Operation Manual Encapsulator B-395 Pro 11593484 en
Table of contents Table of contents 1 2 3 4 5 About this manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2.1 User qualification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2.2 Proper use . . . . . . . . . .
Table of contents 6 7 8 9 10 5.5.3 Installation of the pressure bottle . . . . . . . . . . . . . . . . . . . . . . . . . . . .32 5.6 Option: Concentric nozzle system . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 5.6.1 Mounting of CN nozzles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 5.7 All parts of the Encapsulator B-395 Pro . . . . . . . . . . . . . . . . . . .
1 1 About this manual About this manual This manual describes the Encapsulator B-395 Pro. It provides all information required for its safe operation and to maintain it in good working order. It is addressed to laboratory personnel in particular. If the instrument is used in a manner not specified in this manual, the protection provided by the instrument may be impaired.
2 2 Safety Safety This chapter points out the safety concept of the instrument and contains general rules of behavior and warnings from direct and indirect hazards concerning the use of the product. For the user's safety all safety instructions and the safety messages in the individual chapters shall strictly be observed and followed. Therefore, the manual must always be available to all persons performing the tasks described herein. 2.
2 2.4 Safety Safety warnings and safety signals used in this manual DANGER, WARNING, CAUTION and NOTICE are standardized signal words for identifying risk levels, related to personal injury and property damage. All signal words, which are related to personal injury are accompanied by the general safety sign.
2 Safety Explosive gases, explosive environment Harmful to live-forms Device damage Pressurized gas/air Wear laboratory coat Wear protective goggles Wear protective gloves Additional user information Paragraphs starting with NOTE transport helpful information for working with the device/software or its supplementaries. NOTEs are not related to any kind of hazard or damage (see example below). NOTE Useful tips for the easy operation of the instrument/software.
2 2.5 Safety Product safety Safety warnings in this manual (as described in section 2.4) serve to make the user alert and to avoid hazardous situations emanating from residual dangers by giving appropriate counter measures. However, risks to users, property and the environment can arise when the instrument is damaged, used carelessly or improperly. 2.5.1 General hazards The following safety messages show hazards of general kind which may occur when handling the instrument.
2 Safety Notice Risk of instrument damage by wrong mains supply. • • External mains supply must meet the voltage given on the type plate. Check for sufficient grounding. Notice Risk of damaging labratory glasses or utensils by moving syringe pump unit. • 2.5.2 Do not place any laboratory glasses or other utensils on the Encapsulator. Safety measures Always wear personal protective equipment such as protective eye goggles, protective clothing, and gloves when working with the instrument. 2.5.
2 2.6 Safety General safety rules Responsibility of the operator The head of laboratory is responsible for training his personnel. The operator shall inform the manufacturer without delay of any safety-related incidents which might occur during operation of the instrument. Legal regulations, such as local, state and federal laws applying to the instrument must be strictly followed.
3 3 Technical data Technical data This chapter introduces the reader to the instrument and its specifications. It contains the scope of delivery, technical data, requirements and performance data. 3.1 Scope of application and delivery The Encapsulator B-395 Pro is available • for sterile working conditions in a closed reaction vessel • with one integrated syringe pump. The scope of delivery can only be checked according to the individual delivery note and the listed order numbers.
3 3.1.2 Technical data Standard accessories Table 3-2: Standard accessories Product Order no. Reaction vessel 11057890 Reaction vessel with GMP documentation 11057879 Completely autoclavable reactor made of glass and stainless steel for the sterile production and collection of microcapsules, 2 litre working volume Set of 8 single nozzles 11057918 Set of 8 single nozzles with high precision opening of 0.08, 0.12, 0.15, 0.20, 0.30, 0.45, 0.75 and 1.
3 3.1.3 Technical data Optional accessories Table 3-3: Optional accessories Product Order no. Concentric nozzle set 11058051 Set of 7 external nozzles with high precision opening of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 and 0.9 mm made of stainless steel, incl. 1000 mL pressure bottle 3.1.4 Recommended spare parts Table 3-4: Recommended spare parts 14 Product Order no.
