LI-COR LI-6400 User manual

Using the LI-6400
Portable
Photosynthesis
System
¨-

NOTICE
The information contained in this document is subject to change without notice.
LI-COR MAKES NO WARRANTY OF ANY KIND WITH REGARD TO THIS MATERIAL, INCLUDING,
BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A
PARTICULAR PURPOSE. LI-COR shall not be liable for errors contained herein or for incidental or consequen-
tial damages in connection with the furnishing, performance, or use of this material.
This document contains proprietary information which is protected by copyright. All rights are reserved. No part
of this document may be photocopied, reproduced, or translated to another language without prior written consent
of LI-COR, Inc.
© Copyright 1998,1999, LI-COR, Inc.
Publication Number 9806-122
Printing History:
1st Printing July, 1998 - OPEN Software version 3.2
2nd Printing May, 1999 - OPEN Software version 3.3
Macintosh
¨
is a registered trademark of Apple Computer, Inc.
Printing History
New editions of this manual will incorporate all material since the previous editions. Update packages may be used
between editions which contain replacement and additional pages to be merged into the manual by the user.
The manual printing date indicates its current edition. The printing date changes when a new edition is printed.
(Minor corrections and updates which are incorporated at reprint do not cause the date to change).
U.S. Patent Numbers: 5,332,901 & 5,340,987. Other patents pending in the U.S. and other countries.
LI-COR, Inc. ¥ 4421 Superior Street ¥ Lincoln, Nebraska 68504
Phone: [phone] ¥ FAX: [phone]
Toll-free: 1-800-447-3576 (U.S. & Canada)

Using the LI-6400 iii
Contents
Contents
Welcome to the LI-6400
A Bit of History xi
About this Manual xiii
Part I: The Basics
1 System Description
What it is, what it does, and how it does it
An Open System 1-2
The Flow Schematic 1-4
Equation Summary 1-7
The System Components 1-12
2 Assembling the LI-6400
Putting it all together
Preparations 2-2
Using a Tripod 2-6
6400-01 CO2 Injector Installation 2-7
External Quantum Sensor Installation 2-14
6400-02B LED Light Source Installation 2-15
Powering the LI-6400 2-18
Installing System Software 2-19
3 Guided Tours
Learning how to make it work
Before You Start 3-2
Tour #1: OPEN Overview 3-4
Tour #2: New Measurements Mode Basics 3-14

Contents
iv Using the LI-6400
Tour #3: Controlling Chamber Conditions 3-20
Tour #4: Logging Data 3-41
Tour #5: Configuration Adventures 3-51
Tour #6: Boot Screen and LPL 3-59
4 Making Measurements
The fundamentals of good measurements
Preparation Check Lists 4-2
Some Simple Experiments 4-8
Making Survey Measurements 4-21
Light Response Curves 4-24
CO2 Response Curves 4-30
Matching the Analyzers 4-34
Stability Considerations 4-41
Leaks 4-42
Operational Hints 4-47
Answers to Questions 4-53
Part II: Useful Details
5 Standard Tools
Menus, Editors, and File Dialogs
Standard Menu 5-2
Standard Line Editor 5-5
Standard File Dialog 5-9
Standard Edit 5-13
Hot Keys 5-16
Low Battery Warning 5-17
The Boot Screen 5-18
The LPL Screen 5-20
Power ON Hooks 5-23
6 Real Time Data
Viewing real time data using text and graphics
Text Display 6-2
Real Time Graphics 6-8

