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Operation Manual
Encapsulator
B-395 Pro
11593484
en
Page 2
Page 3
Table of contents
Table of contents
1 About this manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
2 Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
2.1 User qualification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.2 Proper use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.3 Improper use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.4 Safety warnings and safety signals used in this manual . . . . . . . . . . . . . . . . . 7
2.5 Product safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
2.5.1 General hazards. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
2.5.2 Safety measures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.5.3 Built-in safety elements and measures . . . . . . . . . . . . . . . . . . . . . . . . 10
2.6 General safety rules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.7 Disclaimer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
3 Technical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.1 Scope of application and delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.1.1 Standard instrument. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.1.2 Standard accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
3.1.3 Optional accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.1.4 Recommended spare parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.2 Technical data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.3 Materials used. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
4 Description of function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
4.1 Functional principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
4.2 Connections at the Encapsulator B-395 Pro. . . . . . . . . . . . . . . . . . . . . . 18
5 Putting into operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
5.1 Installation site. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
5.2 Installing the Encapsulator B-395 Pro . . . . . . . . . . . . . . . . . . . . . . . . . 20
5.3 Electrical connections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
5.4 Assembling of the reaction vessel . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
5.4.1 Cover plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
5.4.2 Base plate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
5.4.3 Bead collecting flask . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
5.5 Pumping systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
5.5.1 Syringe pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
5.5.2 Pressure bottle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Read this manual carefully before installing and running your system and note the safety precautions
in chapter 2 in particular. Store the manual in the immediate vicinity of the instrument, so that it can be
consulted at any time.
No technical modifications may be made to the instrument without the prior written agreement of
BUCHI. Unauthorized modifications may affect the system safety, the EU conformity or result in accidents.
This manual is copyright. Information from it may not be reproduced, distributed, or used for competitive purposes, nor made available to third parties. The manufacture of any component with the aid of
this manual without prior written agreement is also prohibited.
The English manual is the original language version and serves as basis for all translations
into other languages.
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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 . . . . . . . . . . . . . . . . . . . . . . . . 36
5.8 Final installation check. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
6 Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
6.1 Starting up the instrument. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
6.2 Main screens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
6.3 Menu structure of the control unit . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
6.4 Menu functions of the upper touch screen . . . . . . . . . . . . . . . . . . . . . . 39
6.5 Menu functions of the lower touch screen . . . . . . . . . . . . . . . . . . . . . . . 41
6.5.1 Menu for syringe pump calibration. . . . . . . . . . . . . . . . . . . . . . . . . . . 42
6.5.2 Selecting a calibrated syringe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
6.6 Manual air pressure control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
6.7 Handling the syringe pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
6.7.1 Calibrating the syringe pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
6.7.2 Selecting a pre-calibrated syringe . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
6.8 Practicing with the Encapsulator, using water . . . . . . . . . . . . . . . . . . . . . 46
6.8.1 Using the syringe pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
6.8.2 Using the pressure bottle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
6.9 Practicing with the Encapsulator, using non-sterile alginate solution . . . . . . . . . . 52
6.9.1 Preparation of 1.5 % Na-alginate solution . . . . . . . . . . . . . . . . . . . . . . . 52
6.9.2 Working with the syringe pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
6.9.3 Working with the pressure bottle . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
6.10 Practicing with the Encapsulator, working with the complete reaction vessel . . . . . 58
6.11 Heat sterilization of the reaction vessel . . . . . . . . . . . . . . . . . . . . . . . . 60
6.12 Sterilization of the pressure bottle . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
6.13 Encapsulation procedure for immobilization of micro-organisms in Ca-alginate beads. 61
6.14 Encapsulation protocol for alginate-PLL-alginate membranes . . . . . . . . . . . . . 63
6.15 Theoretical background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
6.15.1 Bead productivity and cell density . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
7 Maintenance and repairs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
7.1 Customer service . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
7.2 Housing condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
7.3 Sealing conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
7.4 Cleaning. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
7.4.1 Cleaning the nozzle after each immobilization run . . . . . . . . . . . . . . . . . . . 72
7.4.2 Cleaning a clogged nozzle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
7.4.3 Cleaning the reaction vessel and the other vessels . . . . . . . . . . . . . . . . . . 73
8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
8.1 Malfunctions and their remedy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
9 Shutdown, storage, transport and disposal . . . . . . . . . . . . . . . . . . . . . . . . 75
9.1 Storage and transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
9.2 Disposal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
10 Declarations and requirements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
10.1 FCC requirements (for USA and Canada) . . . . . . . . . . . . . . . . . . . . . . . 77
10.2 Health and Safety Clearance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
10.3 Declaration of conformity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Table of contents
4 B-395 Pro Operation Manual, Version C
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1 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.
Abbreviations
EPDM Ethylene Propylene Dimonomer
FEP Fluorelastomer
PTFE Polytetrafluoroethylene
1 About this manual
5 B-395 Pro Operation Manual, Version C
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2 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.
2.1 User qualification
The instrument may only be used by laboratory personnel and other persons, who on account of
training and professional experience know the dangers, which can develop when operating the instrument.
Untrained personnel or persons, who are currently being trained, require careful supervision by a qualified person. The present Operation Manual serves as a basis for training.
2 Safety
2.2 Proper use
The Encapsulator B-395 Pro has been designed and built as laboratory instrument.
The Encapsulator B-395 Pro is a semi-automated instrument used for the polymer encapsulation of
chemical substances, bio-molecules, drugs, flavor & fragrances, pigments, extracts, cells and microorganisms under sterile and non-sterile conditions. The bead formation is based on the fact that a
controlled, laminar liquid jet is broken into equally sized beads, if vibrated at an optimal frequency.
The Encapsulator B-395 Pro provides just such controlled conditions to generate beads between 0.15
to 2 mm. The instrument is ideally suited to encapsulate particles < 50 µm.
If the instrument is used with potentially toxic or hazardous substances, it has to be installed inside a
closed fume hood or glove box. In such case, the complete processing and system handling has to
be performed within the ventilated box to avoid toxication and other hazardous situations to the user
and the environment.
2.3 Improper use
Applications not mentioned in section 2.2 are considered to be improper. Applications which do not
comply with the technical data (see section 3 of this manual) are also considered to be improper.
The operator bears the sole risk for any damages or hazards caused by improper use!
The following uses are expressly forbidden:
• Installation or use of the instrument in rooms, which require ex-protected instruments.
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2.4 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.
For your safety it is important to read and fully understand the below table with the different signal
words and their definitions!
Sign Signal word Definition Risk level
DANGER
Indicates a hazardous situation which, if not avoided, will result in
death or serious injury.
2 Safety
★★★★
WARNING
CAUTION
NOTICE
no
Supplementary safety information symbols may be placed in a rectangular panel on the left to the
signal word and the supplementary text (see below example).
Space for
supplementary
safety
information
symbols.
Table of supplementary safety information symbols
The below reference list incorporates all safety information symbols used in this manual and their
meaning.
Indicates a hazardous situation which, if not avoided, could result
in death or serious injury.
Indicates a hazardous situation which, if not avoided, may result
in minor or moderate injury.
Indicates possible property damage, but no
practices related to personal injury.
!
SIGNAL WORD
Supplementary text, describing the kind and level of hazard/risk seriousness.
• List of measures to avoid the herein described, hazard or hazardous situation.
• ...
• ...
(property damage only)
★★★☆
★★☆☆
★☆☆☆
Symbol Meaning
General warning
Electrical hazard
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Explosive gases, explosive environment
Harmful to live-forms
Device damage
Pressurized gas/air
2 Safety
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.
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2.5 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. The user shall observe all listed counter measures in order to achieve and maintain the
lowest possible level of hazard.
Additional warning messages can be found whenever actions and situations described in this manual
are related to situational hazards.
2 Safety
!
Warning
Death or serious injuries by use in explosive environments.
• Do not operate the instrument in explosive environments.
• Do not operate the instrument with explosive gas mixtures.
• Before operation, check all gas connections for correct installation.
• Directly withdraw released gases and gaseous substances by sufficient ventilation.
!
Warning
Pressure increasing 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 section 7.4.
!
Warning
Death or serious injuries by contact with high voltage.
• Only open the housing of the product when machine is switched off and unplugged.
Risk of instrument short-circuits and damage by liquids.
• Do not spill liquids over the instrument or parts of it.
• Wipe off any liquids instantly.
• Ensure a safe positioning of the sample vessel.
• Do not move the instrument when it is loaded with liquid.
• Keep external vibrations away from the instrument.
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Notice
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2 Safety
Risk of instrument damage by wrong mains supply.
• External mains supply must meet the voltage given on the type plate.
• Check for sufficient grounding.
Risk of damaging labratory glasses or utensils by moving syringe pump unit.
• Do not place any laboratory glasses or other utensils on the Encapsulator.
2.5.2 Safety measures
Always wear personal protective equipment such as protective eye goggles, protective clothing, and
gloves when working with the instrument.
2.5.3 Built-in safety elements and measures
High voltage and electrostatic charges
• Safety current limitation.
• Internal grounding to arrest electrostatic charges.
Notice
Notice
Air/Gas
• Over pressure safety valve (opens at 1.5 bar)
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2.6 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.
Duty of maintenance and care
The operator is responsible for the proper condition of instrument at use and that maintenance,
service and repair jobs are performed with care and on schedule by authorized personnel only.
Spare parts to be used
Use only genuine consumables and genuine spare parts for maintenance to assure good system
performance and reliability. Any modifications to the spare parts used are only allowed with the prior
written permission of the manufacturer.
Modifications
Modifications to the instrument are only permitted after prior consultation with and with the written
approval of the manufacturer. Modifications and upgrades shall only be carried out by an authorized
BUCHI technical engineer. The manufacturer will decline any claim resulting from unauthorized modifications.
2 Safety
2.7 Disclaimer
Use and marketing of any material produced with the Encapsulator are in the sole responsibility of the
operator.
