WPI APOLLO 4000 Instruction Manual

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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 — 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.

www.wpiinc.com

061506

World Precision Instruments

APOLLO 4000

INTRODUCTION ............................................................................... A-1

Design Architecture ........................................................................................ A-2

Plug-and-Play Design ..................................................................................... A-2

Free Radical Sensor Technology .................................................................... A-2

Instrument Description ................................................................................... A-3

Unpacking ...................................................................................................... A-5

OPERATING INSTRUCTIONS ...........................................................B-1

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 .......................................................................................... C-1

Calibration by the chemical generation of NO ............................................... C-2

Calibration of NO sensor by decomposition of SNAP .................................... C-7

Calibration of NO sensor using aqueous standards prepared with NO Gas C-15

Measurement of NO ..................................................................................... C-18

Maintenance of NO Sensors ........................................................................ C-20

USING THE APOLLO 4000 TO DETECT OXYGEN ............................ D-1

Initial Set-up .................................................................................................... D-1

Calibration and Use of Oxygen Sensors ........................................................ D-1

Calibration ...................................................................................................... D-2

Probe Structure and Assembly....................................................................... D-8

USING THE APOLLO 4000 TO DETECT HYDROGEN PEROXIDE..... E-1

Initial Set-up ..................................................................................................... E-1

The structure of the HPO sensor .....................................................................E-1

Calibration of the HPO Sensor ........................................................................ E-2

Calibration Procedure......................................................................................E-2

Interference ..................................................................................................... E-4

Maintenance of HPO Sensors ......................................................................... E-4

TROUBLESHOOTING FOR APOLLO 4000 ........................................ F-1

any language, in any form, without prior written permission of World Precision Instruments, Inc.

WORLD PRECISION INSTRUMENTS 3

APPENDIX: BASIC GROUNDING & SHIELDING PRINCIPLES ........ G-1

INTRODUCTION

The APOLLO 4000 is the end result of an extensive three-year research and

development program aimed at designing the

most advanced multifunctional free radical

detection system available. Building on

WPI’s worldwide-recognized expertise in the field of nitric oxide detection and the success of its popular NO detector (the ISO-NO series), WPI’s scientists embarked on an ambitious plan to develop a state-of-the-art free radical detection system incorporating the very

latest digital signal processing (DSP) technology.

APOLLO 4000

INTRODUCTION

The APOLLO 4000 is an optically isolated multi-channel electrode-based free radical analyzer designed specifically for the detection of a variety of redoxreactive species of biomedical importance. The electrochemical (amperometric) detection principle used is similar to that employed in WPI’s popular nitric oxide detection system, the ISO-NO Mark II. However, the APOLLO 4000 incorporates numerous highly advanced design features that enable it to detect a broad range of redox-reactive species with unsurpassed accuracy and sensitivity. 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.

NO sensors used with ISO-NO Mark II are completely compatible with the APOLLO

4000.

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APOLLO 4000

INTRODUCTION

Design Architecture

The APOLLO 4000 is based on an optically isolated 4-channel configuration (a 2- channel version is also available). This design enables simultaneous real-time measurement of NO (or other free radicals) to be performed using up to 4 different electrodes. In addition, each free radical sensing channel also contains an independent channel for temperature measurement.

APOLLO 4000 incorporates a powerful single board computer and proprietary software (apollo.exe) that enables real-time display and data-acquisition of individual channels or any combination of channels. An extensive graphical user interface (GUI) based on a full color LCD monitor allows complete control and programming of all detection and data-acquisition parameters to be made using the standard keyboard and mouse included with the system.

The APOLLO 4000 consists of two functionally independent modules: Front End Converter (FEC) and User Interface (UI). The FEC is an 8-channel data-acquisition module based on a 24-bit A-to-D and 16-bit D-to-A conversion driven by a Digital Signal Processor (DSP). The User Interface is built on a standard PC platform with Windows 2000® operating system. A standard serial port (RS232) provides the communication between the FEC and UI. The system is fully compatible with a standard keyboard and mouse and can be readily interfaced with PC’s, computer networks, printers, and any device that uses Ethernet Tbase-10/100, USB, Serial 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. Each channel is also provided with an independent temperature input port that allows real-time monitoring of temperature using WPI’s appropriate temperature sensors.

Free Radical Sensor Technology

The APOLLO 4000 and its associated free radical sensors can provide fast, accurate, and stable measurements over a wide range of concentrations in both aqueous solutions and in gas mixtures. Its features include a rapid response time,

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APOLLO 4000

INTRODUCTION

high sensitivity and selectivity, ease of use, and versatility unmatched by any other similar instrument.

The detection principles are based on the electrochemical (amperometric) response produced by the various compatible free radical sensors. In summary, the free radical of interest diffuses through a selective membrane covering the sensor and is oxidized at the working electrode, resulting in an electrical (redox) current. The amount of redox current produced is proportional to the free radical concentration in the sample. All of WPI’s free radical sensors are “combination electrodes” in which the sensing and reference electrodes have been combined within a high performance Faraday shield designed to minimize susceptibility to environmental noise. The Apollo software can be programmed to display either redox current ( can also be collected via BNC connectors on the rear panel of the instrument.

APOLLO 4000 is fully compatible with WPI’s extensive range of free radical sensors. Currently this range of sensors includes electrodes for monitoring; nitric oxide, oxygen and hydrogen peroxide. However, new sensors are currently in development. For details on the complete list of compatible sensors please see the latest WPI product catalog, or visit WPI’s website (www.wpiinc.com).

e.g.

, pA) or concentration (

e.g.

, nM). Output from the APOLLO 4000

Instrument Description

Parts List

The package should contain the following items:

Part No. Description

APOLLO4000 Free Radical Analyzer 800408 Standard Keyboard 800407 Standard Mouse 800406 17” LCD monitor 35209 Program CD

— Power cables — Instruction Manual

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APOLLO 4000

INTRODUCTION

DVD / CD-RW

Drive

Temperature

LED (red)

Channels

Temperature

Input

}

Power

Mains

Power

Supply for

Monitor

Sensor

(green)

110V/220V

Switch

Keyboard

LED

mouse

Ethernet

USB

Outputs

Serial

Analog

(BNC)

Video

Sensor Input

Parallel Joystick

Audio

Output

Input

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APOLLO 4000

INTRODUCTION

Unpacking

Upon receipt of this product, make a thorough inspection of the contents and check for possible damage. Missing cartons or obvious damage to cartons should be noted on the delivery receipt before signing. Concealed loss or damage should be reported at once to the carrier and an inspection requested. 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 (unauthorized) by collect freight may be refused. If a return shipment is necessary, use the original container. If the original container is not available, use a suitable substitute that is rigid and of adequate size. Wrap the instrument in paper or plastic surrounded with at least 100 mm (four inches) of shock absorbing material. Please read the section entitled “Claims and Returns” on the Warranty page of this manual.

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APOLLO 4000

INTRODUCTION

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APOLLO 4000

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.

Setting up the APOLLO 4000

1. Place the APOLLO 4000 on a secure, flat surface (

2. Position the LCD display on top of the APOLLO 4000 and connect the

display cable to display output video port located on the rear panel of the APOLLO 4000 (see A-4).

3. Power the LCD display by connecting one end of the cable from the

provided voltage converter (AC to 12V DC) to the LCD display Power input receptacle. Connect the other end of the voltage converter to a matching 3-prong grounded wall receptacle and switch on.

4. Connect the power supply cord to the back of the APOLLO 4000 and plug

the other end into a matching 3-prong grounded wall receptacle (see setup diagram below).

NOTE: Ensure that the red voltage selector switch on the rear panel

(next to the power cable receptacle) is set to the correct voltage — 220 or 110.

e.g.

, laboratory bench).

Switching ON the APOLLO 4000

The unit can be turned on by pressing the “Power” pushbutton on the front panel.

After booting up, the system automatically starts the main application software (APOLLO.exe). If other applications have been used the user can return to Apollo4000 application by double clicking the “Apollo” icon in the right upper corner of Windows® desktop.

During the process of booting up no error messages should appear on the screen. (see Troubleshooting).

Switching OFF the APOLLO 4000

The APOLLO 4000 incorporates a highly advanced single board computer and

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APOLLO 4000

OPERATING INSTRUCTIONS

associated electronics. To turn the unit off, it is therefore only necessary to press the Power button once. It is recommended, however, to close the application programs

before

computer is not responding it may be necessary to press and hold the Power button in for 3 seconds before the unit will turn off (see troubleshooting section).

The alternative method for switching the unit off is to position the mouse cusor on START, click on hold down once, and then choose SHUT DOWN from the pop-up menu. This method will be familiar to Windows

Precautions for handling sensors

pressing the Power button. In some cases when the embedded

users.

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 prevents water and dissolved species such as ions and macromolecules from reaching the electrode surface where they would interfere with normal measurement and poison the electrode surface. When the sensor membrane becomes damaged, sample contents are free to react at the electrode surface. This causes the background current to become very large and/or go off scale (negative or positive depending on the reacting species). The membrane integrity of the sensor can always be checked by ascertaining that the current remains low and stable when the sensor tip is immersed in a 1.0 M saline solution.

Attaching a sensor to the APOLLO 4000

Each channel on the APOLLO 4000 is equipped with two high quality sensor input receptacles. The channel marked “Temp” is for use only with a compatible WPI temperature electrode ( only with one of WPI’s free radical sensors.

e.g.

, ISO-TEMP-2). The channel marked “Sensor” is for use

Fig. B1

attaching a temperature probe or sensor to the APOLLO4000, align the red dot of the instrument with the red dot on the cable connector.

– When

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APOLLO 4000

OPERATING INSTRUCTIONS

NOTE:

The Temp and Sensor inputs are not interchangeable but no damage to the APOLLO 4000 will occur if a sensor/electrode is accidentally inserted into the wrong input receptacle.

To connect a sensor, simply line up the red dot on the metal connector attached to the sensor cable with the red dot on the sensor input receptacle and insert the

cable connector (Fig. B1). An LED (located above each sensor input) will light up

immediately indicating the instrument and sensor are connected and working correctly. The Temp LED is red. The Sensor LED is green. The following LED indications will inform the user of the status of the system:

LED CONDITION INDICATION

Temp LED (red)

Steady RED light Electrode is performing normally

No RED light Electrode is not connected or is damaged

Sensor LED (green)

Steady GREEN light Sensor is performing normally within the

user-selected current range

Intermittent blinking GREEN light Sensor current is outside the user-

selected linear range.

No GREEN light ERROR sensor current range too low.

Sensor error or sensor not connected.

