Manual - Turner Designs
Contents
1. fo In vivo Calibration Using Direct Concentration and a Secondary Standard 1 Insert the in vivo chlorophyll a Trilogy module Turner Part Number 7200 043 2 Turn on the Trilogy fluorometer using the switch located on the back panel 3 Select the Blue module This is indicated on the module label under GUI selection 4 Press OK after verifying that the module loaded matches your selection The default mode loaded is Raw Fluorescence 5 Press Calibrate 6 Choose Run New Calibration 7 Choose the units to be displayed during measurement 8 Insert blank sample and press OK 9 When prompted enter a number which will be correlated to the sample s response You may enter 1 to 5 standards of various concentration levels or calibration values If concentration of sample is known enter the known value This will set the fluorescence response equal to the concentration entered and will give semi quantitative results in subsequent measurements example 5 ug L 137 4 RFU If an arbitrary calibration value is entered the fluorescence response of the sample will be correlated to the new calibration value and subsequent measurements will be scaled proportionally These measurements will only give qualitative results example 500 RFU 137 4 RFU 10 Thoroughly mix sample by inverting or shaking to prevent settlement of algal cells and quickly insert sample 11 After inserting sample immedi2. 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
3. Absorbance Module Performance Chl a Acid 7200 040 485 nm 685 50 nm Photometric Measuring Range Chl a Non Acid 7200 046 485 nm 685 10 nm 0 0 4 0 Ab Chl a in vivo 7200 043 485 nm 685 50 nm Photometric Accuracy Phycocyanin 7200 044 600 nm 640 nm 0 7 Rhodamine WT amp 7290 042 550 nm 610 nm Photometric Precision Phycoerythrin lt 0 5 at 1 Ab Optical Brighteners 7200 047 365 nm 445 15 nm Fluorescein 7200 048 485 nm 540 nm igh gs Crude Oil 7200 063 365 nm 410 600 nm a idl Histamine 7200 049 365nm 450 80 nm p p CDOM amp Ammonium 7200 041 365 nm 430 nm Accommodates 10 x 10 mm square a plastic cuvettes 12 x 75 mm round Turbidity Module tubes and 12 x 35 mm round vials Application Part Number Wavelength Readout a Direct Concentration ug L ppb Turbicity 7200 060 onan etc or Raw Fluorescence RFU Absorbance Module amp Filter Kits Calibration f Description Part Number One to Five point calibration with up to 18 calibrations stored Absorbance Module 7200 050 Light Source amp Detector 560 10 nm Filter Paddle 7200 051 LED and Photodiode 600 10 nm Filter Paddle 7200 052 Blank 750 10 nm Filter Paddle 7200 053 Dimensions 12 92 D x 10 44 W x 8 42 H 32 82 cm D x 26 52 cm W x 21 39 cm H Operating Temperature 60 105 F 15 40 C Reads and subtracts blank Data Output 100 ASCII format through a 9 pin RS 2
4. g above Pate Te Te fele fe e 1 Name and Save the calibration for future ee ea Wea Rea use optional ey ea ea ea ea amamma coarne m Itis recommended that the Solid Standard is now measured and the displayed value noted to enable a quick calibration check prior to later use This completes the Direct Calibration Procedure b Extraction Acidification Single Point and Multi Point Trilogy Fluorometer User s Manual P N 998 7210 Page 22 of 38 TURNER D DESIGNS J Direct Calibration Procedure c Extraction Non Acidification Single Point and Multi Point The Welschmeyer method is a simplified way to measure chlorophyll a without the need for acidification It accurately measures chlorophyll a even in the presence of chlorophyll b and pheophytin However you cannot obtain a pheophytin a measurement with this procedure Using this method you extract your samples according to EPA Method 445 0 but skip the acidification step You still need to calibrate the instrument the first time using a known concentration of pure chlorophyll a in 90 acetone The calibration procedure for the Chlorophyll Non Acidification follows the same steps as for the Direct Calibration mode see Section E however the measurement procedure will prompt for the filtered and solvent volumes K Using the Secondary Standard The following section will describe how to use the Solid S
5. Count The Indicates module Touch here to make display increments by 1 selected on module indicated measurement each time a measurement screen at instrument a Direct Concentration is made turn on calibrated measurement is indicated here Touch here to go to a Measurement Readout selection of help and peas updates each time a new troubleshooting screens 5 2 reading is taken Measurement Buffer holds and displays up to 20 readings Sample 001 and Sample 002 shown Touch here to bring up the Home Screen from other windows Provid Touch a Provides switching TOV es access to ouc i here to initiate between RAW and Settings and Diagnostic calibration routines screens Direct Concentration modes 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 Trilogy Fluorometer User s Manual P N 998 7210 Page 7 of 38 TURNER D DESIGNS ll Fluorometer Operation Fluorescence Optical Module Installation 1 Power the Trilogy OFF 2 Grasp the han