3 3.2 Technical data Technical data Table 3-5: Technical data Encapsulator B-395 Pro Power consumption max. 150 W Connection voltage 100–240 VAC Mains supply voltage fluctuations up to ±10% of the nominal voltage Frequency 50/60 Hz Fuse 3.15 A Dimensions (W×H×D) 32×38×48 cm Weight 11 kg Nozzle diameter of single (= core) nozzles 0.08, 0.12, 0.15, 0.20, 0.30, 0.45, 0.75 and 1.00 mm Nozzle diameter of shell nozzles 0.20, 0.30, 0.40, 0.50, 0.60, 0.70 and 0.90 mm Droplet size range 0.
4 4 Description of function Description of function This chapter explains the basic working principle of the Encapsulator B-395 Pro. It also shows how the instrument is structured and provides a general functional description of its assembly. 4.1 Functional principle The instrument provides the following key functions: Sterile working conditions in a closed reaction vessel –– Sterile containment in an autoclavable reaction vessel.
4 14 Description of function 13 2 11 1 6 5 4 12 7 8 3 10 9 15 17 16 18 19 Figure 4-1: Schematic representation of the Encapsulator B-395 Pro a Syringe pump Syringe Pressure bottle Air Pressure control Pulsation chamber Vibration system Nozzle Electrode Reaction vessel Bypass cup 17 Liquid filter Air filter Electrostatic charge generator Frequency generator Stroboscope lamp Filtration grid Bead collecting flask Magnetic stirrer Waste port B-395 Pro Operat
4 Description of function The main parts of the Encapsulator B-395 Pro are the control unit, with the syringe pump, the electrical and pneumatic systems, and the reaction vessel. All parts of the instrument which are in direct contact with the beads can be sterilized by autoclaving.
5 5 Putting into operation Putting into operation This chapter describes how the instrument has to be installed. It also gives instructions for the initial startup. NOTE Inspect the instrument for damages during unpacking. If necessary, prepare a status report immediately to inform the postal company, railway company or transportation company. Keep the original packaging for future transportation. 5.1 Installation site Put the instrument on a stable, horizontal surface.
5 5.2 Putting into operation Installing the Encapsulator B-395 Pro Place the instrument on the lab bench with convenient access to an AC electrical outlet and to compressed air. Place the instrument in a way that disconnection of the electric supply plug is possible at all times. Installation of the magnetic stirrer, vibration unit and grounding wire Connect the magnetic stirrer, the vibration unit and the grounding wire as shown in figure 5-1 and 5-2. a Air inlet (blue tube 2.6×4.
5 Putting into operation Installation of the air line A 3 m air tube (2.6×4.0 mm) is included with each Encapsulator to connect it to external compressed air or nitrogen. 1. Stick the air tube into the air inlet plug. 2. Attach the other side of the air tube to the external gas supply. 3. Deliver gas to the Encapsulator at 1.5 to 2 bar (23 to 30 psi) when running the instrument. NOTE The integrated pneumatic system (valve and fittings) will tolerate up to 7 bar (100 psi) at the inlet.
5 5.
5 5.4.1 Putting into operation Cover plate The cover plate is delivered with all pieces in place. Before use, wash the cover plate carefully. After each run, disassemble the bead producing unit and the nozzle. Wash with water or appropriate detergent or solvent (according to the nature of the immobilization mixture used), rinse with water and let dry. Be careful not to damage the PTFE membrane while handling the bead producing unit. The other parts should be disassembled only as needed.
5 Putting into operation 5.4.1.1 Bead producing unit and nozzles 1 2 3 4 5 6 a Magnet Holder * b O-Ring (14×1.78) c Pulsation chamber d Pre-filter with 50 µm mesh e O-Ring (3.68×1.78) f Luer Lock g Screw M3×8 h Screw M3×25 i Screw M3×6 *with attached fixation ring and screws M3×5. You can remove the fixation ring for cleaning.
5 Putting into operation 5.4.1.2 Electrode 1 5 a Screw nut M10 (polyamide) b O-Ring (10.82×1.78) c Insulator d Electrode e Connecting piece f Screw M4×12 2 3 4 6 Figure 5-8: Electrode with connecting elements The electrode is attached with either the elongated ring showing downwards or upwards so that the distance between the nozzle and the electrode can be varied.
5 Putting into operation 5.4.1.4 Air filter a HEPA Air filter b O-Ring 3.68×1.78 c Nipple d Silicon tube 5×8 mm 1 2 3 4 Figure 5-10: Airfilter - exploded view (left), assembled view (right) 5.4.1.5 Liquid filter 1 a Liquid filter (e.g. Sartobran 150) Liquid inlet Silicon tube (3×5) 35 mm long Screw M3×12 O-Ring (10.82×1.
