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1. PCX 5100 User s Manual Chapter 6 Fluorescence Waste Detector 100 psi B P Ambient Reagent 2 Reactor Bypass 2 Pressure 100uL Restrictor 2 I Heated Reactor Anti siphon 500uL valves Reagent 1 Bypass 1 Pressure Post Column Restrictor 1 Reagent Press Gauge Filters 2um 1 Reagent 525psi Pump Thermostatted Over pressure Column relief valve and Guard Pre column bio OPA Filter 0 5um baker NaOCI Injector 500 psi Pressure Switch LC Pump Water amp Methanol or K200 amp RG019 PCX 5100 User s Manual Chapter 6 From Reagent 2 Pump Vv y To Reagent 2 Pump Saran tubing Figure 6 7 Post column reagent 2 flow path up to Restrictor 2 includes pressure gauge 2 and prime purge valve 2 PCX 5100 User s Manual Fluorescence Waste Detector Chapter 6 O 100 psi B P M Ambient c Reactor Bypass 2 100uL E E O Q Restrictor 2 dated Resor Anti siphon 500uL O valves A Bypass 1 Post Column Restrictor 1 Press Gauge Column M relief valve and Guard i Pre column Filter 0 5um EL Beaker Injector E 500 psi Pressure Switch E COD RWW 02 a mM Reagent Pump a E D A 525psi 9 Thermostatted 9 Over pressure Reagent 2 Pressure z Reagent 1 Pressure Reagent Filters 2um E E of OPA LC Pump Water amp Methanol or K200 amp RG019 PCX 5100 User s Manual Chapter 6 From Reagent 2 P
2. sss 4 1 glyphosate coooccccconococcccccnaconcncnanannncnnnnno 4 12 Getting to know your PCX 5100 2 2 Glyphosate sien ni asi aiii 1 5 Gradient conditions carbamates oeie sss 4 5 glyphosate zaart anaa 4 15 Index 1 PCX 5100 User s Manual H Hardware 1 6 Hazards ra ia oTt e t Arun 1 6 High background signal 5 13 High column pressure 5 14 High post column pressure 5 13 l Injection full loop sees 3 1 Injector installation ssseeeeeesseeseerenene 3 5 Integrator setup 4 4 4 15 L LC method setup 4 4 4 15 M Maintenance iet ei ede EE een 5 6 Methylamine ssssseassssseessrrresrrrresrrrrnnerernnens 1 4 Mobile phase 4 1 4 12 N Noisy baseline sse 5 14 O Obstruction flow Patata idas 5 13 flowcell ooonnnccnnccoconncnnncccnnocnnnncnnnonos 5 13 Organic solvent in a glyphosate column 5 16 Oxygen permeable sssssss 3 4 P Panel MON ED 2 2 ADR EE Tenn TNR RN 2 5 dri PER 2 8 dm 2 6 Parameter log 5 1 Peak width ccccccoonnccccccccococononononoconononononanos 5 2 Peaks disappear seceesesseseesee 5 14 5 15 o Phthalaldehyde 1 4 3 2 4 2 4 13 EO AIEA AAE N E aa 5 6 PIStON SO al iia iaaa 5 6 Wel En WEE 3 7 Post column conditions 4 6 4 16 Post column interlock 3 4
3. A complete Post column Analysis system for carbamates or glyphosate consists of the following components HPLC binary gradient pump Manual injector or autosampler Pickering Laboratories columns Pickering Laboratories PCX5100 Post Column Derivatization Instrument Pickering Laboratories eluants reagents and standards Fluorescence detector Chart recorder integrator or data system Carbamates PCX 5100 User s Manual Chapter 1 Carbamates a class of highly effective commercial insecticides are used worldwide to protect crops from insect pests Applied directly to food crops such as grains fruit and vegetables carbamates may seep into drinking water sources through agricultural runoff In addition if food crops are harvested too soon after application residues of carbamates and their byproducts may remain in the produce The use of carbamate insecticides has created a requirement for a simple reliable and sensitive method of residue analysis for these compounds found in vegetable matter drinking water and industrial waste water The USEPA Methods 5 and 531 1 and the AOAC International protocol 29 405 describe a direct inject method which employs gradient liquid chromatography with fluorescence detection accomplished by post column hydrolysis and derivatization of the eluted carbamates Me Me Me Me Me N Me Ka N M XL N M N Z NOR SO oR Trace o NT e on I o Zo SMe O Me SMe 1 Aldicarb sulfoxide 2 Aldicarb
4. Step 7 Step 8 Fit the front panel into the post column system make sure the two threaded pins are aligned Note Avoid crimping the electrical cable from the pressure sensing switch Return the inlet and outlet tubings for the HPLC column through the opening at the front of the column oven Partially tighten the nuts with the nut driver Tighten the two bolts at the bottom of post column system then fully tighten the nuts from step 3 Attach the fittings to the pressure regulator Figure 6 2 Reconnect the five electrical cables from the pressure sensing switch two temperature controllers reagent pump on off switch and LED lights Note The electrical cables from the left and right temperature controllers should be connected to their respective sides in the printed circuit board Figure 6 1 Connect inlets and outlets to the post column pumps Install side access panels and top cover Note Installing access panels first will assure a better fit of the top cover 6 3 PCX 5100 User s Manual Chapter 6 Temperature Controller Reactor Temperature Controller Column Heater Pump On Off LED Pressure Sensor Switch EH VW VX Nm Figure 6 1 Electrical connections at the printed circuit board 6 4 PCX 5100 User s Manual Chapter 6 Pressure Regulator Disconnect Figure 6 2 Gas Pressure System 6 5 PCX 5100 User s Manual Chapter 6 Figure 6 3 Bolts in step 7 for diassembly o
5. 3 6 5 Ca A E H L PR A Ier Sr hi 2 1 ng in 150 uL water 14 ppb 25 cm column ONOAKRWODND E Mant eet teat ed Chapter 4 Figure 4 1 Peak Identification Aldicarb sulfoxide Standak Aldicarb sulfone Oxamyl Vydate Methomyl Lannate 3 Hydroxy carbofuran Aldicarb Temik Propoxur Baygon Carbofuran Furadan Carbaryl Sevin 1 Naphthol Methiocarb Mesurol BDMC internal standard 11 12 B TERES MINUTES 4 7 PCX 5100 User s Manual Chapter 4 Methanolic samples on a Cs column using a Water Methanol gradient la 6 b 7 2 10 1 3 8 9 i 5 M 12 T T I T T 10 20 30 40 min Methanolic samples on a C4 column 8 using a Water Acetonitrile gradient 7 2 4 6 8 5 11 1 12 10 3 T T T T 10 20 30 40 min Figure 4 2 Peak Identification Aldicarb sulfoxide Standak Aldicarb sulfone Oxamyl Vydate Methomyl Lannate 3 Hydroxy carbofuran Aldicarb Temik Propoxur Baygon Carbofuran Furadan Carbaryl Sevin 1 Naphthol Methiocarb Mesurol BDMC internal standard Qo EON deo GT c6 Morro e 4 8 System Startup for Carbamate Analysis PCX 5100 User s Manual Chapter 4 Having accomplished the steps in Getting Started you will find some of the following steps to be redundant Use this procedure as a check list to review the logical sequence of tasks and the instrument conditions to be observed prior to
6. Gas Pressure gauge on the front panel should read 2 5 psi in normal operation Gas Out manifold is more than a simple distribution block Each outlet has its own check valve to prevent back flow of gas from the pressurized reagent bottles The manifold also has a safety relief valve that opens at about 12 psi to prevent dangerous over pressurizing of the reagent reservoirs If the input pressure of gas is too low the regulator sticks open and allow the gas to vent from the relief valve rapidly depleting a gas cylinder Column oven door FROM TO O DETECTOR Pump heads WASTE OVER Reagent PRESSURE RELIEF filters Oo O GAS OUT GAS B Thermostatted reactor ON N O O O o OFF O Reagent pump REAGENTS Figure 2 3 2 6 KENT e Internal PCX 5100 User s Manual Chapter 2 Reagent 1 fitting supplies the first pump Usually this reagent is either Hydrolysis Reagent or Oxidizing Reagent Reagent 2 fitting supplies the second pump Usually this reagent is OPA To Detector bulkhead fitting connects to the fluorescence detector with 0 010 inch 0 25mm ID tubing From Detector fitting is where the outlet line of the detector connects This is connected internally to the inlet end of the back pressure regulator Waste fitting is actually the outlet of the back pressure regulator The calibration screw is inside the fitting Normally the pressure is factory set to
7. Power connector ccccccccnccccnncncccnonononcncnonanonan 2 8 Powerewitch uscire rea ege geg 2 8 Pre column filter 2 5 5 11 Preparation OPA reagent 4 2 4 13 Index 2 Index Pressure maximum for safety interlock 2 5 post column ssuusss 2 2 5 13 rating flowcell 3 1 3 5 A seats tant Seca tee facil 2 7 Pressure gauge DE RE 2 3 2 6 post columm nete teet 2 2 CEA 2 2 CE A 2 3 Pressures Interpretation 5 12 Priming reagent pumps 4 8 4 14 Problems common with post column air in reagent pump 5 13 deposit in reactor ssse 5 13 high background noise 5 13 high column pressure 5 14 high post column pressure 5 13 noisy baseline ssesssss 5 14 peaks disappear 5 14 5 15 poor peak shape sesser 5 13 reagent backflows 5 13 reagent pump stops 5 14 Problem prevention eessssss 5 3 Pump duplex eere E AE EE 2 7 R Reactor precautions eseese 5 5 Ready lamp eem ette 2 3 Reagent backflow 1 6 5 13 Reagent filters ssssuuss 2 7 5 11 Reagent precautions sssse 4 13 Reagent prepa
8. Step 1 Remove any stainless steel inlet frit or sinker from the HPLC reservoirs Titanium polymeric or ceramic frits are acceptable but it is better to run without any inlet frits Step 2 Fill eluant reservoir B with RG019 column regenerant 0 005 M KOH Step 3 Fill eluant reservoir A with K200 glyphosate eluant 0 005 M K pH 2 00 phosphate buffer Step 4 Place the filled eluant reservoirs on or near the HPLC pump Step 5 If your HPLC requires it sparge the eluants with helium Do not use stainless Steel frits in the sparging line Do not use continuous sparging with buffers because the composition will change with time Step 6 Prime the HPLC pump by withdrawing at least 30 mL of each solvent from the prime purge port with the priming syringe that is supplied An HPLC pump method can be configured to facilitate this step Consult your HPLC manual Step 7 Close the HPLC prime purge valve and flush the HPLC for at least 30 min without the glyphosate column attached 21 mL min 50 A 50 B Step 8 Stop the HPLC pump Connect guard and column according to Figure 3 1 page 22 Step 9 Start the HPLC pump Pump K200 through the column and system at 0 40 mL min Do not exceed a flow rate of 0 4 mL min for the glyphosate column and guard Continue pumping until the entire post column system is primed The column back pressure should stabilize at approximately 1500 psi 100 bar Post column Reagent Prepar
9. The side access panel can be removed by removing two captive screws along the bottom edge There is an advantage and also a disadvantage to leaving the side panels off With the side panels off you can spot any leakages quickly but it is more dusty because the air flow no longer travels through the foam filter If the side panels were left off vacuum clean the inside as well as outside Soak up spills and leaks with rags paper towels or sponge Clean spill area with a wet towel or water from a squeeze bottle and dry thoroughly Do not spray water directly into the electrical part of the post column pump Chapter 6 Major Repairs Major repairs are usually made on an exchange basis Contact Pickering Laboratories to get a Returned Goods Authorization number before returning any components Write this RGA number prominently on the package and packing slip Generally it is best to return the whole post column system except the reagent reservoirs for repair or recertification This chapter covers disassembly of the post column system Pickering Laboratories recommends disassembly of the post column system only as a last resort for repair Generally chemical problems can be solved with chemistry Please consult Pickering Laboratories before disassembly Warning Disassembly of the post column system should be carried out by qualified service personnel only Ensure that the power cord is disconnected before disassembly of the post colum
10. maximum column heater 2 4 A cte RI Ane 2 4 Temperature controllers 2 3 Temperature display 2 3 Test mixture cooocconnnococccnncnnncnnnnnnnnnnnna no ncnnnnn 3 2 Thiofluor esee 4 2 4 13 Troubleshooting seseeesseessesrresesenesees 5 12 Tubing e TE EE 2 11 3 1 3 4 TEON maria 2 11 U Unpacking nornin ioii 3 3 V Valve EISE EE 2 3 Check iai ez b eR 5 9 safety relief iseer 2 7 VC Yoco recie eee 2 11 VESPO iea a T IRED 3 1 Index W Water drinking ccooooccccnnnnononccncnnccnncnncinnnns 3 1 What to do if Contamination glyphosate column 5 16 Organic solvent in glyphosate column 5 16 Reactors have deposits 5 15 Sodium hydroxide backflows onto a carbamate column 5 15 When priming the pump IS diffiCult teats eto ates 5 17 Index 3
11. 18 1 0 mL min 0 0 0 82 18 inject up to 10 uL methanol 1 0 0 5 0 5 82 18 isocratic 2 0 5 29 28 5 30 70 linear gradient 4 29 01 0 01 0 100 step change 5 29 31 2 0 100 Cleanout 6 31 5 8 82 18 re equilibration 4 5 Post Column Conditions PCX 5100 User s Manual Chapter 4 0840250 column 4 0 mm ID x 250 mm with methanolic samples Step Times min Interval Water MeOH Comment Equil 88 12 0 80 mL min 0 0 2 2 88 12 inject up to 10 uL methanol 1 2 42 40 34 66 linear gradient 2 42 46 4 34 66 isocratic 4 46 1 0 1 0 100 step change 5 46 1 49 2 9 0 100 cleanout 6 49 10 13 88 12 re equilibration 0840250 column 4 0 mm ID x 250 mm with methanolic samples using a water MeCN gradient Step Times min Interval Water MeCN Comment Equil 90 10 0 80 mL min 0 0 2 2 90 10 inject up to 10 uL methanol 1 2 46 44 49 51 linear gradient 2 46 1 0 1 30 70 step change 4 46 1 49 2 9 30 70 cleanout 5 49 10 13 90 10 re equilibration For all the above programs the same post column conditions apply Reagent 1 0 05 N NaOH CB130 Pump 1 0 30 mL min Reactor 1 500 uL at 100 C Reagent 2 OPA amp Thiofluor in pH 9 1 borate buffer Pump 2 0 30 mL min Reactor 2 100 uL at ambient temperature 4 6 PCX 5100 User s Manual 25 ng each in 10 uL methanol 15 cm column 11 Mo 25 ng each in 10 uL methanol 25 cm column 9 2 7 1 3 6 5 8 4 2 ng each in 150 uL water 15 cm column 9
12. 5 RightPanel 22 EET 2 6 WATS LI eric D ah ED 2 7 Rear Panel rU 2 8 COLUMN OVEN ee 2 10 Reagent Hes6rvolts 00 ccce tiene rand nasa cannes dive o o ER aeree na dcc no ek EE 2 11 Specifications 23 65 eh t edi bte EE 2 13 Chapter 3 Installation Site Requirements cic duca Hot tg ten d CL Da te s Fave ia A dod ia 3 1 Standard Analysis System ccccccccceececeeeeeeeeeeeeeaeeeceeeeeceaeeeseaeeeseaeeeseeaeeseeeeessaeeseeeeeessaeeeseaeees 3 1 HPLC system requirements ieii aii aiiai iii 3 1 Space Requirements esssssssssssssssssssss senten nennen trennen nns ss nnns nennen sn sn ns sentes sinis senten 3 1 Electrical Gas Miscellaneous Supplies enne 3 1 Chemicals and reagents required for carbamate analysis 3 2 Supplied by USt aee ina idt ee doe ace ee eee eg eee 3 2 Supplied with the carbamate post column analysis system 3 2 Chemicals and reagents required for glyphosate analysis 3 2 Supplicd by E USEN RET 3 2 Supplied with the glyphosate post column analysis system ooonooncccconccccncccononnnnonanannnnncnnnnos 3 2 leie Ate RE 3 3 Installation of the PGX 5100 vecina ia 3 3 Layout of the HPLC with the PCX BIO 3 3 Note about fittings eege dede oda pen Aide dea dei o ona eed a d a Ra dea d 3 3 Inert Gas He Ar or N amp Reagents Connections oononcoinnnicnnonnnrnnicnncnnnrnn nn ronano narran cc 3 4 Post Column Interlock Connections o oooccincccnnnnccnnnccccnnnnnnn nn cn corran 3 4 Connecting the NetOp a
13. 50 60 Hz 1 5 A grounded supply 200 240 V 50 60 Hz 0 8 A grounded supply Installation overvoltage category II Pollution degree 2 Environmental Indoor use only Altitude up to 2 000 m Ambient temperature 5 40 C Chapter 2 Relative humidity 80 31 C derated to 50 R H 40 C PCX 5100 User s Manual M de Chapter 2 Site Requirements Standard Analysis System HPLC system requirements c AAA Space Requirements Electrical Gas Miscellaneous Supplies Chapter 3 Installation Read all installation instructions and MSDS before operating your post column derivatization instrument and HPLC system Check that you have all the items shown in the Packing List Pickering PCX5100 Post Column Derivatization Instrument Pickering Carbamate and or Glyphosate option package HPLC manual injector or autosampler Binary or more gradient HPLC pump HPLC fluorescence detector Integrator or data system The HPLC pumping system the injector or autosampler the fluorescence detector and the integrator or data system must be supplied by the user e The HPLC pump must be capable of binary gradient elution e The injector should be able to inject a 10uL sample preferably by full loop injection e If drinking water is to be analyzed for carbamate insecticide residues the injector should be able to inject at least 200uL and preferably 400uL e Ifthe system will be used for glyphosate analysis be aware that t
14. and Aminomethylphosponic acid in Environmental Water Monsanto Method Number 86 63 1 1987 Environmental Protection Agency Draft Method 597 Analysis of Glyphosate in Drinking Water by Direct Aqueous Injection LC with Post Column Derivatization Instrumentation e M V Pickering Assembling an HPLC post column system practical considerations LC GC 6 11 1988 994 997 T e M V Pickering Modifying HPLC equipment to tolerate corrosive solutions LCeGC 6 9 1988 800 809 1 J W Dolan and L R Snyder Troubleshooting LC Systems Humana Press Clifton NJ 1989 T Reprints available from Pickering Laboratories Ref 1 PCX 5100 User s Manual Limited Warranty Limited Warranty Instruments Pickering Laboratories Inc Pickering Instruments in standard configuration see Instrument List below are warranted to be free of defects in material and workmanship under normal installation use and maintenance for a period of one year from the date of delivery to the original Customer Pickering will replace or repair without cost any defective items Expendable items such as check valves pistons piston seals and filters are excluded from this warranty In addition physical damage poor quality reagent and sample induced damage and instrument damage due to Customer s misuse are not covered by this warranty Instrument List AO3100 AO3102 EC5100 EC5102 EC5150 PCX5111 CRX400 AT3100 AT3102 EG5100 EG5102 EC5
15. and should be your first consideration Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 1 Step 2 Step 1 Step 2 Step 3 Step 4 Flush the pump with water then shut down the post column system and let the reactor cool Turn off the gas and vent the reservoir Place it at a level below the pump to prevent siphoning of the reagent Remove the right access panel by loosening two captive screws along bottom edge Disconnect the reagent inlet line from the bottom of the pump head Wipe up any spilled liquid Disconnect the outlet tubing from the top of the pump head stabilize the outlet check valve with a 1 2 wrench when removing the fitting to prevent the check valve from moving Remove the pump head from the pump as follows Figure 5 1 with a 5 32 hex wrench remove the two bolts while holding the pump head against the pump housing the pump head is under spring tension Gently pull the pump head straight out from the pump housing in line with the axis of the piston Do not tilt the pump head sideways the piston may break Place the pump head in a beaker of soapy water do not remove the check valves Suspend the beaker in a sonicator bath and turn it on maximum power for 30 minutes Replace the soapy water with deionized water and sonicate for 10 minutes Reconnect the tubings to the pump head stabilize the outlet check valve with a 1 2 wrench when tightening the fitting to prevent the check valve fro