6. 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 Trilogy Fluorometer User s Manual P N 998 7210 Page 13 of 38 TURNER D DESIGNS 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
7. 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 Optical Kit 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 Trilogy Fluorometer User s Manual P N 998 7210 Page 14 of 38 TURNER D DESIGNS 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 Sample Count The Indicates module selected Touch here to make display increments by 1 on module screen at indicated measurement each time a measurement instrument turn on this home screen is is made indicating that calibrated turbidity units are NTU Touch here to go to a selection of help and troubleshooting screens Measurement Readout updates each time a new reading is taken Touch here to bring up Measurement Buffer the Home Screen from holds and displays up to 20 readings Sample 001 thru Sample 003 shown other windows Provides access to Touch here to initiate Pr
8. 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 10 of 38 TURNER D DESIGNS lll Absorbance Operation Absorbance Module Installation 1 2 3 Absorbance Module Installed Power OFF the Trilogy Align the Absorbance Module with the sample compartment Press down to lock the Absorbance Module in place You should hear or feel a click indicating the module has snapped into place Close the lid and power ON the Trilogy Select Absorbance from the list of options on the touch screen Install the filter paddle that corresponds to the wavelength of absorbance for the assay See Figure 7 Filter Paddle Figure 7 Removal 1 2 3 Power OFF the Trilogy before removing the Ab
9. by accident or neglect Your instrument must be installed according to instructions in the User s Manual Damage from corrosion is not covered Damage caused by customer modification of the instrument is not covered e This warranty covers only Turner Designs products and is not extended to equipment used with our products We are not responsible for accidental or consequential damages except in those states where this limitation is not allowed This warranty gives you specific legal rights and you may have other rights which vary from state to state e Damage incurred in shipping is not covered Warranty Service To obtain service during the warranty period the owner shall take the following steps 1 Write email or call the Turner Designs Technical Support department and describe as precisely as possible the nature of the problem Phone 1 877 316 8049 Email support turnerdesigns com 2 Carry out any adjustments or tests as suggested by the Technical Support Department 3 If proper performance is not obtained you will be issued a Return Materials Authorization number RMA to reference Package the unit write the RMA number on the outside of the shipping carton and ship the instrument prepaid to Turner Designs If the failure is covered under the warranty terms the instrument will be repaired and returned free of charge for all customers in the contiguous continental United States For customers outside of the contiguous
10. in the linear range the reading will decrease in direct proportion to the dilution If the reading does not decrease in direct proportion to the dilution or if the reading increases the sample is beyond the linear range of the fluorophore Fluorometer Response Curve 7 7 z Z b Sample Quenching Region Figure 14 Graph showing Linear and Sample Quenching Regions of the ee esin sample s response Sample Con Temperature Considerations Fluorescence is temperature sensitive As the temperature of the sample increases the fluorescence decreases For greatest accuracy record the sample temperature and correct the sensor output for changes in temperature For further information on how temperature light water quality and the physiological state of the algal cells can all affect the measurement of chlorophyll a please refer to the application section of Turner Designs web site at the following URL http www turnerdesigns com esupport understanding html Trilogy Fluorometer User s Manual P N 998 7210 Page 35 of 38 TURNER D DESIGNS Appendix F Chlorophyll a Acidification and Non Acidification calculations When in direct concentration mode the following calculations will be use to calculate corrected chlorophyll a and pheophytin values for the acidification method and the corrected chlorophyll values for the non acidification method Acidification Method I Variables stored during calibration p
