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Measurement of photosynthesis using infrared

gas analyzer

Method · January 2017

DOI: 10.13140/RG.2.2.29626.90561

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Rakesh Pandey Vijay Paul

Indian Council of Agricultural Research ICAR-Indian Agricultural Research Institute (IARI)
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Madan Pal Singh

Indian Agricultural Research Institute
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 Manual of ICAR Sponsored Training Programme on “Physiological Techniques to Analyze the Impact of
Climate Change on Crop Plants” 16-25 January, 2017, Division of Plant Physiology, IARI, New Delhi
Measurement of Photosynthesis using Infrared Gas Analyzer
Rakesh Pandey, Vijay Paul and Madan Pal Singh
Division of Plant Physiology, ICAR- Indian Agricultural Research Institute (IARI), New Delhi-110 012
*E-mail: r_pan_pdcsr@yahoo.co.in
Introduction
Study of changes in the carbon assimilation and
gaseous exchange of plants is important for
understanding the effect of climate change on the
plants. The main parameters related to changing
climate are CO2 and temperature but other important
variables may be light, humidity, pollutants, ozone,
aerosols etc. The measurement of CO 2 in the
different commercially available models of
photosynthesis systems is done mainly with the
enclosure methods. The leaf is enclosed in a chamber
or cuvette and the gaseous exchange (CO2 and H2O)
of the leaf is monitored. Basically, there will be
decrease in CO2 in the cuvette and increase in H2O
due to photosynthesis and transpiration respectively.
What is photosynthesis ‘system’?
The instrument used for measurement of
photosynthesis is not a single instrument in itself. It
is an assembly of instruments for measurement of
CO2, H2O, temperature of air and leaf, flow rate,
PAR, pressure, computer etc. These parameters are
used for calculation of photosynthesis rate,
transpiration rate, stomatal conductance etc. and can
be stored in the computerized data logger. That’s
why it is called Photosynthesis ‘System’ but not
Photosynthesis ‘Meter’. Out of these instruments,
the heart of Photosynthesis system is the CO2, H2O
measuring instrument called Infrared gas analyzer
or IRGA. Therefore Photosynthesis system is many
times simply called IRGA. The recently available
photosynthesis systems have more capabilities and
precision in terms of portability, environmental control
of light, temperature, CO2, RH etc. Fluorescence
attachment can also be integrated for measurement
of photosynthesis and fluorescence of the same leaf.
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Modes of CO2 measurement and types of IRGA
CO2 can be measured in two modes i.e. absolute
and relative or differential mode. In the absolute mode
the reference CO2 is set at zero concentration and
based on it the sample concentration is measured.
The reference cell CO2 is evacuated and is sealed to
maintain zero CO2 concentration. In the differential
mode there is flow of same CO2 concentration in
both the sample and reference cells. Here, the
reference cell is not sealed or not kept at zero CO2
concentration. The decrease in the CO 2
concentration in the sample cell is monitored relative
to the reference cell. Depending upon the air
circulation through the cuvette or the leaf camber,
the CO 2 analyzers are classified into closed,
semiclosed and open systems.
Closed systems
In the closed systems, the air stream is maintained
in a loop i.e. the air is recirculated through the leaf
cuvette. Thus, the chamber conditions change
continuously and the measurement never represent
a truly steady state (Figure 1). Therefore, closed
system is also called transient systems. With time,
the humidity increases in the cuvette due to
transpiration and results in dilution of the mole fraction
of other gases including CO2. This can be controlled
by passing the recirculated air through a portion of
drier. Therefore, it necessitates measurement of flow
rate.
In semi closed system, the CO2 concentration in the
chamber is maintained constant by an input of
external CO2. The inflow of CO 2 from external
source is maintained at a rate equivalent to the rate
of uptake by the leaf so that steady state
photosynthesis is attained. Thus, one important
 Manual of ICAR Sponsored Training Programme on “Physiological Techniques to Analyze the Impact of
Climate Change on Crop Plants” 16-25 January, 2017, Division of Plant Physiology, IARI, New Delhi

Drier systems has increased considerably due to several

Chamber
Pump
IRGA
Figure 1: Diagram of a closed system for photosynthesis
measurement
advantage with semi closed type of system is that
the photosynthesis and transpiration may be studied
at a range of values of CO2 and H2O concentration
by changing the set point values in the system.
For closed systems, the net photosynthesis can be
expressed as
(CO2 initial – CO2 final)
Net photosynthesis =
(Leaf area) x Time
Open system
In open system there is no recirculation of air flow
through the leaf cuvette or chamber the air passes
through the chamber only once. Depending upon the
flow rate – there will be a moderate CO2 depletion
in case of high flow rate and more CO2 depletion
due to low flow rate. Presently the use of open
advantages. Open system operates in the differential
mode (Figure 2). CO2 injection from an external
source can be provided and so that it compensates
Leaf
the depleted CO 2 . Such system is called
compensating differential systems.
For the open system, the net photosynthesis will be
(CO2 reference – CO2 sample)
Net photosynthesis = x (Flow)
(Leaf area)
(Vapour density sample – Vapour density reference)
Transpiration = x (Flow)
(Leaf area)
Principle of working of IRGA
The IRGA works on the principle that
heteroatomic gas molecules (such as CO 2, H2O,
NH3, N2O etc.) absorb infra red radiation in specific
infra-red wavebands and this absorption follows the
Beer-Lambert’s law.
x (Volume)
(-klM)
Therefore,  = 1-e
Where,
k = extinction coefficient at wavelength 
l = radiation path length
M = Molar concentration of CO2 in air
The major absorption band of CO2 is at 4.25 µm
with secondary peaks at 2.66, 2.77 and 14.99 µm.

