infra red gas analyzer photosynthetic apparatus

1. welcome

2. PRINCIPLE, PROCEDURE,
OPERATION, ADVANTAGES
AND DISADVANTAGES OF
IRGA ( PORTABLE
PHOTOSYNTHETIC SYSTEM)
PRESENTED BY
ZUBY GOHAR ANSARI
TAM/ 14- 26

3. Introduction
• In 1971 there were no commercial portable systems;
measurement of photosynthesis in the field was only
possible via mobile or field laboratories.
• Measurement of photosynthesis then required an intricate
knowledge of the infrared gas analyzer, its daily or hourly
calibration, flow meters, properties of the materials used,
and vigilant leak detection.
• Similarly, calculation of CO₂ uptake from measured CO₂
mole fractions, flow rate, pressure, temperature, humidity,
leaf area, etc. would require an intricate knowledge of the
equations and corrections, and probably access to a
mainframe computer.

5. PRINCIPLE
• Infra red gas analyzers (IRGA) are used for the
measurement of a wide range of hetero atomic gas
molecules including CO₂, H₂O, NH₃, CO, SO₂, N₂O, NO
and gaseous hydrocarbons like CH₃.
• Hetero atomic molecules have characteristic absorption
spectrum in the infra red region.
• Therefore absorption of radiation by a specific hetero
atomic molecule is directly proportional to its
concentration in an air sample.

6. • Infra red gas analyzers measure the reduction in
transmission of infra red wavebands caused by the
presence of gas between the radiation source and a
detector.
• The reduction in transmission is a function of the
concentration of the gas.
• The primary role of IRGA is to measure the CO₂
concentration.
• The IRGA is sensitive to detect even a change of one
ppm of CO₂.

7. • The leaf or a plant is enclosed in air tight chamber
and the CO₂ fluxes are determined by measuring the
CO₂ concentration changes in the chamber
atmosphere.
• The major absorption peak of CO₂ is at 4.25µm with
secondary peak at 2.66, 2.77 and 14.99 µm.
• Both water vapour and CO₂ molecules absorb IR
radiation in the 2.7µm range.

8. PROCEDURE
• The portable photosynthesis system is a portable IRGA
and is to operate as an open system to measure the gas
exchange parameters.
• It consists of separate IRGAs to measure CO₂ and H₂O
vapour concentrations, an internal air supply unit and
the necessary software for the computation of gas
exchange parameters .
• Li 6400 uses for independent infrared gas analyzers, 2
each for CO₂ and H₂O.

9. • One pair of C₂O and H₂O analyzers defined as
reference measures the CO₂ and water vapour
concentration in the ambient air that is sent into leaf
chamber.
• Similarly second pair, the analysis chambers
measures the CO₂ and water vapour concentrations
in the air that is coming from the leaf chamber.
• The difference between the reference and the
analysis IRGAs are computed.

10. • Physiological efficiency of green gram genotypes
under moisture stress conditions was measured in
laboratory.
• A leaf is clamped to the leaf chamber.
• The leaf chamber is provided with suitable pads to
clamp an area of 2.5 cm² under airtight conditions.
• Separate tubing is provided to send and withdraw air
from the leaf chamber .
• These tubes are connected to either of the reference or
analysis IRGA for the determination of gas
concentrations.

11. • A quantum sensor is placed inside the leaf chambers
transparent cover to measure the actual light intensity in
PAR range at the leaf surface.
• Blue and red LED (light emitting diode) is fixed on the
top of the leaf chamber.
• The LEDs emit light in the PAR region and the intensity
of which can be fixed and controlled at a required level.
• The light source is capable of providing the
photosynthetically active radiation in the energy range
of 0 to 2000 µ mole m¯² s¯¹.

12. • A CO₂ cartridge normally carrying 8g of pure CO₂
in liquid form is used to get the requisite CO₂
concentration in the leaf chamber.
• The system mixes the ambient air with the CO₂ to
obtain the requisite concentration in the leaf
chamber.
• The path of the ambient air is provided with 2
scrubbers to remove moisture (drierite used as a
desiccant) and CO₂ (soda lime to remove CO₂)

14. IRGA Working Procedure (LI6400)
• Usage of IRGA equipments by students and scientists
often found complicated.
• Here is the operation protocol for easy handling of the
equipments both in green house and field experiments.
STARTING:
1. First charge the batteries one day prior to record data
using IRGA.
2. Load the charged batteries first.
3. Connect the CO₂ tube to the inlet of the instrument.
4. All screws of this instruments must be in tight fitting.

