Aquafluor™ - Photon Systems Instruments
Contents
1. PC with Windows 95 or later e MS Excel 5 0 or later e Atleast 1 available serial port B3 Software Installation 1 Exit out of any programs that are running Insert the CD Disk and select the setup exe file 3 Follow the steps in the setup wizard to install the necessary files 4 When the setup is complete an icon named Spreadsheet Interface Software will be found on the desktop and in the Programs menu list B4 Connecting 1 Using the cable provided connect the 9 pin adapter end of the cable into the available serial port of your computer 2 Plug the opposite end of the cable into the port at the base of the Aquafluor 3 Open the Spreadsheet Interface software Aquafluor User s Manual 28 4 Click on the box to the right of the COM port icon to select the appropriate COM port This is typically COM port 1 or 2 5 Click on Start and the program will open an Excel spreadsheet for the data transfer The indicator boxes to the left should both be green to confirm a good interface connection 6 Follow the directions from Section 3 7 for collecting and downloading data from the Aquafluor Data will appear in the Excel spreadsheet BE SURE to save this data by performing a Save File as after the download has completed B5 Real Time Data Transfer Data can also be transferred directly to the computer after each reading To do so 1 Follow steps 1 6 of Section B4 to create the con2. e If extra cleaning is needed use a mild detergent to dampen the towel for cleaning Aquafluor User s Manual 5 Do not submerge the Aquafluor in water Do not expose the Aquafluor to temperatures outside the specified range of 5 to 40 C or damage may occur to the unit that will not be covered under warranty 2 Quick View Diagrams Aquafluor User s Manual 6 OOOO 5 DOO Aquafluor User s Manual 3 Instrument Operation and Calibration 3 1 Instrument Power Up To turn on the Aquafluor press the lt ON OFF gt button After a 5 second warm up the Aquafluor is ready for operation Pressing the lt ON OFF gt button again will turn the unit off or if left idle for 3 minutes the unit will turn itself off to save battery power A set of new batteries will last for over 1 000 sample readings If the batteries have low power or are not positioned properly the following warning message will be displayed Batt Level gt 20 Caution The contrast of the display can change with temperature and will get lighter as the unit gets colder The contrast can be easily adjusted Press the 1 arrow button to darken the contrast and press the J arrow to lighten the display contrast 3 2 Detection Channel Configuration The Aquafluor has 2 detection channels that are configured as two fluorescent channels or one fluorescent and one turbidity channel You shou
3. The solid secondary standard is useful for checking the reading stability over time and can also be used to recalibrate if needed The Aquafluor is designed with ambient light rejection The black sample compartment cover does NOT need to be closed when calibrating or reading samples This allows for the use of cuvettes with different height dimensions such as the 12x75 mm round glass tubes The orientation and cleanliness of the cuvettes can have an impact on the accuracy of your results Aquafluor User s Manual 10 Refer to Section 4 Sample Analysis Guidelines for information to help insure the best results for your analysis 3 4 1 Assign a Calibration Standard Value This defines the numeric value that you want the standard to read For example if you calibrate with a primary standard that has a concentration of 50 ug L then you will set the value to 50 and the implied unit of measure will be in ug L If you use a Secondary standard to calibrate with you will set this value to a desired relative value and call the units RFU Relative Fluorescent Units 1 Press the lt STD VAL gt button 2 Use the T and 4 arrow buttons to set the standard value Holding down either arrow button down will allow you to change the value using fast scrolling 3 When finished Press the lt ENT gt or lt ESC gt button to accept the value and to return to the Home screen 3 4 2 Perform the Calibration Press the
4. etc The two most commonly used dyes tracers are Fluorescein and Rhodamine WT The Fluorescein dye can be detected using the Blue channel on the Aquafluor and Rhodamine dye is detected using the Green channel The linear detection range for both dyes is 0 4 to 300 PPB active ingredient in potable water For more information on tracer dye use refer to the Application Note section at the following web link http www turnerdesigns com t2 doc appnotes main ht ml Aquafluor User s Manual 35 Appendix C6 _Cyanobacteria Monitoring The Cyanobacteria channel of the Aquafluor continually senses the concentration of either phycocyanin PC or phycoerythrin PE fluorescent pigments unique to Cyanobacteria Phycobilin pigments are a group of accessory pigments unique to Cyanobacteria PC and PE are two phycobilin pigments that also happen to have strong and unique fluorescent signals that can be detected by the Cyanobacteria channel The fluorescence is measured directly using in vivo cyanobacteria detection without extraction or chemical treatment For many types of qualitative work in vivo measurements alone may provide sufficient information For quantitative measurements the in vivo data is calibrated by correlation with other measurements such as cell counts or extracted pigment analysis Aquafluor User s Manual 36
