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1. WETStar User s Guide The user s guide is an evolving document Please check our website periodically for updates If you find sections that are unclear or missing information please let us know WET Labs Inc PO Box 518 Philomath OR 97370 541 929 5650 www wetlabs com WETStar User s Guide WETStar Revision M 13 Jan 2006 WETStar Warranty Standard Warranty This unit is guaranteed against defects in materials and workmanship for two years from the original date of purchase Warranty is void if the factory determines the unit was subjected to abuse or neglect beyond the normal wear and tear of field deployment or in the event the pressure housing has been opened by the customer To return the instrument contact WET Labs for a Return Merchandise Authorization RMA and ship in the original container WET Labs is not responsible for damage to instruments during the return shipment to the factory WET Labs will supply all replacement parts and labor and pay for return via 3 day air shipping in honoring this warranty Annual Servicing Extended Warranty WET Labs will extend the warranty on this unit to five years if it is returned annually for servicing This includes calibration standard maintenance and cleaning Charges associated with this annual service work and shipping are the responsibility of the customer Shipping Requirements for Warranty and Out of warranty Instruments 1 Please retain the original
2. accurate calibration to be done in a lab for all situations The CDOM WET Star therefore is not calibrated to CDOM but rather QSD which has historically been used to establish the response and sensitivity of fluorometers used in CDOM fluorescence applications The absorption and emission characteristics of QSD allow it to be used as a proxy for CDOM An instrument specific Clean Water Offset CWO and 100 ppb QSD measurement standard are provided on the characterization sheet that shipped with your WETStar The conversion of raw counts to QSD equivalent concentration is straightforward using the equation X sample C sample Cora Scale Factor where X samp e Concentration sample of interest Csample raw counts output when measuring a sample of interest counts Cewo Clean Water Offset counts Scale Factor SF CDOM multiplier in ppb QSD counts X standard concentration known QSD equivalent sample Then SF LX standard Csample Cewo For example if the CWO reading was 60 counts and a known concentration of 65 ppb provided a signal of 3500 counts your scale factor would be 65 0 ug l 3500 60 counts 0 018 ppb counts 4 3 Uranine Rhodamine Phycoerythrin WETStar Characterization The characterization procedure is to prepare a dilution series of the dye solution Uranine or fluorescein and RhodamineWT dye also used for the phycoerythrin meter are available from chemical supply dealers The sensitivi
3. flow tube The fluorescence emitted at 90 degrees is synchronously detected at 1 kHz by a silicon photodiode The amplified and demodulated voltage output of the photodiode is provided to the user for connection to a digital voltmeter an A D converter or RS232 input The instrument contains two LEDs doubling the excitation light as well as mirrors and lenses to optimize the instrument s performance WETStar User s Guide WETStar Revision M 13 Jan 2006 7 3 Instrument Operation WET Star meters are available with either analog or digital output Both outputs are proportional to the amount of fluoresced light emitted This value is in turn proportional to the phycocyanin concentration in the sample volume Analog output ranges from 0 5 V and the digital output ranges from 0 4095 counts A four pin bulkhead connector and matching pigtail provide the power ground analog out and analog return or digital signal s The pinouts are clearly described in section 1 2 The analog out and or digital signals can connect to a data acquisition system of your choice WETStar is designed to connect directly to many CTD systems and is compatible with other platforms that can provide power and accept a 0 5 VDC analog signal The digital signal is output to a PC or data logger using the test cable provided 3 1 Connector Check Push the pigtail straight on to the connector sockets without wiggling the pigtail from side to side The connection is