16. case Warning The column heating block may become hotter than 70 C For your safety wear insulating gloves when the column oven is warm Attention La r sistance chauffante de la colonne peut d passer une temp rature de 70 C Pour votre s curit priere de porter des gants isolants lorsque le four de la colonne est chaud Warnung Der Heizblock des S ulenofens k nnte hei er als 70 C werden F r Ihre Sicherheit sollten Sie isolierende Handschuhe tragen wenn der S ulenofen warm ist Atenci n El bloque calefactor de columnas puede estar por encima de 70 C Para su seguridad use guantes aislantes cuando el horno de columnas est caliente Avvertimento Questo blocco che scalda la colonna puo diventare piu caldo di 70 C Per la sua protezione usa guanti insulanti quanto il blocco e la colonna sono caldi Step 1 Shut down the PCX5100 completely and let the reactor cool Disconnect the power cord Remove both access panels Step 2 Remove the five screws holding the top cover Lift the cover off Step 3 Disconnect the liquid connections to the part Step 4 Disconnect the electrical cable Step 5 Remove the screws securing the part to the chassis Remove the part Step 6 Install the new part Reassemble the PCX5100 in reversed order of disassembly 6 2 PCX 5100 User s Manual Chapter 6 Diassembly of Front Panel Note Pickering Laboratories recommends disassembly of the post column system only as a last resort for r
17. caused by a dirty flowcell autosampler or deteriorated samples Test with a second fluorescent detector If a second fluorescent detector is not available use an UV Vis detector set at 330nm absorbance Change the rotor seal of the autosampler or use a manual injector Prepare fresh standards from neat reference material Solution standards even stored in ampoules are not reliable especially when dissolved in acetonitrile What to do if PCX 5100 User s Manual Chapter 5 Peaks disappear or diminish Glyphosate 1 4 5 Iron contamination of column from samples long storage of the column stainless steel inlet frits in the eluant reservoirs or corrosion in system Flush guard and column with the Glyphosate Restore solution Remove stainless steel frits from the eluant reservoirs Clean or replace any corroded parts Glyphosate peak too small or gone but AMPA present Oxidizing reagent too weak too old NaOCI stock solution too old Reactor at wrong temperature Oxidizing reagent too strong AMPA vanishes Reagent pump mis adjusted Using CB910 instead of GA104 for OPA Diluent Reactors or mixing tees have deposits Mineral deposits from hard water samples or reagents can usually be dissolved by pumping 20 nitric acid through the reactor The Pickering pumps and most but not al HPLC pumps will tolerate this Columns and autosamplers probably will not tolerate this a Start HPLC pump at 0 5 mL min H20 for carb
18. in step 5 Remove the pushrod from the pump housing with round end tweezers Inspect for corrosion damage clean or replace the pushrod if necessary Coat the pushrod with a light film of SAE 30 oil and re install with the tweezers Inspect the pump head paying special attention to the cavity for the piston seal Any scratches or irregularities will require replacement of the pump head The whole pump head may be cleaned with soapy water and then with deionized water in a sonicator bath Insert the installation tool blunt end into the retainer assembly and then into the new backup washer and new piston seal When inserting the new piston seal the piston seal should lay flat on a hard surface with the spring side down Insert the tool into the pump head as shown in Figure 5 2 Now the spring side should face the pump head Keep the tool perpendicular with the face of the pump head Press gently and evenly on the edge of the retainer assembly with both thumbs Withdraw the installation tool Seal Back up Washer y i ia Spring side faces this way Compression Ring Piston Seal Figure 5 2 Piston seal replacement with installation tool 5 7 PCX 5100 User s Manual Chapter 5 Optional The piston seal Figure 5 3 in the wash system also requires periodic replacement However it needs not be changed as often as the primary seal Follow steps 6 and 10 if you desire to replace the seal in the wash system Seal Back
19. methanolic samples eseeeesss 4 5 Post Column Condit ONS a a a aea aa a aa eaa A a a nennen nnn nennen nnn nennen 4 6 System Startup for Carbamate Analysis sss enne 4 9 Shutdown Procedures crire aa aaea esee eerte tenni in aa a aa sen tenete rennen 4 11 Short Term Upto 3 days tir ee A Rote i tete amies 4 11 Medium Term Up to 6 days essen eene eene nre AA 4 11 bong Term 7 Gays Or MO coto lacas 4 11 Glyphosate Analysis with the EG5100 sse nnne nnne nennen ns 4 12 Getting Staten orate te n eee oae te et Reds 4 12 HPLC Mobile Phase 4 12 Post Column Reagent Preparation sesssessssssesssesesee senten nennen nnns nnne 4 13 Priming the Reagent Pumps coocoocccccnncoccccnnnononcnononnnnnnnnnn ono n nennen nn 4 14 dT rm onsthe PoX 5 LL EE 4 15 Setting Up the HPLC Fluorescence Detector seseessseessiesriessrrssirnssrrustrnssrnnstnnnrnnnsrnesernnt 4 15 Setting Up the Data Station or Integrator ene 4 15 Setting Up the HPLC Pump Method sse enne enne nnne rennes 4 15 Post Col umn Gohdillons itr RET pte eR edet ote 4 16 The First Chromatogram eene nennen rent ndr sen rene nennen nnns 4 16 Shutdown Procedures cutter ete aite ee oce ert see eU EE Dx Ese aa vero duds 4 16 Short Term Up to 3 days ssssssssssssssssesseeeeen nennen nre en resins nennen inen senes 4 16 Medium Term Up 0 6 days ccoo aii pente ER ede e
20. of low pH samples water samples stabilized with chloroacetic acid or ChlorAC and can accumulate in the ambient reactor Part required ambient reactor Cat No 1100 2927 Step 1 Step 2 Step 3 Step 4 Step 5 Shutdown the post column system and let the reactor cool for at least 30 min Remove both access panels Disconnect two fingertight fittings Figure 6 9 I Right side back of To Detector fitting II Left side lower left Tee Install new ambient reactor Note Do not overtighten the fittings Reinstall side access panels The line fuse is on the back panel in the power inlet module between the cord connector and the power switch Step 1 Step 2 Step 3 Step 4 Remove the cord from the power inlet Use a small flat screwdriver to pry up the fuse holder then pull it out Only use the correct type of fuse for the 120V instrument one each GMA3 type 3A 250V 5 x 20mm fast acting for the 240V instrument two each IEC127 type 1 6A 250V 5 x 20mm fast acting Reinstall the fuse holder and the power cord Troubleshooting Guide Advice Interpretation of Pressures PCX 5100 User s Manual Chapter 5 Rules of Dolan and Snyder see references Rule of One Make one change at a time Rule of Two Confirm the problem before fixing it Substitution Rule Swap in a good part for a questionable one Put it Back If swapping does not fix it put the original back in Write it Down Changes
21. psig swing are normal These pulsations are dampened by the liquids in the Bourdon tubes of the gauges and the flow restrictors packed with diamond particles located on the back of the gauge panel The pulse dampening is very effective as indicated by post column pressure gauge pulsations of less than 10 psig Note Inspect all tubing connections in the post column reaction instrument to ensure there are no leaks Step 5 Wait for the temperatures to come up to setpoint When all the temperatures are stable the Ready lamp lights and the RDY relay switches Refer to your detector manual for setup details Optimum conditions for most detectors are excitation at 330 nm and emission at 465 nm If your detector has a selectable time constant use about 2 seconds Prepare the HPLC data station or integrator and set up a data handling method to accept data from the fluorescence detector Initially an area method without naming peaks is adequate This method should have a peak width of about 20 seconds and data end time of about 20 minutes These are the recommended conditions for glyphosate analysis using the 1954150 column and 1953020 guard column The column temperature is 55 C Step Times min Interval K200 RG019 Comment Equil 100 0 0 40 mL min Maximum 0 0 0 100 0 inject 50 uL 1 0 15 15 100 0 isocratic 2 15 17 2 0 100 step change 3 17 8 12 100 0 re equilibration The exact time of equilibration depends
22. second is a pair of automatic valves that prevent gas pressure from pumping reagents back through the column during extended shutdowns However there are ways that the safety systems can be bypassed accidentally For example residual pressure in the gauges immediately after shutdown will take some time to leak down to zero Follow these procedures to avoid such accidents Never disconnect any fittings between the HPLC pump and the column until the post column system has been shut down and depressurized Any leaking fittings between the HPLC pump and the column can permit backflow in the event of an unattended shutdown When removing the column remove the outlet fitting first Always follow the proper shutdown procedures See Section 4 5 4 Sample and Standard Precautions Reagent Precautions Reactor Precautions Electrical Precautions PCX 5100 User s Manual Chapter 5 l he test mixtures for carbamates and glyphosate are for qualitative use only They are not recommended for calibration purposes Filter all sample through a 0 45um membrane filter Some samples may require even more stringent filtration especially if colloids are present Aqueous samples must always be properly buffered Consult EPA Methods 531 1 or 547 for details For carbamate analysis with methanolic samples inject lt 10uL Large amount of organic solvents can cause peak distortion For small aqueous sample volumes lt 20uL either o
23. sulfone 3 Oxamyl 4 Methomyl Standak Vydate Lannate OR OR OR O Me Me Me Me b N o NP s c op Me ES Me OH Me Me 5 3 Hydroxycarbofuran 6 Aldicarb 7 Propoxur 8 Carbofuran Temik9 Baygon Furadan OR OR OR OH Me Me Me Me SMe Br 9 Carbaryl 10 1 Naphthol 11 Methiocarb 12 BDMC Sevin Mesurol Figure 1 1 Analytes in the Pickering carbamate test mixture 4 Bromo 3 5 dimethylphenyl N methylcarbamate an internal standard HPLC Separation PCX 5100 User s Manual Chapter 1 The general structure of the carbamate insecticides is an N methyl substituted urethane with the variation in the ester moiety The structural formulas for the ten analytes specified in USEPA Method 531 1 are shown in Figure 1 1 including 1 naphthol and BDMC They are listed in the order in which they elute from the Pickering carbamates column All but 1 naphthol 10 contain the N methylcarbamoyl moiety indicated by OR The hydrolysis of carbaryl 9 in the post column reactor also produces 1 naphthol Note that 1 naphthol hydrolyzed from carbaryl and 1 naphthol in the calibration standard are at different retention time This observation is useful for troubleshooting see page 5 13 Each unique R group represents a different commercial product or its metabolite The separation of the carbamates is achieved with the Pickering Bum Cig or Cg column maintained at 42 C and 37 C respectively The chromatographic method r
24. the piston and the scratched piston will in turn cause the primary seal to fail Syringe and Luer adapter gt Figure 3 5 Cut the supplied c flex tubing into 3 sections one 8 inch and two 24 inch sections Connect the 8 inch c flec tubing between the outlet from pump 1 to the inlet of pump 2 The two 24 inch sections are for the connections between the Luer adapter and the inlet of pump 1 and from the outlet of pump 2 to waste 3 7 System Testing amp Verification PCX 5100 User s Manual Chapter 3 Read Chapter 4 System Operation to become familiar with the use of your instrument At the beginning of Chapter 5 there are procedures for the initial system testing The installation is not complete until the initial system tests have been performed satisfactorily The initial system tests consist of Parameter log of pressures temperatures and flows under standard initial conditions Appendix A Chromatogram of test mix using Pickering standard conditions Important If the system will not be used immediately after the installation the system must be shut down properly and the reagents replaced with water methanol Flush the entire system with water methanol as listed in Chapter 4 Shutdown Procedures 3 8 Carbamate Analysis with the EC5100 Getting Started gt gt ARA HPLC Mobile Phase Supplied by User 3D rr Chapter 4 System Operation Important This chapter assumes tha
25. the system with 100 acetonitrile as precipitation of borate salt in the reactor might occur Do not exceed 70 acetonitrile if it will be used as the mobile phase Methanol is recommended as the organic mobile phase for the Pickering Laboratories column and it is less expensive Reagent precipitation problems rarely occur using methanol as the flushing solvent When switching a system between glyphosate and carbamate modes be sure to flush the HPLC and injector with compatible mobile phase before connecting the column Eluants for one analysis will damage the column for the other 5 3 Column Maintenance and Precautions Carbamate Columns Glyphosate MENU iL m Column PCX 5100 User s Manual Chapter 5 Always protect the analytical column by use of the pre column filter and guard column Check for leaks daily at column fittings In particular glyphosate eluants are corrosive e Ifthe column back pressure is high gt 2000psi isolate the source of the high pressure guard analytical column or the 0 5um in line filter Replace items causing the increased back pressure Back pressure from filter and guard should be 200psi During shutdown flush the column with pure methanol Do not store the column in water The analytical column can be back flushed with methanol at 1 mL min to clear partial blockage Do not disassemble or attempt to replace column inlet frit as this will void the column warranty Disconne
26. up Washer Installation Tool Retainer Assy gt d o So lt 0 Eldex Il Ze E Hooked End Blunt End 4 58 ns Compression Ring Piston Seal Figure 5 3 Piston seal replacement in the wash system with installation tool Step 11 Reassemble the pump head and wash system as shown in Figure 5 1 Hold the pump assembly firmly in place and einstall it on the pump in line with the axis of the piston do not tilt the pump head sideways Step 12 Repeat steps 4 11 for reagent 2 side If the pump was removed from the chassis in step 3 replace the pump Reconnect the tubings to the pump head stabilize the outlet check valve with a 1 2 wrench when tightening the fitting to prevent the check valve from moving Relocate the reservoir and turn on the gas Start the PCX5100 and prime the pump 5 8 Check valves Cleaning Check valves Removing Existing Check valves Installing New Check valves PCX 5100 User s Manual Chapter 5 Always work with check valves in a clean area to prevent dust and dirt from entering the pump The check valves are the hexagonal shaped components on the pump head Figure 5 1 Inlet check valves can be distinguished from outlet check valves by the groove on the hexagonal part of the inlet check valve Tools needed for check valve repairs 1 4 amp 1 2 open end wrench 5 32 hex wrench Open end wrenches are not included Cleaning check valves is easy and very effective
27. 100 psi 7 bar Connect a 0 020 inch 0 5mm ID tubing to the waste container here Over Pressure Relief is a safety relief valve that opens in case the post column pressure reaches 525 psi 36 bar This protects the soft fluorocarbon tubing of the reactors from rupture in the event of a blockage in the post column system or other fault Run a tubing from this fitting to a clean dry beaker Any evidence of liquid in this tubing indicates a fault condition A removable panel gives access to the liquid ends of the pump and the reagent filters The panel can be removed by loosening the two captive thumb screws along the bottom edge The duplex reagent pump with a piston wash system is behind the right access panel In normal operation the pump requires no adjustment It is calibrated at the factory to 0 30 mL min for both channels The micrometer knobs on back of the pump adjust the flow rates One full turn of the knob changes approximately the flow rate by about 0 1 mL min Please see page 3 7 before turning on the post column pump The reagent filters are located just downstream of the outlet check valves of the pump The filters and elements are similar in appearance to the pre column filter but they are different The reagent filter element is a 2um frit Cat No 3102 9132 2 7 Rear Panel PCX 5100 User s Manual Chapter 2 l he power connector is a standard IEC 320 type connector Use the appropriate power cord for your loca
28. 152 PCX5112 CHX700 Analytical Columns Pickering s Analytical Columns are warranted to be free of defects in materials and workmanship under normal installation use and maintenance for a period of ninety days from the date of delivery to the original Customer Pickering will replace the Analytical Column under warranty if found defective in material or workmanship However the warranty is void if the Analytical Column was damaged due to Customer s misuse How to Obtain Warranty Service If there is a problem with your Instrument or Analytical Column within the Warranty period notify Pickering immediately at 800 654 3330 if calling from outside U S A use 415 694 6700 If the Instrument or Analytical Column was not purchased directly from Pickering please contact the vendor where it was purchased from Any Instrument part of the Instrument or Analytical Column returned to Pickering for examination or repair shall have Pickering s prior approval call for a Returned Goods Authorization number and be sent prepaid by the Customer Return transportation will be at Pickering s expense if the Instrument part of the Instrument or Analytical Column is found to be defective and under warranty Pickering Laboratories Inc 1951 Colony Street Suite S Mountain View CA 94043 U S A A AMPA aaa i 1 5 4 13 B Back pressure regulator 3 5 Band spreading test ssssss 5 2 BORG ald ici
29. Tubing stainless steel 1 16 OD x 0 010 ID x 30cm Tubing TFE 1 16 OD x 0 011 ID per 3ft 90cm Tubing TFE 1 16 OD x 0 025 ID per 3ft 90cm waste line Nut 1 4 28 x 1 16 Ferrule for 1 4 28 x 1 16 Nut 1 4 28 x 1 8 Ferrule for 1 4 28 x 1 8 Nut Lite Touch for 10 32 1 16 Ferrule Lite Touch for 10 32 1 16 Fuse for PCX5100 120V Fuse for PCX5102 240V References Carbamates e Measurement of N methyl carbamoyloximes and N methyl carbamates in drinking water by direct aqueous injection LC with post column derivatization EPA Method 531 by D L Foerst EPA 600 4 851054 1986 Method 5 revised by T Engels National Pesticide Survey Battelle Columbus Lab 1987 Method 531 1 revised by R L Graves EPA Environmental Monitoring and Support Laboratory Cincinnati 1989 e M W Dong F L Vandemark W M Reuter and M V Pickering Carbamate pesticides analysis by liquid chromatography Amer Environ Lab 2 3 1991 14 27 e K M Hill R H Hollowell and L D Dal Cortivo Determination of N methylcarbamate pesticides in well water by liquid chromatography with post column fluorescence derivatization Anal Chem 56 1984 2465 2475 e H Frister H Meisel and E Schlimme OPA Method Modified by Use of N N Dimethyl 2 mercaptoethylammonium Chloride Fresenius Z Anal Chem 830 1988 631 633 Glyphosate e J E Cowell Analytical Residue Method for N Phosphono methylglycine