11. make indicated measurement a Direct Concentration calibrated measurement is indicated here Measurement Readout updates each time a new reading is taken Measurement Buffer holds and displays up to 20 readings Sample 001 and Sample 002 shown Provides switching between RAW and Direct Concentration modes 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 li
12. 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 Trilogy Fluorometer User s Manual P N 998 7210 Page 17 of 38 TURNER p DESIGNS 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 a Instructions Turn on the Trilogy Sample Screen 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 i fc SAMPLE 001 0 00 RFU d Select Run New Calibration e Select the unit of measurement Insert the calibration blank and touch OK Trilogy Fluorometer User s Manual P N 998 7210 Page 18 of 38 T
13. screw clockwise produces a lower signal 3 Next perform a chlorophyll extraction using the Trilogy Laboratory Fluorometer Spectrophotometer or HPLC to determine the actual chlorophyll a concentration in the sample This will provide the correlation between the solid standard and the actual chlorophyll a concentration Information on doing a chlorophyll extraction can be found on the Turner Designs web site at this URL http www turnerdesigns com t2 doc appnotes 998_9000 html Trilogy Fluorometer User s Manual P N 998 7210 Page 23 of 38 TURNER D DESIGNS 4 Now at any time the Solid Standard can be used to check establish a new correlation between a known equivalent concentration and the current Trilogy reading M Use of the Solid Secondary Standard for Dye Applications The Solid Secondary Standard accessory can also be used to check the fluorometer s stability for dye tracing applications If necessary the Solid Standard can be used to establish a new correlation factor without the need to use a calibration solution each time 1 To use the Solid Standard to establish a correlation between a known dye concentration and the fluorometer reading measure a dye solution of known concentration say 50 ppb and note the Trilogy reading 2 Place the Solid Standard in the Optical Module and turn the adjustment screw to produce the same displayed concentration as in step 1 turning the secondary standard adjust
14. 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 Connection 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 Optical Kit Installed Figure 2 showing Optical Kit installed in preparation for getting to the Home Screen Trilogy Fluorometer User s Manual P N 998 7210 Page 6 of 38 TURNER D DESIGNS Drd co Es Es 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 Sample
15. 32 serial cable at 9600 baud PC Operating System optional if connected to PC Windows 98 or later Power 100 to 240VAC Universal Power Supply included Output 12VDC 0 84A Max Contact us for other applications or wavelengths Specifications subject to change without notice Included Accessories 10 each 10 x 10 mm Methacrylate Cuvettes 5 each 12 x 35 mm Screw Top Glass Vials 50 Well Storage Box Solid Secondary Standard Power Supply 12 mm Round Vial Adaptor CD with User s Guide and Spreadsheet Interface Software Trilogy Fluorometer User s Manual P N 998 7210 Page 33 of 38 TURNER D DESIGNS Appendix D Principles of Fluorescence Principles of Fluorescence Fluorescence is a physical property of certain atoms and molecules It is a molecule s ability to absorb light energy at one wavelength then instantaneously re emit light energy of another usually longer wavelength Each compound that fluoresces has a characteristic excitation wavelength the wavelength of light that it absorbs and a characteristic emission wavelength the wavelength of light that it emits when the molecules relax and return to their ground state These excitation and emission wavelengths or spectra are often referred to as the compound s fluorescence signature Figure 13 shows the key components of a filter fluorometer Digital Readout Light Detector Wavelengths specific to compound Emission Filter Wav
16. Ave Sunnyvale CA 94085 Phone 408 749 0994 FAX 408 749 0998 www turnerdesigns com P N 998 7210 Page 38 of 38