Chamber
IRGA
Flow
Sample
meter Cell
Air
Leaf

IRGA
Reference
Cell

Figure 2: Diagram of an open system for photosynthesis measurement

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 Manual of ICAR Sponsored Training Programme on “Physiological Techniques to Analyze the Impact of
Climate Change on Crop Plants” 16-25 January, 2017, Division of Plant Physiology, IARI, New Delhi
From the above equation, it can be deduced that
sensitivity of IGRA increases with path length of cell.
Parts of IRGA
IRGA consists of following parts - infra red source,
gas cell, detector and filter (Figure 3). The detectors
are designed to produce periodic vibration and its
amplitude depends on CO2 concentration. They are
of two types i.e. series type and parallel or Luft type.
Presently, these have been replaced by solid state
detectors. The available IRGA are factory calibrated
for output. The users can also synchronize the IRGA
by calibrating the IRGA output by absolute or
differential methods. Calibration for measurement
and control of gas flow can also be made with flow
meters. Detailed setup and working can be seen from
the references cited in the end. Control of CO2 and
H2O in leaf chamber can be made by diversion of
flow through the absorbent chemicals. For CO 2
scrubbing soda line is used. It is a mixture of calcium
oxide and sodium hydroxide and converts absorbed
CO 2 to carbonates. Drierite is a hygroscopic
chemical and consists of calcium sulphate. Similarly,
magnesium perchlorate is a hygroscopic chemical
CO2 / H2O in
IR
Source
Gas Cell
Signal
Display
Processing
Figure 3: Parts of IRGA and working setup
21
and can absorb water to about 25% of its weight.
Examples of some photosynthesis systems:
Presently used photosynthesis systems are mostly
of open type as compared to LI-6200 which was a
closed system of LI Cor, USA. The open system
eg. LI-6400 and LI 6800 of LI Cor, CIRAS-2 and
CIRAS-3 of PP-Systems, HCM-1000 and GFS3000
of Walz, LCPro+ and iFL integrated Flurometer and
gas exchange system of ADC and CI340 of CID
Bioscience are some important commercially
available portable photosynthesis systems. These
have following features-
 Non-dispersive infrared gas analyzers for CO2
measurements from 0-3000 μmol mol-1 and H2O
from 0-75 mmol mol-1. In the LI-6400 model,
CO2 and H2O analyzers in are present in the
sensor head for rapid response.
 CO2, light, temperature and humidity can be
controlled manually or automatically. The
temperature control range is 0°C to 50°C. Flow
rates upto 1000 μmol s-1. PAR measurement is
in range 0 to 3000 μmol mol-1.
CO2 / H2O out
Detector
Band pass
filter
 Manual of ICAR Sponsored Training Programme on “Physiological Techniques to Analyze the Impact of
Climate Change on Crop Plants” 16-25 January, 2017, Division of Plant Physiology, IARI, New Delhi

 Leaf Chamber Fluorometer attachment helps in Change in CO2 conc. ΔCO2 μmol CO2 mol-1
measurement of fluorescence and gas exchange (sample - reference)
of the same leaf. Change in H2O conc. ΔH2O mmol H2O mol-1
 The system have good console memory for data (sample - reference)
storage and LCD graphic display with full ASCII Photosynthetic rate Photo μmol CO2 m-2 s-1
keypad is also provided. Conductance to H2O Cond mol H2O m-2 s-1
Measurements and units Intercellular CO2 Ci μmol CO2 mol-1
concentration
A checklist of things that should be done prior to
Transpiration rate Tr mmol H2O m-2 s-1
making measurements is as follows:
Flow rate to the Flow μmol s-1
 During warm up check the air supply, sample cell
temperature values, light and pressure sensor Intercellular CO2 / Ci/Ca -
responses, leaf fan and max flow control. Ambient CO2
 After warm-up check the flow zero, set reference Vapor pressure deficit VpdL kPa
CO2 and H2O, test for leaks and match the based on Leaf temp
IRGAS. Vapor pressure deficit VpdA kPa
based on Air temp
 For taking measurements on the leaf set light
and set flow to desired level. Output of quantum sensor Par μmol m-2 s-1
Temperature in sample cell Tair °C
 The photosynthesis parameters are displayed in
Temperature of leaf Tleaf °C
following units
thermocouple

References
Hall DO, Scurlock HR, Bolhar-Nordenkampf HR, Leegood RC, Long SP (1993). Photosynthesis and production in a
changing environment. pp 464, Chapman and Hall, UK.
Sestak Z, Katsky J, Jarvis PG (1971). Plant Photosynthetic production, manual of methods. pp818, Dr. W Junk NV
Publishers, The Hague.

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