured in this range. The retention order was taken as the principal indication for the chromatographic mode. The separation was found to be governed neither by pure reversed-phase nor by pure normal-phase chromatography, even on monoliths, where large amounts of C6 ligands had been introduced.

Comparison of anion-exchange and hydroxyapatite displacement chromatography for the isolation of whey proteins.

In displacement chromatography, several substances may be isolated and concomitantly concentrated, which makes this separation procedure attractive for the processing of diluted product streams containing a number of high value substances. Here, the suitability of anion-exchange and hydroxyapatite displacement chromatography for the processing of technical dairy whey is investigated. The pH and flow-rate of the carrier, the displacer chemistry and, in case of the apatite, the particle diameter of the stationary phase are considered. As a consequence of the pH sensitivity of the beta-lactoglobulin, one major whey component, apatite displacement chromatography is less then successful in whey separation. At a denaturing carrier pH (> 8.5) the beta-lactoglobulin zone is broad and stretches over the entire displacement train. At a lower carrier pH, previously successful polyanionic displacers do not bring about separation, while low-molecular-mass ones do, but they tend to overrun and thus contaminate the protein zones. In the case of anion-exchange displacement chromatography, polyanions, especially polyacrylic acid (PAA, M(r) 6000), constitute suitable displacers. Here too, a carrier pH of 8.0 is most suited to the separation of the whey proteins. The low-molecular mass-displacer iminodiacetic acid (IDA, M(r) 133.4), on the other hand, displaces only alpha-lactalbumin. The beta-lactoglobulin remains on the column. PAA is used as the displacer to process a dairy whey sample.