4. for uranine meters The linear regression R squared value must be better than 0 9900 16 WETStar User s Guide WETStar Revision M 13 Jan 2006 Appendix A Chlorophyll WETStar Flow Rate Dependence Fluorescent signals from phytoplankton samples exhibit some dependence on the flow rate of the sample water through the measurement chamber Because of WETStar s unique size and flow tube technology providing for a uniform flow rate is highly desirable For this reason we recommend using a small submersible pump with a known flow rate Free flow un pumped measurements are possible but care must be taken to use a steady profiling rate on the order of one meter per second to provide the proper flushing of the flow tube It is important to note that when profiling in free flow mode it is possible for the descent of the profiling package to come to a stop or even reverse direction briefly due to sea state and ship motion Figure 4 shows how the voltage output of the WETsStar can vary with flow rates ranging from about 3 to 30 ml sec A small constant volume pump was placed in line with a closed loop flow system fitted with a throttle valve to control the flow rate It should be noted that the relative response only varies by 15 18 percent over the entire range so that useful data can be obtained even in situations where the flow rate is unknown Flow Rate Dependence Normalized Output Voltages 2 Trendline is derived from 2 1410 da
5. seen when looking straight into the flow tube with the plastic fluorescence test stick inserted 3 3 Analog Signal Output Check Connect analog out socket 2 and analog return socket 4 to a digital multimeter With the flow tube clean and dry the analog output voltage should read approximately 0 05 0 5 VDC Insertion of the fluorescent plastic test stick into the flow tube should produce a signal level at or near saturation 5 VDC WETStar User s Guide WETStar Revision M 13 Jan 2006 9 F 3 E 2 tal Teche 3 4 Digital Signal Output Check The RS 232 output from the digital WETStar is a single column of numbers whose values range between 0 and 4095 counts Connect the test cable to the WETStar Connect the DB 9 connector to a PC running a terminal communications program such as HyperTerminal Set the data rate to 9600 baud 8 data bits 1 stop bit no parity Connect a 9 V battery to the test cable Output should read approximately 40 500 counts Inserting the plastic fluorescent stick should increase the signal to near saturation or 4095 counts Note that RS 232 protocol is limited to a nominal cable length of 5 meters 15 ft WET Labs experience is that these cables can be significantly longer but they should be tested before deployment 3 5 Deployment WETStar can be deployed in either a non pumped flow through mode or a pumped configuration We highly recommend using a pump because as one would expect from
6. shipping material We design the shipping container to meet stringent shipping and insurance requirements and to keep your meter functional 2 To avoid additional repackaging charges use the original box or WET Labs approved container with its custom cut packing foam and anti static bag to return the instrument e fusing alternative container use at least 2 in of foam NOT bubble wrap or Styrofoam peanuts to fully surround the instrument e Minimum repacking charge for WETStars 25 00 3 Clearly mark the RMA number on the outside of your shipping container and on all packing lists 4 Return instruments using 3 day air shipping or better do not ship via ground WETStar User s Guide WETStar Revision M 13 Jan 2006 wer Labs Attention Return Policy for Instruments with Anti fouling Treatment WET Labs cannot accept instruments for servicing or repair that are treated with anti fouling compound s This includes but is not limited to tri butyl tin TBT marine anti fouling paint ablative coatings etc Please ensure any anti fouling treatment has been removed prior to returning instruments to WET Labs for service or repair WETStar User s Guide WETStar Revision M 13 Jan 2006 ll SpecificatiOnS rosita aria 1 1 1 COMME CLOTS ainda 2 1 2 Tee ee 3 1 3 BRETON elastan 3 NTN 5 2 1 Chlorophyll WE TS 3 2 2 MON WE Sra 5 2 3 re WELLS 6 2 4 Rhodamine NETA id 6 25 Phycoerythrin VERS GEL einen ren 6 3
7. the air blank Begin pumping a known volume of culture medium through the fluorometer Check for and clear bubbles Allow the fluorometer to stabilize and record the voltage as the seawater blank Add an aliquot of culture to the blank culture medium with a volumetric pipette The aliquot should be enough that you would expect to see an increase in the fluorometer voltage between 0 5 and 1 volt In the scenario of the WETStar set up for the lower range 0 75 ug l a culture containing approximately 50 ug l chlorophyll and a blank culture medium volume of 400 ml the aliquot would be approximately 25 ml After the reading has stabilized record the voltage and volume added Continue with the serial additions until you saturate the fluorometer Clean the fluorometer after calibrating SBE Pump Digital Multi Meter Outflow Tubing Bulkhead Connector Intake Tubing Figure 5 Instrument setup Field Calibration There are two goals to the field calibration The first is to verify that the lab calibration is valid in the field The second is to detect any changes in the fluorometer over time To achieve these goals chlorophyll a samples should be taken as often and as close as possible to the mooring Record the time that the Niskin bottle containing the sample was tripped Preferably samples would be taken throughout a 24 hr period to investigate the effects of irradiance on in situ fluorescence 20 WETSta