NOTE: WPI strongly recommends that APOLLO 4000 be powered

through a Back-UPS unit to avoid system failure during power loss. It is the responsibility of the user as well to install appropriate antivirus 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 B-3

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. B2. The software automatically recalls the last settings used, therefore the appearance of the screen will depend on how it was last used immediately before being turned off.

In the example shown, the set-up is for a four-channel nitric oxide application with horizonal scale of 6 sec/division and vertical scales 5000 pA/div for channels 1 to 4.

Data for each channel is displayed on the left of the screen. Each data channel display shows the following information:

Fig. B2

1. Zeroed — Relative measurement value (includes any zeroing applied to

signal).

2. Unzeroed — Absolute measurement value (

zeroing) often refered as “background signal”.

3. Temp — Temperature measurement in degrees Celsius.

4. Unit — e.g., pA, nM, etc.

Note: If no temperature sensor is connected there will be no value in the temp

window and no trace in the graph.

i.e.

, true value without

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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. However, in most cases a user familiar with Windows®-based operating programs will be able to operate the

software efficiently with the minimum of instructions.

Fig. B3

File Menu

Fig. B4

—

File Menu

• New

(Fig. B3) consists of typical Windows® commands:

—

starts a new data file

• Open — opens an existing data file

• Save — saves the current experiment with the default name

• Save as — allows the user to chose the name and the path of the saving data file

• Print — prints the screen to the system default printer

• Exit — exits the program and returns to the Windows® desktop.

The standard message (Fig. B4) appears when it is needed. For a detailed explanation of the above commands please refer to a Windows® textbook.

Setup Menu

(Fig. B5) consists of the following menu sub-

commands:

• Number of Channels — Sets the desired number of displayed channels. When One is selected then only the Channel 1 is displayed. Select Two Channels and 1 and 2 are displayed. Select Three channels for 1, 2, and 3. Select Four to display all four channels.

Fig. B5

Setup Menu

WORLD PRECISION INSTRUMENTS B-5

—

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.

NOTE: The default sampling rate is 10 samples per second, sufficient for most applications.

To select a different sampling rate, select OK. The sampling rate change screen (Fig. B7) will appear following confirmation screen (Fig. B8).

which may be undesirable. Therefore the user must carefully

sampling rate before the experiment.

50 samples/sec in increments of 5. It is very important for the user to understand that Windows®-based computers have the limitations of 16,384 pixels per screen and therefore (number of horizontal divisions=10) the maximum horizontal scale value will be limited to an integer of 1,638.4/ sample rate. After the new sample rate is selected the horizontal scale factor is set (for clarification) to the absolute maximum value. For instance, if the sample rate is set to 5 samples/sec the horizontal scale will be set to 327 sec/div. The user can reset it to any lower value (not lower than 1 sec/div).

Sampling rate can be set from 1 to

confirmation by another

The user can cancel the action and continue to work with the current setting. Changing the sampling rate changes the data file structure and it is therefore necessary to close the previous file (Fig. B4). This can interrupt the current experiment

select the

Fig. B6

Fig. B7

Fig. B8

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APOLLO 4000

OPERATING INSTRUCTIONS

• Range. After selecting this submenu the range control screen appears

(Fig. B9). The user must select a current range for each sensor channel within the maximum measured/expected value of the experiment. Proper selection of the measurement range is very important because the

dynamic range of the instrument is limited to approximately 1,000,000. This means that the intrinsic background noise (

i.e.

, “noise floor”) is proportional to the maximum measured value. For example:

If 10 nA range is selected, then the noise floor will be approximately 10 nA divided by 1,000,000 (

Conversely, if 10 µA range is selected then the noise floor will be approximately 10 µA divided by 1,000,000 (

i.e.

, 10 pA).

i.e.

, 10 fA).

If an incorrect range is chosen for any channel, the Apollo 4000 will indicate

Fig. B9

Green (sensor) LED Indication Remedy

Intermittent blinking Current detected by sensor is too high Select a higher range

for the selected range

No Green Light Current detected by sensor is too low Select a lower range

for the selected range

Steady Green Light Sensor is normal and within the selected range No action required

If the user is satisfied with the current setting he can chose work as previously.

this as follows:

Cancel

and

WORLD PRECISION INSTRUMENTS B-7

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 ( 865 mV) on the selected channel to measure nitric oxide.

Oxygen — Automatically configures the poise voltage ( the selected channel to measure oxygen.

Hydrogen Peroxide — Automatically configures the poise voltage ( 400 mV) on the selected channel to measure hydrogen peroxide.

i.e.

, 700 mV) on

i.e.

Fig. B10

Custom — Allows the user to manually set the poise voltage ( (Fig. B11).

Selecting the wrong poise

i.e

., from 0-2000 mV)

voltage will drastically change the results of an experiment and may render any data invalid.

confirmation (Fig. B12) prior to changing any poise voltage.

Hence the program asks for the

Fig. B11

Fig. B12

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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

Conversion

Conversion screen appears (Fig. B13).

submenu in the

Setup

Example: The user defines a new unit called “Custom” with the conversion ratio 1 unit = 186 pA (Fig.B14). If Add Unit button is selected then the new unit name appears in the list for the particular channel (Fig.B15). The user can define as many custom units as needed as well as modify the existing custom ones.

control. The Amplitude Unit

Unit

Fig. B13

Fig. B15

Fig. B14

WORLD PRECISION INSTRUMENTS B-9

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. B16).

• Plot / Analyze toggle control (also the push button in the left bottom corner of the screen) commands the program to start and stop plotting of the incoming data to the screen. In the Analyze mode there is a possibility to measure different parameters of the acquired data defined by Calculate menu with the cursors. The cursors appear when the pointer device (mouse) is moved to a certain position and its left button pushed. There are a total of two cursors. The second cursor appears after the first one when the pointer device left button is released

Fig. B16

Calculate Menu

program can recalculate from the recorded data stream (Fig.B17):

•Value and Delta — The value as well as time parameter of

plotted data is indicated in the appropriate windows (see Fig. B2) when the pointer device moves. Clicking of the pointer device left button fixes the first cursor. While the left button is depressed the first cursor position data is data of the current cursor position. When the left button is released the second cursor is fixed and the difference (delta) between second and first cursors is measured in the approriate amplitude and time windows.

• Samples, Average, Min, Max modes enable the appropriate figure measured minimum or maximum value accordingly.

includes five different self explanatory parameters that the

between

subtracted

the cursors. It is either number of samples, average,

from the

Fig. B17

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APOLLO 4000

OPERATING INSTRUCTIONS

Fig. B18

Fig. B19

Fig. B20

Tools Menu

process.

includes several tools to simplify and improve the acquisition

• Auto Scale. Selecting this option (Fig. B18) allows the user to quickly find the signal within the plotting windows. This option moves the vertical zero position and sets vertical scale to an odd value.

• Zero option subrtacts the current value from the collected data. The channel individually, all together or unzero all of previously “zeroed” channels. The subtracting value is acquired at the moment of selecting the appropriate action. Zero operation can be applied as many times as needed. The unzeroed value is always displayed, too.

• Filter (Fig. B20) applies a moving average digital filter to the upcoming data. It helps to reduce noise and artifacts if desirable. Note that the filter does not remove or substitute data but rather makes the fast transitions of the signal smoother. There are 10 different orders of filtering from 5 samples averaging (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.

Zero

menu (see Fig. B19) allows to reset each

About Menu

current version of the Apollo 4000 software.

WORLD PRECISION INSTRUMENTS B-11

(Fig. B21) shows the

Fig. B21

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. Rightclick a point on the trace where you want the Note to be added (see Fig. B22). This will create a mark at that place. Double-click the created mark. A Note window will open where a message of up to 64 characters can be typed in (see Fig. B23). After the message is entered, close the window. The mark will remain on the trace and can be double-clicked for later viewing. Switch to “PLOT” to observe the data as they are being recorded.

To remove a mark from a trace, delete the message from the Note window. When the Note window is closed, the Note mark will disappear.

Fig. B22

Fig. B23

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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 ( line). However, the sensor signal is not seen. If we apply

Scale

appears as follows (see Fig. B25).

APOLLO 4000

i.e.

, 25°C

Auto

to the channel 1 it

Fig. B24

Fig. B25

WORLD PRECISION INSTRUMENTS B-13

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. The second window has a distinctive yellow background color. Only one second window can be opened at the same time. The size of the second window can be controlled by regular Windows controls ( ).

There are cursors enabled inside the second window and the appropriate value and delta measurements displayed. Please note that the trace on the expanded window is a snapshot of the window you double-clicked and is not updated with time.

Fig. B26

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APOLLO 4000

NITRIC OXIDE DETECTION

USING THE APOLLO 4000 TO DETECT NITRIC OXIDE

Initial Set-up

Attach the nitric oxide sensor to the required sensor input channel on the APOLLO

4000. From the main Setup menu of the software, select the correct poise voltage “Nitric Oxide” and appropriate Range for the selected channel. The electrode must now be calibrated.

Calibration of the NO Sensor

Accurate measurements of NO require an accurate calibration. Three calibration methods are described in this section. The first and most convenient method is based on a simple chemical reaction which generates known amounts of NO (from NO This method can only be used with the 2.0 mm sensor (ISO-NOP).

The second method is based on the decomposition of the S-nitrosothiol NO-donor, SNAP using either Cu(I) as described in Method I or Cu(II) as outlined in Method II, as a catalyst. The NO liberated from SNAP is used to calibrate the sensor.

The third method involves preparing aqueous solutions of NO standards from saturated NO solutions.

The Calibration Kit

Perform the calibration using the NO calibration kit (WPI catalog #5435) which consists of the following items:

Plastic stand with two holes; two glass vials; two silicon septums without holes; two silicon septums with holes and radial slit; one short needle; one long needle.

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. Calibration temperatures from 4 - 40°C can be controlled using an external circulating bath.

WORLD PRECISION INSTRUMENTS C-1

APOLLO 4000

NITRIC OXIDE DETECTION

Calibration by the chemical generation of NO

(Type of NO sensor that can be calibrated with this method: ISONOP)

This method is recommended for use with the 2.0 mm sensor (ISO-NOP). However, because it requires the use of strong acid it cannot be used with other NO sensors.

The first step is to prepare the following two solutions:

Solution #1: 0.1 M H

+ 0.1 M KI

2SO4

To make 500 mL of solution requires:

4.9 g of H2SO4 (2.7 mL of concentrated H2SO4 {18.4 M})

8.3 g of KI

Slowly

add the sulfuric acid to about 400 mL of distilled water while stirring. Then

add the KI and mix; finally add distilled water to a final volume of 500 mL.

