Biocontrol CL-10 Plus User Manual

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CL-10 Plus

User Manual

CL-10 Plus – User Manual 2 / 75 ECPUS vers 0.2 eng

Index

IInnddeexx___________________________________________________________________________________________

_________________________________________________________________________________22

11.. IInnttrroodduuccttiioon

_______________________________________________________________________

_______________________________________________________________________________________44

22.. IInnssttaallllaattiioonn_______________________________________________________________________

_______________________________________________________________________________________55

2.1 . Unpacking and checking. _______________________________________________________________5

2.2 . PC system Configuration._______________________________________________________________5

2.3. System Installation.____________________________________________________________________6

2.3.1 Installation Requirements._________________________________________________________6

2.3.2 Mains voltage setting. ____________________________________________________________6

2.3.3 Connection cables. _______________________________________________________________6

2.3.4 Software Installation and configuration______________________________________________7

2.3.5 Hydraulic connections.____________________________________________________________9

2.3.6 Internal & peristaltic pump tubing’s _______________________________________________10

2.4. CL-10 Plus switching on. _____________________________________________________________11

2.5. Instrument Stabilisation._____________________________________________________________11

2.5.1 Materials.______________________________________________________________________11

2.5.2 Peristaltic starter tube sealing control._____________________________________________11

2.5.3 Electrodes regeneration. _________________________________________________________12

2.5.4 Standby mode (Rest)_____________________________________________________________12

2.6. Instrument stabilisation with wash solution_______________________________________________13

2.6.1 Instrument preparation (Milk application).__________________________________________13

2.6.2 Instrument preparation (Wine application). _________________________________________13

2.6.3 Starting and calibrating __________________________________________________________14

2.7. End of working session. ______________________________________________________________14

2.8. Changing of application within the same working session._________________________________15

33.

TThhee IInnssttrruummeennt

_________________________________________________________________________

___________________________________________________________________1166

3.1 The principle_______________________________________________________________________16

3.2 The Measuring System. ______________________________________________________________16

3.2.1 Instrument layout. ______________________________________________________________16

3.2.2 Titration curve and Buffer Power measurement. _____________________________________17

3.2.3 END POINT measurement. ________________________________________________________20

3.2.4 KINETIC measurement (KIN). ______________________________________________________26

3.2.5 FIXED TIME measurement. ________________________________________________________29

44.. TThhee CCLL--1100 PPlluuss PPrrooggrraam

___________________________________________________

___________________________________________________________________________________3322

4.1. Menu and commands. _________________________________________________________________32

4.2. FILE Menu . ________________________________________________________________________34

4.2.1 Select method __________________________________________________________________34

4.2.2 Create a new method____________________________________________________________35

4.2.3 Edit method____________________________________________________________________35

4.2.4 Add method for Measure unit change; Biochemical Chemistry Application________________39

4.2.5 Clear graph ____________________________________________________________________41

4.2.6 Print graph_____________________________________________________________________41

4.2.7 Method name___________________________________________________________________41

4.3. START UP Menu . ___________________________________________________________________42

4.3.1 Prime Enzyme or F2._____________________________________________________________42

4.3.2 Clean or F3. ____________________________________________________________________43

4.3.3 START UP parameters. ___________________________________________________________43

4.3.4 Run Start up procedure or F4._____________________________________________________43

4.4. ROUTINE Menu._____________________________________________________________________45

4.4.1 Sample (GO icon or F5). __________________________________________________________45

4.4.2 Blank (B icon or F6). _____________________________________________________________46

4.4.3 Calibrate (C icon or F7).__________________________________________________________46

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4.4.4 Stop Measure. __________________________________________________________________46

4.5. WOKLIST Menu _____________________________________________________________________47

4.5.1 Print Worklist __________________________________________________________________47

4.5.2 Edit Worklist ___________________________________________________________________48

4.5.3 Erase Worklist __________________________________________________________________48

4.5.4 Export Worklist _________________________________________________________________48

4.6. SERVICE Menu ._____________________________________________________________________51

4.6.1 Auto increment SAMPLE ID. _______________________________________________________51

4.6.2 Enter rest mode.________________________________________________________________51

4.6.3 Instrument parameters (Access password: default).___________________________________51

4.6.4 Calibrate Temperature.__________________________________________________________53

4.6.5 Communication parameters. ______________________________________________________53

4.6.6 Print Technical Report. __________________________________________________________53

4.6.7 Terminal Emulation _____________________________________________________________54

4.6.8 Electrodes Test 1 and 2.__________________________________________________________56

55.. MMaaiinntteennaannccee aanndd PPrreesseerrvvaattiioonn_______________________________________

_____________________________________________________________________________________5577

5.1 Weekly Maintenance_________________________________________________________________57

5.2 Routine Maintenance ________________________________________________________________57

5.3. Maintenance – rapid guide____________________________________________________________58

5.4. Enzyme Contamination in the reference electrode _______________________________________59

5.5 CL10 preparation for a period of halt (over 2 weeks). ____________________________________61

5.6 Instrument Reactivation _____________________________________________________________61

66.. TTrroouubbllee SShhoooottiinngg.._____________________________________________________________

___________________________________________________________________________________6633

6.1. Electrical Section. __________________________________________________________________63

6.1.1 Instrument does not switch on.____________________________________________________63

6.1.2 Pumps do not operate when the instrument is on.____________________________________63

6.1.3 The analytical unit does not accept commands from the program. ______________________64

6.2. Fluidic Section._______________________________________________________________________65

6.2.1 The Mixer does not fill-in or empty.________________________________________________65

6.2.2 Liquid spills out from the mixing chamber.__________________________________________65

6.2.3 Absence of signal during the measurement. _________________________________________66

6.2.4 Unstable Measurements ._________________________________________________________66

6.3 Electrodes. ________________________________________________________________________66

6.3.1 Control of electrodes performances________________________________________________66

6.3.2 Electrodes replacement. _________________________________________________________68

6.4. CL-10 Plus troubleshooting – rapid guide________________________________________________71

77.. TTHHEE PPIIPPEETTTTEE,, cchhaarraacctteerriissttiiccss,, uussee aanndd mmaaiinntteennaannc

cee..______________________________________________________________________________________7733

7.1 Materials. _________________________________________________________________________73

7.2 Using the Microman Pipette.__________________________________________________________73

7.2.1 How to Mount the Piston. ________________________________________________________73

7.2.2 How to mount the capillary. ______________________________________________________73

7.3 Pipetting.__________________________________________________________________________74

7.3.1 Aspiration._____________________________________________________________________74

7.3.2 Dispensing._____________________________________________________________________74

7.3.3 Simultaneous ejection of the piston and capillary. ___________________________________74

7.4 Pipette Specifications._______________________________________________________________74

7.5 How to Inject The Sample. ___________________________________________________________75

CL-10 Plus – User Manual 4 / 75 ECPUS vers 0.2 eng

CL-10 Plus

1. Introduction

CL-10 Plus instrument is an automatic multi-parametric analyser, working in differential-pH technique for chemical analyses in various type of matrices, from food industry to clinical applications.

Technique characteristics are:

- Operative simplicity on different matrices, also particularly difficult ones.

- Versatility:

o executes the tests using few (from 5 to 1000) µL sample. o in short time (from 15 seconds to 3 minutes).

- Promptness of response of tests made on unstable matrices.

- Use of dedicated and ready to use reagents, of 4-6 months shelf life

Products of common use are listed hereafter, in order to make the reading of the present manual easier. These products will be often mentioned, when required for routine procedures, maintenance and repair.

Product Code Use

Polif Solution GEN718 wash solution, to be used with all Milk and Wine applications

Wash Kit GEN698

wash solution, to be used with all Clinical Chemistry applications, but not with G6PD

Wash Kit GEN699 wash solution, to be used with G6PD Clinical Chemistry application

Diluent Buffer GEN644

to be used with Wine applications (when described in the specific package insert)

Combi Clean GEN674 to be used with Wine Combi Test and pH/Acidity kits

Microman M25 micro-pipette GEN102 to be used with all applications, except Wine Combi Test and pH/Acidity

Electrodes test GEN603

Used to check electrodes performances, regardless the application field (see§4.6.8)

Regenerating Solution GEN518 Used for the Maintenance §5

Strong Regenerating Solution GEN613 Used for the maintenance §5

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2. Installation

The CL10 Plus instrument consists of a thermo stable measuring block which contains two capillary glass electrodes, a mixing chamber, two groups of peristaltic pumps, a differential amplifier and a microprocessor which calculates the analyte concentration or enzyme activity in the sample, and controls all the functions of the instrument. CL-10 Plus is delivered with the fluidics empty (free from liquid) and dry and with the peristaltic pumps unhooked (see § 5.5 figure; the detail of the pump tubing’s). The procedure for the electrodes reactivation and stabilisation takes from 2 to 24 hours (see

§ 2.5.3).

2.1 . Unpacking and checking.

Check the list of content:

1. Instrument CL10 Plus

2. power 230 VAC (110 VAC) - 12 VDC

3. power cable

4. serial cable CL10-PC

5. Plastic bottles ( Kartel type) for the buffer and/or standby solution and for the waste (2x250 ml)

6. Set pump tubing’s (Art. No. SSP112 or SPP112+SPP113)

7. CL-10 Plus Software on CD-ROM and relative Licence

8. CL-10 Plus Operator Manual

9. CL10 Plus Testing Certificate

Instrument Serial Number:

The serial number is located on the back of the instrument; it is the same number written on the CL-10 Plus Testing Certificate .

2.2 . PC system Configuration.

• Pentium PC at 175 MHz or superior

• Microsoft Windows 95/98/2000/Me/XP

• RAM 32 MB or superior

• 6 MB available on the Hard Disk

• floppy disk 3,5”

• Monitor Super VGA with 256 colours

• Video card with 800x 600 pixel

• CD-ROM 4x or superior

• COM1 RS232 9 pin or USB* port

• If USB port is used USB-RS232 converter is required.

(recommended: Targus Replicator type. PA070)

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2.3. System Installation.

2.3.1 Installation Requirements.

The CL10 Plus must be placed away from any vibration source (i.e. centrifuge),

magnetic field and intensive sun light.

It is recommended to maintain the room temperature between 15° and 30°C.

The power of the system (PC + instrument) must be connected to the ground (free

of noise). The power fluctuation must be secured < 5%. If not, it is recommended to connect the CL-10 Plus instrument and the PC to a 500VA stabilizer. In any case, it is recommended to keep the CL10 Plus instrument under UPS (Uninterruptible Power Supply), for protection against accidental power loss.