17. Ea Ea Ea eo Acidification module and confirm by Si ID Ea ee 0 00 FSU c On the home screen touch Calibrate to begin a calibration sequence Trilogy Fluorometer User s Manual P N 998 7210 Page 20 of 38 TURNER D DESIGNS d Select Run New Calibration e Select the unit of measurement f Insert calibration blank and touch OK 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 before Acidification Fb should be inserted Insert sample and touch OK Now insert the Standard after Acidification and touch OK The Fa value will be measured and the ratio of the two readings will be displayed as seen in the next step Trilogy Fluorometer User s Manual P N 998 7210 Page 21 of 38 TURNER D DESIGNS j Ifthe ratio is within the required range touch OK The ratio will be stored in the Trilogy for use in the EPA Method 445 0 for measurement of chlorophyll a and pheophytin a in water k After the calibration is complete either select Proceed with Current Calibration or select Enter More Standards in which case return to
18. 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 TURNER D DESIGNS 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
19. TURNER E DESIGNS Reliable Instruments for an Unreliable World 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 408 749 0994 FAX 408 749 0998 www turnerdesigns com TURNER D DESIGNS This Page Intentionally Left Blank Trilogy Fluorometer User s Manual P N 998 7210 Page 2 of 38 TURNER D DESIGNS Highlights of the Trilogy Fluorometer Sample Chamber accepts snap in Application Modules for Fluorescence Absorbance and Turbidity Multifunctional Compact and Modern Design provides flexibility and precision measurements Spreadsheet Interface Software provides real time display and logging of data to Excel Trilogy saves time by storing Spreadsheet up to 18 named calibrations Automatically calculates and displays Extracted Chl a concentrations using filtered and solvent volumes Color Touch Panel Graphics User Interface provides intuitive ease of use Trilogy Fluorometer User s Manual P N 998 7210 Page 3 of 38 TURNER D DESIGNS 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 Il Fluorometer Operation 8 Touch screen Basics Fluores
20. URNER D DESIGNS 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 After the calibration is complete either select Proceed with Current Calibration or select Enter More Standards in which case return to g above 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 Trilogy Fluorometer User s Manual 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 P N 998 7210 Page 19 of 38 TURNER D DESIGNS F Extracted Chlorophyll Measurements with the Chl a Acidification Fluorescent Module EPA Method 445 0 is a standard method for measuring extracted chlorophyll a and pheophytin a in marine and fresh water alg
21. ae by fluorescence It requires extraction with 90 acetone measurements before and after acidification and some fairly simple calculations to arrive at the chlorophyll a and 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 a in 90 acetone to recalibrate your instrument Liquid Primary and Solid Secondary Chlorophyll a standards 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
22. ase note the Trilogy does not store more than 20 measurements at one time Measurements are not stored between power cycles Trilogy Fluorometer User s Manual P N 998 7210 Page 26 of 38 TURNER D DESIGNS Vil Systems Connections Establish a connection between the Trilogy and a PC or a printer to export data Connect the 9 pin RS 232 serial cable between the Trilogy and the PC or printer The male 9 pin connector attaches to the Trilogy and the female connector attaches to the PC Serial Port connect to the l serial port of your computer Connection Serial Output Functional Check To perform a functional check on the Trilogy Serial Output complete the above for the Trilogy and then complete the following steps note that the Serial Interface Software is not required 1 Connect the Trilogy Serial Port to the PC Serial Port 2 To receive ASCII data from the Trilogy open a HyperTerminal program or some other appropriate data logging software on the PC 3 Open a connection in HyperTerminal with the following COM Port settings Baud Rate 9600 Data Bits 8 Parity None Stop Bits 1 Flow Control None 4 Make a measurement with the Trilogy At the completion of the measurement the concentration value will be displayed on the Trilogy display window and also displayed in the HyperTerminal window This completes the Functional Check of the Trilogy Serial output If you successfully completed these steps the Tr