Solution #2: 50 µM KNO

(or NaNO2)

The recommended method for preparing this solution is to purchase an ion chromatography liquid nitrite standard (NaNO2 or KNO2) 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

KNO2 is extremely hygroscopic and degrades once exposed to atmospheric moisture. It is therefore recommended that if the crystalline reagent is to be used that the reagent packaged under argon be purchased (available from Eastman Kodak Chem #105 7462), and that it be stored in a desiccator. While this will extend the life of the reagent, it will need to be replaced more frequently than will the liquid standard. The standard nitrite solution prepared from this compound should be stored in a gas-tight bottle and refrigerated.

This method of calibration is based on the following reaction:

2KNO2 + 2KI + 2H2SO4 → 2NO + I2 + 2H2O + 2K2SO

(1)

where a known amount of KNO2 is added to produce a known amount of NO. The quantity (and so the concentration) of NO generated can be calculated directly from the stoichiometry if the concentrations of the reactants are known. Since KI and H2SO4 are present in great excess the limiting reagent is KNO2. Experiments

WORLD PRECISION INSTRUMENTSC-2

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 tion will be equal to the amount of KNO be equal to the diluted concentration of KNO

1. Record the value of the sensor current before removing it from the distilled

water in which the tip has been immersed during storage.

2. Immerse the ISO-NOP sensor tip in a strong saline solution (1 M), and after

waiting a few minutes for the current to stabilize record its value. If the current

Figure 4 — calibration setup

and NO is 1:1. Therefore the amount of NO generated in the solu-

added. The final concentration of NO will

in the solution.

Calibration Procedure

is offscale or unstable after several minutes in solution, it is likely that the membrane has been damaged and the sleeve needs to be changed (refer to the section on “Changing the Membrane Sleeve”).

3. Place a magnetic stirring bar into one of the glass vials included in the calibration kit. Add an appropriate volume ( 10 mL) of solution #1.

e.g.

4. Note that the calibration should be carried out at the temperature at which the

Fig. C1

calibration setup

—

measurements of NO are to be made. 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.

WORLD PRECISION INSTRUMENTS C-3

APOLLO 4000

NITRIC OXIDE DETECTION

5. 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.

6. Secure the ISO-NO sensor in an electrode holder or micromanipulator (or use one of the septums included with the start-up kit). Do not push the sensor

tip through the hole — slide the elctrode through the sliced side of the septum. Carefully lower the sensor into the vial sealing the opening

with the septum. The sensor tip should be immersed about 2-3 mm into the solution, and should not be in contact with stir bar. Be very careful when

inserting the sensor not to make contact between the cap and/ or bottom of the jar with the tip of the sensor. This could damage the membrane of the sensor.

7. Wait until the current on the display becomes stable again before continuing.

This may take several minutes if the sensor has undergone a large temperature change.

8. If you feel it is necessary to degas solution #1 prior to calibration, this can be done by inserting one of the long stainless steel needles included with the calibration kit through the septum so that the tip is in the solution. Attach the needle through appropriate tubing to a source of pure argon gas (nitrogen may also be used). Insert one of the short needles included with the kit through the septum such that the needle tip is clearly exposed (not in the solution) inside the vial. The small needle allows gas to escape, thereby avoiding a buildup of pressure. Purge the solution at low pressure (5 psi or less) for 15 minutes.

9. Once purging is complete and the gas source is turned off, remove the purging and pressure relief needles.

10. One should allow a few minutes for the temperature to equilibrate with the water bath again since purging with the gas may have changed the temperature.

11. Once the current has achieved a stable value, record this value.

12. Generally, it is not necessarry to pre-purge the calibration solution, since the NO

decays only very slowly in this solution.

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Creating a Calibration Curve

To create a calibration curve, the user measures the current (pA) generated by the addition of different amounts of KNO

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). For example:

Addition 1:

Add 50 µL of solution #2 to 10 mL of solution #1. Then the amount of NO produced can be calculated by simple dilution factors, as follows:

to the calibration solution.

Fig. C2 —

APOLLO4000 Calibration Output

50 µL of 50 µM KNO

(solution #2) into 10 mL solution #1 = 1:201 dilution.

Hence, amount of NO produced = 50 (µM) ÷ 201 = 0.2487 µM = 249 nM. Likewise:

Addition 2:

100 µL of solution #2 added to the above solution will produce 493 nM NO (

i.e.,

dilution factor =

1:101.5).

The output from the APOLLO4000 will look similar to the example shown in Figure C2: here three sequential additions of KNO2 have been made to solution #1.

From this output a calibration curve can then be created by plotting the changes in current (pA) against the changes in concentration (nM). The slope of this curve indicates the sensitivity of the probe.

[NO] nM Response (pA)

00 249 332 493 746 966 1486

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Once the sensitivity of the probe has been ascertained (in the above example the sensitivity was 1.557 pA / nM) the sensor is ready to use experimentally.

NO2 and NO3 Determination

ISO-NO Calibration Curve Based on Tabulated Data

1600

1200

800

400

0 200 400 600 800 1000

Concentration of NO (nM)

Figure C3 —

APOLLO4000 Calibration Curve

We have recently developed a new reagent-less nitrate-reductor, called the NITRALYZER™. The Nitralyzer is a very useful tool for researchers currently using enzymatic conversion of NO3 to NO2. Researchers who use the Greiss method for NO

determination will also be very interested to learn that using the ISO-NOP (and

the titration method for calibration described above) a detection limit for NO

as low

of 1 nM is routinely possible. This compares to a detection limit using Greiss reagent of approximately 1 µM. Hence, the ISO-NOP is several orders of magnitude more sensitive than the Greiss method. Information on the Nitralyzer is aavailable from WPI.

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Calibration of NO sensor by decomposition of SNAP

This method can be used to calibrated all NO sensors (see Ref. 1: Zhang, “Novel Calibration Method for Nitric Oxide Microsensors by Stoichiometrical Generation of Nitric Oxide from SNAP”

S-nitroso-N-acetyl –D,L-penicillamine (SNAP) is a stable NO containing compound that can be used for quantitative generation of NO in solution. SNAP decomposes to NO and a disulfide by product when dissolved in water. However, the rate of decomposition of the SNAP is very slow. The kinetics controlling the decomposition of SNAP depends on several parameters including, pH, presence of catalyst, temperature and light.

In the procedure described here, SNAP is used in combination with a catalyst, cuprous chloride, to generate known amounts NO in solution, which can then be used to accurately calibrate various NO-sensors. The protocol does not investigate all parameters involved in SNAP decomposition neither is it intended to propose a model by which SNAP is decomposed.

Two methods are described here for the calibration of NO sensors based on decomposition of SNAP. The first method relies on the use of Cu(I) as a catalyst for the 100% conversion of SNAP into NO. This method is extremely accurate but technically more demanding than the second method, which relies on the use of Cu(II) for the partial but quantifiable conversion of SNAP to NO.

Electroanalysis

, 2000, 12: 6).

et al.

Method 1: Calibration by decomposition of a Snitrosothiol compound using Cu(I) as a catalyst

This method of calibration results in the 100% conversion of SNAP to NO. The amount of NO produced, therefore, is based on the final concentration of SNAP.

CAUTION: The described calibration procedure requires the use of cuprous (I) chloride, CuCl, where Cu (I) is the active catalyst for the conversion of SNAP to NO. The calibration curve assumes only the presence of Cu (I) and hence a 100% conversion efficiency of SNAP to NO (see “A novel method to calibrate nitric oxide microsensors by stoichiometrical generation of nitric oxide from SNAP”, X. Zhang, L. Cardosa, M. Broderick, H. Fein, I. R. Davis,

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Electroanalysis

, 2000, 12(6),425-

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 (II) is much less efficient at catalyzing the conversion of SNAP to NO, and this would appear during calibration as an apparent low sensitivity of the electrode to NO.

Since Cu (I) is readily oxidized to Cu (II) special precautions must be taken to keep it in its reduced state prior to any calibration. It is recommended that CuCl be stored under inert conditions and if used in solution then the solution must be degassed with inert gas and absent of all oxygen.

NOTE : If your laboratory is not adequately equipped to satisfy the conditions for storage and use of CuCl please refer to the following section in the manual which describes a similar calibration procedure based on the use of copper (II) sulfate (CuSO4) or cupric (II) chloride CuCl conversion of SNAP to NO.

Getting Started

in which Cu [II] is the active catalyst for the

Figure C4 —

Responses of an NO electrode to the successive additions of 100 nM SNAP into CuCl saturated solution (pH=5.5). Also shown (inset) is the resulting calibration plot.

Prepare the following solutions:

#1—Saturated solution of cuprous chloride: This should be prepared by adding 150 mg CuCl to 500 mL distilled deoxygenated water. The distilled water can be deoxygenated by purging with pure nitrogen or argon gas for 15 min. The saturated CuCl solution will have a concentration of approximately 2.4 mM at room temperature and should be kept in the dark prior to use.

#2—Standard SNAP solution: Dissolve 5 mg EDTA in 250 mL of HPLC pure water (HPLC grade, Sigma). Deoxygenate the solution using the method described above. Add 5.6 mg SNAP to the solution. The Molarity of the SNAP solution ( SNAP f.w.= 220.3) can then be easily calculated. Since the SNAP solution is very sensitive to light and temperature it should be stored in the dark and in a refrigerator until required. (Note: The decomposition of SNAP at low temperature,

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in dark and in absence of trace metal ions proceeds very slowly due to the presence of chelating reagent, EDTA). The standard SNAP solution can then be used for many calibrations of NO probes throughout the day. However, since SNAP will slowly decompose even if stored correctly as described, it is recommended that a fresh standard stock solution of SNAP is prepared at the beginning of everyday. This will ensure an accurate calibration of the NO sensor.

The concentration of SNAP calculated as follows:

Where C is the concentration of SNAP in micromolars, A is the purity of SNAP, W is weight of SNAP in milligrams and V is the volume of the solution in liters. If SNAP purity is 98.5%, hence in the above example the concentration of SNAP is:

[C] = [98.5% x 5.6 / (220.3 x 0.25)] x 1000 = 100.1 µM

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 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. Immerse a NO probe into this solution, and while stirring, allow the background current to decay and stabilize for 3-5 min. As soon as the background current becomes stable start recording the data on the APOLLO4000.

Next inject 3 aliquots containing 5 µL, 10 µL and 20 µL sequentially of the SNAP stock solution (solution #2) into the vial containing cuprous chloride solution. Depending on the required calibration range ( produced) desired, the volumes of SNAP stock solution could be increased to produce a greater concentration of NO. It is recommended that calibration range be kept close to the anticipated experimental concentration of NO.

