WPI APOLLO 4000 Instruction Manual
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WPI APOLLO 4000 Instruction Manual

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World's first fully-integrated Free Radical Analyzer
INSTRUCTION MANUAL
Serial No._____________________
APOLLO 4000
This instrument must not be connected to a local
network nor to the Internet. Do not attach any
peripheral device other than a USB printer. Any
change to the proprietary hard disk registry in this
device — whether by virus or by normally benign
hardware or software installations — may render the
drive or the Apollo software inoperable, requiring the
instrument's return to the factory for reformatting.
File corruption or damage to applications or
operating system caused by such use will not be
covered by the Warranty.
061506
World Precision Instruments
APOLLO 4000
WARNING

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Summary of Contents for WPI APOLLO 4000

  • Page 1 APOLLO 4000 World’s first fully-integrated Free Radical Analyzer INSTRUCTION MANUAL Serial No._____________________ APOLLO 4000 WARNING This instrument must not be connected to a local network nor to the Internet. Do not attach any peripheral device other than a USB printer. Any change to the proprietary hard disk registry in this device —...

  • Page 3: Table Of Contents

    Setting up the APOLLO 4000 ................. B-1 APOLLO.EXE Operating Software ..............B-4 Example of a real signal application ............B-12 USING THE APOLLO 4000 TO DETECT NITRIC OXIDE ....C-1 Initial Set-up ....................C-1 Calibration of the NO Sensor ................C-1 The Calibration Kit ..................

  • Page 5: Introduction

    Using WPI’s extensive range of free radical sensing electrodes the APOLLO 4000 is able to detect nitric oxide, hydrogen peroxide, s-nitrosothiols and oxygen. On-going research at WPI is focusing on expanding the range of detectable species.

  • Page 6: Design Architecture

    Port or Parallel Port communications. Plug-and-Play Design The APOLLO 4000 is designed for use with WPI’s range of free radical sensors. The user simply plugs the required sensor into any one of the input channels located on the instruments main front panel and then selects the detection and acquisition parameters using the integrated software control.

  • Page 7: Instrument Description

    The Apollo software can be programmed to display either redox current ( e.g. , pA) or concentration ( e.g. , nM). Output from the APOLLO 4000 can also be collected via BNC connectors on the rear panel of the instrument.
  • Page 8 APOLLO 4000 INTRODUCTION Channels Temperature DVD / CD-RW Temperature LED (red) Drive Input Sensor Power Sensor Input (green) mouse Analog Mains 110V/220V Outputs Power Switch Ethernet (BNC) Parallel Joystick Serial Keyboard Power Supply for Monitor Audio Video Input Audio Output...

  • Page 9: Unpacking

    Please read the section entitled “Claims and Returns” on the Warranty page of this manual. Returns: Do not return any goods to WPI without obtaining prior approval (RMA # required) and instructions from our Returns Department. Goods returned (unautho- rized) by collect freight may be refused.
  • Page 10 APOLLO 4000 INTRODUCTION WORLD PRECISION INSTRUMENTS...

  • Page 11: Operating Instructions

    OPERATING INSTRUCTIONS OPERATING INSTRUCTIONS To exploit the APOLLO 4000’s capabilities fully, it is very important that the user be aware of the general methods for operating the maintaining the instrument. This will also ensure that the user is able to understand and interpret the readings.
  • Page 12 Precautions for handling sensors The range of free radical sensors offered by WPI vary in their fragility. However, at all times the user must exercise caution to avoid damaging the delicate polymeric membrane covering the end of each sensor. This membrane...
  • Page 13 It is the responsibility of the user as well to install appropriate anti- virus protection software. WPI will not be liable for any loss of data as a result of power loss or virus-attack to the APOLLO 4000 system. WORLD PRECISION INSTRUMENTS...