30. User s Manual PCX 5100 Post column Derivatization Instrument For Carbamate and Glyphosate Determination Pickering Laboratories Inc 1951 Colony Street Suite S Mountain View CA 94043 800 654 3330 International 415 694 6700 FAX 415 968 0749 Cat No UM 5100 Version CE 4 July 1997 O Pickering Laboratories Inc PCX 5100 User s Manual Contents PCX 5100 User s Manual Contents Table of Contents Before You Start Please Read Symbols deere deed etal e a pa e eel oe oe dd oe ade ae Bikey ees BYS 1 Chapter 1 Introduction Post column Analysis 2 rre Senne bn kashan Anin ENEAK aN eii die ALES 1 1 Els 1 2 HPLC Separations xim tmt nete bee dt efe re dde dated des 1 3 Post Golumin BerivatizatiOn 2 ire id eade mae dud d do dea aa ane ed tee 1 4 eljehprrucepe EP 1 5 Post Column Reaction Hardware cccccccccccccccccccceceecueeeesaeaeaeaeaeaeseuesseeseecececeeeeeeeeeeeeeeuaueusananaeaenea 1 6 Chapter 2 The PCX 5100 Components of a System sss ese nenn nennen EADAE Aa titre TAAK LKAA Raitaa 2 1 PCX 5100 Post Column Derivatization Instrument sssssesssesssseessieesiessrressinesrrnssinnssrnssennent 2 1 EC5100 system for carbamate analysis sse 2 1 EG5100 system for glyphosate analysis nennen 2 1 Getting to Know Your PCX 5100 sssssssssssssseeeene nennen teneris inneren resist nein nensis 2 2 Front PANS ai M ENS 2 2 W udzrulee PEDE 2
31. a odo ao 5 11 troubleshooting GUIDE 2 rose tree dd cce tu etta uie ced 5 12 AONE EEEE EM 5 12 Rules of Dolan and Gnvder aaaea aeh Paaie da faaan iadaaa aeaaaee aada nnne ns 5 12 General Procedure for Troubleshooting ooconccccncccnonicccnoncconnnnnnnano canon o nn nano coran o enne 5 12 Interpretation Of PreSSUres vesical bite oio KREN RERS opm 5 12 Most common problems with post column essen ene nennen nnns 5 13 Whatto dO If ons oia Aha E EE 5 15 Chapter 6 Major Repairs EIER 6 1 Removal of Column OVA at ee een tee ei ie deed 6 2 Disassembly of Front Panel 6 3 Appendix A PGX 5100 Parameter Log iii tee etes App A 1 B Recommended Consumables and Spare Parts App B 1 References Limited Warranty Index NEST essen Before You Start Please Read Please Read e Page 3 1 for the HPLC System Requirements especially for glyphosate analysis The HPLC components must be compatible with high pH regenerant Page 2 11 for the safety requirement of coated bottles they must not be substituted Page 4 13 for the calcium hypochlorite caution Instead of calcium hypochlorite use sodium hypochlorite Symbols A note supplies supplementary information which may be helpful or necessary for better understanding of the material The caution calls attention to an operating procedure practice or the like which if not correctly done or adhered to could result in loss of information or damage to or destruct
32. amates column K200 for glyphosate column b Replace both post column reagents with deionized water Run post column pumps for 5 10 min c Stop post column pumps Replace deionized water with 20 nitric acid and run post column pumps for 10 15 min d Reverse the order of washing with water and then replace with the post column reagents Note The washing solution can be stored in Erlenmeyer flasks or spare bottles Pressurizing the washing solution is not necessary Grease deposits can be dissolved by turning off the reagents and pumping methanol through the system Carbamate column only Stronger solvents such as acetone methylene chloride or tetrahydrofuran THF may be needed Silica deposits are too hard to remove Replace the reactor s Carefully clean or replace other components in the flow path You must remove all the silica before the system will work again This will probably entail major repair NaOH backflows onto a carbamate column Do not restart the system Dissolved silica or Cig phase will reprecipitate in the post column reactors or flowcell Most often these additional complications require replacement of both reactor coils Replace the column Call Pickering Laboratories for help PCX 5100 User s Manual Chapter 5 Organic solvent in a glyphosate column e This procedure usually works but may not work every time a b C Shut down the PCX 5100 and remove the analytical and guard columns Flu
33. anual Spare Parts Part Number Appendix B Description 3102 9047 3102 9132 1100 2927 1100 0281 1100 2660 1100 2661 1100 0200 3106 1330 3106 1310 3106 1314 3106 1316 3106 1320 3106 1332 3106 1322 3106 1324 3107 0137 3107 0138 2103 0463 3104 0081 3101 0060 3102 1202 3102 2102 3102 1402 3102 2402 3103 1030 2101 0212 2101 0225 3101 0007 3101 0008 3101 0005 3101 0006 3102 1518 3102 2507 3543 0045 3543 0044 App B 2 Replacement frit 0 5um for pre column filter Replacement frit 2um for reagent filters OPA Reactor 0 011 ID TFE tubing 0 5mL Coil Assembly only no heater Heated Reactor 0 5mL 120V for PCX5100 Heated Reactor 0 5mL 240V for PCX5102 Restrictor for OPA NaOCI amp NaOH reagent with nuts amp ferrules Seal 1 for reagent pump Seal Kit for reagent pump includes 2 seals and seal installation tool Inlet Check Valve for reagent pump Outlet Check Valve for reagent pump Piston sapphire for regular reagent pump Piston sapphire for reagent pump with piston washing system Piston Guide Retainer Liquid End Assembly Reagent Bottle coated 1 liter borosilicate no cap Cap for Reagent Bottle 3107 0137 without tubing Tubing Saran 1 8 OD x 0 063 ID per 3ft 90cm Seal Kit for bypass valve Nut Fingertight for 1 16 plastic tubing Nut male Upchurch type 10 32 1 16 Ferrule Upchurch type 1 16 Nut male Valco type 10 32 1 16 Ferrule Valco type 1 16
34. anual Appendix A Post Column Band Spreading Test Test Conditions LC Eluent 60 40 methanol water Flow rate 1 0 mL min Sample Pickering carbamate test mixture Cat No 1700 0063 10 uL Column TYDE iati ed teet d eet Eh Dre eti ID iiic tet Temperature 42 C Detector Excitation Emission Lamp PMT gain Range Attn Integrator Range Attn Units Multiplier Chart speed Method for calculating peak width Part 1 Column connected directly to the detector Post column pumps off reactor temperature lt 55 C Run Retention time Peak width Average W Part 2 Post column reactors connected to column and detector Post column pumps off normal reactor temperature Run Retention time Peak width Average Wo Ratio Wy Wy ss Operator cuire cere tercer cere res AC App A 3 PCX 5100 User s Manual Appendix A Post Column Delay The post column delay volume is about 600 uL The delay time can be calculated as t 0 5 f 0 1 f fay faa where f is eluent flow rate f is first reagent flow rate and f is second reagent flow rate 0 5 is the volume of the first reactor and 0 1 is the volume of the second App A 4 Appendix B Recommended Consumables and Spare Parts For routine maintenance and minimal interruptions to your operation always keep the neces sary consu
35. ation J PCX 5100 User s Manual Chapter 4 The two derivatization reagents required for glyphosate analysis are a hypochlorite reagent NaOCl and o phthalaldehyde Note During initial installation the reagent bottles lines and pump should first be cleaned and primed with methanol to reduce possible fluorescence background Do not leave caps and lines dangling without a reservoir To fill reservoir transfer caps and lines into a spare bottle or an Erlenmeyer flask filled with deionized water To prepare and pressurize the post column reagents follow this procedure Step 1 Turn off the inert gas Step 2 Thoroughly wash the two reagent reservoirs and then rinse with methanol Wipe down the dip tubes with methanol and a clean cellulose tissue Step 3 Preparation of the Oxidizing Reagent a Pour one bottle 950 mL the Hypochlorite Diluent GA116 directly into the reagent reservoir labeled Oxidizing Reagent Reservoir cap has TFE reagent and gas lines b Add 100 uL of 596 sodium hypochlorite solution Clorox to the diluent The exact amount will depend on the actual hypochlorite concentration of the stock solution When you get your first chromatograms you will be able to adjust the amount to optimize the relative peak areas of glyphosate versus AMPA Figure 4 2 shows a typical response curve c Capthe reservoir close the vent valve and swirl the solution to mix it thoroughly Note The hypochlorite concentration slowly
36. ckground signal caused by Contaminated eluant bacterial growth fingerprints water purifier needs service Contaminated reagent s defective chemicals Reagent backflows into column caused by Not following proper shutdown procedure Not shutting down and depressurizing post column before working on the HPLC Leaking fittings between column and HPLC pump Defective reagent control valves Air in reagent pump or flow conditioners check for Reagent pressure is low Some peaks disappear or change relative intensity Noisy baseline with 2 second period Reagent pressure is low Pump takes too long to come up to pressure Poor peak shape caused by Column worn out Guard column dirty Bad column Deposits in post column flow path Partial obstruction of flowcell Too strong a solvent or too large a sample injected Bad tubing connection wrong style nut too large tubing wrong type union Reagent flow rate s too high Strange injector problems Deposits in reactor caused by Dissolved silica reprecipitating carbamate column NaOH backflow into column Corrosive samples Backflushing a dirty column into the system Contaminated reagents Hard water samples Degradation of Teflon tubing Greasy samples Using calcium hypochlorite as the oxidant in glyphosate determination Preparing your own reagents with poor quality chemicals PCX 5100 User s Manual Chapter 5 High column pressure caused by F
37. criterion of peak width Generally the width at half maximum is the most consistent measure There are several ways to measure it If your computer data station calculates peak width use that The ratio of area over height is a good approximation and some data stations report this number If you are using an integrator that only reports areas you can measure the height in millimeters and calculate a ratio this number has arbitrary units but it is more precise than a direct measurement of the width However you decide record the method and all relevant parameters e g detector sensitivity attenuation chart speed chart scale Step 3 Install the column in the PCX5100 This can be your normal carbamate column or any C18 column in good condition You may have a column reserved especially for quality control or diagnostic purposes and use it here Step 4 Disconnect the detector inlet from the PCX5100 Disconnect the column outlet from the PCX 5100 Connect the detector to the outlet of the column Step 5 Turn ON the HPLC system Set the Pump switch OFF on the PCX5100 Turn ON the main power on the PCX5100 and press the Reset button Immediately lower the temperature setting on the reactor to below 55 C before the reactor can warm up Wait for the column to come to equilibrium Step 6 Collect three chromatograms of the test mixture You should see a single peak around 4 6 minutes The retention times and width parameters sho
38. ct the outlet of the column during the back flush operation Organic contaminants can be washed off the column by first washing with methanol then with dichloromethane Wash again with methanol before use e The column is temperature controlled to reduce baseline shift caused by viscosity changes during gradient formation to reduce back pressure and to improve retention time reproducibility Use the Pickering Laboratories carbamate analysis column which is specifically designed and tested for the separation of carbamates in the EPA Methods e During shutdown flush the column with RG019 for 5 minutes but no more than 10 minutes Do not store the column in the eluant Contamination usually occurs on the guard column Wash it separately from the analytical column This will save much time in the washing and re equilibration Contaminants of special concern iron and other polyvalent cations organic dyes surfactants detergents and lipids They may cause irreversible damage Organic solvents will cause the resin in the column to swell This leads to high back pressure and broadened peaks The column sometimes can be regenerated Use Pickering eluants with the Pickering column as they are designed to work together The PCX5100 has two safety systems to prevent accidental backflow of reagents onto the column The pressure interlock requires that the HPLC pump deliver at least 500psi before the reagent pump can be engaged The
39. d one program for methanolic samples using a water acetonitrile gradient When using a Cg 25 cm column EC5150 the water acetonitrile gradient can be used as a confirmation method The Cig columns operate at 42 C the Cg column at 37 C 1846250 column 4 6 mm ID x 250 mm with aqueous samples Step Times min Interval Water MeOH Comment Equil 100 0 1 0 mL min 0 0 0 100 0 inject up to 400 uL water 1 0 1 7 1 7 100 0 concentrate sample on column 2 1 71 0 01 80 20 step change 3 1 71 45 7 44 25 75 linear gradient 4 45 71 0 01 0 100 step change 5 45 71 50 4 29 0 100 cleanout 6 50 8 12 100 0 re equilibration 1846150 column 4 6 mm ID x 150 mm with aqueous samples Step Times min Interval Water MeOH Comment Equil 100 0 1 0 mL min 0 0 0 100 0 inject up to 200 uL water 1 0 1 1 100 0 concentrate sample on column 2 1 01 0 01 82 18 step change 3 1 01 36 35 30 70 linear gradient 4 36 01 0 01 0 100 step change 5 36 01 38 2 0 100 cleanout 6 38 5 10 100 0 re equilibration 1846250 column 4 6 mm ID x 250 mm with methanolic samples Step Times min Interval Water MeOH Comment Equil 80 20 1 0 mL min 0 0 0 80 20 inject up to 10 uL methanol 1 0 1 1 80 20 isocratic 2 1 44 43 25 75 linear gradient 4 44 01 0 01 0 100 step change 5 44 01 49 5 0 100 cleanout 6 49 5 8 80 20 re equilibration 1846150 column 4 6 mm ID x 150 mm with methanolic samples Step Times min Interval Water MeOH Comment Equil 82
40. dds the following accessories for carbamate analysis 1846150 Carbamate column 4 6mm ID x 150mm 5um Cs with test chromatogram 1846250 Carbamate column 4 6mm ID x 250mm Bum Cs with test chromatogram 18ECGO001 Guard cartridge holder with 3 carbamate guard cartridges 0120 o Phthalaldehyde chromatographic grade crystals 5g CB910 OPA diluent for carbamate pesticide analysis 4 x 950mL 3700 2000 Thiofluor chromatographic grade crystals 2 x 10g CB130 Hydrolysis reagent for carbamate pesticide analysis 4 x 950mL 1700 0063 Carbamate test mixture comprised of 12 components 1 5mL 2 5ug mL one each included with each column 1700 0132 ChlorAC buffer for preservation of aqueous carbamate samples 250mL The EC5150 system is similar to the EC5100 except the two columns are replaced with 0840250 Carbamate column 4 0mm ID x 250mm 5um Cs with test chromatogram The complete system adds the following accessories for glyphosate analysis 1954150 Glyphosate column cation exchange 4mm ID x 150mm 8um K H form with test chromatogram 1953020 Glyphosate guard column cation exchange 3mm ID x 20mm 8um K H form 0120 o Phthalaldehyde chromatographic grade crystals 5g GA104 OPA diluent for glyphosate analysis 4 x 950mL GA116 Hypochlorite diluent for glyphosate analysis 4 x 950mL K200 Eluant for glyphosate analysis 4 x 940mL RG019 Column regenerant for glyphosate analysis 1 x 950mL 3700 2000 Thiofluor chromatographic grade crysta
41. decreases with time This will manifest itself as a change in the relative peak areas of glyphosate and AMPA It will remain usable for several days but we recommend you calibrate daily Caution Do not use calcium hypochlorite in the oxidizing reagent This will cause plugging of the post column reactor The one year warranty does not cover damage caused by calcium hypochlorite based reagents The EPA Draft Method 547 is wrong on this point Ca3 PO4 is insoluble in water 400 Glyphosate 250 O AMPA 200 Peak Area 150 0 100 200 300 400 500 Figure 4 2 uL of 5 NaOCl per L of GA116 Priming the Reagent Pumps PCX 5100 User s Manual Chapter 4 Step 4 Preparation of the OPA Reagent a Pour the contents of one bottle 950 mL of the OPA Diluent Cat No GA104 into the reagent reservoir Save approximately 5 mL for step e b Putthe cap on the bottle open the vent valve and turn on the gas supply Thoroughly de aerate the contents by sparging with inert gas Continue bubbling for at least 10 minutes c Dissolve 100 mg of OPA Cat No O120 in approximately 10 mL of HPLC grade methanol in a clean dry container d Turn offthe gas supply and remove the cap from the bottle Add the OPA solution to the deoxygenated Diluent in the reservoir e Dissolve 2 g of Thiofluor Cat No 3700 2000 in the reserved 5 mL of the OPA Diluent and add into the reservoir Note If Thiofluor is not available pipette 1 mL o
42. e Compare your chromatograms to the test chromatogram supplied with the Pickering column Your chromatograms should not be significantly different If there is a problem see the later portion of this section for troubleshooting Keep copies of your test chromatograms and the Pickering test chromatogram on file Make copies of the blank forms in Appendix A and complete the parameter log on the photocopy Your system should have come with a similar log from factory testing Use the same conditions as for the test chromatogram above Report the pressures for the system equilibrated under initial conditions The pressures reported for Reagent 1 and Reagent 2 should be the maximum swings of the pointers Although the parameters will not be identical to the factory they should be similar Keep a daily log of the four pressures for diagnostic use See page 5 11 Interpretation of Pressures There is also a sheet for you to record the HPLC system parameters Include all the settings for the pump injector detector and integrator Keep copies of this document as it will be very helpful for troubleshooting Typically your conditions for routine analysis will be different than the conditions used for testing You may be using a different sample sample volume standard solution gradient or even column Set up the system for injection of your calibration solution and collect two chromatograms The only standard for comparison is your expectations Fill
43. e column disconnect the outlet fitting first Replace them with a tubing and unions so there are no open lines Glyphosate Analysis with the EG5100 Getting Started HPLC Mobile Phase PCX 5100 User s Manual Chapter 4 Important This section assumes that the PCX 5100 has been installed according to the directions in Chapter 3 Do not operate the instrument until it has been installed properly and you have read and understood the instructions in this section The Pickering Laboratories glyphosate analysis requires two mobile phases K200 eluant and RG019 column regenerant Do not use water or eluant containing any organic modifiers such as methanol acetonitrile etc for the glyphosate column Do not exceed a flow rate of 0 4 mL min To prepare and degas the HPLC mobile phase use this procedure Note Pickering mobile phases are filtered before bottling so it is unnecessary to filter the mobile phases before use Filtering with marginally clean glassware has been known to introduce large amounts of contaminating fluorescent compounds to the mobile phases Always wear gloves for this operation Avoid touching the inside of reservoirs or handling the solvent filter with bare fingers since amino acid contamination present on hands causes high fluorescence background Do not leave caps and lines dangling without a reservoir To fill reservoir transfer caps and lines into a spare bottle or an Erlenmeyer flask filled with deionized water
