IntroductionThe LI-6400/XT can use custom-made chambers for a widevariety of applications. Adapting the LI-6400 for wholeplant canopy gas exchange measurements is discussed inLI-6400 Application Note 2. Another approach using asmaller chamber is described here, along with some generalconsiderations. We complete the example by measuringphotosynthesis of Prostrate Spurge (a weed with tiny leavesgrowing close to the ground) and the respiration rate ofdetached fruit. This application illustrates how othershapes and sizes of chambers can also be attached to thesample IRGA cover plate of the LI-6400 sensor head anddemonstrates the adaptability of the LI-6400 for a variety ofgas exchange configurations.

As a precautionary note, departing from a standard 2 cm ×3 cm LED chamber involves losing control of illuminationand also compromising some humidity and temperaturecontrol. Sometimes modifying the measurement protocolcan avoid the need for a special chamber altogether. Thisnote is for cases where a custom chamber is clearly neces-sary.

Considerations for Building YourOwn ChamberTwo design characteristics of sample chambers are shapeand volume. These characteristics not only affect the sizeand shape of the subject material that can be measured, butalso have a bearing on air flow rates and consequently themagnitude of the reference and sample chamber concentra-tion differences. In addition, as chamber volume increases,lack of control of chamber temperature and humidity maybecome an issue. It may also become necessary to install afan inside the chamber to achieve adequate mixing.

Chamber Volume, Time Constant and MixingFor chamber volumes smaller than about two liters, themixing fan within the sample chamber IRGA providesadequate mixing of the chamber volume. For larger cham-bers, an additional fan should be installed within the

chamber. For a thoroughly mixed, open, flow-throughsystem, the chamber concentration C(t) at time t, is givenby:

C t C C C ee e oft V( ) ( ) ( / )= − − − (1)

Where C0 is the initial chamber concentration, Ce is theincoming concentration, f is the airflow rate and V is thechamber volume. By definition, the time constant τ of thechamber is the time it takes for the chamber concentrationto get to within 1/e or about 63% of the final concentration.Equation 1 shows that τ is given by:

τ =V

f (2)

For example, for a well-mixed chamber of volume 2000cm3 and an airflow rate of 700 μmol/s (15.7 cm3/s), thetime constant is 125 seconds (Fig. 1). By contrast, thestandard 2 cm × 3 cm LED light source chamber has avolume of about 80 cm3 and at a flow of 700 μmol/s, itstime constant is about 4 seconds.

Figure 1. The theoretical change in chamber CO2 concentrationwith time for a 2-liter chamber.

Interfacing Custom Chambersto the LI-6400/XT Sensor Head

Continued on next page

App. Note #3

Chamber CO2 Concentration with TimeChamber Volume = 2000 cm 3, Flow =15.7 cm 3/s

355

360

365

370

375

380

385

0 100 200 300 400 500 600

Time (seconds)

CO

2 C

on

cen

trat

ion

(P

PM

)

63

95%

Starting with an initial chamber concentration of 360 ppm,an incoming flow of 700 μmol/s (15.7 cm3/s) and concen-tration Ce of 380 ppm, the chamber concentration reaches95% of the final concentration after about 380 seconds.

Leaf Area, Flow Rate and CO2 DifferentialsOne of the main reasons for custom-designing a chamberis to increase the amount of plant material that can beenclosed within the chamber, in order to increase theprecision of measuring low rates of exchange (e.g. whenmeasuring plant respiration or low rates of photosynthesis).To find the minimum sample area required to obtain agiven measurement precision, do the following analysis:

Assume:● The CO2 noise is ± 0.1 ppm. (The LI-6400 has a typical

peak-to-peak noise of 0.2 ppm at 350 ppm with 4-second signal averaging).

● We desire a 2% measurement precision.● Minimum flow is 200 μmol/s.

To get a 2% measurement precision of the CO2 concentra-tion differential (difference between reference and samplechamber concentration), the differential should be at least5 ppm (this is obtained by dividing analyzer noise by therequired precision, 0.1/0.02, or 5 ppm).