23. ately press OK to measure 12 If running a multi point calibration repeat step 9 through 11 for each calibrant Blank Subtracting Blanks provide background fluorescence values of samples excluding the fluorophore of interest Subtracting a blank sample from subsequent samples increases accuracy of fluorophore estimates An accurate blank is typically a water sample that has been filtered through a GF F or membrane filter in order to remove the algal cells but to still retain additional dissolved components reget the Solid Secondary Standard To establish a correlation between a known chlorophyll a concentration and the fluorometer reading measure a sample containing algae and note the fluorometer reading 2 Insert the solid standard in the optical module and adjust the solid standard to produce the same reading on the fluorometer as in step 1 Turning the solid standard screw clockwise produces a lower signal 3 Next perform a chlorophyll a extraction using the Trilogy Laboratory Fluorometer to determine the actual chlorophyll a concentration in the sample This will provide the correlation between the solid standard and the actual chlorophyll a concentration 4 The solid secondary standard can now be used to recalibrate and or check the stability of the Trilogy fluorometer Trilogy Fluorometer User s Manual P N 998 7210 Page 37 of 38 TURNER D DESIGNS Trilogy Fluorometer User s Manual Turner Designs 845 W Maude
24. cence 8 Measuring Samples 9 Ill 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 a Acidification 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 Vil 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 Troubleshooti
25. continental United States who purchased equipment from one of our authorized distributors contact the distributor If you purchased directly contact us We will repair the instrument at no charge Customer pays for shipping duties and documentation to Turner Designs Turner Designs pays for return shipment custom duties taxes and fees are the responsibility of the customer Out of Warranty Service Follow steps for Warranty Service as listed above If our Technical Support department can assist you by phone or correspondence we will be glad to at no charge Repair service will be billed on a fixed price basis plus any applicable duties and or taxes Shipment to Turner Designs should be prepaid Your bill will include return shipment freight charges Address for Shipment Turner Designs Inc 845 W Maude Ave Sunnyvale CA 94085 Trilogy Fluorometer User s Manual P N 998 7210 Page 32 of 38 TURNER D DESIGNS Appendix C Specifications Fluorescence Module Performance Minimum Detection Limit Chlorophyll a 0 025 ug L Rhodamine WT 0 01 ppb Linear Range 0 300 ug L Chl a 0 500 ppb RWT Weight Linearity 8 1 Ibs 3 65 kg 0 99R Humidity 75 RH maximum Turbidity Module Performance Warranty Minimum Detection Limit One year 0 05 NTU Linear Range Application Module Filter Wavelengths 0 1 000 NTU Linearity Fluorescence Modules 0 99R oar Part Excitation Emission Application Number Wavelength Wavelength
26. d Trilogy Fluorometer User s Manual P N 998 7210 Page 9 of 38 TURNER D DESIGNS 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
27. dle 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 Optical Kit 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 TURNER D DESIGNS Sample Count The display increments by 1 each time a measurement is made Touch here to go to a selection of help and troubleshooting screens Touch here to bring up the Home Screen from other windows Provides access to Settings and Diagnostic screens Indicates module selected on module screen at instrument turn on Touch here to initiate calibration routines Touch here to
28. e Cause Possible Solution Excel is not installed Make sure Excel is on the PC installed on your PC The software cannot find Excel Wrong COM port selected Open Excel from the Programs Menu on the PC then open the spreadsheet interface software Click STOP then click on the COM button to change the COM port Fluorometer not connected to PC There is an editing process occurring within an Excel cell Trilogy Fluorometer User s Manual P N 998 7210 Check the RS 232 connection between the Trilogy and the PC Wait until all the data is collected before editing the Excel spreadsheet Page 30 of 38 TURNER D DESIGNS Log in as Administrator for the PC then install the software or contact your IT support desk The software The PC allows only does not administrators to install install new software Log in as Administrator and Remove the software and re install The software was not The software installed properly does not open Trilogy Fluorometer User s Manual P N 998 7210 Page 31 of 38 TURNER D DESIGNS Appendix B Warranty and Obtaining Service Warranty Turner Designs warrants the Trilogy and accessories to be free from defects in materials and workmanship under normal use and service for a period of 12 months from the date of shipment from Turner Designs with the following restrictions e Turner Designs is not responsible for replacing parts damaged
29. e 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 Users 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 TURNER D DESIGNS Setup Place
