Chlorophyll - Water Research Center
Contents
1. the sample through a GF F 47 mm filter directly into the 13 x 100 mm disposable culture tube used for analysis Rinse filter holder with acetone prior to filter of the next sample NOTE Do not let the vacuum pressure exceed 1 2 psi or the sample volume will be affected 10 4 3 Wipe the outside of the cuvette dry with a lab wipe and place in the instrument Replace the light cap 10 4 4 Wait about 15 seconds for the reading to stabilize and log the reading Remember because of temperature effects for greatest accuracy read all samples after they have been in the fluorometer for approximately the same length of time 10 4 5 If the display reads OVER it means the sample is too concentrated to be read at the current calibration and is probably over the linear range of the instrument In this case dilute the sample by 75 1 part sample to 3 parts buffered acetone and reread it 10 4 6 Record the fluorescence measurement and the dilution factor if any on the chlorophyll a data sheet LG405 Revision 06 March 2002 Page 5 of 9 Sampling and Analytical Procedures for GLNPO s WQS 11 2 11 3 12 0 12 1 13 0 14 0 14 1 10 4 7 Readings obtained are the actual concentration of extracted chlorophyll a in the cuvette To arrive at the environmental chlorophyll a for each sample corrections for the amount of water filtered and extraction volume of acetone used must be applied as described below CALCU2. a and Pheophytin a in Marine and Freshwater Phytoplankton by Fluorescence In Methods for the Determination of Chemical Substances in Marine and Estuarine Environmental Samples Page 6 of 9 LG405 Revision 06 March 2002 Standard Operating Procedure for In Vitro Determination of Chlorophyll a in Freshwater Phytoplankton by Fluorescence 14 2 14 3 14 4 14 5 Environmental Monitoring and Support Laboratory Office of Research and Development U S EPA Cincimnati OH EPA 600 R 92 121 Arar E J 1994 Evaluation Of A New Fluorometric Technique That Uses Highly Selective Interference Filters For Measuring Chlorophyll a In The Presence Of Chlorophyll b And Pheopigments Environmental Monitoring and Support Laboratory Office of Research and Development U S EPA Cincinnati OH Turner Designs Model 10 AU 005 Field Fluorometer User s Manual November 1992 P N 10 AU 075 Weber C I L A Fay G B Collins D E Rathke and J Tobin 1986 A Review of Methods for the Analysis of Chlorophyll in Periphyton and Plankton of Marine and Freshwater Systems Ohio Sea Grant Program Ohio State University Grant No NA84AA D 00079 54 pp Welschmeyer N 1994 Fluorometric analysis of Chlorophyll a in the presence of Chlorophyll b and pheopigments Limnol Oceanogr 39 1985 1992 LG405 Revision 06 March 2002 Page 7 of 9 Sampling and Analytical Procedures for GLNPO s WQS Analytical Chlorophyll a Calculation Sheet Survey Date Analy
3. to 0 it means the standard concentration is too high for the LOW range Press lt ESC gt change to the MED range on screen 2 42 and return to step 8 2 Similarly if the MED range reads OVER with the Span close to 0 it means the standard concentration is too high for the MED range press lt ESC gt change to the HIGH range on screen 2 42 and return to step 8 2 CALIBRATION OF SECONDARY STANDARDS This procedure only has to be done once for each solid standard These standards are stable for at least two years and can subsequently be used as daily check standards as outlined below Place the solid secondary standard in the instrument so that the low value approximately 4 0 ug L is to be read Replace the light cap Wait 15 seconds for the reading to stabilize Record the reading on the solid standard calibration record sheet Reorient the solid standard so that the high value approximately 30 0 ug L is to be read Replace the light cap Wait 15 seconds for the reading to stabilize and record the reading of the solid standard on the calibration record sheet PROCEDURE Sample Preparation 10 1 1 Add 10 mL of 90 buffered acetone 6 2 to the tube containing the filter Recap tube and invert tube 3 times making sure that the filter is totally submerged in buffered acetone solution 10 1 2 Place each tube in an ultrasonic bath that had been previously filled with ice for 20 minutes 10 1 3 After 20 minute