44. e fitting Use a 7 16 wrench to remove the end of the fitting Remove the filter element The correct filter element is 3102 9047 a 0 5um porosity 0 063 diameter frit It is superficially similar to the reagent filter so take care not to confuse them Wet the new filter element with water and replace the fitting Be careful that the filter element does not fall out of the fitting while installing it Tighten the fitting firmly Reconnect the capillary tubing These filters are located directly above the pump heads inside the PCX5100 cabinet Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Be sure that the PCX5100 is shut down and depressurized before changing a filter Turn off the gas valve and open the vent valves on the reservoirs Place the reservoirs at a level below the pump to prevent siphoning of the reagent Disconnect the tubings from the top and bottom of the filter Use two 7 16 inch wrenches to disassemble the filter Remove the filter element The correct filter element is 3102 9132 a 2um 0 188 inch diameter frit It is superficially similar to the pre column filter so take care not to confuse them Replace the filter element and replace the fitting Be careful that the element does not fall out of the fitting while installing it Tighten the fitting firmly Reconnect the tubings to the filter The arrow on the side of the filter shows the direction of flow The Cig phase can be stripped with repeated injections
45. ecommended for this column is a simple linear water methanol binary gradient that resolves the twelve carbamate products provided in the test standard Figure 1 2 The carbamates elute principally in relation to their relative hydrophobicity Aldicarb sulfone which is minimally hydrophobic elutes early while methiocarb which is more hydrophobic elutes towards the end of the gradient MINUTES 1 Aldicarb sulfoxide Standak 7 Propoxur Baygon 2 Aldicarb sulfone 8 Carbofuran Furadan 3 Oxamyl Vydate 9 Carbaryl Sevin 4 Methomyl Lannate 10 1 Naphthol 5 3 Hydroxy carbofuran 11 Methiocarb Mesurol 6 Aldicarb Temik 12 BDMC internal standard Figure 1 2 2 1 ng in 150 uL water 14 ppb 25 cm C column Post column Derivatization PCX 5100 User s Manual Chapter 1 The separated carbamates are first saponified by sodium hydroxide NaOH at 100 C to release an alcohol carbonate and methylamine In the second post column reaction methylamine reacts with o phthalaldehyde OPA and the nucleophilic Thiofluor or 2 mercaptoethanol to form a highly fluorescent 1 methyl 2 alkylthioisoindole derivative Figure 1 3 This fluorescent derivative provides detection lt 3ng per component on column which meets the method requirements of the EPA Depending on the type of fluorescence detector used detection limits ten times better than the EPA requirements may be obtained The Pickering carbamate post column derivatization
46. eed to provide adequate lengths of capillary tubing to connect HPLC pump and injector to pressure switch 0 010 0 020 inch ID to detector inlet 0 010 inch ID to detector outlet 0 010 0 020 inch ID and to injector outlet 0 007 0 010 inch ID 3 1 Chemicals amp reagents required for carbamate analysis Supplied by User Supplied with the carbamate post column analysis system Chemicals amp reagents required for glyphosate analysis Supplied by User Supplied with the glyphosate post column analysis system PCX 5100 User s Manual Chapter 3 Important These solvents and chemicals must be available in your laboratory before installing your Carbamate Post Column Derivatization Instrument with the HPLC System e HPLC grade methanol from Fisher Scientific J T Baker or Merck Additional filtration is not recommended HPLC grade water also from Fisher Scientific J T Baker or Merck Additional filtration is not recommended Reagents for sample preparation Note Water and methanol even HPLC grade from other vendors may contain traces of amines or ammonia which will react with OPA Thiofluor in the post column system to cause interference Water from laboratory purification systems Milli Q Barnstead etc also may not be acceptable and should be tested for suitability against Fisher HPLC grade water The age of the cartridge the configuration the activated charcoal cartridge should be placed a
47. egulator provides 100 psi 7 bar of back pressure to the fluorometer and prevents outgassing at the flowcell The pressure rating of the flowcell must be at least 110 psi 8 bar so that the flowcell is not damaged If your fluorescence detector has a flowcell pressure rating of less than 100 psi 7 bar contact Pickering Laboratories The back pressure is adjustable with the hex wrench that comes with your system The minimum necessary backpressure is 75 psi 5 bar 3 5 PCX 5100 User s Manual Chapter 3 Step 4 Connect a 0 020 inch ID PTFE tubing to the Waste outlet on the right side of the instrument Place the other end in an appropriately labelled waste container Step 5 Set the excitation and emission of the detector The optimal excitation wavelength is 330nm and maximal emission wavelength is 465nm for most detectors If your detector provides for an emission cutoff filter use a 390nm filter Step 6 Connect the signal cable from the fluorometer to the input terminal of your data station Ensure that the polarity is correct Refer to your HPLC instrument manual Installing the Install the analytical column and guard column in the column heater as follows analytical amp guard columns Step1 One set of PEEK tubing has been provided for installing each length of column Select the column that you wish to install and the corresponding tubing set Step 2 Remove the cover plate from the column block and set it aside Step3 Co
48. emperature controllers should come on but the heaters remain off Turn OFF the Pump switch Press the Reset button on the front This sends power to the heated reactor and column oven Allow the reactor to reach 50 C before turning on the reagent flow Step 4 Ensure that the reagent pump is turned OFF e Press the Reset button e Wait for the temperatures to stabilize 4 9 PCX 5100 User s Manual Chapter 4 The following HPLC system readings should now be displayed Pressure of the pump and PCX 5100 instrument will have dropped by about 100 psi due to the decreased resistance of the post column reactor heated to 100 C Instrument Function HPLC pump delivery HPLC flow column heater temperature post column reactor HPLC pump pressure post column system pressure reagent 1 pressure reagent 2 pressure Reading 82 A water 1896 B methanol 1 0 mL min 42 C 100 C 1200 1500 psi 180 200 psi lt 100 psi lt 100 psi Step 4 Switch the reagent pump on Prime the pump and purge the reagent lines Observe a small increase in the fluorescence signal e Press the Pump switch ON so that the orange stripe becomes visible e Attach the 20 mL plastic syringe to the Luer fitting on Bypass 1 Open the valve 1 turn Draw about 10 mL of reagent into the syringe Close the valve Discard the liquid in the syringe Repeat the operation for Bypass 2 Function eluant composit
49. en close the bypass valve Optional before closing the bypass valve use the syringe to apply pressure until the pressure gauge moves Close the valve while holding pressure on the system h Ifthe pump is not on turn it on i The pressure should come up within a few seconds If it does not begin pulsing within 30 sec repeat steps 4 8 If you can not prime the pump after 2 or 3 applications of vacuum then there is some other problem Check for leaking piston seals dirty check valves loose fittings or other defects Cleaning the Post column System Fittings External Internal PCX 5100 User s Manual Chapter 5 Always wear safety glasses or goggles laboratory coat gloves and other appropriate safety clothing Please read and understand the instructions in the MSDS shipped with the chemicals If the MSDS are missing please contact Pickering Laboratories and we can fax you a copy instantly Clean any leaks from fittings thoroughly with water and dry with paper towels especially if the solution is a buffer or hydroxide Standing salt and hydroxide solution is corrosive Soak up spills with rags paper towels or sponge Clean spill area with a wet towel and dry thoroughly Do not spray water directly into the instrument Vacuum clean the post column system once a year especially the foam filter in the front underside Alternatively the foam filter can be removed cleaned with soap and water air dried and replaced
50. epair Generally chemical problems can be solved with chemistry Please consult Pickering Laboratories before disassembly Tools needed 1 4 open end wrench 5 16 nut driver and a Phillips screw driver Tools are not included Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 Step 9 Shutdown the post column system completely and let the reactor cool for at least 30 min Disconnect the power cord Remove both access panels Remove the five screws holding the top cover two on each side and one on back Lift the cover off Disconnect both inlets and outlets to the post column pumps Disconnect five electrical connections at the printed circuit board Fig 6 1 from two temperature controllers pump on off switch ready LED lights and the pressure sensing switch Note the orientation of the connectors to facilitate reassembly Detach the two fittings on the pressure regulator Figure 6 2 Remove two bolts with an 1 4 open end wrench one on each side at the bottom Figures 6 3 and 6 4 of post column system Remove two nuts Figures 6 3 and 6 4 on inside panel with a nut driver Note Do not remove the two support bars Figure 6 3 The front panel can now be separated from the rest of the post column system Note The inlet and outlet tubings for the HPLC column can be bent slightly to facilitate this final step Reassembly of Front Panel Step 1 Step 2 Step 3 Step 4 Step 5 Step 6
51. er on the dowel pin Do not allow the valve parts to fall out onto a hard surface Step 3 Reassemble the check valve by placing the valve insert in the valve housing using the larger dowel pin 3 16 od Make sure that the valve insert is oriented correctly Step 4 Press a new Kel F seal into the valve housing Step 5 Slide the check valve cartridge into the valve housing making certain the cartridge is oriented correctly Step 6 Press a second Kel F seal into the valve housing The Kel F seal will extend approximately 0 02 0 03 from the valve housing Outlet cartridge detail Note orientation UM Outlet check valve Pump head Inlet check valve BS Inlet cartridge detail Note orientation Figure 5 4 Check valve assemblies S Housing outlet valve Outlet valve insert Inlet valve insert ig Outlet valve cartridge Valve seal Ges gt Valve seal x Inlet valve cartridge l Housing inlet valve Pre column Filter a gt Reagent Filters Ambient Reactor Fuse PCX 5100 User s Manual Chapter 5 The filter is located inside the fitting on the left side of the PCX5100 labelled From Injector Step 1 Step 2 Step 3 Step 4 Step 5 Be sure that the PCX 5100 is shut down and depressurized before changing the filter Disconnect the capillary tubing from th
52. eric nuts and ferrules The low pressure gas and reagent fittings are 1 4 28 x 1 8 inch size These can be used with either flared fittings or reversed ferrule fittings The fitting for the detector waste line is 1 4 28 x 1 16 inch and uses a reversed ferrule type fitting Pickering Laboratories supplies all the matching nuts and ferrules needed for normal assembly Note that fittings and ferrules for the LC and detector are not supplied jm Upchurch Parker Valco style 1 4 28 reversed ferrule Figure 3 2 Note the direction of the ferrules normal for Upchuch Parker amp Valco reversed for low pressure 1 4 28 3 3 Inert Gas No He or Ar amp Reagents Connections PA m AAA Post Column Interlock Connections PCX 5100 User s Manual Chapter 3 Step 1 Using a piece of the tan colored 1 8 inch Saran tubing connect the Gas In port on the right side of the instrument to a supply of inert gas at 45 75 psi 3 5 bar If you are using metal compression fittings be careful not to over tighten as the tubing can collapse or crack Turn on the main gas supply Switch the toggle valve up to the ON position to start gas flow Let the gas system purge for about one minute Switch the toggle valve down to OFF Step 2 Connect the gas lines of the reservoirs to the gas outlet manifold On the OPA reservoir the gas line will be marked with a small piece of black shrink tubing near the bottle cap On the other reservoir the ga
53. es Note HPLC grade mobile phases are filtered before bottling so it is unnecessary to filter the mobile phases before use Filtering with marginally clean glassware has been known to introduce large amounts of contaminating fluorescent compounds to the mobile phases Degassing the mobile phases with an inert gas prior to operation of the PCX5100 system is recommended for optimum performance To prepare and degas the HPLC mobile phase use this procedure Caution Always wear gloves for this operation Avoid touching the inside of reservoirs or handling the solvent filters with bare fingers since amino acid contamination present on hands causes high fluorescence background Do not leave caps and lines dangling without a reservoir To fill reservoir transfer caps and lines into a spare bottle or an Erlenmeyer flask filled with deionized water Step 1 Fill eluant reservoir B with HPLC grade methanol Step 2 Fill eluant reservoir A with HPLC grade water Step 3 Place the filled eluant reservoirs on or near the HPLC pump Step 4 If your HPLC requires it sparge bubble the eluants with helium 4 1 Post column Reagent Preparation J PCX 5100 User s Manual Chapter 4 Step 5 Prime the HPLC pump by withdrawing at least 30 mL of each solvent from the prime purge port with the priming syringe that is supplied An HPLC pump method can be configured to facilitate this step Consult your HPLC manual Step 6 Close the prime
54. f 2 mercaptoethanol If 2 mercaptoethanol is used this step should be performed in the hood since it is volatile and has an unpleasant odor which will permeate the laboratory 2 Mercaptoethanol should be replaced with Thiofluor Pickering Laboratories brand of N N dimethyl 2 mercaptoethylamine hydrochloride a nonvolatile thiol salt f Replace the cap and turn on the gas flow Continue sparging for another minute Close the vent valve Gently swirl the reagent to complete the mixing Caution The preparation of the OPA Diluent by the user is not recommended because sodium borate any grades contains excessive amounts of heavy metal contaminants and insoluble matter These impurities will eventually precipitate in the reactor and flowcell The one year warranty does not cover damage caused by these contaminants Note The OPA reagent is sensitive to air oxidation and degrades over time The PCX 5100 modular system is designed to minimize this oxidation resulting in a minimal loss of OPA reagent due to oxidation When the OPA reagent reservoir is maintained under inert gas pressure the OPA reagent maintains its activity for up to two weeks without significant loss of activity Step 1 Connect a 20 mL plastic syringe to the Luer fitting in the center of one of the prime purge valves Step 2 Open the prime purge valve 1 2 to 1 full turn CCW and let the flow exit into the syringe Step 3 To purge air bubbles from the reservoir line pu
55. f front panel are marked with Nuts in step 8 are marked with Do not remove the support bars marked with 3 6 6 PCX 5100 User s Manual Chapter 6 Figure 6 4 Close up of front panel Bolts in step 7 for diassembly of front panel are marked with Nuts in step 8 are marked with 6 7 PCX 5100 User s Manual Chapter 6 From Reagent 1 Pump 5 To Reagent 1 Pump Teflon tubing Figure 6 5 Post column reagent 1 flow path up to Restrictor 1 includes pressure gauge 1 and prime purge valve 1 6 8 PCX 5100 User s Manual Chapter 6 Fluorescence Waste Detector 100 psi B P Ambient Reagent 2 Reactor Bypass 2 Pressure 100uL O i Restrictor 2 Heated Reactor Anti siphon 500uL valves Reagent 1 Bypass 1 Pressure Post Column Restrictor 1 Reagent Press Gauge E Filters 2um E D mu 1 Reagent Q 525psi Pump Thermostatted Over pressure Column relief valve and Guard m e Es ES SS PA N Pre column Se OPA Filter 0 5 SSC Mer ER Beaker Nao S B Injector 500 psi Pressure Switch LC Pump Water amp Methanol or K200 amp RG019 6 9 PCX 5100 User s Manual Chapter 6 Anti siphon To Reagent 1 Pump r4 Valve From Reagent 1 Teflon tubing Pump Figure 6 6 Post column reagent 1 flow path up to the first mixing Tee includes post column pressure gauge anti siphon valve and the 525 psi over pressure relief valve
56. f position so that the orange stripe is not visible If you are using the optional Remote Off circuit make sure it is in the open or logic 1 state Press the Reset button The Reset lamp should become lit 4 3 Setting Up the HPLC Fluorescence Detector Setting Up the Data Station or Integrator Setting Up the HPLC Pump Method The First Chromatogram PCX 5100 User s Manual Chapter 4 Step 3 Check that the column temperature setting is 42 C and the reactor temperature setting is 100 C Press the button on the corresponding temperature controller to view the setpoint and release it to show the actual process temperature see page 2 3 Step 4 After the temperature of the reactor shows over 50 C turn on the Pump switch The two reagent gauges should begin pulsing with a maximum of about 1 000 1 500 psig Note The pulsating pressure readings of the reagent pumps approximately 500 psig swing are normal These pulsations are dampened by the liquids in the Bourdon tubes of the gauges and the flow restrictors packed with diamond particles located on the back of the gauge panel The pulse dampening is very effective as indicated by post column pressure gauge pulsations of less than 10 psig Note Inspect all tubing connections in the post column instrument to ensure there are no leaks Step 5 Wait for the temperatures to come up to setpoint When all the temperatures are stable the Ready lamp light
57. f the two Pickering columns can be used For volumes up to 500uL only the 25cm column should be used and a gradient delay time should be programmed into the analysis 0 organic to trap the sample onto the head of the column Always wear gloves during the preparation of reagents The Hydrolysis Reagent and Thiofluor cause skin irritation Also fingerprints contaminate reagents The hydrolysis reagent is stable and can be replaced as it is used The OPA reagent is sensitive to air oxidation degrades over time and should be prepared fresh for optimum sensitivity OPA reagent is stable for at least two weeks when properly prepared and pressurized with inert gas Thiofluor is extremely hygroscopic Always keep in a tightly closed container The preparation of the OPA Diluent by the user is not recommended because sodium borate any grades contains excessive amounts of heavy metal contaminants and insoluble matter These impurities will eventually precipitate in the reactor and flowcell The one year warranty does not cover damage caused by these contaminants If you must prepare your own borate buffer for the OPA reagent do not use sodium tetraborate as suggested by the EPA methods Instead use molar equivalents of boric acid and sodium hydroxide because they are available in higher purity ACS grade or better and have very little insoluble matter Do not operate the heated reactor above the boiling point of the eluant unless the back pressure