30. econdary Standard P No 8000 952 with most of the Trilogy fluorescence modules Chlorophyll a Rhodamine WT etc but not UV or Turbidity modules The two main benefits of using the Solid Secondary Standard are 1 It can be used in place of a primary liquid standard once a correlation between a primary standard and the solid standard has been established Figure 11 The Adjustable Secondary Standard includes a fluorescent rod that provides 2 Itcan be used to check the fluorometer stability and or check an extremely stable signal for loss in sensitivity resulting from instrument optical module problems The Solid Secondary Standard provides a very stable fluorescent signal It has an adjustment screw so that you can tune the Solid Standard to provide a signal to match a specific sample It should be noted that each Solid Standard Fluorometer relationship is unique This means that a given Solid Standard can not be used to calibrate multiple fluorometers or modules for identical readings of a given solution L Using the Solid Secondary Standard for in vivo Chlorophyll Applications 1 To establish a correlation between a known chlorophyll concentration and the fluorometer reading measure a sample containing algae and note the fluorometer reading 2 Insert the Solid Standard in the Optical Module and adjust the Solid Standard to produce the same reading on the fluorometer as in step 1 turning the Secondary Standard adjustment
31. elengths created by compound plus stray light Lamp Cuvette or Sample Cell Edition Filter Many wavelengths of light Specific wavelengths of light Figure 13 Lamp The lamp or light emitting diode provides the light energy that excites the compound of interest The lamp actually provides a broader range of light than that which excites the compound This broad light range is illustrated by the many wavelengths of light shown in Figure 13 Trilogy Fluorometer User s Manual P N 998 7210 Page 34 of 38 TURNER D DESIGNS Appendix E Recommended Measurement Practices Linear Range and Quenching The linear range is the concentration range in which the Trilogy output is directly proportional to the concentration of the fluorophore The linear range begins with the smallest detectable concentration and spans to an upper limit concentration that is dependent upon the properties of the fluorescent material the filters used and the path length A non linear relationship is seen at very high concentrations where the fluorescence signal does not increase at a constant rate in comparison to the change in concentration see Figure 4 below At even higher concentrations the fluorescence signal will decrease even though the sample concentrations are continuing to increase This effect is known as signal quenching Linearity may be checked by diluting a sample 1 1 or some other convenient ratio If the sample is still
32. hase of fluorometer Cetana 1 Concentration of standard 1 blank Fluorescence of Blank value F stand 1 B Fluorescence of standard 1 before acidification stand 1 A Fluorescence of standard 1 after acidification m Acidification Ratio Fstanaj1 B8 Fblank Fstanatt A Fblank ll Variables required from the sample analysis phase Fsamp B Fluorescence of sample before acidification Fsamp A Fluorescence of sample after acidification V solvent Volume of solvent used to extract sample Vwater Volume of water filtered lll Interpolation equation used in end calculation of chlorophyll a and pheophytin concentrations Interp s Cstanaj Fsamp B Fotank Fstanaj1 B Folank Interp a Cetanatt Fsamp a Fbiank Fstanaj1 B Folank IV End calculation for corrected chlorophyll and pheophytin a Chlorophyll a concentration Fm Fm 1 Interp s Interp a Vsoivent Vwater Pheophytin a concentration Fm Fm 1 Fm Interp a Interp s Vsoivent Vwater Non Acidification Method I End calculation for corrected chlorophyll a Chlorophyll a concentration Cstanaj F samp Folank Fstanaj Foiank Vsoivent Vwater Trilogy Fluorometer User s Manual P N 998 7210 Page 36 of 38 TURNER D DESIGNS Appendix G In vivo fluorescence measurements using Trilogy In vivo chlorophyll a analysis is the measurement of chlorophyll a fluorescence within a living cel
33. ilogy Serial Output is functioning If you were not able to complete these steps refer to the troubleshooting section of this manual Trilogy Fluorometer User s Manual P N 998 7210 Page 27 of 38 TURNER D DESIGNS VIII Spreadsheet Interface Software Installation Install the Spreadsheet Interface Software SIS to send data to an Excel spreadsheet The Spreadsheet Interface Software requires a PC loaded with Windows 98 or higher an available serial port and Excel Insert the software CD ROM into the CD ROM drive of the PC to initiate the installation program After the installation is complete an icon for the SIS appears on the PC desktop and in the Programs menu Make sure no other program on the PC is already using COM 1 ioii Help Status PEI T COM 1 x MS EXCEL Ready Start Pause Close Figure12 Spreadsheet Interface Software status window showing communications are set up on COM 1 and that the data will be displayed in an EXCEL spreadsheet Excel Spreadsheet Example Turner Designs SAMPLE 001 426 73 RFU SAMPLE 002 24 57 RFU SAMPLE 003 35 49 RFU SAMPLE 004 2 56 RFU Trilogy Fluorometer User s Manual P N 998 7210 Page 28 of 38 TURNER D DESIGNS Appendix A Troubleshooting Fluorescence Troubleshooting Possible Solution A bad calibration error message may occur if the Bad calibration blank is brighter than the standard Compare the error message read
