Izon AUTOMATIC FRACTION COLLECTOR V2 User manual

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USER MANUAL
AUTOMATIC FRACTION
COLLECTOR V2 AFC

Izon Science Ltd. provides this document to its customers with a product purchase to use in the product
operation. This document is copyright protected and any reproduction of the whole or any part of this
document is strictly prohibited, except with the written authorisation of Izon Science Ltd.
The contents of this document are subject to change without notice. All technical information in this
document is for reference purposes only. System configurations and specifications in this document
supersede all previous information received by the purchaser.
Izon Science Ltd. makes no representations that this document is complete, accurate or error-free and
assumes no responsibility and will not be liable for any errors, omissions, damage or loss that might
result from any use of this document, even if the information in the document is followed properly.
Izon products are designed and manufactured under a quality system certified to ISO 13485:2016.
This document is not part of any sales contract between Izon Science Ltd. and a purchaser. This
document shall in no way govern or modify any Terms and Conditions of Sale, which Terms and
Conditions of Sale shall govern all conflicting information between the two documents.
Izon Science Ltd Telephone: [phone]
PO Box 9292 Email: [email protected]
Addington Website: www.izon.com
Christchurch 8024
New Zealand
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TABLE OF CONTENTS
1 Definitions and Writing Conventions.......................................................................................................... 4
2 Safety and Hazards........................................................................................................................................ 6
2.1 Safe Use Requirements and Specifications ...................................................................................................6
2.2 Hazards.......................................................................................................................................................................6
3 Introduction to the Automatic Fraction Collector (AFC) ..................................................................... 10
3.1 Overview................................................................................................................................................................... 10
3.2 Intended Use ............................................................................................................................................................ 11
4 Instrument Specifications ............................................................................................................................12
4.1 Instrument Layout................................................................................................................................................. 12
5 Assembly and Setup Instructions ..............................................................................................................15
5.1 Instrument Power and General Operating Procedures............................................................................ 15
5.2 General Operating Procedures......................................................................................................................... 15
5.3 Assembling and Installing the AFC.................................................................................................................. 15
5.4 Aligning the Carousel ........................................................................................................................................... 16
5.5 Calibrating the Load Cell..................................................................................................................................... 18
5.6 Adjusting Lighting Settings ................................................................................................................................ 18
6 qEV Specifications .........................................................................................................................................19
7 Operating Instructions..................................................................................................................................21
7.1 Setting Up a Run.................................................................................................................................................... 21
7.2 Choosing Run Parameters................................................................................................................................. 22
7.3 Initiating a Run........................................................................................................................................................27
7.4 Completing a Run ................................................................................................................................................. 29
8 Resources........................................................................................................................................................30
8.1 Repair and Servicing of AFC Instrument ........................................................................................................... 30
8.2 Updating the AFC Firmware............................................................................................................................. 30
8.3 Maintenance of AFC Instrument ..................................................................................................................... 32
8.4 Troubleshooting..................................................................................................................................................... 36
Automatic Fraction Collector V2 User Manual 3

DEFINITIONS AND WRITING CONVENTIONS1 /
This symbol indicates general advice on how to improve procedures
or recommends measures to take in specific situations
This symbol indicates where special care should be taken
Earth (ground) terminal
Direct Current (DC)
Voltage polarity of the jack
SETUP Text that appears in pink is used to indicate buttons and
sequences that are used on the AFC touchscreen
Make sure to follow the precautionary statements presented in this guide. Safety and other special
notices will appear in boxes and include the symbols detailed in Table 1.
Table 1: Safety and Hazard Symbols
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TERM DEFINITION
AFC Firmware The firmware that is used on the AFC instrument; the user interacts with
this firmware via the instrument’s touchscreen; see Section 8.
AFC Updater
The software package that is used to update the AFC Firmware. The AFC
Updater is run on a computer with Windows 10 that is connected to the
AFC instrument via a USB cable; see Section 8.