8. the characterization sheet 4 5 6 Noise The noise value is computed from a standard deviation over 60 samples These samples are collected at one second intervals for one minute The smoothing averaging time for these samples is 0 5 seconds A standard deviation is then performed on the 60 samples and the result is the published noise on the characterization sheet The calculated noise must be below 1 5 mV 3 counts 4 5 7 Final Water Blank Test De ionized pure water is introduced into the sample volume The output voltage must be 0 070 0 030 V for most WETStar types Value is recorded on the characterization sheet 4 5 8 Final Standard Test A standard sample specific to the measurement range of the WETStar being tested is placed in the sample volume The output reading is recorded on the instrument specific Characterization Sheet 4 5 9 Voltage and Current Range Verification To verify that the WETStar operates over the entire specified voltage range 7 15 V a voltage sweep test is performed The WETStar is operated over the entire voltage range and the current and operation is observed The total power consumption voltage times current must remain below 500 mW 900 mW for digital over the entire voltage range 4 5 10 Linearity Linearity tests are performed on many WETStars This linearity test consists of a complete Coproporphyrin dilution series for chlorophyll meters a QSD series for CDOM meters and a uranine series
9. ug l This is done at WET Labs using a fluorescent stick and adjusting the electronic gain of the WETStar for a corresponding specific output value As is the case with other fluorometers detailed characterization must be performed by the user to determine the actual zero point and scale factor for his her particular use 4 1 Chlorophyll Characterization To measure the WETStar output voltages for tuning and characterization the analog fluorometer is connected to a 16 bit analog to digital A D converter The A D outputs the voltages in a standard RS 232 serial text format that is collected with a terminal program A spreadsheet is then used to perform calculations on the collected values A Scale Factor is used to convert the fluorescence response of the instrument into chlorophyll a concentration The Scale Factor is determined at WET Labs during a cross calibration using a solid fluorescent standard and a reference fluorometer whose chlorophyll fluorescence response has been characterized in a laboratory using a mono species lab culture of Thalassiosira weissflogii phytoplankton Refer to Appendix B for details on performing a laboratory or field calibration on the WETStar The WETStar sensitivity is adjusted to be within certain limits when a controlled fluorescence standard is introduced into the sample volume The standard is nominally equivalent to 50 ug l of chlorophyll On the WS3S models the output voltage is adjusted for 3 0
10. very snug and it helps to apply a thin coat of silicon grease to the connector sockets Give an additional push to remove any trapped air and seat the pigtail Screw the lock collar securely to the bulkhead connector To remove the pigtail unscrew the lock collar then grasp the body of the pigtail not the wire and pull straight out Many connectors are damaged by rocking the pigtail connector from side to side as they are pulled out 3 2 Electrical Checkout WARNING Do not deliver more than 15 VDC to the WETStar The WETStar is protected against high transient voltage events with a Transient Suppression Device When voltage transients higher than 15 VDC occur the transient voltage suppression device turns on and shunts this voltage to ground If an input power higher than 15 VDC is applied to the WETStar this device will turn on If left in this condition the transient voltage suppression device will fail and damage to the WETStar may occur Electrical checkout of WETStar is straightforward Apply 7 15 VDC to the instrument to provide power to the LEDs and electronics Ensure that positive voltage is applied to V socket 3 and common or ground is applied to the ground socket 1 the large hole in the bulkhead connector A common 9 volt battery makes an ideal power supply for bench testing With the proper voltage applied to sockets the ground socket 1 and V socket 3 the LEDs should illuminate the quartz flow tube This light can be
11. 0 150 V and on the WSIS the output is 1 5 0 150 V 4 2 CDOM Characterization The current substance used to characterize the CDOM WETStar is a quinine sulfate dihydrate QSD solution at a concentration of 100 parts per billion ppb WARNING If you are not experienced or trained in the safe and proper techniques of working with chemicals and acids seek proper assistance The gain of the instrument is adjusted based on the signal level generated from the 100 pbb QSD solution Linearity of the instrument is checked using a dilution series of QSD The sensitivity of individual instruments may vary due to specific requirements of deployment conditions Refer to the Characterization Sheet that accompanied your instrument for precise values regarding range and sensitivity to QSD It is important to note that QSD is used in the characterization of the CDOM WETsStar and does not constitute a calibration of the instrument s response to naturally occurring CDOM WETStar User s Guide WETStar Revision M 13 Jan 2006 13 at TA The CDOM WET Star range and sensitivity is adjusted to be within certain limits when a QSD solution of a certain concentration is introduced into the sample volume The CDOM WETStar output is adjusted by setting the gain at several operational amplifier stages Gain is set with fixed value precision resistors The variable nature of the fluorescence due to naturally occurring CDOM makes it impossible for an
