IZON AFC(V1) Automated Size Exclusion Chromatography User Guide

IZON AFC(V1) Automated Size Exclusion Chromatography

INSTRUCTIONS

IMPORTANT – Read before operating Izon instrumentation

Use of this instrument in a manner not specified by the instructions in this guide may impair the safety protection provided and/or result in impaired performance or damage to the instrument. Do not operate the instrument outside of its rated supply voltages or environmental range.

Read the whole of the AFC(V1) user manual document before unpacking your Izon equipment or proceeding to the steps below.
The full AFC(V1) user manual can be found at support.izon.com/afc-user-manual

Intended Use:
The AFC(V1) 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 use only. Neither the AFC(V1) 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.

Writing Conventions
Make sure that you follow the precautionary statements presented in this guide. Safety and other special notices will appear in boxes and include those listed in Table 1:

| This symbol indicates general advice on how to improve procedures or recommends measures to take in specific situations
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|

This symbol indicates where special care should be taken

| Text that appears in pink is used to indicate buttons and sequences that are used on the AFC(V1) touch screen

INSTRUMENT SPECIFICATIONS

Section 1A: Instrument Layout
The following components are provided in the box.

Components of the AFC(V1) System:

1. AFC(V1) Instrument with attached Nozzle Set and Silicone Waste Tube
2. Nozzle Set (x3)
3. EV Column Mount (x5)
4. Carousel Cover
5. Reversible Carousel
6. Carousel Plate
7. EVoriginal Reservoir
8. 5.0 mL Eppendorf Tube (x15)
9. 2.0 mL Eppendorf 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

Section 1B: 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 single, qEVoriginal, qEV1 and qEV2.
  Figure 2 and Figure 3 show the layout of the reversible carousel. Note that the flush hole does not have a channel for the tube lid and does not have a number associated.
  In addition to the holes for the fraction collection tubes, both sides of the carousel have the following features:A| Central Well
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  B| Central Well Channel
  C| Flush Hole

Section 1C: Instrument Components that are Required but Not Provided

The following components are required for operation of the instrument but are not provided:

• Computer with Windows 10 OS for updating the AFC(V1) operating firmware.
• A qEV column.
• Pipettes and tips.
• Freshly prepared and 0.22 µm filter-sterilized buffer.
• Freshly prepared 0.5 M NaOH solution for cleaning.
• 70% Ethanol solution for cleaning.

SECTION 2: POWERING YOUR INSTRUMENT

Instrument Power and General Operating Procedures

• Plug into earth-grounded protected outlets only.
• 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.

AFC(V1) CALIBRATION

Section 3A: Aligning the Carousel
After the instrument has been assembled on a level surface, the carousel must be aligned with the fluid nozzle.

1. Press the SETUP on the left-hand side of the main screen.
2. Following the screen, choose CAROUSEL > CALIBRATE
3. Press the -/+ buttons (for coarse alignment) and – -/++ buttons (for fine alignment) until the nozzle is positioned directly above the hole in the flush position.
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.

Section 3B: Calibrating 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(V1) 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 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.

AFC(V1) OPERATION

Section 4A: Mounting the Column to the AFC(V1) Tower
The AFC(V1) will only work with RFID-tagged smart qEV columns. Do not attempt to use un-tagged or non-Izon columns with the AFC(V1) instrument.

1. Turn on the instrument by switching on the power button at the rear of the instrument.
2. Check the waste tube to ensure it empties into a sink or waste vessel.
3. Before a column has been inserted or if a column without an RFID tag has been inserted, the tower lighting will be white, and the main screen will display “No Column”. The AFC(V1) instrument records the number of times that each smart column has been used. The columns are intended to be used up to 5 times. A column will become invalid once it has been used 10 times, and the main screen will display “Invalid Column”.
4. Fit the appropriate column mount onto the AFC(V1) tower.
5. If using a single, qEVoriginal, or qEV1, remove the top cap prior to removing the lower luer cap of the column before proceeding to the next step. If using a qEV2 or qEV10, fit the reservoir to the column and ensure the fluid is flowing properly before proceeding to the next step.
6. Insert the column from above into the column mount, then remove the lower cap and carefully dock the column. Ensure that the IZON logo on the column is facing away from the AFC(V1) tower.
7. Turn the column within the column mount until the type of qEV column used is displayed on the touchscreen.
8. Ensure that the connection between the column and the valve is secure to avoid any leakage.
   It is common for a few drops of buffer to be spilled while connecting the column to the luer connector on the valve. To minimize this, make the connection quickly and be sure to wipe up any drops that land on the instrument.

