Turner Designs Trilogy User manual

Trilogy Laboratory Fluorometer
User’s Manual
Version 1.2
September 15, 2010
P/N 998-7210
Turner Designs
845 W. Maude Ave.
Sunnyvale, CA 94085
Phone: [phone]
FAX: [phone]
www.turnerdesigns.com

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Trilogy Fluorometer User’s Manual P/N 998-7210 Page 2 of 38

Highlights of the Trilogy Fluorometer
Multifunctional,
Compact and Modern
Design provides
flexibility and precision
Color Touch-Panel
Graphics User Interface
provides intuitive ease
of use
Sample Chamber accepts
snap-in Application Modules
for Fluorescence,
Absorbance and Turbidity
measurements
Trilogy saves time by storing
up to 18 named calibrations
Automatically calculates and
displays Extracted Chl a
concentrations using filtered
and solvent volumes
Spreadsheet Interface
Software provides real
time display and
logging of data to Excel
Spreadsheet
Trilogy Fluorometer User’s Manual P/N 998-7210 Page 3 of 38

Table of Contents
CHAPTER DESCRIPTION PAGE
Highlights of the Trilogy Fluorometer 3
I. Getting Started 5
Description 5
Unpacking and Inspection 5
Setup 6
Getting to the Home Screen 6
Precautions 7
II. Fluorometer Operation 8
Touch screen Basics (Fluorescence) 8
Measuring Samples 9
III. Absorbance Operation 11
Touch Screen Basics (Absorbance) 11
Measuring Samples 12
IV. Turbidity Operation 14
Touch Screen Basics (Turbidity ) 15
Measuring Samples 15
V. Calibration Overview 16
V. Calibration Overview 17
A. Why Calibrate 17
B. When to Calibrate 17
C. Trilogy Calibration Options, (Fluorescence and Turbidity) 17
D. Trilogy Calibration Procedures 17
E. “Direct Calibration” Procedure – a) Fluorescence and Turbidity Modules, Single Point and Multi Point Cal 18
F. Extracted Chlorophyll Measurements with the Chl a– Acidification Fluorescent Module 20
G. Calibrating and Displaying Corrected Readings for the Chl aAcidification and Non-Acidification Modules 20
H. “Direct Calibration” Procedure – b) Extraction, Acidification, Single Point and Multi Point 20
J. “Direct Calibration” Procedure – c) Extraction, Non-Acidification, Single Point and Multi Point 23
K. Using the Secondary Standard 23
L. Using the Solid Secondary Standard for in vivo Chlorophyll Applications 23
M. Use of the Solid Secondary Standard for Dye Applications: 24
N. Trilogy Calibration Procedure, (Absorbance Module) 24
VI. Touch Screen Basics 26
Touch Screen 26
VII. Systems Connections 27
Serial Output Functional Check 27
VIII. Spreadsheet Interface Software Installation 28
Excel Spreadsheet Example 28
Appendix A - Troubleshooting 29
Fluorescence Troubleshooting 29
Absorbance Troubleshooting 29
Turbidity Troubleshooting 30
Spreadsheet Interface Software Troubleshooting 30
Appendix B - Warranty and Obtaining Service 32
Warranty 32
Out-of-Warranty Service 32
Appendix C – Specifications 33
Appendix D – Principles of Fluorescence 34
Principles of Fluorescence 34
Appendix E – Recommended Measurement Practices 35
Linear Range and Quenching 35
Temperature Considerations 35
Appendix F - Chlorophyll a Acidification and Non-Acidification calculations 36
Appendix G - In vivo fluorescence measurements using Trilogy 37
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I. Getting Started
Description
The Trilogy Laboratory Fluorometer is a compact, multifunctional laboratory instrument that can
be used for making fluorescence, absorbance, and turbidity measurements using the appropriate
snap-in Optical Module. A color touch screen with simple menus makes for an intuitive user
interface.
Fluorescence Modules are available for discrete sample measurement of various fluorescent
materials including chlorophyll (in vivo and extracted), rhodamine, fluorescein, cyanobacteria
pigments, ammonium, CDOM, optical brighteners and other fluorescent compounds.