5. lt CAL gt button 2 Press lt ENT gt to start the calibration 3 Insert your blank sample and press lt ENT gt The Aquafluor will average the reading for 10 seconds and set the blanking zero point 4 Insert the standard sample and press lt ENT gt The reading is averaged for 10 seconds and the Standard Calibration value is set 5 Press lt ENT gt when the calibration is complete to accept the calibration If lt ENT gt is not pressed within 10 seconds you will be asked if you want to abort the calibration Aquafluor User s Manual 1 Press the 1 or J arrow button to abort or accept the calibration respectively If at anytime during steps 1 4 you want to stop the calibration press lt ESC gt This will return you to the Home screen and will default the instrument to the previous calibration 3 5 Instructions for using the Adjustable Secondary Standard 1 Calibrate the Aquafluor with a solution of known concentration 2 Take the adjustable secondary standard from its storage bag Using the 0 05 allen wrench provided loosen the locking screw on the back of the secondary standard by turning it counterclockwise one turn See photo 1 Photo 1 Loosening the locking setscrew on the backside of the adjustable secondary standard 3 Place the adjustable secondary standard in the fluorometer sample compartment with the handle towards the rear of the instrument See photo 2 Photo 2 Placing the adj
6. see Section 2 for diagram Aquafluor User s Manual 4 2 Install the 4 AAA batteries and verify the batteries are positioned correctly 3 Replace the battery panel and press it down into position 4 Hold the battery panel down in place and gently tighten the screw until it is snug Do not over tighten the screw Note If the battery panel is very difficult to press down or remove you can apply a small amount of silicon based o ring grease to lubricate the o ring as needed 1 3 General Information Precautions and Cleaning e The sample compartment cannot accept 10mm square glass or quartz cuvettes because their cuvette wall thickness exceeds the 12 mm maximum outer dimension that the Aquafluor can accept The 10 mm size is the internal dimension of the cuvette and some plastic cuvettes may also be too large e Do not force oversized cuvettes into the sample compartment This can damage the sample compartment If the cuvette does not easily fit down inside the Aquafluor you will need to purchase different cuvettes e Round glass test tubes can be used with an optional adapter that is available Refer to Appendix A5 for cuvette information e Use caution around solvents because they may attack the plastic case of the Aquafluor e Ifasample is accidentally spilled inside the Sample Compartment you can invert the Aquafluor to drain out the excess liquid Then wipe the inside area dry with a clean soft towel or tissue
7. the new fluorometric technique has been proven to provide accurate and precise data over a wide range of water quality ammonium concentration and salinity This method is particularly useful for work in oligotrophic systems where natural ammonium concentrations are commonly in the submicromolar range The following fluorometric method is available at the web link below Holmes R M A Aminot R Kerouel B A Hooker B J Peterson 1999 A simple and precise method for measuring ammonium in marine and freshwater ecosystems Can J Fish Aquat Sci 56 1801 1802 http www turnerdesigns com t2 doc appnotes pdf ammonium pdf Aquafluor User s Manual 34 Appendix C4 Turbidity The Turbidity channel has a detection range of 0 5 to 150 NTU A Primary Turbidity Standard is required for calibration We recommend using a Turbidity standard in the range of 10 to 100 NTU The Polystyrene cuvettes are preferred for best turbidity results Turbidity standards can be purchased from APS Analytical Stds a division of GFS Chemicals When selecting these AMCO Standards select the type for use with non ratio instruments For more details refer to the following web link http www amcoclear com amco index php Appendix C5 Fluorescent Tracer Dyes Fluorescent tracer dyes provide an accurate cost effective method for measuring water flow levels mixing zones time of travel groundwater transport leak detection retention times