2.3.2 Mains voltage setting.

The power converter is set for a range 100 - 230 VAC, automatically. For PC and

printer see producer manual.

2.3.3 Connection cables.

Connect the power to an available plug and to the CL10 PLUS (See picture 1).

Connect the CL10 Plus to the COM1 of the PC, using the serial cable.

Back vision of the instrument

PC + MONITOR

CL10 Plus PRINTER

POWER 12 VDC

VIDEO COM1 LPT1

POWER SUPPLY

SERIAL CABLE

LOCAL POWER SUPPLY POWER SUPPLY POWER SUPPLY 230 VAC 230 VAC 230 VAC (110 VAC) (110 VAC) (110 VAC)

Fig. 1

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2.3.4 Software Installation and configuration

Materials:

- 1 CD-ROM CL10Plus named as follows: U (sequential letter indicating the version)

CDROM_PLUS_05102004 (version release date) or superior.

Procedure .

To install the program on the PC, please follow the instructions:

1. Switch the PC and the Monitor on.

2. Insert the CD-ROM CL10Plus software in the CD-ROM unit.

3. From Explorer, open the CL10Plus vs. 4.3 CD-ROM or superior version.

4. Open the cl10plus vs 4.3 (or sup.) folder and select the Setup.exe icon.

5. Automatically, the program guided installation will start.

6. Follow the instructions on the screen.

7. Once the installation is completed, press OK. Remove the CD-ROM from CD unit.

8. Reboot the PC

For simplifying the access to the operative program: cl10plus.exe, it is recommended to create a shortcut icon on the desktop

Point the mouse anywhere on the desktop

Press the mouse right-hand button and select: new link.

Once the new window appears, search for the following path: C:\cl10\

cl10plus

Select Next, and then End.

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Set up of the software

1. Access to the software icon, on the desktop: cl10plus

2. The following message appears

;

select OK.

3. From Service window in the main menu of CL10Plus program, select Auto increment sample ID to activate the function of start measure by pushing the Enter key of the PC board.

4. Access again to the Service window, select Enter password and type: “default”

5. Access once more to Service, select Instrument parameters

6. In the top left square of the window, select Mixer volume : high

7. In the bottom right square of the same window select Activate rest mode in order to set the rest mode interval between cycles= Period (min) at 120 seconds and the temperature= temp (°C) at 37°C.

8. Press OK.

Methods setting

9. Check that the CL10Plus CD-ROM is in the CD unit.

10. Open the following folder D:\ CD-ROM\ S_Plus_Matrix Name_XXX\CL10Plus vs.4.3.

11. Open the folder containing the name of the application field (Wine or Milk or Bio). There is a letter (example: S) before the field of application. This letter is containing all the information concerning the version number of all methods contained in the CD ROM .

12. From this folder open the next folder called English; There are three more folders inside: Methods_MD, Service_TBL, Tables_TBL

13. Service_TBL, Tables_TBL are not used by the operator, but only by service personnel. Open the Methods_MD folder.

14. Copy all the files contained, do not open them! Paste these files in the following

folder: C:\cl10.

15. Change properties of the pasted files; from read only into archive.

16. Unable the screen saver and the energy save setting, in order to avoid any interference during the working session.

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2.3.5 Hydraulic connections.

1. Hook the peristaltic tubes with lower diameter in position 6 (and 5, if required by the method), without twisting them.

Warning: if tubing’s are twisted, analytical reliability is compromised. To be able to verify the correct tubing’s assessment, mark the thick ends of each tube before hooking it, on the same side along the fusion line and check that, once hooked, it remains marked on the same side.

2. Hook the 4 peristaltic tubes, with higher diameter, in position 1,2,3 and 4 without twisting them. Careful that the ’L’ connections are not pushed to the far end of the silicon but only halfway.

3. Place the bottle in the waste position.

TAMPONE/BUFFER

SCARICO/WASTE

ENZIMI/STARTER

7 8

Fig. 2

CL-10 Plus – User Manual 10 / 75 ECPUS vers 0.2 eng

2.3.6 Internal & peristaltic pump tubing’s

Internal tubing’s are 8 pcs (SPP311); normally they are not considered as consumables, unless accidentally broken or damaged.

Peristaltic tubing’s, or pump tubing’s, are consumables and as such, they must be checked before installation and inspected every three months.

It is recommended the replacement every 3000 cycles, that means in average, 3-5 months of work.

Description of tubing types, and destination:

Pos Description & Packaging

Code

1 Internal Tubing’s set

SPP 311

2 Peristaltic Pump Tubing’s Set

SPP 312

3 Enzyme Starter tubing only mono starter

SPP 313

4 Additional Tubing: sample+starter Wine Combi

1+1

SPP 314

5 Additional Tubing: sample +specific Wine pH/Acidity

1+1

SPP 315

6 Additional Tubing’s: double starter Bio G6PD/6PGD

1+1

SPP 316

SPP 311 : product for all configurations and all applications

SPP 312: product for all configurations and all applications

(pump tubing’s 1-2-3-4, Fig 2 § 2.3.5)

SPP 313: product for all kits other than WCP678-682, BCP 592-596-690 (pump

tubing 6, Fig 2 § 2.3.5)

SPP 314: product for WCP678, only (pump tubing’s 5-6, Fig 2 § 2.3.5)

SPP 315: product for WCP682, only (pump tubing’s 5-6, Fig 2 § 2.3.5)

SPP 316: product for BCP 592-596-690, only

(pump tubing’s 5-6, Fig 2 § 2.3.5)

CL-10 Plus – User Manual 11 / 75 ECPUS vers 0.2 eng

2.4. CL-10 Plus switching on.

Make sure that all the power supply and serial cable are properly connected.

Switch the CL10 Plus on, from the switch placed in the back panel of the instrument.

On (up)

Off (down)

If the light under the instrument lead, in the front panel, does not turn on (green), check all the connections again and the power supply.

2.5. Instrument Stabilisation.

2.5.1 Materials.

EC Polif kit.

Distilled water

2.5.2 Peristaltic starter tube sealing control.

Place (Fig. 2, §6.3.1) a vial containing at least 2 mL distilled water in starter

position and place the needle of the starter tube inside the vial. Run Prime enzyme cycle to fill-in the starter tube. Take both ends of the starter tubing (completely filled-in with water) from the vial

and lift in the air the opposite end to the needle: if the peristaltic starter tube has a

good seal, the level of the water should not move. If the level of the water inside the starter tube comes back, the tubing must be

replaced. When the starter tubing is correctly filled in with water, connect the tube end to the

steel capillary on the mixing chamber stopper.

Warning: the connection of the starter tubing to the top of the mixing chamber is a critical step.

It is very important to connect it gently, taking care to the final position of the yellow ring embracing firmly the silicon of the starter tubing and the steel capillary on top of the mixing chamber.

CL-10 Plus – User Manual 12 / 75 ECPUS vers 0.2 eng

2.5.3 Electrodes regeneration.

NOTE: Regenerating procedure of measuring electrodes is used to reactivate the instrument after a period of halt (installation or after a period of rest); electrodes, after this procedure require a “stabilization” time; it is therefore recommended to operate the Rest Mode procedure immediately after, using wash solution as a buffer.

Electrodes regenerating procedure:

Reconstitute the wash solution, according to the instructions given in the kit.

Place the reconstituted solution in buffer position and an empty bottle in waste position

(Fig. 2, § 2.3.5).

Run 3 Clean cycles.

Replace the bottle containing wash solution with a bottle of Regenerating Solution in

buffer position.

Run 3 Clean cycles.

Wait for 5 minutes.

Replace the Regenerating Solution with a bottle of distilled water and run 2 Clean

cycles.

Replace the bottle of distilled water with reconstituted wash solution, again and run 3

Clean cycles.

Empty the waste bottle.

2.5.4 Standby mode (Rest)

For the CL-10 Plus instrument there are two available different standby mode (REST mode):

1 From the computer, REST mode program ( § 2.3.4).

 From Service window enter Enter Rest Mode command

 Switch the monitor off

 Leave the PC on

 Leave the CL-10 Plus instrument on

ATTENTION: Check if there is enough Wash Solution for all the standby period (the washing cycle automatically runs every 120 minutes and consumes from 4 to 9 mL of wash solution each cycle, depending on the execution method selected).

2 From the instrument, REST mode: the instrument stays at 37°C and the time laps

between consecutive clean cycles is 90 minutes.

 Switch the PC monitor off.

 Switch the CL10 Plus off.

 Allow 5 seconds, then switch the CL10 Plus on again.

 The instrument runs 1 clean cycle every 90 minutes at 37°C.

ATTENTION: Check if there is enough Wash Solution for all the standby period (the washing cycle automatically runs every 90 minutes and consumes from 4 to 9 mL of wash solution each cycle).

ALLOW STANDBY OVERNIGHT, at least; it is not required for 2-3 hours interruption of working session

CL-10 Plus – User Manual 13 / 75 ECPUS vers 0.2 eng

2.6. Instrument stabilisation with wash solution

1. Use reconstituted wash solution and place in buffer position.

2. Empty the waste bottle

3. Enter CL10 Plus program, from File menu select (using Select method command) the execution method (the instrument will automatically set the desired measurement block temperature).

4. Unplug the starter needle (connected to pump 6) from the top of the mixing chamber and place both ends in a vial containing distilled water.

5. Run 1 Clean cycle and wait for temperature stabilisation (the yellow led in the front panel of the instrument will decrease its intensity and turn almost off).

6. Run Start Up procedure setting 5 measure cycles (§ 4.3).

7. The instrument is stable and ready to work when three consecutive measurements have ∆mpH < ± 0.5 mpH, and the D1 of the last two measurements do not differ more than 1 mpH from each other.

2.6.1 Instrument preparation (Milk application). The following procedure (taken as an example) applies to UREA in milk (Art.

No. MCP549).

Replace the reconstituted wash solution with R1 buffer.

Check that the enzyme tubing (connected to pump 6) is plugged to the top of the mixing chamber.

Follow the instructions on the Package Insert of EC Milk Urea.

2.6.2 Instrument preparation (Wine application). The following procedure (taken as an example) applies to EC Wine COMBI

test : pH, total acidity, glucose + fructose (Art. No. WCP678).

Replace the reconstituted wash solution with R1 buffer, reconstituted according to the package insert of the kit.

Check that the enzyme tubing (connected to pump 6) is plugged to the top of the mixing chamber.

Check that the AUX tubing, the longer (connected to pump 5), is plugged to the top of the mixing chamber, too.

Follow the instructions on the Package Insert of EC Wine COMBI test.