(and hence NO produced)

[C] = [A•W/(M•V)]1000

i.e.,

the final amount of NO

in the stock solution is

Immediately following the first addition of SNAP into Solution#1 the current (pA) output from the ISO-NO will be seen to increase rapidly. Within a few seconds the response

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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. Each addition of SNAP corresponds to equivalent NO concentration. The response should be very linear from 10 to 1000 nM.

The sensitivity of the NO probe can be established from the gradient of the response curve. The sensitivity of the ISO-NOP sensor is about 1 pA/nM. Once the slope of the probe has been determined the value can be entered into the APOLLO4000 software program (see previous section) if the user wishes the observe data in concentration mode (

Note: Remember that most NO probes are sensitive to temperature changes. It is therefore recommended that the calibration of a NO sensor is performed at the experimental temperature.

i.e.

, nM, µM).

Method 2: Calibration by decomposition of SNAP using Cu(II) as a catalyst

This method of calibration relies on the use of Cu(II) for the partial but quantifiable conversion of SNAP to NO. This procedure can be used as an alternative to the previous method in which Cu (I) is the active catalyst for the conversion of SNAP to NO. In this procedure Cu(II) is substituted as a catalyst for ease-of-handling.

NOTE: Experimentally it has been shown that Cu(II) is less efficient as a catalyst in the conversion of SNAP to NO ( 60%). The accuracy of the calibration may also be reduced (see discussion).

S-Nitriso-N-acetyl-D,L-penicillamine (SNAP) is a stable NO containing compound that can be used for quantitative generation of NO in solution. SNAP decomposes to NO and a disulfide byproduct when dissolved in water. However, the rate of decomposition is very slow. The kinetics of decomposition for this reagent is a function of several parameters including pH, presence of a catalyst, temperature and light.

In the procedure described here, SNAP is used in combination with a catalyst, copper (II) sulfate (CuSO4) or cupric (II) chloride (CuCl2), to generate a known quantity of NO in solution. Note that this protocol does not investigate the effects of

e.g.,

conversion ratio is reduced to approximately

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all parameters involved in SNAP decomposition nor does it propose a model by which NO is decomposed. The presented procedure provides an empirical estimation of the amount of generated NO based on the molarity of a standard stock solution of SNAP under a controlled set of parameters.

Getting Started

Prepare the following solutions:

Solution #1: Dissolve 5 mg EDTA in 250 mL of water (HPLC grade).

Solution #2: Prepare 250 mL 0.1 M copper (II) sulfate (or cupric (II) chloride) in

distilled water.

Preparing standard SNAP solution:

To prepare the stock solution of SNAP, weigh approximately 5.0 mg +/- 2.0 mg of SNAP and add it to solution #1. Calculate the Molarity of SNAP solution (SNAP f.w. = 220.3). Decomposition of SNAP in the stock solution proceeds very slowly due to the presence of chelating reagent, EDTA. Thus the rate of decomposition is negligible and the stock solution of SNAP remains relatively stable for at least 5 hours if kept in refrigerator.

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. Immerse a NO probe into this solution, and while stirring, allow the background current to stabilize for about 3-5 minutes. As soon as the background current becomes stable start the recording.

Next, sequentially inject three aliquots of SNAP solution, 5 µL, 10 µL, and 20 µL, into the vial containing copper sulfate solution. The current output will rapidly increase upon addition of first aliquot and will reach a plateau within a few seconds. Inject the second aliquot, 10 µL, as soon as the first signal reaches a plateau. Finally add the third aliquot as the second signal reaches its plateau. If aliquots are not added promptly when reaching the previous plateau, the signal will

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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.

Because NO sensors can be calibrated in a linear fashion, the magnitude of every signal should almost double as the volume of SNAP solution added is doubled in the course of the calibration. Use the recorded data to construct a calibration curve. The calibration curve can be simply constructed by plotting the signal output ( every addition of SNAP solution corresponds to a particular NO concentration. This will be discussed below. After the sensitivity of the NO probe is established, the APOLLO4000 software can be programmed to display data in either concentration mode (

e.g.,

in pA) vs. the concentration of SNAP added at that time. Note that

i.e.,

nM, mM) or redox current (

i.e.,

pA, nA).

The standard SNAP solution can be used for the calibration of NO probes throughout the day. Store the solution in the dark and refrigerate when not in use. Prepare a fresh stock solution of SNAP in the beginning of every day to ensure minimal decomposition of SNAP in the stock solution. Concentration of SNAP decreases to 5-10% of its nominal value after approximately 4-5 hours.

NOTE: Remember that most NO probes are sensitive to changes in temperature. It is therefore recommended that the calibration of your sensor is performed at experimental temperature.

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Predicting the level of detectable NO according to the molar ratio of SNAP in the presence of catalyst (Method II)

Experiments have shown that SNAP is decomposed instantaneously under the following set of experimental conditions:

Temperature 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 → 2NO + RS-SR

Theoretically, the concentration of generated NO should be equal to the final concentration of SNAP in the copper sulfate solution in the calibration vial if the decomposition goes to completion and if the generated NO is detected quickly before it is oxidized to nitrite and nitrate.

However, it is expected that the level of detectable NO will be below the theoretical value because the copper sulfate solution was at equilibrium with ambient air, and consequently a portion of the generated NO would have been immediately oxidized to nitrite and nitrate before it was measured by the NO sensor. In addition, it is possible that decomposition of SNAP does not go to completion even in the presence of a catalyst. Results on the kinetics of SNAP decomposition in the presence of a catalyst in an anaerobic environment are published elsewhere (Zhang

et al.

, “Novel Calibration Method for Nitric Oxide Microsensors by Stoichiometrical

Generation of Nitric Oxide from SNAP”,

Our experimental data indicates a conversion efficiency of SNAP to NO of approximately 0.6 ( sensor in a solution, which is at equilibrium with ambient air and at the experimental conditions described above. Hence for each mole of SNAP, 0.6 mole of NO is liberated under the proposed set of parameters. It is assumed the other 40% of SNAP is either not decomposed or a proportion that is decomposed to NO

i.e.,

60%). This result is only applicable for calibration of a NO

Electroanalysis

, 2000, 12: 6).

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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.

3. 20 µL, 40 µL, and 80 µL of SNAP stock were added sequentially into 10 mL of

solution # 2.

4. The current was continuously recorded during the course of the calibration.

5. A standard calibration curve was constructed according to the recorded date.

[SNAP] [NO] = 0.6 X [SNAP] Output Current

232.4 nM 139.4 nM 230 pA

462.0 nM 277.2 nM 488 pA

916.8 nM 550.1 nM 1001 pA

Y = -32.038 + 1.88X

The data from calibration curve indicates that this procedure allows an excellent linear calibration of NO probes (R2= 1). The accuracy of calibration is approximately +/- 10% from mean. The source of error arises most probably from gravimetric measurement of the standard reagent, SNAP. In addition, purity of SNAP as well as partial oxidation of generated NO in the calibration solution could contribute to this error. Such a deviation may not be so important when NO is quantified in biological systems because most often the ability to measure changes in the basal concentration of NO is more significant than measurement of the absolute level of NO.

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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

U.S. Occupational Safety and Health Administration has set a time-weighted average maximum NO value as 25 ppm. That is to say that 25 ppm is cited as the maximum concentration to which workers may be continually exposed. Brief inhalation of concentrations as low as 200 ppm could produce delayed pulmonary edema which may be fatal after an asymptomatic period of up to 48 hours after the initial exposure.

It is therefore critical that the personnel handling the gas be thoroughly familiar with the Material Safety Data Sheet (MSDS) and proper handling procedures.

The precautions recommended

by the gas manufacturer must be followed.

. The

Preparing an NO Standard

This method has the advantage of allowing the user to calibrate NO sensors in the same environment in which the experimental measurements will be made. However, it has the disadvantages of added cost, inconvenience, and greater hazard to the user. All of these factors must be taken into consideration. The setup for preparing a saturated NO aqueous solution is illustrated in Fig. C5.

1. Be certain the fume hood is functioning. See Fig. C5.

2. Make sure that all fittings and connections are secure. The tubing to be used

should not be permeable to NO. We recommend Tygon® tubing if a polymer tubing is to be used; this is permeable to NO but has the best performance compared to other polymer tubing of which we are currently aware. Ideally glass tubing should be used. If Tygon® tubing is used, note that prolonged exposure to NO affects its properties; therefore it is recommended that the tubing be inspected frequently and that it be replaced when it appears to be

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*Lecture bottle of NO (14.2 liters, 98.5%) obtained from Aldrich, catalog #29.556-6; telephone 800558-9160

brittle. The pressure regulator and tee purge adaptor should be stainless steel. This is because nitric oxide is a corrosive gas.

3. Prepare 100 mL of a 10 % (by weight) KOH solution and place it in the

sidearm flask as illustrated above. The flask should be sealed with a stopper through which the tubing passes by means of a Luer fitting to a syringe needle which extends almost to the bottom of the flask. Tubing is used to connect the side arm of the flask to the vial containing the water to be equilibrated with NO. The KOH solution is used to remove other nitrogen oxides from the NO gas.

4. Place 20 mL of distilled (preferably deionized) water in a small glass vial. Seal

the vial with a stopper and insert through the stopper a long syringe needle which extends almost to the base of the vial. Connect this syringe needle to the tubing from the KOH flask as illustrated. Insert an additional shorter syringe needle which should not extend into the solution. This acts as a pressure relief during purging.

Figure C5

Setup for preparing a saturated NO aqueous solution must be in a fume hood with forced ventilation. Nitric oxide is highly toxic and, as illustrated, escapes into the atmosphere during preparation of the standards.

—

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5. 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.

6. Purge the system with argon (or nitrogen) gas for a period of 30 minutes at a

moderate flow rate such that the pressure is maintained at a safe level (1-2 psi). When purging it should be observed that gas is indeed bubbling through the KOH solution as well as the distilled water. After 30 minutes turn off the argon source, and switch the tee purge valve to the correct position for purging with NO from the lecture bottle.

7. Purge the system with NO for 5-10 minutes if using a pure source (longer if the

NO source is not pure). Again make sure that gas is bubbling through both solutions. Warning: NO is now escaping from the pressure relief

needle in the stopper of the distilled water vial. It is imperative that the fume hood be running at maximum capacity with the front panel closed (in the down position).