Next we consider how to generate a 5 ppm CO2 differen-tial. This will obviously depend on the amount of plantmaterial enclosed within the measurement chamber, itsCO2 exchange rate, and the air flow rate through thesystem.

Ignoring the dilution effects of water vapor (see VonCammaerer and Farqhuar, 1981, or LI-6400 Manual, 1999),the gas exchange rate in an open flow through system isgiven by:

A mol m sf mol s CO mol mol

S cm( / / )

( / ) ( / )( )

μμ μ2 2

2 100=

××

Δ(3)

Assuming a fairly low yet practical airflow rate of 200μmol/s, the minimum sample area required to generate a 5ppm CO2 differential is given by:

S cmf mol s CO mol mol

A mol m s( )

( / ) ( / )( / / )

2 22 100

=×

×μ μ

μΔ

(4)

If we wanted to measure a CO2 exchange rate of 0.5 μmol/m2/s with a precision of 2%, the required chamber shouldenclose a minimum leaf area of about 20 cm2.

Other ways we could improve the measurement precisionof low activity material would be to increase the signal

averaging time, or further reduce the flow rate. Forexample, increasing the signal averaging time from 4seconds to 16 seconds would cut the noise in half. De-creasing the flow rate below 100 μmol/s may not be practi-cal however, as this could increase diffusive leaks (seeLI-6400 Manual, page 4-43) and increase the chamber timeconstant. If a 5% measurement precision was acceptable,the 2 cm × 6 cm narrow leaf chamber would be sufficientfor measuring an exchange rate of 0.5 μmol/m2/s.

Figure 2 shows the relationship between flow (μmol/s) andCO2 differentials for various combinations of CO2 assimi-lation rates (A, in μmol/m2/s) and leaf area (S, in cm2). Tofind the expected CO2 differential, multiply the assimila-tion rate expected by the leaf area to be enclosed within thechamber, and follow the appropriate graph line to the airflow rate desired. For example, a 5 cm2 leaf area with a netassimilation rate of 5 μmol/m2/s will generate a CO2 draw-down of 10 ppm when the air flow rate is 250 μmol/s.

Effect of Flow and Leaf Area on CO2

Differentials for Various Assimilation Rates

0.1

1

10

100

1000

0 100 200 300 400 500 600 700 800

Flow (μmol/s)

Del

ta C

O2

(μm

ol/m

ol)

50

25

12.5

100

200

3

LI-6400 CO2 noise (4 second signal averaging)

6

A(umol/m2/s) x S(cm2)

Figure 2. Relationship between flow and CO2 differentials forvarious combinations of CO2 assimilation rates and leaf area.

Leaf Area, Flow Rate and Chamber Humidity ControlEnclosing actively photosynthesizing and transpiring plantmaterial within the measuring chamber will not only lowerthe chamber CO2 concentration, but will also raise humid-ity levels. The primary limitation here is the supply of dryair. If transpiration exceeds the flow rate of dry air, thenchamber humidity will rise and condensation will occur.Lower flow will raise humidity, while a lower transpirationrate requires a lower flow rate of dry air.

Combining equations 1-4 and 1-7 from the LI-6400Manual, stomatal conductance, g (mol/m2/s) can beexpressed as:

2

∴ ≈−⎡

⎣⎢⎤⎦⎥ −

⎡

⎣⎢

⎤

⎦⎥g

f W W

S W Ws r

L s

( )100

1(6)

solving for Ws we get:

WkW W

ksL r=++1

(7)

where

kgS

f=100

If leaf and air temperatures are the same, then the highestvalue of Ws possible before condensation would occurwould be WL. The lowest value possible (at very high flowrates) would be Wr. Figure 3 shows the relationship be-tween airflow and chamber humidity for various multiplesof stomatal conductivity and leaf area. Ws is shown as afraction of the value between Wr and WL.