8. Extracted NH4 UV Chlorophyll Channel Channel Light Source UV LED Blue LED Limit of Detection 0 5u g L Max range 50 uM 300 u g L Temperature N A 0 3PC coefficients Linear A4 Label Designations for Optical Configurations Applications Turbidity UV 420 375 gt 420 Ammonium UV 445 375 445 i lt 2 UV 480 375 480 Po UV 405 375 405 PF Aquafluor User s Manual 26 A5 Cuvette information Cuvette type Required for UV Methacrylate 10 mm Square lt 400 nm 7000 959 plastic height 44 5 works for all mm applications Do NOT use with solvents ie Acetone Preferred for Polystyrene 10 mm Square Turbidity use 7000 957 Plastic height 47 5 will NOT work mm for UV Do NOT use with solvents Required for Borosilicate Extracted Chlor 10 029A glass where solvents are used Requires Adapter PN 8000 932 A6 Optical Configuration for Cyanobacteria Phycocyanin Phycoerythrin Excitation 595 nm 528 nm Optics Limit of 150 cells mL 150 cells mL Detection cells mL cells mL Coefficients Aquafluor User s Manual 27 Appendix B Internal Data B1 Shipping Checklist The Internal Data Logging kit PN 8000 920 contains the following items e Interface cable e Turner Designs Spreadsheet Interface Software on a CD disk Both of these items are necessary for downloading or transferring data from the Aquafluor to a PC B2 Hardware Requirements
9. Standard Value 10 this will give relative reading values that will be proportional to the actual chlorophyll concentration in the water sample The Aqaufluor calibration also requires a Blank sample and the best true blank is the natural water that has been filtered through a GF F or membrane filter in order to remove the algal cells but to still retain any dissolved components However in most cases distilled water is used for the Blank sample since the in vivo readings are semi quantitative Calibration of the Fluorometer 40 E S 5 2 3 8 Measured Chiorophyil Graph C1 For more details on chlorophyll analysis visit Turner Design s webpage at this link http www turnerdesigns com t2 esupport home html Aquafluor User s Manual 32 Appendix C2 Extracted Chlorophyll a In extractive analysis fluorometric measurements are made on solvent extracts from algal cells to determine quantitative concentrations of chlorophyll and pheophytin Acetone methanol or DMSO can be used as the extraction solvent A Primary Chlor a standard is used for calibration and the measurements are made using the 12 x 75 mm glass test tubes The EPA Method 445 is a popular Chlorophyll extractive method that was developed using a Turner Designs Model 10 fluorometer and is published by the United States Environmental Protection Agency The extracted Chlorophyll channel on the Aquafluor requires performing the acidification ste
10. and result in errors Use a clean cuvette for all readings If you are using the same cuvette for your samples it is very important that you thoroughly clean the cuvette between samples A good way to confirm the cuvette cleanliness is to read a blank solution If the reading is higher than the normal blank reading the cuvette is not clean 5 Any bubbles in the sample will effect the readings Take care not to introduce bubbles into samples Remove any bubbles by lightly tapping with your finger on the outside cuvette wall or cover the top of the cuvette and tilt the sample to help dissipate bubbles Aquafluor User s Manual 19 4 2 Positioning Samples The orientation of the cuvette in the sample compartment can give slightly different readings especially for low concentration samples This is due to variations in the walls of the cuvette that are not readily visible to the eye We recommend that the cuvette be marked at the top on one side and positioned in the sample compartment the same way each time for best results Turbidity sample measurements are particularly sensitive to the quality and cleanliness of the cuvette Small scratches or smudges on the cuvette will effect the accuracy of the readings The Polystyrene cuvettes P N 7000 957 give the best Turbidity measurement results due to better quality of the cuvette 4 3 Linear Range and Quenching The linear range is the concentration range in which the readout of
11. the Aquafluor is directly proportional to the concentration of the fluorophore in the sample The linear range begins with the lowest detectable concentration and spans to an upper limit concentration that is dependent on the properties of the fluorescent material and the cuvette optical path length For Rhodamine Fluorescein dye or Chlorophyll in the 10 mm square cuvette the upper limit of linearity is approximately 300 ppb ug L At concentrations above this upper limit the fluorescence reading will not increase at a linear rate in comparison to the change in concentration At concentrations 10 times higher than the upper limit the readings will start to decrease even though the sample concentration is increasing Aquafluor User s Manual 20 This effect is known as sample quenching and is due to light absorption losses in the sample See the Figure graph below When you start to see visual color in the sample this is an indicator that the sample may be above the upper limit of linearity The linearity can be verified by diluting a sample 1 1 or another convenient dilution ratio If the sample is within 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 original sample concentration was above the linear range Cal Standard Value Numeric value assigned to represent the signal level det