CL-10 Plus – User Manual 14 / 75 ECPUS vers 0.2 eng

2.6.3 Starting and calibrating

ATTENTION: Carry out the following measurements in a sequence. If you wait more than 5 minutes between two consecutive measurements, run GO command, without injecting any sample, to renew the buffer in the mixing chamber.

Equilibrate samples at room temperature before testing. Samples must be

homogeneous when assayed.

Run GO cycle or press F5 function key (without injecting any sample).

Repeat the GO cycle and check the stability (according to the package insert in the kit).

a) Offset determination: Execute Blank cycle (F6) once. The blank value must be

in the range of the method package insert.

b) Calibration: inject Calibrator using the Gilson Microman M25 pipette or trough

the AUX tubing (according to the package insert) and run Calibrate cycle (F7) once. If the SLOPE value obtained is OUT OF RANGE, the result will appear in

red (see package insert and § 4.4.3 of this manual); in that case repeat points a) and b).

Blank check: run GO command (F5) without injecting any sample. The result has

to be included between 0 ± allowance claimed in the package insert.

Standard check: inject Calibrator with the Gilson M25 pipette or trough the AUX

tubing (according to the package insert) and run GO command. The result must be within the range expected for the calibrator, from the method package insert ± allowance claimed.

2.7. End of working session.

 At the end of the working session replace R1 buffer with the reconstituted

Wash Solution.

 Replace starter vial (and AUX if used) with a vial containing distilled water

(min 3 mL).

 Run 2 Prime enzyme (F2) and 2 Clean cycles (F3).

 Empty the waste bottle.

 Leave the instrument in standby condition or Rest mode.

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2.8. Changing of application within the same working session.

ATTENTION: Steps 2 and 3 of the following procedure allow recovery of not utilized enzyme, still inside the starter tube (position 6). If the enzyme vial is empty or all the tests are made, it is not necessary to recover the enzyme; it is therefore recommended to neglect steps 2 - 3 and to go from step 1 to 4.

1. Replace R1 buffer, reconstituted as required by the method in use, with the R1 buffer, reconstituted as required by the next method. Leave the enzyme vial (pump 6) of the method in use in its place.

2. From File menu, Select the ENZY_REC_R3 method.

3. Run Sample once using the GO icon or the F5 function key. Press Start Measure and Accept. Wait until the end of the run.

4. Replace the enzyme vial (pump 6) with a vial containing distilled water.

5. Run Prime Enzyme twice using the prime enzyme icon or the F2 function key.

6. Run Clean once using the clean icon or the F3 function key.

7. Wipe the starter needle going to enzyme pump 6 and insert it into the enzyme vial (starter) of the next chosen method.

8. From File menu, Select the next method.

9. Empty the waste bottle.

10. Follow the instructions given in the package insert of the method to use.

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3. The Instrument

3.1 The principle

The differential pH technique is based on the correlation of pH variations, which are measured by two capillary glass electrodes, to the quantity of H+ produced or consumed by a reaction, which is started by adding the necessary enzyme or substrate.

3.2 The Measuring System.

The CL10 Plus is a compact and easy to operate instrument. It is made of a differential amplifier and a microprocessor which calculates the analyte concentration or enzyme activity in the sample, and controls all the functions of the instrument. The mechanical components are:

3.2.1 Instrument layout.

n°1: pump of the Electrode 1

n°2: pump of the Electrode 2

n°3: level pump

n°4: pump of the buffer

n°5: starter pump (AUX)

n°6: starter pump

n°7: electrode 1

n°8: electrode 2

n°9: mixing chamber

TAMPONE/BUFFE

SCARICO/WASTE

ENZIMI/STARTER

7 8

Figure 2 § 2.3.5:

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3.2.2 Titration curve and Buffer Power measurement.

• Titration curve.

Titration is used to determine the amount of acid in a given solution. In this procedure a measured volume of sample is titrated with a solution of a base, usually sodium hydroxide (NaOH), the concentration of which is known. The NaOH is added in a small increments until the acid is neutralised, as determined with an indicator or a pH meter. From the volume of NaOH added at known concentration, it is calculated the concentration of acid in the solution titrated. From the titration of a sample containing weak acids (pK between 3 and 6) additional information are obtained, measuring the pH of the acid after each increment of NaOH added, to the point of neutralisation. A plot of pH of the solution vs. the amount of NaOH added to this point is called ‘titration curve’.

Let us take the titration curve of acetic acid, a typical weak acid. At the beginning, (see figure) before any NaOH is added, the acetic acid is slightly ionised. When aliquots of NaOH are added, the added OH- will combine with free H+ in the solution to form H2O. As soon as free H+ is removed, some of undissociated acetic acid immediately dissociates. At the midpoint of the titration, one-half of the original acetic acid has undergone dissociation, so that the proton-donor concentration will be equal to the proton-acceptor concentration. As the titration is continued by adding further increments of NaOH, the remaining undissociated acetic acid is gradually converted into acetate. The titration curve is a reversible reaction (adding H+ back to the system, acetate is converted back to the initial state).

NaOH added

pK of

Acetic Acid

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• The Buffer Power.

Buffer is an aqueous system that tend to resist to pH changes, once small amount of acid (H+) or base (OH-) are added. A buffer system consists of a weak acid (proton donor) and its conjugate base (proton acceptor), as for example the acetic acid form and acetate form.

The “Buffer Capacity” is defined as:

∆H

(H+ concentration)

= ------------ = --------------------------

∆pH (pH variation)

in which the ∆pH is the pH increment induced by the addition of an amount of base, whereas ∆H+ is the relative change of free H+ in solution. A large “Buffer Capacity” of the medium corresponds to a large amount of base required to achieve a small change in pH.

BUFFER POWER measurement, using CL-10 Plus instrument.

The working buffer, using CL-10 Plus, must provide a known and constant Buffer Power. Sensitivity of the method depends on sample volume, stoichiometry and Buffer Power.

Sample: HCL 100 mM

Buffer Solution: Total Acidity

Stopper: volume of 1225 µL

Temperature: 37°C

Micropipette: Gilson Microman M25

Sample Volume: 10 µL

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PROCEDURE

∗ From the File menu, enter Edit command and select the BETA800_R2.MD method.

Check the parameters (see the following window). If the settings are not the same or the method is not found, contact technical assistance.

∗ Once the settings are controlled, press Esc on your keyboard and from File menu, enter

Select method command. Choose the same method and wait until it is visualised on the

screen

∗ Inject sample (10 µL of HCl 100 mM) in the mixing chamber ∗ Run Sample command or press the GO icon or the F5 function key. Press Start Measure,

then Accept.

Principle.

Step 1: both the electrodes and the mixer are filled with the buffer solution.

Step 2: after 3 seconds (wait time) a sufficient amount of buffer + sample is aspirated into

electrode 2. The buffer solution is maintained in the electrode 1. The pH change due to HCL acidification is instantaneous. 15 seconds are allowed to electrodes stabilisation, then the result is displayed on the screen.

CL-10 Plus – User Manual 20 / 75 ECPUS vers 0.2 eng

Calculation (taken an hypothetical result of 185 mpH).

Dilution: 10 µL of HCL 100 mM are injected into 1225 µL of buffer, the dilution is 1:123. The ∆H+ in the mixer is calculated as follows:

sample volume 10 ∆H+ = sample acid concentration x ------------------------- = 100 x -------- ≈ 0.81 mM final volume 1235

Buffer Power calculation:

∆H

(concentration) 0.81 mM/L

= ------------ = -------------------------- = ------------------- = 4.38 mM/L pH

∆pH (pH variation) 0.185 pH

3.2.3 END POINT measurement.

A practical example: urea determination in milk.

The heart of the differential-pH apparatus are the mixing chamber and the two electrodes:

electr. 1

electr. 2

starter 1

stirrer

Sample

E -E

D 0

Analysis of the steps of measurement:

PHASE ZERO: the mixing chamber and the electrodes are filled with buffer, ready to accept

the sample.

PHASE 1: using the micropipette, inject 25 µL of milk in the mixing chamber; the stirrer

rotates and mixes buffer + milk for some seconds.

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Buffer + Sample

PHASE 2: both electrode pumps n° 1 and n° 2 rotate, taking approximately half of the total

volume of the mixing chamber, filling both the electrodes with buffer + milk.

E -E

At this stage the instrument reads, for 5 seconds, the pH difference between EL 2 - EL 1 = ∆mpH; called D1. The electrodes contain exactly the same solution, the pH difference should be zero (in fact between ± 150 mpH). The signal measured will be subtracted during the following measure.

Example: during the measurement the obtained ∆mpH is 20, the following graph ∆mpH/time

will be displayed

Pump

Pump 2

∆mpH

t (s)

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PHASE 3: the enzyme pump n°6 loads 15-17 µL of urease enzyme in the buffer + milk

solution that still remains into the mixer.

Buffer + Sample + Starter 1

starter 1

PHASE 4: The stirrer homogenises the buffer + milk + enzyme solution and only the pump 2,

of the measure electrode 2, rotates filling the EL 2 with buffer + milk + urease, while in the reference electrode

1 still remains only buffer + milk.

E -E

At this stage the instrument reads, for 30 seconds, the mpH difference between EL 2 and EL 1; but while EL 1 does not modify its pH value because there is no reaction inside, in the EL 2 the urease catalyses the urea hydrolysis:

NH2-CO-NH2 ⎯(urease)→ 2NH3 + CO2 (1) 2NH

+ H2O ↔ 2 NH

+ 2OH-

+ H2O ⎯→ H2CO3 ↔ HCO

+ H+

producing about 1.3 OH- /per each mole of Urea.

Thus, the urea hydrolysis is an alkaline reaction which modifies the pH inside the EL 2: each urea molecule, hydrolysed, increases the pH value (thus the difference EL 2 - EL 1) until all urea in the sample has been fully transformed. In the graph we following the reaction is displayed:

Buffer + Sample

Buffer + Sample + Starter

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The final value of phase 4 is called D2. In our example, milk sample D2 value is 30 ∆mpH. It means that urea in the milk caused a pH variation, D2 - D1 of 10 mpH. But at how many mg/dL of urea correspond to 10 ∆mpH? The answer is quite easy: calculate the amount of [OH-] delivered during the reaction:

[OH-] = β x ∆pH = 11 mM x 0,01 = 0,11 mM

and the urea concentration in the reaction solution:

moles of urea = [OH-] / 1,31 = 0,084 mM.

Dilution factor: 25 µL of milk are injected into 1225 µL of buffer, so the dilution is 1 : 50, then to obtain the urea content in the milk sample we have just to multiply:

Urea = 0,084 x 50 = 4,2 mM = 25,2 mg/dL.