12. Instrument O Nr rara 9 3 1 Connector Check ee 9 Vero aa 9 3 3 Analog Signal Output eve 9 3 4 Digital Signal Output Check nn da 10 3 5 PN 10 30 Data Eg aia 10 e o 11 3 Upkeep and MATE cia 11 4 Characterization ea 13 4 1 Chlorophyll Characterization nica Deia 13 42 GSE GE 200 dd 13 4 3 Uranine Rhodamine Phycoerythrin WETStar Characterization 14 EEE GS EEE E REE 14 43 Final Testing use 15 Appendix A Chlorophyll WETStar Flow Rate Dependence 17 Appendix B Calibration Details for Chlorophyll WETStar 18 Introduction and Cave see 18 Calibration 00 EE ET 18 Appendix References rrnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 21 WETStar User s Guide WETStar Revision M 13 Jan 2006 i WET Labs 1 Specifications Mechanical Size Weight Rated depth Housing Electrical Response time Input Output Current draw Linearity Optical Chlorophyll Dynamic ranges Sensitivity Excitation Emission CDOM Dynamic ranges Sensitivity Excitation Emission Uranine Dynamic range Sensitivity Excitation Emission Rhodamine Excitation Emission Phycoerythrin Excitation Emission Pressure housing 6 7 x 2 7 in 17 1 x 6 9 cm Overall height including bulkhead connector and tubing fittings 10 2 in 25 7 cm in air 1 7 lb 0 8 kg in water 0 25 lb 0 1 kg 600 meters Acetal copolymer 0 17 sec analog 0 125 sec
13. considerations of phytoplankton physiology there is a flow rate dependence of the signal A pump used during calibration and during field work will provide a consistent flow and ensure the highest quality data We supply threaded tubing nipples for the inlet and outlet flow tube ports to aid in plumbing a pump and or water traps If you deploy WETStar in a flow through mode best results will be obtained by lowering the instrument steadily at 0 2 to 1 0 meters per second This is compatible with the descent rate requirements of many small CTDs If the instrument is used in a free flow mode it is important to ensure that the flow tube inlet outlets are seeing a clear water path during descent Since WETStar s size makes it easy to tuck away inside a cage this can present a problem One solution would be to add Tygon tubing to the fittings on the flow tube that are in turn connected to water traps funnel type devices which are mounted with their wide end facing in the direction of deployment If you use a small pump to flush the flow cell the recommended flow rate is in the range of 10 to 30 ml sec Laboratory tests have shown that for phytoplankton cultures increasing the flow rate up to 30 ml sec decreases the signal but slightly improves the signal to noise ratio The flow rate which provides the best signal to noise ratio is 25 ml sec A good pump for this purpose is Sea Bird Electronics SBE 05T which is a small low powered pump wh
14. digital optional 7 15 VDC 0 5 VDC analog 0 4095 counts digital optional lt 40 mA analog lt 80 mA digital optional gt 99 R 0 03 75 ug l standard 0 06 150 ug l optional gt 0 03 ug l 460 nm 695 nm 1000 ppb estuarine waters 250 ppb near coastal waters 100 ppb open ocean waters 0 100 ppb quinine sulfate dihydrate other sensitivities available on request 370 nm 10 nm FWHM 460 nm 120 nm FWHM 0 4000 ug l uranine I ug l uranine 485 nm 532 nm 470 nm 590 nm 525 nm 575 nm Specifications are subject to change without notice WETStar User s Guide WETStar Revision M 13 Jan 2006 1 WET 9 Labs N a al Teco 1 1 Connectors PINS IN LINE CONNECTOR 1 VMG 4 FS WITH G FLS P LOCK RING 4 02 2 IMPULSE OR SEA CON SOCKETS 1 BULKHEAD CONNECTOR ZA A VSG 4 PBCLM HP SS O 2 IMPULSE OR SEA CON ES j4 nu 7 7 77777 3 Figure 1 WETStar bulkhead connector Pinout summary for four pin WETStar connectors Analog Digital Socket Pin Function Socket Pin Function 1 Common ground 1 Ground 2 Analog out 2 Analog 3 V 3 V 4 Analog return 4 RS 232 TX 1 1 6 2 2 6 5 3 3 5 ai 4 IN LINE CONNECTOR _ LOOKING AT PINS LOOKING INTO SOCKETS MFG IMPULSE OR SUBCONN MFG IMPULSE OR SUBCONN P N MCBH 6 MP P N MCIL 6 F LOCK COLLAR MCDLS F NOT SHOWN o
15. elengths DCR J Kitchen 164 H 01 23 02 Correct terminology in section 2 and update H Van Zee Figure 1 DCR 188 04 09 02 Add digital WETStar capabilities DCR 213 S Campos H Van Zee J 10 25 02 Delete reference to Schott glass DCR 246 S Campos K 05 12 03 Add four pin digital connector diagram and M Everett functions DCR 300 L 1 11 05 Add phycoerythrin combine chlorophyll CDOM M Everett uranine Rhodamine into single user s guide DCR 435 M 1 13 06 Clarify warranty statement DCR 481 A Gellatly S Proctor WETStar User s Guide WETStar Revision M 13 Jan 2006