  • Page 14: Apollo.exe Operating Software

    APOLLO 4000 OPERATING INSTRUCTIONS APOLLO.EXE Operating Software The operating software of the APOLLO 4000 is based on a standard Windows ® format, hence many of the software control features will already be familiar to the user. Main Screen The main working screen is shown in Fig.
  • Page 15 APOLLO 4000 OPERATING INSTRUCTIONS Menu System The Apollo 4000 software uses standard Windows ® controls. Help notes will therefore automatically appear when the cursor is positioned on any control function or word. The following section contains a brief description of the programs main menu system.
  • Page 16 APOLLO 4000 OPERATING INSTRUCTIONS • Sample Rate — Selecting this command triggers the following sequence of events: Current sampling rate is indicated (Fig. B6). If this value is satisfactory, the user can chose Cancel and continue to work with the current sampling rate.
  • Page 17 10 µA divided by 1,000,000 ( i.e. , 10 pA). If an incorrect range is chosen for any channel, the Apollo 4000 will indicate this as follows: Fig. B9 Green (sensor) LED Indication...
  • Page 18 APOLLO 4000 OPERATING INSTRUCTIONS • Poise Voltage . This submenu looks (Fig. B10) and works similarly to the Range control. Threre are four choices: Nitric Oxide — Automatically configures the poise voltage ( i.e. , 865 mV) on the selected channel to measure nitric oxide.
  • Page 19 APOLLO 4000 OPERATING INSTRUCTIONS • Unit Conversion. There are four default units of measuring signals: fA, pA, nA and µA. If a new unit needs to be defined chose the Unit Conversion submenu in the Setup control. The Amplitude Unit Conversion screen appears (Fig.
  • Page 20 APOLLO 4000 OPERATING INSTRUCTIONS • On / Off toggle control (also the push button in the left bottom corner of the screen). This menu starts and stops the acquisition of data, including writing to a data file. Before the acquisition starts, the program notifies the user about the sampling rate and maximum horizontal scale factor (Fig.
  • Page 21 (corresponding to filtering level 1) to 50 samples averaging (corresponding to filtering level 10). The user must select the most appropriate filter for the required application. About Menu (Fig. B21) shows the current version of the Apollo 4000 software. Fig. B21 WORLD PRECISION INSTRUMENTS B-11...
  • Page 22 APOLLO 4000 OPERATING INSTRUCTIONS Adding a note: To add a short note to a trace while recording, switch the operating mode from “PLOT” to “ANALYZE” (see page B-10). The data is still recorded in the background although the new incoming data is not plotted. Right- click a point on the trace where you want the Note to be added (see Fig.

  • Page 23: Example Of A Real Signal Application

    APOLLO 4000 OPERATING INSTRUCTIONS Example of a real signal application An example file is loaded in the Apollo 4000 (Fig. B24). Note the temperature sensor is also connected to channel 1 ( i.e. , 25°C line). However, the sensor signal is not seen.
  • Page 24 APOLLO 4000 OPERATING INSTRUCTIONS Fig. B26 demonstrates another option that the software offers: a second plotting window allows the data in any of the channels to be plotted separately for more detailed viewing. To enable the second window the user must double click inside the appropriate channel plot.

  • Page 25: Using The Apollo 4000 To Detect Nitric Oxide

    The chemicals required for the calibration are not provided. NOTE: The NO chamber (WPI #NOCHM) can be used as an alternative to the use of the calibration kit. Designed specifically for use with 2.0 mm electrodes, the chamber can be adapted to other probes.

  • Page 26: Calibration By The Chemical Generation Of No

    (NaNO or KNO ) which may be diluted as appropriate. Standard Nitrite is available from WPI, catalog #7357. Alternatively, crystalline reagent KNO may be used, but the user should note that is extremely hygroscopic and degrades once exposed to atmospheric moisture.
  • Page 27 APOLLO 4000 NITRIC OXIDE DETECTION have demonstrated that the nitric oxide generated from this reaction will persist sufficiently long to calibrate the NO sensor easily and accurately. Since the reaction goes to completion, the equation above states that the ratio between KNO and NO is 1:1.
  • Page 28 APOLLO 4000 NITRIC OXIDE DETECTION Place the stand (and water bath if appropriate) on the magnetic stirrer, and turn on the stirrer so that the bar is stirring at a moderate rate. Secure the ISO-NO sensor in an electrode holder or micromanipulator (or use one of the septums included with the start-up kit).
  • Page 29 APOLLO 4000 NITRIC OXIDE DETECTION Creating a Calibration Curve To create a calibration curve, the user measures the current (pA) generated by the addition of different amounts of KNO to the calibration solution. Once the baseline has been set to zero, generate a known concentration of NO in the solution by adding a known volume of a the NO standard (solution #2).
  • Page 30 APOLLO 4000 NITRIC OXIDE DETECTION Once the sensitivity of the probe has been ascertained ISO-NO Calibration Curve Based on Tabulated Data (in the above example the sensitivity was 1.557 pA / nM) 1600 the sensor is ready to use 1200 experimentally.