Example: Flow = 700 μmol/s, dry incoming air (Wr= 0),Tleaf = 30 °C (WL ≈ 43 mmol/mol), leaf area = 50 cm2, g =0.15 mol/m2/s. Ws would be about halfway between Wrand WL or about 22 mmol/mol. If Wr were 20, then Wswould be about 32 mmol/mol.

Figure 3. Relationship between air flow and chamber humidityfor various multiples of stomatal conductivity and leaf area.

Chamber Temperature ControlThe standard 2 cm × 3 cm LED light source has a volumeof about 80 cm3. Under normal field conditions, thetemperature control range of the LI-6400 is about ±6 °Cfrom the ambient temperature. With a larger chamber, thetemperature control range possible with the standardthermoelectric coolers will be reduced.

Interfacing Custom Chambers to theLI-6400 IRGAFigure 4 shows the LI-6400 sensor head from which thelatching handle, the leaf chamber and the sample IRGA cellcover plate have been removed. The mixing fan can beseen within the sample IRGA cell.

Attaching a custom chamber involves replacing the sampleIRGA cell cover plate with either the mounting plate usedfor the 6400-09 Soil Chamber (part #9864-174) or themounting plate used on the 6400-05 Conifer Chamber(part #9864-157). Figures 5a and 5b show these mountingplates. The Soil Chamber mounting plate is on the righthand side. While either mounting plate will provide asuitable base for attaching a custom chamber, the 9864-174mounting plate provides a larger base area than the 9864-157 plate, and is suitable for larger chambers. A dimen-sional drawing for the 9864-174 mounting plate can befound here.

The air inlet manifolds, attached to the reverse side of theseplates, can be seen in Figure 5b. These manifolds are a little

3

Fig. 4

gf W W

S W

W W

W Ws r

s

L s

L s

=−

−

⎡

⎣⎢

⎤

⎦⎥

−−

−

⎡

⎣

⎢⎢⎢

⎤

⎦

⎥⎥⎥

( )( ) ( )100 1000

10002

(5)

Where f is the airflow rate (μmol/s), S is the leaf area (cm2),Ws is the sample chamber water vapor mole fraction(mmol/mol), Wr is the incoming or reference chamberwater vapor mole fraction (mmol/mol), and WL is thewater vapor mole fraction (mmol/mol) within the leaf airspaces (this value is calculated using the leaf temperaturemeasurement).

Chamber Humidity and Airflow Relations

00 200 400 600 800 1000 1200

Flow (μmol/s)

Ws

(mm

ol/m

ol)

=100

20

7.5

3

1.5

0.5

Wr

WL

0.50.5

g(mol/m2/s)S(cm2)

9864-157 9864-174

Fig. 5a

The air returns to the sample IRGA cell via the largermiddle hole of the mounting cover plate.

Figures 6a and 6b show the 9864-174 Soil Chambermounting plate and the 9864-157 Conifer Chambermounting plate attached to the sample IRGA cell. Theorientation of the three holes (with respect to the sensorhead body) on the conifer chamber mounting plate is atright angle to the orientation of the holes on the soilchamber mounting plate. The difference in orientationdoes not affect circulation or mixing of the chamber air.The air is still propelled out of the two outer holes, andreturns to the sample IRGA cell via the larger central hole.

Figure 7 Shows a relatively large (approximately 2 liters involume) custom chamber made from a piece of Plexiglastubing. The tube diameter is about 14 cm. The chamberwas made by cutting an approximately 13.5 cm length ofthe tube and covering one end of the tube with Propafilm(LI-COR part #250-01885) using double-stick tape. Theother end of the tube was left open. Note the three holes inthe chamber wall the machined flat surface for matchingup against the three holes of the mounting plate. Alsopresent was another small hole (not visible) to which achamber exit tube leading to the Match Valve could beattached (see Matching and Software Considerationsbelow).

Figures 8a and 8b show the Plexiglas tube chamber at-tached to the LI-6400 sensor head using the Soil Chamberand the Conifer Chamber mounting plates.

