Izon qEV User manual

www.izon.com
USER MANUAL
AUTOMATIC FRACTION
COLLECTOR
(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.
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
PO Box 9292
Addington
Christchurch 8024
New Zealand
Telephone: [phone]
Email: [email protected]
Website: www.izon.com
2www.izon.com

CONTENTS
Section 1: Definitions and
Writing Conventions................................................ 4
Section 2: Safety and Hazards........................... 7
Safe Use Requirements
and Specifications.............................................. 7
Hazards .................................................................. 7
Section 3: Introduction to the Automatic
Fraction Collector (AFC)...................................... 10
Overview.............................................................. 10
Intended Use ....................................................... 11
Section 4: Instrument Specifications.............. 12
Instrument Layout............................................ 12
Layout of the Reversible Carousel............. 13
Sample Buffer recommendations:............. 14
Section 5: Assembly and Setup
Instructions............................................................... 15
Instrument Power and General
Operating Procedures.................................... 15
General Operating Procedures................... 15
Assembling and Installing the AFC ........... 15
Aligning the Carousel...................................... 16
Calibrating the Load Cell............................... 18
Adjusting Lighting Settings........................... 18
Section 6: qEV Specifications............................ 19
Section 7: Operating Instructions ................... 22
Choosing run parameters............................ 22
Carousel side selection.................................. 22
Tube type selection......................................... 22
Volume Size Selection.................................... 23
Setting up a run ............................................... 23
Mounting the qEV Column to the
AFC Tower.......................................................... 23
Column Use Counter and Invalid
Columns .............................................................. 25
Adjusting the Collection Schedule ............26
Setting up the Carousel .................................27
Initiating a run .................................................. 28
Flushing the Column.......................................28
Sample Loading and Initiating
Collection ............................................................29
Completing a run............................................. 30
AFC Shut Down................................................ 30
Section 8: Resources ............................................ 31
Repair and Servicing of AFC instrument ...... 31
Updating the AFC Software......................... 31
Requirements for Updating the
AFC Operating Software............................... 31
Downloading and Installing the
AFC Software Updater .................................. 31
Updating the AFC Operating Software.........32
Maintenance of AFC Instrument ............... 33
Calibration Testing .......................................... 33
Surface Decontamination ............................ 33
Replacing the Nozzle...................................... 34
Troubleshooting.................................................37
Automatic Fraction Collector User Manual 3

DEFINITIONS AND WRITING
CONVENTIONS
1
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
www.izon.com
4

Term Definition
AFC Operating Software
The software that is used on the AFC instrument; the user
interacts with this software via the instrument’s touchscreen;
See Section 8.
AFC Software Updater
The software package that is used to update the AFC Operating
Software; it is run on a computer with Windows 10 OS that is
connected to the AFC instrument via a USB cable; See Section 8.
Buffer
A pH-stabilised electrolyte solution used to flush the column and
elute samples. PBS buffer is often used with biological samples
to maintain the functional integrity of sample particles, as it
mimics the pH and osmolarity of blood.
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.
Buffer Volume collection position
The position in which fluid exiting the column will enter the
central well via the central well channel; See Figure 3 and
Figure 4.
Calibration Weight The supplied 10 g weight that is used to calibrate the load cell.
See Section 5.
Column Type The size of the qEV column; the AFC will only work with Izon
qEVsingle, qEVoriginal, qEV1, qEV2, and qEV10 columns.
EVs Extracellular vesicles, including exosomes.
Exosomes A type of extracellular vesicle, typically defined as being between
20 and 100 nm.
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.
Table 2: Terminology Used in this Manual
Automatic Fraction Collector User Manual 5

Term Definition
Fraction Volume The volume of each fraction collected during a run; there are
pre-set fraction volumes for each column type; See Section 7.
Gel Type All qEV columns come with either 35nm or 70nm gel;
See Section 6.
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 base beneath the carousel; See Section 5.
Load Cell
The component of the AFC that measures the weight of each
fraction; the load cell is calibrated using the 10 g calibration
weight; See Section 5.
Nozzle The area from which fluid exits the qEV column.
Nozzle Set The entirety of the nozzle tip, a piece of silicon tubing, and the
adapter used to connect qEV columns is called the nozzle set.
Purified Collection Volume (PCV)
The volume immediately suceeding the buffer volume, containing
particles of interest purified from the loaded sample. The PCV is
different for each column type.
qEV Smart Column
The type of column that is used with the AFC; these columns
are manufactured with an embedded RFID chip; the AFC is not
intended to be used with any other column.
USB Cable The USB 2.0 Type A to USB 2.0 Micro cable (male to male) that is
provided with the AFC instrument.
www.izon.com
6