5 5.4.2 Putting into operation Base plate The base plate serves a dual function with two evacuation ports. One has a filter to retain the beads in the reaction vessel while exchanging encapsulation reagents and the other is a drain valve to harvest the beads without compromising sterility.
5 Putting into operation 5.4.2.1 Bead drain valve 4 3 5 a Plunger b O-Ring (5×1) c Knob for valve d Screw M3×6 e Screw M3×20 f Harvesting valve (BT 14) g O-Ring (18.77×1.78) 6 2 1 7 Figure 5-13: Parts of the bead drain valve 5.4.2.2 Liquid drain system 1 a Liquid drain plate b O-Ring (34.65×1.78) c Screw M3×6 d Filter grid, diameter 35 mm, 100 µm mesh 2 3 4 Figure 5-14: Parts of the liquid drain system NOTE The filter grid shrinks 1 % to 2 % during the first autoclaving.
5 5.4.3 Putting into operation Bead collecting flask After finishing bead production and bead processing, the beads are directly transferred through the bead harvesting valve into the bead collecting flask. The beads can then be transported aseptically to any other container. Figure 5-15 shows the disassembled and assembled bead collecting flask. 1 3 2 5 6 4 7 Figure 5-15: Bead collecting flask for sterile havesting and transportation of the produced beads and capsules.
5 5.5 Putting into operation Pumping systems The Encapsulator B-395 Pro provides two systems for pumping the immobilization mixture: • by volumetric syringe pump • by air pressure from the pressure bottles The syringe pump is mainly used: 1. For small volumes (< 60 mL). 2. Where the liquid flow rate has to be controlled very accurately on every run. 3. When a very low dead volume is needed (approximately 0.5 mL). Pumping with air pressure is recommended: 1. When large volumes (> 60 mL) are needed. 2.
5 5.5.2 Putting into operation Pressure bottle The pressure bottle is an autoclavable container used to pump the immobilization mixture by air pressure. Figure 5-17 shows the different parts of the pressure bottle.
5 5.5.3 Putting into operation Installation of the pressure bottle 1. Assemble and – if needed - autoclave the pressure bottle. 2. Fill the bottle with the immobilization mixture. 3. Attach the silicone tube of the pressure bottle to the luer lock inlet of the bead producing unit. 4. Pass the silicone tube in the liquid regulating flow valve. Squeeze it so that no liquid can pass. 5. Insert the nipple of the air tube into the quick coupling of the air outlet at the control unit.
5 5.6 Putting into operation Option: Concentric nozzle system The concentric nozzle system (CN system) is an optional kit to the single nozzle unit. It is for the production of capsules in a one-step procedure. The system consists of CN bead producing unit, a set of 7 shell nozzles (0.20, 0.30, 0.40, 0.50, 0.60, 0.70 and 0.90 mm) and one pressure bottle of 1000 mL. The shell liquid is pumped by air pressure using the pressure bottle.
5 Putting into operation Figure 5-21: CN bead producing unit with set of 7 shell nozzles. The following nozzle apertures are standard: 0.20, 0.30, 0.4, 0.50, 0.60, 0.70 and 0.90 mm. 1 2 4 3 7 8 5 6 9 10 Figure 5-22: Single parts of the CN bead producing unit a Screw M3×6 b Luer lock female c Pre-filter grid 50 µm mesh, D= 7 mm d O-Ring 14.0×1.78 e Screw M3×8 34 f Screw M3×25 g O-Ring 3.68×1.78 h CN pulsation body i O-Ring 12.42×1.
5 5.6.1 Putting into operation Mounting of CN nozzles 1 2 Put the O-ring 12.42×1.78 in the grove of the CN bead producing unit. Put the inner nozzle (with attached O-ring) into the hole of the CN bead producing unit. There is no thread. The inner nozzle is centered and fixed by the shell nozzle. Exit of the shell liquid O-ring 12.42×1.78 Figure 5-23: Mounting of the inner nozzle Put carefully the shell nozzle over the inner nozzle. Attach the shell nozzle with two screws (M3×6).