PHASE 5: the instrument is automatically washed with buffer and the mixing chamber filled in

again with new buffer.

Two points calibration.

The previous example was useful to present the enzymatic reaction that occurs in the differential-pH apparatus. A number of simplifications have been made: first of all the D1 value obtained for the milk sample is only due to the urea in the sample; in fact this is not true, because a little pH change must be subtracted, due to the addition of the enzyme, which give a contribution to the reading. Moreover, to maintain constant the stoichiometric relation between ∆pH, OH- and urea content it is necessary that a) buffer power is exactly known, b) the enzymatic reaction reaches the End Point in the measuring time, so c) urease enzyme activity remains constant and known, etc. For these reasons, during routine, where not all the approximations made are necessarily true, the operator will run a calibration procedure, before any sample. Hereafter there is an explanation of the meaning of the calibration procedure, in detail:

D2 Value

t (s)

D1 time

(DL1)

D1 Value

D2 time

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1) OFFSET.

First step: run of an analysis cycle without injecting any milk in the mixing chamber and obtaining: D1: the two electrodes pumps (1 and 2) turn and take the mixer content, only buffer; when

the electrodes contain the same solution, their pH difference remains more or less the same, so for 5 seconds it is approx. 20 ∆mpH.

D2: the enzyme pump (6) loads the urease enzyme into the mixer and only the electrode 2

takes the solution buffer + urease, while the electrode 1 contains only buffer; the urease may have a little difference of pH compared to the buffer, due to the enzyme contribution to the reading.

In the graph the reaction is as follows:

During the D2 there is no reaction, but only a measure of the different “acidity” due to the enzyme. So, if you remember the graph of our milk sample, not all the 10 ∆mpH are caused by the urea hydrolysis, 1 ∆mpH is in fact due to the enzyme contribution:

∆mpH

t (s)

(DL1)

D1 time D2 time

∆mpH

t (s)

(DL1)

time

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2) SLOPE.

Second step: run of an analysis cycle injecting 25 µL of urea standard (100 mg/dL): D1: both the electrodes contain the same solution, buffer + standard, and their pH difference

is always approx. the same, 20 ∆mpH.

D2: EL 1 contains buffer + standard, EL 2 buffer + standard + urease enzyme, which

promotes the reaction:

DL1 = D2 - D1 = 30 - 20 = 10 ∆mpH

But, also in this case, not all the 10 ∆mpH are caused by the urea hydrolysis, we have to subtract 1 ∆mpH, the “acidity” of urease: STD 100 mg/dL: 10 - 1 = 9 ∆mpH.

Correcting to 100/9 = 11.11 mg/mpH [slope value].

The instrument, after calibration, will store in the method settings the OFFSET and SLOPE values and calculate the concentration (of the samples unknown) according to the formula

[sample] = [∆mpH sample - offset] * slope

∆mpH

t (s)

(DL1)

D1 time D2 time

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3.2.4 KINETIC measurement (KIN). .

A practical example: Acetyl-cholinesterase determination in whole blood

Sample: Whole Blood

Buffer Solution: Phosphate/Pyro-phosphate (Β = 6.1 mM/L pH)

Stopper: volume 1225 µL

Temperature: 37°C

Micropipette: Gilson Microman M25

Sample Volume: 15 µl of whole blood

Starter: Acetylcholine (92 mM)

Analysis of the measurement steps:

electr. 1

electr. 2

starter 1

stirrer

Sample

E -E

D 0

PHASE ZERO: the mixing chamber and the two electrodes are filled with the same buffer.

PHASE 1: Using the micropipette 15 µL of blood are injected in the mixing chamber; the

stirrer rotates and mixes buffer + blood (which contains Acetyl-cholinesterase) for some seconds. Pumps 1 and 2 rotate and both the electrodes are filled with buffer + blood.

The electrodes contain exactly the same solution, the ∆pH should be zero.

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E -E

In the graph ∆mpH/Time:

PHASE 2: after mixing of buffer + blood, 17 µL of the starter (Acetylcholine) are injected into

the mixing chamber; the EL 2 is filled with buffer + blood + starter whilst EL 1 stays in the solution buffer + blood.

E -E

PHASE 3: the instrument reads, for 70 seconds, the ∆mpH between EL 2 and EL 1. The

calculation of the reaction rate (slope of ∆pH curve) is performed skipping the Lag Phase (40

sec.+ 5 sec. of D1 phase, period in which the reaction is not yet stable ), starting at second

45 until the end of measure phase (second 75).

t (s)

∆mpH

Pump1

Pump 2

Buffer + Sample

Buffer + Sample + Starter

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In the EL 1 there is a small change of pH, only due to the background noise, whereas in EL 2, in addition to the same background noise, the Acetyl cholinesterase in the blood is catalysing the following reaction:

Acetylcholine + H2O Choline + CH3COO- + H+

the Acetylcholine hydrolysis releases hydrogen ions (acidification) and the pH value goes to lower values. In the following graph, the reaction:

Calculation of Acetyl-cholinesterase activity (U/L).

Enzyme activity definition: By international agreement, 1 unit of enzyme activity is defined

as that amount of enzyme causing transformation of 1.0 micromole (µmol) of substrate per

minute, under optimal conditions of measurement. The specific activity is the number of

enzyme units per milligram of protein. The specific activity is a measurement of enzyme

purity: it increases during purification of an enzyme and becomes maximal and constant when the enzyme is in the pure state.

Cholinesterase activity is expressed as micromoles of acetic acid released per minute:

Activity (U/L) = ∆pH/min x

x d x s

mixer volume

where d = ---------------------------

= Buffer Power

sample volume

s = stoichiometry (moles of H

produced / each mole acetylcholine hydrolysed)

and ∆mpH/min is the value obtained during the sample measurement. For example if d = 82.7 (15 µL of blood injected into the chamber with a final volume of 1240 µL), ∆mpH/min = 11.4 and B = 6.1, the Cholinesterase activity is 5494 U/L.

The slope value,

x d, can be entered in the slope field (in method settings), to obtain the

result in U/L.

PHASE 4: the instrument is automatically washed with buffer and the mixer refilled with the

new buffer.

t (s)

∆mpH

Starter

Lag Phase

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When a KINETIC method is used:

• no Offset (Blank step) is required;

• no standardisation (Calibration step) with calibrator is required;

• the slope is defined in the method package insert;

• control samples (haemolysed blood) allow to verify the method performance.

3.2.5 FIXED TIME measurement.

FIXED TIME measurement is normally used for substrate determination with Ist order (i.e. la analyte concentration << Km of the enzyme).

Analysing the steps of the measurement cycle:

electr. 1

electr. 2

starter 1

stirrer

Sample

E -E

D 0

PHASE ZERO: the mixing chamber block and the two electrodes are filled with the same

buffer; the buffer volume in the mixing chamber (SHORT stopper) is 1225 µL PHASE 1: Using the micropipette, the sample is injected in the mixing chamber; the stirrer

rotates and mixes buffer + sample for some seconds and both electrodes are filled with buffer + sample.

The electrodes contain the same solution; the ∆pH should be zero.

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E -E

In the graph, the plot of ∆mpH/time is:

PHASE 2: after mixing of buffer + sample, the starter is injected into the mixing chamber; the

EL 2 is filled with buffer + sample + starter while in the EL 1 still remains buffer + sample.

E -E

PHASE 3: the instrument reads the ∆mpH between EL 2 and EL 1 for some seconds.

While the EL 1 works as reference electrode, in the EL 2 the starter catalyses the reaction.

The result calculation (slope of ∆pH curve) is performed skipping D1 (5 sec.) and the Lag Phase (5 sec.), beginning at second 10 until the end of measure phase is reached.

∆

mpH

t (s)

Pump 1

Pump 2

Buffer + Sample

Buffer + Sample + Sarter

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If higher starter amount is used, the curve (b) will be obtained:

with ∆mpH b is lower than ∆mpH. PHASE 4: the instrument is automatically washed and the mixing chamber refilled with the

new buffer.

Lag Phase

t (s)

Lag Phase

∆

mpH

Starter

t0 t

total measure time

∆mpH

∆

mpH

Starter

t0 t

total measure time

∆mpH

∆mpHb

(b)

t(s)

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4. The CL-10 Plus Program

4.1. Menu and commands.

The following list describes the sections of the figure above:

A) Main menu and submenu If in the Menu or submenu the chose command name starts with an underlined letter, it is possible to activate the command pressing: ALT + the underlined letter, in order to enter the menu (ex. ALT + < f > to access File submenu); press instead the combination CTRL + the underlined letter to open the chosen submenu (example to “S

elect method” press CTRL +

< s >).

B) Toolbar

Contains all the basic icons that allow easy access to the functions:

¾ Select method (select method), ¾ Prime enzyme (fill in/empty enzyme tubing), ¾ Clean (clean cycle), ¾ Run the Start-Up (run the start up procedure, at the beginning of work), ¾ GO (run a measurement), ¾ Blank , ¾ Calibrate, ¾ Stop measure, ¾ Clear graph (cancel all the reactions on the graph) ¾ Print Graph.

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C) pH scale range and screen graph.

• The pH range, extended range is between + 7000 and - 7000 mpH. To select the range

pH scale use arrows <UP> and <DOWN> or the mouse to set the number.

• The base-line is normally between 0 ± 100 mpH. The selected pH range, for centred base-

line, always remains the same (also when the user leaves the program), until the user changes it.

• In the screen graph the measure time (sec.) is on the x-axis; the pH scale is on y-axis (in

this case minimum pH is -560 mpH and maximum is not visualised because under the File window).

• During alkaline reactions (OH-), which increases the pH value, the baseline on the screen

goes to higher values. During reactions which release hydrogen ions (H+, acidification) the baseline on the screen goes to lower values.

D) Temperature Actual measurement temperature is shown only after the start of measuring cycle (after GO command or F5 function key), until then it is displayed the temperature value corresponding to the last measurement made.

E) F) Parameter name It is a "non editable section" from this window (see method editing option at § 4.2.3); the name of the method described in the picture is COMBITEST giving three parameters ( pH, total acidity, glucose + fructose).

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4.2. FILE Menu .

4.2.1 Select method

This function is used to select the desired method for the routine. All default methods are supplied with the installation disk.

Attention: select a method only after choosing the kit intended to be used for the routine; in the package insert of the kit the name of the method (*.MD file) corresponding to the program is specified .

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4.2.2 Create a new method

Using this function, it is possible to create a new method. This function is only used by specialised personnel and not recommended to the user, unless advised by technical assistance.