Buffer Volume (BV)
The volume of liquid that corresponds to the volume before the Purified
Collection Volume (PCV); this volume is collected automatically into the
central well. The buffer volume is different for each column type and may
be different for different resin types in the same column size series.
Calibration Weight The supplied 10 g weight used to calibrate the load cell. See Section 5.
Default Buffer Volume
AFCs have a default buffer volume setting for each column, which can be
adjusted to tailor the volume that is collected. Refer to the appropriate
qEV column user manual for details on selecting the buffer volume that
suits you.
Flush Position The zero position of the carousel; buffer exiting the column
will enter the flush hole and be emptied through the silicone waste tube.
Fraction A quantity of a sample collected from a fractionating separation process.
Fraction Count The number of fractions to be collected during a run; the maximum
number of fractions that can be collected per run is 30.
Fraction Volume The volume of each fraction collected during a run; there are default
fraction volumes for each column type; see Section 7.
Lighting
There are two light modules on the AFC, the “tower” which corresponds
to the vertical portion of the AFC behind the qEV column mount, and the
“halo” which corresponds to the horizontal ring beneath the carousel; see
Section 5.
Purified Collection Volume (PCV)
The volume immediately succeeding the buffer volume, containing
particles of interest purified from the loaded sample. The PCV can be
customised to accommodate different preferences, e.g. to maximise the
recovery of extracellular vesicles, or to maximise protein removal.
Table 2: Terminology Used in this Manual
Automatic Fraction Collector V2 User Manual 5

SAFETY AND HAZARDS2 /
2.1 Safe Use Requirements and Specifications
Table 3: Safe Use Requirements and Specifications
2.2 Hazards
The AFC is a laboratory product. However, if biohazardous samples are present, adhere to current
Good Laboratory Practices (cGLPs) and comply with any local guidelines specific to your laboratory
and location.
Chemical Hazards
The AFC system contains no potentially hazardous chemical materials, however the qEV columns
are delivered containing < 0.1% sodium azide, which is potentially fatal if swallowed or in contact with
skin. When the appropriate qEV column is used on the AFC system, the user is potentially exposed to
a dilute solution of sodium azide from the column and/or waste hose outlet.
SAFE USE REQUIREMENT SPECIFICATION
Operating
Temperature Indoor Use Ambient temperature of 5-25 ˚C
Altitude Up to 2,000 metres above sea level
Relative Humidity 20-80% relative humidity
Power Consumption 15 W
Ingress Protection IP30
Pollution Degree Rating Pollution Degree 2
Power
Supply Unit
(ATS024T- A120)
Input AC
100-240 V
0.58 A
50/60 Hz
Output DC
12 V nom
2 A max current
24 W max power
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Please review the following guidelines and precautions prior to each use of the qEV column, especially if
flushing with an antimicrobial buffer before storage (recommended).
Prevention:
Do not get into eyes, on skin, or on clothing.
Wash skin thoroughly after handling.
Do not eat, drink, or smoke when using this product.
Avoid release of product into the environment.
Wear protective gloves and clothing; follow general laboratory precautions.
Response
IF SWALLOWED: immediately call a POISON CONTROL CENTRE/Doctor/Hospital.
IF ON SKIN: Gently wash with plenty of soap and water and immediately call a POISON CONTROL
CENTRE/Doctor/Hospital.
Remove immediately any contaminated clothing and wash before reuse.
Collect any spillage and dispose of appropriately.
For more information, see the MSDS Documentation for qEV columns: izon.com/resources
Disposal of Biohazardous Material
Be sure to adhere to the following guidelines and comply with any local guidelines specific to your
laboratory and location regarding use and disposal.
General Precautions:
Always wear laboratory gloves, coats, and safety glasses with side shields or goggles.
Keep your hands away from your mouth, nose, and eyes.
Completely protect any cut or abrasion before working with potentially infectious or hazardous material.