Contents
Using the LI-6400 v
7 Environmental Control
How OPEN controls chamber conditions
OpenÕs Control Manager 7-2
Humidity Control 7-7
CO2 Control 7-13
Temperature Control 7-16
Light Control 7-18
8 Light Sensor Considerations
The most important parameter is the hardest to measure
Why Two Sensors? 8-2
Specifying the Source and Sensor 8-3
6400-02 and -02B Light Sources 8-6
Gallium Arsenide Phosphide (GaAsP) Sensor 8-7
9 Data Logging
Storing what you want, where you want, when you want
Basic Concepts 9-2
Getting Started 9-4
Determining What is Logged 9-7
Prompts and Remarks 9-12
AutoPrograms 9-18
AutoProgram Descriptions 9-21
Making Your Own AutoPrograms 9-33
Part III: Working With Files
10 The LPL File System
Managing your data storage space
Files, Directories, and Disks 10-2
Defragmentation 10-5
The Filer 10-6
FilerÕs Directory Operations 10-9
FilerÕs File Operations 10-11
FilerÕs Disk Operations 10-18
Troubleshooting 10-21

Contents
vi Using the LI-6400
11 Downloading Files
Retrieving your data
Connecting the LI-6400 to a Computer 11-3
File Exchange Mode 11-4
Using WinFX 11-4
Using FX for DOS 11-8
Using FX on Macintosh 11-10
Using EW 11-12
Odds and Ends 11-21
Using a Data Capture Program 11-23
12 GraphIt
A tool for viewing and graphing data files
Accessing GraphIt 12-2
Data File Format 12-4
Defining Plots 12-6
Selecting Observations 12-10
Curve Fitting 12-14
Viewing Data 12-17
PlotDef File Format 12-19
Storing and Retrieving Graphics Images 12-19
13 Recomputing Data Files
How to recompute data files
Reasons to Recompute 13-2
A Step By Step Example 13-2
The Details 13-7
Hints 13-14
Part IV: Configuration Issues
14 OPENÕs System Variables
Quantities provided by OPEN
Background Information 14-2
Measured Variables 14-4
Computed Variables 14-9
Time and Logging Variables 14-11
Status Variables 14-12

Contents
Using the LI-6400 vii
Boundary Layer Variables 14-17
List of System Variables 14-19
Band Broadening Correction for Water Vapor 14-23
15 Defining User Variables
Equations, Constants, and Remarks
The ComputeList File 15-2
ComputeList File Format 15-3
Writing and Modifying ComputeList Files 15-7
User Defined Constants and Remarks 15-12
Examples 15-13
Useful Variables and Functions 15-15
The Default ComputeList 15-16
Old Style vs. New Style 15-17
16 Configuration Basics
What you need to know to get by
A Definition of Terms 16-2
Making Configuration Files 16-4
Installation Menu: Behind the Scenes 16-10
Modifying Config Files 16-12
The Reset Menu 16-16
Configuration Command Summary 16-18
17 Using an Energy Balance
Computing what you canÕt measure
The Theory 17-2
Using Energy Balance in OPEN 17-6
Energy Balance And Boundary Layer 17-9
Further Reading 17-11
Part V: Maintenance & Troubleshooting
18 Calibration Issues
How much is good data worth?
CO2 and H2O Analyzers 18-3
Flow meter 18-17

Contents
viii Using the LI-6400
View, Store Zeros & Spans 18-18
Zeroing the Leaf Temperature Thermocouple 18-19
6400-01 CO2 Mixer 18-20
6400-02(B) LED Source 18-24
GaAsP Light Sensors 18-28
Calibration and Configuration File Summary 18-30
19 Maintenance & Service
The care and feeding of your new pet
Chemical Tubes 19-2
6400-03 Batteries 19-8
System Console 19-9
Real Time Clock 19-12
Cables 19-12
The Chamber Handle 19-14
Leaf Temperature Thermocouple 19-18
Leaf Chambers 19-20
LED Source Maintenance 19-22
Match Valve Maintenance 19-23
IRGA Maintenance 19-26
Servicing the External CO2 Source Assembly 19-32
Shipping The LI-6400 19-35
Useful Part Numbers 19-36
20 Troubleshooting
When things go wrong
Power On / Start-up Problems 20-2
Real Time Clock Problems 20-6
New Measurements Mode Warning Messages 20-7
Unreasonable Results 20-9
Pump/Flow Problems 20-13
IRGA Problems 20-14
Match Valve Problems 20-21
6400-01 CO2 Mixer Problems 20-23
Light Source / Sensor Problems 20-30
Chamber Problems 20-33
Finding Leaks 20-35
Soil Chamber Problems 20-39
Useful Information 20-42