4. LATIONS The concentration of chlorophyll a in the lake water sample is calculated by multiplying the results obtained above by 10 mL the extraction volume and dividing this by the volume in mL of the lake water sample that was filtered on the boat If the sample was diluted multiply the reading by 4 Any other dilution factors should be incorporated accordingly All data including readings of the secondary calibration standards should be entered in the electronic Excel 5 0 version of the chlorophyll a data sheet This spreadsheet will automatically perform all calculations A printout of this spreadsheet will then serve as the permanent data record The relative percent difference RPD is calculated according to the equation below rpp Lhigh value low value 199 average value QUALITY CONTROL The following audits are to be performed QC Type Minimum Frequency Calculation Check Daily 10 Laboratory Duplicate Once per batch Relative Percent Difference RPD 25 Field Duplicate Once per batch Relative Percent Difference RPD 25 Field Blank Once per batch 0 00 pg 0 11 ug These limits are interim limits that will be used until there is enough data to calculate performance limits for this procedure WASTE DISPOSAL Follow all laboratory waste disposal guidelines regarding the disposal of acetone solutions REFERENCES Arar Elizabeth J and G B Collins 1992 In Vitro Determination of Chlorophyll
5. Standard Operating Procedure for In Vitro Determination of Chlorophyll a in Freshwater Phytoplankton by Fluorescence LG405 Revision 06 March 2002 Standard Operating Procedure for In Vitro Determination of Chlorophyll a in Freshwater Phytoplankton by Fluorescence TABLE OF CONTENTS Section Number Subject Page 1 0 SCOPE AND APPLICATION srta ens lodos ee as cn To y o a Ad a 1 2 0 SUMMARY OE METHOD lt su eutied ats eciwbn dd RARAS EPA 1 3 0 SAMPLE HANDLING AND PRESERVATION 00000 eee eeeeeee 1 4 0 INTERFERENCES susurra O pote ae nae en SAN 1 5 0 EQUIPMENT REQUIRED oss o5g io a ti hace tee ie 1 6 0 REAGENTS suse seed scan SoS Soule due Meme ee Sree Bale A 2 7 0 PREPARATION OF CHLOROPHYLL a STANDARDS 2 020005 2 8 0 CALIBRATION secos oR he diane Ed AE 3 9 0 CALIBRATION OF SECONDARY STANDARDS 0000000 0 eee eae 4 10 0 PROCEDURE carita a RES 4 11 0 CALCULATIONS sateen Tara di aa A 6 12 0 QUALITY CONTROL 4 wsdl re Gee ea A aia eet eta ata kane ee he ea tial 6 13 0 WASTE IDISROSAL rta O SAS 6 14 0 REFERENCES 1009 eet teen bak dorar bok atan 6 ANALYTICAL CHLOROPHYLL A CALCULATION SHEET 8 10 AU FLUOROMETER SOLID STANDARD CALIBRATION RECORD 9 Disclaimer Mention of trade names or commercial products does not constitute endorsement or recommendation for use Standard Operating Procedure for In Vitro Determination of Chlorophyll a in Freshwater Phytoplankton
6. by Fluorescence 1 0 1 1 1 2 2 0 2 1 2 2 3 0 3 1 3 2 4 0 4 1 4 2 Standard Operating Procedure for In Vitro Determination of Chlorophyll a in Freshwater Phytoplankton by Fluorescence SCOPE AND APPLICATION This method provides a procedure for the fluorometric determination of chlorophyll a in freshwater phytoplankton employing narrow band optical filters This method is based on that originally published by Welschmeyer 1994 and is applicable to waters from the Great Lakes SUMMARY OF METHOD Chlorophyll containing phytoplankton in a measured volume of sample water are concentrated onto a glass fibre filter by low vacuum filtration After sonication pigments are extracted in 90 buffered acetone for 16 to 24 hours at 20 0 C The extracted slurry is removed by filtration and fluorescence of the extract read The concentration in the natural water sample is reported in ug L SAMPLE HANDLING AND PRESERVATION Samples should return from the field frozen and wrapped in aluminum foil Samples should be stored frozen 20 C in the dark until analyzed Filters can be stored frozen for as long as 3 Y weeks without significant loss of chlorophyll a Weber et al 1986 Analysis should be carried out under subdued green light to prevent photodecomposition of chlorophyll a INTERFERENCES Spectral interferences resulting from fluorescence of the accessory pigment chlorophyll b and the chlorophyll a