16. ent These samples when excited by the WETStar internal light source absorb energy in certain regions of the visible spectrum and emit a portion of this energy as fluorescence at longer wavelengths Figure 3 shows a simplified illustration of how the WETStar works LED interference filter interference filter photodiode detector quartz flow tube Figure 3 Light path through WETStar 2 1 Chlorophyll WETStar This WETStar is primarily designed to measure the fluorescence of chlorophyll containing phytoplankton which absorb light of wavelengths between 400 and 520 nm and emit light between 670 and 730 nm The chlorophyll WETStar uses two bright blue LEDs centered at approximately 470 nm and modulated at 1 kHz to provide the excitation Blue interference filters are used to reject the small amount of red light emitted by the LEDs A detector positioned at 90 degrees to the axis of the LED mounts measures the emitted light from the sample volume The approximately 0 25 cm sample volume is defined by the intersection of the excitation light with the field of view of the detector within the quartz flow tube A red interference filter is used to discriminate against the scattered blue excitation light The red fluorescence emitted at 90 degrees is synchronously detected at 1 kHz by a silicon photodiode The amplified and demodulated voltage output of the photodiode is provided to the user for connection to a digital voltmeter an A D convert
17. er or RS232 input The instrument contains two LEDs doubling the excitation light as well as mirrors and lenses to optimize the instrument s performance 2 2 CDOM WETStar The colored dissolved organic matter CDOM WETStar is primarily designed to measure the fluorescence of CDOM which absorbs light in the ultraviolet and emits light in the blue wavelengths The CDOM WETStar uses two UV LEDs centered at approximately 370 nm and modulated at 1 kHz to provide the excitation A detector positioned at 90 degrees to the axis of the LED mounts measures the emitted light from the sample volume The approximately 0 25 cm sample volume is defined by the intersection of the excitation light with the field of view of WETStar User s Guide WETStar Revision M 13 Jan 2006 5 the detector within the quartz flow tube A blue interference filter is used to discriminate against the scattered blue UV excitation light The blue fluorescence is synchronously detected at 1 kHz by a silicon photodiode The amplified and demodulated voltage output of the photodiode is provided to the user for connection to a digital voltmeter an A D converter or RS232 input The instrument contains two LEDs doubling the excitation light as well as mirrors and lenses to optimize the instrument s performance 2 3 Uranine WETStar The uranine fluorescein WETStar uses two bright blue LEDs centered at approximately 470 nm and modulated at 1 kHz to provide the exci
18. g chamber applies a water pressure of at least 50 PSI The rated depth of the WETStar is 600 meters 4 5 2 Mechanical Stability Before final testing the WETStar is subjected to a mechanical stability test This involves subjecting the unit to mild vibration and shock The air water and sample voltages must remain the same before and after the mechanical stability test 4 5 3 Temperature Stability To verify temperature stability the WETStar is immersed in an ice bath The starting temperature is typically 23 30 degrees Celsius and the ending temperature is 1 5 degrees Celsius A voltage sample is collected every 30 seconds with a 0 5 second smoothing Specifications assert that the maximum variation per degree Celsius is 1 25 mV 2 counts 4 5 4 Electronic Stability This value is computed by collecting a sample once per minute for twelve hours or more The smoothing time for this one sample is 0 5 seconds After the data is collected the minimum and maximum values are determined and the difference between these two is divided by the number of hours the test has run The result is the stability value listed on the characterization sheet The stability value must be less than 2 0 mV 3 counts hour WETStar User s Guide WETStar Revision M 13 Jan 2006 15 4 5 5 Full Scale Verification The specified maximum output of the WETStar is nominally five volts or for digital meters 4095 counts The full scale voltage is reported in
