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Manual for the FIAlab-2500 System

1. set delay time start pump to prime lines Valve Delay 5000 sample inject msec Feristaltic Pump Clockwise s 55 Injection Valve Sample Load Delay sec 40 Hardware Settings Optimize Integration Put autosampler in first sample usually a blank Next Sample Delay sec 25 Loop start 4 5000 3 5714905 09 Sample DEF Table 1 std rack 1 Blank 7070 3 514905E 09 Sample DEF Table 2 std rack 1 std 1E 20 010 Figure 4 18 Script Selection 32 Ammonia Phosphate Chloride Iron Dual Channel FLA Contact Closure TTLDAC Setting Peristaltic Pump Injection Valve Spectrometer Create Folder Delay sec End If Email If Insert File Hardware Settings Add Data Loop End Loop Start H Message Next Sample Reset Data Reset T ime Sample Description Save Data Stop Program Text Command lt M Varable FIAlab FIAlab Instruments Leaders in Flow Injection Technology 3 In the script that appears change the mode from safe lock to edit in the top center of the program window s toolbar Then change the initial sample delay to 45seconds and the spectrometer scanning delay to 35seconds Relock the script New Font Save Events Safe Lock Mode Find FIA Templates Sample Definition File Contact Closure Valve Delay 5000 z LL ISEL TTL DAC Setting Feristaltic Pump Peristaltic Pump Clock k Injection Valve ee Valve Sample Spectrometer Put autosampler in
2. Name Mean Response Concentration fit restos rah seta o P lBlank ss 2 387123E 03 01981861 eomm lana 0000 oo A EE En E SS E f HA Standard IA KA Deviation User Notes ample DEF Table 2 std rack 1 OmM 1E 20 010 Sample DEF Table 3 std rack 2 60mM 600100 Sample DEF Table 4 std rack 1 wash 010 Select Loca Local Maimun Maximum 1 True 1 pannel Selection settings Click to save data Auto Refresh ocd Mamm TE TP Show Concentration Local Maximum Show Data Labels Save Data Load Data Add Data Report Refresh y Figure 4 33 Example RSD from precision test 44 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 5 0 Several Common FIA Methods The methods listed are not the only available there are many additional methods for these as well as other analytes Make sure that the standards you create fully encompass the range of concentrations you expect to see in your samples At absolute minimum four standards including 0 should be used A quality control QC solution is also recommended and measured every N th e g 12 sample This QC sample 1s used to correct for any drift in response caused for example by degradation of the Cadmium column or shifts in room temperature during a long run Many FIA methods are subject to effects of interfering analytes which can either enhance or suppress the colorimetric response and result in erroneous readings It is c
3. 5 Transfer to a plastic bottle Reagent 2 Azomethine H Solution use hood 1 Measure out the following into a 1 liter beaker 5 g ascorbic acid 2 25 g Azomethine H monosodium salt monohydrate 2 Add 600 mL distilled water and stir to dissolve Reagent 2 stays good for 3 5 days Keep refrigerated in the dark 56 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 5 0 9 Silica Soluble silica species react with molybdate under acidic conditions to form a yellow silicamolybdate complex This complex is subsequently reduced with ANSA 1 amino 2 napthol 4 sulfonic acid and bisulfite to form a heteroploy blue complex which has an absorbance maximum at 820 nm Comments A heater is necessary and should be set to 60C The flowrate of the pump should be set to 55C Recommended wavelengths 800 nm for the primary and 300 nm for reference Make the sample loop from 10 25 4cm of 0 03 0 75mm ID tubing To some degree phosphate creates a positive interferes with the silica assay Carrier DI Water The quality of the DI water should be watched since silica is often encountered as a contaminant in water Silica contamination typically appears as the filters on the DI apparatus approach the end of their life cycle Reagent 1 Indicator solution 250 mL of 0 02 M Ammonium Molybdate solution 750 mL Degassed DI Water A volume of 1 M Hydrochloric Acid corresponding to 0 33 mol acid equivalents For example 3
4. 6 0 4 Spectrometer Wavelengths Each assay product has a unique absorption spectrum FIAlab software allows monitoring of up to four wavelengths along this spectrum simultaneously To look at low concentrations a wavelength with a high absorbance should be chosen To look at high concentrations a wavelength with a low absorbance should be chosen Choosing different wavelengths allows for an increased usable concentration range per run To optimize your run using monitored wavelengths 1 Observe the reaction product s spectra a Set up the assay as usual and run a high concentration standard When the sample zone reaches the flow cell stop the program Go to the spectrometer graphing tab make sure absorbance is selected in the bottom of this window and take a single scan The spectra will appear in the upper half of the screen 2 Select the wavelengths a Select the desired wavelengths from the spectra Generally three wavelengths are chosen to monitor for absorption signal and a fourth is chosen to monitor as a reference wavelength The reaction product should not absorb at the reference wavelength and adequate light should be available If these two conditions are met the reference wavelength will monitor noise throughout the system and its signal we be subtracted from the signal of the other three channels giving smoother peaks The signal subtraction is performed via the hardware settings use wavelength 4 as reference command Spectromet
5. 7 6cm of 0 03 0 75mm ID green tubing Recommended wavelengths 510 primary and 650 reference Carrier DI Water 1 Liter DI Water with surfactant 4 drops of dishwashing liquid or Brij per 500 ml Reagent 1 1 10 phenanthroline chelation chemistry AQUANAL plus reagent kit for iron analysis is used and is based on 1 10 phenanthroline chelation chemistry to Fe This Fe complex has an absorbance maximum around 510nm Source Look up product number 37404 or 37444 at http www sigmaaldrich com Dilute reagent by mixing one drop per one ml of DI water Reagent 2 Not used Plug R2 port with Teflon Plug Iron Standard 500ml 1 4784 500 Iron standard Source 727 524 7732 sales exaxol com www exaxol com Alternative A common alternative Iron method is based on Ferrozine For use with Iron II and total dissolved iron especially for ultra low level lt 100 nmol l consider using the method described in this paper COLORIMETRIC FLOW INJECTION ANALYSIS OF DISSOLVED IRON IN HIGH DOC WATERS MICHAEL J PULLIN1 and STEPHEN E CABANISS Department of Chemistry and Water Resources Research Institute Kent State University Kent OH 44242 USA 53 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 5 0 6 Chloride A heater 1s not necessary The flowrate of the pump should be set to 55 for Chloride The FIA LOV connections B should be bridged by a simple tubing no Cadmium Column Recom
6. Leaders in Flow Injection Technology 4 0 3B Analyzing the Results 1 From the software s main toolbar click on the analysis button THAlab for Windows Sii Instruments Options About 4 11 2013 6 46 01 PM Spectrometer Autosampler Frogram Figure 4 31 Analysis Button 2 Click on the plots tab in the analysis window and select channel 1 in the lower right hand corner Observe the peak shapes right click and drag to zoom in Peaks should be smooth for all measurments containing blue dye Absolute values of absorbance should be close to 0 4AU for the 60uM standard ktoplExample Data Precisi Report Generation Format Advanced Options pe e a an O AA ee Time sec 211212345067 amp 9 W411 12 15 Auto Refresh Show Concentration Local Maximum vw Show Data Labels Save Data Load Data Add Data Refresh it Figure 4 32 Example plots from precision test 43 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 3 Select the summary tab in the analysis window In the bottom right hand corner select Local Maximum from the pull down menu Observe the standard deviation Std value for the 60uM erioglaucine A good value is 1 5 or less 4 Save the data by clicking on the button in the bottom left of the analysis window C AUsers FIALab Andrea Desktop Example Data Precision Test dat Report Generation Format Advanced Options Summary Tab Day Summary
7. Set the pump rate to 50 in the upper left hand corner of the screen and switch the valve from the load to the inject position by clicking on the valve icon Figure 4 1 FIAlab screen with pump rate selected for flow rate test 2 Place the carrier reagent 1 and reagent 2 lines coming from the bottom of the peristaltic pump into a large 250ml container of deionized DI water At this point the pump s tension arm should be loose 3 Geta container for waste and into it insert the tubing coming out of the exit top port of the flow cell low cell outlet a Figure 4 2 Initial system setup for flow rate test 22 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 4 Start the pump by clicking on its icon in the FIA lab screen and while it runs lift the tension up and over the bottom of the peristaltic pump plate Manually tighten by screwing the tension arm against the plate until there is smooth flow of water through each of the carrier reagent 1 and reagent 2 lines 5 Once flow 1s smooth to prime the system let the pump run for an additional 43sec Stop the pump by clicking on its icon in the FIA lab screen Pump Plate mi ighten the tension arm against the bottom of the pump plate until lines draw DI Water smoothly Figure 4 3 Tightened peristaltic pump 6 Place 10ml of DI water into a thin graduated cylinder and then move the carrier line from the large container of DI wat
8. Single Channel Edit Selected File E Multi Channel 4 In WC Alleah fee Alimman ieradas areas r Create New Sample Df i ee Freq of OC Drittcor Sampling O Pos of OC Drittcor Sample la Create New File Figure 4 28 Final sample definition window for precision test with AIM autosampler 5 Close out of the window and start the program 40 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 4 0 3A C Creating the Sample Definition File Manual Operation Since there is no autosampler involved positions of samples are irrelevant 1 Make 40ml of a 60uM Erioglaucine solution and use extra DI Water to serve as a blank Ostd and wash 2 In manual operation the computer doesn t recognize the number of repeats column in the sample definition file Therefore each measurement must have its own line in the file Create a new file from the sample definition window as done in the linearity test and name it Precision Sample File Manual Edit the file so that it matches that shown below Save and close out of the file rack 1 Blank E 0 0 1 0 0 d rack O OuM 0 0 0 0 1 0 0 d rack 60 OuM 6 0 0 D rack 60 OuM 6 0 0 D d rack 60 OuM 60 0 0 D rack 60 Qum 60 0 0 D rack 60 OuM 60 0 0 D rack 60 OuM 6 0 0 D rack 60 OuM 60 0 0 0 d rack 60 OuM 6 0 0 D d rack 60 OuM 60 O d rack 60 OuM 60 O