Contents
Using the LI-6400 ix
21 Diagnostics and Utilities
Useful programs
Diagnostics & Tests Menu 21-2
/Sys/Utility Programs 21-10
Part VI: Programming
22 Programming with LPL
An introduction to the LI-6400Õs programming language
Overview of LPL 22-2
Making Objects 22-7
Functions 22-12
Pointers 22-17
Public and Static 22-22
Compiler Directives 22-25
23 LPL Topics
Programming with LPL
Stack Control 23-2
Conditionals and Loops 23-4
Array Operations 23-6
Math Functions 23-18
Display Control 23-23
Keyboard Control 23-29
Clock 23-31
Event Handling 23-33
The Function Keys 23-37
I/O Programming 23-39
File System 23-46
Menus and Editors 23-51
Graphics 23-56
RS-232 Communications 23-64
Analog Measurements 23-66
Analog Output Control (D/A) 23-73
Digital I/O 23-75
Application Tools 23-78
Battery and Power 23-82

Contents
x Using the LI-6400
24 LPL Reference
Keyword Summary
Syntax Summaries 24-2
Definitions 24-11
The Reference 24-16
25 AutoProgramming Reference
Making them do what you what them to do
Autoprogram Format 25-2
Some AutoProgram Listings 25-4
Useful AutoProgram Commands 25-12
AutoPrograms and the Control Manager 25-19
Low Level Control Tools 25-19
26 Customizing Open
ÒCry ÔHavocÕ - and let slip the dogs of warÓ
Using PATCH= 26-2
Useful Variables 26-4
OpenÕs Hooks 26-7
New Style ComputeList Files 26-16
Using Spare Channels 26-22
INDEX

Using the LI-6400 xi
Welcome to the LI-6400
A Bit of History
The LI-6400 is LI-CORÕs third generation gas exchange system. In the begin-
ning was the LI-6000; it used a third party CO
2
analyzer having the novel (for
1983) features of small size, light weight, and low power. Photosynthesis was
computed by measuring the rate of change of CO
2
with time of a leaf enclosed
in a relatively large chamber - a closed system. The LI-6000 was limited by
the signal noise of the analyzer (1-2 µmol mol
-1
) and an unfortunate choice
of method for computing slopes. These problems were corrected in 1986 in
the LI-6200, which sported LI-CORÕs first CO
2
analyzer and much improved
software. It was still a closed system for CO
2
, but had the potential for steady-
state transpiration measurements. The steadiness of the state, however, de-
pended heavily on a motivated, attentive operator continually adjusting a
knob.
While the LI-6200 was fairly well suited for survey measurements, a growing
number of customers (and potential customers) were looking to answer deep-
er questions. The deeper answers could be found in response curves, and that
meant using a system that could control the environmental quantities impor-
tant to photosynthesis: CO
2
, light, humidity, and temperature. Several inno-
vators attempted to do various response curves with the LI-6200, with
varying degrees of success. Meanwhile, we were having a very hard time in
our attempts to transform that instrument into a next-generation system.
By 1990, having abandoned the idea of enhancing the LI-6200, we restarted
with the question: ÒWhat would the ideal gas exchange system be like?Ó It
would do response curves, of course, so it would need the ability to control
chamber conditions. To us, ideal response curves meant labor-free response
curves, so manual controls were out, computer controls were in. Response
curves should be seen, not imagined, so we needed built-in graphical capabil-
ity. The ideal system would also do survey measurements: you should be able
to carry it around, clamp onto a leaf using only one hand (our previous sys-
tems needed two or three), and be done with the measurement quickly. Ques-

