| 1. |
- Gustavsson, Per-Erik, et al.
(författare)
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Direct measurements of convective fluid velocities in superporous agarose beads
- 1998
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 795:2, s. 199-210
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Tidskriftsartikel (refereegranskat)abstract
- Superporous agarose beads contain two sets of pores, diffusion pores and so-called superpores or flow pores, in which the chromatographic flow can transport substances to the interior of each individual bead [Gustavsson and Larsson, J. Chromatogr. A 734 (1996) 231]. The existence of pore flow may be proven indirectly by the chromatographic performance of beads but it has never been directly demonstrated in a chromatographic bed. In this report, pore flow was directly measured by following the movement of micro-particles (dyed yeast cells) in a packed bed. The passage of the micro-particles through the superpores and through the interstitial pores was followed by a microscope/video camera focused on beads which were situated four layers from the glass wall. The video recordings were subsequently used to determine the convective fluid velocities in both the superpores and the interstitial pores. Experiments were carried out with three different bead size ranges, all of which contained superporous beads having an average superpore diameter of 30 mu m. The superpore fluid velocity as % of interstitial fluid velocity was determined to be 2-5% for columns packed with 300-500-mu m beads (3% average value), 6-12% for columns packed with 180-300 mu m beads (7% average value) and 11-24% for columns packed with 106-180-mu m beads (17% average value). These data were compared to and found to agree with theoretically calculated values based on the Kozeny-Carman equation. In order to observe and accurately measure fluid velocities within a chromatographic bed, special techniques were adopted. Also, precautions were made to ensure that the experimental conditions used were representative of normal chromatography runs. (C) 1998 Elsevier Science B.V.
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| 2. |
- Gustavsson, Per-Erik, et al.
(författare)
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Superporous agarose beads as a hydrophobic interaction chromatography support
- 1999
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 830:2, s. 275-284
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Tidskriftsartikel (refereegranskat)abstract
- Superporous agarose beads were used as a support for hydrophobic interaction chromatography. These beads have large connecting flow pores in addition to their normal diffusion pores. The flow pores, which are approximately one fifth of the overall diameter of the superporous agarose beads, were earlier shown to give the beads improved mass transfer properties relative to homogeneous agarose beads (Gustavsson and Larsson, J. Chromatogr. A, 734 (1996) 231-240). Superporous agarose beads and homogeneous agarose beads of the same particle size range (106-180 mu m) were derivatized with phenyl groups. The properties of the superporous beads were then compared with the homogeneous beads in the separation of a mixture of three model proteins (ribonuclease A, lysozyme and bovine serum albumin) at various superficial flow velocities from 30 to 600 cm/h. The superporous beads gave satisfactory separation at flow velocities five times higher than was possible for homogeneous beads. The performance of the two types of beads was also compared in the purification of lactate dehydrogenase from a beef heart extract at a superficial flow velocity of 150 cm/h. The superporous beads performed considerably better, leading to twice the purification factor and twice the concentration of the desired product. The results were interpreted using the theoretical treatment given by Carta and Rodrigues (Carta and Rodrigues, Chem. Eng. Sci., 48 (1993) 3927). (C) 1999 Elsevier Science B.V. All rights reserved.
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| 3. |
- Palsson, E, et al.
(författare)
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Theories of chromatographic efficiency applied to expanded beds
- 2001
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 912:2, s. 235-248
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Tidskriftsartikel (refereegranskat)abstract
- Various quantities such as plate height (HETP), number of plates (N), axial dispersion coefficient (D-ax) and Bodenstein number (Bo) are used to describe the efficiency of, and dispersion in chromatographic columns. Different quantities highlight different aspects of the performance. Due to the expansion of expanded-bed columns, the information contained in some of these quantities is not the same for expanded beds as for packed beds. In this article the mentioned quantities are described and discussed both theoretically and related to experimental data. It is concluded that they are often used in a confusing way. Quantities modified to be more informative when comparing beds of different expansions are developed (N-EB =N/expansion(2) and HETPEB=HETP . bed expansion) and recommendations of which quantity to use in what situation are given. (C) 2001 Published by Elsevier Science B.V.
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| 4. |
- Gustavsson, Per-Erik, et al.
(författare)
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Purification of plasmid DNA with a new type of anion-exchange beads having a non-charged surface
- 2004
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1038:1-2, s. 131-140
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Tidskriftsartikel (refereegranskat)abstract
- We have prepared a new type of anion exchanger, which effectively discriminates between RNA and plasmid DNA. The material is based on a Sephacryl S-500 HR matrix provided with quartenary amine anion-exchange groups. A distinguishing feature of the beads is that a thin (2-3 mum) outer layer of the beads lacks ion-exchange groups. In the synthesis of these beads the vinyl groups in the outer layer of vinylalkyl substituted Sephacryl S-500 HR beads are reacted with bromine. The resulting layer of bromoalkyl groups are hydrolysed, creating an inert outer layer of hydroxyalkyl groups. Finally, bromination and trimethylamine reactions of the inner vinyl groups provide the beads with a core of cationic groups. Large plasmid molecules will not bind to such beads since they are too large to enter the pores and therefore cannot come into contact with the charged matrix in the inner parts of the beads. RNA and protein molecules present in a cleared lysate, on the other hand, readily enter the pores and become adsorbed. A two-column strategy was developed for plasmid purification (recombinant pBluescript, 5.9 kilo base pairs, kbp). The first column was packed with the restricted access anion-exchanger beads (lid beads) and the second column with normal ion-exchange material (same ligand density as the lid beads). Diluted (3 x), cleared lysate was pumped through the tandem columns. The first column was subsequently disconnected from the system and the purified plasmid adsorbed on the second column was eluted in a concentrated form (6x) and with 89% recovery. The two-column procedure removed 99.5% of the RNA and 96% of the proteins. (C) 2004 Elsevier B.V. All rights reserved.
