5.4 Experimental Methods
5.4.2 Characterization of Bulk Packings
Macroscopic Compression-Relaxation Experiments
The analysis of the macroscopic packing behavior was carried out using SEP particles.
The agarose-based media was chosen because it is a little softer than the methacrylic media and, hence, assured a well detectable amount of compression of the packing at that scale. Moreover, the packed bed was divided into seven equally sized sections by
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inserting small layers of Blue Sepharose 6 FF®which basically is the SEP resin dyed with Cibacron Blue. The resulting section thickness and the blue layer thickness were about 4 and 0.5 mm, respectively. The total packed bed height was 31 mm. A photograph of the column containing the different layers is shown in Figure 5.1 B.
The changes in bed height and height of each section due to either hydrodynamic or mechanical load were recorded by the camera for both experiments (see Figure 5.2).
From the resulting photographs, the packed bed compression and relaxation was cal-culated by automated image analysis using the public domain image analysis software ImageJ and Matlab. Therein, the changes in packing section height were proportional to the changes of the number of pixels representing a packing section. Using the cam-era macro objective, the resolution of each of the packing sections was high enough (>
200000 pixels) so that even small changes in the section heights were represented by a sufficiently large amount of pixels. The compression is defined as the ratio of change in section height and initial section height according to Equation (3.72). The packing measurements were repeated five times with freshly packed columns.
Flow Compression. During flow compression experiments, the flow rate was increased every 2 min by 0.1 kg h-1 up to 2 kg h-1 — being equal to a superficial flow velocity of 2763 cm h-1 for the 9.6 mm i.d. column — and decreased afterwards by the same rate. The height of the sections was measured at each flow step. This high superficial velocity beyond velocities typically recommended by the manufacturer for large-scale applications was necessary to exert large enough packed bed compression in order to obtain meaningful experimental data.
Mechanical Compression. The analysis of the macroscopic packed bed behavior dur-ing uniaxial mechanical compression was carried out by lowerdur-ing the plunger manually by 20 mm min-1 in absence of any external fluid flow application. The height of the sections was measured at every 1/4 turn of the screw thread by which the plunger was lowered. Columns packed with wet as well as semi-dry particles were investigated. In the former packing process, the particles were first gravity settled from resin slurry and afterwards compressed mechanically by lowering the plunger. The column inlet valve was then closed so that excess fluid left the column through the outlet. The particles of the packing were still surrounded by fluid. In the latter packing process, the inter-particle fluid was drained by gravity first. Then, most of the fluid contained in the
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interparticle void space was removed by a nearly stagnant flow of about one column volume of air through the column. This resulted in a semi-dry gravity settled packed bed. However, the residual moisture content of the resin particles was not quantified because this parameter was not experimentally accessible.
Microscopic Particle Measurements
The analysis of the microscopic column packing behavior was carried out using Toy-opearl AF-Amino-650M® (kindly provided by Tosoh Bioscience, Stuttgart, Germany).
Toyopearl AF-Amino 650M® is an affinity chromatography resin made of spherical methacrylic polymer. According to the vendor, over 80% of the wet particles are within a range of 40-90 µm with a mean diameter of 65 µm.
Fluorescent Particle Labeling. Toyopearl AF-Amino-650M and Toyopearl SP-650C particles were labeled with fluorescent dyes according to the suppliers labeling protocol [Johnson and Spence 2010] in order to increase contrast and visibility during fluores-cence microscopy measurements. As fluorescent dyes O-10465, O-6185, F-2181, and A-30052 (see Appendix Table A.3) were used. Depending on the reactive group of the chromatographic particles and the dye molecules, slightly different labeling protocols were developed. Detailed information about the labeling process is given in Appendix Chapter C.1.
Confocal Laser Scanning Microscopy (CSLM). Confocal laser scanning microscopy (CSLM) (LSM510 META, Carl Zeiss, MicroImaging GmbH, Jena, Germany) was used to analyze the packing structure, i.e. the particle deformation and rearrangement during mechanical and hydrodynamic load at the particle scale. To use the limited space below the microscopic lens, a microfluidic flow cell was designed (see Appendix Figure B.2) and packed with dyed Toyopearl AF-Amino 650M particles. This aminated resin was optically denser than the resin SEP and therefore more suitable for microscopic analysis.
The microfluidic device was further equipped with a water pool in which the objective lenses needed to be submerged duing measurements to reduce optical refraction of the laser beam. Measurements were carried out using an Argon laser at 488 nm extinction wavelength. The particles were labeled with fluorescent A-30052 and F2182 dyes (see Appendix Table A.3 and Appendix Chapter C.1 for details).
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UV-Light Microscopy. Fluorescent particles were mixed with the unlabeled ones at a ratio of 1:5 (v/v) and packed into the column. The column was then positioned hori-zontally on the stage of an optical microscope type Axioplan equipped with a 20x/0.45 objective (Carl Zeiss, Göttingen, Germany) (see Figure 5.4).
By applying fluorescence microscopy (see Figure 5.4 B), the emitted light of the la-beled beads illuminated the packing from inside and made several layers of the packing structure visible. The pressure-dependent packing structure was then photographed and post-processed using the software AxioVision version 4.7.2 (Carl Zeiss, Göttingen, Germany) and ImageJ.
Micro Computer Tomography. Measurements of the inner particle packing structure were carried out using micro computer tomography (Xradia Versa XRM-500, Carl Zeiss, Oberkochen, Germany) at different energy levels (40-60 keV) at the TUM Institute of Medical Engineering (IMETUM). The X-ray attenuation can be calculated according to Lambert Beer’s law
IA
I0
= 1−e(−µρx), (5.2)
where IA and I0 are the absorbed and incident photon intensities, (µ/ρ) is the mass attenuation coefficient (units cm2 g-1) andx is the mass thickness of the probe (x=ρD in g cm-2 and D the diameter of the probe). From Equation (5.2) it gets clear that the fundamental basis of the attenuation is the number of atoms encountered by the X-ray beam. The mass attenuation coefficient(µ/ρ)is a material property and a strong function of the atomic number Z as well as the X-ray wavelength λ (inverse of energy) that can be described by the relationshipµ/ρ ∼Zmλn wheremequals three or four and n equals (approximately) three [Stock 2009, pp. 9-21].
Chromatographic packings of agrose- and methacrylate-based particles were prepared in small columns made of a 1 ml B Braun Omnifix® Solo polypropylene syringe (Carl Roth GmbH, Karlsruhe, Germany) with inner diameter of 4.6 mm. The packing height was adjusted to 5 mm and not compressed prior to measurements. The following samples were analyzed:
• Dry Toyopearl SuperQ-650C particles; preparation: Washing particles in pure water and incubation in 0.1 M calcium cloride solition for 1 h, drying overnight at 60 ℃ in a drying oven (see Appendix Tables A.5 and A.2).
• Wet Sepharose 6FF and Toyopearl SuperQ-650C particles; preparation: Washing
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Microscope
stage Micro column
Outlet
Inlet
Holding
Packed column Objective
lenses
A
B
Figure 5.4.: UVM measurements of the packing behavior. (A) CAD drawing of the setup of the micro column fixed on the microscope stage. (B) View of the column during operation and illumination of the packing by UV light.
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particles in pure water and incubation in the following contrast agent solutions (see Appendix Table A.2): 0.05 m% iodine in ethanol solution, 1 m% iodine in ethanol solution, 5 m% iodine in ethanol solution, 0.1 m% PTA solution, 10 m%
PTA solution, pure Imeron400 solution and 10 m% Imeron400 solution