19. ich has an adjustable motor speed so that flow rate can be precisely controlled Flow rate dependence for chlorophyll WETsStars is discussed further in section 5 Note that while CDOM does not display the physiological response behavior of phytoplankton flow rates that are very low or very high may prove problematic UV bleaching can occur at very low rates and the instrument s response may be limiting at very high rates 3 6 Data Collection Analog WETStars must be connected to a host system that will receive the analog voltage output and digitize it Many oceanographic instruments such as CTDs radiometers and data loggers are equipped with analog input channels and carry on board A D converters 10 WETStar User s Guide WETStar Revision M 13 Jan 2006 WET Adding the instrument to a CTD or other host solves several other problems Since the data is merged with the CTD data correlating the WETStar output with depth or time is done automatically If one is building a logger or interface it will be necessary to provide some pressure or time reference to stamp the fluorescence data tying it to the rest of the physical data Analog WETStar output is limited to a current of 10 mA or less Its output impedance is approximately 500 ohms that effectively limits the drive current Therefore the electrical signal will degrade over a long electrical wire due to the electrical resistance of the cable For best results the analog signal
20. ion is already familiar with the spectrophotometric extraction method for determining chlorophyll concentration Calibration should be a two phase process The first phase a serial addition procedure occurs in the lab the second a simple correlation procedure in the field 18 WETStar User s Guide WETStar Revision M 13 Jan 2006 WET OG Labs Lab Calibration Materials needed e WETStar fluorometer e large tub for immersing instruments e SBE pump e lab notebook e cables for fluorometer and pump e DC power supply e tubing for fluorometer e digital voltmeter inlet e ring stand w 2 large clamps outlet e 50 100 and 500 ml graduated cylinders e thermometer e phytoplankton culture e scintillation vials w 10 ml 90 e stopwatch acetone e set of volumetric pipettes e some 500 ml beakers 5ml e GF F filters 10 ml e forceps 25 ml e filter rig w pump tubing traps e pipette bulb It is critical that the entire calibration process be performed under non varying conditions Changes in temperature or light will affect the in vivo fluorescence of the phytoplankton culture and possibly the instrument It is recommended that the room be dimly lit and that the culture be allowed to sit in the calibration area for 30 minutes prior to use The instrument should be equilibrated to calibration temperature for at least 4 hours Obtain a culture of late logarithmic phase phytoplankton and some of the culture media Use a species of phyto
21. m Figure 2 Optional 6 pin digital WETStar connector Pinout summary for optional digital WETStar connectors Pin Socket Function 1 Power ground 2 RS 232 RX 3 Analog ground 4 Vin 5 RS 232 TX 6 Analog output Input power of 7 15 VDC is applied to pin 4 The power supply current returns through the common ground pin Data is sent out the serial output pin pin 5 2 WETStar User s Guide WETStar Revision M 13 Jan 2006 1 2 Test Cable Some Digital units ship with a test cable that allows you to supply power to the WETsStar and provides a DB9 serial connector for RS232 output 1 3 Delivered Items The standard WETStar delivery includes e Tubing nipples that allow a small pump to be connected to the instrument e small stick of fluorescing plastic material for functionality checks o pink stick for chlorophyll Rhodamine and phycoerythrin o blue stick for CDOM o yellow stick for Uranine e this manual e instrument specific characterization sheet e pigtail lead with mating connector except some digital units WETStar User s Guide WETStar Revision M 13 Jan 2006 3 2 Theory of Operation The WETStar miniature fluorometer allows the user to measure relative chlorophyll CDOM or other concentrations by directly measuring the amount of fluorescence emission from a given sample of water The sample media is pumped through a quartz tube mounted through the long axis of the instrum
22. ng the voltages from the instrument obtained from a dilution or addition series of a phytoplankton culture of known concentration creating a linear regression of the recorded voltages against chlorophyll concentration and then obtaining a calibration coefficient Problems arise from the fact that the optical properties of phytoplankton are functions of size shape pigmentation taxonomic composition photo adaptation and physiological status For example exposure to supersaturating light will cause an immediate time scale of seconds depression in fluorescence with any change in chlorophyll concentration happening very much slower time scale of hours Kiefer 1973 Cullen et al 1988 However since it is unreasonable to calibrate a fluorometer with every species of phytoplankton at all different physiological states one has to simply be aware of the problems and go forth Thus any conversions of in situ fluorometry into chlorophyll a concentration are estimates at best and guesses at worst Over the time scale of a mooring deployment a fluorometer will probably estimate chlorophyll a within a factor of 2 Lorenzen 1966 Calibration Protocol The following procedures involve using some improvisational techniques and equipment For example the Sea Bird SBE 5 pump is designed for submersible work and may prove difficult or impossible for some people to use in the way described This calibration procedure also assumes the person performing calibrat