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3 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.
NOTE
For additional information about the listed products, see www.buchi.com or contact your local dealer.
3 Technical data
3.1.1 Standard instrument
Table 3-1: Standard instrument
Product Order no.
Encapsulator B-395 Pro
50 – 60 Hz, 100 – 240 V
Encapsulator B-395 Pro
50 – 60 Hz, 100 – 240 V
with GMP documentation
Complete Encapsulator B-395 Pro system for sterile procedures with integrated syringe pump, magnetic stirrer and
closed reaction vessel.
11058220
11058230
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3.1.2 Standard accessories
3 Technical data
Table 3-2: Standard accessories
Product Order no.
Reaction vessel 11057890
Reaction vessel
11057879
with GMP documentation
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 1.00 mm, made of stainless steel 316L including nozzle rack
Pressure bottle 500 mL 11058190
Pressure bottle 1000 mL 11058191
Glass bottles with fittings, tubes and air
filter, working pressure up to 1.5 bar,
autoclavable
Grounding set 11058189
Operation Manual English 11593484
13 B-395 Pro Operation Manual, Version C
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3.1.3 Optional accessories
3.1.4 Recommended spare parts
3 Technical data
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
Table 3-4: Recommended spare parts
Product Order no.
O-ring set for single nozzle 11057954
O-ring set for concentric nozzle 11057955
O-ring set for reaction vessel 11057970
Pre-filters for nozzle,
diameter 7 mm (10 pcs.)
Drain filters for reaction vessel,
diameter 35 mm (10 pcs.)
11057957
11057958
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3.2 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 0.15 to 2.00 mm
Vibration frequency 40 to 6,000 Hz
Electrode tension 250 to 2,500 V
Syringe pump rate 0.01 to 50 mL/min
3 Technical data
Pump rate by air pressure 0.5 to 200 mL/min
Maximal allowed air pressure in the system 1.5 bar
Reactor gross volume 4.5 liter
Reactor working volume 2 liter
Parts in contact with medium autoclavable
Sterile working conditions full
Overvoltage category II
Pollution degree 2
Environmental conditions:
Temperature 5–40 °C for indoor use only
Altitude up to 2000 m
Max. relative humidity (curve parameter) Maximum relative humidity 80 % up to 31 °C, then
Table 3-6: Material and Approvals
Material in contact with sample stainless steel, silicone, glass, FEP, PTFE
Approvals CE, CSA
3.3 Materials used
decreasing linearly to 50 % relative humidity at 40 °C
Table 3-7: Materials used
Component Material description
Reactor Stainless steel, 3.3 boroscillate glass, FEP, PTFE
sealings: silicone, EPDM
Nozzles Stainless steel, sealings: EPDM
Pressure bottle Stainless steel, 3.3 boroscillate glass, FEP, PTFE
sealings: silicone, EPDM
15 B-395 Pro Operation Manual, Version C
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4 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.
Reproducible bead size from one production to the next
– Adjustable parameters (nozzle size, liquid flow rate and vibration freequency) determine bead
size.
4 Description of function
Reproducible bead formation
– In the range of 0.15 mm to 2.0 mm.
High bead size uniformity
– Due to the integrated Electrostatic Dispersion Unit (EDU); approximately 5 % relative standard
deviation of bead size using pure alginate.
Immediate process control
– Visual monitoring in the light of a stroboscope lamp.
High cell viability
– Bead formation technique is at low shear stress and under physiological conditions, thus
resulting in high cell survival.
Batch size
– When using syringes the batch size is of 2 mL to 60 mL and the dead volume is approximately
0.5 mL.
dead volume is approximately 2 mL.
Set of 8 single nozzles
– The 8 nozzle sizes of 0.08, 0.12, 0.15, 0.20, 0.30, 0.45, 0.75 and 1.0 mm cover the bead size
range of approximately 0.15 mm to 2.0 mm.
Delivery of the polymere mixture
– By the integrated syringe pump or by air pressure with flow rates from 70 mL/h (0.08 mm
nozzle) to 2’500 mL/h (1.0 mm nozzle).
When using air pressure for pumping the batch size is of 5 mL to 1’000 mL and the
High bead production
– Up to 6000 beads are produced per second depending on encapsulation conditions and
polymer mixture.
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4 Description of function
14
13
2
1
11
5
6
12
4
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 B-395 Pro Operation Manual, Version C
Liquid filter
Air filter
Electrostatic charge generator
Frequency generator
Stroboscope lamp
Filtration grid
Bead collecting flask
Magnetic stirrer
Waste port
Page 18
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.
The product to be encapsulated (cells, microorganisms, or other biologicals and chemicals) is mixed
with an encapsulating polymer (typically alginate) and the mixture put into a syringe or a pressure bottle , see figure 4-1. The polymer-product mixture is forced into the pulsation chamber
by either a syringe pump a or by air pressure . The liquid then passes through a precisely drilled
nozzle and separates into equal size droplets on exiting the nozzle. These droplets pass through an
electrical field between the nozzle and the electrode resulting in a surface charge. Electrostatic
repulsion forces disperse the beads as they drop to the hardening solution.
Bead size
The bead size is controlled by several parameters including the vibration frequency, amplitude, nozzle
size, flow rate, and physical properties of the polymer-product mixture. In general, the bead diameter
of Ca-alginate beads is twice the nozzle diameter. But, by varying the jet velocity and the vibration
frequency, the range can be adjusted by about ±15 %.
Optimal parameters for bead formation are indicated by visualization of real-time bead formation in the
light of a stroboscope lamp . When optimal parameters are reached, a standing chain of droplets is
clearly visible. Once established, the optimal parameters can be preset for subsequent bead production runs with the same encapsulating polymer-product mixture. Poorly formed beads, which occur at
the beginning and end of production runs, are intercepted by the bypass cup .
Depending on several variables, 50 to 5000 beads are generated per second and collected in a
hardening solution within the reaction vessel . Solutions in the reaction vessel are continuously
mixed by a magnetic stir bar to prevent bead clumping. In addition, the reaction vessel and/or
solution must be electrically grounded. At the conclusion of the production run, the hardening solution
is drained off (waste port ), while the beads are retained by a filtration grid . Washing solutions,
or other reaction solutions, are added aseptically through a sterile filter . The beads can be further
processed into microcapsules, or transferred to the bead collecting flask .
4.2 Connections at the Encapsulator B-395 Pro
Front connections (See figure 5-2)
• Main switch
• Air out
• Voltage
• Ground
Rear connections (See figure 5-1)
• Electric supply
• Air inlet
• Magnetic stirrer
• Vibration
• Optional plug
18 B-395 Pro Operation Manual, Version C
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5 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. Consider the maximum product dimensions and
weight. The instrument must be set up in such a way that the main switch and the mains plug are
easily accessible at all times.
Obtain the environmental conditions as described in section 3.2 “Technical data”.
5 Putting into operation
!
Warning
Death or serious injuries by use in explosive environments.
• Do not operate the instrument in explosive environments.
!
Warning
Death or serious poisoning by contact or incorporation of harmful substances.
• Wear safety goggles.
• Wear safety gloves.
• Wear a laboratory coat.
• Clean the instrument and all accessories thoroughly to remove possibly dangerous
substances.
• Do not clean dusty parts with compressed air.
• Store the instrument and its accessories at a dry place.
19 B-395 Pro Operation Manual, Version C
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5.2 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.
1
3
2
5 Putting into operation
a Air inlet (blue tube 2.6×4.0 mm)
Electric supply socket with inte-
grated fuse
4
5
Optional socket
Socket for magnetic stirrer
Socket for vibration unit
Figure 5-1: Rear view of the control unit
All controlling systems for bead production are incorporated in the control unit. Vibration frequency,
pump speed, light intensity, electrostatic dispersion and magnetic stirrer speed are controlled on the
two touch screens. Air pressure is regulated with the pressure regulating valve. The integrated stroboscope lamp allows real time jet breakup control. The vibration unit is attached to the control unit on the
rear panel by a wire. The reaction vessel is attached to the reactor holder with two screws.
a Syringe pump
Reactor holder
Vibration unit
Upper touch screen (vibration
frequency & electrode)
Lower touch screen (syringe
pump, magnetic stirrer control &
pressure indication)
Pressure regulating valve
Stroboscope lamp
Mains switch
Magnetic stirrer
Air outlet
EDU (Voltage outlet)
Plug for grounding wire
LIquid flow regulating valve
13
11
10
1
3
7
2
4
5
6
12
9
Figure 5-2: Front view of the control unit
8
20 B-395 Pro Operation Manual, Version C
Page 21
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.
An over pressure safety valve, which opens at 1.5 bar, is incorporated after the pressure regulating
valve, so that the maximum air pressure at the air outlet is 1.5 bar. However the working range is 0 to
1 bar.
5.3 Electrical connections
5 Putting into operation
Verify that the electrical requirement of the unit, stated on the type plate of the control unit, corresponds to voltage of your local electrical network. Connect the power plug of the Encapsulator to the
mains supply.
Caution
!
Risk of instrument damage by wrong mains supply.
• External mains supply must meet the voltage given on the type plate.
• Check for sufficient grounding.
• Additional electrical safety measures such as residual current brakers may be necessary to
meet local laws and regulations! External connections and extension lines must be provided
with an earthed conductor lead (3-pole couplings, cord or plug equipment). All used power
cords shall be equipped with molded plugs only to avoid risks due to unobservant defective
wiring.