58. fter the ion exchange resin cartridge and the quality of the feed source determine acceptability Water from qualified purification systems should be monitored on a regular basis and proper maintenance procedures should be followed strictly Pickering Laboratories supplies the following reagents for system start up Additional reagents should be ordered to replenish the initial supply Hydrolysis Reagent 0 05M sodium hydroxide Cat No CB130 4 x 950mL e OPA Diluent 0 05M sodium borate buffer solution Cat No CB910 4 x 950mL o Phthalaldehyde 5g chromatographic grade crystals Cat No O120 Carbamate Test Mixture Cat No 1700 0063 2 x 1 5mL Thiofluor Cat No 3700 2000 2 x 10g chromatographic grade crystals ChlorAC buffer Cat No 1700 0063 for preservation of aqueous samples 250mL e 5 Sodium hypochlorite for preparing oxidizing reagent or Clorox from local grocery stores Pickering Laboratories supplies the following reagents for system start up Additional reagents should be ordered to replenish the initial supply Glyphosate Eluant Cat No K200 4 x 950mL Glyphosate Column Regenerant Cat No RG019 1 x 950mL Hypochlorite Diluent Cat No GA116 4 x 950mL OPA Diluent sodium borate buffer solution Cat No GA104 4 x 950mL o Phthalaldehyde 5g Cat No 0120 Thiofluor 2 x 10g Cat No 3700 2000 Glyphosate test mixture Cat No 1700 0080 1 5mL RESTORE Cat No 1700 0140 for removal of
59. he column regenerant is strongly alkaline Any polymers or other materials in the HPLC pump injector needle seat and detector must be compatible For example the standard rotor seal in Rheodyne injector valves is Vespel polyimide which is not recommended at pH 2 9 a Tefzel or PEEK rotor seal must be installed e The PCX5100 contains an integral back pressure regulator for the detector waste line The pressure rating of the detector flowcell must be gt 110 psi 7 5 bar If your detector flowcell is rated lower consult Pickering Laboratories e Because the OPA reaction is extremely sensitive the HPLC system must be thoroughly clean before using it with the PCX5100 Pay special attention to the cleanliness of eluant reservoirs and delivery tubings Space requirements for the entire HPLC system are determined by the brand of HPLC pump and detector in use Minimum benchtop space required for a standard system is approximately 5 feet 150cm long by 2 feet 60cm deep In addition to the outlets required for the HPLC system one grounded outlet will be needed Nitrogen helium or argon in order of preference is required to pressurize the reagent reservoirs The PCX5100 requires gas pressure of 45 75 psi 3 5 bar at the gas inlet An adaptor from the gas regulator to 1 8 inch OD tubing is required To minimize oxidation of the OPA reagent use oxygen impermeable tubing for the entire gas supply line metal or Saran The user will n
60. he module is reset 2 the pump switch is ON 3 the reactor is within 2 of its set temperature and 4 the column heater is within 2 of its set temperature This lamp shows the state of the ready relay outputs on the rear panel 2 4 PCX 5100 User s Manual Chapter 2 Left Panel The liquid connections to the pressure switch are labelled From LC Pump and To Injector The pressure switch is part of the safety interlock system The maximum pressure rating for this switch is 4 500 psi 310 bar The switch requires 500 psi 34 bar before the module can be reset Ke Caution Set the maximum pressure of the HPLC to no more than 4 500psi 310bar Lae e The liquid connection From Injector is also the pre column filter The filter element is a 0 5um frit Cat No 3102 9047 e Aremovable panel gives access to the electrical connections of the reagent pump and the thermostatted reactor and the flow adjustment knobs of the post column pump It is removed by loosening the two captive thumb screws along the bottom edge FROM INJECTOR TO INJECTOR FROM LC PUMP Thumb screw Figure 2 2 Sc Q 2 5 Right Panel PCX 5100 User s Manual Chapter 2 Gas In fitting is where inert gas enters the system for pressurizing the reagent reservoirs The internal gas regulator requires 45 75 psi 3 5 bar to function properly Gas is controlled by the toggle valve Lever UP pressurizes the manifold The
61. ilter is clogged replace the frit Guard column is clogged replace it Worn HPLC pump seal or worn injector rotor seal Unfiltered samples Particulate matter in eluant reservoirs Post column pressure is high Column is damaged replace it Organic solvent in glyphosate column wash column Noisy baseline check for Is there a pattern or rhythm in the noise Match the frequency of the noise to one of the pumps The Pickering pump has a 2 second period Most HPLC pumps have a period of 5 30 seconds The problem is related to the pump with the matching frequency If the noise is random check your detector If the background signal is also elevated check for chemical contamination or an error in formulation OPA reagent is too old or oxidized Reagent pump stops or delivers wrong flow rate Check pump setting Check reagent pressurization Lubricate pump LDC Minipump only Check pump seal for leakage Do not open the restrictor It is supposed to be full of gray green powder Peaks disappear or diminish Carbamates 1 All disappear except 1 naphthol and carbaryl OPA reagent expired Error in preparing OPA reagent no thiol no OPA wrong pH Reagent 2 pump air locked All disappear except 1 naphthol Out of Hydrolysis Reagent Reagent 1 pump air locked Some peaks small or missing others normal size Reactor at wrong temperature Mis adjusted reagent pumps Error in preparing a reagent All peaks diminish
62. in access to it There is space for one analytical and guard column The heater block is slotted to receive the analytical column e The last part of the lead in capillary is embedded in the heating block to preheat the eluant for a more uniform temperature within the column The lead in capillary is 0 007 inch ID to minimize loss of efficiency Warning The column heating block may become hotter than 70 C For your safety wear insulating gloves when the column oven is warm Attention La r sistance chauffante de la colonne peut d passer une temp rature de 70 C Pour votre s curit priere de porter des gants isolants lorsque le four de la colonne est chaud Warnung Der Heizblock des S ulenofens k nnte heiBer als 70 C werden F r Ihre Sicherheit sollten Sie isolierende Handschuhe tragen wenn der S ulenofen warm ist Atenci n El bloque calefactor de columnas puede estar por encima de 70 C Para su seguridad use guantes aislantes cuando el horno de columnas est caliente Avvertimento Il blocco della colonna potr diventare molto caldo e superare ai 70 C Per la sua protezione usa guanti con insulazione per questa applicazione Reagent Reservoirs PCX 5100 User s Manual Chapter 2 The PCX5100 includes two pressurized reagent reservoirs The one with TFE tubing on the cap is used for either Hydrolysis Reagent or Oxidizing Reagent The one with tan colored Saran tubing on the cap is used for OPA Reagent Warning For
63. injecting the carbamate sample for analysis Step 1 Turn on the HPLC system detector injector and data system Start the HPLC pump with 100 methanol If the 15 cm carbamate column is installed the following readings should be observed on the PCX5100 instrument and LC system The LC pump pressure will be higher if you are using the 25 cm column Instrument Function HPLC pump delivery HPLC flow column heater temperature post column reactor HPLC pump pressure post column system pressure reagent 1 pressure reagent 2 pressure Reading 100 B methanol 1 0 mL min OFF OFF 850 1050 psi 180 190 psi 100 psi 100 psi Step 2 Ifthe above readings are displayed set the HPLC system to deliver 8296 water and 18 methanol After approximately 10 minutes the PCX5100 instrument and HPLC system should display the following readings The 82 water 18 methanol mixture has a higher viscosity than methanol alone and will result in higher pump and system pressures The following conditions are typical of a system without post column reagent flow Instrument Function HPLC pump delivery HPLC flow column heater temperature post column reactor HPLC pump pressure post column system pressure reagent 1 pressure reagent 2 pressure Reading 82 A water 18 B methanol 1 0 mL min OFF OFF 1800 2200 psi 300 325 psi lt 100 psi lt 100 psi Step 3 Turn ON the main power switch The lights on the t
64. instrument when used with an HPLC binary gradient pump fluorescence detector and recorder or integrator will meet or exceed EPA requirements High sensitivity detection limits of 0 1 0 5ng or 0 2 1ppb levels for drinking water can be routinely achieved Selectivity specificity only N methylcarbamates and N methyl carbamoyloximes plus components reactive to OPA under the specified operating conditions are detected Minimum sample preparation drinking water can be directly injected into the HPLC after filtration No pre extraction or sample cleanup is required e The analysis is easily automated for unattended analyses with the addition of an autosampler O t OH na Ho ge CHaNHp RLOH CO RO NH 2 KINAG 3 100 C Carbamate SCHoCH3N CH3 2 CHO CH3z 9 NCHo2CH2SH ZA C Pp CH3 OPA Fluorescent Isoindole Figure 1 3 Glyphosate PCX 5100 User s Manual Chapter 1 Glyphosate N Phosphonomethylglycine Rodeo Roundup is a broad spectrum herbicide Its wide use in agriculture can result in its presence in ground water A sensitive analytical technique has been developed to monitor levels of glyphosate and its principal metabolite aminomethylphosphonic acid AMPA This method is an improved version of USEPA Draft Method 547 AMPA Glyphosate E RME 5 1 10 20 Minutes Figure 1 4 Glyphosate and AMPA are separated on a strong cation exchange column fully sulfo
65. ion eluant flow rate column heater temperature reactor temperature HPLC pump pressure post column system pressure reagent 1 pressure reagent 2 pressure Reading 82 A water 1896 B methanol 1 0 mL min 42 C 100 C 1300 1600 psi 210 250 psi max 600 1100 pulsating max 600 1100 pulsating As the OPA reagent appears in the detector you should see an increase in the baseline signal typically 50 100 When all pressures are stable and the fluorescence detector baseline is flat the system is ready for injection of a carbamate sample 4 10 Shutdown Procedures Short Term Up to 3 days J Medium Term Up to 6 days ee Long Term 7 days or more PCX 5100 User s Manual Chapter 4 Upon completion of the analyses use one of the following three procedures to shut down the PCX 5100 system properly These procedures can prevent potential column damage reaction coil blockage high background fluorescence reagent precipitation or other problems Turn off the PCX 5100 either manually or via the Remote Off function e Set the HPLC pump at 1 mL min of methanol to flush the system until the detector baseline drops e Set the HPLC pump to lt 0 1 mL min methanol e Turn off the detector lamp You may also program a slowdown method to accomplish all the above steps Step Time min MeOH Flow mL min 0 0 100 0 02 1 5 100 0 02 2 5 1 100 1 3 15 100 1 4 15 1 100 0 02 Note The automatic va
66. ion of part or all of the equipment Do not proceed beyond a caution sign until the indicated conditions are fully understood and met BYS 1 PCX 5100 User s Manual Before You Start The following symbols appear on the PCX5100 or its accessories This warning sign denotes a hazard It calls attention to a procedure practice or the like which if not correctly done or adhered to could result in injury or loss of life Do not proceed beyond a warning sign until the indicated conditions are fully understood and met Ce symbole est un signal de danger II indique qu une manipulation si elle n est pas respect e ou effectu e correctement risque d entrainer des blessures voir la mort Dieses Warnsymbol kennzeichnet eine Gefahr Es macht aufmerksam auf einen Vorgang eine Handhabung oder ein Vorhaben die bei unkorrektem Befolgen der Vorschriften zu einer Verletzung oder einer lebensgef hrlichen Situation f hren k nnen El signo de atenci n indica un riesgo Requiere atenci n sobre un procedimiento pr ctica o similar que si no se ejecuta correctamente o se sigue minuciosamente prod a producir heridas o muerte No contin e a partir de un signo de atenci n hasta que no se hayan entendido y alcanzado completamente las condiciones indicadas Questo avvettimento informa del pericolo Molto attenzione riguardante il moto di usare questa macchina molto inportante altrimenti risulter danni e anche morte Non continuare di pi
67. l wall outlet and electrical code The 120V version comes with a standard North American cord set The 240V version comes with a cord set used in much of continental Europe France Germany Benelux etc or your local reseller may have provided the correct local cord set If your local power outlets are different you will need to obtain the appropriate grounded cord set e The main power switch is located in the power connector assembly e The fuse holder is located in the power connector assembly To change the fuse first remove the power cord from the connector Carefully pry out the fuse clip with a small screwdriver Replace with the specified type fuse For 120V systems PCX5100 use a fast acting 3A 250V 5 x 20mm fuse type GMA3 Cat No 3543 0045 For 240V systems PCX5102 use a fast acting 1 6A 250V 5 x 20mm fuse meeting IEC127 specifications Cat No 3543 0044 Warning Ensure that the power cord is disconnected before replacing a fuse Use only the specified type fuse Attention Assurez vous que le cable secteur n est pas connect avant de changer un fusible Warnung Sicherungen d rfen nur bei nicht angeschlossenem Netzkabel ersetzt oder gewechselt werden Cuidado Aseg rese que el cable de red est desconectado antes de instalar o cambiar un fusible Attenzione Assicuratevi che il cavo di alimentazione sia scollegato prima di installare o sostituire un fusible Waarschuwing Zorg dat de voedingskabel
68. losgekoppeld is voordat een zekering wordt geplaatst of vervangen Avvertimento Fare atenzione che la corda del voltaggio sia staccata prima di cambiare valvole Usa solo valvole di capacit precisata dalla fattoria 2 8 PCX 5100 User s Manual Chapter 2 1 fuse for 120V systems T 2 fuses for 240V systems Ce Fuse Clip Figure 2 4 2 9 Column Oven PCX 5100 User s Manual Chapter 2 The ready relay and remote off circuits connect via the removable terminal block From left to right the terminals are 1 Ground 2 Remote off input 3 Ready out relay normally closed RDY NC 4 Ready out relay common RDY C 5 Ready out relay normally open RDY NO Remote Off Ground e The remote off input when momentarily grounded will turn off the reagent pump column oven and heated reactor It can be actuated by a relay contact closure TTL logic or other device capable of sinking 100uA to ground The maximum input voltage Figure 2 5 is 10V If held to ground the remote off will prevent the Reset button from working e The ready out relay actuates when the module becomes ready When the module is not ready the normally closed circuit is connected to the common when the module is ready the normally open circuit is connected to the common The relay contacts are rated at 0 5A and 100 VDC Column oven is located on the upper right side of the instrument Simply lift the hinged lid to ga
69. ls 2 x 10g 1700 0080 Glyphosate test mixture containing 2 5ug mL each glyphosate amp AMPA 1 5mL 1700 0140 RESTORE for removal of metal ion contamination from glyphosate ion exchange column and guard 250mL 2 1 PCX 5100 User s Manual Chapter 2 Getting to Know Post column pressure gauge measures the liquid pressure at the first mixing tee This Your PCX 5100 is effectively the pressure on the heated reactor This gauge indicates pressure when Front Panel liquid is flowing through the system Reagent 1 pressure gauge measures the pressure of the reagent at the outlet of the reagent pump This gauge is an integral part of the pulse dampening system When the reagent pump is on the needle swings over a range of about 200 psi in time with the pump piston This gauge is downstream of the reagent filter and upstream of the bypass valve and restrictor PICKERING LABORATORIES POWER PCX5100 POST COLUMN REACTION MODULE l 1500 N Y 1000 2000 POST COLUMN REAGENT 1 REAGENT 2 PRESSURE PRESSURE PRESSURE GAS PRESSURE BYPASS 1 BYPASS 2 Figure 2 1 2 2 PCX 5100 User s Manual Chapter 2 Reagent 2 Pressure gauge operates the same as the Reagent 1 Pressure gauge Gas Pressure gauge shows the pressure of inert gas supplied to the gas manifold In normal operation it shows 2 5 psi Bypass 1 valve is used to purge and prime the first reagent pump Attach the 20mL syringe to the Luer fitting in the center
70. lves prevent reagents from back flowing onto the column The inert gas should be left on to preserve the OPA reagent e Turn off the PCX 5100 either manually or via the Remote Off function e Set the HPLC pump at 1 mL min of methanol to flush the system until the reactor temperature drops below 60 C This takes about an hour e Disconnect the outlet of the detector at the From Detector fitting relieving pressure on the post column system Relieve the pressure in the reagent gauges by briefly opening the bypass valves e Let the system drain for 1 2 minutes e Turn off the fluorescence detector and HPLC pump Caution The medium term shutdown should be performed prior to any work on the HPLC or PCX 5100 Failure to do so could defeat the safety systems e Set the HPLC to pump methanol at 1 mL min Turn off the reagent pump Set the reactor temperature to lt 60 C Turn off the gas at the toggle valve and vent the reservoirs Replace both reagents with water and draw 10 mL through each prime purge valve Replace the water with water methanol 1 1 Turn the reagent pump on and flush the system until the temperature of the reactor has fallen below 60 C Turn off the PCX 5100 Relieve the pressure in the reagent gauges by briefly opening the bypass valves Let the system drain for 1 2 minutes Turn off the inert gas source Turn off the HPLC system Remove the column and guard column and plug them When removing th
71. m moving Replace the access panel Relocate the reservoir and turn on the gas Start the post column system and prime the pump Follow steps 1 4 from the Cleaning Check valves section To prevent the internal components of the valve from falling out upon removal keep the pump head in its normal position when removing the inlet check valve turn the pump head upside down when removing the outlet check valve Remove the check valves with a 1 2 wrench counter clockwise After removal keep the valve oriented so the translucent washers faces upwards Unscrew the shipping nut and remove the metal shipping washer Insert the new inlet check valve into the pump head and tighten by hand until just finger tight Tighten with a 1 2 wrench 1 8 to 1 4 turn more Do not overtighten The sapphire seats may crack Turn the pump head upside down insert the new outlet check valve into the pump head and tighten by hand until just finger tight Tighten with a 1 2 wrench 1 8 to 1 4 turn more Do not overtighten Follow step 7 from the Cleaning Check valves section 5 9 PCX 5100 User s Manual Chapter 5 Rebuilding Step 1 Remove the check valve cartridge from its housing Figure 5 4 Check valves Step 2 Insert the smaller dowel pin provided 1 8 od x 1 1 4 long into the hexagonal end of the valve and press out the internal components of the check valve assembly using a steady pressure Do not hammer parts through with the dowel pin or hamm