34. ing of the standard and the blank in the Raw mode When direct fluorescence readings do not Erratic reading produce expected values review the standard value entered during the calibration The number of the standard value should correspond to the actual concentration of the standard After calibration the blank value is automatically Negative values subtracted from subsequent readings A negative reading can occur if a sample reading is less than the blank This may also indicate that the module is not properly snapped into place Press down on the module and listen for a Click indicating the module has properly been installed Check the excitation and emission wavelengths of the analyte against the specifications of the Low readings Fluorescence Optical Application Module in use Different analytes require different Optical Application Modules A wet cuvette or spill could contaminate the High cuvette holder and increase the background background signal Carefully clean the cuvette holder with 70 ethanol Absorbance Troubleshooting Possible Solution Many absorbance assays do not produce a Non linear linear response but instead produce a sigmoidal response or pseudo sigmoidal response Refer to the Application Note for the assay for more information Check the filter installed in the Absorbance Low readings Module and make sure it is the correct filter for the assay View the Calibration details from the Tools menu I
35. l The advantage of this type of analysis is that it is quick and simple and does not require special sample preparation or extraction It allows the user to measure a large number of samples in the field However without comparisons to extractive analysis in vivo readings are qualitative in nature There are two modes for making in vivo measurements The first utilizes the RAW fluorescence mode the second requires calibrating to a secondary standard No calibration is necessary for making in vivo measurements in the raw mode In vivo measurements using the RAW mode Insert the in vivo chlorophyll a Trilogy module Turner Part Number 7200 043 Turn on the Trilogy fluorometer using the switch located on the back panel 3 Select the Blue module This is indicated on the module label under GUI selection Press OK after verifying that the module loaded matches your selection The default mode loaded is Raw Fluorescence 5 Before measurements are made it is advisable to filter a sample and measure the filtrate to obtain a blank or Background fluorescence reading for a given location 6 Thoroughly mix the sample by inverting or shaking to prevent settlement of algal cells and quickly insert sample 7 Close the lid and press the Measure Fluorescence button 8 Subsequent readings will indicate relative changes in concentration levels These readings will be presented in Relative Fluorescence Units of measure RFU
36. ment screw clockwise reduces the displayed concentration Comprehensive information on dye trace measurements can be found at the following Turner Designs URL http www turnerdesigns com t2 doc appnotes main html fluorescent N Trilogy Calibration Procedure Absorbance Module Calibrate the Trilogy after powering up and after changing filters For best results calibrate the Trilogy and Absorbance Optical Module immediately before reading a series of samples Instructions Sample Screen a Turnon the Trilogy b Touch Absorbance to select the Absorbance Application Module and calibration firmware and confirm by touching OK c On the home screen touch Calibrate to update the blank Trilogy Fluorometer User s Manual P N 998 7210 Page 24 of 38 TURNER D DESIGNS d Insert a sample cuvette containing your blank sample and touch OK to complete the calibration update Sample ID SAMPLE 001 0 000 Ab e When the blanking is complete the display will revert to the Absorbance Home Measurement Screen f This completes the Absorbance calibration procedure and the Trilogy is now ready to make Absorbance measurements Trilogy Fluorometer User s Manual P N 998 7210 Page 25 of 38 TURNER D DESIGNS VI Touch Screen Basics Touch Screen The touch screen provides a user friendly method to operate the Trilogy The touch screen is sen