21 B-395 Pro Operation Manual, Version C
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5.4 Assembling of the reaction vessel
1
5
3
9
4
2
8
10
11
16
18
15
12
13
14
17
20
19
6
7
The Reaction vessel forms a closed, autoclavable unit in which the beads are formed under sterile
conditions and can be further processed if neede
5 Putting into operation
a Liquid filter
Connection to electrode
Screw M4×10
Flange
Glass cylinder
O-Ring (6×2) for gap control
Harvesting valve
Plastic clamp
Silicon tube (6×9) of drain
line
Air filter
Bead producing unit
Bypass knob
Syringe
Cover plate
Bypass cup
Support bar
Filter of drain line
Flat silicone fitting
Base plate
t Foot
Figure 5-3: General view of the reaction vessel
The reaction vessel‘s main parts are:
1. Stainless steel cover plate with electrode, bypass, liquid inlet, and air exchange filter
2. Bead producing unit
3. Nozzle
4. Glass cylinder
5. Stainless steel base plate with bead and drain valve
6. Bead collecting flask
22 B-395 Pro Operation Manual, Version C
Page 23
5.4.1 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. Wash with detergent, rinse with water and
let dry.
When reassembling, check the integrity of the flat fittings and o-ring seals – replace if needed.
1
2
5 Putting into operation
a Front notch for alignment
Groove for fitting
Nozzle
5
6
Liquid inlet
Electrode
Screw M4×12
Bead bypass
3
4
Figure 5-4: Bottom view of mounted cover plate
1
7
a O-Ring 117.1×3.53
Air inlet
O-Ring 29.87×1.78
Liquid inlet
2
3
4
Figure 5-5: Bottom view (left) and top view (right) of cover plate
23 B-395 Pro Operation Manual, Version C
Page 24
5.4.1.1 Bead producing unit and nozzles
2
3
5 Putting into operation
a Magnet Holder *
41
5 6
O-Ring (14×1.78)
Pulsation chamber
Pre-filter with 50 µm mesh
O-Ring (3.68×1.78)
Luer Lock
Screw M3×8
Screw M3×25
Screw M3×6
*with attached fixation ring
and screws M3×5.
You can remove the fixation
ring for cleaning.
Do not remove either the
magnet or the glass fiber
reinforced PTFE membranes!
7
Figure 5-6: Parts of the bead production unit
98
A high quality nozzle is crucial for homogenous bead production. The holes of the Encapsulator
nozzles are precisely drilled using the newest technology. Every Encapsulator B-395 Pro is delivered
with a set of 8 nozzles; nozzle aperture sizes are 80, 120, 150, 200, 300, 450, 750 µm and 1.0 mm.
They are made completely of stainless steel.
Figure 5-7: Set of 8 nozzles on the nozzle rack
The nozzle rack contains 8 nozzles (80, 120, 150, 200, 300, 450, 750 µm, and 1.0 mm). The size of
the O-ring is 4.47×1.78.
24 B-395 Pro Operation Manual, Version C
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5.4.1.2 Electrode
8
4
765
1
2
3
4
Figure 5-8: Electrode with connecting elements
5 Putting into operation
a Screw nut M10 (polyamide)
O-Ring (10.82×1.78)
Insulator
Electrode
Connecting piece
5
Screw M4×12
6
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. The short distance between the
nozzle and the electrode is recommended during the production of small beads and if solutions of low
viscosity are used. The long distance is recommended during the production of large beads (approximately > 800 µm). The separation of the bead from the liquid jet should happen inside of the ring of
the electrode, where the electrostatic field is highest, or secondarily, in the space between the electrode and the nozzle, depending on the properties of your material.
5.4.1.3 Bead bypass system
The bead bypass system is used at the beginning and end of the encapsulation run to eliminate
unwanted beads produced by an unstable stream.
31 2
a Cup
Knob
Screw M5×8
Screw M3×6
Side axis
Main axis
Clip
O-Ring (6.1×1.6)
Figure 5-9: Bead bypass system - exploded view (left), assembled view (right)
25 B-395 Pro Operation Manual, Version C
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5.4.1.4 Air filter
5
4
6
1
432
Figure 5-10: Airfilter - exploded view (left), assembled view (right)
5.4.1.5 Liquid filter
1
2
5 Putting into operation
a HEPA Air filter
O-Ring
3.68×1.78
Nipple
Silicon tube
5×8 mm
a Liquid filter (e.g.
Sartobran 150)
Liquid inlet
Silicon tube (3×5)
35 mm long
Screw M3×12
O-Ring
(10.82×1.78)
Silicon tube
(10×14), 40 mm
long
3
Figure 5-11: Liquid filter - exploded view (left), assembled view (right)
26 B-395 Pro Operation Manual, Version C
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5.4.2 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.
2
1
5 Putting into operation
a Flat silicone fitting
Bead drain valve
Support bar
3
4
5
O-Ring (6×2) for gap control
Filter for liquid drain
Front notch for alignment
Liquid drain outlet
Base plate
Bead drain valve
Foot
6
8
7
Figure 5-12: Base plate top view (above) and bottom view (below)
9
10
27 B-395 Pro Operation Manual, Version C
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5.4.2.1 Bead drain valve
4
3
2
5 Putting into operation
a Plunger
O-Ring (5×1)
Knob for valve
Screw M3×6
5
Screw M3×20
Harvesting valve (BT 14)
O-Ring (18.77×1.78)
6
1
Figure 5-13: Parts of the bead drain valve
5.4.2.2 Liquid drain system
7
a Liquid drain plate
O-Ring (34.65×1.78)
Screw M3×6
Filter grid, diameter
1
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. Thereafter, its dimensions remain stable.
28 B-395 Pro Operation Manual, Version C
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5.4.3 Bead collecting flask
6 7
5
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.
5 Putting into operation
1 4
Figure 5-15: Bead collecting flask for sterile havesting and transportation of the produced
beads and capsules. The bead collecting flask is attached to the bead harvesting valve of
the reaction vessel.
a 250 mL flask
Air filter, see Fig. 5-10
Tube clamp
Silicone tube 10×14
2
Plate of collecting flask
O-Ring (31.42×2.62)
Cap with hole
3
29 B-395 Pro Operation Manual, Version C
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5.5 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. When high flow rates are to be used for producing large beads, as would be the case when using
nozzles > 300 µm.
5 Putting into operation
Both systems may be used with the concentric nozzle system. The core liquid is pumped with the
syringe pump and the shell liquid is pumped with air pressure. Of course, you may also use two air
pressure bottles by splitting the air line to the bottles with a “T” or “Y” connector.
5.5.1 Syringe pump
The syringe pump is used as a volumetric delivery system. It is a pump that very accurately delivers
the immobilization mixture. Most brands of plastic syringes can be used. (The use of glass syringes
is not recommended!) Each syringe type can be individually calibrated using the integrated syringe
calibration system (see chapter 6 “Operation”). The availability of pre-sterilized syringes makes aseptic
handling more convenient.
The pumping rate can vary from 0.01 mL/min to 50 mL/min depending on the syringe size.
a
Figure 5-16: Syringe pump
The syringe is attached to the bead producing unit with the luer lock fitting. The piston of the syringe is
pushed by the moving arm of the syringe pump a.
30 B-395 Pro Operation Manual, Version C
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5.5.2 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 Putting into operation
1 3 42
5
89
Figure 5-17: Pressure bottle with HEPA filter for sterile pumping of the immobilization mixture with air pressure
a Pressure stable flask of 500 mL or 1,000 mL
HEPA air filter
PTFE tube (4×6)
Silicone tube for liquid (4×7)
Silicone tube for air (5×8)
Luer lock male, 4.8 mm ID
Nipple for quick coupling
Two port cap
Cap with PTFE fitting for 6 mm tubes
67
The air passes through a silicone tube with an inner diameter of 5 mm (5×8 mm). The Hepa-filter
prevents contamination of the sterile immobilization mixture and should be replaced according to the
manufacturer’s instructions or if signs of reduced air passage are noticeable.
The liquid passes from the inside of the bottle through a PTFE tube (3×6 mm) to the silicone tube
outside of the bottle. This silicone tube is attached to the bead producing unit with the luer lock male .
31 B-395 Pro Operation Manual, Version C
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5.5.3 Installation of the pressure bottle
5 Putting into operation
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 g of the air tube into the
quick coupling of the air outlet at the control
unit.
Figure 5-18: Installed pressure bottle
32 B-395 Pro Operation Manual, Version C
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5.6 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.
The main parts of the concentric nozzle unit are (refer to figure 5-20):
• the nozzle pair with shell a and core nozzle.
• CN bead producing unit with CN pulsation body and magnet
holder .
5 Putting into operation
Figure 5-19: Capsule formation
Figure 5-20: Schematic description of the concentric nozzle system
a Shell nozzle
Core nozzle
CN pulsation body
Magnet holder
Luer connection, for
core liquid
Luer connection, for
shell liquid
Vibration unit
Carrier plate
33 B-395 Pro Operation Manual, Version C
Page 34
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 4
2
Figure 5-22: Single parts of the CN bead producing unit
3
7
8
5 6
9
10
a Screw M3×6
Luer lock female
Pre-filter grid 50 µm mesh, D= 7 mm
O-Ring 14.0×1.78
Screw M3×8
Screw M3×25
O-Ring 3.68×1.78
CN pulsation body
O-Ring 12.42×1.78
CN membrane holder
34 B-395 Pro Operation Manual, Version C
Page 35
5.6.1 Mounting of CN nozzles
2
Figure 5-23: Mounting of the inner nozzle
5 Putting into operation
Put the O-ring 12.42×1.78 in the grove of the CN
1
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.
a Exit of the shell liquid
O-ring 12.42×1.78
Put carefully the shell nozzle over the inner
nozzle. Attach the shell nozzle with two screws
(M3×6). The shell nozzle centers and fixes the
inner nozzle.
Figure 5-24: Mounting of the shell nozzle
Figure 5-25: Installation of the CN system with one syringe pump and one pressure bottle
35 B-395 Pro Operation Manual, Version C
Page 36
5.7 All parts of the Encapsulator B-395 Pro
5 Putting into operation
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.
• Inspect all glass components for damage.
• Check all other electrical connections for proper connection, such as optional or external components, e.g. magnetic stirrer, vibration unit, syringe pump cabling.
36 B-395 Pro Operation Manual, Version C
Page 37
6 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
• 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.