8. After the time in step 7 has elapsed turn off the NO source.

9. Immediately remove the two needles from the distilled water vial.

10. Set the tee purge valve for purging with argon (or nitrogen) gas, and turn on

the argon source. Purge the system for 5-10 minutes at a moderate flow rate. Gas should be bubbling through the KOH and then escaping from the flask into the atmosphere. Again be sure that the fume hood is ventilating well.

11. Turn off the argon (or nitrogen) source, and allow the fume hood to continue to

ventilate for 10-15 minutes so as to ensure that all traces of NO gas are removed from the atmosphere.

12. The solution of distilled water should now be saturated with NO. The

concentration of NO produced by this saturation is dependent upon the temperature. At 0° C, the concentration is approximately 3.3 mM, and at 20°C the concentration is approximately 1.91 mM.

13. Dilutions of known concentration can be prepared from this saturated solution In preparing a dilution, be careful not to unseal the vial, for this exposes the solution to atmospheric oxygen.

Once the dilutions are prepared, it is a simple matter to calibrate the instrument.

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Measurement of NO

It is impossible to outline in detail how to use NO sensors to measure NO in every experimental set up the user may encounter. There are, however, some guiding principles of which the user should be aware to exploit fully the capabilities of the technology. These are outlined below.

NO Delivery

For any measurement of NO to be made, the NO must reach the sensor surface so it can react on the surface of the electrode. This point is of particular concern because in many experiments the lifetime of NO is quite short. This is especially so in biological systems where compounds such as hemoglobin can reduce the halflife of NO to less than a second. It is therefore very important that the experimental set up is designed so as to maximize delivery of NO to the sensor. In particular, the tip of the sensor should be placed as close as possible to the site of release of NO.

Durability and Handling

The user must excercise caution when handling any NO sensor to avoid actions which could damage the sensor tip. The sensor membrane and membrane coatings are extremely delicate and improper handling will lead to damage.

Environmental Influences

There are two environmental parameters to which NO sensors are quite sensitive: temperature and electrical interference, both of which are discussed in greater detail below.

Temperature

Note that the sensitivity of the NO sensor is temperature-dependent. This is due to the effects of temperature on the partial pressure of NO in either liquid or gas samples, on the permeability of the membrane or coatings, and on the conductivities of various circuit components. It is therefore recommended that any calibration is performed at the same temperature as the experiment.

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

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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. The magnitude of this external noise depends on the environment of the laboratory. If the interference introduced by the electrical signals in the environment is large, the first step towards eliminating it is to ground and shield the system properly. See the section on Grounding and Shielding Procedure for the Apollo 4000.

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Maintenance of NO Sensors

The various NO sensors, if well cared for, will require very little maintenance.

Maintenance of the ISO-NOP

When the ISO-NOP sensor is not being used it should be left connected to the APOLLO 4000 in tip suspended in distilled water. The basic structure of the ISO-NOP sensor is quite simple (see Fig. C6). It consists of an internal NO-sensing working/counter electrode combination. This electrode fits inside a disposable protective stainless steel sleeve (WPI #5436) separated from the external environment by a gas permeable polymeric membrane covering the end of the stainless sleeve. The other end of the sleeve is flanged. The locking cap is used to attach the sleeve to the probe handle.

position (or to Pre-Polarizer NSA-1, NSA-2 or NSA-3) with the

which must contain fresh electrolyte

(WPI #7325), and is

When the sensor is fully assembled (

i.e.,

with locking cap and sleeve in place) the internal electrode should be seen to press gently against the polymeric membrane, which will then be slightly stretched. This ensures that the electrolyte diffusion layer will be as thin as possible, which is necessary to minimize sensor response time. Once a membrane is stretched it is permanently deformed and cannot usually be reused if the sleeve is removed from the electrode. Four additional membrane sleeves accompany the ISO-NOP in the start-up kit, together with a MicroFil™ electrolyte filling needle (WPI #MF28G67-5) and 1 mL syringe. Additional sleeves

stainless steel

gas permeable

membrane

combination

working / counter

electrode

sleeve

locking cap

probe handle

connection to meter

Figure C6

Basic probe structure of ISONO sensor

—

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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. After each use the membrane should be cleaned by suspending the tip in distilled water for 20-30 minutes to dissolve salts and remove particles, which may have accumulated on the membrane. If the probe was used in a protein rich solution the tip should first be soaked in a protease solution for several minutes to remove protein build-up, and then in distilled water. Enzymatic detergent ( #7363) can also be used. The membrane sleeves can also be sterilized chemically using an appropriate disinfectant ( matter can be removed by briefly immersing the tip in an acid or base solution (at times both may be necessary) for 10 seconds. A good indication of a dirty membrane sleeve is a sluggish response or an unusually low sensitivity. If these problems are not rectified by the above cleaning procedures then the membrane sleeve should be replaced.

e.g.,

Cidex, WPI #7364). Accumulated organic

The probe cannot be used in organic

solvents.

e.g.,

Enzol, WPI

Replacing a Membrane Sleeve

All membrane sleeves will eventually have to be replaced by the user. The procedure for doing so is simple and straightforward.

1. Unscrew the locking cap from the handle.

2. Hold the stainless steel sleeve and remove it with the locking cap from the

internal electrode assembly, being careful not to bend the electrode assembly when doing so.

3. Rinse the internal electrode with distilled water (particularly the tip) and let it

soak for at least 15 minutes. Be careful not to let water get into the handle. The current on the ISO-NO meter should go offscale when the electrode is being rinsed.

4. Gently dry the electrode with a soft tissue (Kimwipes). Be sure to dry

thoroughly the flat surface at the tip of the electrode. After drying the current should stabilize fairly quickly to a low value ( the electrode is in good working order.

e.g.,

0 - 20 pA). If this occurs then

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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.

7. Dip the internal electrode 1-2 cm into the electrolyte provided in the ISO-NO

start-up kit, the current should go offscale during this. Using the MicroFil™ nonmetallic syringe needle and 1 mL plastic syringe, supplied, inject approximately 100 microliters of electrolyte directly into the new sleeve. The MicroFil supplied should be less than the length of the sleeve, so that it will not puncture the delicate membrane at the tip of the sleeve during injection. If the MicroFil is longer than the sleeve it can be cut to the correct length.

8. Slowly and smoothly insert the electrode into the sleeve, and screw the locking

cap into the handle. The electrode should be observed to press gently against the membrane.

9. The current displayed on the meter at this time will be high or offscale.

10. Suspend the tip of the new assembled probe in distilled water.

11. After 10-15 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.

12. The integrity of the new membrane can be determined by immersing the probe

tip into a strong saline solution (1 M). If the current observed, after a few minutes in the saline solution, increases dramatically or is offscale then the membrane integrity is not good and a new membrane will have to be fitted.

13. When the ISO-NOP is not being used it should be stored with the tip

suspended in distilled water.

Additional membrane kits (WPI #5436) may be purchased separately.

Maintenance of Nitric Oxide Microsensors

WPI’s Nitric Oxide microsensors are maintenance-free consumable sensors that are warranted against defect for 30 days from the date of purchase. The following information should increase the lifetime of the sensor:

WORLD PRECISION INSTRUMENTSC-22

APOLLO 4000

NITRIC OXIDE DETECTION

Tip care

The surface of the sensor tip is very sensitive and is covered with a unique layer of propriety selective membranes. The tip of the sensor should never be handled as this will damage the membranes and compromise the electrode’s selectivity for NO. During use the electrode should be held securely, preferably using a micromanipulator or other similar device that permits accurate positioning.

The electrode should be cleaned periodically in distilled water and dried using soft tissue paper. Organic contamination can be removed using a mild enzymatic detergent such as ENZOL (WPI part # 7363)

Sterilization

The following methods can be used to sterilize the sensor:

• Ethylene oxide

• Cidex solution (WPI part # 7364)

Storage

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 immediate use when required.

WORLD PRECISION INSTRUMENTS C-23

APOLLO 4000

NITRIC OXIDE DETECTION

WORLD PRECISION INSTRUMENTSC-24

APOLLO 4000

OXYGEN DETECTION

USING THE APOLLO 4000 TO

DETECT OXYGEN

Initial Set-up

Attach the oxygen sensor to the required sensor input channel on the APOLLO

4000. From the main Setup menu of the software select the correct poise voltage “Oxygen” and appropriate Range for the selected channel. The electrode must now be calibrated.

Calibration and Use of Oxygen Sensors

NOTE: Before using the ISO‑OXY‑2 dissolved oxygen sensor for the ﬁrst time, connect the sensor to the APOLLO 4000 (power ON) overnight.

The ISO-OXY-2 sensor provides accurate, stable and electrically isolated oxygen measurements. With a probe tip diameter of just 2 mm and low oxygen consumption, ISO-OXY-2 is ideal for making measurements in small sample volumes. Other features include a fast response time and a sturdy stainless steel probe body.

The ISO-OXY-2 in combination with APOLLO 4000 amperometrically measures the concentration of oxygen in aqueous solutions and gas mixtures. ISO-OXY-2 is not suited for long-term continuous gas measurements, as it needs to be calibrated on a daily basis. Measurements can be displayed either as a percentage of atmospheric pressure, parts per million (ppm), or as a redox current in nanoamperes (nA). The ISO-OXY-2 houses a platinum working electrode and a silver counter \ reference electrode inside a stainless steel sleeve. A gas permeable polymer membrane ﬁts over the end of the sleeve which allows oxygen to pass while blocking liquids, ions and particulate matter. Oxygen diffuses through the membrane and is reduced at the platinum cathode which is held at -0.7 V when the instrument is on. This results in an electrical current being generated, the magnitude of which is determined by the rate of diffusion to the electrode which is proportional to the partial pressure of oxygen outside the membrane. Thus the current serves as a measure of the partial pressure of oxygen.

ISO-OXY-2 comes ready to use. 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. Once the current stabilizes the user may then calibrate the instrument.

WORLD PRECISION INSTRUMENTS D-1

APOLLO 4000

OXYGEN DETECTION

Calibration

For accurate results the sensor probe should be calibrated as closely as is possible to the temperature at which the measurement is to be made.

Polarization

The user must select the correct poise voltage potential for oxygen applied to the electrode (Fig. B10). After initially connecting the oxygen sensor to the Apollo 4000 in ON position, the probe current will be high — approximately 10,000 pA. The current will decrease and settle to a stable value after a period of time — usually six to eight hours. The ISO-OXY-2 should always remain connected to the 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.

