A LAZAR LABORATORIES HANDBOOK DISSOLVED OXYGEN PRIMER Answers to your basic dissolved oxygen measurement questions and helpful hints on how to optimize measuring dissolved oxygen. Copyright Lazar Research Laboratories, Inc. 2000-2004
Lazar Research Laboratories, Inc. Dissolved Oxygen Measurement Primer 2 About Lazar Research Laboratories, Inc. Lazar Research Laboratories, Inc. have been coaching researchers on measuring dissolved oxygen for over 27 years. Our knowledge has helped researchers in all scientific arenas including biotechnology, biomedical research, microchemistry, chemical engineering, environmental science, geology and more. Our family of standard and micro dissolved oxygen electrodes are the premier product for measuring DO in various samples in all sizes down to one micro liter. Free Consultation Got any dissolved oxygen questions? Call us at 1-800-824-2066 (1-323-931-1204 international), fax us at 1-323-931-1204, or email us at service@lazarlab.com and we will put our 24 years of experience in the dissolved oxygen field at your disposal. There is no cost or obligation for this service. Contact Lazar Research Laboratories, Inc. Email: service@lazarlab.com Website: http://www.lazarlab.com Fax: 1-323-931-1434 Phone: 1-323-931-1204 1-800-824-2066 (toll free in the U.S. only) Address: Lazar Research Laboratories, Inc. 731 North Labrea Avenue, Building 5 Los Angeles, California 90038 U.S.A.
Lazar Research Laboratories, Inc. Dissolved Oxygen Measurement Primer 3 TABLE OF CONTENTS 1. Comparing the dissolved oxygen electrode to the Winkler colorimetric method 2. How an oxygen electrode works A. Clark polarographic oxygen electrode B. Galvanic oxygen electrode 3. Zeroing and calibrating an oxygen electrode 4. Effect of temperature, atmospheric pressure, salinity 5. Effect of stirring 6. Oxygen electrode maintenance 7. Oxygen electrode that can be used with ph/mv meter, chart recorder, and PC 8. Standard size oxygen electrode 9. Micro oxygen electrode 10. Needle oxygen electrode 11. Micro flow through oxygen electrode 12. Micro biological po2 monitoring system 13. Oxygen electrodes for fermentors and bioreactors 14. Ordering contact information
Lazar Research Laboratories, Inc. Dissolved Oxygen Measurement Primer 4 1. COMPARING THE OXYGEN ELECTRODE TO THE WINKLER COLORIMETRIC METHOD The Winkler method, which involves measuring a color change in a sample when oxygen reacts with a special reagent, is inherently inaccurate for several reasons. The method involves adding a special reagent and waiting for the oxygen-reagent reaction to come to completion resulting in a yellowish tint. The amount of dissolved oxygen is related to the intensity of the yellow color. The color can be quantified by using a color comparison chart with the naked eye or by measuring the color with a colorimeter. The results can be affected by the subjective color comparison from one person to the next when using a color comparison chart, or by residual color or turbidity in the sample which affect the colorimeter results. The tedious setup procedure using the special reagent can also introduce errors due to user carelessness. Besides being inaccurate, the Winkler method is often very tedious especially when doing a large series of samples. Another factor which adds to the inaccuracy of this method is that the sample is first taken from the source (river, lake, etc.) and put into a sample container which is capped to prevent oxygen in the sample from escaping or oxygen from the atmosphere from entering the sample. The sample then has to be processed with the Winkler reagent prior to measuring the yellow tint. The removal of the water sample from the source prior to measurement can cause a rapid change in the oxygen level due to exposure to the atmosphere giving another cause for inaccuracy. With the electrode method, dissolved oxygen is measured directly at the source without having to take a sample and risking that the sample will either absorb or lose oxygen from the atmosphere. Since the oxygen electrode measurement does not depend on any change in color, this measurement is free of any interferences from other color bearing components in the water or from any effect of turbidity. Furthermore, the electrode method does not involve any introduction of reagents which are dependent on operator error and reagent aging. The readout from the oxygen meter is a quantitative digital concentration reading as opposed to a qualitative color comparison. The Lazar Research Standard Dissolved Oxygen Probe