The Absorbance Module accepts interchangeable filter paddles so measurements can be made
at different wavelengths in order to identify or place a sample in a particular class of compounds.
The standard filter paddle wavelengths/bandwidths are: 560/10; 600/10 and 750/10 nm.
The Turbidity Module uses an Infrared (IR) LED with a wavelength of 850 nm as required for
reference method: ISO 7027/DIN EN 27027, “Water Quality – Determination of Turbidity”. Using
Infrared allows Turbidity to be measured at wavelengths that are not normally absorbed by
organic matter thereby reducing susceptibility to interference by sample color.
When properly calibrated, the Trilogy Fluorometer will read out the actual concentration of the
solution. Optical Modules contain the necessary light source and filters for the relevant
application
Unpacking and Inspection
Upon receiving the Trilogy, please inspect it carefully and make certain all accessories are
present. Refer to the checklist shipped with the instrument for order-specific items.
A typical Trilogy shipment includes:
- Checklist
- Trilogy Laboratory Fluorometer
- CD – Trilogy, includes files for: User’s Manual (pdf); Quick Start Guide (pdf);
Spreadsheet Interface Software (pdf)
- Solid Secondary Standard, (not intended for use with UV or Turbidity optical modules)
- Power Supply Kit
- RS-232 Cable
- Quick Start Guide, (hardcopy)
- 10 x 10 mm Methacrylate Cuvettes, (quantity of 10)
- 12 x 35 mm Glass Round Vial, (quantity of 5)
- 12 mm Round Adaptor
- 50-Well Microtube Storage Box
- Optical Module(s) as ordered
Fluorescence Module
Absorbance Module
Turbidity Module
Trilogy Fluorometer User’s Manual P/N 998-7210 Page 5 of 38

Setup
Place the Trilogy Fluorometer on a flat, level surface. Allow at least 6 inches (16 cm) of clearance
above the instrument to open and close the lid. Position the instrument so that the touch screen
faces you .
Power Supply
Connect the power supply into the power connection of the instrument, see Figure 1, and plug it
into a wall outlet. See Specifications for power requirements.
Figure 1.
Getting to the Home Screen
1. With the unit turned off, lift the lid and insert an optical module into position, (see Fig 2).
Then press down on the module until you hear it snap into place. Be sure to close the
fluorometer lid so that the Trilogy power on step will complete successfully.
2. Turn on the On/Off switch located on the back of the Trilogy, see Figure 1. Verify the display
becomes active, and shows the module selection screen, see Figure 3 – Module Selection.
Figure 2 showing Optical Kit
installed in preparation for getting to
the Home Screen.
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Figure 3 Module Selection Screen
3. Select the module inserted, and touch “OK” on the confirming screen.
4. When the Home Screen is displayed (Fig. 4), you are now ready to use Trilogy in its Raw
Mode (measurements are relative) or calibrate the Trilogy and the snapped-in Optical
Application Module to make quantitative measurements.
Figure 4: Trilogy Home Screen Display for Fluorescence Module Configuration.
Precautions
The Trilogy is intended for indoor use only. Wipe up spills immediately. The Trilogy contains
sensitive optical components and precision-aligned mechanical assemblies. Avoid rough
handling. Do not leave the lid open for extended periods of time. Turn OFF the Trilogy to change
or install Optical Application Modules.
Note: After 20 minutes without activity or user stimulation, the Touch Screen hibernates. Lightly
touch the screen to activate.
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II. Fluorometer Operation
Fluorescence Optical Module
Installation
1. Power the Trilogy OFF
2. Grasp the handle of the Optical Application Module and align the kit with the sample
compartment.
3. Press down firmly to lock the Optical Application Module in place. You should hear or
feel a click indicating the module has snapped into place. (See Figure 5.)
4. Close the lid and power ON the Trilogy. Use the touch screen to identify the type of
Optical Application Module installed.
Figure 5.
