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Ultimate BOD header graphic

Calibration of DO meter

Calibration of the DO meter is arguably the most critical aspect of BOD testing, since we measure the consumption of dissolved oxygen (DO). While calibration may be the center of the DO measurement universe, calibration is in turn impacted by both temperature and barometric pressure.

Understanding the relationship between pressure, temperature and oxygen saturation is critical in consistently obtaining quality BOD data. In the absence of this understanding, we encounter problems with supersaturation and blanks.

Calibration, pressure and temperature are inter-related.

Barometric pressure and temperature affect calibration. Accurate calibration is critical to obtaining quality data.

Supersaturation
  • Sample initial DO > 9.0 mg/L (in most cases).
  • Sample initial DO > saturation point (defined by temperature & pressure).
Blanks
  • Blanks can — and typically do — fail simply due to calibration problems.
  • Atmospheric pressure changes, including both low and high pressure systems can significantly affect blank results over the five-day incubation period.

A word about Winkler

Winkler remains the "gold" standard of calibration techniques. Winkler is based on chemistry, which means, as long as your reagents are fresh and prepared properly and accurately, your calibration will be accurate. We do see differences when Winkler is compared to Probe technology but the differences are in absolute DO measurements; critical QC results are the same.

Winkler was, at one point, the best available calibration technique. The reality is, however, that advances in probe technology combined with the simple fact that Winkler calibration is time and labor intensive, are slowly contributing to Winkler's demise. Certainly, if Winkler works for you, and you have the time required, by all means continue to use it. Alternatively, rest assured that you can obtain consistent and reliable calibrations using a DO probe.

The keys to successful Winkler calibration are:

  • be sure that dilution water is air saturated;
  • use fresh reagents;
  • standardize titrant;
  • perform Winkler titration;
  • check Winkler result against theoretical saturation;
  • set meter; and
  • document your procedure.

Apples to apples or Apples to oranges

Apples-to-apples graphic

For optimal calibration results, samples must be treated exactly as calibration.

For many years, we have referred to WSA calibration as a case of "apples to oranges" since the probe sits in air for calibration but is immersed in sample (or dilution water) during sample analysis. This is distinctly different from the WATER (saturated air) calibration in which the probe is always immersed in sample (or dilution water). Historically, this difference has been the root cause of many a blank failure.

At the heart of this issue is physics. We know we can easily saturate water with oxygen by simply shaking the water in an environment that offers sufficient exposure to oxygen in the air. This is best accomplished using a large container which is half full of dilution water, offering sufficient headspace for air (and thus oxygen). Maintaining a water-saturated air (WSA) environment, however, is a far more difficult proposition.

Consider an air calibration performed in the winter. It's quite possible that the atmosphere in a BOD bottle containing a little water, and sealed with the probe, represents 100 percent saturation (100 percent relative humidity). This is usually evidenced by the formation of condensation droplets on the probe tip. What's the first thing you do? Remove the probe from the bottle and either shake or wipe off the probe tip to remove the drops? Of course but what have you done to the water saturated air in the bottle? If your lab is like most, the room air likely has a humidity of about 50 percent (or less). Removing the probe from the bottle causes a draft effect, which pulls the water-saturated air out of the bottle to be replaced by air of much lower relative humidity.

Apples-to-oranges graphic

With water-saturated AIR calibration, samples and calibration are treated differently - the probe is in air for calibration, but submersed for sample analysis.

Now comes the physics lesson. So you seal up the bottle again by replacing the probe. Physics pop quiz time: How long does it take for the air in the bottle to become saturated with water again. Here's a tip, it doesn't occur in just a few minutes. Frankly, this "passive saturation" can take hours, depending on barometric pressure, the room temperature, the amount of water in the bottle and the amount of headspace of the bottle (i.e., physics). Bottom line: There are just too many variables, and that's exactly why your blanks can deplete 0.3 mg/L one day and "create" 0.3 mg/L the next day. That's why we haven't been huge fans of WSA calibration. But things are changing - technology is improving.

Update on ASW Calibration: With the introduction of the luminescence technology (RDO, LDO), we have observed far fewer calibration problems related to WSA (probe in air) calibration. We maintain, however, that if you do experience calibration problems, the ASW calibration approach is both valid and represents an "apple to apples" situation.

Chemists at the State Lab of Hygiene performed AIR and WATER calibrations in succession with the same sample, DO meter and probe. They noted that the AIR calibration result was higher by 0.15 mg/L (8.68 vs. 8.53). Note that 8.68 divided by 1.023 equals 8.48, which represents the true saturation of oxygen at 742 mm pressure and 22.2° C. The factor 1.023 (102.3%) represents a correction factor used to adjust results obtained following AIR calibration to account for difference in temperature equilibration time. The calibration for new technology (dual thermistor) probes is 102.3% and 101.7% for older polarographic probes.

Bottom line: the value shown after air calibration will be slightly high but will read correctly if immediately placed in water.