Lazar Research Laboratories, Inc. Dissolved Oxygen Measurement Primer 5 2. HOW AN OXYGEN ELECTRODE WORKS All oxygen electrodes are based on a simple electrochemical reaction that occurs in water when oxygen is present. The oxygen reacts with water at an electrode surface producing hydroxyl ion. A. THE CLARK POLAROGRAPHIC OXYGEN ELECTRODE Oxygen is reduced at a cathode by receiving electrons and is converted into hydroxyl ion. This reaction occurs at the cathode and is the basis of the Clark type oxygen electrode. In the Clark electrode the cathode is made out of platinum and the anode is made out of silver. The anode is necessary to complete the electrical circuit so that electrons are available at the cathode to reduce the oxygen. In order to prevent other electrochemical reactions from interfering with the oxygen reaction and giving a false signal at the cathode, the anode and cathode are usually inside an electrode body which is covered with a semipermeable membrane. The job of the membrane is to prevent most components present in water except for oxygen from entering the electrode interior. The membrane is what gives the oxygen electrode great selectivity for oxygen with hardly any interferences. The Clark electrode is based on a polarographic principle where the current running between the cathode and anode is generated by imposing a known voltage difference (potential) between these two electrodes. The consumption of electrons at the cathode during the oxygen reaction, results in a small current between the anode and the cathode. This current is directly related to the concentration of dissolved oxygen in the water sample being measured. The current which is in the low nanoampere range (1 billionth of an amp) is converted into a voltage output by a special current amplifier and is then read using a digital voltmeter or chart recorder. The voltage output can be calibrated to directly indicate the concentration of dissolved oxygen in the sample. B. THE GALVANIC OXYGEN ELECTRODE Another common type of oxygen electrode, called the galvanic oxygen electrode uses a similar principle where oxygen is reduced at a cathode, but instead of having an electronic circuit produce a voltage difference between the cathode and anode, the two electrodes have dissimilar electrode potentials which produces the voltage (potential) difference. This is similar to the operation of a standard battery. The cathode in this case is usually silver or an alloy of silver, and the anode is composed of lead. The current produced at the cathode is identical to the current produced at the Clark type electrode. The current is again amplified and converted to a voltage to be displayed on a digital voltmeter or chart recorder.
Lazar Research Laboratories, Inc. Dissolved Oxygen Measurement Primer 6 3. ZEROING AND CALIBRATING AN OXYGEN ELECTRODE In an ideal electrode system for measurement of dissolved oxygen the residual current present at the cathode should be zero when the dissolved oxygen concentration is zero. For practical oxygen electrodes, either Clark polarographic type or galvanic there is always a small amount of residual current present even when the dissolved oxygen level is zero. In order to compensate for this residual current, there is an adjustment available on most oxygen amplifiers (which amplify the current signal and convert it to a voltage output) where the zero baseline can be adjusted so when the oxygen level is zero the output from the amplifier will also read zero. The zero adjustment is done by dipping the oxygen electrode into an environment which is totally devoid of oxygen and adjusting the amplifier output to zero. The calibration control on an oxygen meter (CAL or span) adjusts the amplifier output to correspond to the actual value of dissolved oxygen in a standard being measured. This calibration is accomplished by exposing the oxygen electrode to a known value of dissolved oxygen and then adjusting the CAL control so that oxygen value is read at the output of the amplifier or the digital voltmeter connected to the amplifier. A simple practical method to calibrate an oxygen electrode is to expose it to atmospheric oxygen or a solution of water in equilibrium with atmospheric oxygen. The calibration point is then set to correspond to the known saturation value of oxygen dissolved in water which is in equilibrium with air. There are tables available which give the saturation value in air for various temperatures and atmospheric pressures.
Lazar Research Laboratories, Inc. Dissolved Oxygen Measurement Primer 7 4. EFFECT OF TEMPERATURE, ATMOSPHERIC PRESSURE, AND SALINITY The effect of temperature is most important because the reaction which takes place at the cathode where oxygen reacts to produce a current is directly dependent on the temperature of the water which is being measured. Electronic amplifiers which convert the current signal to a voltage output can have an adjustment potentiometer which is set to correspond to the temperature of the water being measured (temperature needs to be measured separately). An alternate approach which is usually done in most modern oxygen meters is to have an automatic temperature adjustment where a thermister is embedded at the tip of the oxygen probe which changes resistance with temperature. The thermister is connected to the amplifier circuit and changes the output signal corresponding to the temperature of the sample. Atmospheric pressure is important, but to a lesser degree than temperature, because changes in atmospheric pressure will effect the amount of oxygen which is dissolved in the water. The effect of atmospheric pressure can be compensated for in the initial calibration of the oxygen electrode-amplifier where the saturation of oxygen is established based on well defined tables which give different saturation values as a function of atmospheric pressure. When measuring dissolved oxygen in sea water the salinity of the water also becomes a factor. Salinity level in water will affect the amount of oxygen which can dissolve in the water.
