• Inspect the complete system for eluent leakage.
• The system can be left filled with buffer overnight. If you are not using the separation unit for a few days, wash the flow path with distilled water. Remove the column and the pH electrode (if applicable). Replace the column by a bypass capillary and fit the pH dummy electrode. Then wash the system with 20% ethanol and store it in 20% ethanol. Make sure that all tubing and all flow paths used are rinsed.

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Refer to the Calibrate the pH electrode procedure below.

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• Check for leakage. If there is a sign of liquid leaking between the pump head and the housing side panel or increased or decreased volume of rinsing solution, run the Replace the piston seals procedure.
• When changing eluent, it is important to remove trapped air and run the Purge the pump procedure. If there is still air in the inlet tubing, stop and run the Removing trapped air bubbles from the pump procedure.
  
  Note: If air is allowed to enter the columns, their performance can be heavily altered or destroyed.

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Check the inlet filters visually and replace them if necessary.

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• Change rinsing solution. Always use 20% ethanol as rinsing solution.
• If the volume of rinsing solution in the storage bottle has increased, it can be an indication of internal pump leakage. Run the Replace the piston seals procedure.
• If the volume of rinsing solution in the storage bottle has decreased significantly, check if the rinsing system connectors are mounted properly.
• If the rinsing system connectors are not leaking, the rinsing membranes or piston seals may be leaking. Run the Replace the membranes and piston seals procedure.

 

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Run the Cleaning the system surface procedure.

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Check that all fans are running.

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Check that flow restrictor generates the following back-pressure:
FR-904: 0.4 ±0.05 MPa
FR-902: 0.2 ±0.05 MPa

Check the back-pressure as follows:

1
Disconnect the flow restrictor.

2
Connect a capillary (approx. 1 m, i.d. 1 mm) to a free port in valve V2. Set the valve manually to this port. Put the open end in a waste container.

3
Run the pump at 10 ml/min with water. Note the back-pressure (Bp1) on the pump display, or in the Run Data window.

4
Connect the flow restrictor to the open end of the capillary (observe the IN marking). Put the flow restrictor in the waste container.

5
Run the pump at 10 ml/min with water. Note the back-pressure (Bp2) on the pump display, or in the Run Data window.

6
Calculate the back-pressure generated by the flow restrictor. Replace it if it is not within limit.

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Run the Check the UV monitor procedure

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Run the Check the UPC monitor procedure

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Run the Cleaning the flow cell and optical fiber connectors procedure below.

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Refer to the Clean the flow cells procedure below

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Check that the mixer chamber is clean and without damage. Check the tubing connectors. Replace if required.

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Check for external or internal leakage. Run the Replace channel plate and distribution plate procedure yearly or when required.

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Replace the complete mixing chamber on a regular basis.

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• Run the Replace piston seals and piston procedure.
• Run the Clean or replace the inlet and outlet check valves procedure.
• Calibrate the pressure sensor.

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• Run the Clean the flow cell procedure.
• Calibrate the temperature sensor when changing conductivity cell.
• Enter a new cell constant when changing conductivity cell.
• Calibrate the cell constant if specific conductivity with high accuracy is measured.

 

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• Run the Clean the conductivity flow cell procedure.
• Run the Clean the pH electrode flow cell procedure.

 

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Replace the on-line filter.

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A replacement kit, Valve Kit INV-907, is available, see Spare parts recommended to keep on site in the Spare Parts section under the Related Products tab for code no.

1
Ensure that the valve is in position 1 and then disconnect it from the pump.

2
Remove the 4 screws on the front using the supplied 3 mm Allen key. Loosen each one equally in turn so the distribution plate comes off parallel to the valve body.

3
Slide the screws out.

4
Remove the distribution plate containing the ports.

5
Remove the old channel plate and insert a new one.

6
Remount a new distribution plate so that the text 3 is horizontal and to the right of the central tubing connection. Using the Allen key, tighten the 4 screws in turn, a little at a time, until the distribution plate is fixed to the valve body.

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1
Select menu Check and press OK.