23. plankton or at least genera that you are likely to encounter in the field If time energy and materials allow use two species of different groups e g a diatom and a chrysophyte for comparison purposes Absolute concentration of chlorophyll a in the culture should be no more than 50 ug l you can start to see color by eye at about 20 ug l at which point the culture is starting to slow down and enter the stationary phase Filter approximately 50 100 ml onto a GF F filter for spectrophotometric chlorophyll determination You should be able to easily see color on the filter If you can t filter more Place the filter into 10 ml of 90 percent acetone and store in a freezer for 24 hrs Record the optical density of the solution at 750 664 647 and 630 nm Calculate pigment concentrations by Ca 11 85 0D664 1 54 0D647 0 08 0D630 Cp 21 03 OD6q7 5 43 ODes4 2 66 0D630 Ce 24 52 0D630 5 1 67 0D664 7 60 0D647 where a b and c denote chlorophyll a b and c respectively and all optical densities have had the 750 nm signal subtracted WETStar User s Guide WETStar Revision M 13 Jan 2006 19 WET A y a Tacna Set up the WETStar fluorometer and pump on a ring stand according to Figure 5 Provide power to the fluorometer and pump via a DC power supply Record all voltages from the fluorometer with a voltmeter Power up the fluorometer and let it warm up for 10 minutes At the end of this period record the voltage as
24. r User s Guide WETStar Revision M 13 Jan 2006 E g 3 3 gt tay Appendix C References Cullen J J C M Yentsch T L Cucci and H L MacIntyre 1988 Autofluorescence and other optical properties as tools in biological oceanography In Ocean Optics VIII Proc SPIE 149 156 Cullen J J and M R Lewis 1995 Biological processes and optical measurements near the sea surface Some issues relevant to remote sensing J Geophys Res 100 C7 13 255 13 266 Lorenzen C J 1966 A method for the continuous measurement of in vivo chlorophyll concentrations Deep Sea Res 13 223 227 Marra J and C Langdon 1993 An evaluation of an in situ fluorometer for the estimation of chlorophyll a Tech Rep LDEO 93 1 Lamont Doherty Earth Observatory WETStar User s Guide WETStar Revision M 13 Jan 2006 21 WET Labs on hal ratories AN Pr mar Techn WETStar User s Guide Revision History Revision Date Revision Description Originator A 10 07 99 Begin revision tracking H Van Zee B 01 03 00 Change Specifications Delete section 3 3 DCR C Moore 6 C 01 06 00 Update document DCR 8 D Hankins D 03 29 00 Reorder chapters for consistency DCR 19 H Van Zee E 12 12 00 Update document and illustrations DCR 75 D Hankins H Van Zee F 03 12 01 Correct Copro standard preparation DCR 90 D Hankins G 11 26 01 Revise references to excitation wav
25. should be fed directly into an A D converter and the digital signal should be sent up the cable One such option is to use WET Lab s DH 4 data logger a sub surface data logging system that can handle up to three analog signals simultaneously as well as two digital signals if necessary 3 7 Data Analysis Because of the varied environments in which each user will work it is important to perform characterizations using similar seawater as you expect to encounter in sifu 3 8 Upkeep and Maintenance WETsStar is a very compact instrument and its maintenance can be easily overlooked However the miniature fluorometer is a precision instrument and does require a minimum of routine upkeep After each cast or exposure of the instrument to natural water flush the instrument with clean fresh water paying careful attention to the flow tube Soapy water will cut any grease or oil accumulation The tube is high quality quartz that can easily be broken or scratched so use caution Do not use a dowel or stiff brush in the tube A long cotton swab works nicely for cleaning the tube At the end of an experiment the instrument should be rinsed thoroughly air dried and stored in a cool dry place Solvents such as methanol may also be used to clean the tube WETStar User s Guide WETStar Revision M 13 Jan 2006 11 4 Characterization The chlorophyll WET Star is typically configured for one of two measurement ranges 0 03 75 0 ug l or 0 06 150
26. ta collected from three a e 1 05 fluorometers 2 1 00 095 0 90 0 00 5 00 10 00 15 00 20 00 25 00 30 00 Flow Rate ml sec Figure 4 Flow rate dependence for Thalassiosira weissflogii Output voltages are normalized to the voltage value recorded at 24 ml sec Using the same flow rate in the field that was used during one s calibration will ensure consistent results WETStar User s Guide WETStar Revision M 13 Jan 2006 17 Appendix B Calibration Details for Chlorophyll WETStar This section is provided by Richard Davis Oregon State University WETsStar is shipped pre configured to provide accurate linear response over one of two dynamic ranges 0 03 75 0 or 0 06 150 ug l However because of myriad calibration techniques and different properties of the natural waters from which blanks will be prepared it is important that an experiment specific calibration is done before and after each major cruise or event The key to obtaining high quality data is to determine the instrument s response to the conditions that will be found in the field Because of the many different applications involving fluorometric chlorophyll determinations detailed calibration of the instrument must be done by the user Introduction and Caveats The purpose of calibrating an in situ fluorometer is to be able to convert its in water signal to an absolute value of chlorophyll a In theory this should be a simple process of measuri