4.2.3 Edit method

Using this function, it is possible to modify parameters of methods and possibly to insert new values for upgrades, as described in the package insert of the kits. Moreover, to change the user unit upon needs. Access the Edit Method command from file menu and follow the instructions:

∗ From File menu, select Edit Method command, then select the method chosen

(file.MD).

∗ If the version number of the method (*.MD) does not correspond to the same in the

package insert, contact costumer service.

∗ Modify the method parameters, as described, then select SAVE to confirm the

changes.

Figure 1

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∗ the new method, containing the modified parameters, is not overwritten to the previous

one, instead, it will be named with the Short Name field content. Rename the file (see picture 1 in the previous page) using the right hand button of the mouse, as described in the package insert, give the same release number (R): access to Edit Method and delete the original *MD file.

Attention: Do not change the method settings during an execution command (GO/Clean/Prime Enzyme/Blank/Calibrate); the instrument could halt immediately and loose the communication with the software; in this case it is necessary to reboot the PC and the instrument before starting the routine.

Description of method fields (in Edit method)

¾ The Method definition panel: Short Name: method name; is giving the name to the file, by default (max 10 digits). Description: name/ method description (max 10 digits). Execution: different kind of execution modes are available: End Point, Fixed Time,

Kinetics and Beta, as described in § 3.3.

STD units: calibrators concentrations, expressed in standard unit (SI), according to the

international standards (mM, µM, meq./L).

User units: used to obtain different measure units from standard units. Cnv factor: used to convert standard units (calibrator units) into User units.

Conversion of standard units into other units (user units):

Insert the new measure units in the User units field and insert the conversion factor (Cnv.factor) in the relative field and select Save. The software makes the following transformation:

Result (user unit)= Cnv. Factor x Result (STD unit);

Example for Glucose + Fructose :

STD units: mM User units: g/L Cnv factor: 0.18

The STD value is 200 mM; Result STD (g/L) = 0.18 x 200 (mM) = 36 (g/L)

Therefore, the same result (200mM), after user unit and conversion factor (cvn factor) have been introduced, becomes 36 g/L.

Note:

To return to standard units, delete the User units content and press Save. Automatically, the program gives the next results back in standard units.

¾ Execution parameters panel: Temperature (°C): defines the temperature of each method execution. Measure Time (sec.): total measure time from the selection of GO command (or F5 function

key) to the end of the measure and the start of cleaning (see § 3.2.3)

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Wait time (sec.): measure time of D1 phase (see § 3.2.3) Lag Time (sec.): this field is only used for execution of KINETICS and FIXED TIME.

(see. § 3.3.3-3.3.4).

¾ Offset parameters panel:

The Offset value is automatically set during the Blank measure. The value must be within the range defined by Minimum offset and Maximum offset and it can be a positive or negative number depending on the method.

During Blank measurement:

If the Offset value stays within Minimum offset and Maximum offset range, the result on the screen will be displayed in green. This result is saved in the method settings, in the Offset field.

If the Offset value is not within Minimum offset and Maximum offset range, the result on the screen will be displayed in red.

This result is not saved in the method settings.

¾ Slope parameters panel

The Slope value is automatically set during the Calibrate measure. The value must be within the range defined by Minimum slope and Maximum slope and it can be a positive or negative number depending on the method.

During Calibration measurement:

If the Slope value stays within Minimum slope and Maximum slope,

the result on the screen will be displayed in green. This result is saved in the method settings, in the Slope field .

If the Slope value is not within Minimum slope and Maximum slope,

the result on the screen will be displayed in red.

This result is not

saved in the method settings.

Significance of slope parameter: The slope corresponds to the gradient of calibration linear regression of the method. It is defined during calibration procedure, according to the following equation:

Slope= Calibrator concentration / Calibrator Signal

The slope is then used by the program itself during samples analysis, according to the following equation, to relate sample signal to its concentration:

Sample concentration = Slope x Sample signal

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¾ Standard field. Standard value is the calibrator concentration declared by the producer (), used to calibrate the method and define the slope factor.

¾ Results Panel. “Normal Values” of typical samples for a defined method are contained within the range Min Result and Max Result. This range may be changed by the operator, according to the needs.

If results are outside this range, they will be outlined in red on the screen.

¾ Quality Control Panel. This function is idle.

¾ Instrument initialisation string field. The ‘String’ (instrument initialization string) is a code that regulates the peristaltic pumps and the stirrer bar movements. These movements represent a measure cycle and a cleaning cycle for a specific method. Each method is characterized by only one dedicated string (or code); vice versa, the same string can characterize few methods. The string is specified inside the Kit package insert. The string can be visualized, created, modified trough a program (TBL Builder), different from the working program (CL-10Plus vs 4.3) and normally managed only by specialists.

¾ Prev/Next buttons. "Prev/Next " commands allow visualizing the settings of the second and, possibly, the third parameter if the method contains more than one of them, (ex, COMBI TEST METHOD contains the following parameters: pH, total acidity , glucose + fructose).

¾ Add method button. This function can be used to increase the options of a method; in the next § 4.2.4 a very important example is made for using this function in Biochemical Chemistry applications

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4.2.4 Add method for Measure unit change; Biochemical Chemistry Application

Transformation from U/L (unit/Liter) to U/g Hb (unit/Gram of Haemoglobin) for the following methods:

1. Glucose-6-phosphaeo dehydrogenase (G6PD)

2. Pyruvate Kinase (PK)

3. Acethylcholinestarase (Ache)

4. Butirrhylcholinesterase (Buche)

The default measure unit for erythrocyte enzymes concentration (G6PD, PK, ACHE, BUCHE) used by CL-10 Plus is U/L. However, it is possible to express the erythrocyte enzymes concentration also in U/g Hb, directly from CL-10 Plus Software, modifying the method (file MD).

For this function it is crucial to know the Haemoglobin concentration in the blood sample, then proceed as follows.

Example: G6PD in figure 1:

- From File menu select Edit Method and then G6PD_R1.MD. The method on the screen is

the method chosen for the measurement of the desired analyte (concentration units are U/L). Information hereafter given are the same in the package insert of the kit (EC Bio G6PD; Appendix B- method settings).

Figure 1

- Select Add method and fill in the settings as in Figure 2.

Figura 2

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- Select Add method again and fill in the settings as in Figure 3

- A third method is added. This gives the information for calculate results in U/g Hb.

Figure 3

- After the third method has been added, select Save, so that the changes are saved on a

new file, leaving unchanged the original file (G6PD_R1.MD); the method, with the new settings, will be saved as reported in the Short Name field (G6PD).

- Select Edit method and rename the new file G6PD as G6PD_HbR1.MD

(right hand button of the mouse placed on the document: Rename command).

- From File menu enter Select method and choose G6PD_HbR1.MD. Start the working

session, following instructions on the package insert.

- At the start of each measurement (GO command or F5 function key), it will appear a

window (Figure 4) in the same window as for the sample id and sample name, called Hb g% value, where it is supposed to be added the haemoglobin value for that sample. This value must be manually introduced by the operator at this stage for each sample, in order

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to transform the U/L into unit/gram of Hb. Results in the worklist will be visualized in both units (U/L and U/g di Hb).

Figure 4

Hb value activate for calculation U/gHb Hb value unactivate. Concentration given in U/L

4.2.5 Clear graph

This function is accessible trough the Clear Graph command from File menu or can also be

selected by the icon

4.2.6 Print graph

This function is accessible trough the print graph command from File menu or can also be

selected by the

icon.

4.2.7 Method name

It is possible to make a quick select method in the list of the last five methods used, accessing from File menu .

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4.3. START UP Menu .

Use the commands on this section at the beginning of the daily routine, as described in the package insert of the method chosen.

Shortcut keys

can be used to carry out menu commands in rapidity. Shortcut key combinations are listed to the right of a menu item. Instead of opening the menu and choosing a command, you simply press the key combination.

SHORTCUT KEYS LIST

F2 Prime Enzyme F3 Clean F4 Run Start Up F5 Sample F6 Blank F7 Calibrate F8 Rest Mode

4.3.1 Prime Enzyme or F2.

This function is used to fill-in the starter line (peristaltic pumps n° 5 and n° 6). The pumps rotate clockwise until the starter tubes around them will be completely full and ready to dispense the product in the mixing chamber at the next step.

The icon for the command is

; the function key is F2

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4.3.2 Clean or F3.

This function allows to fill-in the fluidic circuit with the buffer solution before starting sample measurements after standby conditions or at the instrument installation or at method execution change. For thorough washing of the complete fluidics this command is run twice;

The icon for the command is ; the function key is F3

4.3.3 START UP parameters.

This function allows definition of parameters for the start-up program and, by default, it has the setting described in the figure above.

Number of Start- up cycles: it allows to set the number of automatic measurements (GO) cycles (range from 3 to 9999).

Time interval between cycles: it allows to set the time lag (range between 0 and 120 seconds) between two consecutive cycles.

Maximum Error (mpH): it allows to set the maximum error allowed, i.e. the maximum ∆mpH (possible range between 0.2 and 10.0 mpH). The last three consecutive GO results of the Start-up must not exceed the Maximum Error value.

4.3.4 Run Start up procedure or F4.

This command runs sequential measure cycles, automatically; using the information given in the Start up parameters section. This procedure provides information about instrument stability and is normally used at the beginning of the working session, if advised in the package insert. The operator’s presence is not required, during this job, because at the end a window will register the result and give information on how to proceed.

The icon for the Run Start Up procedure command is

the function key is F4.

If, at the end of the start up procedure, the conditions described in the Start up parameters are not fulfilled, an error message will appear, asking to repeat the startup procedure (see picture underneath).

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∗ First case (Maximum error =0.5):

The startup procedure passed

∗ Second case (Maximum error =0.5):

The startup procedure failed

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4.4. ROUTINE Menu.

Routine menu is used for the method execution.

4.4.1 Sample (GO icon or F5).

Following the package insert instructions, at the stage of sample analysis, after the

calibration procedure and control, inject the sample, then select

icon or press F5 function

key. Samples can be identified:

After the GO command is given, before starting the measurement, a window appears (see pictures underneath); in the Sample ID and Sample Name fields identify the sample, then select Start Measure to start the measurement and Accept to confirm the inserted data.

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Use the option of automatic increment (from Service menu select Auto increment sample ID box, see § 4.6.1), in the Sample ID field the program automatically inserts a sequential number at each measure. After the measurement, the results obtained are saved in the WORKLIST, stored by date (see § 4.5).