5 5.7 Putting into operation All parts of the Encapsulator B-395 Pro Figure 5-26: Picture of all parts of the Encapsulator B-395 Pro 5.8 Final installation check This check has to be carried out after every installation and prior to the first encapsulation process. All connected supply media (e.g. mains voltage and gas pressure) must match the technical data of the installed system or system set-up. • • 36 Inspect all glass components for damage.
6 6 Operation Operation This chapter gives examples of typical instrument applications and instructions on how to operate the instrument properly and safely. See also section 2.5 “Product safety” for general warnings. 6.1 Starting up the instrument • • • 6.2 Make sure the Encapsulator B-395 Pro is properly connected to the mains supply. Carry out a final installation check (see section 5.8) before every bead production. Switch on the Encapsulator B-395 Pro. The system runs an internal check.
6 6.3 Operation Menu structure of the control unit The figure below shows a schematic overview of all menus of the Encapsulator B-395 Pro, each with the available functionality. Upper Screen - main menu Frequency On/Off Electrode On/Off Store function Set Freq. | Set Elect.
6 6.4 Operation Menu functions of the upper touch screen Vibration (frequency and amplitude), electrostatic dispersion (voltage), and light intensity of the stroboscope lamp are controlled on the upper touch screen. When the Encapsulator is switched on, the touch screen runs an initialization program for few seconds. Then the screen shows the start menu (Screen 6.3) with three sub-parts (see screen 6-4 to 6-6) for frequency, electrode, and more options concerning frequency and light intensity.
6 Operation The light intensity of the stroboscope lamp and amplitude ( = intensity) of the vibration can be set from 1 to 9. Above a frequency of 1500 Hz the amplitude can be set from 1 to 12. By increasing the amplitude the vibration becomes stronger. Values above 3 are mainly for solutions with viscosity > 100 mPa s. Pressing on the (+) and (-) buttons will immediately change the parameters. Pressing the “Esc”-button will cause a return to the start menu and the set value will be kept.
6 6.5 Operation Menu functions of the lower touch screen Syringe pump (pump speed and calibration) and magnetic stirrer are controlled on the lower touch screen. When the Encapsulator is switched on, the touch screen runs an initialization program for few seconds. Then the screen shows the start menu with two sub-parts (see figure 6-7 to 6-10). 1 5 2 6 3 4 a On/off switch for magnetic stirrer control. b Button for passing to screen 6-9 for setting magnetic stirrer speed .
6 Operation Pressing on the (+) and (–) buttons will change the pumping rate. Pressing “Esc” will return you to the start menu and the set value will be kept. Pressing the “cal mL/min” button, while the pump is running, will open screen 6-11 and allows you to calibrate the current syringe. If the pump is stopped, the screen will ask you to select a calibrated syringe. Screen 6-10: Speed regulation of the syringe pump 6.5.
6 Operation Pressing on the (+) and (–) buttons for entering the liquid pumped for 1 min. Then press button “store”. You are forwarded to screen 6-14. Screen 6-13: Syringe pump calibration - setting the pumped liquid Press “Yes” to store the values. You are forwarded to the start menu. The syringe pump will now run with the new calibration after pressing the “on” button of the pump control. Screen 6-14: Syringe pump calibration - store value 6.5.2 Selecting a calibrated syringe Stop the pump.
6 6.6 Operation Manual air pressure control In the control unit the pressure is manually controlled by the pressure regulating valve, integrated in the front panel of the control unit (see figure 6-2). Set the air pressure at a value which is 0.2 to 0.3 bar higher then the maximal air pressure needed during the encapsulation procedure; but not higher then 1 bar. Turning the knob of the pressure regulating valve clockwise increases the pressure, counterclockwise decreases the pressure.
6 6.7 Operation Handling the syringe pump When the syringe pump is used the first time after switching on the control unit, press the “home” button on the lower touch screen to move the pump arm back. Let the arm move completely back until it touches the end knob microswitch (see figure 6-3) where it stops itself. In this way, the computer of the control unit recognizes the exact position of the syringe arm.
6 6.8 Operation Practicing with the Encapsulator, using water Before working with encapsulation polymers, use water for practicing with the Encapsulator to become familiar with the effects of the controls. Take the cover plate of the reaction vessel, attach the bead producing unit and a 200 µm or 300 µm nozzle to it. Place the assembled cover plate on the control unit. Attach it with the two thumb screws. Put the vibration unit on the bead producing unit. Place a large beaker (approx.