Lazar Research Laboratories, Inc. Dissolved Oxygen Measurement Primer 8 5. EFFECT OF STIRRING Stirring can have an effect on the dissolved oxygen reading with a dissolved oxygen electrode. The reason for this is that a very small amount of oxygen is actually consumed by the electrode as it measures the concentration of dissolved oxygen. This oxygen consumption produces a depletion of oxygen at the membrane surface as compared to the oxygen level in the water sample. In other words, the oxygen concentration found at the membrane which is in direct contact with the oxygen electrode can be lower than the oxygen level in the sample itself. Stirring the sample reduces this effect whereby the oxygen level at the membrane now becomes equal to that of the sample. Stirring can be accomplished by using a stirrer or by measuring oxygen in water which is moving or flowing. Often the water sample being measured is not totally still but there is turbulence present. The turbulence can act as a stirring mechanism so that external stirring is not needed. Another instance of a natural stirring mechanism is measuring oxygen in a pipe where liquid is flowing by. In instances where stirring cannot be accomplished such as in very small samples, the effect of oxygen depletion can be compensated for in the calibration procedure.
Lazar Research Laboratories, Inc. Dissolved Oxygen Measurement Primer 9 6. OXYGEN ELECTRODE MAINTENANCE In order to get the most accurate readings with an oxygen electrode certain maintenance procedures need to be carried out. The semipermeable membrane on the oxygen electrode is usually constructed of a fluoropolymer and is susceptible to tearing, pinholes, scratching etc. It is important to periodically check the membrane visually to make sure that the membrane is still in good condition. If any type of damage is seen, it is advisable to replace the membrane. A membrane which is leaking can produce very erratic oxygen readings. Another expendable item in oxygen electrodes is the internal filling solution. This solution is an elecrolyte which maintains solution conductivity between the cathode and anode. This solution should be replaced periodically in order to maintain the oxygen electrodes at optimal condition. Some manufacturers of oxygen electrodes also recommend a periodic polishing of the cathode using a very fine emery paper. The polishing removes any metal oxide films which can reduce the overall signal output of the electrode. When and if to polish is something to ask the manufacturer of the oxygen electrode. Recalibration of the oxygen electrode should also be done periodically to make sure that the electrode is still within calibration. Calibration intervals depend somewhat on the type of oxygen electrode and the application it is being used for. In most instances an air calibration can be done which does not require any special reagents or other items. Zeroing of the oxygen electrode is usually done at the factory for most oxygen electrodes and needs to be done by the user on an infrequent basis. Zeroing is usually done by adding a chemical such as sodium bisulfite to water. The sodium bisulfite reacts with oxygen depleting the water of any oxygen. Another convenient method for zeroing which can be done in a laboratory is to use a gas like nitrogen (N2) which is bubbled through a water sample removing the dissolved oxygen by gas displacement. Maintenance procedures for portable oxygen meters should include a check of the batteries. If the batteries read a low voltage they should either be replaced or recharged (for rechargeable batteries). Low battery voltage can give erroneous and erratic oxygen readings.