4.4.2 Blank (B icon or F6).

The BLANK step is the first stage of calibration procedure and it is required to define the Offset; this value is saved in the method parameters. The method parameters can be

visualised trough the Edit method function (see.§ 4.2.3). The result must be in the range given in the method (Minimum offset e Maximum offset, §.

4.2.3), in order to be automatically saved, by the program.

The icon for this command is

or F6 function key.

4.4.3 Calibrate (C icon or F7).

This function, necessarily associated to the injection of a known concentration solution in the mixing chamber (calibrator), defines the Slope value (second step of the calibration procedure) and the value is automatically saved in the method parameters (if it is within the range described in the method by Minimum Slope and Maximum Slope, §. 4.2.3). The method parameters can be visualised trough the E

dit method function (see.§ 4.2.3).

The icon for this command is or F7 function key.

4.4.4 Stop Measure.

This function allows to stop immediately the measurement, without waiting to the end of the time set.

The icon for this command is . This function is enabled only after the D1 reading.

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4.5. WOKLIST Menu

The WORKLIST menu contains the commands for printing, modifying, exporting and deleting results of working sessions, stored in the archive.

4.5.1 Print Worklist

If the PC is equipped with a printer, through this command it is possible to produce the printout of data stored in the archive. The paper layout of the printed results can be customised, in the following way:

1. Exit the CL-10 Plus program

2. Opne the CL-10 plus (C:\cl10 plus) folder

3. Open the ‘’rptstruct.ini’ file (open as text file .txt)

4. The text field under the following headers (ONLY the text field) can be modified:

• [HEADER_1]

• [HEADER_2]

• [HEADER_3]

• [HEADER_1]

• [PAGEFOOTER] you can ONLY change:

i. LeftFooter = SpA

ii. RightFooter = CL 10 Plus

rptstruct.ini

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4.5.2 Edit Worklist

It is used to modify the Sample ID and/or Sample Name inside the Worklist, and to visualise specific entries.

If you wish to plot the graph of a measurement or sequence of measurements, visualize the entry (from File Position) and select ‘Plot Entry’ key, make the same for all the data to be plotted. Once all the desired data have been plotted, select ‘Done’ key to close the window.

4.5.3 Erase Worklist

This command is used to delete completely a working session from the database.

4.5.4 Export Worklist

This function is used to export data into text (.txt) or excel (.xls) format. This format (excel) enables the visualisation, editing and data transfer into other databases. To export into excel format, follow the procedure:

1. Select Export Worklist command:

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2. Select the worklist (W970729.DAT) corresponding to the desired working day (29th July, 1997):

3. select the boxes: Use semicolon to separate fields and Add title to export file

4. Press ‘Select’.

5. The following window appears: Select “find:” enter the destination folder

6. Change file type: file type from .txt to All Files ( * .*)

7. Give the name to the file from export to desired name followed by .xls extension

8. Press OK; follows the information:

select OK again to exit.

9. Data will be saved in excel format and can be visualised as follows:

10. Open the excel program from Start menu and open the saved file; The Auto- composition text Import will appear

11. Select the option limited and continue clicking on Next >

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12. Trough the separation fields it is possible to select the columns width; the separation symbol is: “;”

13. During the third and last step you can decide which column to transfer or delete, which format to give. After these steps you can select the Finish command to close the data formatting

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4.6. SERVICE Menu .

Service menu contains all possible profiles defining the working configuration.

4.6.1 Auto increment SAMPLE ID.

The Auto Increment Sample ID function, once activate (the box is selected with √), allows to obtain an automatic sequential number every analysis (also see §.4.4.1)

4.6.2 Enter rest mode.

This command activates the standby mode for the instrument halt period (normally at least 8-10 hours, for longer periods than 2 weeks, see § 5.5). With the rest program in active mode, one measure cycle of the method visualized on the screen is run and it is repeated at intervals and temperature defined in Instrument parameters (§ 4.6.3). Enter rest mode command remains blinded in case it is not activate from Instrument parameters (§ 4.6.3).

4.6.3 Instrument parameters (Access password: default).

This command defines working and Rest mode instrument configuration.

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• Mixer volume: to select the mixing chamber volume (where the reaction takes place).

Only one is the correct option:

a) High Volume = 1,2 mL

All methods use this volume setting. All the other options (medium volume, low volume) refer to previous CL-10 versions.

• Sample Pump Timing: This function is not active.

• Misc. parameters. This function is not active.

• Rest parameters: This function is used to define rest mode parameters (§ 4.6.2)

∗ temperature: Temp.(°C); normally set at 37°C (in any case between 20 - 40 °C) ∗ time interval between consecutive cycles: Period (min); normally set at 90 min ∗ the cycle type used in the rest mode is the same displayed on the program.

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4.6.4 Calibrate Temperature.

This command is only used by technical personnel. CL-10 Plus contains in the hardware all information for the right temperature during the working session.

To check that this setting is not idle: From Explorer Enter CL10 folder and open the text file (.txt): C:\cl10\cl10.ini:

cl10.ini

Under the Header [OPERATIONS], verify that TemperatureOffset = 0. Digit zero if a different value is displayed. From File menu select Save and close the window.

Once this is made, it is possible to change the PC without affecting the temperature offset; in fact this parameter remains completely independent from the PC used and it remains stored in the instrument hardware.

4.6.5 Communication parameters.

This window is used if it is required to configure and select the serial port (RS 232) used by CL10 Plus. The preferential channel is COM1, the alternative one is COM2, in case COM 1 is engaged in other jobs. COM3 and COM4 are not enabled. Concerning Baud Rate (9600), Data bits (8 bit), Stop bits (1 stop), Parity (None), the respective values must be as described in the picture; in any case the same as the interface values used by the PC.

4.6.6 Print Technical Report.

This command is used when required to check the instrument performance of a working session. The function is only used by technical personnel from , unless the CL-10 operator is addressed to cooperate. The printing procedure is the same as Print Worklist command (see § 4.5.1); although the printout has a different structure and a number of additional information (D1, D2, ∆mpH) compared to Print Worklist, allowing technical specialised personnel to detect possible causes of faults.

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4.6.7 Terminal Emulation

This command is mainly used by technical specialised personnel, to investigate upon possible faults. However, it is useful to the CL-10 Operator, also in two cases:

1. peristaltic pumps working performance check (hall sensors)

2. stirrer motor working performances (it manages the stirrer bar rotation in the mixing chamber, for obtaining homogeneous solutions).

1. peristaltic pumps working performance (hall sensors):

¾ From Service window enter Terminal Emulation command ¾ Digit ’*’ (asterisk) and wait for the symbols to appear as in the window

underneath, figure 1:

Figure 1

¾ Digit ‘+!‘ in sequence: all the numbers and respective commands appear.

Digit ‘5’ (ref: tst Hall sensors); on the screen appear numbers in sequence, as descbed in figure 3.

Figure 2

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Figure 3

¾ If the sequence runs to the end and then the instrument stops,

the test is positive

(meaning that no error is detected in hall sensors routine).

¾ If one of the sensors does not respond to the test, the sequence is interrupted

and the inefficient rotor stays under continuous rotation. This implies that the test is negative and repair or replacement of peristaltic pump set is required

(§ 6.1.2)

2. control of correct stirrer motor operation

ATTENTION: in case the sensors have passed peristaltic pumps working performances check, it is required to exit the Terminal Emulation and CL-10 working program, before accessing another Terminal Emulation command.

Switch CL-10 Plus off and on again, then access the working program and enter Terminal Emulation mode; then procede as follows:

¾ Digit ’*’ (asterisk) and wait for the screen to be filled with symbols, as in figure 1

¾ Digit ‘+!‘ in a sequence: all the numbers and respective commands appear.

Digit ‘7’ (ref: tst Stirrer); on the screen a column of numbers in a sequence will appear, spaced by a column of zero.

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When the column of numbers appears, it is possible to detect the stirrer bar inside the measuring chamber, by looking inside or inserting from the top a micropipette (Microman M25) tip and ‘feeling’ the collisions of the bar with the tip.

4.6.8 Electrodes Test 1 and 2.

This command is used to check the electrodes performances. Normally it is used by service personnel, only, unless directly advised the CL- 10 user to proceed.

For proceeding to the test select TEST-EL R3.MD method from Select method and operate following instructions from an representative and from package insert instructions: Art. No. GEN 603.

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5. Maintenance and Preservation

5.1 Weekly Maintenance

Run the following procedure:

→ at least 12 hours before the working session, on a weekly base, or → when the Blank value, during calibration procedure, is beyond the limits accepted by the

method, or

→ after changing any fluidic component (pump tubing’s, internal tubing’s, parts of mixing

chamber, etc).

1. Start the CL10 Plus program.

2. Replace the wash solution with REGENERATING SOLUTION.

3. Check that in starter position there is a vial containing distilled water.

4. Run 1 Prime enzyme and 2 Clean.

5. Wait until completion of cycles and 5 minutes more.

6. Replace the REGENERATING SOLUTION with reconstituted wash solution.

7. Run 1 Prime enzyme and 2 Clean.

8. Leave the instrument in Rest mode (see § 4.6.2).

5.2 Routine Maintenance

Run the following procedure:

→ regularly every 3 months → at least 1 day before working session, after extended period halt → when the ‘enzyme Contamination’ occurs (see. § 5.4 )

1. Start the CL10 Plus program.

2. Replace the wash solution with STRONG REGENERATING SOLUTION.

3. Check that in starter position there is a vial containing distilled water.

4. Run 1 Prime enzyme and 2 Clean.

5. Wait until completion of cycles and 3 minutes more.

6. Replace the STRONG REGENERATING SOLUTION with distilled water.

7. Run 1 Prime enzyme and 2 Clean.

8. Replace the distilled water with reconstituted wash solution.

9. Run 2 Clean cycles.

10. Leave the instrument in Rest mode (see § 4.6.2).

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5.3. Maintenance – rapid guide

WHEN CHECK DESCRIPTION SOLUTION

1) Unplug the pump tubing’s from their respective pumps and check if they are marked, thinner, and/or lost their original shape. Compare the tubing’s length with a new set (it is recommended to always keep a new set in stock, since an accidental brake can occur at any time. In that case the liquid is spread on the instrument and electric interference is possible, as well as (in the worst case), permanent corruption of the system.

2) Observe if, during measurement the liquid inside the coils behind the chamber, stops when the pumps stop, or flows continuously. If the flow continues during the measure time, the tubing’s are not in proper sealing conditions.

ONCE / month Consumption of

peristaltic pump tubing’s (SPP312-316)

3) Observe if during the wash cycle (Clean or F3) the liquid comes up to the top of the mixing chamber. If not, tubing’s are not in proper sealing conditions.