6 Operation Table 6-1: Determination of the working field (with syringe pump) Nozzle size: Pumping Speed mL/min Syringe size: Clear Bead Chain without Electrostatic Tension Clear Bead Chain with Electrostatic Tension Lowest Frequency Electrostatic Tension Nozzle size: Pumping Speed mL/min 47 Highest Frequency Lowest Frequency Highest Frequency Syringe size: Clear Bead Chain without Electrostatic Tension Clear Bead Chain with Electrostatic Tension Lowest Frequency Electrostatic Tension Hi
6 Operation 4. Set the pumping speed and the vibration frequency to the values you determined that creates a good, clear bead chain. Activate the electrostatic dispersion unit at 300 V and increase the voltage by steps of 100 V until the one-dimensional bead chain is transformed into a funnel-like, multi-line stream. The higher the electrostatic charge, the earlier the bead chain will separate.
6 6.8.2 Operation Using the pressure bottle 1. Assemble the bead producing unit, screw the 0.30 mm single nozzle to the bead producing unit and attach all on the cover plate with the screw (M3×25). Place the vibration unit on the bead producing unit. Connect the electrode with the red wire to the electrostatic dispersion unit (EDU). 2. Fill the pressure bottle with 200 to 300 mL distilled water and screw on the assembled cap.
6 Operation Table 6-2: Determination of the working field (with pressure bottle) Nozzle size: Air pressure Clear bead chain without electrostatic tension Clear bead chain with electrostatic tension Lowest frequency Electrostatic Voltage Highest frequency Lowest frequency Highest frequency Nozzle size: Air pressure 50 Clear bead chain without electrostatic tension Clear bead chain with electrostatic tension Lowest frequency Electrostatic Voltage Highest frequency Lowest frequency Highest fr
6 Operation 7. Set the liquid flow rate and the vibration frequency to a value where a clear bead chain is obtained. Activate the electrostatic dispersion unit at 300 V and increase the tension stepwise by 100 V until the one-dimensional liquid jet is transformed into a funnel-like multi-line stream. The higher the electrostatic charge the earlier the bead chain is separated. This prevents the beads from hitting each other in flight, and from hitting each other as they enter the hardening solution.
6 6.9 Operation Practicing with the Encapsulator, using non-sterile alginate solution After getting comfortable with the bead formation controls, perform test runs with non-sterile alginate solutions. Sodium alginate is the most commonly used polymer, but there are others in use with varying properties. We recommend the low viscosity grade alginate. The alginate concentration strongly influences the viscosity and this in turn influences the pressure drop in the nozzle.
6 6.9.2 Operation Working with the syringe pump 1. Attach a 200 µm or 300 µm nozzle to the bead producing unit. Place the assembled cover plate on the control unit. Attach it with the two thumb screws. Put the vibration unit on the bead producing unit. Connect the electrode with the red wire to the electrostatic dispersion unit (EDU). Put the magnetic stirrer below the nozzle and a large beaker on the stirrer. Fill the beaker with 100 mM CaCl2 so that at least 2 cm (approx.
6 Operation Table 6-4: Encapsulator trial test work sheet (syringe pump) Syringe size [mL] Nozzle size [µm] Alginate concentr. [%] Pumping speed [mL/min] Vibration frequency [Hz] Amplitude Approximate bead size [µm] Homogeneity [%] Comments 6. Inspect the beads under a microscope with a micrometer scale eyepiece and record your observations of diameter, uniformity and shape in table 6-4. 7. Repeat this procedure for each change in process parameters.
6 Operation Table 6-5: Determination of the working field Nozzle size: Alginate concentration: Syringe size: Pumping speed [mL/min] Clear bead chain without electrostatic tension Clear bead chain with electrostatic tension Lowest frequency Electrostatic tension Highest frequency Lowest frequency Nozzle size: Alginate concentration: Syringe size: Pumping speed [mL/min] Clear bead chain without electrostatic tension Clear bead chain with electrostatic tension Lowest frequency Electrostatic
6 6.9.3 Operation Working with the pressure bottle 1. Attach a 200 µm or 300 µm nozzle to the bead producing unit. Place the assembled cover plate on the control unit. Attach it with the two thumb screws. Put the vibration unit on the bead producing unit. Connect the electrode with the red wire to the electrostatic dispersion unit (EDU). Put the magnetic stirrer below the nozzle and a large beaker on the stirrer. Fill the beaker with 100 mM CaCl2 so that at least 2 cm (approx.