  • Page 31: Calibration Of No Sensor By Decomposition Of Snap

    APOLLO 4000 NITRIC OXIDE DETECTION Calibration of NO sensor by decomposition of SNAP This method can be used to calibrated all NO sensors (see Ref. 1: Zhang, et al. , “Novel Calibration Method for Nitric Oxide Microsensors by Stoichiometrical Generation of Nitric Oxide from SNAP” Electroanalysis , 2000, 12: 6).
  • Page 32 APOLLO 4000 NITRIC OXIDE DETECTION 428). However, in the presence of oxygen Cu (I) is readily oxidized to Cu (II). This will happen naturally if the compound is exposed to air and/or there is inadequate storage of CuCl. The oxidation product Cu...
  • Page 33 Note: The purity of SNAP used is extremely important to ensure an accurate calibration. We recommend the use of high grade SNAP with minimal purity of 98% or better. SNAP can be purchased from WPI in various amounts. Calibration Procedure Within a nitrogen or argon environment, place 10.0 mL of solution #1 (CuCl) in a...
  • Page 34 APOLLO 4000 NITRIC OXIDE DETECTION will reach a plateau and the second aliquot of SNAP can then be added. Successive additions of the remaining aliquots of SNAP can be made in a similar way. A calibration curve can be constructed by plotting the signal output (pA) vs concentration (nM) of SNAP.
  • Page 35 Note: The purity of standard reagent, SNAP, is very important for the reported data. Use high grade SNAP with minimal purity of 95% or better. SNAP can be purchased from WPI (catalog # SNAP50, SNAP100, SNAP500). Calibration Procedure Place 10.0 mL of solution #2 in a 20 mL vial (supplied in the calibration kit). Drop a small stirring bar into the solution, and place the vial on the top of a magnetic stirring plate.
  • Page 36 APOLLO 4000 NITRIC OXIDE DETECTION slowly decline because generated NO is quickly oxidized to nitrite and nitrate which will not be detected by the probe. Note: You can change the volume of injected aliquots according to the concentration of SNAP stock solution. Decrease the volume of aliquot if the electrode is very sensitive or increase the volume of aliquot if the electrode is less sensitive.
  • Page 37 25°C Catalyst solution 0.1M copper sulfate SNAP WPI, 98% purity. Fresh stock solution with 5 mg/250 mL solution EDTA added. Copper sulfate is at equilibrium with ambient air (aerobic conditions). SNAP (RSNO) decomposes to NO and a disulfide byproduct according to the following equation: 2RSNO →...
  • Page 38 APOLLO 4000 NITRIC OXIDE DETECTION is subsequently oxidized immediately before it is detected by the NO sensor. Example for creating a calibration curve and related computations 1. SNAP weight = 6.4 mg. 2. SNAP was dissolved in 250 mL solution #1 to obtain the standard stock solution.

  • Page 39: Calibration Of No Sensor Using Aqueous Standards Prepared With No Gas

    APOLLO 4000 NITRIC OXIDE DETECTION Calibration of NO sensor using aqueous standards prepared with NO Gas The following method can be used with all NO sensors. WARNING: Nitric oxide must be handled only in a well-ventilated area, usually a laboratory fume hood with forced ventilation . The U.S.
  • Page 40 APOLLO 4000 NITRIC OXIDE DETECTION *Lecture bottle of NO (14.2 liters, 98.5%) obtained from Aldrich, catalog #29.556-6; telephone 800- 558-9160 Figure C5 — brittle. The pressure regulator and tee purge adaptor should be stainless Setup for preparing a saturated NO steel.
  • Page 41 APOLLO 4000 NITRIC OXIDE DETECTION Place the distilled water vial in an ice-water bath. Reducing the temperature increases the solubility of NO in solution. Thus when the solution is used at room temperature you will be assured of a saturated NO solution.