72. mables and spare parts available Carbamate Reagents Part Number Description O120 o Phthalaldehyde Chromatographic Grade crystals 5 g 3700 2000 Thiofluor Chromatographic Grade crystals 10 g CB910 OPA Diluent for Carbamate Pesticide Analysis 4 x 950 mL CB130 Hydrolysis Reagent for Carbamate Pesticide Analysis 4 x 950 mL 1700 0063 Carbamate Test Mixture qualitative sample 12 components 1 5 mL 2 5 ug mL 1700 0132 ChlorAC Buffer for preservation of aqueous carbamate samples 250 mL Glyphosate Reagents Part Number Description O120 o Phthalaldehyde Chromatographic Grade crystals 5 g 3700 2000 Thiofluor Chromatographic Grade crystals 10 g GA104 OPA Diluent for glyphosate analysis 4 x 950 mL GA116 Hypochlorite Diluent for glyphosate analysis 4 x 950 mL K200 Eluent for glyphosate analysis 4 x 950 mL RG019 Column Regenerant for glyphosate analysis 4 x 950 mL 1700 0080 Test mixture 2 5 ug mL each glyphosate and AMPA 1 5 mL 1700 0140 RESTORE for removal of metal ion contamination from glyphosate column and guard 250 mL Columns amp Guards Part Number Description 0840250 C Carbamate column 4 0 mm ID x 250 mm 1846150 C Carbamate column 4 6 mm ID x 150 mm 1846250 C Carbamate column 4 6 mm ID x 250 mm 18ECGO002 Replacement Carbamate Guard Cartridges Qty 2 1954150 Glyphosate column 4 0 mm ID x 150 mm 1953020 Glyphosate guard column 3 0 mm ID x 20 mm App B 1 PCX 5100 User s M
73. metal ion contamination from guard amp column 3 2 Unpacking Installation of the PCX5100 Layout of the HPLC with the PCX5100 Note about fittings PCX 5100 User s Manual Chapter 3 The PCX5100 instrument is shipped in two cartons Report any carton damage to the carrier Unpack both cartons and review the contents using the Packing List to ensure that your order is complete If any items are missing immediately contact Pickering Laboratories at 415 694 6700 or by fax at 415 968 0749 Complete and mail the warranty registration included in this manual so that you will receive instrument and method updates from Pickering Laboratories The PCX5100 system flows left to right The connection to the LC pump and injector are on the left The connections to the detector are on the right The connections for the gas lines and reagent lines are on the right You will need ca 4 inches 10cm clearance on either side of the PCX5100 to make these connections The column oven is in the upper right side of the instrument the oven door swings up about 5 inches 12cm HPLC Fluorescence Autosampler Detector Figure 3 1 The PCX5100 uses several styles of fittings The external high pressure fittings are all 10 32 x 1 16 inch Upchurch style These fittings are compatible with Valco Parker CPI Swagelok or various of the polym
74. mp head or reagent gauge syringe suction may be applied Draw liquid until no bubbles come through Step 4 Close the valve remove the syringe and wash the Luer fitting with a little water Step 5 Repeat the process for the other valve If priming the reagent pump is difficult see Chapter 5 Troubleshooting Guide p 5 16 Turn on the PCX 5100 Setting up the Fluorescence Detector Setting up the Data Station or Integrator Setting up the HPLC Pump Method PCX 5100 User s Manual Chapter 4 Step 1 The HPLC pump should be on and pumping K200 at this time If not turn on the pump and wait until at least 500 psi 35 bar of pressure develops Step 2 Turn on the main power at the rear of the PCX 5100 The temperature controllers should turn on Push the Pump switch to OFF so that the orange stripe is not visible If you are using the optional Remote Off circuit make sure it is in the open or logic 1 state Press the Reset button The Reset lamp illuminates Step 3 Check that the column temperature setting is 55 C and the reactor temperature setting is 36 C Press the button on the corresponding temperature controller to view the setpoint and release it to show the actual process temperature see page 2 3 Step 4 Turn on the Pump switch The two reagent gauges should begin pulsing with a maximum of about 600 1000 psi Note The pulsating pressure readings of the reagent pumps approximately 500
75. n system Operation of the instrument with the top cover off will expose hazardous live voltages Le d montage du systeme poste colonne ne doit tre effectu que par du personnel qualifi S assurer que le cable lectrique est d branch avant de d monter le systeme Danger d lectrocution en cas d utilisation du syst me sans couvercle Warnung Reparaturen die nur mit Eingriffen in das System durchgef hrt werden k nnen sollten ausschlieBlich durch ausgebildete Servicetechniker durchgef hrt werden Stellen Sie sicher daB vor dem Offnen des Geh uses das Ger t vom Netz getrennt wird Die Inbetriebnahme des Ger tes mit offenem Geh use kann lebensgef hrlich sein Atenci n El desmontaje del sistema post columna s lo puede ser efectuado por personal cualificado Aseg rese que el cable de alimentaci n est desconectado antes de desmontar el sistema post columna La operaci n del instrumento sin la cubierta superior expondr a voltajes activos peligrosos Avvertimento Disunire il sistema della colonna dovrebbe essere fatto solamente da personale technico Deve essere sicuro che questa macchina sia spenta e il voltaggio staccato prima di disunire la colonna e altri pezzi Usare questa macchina senza essere completamente chiusa pericoloso perch il voltaggio in posizione che facilmente viene in contatto con il technico 6 1 PCX 5100 User s Manual Chapter 6 Removal of the column oven will require removal of the outer
76. nated cross linked polystyrene mixed K H form The eluant is a phosphate buffer with K 0 05M and H 0 10M After isocratic separation the column is regenerated with dilute KOH then re equilibrated with eluant Figure 1 4 Fluorometric detection follows a two stage post column reaction In the first stage glyphosate is oxidized by hypochlorite to glycine In the second stage glycine reacts with o phthalaldehyde and Thiofluor or 2 mercaptoethanol at pH 9 10 to produce a highly fluorescent isoindole AMPA does not need the initial oxidation to react with OPA Figure 1 5 indeed oxidation reduces its fluorescent yield OC NH ZPO ocr M ON Glyphosate Glycine Thiofluor SCH CH N CH CHO CH NCH CH SH Z 2 CHO R NH EES OPA Fluorescent Isoindole Where R CH COC for glycine CH PO for AMPA Figure 1 5 Post column Hardware PCX 5100 User s Manual Chapter 1 At its minimum a post column reaction instrument consists of a pulse free reagent pumping system a mixer to combine the flows of reagent and eluate and a continuous flow reactor To perform the carbamate and glyphosate procedures you need two post column systems in series The Pickering design Figure 1 6 uses a single piston reagent pump to deliver the reagent Pulses are eliminated by the combination of a gauge followed by a packed bed restrictor The pulses are absorbed by the mechanical action of the Bourdon tube inside the ga
77. nes dangling without a reservoir because this will cause fluorescent contamination Note Ensure that the reagent outlet and gas lines connected to the OPA reservoir are Saran amber color Saran tubing is necessary because of its low permeability to oxygen Step2 Place the small tray in a convenient location and put the reservoirs in it Note The reagent reservoirs are specially coated with a protective polymer to ensure operator safety if the reservoirs should become over pressurized Non coated bottles must not be substituted in the PCX5100 system Replacement or 2 L reagent reservoirs may be ordered directly from Pickering Laboratories Step 1 Install the HPLC fluorescence detector referring to the manufacturer s manual supplied with the instrument Step 2 Connect the inlet of the detector flowcell to the bulkhead union on the right side of the instrument labelled To Detector Use 1 16 inch x 0 010 inch ID capillary tubing Note to Hewlett Packard 1046A end users Replace the 0 12mm ID inlet tubing red and heat exchanger from the left side of the detector to the flowcell behind the front panel of the detector with a 0 25mm ID tubing HP Cat No 79881 67302 or Pickering Cat No 3110 6045 blue tubing to reduce the back pressure Step3 Connect a 1 16 inch x 0 020 inch ID tubing from the outlet of the flowcell to the fitting labelled From Detector using a 1 4 28 nut with a 1 16 inch reversed ferrule Caution The internal r
78. nnect the outlet of the guard column to the inlet of the analytical column Step 4 Loosely fit the inlet of the guard column to the eluent heat exchanger Carefully lay the analytical column into its slot in the heating block Tighten the connections Step5 Aloose end of tubing in the column oven leads to the first mixing tee Attach the loose end to the outlet of the analytical column Step 6 Replace the cover plate Analytical column 15cm CXXS HO Analytical column 25cm PEEK tubing Column heater block Eluent pre heat Guard column Figure 3 3 3 6 Reagent Pump Piston wash NEM SE Eu mma System 3 PCX 5100 User s Manual Chapter 3 Off wm f 2 From Heat exchange Tubing Flow To Column Figure 3 4 Direction of Carbamate guard column The reagent pumps have been calibrated to 0 30 mL min at the factory and should not need further adjustment Important Before starting the post column pump connect the piston wash system as illustrated in Figure 3 5 Flush periodically with 80 20 water methanol 4mL each time at least twice a day The piston wash system is designed to flush the back end of the primary seal which significantly extends seal life However if a pump with a piston wash system is used without liquid in the piston wash system the secondary seal will wear out quickly because it is dry It can then scratch
79. o the bulkhead fitting Reagent 2 and connect the Hydrolysis or Oxidizing reagent to the bulkhead fitting Reagent 1 Use 1 4 28 nuts and reversed ferrules e The reservoir cap has a built in vent valve The large white knob is the valve pull it up for CLOSED and push it down for OPEN If the gas is turned on opening the vent valve will sparge the reagent Closing the valve will pressurize the reservoir this is the normal operating position In the center of the knob there is a 1 4 28 fitting you may optionally connect a tube here to carry vapors to an exhaust vent When changing reagent first turn off the gas with the toggle valve on the PCX5100 Then vent the reagent bottle by pushing down the valve Now you can safely remove the cap It is convenient to have extra bottles so that you can simply transfer the cap without setting it down and risking contamination PCX 5100 User s Manual Chapter 2 Clear reagent tubing with shut off valve Gas REAGENT REAGENT Figure 2 6 Specifications Wetted Materials PCX5100 PCX5110 Ratings PCX 5100 User s Manual 316 Stainless steel Teflon FEP Saran PVDC Hastelloy C Kel F CTFE PEEK Borosilicate glass Sapphire UHMW polyethylene 316 Stainless steel Viton Teflon FEP Saran PVDC Kel F CTFE PEEK Tefzel ETFE Borosilicate glass Sapphire UHMW polyethylene Dimensions 32 cm x 27 5 cm x 45 cm hx w x d Weight 15 kg Electrical Power 100 120 V
80. of the knob Open the valve by turning it counterclockwise about one turn Apply suction with the syringe to draw reagent through the pump Use strong suction to remove bubbles from the Reagent 1 Pressure gauge from the reagent pump or from the reagent supply line Close the valve by turning it clockwise only gentle pressure is needed to close the valve Keep the Luer fitting clean by rinsing it with water after use Bypass 2 valve is used to purge and prime the second reagent pump and it operates the same as Bypass 1 valve Reset button enables the controls for the heated reactor column oven reagent pump and ready relay The interlock system requires the pressure switch to sense over 500 psi before the PCX5100 can be enabled This means that the LC pump must be running before pressing Reset can start the PCX5100 The Remote Off circuit on the rear panel must also be open if connected to the LC When reset the corresponding indicator lamp lights e Pump switch controls power to the reagent pump The ON position is indicated by a visible orange stripe To power the pump the switch must be in the ON position and the reset indicator lamp must be ON The pump indicator lamp lights when the pump is ON e Temperature Controllers The four digit display normally shows the process temperature to the nearest degree It also displays the temperature setpoint or the Option Function list There are four buttons below the digi
81. on the internal volume of your HPLC When the baseline and column pressure are stable for two minutes the column has been re equilibrated PCX 5100 User s Manual Chapter 4 Post column Reagent 1 100 uL of 5 NaOCl Clorox in GA116 Diluent Conditions Pump 1 0 30 mL min Reactor 1 500 uL at 36 C Reagent2 o Phthalaldehyde and Thiofluor in GA104 borate buffer Pump 2 0 30 mL min Reactor 2 100 uL at ambient temperature The First Allow the column to equilibrate for about 20 minutes under initial conditions Inject Chromatogram 10uL of Glyphosate Text Mixture and collect the first chromatogram Shutdown Upon completion of the analyses use one of the following three procedures to shut down Procedures the EG5100 system properly These procedures can prevent potential column damage reaction coil blockage high background fluorescence reagent precipitation or other problems Short Term Turn off the PCX 5100 either manually or via the Remote Off function Upto3 days Continue the HPLC pump at 0 40 mL min of K200 to flush the system until the detector baseline drops e Set the HPLC pump to lt 0 1 mL min K200 so that the column pressure is below 500 psi 35 bar e Turn off the detector lamp You may also program a slowdown method to accomplish all the above steps Step Time min K200 Flow mL min 0 0 100 0 02 1 5 100 0 02 2 5 5 100 0 25 3 15 100 0 25 4 15 1 100 0 02 3 Note The automatic valves prevent reagents f
82. or and the inlet lines of the PCX 5100 with K200 and RG019 for at least 30 min at gt 1 mL min without the glyphosate column and guard attached Do not allow methanol into the glyphosate column Change the reagents from CB130 and CB910 to GA116 and GA104 The buffering capacity of CB910 is inadequate to neutralize K200 so you must use GA104 Turn off the HPLC pump Install the glyphosate column and guard Change HPLC program and start the HPLC pump to a maximum of 0 4 mL min of K200 Turn on the PCX 5100 and press the Reset button Immediately before the reactor can heat up lower the temperature setting from 100 C to 36 C Set the column temperature to 55 C Prime the reagent pumps by drawing 10 20 mL through the bypass valves Start the reagent pumps Allow the system to equilibrate and flush itself for at least one hour before using it to collect data Perform the medium term shutdown Remove the glyphosate column and guard column and plug them When removing the column disconnect the outlet fitting first Change the HPLC eluants from K200 and RG019 to water and methanol Flush the HPLC pump injector and the inlet lines of the PCX 5100 with methanol without the carbamate column and guard attached for at least 30 min Do not allow either of the glyphosate eluants onto the carbamate column Turn off the HPLC pump Install the carbamate column and guard Change HPLC program and start the HPLC pump Turn on the PCX 5100 and press
83. or modifications incidents Crystal Ball Preventive maintenance saves more time in the long run Buffer Rule Remove buffers from LC when not in use General Procedure for Troubleshooting Examine the system front to back Fix all leakages Verify that all settings eluants reagents valves etc are according to specifications Have there been any changes in the system Compare against reference conditions standard sample column parameter log as appropriate e Gather information observations manuals books technical assistance Test your conclusions about the nature of the problem e Start working The most useful diagnostic tool is a pressure log Note that it is important to record all four pressures under initial conditions Each permutation indicates a specific problem Condition Column Post Column Reagent 1 Reagent 2 Normal 1200 250 1500 1500 Pre column filter blocked T SS Heated reactor obstructed T T T x Ambient reactor obstructed T T T 7 Reagent 1 not pumping 4 y Reagent 2 not pumping l y Restrictor 1 blocked T Restrictor 2 blocked E T Most Common Problems with Post column PCX 5100 User s Manual Chapter 5 High post column pressure caused by obstruction of flow path by deposits over tightened fittings pinching a Teflon tube closed obstruction of detector flowcell heat exchanger in detector is too restrictive defective back pressure regulator High ba
84. orth of your analytical results The one year warranty does not cover damage caused by poor quality reagents and eluants not purchased from Pickering Laboratories Use the proper start up and shutdown procedures consistently see Section 4 Frequently observe the pressures and check for leaks You should be able to identify a problem before it becomes serious Keep a daily log of the four pressures Avoid touching the interior of the mobile phase reservoirs and the dip tubes with your fingers Amino acids in fingerprints will cause contamination Gloves are suggested Do not leave caps and lines dangling without a reservoir To fill reservoir transfer caps and lines into a spare bottle or an Erlenmeyer flask filled with deionized water Use HPLC grade methanol and water Fisher Scientific JT Baker or Merck for carbamate analysis to avoid problems with baseline drift spurious peaks and noise Use bottled HPLC grade water if possible Fisher Scientific JT Baker or Merck especially during the initial system start up If water from a water purification system is used ensure the system has an activated charcoal unit to eliminate organics and that the charcoal cartridge is placed after the ion exchange cartridges Many ion exchange resins leach out OPA positive contaminates that cause unacceptable fluorescence background The water in the solvent reservoir should be changed every 3 to 4 days to prevent possible bacterial growth Avoid purging
85. out the parameter log for your initial conditions if they are different than the Pickering standard conditions Record all the LC settings for your method Keep copies of these chromatograms and logs for future use We suggest posting this information near your instrument 5 1 Post Column Band Spreading Test PCX 5100 User s Manual Chapter 5 This test measures the amount of increased band spreading due to the post column system It exploits the fact that 1 naphthol is fluorescent without derivatization The general scheme is to analyze the test mixture with the post column reagents turned off First test with the column connected directly to the detector bypassing the PCX5100 second test with the column effluent flowing through the post column reactors This test is best performed on a system that has been shut down and at least partially cooled down A normal carbamate analyzer works for this test If you have a glyphosate analyzer you will need to change the eluants and column Step 1 Conditions Isocratic program 40 water 60 methanol Flow rate 1 0 mL min Sample 10uL of Carbamate Test Mix 1700 0063 Detector excitation 330nm emission 465nm The integrator or data system should be set up similarly to normal conditions but the fluorescence of the 1 naphthol will only be 20 as much as the same amount under post column conditions the alkaline PC conditions enhance the native fluorescence Step 2 You need to decide on a