Zero (Oxygen) Point Calibration

After polarization of the ISO-OXY-2 as described above, a calibration for zero percent oxygen may be carried out in pure nitrogen gas or in water saturated with nitrogen. With stirring, the complete saturation of water with nitrogen may take more than ten minutes. Calibration in pure nitrogen gas is much faster and generally considered more reliable. A plastic calibration bottle (Fig. D1) is supplied with the utility kit. Connect a plastic tube (1) from the side tube to a pure nitrogen gas source at a low pressure (less than 5 psi) and purge the bottle with nitrogen gas. Insert the ISO-OXY-2 (2) into the bottle through the top vent hole on the bottle cap (3). The current should be observed to drop rapidly (a few seconds) to a stable value. If the current value is not zero it must be zeroed using the software.

Fig. D1

Scale Factor Calibration

After the instrument has been zeroed (at 0% oxygen) using the software, it must be calibrated by measuring at least one more known concentration of oxygen.

WORLD PRECISION INSTRUMENTSD-2

Fig. D2 — Oxygen calibration trace in nA.

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 100 % O2.

Alternatively, air can be used as the calibration standard but since water vapor does affect the probe reading it is best to use dry air unless the ambient humidity is accurately known. Dry air can be obtained by passing room air through a column containing a solid drying agent such as silica gel or calcium chloride and then into the calibration bottle for calibration. Ambient humidity may cause a calibration error of as much as 1% O2. The user may judge if this is acceptable.

WORLD PRECISION INSTRUMENTS D-3

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 100% oxygen environment a reading of 100 means that the partial pressure of oxygen is 1 atm (760 mm Hg). A reading of 21 means that the partial pressure of oxygen is 0.21 atm (160 mm Hg).

Fig. D2 shows a record of the calibration procedure described above. Before point 1 the record displays the background current of the oxygen sensor in air. This current value may vary from sensor to sensor. At point 1 the sensor is exposed to 0 % Oxygen. At point 2 the background is zeroed using the software. At point 3 and point 4 the sensor is exposed to air (21% oxygen) and 100% oxygen, correspondingly. By using the current at 0% (0 nM) and 100% (236 nA), a two-point calibration curve is built and the slope is determined at 2.36 nA/%. Alternatively, a three-point calibration can be implemented by adding the information for the current of the sensor in air and by using linear regression software.

Fig. D3 — Creating a new unit (% oxygen).

To present the data in % oxygen you may use the Setup/Unit conversion option from the Menu. A new unit for the oxygen-measuring channel must be created by setting up 2360 pA to correspond to 1 % oxygen (Fig. D3). Switch to % unit as shown in Fig. D4 and all recorded data as well as incoming data will be displayed in % oxygen unit.

Note: After the calibration the trace should not be re-zeroed as this will invalidate the calibration.

WORLD PRECISION INSTRUMENTSD-4

APOLLO 4000

OXYGEN DETECTION

Fig. D4 — Oxygen calibration trace in % oxygen.

WORLD PRECISION INSTRUMENTS D-5

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. Aerate, for a few minutes, by bubbling air through the side arm of the bottle at a low pressure using a simple aquarium aeration pump. The scale reading should be allowed to settle to a stable reading. Dissolved oxygen calibration is corrected for the effect of water vapor by the following equations :

(1) pO2 = 21% × (1 - pH2O)

(2) pO2 = 21% × (1 -p'H2O/760)

APOLLO 4000

OXYGEN DETECTION

where pH2O and p'H2O are the partial pressure of water vapor at standard atmospheric pressure in atmospheres and in mm Hg, respectively (see Table 3).

For example, the pH2O in water- saturated air at 24 deg is 22 mm Hg (see Table 3). Therefore the pO2 = 21% x (1 - 22/760) = 20.4 %. Note that for purposes of oxygen measurements liquid water is considered to be “water-saturated air”. The values of water vapor pressures at different temperatures can be found in Table 3.

You may obtain zero % oxygen concentration by adding to your solution of several mg Na2S2O3 per 20 mL of solution.

To present dissolved oxygen concentration in parts per million (ppm), refer to Table 1a. This table gives the solubility of oxygen in water at different temperatures at an ambient pressure of 1 atm. If the solution temperature is 25°C, for example, the oxygen concentration when the probe is in water is 8.4 ppm. You do not need to correct for the water-vapor effect for a ppm calibration since the values in Table 1a are obtained in “water-saturated air” at an atmospheric pressure of 760 mm Hg.

The unit ppm is equivalent to mg/L. This is illustrated as follows. The solubility of oxygen in water at 0 deg according to the Merck index is 4.889 mL per 100 mL. Using the ideal gas law we can calculate the number of moles of oxygen present in 100 mL :

PV = nRT

n = P*V/R*T

n = (0.21)*(4.889x10-3) / (0.08206)*(273)

n = 45.8 x 10-6 moles

Where P is the partial pressure of oxygen, V is the volume of oxygen, n is the number of moles of oxygen, R is the universal gas constant, and T is the absolute temperature. From the number of moles of oxygen we can calculate the number of grams of oxygen :

45.8 x 10-6 mol * 32 g / mol

1.46 x 10-3 g

Therefore there will be (1.46 x 10-3 g / 0.1 L) 14.6 mg of oxygen per liter. Since 1 L of water has a mass of 1000 g, and there are 1 million mg in 1000 g, the concentration in ppm shall be:

(14.6 x 10-3 g/L) / (1000 g/L) = 14.6 ppm

WORLD PRECISION INSTRUMENTSD-6

APOLLO 4000

OXYGEN DETECTION

Note that this value corresponds to that given in Table 1a.

For accurate results the temperature of the water sample and the ﬂuid being tested should be identical. They should be continuously stirred using a magnetic stirrer.

When measuring ﬂuid samples for dissolved oxygen, periodically rinse the exterior of the probe with distilled water, blot the membrane dry and recheck the electrode’s calibration as described above.

Creating a Calibration Curve

To create a calibration curve, the user measures the current (nA) of the sensor at various concentrations of oxygen. The calibration could be either a two-point or three-point calibration, including measurements at 0% oxygen, 100% oxygen or air (21% oxygen) with the corresponding correction for water vapor in case of aqueous measurements (see Table 3).

Note: For absolute measurements in % and ppm units, zeroing function should not be used after the initial zeroing.

After calibration the user can select to show the oxygen concentration in % or ppm units. The conversion factor %-to-nA or ppm-to-nA (sensitivity of the probe) is determined from the slope of the calibration curve and must be typed into the corresponding ﬁeld (see Fig. B15). Only after selecting ppm or % unit will oxygen concentration be displayed.

Calibration Method for O2 Measurements in Living Tissue or Blood

The APOLLO 4000 and ISO-OXY-2 probe may be used extensively in applications involving O2 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. Although it is normal to think in terms of dissolved oxygen concentration, it is actually more appropriate to deﬁne oxygen in solution in terms of activity, since this is the “effective concentration”. For example, in distilled water the activity coefﬁcient, γc, is close to unity; but in solutions with high salt concentration the activity coefﬁcient is different from unity and concentration and activity of dissolved oxygen are no longer equal: the oxygen concentration falling with salt concentration increase, while activity remains constant. For a membrane-covered oxygen electrode this is an important effect

WORLD PRECISION INSTRUMENTS D-7

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.

Thus, if it is necessary to have a measure of dissolved oxygen in terms of concentration, then the calibration is somewhat more complicated since the relationship between activity and concentration may change with the change of salt concentration in the samples. The activity coefﬁcient, a ratio of the activity to the concentration, generally cannot be predicted and one must rely on empirical determinations since the compositions of living ﬂuids such as blood are extremly complicated. One may directly use the ﬂuid to be tested as a “solvent” to prepare a calibration stantard. Alternatively, one may use the Bunsen absorption coefﬁcient, α, to calculate oxygen concentration in blood in terms of the results with the oxygen electrode. The equation is:

where K is a conversion factor depending on the unit of pressure chosen (1 for atm), pt and pH2O are the total pressure of gas and the partial pressures of water, respectively. pO2 is the partial pressure of oxygen in blood obtained from the measurements with the oxygen electrode. Bunsen Coefﬁcients for solubility of oxygen in plasma and blood can be found in Table 4. However, it is very important

to calibrate at the same temperature as that of the measurement site.

Probe Structure and Assembly

Fig. D5 shows the principal components of an ISO-OXY-2. A gas permeable membrane (3) is cemented to the tip of the outer stainless steel sleeve (1). The interior of the probe is a slender wand containing the platinum cathode and silver counter / reference electrode inside the sleeve. The wand is permanently mounted in the probe’s plastic handle (2). After electrolyte has been deposited inside the sleeve (see below), the wand is slowly inserted into the sleeve and secured by screwing the sleeve cap (4) gently into the probe handle. If the membrane on the stainless steel sleeve becomes damaged, the entire sleeve must be replaced. Two spare sleeves are provided with the ISO2 kit, and the user may purchase more. Should the user wish to sterilize the probe this should be done with alchohol or some other chemical for an assembled probe. The membrane sleeves can be

WORLD PRECISION INSTRUMENTSD-8

Fig. D5

— Principal components of ISO-OXY-2 electrode.

APOLLO 4000

OXYGEN DETECTION

autoclaved seperately from the probe should the user desire to do this and then place the sterilized membrane sleeve onto the probe.

Changing the membrane sleeve

All membrane sleeves replacing will eventually have to be done by the user. The procedure for

doing is simple and straightforward.

1. Unscrew the locking cap from the handle.

2. Hold the stainless steel sleeve and remove it with the locking cap from the

internal electrode, being careful not to bend the electrode assembly when doing so.

3. Rinse the internal electrode with distilled water (particularly the tip) and let it soak for at least 15 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.

4. Gently dry the electrode with a soft tissue (Kimwipes). Be sure to dry thoroughly the ﬂat surface at the tip of the electrode. After drying the current should stabilize fairly quickly to a low value (e.g., 0 - 20 pA). If this occurs, then the electrode is in good working order.

5. If the electrode is not clean, repeat steps 3 and 4.

6. Remove the locking cap from the old used sleeve, and gently slide it onto

the new replacement sleeve.

WORLD PRECISION INSTRUMENTS D-9

APOLLO 4000

OXYGEN DETECTION

less than the length of the sleeve, so that it will not puncture the delicate membrane at the tip of the sleeve during injection. If the MicroFil is longer than the sleeve it can be cut to the correct length.

8. Slowly and smoothly insert the electrode into the sleeve, and screw the locking cap into the handle. The electrode should be observed to press gently against the membrane.

9. The current at this time will be high or offscale.

10. Suspend the tip of the new assembled probe in PBS buffer solution.

11. After 10-15 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.

12. If the current observed, after a few minutes in the saline solution,

increases dramatically or is offscale, then the membrane integrity is not good and a new membrane will have to be ﬁtted.

13. When the ISO-OXY-2 is not being used it should be stored with the tip

suspended in distilled water.