SAFETY AND HAZARDS2
Safe Use Requirements and Specifications
Table 3: Safe Use Requirements and Specifications
Hazards
The AFC is a laboratory product. However, if biohazardous samples are present, adhere to current Good
Laboratory Practices (cGLP) and comply with any local guidelines specific to your laboratory and location.
Disposal of Biohazardous Material
The AFC system contains no potentially hazardous biological materials, however the qEV columns are
delivered containing < 0.1% sodium azide, which is potentially fatal if swallowed or in contact with skin.
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.
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
Automatic Fraction Collector User Manual 7

– 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 Izon qEV columns: izon.com/sds
Sodium Azide can be fatal if swallowed or in contact with skin. It can cause damage to neurological
organs through prolonged or repeated exposure. It is very toxic to aquatic life with long lasting effects.
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).
Dispose of the following potentially contaminated materials in accordance with laboratory local,
regional, and national regulations:
– Biological Samples
www.izon.com
8

– Reagents
– Used reaction vessels or other consumables that may be contaminated
Chemical Hazards
The AFC system contains no potentially hazardous chemical materials.
However, 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. Refer to page
7 of this manual under the heading “Disposal of Biohazardous Material” for guidlines and precautions
to mitigate the risk of exposure to Sodium Azide.
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 postition. 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.
Use only the power supply unit provided with this product. Specification of unit is detailed in Table 3.
The AFC system is to be installed indoors and positioned away from any 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 Science 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
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 Science representative for more information.
Automatic Fraction Collector User Manual 9

INTRODUCTION TO THE AUTOMATIC
FRACTION COLLECTOR (AFC)
3
Overview
Isolation of extracellular vesicles (EVs) using the combination of qEV size-exclusion columns and the
Automatic Fraction Collector (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
the equilibration, PCV elution, washout, and regeneration of the columns.
The AFC manages the collection of fractions from the column by automatically differentiating between
the buffer volume (pink area in Fig. 1) and the volume of the fractions containing EVs. The buffer volume
does not contain any EVs and is emptied into the central well of the carousel. The type of qEV column
used in each run will determine the volume of liquid that is emptied into 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
Fig. 1).
10 www.izon.com

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.
Intended Use:
The AFC is used to automate fraction collection of Izon’s qEV size-exclusion columns.
The instrument is intended to be used 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 (GLP) be followed to
ensure reliable analyses.
Figure 1: Typical elution profile for the qEVoriginal. Larger extracellular vesicles (EVs) and exosomes elute first, while smaller
vesicles and protein complexes elute later; 500 L of bovine plasma on a qEVoriginal; concentration of EVs measured with a
qNano and TRPS; concentration of proteins measured with spectrophotometry.
*Volumes are labelled as the highest volume in that sample i.e label “0.5” refers to the volume from 0.0-0.5 mL after the buffer
volume, label “1.5” refers to the volume from 1.0-1.5 mL after the buffer volume and so on.
Volume (mL)
0.25
0.15
0.30
0.20
0.10
0.05
0.00
1.4
1.0
1.6
1.2
0.8
0.6
0.4
0.0
0.2
Concentration (EVs/mL x 1010)
Plasma protein (Abs 280 nm)
Buffer Volume PCV
0.5 1.5 6.55.54.53.52.5
Automatic Fraction Collector User Manual 11

1AFC Instrument with attached nozzle set and silicone waste tube
2Nozzle Set (x3)
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
Instrument Layout
The following components are provided in the box.
Table 4: Components of the AFC System
www.izon.com
12

Figure 2: Components of the AFC System
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. This side is for use with the qEV10 ONLY.
– One side has thirteen small holes and uses either 1.5 mL or 2.0 mL collection tubes. This side can be
used with the qEVsingle, qEVoriginal, qEV1, or qEV2.
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.
In addition to the holes for the collection tubes, both sides of the carousel have the following features:
Automatic Fraction Collector User Manual 13