Welcome to the LI-6400
A Bit of History
xii Using the LI-6400
tions of weight, power, and compactness thus emerged. Could the capabilities
of a laboratory system (response curves) be squeezed into a field system (sur-
vey measurements)?
Challenges loomed, two of them formidable: CO
2
and H
2
O control is done
best by having gas analyzers right in the leaf chamber, but suitable IRGAs did
not exist. Secondly, chamber CO
2
could in principle be controlled by mixing
scrubbed air and pure CO
2
(available in very portable 12 gram cylinders), but
again, a suitable mixing device did not exist. So, we set about inventing them.
Five years and one name change
1
later, we shipped the first LI-6400. And
many more since then. We trust that this instrument will serve you well, and
stand ready with support and help as you put it to work. Welcome to the
LI-6400.
1.
It started out as the LI-6300.

Welcome to the LI-6400
About this Manual
Using the LI-6400 xiii
About this Manual
When confronted with a new instruction manual, most people turn to the sec-
tion named ÒAbout this manualÓ for one simple reason: to find out how much
they can skip. Since this manual has 26 chapters, you are probably eager to
skip most of them.
YouÕre in luck. You can.
Where to Start?
Select the category that best describes you, and follow the suggested itiner-
ary.
¥ New to the LI-6400; new to gas exchange; and fairly impatient
Chapter 2: Assembling the system.
Chapter 3 (The minimum for learning the software is):
Tour #1, stop after Step 5.
Tour #2, the whole thing.
Tour #3, Experiments 1, 2, and 5.
Chapter 4: Do the check lists, and the simple experiments.
Having measured some leaves (and satisfied your impatience), you might
want to go back and pick up the parts you skipped in Chapters 3 and 4.
¥ New to the LI-6400; new to gas exchange; methodical and thorough
Work through Chapters 1, 2, 3, and 4. YouÕll understand the LI-6400 and the
fundamentals of making measurements fairly well by the end.
¥ New to the LI-6400, experienced at gas exchange measurements
Skim Chapter 1.
Chapter 2: Assembling the system.
Chapter 3: Tours 1 through 5.
Chapter 4: The check lists are important; pick and choose after that.
¥ Experienced with the LI-6400
Consider working through Tour #3 in Chapter 3; youÕll improve your under-
standing and Òfeel forÓ how the LI-6400 does chamber control. Note the
check lists at the start of Chapter 4, and the de-emphasis on zeroing and span
setting. Finally, a Troubleshooting Guide (Chapter 20) is now available.
The Old Manuals
If you are not a new LI-6400 user, you may be wondering how Using the
LI-6400 relates to the original documentation, The LI-6400 Primer and The
LI-6400 Technical Reference . If you have these original manuals, you can
safely get rid of them, because Using the LI-6400 includes the relevant prior
material, and adds much new material.

Welcome to the LI-6400
About this Manual
xiv Using the LI-6400
Electronic version This manual is also available as an Adobe¨ Acrobat¨ file, on CD and from
our web site (www.licor.com). All cross references in the text, table of con-
tents, and index are hyper-linked, allowing one-click access. (Version 3.0 of
Adobe¨ Acrobat¨ Reader is required. You can download this software for
no charge from www.adobe.com)

Part I
The Basics

Using the LI-6400 1-1
1
System Description
What it is, what it does, and how it does it
ANOPEN SYSTEM 1-2
Measuring Differentials 1-2
The Air Supply 1-3
Leaks 1-3
THE FLOW SCHEMATIC 1-4
Matching the IRGAs 1-6
EQUATION SUMMARY 1-7
Transpiration 1-7
Total Conductance to Water Vapor 1-8
Stomatal Conductance to Water Vapor 1-9
Net Photosynthesis 1-9
Intercellular CO2 1-10
Everything Else 1-11
Summary of Symbols 1-11
THE SYSTEM COMPONENTS 1-12
The Standard Parts 1-13
The Optional Accessories 1-14

