Amersham UPC-900 Operation & User’s Manual

Text of Amersham UPC-900 User Guide:

• Amersham UPC-900, 5.3 Error messages If the suggested actions do not correct the fault, call Amersham Biosciences. Message Action 1 Perform a new start-up. The preceding message may tell more about the cause. 2 If not, call service. 1 Wrong value for averaging time set. See section 3.11 on how to set the averaging time relative to peak width. 1 Call for Service. 1 Connect the lamp, or call for service. 1 Check connection between optical unit and monitor. 2 Check that lamp and filter position correspond. 3 Change lamp. If warning persists, call for service. 1 If used in cold room, additional warm up might be needed.

• Amersham UPC-900, Storage of the pH electrode The pH electrode should always be stored in a 1:1 mixture of pH 4 buffer and 2 M KNO 3 when not in use. When the pH electrode is removed from the flow cell, the dummy electrode (supplied) can be inserted in the flow path. Electrode regeneration: If the electrode has dried out, immerse the lower end of the electrode in buffer with a 1:1 mixture of pH 4 buffer and 2 M KNO 3 overnight. 3.15 Restart after power failure If the power supply to the module is interrupted, it automatically restarts when pow

• Amersham UPC-900, 41 B.2.7 Setup adjust cell constant After replacing the flow cell, the cell constant has to be set. (The cell constant is shown on the packaging). 1 Select sub menu Setup Cond and press OK. 2 Select sub menu Setup Adjust Cell Const and press OK. The current cell constant is shown. 3 A warning message is shown until confirmed by pressing OK. 4 The current cell constant value is displayed as default. Enter the new cell constant as read from the packaging and press OK. The range is 0.1-300.0 cm -1 . When entering the cell constant from UNICORN, select System Control:System:Calibrate

• Amersham UPC-900, Monitor UPC-900 User Manual 18-1125-55 Edition AE 22 4 Maintenance 4 Maintenance 4.1 Periodic maintenance Interval Action (see procedures below) Every 3 months Check the monitor Every 6 months Change the pH electrode or more often if required Clean the UV flow cell When required Clean the conductivity cell Clean the pH electrode 4.2 Cleaning and checking the module Wipe the monitor housing regularly with a damp cloth. Let the monitor dry completely before use. Lamp intensity 1 Select menu Check and press OK. 2 Select menu Check Lamp Intensity. If: R <300 mV for 254 nm R <150 mV for 280 nm R <150 mV for 214 nm, replace the lamp according to Sect

• Amersham UPC-900, Fault Action Ghost peaks appear in the gradient profile 1 A charged sample has been detected (e.g. protein). 2 Air bubbles are passing through the flow cell. Check for loose tubing connections. If necessary, use a flow restrictor after the conductivity flow cell. Other problems Error in external pH or Cond. 1 Check the chart recorder in accordance with its manual. chart recorder 2 Test the recorder function and input voltage, which should be 1 V full scale. 3 Check the conductivity scaling and pH scaling, see section 3.8 Setting the Cond analogue output and section B.1.4

• Amersham UPC-900, 3.5 Reading the UV absorbance value The main menu 1 shows the absorbance value with four digits for the chosen wavelength. This menu is reached from any other menu by pressing ESC. If the lamp is off, Lamp Off is displayed instead. 3.6 Autozeroing UV The autozero function in the UV part of the module sets the detected absorbance to zero when OK is pressed. Using autozero is recommended at the start before the sample is injected. 1 From main menu 1, turn the dial one step clockwise to select Autozero UV, then press OK. The normal absorbance value display is then shown. In UNICORN, Autozero is set with the instruction AutoZeroUV in System Control:Manual:Alarm&Mo

• Amersham UPC-900, 9 This text disappears when the reading is stable and the following text is then shown: 10 Adjust the pH value in the display using the dial so that it corresponds to the known pH value of the second buffer solution and press OK. 11 When the two point calibration is performed successfully, the following text is shown for one second: 12 From the Calibrate pH Buffer 2 menu, turn the dial one step clockwise to access sub menu Calibrated Electrode Slope. This menu shows the slope of the calibration curve, where 100% corresponds to

• 5 Trouble-shooting 5.1 General When contacting Amersham Biosciences for support, state the program version of the module, which is shown for 2 seconds during start-up. 5.2 Faults and actions If the suggested actions do not correct the fault, call Amersham Biosciences. UV measurement No text on the front display 1 Check that the mains cable is connected and t

• 4.5 Cleaning the conductivity flow cell in-place Remove the pH electrode and install the dummy electrode in the pH flow cell. Pump a cleaning or sanitising agent through the flow cell. The standard recommendation is to pump 1 M NaOH for 30 minutes and then wash out with buffer. 4.6 Cleaning the conductivity flow cell off-line If the conductivity measurements are not comparable to previous r