Wash your hands thoroughly with soap and water after working with any potentially infectious or hazardous
material before leaving the laboratory.
Remove watches and jewellery before working at the bench.
The use of contact lenses is not recommended due to complications that may arise during emergency eye-
wash procedures.
Before leaving the laboratory, remove protective clothing.
Do not use a gloved hand to write, answer the telephone, turn on a light switch, or physically engage
people without gloves.
Change gloves frequently.
Remove gloves immediately when they are visibly contaminated.
Do not expose materials that cannot be properly decontaminated to potentially infectious or
hazardous material.
Upon completion of the tasks involving potentially infectious or hazardous materials, decontaminate the work
area with an appropriate disinfectant or cleaning solution (1:10 dilution of household bleach is recommended).
Automatic Fraction Collector V2 User Manual 7

Dispose of the following potentially contaminated materials in accordance with laboratory local,
regional, and national regulations:
Biological samples
Reagents
Used reaction vessels or other consumables that may be contaminated
Explosive or Flammability Hazards
The AFC poses no uncommon hazard related to flammability or explosion when used in a proper
manner as specified by Izon Science Ltd.
Mechanical Hazards
The carousel is capable of moving during sample collection, calibration, and when the unit returns
to the home position. Please keep fingers and loose clothing clear, and refrain from removing the
carousel cover when the unit is in operation.
Electrical Hazards
The AFC poses no uncommon electrical hazard to operators if installed and operated properly
without physical modification and connected to a power source of proper specification.
The user is protected from hazardous voltage with power adaptor double insulation.
Touching exposed USB connection on back of unit without electrostatic discharge (ESD) precaution
may lead to temporary fault. Please avoid contacting USB port unless updating unit. In the event of
an ESD event that causes a temporary fault, please switch off the unit and leave for 1 minute before
resuming operation
Use only the power supply unit provided with this product. Specification of unit is detailed in Table 3.
Use as directed: install indoors and avoid contact between the AFC and spilled fluids.
Transport
Before moving or shipping the AFC, decontamination procedures must be performed. Always move or
ship the AFC with the supplied packaging materials which will protect the instrument from damage. If
appropriate containers cannot be found, contact your local Izon office.
Storage
The AFC system can be stored under the following conditions:
Temperature range: 0 to 60 ˚C
Relative humidity: maximum 80%
Disposal
The AFC system contains electronic or electrical materials; it should be disposed of as unsorted waste and
must be collected separately, according to the European Union Directive: Waste Electrical and Electronic
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Equipment – WEEE Directive. The user is fully responsible for ensuring that the obsolete equipment
and/or consumables are recycled or disposed of in accordance with this and/or any other relevant
laws and regulations in the countries where the instrument is being recycled or disposed of.
Alternatively, obsolete equipment and/or consumables may be returned to your local Izon Science
representative to be processed appropriately. Contact your local Izon representative for more
information.
Automatic Fraction Collector V2 User Manual 9

INTRODUCTION TO THE AUTOMATIC
FRACTION COLLECTOR AFC
3 /
3.1 Overview
Isolation of extracellular vesicles (EVs) using the combination of qEV size-exclusion columns and
AFC increases uniformity of collected volumes, decreases experimental run-time, and eliminates
many of the inaccuracies that are inherent in the manual collection of the PCV. The AFC
offers users the ability to scale up EV isolation through automation of several steps, including
equilibration, PCV elution, cleaning, and regeneration of the columns.
The AFC manages the collection of fractions from the column by automatically separating the
buffer volume (pink area in Figure. 1) and the volume of the fractions containing EVs. The buffer
volume is collected in the central well of the carousel. The type of qEV column used in each run
will determine the volume of liquid that is collected in the central well. Once the buffer volume has
been collected, the instrument will begin collecting a predetermined number of fractions of a given
volume as set by the user. The fractions after the buffer volume correspond to the PCV (blue zone
in Figure. 1).
