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Sökning: L773:0021 9673

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61.
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62.
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63.
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64.
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65.
  • Gräslund, Torbjörn, et al. (författare)
  • Strategy for highly selective ion-exchange capture using a charge-polarizing fusion partner
  • 2002
  • Ingår i: Journal of Chromatography A. - 0021-9673 .- 1873-3778. ; 942:1-2, s. 157-166
  • Tidskriftsartikel (refereegranskat)abstract
    • To achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography, a novel basic protein domain is used as a purification handle. The proteolytic instability usually encountered for basic peptide tags is avoided by the use of a highly constrained α-helical domain based on staphylococcal protein A into which positively charged amino acids have been introduced. Here we show that this domain, consisting of 58 amino acids with a calculated isoelectric point (pI) of 10.5, can be used to efficiently capture different fused target proteins, such as a bacterial DNA polymerase (Klenow fragment), a viral protease (3C) and a fungal lipase (Cutinase). In contrast to standard cation-exchange chromatography, efficient capture can be achieved also at a pH value higher than the pI of the fusion protein, demonstrated here by Zbasic-Klenow polymerase (pI≈5.8) and ZZ-Cutinase-Zbasic (pI≈7.2) both purified at a pH of 7.5. These results show that the Zbasic domain is able to confer a regional concentration of positive charge on the fusion protein even at a relatively high pH. Hence, the data suggest that this domain could be used for highly efficient and selective capture of target proteins at conditions where most host-cell proteins do not bind to the chromatographic resin. The obtained purity after this one-step procedure suggests that the strategy could be an alternative to standard affinity chromatography. Methods for site-specific proteolysis of the fusion proteins to release native target proteins are also discussed.
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66.
  • Gröning, M., et al. (författare)
  • Headspace solid-phase microextraction - a tool for new insights into the long-term thermo-oxidation mechanism of polyamide 6.6
  • 2001
  • Ingår i: Journal of Chromatography A. - 0021-9673 .- 1873-3778. ; 932:02-jan, s. 1-11
  • Tidskriftsartikel (refereegranskat)abstract
    • Low-molecular-mass products formed. during thermo-oxidation of polyamide 6.6 at 100 degreesC were extracted by headspace solid-phase microextraction and identified by GC-MS. A total of 18 degradation products of polyamide 6.6 were identified. In addition some low-molecular-mass products originating from the lubricants were detected. The identified degradation products were categorized into four groups where compounds within each group contain the same structural feature. In groups A, B and C several new thermo-oxidation products of polyamide 6.6 were identified including cyclic imides, pyridines and structural fragments from the original polyamide chain. 1-Pentyl-2,5-pyrrolidinedione (pentylsuccinimide) showed the largest increase in abundance during oxidation. The cyclopentanones in group D were already present in the un-aged material. Their amounts decreased during ageing and they are thus not formed during thermo-oxidation of polyamide 6.6 at 100 degreesC. The identified thermo-oxidation products can be formed as a result of extensive oxidation of the hexamethylenediamine unit in the polyamide backbone. The degradation products pattern shows that the long-term thermo-oxidative degradation, just like thermal degradation and photo-oxidation of polyamide 6.6, starts at the N-vicinal methylene groups.
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67.
  • Hakkarainen, Minna (författare)
  • Qualitative and quantitative solid-phase microextraction gas chromatographic-mass spectrometric determination of the low-molecular-mass compounds released from poly(vinyl chloride)/polycaprolactone-polycarbonate during ageing
  • 2003
  • Ingår i: Journal of Chromatography A. - 0021-9673 .- 1873-3778. ; 1010:1, s. 9-16
  • Tidskriftsartikel (refereegranskat)abstract
    • A solid-phase microextraction (SPME) method was developed to quantitatively determine the amount of 6-hydroxyhexanoic acid in aqueous solutions. The SPME method in combination with GC-MS was then applied to identify and quantify the low-molecular-mass compounds migrating from a new poly(vinyl chloride) (PVC) material, PVC/polycaprolactone-polycarbonate (PCL-PC) during ageing in water. It was shown that only a small amount of 6-hydroxyhexanoic acid, the final hydrolysis product of PCL-PC, migrated from the blend during ageing at 37 and 70 degreesC. If, however, the temperature was raised to 100 degreesC rapid hydrolysis of PCL-PC resulted. In addition to 6-hydroxyhexanoic acid, 6-hydroxyhexanoic acid dimer, caprolactone, different carboxylic acids, acetophenone and phenol were identified. SPME-GC-MS was also applied to monitor the low-molecular-mass compounds migrating from the PVC/PCL-PC blend during thermo-oxidation.
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68.
  • Hedeland, Ylva, et al. (författare)
  • Chiral separation of amines with N-benzoxycarbonylglycyl-L-proline as selector in non-aqueous capillary electrophoresis using methanol and 1,2-dichloroethane in the background electrolyte
  • 2003
  • Ingår i: Journal of Chromatography A. - 0021-9673 .- 1873-3778. ; 984:2, s. 261-271
  • Tidskriftsartikel (refereegranskat)abstract
    • N-Benzoxycarbonylglycyl-L-proline (L-ZGP) has been introduced as a chiral selector for enantioseparation of amines in non-aqueous capillary electrophoresis. Methanol mixed with different proportions of dichloromethane, 1,2-dichloroethane or 2-propanol containing L-ZGP and ammonium acetate was used as the background electrolyte. Enantioseparation of different types of pharmacologically active amines was performed, e.g. the local anaesthetic bupivacaine and the beta-adrenoceptor blocking agent pindolol. Addition of the solvents (dichloromethane, 1,2-dichloroethane or 2-propanol) gave an improved chiral separation partly due to a distinct decrease in the electroosmotic flow. The use of 1,2-dichloroethane in the background electrolyte gave higher precision in migration time (RSD 2.2%) compared to the systems containing dichloromethane. An enantiomeric separation of mepivacaine was performed within 72 s by use of short-end injection with an effective capillary length of 8.5 cm.
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69.
  • Henriksson, Hongbin, et al. (författare)
  • Cellobiohydrolase 58 (P.c. Cel 7D) is complementary to the homologous CBHI (T.r. Cel 7A) in enantioseparations
  • 2000
  • Ingår i: Journal of Chromatography A. - 0021-9673 .- 1873-3778. ; 898:1, s. 63-74
  • Tidskriftsartikel (refereegranskat)abstract
    • Cellobiohydrolase 58 (EC 3.2.1.91, pc. Cel 7D) from Phanerochaete chrysosporium was immobilized on silica and the resulting material, CBH 58-silica, was then used as a chiral stationary phase (CSP) in liquid chromatographic separations of enantiomers. The enantioselectivities obtained on CBH 58-silica were compared with those on CBH I-silica (a phase based on a corresponding cellulase from Trichoderma reesei). CBH 58-silica displayed higher selectivity than CBH I-silica for the more hydrophilic compounds, such as atenolol and metoprolol, although great similarities in chiral separation of beta -adrenergic antagonists were found between the two phases. None of the acidic compounds tested could be resolved on the CBH 58 phase. Moreover, the solutes were retained more on the CBH 58 phase in general, indicating an improved application potential in bioanalysis. Addition of cellobiose or lactose, both of which are inhibitors of cellulases, To the mobile phase impaired the enantioselectivity, indicating an overlap of the enantioselective and catalytic sites. The chiral analytes also functioned as competitive inhibitors and their inhibition constants were determined.
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70.
  • Horváth, E., et al. (författare)
  • Investigation of mandelic acid bonding on Pirkle type chromatographic stationary phases by Raman spectroscopy
  • 2000
  • Ingår i: Journal of Chromatography A. - 0021-9673 .- 1873-3778. ; 893:1, s. 37-46
  • Tidskriftsartikel (refereegranskat)abstract
    • The bonding of mandelic acid enantiomers has been studied on benzene-leucine, dinitrobenzene-leucine and dinitrobenzene-phenylalanine type chiral stationary phases connected to zeolite A supports. The π-donor, π-acceptor and H-bonding interactions responsible for diastereomer pair formations can be studied under quasi in situ chromatographic conditions by Fourier transform Raman and surface enhanced Raman spectroscopic techniques. Structural differences between diastereomer pairs result in observable spectral differences at a phase load of approx. 50%. It was shown that the decreasing π-acceptor character of the phase is associated with its increasing capability of H-bond formation. Correlating spectral data to chromatographic results it can be concluded that, in addition to H-bonding as well as to π-donor-π-acceptor interactions, steric hindrances due to bulky moieties of either the stationary phase or the analyte molecules are of importance in successful separations.
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