Lazar Research Laboratories, Inc. Dissolved Oxygen Measurement Primer 10 7. OXYGEN ELECTRODES WHICH CAN BE USED WITH A ph/mv METER OR CHART RECORDER Oxygen electrodes requires an amplifier circuit to read oxygen concentration and cannot be used directly with a regular ph/mv meter or chart recorder. Most manufacturers incorporate the oxygen electrode as part of an oxygen instrument which often costs many times the price of a ph/mv meter. Oxygen measurements are therefore normally restricted to users with a large enough budget to be able to afford a dedicated oxygen instrument which incorporates the oxygen electrode, amplifier circuit, and readout device. Lazar Research Laboratories, Inc. has developed a better solution to the high expense of dissolved oxygen meters. We have included the amplifier circuit directly with the dissolved oxygen probe, and made the output compatible with any standard ph/mv meter or chart recorder. Instead of using an expensive oxygen readout device, you can use the oxygen electrode with the most common instrument in any laboratory, namely a ph meter. Using the oxygen electrode now becomes no more complicated or expensive than using a standard ph electrode. Our family of oxygen electrodes can all be used with any laboratory or portable ph/mv meter, various chart recorders, and even directly with a PC having an A to D board. Given below is a description of the Lazar dissolved oxygen family which includes a standard size oxygen electrode for measuring water quality in lakes, rivers, the ocean etc, and also measuring oxygen in BOD bottles directly. Other oxygen electrodes includes a micro model for measuring dissolved oxygen in very small samples down to 20 microliters including 96 well plates, micro centrifuge tubes, petri dishes etc; a needle oxygen probe for measuring tissue samples; and a micro flow through oxygen electrode for measuring oxygen in micro flowing streams in HPLC, FIA, and organ perfusion. Lazar also sells a broad selection of dissolved oxygen electrodes for fermentation and bioreactors.
Lazar Research Laboratories, Inc. Dissolved Oxygen Measurement Primer 11 8. STANDARD SIZE DISSOLVED OXYGEN PROBES FEATURES USE WITH ANY ph METER. Probe can be plugged directly into any standard ph meter for dissolved oxygen measurements. USE WITH PC COMPUTER. Use with Lazar low cost datalogger for monitoring and recording with your PC. COVERS FULL DO RANGE. Electrode can measure dissolved oxygen from 0 to 20 ppm or 0 to 150% saturation. ELIMINATES WINKLER METHOD. Probe measures dissolved oxygen directly without involved wet chemistry or titrations needed with the Winkler method. BOD MEASUREMENTS. BOD probe fits directly into BOD bottle for 5 day or continuous BOD measurements. PORTABLE. On-the-spot field measurements of dissolved oxygen can be made without a trip back to the laboratory. RAPID RESPONSE. Typical response time at room temperature is 12 seconds. USE FOR BOTH BOD MEASUREMENTS IN THE LAB OR FOR FIELD USE. SPECIFICATIONS Oxygen Range: 0 to 20 ppm dissolved oxygen Accuracy: +/- 0.2 ppm at calibration temperature Temperature Range: 0 to 50 degrees C Response Time: Response time at 25 C is 12 seconds for 90% of reading. Connector: Available in either U.S. standard or BNC connector (please specify when ordering). Power: 9 volt disposable battery or 110/220 AC. Sample Size: Minimum sample size is about 2 ml.
Lazar Research Laboratories, Inc. Dissolved Oxygen Measurement Primer 12 ORDERING INFORMATION DO-166 OXYGEN ELECTRODE including spare membranes and bottle of filling solution. Can be used for both BOD and field measurements. Cat. No. DO-166-4 with 4 ft. cable Cat. No. DO-166-15 with 15 ft. cable Cat. No. DO-166-25 with 25 ft. cable Cat. No. SM-166 Spare DO Membranes- Package of 10 membranes Cat. No. SF-166 Filling Solution (2 ounce bottle of filling solution)
Lazar Research Laboratories, Inc. Dissolved Oxygen Measurement Primer 13 9. MICRO DISSOLVED OXYGEN ELECTRODE 96 well plates micro test tubes serum cups capillary tubes NMR tubes microcentrifuge tubes ADVANTAGES MEASURE ph IN 1/5 OF A DROP. This electrode enables you to measure samples as small as 10 microliters (1/5 of a drop). USE WITH ANY ph METER. The micro po2 electrode utilizes a self contained amplifier circuit which makes it useable with any standard ph/mv meter for dissolved oxygen measurements. The po2 electrode can be plugged directly into any ph meter and is available with all standard ph meter connectors including BNC, U.S. standard, Radiometer(R), and Metrohm (R). USE WITH CHART RECORDER. Output voltage of 0 to 2 volts DC allows connection to any standard chart recorder for continuous monitoring of po2 level. USE WITH PC COMPUTER. Can be used with any PC that has an A to D data card, or use the Lazar low cost A to D datalogger which plugs into the serial port of a PC. PORLAROGRAPHIC PRINCIPLE. The po2 electrode works on polarographic principle where oxygen is reduced at a tiny cathode sensor which is covered with a semipermeable membrane. EXTREMELY SMALL SENSOR SIZE. Tip diameter of the micro po2 electrode is only 2 mm. LOW IMMERSION DEPTH. Immersion depth for the micro po2 electrode is less than 1 mm. *CALIBRATE IN CONVENIENT UNITS. The micro po2 probe can be calibrated in mm Hg po2 partial pressure, % oxygen, or ppm dissolved oxygen. BROAD po2 CONCENTRATION RANGE. The micro po2 has a range from 0 to 100% oxygen or 0 to 760 mm Hg po2 partial pressure. LOW O2 DETECTION LIMIT. The micro po2 has a detection limit of 1 mm Hg po2 partial pressure. BROAD TEMPERATURE RANGE. Temperature range is 0 to 60 oc.