9. d rack wash 7 0 0 rack std4 40 0 0 1 unknownl unknown i unknowns unknown4 unknowns cocooocosoas 0 OPRPRRRRPRRARAR eae a unknown unknown unknown unknowns unknownLo Figure 4 29 Precision sample file for manual operation cf ffs ffi fal a G a G Ga Ga Ga Soo oeqeqcea 41 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 3 Select the file just created from the sample definition file window as done in the linearity test Enter 13 in the total number of vials box and hit refresh Sample Definition File File Edit Forrnat Motepad Sample Definition File oo Sample File Manual cz a File Name Sample Definition Table View _ Rack Position Sample Name Conc Start Time hts Period hrs ICE r ON as Y AO ES PARIS CET CS CI CIR CE ETT PRI CTI CIS CI CE CE A QPE EE O uo ECO CC CC Select Existing File i Single Channel Total Number of Vials C Multi Channel Edit Selected File Select Redo File C Program Files x86 1FlAlab Instruments In ALA lnh fer Afindasomtenda arse Create New Sample De e File Click here to update andards f4 Freq of OC Drittcor sampling lo samples 1180 Pos of OC DOrittcor sample 4 Create New File Figure 4 30 Final sample definition window for precision test in manual operation 4 Close out of the window and start the program 42 FIAlab Instruments FIAlab
10. 5 6 grams Cadmium granules 30 80 mesh for a 1cc column 0 5mm ID by 5cm long 2 solution of Cupric Sulfate 2 grams in 100 ml water Acetone as needed 1N HCL as needed Place cadmium in a 100 ml beaker and wash with 25 ml of acetone followed by a 25 ml wash of water CAUTION Cadmium is very toxic and all washes should be collected and disposed carefully in accordance to regulations Wear gloves and wash thoroughly after handling Expose a fresh surface on the cadmium by washing the granules twice with 25 ml of IN HCL Follow acid washings with three water rinses Add 50 ml of the 2 cupric sulfate solution to the cadmium and stir gently for 5 minutes Repeat with a fresh addition of cupric sulfate solution Cadmium should be a dark gray black in color Follow with 3 5 DI water rinses Decant off any fine suspended matter Pack column with copperized cadmium in water making sure that all air bubbles are removed Store column in the ammonium chloride buffer 47 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 5 0 2 Ammonia Water Ammonia Water Bath Heater should be turned on to 60 C The flowrate of the pump should be set to 45 55 The FIA LOV connections B should be bridged by short green tubing Recommended wavelengths 650 nm primary and 525 nm reference 825 nm can also be used as the reference For most Ammonia assays gt 0 5 ppm make the sample loop from 3 7 6cm of 0 03 0 75mm ID green
11. DC y F A Power Suppl E ou 2 omputer s Native A A A serial Port 4 A Figure 2 1 FIAlab 2500 back panel and hookup to computer 4 Wind the Tygon peristaltic pump tubes around the wheel of the pump so that they are held in place by the tubing stops The pump tube connecting to the S port on the LOV should be wound around the pump channel marked S the pump tube connecting to the C port on the LOV should be wound around the pump channel marked C the pump tube connecting to the R1 port on the LOV should be wound around the pump channel marked R1 and the pump tube connecting to the R2 port on the LOV should be wound around the pump channel marked R2 A Pi Figure 2 2 Peristaltic pump tubing connections FIAlab Instruments FIAlab Leaders in Flow Injection Technology 2 0 2 FIA LOV Manifold Figure 2 3 Classical FIA Configuration The FIA Lab On Valve Manifold is used to organize the fluidic connections for your FIAlab 2500 Figure 2 3 shows the classical FIA layout Figure 2 4 shows the equivalent FIA Integrated Manifold and Table 2 1 describes specific fitting connections s V lea ample Loop _ E a Aa Figure 2 4 FIA ee Manifold FIAlab Instruments FIAlab Leaders in Flow Injection Technology LOV Ports M1 to M1 Miximg Coil 1 Recommend 69 175cm clear tubing of 0 03 0 7
12. File ES esktop Precision Sample File Cetac csv lt a File Name Sample Definition T able View Rack Position_ Sample Name Conc Start Time his Period hrs 0 1 0 LPI std rack 1 Blan stdrack 2 60 0uM 60 0 10 0 cog Set to 4 for Select Existing File Clear File Name Single Channel Total Number of Vials lt a precision test ee Edit Selected File MEAR OS Program Cta riruments Create New Sample Definit 4 Freq of OC Driftcor Sampling fo y Samples 180 Pos of QC Driftcor Sample 4 y Create New File Figure 4 26 Final sample definition window for precision test with Cetac autosampler 5 Close out of the window and start the program 38 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 4 0 3A 2 Creating the Sample Definition File AIM Autosampler The standard tubes of AIM autosamplers hold up to 10ml of solution The solution made for the precision test must be split between three standard vials to account for the number of measurements required for the test 1 Make 40ml of a 60uM Erioglaucine solution and use it to fill three AIM standard tubes Put DI Water in two other standard tubes one to serve as a blank and Ostd and one to serve as a wash 2 Place the vials into the standard rack in the following order Position 1 Blank Ostd Position 2 60uM Erioglaucine Standard Position 3 60uM Erioglaucine Stan
13. accurate absorbance measurements in the flow cell Solution By putting a 20 PSI pressure restrictor see PN P 785 at www upchurch com on the flow cell output line and running the pump at 65 the resulting overpressure prevents gas from coming out of solution In this fashion Olsen phosphate can then be run like other phosphate extractions e g Bray Bray Phosphate The carrier and reagent mixture wavelength selection and configuration is the same as for Ortho Phosphate Alternatively Bray phosphate may be run using the exact configuration as described above for Olsen 51 FIAlab Instruments FIAlab Leaders in Flow Injection Technology Total Phosphate TP A heater is not always necessary but may be turned on to 50C for increased sensitivity The flowrate of the pump should be set to 50 The FIA LOV connections B should be bridged by a simple tubing no Cadmium Column Recommended wavelengths either 660 or 860 nm for the primary and 490 reference For most TP assays make the sample loop from 3 7 6cm of 0 03 0 75mm ID green tubing Carrier DI Water and H SO acid 1 Liter Degassed DI Water Recommend carrier line to be DI water Add 60 mL conc HSO acid 36N ACS grade Sigma Aldrich 38 337 6 The acid is to match the total phosphate matrix For low concentrations the carrier should be identical to the standard 0 blank Reagent 1 Sample Carrier Stream of 6 mM Ammonium Molybdate 10 0 grams Ammo
14. cell for concentrations consistently greater than 100 ppm Recommended monitored wavelength is 500 nm though any wavelength from 400 to 800 nm will work Critical Do not use a reference wavelength Sulfur can be added to the reagent to seed the precipitate This is necessary for the lower concentrations Sulfate 16 std200 U4 std100 Blank std0 D 81 5 137 3 193 1 248 9 304 6 360 4 Time sec Above plot shows response for Sulfate concentrations of 0 50 100 200 ppm Carrier Matrix Match Matrix Match If samples are water then use water as carrier If soil extract then typically add KCL in proportion with the extracted samples e g 12 g KCL per liter of water The matrix matching isn t critical if samples have greater than 100 ppm Sulfate concentration Reagent 1 Barium Chloride HCL Sulfur for one liter 30 0 grams BACI Dihydride 10 ml Hydrochloric acid 0 004 grams Sulfur of DI Water this is not needed for sample sulfate conc gt 10 ppm 1 Liter DI Water Stir until dissolved Reagent 2 No Reagent 2 plug this channel port 55 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 5 0 8 Boron Boron reacts with azomethine H to form a colored complex which absorbs at 430 nm The increase in absorbance is proportional to the boron concentration The following method should work for concentrations from 0 2 ppm to 15 ppm For higher concentrations than this increase pump spe
15. delay time start pump to prime lines Message Valve Delay 5000 sample inject msec Next Sample Peristaltic Pump Clockwise 55 Reset Data Iniecti Reset Time njection Valve Sample Load si Sample Description Delay sec 40 o Save Data Hardware Settings Optimize Integration Stop Program Text Command lt N gt Put autosampler in first sample usually a blank Variable Next Sample Delay sec 25 LoopStart 5000 3 57486E 09 Sample DEF Table 1 std rack 1 Blank 2010 3 57486E 09 Sample DEF Table 2 std rack 1 std1 1E 20 010 LM D Start Stop Emergency Figure 4 11 Opening the sample definition file window 28 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 3 In the sample definition window that appears make sure that the file edit format that is selected is Notepad and that the system selected 1s single channel Hit Create New File Sample Definition File File Edit Format Notepad Y Notepad Excel top precision sample file txt Sample Definition Table View Check k Position Sample Name Conc Start Time hrs Period hrs 1 0 1 0 Notepad Blank std rack 2 60uM 60 0 10 0 gt es es System Select Existing File Clear File Name Single Channel Select Single Channel Unlock P Multi Channel Edit Selected File Refresh Select Redo File WProgram Files FlAlab Instruments Inc FlAlab Enr Ulin daverm enda an
16. first sample nal a blank Next sample Delay sec 25 Gi change to 45 Change to 45 Loop start g 5000 inject sample load next sample autosampler wash Analyte New Sample Next Sample Injection valve Sample Inject Delay sec 3 o Sample Description perform reference scan and start absorbance scans save Data Stop Prograrn spectrometer Reference al Text Command lt M spectrometer Abgo rb gu Variable Delay see 20 LA Ch spectrometer Sta Scanning Refresh plots and update concentrations 3 574918E 09 Sample DEF Table 1 std rack 1 Blank 4010 3 074918E 09 Sample DEF Table 2 std rack 1 0 0uM 1E 20 01 0 Figure 4 19 Script Modifications 4 Ifusing an autosampler connect the sample line coming from the bottom of the FIAlab 2500 s peristaltic pump to the sampler s probe tubing using two P 286 nuts two P 200 ferrules and a PEEK union Autosampler Probe Tubing 2 ample Line tubing from peristaltic pump Figure 4 20 Autosampler Fluidic Connection 33 FIAlab Instruments F TA lab 5 6 1 8 Leaders in Flow Injection Technology Also 1f using an autosampler click on Sample Definition File in the upper right hand corner of the program window and make sure that Use Sample Definition File is checked and that User Sample Prompt is unchecked For manual operation click on Sample Definition File in the upper right hand corner of the program window and make
17. most sample loops nr 6 y Figure 6 1 LOVs with sample loop lengths of 3 7 6cm 6 15 2cm and 12 30 5cm respectively Increasing the sample loop volume will increase signal only up to a certain point Beyond that point peaks will grow wider and progressively more flat topped but peak height itself will no longer increase 58 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 6 0 2 Extended Range Option Figure 6 2 FIAlab 2500 configured for extended range If you wish to measure high concentration samples and low concentration samples within the same run the extended range option may prove the best solution This configuration requires an extra spectrometer bifurcated cable and both flow cells 1cm and 10cm that came with the unit One spectrometer will read the signal from the low concentration samples on the 10cm path flow cell and the other spectrometer will read the signal from the high concentration samples on the 1cm flow cell Each spectrometer reads two wavelengths As an example of how the analysis works Spectrometer is connected via fiber optic cable to the 1cm flow cell and is set to measure wavelength 1 at 540nm and wavelength 2 at 600nm The 1cm flow cell yields the best signal for high concentration samples In the analysis window the calibration for these high samples corresponds to channel and channel 2 Low concentration samples should be taken out of these c