Fig. 7

9864-157

9864-158

9864-174

9864-032Fig. 5b

4

Fig. 6a

Fig. 6b

Fig. 8a

different from each other, and from the inlet manifold usedwith the other standard chambers. The correct air inletmanifold must be used with each type of mounting plate(part # 9864-158 for the Conifer Chamber mounting plateand part #9864-032 for the Soil Chamber mounting plate).During operation, the mixing fan pushes air out of the twoouter holes of the mounting plate.

Although in this design a piece of Plexiglas tube was usedto make an open-ended cylindrical chamber, the user isfree to employ any shape that is appropriate for a particularapplication.

Chamber Material and Sorption/Desorption of Water VaporAn important consideration in chamber design is thechamber material and its water sorption/desorptionproperties. Water vapor tends to sorb and desorb slowly onmost types of surfaces, and may require long periods oftime (approximately one hour was needed for the chambershown in Figure 7) to establish equilibrium. If adequatetime is not allowed for the water vapor readings to stabilize,then transient sorption/desorption effects can cause theapparent leaf transpiration rates to appear smaller or largerthan they actually are. Therefore, stomatal conductancereadings (and values such as intercellular CO2 concentra-tion) will be in error. To minimize these effects the cham-ber walls can be coated with Teflon tape (LI-COR part#212-02314). In the above chamber example, stomatalconductance values were not of interest and the chamberwalls were not coated to minimize water sorption effects.

Matching and SoftwareConsiderationsWhen taking open, flow-through measurements, chambervolume is not used in calculations (see Equation 3), so thestandard configuration in Open is fine. The only changeneeded is the protocol used to match the analyzers. For alarge chamber such as the one described above, the cham-ber time constant even at a high flow rate of 700 μmol/s isseveral minutes (Fig. 1). This means the sample chamberconcentration may take a long time to stabilize, whichcould cause problems during matching. The simplest way

to deal with this is to turn off the mixing fan (Menu level 3,f3) so that the sample chamber air does not come back intothe sample IRGA cell. In this mode, the air flowing throughthe reference IRGA will also flush out the sample IRGA,allowing a matching of the IRGAs (under menu level 1, f5)without having to wait for the sample chamber to reachequilibrium concentration. However, it is important to turnthe mixing fan on again after matching is complete. An-other way to do this is to setup an Autoprogram to turn offthe mixing fan and enter the Match Mode. This can bedone by either building a new Autoprogram (under UtilityMenu) or editing an existing Autoprogram. When editingan existing Autoprogram, insert LPL Set Fan Speed state-ments before and after an LPL Match statement as shownbelow:

0 LPSetFanOSF

LPMatch

2 LPSetFanOSF

When building a new Autoprogram, the steps shouldinclude:

Set Control (choose fan speed, and turn fan offwithout prompting)MatchSet Control (choose fan speed, and turn fan onwithout prompting)

Autoprograms are described in Chapter 9 of the LI-6400Manual.

With respect to matching, the Plexiglas chamber describedabove had a chamber exit tube leading to the Match Valve.The main purpose of this exit tube was to provide a routefor the sample air stream to escape when the chamber wassealed. This occurred during fruit respiration measure-ments when the chamber was placed on a flat surface (seebelow). When using the chamber over soil, the exit tubewas not necessary, because soil is porous and the chamberdid not seal well against the ground. With a completelysealed chamber design, a chamber exit tube should alwaysbe installed, even though it may not be used for matchingIRGAs.

Measurement Examples

Effect of Temperature on Fruit RespirationFigure 9 Shows the Plexiglas custom chamber being usedfor measuring the respiration rate of a banana. Although itis not immediately apparent from Figure 9, the top end ofthe Plexiglas tube is covered with Propafilm. The lower

Fig. 8b

5

end of the Plexiglas tube remained open-ended. Note thatthe standard leaf thermocouple has been replaced with alonger “E-type” thermocouple wire which has been tapedon the fruit for measuring its skin temperature. Thebanana had a surface area of about 220 cm2. The CO2mixer was used to set the reference CO2 concentration to400 μmol/mol. Temperature was raised by activating thethermoelectric coolers in New Measurements (menu level2, f4).