Lazar Research Laboratories, Inc. Dissolved Oxygen Measurement Primer 14 APPLICATION AREAS Biotechnology Biological sciences Medicine Physiology Environmental studies Micro chemical analysis Forensic science Pharmaceuticals Radioactive samples NMR samples ORDERING INFORMATION Cat. No. DO-166MT-1 micro po2 probe. Comes complete with po2 electrode, internal amplifier circuit, connector cable to ph meter or PC, spare membranes, and spare filling solution. Specify type of connector needed for ph meter being used (BNC, US Standard, Radiometer(R), Metrohm(R), other) when ordering. Cat. No. SM-166 Spare DO Membranes - Package of 10 membranes. Cat. No. SF-166 Filling Solution - 2 ounce bottle of filling solution.
Lazar Research Laboratories, Inc. Dissolved Oxygen Measurement Primer 15 10. MICRO NEEDLE po2 PROBE YOU CAN NOW MEASURE po2 IN TISSUE SAMPLES The needle probe inserts directly into tissue samples for oxygen concentration measurements in various kinds of tissues including various plant and animal tissues and cell cultures. USE WITH ANY ph METER. The needle po2 electrode utilizes a self contained amplifier circuit which makes it useable with any standard ph/mv meter for dissolved oxygen measurements. The po2 needle probe can be plugged directly into any ph meter and is available with all standard ph meter connectors including BNC, U.S. standard, Radiometer(R), and Metrohm (R). USE WITH CHART RECORDER. Output voltage of 0 to 2 volts DC allows connection to any standard chart recorder for continuous monitoring of po2 level. USE WITH PC COMPUTER. Can be used with any PC that has an A to D data card, or use the Lazar low cost A to D datalogger which plugs into the serial port of a PC. PORLAROGRAPHIC PRINCIPLE. po2 needle probe works on polarographic principle where oxygen is reduced at a tiny cathode sensor housed inside a hypodermic needle. po2 DEPTH PROFILES. Determine po2 as a function of tissue depth. SMALL SENSOR SIZE. The po2 needle diameter is 16 gauge. LONG NEEDLE LENGTH. Needle length is 38 mm giving it ample size to insert into thick tissue samples. CALIBRATE IN CONVENIENT UNITS. The micro po2 probe can be calibrated in mm Hg po2 partial pressure, % oxygen, or ppm dissolved oxygen. BROAD po2 CONCENTRATION RANGE. The micro po2 has a range from 0 to 100% oxygen or 0 to 760 mm Hg po2 partial pressure. LOW O2 DETECTION LIMIT. The micro po2 has a detection limit of 1 mm Hg po2 partial pressure. BROAD TEMPERATURE RANGE. Temperature range is 0 to 50 oc. ORDERING INFORMATION Cat. No. DO-166NP po2 needle probe. Comes complete with needle po2 electrode, electronic amplifier circuit, and connector cable to fit ph meter. Specify type of connector needed for ph meter being used (BNC, US Standard, Radiometer(R), Metrohm(R), other) when ordering.