6 Operation NOTE The stronger the circular dispersal of the bead stream the better is the bead homogeneity. This does not only depend on the electrostatic tension, but the liquid flow rate and the vibration frequency are also factors. Ideally, the bead should separate from the liquid jet within the electrostatic field between the nozzle and the end of the electrode. 8.
6 6.10 Operation Practicing with the Encapsulator, working with the complete reaction vessel After getting comfortable with Ca-alginate bead formation, perform test runs with the complete reaction vessel to simulate sterile working conditions, but by using non-sterile alginate solution.
6 Operation 6. As soon as the bead chain is stable, move the collection cup out of the way of the liquid jet to start the actual bead production process. The bead stream should disperse 3 to 10 cm (approximately 1” to 4”) below the electrode. An optimal distance is about 5 cm (2”) below the electrode. To achieve this goal you will need to adjust the electrostatic tension and possibly slightly fine tune the vibration frequency and pumping speed. 7. Note and record the exact process parameters.
6 6.11 Operation Heat sterilization of the reaction vessel 1. Prepare the Reaction Vessel according to section 5.4. 2. Add 2 to 5 mL of water into the reaction vessel. 3.
6 6.13 Operation Encapsulation procedure for immobilization of micro-organisms in Ca-alginate beads In this section, a simple but well established method is described for the immobilization of microorganisms in Ca-alginate beads. The stability of these beads depends not only on the alginate type used, but on the future culture conditions. The Ca ions in the hardening solution substitute for the Na ions in the droplets causing the alginate beads to harden (this is a reversible reaction).
6 Operation 8. As soon as the bead chain is stable, move the collection cup out of the way of the liquid jet to start the actual bead production process. Verify that the bead stream is dispersed 3 to 10 cm (approximately 1” to 4”) below the electrode. An optimal distance is about 5 cm (2”) below the electrode. Adjust the electrostatic voltage to achieve this goal. 9. Record the exact process parameters while monitoring the bead production. 10.
6 6.14 Operation Encapsulation protocol for alginate-PLL-alginate membranes The Alginate-Polylysine-Alginate-membrane is a well established encapsulation system for animal cells first described by Lim and Sun1.Below is a well tried protocol. Required solutions 1. 1.5 % Alginate solution: 1.5 % low viscositiy alginate in MOPS washing-buffer adjust pH to 7.0 at 25°C sterile filtration through a 0.2 µm filter 2. Polymerization solution: 10 mM MOPS (Morpholinopropanesulfonic acid) 100 mM CaCl2 pH = 7.
6 Operation Procedure 1. Prepare the reaction vessel and autoclave it as described in section 6.10 and 6.11. 2. Prepare all solutions and labware. 3. Fill the autoclaved reaction vessel with 225 mL polymerization solution. 4. A cell culture with ca. 6×106 cells (or according to personal need) is centrifuged and the pellet is re-suspended in 2 mL sterile MOPS washing buffer and mixed with 10 mL 1.5 % sodium-alginate solution. Give care, that no or only few air bubbles are introduced during mixing. 5.
6 Operation Special recommendations for cells For dividing cells - dissolve the core alginate, then maintain Na/Ca-ratio >20:1 in the culture medium so that the core will not re-solidify. For resting cells – you can maintain the alginate core structure gelated and you can use even Ba2+, a stronger gelating ion than Ca2+. Ba-alginate is extremely stable and withstands dissolution by 50 mM citrate-solution for days. Also see: Gröhn P. et al. 1994.
6 6.15 Operation Theoretical background Equ. 1: When a laminar jet is mechanically disturbed at the frequency ƒ, beads of uniform size are formed1. The optimal wavelength λopt for breakup, according to Weber2 is given by: Equ. 2: where: D = nozzle diameter η = dynamic viscosity [Pa s] ρ = density [kg/m3] (ca. 1000 kg/m3 for alginate solutions) σ = surface tension [N/m] (ca.
6 Operation Figure 6-5: Influence of the liquid jet velocity and the nozzle diameter on flow rate, as calculated by Equation 4. Figure 6-6 shows the correlation between the vibration frequency and the bead diameter for five different flow rates as calculated by equation 4. Lower flow rates, which corresponds to lower pumping rates, produce smaller beads. Higher vibration frequencies produce smaller beads also.