Common sources of error in the water-saturated air (WSA) calibration

  • Temperature: Thermistor must equilibrate to air temperature in the BOD bottle before calibration. It takes time for the probe equilibrate initially after removing the droplets from the tip. It also takes longer to equilibrate in air than water. That's why some have more success with the ASW approach.
  • Not allowing the air to become saturated with water — it takes at least 30 minutes.
  • Not allowing the meter and probe to warm-up for at least 30 minutes.
  • Poor DO probe maintenance
    • not changing the membrane regularly,
    • failure to inspect and remove sulfide deposits from anode & cathode.
  • Neglecting to check (and possibly re-calibrate) the on-board barometer.

Keys to successful water-saturated air (WSA) calibration

Your calibrations will likely work even if you don't wait for the air to be 100 percent saturated with water as long as you do your calibration the same everyday.

  • Consistency is the key.
  • Meter warm up time (at least 30 mins).
  • How droplets removed from the probe tip (shake or dab).
  • Amount of water in the BOD bottle (~1 inch).
  • How long you let the probe sit in the BOD bottle or the calibration chamber before calibration ( ≥30 minutes).
  • Consistent temperature conditions in lab.
  • Must be consistent from day zero to day five.
  • Get into a routine and stick with it.
  • How important is consistency?

One operator's SOP for consistency

Source: Joe Flannigan, Blanchardville Wastewater Treatment Plant; former Lab-of-the-Year winner.

  • 30 minute warm-up for meter.
  • Allow one-hour in bottle after wiping probe tip.
  • New membrane every two weeks.
  • Result: Has successfully met blank requirements for several years.

Conclusions: air (WSA) vs. water (ASW) calibration

  • There is a difference when calibrating in air vs. measuring samples in water.
  • Manufacturers often program a correction factor to account for the difference between oxygen diffusion in air vs. water.
  • This is often seen as a saturation of 102.3% (polarographic) when calibrating in air vs. calibrating in water.
  • Whatever works, is the best technique.

Correct calibration protocols

Water-saturated air (WSA)

Video: How to calibrate using WSA technique

Click to watch a video showing how to calibrate by the WSA protocol. [VIDEO Length 3:23]

  • Place the probe in a BOD bottle containing about 3 cm of water.
  • Shake BOD bottle prior to inserting probe to assure saturation. We recommend leaving the stirrer on (although manufacturer says it's not necessary) — it speeds up equilibration.
  • The probe may need to sit in the bottle for 30-35 minutes in order to match the temperature of the air.
  • Determine barometric pressure and adjust meter's internal barometer as necessary.
  • Check the temperature of the air (in the bottle) to be sure the probe thermistor is working correctly.
  • Use the meter's auto-calibration function to calibrate the probe and meter.

Air-saturated water (ASW)

Video: How to calibrate using ASW technique

Click to watch a video showing how to calibrate by the ASW protocol. [VIDEO Length 3:07]

  • Place the probe in a BOD bottle filled with air-saturated (well-shaken) water.
  • Leave probe in the water with stirrer operating long enough for the probe temperature to equalize with the water temperature.
  • Determine barometric pressure and adjust meters internal barometer as necessary.
  • Check the temperature of source water to be sure the probe thermistor is working correctly.
  • Use a detailed DO saturation table to determine the theoretical DO concentration.
  • Adjust the meter to read the DO concentration determined from the saturation table.

Summary of calibration techniques

  • The Winkler calibration takes longer than the other calibration techniques with no net gain in quality.
  • Calibration with air-saturated water takes less time because the probe's temperature equilibrates quicker in water than air. This is because water is a more effective heat sink than air.
  • You don't have to worry about dealing with droplets on the probe tip when calibrating in air-saturated water.
  • All three methods work. The results of the seed control and GGA were the same even though the IDOs and DOs were different.
  • Consistency is the key to good results regardless of calibration technique.
  • Whatever works for you, is the best technique.

How to know DO measurements are accurate

In order to know DO measurements are accurate, you need a "known" standard. If you have a standard which you know to be a specified dissolved oxygen concentration, and you measure that standard and obtain a reading equal to (or acceptably close to) that true value, then you have documentation to support the ability to obtain accurate measurements.

We also understand that labs have many QC responsibilities already, so the best part of all this is that you can analyze a known standard without having to add any additional analyses. This is because, the initial DO reading of your calibration blank is a known standard.

Remember: Pressure ± 5 mm translates to ± 0.06 mg/L DO.
Temperature ± 0.5 °C translates to ± 0.1 mg/L DO.

Summary of process

  • Prepare air-saturated (by shaking) dilution water.
  • Get some basic physical data.
    • Temperature
    • Absolute barometric pressure
  • Compare apples to apples. Has the pressure value been corrected to sea level (most are)? If so, you'll need to "uncorrect" it.
  • Make adjustments as necessary.
  • Use physical data to determine standard "true" value (use a DO saturation table).
  • Determine theoretical true value for oxygen in mg/L (i.e. saturation point).
  • If measured value = True value ± 0.2 mg/L; calibration is accurate.

Copyright 2006. University of Wisconsin Board of Regents.
Unauthorized use prohibited without the expressed written consent of the UW, State Laboratory of Hygiene and the DNR Laboratory Certification & Registration Program.

Last revised: Wednesday August 22 2012