12. Aquafluor Handheld Fluorometer and Turbidimeter User s Manual Dated September 2004 Version 1 3 P N 998 0851 845 W Maude Avenue Sunnyvale CA 94085 ai 408 749 0994 FAX 408 749 0996 wew tumerdesigns cam Toll free 1 877 316 8049 TURNER DESIGNS Table of Contents 1 Introduction 1 DeSCription 4 1 2 Inspection and Setup ee eeeeeeeees 4 1 3 General Information Precautions amp Cleaning 2 Quick View Diagrams 0 000 ce eeeeeeeeneteeeeees 6 3 Instrument Operation and Calibration 3 1 Instrument Power 11 8 3 2 Detection Channel Configuration 0 00 8 3 3 Calibration 1 9 3 4 Calibration Procedure oes 10 3 5 Instructions for using the Adjustable 12 Secondary Standard 3 6 Sample 818186 3 7 Diagnostic Information 3 8 Internal Data Logging IDL 3 8 1 Activate Data Logging s es 15 3 8 2 Download 8 15 3 8 3 Erase Data ets 3 9 Display Screen 1000 17 4 Sample Analysis Guidelines 4 1 Handling Samples 0 eeeeeeeeeeeeees 4 2 Positioning Samples 4 3 Linear Range and Quenching 44 Temperature Considerations 0 ee 21 45 Data Quality Sare 22 5 Warranty 5 2 Warranty Service 5 3 Out of Warranty Service Aquafluor User s Manual 2 Appendices A Instrument Specifications Al General Specifications eee 25 A2 A4 Optical Configurations seseseeseses 25 AS Cuvette Information se sess
13. WN gt A Press lt DIAG gt then lt ENT gt to toggle between Logging Poi Left XXX d an Reading Cal Soln Calibration Completed lt ENT gt FS Blk X XX If lt ENT gt is not pressed FS STD XX XX within 5 seconds this screen will appear Abort Cal lt UP gt Yes lt DOWN gt No Aquafluor User s Manual 17 Press lt DATA gt Datalogger lt DATA gt to toggle Press lt DATA gt 1X Press lt DATA gt 2X Press lt DATA gt 3X Download data Erase Data 5X lt ENTS gt to start 5X lt ENT gt to start Status Stop lt ENT gt to toggle lt ENT gt Download data Erase Data All downloaded All data erased If there is no data logged this screen will appear Status Logging lt ENT gt to toggle Data logger has no valid data Aquafluor User s Manual 18 4 Sample Analysis Guidelines 4 1 Handling Samples 1 Take care not to spill samples into the sample chamber Wipe up any spills promptly 2 The cuvette MUST BE DRY on the outside when taking readings Any Moisture or condensation on the outside of the cuvette can effect the reading 3 Fill the cuvette with at least 2mL solution volume or at least 50 full Significant error in the readings can result if the cuvette contains less than this minimum volume 4 The Aquafluor is very sensitive and even small amounts of material from a previous sample may contaminate the sample
14. and to help decide on the best action to resolve the issue as quickly as possible 5 3 Out of Warranty Service Proceed exactly as for Warranty Service above If our Technical Support 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 Shipment to Turner Designs should be prepaid Your bill will include return shipment freight charges Address for Shipment Turner Designs 845 W Maude Ave Sunnyvale CA 94085 Aquafluor User s Manual 24 Appendix A Instrument Specifications Al General Specifications Specification Description Size 1 75 x 3 5 x 7 25 4 45cm x 8 9cm x 18 4cm 3907 0 4ke Dynamic range 3 orders of magnitude LCD Display 2 x 16 characters Case Meets IP 67 Standard dustproof and waterproof Temperature 41 104 F 5 40 C Error message Low battery High blank A2 Optical Configurations for in vivo Chlorophyll Rhodamine and Turbidity in vivo Chlor Rhodamine Turbidity Channel Channel Channel Light Source Blue LED Green LED Green LED Excitation 460420nm 540420nm 515 10nm Optics Emission gt 665nm gt 570nm 515 10nm B eee a Limit of 0 25ug 1 0 4ppb 0 5NTU gt 300 ppb gt 300ppb gt 150 NTU Temperature 1 4 C 0 026 C N A coefficients Linear Exponential Aquafluor User s Manual 25 A3 Optical Configurations for Ammonium and Extracted Chlorophyll Ammonium