18. sure that both Use Sample Definition File and User Sample Prompt are checked When the script is run in manual mode it will give a prompt to the user when it is time to put the sample line in the next vial Make sure the new sample has fully passed through the sample loop and out the FIA LOV s waste port before clicking ok Hit start in the bottom of the program window New Font SaveEvents SafeLockMode Find FLA Template set delay time start pump to prime Always check P Y Use Sample Definition File Contact Closure Valve Delay 5000 sample inject msec Select Edit Create File re peral Peristaltic Pump Clackwisels 55 Injection Vawe Injection Valve Sample Load User Sample Prompt Delay sec 40 inr l Spectrometer Hardware Settings Optimize Integration Put autosampler in first sample usually a blank Creste Folder Next Sample Delay sec Delay sec 25 Loopstart 5000 Hardware Settings inject sample load next sample Add Data autosampler wash ee E i Analyte New Sample ee 4 Next sample Next Sample Injection Valve Sample Inject Reset Data Delay sec 3 Reset Time Sample Description save Data perform reference scan and start absorbance scans Stop Program spectrometer Reference scan Text Command lt N gt Spectrometer Absorbance Scanning Variable Delay sec 20 spectrometer Stop Scanning Refresh plots and update concentration a 3 574918E 09
19. tubing Low Concentration Assays For low concentrations below 0 5 ppm consider using the 10 cm flow cell Also for lower concentrations 01 to 2 ppm use a longer sample loop e g 12 30 5cm of 03 0 75mm ID tubing For the low end of this range care must be taken to use clean glassware and extended washouts of tubing on the system to prevent crossover contamination Carrier De ionized DI water Reagent 1 Hypochloride Solution 10 mL of a 6 Sodium hypochloride solution common household bleach 5 grams NaOH s Balance Degassed DI water 1 L Place a 10ml solution of bleach into a 1 liter volumetric flask and fill with 700 mL of degassed DI water Dissolve all other chemicals and fill flask to the 1 liter mark Reagent 1 degrades with time should be prepared daily Note If bubbles become an issue sticking in the flow cell and other parts of the manifold then add 1 gram Brij 35 detergent to Reagent 1 Reagent 2 Salicylate Catalyst Solution 100 grams Sodium Salicylate 160 11 FW 0 40 grams Sodium Nitroferricyanide III dihydrate 297 95 FW catalyst 5 grams NaOH s Balance Degassed DI water 1 L Place the sodium salicylate into a 1 liter volumetric flask and mix with 700 mL of degassed DI water until dissolved Add the sodium nitroferricyanide and mix until dissolved Add NaOH to adjust the pH to the 12 0 range Add the Brij 35 mix and fill flask to the mark Transfer solution into a dark airtight
20. weekly since this solution is unstable Phosphate Standard 100ml ICPHO 100 Phosphate standard Source 727 524 7732 sales exaxol com www exaxol com 50 FIAlab Instruments FIAlab Leaders in Flow Injection Technology Olsen Sodium Bicarb Phosphate The carrier and reagent mixture is the same as for Ortho Phosphate previous page Due to common brown coloring in samples Olsen phosphate must usually be run using 880 nm for the primary and 925 nm for the reference wavelength The flowrate of the pump should be set to 65 The sample loop should be made of 3 7 6cm of 0 02 0 50mm ID orange tubing Heat is typically set to 45C For lower concentrations Commonly occurring in soil samples the 10 cm flow cell is recommended Do not increase the sample loop size Important Note The provided LS1 light source has a very high output at around 700 nm This can cause saturation and bleed over of the detectors when the spectrometer is optimized for the Olsen wavelengths 880 925nm For this reason please insert an Edmund Optics TS Longpass filter included with the FIAlab Olsen Kit into the slit of the LS1 lamp Special Notes for Olsen Phosphate Problem Olsen Sodium Bicarb extracted samples contain high quantities of sodium bicarbonate which normally causes large number of bubbles foam to be created when the sample comes in contact with the acidic reagents This will cause significant problems with
21. 20 Figure 4 14 Linearit test sample definition file 30 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 7 From the lower left hand corner of the autosampler screen hit Select Existing File and then go to the location the file is saved in Make sure All files 1s selected so that the appropriate file will appear can be chosen 1 Tes y BE Desktop Organize v New folder YX Favorites f FlAlab for Windows ME Desktop Shortcut Shortcut lg Downloads L 2 19 KB 2 09 KB T Nun y EE Recent Places FlAlab Pumper FlAlab for Windows po A l File folder _ s A Libraries Documents LOV Research Release FlAlab Lite d Music File folder File folder t Pictures RE TE Release FlAlab Pumper ee ate wre bo sio E Videos CSV File File folder 8 32 KB jE Computer blue dye linearity test csv LOV test script wash fia CSVFil dl 0s c 8 36 KB DVD RW Drive D AP PA x Comma delimited files csv gt All files Figure 4 15 Selecting the sample definition file File name blue dye fia 8 After selecting the file its name will appear at the top of the sample definition file window In the total number of vials box type 6 and then hit refresh The sample definition file window should now look as it does below Sample Definition Fil File Edit Format Notepad m Sample Definition File pl dye linearity test csy lt a File Na
22. 30 mL of 1 000 N HCl or 333 mL of 0 990 N HCI 0 02 M Ammonium Molybdate solution 25 0 grams Ammonium molybdate tetra hydrate 1235 81 FW Dissolve in water under gentle warming and dilute to 250 mL Filter Make sure the filter is free of silica do not use glass filters Adjust pH to 7 8 with silica free NaOH approx 7 8 mL of 50 NaOH Store in a plastic container Reagent 2 Reducing Agent amp Phosphate Suppressant solution 250 mL of 0 15 M Oxalic Acid solution 750 mL of 0 23 M ascorbic acid solution with 1 3 mL sodium dodecyl sulfate solution 0 15 M Oxalic Acid solution 18 75 grams Oxalic acid dihydrate 126 07 FW Dissolve in water and dilute to 250 mL Store in a plastic container 0 23 M Ascorbic Acid with 1 3 g L Sodium Dodecyl Sulfate 30 grams Ascorbic acid 176 12 FW plus 1 grams Sodium dodecyl sulfate 288 38 FW surfactant Sigma Aldrich 436143 25G 0 75 Liter Degassed DI Water Place the ascorbic acid into a 1 liter container and mix with 500 cc of DI water until dissolved Add the sodium dodecyl sulfate and mix slowly prevent foaming until dissolved Fill the container to 750 mL Transfer solution into an airtight light sensitive glass bottle for maximum longevity Minimize exposure to air and prepare fresh weekly since this solution is unstable 57 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 6 0 Assay Optimization There are a number of ways to optimize your run Whether you ne
23. 5mm ID coiled M2 to M2 Miximg Coil 2 or heating coil Recommend 69 175cm clear tubing of 0 03 0 75mm ID coiled This coil may be replaced by a flow through heater coil of the same length Turn the heater off when heating isn t required One end of mixing coil 2 or heater coil will attach to the lower port on the flow cell B to B Bridge or Cadmium Column For Nitrate assays use a Cadmium Column here else connect B to B with 3 5 8 9cm clear tubing of 0 03 0 75mm ID first reagent to mix with sample second reagent to mix with sample Carrier Peristaltic pump tubing connects to carrier that pushes sample through system Usually bubble free water but must match sample matrix S Sample Connects to autosampler through peristaltic pump tubing If an autosampler is not used put sample line into appropriate vial as needed Connects to waste SL Sample Loop Length and ID adjustable to alter concentration range of assay Standard for 1cm flow cell 3 7 6cm of 0 03 0 75mm ID clear tubing Table 2 1 FIA Lab On Valve Manifold Connections Notes on common assay manifold configurations can be found in section 5 0 To change Teflon tubing on the LOV manifold 1 Remove the used or unwanted tubing by unscrewing the appropriate fittings on the FIA LOV 2 Slide a P 286 1 16 14 28 nut onto one end of the new tubing and follow with a P 200 1 16 ferrule cone pointed towards the nut 3 Sc
24. 7 6cm of 0 03 0 75mm ID green tubing Low Concentration Assays For low concentrations below 1 0 ppm consider using the 10 cm flow cell Increase the sample loop to 18 45 7cm of 0 03 0 75mm ID green tubing Adjustments to the selected wavelengths may be necessary to compensate for any natural color in the samples Carrier DI Water 1 Liter Degassed DI Water Reagent 1 6mM Ammonium Molybdate 10 0 grams Ammonium molybdate tetra hydrate 1235 81 FW 0 2 grams Antimony Potassium Tartrate half hydrate 333 94 FW catalyst 40 mL conc H SO acid 36N ACS grade Sigma Aldrich 38 337 6 1 Liter Degassed DI Water Place acid into 800mL of DI water mix and let cool to room temperature Add molybdate and antimony potassium tartrate and mix until dissolved Fill flask to the mark Transfer solution into a dark and airtight glass bottle for maximum longevity This solution is stable for several weeks Reagent 2 Reagent Carrier Stream of 300mM Ascorbic Acid 30 grams Ascorbic acid 176 12 FW 1 0 grams Sodium dodecyl sulfate 288 38 FW surfactant Sigma Aldrich 436143 25G 1 Liter Degassed DI Water Place the ascorbic acid into a 1 liter volumetric flask and mix with 600 cc of DI water until dissolved Add the sodium dodecyl sulfate and mix slowly prevent foaming until dissolved Fill flask to the mark Transfer solution into an airtight light sensitive glass bottle for maximum longevity Minimize exposure to air and prepare fresh
25. FIAlab Instruments Leaders in Flow Injection Technology Manual for the FIAlab 2500 System Version 2 0 FIAlab Instruments FIAlab Leaders in Flow Injection Technology Table of Contents 1 0 INTRODUCTION cocos iii cana 3 20 PHYSICAL SYSTEM SETUP lt a da 4 OL PALA DONT oee E a 5 2 We NANO ee EEE E E E E 6 ZA Os ANPE e E A E elem oie tens seni aende 8 PRIR Fea TON MT E as recesses E nc a inser tes EE E osetia EE E EEE E EE EE 10 OO co IB 61 8 i lie OR EEE EE 11 SA OK IBER OPE CABLES ee ee ne een eC See vee eee Teen entre ee 13 AER cece sos e ce cn oe pe cha A E 14 3 0 LOGGING ONTO THE SYSTEM siii 16 A BO UNI aac reido 17 SA SEC A n 18 A POS AMP ER eoc EET AOE E ETTE do ceedleade ned ceaw T EEEE TaN 20 4 0 INITIAL TES TS ondaa AE Aa aa aai 22 AO MRO IRA lee DISS eorr a EAEE E AEN 22 402 BLUE DYE LINEAR EST unio pie AGEE 25 40 3 BLUE DYR PRECISION TES Tio asic s 37 5 0 SEVERAL COMMON FIA META O DSi ai 45 5 0 1 NORATE NITRITE escusa idad 46 0 2 AMMONIA datada llas rodeos 48 5 0 3 TOTAL KJELDAHL NITROGEN TKN cccccccccceecccssccceecccceccceecccsscseusscsesecseecsescseuscesusesseecseeeceseeessuseseuseseues 49 O eee ents ses Sees geee Sees ac ods nob Banat a TA 50 By Roos sss ess secs ese Se seo ais nese AE T eee te tne snes Seco poset Seine ac oe ANE EE EEE E 53 BY A 54 A A siete Se aoe cess Sin ees ne es Deron A 55 MS BORON tarotista OEE 56 A O SS ee ee ee vee ere ee eee 57 60 ASSAY OPTIMIZATION od osccccseecc