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The AFC measures the volume of each fraction by weight as it fills with liquid eluting from the column.
When the designated threshold weight is reached, the fluid flow is automatically stopped to prevent
dripping of fluid during repositioning of the carousel.
The type of qEV column used in each run will depend on the user’s preferences and research
applications. For more information on which column to use, see Section 6: qEV Specifications and for
more information on choosing a collection schedule, see Section 7: Choosing Run Parameters.
3.2 Intended Use
The AFC is used to automate fraction collection of Izon’s qEV columns.
The instrument is intended for use in research laboratories by professional personnel for research purposes
only. Neither the AFC nor the qEV column are intended for diagnostic purposes and should not be used to
make treatment decisions.
For verification of the entire system, it is recommended that Good Laboratory Practices (GLPs) be followed
to ensure reliable analyses.
Figure 1: Typical elution profile for a qEVoriginal/35 nm Gen 2 column with 0.5 mL of human plasma loaded; proteins elute in
a later volume than extracellular vesicles (EVs) and similarly sized particles >60 nm. The particle concentration was measured
using an Exoid and protein levels by bicinchoninic acid (BCA) assay.
16
14
0
2
6
8
4
0.4 1.2 2.0 3.6 4.4 5.2 6.0 7.66.8 8.42.8
12
10
Volume (mL)
Concentration of EVs (and similarly sized particles >60 nm)/mL
% of total loaded protein
Default
Buffer
Volume
(2.9 mL)
Default
PCV
0.0E+00
5.0E+10
1.0E+11
3.5E+11
3.0E+11
2.5E+11
2.0E+11
1.5E+11
Automatic Fraction Collector V2 User Manual 11

1AFC Instrument with attached Nozzle Set and Silicone Waste Tube
2Nozzle Set (x3) - The entirety of the nozzle tip, silicone tubing, and the adaptor
used to connect qEV columns
3qEV Column Mount (x5)
4Carousel Cover
5Reversible Carousel
6Carousel Plate
7qEVoriginal Reservoir
85.0 mL Collection Tube (x15)
92.0 mL Collection Tube (x30)
10 12 Volt Power Supply
11 Power Lead
12 USB 2.0 Type A to USB 2.0 Micro Cable (male to male)
13 10 g Calibration Weight
INSTRUMENT SPECIFICATIONS4 /
4.1 Instrument Layout
The following components are provided in the box.
Table 4: Components of the AFC System
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Layout of the Reversible Carousel
The carousel is reversible and can be used in two orientations:
One side has seven large holes and uses 5 mL collection tubes.
One side has thirteen small holes and uses either 1.5 mL or 2.0 mL collection tubes. This side cannot
be used with the qEV10.
qEV100 is not compatible with the AFC.
Note: some collection tube brands will not fit properly into the AFC carousel. We recommend ordering
2 mL and 5 mL Axygen tubes (Product codes: MCT-200-C; MCT-500-C, respectively).
Figure 3 and Figure 4 show the layout of the reversible carousel. Notice that the flush hole does not
have a channel for the tube lid.
Figure 2: Components of the AFC system.
Automatic Fraction Collector V2 User Manual 13

The following components are required for operation and/or maintenance of the instrument but are
not provided:
Computer with Windows 10 for updating the AFC operating firmware.
A qEV column.
Pipettes and tips.
Freshly prepared, filtered (0.22 m) and degassed buffer. Refer to your qEV user manual for more
information about buffers.
Freshly prepared 0.5 M NaOH solution for column cleaning.
70% ethanol solution for cleaning.
ACentral Well
BCentral Well Channel
CFlush Hole
Figure 3: Layout of the carousel with large holes. Figure 4: Layout of the carousel with small holes.
C
B
AA
C
B
In addition to the holes for the collection tubes, both sides of the carousel have the following features:
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ASSEMBLY AND SETUP INSTRUCTIONS5 /
5.1 Instrument Power and General Operating Procedures
Make sure the power supply box is positioned away from fluids.