15. 5. Connect the CO₂ tube in a proper way. Connect this
tube very tightly otherwise it shows leak (- ppm) in
display.
6. the 2nd edge of this tube was kept in empty thermocol
box and enclosed for uniform entry of air into the tube.
7. Switch “ON” the instrument.
8. Displays shows –
A. Welcome to loading open system.
B. Starting net working.
C. It shows the fluorescence + WUE X m1 –
press “enter”.
D. Is the chamber IRGA connected Y/S – Yes
– press “Y”.
9. Open the IRGA leaf chamber one time and close it.

16. 10. Select ‘New measurements’ press (F4).
11. In display select ‘open log file’ press (F1).
A. Give file name and press “enter”.
B. Next – give sub file name and press
“enter”.
C. Give date and press “enter”.
12. Next – CO₂ matching.
A. Select Match (F5)
B. Wait up to we get equal values of
reference CO₂ and sample CO₂.
C. If we need close matching press ‘Match
IRGA’ (F5) after that press “exit” (F1).

17. 13. In display set the rows - m, n, c and 9.
A. If we want ‘m row’ – press ‘ m alphabet’.
B. If we want ‘ n row’ – press ‘ n alphabet’.
C. If we want ‘ c row’ – press ‘ c alphabet’ it is
already exist.
D. If we want ‘ 9 row’ – press ‘ 9 number’.
14. In this condition wait for 15-20 min. for warming of
instrument (before inserting the leaf in IRGA chamber).
15. Leaf should not fold in IRGA chamber. If leaf get
folds it shows negative readings. Leaf should not have
any moisture and dust before inserting leaf.

18. 16. Insert the leaf in IRGA chamber.
A. Give the ‘Dark pulse’ (F3).
B. Press ‘zero’ getting ‘zero’ row.
C. Before going to next step, see the ‘F’ value
must be stable and df/dt value is ˂5.
D. Select DOFoFm – (F3).
17. Select row no : 9 : press ‘Actinic On’ (F4).
18. Select row no : 8: press ‘Define Actinic’ (F3).
A. It shows ‘Actinic Definition – press “enter”.
B. Type 1000 ( PAR value 1000) press “enter”.

19. 19. Select ‘zero’ row.
A. Before going to next step, see them, ‘F’ value must be
stable and df/dt value is <5
B. Select DOFsFoFm – (F4).
20. If we want fluorescence value select ‘O’ alphabet and note down the
Fv’/Fm’ value.
21. Now note down the IRGA readings (photosynthetic rate,
transpiration rate, stomatal conductance).
22. Before taking next reading ‘Actinic is in OFF’(F4). Do as above for
taking every next reading.
23.time taken for each reading is 10-20 min.

20. 24. After taking of readings IRGA chamber must be in
open conditions (loose the screw).
25. Replace the fluorescence chamber foam (white foam)
at the time of entire damage.
SHUTDOWN:
1. In every shutdown process ‘Actinic’ must be in “Off”
condition.
2. Press ‘Escape button’.
3. Select ‘Utility menu – F5’.

21. 4. Coming down using down arrow.
5. Select ‘Sleep’.
6. Give ‘Enter’.
7. It shows – Ok to sleep Y/N.
A. Press Yes – ‘Y’ alphabet.
8. Switch off the system.
9. Disconnect the CO₂ tube.
10. Keep batteries for charging.

22. PARAMETERS RECORDED FROM IRGA:
1. Photosynthetic rate (photo): µmole CO₂ m²/sec.
2. Stomatal conductance (cond): mole H₂O m²/sec.
3. Transpiration rate (Trmmol): m. mole H₂O m²/sec.
4. Intercellular CO₂ concentration (Ci): µmole CO₂
mole¯¹.
5. Chlorophyll fluorescence (Fv’/ Fm’ values)
Where, Fv’= Variable fluorescence; Fm’= Maximum
fluorescence.