Replace all pump tubing’s with a new set, not only the damaged ones.

Every day Check the efficiency of

level pump (pump 3).

At the end of each cycle, pump 3 aspirates liquid from the top of the mixing chamber, in the tube, the last two centimetres are only air (or bubbles). If not, report to technical assistance.

Report the problem to technical assistance

During working session

Place the reaction graph properly inside the maximum and minimum limits.

The D1 (first few seconds of the reaction) must be a flat line and must overlap to the following reaction one (Figure 1). If the line is not flat (Figure 2), there is a possible contamination in the reference electrode, called Enzyme Contamination; see § 5.4

If the line is flat but does not overlap to the next cycle one, there is possibly a cloth in the fluidics or enzyme contamination in the reference electrode

see § 5 Figure1-2

see § 6.2.3

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5.4. Enz yme Contamination in the reference electrode

When Check problem description solution

Every working session

Enzyme Contamination in the reference electrode

If there is a D1 drift, like in Figure 2, an electrode contamination is

possible (in case of urea, the correct description of the problem

is urease contamination).

The problem affects the method performances mainly at high concentrations (Figure 2).

The result is a set of unreliable data, low repeatability and frequent failure of calibration/controls checks.

Using a Pasteur pipette, empty, reaching the bottom of mixing chamber, it from the top.

Remove the enzyme tubing from the top pf the mixing chamber

Fill in the chamber with 1 mL of HCL 0,1M and run a

measurement (GO or F5)

Inject 1 mL milk (any kind) and

run 3 Clean cycles (F3)

Reconnect the enzyme tube to the

top of the mixing chamber

Proceed to the working session, according to the package insert of

the kit.

Every working session

Kind of measure ‘end point’ see § 3.3.2

The curve, at the end of the reaction, has a final slope=0. If not, obtained results are neither trustful nor repeatable.

The reason is:

1) old peristaltic pump tubing’s, with no optimal sealing properties or

2) a blockage in the system.

Replace peristaltic pump tubing’s.

Check if the stirrer bar, inside the mixing chamber, is rotating (insert the Microman M25 tip in the chamber at the end of the measurement and ‘feel’ the bar movements.

The enzyme could be damaged or expired; replace with a new vial from the same kit or another kit of the same Lot n°.

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Figure 1

Figure 2.

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5.5 CL10 preparation for a period of halt (over 2 weeks).

Replace the reconstituted wash solution with distilled water, in buffer position. Check that in the starter position (connected to pump 6) and, in case, in AUX starter (connected to pump 5) there is a vial with distilled water.

1. Run two Prime enzyme cycles selecting prime enzyme icon or F2 function key.

2. Run three Clean cycles using clean icon or F3 function key.

3. Remove distilled water vials both from buffer and starter positions

4. Empty the fluidics repeating the sequence 1) and 2) and empty the waste bottle.

5. Unhook all 6 peristaltic pumps tubing’s from their seats around the peristaltic pumps and leave them as described in picture underneath.

6. Exit the CL-10 Plus program (select Exit from File menu).

7. Switch the instrument and the PC off.

8. Close the protective Plexiglas shield.

5.6 Instrument Reactivation

1. Hook the 5 or 6 pump tubes in their seats, without twisting them and avoiding knots

that may prevent the correct fluids circulation in the system.

2. Place the reconstituted wash solution in buffer position and an empty bottle in the

waste position.

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3. Switch CL-10 Plus on from the instrument rear panel.

4. Wait for about 2 minutes to the end of the first standby cycle.

5. Enter the CL-10 Plus working program.

6. Run three Clean cycles using the clean icon or F3 function key.

7. Replace the wash solution with REGENERATING SOLUTION in buffer position.

8. Place a vial containing distilled water (2 mL minimum) in starter position and insert the

starter tube needle (connected to pump 6) and, in case, also the AUX starter needle (connected to pump 5).

9. Run two Prime enzyme cycles selecting the prime enzyme icon or F2 function key.

10. Run Clean three cycles using clean icon or F3 function key.

11. Wait for 5 min.

12. Replace the REGENERATING SOLUTION with the wash solution in buffer position

and run three Clean cycles.

13. Leave the instrument in Standby mode for at least 24 hours before the working

session.

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6. Trouble Shooting

6.1. Electrical Section.

6.1.1 Instrument does not switch on.

The green led under the Plexiglas cover does not have the light on. Check that the electrical connections are properly plugged in (the cable from the analytical unit to the power converter and the power converter to the electrical source).

6.1.2 Pumps do not operate when the instrument is on.

CL10 plus peristaltic pumps group (Code SPP 320) is made of three black rotors on a metal support; underneath, there is an electromagnetic field that manages the rotations. It may happen that an electrical interference (some liquid spilled on a rotor and slipping inside the circuit) damages the rotor- circuit communication or a whole set of pumps is not responding. Vice versa, it may happen that one of the rotors, once activated, is never stopping, regardless the method selected or the command given. In order to understand if the problem is given by an electrical fault, it is recommended to use Terminal Emulation (see § 4.6.7) command. In case a problem is found to the group of pumps, replacement of the part is necessary.

Replacement of peristaltic pumps group

Empty the system (§5.5). Unscrew the set of pumps from its place on the chassis and check if the motor flat cable is correctly connected to it (Fig. 1) (if traces of salts and dirt are found, remove using a moistened cloth).

FIGURE 1: CL10 PERISTALTIC PUMP SET

A CHASSY B LEFT PERISTALTIC PUMPS GROUP C RIGHT PERISTALTIC PUMPS GROUP D ALLEN STEEL SCREW E MOTOR FLAT CABLE

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Close the panel, fix the pumps into their seats and switch the instrument on. If the problem persists, ask for technical assistance.

6.1.3 The analytical unit does not accept commands from the program.

• The “CL10 Protocol Error” following box appears on the screen.

• Check that the instrument is on

• Check that the CL-10TBL_BUILDER program is off (if not, turn it off)

• Check the connections between the serial port RS 232 (COM 1 in the PC) and the

analyser; if the instrument is connected to the wrong serial port (COM 2), connect it to the serial port COM 1, or select the COM 2 port from Communication Parameters in SERVICE menu (see following windows):

• If the problem persists, ask for technical assistance.

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6.2. Fluidic Section.

6.2.1 The Mixer does not fill-in or empty.

• Check that there are no broken, chocked or obstructed peristaltic tubes

• Check that the connections to the pumps are not inverted (electrode 1 connected to

pump 1, electrode 2 connected to pump 2).

• Place a bottle of distilled water in buffer position and manually turn the buffer pump

(pump 4) clockwise; check that the water is correctly loaded and reaches the mixer. If any defect is found replace the pump tube and inspect the fluidic line.

• When the mixing chamber is full, manually rotate the pump of the Electrode n.1

(pump 1) clockwise and empty the Mixer.

• If the mixing chamber does not empty, check if tubes are broken or obstructed and if

there is any leakage.

• Fill in the mixing chamber and repeat the last step with the pump of Electrode n. 2

(pump 2).

• Turning pump n.4, fill the mixing chamber up to the top. Manually rotating the Level

pump n.3 clockwise, check that it levels.

• Carry out a measurement (Sample command) and check that the magnetic stirrer inside

the mixing chamber start rotating at the beginning of the measurement for approx. 4 seconds. If you cannot hear it, insert a pipette tip in the chamber, you should feel the collision with the bar.

• If not, ask for technical service.

6.2.2 Liquid spills out from the mixing chamber.

If some liquid spills out from the mixing chamber during the cleaning, it is possible that the fluidics have an obstruction. In this case follow the procedure to ‘release’ electrodes channels from sediments, obstructions, etc:

1. Place the wash solution (Polif kit ) in place of the working buffer,

2. Run 2 Clean cycles using clean icon or F3 function key,

3. Unplug the pump 1 tubing from its place around the rotor,

4. Take the connection between the pump tubing and the waste off,

5. Connect a 10mL syringe to the loose end of the pump tubing and pull/push the piston

for few seconds.

ATTENTION: during this phase, pushing the piston, some liquid could flow out the top of the mixing chamber. Therefore, it is advisable, with the hand, to keep a tissue on the black stopper of the chamber and push the piston gently.

6. Run 2 Clean cycles using clean icon or F3 function key

7. Repeat the same procedure with electrode 2 (pump 2).

8. Run 4 Clean cycles using clean icon or F3 function key and leave the instrument in

standby until the following working session.

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6.2.3 Absence of signal during the measurement.

• Check that the cable plugs of the two electrodes are inserted correctly.

• Check that the Starter is injected correctly into the stopper of the mixing chamber.

• Using a metal wire inspect that the starter course and the steel capillary on the stopper

are free from dirt and deposits.

• Call for technical assistance

6.2.4 Unstable Measurements .

In case the measurements are unstable (The D1 of consecutive measurements fluctuates more than 0,5 mpH) carry out the following instrument checks:

• The Fluidic courses are clean (see also § 6.2.2).

• The Starter is injected correctly

• The Level pump (pump 3) is working correctly

• Stirrer bar inside mixing chamber rotates (see also § 6.2.1)

• The mixing chamber is free from deposits, rust, salts.

If the problem persists, call for technical assistance.

6.3 Electrodes.

6.3.1 Control of electrodes performances

The following procedure is used and recommended by in order to control electrodes performances; stability, linearity and promptness of response. This procedure is called: Electrodes Test.

If the instrument performances are not good, it is important to exclude a fluidic problem (old and overrun tubes, etc) then run the maintenance (§ 5.2/5.3). If performances stay unchanged and the ‘electrodes test’ is not passed

, replace the damaged electrode or both

(the test detects which electrode is not performing properly).

Required instrumentation

KIT: Electrodes Test Art n° GEN603 Micropipette: Gilson Microman M25 Injection volume: 24 µL Report: Electrodes performance CL-10 Plus Report

Test Electrodes Procedure

From File menu, trough Select method select: TEST-EL R3.MD

Attention: Check the version number (R3= release 3) to be the same in the program as in the package insert of the kit, if not, call technical assistance before proceeding.

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1. Replace the reconstituted wash solution with R1 buffer of Electrode test kit.

2. Disconnect the starter tube (B, Figure 2) from the steel capillary (A) and insert it in a vial containing distilled water (the same where also the other end C, is placed)

3. Follow the instructions of package insert and fill in the form: Report Electrodes performance CL-10 Plus (available upon request and included in the kit GEN603).

FIGURE 2: STARTER line

4. If necessary, print the graph of the test (use the Print Graph icon), after placing the limits at +/-100 from the symmetry line of the graph.