2
Select menu Check Lamp Intensity.

Note: R 215.5 S 214.7mV

If:
R<300mV for 254 nm,
R<150mV for 280 nm, or
R<150mV for 214 nm,

replace the lamp assembly according to the Changing the lamp assembly procedure (optional), or contact your your local GE Healthcare service representative for lamp replacement.

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1
Select menu Check and press OK.

2
Select menu Check Lamp Run Time.

Note: Hg 2300h Zn 340h

• The lifetime of a Hg lamp at 254 nm is typically 7000 hours in room temperature (in coldroom, typically 2000 h).
• The lifetime of a Hg lamp at 280 nm is typically 3500 hours in room temperature.
• The lifetime of a Zn lamp is typically 2000 hours in room temperature.

When necessary, replace the lamp assembly according to Changing the lamp assembly procedure (optional), or contact your your local GE Healthcare service representative for lamp replacement.

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The internal absorbance value for autozero can be checked to test the consistency of buffers.

1
Select menu Check and press OK.

2
Select menu Check Autozero. The autozero absorbance value for the wavelength used is shown.

Note: AZ 0.0001 AU

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Remove the pH electrode and install the dummy electrode in the pH flow cell.

Pump a cleaning or sanitizing agent through the flow cells. The standard recommendation is to pump 1 M NaOH for 30 minutes and then wash out with buffer.

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Pump a cleaning or sanitising agent through the flow cell. The standard recommendation is to pump 1 M NaOH for 30 minutes and then wash out with buffer.

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A clean flow cell is essential for ensuring the correct operation of the UV monitor.

1
Connect a syringe to the inlet of the flow cell and squirt distilled water through the cell in small amounts. Then fill the syringe with a 10% surface active detergent solution like Decon 90, Deconex 11, RBS 25 or equivalent, and continue to squirt five more times.

2
After five squirts, leave the detergent solution in the flow cell for at least 20 minutes.

3
Pump the remaining detergent solution through the flow cell.

4
Rinse the syringe and then flush the flow cell with distilled water (10 ml).

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Pump a cleaning or sanitising agent through the flow cell. The standard recommendation is to pump 1 M NaOH for 30 minutes and then wash out with buffer.

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If the conductivity measurements are not comparable to previous results, the electrodes in the flow cell may be contaminated and require cleaning. To clean the flow cell:

1
Pump 15 ml of 1 M NaOH at 1 ml/min through the flow cell either by using a pump or a syringe.

2
Leave it for 15 minutes.

3
Rinse thoroughly with 50 ml distilled water.

Note: If the flow cell is totally blocked, the blockage can be removed using a needle or a wire with a diameter less than 0.8 mm.

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1
Use a screwdriver to detach the end plate by removing one and loosening the other of the two holding screws on the lamp housing to be removed.

2
Slide the lamp housing off the filter housing.

3
Detach the end plate, as in step 1 above, from the lamp housing to be fitted to the optical unit.

4
Slide the lamp housing onto the filter housing. The lamp and signal cables should be on the same side. As you slide the lamp housing into position, depress the two pressure pads on the filter housing in sequence to facilitate the installation.

5
Refit the lamp housing end plate.

6
Slide the lamp housing firmly into place. There will be a faint click when the housing is positioned correctly. The Hg lamp housing can take up two positions, one for 280 nm, marked by on the filter housing, and the other marked by for all other wavelengths. The Zn lamp housing has only one position.

7
Set the wavelength to be used by selecting lamp position (indicated by a dot on the lamp housing) in combination with the appropriate filter, i.e. the dot on the lamp housing should be adjacent to the symbol on the filter housing corresponding to the symbol on the filter wheel for the filter to be used. A click will indicate that the filter is in position.

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Cleaning the flow cell

A clean flow cell and optical connectors are essential for ensuring the correct operation of the UV-monitor.

1
Connect a syringe to the inlet of the flow cell and squirt distilled water through the cell in small amounts. Then fill the syringe with a 10% surface active detergent solution like Decon 90, Deconex 11, RBS 25 or equivalent, and squirt five times.

2
After five squirts, leave the detergent solution in the flow cell for at least 20 minutes.

3
Pump the remaining detergent solution through the flow cell.

4
Rinse the syringe and flush the cell with distilled water (10 ml).

Cleaning the optical fiber connectors

Wipe with 30% isopropanol on lens paper.