Clean the Membrane

The membrane sleeve itself requires very little maintenance. The primary concern is to avoid to damage the membrane and to keep it as clean as possible. After each use, the membrane should be cleaned by suspending the tip in distilled water for 20-30 minutes to dissolve salts and remove particles ,which may have accumulated on the membrane. If the probe was used in a protein rich solution the tip should ﬁrst be soaked in a protease solution for several minutes to remove protein build-up, and then in distilled water. Enzymatic detergent (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). Accumulated organic matter can be removed by brieﬂy immersing the tip in an acid or base solution (at times both may be necessary) for 10 seconds. A good indication of a dirty membrane sleeve is a sluggish response or an unusually low sensitivity. If these problems are not rectiﬁed by the above cleaning procedures then the membrane sleeve should be replaced. The probe cannot be used in organic solvents.

WORLD PRECISION INSTRUMENTSD-10

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. As such, it is recommended to use the following guidelines to maximize the life of the electrode:

If the oxygen electrode is being used on a daily basis, it’s recommended that the instrument be left ON with the electrode connected to maintain polarization (see “Polarization” on page D-2). However; if the electrode is not to be used for a period of more than 2-3 days, it’s recommended that the electrode be disconnected from the instrument, and stored with the tip immersed in distilled water. This practice will reduce the possibility of a gradual reduction of electrode surface activity (as discussed above) under long term polarization. If the electrode will not be used for a long period of time (several months), refer to the section titled “Storage” below.

Handling Precaution

When passing the O2 probe through small holes, gaskets or O-rings, allow sufﬁcient clearance so that the probe tip is not damaged by abrasion. The probe tip should slide through openings easily before sealing the probe shaft to assure an air-tight ﬁt.

Storage

For long term storage (several months) unscrew and remove the sleeve from the probe handle, rinse the electrode tip and the sleeve with distilled water. When both are dry, replace sleeve to protect probe wand but do not screw the sleeve completely onto the handle.

WORLD PRECISION INSTRUMENTS D-11

APOLLO 4000

OXYGEN DETECTION

Table 1a

°F °C ppm °F °C ppm

32 0 14.6 66 19 9.4 34 1 14.2 68 20 9.2 35 2 13.8 70 21 9.0 37 3 13.5 72 22 8.8 39 4 13.1 73 23 8.7 41 5 12.8 75 24 8.5 43 6 12.5 77 25 8.4 45 7 12.2 79 26 8.2 46 8 11.9 81 27 8.1 48 9 11.6 82 28 7.9 50 10 11.3 84 29 7.8 52 11 11.1 86 30 7.6 54 12 10.8 88 31 7.5 55 13 10.6 90 32 7.4 57 14 10.4 91 33 7.3 59 15 10.2 93 34 7.2 61 16 10.0 95 35 7.1 63 17 9.7 97 36 7.0

64 18 9.5 99 37 6.9

Table 1b

Chloride Concentration of Seawater

°C 5 g/l 10 g/l 15 g/l 20 g/l

0 13.8 13.0 12.1 11.3

1 13.4 12.6 11.8 11.0 2 13.1 12.3 11.5 10.8 3 12.7 12.0 11.2 10.5 4 12.4 11.7 11.0 10.3 5 12.1 11.4 10.7 10.0 6 11.8 11.1 10.5 9.8 7 11.5 10.9 10.2 9.6 8 11.2 10.6 10.0 9.4 9 11.0 10.4 9.8 9.2 10 10.7 10.1 9.6 9.0 11 10.5 9.9 9.4 8.8 12 10.3 9.7 9.2 8.6 13 10.1 9.5 9.0 8.5 14 9.9 9.3 8.8 8.3 15 9.7 9.1 8.6 8.1 16 9.5 9.0 8.5 8.0 17 9.3 8.8 8.3 7.8 18 9.1 8.6 8.2 7.7 20 8.7 8.3 7.9 7.4 21 8.6 8.1 7.7 7.3 22 8.4 8.0 7.6 7.1 23 8.3 7.9 7.4 7.0 24 8.1 7.7 7.3 6.9 25 8.0 7.6 7.2 6.7 26 7.8 7.4 7.2 6.7 27 7.7 7.3 6.9 6.5 28 7.5 7.1 6.8 6.4 30 7.3 6.9 6.5 6.1

Table 1a:

Solubility of oxygen in parts per million (ppm) in fresh water at different temperatures, in equilibrium with air at barometric pressure of 760 mm Hg (101.3 kPa) and oxygen partial pressure of 159 mm Hg (21.1 kPa).

Table 1b:

Solubility of oxygen (milligrams/liter) in seawater of different salinities, in equilibrium with air at barometric pressure of 760 mm Hg (101.3 kPa) and oxygen partial pressure of 159 mm Hg (21.2 kPa).

WORLD PRECISION INSTRUMENTSD-12

APOLLO 4000

OXYGEN DETECTION

Table 2:

Oxygen solubility

obtained from

Table 1a or

Table 1b should

be corrected

if barometric

pressure is different

than 760 mm Hg or

at altitudes other

than sea level.

Table 2

Altitude Pressure Solubility (feet) (mm Hg) Correction

Factor

-540 775 1.02 Sea Level 760 1.00 500 746 0.98 1000 732 0.96 1500 720 0.95

2000 707 0.93 2500 694 0.91 3000 681 0.90 3500 668 0.88 4000 656 0.86

4500 644 0.85 5000 632 0.83 5500 621 0.82 6000 609 0.80

Table 3

Water-vapor partial pressure in mm Hg:

Temp. Pv Temp. Pv °C mm Hg °C mm Hg

0 5 20 18

2 5 22 20 4 6 24 22 6 7 26 25 8 8 28 28 10 9 30 32 12 11 32 36 14 12 34 40 16 14 36 45 18 16 38 50 40 55

WORLD PRECISION INSTRUMENTS D-13

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 5 g 10 g 15 g 20 g

15 0.0302 0.0310 0.0312 0.0316 0.0323 20 0.0277 0.0282 0.0284 0.0287 0.0293 25 0.0257 0.0261 0.0263 0.0265 0.0271 28 0.0246 0.0249 0.0251 0.0253 0.0259 30 0.0238 0.0241 0.0243 0.0245 0.0251 35 0.0220 0.0226 0.0227 0.0229 0.0234 37 0.0214 0.0220 0.0221 0.0223 0.0228 40 0.0208 0.0221 0.0212 0.0214 0.0219

WORLD PRECISION INSTRUMENTSD-14

APOLLO 4000

HYDROGEN PEROXIDE DETECTION

USING THE APOLLO 4000 TO

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.

The structure of the HPO sensor

The basic structure of the ISO-HPO-2 sensor is identical to that of the ISO-NOP nitric oxide sensor (see Fig. C6), although there are signiﬁcant differences in the actual type of materials used.

It consists of an internal working/counter electrode combination. This electrode ﬁts inside a disposable stainless steel sleeve (WPI#600012) which must contain fresh reﬁllable electrolyte (WPI#100042), and is separated from the external environment by membrane covering the end of the stainless sleeve. The other end of the sleeve is ﬂanged. The locking cap is used to attach the sleeve to the probe handle. When the sensor is fully assembled ,the internal electrode should be press gently against the membrane, which will then be slightly stretched. This ensures that the electrolyte diffusion layer will be as thin as possible, which is necessary to minimize sensor response time.

Once a membrane is stretched, it is permanently deformed and cannot usually be reused if the sleeve is removed from the electrode. Four additional membrane sleeves (WPI#600012), a MicroFil™ electrolyte ﬁlling needle (WPI#MF28G67-5) and 1 mL syringe are provided in the ISO-HPO startup-kit (WPI#600011).

With proper care and by following the instructions below, a membrane sleeve should last more than one month.

WORLD PRECISION INSTRUMENTS E-1

APOLLO 4000

HYDROGEN PEROXIDE DETECTION

Calibration of the HPO Sensor

Hydrogen Peroxide (H2O2) is a very important product in the biological system. The determination of H2O2 requires an accurate method of calibration. Amperometeric (electrochemical) determination using the Apollo is a very reliable method to measure H2O2. The Apollo measures the amount of H2O2 oxidized on the surface of the sensor using a poise voltage of +400 mV. The oxidation of H2O2 at the sensor surface produces a small current (pA), which is detected by the Apollo. The amount of current produced is linearly proportional to amount of H2O2 in the experiment.

Items Required

• Plastic stand with two holes

• One glass vial

• 1.0mM H2O2 standard solution (available from WPI)

• 0.01M PBS buffer solution (PBS buffer tablets available from WPI)

NOTE: The multi-port measurement chambers (WPI#NOCHM or #NOCHM-4) can be used as an alternative calibration kit, speciﬁcally for use at different temperature condition. Calibration temperatures from 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.

2. Add an appropriate volume (e.g.,10 mL) of PBS buffer solution (0.01M) in glass vial. Turn on the stirrer so that the bar is stirring in moderate rate.

3. Remove the sensor from the electrolyte solution in which the tip has been immersed during storage. Immerse the ISO-HPO-2 sensor tip in PBS buffer solution (0.01M), The sensor tip should be immersed about 0.3-

0.5mm into the solution, and should not be touched by stir bar.

4. Record the current value. If the current is offscale or unstable after a half hour in solution, it is likely that the membrane has been damaged and the sleeve needs to be changed (refer to the section on“Changing the Sleeve”).

WORLD PRECISION INSTRUMENTSE-2

APOLLO 4000

-100

100

300

500

700

900

1100

1300

-30 20 70 120 170

time (sec)

current (pA)

0.5 µM

1 µM

2 µM

4 µM

8 µM

y = 73.296x + 6.4857

R2 = 0.9988

100

200

300

400

500

600

700

0 2 4 6 8 10

Concentration of H2O2 (µM)

Current (pA)

HYDROGEN PEROXIDE DETECTION

NOTE: The calibration should be carried out at the temperature at which the samples of H2O2 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.

5. Once the current has achieved a stable value. It is ready for calibration.

Creating a calibration curve

To create a calibration curve, the user measures the current (pA) generated by the addition of different amounts of H2O2 to the calibration solution.

1. After the current is stable, zero the baseline.

2. Add a known volume of the H2O2 standard

solution to the PBS buffer solution.

Figure E1

— H2O2 calibration output.

Figure E2 — ISO-HPO-2 calibration curve based on Figure 1

WORLD PRECISION INSTRUMENTS E-3

For example, add 0, 5, 10, 20, 40, 80mL H2O2

standard solution (1.0mM) into 10mL PBS buffer solution.