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| 5. |
- Tiainen, Peter, et al.
(författare)
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High-capacity composite adsorbents for nucleic acids.
- 2011
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Ingår i: Journal of chromatography. A. - : Elsevier BV. - 1873-3778 .- 0021-9673. ; 1218:31, s. 5235-5240
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Tidskriftsartikel (refereegranskat)abstract
- Cytopore is a bead-shaped, macroporous and easily compressible cellulose-based anion-exchange material intended for cultivation of anchor-dependent animal cells. Reticulated vitreous carbon (RVC) is a strong, non-compressible, high voidage (97%) matrix material that can be cut to desired geometrical shapes. Cytopore and RVC were combined to cylindrical composites (25mm 10mm) fitted inside chromatography columns. The composite combined the advantageous properties of both its constituents, making it suitable for column chromatography. The composite could withstand very high flow rates without compaction of the bed (>25column volumes/min; 4000cmh(-1)). Chromatography runs with tracers showed a low HETP value (0.3mm), suggesting that pore flow was in operation. The dynamic binding capacities (10% breakthrough) per gram of dry weight Cytopore were determined for several compounds including DNA and RNA and were found to be 240-370mg/g. The composite was used to isolate pUC 18-type plasmids from a cleared alkaline lysate in a good yield. Confocal microscopy studies showed that plasmids were bound not only to the surface of the Cytopore material but also within the matrix walls, thus offering an explanation to the very high binding capacities observed. The concept of using a composite prepared from a mechanically weak, high-binding material and a strong scaffold material may be applied to other systems as well.
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| 6. |
- Tiainen, Peter, et al.
(författare)
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Plasmid purification using non-porous anion-exchange silica fibres
- 2007
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1149:2, s. 158-168
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Tidskriftsartikel (refereegranskat)abstract
- A new type of fibre-based anion-exchange material for plasmid purification was developed. The basic material consisted of non-porous silica fibres with a mean diameter of 1.5 mu m and a surface area of 2.4 m(2) g(-1). The fibre surface was provided with several types of ligands, either by adsorption of polymers (chitosan or poly(ethyleneinune)) or by polymerization of amine-containing acrylic monomers onto a propyl methacrylatesilanized surface. The resulting polymer layers contained primary, tertiary or quaternary amines as ion-exchange groups. The packing density could be varied considerably, 9-34% (v/v). The loose packing structure provided excellent flow properties suitable for high-speed operations. The best overall performance was shown by silica fibres provided with tertiary amine polymers, having a plasmid-binding capacity of 0.9 mg ml(-1) (pre-purified plasmid) and a plasmid recovery of 62% (performance data remained stable though several adsorption cycles). The high flow rates possible with the fibre material made it especially useful when large volumes of cleared lysate were processed. The columns could be operated with retention of their adsorption properties at speeds of up to 1800 cm h(-1) equivalent to 0.5 column volumes per minute. The binding capacity was found to be lower than anticipated from the design of the fibres. Fluorescence imaging showing individual plasmid molecules indicated the fibre population to be heterogeneous with respect to plasmid adsorption, some fibres displaying poor binding properties. Possible reasons for this heterogeneity are discussed. (c) 2007 Elsevier B.V. All rights reserved.
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| 7. |
- Tiainen, Peter, et al.
(författare)
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Superporous agarose anion exchangers for plasmid isolation
- 2007
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1138:1-2, s. 84-94
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Tidskriftsartikel (refereegranskat)abstract
- Superporous agarose beads have wide, connecting flow pores allowing large molecules such as plasmids to be transported into the interior of the beads by convective flow. The pore walls provide additional surface for plasmid binding thus increasing the binding capacity of the adsorbent. Novel superporous agarose anion exchangers have been prepared, differing with respect to bead diameter, superpore diameter and type of anion-exchange functional group (poly(ethyleneimine) and quaternary amine). The plasmid binding capacities were obtained from breakthrough curves and compared with the binding capacity of homogeneous agarose beads of the same particle size. Significantly, the smaller diameter superporous agarose beads were found to have four to five times higher plasmid binding capacity than the corresponding homogeneous agarose beads. The experimentally determined plasmid binding capacity was compared with the theoretically calculated surface area for each adsorbent and fair agreement was found. Confocal microscopy studies of beads with adsorbed, fluorescently labelled plasmids aided in the interpretation of the results. Superporous poly(ethyleneimine)-substituted beads with a high ion capacity (230 mu mol/ml) showed a plasmid binding of 3-4 mg/ml adsorbent. Superporous quaternary amine-substituted beads had a lower ion capacity (81 mu mol/ml) and showed a correspondingly lower plasmid binding capacity (1-2 mg/ml adsorbent). In spite of the lower capacity, the beads with quaternary amine ligand were preferred, due to their much better plasmid recovery (70-100% recovery). Interestingly, both capacity and recovery was improved when the plasmid adsorption step was carried out in the presence of a moderate salt concentration. The most suitable superporous bead type (45-75 mu m diameter beads; 4 mu m superpores; quaternary amine ligand) was chosen for the capture of plasmid DNA from a clarified alkaline lysate. Two strategies were evaluated, one with and one without enzymatic digestion of RNA. The strategy without RNase gave high plasmid recovery, quantitative removal of protein and a 70% reduction in RNA. (c) 2006 Elsevier B.V. All rights reserved.
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