6.2 Main screens
All controlling systems for bead production are incorporated in the control unit. Vibration, pump speed,
light intensity and electrostatic dispersion are controlled on two touch screens. Air pressure is regulated with the pressure regulating valve. The integrated stroboscope lamp allows real time jet breakup
control.
After the internal system check the two touch screens show the following main screens:
6 Operation
Screen 6-1: Upper touch screen
The upper touch screen is for the control of vibration
frequency and electrode tension.
The lower touch screen is for the control of the syringe pump and the magnetic stirrer speed.
The air pressure is also indicated on this screen,
however it is controlled manually through the pressure regulating valve.
Screen 6-2: Lower touch screen
NOTE
Icons with a thick bar at the bottom e.g.
37 B-395 Pro Operation Manual, Version C
on
activate/stop a process or lead to another screen.
off
Page 38
6.3 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 -mainmenu
FrequencyOn/Off
ElectrodeOn/Off
Store function
Set Freq. | Set Elect. | More
6 Operation
Frequency[Hz]
FrequencyOn/Off
Set Frequency
Syringe Pump Calibration
Syringe Pump Calibration
Start Timer: Collection of Liquid
for 60 sec.
LowerScreen -mainmenu
Set Pump | Set Stirrer
Syringe Pump
Pump On/Off
Set PumpingRate
Cal ml/min
Select Syringetyp e
Electrode [V]
ElectrodeOn/Off
Set Electrode
Pump On/Off
StirrerOn/Off
Home/Turbo button
Pressure value
Stirrer[%]
StirrerOn/Off
Set StirrerSpeed
more Frequency
Set Light Intensity
Set Amplitude
Syringe Pump Calibration
Set Pumped Liquid
Store
Syringe Pump Calibration
Store valueYes/No
Figure 6-1: Menu structure of the control unit
38 B-395 Pro Operation Manual, Version C
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6.4 Menu functions of the upper touch screen
21
3 4
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.
a On/off switch for frequency control.
Indication of control parameter and status of control
(value or off).
Button for passing to screen 6-4 for setting frequency
parameters.
Button for storing set values: press twice within one
second. A sound indicates that the values are stored.
On/off switch for electrode control.
5 6
7
Button for passing to screen 6-5 for setting electrode
parameters.
Button for passing to screen 6-6 for setting more
frequency parameters.
6 Operation
Screen 6-3: Start menu of the upper touch screen
Screen 6-4: Frequency regulation
Screen 6-5: Electrostatic dispersion unit
The frequency regulation generates the appropriate electric oscillation in the vibration unit. Pressing on the (+) and
(–) buttons will change the frequency. Pressing the “on/off”
button activates or deactivates frequency. Pressing “Esc”
will return you to the start menu and the set value will be
kept.
The electrostatic dispersion unit is used to charge the
surface of the beads. The repulsion forces induced by the
equally charged surfaces prevent the beads from hitting
each other in flight, and from hitting each other as they enter
the hardening solution. The applied voltage often lies in the
range of 500 to 2000 V, depending primarily on the bead
size and the liquid flow velocity. In this way, the Encapsulator
B-395 Pro can routinely generate bead batches with homogeneity greater than 95 %.
Pressing on the (+) and (–) buttons changes the electrostatic
dispersion parameter. The system needs few moments to
reach the set value. Pressing “Esc” will return you to the
start menu and the set value will be kept.
39 B-395 Pro Operation Manual, Version C
Page 40
Screen 6-6: More options concerning amplitude of vibration
and light Intensity of the stroboscope lamp.
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.
40 B-395 Pro Operation Manual, Version C
Page 41
6.5 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).
6 Operation
1
5 7
Screen 6-7: Start menu of the lower touch screen
6
2
3
8
a On/off switch for magnetic stirrer control.
4
Button for passing to screen 6-9 for setting magnetic
stirrer speed .
Button for sending back syringe pump arm.
This button is only visible, if the arm is not already in
the “home-position”. When the pump is advancing,
then this button becomes the “turbo”-button, see
screen 6-8.
Button for storing set values: press twice within one
second. A sound indicates that the values are stored.
On/off Switch for syringe pump control.
Indication of control parameter and status of control
(value or off).
Button for passing to screen 6-10 for setting syringe
pump parameters.
Indication of the pressure value at the air outlet from 0
to 1000 mbar.
9
Pressing the "turbo" button will double the current
pumping rate.
Screen 6-8: Lower touch screen with turbo button
Screen 6-9: Speed regulation of the magnetic stirrer
Pressing on the (+) and (–) buttons will change the stirrer
speed. Pressing “Esc” will return you to the start menu
and the set value will be kept.
NOTE
The values are arbitrary values but reproducible and do
not correspond to rpm.
41 B-395 Pro Operation Manual, Version C
Page 42
Screen 6-10: Speed regulation of the syringe pump
6.5.1 Menu for syringe pump calibration
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.
Select appropriate syringe volume by pressing the corresponding button. You are forwarded to screen 6-12
(if the pump is running) or to the main screen (if the pump
is stopped).
Screen 6-11: Syringe pump calibration - selecting syringe
type
Screen 6-12: Syringe pump calibration - timer
Pressing button “on” starts timer. The timer counts down
for 1 minute from 60 to 0 sec. During this time the liquid
from the jet is collected in a pre-weighted vessel. The
three last seconds are announced by a short tone. One
second later the syringe pump stops and you are forwarded to screen 6-13.
42 B-395 Pro Operation Manual, Version C
Page 43
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:
liquid
Screen 6-14: Syringe pump calibration - store value
Syringe pump calibration - setting the pumped
6.5.2 Selecting a calibrated syringe
Stop the pump. Press on screen 6-10 the “cal mL/min” button. You are forwarded to screen 6-15.
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.
Select the appropriate syringe by pressing on the item.
The syringe is stored and you are forwarded to the start
menu.
NOTE
This screen is only accessible from the start menu, if the
pump is stopped.
Sreen 6-15: Selecting the syringe size
43 B-395 Pro Operation Manual, Version C
Page 44
6.6 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. The knob of the pressure regulating valve has two positions. If it
is pushed in, it is locked, if it is pulled out, it is unlocked. Turning the knob counter-clockwise reduces
the pressure via the self-venting system in the valve. The pressure is indicated on the touchscreen (see
screen 6-7).
NOTE
• The pressure of air or nitrogen entering the control unit on the rear panel of the Encapsulator
should be below 7 bar (100 psi). The prefered range is between 1.5 and 2 bar (20 to 30 psi).
• Be aware that the pressure regulation system reacts relatively slowly, because the displacement
of air in or out through the constriction valve is delayed.
• Do not leave the gas supply line on when the Encapsulator is not being used. The self-venting
system in the valve would drain the gas tank.
6 Operation
• The maximum pressure at the air outlet is 1.5 bar (20 psi). This value is controlled by an incorporated overpressure safety valve, which opens at 1.5 bar. However the working range is from 0 to
1 bar.
Figure 6-2: Air pressure regulating system for manual
air pressure control - turning the pressure regulating
valve clockwise increases the pressure.
44 B-395 Pro Operation Manual, Version C
Page 45
6.7 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. Attach the filled syringe
(we recommend using plastic syringes with a luer lock system) to the bead producing unit.
Let the syringe arm move forward by starting the pump (see screen 6-10). By pressing the “turbo”
button, the arm moves at double speed. You might increase this forward speed by temporarily setting
higher pumping rates. Reduce the speed as the pump arm approaches the syringe plunger. Stop the
pump as the arm touches the syringe plunger. Set the desired liquid flow rate (see screen 6-10). Start
pumping by pressing the on/off button. To prime the system, press the “turbo” button – the pump
moves at double speed – until a continuous liquid jet is formed at the nozzle, then press again on the
“turbo” button to move back to the preset value. If needed, adjust the pumping speed to get clearly
separated beads in the light of the stroboscope.
6 Operation
Figure 6-3: Syringe pump end knob
6.7.1 Calibrating the syringe pump
Fill the syringe with water or with the immobilization mixture and attach it to the bead producing unit.
Weigh a container for collecting the liquid pumped for 1 minute and start pumping. Set the pumping
rate to obtain good bead formation. Start the calibration procedure by pressing the “cal mL/min”
button (see screen 6-10). Choose the appropriate syringe size (see screen 6-11).
Press the “on/off” button (see screen 6-12) and collect the liquid coming from the nozzle in the preweighed container for 60 seconds. The last three seconds are announced by a sound. One second
after the last sound, the pump stops automatically. Weight the pumped liquid. Insert the value (see
screen 6-13) and store the value. This type of syringe is now calibrated.
6.7.2 Selecting a pre-calibrated syringe
The calibrated syringe types can be recalled as needed. Stop the pump, press the “cal mL/min”
button (see screen 6-10) and you will be forwarded to screen 6-15. Select the appropriate syringe and
you will be forwarded to the start menu. The selection is done.
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6.8 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. 600 mL) under the nozzle. Connect the electrode with the red wire to
the electrostatic dispersion unit (EDU).
6.8.1 Using the syringe pump
1. Fill a 60 mL syringe with distilled water and install it as described in section 5.5. Set the syringe
pump speed to 4 mL/min. Activate the vibration control system and set the vibration at 1500 Hz.
Activate the syringe pump. The water will flow in large drops from the nozzle. Increase the
pumping speed until a continuous liquid jet is formed. Change the pumping speed and observe
the bead chain in the light of the stroboscope. The proper working condition is when the beads
within the bead chain are clearly separated over a length of several centimeters, 3 to 5 mm below
the nozzle. Note the vibration, voltage and syringe pump settings before you stop the pump.
6 Operation
NOTE
If you have difficulty seeing the bead chain, reduce the amount of light around the Encapsulator and
look from a distance of 20 to 30 cm (8” to 12”) into the liquid jet so that the black frame of the stroboscope is directly behind the stream.
