Akimbekov N.Sh.¹, Heras C.O.², Digel I.E.², Zhubanova A.A.¹

¹Al-Farabi Kazakh National University, Kazakhstan

²Aachen University of Applied Sciences, Germany

Flow-through column design for elimination biological liquids

Column-based experiments are of great importance since they probably represent the most convenient and efficient way of future medical application of the carbonized materials.

The activated carbon (AC) on the basis of rice shells was used during the experiment. The sample was carbonized according to the procedure developed at the Laboratory of Hybrid Technologies in the Institute of Combustion Problems, Almaty, Kazakhstan and all experiments were done in Biomedical Engineering laboratories of Aachen University of Applied Sciences, Julich, Germany.

Flow-through column experiments are intended to provide a more realistic simulation of dynamic conditions and to quantify the movement of the desired materials relative to the packed column. The basic experiment is completed by passing a liquid (hemoglobin, BSA (bovine serum albumin) and LPS (lipopolysaccharide) with certain concentration of the material of interest through a column packed with activated carbon. The design of the experiment was complicated due to the intrinsic characteristics of the Activated carbon. Its fragility during handling made it difficult to pack into the column, and different attempts were done, however, it was necessary to avoid any kind of air bubble interface in the packed column, as it would again interfere with the procedure. This was rather difficult to archive, the first attempts were full of air bubbles, as the column used was open in the middle in an attempt to introduce the material without having to crush it, or damage it unnecessarily, as it was stated before, if the material was crushed, dust contents are liberated and the overall performance of the experiment is reduced, furthermore, the experiment can’t be repeated in the same conditions.

For the column design a Serologische Rotilabo^®- Einmalspritzen (50 mL) from Carl Roth GmbH, the design was consistent with the amount of activated carbon planned for this experiment. The only disadvantage is the fragility of the syringe, as it can be easily broken. Also the diameter from the inlet was 3.5 mm, since the medium size of activated carbon particle is 1 mm x 4 mm, the first attempt was done cutting the syringe by a half, the inner diameter of 2 centimeters allowed an easier filling of the syringe, however air bubbles were introduced between the activated carbon and the walls of the syringe, moreover, some liquid was drop once both sides were sealed together.

The amount of activated carbon per column had some variations from column to column, since the process was at some point random filling, each of the particles occupied a site in the column, and the form of the activated carbon is not regular.

The amounts of carbon variation was low with a standard deviation of 0.06 g and an average of 10,9 g.

Figure 1. Column experiment setup

The syringe volume was 30 см³, with a standard deviation of 1 mL, again, the volume of liquid obtained after each experiment was variable because it depends on the amount of activated carbon introduced into the column. The values were obtained after the experiment was done. In order to obtain the adsorbed amount, the column was opened and the solution was decanted overnight with its amount measured afterwards. As the activated carbon possesses a huge surface area and it’s pore network is capable of trapping water molecules, to measure the weight it was necessary to dry the Activated Carbon afterwards. The columns were cut by half and the AC taken out. They were dried for 3 hours at 255 °C. Since the activated carbon was not going to be used again for this experimental setup, the temperature and time was not an important variable since the reactivation of the carbon was not part of this study.

The experimental setup for the column was done. The time points used during the hemoglobin, BSA and LPS measurements were established at one measurement each 10 min, from 0 to 240 minutes. The concentrations used for all the adsorbates correspond to the ones, since the column experiment can give some information about the dynamics of the activated carbon, Breakthrough curves are used to find those values. The column was connected to the pump and the d was replaced with PBS (pH 7.4, 280 milliosmole), 200 mL of PBS were introduced to the column with a rate of 100 mL/h, by doing that, the experiment was started knowing that there was only PBS as filling volume in the column. The stock solution was in the meantime already prepared in the pump, so once it started the solution was directly in contact to the Activated Carbon in the column.

At t=0 the pump was injecting the solution into the column. After 10 minutes the first measurement was taken, with a mean volume of 22.45 mL per column, it was expected that in 15 minutes the whole volume of the column was replaced by the stock solution and therefore the adsorption process was in motion. By taking the first value before that threshold was archived, some information could be obtained regarding the dynamics of adsorption. Theoretically the curves obtained from such experimental setup correspond to the following figure:

Figure 2. Solution exchange in the column experiment

At time t=170, the solution was changed, stock solution (with the corresponding concentration for each experiment) was changed with PBS buffer (pH 7.4, 280 milliosmole), as it’s shown in the figure, the purpose of this exchange in the solutions was to find out how was the material reducing the concentration of the solute, if it was because there was an adsorption process going on and the material was therefore removed either by either relatively irreversible physisorption or chemisorption, or it was just working as a chromatographic column, which of course would, after some time (presumably the one that takes for the contents of the solution to be exchanged completely, or 15 minutes) give us an increase or peak in the concentration on the output of the column. This would be shown after the t=170 in the graph.

Also, this approach would give information on the rate of sorption for the last part of the experiment, as again there will be a progressive dilution in the solution that would arouse a change in the shape of the curve and a negative slope. For the activated carbon to work as adsorbate there should be a concentration reduction that would appear as a decrement in plateau part of the graphic compared to the initial concentration, the rate of adsorption depends on how that reduction was observed and how big was it. This is important since a column experiment is an open circuit experiment in the sense that the solution flows through the column with a certain velocity, depending on the properties of the activated carbon is how the uptake of solute is going to take place and if it remains there once solution is changed. Otherwise there would be a point at which the out coming solution would show an increment in the concentration.

References:

1. Endotoxin removal from protein solutions. Dagmar Petsch, Friedrich Birger Anspach. s.l. : Journal of Biotechnology, 2000, Vol. 76, pp. 97–119.

2. Bacterial LPS: a mediator of inflammation. Pabst M. J., Johnston R. B. Amsterdam : Handbook of inflammation, 1989, Vol. 6.

3. Tushev, Georgi. Carbonized Materials for Lipopolysaccharides Removal. Juelich : Lab Cell-Biophysics, Lab Medical and Molecular Biology.