7. degradation product pheophytin a can result in overestimation of chlorophyll a concentrations However the highly selective optical filters used in this method minimize these interferences Previous work with this method has shown maximum interferences from chlorophyll b and pheophytin a to be 6 and 10 respectively Welschmeyer 1994 Arar 1994 LG405 Revision 06 March 2002 Page 1 of 9 Sampling and Analytical Procedures for GLNPO s WQS 5 0 5 1 6 0 6 1 6 2 7 0 7 1 7 2 EQUIPMENT REQUIRED Turner Designs Digital Fluorometer equipped with 10 113 Excitation Filter 436 nm and 10 115 Emission Filter 680 nm high intensity F4T 5 blue lamp Plastic Filter Funnel Gelman 300 mL with magnetic base Vacuum System 1 4 psi GF F Filters Whatman 47 mm 16 x 100 mm Screw Cap Culture Tubes 13 x 100 mm Culture Tubes Disposable 250 mL Filter Flask with Sidearm Nalgene Tubing 1 200 mL Volumetric Flask 5 100 mL Volumetric Flasks 1 50 mL Volumetric Flask Aluminum Foil Parafilm Spectrophotometer Disposable Glass Pipettes 20 0 pg L Chlorophyll a Calibration Standard available from Turner Designs Solid secondary Chlorophyll a Standards available from Turner Designs High Purity Grade Acetone 1 L Magnesium Carbonate Filter Forceps Vacuum Source REAGENTS Saturated Magnesium Carbonate Solution Add 10 grams magnesium carbonate to 1000 mL of de ionized water The solution is allowed to set
8. le TC on screen 2 11 cycles from 1 to 8 seconds and assuming the Blank 1s less than 200 press lt 0 gt When FINISHED appears press lt ESC gt 8 3 3 Remove the blank Set cuvette aside Run Calibration Standard Solution 8 4 1 On screen 2 0 press lt 3 gt to bring up screen 2 3 8 4 2 FS is the maximum concentration or relative fluorescence you will be able to read on a particular range and it is not necessary or likely that FS match the value of the calibration standard For example if you are calibrating on the MED range with a 25 g L standard LG405 Revision 06 March 2002 Page 3 of 9 Sampling and Analytical Procedures for GLNPO s WQS 9 0 9 1 9 2 9 3 9 4 9 5 10 0 10 2 you would want to adjust the Span until the FS for the MED range is approximately 30 meaning you will be able to read samples up to 30 g L on the MED range 8 43 Filla clean 13 mm culture tube with the chlorophyll a standard Put the standard in the sample chamber and replace the light cap Adjust the Span using the UP and DOWN arrows until the FS readings for each range are satisfactory Pressing the UP arrow increases Span sensitivity but decreases FS Pressing the DOWN arrow decreases Span but increases FS Wait until readings are stable TC on screen cycles from 1 to 8 sec then press lt gt When FINISHED appears press lt ESC gt 8 4 4 Ifthe LOW range reads OVER with the Span close
9. ould be performed once prior to the analysis of samples from each survey Allow the instrument to warm up for at least 15 minutes Have ready a blank of 90 buffered acetone and the calibration standard These are to be maintained at room temperature 23 25 C a water bath is recommended Start Calibration 8 2 1 To begin calibration from the Main Menu press lt 2 gt to access screen 2 0 Calibration 8 2 2 Set the concentration range control to MAN From screen 2 0 press lt 4 gt to bring up screen 2 4 then lt 3 gt to bring up screen 2 43 set conc range control and press lt ENT gt to toggle 8 2 3 Change to the desired concentration range i e HIGH from screen 2 4 press lt 2 gt to bring up screen 2 42 change concentration range and press lt ENT gt to toggle Return to screen 2 0 8 2 4 From screen 2 0 press lt 2 gt to access screen 2 2 standard solution concentration Enter the actual concentration of the calibration standard e g 155 g L Run Blank 8 3 1 Torun blank press lt 1 gt to access screen 2 1 Make sure 2 on screen 2 1 reads YES Then from screen 2 1 press lt 1 gt to call up screen 2 11 Because temperature affects fluorescence do not allow the blank to remain in the instrument any longer than necessary for a stable reading 8 3 2 Filla clean 13 mm culture tube with the blank of 90 acetone Put the blank in the sample chamber and replace the light cap After the Blank reading is stab