26. IAlab 2 0 5 Light Source 2 0 54 HL 2000 LL Figure 2 12 Ocean Optics HL 2000 LL Light Source To setup the HL 2000 LL 1 The HL 2000 LL comes with an optional attenuator which can be attached to its SMA connection If when running the system you experience high light levels or very low integration times attach the attenuator by screwing it onto the light source Figure 2 13 Attaching the attenuator to the HL 2000 LL ight Source SMA onnection 2 Place the HL 2000 LL into the left side of the light bracket so that its rubber feet line up with the four holes on the interior of the bracket Make sure the power switch on the light source protrudes out of the bracket s back 3 Screw the HL 2000 LL into the bracket using the four provided M3 screws Tighten each screw loosely at first and then tighten all HL 2000 LL Ser No 004800577 Mounting Holes A on Internal Plate Mounting Holes in Center of Rubber Feet t Left Side of Light Bracket Figure 2 14 Installing an Ocean Optics HL 2000 LL light source onto a FIAlab 2500 light bracket 4 Reattach the light bracket to the back of the instrument and plug the HL 2000 LL into the wall 11 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 2 0 5B DCON LED Figure 2 15 FIAlab Instruments DCON LED To connect the DCON LED 1 Unscrew the cap of the DCON LED and attach it onto the SMA connector on the left side of the fl
27. Sample DEF Table 1 st tart cb P Ll here 3 574915E 09 Sample DEF Table 2 std rack 10 0 20010 Figure 4 21 Starting the program As the lines begin to prime watch the flow cell for any bubbles that may get stuck Attempt to dislodge them by knocking on the flow cell if necessary 34 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 4 0 2F Analyzing the Results 1 From the software s main toolbar click on the analysis button Instruments Options About 4 11 2013 6 46 01 PM XYZ 4 1010 erf Program Figure 4 22 Analysis Button 2 Click on the plots tab in the analysis window and select channel in the lower right hand corner Observe the peak shapes Peaks should be smooth for all samples containing blue dye Absolute values of absorbance should range from close to 0 2absorbance units AU for the 30uM standard to close to 0 4AU for the 60uM standard CAUsers FIALab Andrea Desktop Example Data Linearity Testda Report Generation Format Advanced Options Summary j Time sec a 2 Gis cid Auto Refresh Show Concentration Show Data Labels ort Fe tresh Figure 4 23 Example plots from linearity test 35 FIAlab FIAlab Instruments Leaders in Flow Injection Technology gt 3 Select the calibration tab in the analysis windo
28. alibration curves by changing their true concentration to a in the analysis window at the end of a run 59 FIAlab Instruments FIAlab Leaders in Flow Injection Technology Spectrometer 2 is connected via fiber optic cable to the 10cm flow cell and is set to measure wavelength 3 at 540nm and wavelength 4 at 600nm The 10cm flow cell yields the best signal for low concentration samples In the analysis window the calibration for these low samples corresponds to channel 3 and channel 4 High concentration samples should be taken out of these calibration curves by changing their true concentration to a in the analysis window at the end of a run To set up the extended range option 1 Mount a 10cm flow cell in addition to the 1cm flow cell on your instrument 2 Remove the waste line from the 1cm flow cell and in its place put a piece of tubing linking its outlet to the inlet of the 10cm flow cell Attach a waste line to the 10cm flow cell outlet ubing from uppe exit port of 1st ow cell to lower entrance port of 2nd flow cell i Figure 6 3 Flow cell configuration in extended range option 3 Plug both spectrometers into the computer open the spectrometer setup tab and check the box below that says use multiple spectrometers Click Omnidriver on the right hand side and hit Logon 4 Configure one of the spectrometers in the spectrometer setup tab a Under Selected Spectrometer pick Master b U
29. also physically listed on the bottom of the spectrometer Spectrometer Graphing Spectrometer Setup USB DT Lamp ignore if not using a USB DT Lamp Sample Rate Hz Settings applied to all spectrometers Detectors to Average 1 3 5 Samples to Average n a F p Visible Lamp Intensity i 0 i Stream data to file Plotting Threshold ents Tice pO j Use wavelength 4 as reference for Master Spec fio C DODriver Turn Off Reference Warnings Omnidriver beta M USB DT On Visible Lamp On oo Lamp On Serial number will sad Optimize Integration show here after logon Time Offset Absorbance Y Auto Dark Correction Settings for specific spectrometers Use for fluorescence return age when absorbance measurement is commanded Normally Fy be unchecked Defaults Selected Spectrometer Master M Enabled Serial Number USB4H11844 B Use Multiple Spectrometers Scale Response a ormally should be 1 0 integration Time mse efficients will be listed in these boxes 5 Q et Loetticents First Coefficient Second Coefficient Third Coefficient Intercept p 2174768 pe D6 P 687645E 10 i 3109 Es i gt Reference Voltage Absorbance Single Scan Repeated Scan Scan to File Exit Figure 3 7 Spectrometer Coefficients and Serial Number 5 The user must logon to the spectrometer from the spectrometer window each time the software is opened 19 FIAlab FIAlab Instruments Leaders in Flow Injection Technolog
30. b Leaders in Flow Injection Technology 2 0 4 Spectrometer Figure 2 10 Ocean Optics USB4000 Spectrometer To install the spectrometer onto the instrument s light bracket execute the following steps 1 Unscrew the top two screws on the instrument s back panel and slide the light bracket off of the instrument 2 Place the spectrometer into the rectangular slot on the right side of the light bracket so that two of the mounting holes on the spectrometer line up with two of the holes on the bracket s bottom and the USB port on the spectrometer protrudes out of the bracket s back 3 Screw the spectrometer into the bracket using two 4 40 3 8 pan head screws Tighten each screw loosely at first and then tighten both S Set Back Panel Screws LN DE lt lt lt 2 B Ni Back Side of JLight Bracket USB Port y _ Spectrometer Side of Light Bracket g t j Light Bracket Alignment Holes Figure 2 11 Installing an Ocean Optics USB4000 spectrometer onto a FIAlab 2500 light bracket 4 Do not reattach the light bracket 1f using an HL 2000 LL as a light source The HL 2000 LL will also need to be installed on the bracket If not using an HL 2000 LL reattach the light bracket to the instrument s back panel Once the light bracket is reattached plug the spectrometer into the computer using the provided USB cable 10 FIAlab Instruments Leaders in Flow Injection Technology F
31. configuration 1 Turn on the FIAlab 2500 and the autosampler via the switches on the back of each unit 2 Open the FIAlab for Windows software and in the main tool bar under systems make sure that FIAlab 2500 is checked under detectors that spectrometer is checked and under accessories that autosampler is checked If you have additional detectors or accessories make sure these items are checked as well FIAlab for Windows 5 0 Options About 1 15 2013 3 21 28 PM Systems gt FIAlab 2500 SFIAInh for Windows al Detectors FIAlab 3000 FIAlab 3500 MicroSIA Accessories CALM Systems gt a Detectors gt Y Spectrometer Analysis FlAlab for Windows 5 0 Instruments Options About 1 15 2013 3 24 46 PM Systems gt XYZ 444 Lui Detectors L L 1010 ue Analysis Accessories b Polarimeter A D Card LabJack Accessories rl v Autosampler Heater Control Modbus Figure 3 1 Complete setup from software s main toolbar 16 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 3 0 1 FIAlab 2500 unit 1 Click on the FIAlab button in the software s main toolbar Instruments paa About 1 15 2013 4 56 30 PM as g ser f NA Program ES Figure 3 2 FIAlab button on software s main toolbar 2 Make sure that Serial Port 1 is selected in the upper left hand corner of the FIAlab window that has just popped up 3 Hit logon i
32. ctions and time delays between each injection The standard template will need to be edited according to the instructions below so that the 6 computer knows what to expect during the linearity test a b c d e g Field 1 the first column entry in the line is the Rack name For all six entries in the blue dye test assay this should read std rack Field 2 the second column entry in the line is the Sample Position Each rack has positions labeled one through num where num is the capacity of the rack named in field 1 For entries in the blue dye test assay this should read going down the column 1 1 2 3 4 5 Field 3 the third column entry in the line is the Sample name The name is arbitrary However having multiple samples with the same name will cause the software to group these samples and give such statistical information on the Analysis page such as the mean and standard deviations For all entries in the blue dye test assay this should read going down the column blank OuM 30uM 45uM 60uM wash Field 4 the fourth column entry in the line is them nominal concentration This field is used if the specific defined vial is a standard If the vial contains an unknown this field should have a question mark A blank should have concentration B Use 0 0 to include a blank in the calibration curve 1 e standard concentration of 0 0 For all entries in the blue dye test assay this should
33. dard Position 4 60uM Erioglaucine Standard Position 5 wash 3 Create a new file from the sample definition window as done in the linearity test and name it Precision Sample File AIM Edit the file so that it matches that shown below Save and close out of the file File Edit Format View Help rack 1 Blank i rack 1 0 0uM rack 60 0uM d rack 2 60 0uM d rack 60 OuM rack wash 7 unknownl unknown unknowns unknown unknowns unknowns unknown unknowns unknowns unknown Figure 4 27 Precision sample file for AIM autosampler SOS SoS Se G O ooo Ooa H oooococcccoww cooocescso oooccoocecoce Loto ga bn P u hJ es HHHHHHHHH ao H D O O pl D fal jad fal fl pul pol pel pul ps 39 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 4 Select the file just created from the sample definition file window as done in the linearity test Enter 6 in the total number of vials box and hit refresh Sample Definition Fil File Edit Format Notepad sample Definition File C UsereSFlALab AndreaSD esktop Precision S ample File AlN cay dea j File Name EN Sample Definition Table View Tra Postion SanpleName cons Stat Time vs Num ot Repeats Period vs SC Lc CIONES CIONES CONEA std rack 2 RA std rack d4 J60 duM e o a a o PE y System Select Existing File