7 Maintenance and repairs Table 6-7: Optimal working conditions for the Encapsulator determined with alginate solution Nozzle diameter [µm] Flow rate * [mL/min] Frequency interval ** Amplitude Air pressure [bar] 1.0 mm 30 to 40 40 to 220 Hz 2 to 6 0.3 to 0.6 750 µm 19 to 25 40 to 300 Hz 2 to 5 0.3 to 0.5 450 µm 9 to 14 150 to 450 Hz 2 to 5 0.3 to 0.5 300 µm 5.5 to 7 400 to 800 Hz 1 to 3 0.3 to 0.5 200 µm 3.5 to 4.5 600 to 1200 Hz 1 to 3 0.4 to 0.6 150 µm 2.3 to 2.
6 Operation Figure 6-7: Amount of beads with a diameter of 0.3 to 0.6 mm formed from 1 mL of immobilization mixture. Figure 6-8: Amount of beads with a diameter of 0.6 to 1.1 mm formed from 1 mL of immobilization mixture.
6 Operation Figure 6-9: Amount of cells per bead made from different cell concentrations for bead diameters of 0.3 to 0.6 mm. Figure 6-10: Amount of cells per bead made from different cell concentrations for bead diameters of 0.6 to 1.1 mm.
7 7 Maintenance and repairs Maintenance and repairs This chapter gives instructions on maintenance work to be performed in order to keep the instrument in good and safe working condition. All maintenance and repair work requiring the opening or removal of the instrument housing must be carried out by trained personnel and only with the tools provided for this purpose.
7 7.4 Maintenance and repairs Cleaning ! Warning Pressure increasement in the inlet-system due to clogged nozzles. Bursting of the inlet system. Death or serious poisoning by contact or incorporation of harmful substances at use. • Clean nozzle immediately after use, see following section. Wear laboratory coat Wear protective goggles Wear protective gloves 7.4.
7 Maintenance and repairs Figure 7-1: Cleaning procedure of the nozzle –– –– –– –– Take a syringe containing air on top and water on the bottom. Push the air through the nozzle (left figure). Push the water through the nozzle immediately afterwards (right figure). Examine the nozzle tip under a stereoscopic microscope to make sure the passage is clear and clean. NOTE If lipophilic immobilization solutions were used, then use appropriate solvents for cleaning.
8 8 8.1 Troubleshooting Troubleshooting Malfunctions and their remedy The table below lists possible operating errors and their cause. As remedy set the parameter stepwise in the opposite direction or fix the missing part. Table 8-1: Possible cause Problem Possible cause Unstable liquid stream The liquid flow rate is too low. The nozzle is not adequately cleaned (frequent cause). The frequency is too high. The amplitude is too high. Unstable bead chain The frequency is too high or too low.
9 9 Shutdown, storage, transport and disposal Shutdown, storage, transport and disposal This chapter instructs how to shut down and to pack the instrument for storage or transport. Specifications for storage and shipping conditions can also be found listed here. 9.1 Storage and transport Switch off the instrument and remove the power cord. To disassemble the Encapsulator B-395 Pro follow the installation instructions in section 5 in reverse order.
9 9.2 Shutdown, storage, transport and disposal Disposal For instrument disposal in an environmentally friendly manner, a list of materials is given in chapter 3.3. This helps to ensure that the components can be separated and recycled correctly. You have to follow valid regional and local laws concerning disposal.
10 10 10.1 Declarations and requirements Declarations and requirements FCC requirements (for USA and Canada) English: This equipment has been tested and found to comply with the limits for a Class A digital device, pursuant to both Part 15 of the FCC Rules and the radio interference regulations of the Canadian Department of Communications. These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment.
10 10.2 Declarations and requirements Health and Safety Clearance Health and Safety Clearance Declaration concerning safety, potential hazards and safe disposal of waste. For the safety and health of our staff, laws and regulations regarding the handling of dangerous goods, occupational health and safety regulations, safety at work laws and regulations regarding safe disposal of waste, e.g.
10 10.
BÜCHI Labortechnik AG CH-9230 Flawil 1/Switzerland T +41 71 394 63 63 F +41 71 394 65 65 www.buchi.