• Amersham UPC-900, 34 A Reference information Reference information A Description A.1 Module Monitor UPC-900 is an on-line monitor for measurement of UV- absorption, pH (optional in ÄKTA FPLC) and conductivity. The monitor can work with standard glass pH electrodes with a built in liquid-filled reference electrode and a BNC connector. Conductivity measurement has a dynamic range from 1 µS to 999.9 mS/cm and is suitable for a wide range of applications. Connector/switch Function UniNet 1 Computer network Analogue out 0-1 V Chart recorder outputs, 3 channels 0–1.0 V Optical Unit UV signal from optical unit pH-Probe Connection to pH electrode, standard BNC socket pH-Groun

• Amersham UPC-900, 2.3 Installing the optical unit Installing the holder 1 Hook the holder into the slot on the right hand side of the module. Secure it by pushing up the slide clamp. Flow cell installation There is one analytical (5 mm) and one preparative (2 mm) flow cell available (stand-alone units only include the preparative flow cell). Both cells are installed in the same way, as described below: 1 Remove the red protective plugs from the detector housing and the flow cell. 2 Insert the flow cell into the detector housing from above. Note: The flow cell can only be placed in one correct position. 3 Secure the flow cell by turning the locking nut unt

• Amersham UPC-900, 49 B.5 Menu overview B.5 Menu overview (continued) C Technical specifications The full specifications apply only after at least 1 hour warm-up. C.1 Operating data UV measurement Absorbance range 0.01 - 5 AU (full scale) Autozero range -0.2 - 2.0 AU Baseline adjust Adjustable 0-100% of full scale Wavelengths Hg lamp, fixed 254 and 280 by changing filter 313, 365, 405, 436 and 546 nm Zn lamp 214 nm Linearity, 5 mm cell <3% up to 2 AU at 254 nm <5% up to 1 AU at 280 nm Static noise short term 40x10 -6 AU at 254 nm (typically 6x10 -6 AU at 254 nm) long term 40x10 -6 AU at 254 nm Static drift ±100x10 -6 AU/h a

• Amersham UPC-900, Temperature sensor accuracy ±2.0 °C drift ±0.5 °C per 10 h Flow rate sensitivity ±1% within 0–100 ml/min Conductivity flow cell Flow rate 0 - 100 ml/min Max. pressure 5 MPa (50 bar, 725 psi) Generated backpressure Max. 0.01 MPa at 100 ml/min pH measurement pH range 0 to 14 (spec. valid between 2 and 12) Accuracy temperature compensated ±0.1 pH within +4 to +40 °C not temperature 50 Monitor UPC-900 User Manual 18-1125-55 Edition AE C Reference information

• Amersham UPC-900, 45 B.2.14 Setup display angle Sets the display angle to compensate for different viewing heights. 1 Select main menu Setup and press OK. 2 Select sub menu Set Display Angle and press OK. 3 Select viewing angle ( ->\ Up, ->| Mid or ->/ Down). B.3 Setting and using the alarm timer You can set the alarm function to either a fixed alarm time or use a count-down timer. You cannot set both an alarm time and the count-down timer. The default or current value is shown in parentheses. 1 Select main men

• 36 A Reference information A.3 pH electrode The pH electrode is of the sealed combination double junction type. It contains a sealed Ag/AgCl reference which cannot be refilled, an internal electrolyte bridge of 4 M KCl saturated with Ag/AgCl, an outer electrolyte bridge of 1 M KNO 3 , an annular ceramic reference junction and a low profile pH membrane. The pH electrode is delivered

• Amersham UPC-900, Monitor UPC-900 User Manual 18-1125-55 Edition AE 6 2 Installation Connection to the column 1 Fix the optical unit directly under the column if possible. Note: Always position the optical unit with the filter wheel cover facing upwards. 2a Connect the column outlet tubing directly onto the top (a) of the optical unit for the 5 mm flow cell using a fingertight connector. 2b Connect the column outlet tubing onto the bottom (b) of the optical unit for the 2 mm flow cell using a fingertight connector. Note: The inlet port of the 5 mm UV cell is above the optical unit. The inlet port of the 2 mm UV cell is below the optical unit. 3 Screw to fingertightness. 4 Connect

• Amersham UPC-900, 3.3 Main menu overview Main menu 1. This menu is accessed from all positions by pressing ESC. Lamp control. This menu is accessed by turning the dial one step counter-clockwise from main menu 1. It is used to manually switch the lamp on/off. Autozero. Used to zero the UV signal from the monitor. Eventmark. Used to set eventmarks on the UV curve. Main menu 2. Used to display pH, temperature (in the conductivity flow cell) and conductivity in mS/cm as well as in percentage of full range. Parameter menus. Used to set measurement parameters for Cond, p