34. e Plotting IV only last 100 4 only last 100 Figure 4 8 Picture of single scan graphs for USB4000UV V IS left and USB4000VIS NIR right 4 Ifthe peak has a very low less than 10 000 count maximum there is likely a bubble in the flow cell Try to dislodge the bubble by hand or by flowing water through the system and manipulating the pump rate Then take the single scan again 26 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 4 0 2C Organizing Standards in an Autosampler If not using an autosampler keep the blank wash and Erioglaucine standards in the containers they were made in and skip this section 1 Take five of the autosampler s standard tubes and into one pour your blank standardO into another your wash and into the other three the 30uM 45uM and 60uM Erioglaucine standards Label each standard tube with its contents Make sure if using Cetac standard tubes the fluid level is above the 10ml mark at minimum and fill AIM standard tubes at least halfway 2 Each autosampler rack has set positions Place the solutions in the autosampler s standard rack in the following order Position 1 Blank Position 2 30uM Erioglaucine Standard Position 3 45uM Erioglaucine Standard Position 4 60uM Erioglaucine Standard Position 5 Wash Position1 Position 3 Position 5 Figure 4 9 Standard rack configuration for Cetac ASX 260 autosampl
35. e assays 0 5 to 100 ppm make the sample loop from 3 7 6cm of 02 0 50mm ID orange tubing and use a 1 cm flowcell Low Concentration Assays For low concentrations below 0 5 ppm consider using a 5 10 cm flow cell Also for lower concentrations use a longer sample loop e g 6 15 2cm of green tubing which is 03 0 75mm ID Carrier DI Water Reagent 1 1 6 M Ammonium Chloride Buffer for Nitrate only 43 grams Ammonium chloride 500 ml TOTAL Balance with Degassed DI water 4 drops of dishwashing liquid or Brij per 500 ml Note For Nitrite plug this port with an included Teflon Plug Reagent 2 Colorimetric Sulfanilamide Solution LC13280 Color reagent for Nitrate From Fisher Sci search for LC13280 at www fishersci com This reagent works for Nitrate and Nitrite Alternative Reagent 2 20 grams Sulfanilamide 172 21 FW 50 ml 85 Conc Phosphoric Acid H3PQ 0 50 grams N 1 naphthylethylenediamine dihydrochloride 259 18 FW 500 ml TOTAL Balance with Degassed DI water Mix well and store in dark bottle Cadmium Column Cadmium column can be prepared packed by user see next page or purchased ready for use from FIAlab Instruments Inc Nitrate Nitrite Standards 100ml ICNAT 100 Nitrate standard 100ml ICNIT 100 Nitrite standard Exaxol 727 524 7732 sales exaxol com www exaxol com 46 FIAlab Instruments FIAlab Leaders in Flow Injection Technology Cadmium Column Production
36. e coe caceacccesscccceeecsealecnaceecsscswesccacseasdssesvaicaepick caccoisaacnauicaceesecwenlesvaeGecsecscesccasesee 58 LAMPE LOA nee a a Ta SNe ene eee Seen wee Penne ee eer ee eee een ere 58 6 0 2 EXTENDED RANGE OPTION custodia 59 OS REACTION MANIPULATION callo dcolaiaie 61 6 0 4 SPECTROMETER WAVELENGTHG csccsccscosceccecceccsccececceccscescscecceceecescescestecescecesescescescescsscscescscescesescescescuces 62 7 0 MAINTENANCE esecscscsesesesesesesesescsecscscseseseseseseceseososoecsoseseseseseseseceseososcseseseseseseseseseseseososcsesesesesesesesesesssesscsessso 63 LOL PERSTALTIC PUMP TUBE areni a e aae a a aea e a e diet scosuesedesediaateatd 63 70 2 INJECTION VALVE ROTOR oesie AA O a E i 64 SO NOT S ON Plica 65 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 1 0 Introduction The FIAlab 2500 system is designed to be a user friendly reliable laboratory workhorse for serial assays A large number of colorimetric procedures have been automated by FIA and the FIAlab 2500 will handle the majority of these FIA based colorimetric assays have been used for analysis of water foodstuffs pharmaceuticals soils and fertilizers In the area of plant soil and water analysis phosphate nitrate nitrite chloride and ammonia are routinely analyzed by flow injection in many laboratories This manual will provide simple instructions on how to configure and run a single channel FIAlab 2500 unit The manual w
37. ed to 50 though this will also increase the limit of detection Warning the reagents in this assay have a very strong and possibly hazardous odor It is recommended that preparation and use be performed in a well ventilated location or under a fume hood The Boron assay color development is very slow The M2 coil should be twice as long as standard use two coils in series both submerged in a water bath set to65C The M1 coil should be the standard length The peristaltic pump speed should be set to 25 and a sample loop of 12 30 5cm utilized The timing in the script should be adjusted accordingly A 10 cm flow cell is required Use 430 nm as the primary wavelength and 480 nm as the reference The pump speed can be increased to 45 for sample batches of Boron concentrations gt 5 ppm Boron samples should be taken in plastic bottles or alkali resistant boron free glassware No preservation 1s required Samples should be analyzed within 28 days Carrier Matrix Match Matrix Match If samples are water then use water as carrier The salinity should be matched if applicable Reagent 1 Buffer Masking Solution 1 Measure out the following into a 1 liter beaker 200 g ammonium acetate 20 g tetrasodium salt of EDTA 8 0 g disodium salt of NTA nitrilotriacetic acid 2 Add 500 mL distilled water and stir to dissolve 3 Add 100 mL concentrated acetic acid use hood 4 Add stir bar and mix well until the solution becomes transparent
38. ed to cover a greater concentration range or need an improved signal the FIAlab s flexibility can provide the solution 6 0 1 Sample Loop The internal dimensions of the sample loop on the FIA LOV determine the quantity of sample that is injected for reaction A larger sample loop will introduce a greater volume of sample and thus result in more reaction product yielding a higher signal Because of this relationship long sample loops are advantageous when targeting low concentrations while short sample loops are vital to high concentrations A 3 7 6cm piece of 0 03 0 75mm ID tubing is generally used for 1cm flow cells while a 6 15 2cm piece of the same tubing is used for 10cm flow cells If the sample loop is made longer than 6 15 2cm then the valve delay in the script must be modified To change the sample loop 1 Remove the current sample loop by unscrewing it from the SL ports on the LOV 2 Cuta piece of tubing to the desired length and slide a P 286 nut and P 200 ferrule on each end 3 Screw both ends into the sample ports on the LOV 4 Ifthe tubing is longer than 6 15 2cm adjust the valve delay near the top of the script The valve delay tells the valve how long to remain in the inject position and it is essential that the valve stays in this position long enough for the entire sample to flush through The valve delay maximum is 12000 msec 12 seconds however the length of time is more than adequate to flush
39. ell 2 Attach the heater s coil to the FIAlab 2500 by placing a P 286 nut and P 200 ferrule on either end of the heater tubing and screwing it into the M2 port on the LOV Push the tubing into the M2 port as far as it will comfortably go and tighten 3 Place a P 286 nut and P 200 ferrule on the other end of the heater coil and screw this into the entry lower port on the flow cell Do not push tubing into the port more than 0 5 cm to prevent obstruction of the light path o Flow Cell s fey TO FIA LOV s Entry Port M2 Port Figure 2 20 FT Heater s fluidic connections 14 FIAlab Instruments FIAlab Leaders in Flow Injection Technology To control the Heater FT 1 Turn on the heater via the switch in the back 2 Set the heater to the appropriate temperature C by using the arrow keys on the right side of the display The green number represents the set point temperature the red number the actual temperature of the heater s heating rod Start the assay once the red number has stabilized when both the red and green numbers are the same A Real Time Temperature of Heating Rod Heating Rod D2 l z EZ TO y a WATLOW t Figure 2 21 Controlling the FT Heater 15 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 3 0 Logging onto the System In the desktop computer provided by FIAlab all software settings should already be set to your specific
40. er above and AIM 3200 autosampler below A Cetac ASX 520 autosampler has standard positions 1 10 in line with one another from left to right 27 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 4 0 2D Creating the Sample Definition File 1 From the software s main toolbar click on the program button FIALab for Windows 5 44 1010 _ Program _ Figure 4 10 Program Button 2 Inthe upper right hand corner of the window that appears select sample definition file and then hit Select Edit Create File This will open the sample definition file window C Program Files FlAlab Instruments Inc FIAlab for Windows Method Files fia Generic fia New Font Save Events SafeLockMode Find FIA Templates Sample Definition File Test Assay e g Blue Dye Use Sample Definition File Pie a a Select Edit Create File Peristaltic Pump FIA Template For FlAlab 2500 System Injection Valve Global Logon logon to all components User Sa t pmr Sample Description load sample description file Spectrometer Injection Valve Sample Load Optimize_FlAlab2500 Create Folder Set Wavelengths Delay sec Hardware Settings Wavelength 1 nm 620 Sd Hardware Settings Wavelength 2 nm 630 if Hardware Settings Wavelength 3 nm 610 Insert File Hardware Settings Wavelength 4 nm 540 Hardware Settings Hardware Settings Use Wavelength 4 as Reference Add Data Loop End i f E E Loop Start set
41. er r Spectrometer Setup Select Wavelength H e 377 Master vw 540 Optimum wavelength Master for low concentrations w Master v o z s 2 Master 630 wo Status Wavelength nm mouse X position mouse Y position Realtime Plotting M only last 100 datapoints 2 Time sec Figure 6 4 Blue dye absorbance spectra 62 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 7 0 Maintenance 7 0 1 Peristaltic Pump Tubes Peristaltic pump tubing should be replaced every 4 6 weeks at minimum and more often depending on use Using worn out tubing will lead to inconsistent and slow flow rates throwing off accuracy and script timing 1 Insert green PEEK tubing into one end of each of 4 pieces of 2 stop 1 02mm ID Tygon tubing PEEK should stick in approximately 3 4mm Cut the green PEEK tubing so that a little less than a cm sticks out and 1s unsheathed by the pump tube 2 Slide a P 330 PEEK nut followed by a P 300 Tefzel ferrule onto the side of pump tubing where PEEK is inserted 3 Cut four 12 30 5cm pieces of 0 03 0 75mm ID Teflon tubing if using an autosampler its probe tubing counts as one piece regardless of length and stick one piece into the free end of each of the four peristaltic pump tubes Pinch the Tygon pump tubing in order to fit the Teflon inside Do not pinch the Teflon tubing each Teflon tube should stick into the pump tube b