Instrument housing

Wipe the instrument housing regularly with a damp cloth. Let the instrument dry completely before use.

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1
Select menu Check, press OK.

2
Select menu Check Lamp Intensity.

Check lamp Intensity
210nm 85%
300nm 95%

If the lamp intensity is < 20%, contact your local GE Healthcare service representative for lamp replacement or change of internal optical fiber.

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1
Select menu Check, press OK.

2
Select menu Check Lamp Run Time.

Check Lamp Run Time 200h

If the lamp on time is > 4000 hours, contact your local GE Healthcare service representative for lamp replacement.

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The flip time is the time the monochromator stepper motor has been in operation.

1
Select menu Check, press OK.

2
Select menu Check Flip Time.

Check Flip Time 50h

If the flip time is > 2000 hours, contact your local GE Healthcare service representative for maintenance.

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This menu shows the set path length and the serial number of the UV flow cell.

1
Select menu Check and press OK.

2
Select menu UV Flow Cell.

UV Flow Cell
(- -, - -mm, SN - - - - -)

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The system surface should be kept dry and clean to maintain proper safety and function of the system. Chemical stains and dust should be removed.

To clean the system surface:

1
Wipe the surface regularly with a damp cloth.

2
To remove stains, spray the system with 20% ethanol and wipe off the excess.

Note: Be careful not to bend any tubing.

3
Let the system dry completely before using it.

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During routine operation, the presence of air bubbles in the pump heads is seen as an erratic flow, a noisy detector signal or an irregular pressure recording. With air bubbles trapped in the pump, the pressure reading on the display will fluctuate considerably.

To remove the air from the pump:

1.
If the air has accumulated because of a leaking connector, correct the fault.

2.
Ensure that the inlet filter is fully immersed in eluent.

3.
Run the Purge the pump procedure.

If the problem remains, try to remove the air bubbles in accordance with the method described below. Use 100% methanol.

1.
Run at 40 ml/min for P-901, or 4 ml/min for P-903.

2.
Continue to run for 10 minutes, or until the air bubble is removed.

3.
Run the Change the eluent procedure.

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1
Fill a reservoir flask with distilled water. Immerse the inlet tubings of both pump modules, with filters, in the water.

Note: Never place the reservoir flask below the level of the pump inlet.

2
Connect a male Luer syringe of about 30 ml to the open end of the purge tubing.

3
Connect the male Luer connector at the other end of the purge tubing to the left purge valve at pump module module A.

4
Turn the purge valve counterclockwise half a turn to open it and slowly draw eluent to the syringe.

5
When fluid starts to enter the syringe continue to draw a few milliliters before closing the purge valve. Check that there is no air left in the inlet tubing.

6
Repeat steps 3 to 5 for pump module B, if fitted.

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Faulty operation of the check valves is usually indicated by irregular flow, very low flow or unstable pressure traces. Probable causes of this are air or dirt in a check valve preventing it from closing to seal and hold the pressure.

Record the pressure according to instructions, checking the pump pressure at the end of this document and identify the faulty check valve by observing which pump head is delivering the flow according to check menu at the end of this document.

Try to clean the check valves in–place on the pump head by pumping distilled water at 2 ml/min for 2 minutes. This also prevents precipitation of crystals. The pump 100% methanol for approximately 10 minutes. If this does not correct the problem, follow the instructions for removing and then cleaning the valves.

Note: Change solvent to distilled water and flush out all salt before removing the check valves.

Tools required: A 13 mm, a 18 mm and a 1/4 inch wrench.

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Before removing the check valves, move all input buffers bottles below the level of the pump heads, to prevent siphoning.

1
If the condition of the check valve is not improved by in–place cleaning, disconnect and remove the inlet manifold and outlet tubing.

2
Use the 13 mm wrench (inlet check valve) or the 18 mm wrench (outlet check valves) to remove the valve from the pump head.

3
Immerse the complete valve in methanol and place in an ultrasonic bath for some minutes.

Then repeat the ultrasonic bath with distilled water.