15. easurements with the Aquafluor use the secondary standard to verify the Aquafluor calibration If the secondary standard value has changed by more than 10 of its assigned value then the Aquafluor should be recalibrated using the Secondary Standard 3 6 Sample Analysis 1 Insert your sample The orientation and cleanliness of the cuvettes can have an impact on the accuracy of your results Refer to Section 4 for more details 2 Press either lt READ gt button The instrument will measure and average the fluorescence signal for 5 seconds 3 The reading result will be displayed on the top line of the Home screen 4 The top left corner will then display WAIT for 5 seconds Once WAIT disappears another sample reading can be performed 3 7 Diagnostic Information 1 Press lt DIAG gt to access the diagnostic screens 2 The first screen shows the number of data points available for internal data logging 3 Press lt ENT gt to toggle to the FS Full Scale values for the blank Blk and standard STD calibration points The STD value should be at least 3 times larger than the Blk value to insure consistent results This is often referred to as the Standard to Blank Ratio Aquafluor User s Manual 14 4 Press lt ESC gt when finished to return to the Home screen 3 8 Internal Data Logging IDL The Internal Data Logging option allows the downloading of stored data from the Aquafluor For further
16. eded fran the Solid Secondary Calibration Standard Blanking Level Blarking is done uanga filtered sarnple of the water being tested Distiled water Btsnk Caiteation 4 can be used for in vivo dad C meal N chlorophyll measurements Since readings are semi 4 Quanbtative ap p or Valid Calibration Blank Ratio 6303100 a Blanking Level Figure 1 Linearity and Calibration Response Curve Aquafluor User s Manual 21 4 4 Temperature Considerations Fluorescence is temperature sensitive As the temperature of the sample increases the fluorescence decreases For best accuracy make sure your samples are at the same temperature as the calibration standard Some temperature coefficients examples are Rhodamine dye is approx 2 6 per degree C and Fluorescein dye is 0 36 per degree C 4 5 Data Quality The Aquafluor is only as accurate as the standards that are used to calibrate it This is why it is important to take care when preparing standards samples and blank One should follow good laboratory practices when preparing all solutions and samples 5 Warranty 5 1 Terms Turner Designs warrants the Aquafluor Fluorometer and accessories to be free from defects in materials and workmanship under normal use and service for a period of one year from the time of initial purchase with the following restrictions The instrument and accessories must be installed powered and operated i
17. essseeseessssresseeresee 27 B Internal Data Logging B1 Shipping Checklist eee 28 B2 Hardware Requirements 0 0 0 eee 28 B3 Installation B4 Connecting a BS Real Time Data Transfer 29 B6 IDL 0000 29 C Applications C1 In Vivo 021100 C2 Extracted Chlorophyll C3 Ammonium Detection 2 CA Turbidity esc csc 55 C5 Fluorescent Tracer 1268 35 C6 Cyanobacteria 1 0108 36 Aquafluor User s Manual 1 Introduction 1 1 Description The Aquafluor isa lightweight handheld fluorometer turbidimeter Its dual channel capability allows the user to measure either fluorescence or turbidity in one sample The Aquafluor can be configured for any two channels as follows in vivo chlorophyll a cyanobacteria phycocyanin or phycoerythrin turbidity Rhodamine WT fluorescein ammonium and extracted chlorophyll a 1 2 Inspection and Setup 1 2 1 Inspection Upon receiving your instrument please inspect everything carefully and make sure all accessories are present All shipments include e The Aquafluor e The User s Manual e 4 AAA batteries e Cuvettes Qty 4 See Appendix A5 e Storage Pouch 1 2 2 Setup Before the Aquafluor can be used the supplied batteries must be installed 1 On the backside of the instrument remove the battery panel by loosening the center screw and then gently pull on the screw There is some resistance due to the o ring used for watertight sealing
18. information see Appendix B The Aquafluor can log up to 1000 data points The DATA screens control logging downloading and erasing the data as described below 3 8 1 Activate Data Logging 1 Press the lt DATA gt button 2 times 2 Press lt ENT gt to toggle between logging and stop status 3 Press lt ESC gt when finished to return to the Home screen 3 8 2 Download Data 1 Connect the Aquafluor to the serial port of your computer 2 Open the Turner Designs Interface Software See Appendix B for computer requirements and installation 3 Press the lt DATA gt button 3 times 4 Press lt ENT gt 5 times to start the data download 5 Press lt ESC gt when finished to return to the Home screen Aquafluor User s Manual 15 3 8 3 Erase Data Press the lt DATA gt button 4 times 2 Press lt ENT gt 5 times to erase all logged data 3 Press lt ESC gt when finished to return to the Home screen Aquafluor User s Manual 16 3 8 Display Screen Flowchart T r Desig TD HH1B 0500 5 5 second warm up Indicates which channel Fluorescence reading isactive A Indicates activity of Home Screen IDL Press lt READ gt Press lt A B gt to toggle Press lt CAL gt between READING Calib A lt ENT gt to start A Insert Blank then press lt ENT gt Press lt STD VAL gt Insert Cal Soln Cal Val XXX then press lt ENT gt lt UP DO