Lazar Research Laboratories, Inc. Dissolved Oxygen Measurement Primer 16 11. MICRO FLOW THROUGH po2 AND pco2 ELECTRODES FEATURES USE IN BIOREACTOR, CELL CULTURE, AND ORGAN PERFUSION. The po2 micro flow through system (180 microliter internal volume) includes a special geometry dissolved oxygen electrode housed within a micro flow cell and is ideally suited for monitoring micro flowing streams in biotechnology, organ perfusion, cell culture, and HPLC. The po2 probe includes an internal preamplifier making it useable with any ph/mv meter. A micro stir bar is also included in the flow cell. The pco2 micro flow through system has a flow cell volume of only 50 microliters. ATTACHES DIRECTLY TO SILASTIC TUBING. End fittings on po2 & pco2 probes attach directly to standard micro silastic tubing. USE WITH ANY ph METER. The flow through po2 and pco2 electrodes plug directly into any standard ph/mv meter. Electrodes are available with all standard ph meter connectors including BNC, U.S. standard, Radiometer(R), and Metrohm(R). USE WITH CHART RECORDER. Output voltage from 0 to 2 volts allows connection of both po2 and pco2 flow through electrodes to any standard chart recorder for continuous monitoring of po2 and pco2 levels. USE WITH PC COMPUTER. Can be used with any PC that has an A to D data card, or use the Lazar low cost A to D datalogger which plugs into the serial port of a PC. ORDERING INFORMATION Model DO-166FT micro flow through po2 electrode. Comes complete with micro flow through po2 probe, micro flow through cell with input and output ports, silastic tubing for port connections, electronic amplifier circuit, and connector cable to fit ph meter. Specify type of connector needed for ph meter being used (BNC, US Standard, Radiometer(R), Metrohm(R), other) when ordering. Model GS-136CO-1 micro flow through pco2 electrode. Comes complete with micro flow through pco2 probe, micro flow through cell with input and output ports, silastic tubing for port connections, and connector cable to fit ph meter. Specify type of connector needed for ph meter being used (BNC, US Standard, Radiometer(R), Metrohm(R), other) when ordering. Model PP-166 peristaltic pump. A variable speed peristaltic pump with flow rates from 0 to 25 ml/min.
Lazar Research Laboratories, Inc. Dissolved Oxygen Measurement Primer 17 12. COMPLETE MICRO BIO po2 MONITORING SYSTEM You can now continuously monitor oxygen uptake or oxygen evolution of cells or tissue samples. Output from oxygen probe can be fed directly to chart recorder for plotting oxygen uptake or evolution curves. SYSTEM INCLUDES THE FOLLOWING: 1. Specially designed micro po2 electrode with built in amplifier. 2. micro chamber with variable volume from 200 to 1000 microliters 3. micro stir bar for stirring sample (when needed) 4. Miniature magnetic stirrer 5. Sealed septum sample injection system using syringe 6. Digital ph/mv meter for monitoring po2 and pco2 level 7. Optional pco2 electrode from monitoring pco2 level ORDERING INFORMATION Model RMS-166 bio po2 monitoring system including micro po2 electrode, micro variable volume sample chamber, septum sample injection system, digital ph/mv meter, micro magnetic stir bar, and miniature magnetic stirrer. Model GS-136-CO pco2 electrode designed to be used with sample chamber above.
Lazar Research Laboratories, Inc. Dissolved Oxygen Measurement Primer 18 13. OXYGEN ELECTRODES FOR FERMENTATION AND BIOREACTORS Please refer to the following web page for our extensive line of stainless steel and glass dissolved oxygen electrodes for fermentation and bioreactors. http://www.lazarlab.com/ferment.htm
Lazar Research Laboratories, Inc. Dissolved Oxygen Measurement Primer 19 14. HOW TO PLACE AN ORDER OR REQUEST PRICING INFORMATION by phone: 1-800-824-2066 in the U.S. 1-323-931-1204 International by email: service@lazarlab.com by fax: 1-323-931-1434 by mail: Lazar Research Laboratories, Inc. 731 North LaBrea Avenue, Suite 5 Los Angeles, CA 90038 U.S.A. Note: Specifications and prices subject to change without notice To get a brochure on the Lazar micro ion electrodes contact us via email at service@lazarlab.com giving us your fax number and request brochure FIS. METHODS OF PAYMENT net 30 to companies with recognized credit rating Visa or Mastercharge COD, check, money order Note: Specifications and prices subject to change without notice To get a full brochure on the micro ph electrode including pictures contact Lazar Research Labs, Inc. at service@lazarlab.com giving your fax number and asking for brochure FPHR.