2. Start the pump again and depress the “turbo” button – the pump will move at double speed and
a continuous liquid jet will be formed. Release the “turbo” button and the jet will soon stabilize
at the preset flow rate. The “turbo” button is very helpful priming the system when using viscous
polymer solutions and in dislodging small occlusions that impede the flow.
3. Increase the vibration frequency until the bead chain becomes unstable and then increase the
pumping speed until a good bead chain is restored. Repeat this procedure in the opposite
direction by decreasing the pumping speed and then decreasing the vibration frequency. After
performing this exercise a few times, you will become familiar with the relationship between these
two parameters. Insert the values you have determined for the optimal bead chain in table 6-1.
NOTE
The pumping speed and the vibration frequency influence each other within a given working range.
The working range itself is mainly determined by the nozzle diameter and the viscosity of the polymer
mixture.
General Rules:
• Higher frequencies generate smaller bead sizes.
• Lower liquid flow rates generate smaller bead sizes.
• The smaller the beads the lower the electrostatic voltage needed to separate the bead
stream.
• Smaller nozzles generate smaller bead sizes. The final bead diameter will be approximately 2 times the nozzle size.
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Table 6-1: Determination of the working field (with syringe pump)
Nozzle size: Syringe size:
6 Operation
Pumping Speed
mL/min
Clear Bead Chain
without Electrostatic Tension
Lowest
Frequency
Highest
Frequency
Clear Bead Chain
with Electrostatic Tension
Electrostatic
Tension
Lowest
Frequency
Highest
Frequency
Nozzle size: Syringe size:
Pumping Speed
mL/min
Clear Bead Chain
without Electrostatic Tension
Lowest
Frequency
Highest
Frequency
Clear Bead Chain
with Electrostatic Tension
Electrostatic
Tension
Lowest
Frequency
Highest
Frequency
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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.
This prevents the beads from hitting each other in flight and from hitting each other as they enter
the hardening solution since they are now like charged particles that repel each other. With this
exclusive feature, the Encapsulator can routinely generate bead batches with homogeneity greater
than 95 %.
5. Change the vibration frequency and the pumping speed and observe their influence on the
electrostatic voltage needed to generate the bead stream separation. The use of the electrostatic
dispersion unit enlarges the working range.
It can happen that after some time the beads no longer enter the receiving beaker or actually
jump out of it. This is due to the fact that electrostatic charges have accumulated in the electrically
isolated beaker. To avoid this phenomenon, place the supplied stainless steel clip of the grounding
wire over the edge of the beaker so it extends into the receiving liquid and connect the other end
of the green-yellow wire to the grounding plug on the front panel of the control unit (see figure 6-4).
If you work with the complete reaction vessel, then the electrostatic charges will be automatically
eliminated without the need of the grounding wire.
Figure 6-4: Grounding the open polymerization bath
6. Change the amplitude of the vibration and you will observe only slight changes in the bead chain.
In general, values between 1 and 3 are optimal for low viscous solutions. If immobilization mixtures
with high viscosity (> 150 mPa s) are used, values higher than 3 might be more appropriate.
7. Repeat this experiment with another nozzle size.
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6.8.2 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. Pass
the silicon tube (4×7 mm) between the blades of the flow regulating valve and attach the male luer
lock fitting of the silicon tube to the female luer lock fitting of the bead producing unit. Squeeze the
valve by turning the knob clock wise so that the silicon tube is closed.
3. Open the external pressurized air supply. The air inlet pressure is optimally at 1.5 to 2 bar (20 to 30
psi). However the system tolerates air inlet pressures of up to 7 bar (100 psi).
4. Set the air outlet pressure to 0.2 bar with the pressure regulating valve. Check the readout periodically to verify that the air pressure still corresponds to the set value. Activate the vibration control
system and set the frequency at 800 Hz.
5. Open the flow regulating valve by turning the knob counter-clock wise until the water flows
through the silicone tubing and the bead producing unit to the nozzle where it forms a continuous
liquid jet. Adjust the liquid flow and/or the frequency to obtain a good bead chain in the light of the
stroboscope lamp. The desired setting is when the beads within the bead chain are clearly separated for several centimeters, starting 3 to 5 mm below the nozzle. Record the position of the flow
regulating valve for this desired setting.
6 Operation
6. Increase the vibration frequency until the bead chain becomes unstable. Then increase the liquid
flow rate by slowly increasing the air pressure until a uniform bead chain is restored. Repeat this in
the opposite direction by decreasing the flow rate and compensating by decreasing the vibration
frequency. This may be done until you become familiar with the relationship between these two
settings. Record the values in table 6-2.
NOTE
• The liquid flow rate and the vibration frequency influence each other within a given working range.
The working range itself is mainly determined by the nozzle diameter and the viscosity of the polymer
mixture.
• An air pressure setting between 0.05 to 0.15 bar is sufficient to pump distilled water. Greater
working pressures indicate problems such as a clogged nozzle.
General Rules:
• Higher frequencies generate smaller bead sizes.
• Lower liquid flow rates generate smaller bead sizes.
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Table 6-2: Determination of the working field (with pressure bottle)
Nozzle size:
6 Operation
Air pressure Clear bead chain without
electrostatic tension
Lowest
frequency
Highest
frequency
Clear bead chain with
electrostatic tension
Electrostatic
Voltage
Lowest
frequency
Highest
frequency
Nozzle size:
Air pressure Clear bead chain without
electrostatic tension
Lowest
frequency
Highest
frequency
Clear bead chain with
electrostatic tension
Electrostatic
Voltage
Lowest
frequency
Highest
frequency
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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. Therefore the
Encapsulator can routinely generate bead batches with homogeneity greater than 95 %. If nothing
happens, check that the electrode is connected to the control unit.
8. Change the vibration frequency and the flow rate and observe their influence on the electrostatic
tension needed to generate a jet separation. The use of electrostatic tension enlarges the working
range.
It can happen that after some time, the beads no longer enter, or actually jump out of the beaker.
This is due to the fact that electrostatic charges have accumulated in the electrically isolated
beaker. To avoid this phenomenon, place the supplied stainless steel clip of the grounding wire
over the edge of the beaker so it extends into the receiving liquid and connect the green-yellow
wire to the grounding plug on the front panel of the control unit. If you work with the complete
reaction vessel, then the electrostatic charges will be automatically eliminated without the need for
the grounding wire.
General Rule:
The larger the beads, the higher the electrostatic voltage needed to seperate the jet.
9. Change the amplitude of the vibration. You will observe only slight changes of the bead chain.
Very often values between 1 and 3 are optimal for low viscous solutions. If using immobilization
mixtures with rather high viscosity (> 150 mPa s), values higher than 3 might be more appropriate.
10. Repeat this procedure with another nozzle size.
General Rule:
• Smaller nozzles generate smaller bead sizes.
• The final bead diameter will be approximately 2 times the nozzle size.
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6 Operation
6.9 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. Therefore,
the concentration of alginate solution is a function of the nozzle diameter (see the following table).
Table 6-3: Recommended alignate concentration (based on dry weight) for different nozzle diameters
Nozzle diameter Concentration of low viscosity grade alginate
Working range Recommended concentration
80 to 120 µm 0.75 to 1.4 % 1.1 to 1.2 %
120 to 200 µm 1.0 to 1.6 % 1.3 to 1.4 %
200 to 300 µm 1.2 to 1.8 % 1.5 to 1.6 %
300 to 500 µm 1.5 to 2.5 % 1.8 to 2.0 %
NOTE
Under normal storage conditions the alginate powder contains 10 - 12 % water. Therefore we refer to
the alginate concentration on a dry weight base.
6.9.1 Preparation of 1.5 % Na-alginate solution
1. Take a 400 mL beaker and weigh in 3.3 g Na-alginate powder of low viscosity grade.
2. Add 200 mL of deionized water and mix vigorously with a laboratory mixer for 1 to 2 minutes.
3. Alginate has the tendency to get lumpy. Remove the alginate lumps from the beaker and the mixer
blades with a spatula and mix again for 1 to 2 minutes. If lumps remain in the liquid, repeat mixing.
4. Then let the mixture stand so that the trapped air bubbles will escape from the liquid.
5. If needed, de-gas the mixture under reduced pressure.
6. Dissolution of alginate with a magnetic stirrer takes much more time and should be done overnight.
NOTE
Alginate solutions will support the growth of microorganisms and are stable for about 2 weeks in a
refrigerator. An indication of microbial contamination is reduction of the mixture’s viscosity. Alginate
solutions can be stored for much longer time, even at room temperature, if sterilized or if preservatives are added, like 0.05 % NaN3 .
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6.9.2 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. ¾”) is filled with the polymerization liquid. Put a
magnetic stir bar in the beaker and adjust the stirrer, so that a slight vortex is visible. A vortex in
the liquid will create shear forces which may deform the beads. It is best to use a stir bar without
a spin ring (supplied) because the spin ring will raise up the stir bar and may crush the beads
beneath it. Also, place the grounded clip over the edge of the beaker and into the liquid. At this
time, either cover the beaker with a plate (petri dish) or move it and the stirrer out of the way and
position another beaker with water in it (and the grounding clip) under the nozzle in its place.
2. Fill a 60 mL syringe with the above 1.5 % alginate solution and install it on the Encapsulator.
3. Activate the vibration control system and set the vibration frequency at 1200 Hz for the 200 µm
nozzle or at 900 Hz for the 300 µm nozzle. Activate the syringe pump and set the pumping speed
to 5 mL/min for the 200 µm nozzle or 8 mL/min for the 300 µm nozzle. Depress the “turbo” button
until a continuous liquid jet is formed. Release the “turbo” button and the jet will soon stabilize at
the preset flow rate. Adjust the pumping speed and/or the frequency to obtain a clear bead chain
below the electrode.
6 Operation
4. Activate the electrostatic dispersion unit at 500 V. Increase the voltage by steps of 100 V to get a
circular dispersal of the bead stream 3 to 10 cm (1” to 4”) after the electrode. An optimal distance
is about 5 cm (approximately 2”) below the electrode. If nothing happens, verify that the electrode
is connected to the control unit.