  • Page 42: Measurement Of No

    Electrical Interference Although nitric oxide monitoring using the Apollo 4000 involves the measurement of extremely small currents ( e.g., less than 1 pA), the intrinsic noise level of the C-18...
  • Page 43 APOLLO 4000 NITRIC OXIDE DETECTION Apollo 4000 and NO sensors is sufficiently low so as to provide accurate measurements of nitric oxide. However various external electrical sources may couple to the system and produce large extraneous signals in the output record.

  • Page 44: Maintenance Of No Sensors

    When the ISO-NOP sensor is not being used it should be left connected to the APOLLO 4000 in ON position (or to Pre-Polarizer NSA-1, NSA-2 or NSA-3) with the tip suspended in distilled water. The basic structure of the ISO-NOP sensor is quite simple (see Fig.
  • Page 45 APOLLO 4000 NITRIC OXIDE DETECTION can also be purchased separately (WPI #5436). With proper care and by following the instructions below a membrane sleeve should last more than one month. Cleaning the Membrane The membrane sleeve itself requires very little maintenance. The primary concern is to avoid damage to the membrane and to keep it as clean as possible.
  • Page 46 NITRIC OXIDE DETECTION 5. If the electrode is not clean, repeat steps 3 and 4. If necessary the ISO-NOP Rejuvenator (WPI #JUV) can be used restore sensitivity of an old electrode (contact WPI for assistance). 6. Remove the locking cap from the old used sleeve, and gently slide it onto the new replacement sleeve.
  • Page 47 NO microsensors should be stored dry in a cool place away from direct sunlight. They can also be left attached to an ISO-NO Activator (WPI part # NSA-1, NSA-2, NSA-3). The Activator maintains the sensor in a polarized state, ready for immedi- ate use when required.
  • Page 48 APOLLO 4000 NITRIC OXIDE DETECTION C-24 WORLD PRECISION INSTRUMENTS...

  • Page 49: Using The Apollo 4000 To Detect Oxygen

    All the user must do is attach the sensor to the APollo 4000, turn the power on and wait for the current to decay to a stable value. this usually takes several hours. the current can be monitored directly on the APollo 4000.

  • Page 50: Calibration

    APollo 4000. When the APollo 4000 is turned off it no longer applies a polarizing voltage to the electrode Hence, it may then take several hours for the background current of the electrode to become stable once the APollo 4000 is switched back on again.
  • Page 51 APOLLO 4000 OXYGEN DETECTION Gas Phase Measurements Probe calibration for gas phase measurements can be accomplished using the calibration bottle,as described above for zeroing the instrument with nitrogen, using a tank of known oxygen composition, for example 00 % o...
  • Page 52 APOLLO 4000 OXYGEN DETECTION the physical interpretation of the % oxygen is the percentage of atmospheric pressure that the oxygen present exerts. For example, in a 00% oxygen environment a reading of 00 means that the partial pressure of oxygen is  atm (760 mm Hg). A reading of 2 means that the partial pressure of oxygen is 0.2 atm (60...
  • Page 53 APOLLO 4000 OXYGEN DETECTION Fig. D4 — Oxygen calibration trace in % oxygen. Aqueous measurements For aqueous calibration, fill the calibration bottle with distilled water to approximately two thirds of its full volume. immerse the probe tip into the water via the top hole.
  • Page 54 APOLLO 4000 OXYGEN DETECTION where pH o and p'H o are the partial pressure of water vapor at standard atmospheric pressure in atmospheres and in mm Hg, respectively (see Table 3). For example, the pH o in water- saturated air at 24 deg is 22 mm Hg (see Table 3).
  • Page 55 Calibration Method for O Measurements in Living Tissue or Blood the APollo 4000 and iso-oXY-2 probe may be used extensively in applications involving o measurements in vitro or in vivo in living tissue or fluids such as blood. You may still use the calibration procedure in this manual for these measurements since a membrane-covered amperometric oxygen electrode will always measure oxygen activity, not concentration.