23. ADVANTAGES
Three advantages to the closed IRGA system:
1. It is compact and light-weight.
2. Comparatively low-priced, and relatively simple to
calibrate and operate.
3. This makes it an appropriate instrument to use in
secondary and undergraduate field courses.

24. DISADVANTAGES
• There are two major disadvantages to using a closed IRGA system:
• Photosynthesis measurements must be made within a few seconds
after closing the leaf chamber: and the operator has limited control
over environmental conditions within the chamber.
• Once the leaf is sealed in the chamber, CO₂ concentration in the
leaf chamber- is continually decreasing.
• Consequently, if the leaf has a high photosynthetic rate, resulting
in a rapid reduction of the chamber CO₂ concentration,
measurements must be made quickly to avoid the possibility of a
direct effect of low CO₂ concentration on photosynthesis.

25. • This limits the amount of time one may allow for a
leaf to adjust to a particular experimental condition
(light level, temperature, etc.).
• This problem may be partially overcome in some
closed systems by using an external flow switch
which allows the operator to open the system and
draw outside air into the chamber while the leaf
acclimates prior to beginning measurements.
• The second limitation concerns the control of
temperature and relative humidity within the
chamber during measurement.

26. • Because the closed system was designed to be
portable.
• It typically does not include heat-exchange devices
for maintenance of constant air temperatures within
the chamber.
• In addition, the air stream cannot be consistently
humidified to a desired level.
• whereas steady state humidity control is commonly
a part of open systems.

27. • ARTICLE
• 1.Analysis of leakage in IRGA’s leaf chambers of open gas
exchange systems: quantification and its effects in
photosynthesis parameterization (J. Flexas et al.)
• The measurement of the response of net photosynthesis to leaf
internal CO2 (i.e. A–Ci curves) is widely used for ecophysiological
studies. Most studies did not consider CO2 exchange between the
chamber and the surrounding air, especially at the two extremes
of A–Ci curves, where large CO2 gradients are created, leading to
erroneous estimations of A and Ci.
• A quantitative analysis of CO2 leakage in the chamber of a
portable open gas exchange system (Li-6400, LI-COR Inc., NE,
USA) was performed.
• In an empty chamber, the measured CO2 leakage was similar to
that calculated using the manufacturer’s equations.

28. • However, in the presence of a photosynthetically inactive leaf,
the magnitude of leakage was substantially decreased, although
still significant.
• These results, together with the analysis of the effects of
chamber size, tightness, flow rate, and gasket material, suggest
that the leakage is larger at the interface between the gaskets
than through the gaskets.
• This differential leakage rate affects the parameterization by
photosynthesis models.
• The magnitude of these errors was assessed in tobacco plants.
• The results showed that leakage results in a 10% overestimation
of the leaf maximum capacity for carboxylation (Vc,max) and a
40% overestimation of day respiration (Rl).

29. • Using the manufacturer’s equations resulted in
larger, non-realistic corrections of the true values.
• The photosynthetic response to CO2 concentrations
at the chloroplast (i.e. A–Cc curves) was
significantly less affected by leakage than A–Ci
curves.
• Therefore, photosynthetic parameterization can be
improved by: (i) correcting A and Ci values for
chamber leakage estimated using a
photosynthetically inactive leaf; and
(ii) using A–Cc instead of A–Ci curves.

30. • 2. Measurement of leaf and canopy photosynthetic
C02 exchange in the field (S.P. Long et al.)
• The principles and limitations of leaf gas exchange
measurements in portable gas exchange systems .
• Attention is given to the design and developments in infrared gas
analyzers used in portable systems, and the basic structure of
single and dual beam instruments is presented.
• The significance of flow measurement in these systems and the
principles of thermal mass flow measurement are illustrated.
• Considerations of leaf area measurement, chamber design and
choice of materials are outlined.

31. • Two specific developments in field gas exchange systems are described and
their significance in field measurements is illustrated with examples.
• (1) An integrating sphere leaf chamber for the determination of the quantum
yield of photosynthesis, on the basis of absorbed light, is explained.
• The significance of this approach is illustrated by a comparison of data for
contrasting leaves plotted on an absorbed and incident light basis.
• This measurement of light-limited photosynthesis is also critical in
understanding the contribution of shaded leaves to canopy photosynthesis.
• (2) A system for the measurement of canopy photosynthesis from arable
crops and low stature natural vegetation is described.