42. er Setup a Setup Tab USB DT Lamp ignore if not using a USB DT Lamp Sample Rate Hz Detectors to Average 1 3 5 Samples to Average H i b Visible Lamp Intensity i 0 Stream data to file Plotting Threshold cents IN Lona enii Poo j Use wavelength 4 as reference for Master Spec fio C DODriver Turn Off Reference Warnings Omnidriver beta Offset Absorbance V Auto Dark Correction i USB DT On M Visible Lamp O oo Lamp On Select Omnidriver Shutter Open Optimize Integration Time Settings applied to all spectrometers Settings for specific spectrometers Use for fluorescence returns voltage when absorbance measurement is commanded Normally this should be unchecked Defaults Selected Spectrometer Master Y V Enabled Serial Number First vailable m Use Multiple Spectrometers Scale Response Normally should be 1 0 Get Coefficents j Second Coefficient Third Coefficient Intercept 5 83E 06 i 69E 10 a 09 DOS l Voltage Absorbance Single Scan Repeated Scan Scan to File Exit Figure 3 6 Spectrometer Setup tab with Omnidriver selected Integration Time msec 18 FIAlab FIAlab Instruments Leaders in Flow Injection Technology 4 Once logged on verify that both the spectrometer serial number and the coefficients listed on the spectrometer setup tab match the serial number and coefficients listed on the paper insert that came with your spectrometer The serial number is
43. er to the cylinder 7 Set a timer for 1 00 minute and start the pump again by clicking on its icon After the minute has passed stop the pump and remove the carrier line from the cylinder Take the current level of water in the cylinder and subtract it from the original 10ml volume to get the carrier line s flow rate per minute 8 Place the carrier line back into the large container of DI water and refill the cylinder to the 10ml mark 9 Repeat steps 7 and 8 for both reagent 1 and reagent 2 lines making sure to measure the flow rate of only one line at a time 4 Fill cylinder to the 10ml mark before jinserting tubing Only put one line linto the cylinder at a time Figure 4 4 Graduated cylinder for flow rate test 23 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 10 The instrument has passed the flow rate test if the rate on each of the measured lines is about 2ml min More importantly all three lines must draw at similar flow rates a total span of 0 2ml or less Ifany line draws below 1 9ml min or above 2 3ml min OR if flow rates are inconsistent between lines the tension arm on the pump may have to be readjusted and the test ran again 24 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 4 0 2 Blue Dye Linearity Test 4 0 2A System Setup 1 At this point the FIAlab 2500 spectrometer and autosampler should all be communicating with the FIAlab software The pu
44. glass bottle for maximum longevity Prepare fresh weekly due to limited storage life Ammonia Standard 100ml ICAMU 100 Ammonia standard Source 727 524 7732 sales exaxol com www exaxol com 48 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 5 0 3 Total Kjeldahl Nitrogen TKN Water Bath Heater should be turned on to 60 C The flowrate of the pump should be set to 45 The FIA LOV connections B should be bridged by short green tubing Recommended wavelengths 650 primary and 525 reference For most TKN assays make the sample loop from 6 15 2cm of 0 03 0 75mm ID green tubing For Macro TKNs dilute sample 5 fold with De ionized DI water Use all PEEK fittings in LOV manifold normal fittings may be damaged by high NaOH content Low Concentration Assays For low concentrations below 10 0 ppm consider using a 5 10 cm flow cell Increase the sample loop to 12 30 5cm of 0 03 0 75mm ID tubing Also carrier matrix matching becomes critical The carrier should be identical with the 0 standard 0 Carrier For high concentrations De ionized DI water will work use the 1 cm flow cell For low concentrations the carrier should be identical with the standard 0 blank See discussion above Reagent 1 Hypochloride Solution 10 mL of a 6 Sodium hypochloride solution common household bleach 50 grams NaOH s note 10 fold increase of NaOH over water Ammonia assays Balance Degassed DI wate
45. ill cover the basic operation of the FIAlab 2500 system and usage of the FIAlab for Windows software This manual also includes methods and brief technical discussions of the specific chemistries however for a more detailed discussion regarding the science of FIA please contact FIAlab An informative tutorial CD on FIA and SIA is available free upon request For additional information on the FIAlab for Windows software please review the separate FIAlab for Window Software user s manual Note On the computer that accompanied your instrument the software and software drivers have already been installed prior to shipping There is no need to install FIAlab or Ocean Optics software as described in this manual All proper software settings have already been made Installation instructions are provided for cases where software needs to be reinstalled e g in connection with a computer update The computer regional settings found in MS Windows control panel Regional Settings should be set up such that numerical values use periods as used in the USA instead of commas for the decimal location For example 10 Y should be written as 10 5 not 10 5 Make sure that this convention is utilized and is also set up in Regional Settings this way FIAlab Instruments FIAlab Leaders in Flow Injection Technology 2 0 Physical System Setup The FIAlab 2500 system is most commonly used for colorimetric assays This section of the manual show
46. in section 7 0 5 Plug the autosampler in with its included power supply and cord It is critical that the tubing between the Autosampler probe and the FIAlab system is just long enough to allow free motion of the probe to each corner of the autosampler s sample racks FIAlab Instruments FIAlab Leaders in Flow Injection Technology 2 0 3B AIM 3200 3300 Figure 2 8 Front view of AIM 3200 autosampler To setup an AIM autosampler 1 Setup the autosampler according to the manufacturer s instructions If there are multiple probes included with the autosampler choose the probe that uses 0 03 0 75mm ID Teflon tubing 2 The assembled autosampler should be placed to the left of the FIAlab 2500 unit It should be in close proximity to the FIAlab so that the sample line from the probe to the FIAlab can be as short as possible 3 Attach the AIM autosampler to the computer by connecting the serial cable that came with the autosampler from the autosampler s HOST port to the COM3 port on the back of the computer d t kE 3 15AT Autosampler connects to omputer here 1 il Figure 2 9 AIM autosampler side view and hookup to computer 4 Cut the autosampler s Teflon probe tubing to the appropriate length and connect it to the peristaltic pump tubing of the S channel by either method described in section 7 0 5 Plug the autosampler in with its included power cord FIAlab Instruments FIAla
47. ion file 4 0 3A 1 Creating the Sample Definition File Cetac Autosampler The standard tubes of Cetac autosamplers can hold up to 50ml of solution so all analyses necessary from this run can be drawn from the same vial 1 Make 40ml of a 60uM Erioglaucine solution and pour into a Cetac standard tube Put DI Water in two other standard tubes one to serve as a blank and Ostd and one to serve as a wash 2 Place the vials into the standard rack in the following order Position 1 Blank Ostd Position 2 60uM Erioglaucine Standard Position 3 wash 3 Create a new file from the sample definition window as done in the linearity test and name it Precision Sample File Cetac Edit the file so that 1t matches that shown below Save and close out of the file Blank B 0 0 1 0 0 O OuM 0 0 0 0 1 60 OuM 60 0 0 10 0 wash 7 0 0 1 0 0 std4 40 O 1 Q unknowni 0 0 unknownz o unknowns o unknownd unknowns unknowns unknown unknowns D 0 unknown9 0 O unknown 7 0 0 1 0 0 op o o o o o ol D 0 ig ta ta taj ia ia ta ta ia H a HHH HEHEHE HO a OPSPPOPO 37 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 4 Select the file just created from the sample definition file window as done in the linearity test Enter 4 in the total number of vials box and hit refresh Sample Definition File File Edit Format Notepad Sample Definition
48. me Sample Definition Table View Start Time hrs Period hrs std rack 1 Blank 0 1 0 std rack 2 30uM 30 0 1 0 std rack Dub 60 0 1 0 System Set to 6 for Select Existing File Clear File Name Single Channel Total Number of Vials Q SAA Multi Channel Ai Fie oe Select Redo File Program FilesiFlAlab Instruments Inc FlAlab Far Wlindaurmicada ansi Create New Sample D qfiniki 4 Freq of QC Driftcor Sampling Samples 190 Pos of QC Driftcor Sample Create New File Figure 4 16 Complete linearity test sample definition file 31 FIAlab Instruments Leaders in Flow Injection Technology 4 0 2E Running the Method FIAlab 1 Ifnot already open open the program window by clicking on the program button in the software s main toolbar toon E Instruments Options About 4 11 2015 4 28 17 PM Figure 4 17 Program Button 2 In the top of the program window go to FIA templates then FIA and select Generic Indows Test Assay e q Blue Dye FIA Template For FlAlab 2500 System Global Logon logon to all components sample Description load sample description file Injection Valve Sample Load Optimize _FlAlab2500 oet Wavelengths Hardware settings Wavelength 1 nm 620 Hardware Settings Wavelength e fam 630 Hardware Settings Wavelength 3 fam 610 Hardware Settings Wavelength 4 nm 540 Hardware Settings Use Wavelength 4 as Reference
49. mended wavelengths 480 primary and 650 reference For typical Chloride assays 0 5 to 100 ppm make the sample loop from 3 7 6cm of 0 03 0 75mm ID green tubing Carrier DI Water 1 Liter DI Water Reagent 1 Hg SCN 2 Fe IID Labchem s Color Reagent Cat No LC13260 Go to http www labchem net catalog search_catalog asp type in LC13260 under catalog and click search Alternative source Chloride Color Reagent Technicon No T01 0352 Additional information can be found at http www epa gov glnpo Immb methods methd140 pdf Note If bubbles become an issue sticking in the flow cell and other parts of the manifold then add 1 gram Brij 35 detergent to Reagent 1 Reagent 2 Not used Plug R2 port with Teflon Plug Chloride Standard 100ml ICCHL 100 Chloride standard Source 727 524 7732 sales exaxol com www exaxol com 54 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 5 0 7 Sulfate Sulfate in the sample is precipitated with acidified barium chloride This precipitate scatters light producing a signal proportional to sulfate concentration The following method should work for concentrations from 10 ppm to 200 ppm Please inquire for suggestions on how to measure lower or higher concentrations than these ranges For most Sulfate assays make the sample loop from 12 30 5cm of 0 03 0 75mm ID tubing Use a 10 cm flow cell for concentrations of 10 ppm to 200 ppm Use a 1 cm flow