86. pecification Sp cification du fusible Spezifikation der Sicherung Fusible Valvole Specificazioni Protective Ground Prise de terre Erdung Masa de protecci n Protezioni a terra BYS 4 Before You Start Post column Analysis Chapter 1 Introduction High performance liquid chromatography HPLC with post column derivatization is a technique for rendering analytes more detectable than they would otherwise be in their native forms Post column derivatization can give improved sensitivity or better selectivity reduction of interference leading to lower detection limits The Pickering Laboratories PCX5100 was developed to facilitate the determination of carbamate insecticides 5um Cig column meeting or exceeding performance requirements for precision and accuracy of U S Environmental Protection Agency USEPA Method 531 1 and the AOAC International Protocol 29 405 and the herbicide glyphosate Bum sulfonated cation exchange column meeting or exceeding performance requirements for precision and accuracy of USEPA Draft Method 547 In addition there are a number of carbamate pesticide compounds employed worldwide which are not included in the 10 compounds mandated by USEPA Method 531 1 and AOAC Protocol 29 405 The Pickering Laboratories PCX5150 5um Cg column can separate as many as 23 compounds The Cg column can also be used as a confirmation column when using a water acetonitrile gradient instead of a water methanol gradient
87. pump Ordinary priming will not always remove the bubble especially if it is caught inside the piston seal or inside one of the gauges There are two ways to overcome this The first and simplest way is to first prime the pump with a low surface tension liquid then change to the normal reagent Degassed methanol will work well Simply use the priming syringe to draw about 5 mL of methanol through the pump then draw 10 mL of reagent through the pump If there is air in a gauge the pump may take several minutes to come up to final pressure The second and more thorough method is vacuum priming You will need a liquid shut off valve Pickering 3104 0050 or Omnifit 1101 or equivalent to perform this Newer PCX5100 April 94 has built in liquid shut off valves Start with Step 2 a Disconnect the reagent reservoir and install the liquid shut off valve in the reagent supply tubing b This can be performed with the pump on or off it makes little difference c Connectthe 20 mL priming syringe to the bypass Open the Bypass valve Draw liquid until no bubbles come through d Close the liquid shut off valve Empty the syringe and reconnect it to the bypass e Pull a vacuum with the syringe Hold the vacuum until no more bubbles come out This causes the trapped bubbles to expand f While still holding a vacuum open the liquid shut off valve This sweeps the expanded bubbles out g Wait until about 5 mL of liquid has collected th
88. purge valve Step 7 Start the HPLC pump Pump methanol through the column and system at 1 0 mL min Continue pumping until the entire post column system is primed The column back pressure should stabilize at approximately 700 psi 50 bar for the 15 cm column or 1200 psi 80 bar for the 25 cm column The two derivatization reagents required for carbamate analysis are a hydrolysis reagent NaOH and o phthalaldehyde reagent Note During initial installation the reagent bottles lines and pump should first be cleaned and primed with methanol to reduce possible fluorescence background To prepare and pressurize the post column reagents follow this procedure Step 1 Turn off the inert gas Step 2 Thoroughly wash the two reagent reservoirs and then rinse with methanol Wipe down the dip tubes with methanol and a clean cellulose tissue Step 3 The hydrolysis reagent does not require preparation Pour the hydrolysis reagent Cat No CB130 directly into the reagent reservoir labeled Hydrolysis Reagent Hydrolysis reagent reservoir cap has TFE lines Put the cap on the reservoir Close the vent valve Note The preparation of the Hydrolysis Reagent by the user is not recommended because it is hard to obtain NaOH of adequate purity Step 4 Preparation of the OPA Reagent a Pour the contents of one bottle 950 mL of the OPA Diluent Cat No CB910 into the reagent reservoir Save approximately 5 mL for step 4e b Putthe cap on
89. r a programmed shut down after the last sample The interlock turns off the reagent pumps to prevent backflow of reagents into the column which dissolves the silica and ruins the column The interlock also defaults to OFF when a power loss occurs The PCX5100 instrument does not automatically turn on as the eluent pressure rises above 500 psi Press the RESET button to enable the instrument 1 Caution Operating the reagent pumps when the HPLC pump is not producing eluent flow can pump reagent into the analytical column causing irreversible damage 2 Maintaining high temperature in the post column reactor when there is no HPLC eluent flow can cause precipitation and complete blockage of the post column reactor 8 4 Connecting the injector Reagent Reservoirs J HPLC Detector Installation e PCX 5100 User s Manual Chapter 3 Connect the outlet of the injector or autosampler to the bulkhead fitting on the left side of the instrument labelled From Injector Use 1 16 inch x 0 007 inch ID or 1 16 inch x 0 010 inch ID capillary tubing This fitting contains a replaceable 0 5um filter element Step 1 Assume that new reservoirs have not been cleaned Wash the bottle with laboratory detergent and hot water Rinse with methanol then with deionized water Wipe down the dip tubes on the caps with methanol and a clean cellulose tissue Avoid touching the tubings or the interior of the reservoir with your skin and do not leave caps and li
90. ra 5 5 Bottles coated iniiai 2 11 3 5 C Calcium hypochlorite 4 13 Carbamate analysis ssss 4 1 Carbamate column sesssss 2 1 Garbamiales ie aie Hase rs 1 2 Cation exchange esse 1 5 Changing carbamate to glyphosate 4 18 Changing glyphosate to carbamate 4 18 Check valve Cleaning Changing 5 9 CIO usc rer ers 3 2 4 13 4 16 Coated bottles uusssss 2 11 3 5 Column carbamate coocccccncnnconconcncnnncnnnnnnnnnnnnnnnnnns 2 1 glyphosate sasssseesrreseerressrrrrnesrernee 2 1 installation sseesssseees 3 6 maintenance sssssssee 5 4 OVEN E 2 10 CONNECTIONS iniciaran 3 4 D Damage calcium hypochlorite 4 13 Deposits in reactor enesenn 5 15 Detecta tia 2 7 installation eene 3 5 SG p i ci eed 4 4 4 15 Dimethyl 2 mercaptoethylamine 4 8 4 14 E Electrical systems 3 1 5 5 Equilibration s uicti es 4 16 Index F Filter TA 2 5 5 11 reagent EEN 2 7 5 11 First chromatogram 4 4 4 16 ilic 3 3 detector ite ao itum 2 7 RECEDIT 2 6 reagent eterne tet Ie tr en nuu 2 7 Waste eiie exe 2 7 IUS soot tetra ioa hs 2 8 5 11 G ad S isa didi edt lait tare tede dg 3 1 LUDINO iue eie dd 3 4 Getting started carbamates
91. raia areata enan raa inaapi a a anna 3 5 Reagent Reservolrs iui suicide dandi E js dir E du e a O dk 3 5 HPLC Detector Installation ssssssssssssseseeeneennnne nennen nennen nennen nnn senes 3 5 Installing the analytical and guard columms esee 3 6 Reagent Pump Piston wash ssssssssssssseeeeese eene nennen nnn nnne nennen inen nnne 3 7 System Testing and Verification sss eene en nennen nsns 3 8 PCX 5100 User s Manual Contents Chapter 4 System Operation Carbamate Analysis with the EC5100 ssssssssssssssssseseee eene enne nentes nnne 4 1 Getting E Lut 4 1 HPEC Mobile Phase i eet iet e eee nim ete P e ede 4 1 Post Column Reagent Preparation sssssssssssssssseeeee enne nennen 4 2 Priming the Reagent PUMPS Seriese tenter hex bote EEE e pee td 4 3 E o ie LR EE 4 3 Setting Up the HPLC Fluorescence Detector sse 4 4 Setting Up the Data Station or Integrator enne 4 4 Setting Up the HPLC Pump Method aan ae aagana enne enne nc 4 4 The First Chromatogram nennen enhn nensis nene tenni nnns nennen nnns sen nenas 4 4 Gradient Conditions for Carbamate Analysis sss eene nnne 4 5 1846250 column 4 6 x 250 mm with aqueous samples esse 4 5 1846150 column 4 6 x 150 mm with aqueous samples sese 4 5 1846250 column 4 6 x 250 mm with methanolic samples sesseessss 4 5 1846150 column 4 6 x 150 mm with
92. ration 4 2 4 13 hypochlorite 3 2 4 13 4 16 NAO rst ni oe 25 OPA reagent ssnnssssesnneneeenenen 4 2 4 13 oxidizing reagent 3 2 4 13 4 16 Reagent reservoirs 2 11 3 5 Remote Control 2 10 Repairs malor creena seirak ra inaa 6 1 Requirements chemicals senrose s EEEE 3 2 EG System nm ede e 3 1 dire EHE 3 1 Cleri R 3 1 Reservoirs cooccccccccnoconnncncnocnnoncncnonanonnn 2 11 3 5 Reset button r sentein eike einai 2 3 Rheodyne injector secese 3 1 Routine maintenance 5 1 PCX 5100 User s Manual S Safety interlock system 2 5 Safety switch eet enis diera iedera aaen aa ae drana 2 5 Safety systems ssssssssssssss 1 6 Sample precautions s 5 5 Saran bing 2 11 3 1 3 4 Seal changing EE 5 6 Shutdown procedures 4 11 4 16 Site requirements neeeeeeeeeneeneeenee 3 1 Sodium borate s eener eresse 5 5 Sodium bwdroside 5 5 Sodium hypochlorite 3 2 4 13 4 16 Sodium tetraborate ssesseeeeee ereere 5 5 Stability OPA reagent 4 3 4 14 System carbamate analysis 1 2 glyphosate analysis 1 5 System startup carbamate 4 9 System testing sssssss 3 8 5 1 T Tetzel zeit e dra ids 3 1 Temperature
93. regulator is connected to the waste line of the detector Boiling inside the reactor causes precipitates to form Do not operate the reactor above 130 C This can weaken and deform the PTFE tubing Do not operate with a post column pressure above 600 psi Always use the correct fuse Do not alter the programming of the two temperature controllers If you accidentally change the programming contact Pickering Laboratories for advice 5 5 Routine Maintenance Reagent Pump PCX 5100 User s Manual Chapter 5 The PCX5100 uses a custom made Eldex reagent pump with piston wash The piston seals require periodical replacement The length of service to be expected from the seal depends on a wide variety of factors including if proper shutdown procedures were followed how often the system was turned on and off and if the piston wash is wetted It is critically important that the seal be replaced immediately upon failure or better yet before failure because the reagent can leak into the mechanical housing of the pump and cause corrosion When a leak occurs you may notice fluid on the side of the pump However a leak may not always be visible particularly at slow flow rates A litmus paper can be placed up the drain slot both sides of the pump the litmus paper should be removed periodically and checked for color changes to see if leakage has occurred You will need to purchase a seal kit 3106 1310 including two seals two back up washer
94. rom back flowing onto the column The inert gas should be left on to preserve the OPA reagent Medium Term Turn off the PCX 5100 either manually or via the Remote Off function Up to 6 days Set the HPLC pump to 0 40 mL min of RG019 for 5 min Excessive flushing will require an equally excessive re equilibration when you start up again Disconnect the outlet of the detector at the From Detector fitting relieving pressure on the post column system Relieve the pressure in the reagent gauges by briefly opening the bypass valves Let the system drain for 1 2 min e Turn off the fluorescence detector and HPLC pump So Caution The medium term shutdown should be performed prior to any work on the cuu d HPLC or PCX 5100 Failure to do so could defeat the safety systems 4 16 Long Term 7 days or more PCX 5100 User s Manual Chapter 4 Turn off the reagent and HPLC pumps Turn off the gas at the toggle valve and vent the reservoirs Replace both reagents with water and draw 10 mL through each prime purge valve Replace the water with water methanol 1 1 Set the HPLC to pump 100 RG019 at 0 4 mL min Turn on the reagent pumps and flush for 5 minutes Turn off the PCX5100 and the HPLC pump Relieve the pressure in the reagent gauges by briefly opening the bypass valves Let the system drain for 1 2 min Turn off the inert gas source Remove the column and guard column and plug them When removing the column di
95. s a 5 32 hex wrench and a seal installation tool Extra seals and a preventive maintenance kit may also be ordered from Pickering Laboratories see Appendix B Additional tools needed for piston seal change are 1 4 and 1 2 open end wrench Open end wrenches can be purchased at local hardware stores Step 1 Flush the Eldex pump with water then shut down the PCX5100 and let the reactor cool Turn off the gas valve and vent the reservoirs Place them at a level below the pump to prevent siphoning of the reagents Step 2 Remove right access panel by loosening two captive screws along bottom edge Step 3 Disconnect the reagent inlet line from the bottom of the pump head it is best to repair one pump head at a time Wipe up any spilled liquid Disconnect the outlet tubing from the top of the pump head stabilize the outlet check valve with a 1 2 wrench when removing the 1 16 fitting to prevent the check valve from moving Optional but highly recommended remove the pump from the chassis by 1 loosening the two captive screws and 2 unplugging the electrical connector Step 4 Remove the pump head from the pump as follows Figure 5 1 with a 5 32 hex wrench remove the two bolts while holding the pump head against the pump housing the pump head is under spring tension Gently pull the pump head straight out from the pump housing in line with the axis of the piston Do not tilt the pump head sideways the piston may break 1 6 1 Ou
96. s and the RDY relay switches Refer to your HPLC manual for setup details Optimum conditions for most detectors are excitation at 330 nm and emission at 465 nm If your detector has a selectable time constant use about 2 seconds Prepare the HPLC data station or integrator and set up a data handling method to accept data from the fluorescence detector Initially an area method without naming peaks is adequate This method should have a peak width of about 10 seconds and data end time of about 27 minutes for the 15 cm column or a data end time of about 45 minutes for the 25 cm column Pickering Laboratories recommends various gradient conditions depending on the column and type of sample For the purposes of testing and set up use the 15 cm column with the 4th gradient on the next page Note that the exact time of equilibration depends on the internal volume of your HPLC When the column pressure is stable for at least one minute the column has been re equilibrated Allow the column to equilibrate for about ten minutes under initial conditions Inject 10 uL of Carbamate Test Mixture and collect the first chromatogram 4 4 Gradient Conditions for Carbamate Analysis PCX 5100 User s Manual Chapter 4 There are six possible gradients two for the Cig 15 cm column EC5100 two for the Cig 25 cm column EC5100 and two for the Cg 25 cm column EC5150 Two programs are for aqueous samples three programs for methanolic samples an
97. s line is dyed blue Each of these tubings has a 1 4 28 nut and ferrule already in place Plug any unused outlets of the manifold Note Two types of gas tubing are used in this system Saran and PTFE a fluorocarbon material The Saran tubing is impermeable to oxygen and is used for the OPA reagent reservoirs to prevent degradation of the OPA Fluorocarbon tubing is 1000 times more oxygen permeable than Saran tubing However Saran tubing is not compatible with NaOH Step3 Connect the reagent lines of the reservoirs to the reagent inlets on the right side of the instrument Connect the OPA reagent to port 2 Connect the other reagent to port 1 Each of these tubings has a 1 4 28 nut and ferrule already in place Important The reagent lines and gas lines are not interchangeable Step 1 Using 1 16 inch x 0 020 inch ID capillary tubing connect the outlet of the HPLC pump directly to the pressure switch on the left side of the instrument The connection is labelled From Pump Step 2 Using 1 16 inch x 0 020 inch ID capillary tubing connect the pressure switch to the inlet of the injector or autosampler Use the fitting labelled To Injector Note on usage The post column interlock monitors the eluent pump pressure and turns OFF electrical power to the post column reactor column oven and reagent pump when the eluent pressure drops below 500 psig This pressure decrease may occur due to eluent pump malfunction empty reservoirs o
98. sconnect the outlet fitting first Replace them with a tubing and unions so there are no open lines Replace the eluants with waterand draw 10 mL through the HPLC prime purge valve Replace the water with water methanol 1 1 Turn on the HPLC pump for 15 minutes Turn off the HPLC system Combination Carbamate Glyphosate Systems Changing from carbamate to glyphosate Changing from glyphosate to carbamate PCX 5100 User s Manual Chapter 4 The PCX 5100 can be used for either carbamate or glyphosate analysis To change from one to the other you will need to change the reagents column eluants and temperatures Refer to the instructions above for the details ee Please read page 3 1 for the HPLC System Requirements for glyphosate analysis The HPLC components must be compatible with high pH regenerant Because the reactor is so slow to cool this is best performed first thing in the morning after the system has been cooling off overnight Perform the medium term shutdown at the end of the day before the conversion You may change the reactor temperature to 36 C and column temperature to 55 C at this time or wait until the next day Remove the carbamate column and guard column and plug them When removing the column disconnect the outlet fitting first Remove any stainless steel inlet frits or sinkers from the HPLC reservoirs Change the HPLC eluants from water and methanol to K200 and RG019 Flush the HPLC pump inject
99. se contaminants Note The Hydrolysis reagent remains stable indefinitely The OPA reagent is sensitive to air oxidation and degrades over time The PCX5100 modular system is designed to minimize this oxidation resulting in a minimal loss of OPA reagent due to oxidation When the OPA reagent reservoir is maintained under inert gas pressure the OPA reagent maintains its activity for up to two weeks without significant loss of activity Step 1 Ensure that the reagent and gas supply tubes for the reservoirs are connected to their proper fittings on the right side of the instrument Step 2 Connect a 20 mL disposable syringe to the Luer fitting in the center of one of the prime purge valves Step 3 Open the prime purge valve 1 2 to 1 full turn CCW and let the flow exit into the syringe Step 4 To purge air bubbles from the reservoir line pump head or reagent gauge syringe suction may be applied Draw liquid until no bubbles come through Step 5 Close the valve remove the syringe and wash the Luer fitting with a little water Step 6 Repeat the process for the other valve If priming the reagent pump is difficult see Chapter 5 Troubleshooting Guide p 5 16 Step 1 The HPLC pump should be on and pumping methanol through the column at this time If not turn on the pump and wait until at least 500 psi 35 bar of pressure develops Step 2 Turn on the main power at the rear of the PCX5100 Push the Pump switch onto the of