To prevent heat build-up, do not cover the power supply box.
Position unit so it can be quickly and easily disconnected from the mains power.
Check the local supply meets the AC input requirement given in the specification.
Izon instruments are only to be operated with Izon supplied leads and power supplies. Failure to use
the correct power supply may result in invalid operation.
Make sure the power supply is placed away from and to the rear of the instrument to avoid coming into
contact with any spills or fluid.
5.2 General Operating Procedures
For indoor use only and to be used within the rated conditions noted in system specifications.
Take care not to spill any fluids on electrical parts during operation.
5.3 Assembling and Installing the AFC
1. Unpack the AFC and box contents.
We recommend that you save the box and packaging materials in case the instrument needs to be
returned for servicing.
If you choose not to save the materials, please recycle them wherever cardboard recycling services
are provided.
The AFC has a sensitive weight scale and should not be installed near any equipment that causes
vibrations, such as centrifuges, culture and plate shakers, vortex mixers, sonication devices, etc.
as this could negatively impact the load cell measurements.
Automatic Fraction Collector V2 User Manual 15

2. Place the AFC onto a stable and level laboratory bench.
3. Ensure that the clear silicone waste tube of the AFC exits from the rear or side of the instrument
and empties into a sink or waste vessel.
4. Connect the power lead to the 12 Volt power supply.
5. Plug the 12 Volt power supply into the rear of the instrument.
6. Plug the power lead into a wall socket.
7. Turn on the instrument using the power switch at the rear. The screen will light up briefly showing
the IZON logo and the carousel will rotate to its zero position. Ensure the nozzle set is in place, as
shown in Figure 5.
Figure 5: AFC nozzle set correctly in place.
5.4 Aligning the Carousel
After the instrument has been assembled on a level surface, the carousel must be aligned with the fluid
nozzle. If the fluid nozzle is already above the flush hole (See Figure 7), then this step may be skipped.
1. Press Settings on the left-hand side of the home screen as shown in Figure 6:
Figure 6: Press Settings on the home screen.
2. Select Carousel Calibration.
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3. Press the </> buttons (for fine alignment) and <</>> buttons (for coarse alignment) until the nozzle is
positioned directly above the hole in the flush position as shown in Figure 7.
Figure 7: The nozzle tip centred over the flush hole. Note that for the side of the carousel that holds 2 mL tubes, the flush hole will
be on the right of the central well channel. For the other side of the carousel that holds 5 mL tubes, the flush hole is on the left of
the central well channel.
4. Once the flush hole has been aligned with the nozzle, press Set Zero. Press Back to return to
the home screen.
Improper calibration of the carousel can cause drops from the nozzle to land on the carousel or miss
collection tubes. If this occurs, simply recalibrate the carousel. For more information, please refer to
the Troubleshooting Guide.
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5.5 Calibrating the Load Cell
The AFC load cell measures the weight of each fraction to determine its volume and ensures uniform
volumes across all fractions. Calibration of the load cell is performed using the supplied 10 g weight.
Calibration of the Load Cell
1. From the home screen, press Settings > Scale Calibration > Start Calibration, and follow the
instructions on the screen.
2. When instructed, remove the carousel and place the carousel cover onto the AFC and press OK.
3. The lighting will change to red to indicate that the instrument is measuring the zero weight.
Once the measurement process has started, the user should not bump or interfere with the unit
until the lighting has changed back to green to indicate that the process is complete. Any interference
will require the user to restart the calibration process.
4. Once the measurement is complete, the lighting will change to yellow and the screen will instruct
the user to place the 10 g calibration weight onto the centre of the scale/carousel plate. Remove
the carousel cover to place the weight.
5. Replace the carousel cover. Ensure that the weight is completely still BEFORE pressing OK to
continue.