50. mp should be clamped and the lines should be primed from the flow rate test If you have not logged onto the system s components please see section 3 If the lines are not primed place carrier reagentl and reagent 2 lines into a large container of DI water 250ml and run the pump for 43 seconds making sure the pump is clamped and the lines all draw from the container smoothly 2 Turn on the light source 3 Place the line coming from the LOV s waste W port into the waste container used in the flow rate test The line coming from the top of the flow cell should also be in this container i A LOV s Waste W port o 4 MN Figure 4 5 LOV s waste port 4 With the system primed check the flow cell for any bubbles that may be stuck in the corners of the fluidic path If bubbles are present try to dislodge them physically by knocking on the flow cell or by running the pump at a variety of speeds Figure 4 6 Air bubble caught in corner of flow cell s fluidic path 5 Make 30uM 45uM and 60uM standard solutions of Erioglaucine Sigma p n 861146 Make at least 15ml of each standard Also make a blank 0 standard and a wash with using DI water 25 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 4 0 2B Initial Spectrometer Scans 1 Turn off the light source and open the spectrometer window from the button on the software s main toolbar The spectrometer should already be com
51. municating with the software from the steps taken in section 3 Ifusing any model other than a USB4000 UV VIS or USB4000 VIS NIR and you have closed out of the software since initially setting up the system you will need to logon to the spectrometer again from the lower left hand corner of the spectrometer window 2 Under the spectrometer graphing tab with the light source off make sure the voltage button on the bottom of the window is depressed and click on dark scan in the lower left hand corner A jagged line fluctuating roughly around O voltage counts should appear in the top graph Jeon i A A Hll i mouse X position mouse Y position Realtime Plotting V only last 100 datapoints 0 0 Dark Scan Button Logon Time sec Figure 4 7 Picture of dark scan graph centered around 0 volts 3 Turn on the light source make sure the voltage button is still depressed and hit single scan on the bottom toolbar of the window The spectrum that appears should be a broad peak For USB4000UV VIS and USB4000 VIS NIR models the peak should reach its maximum between 600nm and 700nm and should max out between 30 000 to 60 000 voltage counts Spectrometer Setup al alele aj 7 lal 50000 40000 30000 20000 10000 0 10000 Voltage Cnts Wavelength nm mouse X position lth Hi aala gl H all mouse Y position mouse Y position Realtime Plotting Realtim
52. n the lower left hand corner of this window If successful after a few seconds the upper left hand corner of the window should read FIAlab 2500 Detected and Ready Do not close out of this screen Serial Port 1 Ni er of C 5 Simplified Interface Pug Pred et to Serial Port 1 AUKS gt Flow Cell Figure 3 3 FIAlab window with settings and logon message 4 Verify that the system is communicating by clicking on the peristaltic pump icon The pump should start turning Click the pump icon again to stop the pump 5 Click on the injection valve icon The valve should switch positions from load to inject you should be able to hear this Click on the icon again to return the valve to the load position Serial Port1 Number of Channels 1 Additional Components Simplified Interface Pump Speed MMS Flow Cell Figure 3 4 Description of icons in FIAlab window 17 FIAlab FIAlab Instruments Leaders in Flow Injection Technology 3 0 2 Spectrometer 1 Click on the Spectrometer button in the software s main toolbar FlAlab for Windows 5 0 Instruments Options About 4 10 2015 9 14 05 AM LEF Spectrometer YZ Button Figure 3 5 Spectrometer button on software s main toolbar 2 On the right hand side of the setup tab in the spectrometer window make sure Omnidriver beta is selected 3 Hit logon in the bottom left hand corner of the setup window Spectrometer Graphing Spectromet
53. nder Serial Number pick one of the spectrometer s serial numbers and make note of which you choose Also make note of which flow cell it connects to c Check the enabled box 5 Configure the other spectrometer in the setup tab a Under Selected Spectrometer pick Slave 1 b Under Serial Number pick the other available serial number c Check the enabled box 6 Goto the Spectrometer Graphing Tab and under Select Wavelength set the top two wavelengths pull down menus to Master 7 Set the bottom two wavelengths pull down menu to Slave 1 8 Exit the software to save your settings and reopen 9 Attach the single end of the bifurcated fiber to your light source and one of each of the bifurcated ends to the 1cm and 10cm flow cell respectively 10 Attach one spectrometer to the SMA connector on the right side of the 1cm flow cell via fiber optic cable 11 Attach one spectrometer to the SMA connector on the right side of the 10cm flow cell via fiber optic cable 12 In the script set the wavelengths you wish the spectrometers to read at 13 Increase the delay while the spectrometer is scanning to give the peak adequate time to clear both flow cells When the script has finished the calibration and data on channels 1 and 2 correspond to the master spectrometer and the calibration and data on channels 3 and 4 correspond to slave 1 60 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 6 0 3 Reaction Ma
54. nipulation Various parameters adjustable on the FIAlab will alter the extent of reaction and change the magnitude of the absorbance signal Three common methods are as follows and can be used alone or in conjunction with one another 1 Pump rate a Increasing the pump rate will give less time for the reaction to complete before the sample segment enters the flow cell In general because of this phenomenon absorbance values with an increased pump rate read lower whereas absorbance signals with an decreased pump rate read higher 2 Mixing coils a Similar to the pump rate the length and volume of mixing coils changes the residence time of the sample before it enters the flow cell A longer coil means the sample will take longer to get to the flow cell and the extent of reaction has increased by the time it is detected A shortened mixing coil has the opposite effect 3 Reaction temperature a For many reactions the reaction rate is greatly dependent on temperature To increase the concentration of reaction products raise the temperature on the FT Heater To decrease the concentration lower the temperature Making the mixing coil too short will lead to inadequate time for smooth dispersion of the sample zone and will read as choppy peaks Making the mixing coil too long will lead to excessive dilution of the sample zone Y Do not set the heater above 70C 61 FIAlab Instruments FIAlab Leaders in Flow Injection Technology
55. nium molybdate tetra hydrate 1235 81 FW Sigma Aldrich 22 123 6 0 2 grams Antimony Potassium Tartrate half hydrate 333 94 FW catalyst Sigma Aldrich 38 337 6 1 Liter Degassed DI Water Add molybdate and antimony potassium tartrate to 800 mL DI water and mix until dissolved Fill flask to the mark with DI water Transfer solution into a dark and airtight glass bottle for maximum longevity This solution is stable for several weeks Reagent 2 Reagent Carrier Stream of 300mM Ascorbic Acid 30 grams Ascorbic acid 176 12 FW 1 0 grams Sodium dodecyl sulfate 288 38 FW surfactant Sigma Aldrich 436143 25G 1 Liter Degassed DI Water Place the ascorbic acid into a 1 liter volumetric flask and mix with 600 cc of DI water until dissolved Add the sodium dodecyl sulfate and mix slowly prevent foaming until dissolved Fill flask to the mark Transfer solution into an airtight light sensitive glass bottle for maximum longevity Minimize exposure to air and prepare fresh weekly since this solution is unstable Phosphate Standard 100ml ICPHO 100 Phosphate standard Source 727 524 7732 sales exaxol com www exaxol com 52 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 5 0 5 Iron A heater is not necessary The flowrate of the pump should be set to 55 for Iron The FIA LOV connections B should be bridged by a simple tubing no Cadmium Column For typical Iron assays 5 to 100 ppm make the sample loop from 3
56. ow cell 2 Place the LED s bulb and socket up and inside the cap and screw the LED s base up onto the cap Screw the base onto the cap until just finger tight to secure the bulb in place LED Anode to Red Wire ie Bulb LED Cap SN Wij pw VO We LED Cathode to Black Wire FA A PA Figure 2 16 Attaching a DCON LED to an SMA Z flow cell 12 FIAlab Instruments Leaders in Flow Injection Technology 2 0 6 Fiber Optic Cables Figure 2 17 FIAlab Instruments P600 20 fiber optic cables To attach the fiber optic cables 1 Connect one end of a fiber optic cable to the SMA connector on the right side of the flow cell Connect the other end of this fiber to the SMA connector on the spectrometer 2 If using a HL 2000 LL connect the other fiber optic cable between the SMA connector on it and the SMA connector on the left side of the flow cell low Cell SMA onnector a ight Source Figure 2 18 Attaching fiber optic cables to flow cell and spectrometer light source 13 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 2 0 7 FT Heater Figure 2 19 FIAlab Instruments FT Heater If your system came with a heater its heated coil will be used in place of mixing coil 2 To install the Heater FT 1 Unscrew mixing coil 2 by removing one end the coil from the FIA LOVs M2 port Remove the other end of the coil from the entry lower port of the flow c
57. r 1 L Place a 10ml solution of bleach into a 1 liter volumetric flask and fill with 700 mL of degassed DI water Dissolve all other chemicals and fill flask to the 1 liter mark Reagent 2 Salicylate Catalyst Solution 100 grams Sodium Salicylate 160 11 FW 0 40 grams Sodium Nitroferricyanide III dihydrate 297 95 FW catalyst 5 grams NaOH s Balance Degassed DI water 1 L Place the sodium salicylate into a 1 liter volumetric flask and mix with 700 mL of degassed DI water until dissolved Add the sodium nitroferricyanide and mix until dissolved Add NaOH to adjust the pH to the 12 0 range Add the Brij 35 mix and fill flask to the mark Transfer solution into a dark airtight glass bottle for maximum longevity Prepare fresh weekly due to limited storage life Ammonia Standard 100ml ICAMU 100 Ammonia standard Source 727 524 7732 sales exaxol com www exaxol com Standards must be made in TKN matrix and treated identical diluted same way as samples 49 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 5 0 4 Phosphate Ortho Phosphate A heater is not always necessary but may be turned on to 50C for increased sensitivity The flowrate of the pump should be set to 50 for Phosphate The FIA LOV connections B should be bridged by a simple tubing no Cadmium Column Recommended wavelengths either 660 or 860 nm for the primary and 490 reference For most Phosphate assays make the sample loop from 3
58. read going down the column B 0 0 30 0 45 0 60 0 Field 5 the fifth column entry in the line is the time to measure hours This is the time in hours after the start of the run that the first measurement will be made When multiple samples have the same start time the software will start at the first sample with this time and work its way down The values in this column should be set to zero for all entries in the blue dye test Field 6 the sixth column entry in the line is the number of measurements The specified sample will be measured this number of times The values in this column should be set to one for all entries in this blue dye test Field 7 the seventh column entry in the line is the period between measurements in hours The values in this column should be set to zero for all entries in the blue dye test When finished editing the sample file should be identical to that shown below Use the Save As command to save this file in a known location and exit out of the screen blue dye linearity test csv Notepad Edit Format View Help A o o a fal ooo HOGD Wows 999999999 lt JooossossossoscosscooSsonHE o ES 15 E BEES OOOE HHHHHHHHH cn oo999S999 29999999999900000000 RRR ERR eR RR Joab fash a jad fash pod fk fos foal fash fab ful foo fash ful fh feck ful fu Jud PA a a EE pl pl pl pl D D P Y P P Y tJ KES EES KES BES KES RES WES KES BES EEN ooocooccececsa unknown