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 he liquid flow rate and the vibration frequency are
also factors. They influence the way that the bead is separated from the liquid jet within the electrostatic field between the nozzle and the end of the electrode. Smaller beads are often separated from
the liquid jet nearer to the nozzle than larger beads.
5. As soon as a symmetrical and stable dispersal pattern is obtained, exchange the beaker with
the beaker containing polymerization solution. Collect the beads for about 1 minute. Record the
process parameters in table 6-4 while the beads are accumulating. Cover the beaker (or exchange
it with the previous beaker containing waste) and stop the bead production by turning off the
syringe pump, vibration control and electrostatic voltage.
NOTE
Clean the nozzle thoroughly immediately after each run with distilled water to avoid nozzle clogging or
partial occlusion due to dried out polymer mixture.
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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.
NOTE
When producing small beads with a diameter <500 µm, it may occur that their shape is not spherical
but somewhat oval. This is mainly due to the surface tension of the polymerization solution. A very
critical point for the bead is it‘s entrance into the polymerization solution. If the surface tension is
high, then the bead is partially held back at the surface and polymerization starts before the bead can
regain a round shape. This problem can be eliminated by adding a small quantity of surfactant like
Tween 20 to the polymerization mixture.
8. Compare the influence of the electrostatic dispersion unit by collecting beads at the same vibration frequency and pumping rate with and without the electrostatic function turned on.
9. Determine the working field by stepwise changing the pumping speed from the lowest liquid flow
rate which just creates a continuous liquid jet up to a flow rate where a clear bead chain is no
longer visible at any vibration frequency. Note the corresponding lowest and highest frequencies in
table 6-5.
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Table 6-5: Determination of the working field
Nozzle size: Alginate concentration: Syringe size:
6 Operation
Pumping speed
[mL/min]
Clear bead chain without
electrostatic tension
Lowest
frequency
Highest
frequency
Clear bead chain with
electrostatic tension
Electrostatic
tension
Lowest
frequency
Highest
frequency
Nozzle size: Alginate concentration: Syringe size:
Pumping speed
[mL/min]
Clear bead chain without
electrostatic tension
Lowest
frequency
Highest
frequency
Clear bead chain with
electrostatic tension
Electrostatic
tension
Lowest
frequency
Highest
frequency
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6.9.3 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. ¾”) is filled with the polymerization liquid. Put a
magnetic stir bar in the beaker and adjust the stirrer, so that slight vortex is visible. Also, place the
grounded clip over the edge of the beaker and into the liquid. At this time, either cover the beaker
with a plate (petri dish) or move it and the stirrer out of the way and position another beaker with
water in it (and the grounding clip) under the nozzle in its place.
2. Fill the pressure bottle with the above described 1.5 % alginate solution and screw on the
assembled cap. Pass the silicon tube (4×7 mm) between the blades of the liquid flow regulating
valve and attach the male luer lock fitting of the silicon tube to the female Luer lock fitting of the
bead producing unit. Squeeze the valve by turning the knob clock wise so that the silicon tube is
closed.
3. Open the external pressurized air supply. The air inlet pressure is optimally at 1.5 to 2 bar (20 to 30
psi). However the system tolerates air inlet pressures of up to 7 bar (100 psi).
4. Set the air pressure to 0.4 bar at the pressure regulation system. Check the readout periodically to
verify that the air pressure still corresponds to the set value. Activate the vibration control system
and set the vibration frequency at 1100 Hz for the 200 µm nozzle and at 800 Hz for the 300 µm
nozzle.
6 Operation
5. Open the flow regulating valve by turning the knob counter-clock wise until the liquid flows through
the silicone tubing and the bead producing unit to the nozzle where it forms a continuous liquid jet.
Adjust the liquid flow and/or the frequency to obtain a good bead chain in the light of the stroboscope lamp. The desired setting is when the beads within the bead chain are clearly separated for
several centimetres, starting 3 to 5 mm below the nozzle. Record the position of the flow regulating valve for this desired setting.
6. Increase the vibration frequency until the bead chain becomes unstable. Then increase the liquid
flow rate by slowly increasing the air pressure or by slowly opening the flow regulating valve until
a uniform bead chain is restored. Repeat this in the opposite direction by decreasing the flow rate
and compensating by decreasing the vibration frequency. This may be done until you become
familiar with the relationship between these two settings. Record the values in table 6-5.
NOTE
An air pressure setting from 0.1 to 0.8 bar is generally sufficient to pump the polymer mixture.
Working pressures greater than 1.0 bar should be avoided and are indicative of problems such as:
• Clogged nozzle,
• Overly viscous polymer mixture,
• Under sized nozzle for the polymer mixture in use.
7. Activate the electrostatic dispersion unit at 500 V. Increase the voltage by steps of 100 V to get a
circular dispersal of the bead stream 3 to 10 cm (1” to 4”) after the electrode. An optimal distance
is about 5 cm (approx. 2”) below the electrode.
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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. As soon as a symmetrical and stable dispersal is obtained, remove the plate from the beaker
filled with polymerization solution or replace the beaker of water with the beaker of polymerization
solution and the stir plate (and the grounding forceps), and collect the beads for about 1 minute.
Record the process parameters in table 6-5 while the beads are accumulating. Cover or switch
the beaker and stop the bead production by turning off the electrostatic voltage, air pressure
control and vibration control.
NOTE
Clean the nozzle thoroughly immediately after each run with distilled water to avoid nozzle clogging or
partial occlusion due to dried out polymer mixture.
9. Check the beads under a microscope with a micrometer scale eyepiece, and record your observations of diameter, uniformity and shape in table 6-6.
10. Repeat this process for each change in process parameters.
Table 6-6: Encapsulator trial test work sheet (pressure bottle)
Nozzle size [µm]
Alginate concentr. [%]
Position of the flow regulating valve
Vibration frequency [Hz]
Amplitude
Approximate bead size [µm]
Homogeneity [%]
Comments
NOTE
When producing small beads with a diameter <500 µm, it may occur that their shape is not spherical
but somewhat oval. This is mainly due to the surface tension of the polymerization solution. A very
critical point for the bead is it‘s entrance into the polymerization solution. If the surface tension is
high, then the bead is partially held back at the surface and polymerization starts before the bead can
regain a round shape. This problem can be eliminated by adding a small quantity of surfactant like
Tween 20 to the polymerization mixture.
11. Compare the influence of the electrostatic tension by collecting beads at the same vibration
frequency and pumping rate with and without the electrostatic tension function turned on.
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6 Operation
6.10 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.
Sterile working conditions have the additional difficulty that it is often impossible to modify something
in the autoclaved reaction vessel without losing sterility, like if a piece was forgotten or assembled
in the wrong way. Therefore, it is important that the reaction vessel is properly prepared before
sterilization. Follow section 5 for the assembly and sterilization of the reaction vessel. If you change
something, note it in a separate procedure.
1. Prepare all the needed encapsulation reagents (as an example, for animal cell encapsulation, see
section 6.14).
For this run: 60 mL 1.5 % alginate solution
500 mL 100 mM CaCl2 polymerization solution
600 mL 0.9 % NaCl + 10 mM CaCl2 washing solution
Assemble a pressure bottle.
2. Take the autoclaved reaction vessel and attach it to the control unit.
Verify that the silicone tubing at the liquid drain port of the reactor base plate is closed with a
clamp. Make sure that the bead collecting valve at the reactor vessel is closed, that the magnetic
stirrer is below the reactor and that the stirrer bar is above the magnetic stirrer.
– Connect the electrostatic dispersion unit with the red wire to the reaction vessel.
– Move the collection cup of the bead bypass below the nozzle.
3. Place 500 mL of polymerization solution in the pressure bottle. Attach the silicone tubing to the
liquid membrane filter.
Connect the pressure bottle to the air outlet of the control unit. Switch on the control unit. Set the
air pressure at 0.3 to 0.7 bar (4 to 10 psi). When the desired amount of liquid is pumped, release
the air pressure.
NOTE
The amount of hardening solution should be 8 to 10 times the volume of the polymer mixture. The
polymerization solution should have a height of at least 2 cm (approx. ¾“) in the reaction vessel (a
minimum of 200 mL).
4. Set the vibration frequency, the electrostatic tension and – if the syringe pump is used – the
pumping rate to the appropriate values as previously determined. Set the speed of the magnetic
stirrer so that a vortex is just visible.
5. Fill a 60 mL syringe with the alginate/sample solution and attach it to the Encapsulator. Activate
the vibration control system and set the vibration frequency at values predetermined in section
6-9. Activate the syringe pump and set the pumping speed as previously determined. Press the
“turbo” button until a continuous liquid jet is formed. Activate the electrostatic dispersion system. If
needed, adjust the pumping speed and/or the frequency to obtain a clear bead chain down to the
collection cup.
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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. Inspect the actual bead production in the light of
the stroboscope lamp to verify the settings.
8. Shortly before the syringe plunger is fully depressed, move the collection cup of the bypass back
into the bead stream. This will prevent that large droplets, which are formed at the end of the
production process, contaminate the homogeneous collection. Stop the pump. Turn off the electrostatic dispersion unit and the vibration control system. Or press "home" to move back the arm
of the syringe pump. This will also deactivate the electrostatic dispersion unit and the vibration.
9. Let the beads harden for 5 minutes.
10. To drain the hardening solution from the reaction vessel, slowly open the drain clamp so that it will
take 1 to 2 minutes to drain 500 mL. Turn off the magnetic stirrer when about ¾ of the liquid has
drained. Close the drain clamp as soon as the liquid level reaches the settled beads.
NOTE
Always leave the beads slightly covered with solution to prevent clumping.
11. Fill the bottle of the liquid transfer set with 400 mL of washing solution and pump it into the reac-
tion vessel. Restart the magnetic stirrer as soon as the magnetic stir bar is covered with liquid
so the beads are not damaged. Wash the beads for 5 minutes and then drain off the liquid as
described above (item 10).