6. The lighting will then change to red to indicate that the instrument is measuring the
calibration weight.
7. Once the load cell calibration is complete, the lighting will change to green if the calibration was
successful, press OK to return to the setup menu.
8. Press Back to return to the home screen.
The scale should be calibrated before the first use of the AFC, if the unit has been shifted, after every
firmware update, or if the AFC is measuring inconsistent fraction volumes during runs. For more
information, please refer to the Troubleshooting Guide.
5.6 Adjusting Lighting Settings
The user can choose whether to leave the tower and halo lights on or off through the Setup Menu.
1. From the home screen, press Settings > Lighting.
2. On the following screen, select or deselect the tower or halo lighting modules.
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Choosing the right qEV column to run will depend on the volume of the sample and the target
isolation range of the analyte.
Table 5: qEV Legacy Column Specifications
COLUMN TYPE qEVSINGLE
LEGACY
qEVORIGINAL
LEGACY
qEV2
LEGACY
qEV10
LEGACY
Optimal Sample Loading Volumea0.15 mL 0.5 mL 2 mL 10 mL
Reusability No, single use Yes, up to 5
times
Yes, up to 5
times
Yes, up to 5
times
Column Volume 3.5 mL 9.6 mL 45.1 mL 69.3 mL
Buffer VolumebqEV/35
0.8 mL
qEV/70
1.0cmL 2.7 mL 14.1 mL 21.4 mL
Flush Volume 7.0 mL 20.0 mL 90.0 mL 140.0 mL
Optimal Fraction Size 0.2 mL 0.5 mL 2.0 mL 5.0 mL
0.5 M NaOH required per clean N/A 10 mL 2 mL 5 mL
Buffer required per clean N/A 20.0 mL 180.0 mL 280.0 mL
Buffer reservoir required No Yes Yes Yes
Collection tube size 1.5 or 2 mL 1.5, 2 or 5 mL 1.5 or 2 mL 5 mL
qEV SPECIFICATIONS6 /
a / Loading a lesser volume than the optimal sample loading volume will result in higher sample dilution, while loading a higher
volume can overload the column matrix and cause contamination of the purified sample in the PCV.
b / Buffer volumes stated are exclusive of an additional 0.284 mL from the volume of buffer inside the nozzle set.
c / EVs and similarly sized particles have been detected from 0.8 mL on a qEVsingle Legacy column (70 nm Series), however the
concentration is extremely low and may cause significant sample dilution if included.
Automatic Fraction Collector V2 User Manual 19

Table 6: qEV Gen 2 Column Specifications
a /Loading a lesser volume than the optimal sample loading volume will result in higher sample dilution, while loading a
higher volume can overload the column matrix and cause contamination of the purified sample in the PCV.
b /Buffer volumes stated are exclusive of an additional 0.284 mL from the volume of buffer inside the nozzle set.
COLUMN TYPE qEVSINGLE
GEN 2
qEVORIGINAL
GEN 2 qEV1 qEV10 GEN 2
Optimal Sample Loading Volume0.15 mL 0.5 mL 1 mL 10 mL
Reusability No, single use Yes, up to 5
times
Yes, up to 5
times Yes, up to 5 times
Column Volume 3.0 mL 8.5 mL 13.5 mL 69.3 mL
Buffer Volume0.7 mL 2.5 mL 4.0 mL qEV/35
22.9 mL
qEV/70
23.2 mL
Flush Volume 6.0 mL 17 mL 27 mL 140 mL
Optimal Fraction Size 0.17 mL 0.4 mL 0.7 mL 5 mL
0.5 M NaOH required per clean N/A 8.5 mL 13.5 mL 20 mL
Buffer required per clean N/A 17 mL 27 mL 280 mL
Buffer reservoir required No Yes Yes Yes
Collection tube size 1.5 or 2 mL 1.5, 2 or 5 mL 1.5, 2 or 5 mL 5 mL
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