  • Page 56: Probe Structure And Assembly

    APOLLO 4000 OXYGEN DETECTION since an oxygen detector only responds to the difference in activity across the membrane rather than the concentration difference. so in samples containing an electrolyte, while the oxygen concentration falls with increasing salt concentration the probe current remains constant.
  • Page 57 3. rinse the internal electrode with distilled water (particularly the tip) and let it soak for at least 5 minutes. Be careful not to let water get into the handle. the current on the APollo 4000 should go offscale when the electrode is being rinsed.
  • Page 58 (e.g., enzol, WPi #7363) can also be used. the membrane sleeves can also be sterilized chemically using an appropriate disinfectant (e.g., cidex, WPi #7364).
  • Page 59 APOLLO 4000 OXYGEN DETECTION Care of the Electrode the reduction of oxygen and other trace impurities causes a decrease in the surface activity of the working electrode. this phenomenon is referred to as “poisoning”, and over time has the effect of gradually reducing the electrode’s capability to generate a sufficient redox current.
  • Page 60 APOLLO 4000 OXYGEN DETECTION Table 1a Table 1a: solubility of oxygen °F °C °F °C in parts per million 4.6 4.2 (ppm) in fresh 3.8 water at different 3.5 temperatures, in 3. 2.8 equilibrium with 2.5 air at barometric 2.2 pressure of .9...
  • Page 61 APOLLO 4000 OXYGEN DETECTION Table 2: Table 2 oxygen solubility Altitude Pressure Solubility obtained from (feet) (mm Hg) Correction table a or Factor table b should be corrected -540 .02 if barometric sea level .00 pressure is different 0.98 000 0.96...
  • Page 62 APOLLO 4000 OXYGEN DETECTION Table 4 Bunsen Coefficients (α) for Solubility of Oxygen in Plasma and Blood Temp –––––––––––– Blood Hb g/100 mL –––––––––––– °C Plasma 10 g 15 g 20 g 0.0302 0.030 0.032 0.036 0.0323 0.0277 0.0282 0.0284 0.0287...

  • Page 63: Using The Apollo 4000 To Detect Hydrogen Peroxide

    DETECT HyDrOGEN PErOxIDE Initial Set-up Attach the HPo sensor to the required input channel of the APollo 4000. From the main setup menu of the software select the correct poise voltage “Hydrogen Peroxide” and appropriate range for the selected channel. the electrode must now be calibrated.

  • Page 64: Calibration Of The Hpo Sensor

    4 - 40°c can be controlled using an external circulating bath (contact WPi for information). Calibration Procedure 1. turn on the instrument Apollo 4000, set all the parameters that are needed in experiment. select the poise voltage for hydrogen peroxide in the software.
  • Page 65 APOLLO 4000 HYDROGEN PEROXIDE DETECTION note: the calibration should be carried out at the temperature at which the samples of H are to be measured. this can be accomplished by placing the vial and stand in a water bath at the appropriate temperature, and allowing the temperature of the solution in the bottle to equilibrate with the water bath.

  • Page 66: Interference

    When the iso-HPo- sensor is not being used (and for short-term storage) it should be connected to the APollo 4000 with power on and with the tip suspended in distilled water. tthis will keep the sensor polarized and ready for immediate use.
  • Page 67 5. if the electrode is not clean, repeat steps 3 and 4. if necessary the iso- noP rejuvenator (WPi #JuV) can be used to restore sensitivity of an old electrode (contact WPi for assistance).
  • Page 68 APOLLO 4000 HYDROGEN PEROXIDE DETECTION 10. suspend the tip of the new assembled probe in PBs buffer solution. 11. After 0-5 minutes the current should no longer be offscale and will gradually decrease with time. it may take several hours for the sensor current to reach a low stable value, at which time it will be ready for use.

  • Page 69: Troubleshooting For Apollo 4000

    The membrane may be damaged. Replace the membrane sleeve. c) The electrode has just been attached to the Apollo 4000. This is normal. 2) The sensor is unresponsive; current is close a) Make certain that the contents of the...
  • Page 70 APOLLO 4000 TROUBLESHOOTING WORLD PRECISION INSTRUMENTS...

  • Page 71: Appendix: Basic Grounding & Shielding Principles

    APPENDIX: BASIC GROUNDING & SHIELDING PRINCIPLES While the current model of the Apollo 4000 is protected against EMI it may still be necessary under very noisy conditions to provide additional shielding. Enclosing the system in a iron shield (Faraday cage) is the best way to shield against stray electric fields.
  • Page 73 WPI shall not be liable for any damage to data or property that may be caused directly or which the customer indirectly by use of this product.

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