19. ld identify the configuration by looking at the label on the back side of the Aquafluor Refer to the table in Appendix A4 to confirm the correct configuration for your application The appropriate channel is selected by pressing the lt A B gt button to toggle between the 2 channels The display will show a label in the lower left corner of the Home screen to identify which channel is activated Aquafluor User s Manual 8 3 3 Calibration Overview The Aquafluor can be calibrated using Primary or Secondary standards A Primary Standard is one that contains the same fluorescent material that you are measuring in your unknown samples The Aquafluor will give an actual quantitative concentration reading when a primary standard of known concentration is used for calibration The standard and samples must be in the linear detection range to get accurate quantitative results Refer to Section 4 3 for more details about the linear range and quenching of the samples For some applications Secondary Standards are used for calibration A secondary standard is one that contains a different type of fluorescent material than your samples The in vivo Chlorophyll application is an example where a Secondary Standard is often used for the calibration and we recommend using the Solid Secondary Standard PN 8000 950 Refer to Appendix C for details on Chlorophyll applications and calibration This appendix also has details about the Ammonium a
20. n compliance with the directions in this Aguafluor User s Manual and directions accompanying the accessories Aquafluor User s Manual 22 Damage resulting from measurement of samples found to be incompatible with the materials used in the sample system or resulting from contact with corrosive materials or atmosphere is not covered Damage caused by modification of the instrument by the customer is not covered Damage incurred in shipping is not covered 5 2 Warranty Service To obtain service during the warranty period the owner shall take the following steps 1 Write or call the Turner Designs Technical Support and describe as precisely as possible the symptoms or nature of the problem Phone 408 749 0994 Email support turnerdesigns com 2 Carry out any adjustments or tests as suggested by Technical Support 3 If proper performance is not obtained a Return Authorization number RMA will be issued to you Ship the instrument prepaid to Turner Designs with the RMA number referenced and include your complete shipping address and phone number inside The instrument will be repaired at no charge but for customers outside of the United States any shipment or documentation charges will be billed at cost Aquafluor User s Manual 23 NOTE The instrument or accessories should not be returned without first contacting Turner Designs Prior correspondence is needed to understand the nature of the problem
21. nd Turbidity applications When a Secondary Standard is used for calibration the Aquafluor will give relative sample readings that are proportional to the measured fluorescence In some cases these relative sample readings will be correlated back to actual concentrations that are determined later For example this is commonly done for in vivo Chlorophyll monitoring applications as described in Appendix Cl Aquafluor User s Manual 9 For Dye tracing applications the calibration is normally performed with a primary standard made from the same dye that is being used for the testing The primary standard will either be made to a known concentration typically in ppb or ug L units or to a known dilution factor Ideally the Primary Standard and Blank samples used for calibrating will be made with the same water the tests are being performed in For more details on this and tracer dye use please refer to the Application Note A Practical Guide to Flow Measurement at the following web link http www turnerdesigns com t2 doc appnotes 3 4 Calibration procedure It is recommended for best accuracy that you always calibrate before performing your sample analysis The Aquafluor will save the calibration settings for each channel until a new calibration is performed If the temperature of your samples or the Aquafluor changes significantly the readings may show a small shift and in this case you should consider recalibrating
22. nection between the Aquafluor and your computer 2 Insert a sample and press the lt READ gt button When the reading is finished the results will automatically transfer to the active Excel spreadsheet B6 IDL Troubleshooting Difficulties can arise when parameters are set incorrectly or improper cable connections Here are some common solutions 1 Box to the left of the COM port is red This means that the COM port is not available Causes a Another software program such as palm pilot hot sync could be using the COM port making it unavailable Make sure to Aquafluor User s Manual 29 close all programs of this type before opening the Spreadsheet Interface software b The port selected is incorrect Follow step 4 in section B4 above to choose the correct COM port 2 All lights are green but no data transferred even though the instrument says All data downloaded a The connection between the instrument and the computer is bad Check and tighten the cable connections Make sure both ends of the cable are plugged in tightly Aquafluor User s Manual 30 Appendix C1 In Vivo Chlorophyll In vivo chlorophyll analysis is the fluorescent detection of chlorophyll a in algal cells in water In this technique the excitation light from the fluorometer passes through the untreated sample of water and causes the chlorophyll a within the cells to fluoresce Environmental conditions presence of interfe