10. s return sample tube to freezer 20 0 C to steep for 16 to 24 hours Instrument Preparation 10 2 1 Samples should all be maintained at room temperature during analysis Page 4 of 9 LG405 Revision 06 March 2002 Standard Operating Procedure for In Vitro Determination of Chlorophyll a in Freshwater Phytoplankton by Fluorescence 10 3 10 4 10 2 2 Allow the fluorometer to warm up for at least 15 minutes prior to analysis 10 2 3 Access screen 2 43 by pressing successively lt 2 gt lt 4 gt and lt 3 gt Set the concentration range control to AUTO using lt ENT gt to toggle Daily Check Standard 10 3 1 Place the solid secondary standard in the instrument so that the low value is to be read Replace the light cap 10 3 2 Wait 15 seconds for the reading to stabilize and log the reading on the chlorophyll a data sheet 10 3 3 Reorient the solid standard so that the high value is to be read Replace the light cap 10 3 4 Wait 15 seconds for the reading to stabilize and log the reading on the chlorophyll a data sheet 10 3 5 Daily check standard values should be 10 of their previously determined values If they are not the instrument may have to be recalibrated Speak to the Biology Team Leader before proceeding with sample analysis Sample Analysis 10 4 1 Invert the sample test tube 4 times to mix 10 4 2 Using the filter flask with a sidearm attached to a vacuum unit filter the entire contents of
11. tle for a minimum of 24 hours Only the clear powder free solution is used during subsequent steps 90 v v Buffered Acetone Add 100 mL of the Magnesium Carbonate solution 6 1 to 900 mL of acetone in a 1 L volumetric flask PREPARATION OF CHLOROPHYLL a STANDARDS Primary standards of chlorophyll a in 90 acetone will be ordered from Turner Designs 845 W Maude Avenue Sunnyvale CA 94086 408 749 0994 If stored at 20 C in the dark standards are good for one month from their receipt from Turner Designs 7 1 1 Standards come in an ampule which is broken and poured directly into the cuvette Approximate concentration is 20 0 pg L Actual concentration varies by lot and is listed on a certificate of analysis to three significant figures Solid secondary standards also available from Turner Designs will be used for daily calibration check standards This standard is valid for two years Page 2 of 9 LG405 Revision 06 March 2002 Standard Operating Procedure for In Vitro Determination of Chlorophyll a in Freshwater Phytoplankton by Fluorescence 8 0 8 1 8 2 8 3 8 4 7 2 1 Secondary standards come sealed in a holder that fits directly into the 10 AU s 13 mm test tube adaptor The holder contains two ready to use fluorometric standard concentrations approximately 30 0 ug L and approximately 4 0 ug L CALIBRATION The following instructions apply to the Turner Designs 10 AU digital fluorometer Calibration sh
12. zed Lake Analyst Solid Secondary low Solid Secondary high Dilution Factor Volume Filtered Extract Volume Chlorophyll a Chlorophyll a ug L Sample ID 1 2 3 4 mL mL Analysis Reading Calculated Result Page 8 of 9 LG405 Revision 06 March 2002 Standard Operating Procedure for In Vitro Determination of Chlorophyll a in Freshwater Phytoplankton by Fluorescence 10 AU Fluorometer Solid Standard Calibration Record Instrument Serial Number Date Standard Received Date Analyst L reading H Reading LG405 Revision 06 March 2002 Page 9 of 9
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