• Amersham UPC-900, 40 B Reference information before adjusting the cell constant (see section B.2.2). 1 Prepare a calibration solution of 1.00 M NaCl, 58.44 g/l. Let the solution stand until it is at room temperature. This is important for exact measurements. 2 Fill the flow cell completely with the calibration solution by pumping at least 15 ml through the cell with a syringe. 3 Stop the flow and wait 15 minutes until the temperature is constant in the range 20–30 °C. 4 Read the conductivity value displayed and compare it with the theoretical value from the graph below at the temperatur

• Amersham UPC-900, 4.8 Cleaning the pH electrode Note: The pH electrode has a limited lifetime and should be replaced every six months, or when the response time is slow or the slope is out of range (<80%). Use one of the following procedures to clean the electrode to improve the response: • Salt deposits: Dissolve the deposit by immersing the electrode first in 0.1 M HCl, then in 0.1 M NaOH, and again in 0.1 M HCl. Each immersion is for a 5 minute period. Rinse the electrode tip in distilled water. • Oil or grease films: Wash the electrode tip in a liquid detergent and water. If the film is known to be soluble in a particular organic solvent, wash with this solvent. Rinse the ele

• Amersham UPC-900, 3.13 Reading pH, temperature and conductivity values The main operating menu 1 shows UV absorption, conductivity in percentage of full range and pH. If the pH value is not stable or is changing, an asterisk is displayed, i.e. pH*. This menu is reached from any other menu by pressing ESC. If temperature compensation is switched on, the display will show Cond% T c instead of Cond%, see section B.2.4 Setup conductivity temperature compensation in Reference information. By turning the dial three steps clockwise, main operating menu 2 is shown instead. This display shows pH, temperature (in the conducti

• 2 Press OK to access the settings menu. The current setting is displayed. Press OK. 3 Set the desired full range value. Press OK to acknowledge. 4 Turn the dial clockwise to access the settings menu. The current setting is displayed. Press OK. 5 Set the desired zero level value. Press OK to acknowledge. Press ESC twice to return to menu Set Parameters. The module has a

• Amersham UPC-900, Monitor UPC-900 User Manual 18-1125-55 Edition AE 51 Reference information D compensated ±0.2 pH within +15 to +25 °C ±0.5 pH within +4 to +15 °C and +25 to +40 °C Response time <10 s (0 - 95% of step) Long term stability Dev. max. 0.02 pH/h (measured at pH 4.0) Flow rate sensitivity Dev. max. 0.1 pH units pH flow cell Flow rate 0.1 - 100 ml/min Max. pressure 0.5 MPa (5 bar, 72 psi) Generated back-pressure Max. 0.02 MPa (0.2 bar, 2.9 psi) at 100 ml/min C.2 Physical data Control Stand-alone or from a PC with UNICORN version 3.0 or higher, via UniNet 1 connection. Degree of protection housing IP 20, IP 21 flow cells I

• Amersham UPC-900, 1 Conductivity cell constant is out of range. 2 Wrong solution used during calibration. Use 1.00 M NaCl and recalibrate. 3 Air in conductivity cell during calibration. Flush the flow cell with calibration solution and recalibrate. 4 Dirty conductivity cell. Clean the flow cell and recalibrate. 5 If the problem remains, change the conductivity cell. 1 The key was pressed during self-test, or is faulty. 2 Switch off the module. 3 Switch on the module. 1 Switch off the module. 2 Check all connections. 3 Switch on the module. 1 Switch off the module. 2 Check all UniNet 1 and UniNet 2 connections. 3 Switch on the module. 1 Switch off the module. 2 Check

• 3.11 Filtering noise in the UV signal To filter the noise in the UV-signal, a moving average filter is used. The averaging time is the time interval used for calculating the moving average of the absorbance signal. A long averaging time will smooth out noise efficiently, but it will also distort the peaks. Peaks narrower than the minimum peak width value according

• Amersham UPC-900, 1 Press OK to accept the change. 2 Press ESC to skip the change. 1 The difference between 0 and 100% must be at least 0.1 mS/cm. 2 Increase the span between zero and full scale setting. See section 3.8. 1 Check that the conductivity cell is connected. 2 Recalibrate temperature. 3 If problem remains, replace the conductivity cell. 1 Check the pH electrode connection. 2 Clean the pH electrode. 3 If problem remains, change the pH electrode. 1 Factory calibration for pH is lost. The monitor can still be used but may not meet specifications for pH measurement. 2 Call for service. 1 Factory calibration for c

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