The following components are required for operation and/or maintenance of the instrument but are
not provided:
–Computer with Windows 10 OS for updating the AFC operating firmware.
–A qEV column.
–Pipettes and tips.
–Freshly prepared and 0.22 m filter-sterilised buffer.
–Freshly prepared 0.5 M NaOH solution for cleaning.
–70% Ethanol solution for cleaning.
Sample Buffer Recommendations:
– Buffer temperature should be within the operational temperature of 18-24˚C (65-75˚F).
– Use a buffer with an ionic strength of 0.15 M or greater to avoid any unwanted ionic interactions
between the solute molecule and the matrix.
– qEV columns come equilibrated in filtered PBS containing < 0.1% w/v sodium azide.
– To maintain the functionality of EVs, the buffer used with the columns should be of the same ionic
strength and pH as the original sample buffer.
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
www.izon.com
14

ASSEMBLY AND SETUP INSTRUCTIONS5
Instrument Power and General Operating Procedures
– Make sure 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 the instrument and to the rear of the
instrument to avoid coming into contact with any spills or fluid.
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.
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 User Manual 15

2. Place the AFC onto a stable and level laboratory bench.
3. Ensure that the clear silicon waste tube of the AFC exits from the rear or side of the instrument and
empties into a sink or waste bottle.
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 valve is in place, as shown
Figure 5 below:
Figure 5: AFC valve in position
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 SETUP on the left-hand side of the main screen as shown in Figure 6 below:
Figure 6: Press SETUP on the main menu screen
www.izon.com
16

2. Following the screen, choose CAROUSEL > CALIBRATE
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 below.
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. You will then return to
the main setup 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.
Automatic Fraction Collector User Manual 17

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 main menu, press SETUP > SCALE > CALIBRATE, and follow the instructions on the screen.
2. When instructed, place an empty carousel onto the AFC and press OK.
3. The lighting will change to red to indicate that the instrument is measuring the weight of the carousel.
Once the measurement process has started, the user should not bump or interfere with the carousel
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 green and the screen will instruct
the user to place the 10 g calibration weight onto the centre of the empty carousel.
5. 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 user will be taken back to the setup menu.
8. Press EXIT to return to the main screen.
Calibration of the load cell should be completed on first use, after every software update,
and if your fraction volumes are being measured inconsistently. For more information,
please refer to the Troubleshooting Guide.
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 main menu, press SETUP > LIGHTING.
2. On the following screen, select or deselect the tower or halo lighting modules.
www.izon.com
18

Column Type qEVsingle qEVoriginal qEV2 qEV10
Optimal Sample Loading
Volume150 L 500 L 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 VolumeqEV/35
0.8 mL
qEV/70
1.0 mL 2.7 mL 14.1 mL 21.4 mL
Flush Volume 3.5-4.0 mL 20.0 mL 60.0 mL 120.0 mL
Optimal Fraction Size 200 L 500 L 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 120.0 mL 240.0 mL
Buffer reservoir required No Yes Yes Ye s
Collection tube size 1.5 or 2.0 mL 1.5 or 2.0 mL 1.5 or 2.0 mL 5.0 mL
qEV SPECIFICATIONS6
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
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.
Buffer volumes stated are exclusive of an additional 0.25 mL from the buffer volume inside the nozzle set.
EVs and similarly sized particles have been detected from 0.8 mL on a qEVsingle/70 column, however the concentration is
extremely low and may cause significant sample dilution if included.
Automatic Fraction Collector User Manual 19

Table 6: qEV Gen 2 Column Specifications
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.
Buffer volumes stated are exclusive of an additional 0.25 mL from the buffer volume inside the nozzle set.
Column Type qEVoriginal qEV1
Optimal Sample Loading
Volume500 L 1 mL
Reusability Yes, up to 5
times
Yes, up to 5
times
Column Volume 8.5 mL 13.5 mL
Buffer Volume2.5 mL 4.0 mL
Flush Volume 17 mL 27 mL
Optimal Fraction Size 400 L 700 L
0.5 M NaOH required per clean 8.5 mL 13.5 mL
Buffer required per clean 17 mL 27 mL
Buffer reservoir required Yes Yes
Collection tube size 1.5 or 2.0 mL 1.5 or 2.0 mL
www.izon.com
20