4
Refit the check valves. The inlet check valve (with a lip for the manifold and a larger diameter opening) is fitted to the side marked IN of the pump head. Tighten the valves until fully finger-tight and then use the wrench to tighten a further 1/3rd (110^o) of a turn. Do not overtighten the valves since damage to the internal components can occur.

5
Refit the outlet tubing and the inlet manifold.

6
Purge the pump carefully and check that the pumping action has been corrected.

Note: Check valves have precision matched components and should only be disassembled further by a trained service engineer. If the problem cannot be corrected, the valve should be replaced completely.

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To check the pump function, a recording of the pressure can be made or by checking the pressure in UNICORN. This pressure recording is more sensitive than the reading on the display. By observing the piston stroke indicator in the Check menu together with the pressure trace, the pump head which is functioning abnormally can be identified.

There can be several causes of an abnormal pressure recording, for example:

• air trapped in the pump heads
• partially blocked solvent filters
• leaking connections
• piston seal leakage
• check valve malfunction
• piston damaged

Check menu

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To enable trouble shooting it is possible to check which pump module head that delivers flow.

1
Select main menu Check, press OK.

2
The display shows the status of the pistons for both pump modules.

A: Left means that the left pump head is delivering flow in the A pump and

B: Right means that the right pump head in delivering flow in the B pump. At the changing point both are displayed. The A pump is closest to the front panel.

Note: 
Check Heads 2.30MPa
A: Left
B: Right

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1
Select main menu Check, press OK.

2
Select sub menu Check Total Run Time. The display shows the accumulated operation time (Run and Hold mode) for the A and B pumps.

Note:
Check Total Run Time
A: 50 h
B: 20 h

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1
Select main menu Check, press OK.

2
Select sub menu Check Piston Strokes. The display shows the accumulated number of piston strokes for the A and B pumps.

Note:
Check Piston Strokes
11374, 2398

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1
Ensure that the pump is at zero pressure.

2
Select main menu Check, press OK.

3
Select sub menu Calib Press Offset, press OK. “Working, please wait ...” is displayed until the calibration is completed.

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Service information relevant to the instrument can be checked. Information may not be available in all menus.

1
Select main menu Check, press OK.

2
Select sub menu Check Service Mode, press OK.

3
The service telephone number is displayed¹. Turn the selection dial clockwise to select next sub menu.

4
The service contract number is displayed¹. Turn the selection dial clockwise to select next sub menu.

5
The instrument serial number is displayed. Turn the selection dial clockwise to select next sub menu.

6
Instrument name and software version are displayed. Turn the selection dial clockwise to select next sub menu.

7
The date of the last service is displayed. Turn the selection dial clockwise to select next sub menu.

8
A test of the instrument buzzer is performed, press OK.

¹Not always pre-programmed. Depends on customer-specific contract.

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When changing from one eluent to another, it is extremely important that the two eluents are totally miscible with one another. If the two eluents are immiscible, the pump should be flushed first with an intermediate liquid, which is miscible with both eluents. Failure to do this will cause a wrong flow of eluent from the pump.

When changing from a salt-containing buffer to an organic solvent, use water as the intermediate liquid to prevent precipitation.

1
Stop the pump by setting it in Pause mode.

2
Transfer the inlet tubing into the new eluent or into the intermediate liquid.

3
Run the pump at a flow rate and time as specified in the table.

Pump	Flow rate	Time
P-901	40 ml/min	10 minutes
P-903	4 ml/min	10 minutes

4
Stop the pump. If an intermediate liquid is being used, transfer the inlet tubing into the final eluent and repeat step 3 with the new eluent.

In UNICORN, select instruction PumpWash in System Control:Manual:Pump.

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The piston seal should be run-in using 100% methanol.

1
Ensure that the reservoir is filled with sufficient eluent. Immerse the inlet tubing in the eluent. The reservoir should be placed at least 30 cm above the pump inlet.

2
Connect a male Luer syringe of about 30 ml to the open end of the purge tubing.

3
Connect the male Luer connector at the other end of the purge tubing to the left purge valve at pump module module A.

4
Turn the purge valve counter-clockwise half a turn to open it and slowly draw eluent to the syringe.

5
When fluid starts to enter the syringe continue to draw a few milliliters before closing the purge valve. Check that there is no air left in the inlet tubing.