Removal
1. Power OFF the Trilogy before removing the Optical Application Module
2. Grasp the handle and gently pull up to release the kit from the sample compartment.
Touch screen Basics (Fluorescence)
Home Screen
The "Home" screen appears after confirmation of the Optical Application Module. The "Home"
screen provides orientation for the multiple functions of the Trilogy. From the "Home" screen,
select "Calibrate," "Tools," "Mode," or "Help." The "Home" screen is also the measurement
screen. (See over for Figure 6 showing Home Screen with callouts).
Trilogy Fluorometer User’s Manual P/N 998-7210 Page 8 of 38

Figure 6 – Trilogy Home Screen for Fluorescence Module Configuration.
Calibration
Refer to Section V.
Measuring Samples
There are two measurement modes available on the Trilogy when using the Fluorescence
Module:
Raw Fluorescence Mode – No calibration required
Direct Concentration Mode – Calibration required (see Section V)
Touch “Mode” on the Home Screen to select the measurement mode.
1. Raw Fluorescence Mode: The Raw Fluorescence Mode should be used for qualitative
measurement, relative changes in fluorescence rather than absolute concentration values.
Readings are displayed in Relative Fluorescence Units (RFU).
2. Direct Concentration Mode: The Direct Concentration mode makes absolute measurements
based on a calibration; see Section V for the Calibration Procedure.
The Trilogy accommodates 10 x 10 mm methacrylate and polystyrene cuvettes (minimum 2 mL
volume). Use 12 x 35 mm or 12 x 75 mm glass test tubes for measuring solvent applications,
such as extracted chlorophyll, and use methacrylate cuvettes for UV water applications.
1. Open the lid of the Trilogy and insert the cuvette. Close the lid.
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2. Touch "Sample ID" to name your sample (optional).
3. Using the keypad, enter the sample name into the name field and touch "Save" to
save the sample ID.
4. Touch "Measure Fluorescence" to make a measurement. The Trilogy will measure
the sample for 6 seconds and report the average reading for the sample.
The Trilogy reports data on the "Home" screen and displays the results for the most recent 20
measurements. Use the arrow keys to scroll through the most recent measurements. The data
automatically exports to a printer or PC when properly connected, (see Section VII). Please note
the Trilogy does not store more than 20 measurements at one time. If more than 20 readings are
taken, the oldest reading will be overwritten. Measurements are not stored between power
cycles.
Tools (Fluorescence)
Touch the “Tools” key to access "Settings."
Tools - Settings
View Cal Details
Touch "View Cal Details" to see information on the current calibration for Direct Concentration
Mode. "View Cal Details" specifically provides information on the raw fluorescence for each
standard and the blank as well as the unit of measure and the Optical Application Module.
Continuous Sampling
The Continuous Sampling feature enables repeat measurements at user-defined intervals.
1. Touch "Continuous Sampling" and turn the feature ON. Highlight the frequency of
measurement and the number of total measurements. The maximum number of total
measurements is 9999.
2. Touch "OK" to return to the "Home" screen.
3. Connect the Trilogy to a printer or a PC to collect the data. Touching the screen
repeatedly causes an early-abort of Continuous Sampling measurements.
Measurement Tip:
On the Home Screen, touch Settings then touch the
Lid Start key to turn the feature ON. When the Lid
Start feature is ON, measurement begins as soon as
the lid closes. The lid start feature allows for immediate
measurement and eliminates the need to touch the
"Measure" key. Also, the touch screen does not
hibernate when Lid Start is ON.
Return to the Lid Start key under the Settings menu
to turn the feature OFF.
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III. Absorbance Operation
Absorbance Module
Installation
1. Power OFF the Trilogy.
2. Align the Absorbance Module with the sample compartment.
3. Press down to lock the Absorbance Module in place. You should hear or feel a click
indicating the module has snapped into place.
4. Close the lid and power ON the Trilogy. Select "Absorbance" from the list of options
on the touch screen.
5. Install the filter paddle that corresponds to the wavelength of absorbance for the
assay. (See Figure 7.)
Figure 7.
Removal
1. Power OFF the Trilogy before removing the Absorbance Module.
2. Grasp the handle and gently pull up to release it from the sample compartment.
3. Close the lid to protect the light detector from exposure to ambient light. Alternatively,
install an Optical Application Module.