37. ng 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 Trilogy Fluorometer User s Manual P N 998 7210 Page 4 of 38 TURNER D DESIGNS 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 Th
38. nstall the proper filter and use the ultrapure Bad Calibration water in a clean cuvette to update the zero Error Message Check the Calibration details from the Tools menu Trilogy Fluorometer User s Manual P N 998 7210 Page 29 of 38 TURNER D DESIGNS Turbidity Troubleshooting Possible Solution Trilogy readings do not agree with other Turbidity meters The turbidity readings change each time a reading is taken My turbidity readings seem to be different when re calibrated with a new primary standard Calibrate both meters with the same calibration standard solution If meters still display significantly different readings it may be that the second turbidity meter does not make an IR measurement and the sample contains interference colors This is normal Particles in a liquid sample do not remain in the same position and these position changes affect the scattering of the light and therefore the turbidity reading Formazine standards form the basis of all turbidity measurements and they are very susceptible to aging ISO 7027 recommendation specifies that the 4 000 NTU Formazine solution can be kept for only 4 weeks For consistent readings calibrate with current standards Spreadsheet Interface Software Troubleshooting Excel does not open Excel does not open Both green lights are on but data does not appear in Excel New data does not report to Excel Symptom Possibl
39. ovides switching Settings and Diagnostic calibration routines between RAW and Direct screens Concentration modes 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 Trilogy Fluorometer User s Manual P N 998 7210 Page 15 of 38 TURNER D DESIGNS 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 repor
40. sitive to the light pressure of a fingertip It is not necessary to use a stylus After 20 minutes without activity or user stimulation the touch screen hibernates Lightly touch the screen once to reactivate To select a function touch the button corresponding to the function once Tools Touch the Tools key to access Settings and Diagnostics Settings Contrast Touch the Contrast key to increase or decrease the brightness of the touch screen and enhance visibility The arrows increase or decrease contrast Touch the Home key to save the adjustment and return to the Home screen Reset The Reset button restarts the Trilogy Normal operation does not require this feature The Reset feature erases data displayed on the Home screen Lid Start Touch the Lid Start key to turn the feature ON While the Lid Start feature is ON measurement begins as soon as the lid closes and the touch screen does not hibernate The Lid Start feature allows for immediate measurement and eliminates the need to touch the Measure key Return to the Lid Start key under the Settings menu to turn the feature OFF Diagnostics Touch Screen Calibration The Diagnostics menu contains a method for screen calibration Although the touch screen is calibrated at the factory it may need re calibration over time Device Configuration The Device Configuration key contains useful information on firmware revisions and instrument setup General Ple
41. sorbance Module Grasp the handle and gently pull up to release it from the sample compartment 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 Trilogy Fluorometer User s Manual P N 998 7210 Page 11 of 38 TURNER D DESIGNS 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 Logio T Where Zz zero b baseline s signal Sample Count The display increments by 1 each time a measurement is made Touch here to go to a selection of help and troubleshooting screens Touch here to bring up the Home Screen from other windows Provides access to Settings and Diagnostic screens Indicates module selected on module screen at instrument turn on 29 590 Touch here to initiate calibration routines Figure 8 Home Screen When Using Absorbance Module Calibration See Section V Mea
42. suring Samples Touch here to make indicated measurement display shows that Absorbance Units Ab have been selected Measurement Readout updates each time a new reading is taken Measurement buffer holds and displays up to 20 readings Sample 001 thru Sample 003 shown Touch Mode to select measurement units Ab or T 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 Trilogy Fluorometer User s Manual P N 998 7210 Page 12 of 38 TURNER D DESIGNS 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
43. ts 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
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