Section 4B: Adjusting the collection schedule

Once the appropriate column has been selected and mounted to the AFC(V1) tower, the collection schedule must be set.

1. From the main screen, press COLLECTION SCHEDULE.
2. From this screen, you can adjust the fraction count, fraction size, and buffer volume for collection. There are several fraction size options available depending on the column type. Press -/+ to choose your collection count, collection size, and buffer volume.
3. Press X to save your parameters and return to the main menu.
4. When you are ready to start your collection, press START COLLECTION and review your collection parameters.
5. Press YES to proceed to carousel setup or NO to return to the main menu.

Section 4C: Setting up the Carousel
Once you have defined your collection parameters, you are ready to prepare the carousel for collection.

1. Load the collection tubes onto the carousel in the appropriate orientation.
2. Ensure that you have loaded enough tubes for your collection protocol.
3. Gently place the carousel onto the carousel plate. Ensure that the carousel alignment hole engages with the raised carousel alignment pin on the carousel plate.
   Be sure to use the correct collection tubes for your fraction sizes. If you have chosen 2.0 mL fraction sizes for the qEV2, then 2.0 mL collection tubes must be used. Always load the required tubes into the carousel with the lids facing inwards. Ensure that the lids of the tubes do not make contact with the carousel cover.

Section 4D: Flushing the Column

1. The next screen will ask you to mount the column reservoir. If using a qEVoriginal, qEV1, qEV2, or qEV10 column, mount the reservoir. If using a qEV single, a column reservoir is not needed.
2. Press OK to continue.
3. The next screen will ask you whether you would like to flush the column. We recommend flushing of all columns with fresh, filtered PBS buffer.
   • Press YES if you would like to flush the column and ensure that you have the appropriate amount of buffer for the column type.
   • Press NO if the column has already been flushed. The next screen will ask you to prime the column.
4. While the AFC(V1) is in flushing/priming mode, the carousel will be in the flush position and the buffer will flow out of the column and through the flush hole in the carousel, to be pumped out through the waste tube. Press OK to continue.
5. Press OK when the column has been flushed/primed with the recommended volume of buffer.
6. The next screen will prompt the user to place the carousel cover over the
   AFC(V1). The cover protects the carousel from being inadvertently bumped during operation. Press OK to continue.

Section 4E: Sample Loading and Initiating Collection

1. The next screen will ask you to have sufficient buffer available to run the column and to load the sample. Ensure that there is no residual buffer left on the top of the column (remove any excess with a pipette) before adding the sample. Press OK to continue.
   It is important to only load the sample at this point. Adding buffer at the same time or before the sample has fully entered the frit of the column will result in incorrect sample loading.

2. Press OK to continue. The column will start running and the carousel will move to the buffer collection position. The buffer volume will then begin collecting into the central well of the carousel and the lighting will change to red to indicate that the process has started.

3. When the sample has fully entered the upper frit of the column, top up the column/reservoir with the correct amount of buffer to resume collection of the buffer volume.

4. When the buffer volume has been collected, the carousel will move to position 1, collect the specified volume and then progressively move to collect each successive individual fraction. If more fraction tubes are required than the fully loaded carousel, the user will be instructed when to remove the cover and carousel, replace the tube positions with fresh tubes and continue the run.

Section 4F: Completing a run
After a run has been completed, it is important to clean the column to ensure there is no carryover between samples. The AFC(V1) will guide the user through this process via on-screen instructions.

1. When the final fraction has been collected, the display lighting will turn green and the carousel will return to the waste position. The carousel cover and the collection tubes can now be safely removed.
2. If using a qEVsingle, pressing OK will return the user to the home screen as these are not reusable. For all other column types the user will be asked if they wish to clean the column.
3. Select NO to return to the main screen and begin another run without cleaning. This is not recommended.
4. Select YES to be guided through the cleaning process before being returned to the home screen.

Section 4G: AFC(V1) Shut Down

1. Switch off the AFC(V1) at the rear of the instrument.
   The nozzle should be flushed thoroughly with a 70% ethanol solution after the last run of the day to prevent the growth of bacteria within the tubing.

SYSTEM MAINTENANCE

Section 5A: Repair and Servicing of AFC(V1) instrument

• All replacement parts must be obtained via Izon Science Ltd.
• Contact your regional Izon Science representative for replacement parts.
  We recommend that you save the box and packaging materials in case the instrument needs to be returned for servicing.

References

• AFC(V1) User Manual

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