Unreasonable Results

The first indicator of trouble to novices at gas exchange will typically be those values the user is paying attention to: photosynthesis, conductance, Ci, etc. Sometimes, the tendency is to blame the computer (“This thing is computing crazy numbers for photosynthesis!”). However, the program is doing exactly what it has been told to do (that being the abominable nature of computers); crazy numbers come from crazy inputs. You will need to look behind the computations and determine which input is crazy and why.

Photosynthetic Failures

Photosynthetic rate (1‑15) is primarily based upon a) the difference between sample and reference CO2 readings and b) flow rate, so look at those three variables (CO2R_μml, CO2S_μml, and Flow_μml) to determine which, if any, is causing problems. There is also a dilution correction, so wild values for H2OR_mml and/or H2OS_mml can also have an effect.

Unstable Photosynthetic Rates

If photosynthesis seems to be jumping around, try these suggestions:

  1. Are you just impatient?
  2. Bear in mind that right after a change in input conditions (such as a substantial change in the CO2 mixer setting), there will be a short period (up to 1 or 2 minutes) where the photosynthetic rate may be nonsense, since both IRGAs are coming to new equilibrium values.
  3. What’s the magnitude of the variation?
  4. There will always be some variation in the displayed value of any measured or computed quantity. Is the variation you see excessive? Is it due to the normal noise in the analyzers? Remember that at low rates, the noise in the CO2 differential (typically 0.4 ppm) will become more and more significant. (So: is the variation in ΔCO2 greater than 0.4 ppm?).
  5. Watch those flow rates
  6. For purposes of troubleshooting, operate in fixed flow mode, and set the flow to about 500 μmol s-1. If you are operating in constant VPD mode, or constant RH, you could be having problems by asking for a humidity value that cannot be achieved given the flow limitations and transpiration rate of the leaf. For example, if you have asked for 80% RH, and have a stressed leaf with nearly closed stomata, and are using very dry input air, your flow rate will go to zero (or about 30 μmol s-1 with a CO2 mixer installed) while the system waits in vain for the leaf to raise the humidity to what you asked for. Meanwhile, the computed photosynthetic rate will be quite unstable, being the product of a growing CO2 differential and a near-zero flow rate.
  7. If the photosynthetic rate is low, try operating at a low fixed flow rate (such as 100 μmol s-1); this will a) keep the flow rate stable, and b) make the CO2 differential as large as possible. See Dealing With Low Rates for other suggestions.
  8. Is the input stable?
  9. Watch reference CO2 (CO2R_μml) for 15 seconds. How much does it vary? Expect variations near 0.2 μmol mol-1 at ambient concentrations. If it is much more than that, you may have problems. An open system such as the LI-6400 depends upon stable inputs. Because air flows through the sample and reference cells at different flow rates, and because there are differing volumes involved, any fluctuation in the input will show up in the sample and reference at different times, causing the differential value to oscillate.
  10. If using the CO2 Mixer: Set it to control reference concentration (R), as this will ensure that the changes aren’t coming from the system’s attempts to maintain a particular concentration in the leaf chamber, and make sure the soda lime adjustment knob is on full scrub. (To test the mixer stability, Instability).
  11. Not using the CO2 Mixer: A larger buffer volume is probably called for. See Air Supply Considerations. Or, there may be moving debris in the sample cell. If so, it will affect both CO2S_μml and H2OS_mml. See Unstable CO2 and/or H2O.
  12. Is the sample cell stable?
  13. If the reference values are stable, but the sample values aren’t, try testing for a leak in the chamber. See Sensor Head Leaks.

Photosynthesis is stable, but “can’t be right”

An example of this problem would be negative, low, or ridiculously high photosynthetic rates on fully illuminated leaf of a well-watered, healthy plant.

  1. Check the chamber conditions
  2. Is CO2 where you’d like it to be, or is it near zero? (A common mistake: not using the mixer, forgetting to change the scrub tube setting after testing the IRGAs’ zero, and not noticing the absence of CO2 in the reference air.)
  3. Is the light what you think it is, or did you forget to turn on the LED source?
  4. Check other inputs
  5. Are you using the correct leaf area? You want the one-sided leaf area that is enclosed within the chamber. Is the flow rate (display line b) OK? It is typically between 200 and 700 μmol s-1. Is the pressure sensor OK (display line g)? Typical values: 100 kPa near sea level, 97 kPa at 1000 ft., 83 kPa at 5000 ft., etc.

Questionable Conductances

Since conductances are usually between 0 and 1 mol m-2 s-1, you might not notice problems with this value unless it makes intercellular CO2 be negative (next section), or conductance itself is negative.

  1. Leaf Area
  2. If the value of leaf area that is being used is much too low, the leaf conductance will exceed the boundary layer conductance, and the stomatal conductance will become very large, eventually becoming negative (equation 1‑9.
  3. Match problem
  4. Compare the sample and reference water IRGAs. Are they well matched? If sample is lower than reference (meaning transpiration is negative), that is a clear sign they aren’t well matched.
  5. Leaf temperature
  6. Transpiration does not depend on the leaf temperature measurement, but conductance does. If the transpiration number appears OK, but the conductance doesn’t, leaf temperature might be the reason. Is the sensor broken? Is it making good contact with the leaf? Is it well zeroed?

Negative Boundary Layer Conductance

Boundary layer conductance is normally computed from a lookup table based on leaf area and fan speed (see Boundary Layer Variables on page 14-19 in the instruction manual). If you change to a chamber that allows large leaf areas, but use the standard 2x3 chamber lookup table, you can get negative boundary layer conductances as an artifact of extrapolating that table’s data. The remedy is to use the appropriate lookup table, or use a constant value. If you’ve already logged the data, you can recompute (Chapter 13 in the instruction manual) using an appropriate boundary layer value.

Impossible Ci’s

The intercellular CO2 value is essentially the ratio of photosynthetic rate to conductance (equation 1‑18). The typical problem is that Ci is too low or negative. Conductance is generally the culprit, but here are three things to check:

  1. Transient condition?
  2. Very low Ci’s can be real, especially for short time periods. Example: take a low light leaf into bright light. The plant’s photosynthetic biochemistry responds much faster than do the stomata, so until the stomata can open wider, CO2 is consumed within the leaf, and Ci will be low. (Negative Ci’s, of course, cannot be real).
  3. Photosynthesis too high?
  4. If the value of photosynthesis is too high, Ci will be too low. The primary culprit: mismatched IRGAs.
  5. Conductance too low?
  6. If something is making the value of conductance too low, that will drive Ci down low or negative. Possible reasons:
  • Poorly matched IRGAs
  • Is the match valve working ok?
  • A terrible water calibration
  • If you zeroed with wet air (bad desiccant?), all subsequent water readings will be too low, making conductance too low.
  • Bad leaf temperature measurement or computation
  • If leaf temperature is too high, conductance will be too low. If you are measuring leaf temperature, is the thermocouple working? Is there good contact? Is it well zeroed (page 18-24 in the instruction manual)? If you are computing leaf temperature (energy balance), do the values seem reasonable? Do you have the right light source specified?