12. Pump in the final 200 mL of washing solution into the reaction vessel. Re-suspend the beads by
turning on the magnetic stirrer.
13. Open the bead collecting valve and let the beads flow into the bead collecting flask. If beads
remain in the reaction vessel, let liquid flow back from the bead collecting flask into the reaction
vessel by raising the bead collecting flask higher than the reaction vessel drain. Re-suspend the
remaining beads with this liquid and transfer it back into the bead collecting flask by lowering it
below the reaction vessel..
14. Close the silicone tube from the reaction vessel to the bead collecting flask with the clamp.
Disconnect the bead collecting flask from the reaction vessel and check the beads for quality
under a microscope.
15. Immediately after the end of the bead production process fill a syringe with distilled water, attach it
to the bead producing unit and flush the nozzle to avoid having the polymer dry and clogging the
system thus creating a maintenance problem. Clean the reaction vessel thoroughly, including the
various in and out ports.
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6.11 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. Verify that the following items are connected or prepared correctly:
• The proper sized nozzle
• The electrode centered below the nozzle
• Does the luer lock plug close the bead producing unit?
• Are the air and liquid filters attached?
• Is a magnetic stirrer bar in the reaction vessel?
• Is the bead collecting flask attached?
• Is the passage from the reaction vessel to the bead collecting flask open (open bead drain
valve)?
• Is the drain tubing closed with a clamp?
• Is the luer lock of the bead producing unit closed with a luer lock stopper?
4. Put the assembled reaction vessel in the autoclave and steam sterilize at 121°C for 20 minutes or
according to your protocol.
6 Operation
5. After autoclaving, remove the hot reaction vessel from the autoclave as soon as possible to avoid
water condensation in the air filter. Because a completely wetted air filter will no longer let air
pass, it will be difficult to drain the liquids from the reaction vessel, due to the negative pressure
created in the reaction vessel. To test the air filter’s condition, attach a 60 mL syringe to the filter.
When moving the syringe piston back and forth, only a slight resistance should be felt.
6. Close the bead collecting valve only after the reaction vessel has cooled down to room temperature.
6.12 Sterilization of the pressure bottle
1. Assemble the pressure bottle and close the luer lock fitting with a luer lock stopper.
2. Add 1 to 2 mL of water into the pressure bottle. Place the assembled pressure bottle in the autoclave and steam sterilize at 121°C for 20 minutes or according to your protocol.
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6.13 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). If more resistant beads
are required, because some culture media have ingredients that will slowly dissolve the beads, Ca ions
may be replaced by Ba ions, which have a stronger affinity to alginate than Ca ions and the resultant
Ba-alginate beads will be more stable.
Sterilization of alginate solutions is best accomplished by sterile membrane filtration (0.2 µm). Heat
sterilization tends to partially degrade the alginate and unpredictably changes the viscosity and the
polymerization capacity.
For the encapsulation of animal cells, it is recommended to use another protocol because the three
dimensional structure of the Ca-alginate hinders the formation of the new cell membrane during cell
division. For dividing cells, capsules are better suited. A procedure for the production of alginate-PLL
capsules is described in section 6-14.
1. Prepare all needed materials as described in section 6-10; reaction vessel, pressure bottle, 60 mL
syringe, beakers, graduated cylinders, etc. Autoclave the reaction vessel.
2. Prepare all needed encapsulation reagents.
For this run: 50 mL 1.5 % alginate solution, low viscosity grade, sterile filtered
500 mL 100 mM CaCl2 polymerization solution (non-sterile)
600 mL 0.9 % NaCl + 10 mM CaCl2 washing solution (non-sterile)
3. Switch on the control unit. Set the vibration frequency, the electrostatic tension and the pumping
rate to the appropriate values as previously determined.
4. Place 500 mL of polymerization solution in the pressure bottle and close it. Attach the silicone
tubing to the liquid membrane filter. Pump the polymerization solution into the reaction vessel.
5. Detach the silicone tubing from the membrane filter and place the reaction vessel in a sterile
biological hood.
6. Prepare 10 mL of concentrated microorganism suspension. The suspension should be free of
bi- or trivalent cations (e.g. Ca, Mg, Al, Fe) or contain them in a very low concentration, to avoid
preliminary polymerization reactions with the alginate. Chose the desired microorganism concentration the way that it is in the final polymer mixture < 1010 cells/mL (for animal cells <107 cells/
mL). Carefully mix the 10 mL microorganism suspension with 50 mL 1.5 % sterile alginate solution
gently to minimize the formation of air bubbles.
7. Fill a sterile 60 mL syringe with the polymer-product mixture aseptically. Attach the syringe to the
bead producing unit. Attach the reaction vessel (with the attached syringe) to the control unit.
Advance the syringe pump arm, so that it touches the plunger. Start the magnetic stirrer so that a
slight vortex is visible. Activate the vibration and the syringe pump. Press the “turbo” button until
a continuous liquid jet is formed. Activate the electrostatic dispersion unit. If needed, modify the
pumping speed or/and the frequency to obtain a clear bead chain down to the collection cup.
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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. Shortly before the syringe plunger is fully depressed, move the collection cup back into the bead
stream. Stop the pump.
Turn off the electrostatic dispersion unit and the vibration control system.
11. Let the beads harden for 5 minutes.
12. To drain the hardening solution from the reaction vessel, slowly open the drain clamp so that it will
take 1 to 2 minutes to drain 500 mL. Turn off the magnetic stirrer when about ¾ of the liquid has
drained. Close the drain clamp as soon as the liquid level reaches the settled beads.
NOTE
Always leave the beads slightly covered with solution to prevent clumping.
13. Fill the pressure bottle with 400 mL of washing solution and pump it into the reaction vessel.
Restart the magnetic stirrer as soon as the magnetic stir bar is covered with liquid so the beads
are not damaged. Wash the beads for 5 minutes and then drain off the liquid as described above
(item 12).
14. Pump the final 200 mL of washing solution into the reaction vessel. Re-suspend the beads by
turning on the magnetic stirrer.
15. Open the bead collecting valve and let the beads flow into the bead collecting flask.
16. Close the silicone tube from the reaction vessel to the bead collecting flask with the clump.
Disconnect the bead collecting flask from the reaction vessel and check the beads for quality
under a microscope.
17. Immediately after the end of the bead production process fill a syringe with distilled water, attach it
to the bead producing unit and flush the nozzle to avoid having the polymer dry and clogging the
system thus creating a maintenance problem. Clean the reaction vessel.
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6.14 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 CaCl
pH = 7.2 at 25°C
3. PLL solution: 0.05 % Poly-L-lysine MG 15’000-30’000 in MOPS washing buffer
2
6 Operation
4. MOPS washing buffer: 10 mM MOPS (Morpholinopropanesulfonic acid)
0.85 % NaCl
pH = 7.2 at 25°C
5. 0.03 % alginate solution: 2 mL of 1.5 % alginate solutio.
+ 98 mL MOPS washing-buffer.
6. Depolymerization solution: 50 mM Na3-Citrate
0.45 % NaCl
10 mM MOPS
pH = 7.2 at 25°C
For one encapsulation of 12 mL polymer-product mixture the following is needed:
• 12 mL 1.5 % alginate solution (sterile filtered)
• 100 mL 0.03 % alginate solution (not sterile)
• 225 mL Polymerization solution (not sterile)
• 75 mL PLL Solution (not sterile)
• 900 mL MOPS Washing buffer (not sterile)
• 200 mL Depolymerization solution (not sterile)
1
Lim F. and Sun A.M. 1980. Microencapsulated Islets as Bioartificial Pancreas. Science 210: p.908-910.
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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. Fill a 20 mL syringe with the cell-alginate suspension and attach the syringe to the reaction vessel
in a laminar air hood.
6. Fix the reaction vessel to the Encapsulator control unit, which is placed on the bench.
7. Start bead formation with previously established parameters.
8. Allow for bead hardening for 5 minutes, then stop stirrer and drain off polymerization solution.
NOTE
The beads and later the capsules should always be covered by a small amount of liquid to prevent
clumping, otherwise re-suspension of the beads and capsules would become difficult and the
membrane might be damaged.
9. Pump in 75 mL 0.05 % PLL solution and let form the PLL-alginate membrane for 10 minutes.
10. Drain of the 0.05 % PLL-solution.
11. Pump in 150 mL MOPS washing buffer, stir for 1 min and then drain off.
12. Pump in another 150 mL MOPS washing buffer, stir for 5 min and then drain off the buffer.
13. Pump in 100 mL 0.03 % alginate solution and allow 5 minutes stirring for the formation of the
outer alginate membrane, then drain off the alginate solution.
14. Pump in 150 mL MOPS washing buffer, stir for 1 min and then drain off the buffer.
15. Pump in 150 mL depolymerization solution and stir approx. 10 minutes to dissolve the alginate of
the bead core. Appropriate dissolution time is dependent on the molecular weight and purity of the
alginate, and the susceptibility of the encapsulation product to the depolymerization solution.
16. Drain off the depolymerization solution.
17. Pump in 150 mL MOPS washing buffer, resuspend the capsules and transfer them into the bead
collecting flask.
18. Transfer the capsules in culture medium and cultivate them.
NOTE
Dissolved alginate diffuses out slowly. Depending on the alginate in use and the thickness of the
capsule membrane, it can take up to 2 h for substantial amounts to leave the capsule. To maximize
removal of the core you can:
• Extend the extraction time in MOPS or in a culture medium without bivalent ions.
• Cultivate the cells in a medium containing < 50 mg/l of Ca ions.
• Cultivate the cells in a medium with a ratio of monovalent ions (Na+, K+) to bivalent ions
(Ca2+, Mg2+) between 20:1 and 50:1.
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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. Large-scale production of Ba2+ alginate-coated islets of Langerhans for
immunoisolation. Exp. Clin. Endocrinol. 102: p.380-387.