100. sh out all organic solvents from the LC and injector Backflush both columns with K200 glyphosate eluant Use a very slow flow rate so that the back pressure does not exceed 2000 psi Keep flushing until the pressure drops Keep raising the flow rate until the pressure is normal at 0 40 mL min and 55 C Reinstall the analytical in reversed direction and guard column and test them Glyphosate peak is a doublet Add 2 4 uL Glyphosate RESTORE Cat No 1700 0140 to the sample before injections Glyphosate and AMPA peaks are late and broad due to contamination Usually only the guard column is contaminated We suggest you buy a spare guard column to minimize down time Wash guard in reversed direction with Glyphosate RESTORE a b Remove the analytical column after ensuring no residual post column pressure Reverse the guard column and pump RESTORE through the guard at 0 4 mL min for a minimum of 15 min directing the effluent to waste Pump K200 eluant through the guard long enough to displace RESTORE Reconnect the column and guard in the normal directions and restart the HPLC and post column systems PCX 5100 User s Manual Chapter 5 When Priming the Reagent Pump is Difficult Sometimes the reagent pump may be very difficult to prime This can happen after a pump has been shipped serviced stored for a long time or after putting a new bottle of reagent on Almost always this is due to a trapped bubble inside the
101. si Pump o Thermostatted Over pressure Column relief valve and Guard dL e as CH Pi ES P s j Pre column Naom OPA Filter 0 5um ES Beaker NaOC E Injector 500 psi Pressure Switch LC Pump Water amp Methanol or K200 amp RG019 PCX 5100 User s Manual Chapter 6 YG de Appendix A PCX 5100 Parameter Log Reagent Pump Calibration Pump 1 Tested weree NA DAE eE EEEN App A 1 PCX 5100 User s Manual Appendix A Analytical Conditions EG Program iue Initial Composition seeenee Initial Flow Rate Gradient Program Column notte I ard Ty pea c etie egeta ID eere e Temperature Injector Autosampler eere Sample aet aei eei E eee Pera Volume Solvent uet cenas Manual Auto Other Parameters au ted cete ete ete DOTS A te Excitation Bandit Emission Band iie nta Hange eset PME sten Lali Resp time Integrator or Data Station sess Range or Scale UNES e net Attenuation neneeese Chart Speed susus Peak Threshold VfL EE RUN IMG artesian eet haves Operator Date esse Reference App A 2 PCX 5100 User s M
102. t eene 4 16 Long Term 7 days or more 4 17 Combination Carbamate Glyphosate Systems sse 4 18 Changing from carbamate to olvphosate nnns 4 18 Changing from glyphosate to carbamate sss enne 4 18 PCX 5100 User s Manual Contents Chapter 5 Routine Maintenance and Troubleshooting Initial System Testing eerie meet tela tede n ERR ie Rb PO eR e ad 5 1 Test E lge ee ET EE 5 1 Parameter LOG tec iiec tree eet ede ven teak eves tu edi pde ced chen gena o te enki ede idu eae deed 5 1 Test Chromatogram and Parameter Log for User Defined Conditions 5 1 Post Column Band Spreading Test sssssssssssssssssseseeeee enne 5 2 Precautions and Problem PDreventton nennen nennen nmn 5 3 CA AN 5 3 Mobile Phases sis 4 d RE Nn eei 5 3 Column Maintenance and Precautions oocnncccnnonicinnccccnnnnnnanonnncnn cnn ano nn nc n nr eene entente 5 4 Sample and Standard Precautions sse nnns 5 5 Reagent Precautloris tan asc ceat ete e ee reip en tope d jeu d eed crea 5 5 Reactor Precautions rer mite ERO HV AR BRE ua ERRARE ER MESURER RR 5 5 Electrical PrecautiOris iint tecto ib ete dra Sup lr ordo 5 5 Routine Maintenance 5 6 Reagent Pump dee cte n e e e te ex e ten t dte ne t e en ee Ren 5 6 Pisto Seals tes OE eb e d ei REN eR er dm e E 5 6 Glieck ValVv6s 1 te eee egit a ie div Eo derent e e eO Fa pet 5 9 Pres Columna Ple sacas 5 11 Reagent Elle Sit ad idas 5 11 US Ce a
103. t the PCX5100 has been installed according to the directions in Chapter 3 Do not operate the instrument until it has been properly installed and you have read and understood the instructions in this section and the Material Safety Data Sheets MSDS for important safety information about the chemical reagents You may use either the 15 cm or 25 cm column but for the purposes of initial testing and training install the 15 cm column Later you may change to the other column The Pickering Laboratories carbamate analysis requires two mobile phases HPLC grade water and HPLC grade methanol You may choose to use other conditions or eluants for your routine analysis but during installation and checkout we recommend you use these standard conditions e HPLC grade methanol Fisher Scientific JT Baker or Merck must be used e HPLC grade water Fisher Scientific JT Baker or Merck or high quality purified and deionized water may be used instead For initial startup use HPLC grade bottled water to verify system performance Water from some laboratory purification systems Milli Q Barnstead etc may not be acceptable for high sensitivity carbamate analysis The age of the cartridge configuration system maintenance and quality of the feed source will determine acceptability Ensure that an activated charcoal cartridge for the elimination of organics is present in the purification system Place this cartridge after the ion exchange resin cartridg
104. tal display p V To view the setpoint press the button the display will show the setpoint in flashing numbers as long as the button is held down To change the setpoint press and hold the button and press the A button to increase or press the V button to decrease The process temperature display will return when you release the buttons 2 3 AA IS AA PCX 5100 User s Manual Chapter 2 E e Caution Do not press the p button The p button is used by the factory to program the controller Do not press the p button If you should press the p button by accident it is half way recessed and requires extra efforts to activate it press the p button a second time and the process temperature will reappear There are some auxiliary displays The green LED lamp located on the bottom right will light when heat is being applied There are three LED lamps located just to the left of the numerical display They indicate whether the process temperature is above within or below the setpoint range The red lamp is lit when the temperature is not ready The recommended maximum temperature for the column heater is 100 C A thermal safety switch shuts off the heater at ca 110 C The recommended maximum temperature for the heated reactor is 130 C Above this temperature the reaction coil begins to lose strength A thermal safety switch shuts off the heater at ca 150 C Ready lamp lights when all these conditions are met 1 t
105. the Reset button Immediately before the column oven can heat up lower the temperature setting from 55 C to 42 C Set the reactor temperature to 100 C Change the reagents from GA116 and GA104 to CB130 and CB910 Prime the reagent pumps by drawing 10 20 mL through the bypass valves Start the reagent pumps Allow the system to equilibrate and flush itself for at least one hour before using it to collect data 4 18 Initial System Testing Test Chromatogram Parameter Log Test Chromatogram amp Parameter Log for User Defined Conditions Chapter 5 Routine Maintenance amp Troubleshooting Your Pickering PCX5100 will require some routine maintenance to stay in top condition Ordinarily little maintenance is needed beyond good operating procedures The initial system testing is part of the installation process Part of this testing is to establish standard conditions so that you can return to them for diagnostic purposes in the event of later problems Set up the HPLC and the PCX5100 as recommended in Chapter 4 For carbamate analysis use the gradient for methanolic samples with either 15 or 25 cm column The sample should be 10uL of Carbamate Test Mixture 1700 0063 For glyphosate analysis the sample is 10uL of Glyphosate Test Mixture 1700 0080 These conditions are close approximations to those used by Pickering for column and instrument testing Collect two chromatograms to be sure that the system is stable and repeatabl
106. the bottle open the vent valve and turn on the gas supply Thoroughly de aerate the contents by sparging with inert gas Continue bubbling for at least 10 minutes c Dissolve 100 mg of OPA Cat No O120 in approximately 10 mL of HPLC grade methanol in a clean dry container d Turn off the gas supply and remove the cap from the bottle Add the OPA solution to the deoxygenated Diluent in the reservoir e Dissolve 2 g of Thiofluor Cat No 3700 2000 in the reserved 5 mL of the OPA Diluent and add into the reservoir 4 2 Priming the Reagent Pumps Turn on the PCX 5100 PCX 5100 User s Manual Chapter 4 Note If Thiofluor is not available pipette 1 mL of 2 mercaptoethanol Handling of 2 mercaptoethanol should be in the hood since it is volatile and has an unpleasant odor which will permeate the laboratory 2 Mercaptoethanol should be replaced with Thiofluor Pickering Laboratories brand of N N dimethyl 2 mercaptoethylamine hydrochloride a non volatile thiol salt f Replace the cap and turn on the gas flow Continue sparging for another minute Close the vent valve Gently swirl the reagent to complete the mixing Note The preparation of the OPA Diluent by the user is not recommended because sodium borate any grades contains excessive amounts of heavy metal contaminants and insoluble matter These impurities will eventually precipitate in the reactor and flowcell The one year warranty does not cover damage caused by the
107. tire system turns itself off Second check valves in the reagent delivery system prevent reagents from siphoning when the pump is off PCX 5100 User s Manual Chapter 1 Fluorescence Waste Detector O 100 psi B P D ad l Ambient 2 Reagent 2 Reactor e Bypass 2 Pressure 100uL o E No en Q Restrictor 2 Heated eate Reactor Anti siphon 500uL C3 valves Reagent 1 lt Ga Bypass 1 Pressure r aD Post Column Restrictor 1 Reagent Press Gauge E Filters 24m E 0 Reagent Cl 525psi Pump Thermostatted O Over pressure Column relief valve and Guard d eb eE 5 Pre column Naon OPA Filter 0 5um i Back ec Injector 6 500 psi Pressure Switch LC Pump Water amp Methanol or K200 amp RG019 Figure 1 6 The Pickering post column system is depicted inside the dotted box 1 7 PCX 5100 User s Manual Chapter 1 YG de System Components PCX5100 Post Column Instrument EC5100 system for carbamate analysis EC5150 carbamate expanded resolution EG5100 system for glyphosate analysis Chapter 2 The PCX5100 The PCX5100 is available for 120V or 240V operation and is shipped completely assembled calibrated and tested The PCX5100 consists of a duplex reagent pump heated and ambient reactors column heater backflow and over pressure safety devices filters and flow conditioners reagent reservoirs Saran gas tubing and other accessories The complete system a
108. tlet Check valve Piston Spring Retainer Assy o mmm 4 Compression Ring NOR CID Check valve Figure 5 1 Pump assembly with wash system Pump assembly with wash system 1 Back up Washer 2 Piston Seal 3 Wash Oylinder 4 Inlet Port of the Wash System 5 Outlet Port of the Wash System 6 O ring 5 6 PCX 5100 User s Manual Chapter 5 Step 5 Step 6 Step 7 Step 8 Step 9 Step 10 Installation Tool Retainer Assy Hooked End Blunt End 4 i Inspect the piston If the piston has scratches on the sapphire or significant corrosion on the stainless steel piston holder it should be replaced Clean any deposits on the sapphire with soapy water deionized water and then methanol If the sapphire cannot be cleaned the piston should be replaced Set the cleaned piston in a safe place Remove the retainer assembly from the pump head Insert the hooked end of the installation tool into the pump head through the back up washer and piston seal Discard the seal but the back up washer is reusable Do not scratch the walls of the seal cavity with the tool Inspect the retainer Clean the retainer if necessary If there is evidence of wear or the piston does not fit snugly in the retainer or the retainer grips the piston too tightly the retainer should be replaced This step is only necessary if there are signs of corrosion on the stainless steel piston holder
109. u affinch le condizioni e instruzioni sono completamente chiare BYS 2 PCX 5100 User s Manual Before You Start This warning sign denotes a hot surface a high temperature hazard It calls attention to a column heating block hotter than 70 C For your safety wear insulating gloves when the column oven is warm Ce symbole indique une surface br lante Il signifie que la r sistance chauffante de la colonne a atteint une temp rature sup rieure 70 C Pour votre s curit pri re de porter des gants isolants Dieses Warnsymbol kennzeichnet eine heiBe Oberfl che oder eine Gefahr durch hohe Temperaturen Es macht aufmerksam auf den Heizblock des Saulenofens der hei er als 70 C sein kann Zu Ihrer Sicherheit sollten Sie isolierende Handschuhe tragen wenn der Saulenofen warm ist Este signo de atenci n indica una superficie caliente un riesgo de alta temperatura Pide atenci n sobre un bloque calefactor de columnas por encima de 70 C Para su seguridad use guantes aislantes cuando el horno de columnas est caliente Questo avvettimento informa della temperatura molto alta che possibilmente potrebbe bruciare Molto attenzione necesaria specialmente al blocco caldo della colonna che superiore ai 70 C Per essere protetti necesario usare guanti insulanti per questa applicazione BYS 3 PCX 5100 User s Manual Power On En marche An Escendido Acceso Power Off teint Aus Apagado Spento Fuse S
110. uge and then released through the restrictor The mixing device is simply a steel tee fitting with a 0 010 inch bore The continuous flow reactor is a length of 0 011 inch ID Teflon capillary There are of course many refinements in a practical instrument First the reaction temperature may need to be controlled as is the case for hydrolysis of carbamates Elevated temperatures then require a back pressure regulator to suppress boiling inside the heated reactor The Pickering design also includes a gauge to monitor pressure at the first mixing tee which is also the pressure at the first reactor For the convenience of operation bypass valves are provided for priming or purging the reagent pumps Another refinement is the use of pressurized reagent reservoirs allowing the pump to operate more precisely at low flow rates and also provides an inert atmosphere to protect air sensitive reagents Safety systems have also been incorporated into the design Two greatest hazards to post column systems are rupture of the reactor because of the excessive pressure and the back flow of caustic reagent onto the analytical column The first hazard is managed by providing a relief valve that opens at about 525 psi 36 bar and diverts flow away from the reactor Two devices protect against reagent back flow To ensure flow through the column during operation a pressure switch upstream of the analytical column must detect at least 500 psi 34 bar or else the en
111. uld agree within 2 If not keep trying until you have three consistent runs in sequence The precision of the height and area is not important for this test Calculate the average width Wd Step 7 Turn OFF the LC pump Restore the normal post column connections Turn the LC pump back ON and press the Reset button Set the reactor to the normal operating temperature 100 C Wait for the entire system to equilibrate about 15 min 5 2 Precautions amp Problem Prevention General a AAN Mobile Phase PCX 5100 User s Manual Chapter 5 Step 8 Collect three consistent chromatograms with the effluant passing through the post column reactors Calculate the average width Wpc Step 9 The ratio of Wd Wpc should be 0 80 to 0 85 for a post column system in good condition If the ratio falls below 0 75 the PCX5100 needs either cleaning or repair If there is band spreading elsewhere in the system column injector detector the ratio will be artificially high so be certain that the rest of the system is operating properly You can also check for column degradation by comparing the historical value of Wd to the current one for that column Be sure that the conditions are consistent when making this comparison Step 10 Keep records of the test results and conditions for future comparison Use Pickering Laboratories reagents and eluants The quality of the chemicals is excellent and the cost is low relative to the w
112. ump Vv Le To Reagent 2 Pump Saran tubing Figure 6 8 Post column reagent 2 flow path up to the second mixing Tee includes anti siphon valve PCX 5100 User s Manual Chapter 6 Fluorescence Waste Detector i 100 psi B P Ambient Reagent 2 meara Bypass 2 Pressure 100uL Restrictor 2 I Heated Reactor Anti siphon 500uL valves Reagent 1 Bypass 1 Pressure Post Column Restrictor 1 Reagent Press Gauge Filters 2m 1 Reagent 525psi Pump Thermostatted Over pressure Column relief valve and Guard I Pre column Naom OPA Filter 0 5 ME Beaker NaOCl Injector 500 psi Pressure Switch LC Pump Water amp Methanol or K200 amp RG019 PCX 5100 User s Manual Chapter 6 To Column M c From Column To Heated Reactor Ambient Reactor From Heated Reactor Figure 6 9 HPLC column and post column flow path includes pressure sensing switch inlet filter first mixing Tee inlet and outlet tubings for the heated reactor second mixing Tee ambient reactor outlet from post column to detector and built in 100 psi back pressure regulator 6 16 PCX 5100 User s Manual Chapter 6 Fluorescence Waste Detector Ambient Reactor 100uL Reagent 2 Bypass 2 Pressure Restrictor 2 Heated Reactor 500uL Anti siphon valves Reagent 1 Bypass 1 Pressure MES Post Column Restrictor 1 Reagent Press Gauge E Filters 2um E mu 1 Reagent 525p
113. your safety the bottles are coated with a tough plastic film and are rated to a maximum of 15 psig 1 bar Do not use uncoated bottles Attention Pour votre s curit les bouteilles sont recouvertes d un film de plastique dur et sont calibr es un maximum de 15 psig 1 bar Ne pas utiliser les bouteilles non recouvertes Warnung F r Ihre Sicherheit wurden die Reagenzienflaschen mit einem festen Schutz berzug aus Kunststoff versehen Die Flaschen sind bis max 1 bar 15 psig zugelassen Flaschen mit besch digtem Schutz berzug d rfen nicht mehr benutzt werden Verwenden Sie keine Flaschen ohne Schutz berzug Atenci n Para su seguridad las botellas est n recubiertas con una resistente pel cula pl stica y est n constrastadas a 15 psig 1 bar No utilice botellas sin recubrimiento Avvertimento Per la sua protezione le bottiglie sono construite forti con un percentuale du plastica e sono usabili per un massimo di una Bar 15 psi Non usare bottiglie normali Gas inlet tubing on the reservoirs is marked with a black band for Saran tubing and is dyed blue for TFE tubing Just under the cap there is a pinhole drilled in the gas tubing to prevent liquid from creeping up the gas line in case of a slow leak in the gas system Connect the gas tubing to the gas manifold using 1 4 28 nuts and reversed ferrules Reagent tubing is neither marked nor dyed and has a shut off valve Cat No 3104 0050 Connect the OPA reagent t

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