The output from the H2O2 will like similar to the example shown in Fig. E1.

From this output, a calibration curve (Fig. E2) can be created by plotting the changes in current (pA) against the changes in concentration (mM). The slope of this curve indicates sensitivity of the probe.

APOLLO 4000

HYDROGEN PEROXIDE DETECTION

Interference

Temperature

The background current of the sensor will usually increase with increasing temperature of the experiment. Although, the sensitivity of the sensor does not change signiﬁcantly within the range 20-37°C. It is therefore recommended that any calibration should be performed at the same temperature as the experiment.

Between pH 3-10, changing the pH of the solution does not affect the sensitivity of the HPO sensor. However if the pH is below 3.0, the noise of the sensor will increase. At pH 10.0 and higher, the response of the sensor will diminish signiﬁcantly.

Maintenance of HPO Sensors

When the ISO-HPO-2 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.

Clean the Membrane

The membrane sleeve itself requires very little maintenance. The primary concern is to avoid to damage the membrane and to keep it as clean as possible. After each use, the membrane should be cleaned by suspending the tip in distilled water for 20-30 minutes to dissolve salts and remove particles ,which may have accumulated on the membrane. If the probe was used in a protein rich solution the tip should ﬁrst be soaked in a protease solution for several minutes to remove protein build-up, and then in distilled water. Enzymatic detergent (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). Accumulated organic matter can be removed by brieﬂy immersing the tip in an acid or base solution (at times both may be necessary) for 10 seconds. A good indication of a dirty membrane sleeve is a sluggish response or an unusually low sensitivity. If these problems are not rectiﬁed by the above cleaning procedures then the membrane sleeve should be replaced. The probe cannot be used in organic solvents.

WORLD PRECISION INSTRUMENTSE-4

APOLLO 4000

HYDROGEN PEROXIDE DETECTION

Changing the Membrane Sleeve

All membrane sleeves replacing will eventually have to be done by the user. The procedure for doing is simple and straightforward.

1. Unscrew the locking cap from the handle.

2. Hold the stainless steel sleeve and remove it with the locking cap from the

internal electrode, being careful not to bend the electrode assembly when doing so.

3. Rinse the internal electrode with distilled water (particularly the tip) and let it soak for at least 15 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.

4. Gently dry the electrode with a soft tissue (Kimwipes). Be sure to dry thoroughly the ﬂat surface at the tip of the electrode. After drying the current should stabilize fairly quickly to a low value ( e.g., 0 - 20 pA). If this occurs, then the electrode is in good working order.

5. If the electrode is not clean, repeat steps 3 and 4. If necessary the ISONOP Rejuvenator (WPI #JUV) can be used to 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.

7. Dip the internal electrode 1-2 cm into the electrolyte , the current should go offscale during this. Using the MicroFil™ non-metallic syringe needle and 1 ml plastic syringe, supplied, inject approximately 100 uL of electrolyte directly into the new sleeve. The MicroFil supplied should be less than the length of the sleeve, so that it will not puncture the delicate membrane at the tip of the sleeve during injection. If the MicroFil is longer than the sleeve it can be cut to the correct length.

8. Slowly and smoothly insert the electrode into the sleeve, and screw the locking cap into the handle. The electrode should be observed to press gently against the membrane.

9. The current at this time will be high or offscale.

WORLD PRECISION INSTRUMENTS E-5

APOLLO 4000

HYDROGEN PEROXIDE DETECTION

10. Suspend the tip of the new assembled probe in PBS buffer solution.

11. After 10-15 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.

12. If the current observed, after a few minutes in the saline solution, increases dramatically or is offscale, then the membrane integrity is not good and a new membrane will have to be ﬁtted.

Storage

Store the electrode with its tip immersed in electrolyte solution in the sealed vial provided with the electrode.

WORLD PRECISION INSTRUMENTSE-6

APOLLO 4000

TROUBLESHOOTING

Troubleshooting for APOLLO 4000

Problem Solution

1) The current reads too high or offscale on the display.

2) The sensor is unresponsive; current is close to zero and/or there is no response to NO/ HPO/O2 during calibration.

a) If the membrane sleeve has just been

changed, this is normal. Refer to the section “Replacing a Membrane Sleeve.”

b) The membrane may be damaged. Replace

the membrane sleeve.

c) The electrode has just been attached to the

Apollo 4000. This is normal.

a) Make certain that the contents of the

solutions used in the calibration procedure are correct.

b) The membrane may be heavily

contaminated. Clean the membrane as described in the section, “Cleaning the Membrane.”

c) The electrolyte inside the sleeve may no

longer be functioning. Refer to the procedure “Replacing a Membrane Sleeve”.

d) Open circuit in cable. Check cable

connections.

3) The baseline is noisy and/or drifts.

WORLD PRECISION INSTRUMENTS F-1

a) Make sure that the experimental set up is well

grounded and shielded as outlined in the appendix on “Grounding and Shielding”.

b) Check that the electrode has not been

damaged. If it has, replace the sleeve or fit a new sensor.

APOLLO 4000

TROUBLESHOOTING

WORLD PRECISION INSTRUMENTSF-2

APOLLO 4000

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. The best Faraday cage is constructed of solid sheet metal, but in NO measurements it is usually necessary to see the measurement system. The alternative is to use copper screening but it must be soldered completely along any joining seams. Place all the instruments and the sample into a Faraday cage

It may not always be possible to put the whole measurement system in a Faraday cage for shielding, as for example with a flow-through system when the probe is immersed into the effluent of a perfusion system or placed directly into the vein of the heart of an animal. In this case grounding the external bathing fluid, vein, or tissue with a stainless steel needle or Ag/AgCl reference electrode will often help significantly. If pumps or other electrical instruments are to be used in a flowthrough system, the associated equipment or instruments should be grounded as well. Use a common ground for all equipment in the experiment.

grounded

After careful grounding and shielding of the electronic equipment and the probe system, sometimes it is found that movement of people in the immediate vicinity causes current fluctuations. These are due to variations in the resulting stray capacitance. There are several ways to minimize these effects. When the measurements are made addition, the operator may need to be grounded because large static charges can be generated by the operator’s body. Wrist straps connected to ground the operator may be helpful.

WORLD PRECISION INSTRUMENTS G-1

in vivo

, it is good practice to ground the animals. In

* Electrodes, batteries and other consumable parts are warranted for 30 days only from the date on which the customer receives these items.

APOLLO 4000

Warranty

WPI (World Precision Instruments, Inc.) warrants to the original purchaser that this equipment, including its components and parts, shall be free from defects in material and workmanship for a period of one year* from the date of receipt. WPI’s obligation under this warranty shall be limited to repair or replacement, at WPI’s option, of the equipment or defective components or parts upon receipt thereof f.o.b. WPI, Sarasota, Florida U.S.A. Return of a repaired instrument shall be f.o.b. Sarasota.

The above warranty is contingent upon normal usage and does not cover products which have been modified without WPI’s approval or which have been subjected to unusual physical or electrical stress or on which the original identification marks have been removed or altered. The above warranty will not apply if adjustment, repair or parts replacement is required because of accident, neglect, misuse, failure of electric power, air conditioning, humidity control, or causes other than normal and ordinary usage.

To the extent that any of its equipment is furnished by a manufacturer other than WPI, the foregoing warranty shall be applicable only to the extent of the warranty furnished by such other manufacturer. This warranty will not apply to appearance terms, such as knobs, handles, dials or the like.

WPI makes no warranty of any kind, express or implied or statutory, including without limitation any warranties of merchantability and/or fitness for a particular purpose. WPI shall not be liable for any damages, whether direct, indirect, special or consequential arising from a failure of this product to operate in the manner desired by the user. WPI shall not be liable for any damage to data or property that may be caused directly or indirectly by use of this product.

Claims and Returns

• Inspect all shipments upon receipt. Missing cartons or obvious damage to cartons should be noted on the delivery receipt before signing. Concealed loss or damage should be reported at once to the carrier and an inspection requested. All claims for shortage or damage must be made within 10 days after receipt of shipment. Claims for lost shipments must be made within 30 days of invoice or other notification of shipment. Please save damaged or pilfered cartons until claim settles. In some instances, photographic documentation may be required. Some items are time sensitive; WPI assumes no extended warranty or any liability for use beyond the date specified on the container.

• WPI cannot be held responsible for items damaged in shipment en route to us. Please enclose merchandise in its original shipping container to avoid damage from handling. We recommend that you insure merchandise when shipping. The customer is responsible for paying shipping expenses including adequate insurance on all items returned.

• Do not return any goods to WPI without obtaining prior approval and instructions (RMA#) from our returns department. Goods returned unauthorized or by collect freight may be refused. The RMA# must be clearly displayed on the outside of the box, or the package will not be accepted. Please contact the RMA department for a request form.

• Goods returned for repair must be reasonably clean and free of hazardous materials.

• A handling fee is charged for goods returned for exchange or credit. This fee may add up to 25% of the sale price depending on the condition of the item. Goods ordered in error are also subject to the handling fee.

• Equipment which was built as a special order cannot be returned.

• Always refer to the RMA# when contacting WPI to obtain a status of your returned item.

• For any other issues regarding a claim or return, please contact the RMA department.

Warning: This equipment is not designed or intended for use on humans.

World Precision Instruments, Inc.

International Trade Center, 175 Sarasota Center Blvd., Sarasota FL 34240-9258

UK: Astonbury Farm Business Centre • Aston, Stevenage, Hertfordshire SG2 7EG • Tel: 01438-880025 • Fax: 01438-880026 • E-mail: wpiuk@wpi-europe.com

WORLD PRECISION INSTRUMENTS 5

Germany: Liegnitzer Str. 15, D-10999 Berlin • Tel: 030-6188845 • Fax: 030-6188670 • E-mail: wpide@wpi-europe.com

Tel: 941-371-1003 • Fax: 941-377-5428 • E-mail: sales@wpiinc.com

WPI APOLLO 4000 Instruction Manual

Specifications and Main Features

Frequently Asked Questions

User Manual

Contents

INTRODUCTION

Design Architecture

Plug-and-Play Design

Free Radical Sensor Technology

Instrument Description

Unpacking

USING THE APOLLO 4000 TO DETECT NITRIC OXIDE

Initial Set-up

Calibration of the NO Sensor

The Calibration Kit

Calibration by the chemical generation of NO

Calibration of NO sensor by decomposition of SNAP

Calibration of NO sensor using aqueous standards prepared with NO Gas

Measurement of NO

Maintenance of NO Sensors

Initial Set-up

Calibration and Use of Oxygen Sensors

Calibration

Troubleshooting for APOLLO 4000