With a flow rate of around 700 μmol/s, the respiringbanana generated a delta CO2 concentration of over 300μmol/mol. The measurement was also repeated on a pearwith a surface area of about 175 cm2. Figure 10 shows therespiration rate of a banana and pear as affected by the fruitskin temperature.

The advantage of using a large chamber to measure lowrates of exchange was evident in that even with respirationrates of less than 2 μmol/m2/s and flow rates of 700 μmol/s,the large area of the pear generated CO2 deltas of over 50μmol/mol. This provided stable, high-precision measure-ments of the respiration rate.

Fruit Respiration vs. Temperature

0

2

4

6

8

10

12

14

16 18 20 22 24 26 28 30

Temperature (oC)R

esp

irat

ion

(u

mo

l CO

2/m

2 /s)

Banana

Pear

Figure 10. Respiration rate of a banana and pear as affected bythe fruit skin temperature.

Measurement of Photosynthetic Rate of ProstrateSpurge (Euphorbia prostrata)Figure 11 shows the Plexiglas chamber being used tomeasure the photosynthetic rate of Prostrate Spurge, acreeping weed. An airflow of 700 μmol/s and an incomingCO2 concentration of around 400 μmol/mol were em-ployed. The chamber area was 154 cm2 and photosynthesiswas calculated on a unit ground area basis for this exercise.The measurements were taken on a cloudy day, so that lightlevels varied as clouds moved across the path of the sun-light.

Figure 11. Plexiglass chamber measuring Prostrate Spurge.

Fig. 9

6

Figure 12 shows the photosynthetic rate of the ProstrateSpurge as affected by sunlight. Placing a cardboard boxover the chamber resulted in zero light level, and providedan estimate of the soil and plant respiration components(about 2 μmol/m2/s), which were subtracted prior toplotting Figure 12.

Photosynthesis of Prostrate Spurge Weed

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0 500 1000 1500 2000 2500

PAR (umol/m2/s)

Ph

oto

syn

thes

is (

um

ol C

O2/

m2 /s

)

Figure 12. Photosynthetic rate of Prostrate Spurge as affectedby sunlight.

This chamber was also used to measure the photosytheticrate of an actively growing grass sward (see Figure 13). Thegrass had photosynthetic rates of around 15 μmol/m2/s (ona unit ground area basis). However, even with an airflowrate of 700 μmol/s fully diverted through the desiccant, thesystem was unable to control chamber humidity. Figure 13shows the condensation on the chamber walls during thegrass sward measurement. A smaller volume chamberwould have been more appropriate for measuring photo-synthesis of actively growing grass.

Figure 13. Chamber measuring photosynthetic rate of grasssward.

ConclusionsThe LI-6400 can be easily adapted for a variety of measure-ments. Chambers ranging in size from the 1 cm diameterArabidopsis leaf chamber to the one-liter Soil Chamber areavailable from LI-COR. Also of interest may be LI-6400Application Note 2 which describes how to set up a canopychamber. For chambers in between these sizes, it is rela-tively simple to use the mounting plates for the 6400-09Soil Chamber or the 6400-05 Conifer Chamber for attach-ing almost any unique chamber design.

References1. Caemmerer, S Von, and Farquhar, G.D. 1981. Some

relationships between the biochemistry of photosynthe-sis and gas exchange of leaves. Planta, 153:376-387.

2. LI-6400 Manual, 1999. LI-COR, Inc.

7

979-06952Rev. 1, 06/16

© 2016, LI-COR, Inc. LI-COR and SoilFluxPro are trademarks or registered trademarks of LI-COR, Inc.All other trademarks belong to their respective owners.

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