6
Repeat steps 3 to 5 for pump module B, if fitted.

7
Check that the outlet tubings are not blocked.

8
Connect a thin capillary or a column that will give sufficient back pressure.

9
Run at the following flow rates for 15 minutes:
P-901 1 ml/min (or 2 ml/min 50%B)
P-903 0.1 ml/min (or 0.2 ml/min 50%B)

10a
For P-901, run at 20 ml/min (or 40 ml/min 50%B) at a backpressure of 2–5 MPa for 15 minutes.

10b
For P-903, run at 2 ml/min (or 4 ml/min 50%B) at a backpressure of 5–10 MPa for 2 hours, or longer if possible (e.g. overnight).

11
Finally, run the Change the eluent procedure.

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If there are signs of liquid leaking between the pump head and the housing side panel or the volume of the rinsing solution has increased or decreased, replace the piston seal of the leaking pump head.

CAUTION! Do not disassemble the pump head unless there is good reason to believe that the seal is leaking. Always ensure that sufficient spare components are available before attempting to replace the piston seal. It is not possible to reinstall a used piston seal after removal.

Note: The power must be switched OFF when removing and refitting the pump heads.

Note: Always replace the piston seals on both pump heads at the same time. An even better practice is to replace all four piston seals.

Spare parts and tools required:
Seal kit containing:
2 or 4 piston seals
2 or 4 rinse membranes
1/4 inch wrench (supplied with the pump)
3 mm allen key (supplied with the pump)
Screwdriver (supplied with the pump)

Note: After a new seal has been installed, the pump should be run in. Run the Running-in a new piston seal procedure.

Note: Before disassembling the pump heads move all input buffers bottles below the level of the pump heads to prevent siphoning.

CAUTION! Read the following instructions carefully. The individual parts of the pump head can be assembled incorrectly. Take care to ensure that the orientation of each part is correct before continuing with the next instruction.

1
Switch off the pump at the mains power switch on the back panel. Remove the piston seal rinsing system. The connectors are simple plugin fittings.

2
Completely loosen the tubing connector on the outlet valve.

3
If the pump is equipped with Switch Valves SV-903, first remove the switchvalves and disconnect the tubing. Unscrew the two white knurled screws under the pump to release the support bracket locking the inlet manifold into the inlet valve. Gently lower and remove the complete manifold.

4
Using the Allen key, unscrew and completely remove one of the two Allen screws locking the pump head in position.

5
When unscrewing the second locking screw, push firmly on the front face of the pump head to compensate for the pressure of the piston return spring. Hold the pump head firmly to prevent it from twisting. Remove the second screw and, without allowing the pump head to twist sideways, carefully pull it out.

6
Place the pump head face down on the bench. Pull out the piston together with the return spring.

7
Inspect the piston and return spring for sign of damage. If damaged, they should be replaced.

8
Wipe the piston with a clean cloth. If salt solutions have been used the piston may be slightly corroded. This corrosion can be removed with a rubber eraser. If it cannot be wiped or rubbed clean, scrape off any deposits with a scalpel or razor blade. Inspect the piston with a magnifying glass for scratches. Replace with a new piston if any scratches or cracks are found.

9
Remove the two screws securing the drain plate and the rinse chamber. Remove and discard the rinsing membrane. Remove the rinse chamber. For P-903, remove also the support washer.

10
Gently withdraw the piston seal. Discard the used seal.

11
The pump head, rinse chamber and drain plate should be carefully rinsed or cleaned in an ultrasonic bath, if available. If dirt can be seen on any surfaces, the inlet and outlet check valves should be removed and cleaned separately. Run the Clean the inlet and outlet check valves procedure.

12
Wet the new seal slightly and place it in the hole on the pump head and press it down into position with a hard flat object For P-903, refit the support washer on top of the new seal.

13
With the pump head still facing downwards on the bench, place the rinse chamber onto the head with the rinse ports in line with the inlet and outlet check-valves. The conical depression in the rinse chamber should be facing upwards, ready to accept the new rinsing membrane. Fit the rinsing membrane with the conical face downwards.