23. p to correct for the pheophytin This is referred to as the corrected chlor a method in section 12 2 of the 445 method Also in section 10 1 of the EPA 445 method the calculation for the F is not required because it will always be equal to when using the Aquafluor The EPA 445 method is available at the following link http www epa gov nerlcowww ordmeth htm Appendix C3 Ammonium Detection Accurate determination of ammonium in aquatic environments is a critical measurement when investigating Nitrogen cycling and nutrient dynamics Historically methods for ammonium determination have been a source of frustration within the scientific community due to the lack of a simple accurate and affordable method particularly for measurements in the submicromolar range Aquafluor User s Manual 33 The new ammonium technique offers researchers and technicians an excellent alternative to the existing colormetric ideophenol blue method Benefits of the fluorometric method include e Sensitivity Detection in the submicromolar range e Accuracy More accurate than previous methods for low ammonium concentration samples e Simple Requires only one mixed non toxic reagent and no special equipment other than a fluorometer e Non toxic Reagents OPA sodium sulfite and sodium borate The colometric ideophenol blue method is susceptible to inconsistent results particularly with submicromolar ammonium concentrations Whereas
24. ring compounds cellular physiology morphology and light history can influence the relationship between the in vivo fluorescence and the actual concentration of chlorophyll a in the sample These factors cause in vivo fluorescence to be a semi quantitative tool Despite its semi quantitative nature in vivo fluorescence data can supply valuable information on the spatial and temporal distribution of chlorophyll concentrations quickly and easily To obtain quantitative data the in vivo fluorescence data must be correlated with extracted chlorophyll a data that can be obtained through the extraction and measurement of the pigment from grab samples on a laboratory fluorometer spectrophotometer or HPLC When collecting grab samples for chlorophyll extraction the in vivo reading must be noted at the same time the sample is collected Several samples should be collected within each niche or environment Once the chlorophyll concentration has been determined through extraction the concentration should be correlated with the corresponding in vivo value as shown in Graph C1 below We recommend using the Solid Secondary Standard to calibrate the Aquafluor for in vivo chlorophyll use Aquafluor User s Manual 31 The Solid Standard simulates the in vivo fluorescence of a 10 ug L marine diatom culture This should be viewed only as a ballpark estimate of actual chlorophyll concentration For this reason if you assign the Cal
25. ustable secondary standard in the Aquafluor Aquafluor User s Manual 12 Close the lid Wait 15 seconds then read the value Record the value of the secondary standard Open the lid and use the 3 32 allen wrench provided to adjust the attenuation screw through the hole at the top of the secondary standard to increase or decrease the value INNA displayed on the screen Turning the screw counterclockwise will increase the reading See Photo 3 Photo 3 Adjusting the value on the secondary standard by turning the attenuation screw 8 Repeat steps 3 6 until the secondary standard reads the concentration value of interest Record the secondary standard reading for future reference 9 At this point remove the solid standard and turn the locking setscrew clockwise until it just makes contact with the attenuation screw DO NOT OVERTIGHTEN 10 Now you can recalibrate the Aquafluor using the value of the secondary standard obtained in step 8 11 Proceed with analyzing your samples 12 You may use your adjustable secondary standard at any time to check the stability of the fluorometer Simply insert your standard to read the value The value should be similar to what was previously obtained in step 8 above Aquafluor User s Manual 13 13 When the secondary standard is not in use store it in its protective bag to prevent dust collecting on the optical fiber On a daily basis before making m
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