27. tation Blue green interference filters are used to reject the small amount of red light emitted by the LEDs and produce the 485 nm excitation light A detector positioned at 90 degrees to the axis of the LED mounts measures the emitted light from the sample volume The approximately 0 25 cm sample volume is defined by the intersection of the excitation light with the field of view of the detector within the quartz flow tube A green interference filter is used to discriminate against the scattered blue excitation light The green fluorescence emitted at 90 degrees is synchronously detected at 1 kHz by a silicon photodiode The amplified and demodulated voltage output of the photodiode is provided to the user for connection to a digital voltmeter or an A D converter The instrument contains two LEDs doubling the excitation light as well as mirrors and lenses to optimize the instrument s performance 2 4 Rhodamine WETStar This WETStar is primarily designed to measure the fluorescence of rhodamineWT dye which absorbs light of wavelengths between 470 and 550 nm and emits light around 590 nm The rhodamine WETStar uses two bright blue LEDs centered at approximately 470 nm and modulated at 1 kHz to provide the excitation Blue interference filters are used to reject the small amount of red light emitted by the LEDs A detector positioned at 90 degrees to the axis of the LED mounts measures the emitted light from the sample volume The approximatel
28. ty of the WETStar is adjusted such that the slope of the linear regression and range of the dilution series is appropriate to the range requested by the user 4 4 Gain Adjustment The output is adjusted by setting the gain in several operational amplifier stages in the WETStar Gain or sensitivity is set with fixed value resistors 14 WETStar User s Guide WETStar Revision M 13 Jan 2006 4 4 1 Pure Water Blank Pure de ionized water is used to set the zero voltage of the WETStar This zero voltage is set for approximately 0 070 0 030 V on most WETStar models The WETStar employs an offset voltage circuit Water blank is adjusted with two fixed voltage divider resistors at the factory 4 4 2 Response Time Time Constant The specified time constant for the analog WETStar is 0 167 seconds 0 125 for the digital This time constant is the RC value computed by 1 RC To verify the time constant the step response is observed on an oscilloscope A sample is introduced that produces a full scale reading The sample is then quickly removed and the decay is observed on the oscilloscope The output voltage must reach a value of 66 percent of the original within 0 167 or 0 125 for digital seconds A nominally full scale output is obtained after six time constants Final Testing 4 5 1 Pressure To ensure the integrity of the housing and seals the WETStar is subjected to a wet hyperbaric test before final testing The testin
29. y 0 25 cm sample volume is defined by the intersection of the excitation light with the field of view of the detector within the quartz flow tube A orange interference filter is used to discriminate against the scattered blue excitation light The orange fluorescence emitted at 90 degrees is synchronously detected at 1 kHz by a silicon photodiode The amplified and demodulated voltage output of the photodiode is provided to the user for connection to a digital voltmeter an A D converter or RS232 input The instrument contains two LEDs doubling the excitation light as well as mirrors and lenses to optimize the instrument s performance 2 5 Phycoerythrin WETStar This WETStar is primarily designed to measure the fluorescence due to the presence of phycoerythrin pigment in cyanobacteria with excitation and emission wavelengths of approximately 520 and 570 nm respectively The phycoerythrin WETStar uses two green LEDs centered at approximately 520 nm and modulated at 1 kHz to provide the excitation Interference filters are used to filter out of band light emitted by the LEDs A detector positioned at 90 degrees to the axis of the LED mounts measures the emitted light from the sample volume The approximately 0 25 cm sample volume is defined by the intersection of the 6 WETStar User s Guide WETStar Revision M 13 Jan 2006 WET Og Labs y at Ta excitation light with the field of view of the detector within the quartz

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