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Electrode test performances evaluations

A performing electrode must fulfil the following requirements:

∗ The max difference between the D1’s is < 1.5 mpH. ∗ Test Electrodes Blanks: the three segments, R1f, R2f and R3f differ < 1 mpH between

max and min.

∗ Test Electrode with HCl: R3f is within range 87-93 ∗ (R1f/R3) x 100 ≥ 45%. ∗ The third segment R3 has a drift <1 mpH: R3f - R3i < 1.5 mpH.

6.3.2 Electrodes replacement.

The instrument is delivered with two glass electrodes. The average lifetime of each electrode depends on working and maintenance conditions. Electrodes are covered by 24 months warranty from delivery. If the instrument has damaged or old electrodes (check as described in § 6.3.1), follow the instructions hereafter or call technical assistance (compulsory if electrodes are under warranty):

Place a bottle of distilled water in buffer position on the instrument.

Run 3 Clean cycles using the clean icon or F3 function key.

Replace the bottle of distilled water with an empty bottle and run 3 Clean cycles. The system will discharge all the water and flush inside only air, to be ready for the next steps.

Remove the steel cover of the mixing chamber (Fig. 4, part A) by unscrewing the black stopper on the top (Fig. 4, part B) and the o-ring underneath (Fig. 4, part C).

Unscrew the electrodes holder (Fig. 3, part E).

Unplug the electrodes cables from their seats on the chassis (Fig. 3, part F) and silicon coils from the back of the electrodes (Fig. 3, part C) .

Gently pull the electrode/s from its/their housing/s.

Transfer the silicon “tip/s” (Fig. 3, part B), if not damaged, on the new electrode/s.

Connect the electrodes fluidics, trough the silicon tubing to the coils (as described in Fig.2 § 2.3.5) of the respective pumps; electrode 1 to peristaltic pump 1 and electrode 2 to peristaltic pump 2.

10.

Insert both electrodes in the measuring chamber body (Fig. 3, part A).

Attention

: do not push the electrodes too hard, but fix them properly, in order to touch the front Plexiglas

panel of the mixing chamber (Fig. 4, part D) gently.

11.

The electrode cable (Fig. 3, part F) must be sitting over the silicon tubing’s, in order to avoid any possible electrical fault in case of liquid spillage from the silicon tubing’s.

12.

Push gently the electrodes holder (Fig. 3, part G) towards the electrodes; fix it using the screw.

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FIGURE 3: Electrodes Installation

A MEASURING CHAMBER B SILICON TIPS C FLUIDIC’S D GLASS ELECTRODES E ELECTRODES HOLDER SCREW F ELECTRODE CABLE G ELECTRODES HOLDER

A STEEL COVER B BLACK STOPPER C O’RING D FRONT PANEL PLEXIGLASS

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FIGURE 4: MIXING CHAMBER

FIGURE 5: PERISTALTIC PUMP GROUP

A CHASSIS B LEFT PERUISTALTIC PUMP GROUP C RIGHT PERISTALTIC PUMP GROUP D EXHAGONAL SCREW E FLAT CABLE

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6.4. CL-10 Plus troubleshooting – rapid guide

SYMPTOM CAUSE SOLUTION

The green lamp on the front does not switch on

The switch on the back is in OFF position

The power supply is disconnected

The internal fuse is blown

Turn the switch on the back to the position ON

Connect the power supply

Unplug the power supply for 5 minutes

and plug back in (restorable fuse). The program does not start and does not accept commands

The serial port is not connected Connect the serial port to the analyser

The read out temperature is 99° centigrade

The flat cable is not connected Connect flat cable or control that it is

connected correctly

During D1 and/or D2 reading, values beyond +/- 800 are obtained: Saturation effect

buffer pump (pump 4) tube is broken and leaks.

method settings are wrong (check from Edit method).

Replace the broken tube

Compare method settings with the

package insert settings and change if

necessary.

If there are differences call technical

support.

No enzymatic reaction is observed during calibration

The starter tube has an occlusion

The starter tube is broken/old and seals not properly

With the starter tube full of water,

disconnect both ends and run F2

(Prime enzyme) once. If the liquid

inside is not flowing, change the

peristaltic pump of the tube, if the

problem persist change all the

components.

Replace peristaltic pump of the tube

(pump 6)

During the routine, some liquid overflows from the black stopper (Figure 4, part B, page 70)

One of the electrodes has the channel blocked by sediments

One of the peristaltic pump tubing’s (pumps 1,2 3) is blocked, broken, damaged

see § 6.2.2

Inspect all the fluidics and replace the

damaged tube

During Start up procedure the measurements are very unstable (D1 and ∆mpH fluctuate more than it is allowed in the package insert of the method used).

The instrument has not reached working temperature, yet

The instrument has not been used for a long time

There is a contamination (see Enzyme contamination) in the system.

Wait for equilibrium temperature

see § 5.4 for activation procedure

See charter 5

Unstable sample measurements

Stirred blocked

Instrument not in use for a long period

Deposits in mixer (deposits, salts crystallization)

Peristaltic tubes of pump 1 or 2 or 3 are damaged

see § 6.2

see § 5.4 for activation procedure

Empty the system, inspect the inside

of mixing chamber (see § 6.3.2 how to

look inside the chamber) and if

necessary clean with ethanol

Inspect and replace the tube

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SYMPTOM CAUSE SOLUTION

Blanks regular, the control or the sample do not react

The starter doesn’t reach the mixing chamber

initialise has not been carried out (Prime enzyme or F2)

The starter needle is not correctly inserted into the starter vial

The starter has not been restored

The starter course is blocked

Inspect the starter course and if

necessary change damaged parts

Carry out initialise cycle

(Prime Enzyme or F2)

Insert the needle in the vial and run

Prime Enzyme or F2 cycle

Replace the starter vial and run one

Prime Enzyme or F2 cycle

Inspect and if necessary change

damaged parts

High un-stability/D1 jumping values

There is a contamination (see Enzyme contamination) in the system

One of the electrodes has the channel blocked by sediments

liquid leakage from one of the electrodes

Damaged CPU mother board

See chapter 5

see § 5.1 3 and § 6.2.2

Check that the coils behind the

electrodes are correctly placed, are not

damaged or cut

Replace CPU or ask technical

assistance for repair .

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7. THE PIPETTE, characteristics, use and maintenance.

7.1 Materials.

Because of the nature and quality of the materials used, the Gilson MICROMAN requires no lubrication or maintenance.

7.2 Using the Microman Pipette.

To adjust the volume, turn the lower part of the push button. The upper part is independent and therefore prevents accidental alteration of the volume when the pipette is in use.

Read the volume from the top to bottom in microliters. The numbers written in black represent microliters and those written in red represent tenths of microliters.

The following procedures are recommended to obtain the maximum accuracy when setting a new volume:

•To set a volume below the initial value shown on the volume, come slowly to the new value

making sure not to pass the required volume.

•To set a volume above the initial value shown on the volume, pass the required volume by

one-third of a turn and then come back slowly to the new value making sure not to pass the required volume.

7.2.1 How to Mount the Piston.

The piston consist of:

- a sealing tip,

- a mounting stem.

Handle the piston with care in order to not damage the sealing tip.

Press the push button of the Pipette to the second stop. The clamp appears and opens.

Select a piston and slide the mounting stem fully into the clamp. Release the push button.

7.2.2 How to mount the capillary.

Slide the mounted piston into the capillary (colour matching the push button). Gently push the capillary back until it snaps onto the pipette shaft.

IF THE CAPILLARY TENDS TO SLIP OFF THE SHAFT, CLEAN THE SHAFT WITH TISSUE PAPER AND ALCOHOL.

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7.3 Pipetting.

BEFORE PIPETTING, FIRMLY PRESS THE PUSH BUTTON TO THE FIRST STOP. THIS CORRECTS THE POSITION OF THE MOUNTING STEM.

The pipette is now calibrated and the sampling will be accurate and reliable.

7.3.1 Aspiration.

-Press the push button to the first stop.

-Immerse the capillary 3 or 4 mm into the sample liquid.

-Release the push button slowly to draw up the sample.

-Wipe any fluid from the outside of the capillary, taking care not to touch the tip of the capillary.

7.3.2 Dispensing.

-Place the capillary end against the inside wall of the vessel and press the push-on button slowly to the first stop.

-Move the capillary away from the side wall, keeping the push button pressed, then withdraw the pipette from the vessel.

-Release the push button.

7.3.3 Simultaneous ejection of the piston and capillary.

The pipette has a second stop, located after the first one, which allows the simultaneous ejection of the piston and capillary. Press the push button of the pipette to the first stop and, pressing harder, to the second stop. At this stage both piston and capillary are ejected.

7.4 Pipette Specifications.

ACCURACY

(mean error)

PRECISION (repeatability)

MODEL VOLUME

µL

ABSOLUTE µL

RELATIVE %

ABSOLUTE S.D. µL

RELATIVE C.V. %

M25 Min

Max

3 5 10 20 25

±0.15 ±0.15 ±0.17 ±0.20 ±0.25

±5.0 ±3.0 ±1.7 ±1.0 ±1.0

≤0.06 ≤0.06 ≤0.08 ≤0.10 ≤0.10

≤2.0 ≤1.2 ≤0.8 ≤0.5 ≤0.4

CL-10 Plus – User Manual 75 / 75 ECPUS vers 0.2 eng

1 - Specifications were obtained by a gravimetric method with the temperature stabilised between 21 and 22 °C. The values given take into account all variables, error included, both normal warning of the handle, and the changing of the capillaries and pistons.

2 - Repeatability defined by dispensing 30 measures with distilled water, with a single instrument fitted with Gilson capillaries and pistons.

7.5 How to Inject The Sample.

- Check the volume set on pipette.

- Aspirate slowly the sample taking care not to have air bubbles into the capillary.

- Wipe with laboratory paper any fluid outside the capillary.

- Insert the capillary into the CL10 measuring chamber, until gently touching the bottom and

push with moderate speed the push button. Do not release the push button before having completely lifted the pipette from the measuring chamber.

ATTENTION:

- AVOID INSERTING THE CAPILLARY FORCEFULLY INTO THE MEASURING CHAMBER.

IT COULD DAMAGE OR AFFECT THE PIPETTING PERFORMANCES.

- REPLACE THE CAPILLARY AND PISTON IF YOU NOTICE SOME LIQUID INTO THE

CAPILLARY, BEYOND THE PISTON TIP.

- AFTER EVERY INJECTION WIPE THE CAPILLARY, OTHERWISE YOU MAY

CONTAMINATE THE NEXT SAMPLE.

Biocontrol CL-10 Plus User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual