The use of confocal laser scanning microscopy to study the transport of biomacromolecules in a macroporous support

Anuradha Subramanian, Jennifer Hommerding

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Large-pore materials or supports resembling polymer conduits are used as packing material in chromatographic operations. Our ongoing research has shown that, when modified with peptides or ligands, chitosan beads that are 800 μm in diameter and have 3.5% solids can be used as matrices in bioseparations. The goal of the present study is to evaluate the transport properties of biomolecules in the modified chitosan beaded matrices. Batch uptake experiments with fluorescently tagged pure human IgG, human IgA and human IgM were conducted to visualize the distribution of binding sites throughout the bead as well as to evaluate restrictions to diffusion, if any, within the support. The chromatographic performance of the macrobeads was first assessed by the classical height equivalent of a theoretical plate HETP analysis. The independence of HETP on linear flow rates studied suggests that a likely mode of solute transport within the macrobeads may be a combination of convection and diffusion-convective components. By using fluorescent-tagged immunoglobulins, the penetration of the adsorbent particle at different times and different levels of saturation was visually observed. The profiles obtained from dynamic experiments were compared to the profiles obtained from finite bath experiments. With an increase in the incubation time, the degree of penetration increased and the bead interior was saturated with FITC immunoglobulins at the end point of the finite bath experiment. In the dynamic uptake experiment, the degree of penetration was found to be a function of the linear velocity and level of breakthrough. The penetration of the bead radius, at times lower than the predicted diffusion time, suggests that the mode of transport in the chitosan beads is governed by a combination of convective and diffusive forces.

Original languageEnglish (US)
Pages (from-to)89-97
Number of pages9
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume818
Issue number1 SPEC. ISS.
DOIs
StatePublished - Apr 15 2005

Fingerprint

Chitosan
Confocal Microscopy
Microscopic examination
Scanning
Baths
Lasers
Immunoglobulins
Convection
Fluorescein-5-isothiocyanate
Experiments
Immunoglobulin A
Immunoglobulin M
Polymers
Solute transport
Immunoglobulin G
Binding Sites
Ligands
Biomolecules
Peptides
Transport properties

Keywords

  • Biomolecules
  • Confocal microscopy
  • Convection
  • Diffusion
  • FITC
  • Protein transport

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry
  • Cell Biology

Cite this

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abstract = "Large-pore materials or supports resembling polymer conduits are used as packing material in chromatographic operations. Our ongoing research has shown that, when modified with peptides or ligands, chitosan beads that are 800 μm in diameter and have 3.5{\%} solids can be used as matrices in bioseparations. The goal of the present study is to evaluate the transport properties of biomolecules in the modified chitosan beaded matrices. Batch uptake experiments with fluorescently tagged pure human IgG, human IgA and human IgM were conducted to visualize the distribution of binding sites throughout the bead as well as to evaluate restrictions to diffusion, if any, within the support. The chromatographic performance of the macrobeads was first assessed by the classical height equivalent of a theoretical plate HETP analysis. The independence of HETP on linear flow rates studied suggests that a likely mode of solute transport within the macrobeads may be a combination of convection and diffusion-convective components. By using fluorescent-tagged immunoglobulins, the penetration of the adsorbent particle at different times and different levels of saturation was visually observed. The profiles obtained from dynamic experiments were compared to the profiles obtained from finite bath experiments. With an increase in the incubation time, the degree of penetration increased and the bead interior was saturated with FITC immunoglobulins at the end point of the finite bath experiment. In the dynamic uptake experiment, the degree of penetration was found to be a function of the linear velocity and level of breakthrough. The penetration of the bead radius, at times lower than the predicted diffusion time, suggests that the mode of transport in the chitosan beads is governed by a combination of convective and diffusive forces.",
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