Touch Screen Basics (Absorbance)
Home Screen
The "Home" screen appears after confirming the photometer operation by installing the
Absorbance Module. From the "Home" screen, select "Calibrate," "Tools," "Mode" or "Help." The
"Home" screen is also the measurement screen. (see Figure 8 on next page)
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Mode
Touch "Mode" to select the unit of measure for absorbance. The available options include
Absorbance units (Ab) and Transmittance (%T).
The following formulas describe the method of the Trilogy Absorbance Module for measuring %
transmittance and absorbance:
%T = [(s-z) / (b-z)] * 100
Ab = 2 - Log10 * (%T)
Where,
z = zero
b = baseline
s = signal
Figure 8: Home Screen When Using Absorbance Module
Calibration
See Section V.
Measuring Samples
The Absorbance Module accommodates 10 x 10 mm methacrylate and polystyrene cuvettes as
well as glass cuvettes (minimum 2 mL volume).
1. Open the lid and insert the cuvette. Close the lid.
2. Touch "Sample ID" to name your sample (optional). Using the keypad, enter the
sample name into the name field and touch "Save" to save the sample ID.
3. Touch "Measure Absorbance" to make a measurement. The Trilogy will measure the
sample for 6 seconds and report the average reading for the sample.
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The Trilogy reports data on the "Home" screen and displays the results for the most recent 20
measurements. Use the arrow keys to scroll through the most recent measurements. The data
exports to a printer or PC if properly connected, (see Section VII).
Please note the Trilogy does not store more than 20 measurements at one time. If more than 20
readings are taken, the oldest reading will be overwritten. Measurements are not stored between
power cycles.
Tools - Settings
Touch the "Tools" key to access "Settings."
View Cal Details
Touch "View Cal Details" to see information on the current calibration for the baseline and the
zero.
Continuous Sampling
The Continuous Sampling feature enables repeat measurements at user-defined intervals.
1. Touch "Continuous Sampling" and turn the feature ON.
2. Highlight the frequency of measurement and the number of total measurements. The maximum
number of total measurements is 9999.
3. Touch "OK" to return to the "Home" screen.
4. Connect the Trilogy to a printer or a PC to collect the data obtained during Continuous
Sampling. Touching the screen repeatedly causes an early-abort of Continuous Sampling
measurements.
Measurement Tip:
On the Home Screen, touch Settings then touch
the Lid Start key to turn the feature ON. When
the Lid Start feature is ON, measurement begins
as soon as the lid closes. The lid start feature
allows for immediate measurement and eliminates
the need to touch the "Measure" key. Also, the
touch screen does not hibernate when Lid Start is
ON.
Return to the Lid Start key under the Settings
menu to turn the feature OFF.
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IV. Turbidity Operation
Turbidity Optical Module
Installation
1. Power the Trilogy OFF
2. Grasp the handle of the Optical Application Module and align the kit with the sample
compartment.
3. Press down firmly to lock the Optical Application Module in place. You will hear or
feel a click indicating the module has snapped into place. (See Figure 9.)
4. Close the lid and power ON the Trilogy. Use the touch screen to identify the type of
Optical Application Module installed.
Figure 9.
Removal
1. Power OFF the Trilogy before removing the Optical Application Module
2. Grasp the handle and gently pull up to release the kit from the sample compartment.
Touch Screen Basics
See next page
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Touch Screen Basics (Turbidity )
Home Screen
The "Home" screen appears after confirmation of the Optical Application Module. The "Home"
screen provides orientation for the multiple functions of the Trilogy. From the "Home" screen,
select "Calibrate," "Tools," "Mode," or "Help." The "Home" screen is also the measurement
screen.
Figure 10: Home Screen When Using Turbidity Module
Calibration
Refer to Section V.
Measuring Samples
There are two measurement modes available on the Trilogy when using the Turbidity Module:
Raw Mode – No calibration required
Direct Concentration Mode – Calibration required – See Section V
Touch “Mode” on the Home Screen to select the measurement mode.
1. Raw Mode: The Raw Mode should be used for qualitative measurement, relative changes in
fluorescence rather than absolute concentration values. Readings are displayed in Relative
Fluorescence Units (RFU).