System Description
1-2 Using the LI-6400
1
This chapter acquaints you with the LI-6400Õs operating principle, major
components, and equations.
An Open System
Measuring Differentials
The LI-6400 is an open system, which means that measurements of photosyn-
thesis and transpiration are based on the differences in CO2and H2O in an air
stream that is flowing through the leaf cuvette (Figure 1-1).
Sample IRGA
Reference IRGA
Flow x DCO2
Area
Photo = Trans = Flow x DH2O
Area
Figure 1-1. In an open system, photosynthesis and transpiration are computed
from the differences in CO2and H2O between in-chamber conditions and pre-
chamber conditions. The equations are given in Equation Summary on page 1-7.
Sample IRGA
Reference IRGA
Flow
Meter
Flow
Meter
Traditional Open System
LI-6400
Console
Console
Cuvette
Cuvette

System Description
An Open System
Using the LI-6400 1-3
The LI-6400 improves upon traditional open systems by having the gas ana-
lyzers in the sensor head. This eliminates plumbing-related time delays, and
allows tight control for responding to leaf changes. For example, if stomata
close, the control system immediately detects the drop in water vapor and can
compensate. Similarly, a sudden change in light level will cause an immedi-
ate change in photosynthetic rate, which will be detected as a change in the
CO2concentration. The speed of detection is not a function of the systemÕs
flow rate, as in traditional systems, since the sample IRGA is in the cuvette.
There is a second advantage of having the IRGAs in the sensor head. The tra-
ditional system has the potential for concentration changes (because of water
sorption and CO2diffusion) as the air moves from the reference IRGA to the
chamber, and again from the chamber back to the sample IRGA. This is not
a problem for the LI-6400, because the IRGA measurements are made after
the air has travelled through the tubing.
The Air Supply
One strength of an open system is that the incoming air stream can be condi-
tioned. That is, its humidity, CO2concentration, temperature, etc. can be es-
tablished by some means prior to entering the system.
Regardless of what is done to the incoming air, however, one thing is crucial:
This is especially true for CO2, where the potential for fluctuations is huge
(your breath, for example, is probably about 50,000 µmol mol-1). Fluctua-
tions in concentration will cause concentrations differences to be very erratic.
Leaks
ÒPressure inside the leaf chamber is slightly above ambient to ensure that
leaks are outward, so outside air does not enter the chamber and affect the
CO2and H2O concentrations.Ó This argument for the advantage of an open
system has been made for a long time, and itÕs true - as far as it goes. There
is another process at work, however: diffusion. CO2will always diffuse from
higher concentrations toward lower, even against a pressure gradient, and is
only limited by the material through which it is moving. The black neoprene
gaskets that we use on the LI-6400 (except for the light source) have the low-
est diffusivity to CO2of all the gasket material we have tested, but itÕs not
perfect. Diffusion effects are measurable at low flow rates when there is a
Incoming concentrations must be stable.

System Description
The Flow Schematic
1-4 Using the LI-6400
1
large concentration difference between chamber and ambient. See Diffusion
Leaks on page 4-43 for more details.
The Flow Schematic
The LI-6400 provides mechanisms for modifying the incoming airÕs CO2and
H2O concentrations (Figure 1-2 on page 1-5). There are chemical tubes for
scrubbing CO2and H2O, and air can be diverted through these tubes in any
proportion desired. CO2, however, is best controlled by scrubbing all of it
from the incoming air, and using the 6400-01 CO2mixer to inject just enough
CO2to provide a stable concentration at the desired value. If the 6400-01 is
not part of your system, you will need to use a buffer volume. For a more
complete discussion of buffer volumes, see Air Supply Considerations on
page 4-47.
Humidity control in the leaf cuvette is achieved by regulating the flow rate
that is going though the cuvette. In units without a CO2mixer, the pump
speed controls the flow, and in units with a CO2mixer, a flow diverter regu-
lates this flow.