# Hands on an infrared gas analyzer (IRGA) to measure photosynthesis

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1 University of Toledo - Dept of Envir. Sci., EEES 6660/8960: Biophysical Processes of Ecosystems, Spring 2010 Hands on an infrared gas analyzer (IRGA) to measure photosynthesis Burkhard Wilske for Jiquan Chen, BO 3008, February 8, 2010 Basics of measurements using light (electromagnetic radiation) Basics of measuring gas (e.g., CO 2 ) Reminder: photosynthesis Keep in mind what we considered so far while getting your hands on the measurement system

2 Infrared gas analyzer Basics: Absorption bands of CO 2 and H 2 O See e.g.: Carbon Dioxide Infra-Red Gas Analysis: EFFECTS OF WATER VAPOUR PARKINSON J. Exp. Bot..1971; 22:

3 Diagram of Beer Lambert absorption of a beam of light as it travels through a cuvette of width l. I 0 and I are the intensity (or power) of the incident light and the transmitted light

4 absorption (A) is proportional to the light path length A ~ c (concentration of absorbing species in the material) for gases absorption A is A = -ln (I / I 0 ) and A = ε l c ε absorption coefficient of substance l path length c concentration of substance

5 What you know: ε, l, A IR source IR detector (A) gas in gas out

6 IR source IR detector (A) gas in gas has passed an empty cuvette temporally or spatially separated (differential) measurement of gas has passed a cuvette with leaf IR source IR detector (A) gas in

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9 5 5 / volume 5 / volume / time concentration c (note: you start calculating from a mixing ratio) rate flux

10 What confines gas? Gay-Lussac p1/t1 = p2/t2 p/t = const Boyle (Charles) p1*v1 = p2*v2 Combined gas law (p1*v1)/t1 = (p2*v2)/t2 Avogadro (p1*v1)/(t1*n1) = (p2*v2)/(t2*n2) p*v = const (p*v) / T = const V / n = const Avogadro 22.4 L is the V that 1 mole of a gas takes at standard conditions Boltzmann R = J / K mol (universal gas constant) Ideal gas law p*v = n*r*t

11 Look around and play the gas laws Hot air balloon: Cooking: Car tire: more gas (n) at the same V increases pressure and keeps your tire in shape with the same air (n) you tires may be flat in winter if tires at maximum pressure run hot (T) in the summer they burst

12 Which standard? Mol volume of an ideal gas L 24.45L at standard ambient temperature and pressure (298 K, 1 atm) IUPAC standard: a temperature of 0 C ( K, 32 F) and an absolute pressure of 100 kpa ( psi, atm) NIST standard: a temperature of 20 C ( K, 68 F) and an absolute pressure of kpa ( psi, 1 atm) Mol volume of real gases see tables

13 Calculate photosynthesis from raw data? Ref cell(400) Sam cell(390) = 10 µmol / mol assume standard conditions (1 mol takes L) (10 µmol / mol) * (1 L / min air flow) / ( L / mol) = x µmol CO 2 /min link to leaf (((x µmol CO 2 / min) / 60 s) / leaf area) * multiplier square meter net photosynthesis = x µmol CO 2 / m 2 s

14 Photosynthesis: removes CO 2 from the atmosphere and first of all decreases CO 2 conc. inside the leaf Ci CO 2 conc. inside leaf

15 CO 2 supply to photosynthesis compartments gradients bottlenecks atmosphere substomatal cavity cell chloroplast (stroma) CO 2 mixing ratio CO 2 mixing ratio Calvin cycle diffusion along gradient

16 Photosynthesis (or the rate of) is limited by the two factors light and the carboxylation at Rubisco light provides the energy works like an accelerator carboxylation can process more substrate than supplied based on the current CO 2 mixing ratio in the atmosphere

17 Photosynthesis (or the rate of) is further influenced by Temperature (T), air humidity (RH), water availability at the root T affects enzymatic processes, (transpiration), gas diffusion plants photosynthesis measurements show optimum curves relative to temperature RH and water availability at the root affect transpiration, stomata regulation

18 What do you want to know from plant photosynthesis? How much? Net Photosynthesis (assimilation A) In the night? Dark respiration (or Respiration R) Light dependence? Light compensation / saturation Light curve measure at different light intensity Temperature dependence? Temperature curve measure at different... Carbon dioxide dependence? CO 2 curve measure at different... (A-Ci curve) Efficiency? Light use efficiency (LUE) µmol m -2 s -1 CO 2 / µmol photons m -2 s -1 (PAR) Water use efficiency (WUE) µmol m -2 s -1 CO 2 / mmol m -2 s -1 H 2 O Stress? Depression in photosynthesis due to too much heat, light, too less heat, water

19 Conditioned air is pumped into cuvette flow is controlled flow restrictors partition the air stream: 75% into the sample and 25% to the reference cell dual IR analyzers monitor CO2 and H2O of the two air streams calculate CO2 and H2O as the difference between cells

20 LI-6400 has the gas analyzers in the sensor head sensor head has 2 complete, dual path, non-dispersive infrared analyzers, both measuring absolute concentrations of CO2 and H2O open path design: optical bench of sample analyzer open to the leaf chamber mixing volume leaf dynamics are measured in real time: no return tubing between the leaf chamber and console no time delays confounding correlations between gas exchange and changes in drivers absence of time delays fast, automatic control of humidity at user-defined set points even when the transpiration rate is changing absence of return tubing to analyzers eliminates equilibration times due to water vapor sorption on the tubing walls Fan in the mixing volume of the optical bench pushes air through inlet ports into the upper and lower sections of the leaf chamber. Fan draws air from the leaf chamber through a central flow path. The system console combines a data acquisition system with a high speed computer. All computed variables are calculated and displayed in real time.

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22 Automated CO 2 Control 12g CO 2 cartridges provide up to 8 hours of operation Easy to generate A-Ci curves CO 2 can be adjusted at any level from µmol m -2 s -1 Automated Light Control easy to generate light response curves light can be adjusted at any level 0 >2000 µmol m -2 s -1 Automated Temperature Control the temperature of the chamber block is controlled by Peltier coolers to any level within +- 6 C of the air temperature Automated Humidity Control humidity controlled by automated delivery of conditioned air to leaf chamber accurate measurement without time delays

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24 Basic steps of operation Refresh chemicals (Drierite, Soda lime) (system was designed to use fingers, no tools (pliers! etc.) should be required) Select chamber type (Standard (default), LED, Soil, Fluorescence ) (get out from every menu w/o change: press escape ) Calibrate 1. flowmeter, 2. CO2 zero, 3. H2O zero (see mixing ratios CO 2, H 2 O; wait for stable readings; H 2 O takes more time than CO 2 ) New Measurements Open log file, assign name, start measurements maneuver through the screen using the red arrows (up down left right) with label change the menu options at the bottom line of screen (see current ambient conditions w/o leaf: PAR, T, RH ) (right of escape, press 2:) control conditions PAR, T, RH, CO 2 (set not to far from ambient, keep in mind what the system can and cannot do ) (right of escape, press 5:) automatic measurements Autolog (w/o control ( for incident photosynthesis) or one-value control for PAR, T, RH, CO 2 ; for leaf enclosure times > 3 min control at least T Light curve (control constant T, RH, CO 2 and choose a range of light levels) A-Ci curve (set control to (optimum) PAR, T, RH and choose range of CO 2 mix ratios) repeatedly check battery voltage and see that you have enough power especially for using the controls

25 apart from the library and original papers you can find, for example to download LI-6400 Instruction Manual (read e.g., version 5 pages 4-1 to 4- Making Measurements) units questions?

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