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2. Direct Concentration Mode: The Direct Concentration mode makes absolute measurements
based on a calibration. Readings are displayed in Nephelometric Turbidity Units (NTU). (see
Section V for the Calibration Procedure)
Use polystyrene cuvettes for measuring turbidity.
1. Open the lid of the Trilogy and insert the cuvette. Close the lid.
2. Touch "Sample ID" to name your sample (optional).
3. Using the keypad, enter the sample name into the name field and touch "Save" to
save the sample ID.
4. Touch "Measure Turbidity" to make a measurement. The Trilogy will measure the
sample for 6 seconds and report the average reading for the sample.
The Trilogy reports data on the "Home" screen and displays the results for the most recent 20
measurements. Use the arrow keys to scroll through the most recent measurements. The data
automatically exports to a printer or PC when properly connected, (see Section VII). Please note
the Trilogy does not store more than 20 measurements at one time. If more than 20 readings are
taken, the oldest reading will be overwritten. Measurements are not stored between power
cycles.
Tools (Turbidity)
Touch the “Tools” key to access "Settings."
Tools - Settings
View Cal Details
Touch "View Cal Details" to see information on the current calibration for Direct Concentration
Mode. "View Cal Details" specifically provides information on the raw fluorescence for each
standard and the blank as well as the unit of measure and the Optical Application Module.
Continuous Sampling
The Continuous Sampling feature enables repeat measurements at user-defined intervals.
1. Touch "Continuous Sampling" and turn the feature ON. Highlight the frequency of
measurement and the number of total measurements. The maximum number of total
measurements is 9999.
2. Touch "OK" to return to the "Home" screen.
3. Connect the Trilogy to a printer or a PC to collect the data. Touching the screen
repeatedly causes an early-abort of Continuous Sampling measurements.
Measurement Tip:
On the Home Screen, touch Settings then touch
the Lid Start key to turn the feature ON. When the
Lid Start feature is ON, measurement begins as
soon as the lid closes. The lid start feature allows
for immediate measurement and eliminates the
need to touch the "Measure" key. Also, the touch
screen does not hibernate when Lid Start is ON.
Return to the Lid Start key under the Settings
menu to turn the feature OFF.
Trilogy Fluorometer User’s Manual P/N 998-7210 Page 16 of 38

V. Calibration Overview
A. Why Calibrate
When calibrated, the Trilogy converts the fluorescence signal to concentration units based on the
standard(s) used for the calibration. This saves you from having to perform any calculations.
B. When to Calibrate
-For greatest accuracy, calibrate or check the calibration using a secondary solid
standard before running a new batch of samples.
-Recalibrate if the ambient temperature changes by +/- 10°C.
-Recalibrate after changing Optical Application Modules or if you make measurements
on a new analyte.
-Verify the need to calibrate by reading a stable, known concentration standard
immediately after calibration and again every few hours to see if readings have
changed significantly. Recalibrate when the accuracy becomes unacceptable for your
study.
C. Trilogy Calibration Options, (Fluorescence and Turbidity)
There are two measurement modes available on the Trilogy when using either the Fluorescence
or Turbidity Modules, (see section K for Absorbance Module Calibration Procedure):
Raw Fluorescence Mode – No calibration required
Direct Concentration Mode – Calibration required
1. Raw Fluorescence Mode: The Raw Fluorescence Mode should be used for qualitative
measurement, relative changes in fluorescence rather than absolute concentration values.
Readings are displayed in Relative Fluorescence Units (RFU).
2. Direct Concentration Calibration: Direct Concentrations can be calibrated by using single or
multi-point calibrations. In multi-point calibrations, up to five standards and a blank can be read
generating a calibration curve for increased accuracy. The software uses these points to
calculate direct concentrations. The Trilogy will display the actual concentration of your samples
in units that were chosen during calibration.