59. rew the Teflon tube into the appropriate LOV port s pushing the tubing in as far as it will comfortably go 4 Gently tug on the tubing once screwed in to make sure it is secure Ferrule Nut V Figure 2 5 Basic tubing connection To change peristaltic pump tubing please see section 7 0 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 2 0 3 Autosampler 2 0 3A Cetac ASX 260 520 Figure 2 6 Front view of Cetac ASX 260 autosampler To setup a Cetac autosampler 1 Setup the autosampler according to the manufacturer s instructions If there are multiple probes included with the autosampler choose the probe that uses 0 03 0 75mm ID Teflon tubing 2 The assembled autosampler should be placed to the left of the FIAlab 2500 unit It should be in close proximity to the FIAlab so that the sample line from the probe to the FIAlab can be as short as possible 3 Attach the Cetac autosampler to the computer by connecting the 9 pin female to female serial cable that came with the autosampler from the autosampler s COM 1 port to the COM3 port on the back of the computer A Autosampler connects Gu E o computer here Figure 2 7 Cetac autosampler back panel and hookup to computer 4 Cut the autosampler s Teflon probe tubing to the appropriate length and connect it to the free end of the peristaltic pump tubing on the S channel by either method described
60. ritical that the user understands the underlying chemistry and ensures that either there are no interfering components in the sample or the interfering components are compensated for by adding in equal amounts to the standards Also note that the recommended wavelength selections for each method described below are automatically set based on the selected script from the FIA Template library not all methods have templates 40 1 1 3 Ak 1 2 lh u5 ry 0 A U5 A ES 16 M U3 lt 08 A aA A 4 k 44 0 6 ft al f hu A i 0 ae 4 la i q A A A A 4 A 44 A 7 1 B Ji 4 ta EER A 0 2 BlankDriftcor y 1 A a chk std a2 4 14 F f 0 0 ammm 2280 n ab jab A TS nt ab o ue 50 100 150 200 250 300 350 400 450 500 Time sec Figure 5 0 1 Plot showing the beginning of a Nitrate run The peaks between 150 and 300 seconds make up the calibration curve 45 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 5 0 1 Nitrate Nitrite To convert the manifold to process Nitrate connect a Cadmium column between the FIA LOV Manifold ports labeled B If there is a heater installed on the unit the heater should be turned off or the coil removed from the heated waterbath Two reagents are utilized for Nitrate and one for Nitrite The flowrate of the pump should be set to 50 60 Recommended wavelengths 540 primary and 650 reference For typical Nitrate Nitrit
61. s Create New Sample DefinitionT emplate File Standards 4 Freq of QC Driftcor Samplin fo Create New File q ping e Samples 180 Pos of QC Driftcor Sample 4 y Hit Create New File Figure 4 12 The sample definition file window 4 Inthe window that appears name the file blue dye linearity test and hit open When the software then asks if you want to create the file click yes A standard template in notepad with your file name will appear Edit Format View Help rack Blank E 0 0 1 0 0 rack std 0 0 0 0 1 0 0 1 rack std 10 0 0 1 O 1 rack i std 20 D 1 0 d rack stda 40 O 1 i unknown 7 0 unknown 7 0 unknowns 7 0 unknown4 0 unknowns 7 0 unknowns 2 0 unknown 7 0 unknowns unknown9 unknowml0 unknownll unknown unknowr 3 unknownla unknownl5 unknownl6 unknownl7 unknown1g8 unknown a unknown20 m ooo SI cero 999999999 ooS9S9999999000000000 HHHHHHHHH eou u u u u u eee O ag lea ak wah al a HHHHHHHHHH PA a fl pl pl fl pl po pd pd fl pd pd pd pd pd pd pl AE O IS E E ES iad ta ES o H Figure 4 13 Standard sample definition file template 29 FIAlab Instruments F TA lab Leaders in Flow Injection Technology 5 The sample definition file contains one line per sample or standard defining the sample name position in the autosampler concentration 1f it is a standard number of replicate inje
62. s the basic system setup for this type of assay Upon unpacking your FIAlab system configured for absorbance measurements you should find 1 Basic FIAlab 2500 unit with FIA LOV 1 Desktop Computer pre configured with FIAlab for Windows software 1 Autosampler optional 1 USB4000UV VIS or USB4000VIS NIR Spectrometer other types available 1 Tungsten light source LED light sources also available 2 SMA terminated fiber optic cables 1 Heater FT optional 1 Fittings and tubing kit which has been used to plumb your instrument 1 USB stick with a backup copy of the software and electronic copies of the FIAlab 2500 and FIAlab for Windows manual as well as quality control test data on your specific instrument FIAlab Instruments FIAlab Leaders in Flow Injection Technology 2 0 1 FIAlab 2500 Unit To setup the FIAlab 2500 unit 1 Make sure the switch on the back of the FIAlab 2500 is in the off position 2 Using the supplied serial cable connect the FIAlab 2500 s IN port to the computer s native COM 1 serial port 3 Plug the 24 VDC power supply into the FIAlab 2500 y Fi D 4 4 ge HEHE t HHHH gt Al k e a o JIN Connects FIAlab 2500 y E a O ye A r tel Not Used f All TA i 7 OUT A OUT B Can be usedlg gt Ito daisy chain multiple RBCS on off switch jFlAlab 2500s or to connect Sa oe q dd junit to FIAlab accessories o a o Y eaer o t EN
63. trite in seawater the carrier must have salinity content similar to the samples The extent of the similarity is dependent upon the concentration of the nitrite The lower the levels to measure the more critical it becomes for accurate matrix matching To test for matrix affects run some sample blanks through 0 concentration of the analyte being tested Matrix mismatch is suspect when negative or positive peaks appear Perfect matrix matching is generally not possible Several iterations may be required to determine the required degree of closeness for satisfactory results Again the lower the levels to measure the more critical it becomes for accurate matrix matching Interferences Many FIA methods are subject to effects of interfering analytes which can either enhance or suppress the colorimetric response and result in erroneous readings It is critical that the user understands the underlying chemistry and ensures that either there are no interfering components in the sample or the interfering components are compensated for e g added in equal amounts to the standards Surfactants In most assays it is important that surfactant be added to either reagent 1 or reagent 2 The type of surfactant is dependent upon the type of assay which is discussed in section 5 0 Sodium dodecyl sulfate 288 38 FW surfactant Sigma Aldrich 436143 25G works for most assays 65
64. w In the bottom right hand corner select Local Maximum from the pull down menu Then select 1 Order Polynomial under Fitting Coefficients near the left side Observe the R Square value also on the left side of the window 4 Save the data by clicking on the button in the bottom left of the analysis window Report Generation Format Advanced Options Plots Data alibration Tab pooo Standards Name Response Concentration fit Concentration T ruth Peak Time sec gt Blank 2 387123E 03 0 1981861 14 35305 std 3 868855E 03 0 4028856 21 34415 29 03063 45 3272 60 23928 21 44147 1 372508 Fitting Coefficients fi st Order Polynomial v a Coefficient A Correct for Drift 9 525391 E 04 r Spike Baseline Removal Coefficient B 7 238572E 03 Coefficient C 0 R Square 0 9993572 R Square alue 20 40 Select Local Calibration Set n Concentration Maximum i Refresh Standards PO M Auto Refresh Show Concentration Local Maximum y M Show Data Labels Response F R mE Figure 4 24 Example calibration from linearity test 36 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 4 0 3 Blue Dye Precision Test The initial setup and script are the same for the blue dye precision test as they were for the linearity test The only thing that needs to be changed is the sample definit
65. y 3 0 3 Autosampler 1 From the software s main toolbar click on the autosampler button FIAlab for Windows 5 0 Instruments Options About 4 10 2013 9 17 05 AM Button za A ES Figure 3 8 Autosampler Button 2 Make sure that Serial Port 3 is selected in the upper left hand corner of the autosampler window 3 Inthe autosampler type drop down menu make sure that the appropriate model is selected 4 Inthe Sub address menu make sure that Comm is selected Goto Sample Send Check Status i Get Position Pump On Pump Off Get Error Down Po PEN Autosampler Type Cetac ASX 260 520 A Select Appropriate Mode Sub address Comm gt lt Set to Commie Sample Definition File Logon Complete Figure 3 9 Autosampler window settings 20 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 5 Hit Logon in the autosampler window s lower left hand corner The message should read Logon Complete and you should be able to hear the autosampler initialize ca Autosampler a Serial Port 3 Commands Check Status Pump On Pump Off Autosampler Type Cetac 45 260520 Y Logon Complete Figure 3 10 Autosampler logon message 21 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 4 0 Initial Tests 4 0 1 Flow rate test 1 Ifnot already done log onto the instrument from the FIAlab screen of the software
66. y about 2 3mm e t ygon Pump eflon Figure 7 1 Peristaltic Pump Tubing 4 Screw the peristaltic pump tubing into the appropriate ports on the LOV S C R1 and R2 by placing the peek end of tubing into the LOV and tightening the nut and ferrule PEEK tubing inside a gt Figure 7 2 Inserting pump tubing in LOV 5 Gently tug on the tubing to check that it is secure 63 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 7 0 2 Injection Valve Rotor The injection valve rotor should be replaced every 6 months to prevent shedding which can clog the system and lead to inaccuracy 1 Use a 7 64 hexagonal wrench to carefully remove the FIA LOV Tubing may be removed from the LOV prior to this however it is unnecessary if the technician is careful 2 Remove and replace the exposed rotor disc A Figure 7 3 Valve Rotor Disc 3 Screw the LOV back onto the instrument To prevent cracking gently tighten all three screws before firmly tightening any Figure 7 4 Gently tightening all LOV screws 64 FIAlab Instruments FIAlab Leaders in Flow Injection Technology 3 0 Notes on FIA Matrix Matching Probably one of the most common problems encountered in FIA occurs when the matrix is not adequately matched to the samples In other words the carrier is not similar enough to the samples in terms of color pH and index of refraction For instance measuring ni

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