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6.15 Theoretical background
Equ. 1:
When a laminar jet is mechanically disturbed at the frequency ƒ,
beads of uniform size are formed1. The optimal wavelength λ
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. 55×10-3 N/m for alginate solutions)
opt
6 Operation
for
λ
is the optimal wavelength to get the best bead formation for the given nozzle diameter and the
opt
viscosity of the encapsulation mixture. It is possible to change λ
by 30 % and still achieve a good
opt
bead formation.
The diameter of a bead = d [m] can be calculated with the flow rate = V’ [m3/s] and the frequency of
the pulsation ƒaccording to:
Equ. 3:
The jet velocity = v [m/s] and the nozzle diameter = D [m] are correlated to the flow rate (V’) according
to:
Equ. 4:
Figure 6-5 shows the dependence of the flow rate to the jet velocity and the nozzle diameter as calculated by Equation 4. Because the liquid must flow laminarly the working range of the jet velocity will
normally lay between 1.5 and 2.5 m/s, depending on the liquid viscosity and the nozzle diameter.
1
Lord Rayleigh 1878. Proc. London Math. Soc. 10:4.
2
Weber C. 1936. Zeitschrift für angewandte Mathematik und Mechanik. 11:136.
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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.
Figure 6-6: Influence of the vibration frequency and the flow rate on the bead diameter as calculated by Equation 4.
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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 2.8 800 to 1800 Hz 1 to 3 0.4 to 0.6
120 µm 1.5 to 1.8 1000 to 2500 Hz 1 to 4 0.5 to 0.7
80 µm 1.1 to 1.3 1300 to 3000 Hz 1 to 4 0.5 to 0.7
* Tested with 2 % low viscosity grade alginate solution for 750 µm and 1.0 mm nozzle, with 1.5 %
alginate solution for the 150 to 500 µm nozzle and with 1.2 % alginate solution for the 80 and 120 µm
nozzles.
**Upper values with application of high voltage.
NOTE
For solutions with a viscosity different from the tested one, it can be said that:
• the higher the viscosity the higher the minimal jet velocity
• the higher the viscosity the higher the working flow rate
• the higher the viscosity the lower the optimal frequency
• the higher the viscosity the larger the beads
6.15.1 Bead productivity and cell density
Figures 6-7 and 6-8 indicate the amount of beads formed from 1 mL of liquid. About 30,000 beads
with a diameter of 0.4 mm will be formed, but only 2,000 with a diameter of 1 mm.
Figures 6-9 and 6-10 indicate the number of cells which are encapsulated in one bead for a given
cell density and bead diameter. These figures may help you select the appropriate cell density in the
immobilization mixture. For example, if the immobilization mixture contains 1×106 cells per mL, then
about 33 cells are, on average, in each 0.4 mm bead, but, about 520 cells will be in each 1 mm bead.
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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.
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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.
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7 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.
NOTE
Use only genuine consumables and spare parts for any maintenance and repair work in order
to assure warranty and continued system performance. Any modifications of the Encapsulator
B-395 Pro or parts of it need prior written permission of the manufacturer.
7.1 Customer service
Only authorized service personnel are allowed to perform repair work on the instrument. Authorization requires a comprehensive technical training and knowledge of possible dangers which might arise
when working at the instrument. Such training and knowledge can only be provided by BUCHI.
7 Maintenance and repairs
Addresses of official BUCHI customer service offices are given on the BUCHI website under:
www.buchi.com. If malfunctions occur on your instrument or you have technical questions or application problems, contact one of these offices.
The customer service offers the following:
• Spare part delivery
• Repairs
• Technical advice
7.2 Housing condition
Check the housing for visible defects (switches, plugs, cracks) and clean it regularly with a damp cloth.
The Encapsulator control unit should be handled as with any other piece of electrical equipment. The
front panel is covered by a polyamide sheet, so that it may be cleaned with a mild detergent solution
or alcohol.
7.3 Sealing conditions
It is recommended to check the integrity of the sealings on a regular base. Gaskets, O-rings and silicone tubing should be replaced periodically (approximately once per year). Check all parts before use
and replace if needed.
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7.4 Cleaning
7 Maintenance and repairs
!
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
7.4.1 Cleaning the nozzle after each immobilization run
It is critical to clean the nozzle immediately after use so that the encapsulation medium (alginate, etc.)
will not dry and clog the system.
1. Leave the nozzle in place on the bead producing unit.
2. Attach a 20 mL or 60 mL syringe to the bead producing unit and inject 20 to 60 mL of distilled
water or of the solvent used for the encapsulation polymer.
3. If needed unscrew the nozzle from the bead producing unit, rinse with deionized water (see
figure 7-1) or an appropriate solvent and dry the nozzle with a flush of air.
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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. Do not use
acid solution for alginate, as this would create a precipitate.
7.4.2 Cleaning a clogged nozzle
Unscrew the nozzle. Pass air or water through the nozzle as shown in figure 7-1.
If the nozzle tip is not clear, soak the nozzle in water, the appropriate solvent, in 1N NaOH or 1N
sulfuric acid (never use HCl) according to the encapsulation mixture for 1 hour at room temperature,
with periodic agitation. Sonication of full stainless steel nozzles is also a helpful procedure. Wear
appropriate protection equipment. Rinse with distilled water, with air and let dry.
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. Do not use
acid solution for alginate, as this would create a precipitate.
7.4.3 Cleaning the reaction vessel and the other vessels
Disassemble the reaction vessel. However the magnet holder should not be disassembled!
Disassemble the liquid transfer system.
Wash all parts, except the air filters, with a mild detergent solution, 0.01N NaOH or 0.01N sulfuric acid
(never use HCl) as appropriate.
Rinse thoroughly with hot water, then with distilled water and let dry.
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8 Troubleshooting
8.1 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.
Unstable bead chain The frequency is too high or too low.
8 Troubleshooting
The nozzle is not adequately cleaned (frequent cause).
The frequency is too high.
The amplitude is too high.
The liquid flow rate is too high or too low.
The Nozzle is not adequately cleaned.
The amplitude is too low or too high.
Non-homogenous bead-size-distribution The liquid flow rate is too high.
The frequency is too high.
The electrostatic tension is too low.
The immobilization mixture is a non-Newtonian liquid, making it
difficult to extrude or to prill.
The bead chain does not separate The electrode is not connected to the control unit.
The electrical tension is too low.
The electrode is not on.
Beads are not visible in the strobo light The vibration unit is not activated.
The vibration unit is not put on the bead producing unit.
The vibration frequency is too low or too high.
The viscosity of the immobilization mixture is too high.
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9 Shutdown, storage, transport and disposal
9 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. Remove all liquids and dusty residues
before packaging the instrument.
!
Warning
Death or serious poisoning by contact or incorporation of harmful substances.
• Wear safety goggles
• Wear safety gloves
• Wear a laboratory coat
• Clean the instrument and all accessories thoroughly to remove possibly dangerous substances
• Do not clean dusty parts with compressed air
• Store the instrument and its accessories at a dry place in its original packaging
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9.2 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. For help, please contact your
local authorities!
NOTE
When returning the instrument to the manufacturer for repair work, please copy and complete the
health and safety clearance form in section 10.2 and enclose it with the instrument.
9 Shutdown, storage, transport and disposal
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10 Declarations and requirements
10.1 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.
This equipment generates, uses and can radiate radio frequency energy and, if not installed and used
in accordance with the instruction manual, may cause harmful interference to radio communications.
Operation of this equipment in a residential area is likely to cause harmful interference in which case
the user will be required to correct the interference at his own expense.
Français:
Cet appareil a été testé et s’est avéré conforme aux limites prévues pour les appareils numériques
de classe A et à la partie 15 des réglementations FCC ainsi qu’à la réglementation des interférences
radio du Canadian Department of Communications. Ces limites sont destinées à fournir une protection adéquate contre les interférences néfastes lorsque l’appareil est utilisé dans un environnement
commercial.
Cet appareil génère, utilise et peut irradier une énergie à fréquence radioélectrique, il est en outre
susceptible d’engendrer des interférences avec les communications radio, s’il n’est pas installé et
utilisé conformément aux instructions du mode d’emploi. L’utilisation de cet appareil dans les zones
résidentielles peut causer des interférences néfastes, auquel cas l’exploitant sera amené à prendre les
dispositions utiles pour palier aux interférences à ses propres frais.
10 Declarations and requirements
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10.2 Health and Safety Clearance
Health and Safety Clearance
10 Declarations and requirements
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. chemical waste, chemical residue or
solvent, require that this form must be duly completed and signed when equipment or
defective parts were delivered to our premises.
Instruments or parts will not be accepted if this declaration is not present.
Equipment
Model: Part/Instrument no.:
1.A Declaration for non dangerous goods
We assure that the returned equipment
has not been used in the laboratory and is new
was not in contact with toxic, corrosive, biologically active, explosive, radioactive or
other dangerous matters.
is free of contamination. The solvents or residues of pumped media have been
drained.
1.B Declaration for dangerous goods
List of dangerous substances in contact with the equipment:
Chemical, substance Danger classification
We assure for the returned equipment that
all substances, toxic, corrosive, biologically active, explosive, radioactive or
dangerous in any way which have pumped or been in contact with the equipment
are listed above.
the equipment has been cleaned, decontaminated, sterilized inside and outside and
all inlet and outlet ports of the equipment have been sealed.
2. Final Declaration
We hereby declare that
- we know all about the substances which have been in contact with the equipment
and all questions have been answered correctly
- we have taken all measures to prevent any potential risks with the delivered
equipment.
Company name or stamp:
Place, date:
Name (print), job title (print):
Signature:
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10.3 Declaration of conformity
10 Declarations and requirements
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BÜCHI Labortechnik AG
CH-9230 Flawil 1/Switzerland
T +41 71 394 63 63
F +41 71 394 65 65
www.buchi.com Quality in your hands
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