Manufacturing of agarose-based chromatographic adsorbents – Effect of ionic strength and cooling conditions on particle structure and mechanical strength

Abstract

The effect of ionic strength of agarose solution and quenching temperature of the emulsion on the structure and mechanical strength of agarose-based chromatographic adsorbents was investigated. Solutions of agarose containing different amounts of NaCl were emulsified at elevated temperature in mineral oil using a high-shear mixer. The hot emulsion was quenched at different temperatures leading to the gelation of agarose and formation of soft particles. Analysis of Atomic Force Microscopy (AFM) images of particle surfaces shows that pore size of particles increases with ionic strength and/or high quenching temperature. Additionally it has been found that the compressive strength of particles measured by micromanipulation also increases with ionic strength of the emulsion and/or high quenching temperature but these two parameters have practically no significant effect on the resulting particle size and /particle size distribution. Results from both characterization methods were compared with Sepharose 4B, a commercial agarose-based adsorbent. This is the first report examining the effect of ionic strength and cooling conditions during manufacturing, on the microstructure of micron-sized agarose beads for bioseparation.Keywords: agarose beads; microstructure; pore size; mechanical properties; AFM; micromanipulation 2 IntroductionAgarose is one of the two constituents of agar (the other being agaropectin) which is extracted from red algae [1]. It is a linear polysaccharide consisting of 1, 3-linked β-D-galactopyranose and 1, 4-linked 3, 6-anhydro-α-L-galactopyranose, while agaropectin is a more complicated polysaccharide containing sulphuric and uronic acid residues [2]. The gelation of agarose involves a change from a random coil in solution to a double helix in the initial stages of gelation and then to bundles of double helices in the final stage [3]. The gel is formed when an infinite extensive three-dimensional network of agarose fibres (consisting of helices) develops [4]. At this state the material is characterized by a co-continuous macroporous morphology with polymer-rich regions (fibres) and solvent-rich regions (pores holding water), and therefore the gelation can be interpreted as a phase separation [5].Because of its physical and chemical stability, neutral charge, hydrophilic character, open structure [6, 7] and biocompatibility, agarose has been extensively used in chromatographic separation of biomolecules [8], cell microencapsulation [9] and food additives [10]. Following functionalization with the appropriate chemistry a selective porous adsorbent can be produced for the purification of biomolecules both in packed beds and, after inclusion of densifiers, in fluidized beds [11]. The efficiency of purification depends both on macro-and microscopic properties of adsorbent. Macroscopic properties such as particle size/size distribution, shape and mechanical strength have a critical effect on throughput, pressure drop and mechanical stability of adsorbent [12]. Particles must also be sufficientl...

Precise control of agarose media pore structure by regulating cooling rate

“…After the fibers mature development, the lower cooling rate will not affect the pore structure. The trends in mean pore size and pore size uniformity of the agarose media as a function of cooling rate are in accordance with previous literatures [13,14]. However, our results are more accurate and finer changes could be distinguished than in other studies.…”

“…Loannidis et al. quenched the hot agarose emulsion at different temperature leading to the gelation of the agarose and the formation of soft particles. Characterization by atomic force microscopy (AFM) of the particle surfaces showed that the particle pore size increased when subjected to high quenching temperatures.…”

“…The analysis by TEM indicated that the rapidly cooled gels appeared to display significantly "tighter" and more homogeneous pore structure with smaller average pore size than the corresponding gels cooled slowly. Loannidis et al [14] quenched the hot agarose emulsion at different temperature leading to the gelation of the agarose and the formation of soft particles. Characterization by atomic force microscopy (AFM) of the particle surfaces showed that the particle pore size increased when subjected to high quenching temperatures.…”

Agarose particle-templated porous bacterial cellulose and its application in cartilage growth in vitro

“…Commercial agarose powder is manufactured using a range of different methods, including spray drying, suspension gelation and membrane emulsification and is generally polydisperse [20,21]. To create agarose microparticles, we used a fluidic system that is similar to an approach described for interfacial polymeriza-tions in liquid prepolymer droplets [19].…”

Evaluation of techniques used for visualisation of hydrogel morphology and determination of pore size distributions

“…Agarose hydrogels form upon cooling, and the final gel structure and pore size distribution are affected by the cooling rate. 38,39 In the current study, the gel was formed with rapid cooling over 30 min from 72 to 30 °C, followed by a very slow cooling phase until the hydrogel reached 22 °C (the cooling curve was previously published 9 ). Separate agarose gels prepared from agarose solutions of various concentrations (0.38–2.0% w/v) are referred to as agarose gels of the respective concentration.…”