14
Place the drain plate on top of the assembly. Use the two screws to lock the complete assembly together.

Note: Align the drainage hole in the drainage plate with the inlet check valve (the opposite side of the pump head marked OUT/UP).

15
Wipe clean the piston and remove all finger prints. Wet the piston and then insert it into the return spring. With the pump head facing downwards on the bench, insert the piston into the pump head by pushing it gently but firmly vertically downwards into the seal.

CAUTION! Do not push the piston at an angle to the head and DO NOT twist the piston.

16
Turn the head so that the inlet valve and drainage hole are facing downwards and the text UP/OUT on the pump head is facing upwards. Mount the complete pump head over the locating pins on the front panel. Locate the metal end of the piston and the spring towards the drive cam.

Hold the pump head firmly against the side panel of the housing with one hand. Do not allow the assembly to twist under pressure from the return spring. Using the Allen key, fit and tighten one of the Allen screws. Fit and tighten the remaining Allen screw.

17
Reconnect the outlet tubing to the outlet check valve and the manifold block as described above.

18
Reconnect the inlet manifold and, if used, the switch valves.

19
Refit the tubing of the piston seal rinse system.

20
The pump should now be purged and the new piston seal carefully run-in as described in the Running-in a new piston seal procedure.

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A good laboratory routine is to calibrate the instrument once a day, when the electrode is replaced and if the ambient temperature is changed. The pH monitor is calibrated using standard buffer solutions in a two point calibration. The two buffer solutions can have any pH value as long as the difference between them is at least 1 pH unit. Calibration can also be performed from UNICORN. In UNICORN select System Control: System: Calibrate. Select the pH monitor. The calibration procedure can be done with the pH electrode either fitted in or removed from the flow cell.

Calibrating with the electrode outside the flow cell

When calibrating the electrode out of the flow cell and changing from one buffer to another, rinse the electrode tip with distilled water and dab it carefully with a soft tissue to absorb the remaining water. Do NOT wipe the electrode as this may charge it and give unstable readings.

The steps below describe the procedure used with the electrode removed from the flow cell.

Note: The user interface of the monitor must be unlocked if connected to a UNICORN control system.

1
Remove the pH electrode from the flow cell and immerse the electrode in the first standard buffer solution (normally pH 7.0).

2
Select main menu Calibrate pH. The display shows the current low and high calibrated pH value. Press OK.

3
Select sub menu Calib pH Buffer 1, press OK. When the pH value has stabilized, the Please wait message will disappear.

4
Adjust the pH value in the display using the dial, so that it corresponds to the known pH value of the 1:st buffer solution, press OK. The sub menu Calib pH Buffer 2 is shown.

5
Rinse the electrode tip with distilled water and then immerse the electrode in the 2:nd standard buffer solution (e.g. 4.0 or 9.0), press OK.

6
When the pH value has stabilized, the Please wait message will disappear.

7
Adjust the pH value in the display using the dial, so that it corresponds to the known pH value of the 2:nd buffer solution, press OK.

8
The sub menu Calibrated Electrode Slope shows the slope of the calibration curve where 100% corresponds to 59.16 mV per pH step at 25 °C. The asymmetry potential at pH 7 is shown as a mV value. Press ESC to return to the main menu.

9
Before use, rinse the electrode using distilled water.

A new electrode has a slope of, typically, 95–102% and an asymmetry potential within ±30 mV. As the electrode ages the slope decreases and the asymmetry potential increases.

As a rule, when an electrode has an asymmetry potential outside of ±60 mV and a slope lower than 80%, and no improvement can be achieved by cleaning, it should be replaced.

An electrode is still usable at lower slopes and higher asymmetry potentials but the response will be slower and the accuracy diminished.

Calibrating with the electrode in the flow cell

When calibrating with the electrode fitted in the flow cell in ÄKTApurifier, follow the above procedure. Before adjusting the pH monitor, ensure that the pH has stabilized. Leave the pump running while calibrating. Switch to the other standard buffer solution and repeat the procedure. For a description of calibration from UNICORN with the electrode fitted in the flow cell, see section 6.6 in UNICORN User Manual.

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