D. Trilogy Calibration Procedures
1. Raw Fluorescence Mode: A calibration is not necessary to measure fluorescence with the
Trilogy. Simply use the Raw Fluorescence Mode to obtain the fluorescence value of a sample
in Relative Fluorescence Units (RFU). Use a standard curve to determine the concentration
of the analyte in the samples. The Trilogy does not manipulate data while operating in the
Raw Fluorescence Mode. It is not necessary to zero the Trilogy for use in the Raw
Fluorescence Mode; however a blank sample should be run to determine background
fluorescence. A solid secondary standard may be used to verify instrument stability and
function (see section J).
2. Direct Calibration Mode: The Direct Concentration Mode requires a calibration with one
blank solution and at least one standard solution. The following procedure applies to all the
fluorescence modules with the exception of the Chl Acidification and Non-Acidification
modules. (There are separate procedures for these two exceptions). The procedure requires
the use of at least one calibration standard, (Fluorescein, Rhodamine WT, etc.). Up to 5
standard solutions can be used for a Multi Point Calibration. Calibrations can be given a
name and stored for future use.
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E. “Direct Calibration” Procedure – a) Fluorescence and Turbidity
Modules, Single Point and Multi Point Cal
The following procedure applies to Trilogy and the Fluorescence or Turbidity Modules. See page
20 for procedures to calibrate the Chl Acidification and Non-Acidification Modules.
Instructions Sample Screen
a) Turn on the Trilogy
b) Select the module/application to be
calibrated and confirm by touching “OK”.
Note: Refer to the module label to confirm
which module is in use.
c) On the home screen, touch "Calibrate" to
begin a calibration sequence
d) Select “Run New Calibration”
e) Select the unit of measurement
f) Insert the calibration “blank” and touch
“OK”
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g) Enter the concentration for the first
Standard. If using the Turner Designs
Chlorophyll a Standard, this will be the
concentration data supplied with the
Standard. If doing multi point calibrations,
be sure to use Standards in order of
increasing concentration.
h) Follow the screen prompt indicating that
the standard should be inserted, touch OK
i) After the calibration is complete, either
select “Proceed with Current
Calibration” or select “Enter More
Standards”, in which case, return to “g”
above.
j) Name and Save the calibration for future
use (optional).
k) Subsequent readings in the Direct
Concentration mode should reflect the
actual concentration of the analyte.
l) Confirm successful completion of the
calibration by measuring the same
Standard. The displayed concentration
should equal the value used in step “g”
above. Optionally, the Solid Secondary
Standard could now be adjusted to give the
same reading for future calibrations, see
Section K.
This completes the “Direct Calibration”
Procedure for the Fluorescence and Turbidity
Modules.
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F. Extracted Chlorophyll Measurements with the Chl a– Acidification
Fluorescent Module
EPA Method 445.0 is a standard method for measuring extracted chlorophyll aand pheophytin a
in marine and fresh water algae by fluorescence. It requires extraction with 90% acetone,
measurements before and after acidification and some fairly simple calculations to arrive at the
chlorophyll aand pheophytin concentrations (see appendix F). Method 445.0 is detailed and
straightforward. (If high concentrations of pure chlorophyll b are present, see the next paragraph).
A known concentration of pure chlorophyll a(as a standard) is required at least the first time you
calibrate Trilogy. For greatest accuracy however, we recommend that you periodically (once
every few months) use a known concentration of pure chlorophyll ain 90% acetone to recalibrate
your instrument. (Liquid Primary and Solid Secondary Chlorophyll astandards are available from
Turner Designs).
G. Calibrating and Displaying Corrected Readings for the Chl a
Acidification and Non-Acidification Modules
The Trilogy software can correct displayed chlorophyll readings for filtered and solvent volumes
used during the calibration and measurement cycles. Be sure to touch either “Chl A” or “Chl
NA” selections in the module selection screen so that the appropriate corrections are applied to
measured data. This results in displayed readings that correspond to the actual sample
concentration.
H. “Direct Calibration” Procedure – b) Extraction, Acidification, Single
Point and Multi Point
Instructions Sample Screen
a) Turn on the Trilogy
b) Touch “Chl – A” to select the Chlorophyll a
Acidification module and confirm by
touching “OK”
c) On the home screen, touch "Calibrate" to
begin a calibration sequence
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