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1. O Tali 7 Te E 2 6 PCX5200 User s Manual Chapter 2 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 pri re 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 Il blocco della colonna potr diventare molto caldo e superare ai 70 C Per la sua protezione usa guanti con insulazione per questa applicazione 2 7 PCX5200 User s Manual Chapter 2 Reagent Reservoirs The PCX5200 includes one pressurized reagent reservoir for the one reagent system and two for the two reagent system Warning For your safety the bottles are coated with a tough plastic film and are rated to a maximum of
2. POWER CX 5200 na COLUMN EACTOR REA psh es f PICKERING PUMP TEMP TEMP i LABORATORIES PA et POX 52 Ber eo SES PostColumn Derivatizer mil een Cosi Lor Autosampler Detector REAGENT 1 PRESSURE PRESSURE PRESSURE INJECTOR PRESSURE 1 BYPASSI RELIEF DETECTOR Figure 3 1 3 2 Note About Fittings Inert Gas 3 Reagent Reservoir Connections PCX5200 User s Manual Chapter 3 The PCX5200 uses three styles of fittings 1 The high pressure fittings are 10 32 x 1 16 inch Upchurch style These fittings are compatible with Valco Parker CPI Swagelok or various of the polymeric nuts and ferrules The Valco Fittings can be indentified by a slightly longer fitting and in most cases they are found in areas that are labled Valco ex restrictors 2 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 3 The fittings for the over pressure relief valve and external back pressure regulator are 1 4 28 x 1 16 inch and use reversed ferrule type fittings 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 gt Upchurch Parker Valco style 1 4 28 reversed ferrule Figure 3 2 Note the direction of the ferrules normal for Upchuch Parke
3. 2 8 3 3 Rebuilding check valves 4 8 Removing check valves 4 7 PVC GCE VOI e e rege 3 3 Reversed ferrule sess 9 9 Reagent PUMP ina 4 3 Rotor seal en 3 1 Routine maintenance 4 3 ee 2 4 2 21 Index 2 Index S Saran tUDINE airada 3 3 Saving a Preset program 2 15 Seal replacemen reagent pump 4 3 DNULAOWN essa cis 3 10 Slowdown program 3 10 Space requirements e nenen eee 3 2 Specifications ane ene en eee eee venes 2 20 DPI cleaning nenen ecboctteeesaiabedis 4 15 STATUS k y cR 2 10 System testing gu ie esas 3 9 T Tefzel rotor seal eee sene e ee 3 1 TEMP K e N 2 10 Troubleshooting guide 4 12 V Vaccum priming i 4 13 Vespel rotor seal ena ee ena een ase 3 1 W Warranty registration 3 1 Wetted materials 2 20 VYhen priming the reagent pump is difficult 4 13
4. 4 7 Interpretation of pressures 4 14 K DA 2 10 Keypad examples se 2 13 L Layout of HPLC PCX5200 3 2 LED light emitting diode 2 11 2 12 Liquid connectonsg ua aaa aaa ua aa une nene e va senss 2 1 LCD liquid crystal display 2 10 Index 1 PCX5200 User s Manual M Main power enn 2 4 Maximum temperature 2 12 Miscellaneous supplies 3 2 NDI gg 3 1 O Over pressure relief 2 1 P PEER ACIDOS nana 3 3 Piston wash system 2 3 3 8 Post column derivatization 1 1 Post column panel oonss 2 1 Pre column filter seen 4 9 Preset programs eere 2 13 Pressure CAUCE surreali 1 2 Pressure interlock 3 5 Pressure interlock connections 3 4 Pressurized reservoir 1 2 Priming the reagent pump 9 9 PRESET key eta 2 18 Iura E 2 18 PUMP Eege Eege 2 10 Pump seal replacement 4 3 R Radio frequency ena ena ion eses 2 21 Reactor precautions 00 4 2 Reagent filter ann nene eee neo n enes 4 10 Reagent PUMP idas 3 8 Reagent reservoir
5. Tested DV nennen Dale anne aa App A 1 PCX 5200 User s Manual Appendix A Analytical Conditions LG dpi Mc Initial COMPOSstioni ssaa Initial Flow Rate Gradient Program CO Le le cade IPs Denn Temperature Injector Autosampler nenen enen wipe VOlUme ss SONEN ae Manual Auto Ai hh DOTA EXCMO casada BANOE siii arca Emission BANO Pr Ranger PMT sia Lampen Resp time Integrator or Data Station Range or Scale Biet EE Attenuation Chart Speed Peak Threshold Va To irn ac c RUNAS aid onto nt Operator u A asi ana Reference App A 2 PCX 5200 User s Manual 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 NP iui Dirige 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 x 55
6. Internal Figure 2 3 Post column pump amp piston wash system PCX5200 User s Manual Chapter 2 e On the Reagent 1 flow conditioner panel the 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 800 psi in time with the pump piston e The Bypass 1 valve is used to purge and prime the first reagent pump Attach the 20 mL syringe to the Luer fitting in the center 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 e If applicable the Reagent 2 pressure gauge operates the same as the Reagent 1 pressure gauge e The Bypass 2 valve is used to purge and prime the second reagent pump and it operates the same as the Bypass 1 valve e The three removable front panels gives access to the liquid ends of the pump reagent filters restrictors the thermostatted reactor electrical connections for the reagent pump and the flow adjustment knobs of the post column pump The two reagent panels can
7. stainless steel fitting to the pressure gauge tee with a 1 4 wrench 1 8 to 1 4 turn beyond finger tight r J Reagent Filter Figure 4 6 Flow conditioner and 2 um reagent filter 4 10 Ambient Reactor not applicable to single reagent PCX5200 Heated Reactor PCX5200 User s Manual Chapter 4 The Ca phase can be hydrolyzed with repeated injections 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 1 Shutdown the post column system and let the reactor cool for at least 30 min 2 Remove the post column panel by loosening the two captive thumb screws 3 Disconnect two fingertight fittings Back side of To Detector fitting Trace the ambient reactor coil to the Reagent 2 Tee and disconnect 4 Install new ambient reactor Do not overtighten the fittings 5 Start the LC pump and inspect for leaks 6 Reinstall the post column panel Calcium and magnesium hydroxide precipitates due to hard water and fats and waxes of plant origin are two main causes of clogging in the carbamate analysis systems For the end user running a Trione ninhydrin system the most likely clogging agent is the precipitation of hydrindantin from expired Trione ninhydrin solution Please read the tr
8. 250 V 5 x 20 mm fast acting for the 240 V instrument two each IEC127 type 1 6 A 250 V 5 x 20 mm fast acting 4 Reinstall the fuse holder and the power cord Rules of Dolan and Snyder see references e Rule of One Make one change at a time e Rule of Two Confirm the problem before fixing it e Substitution Rule Swap in a good part for a questionable one e Put it Back If swapping does not fix it put the original back in e Write it Down Changes or modifications incidents e Crystal Ball Preventive maintenance saves more time in the long run e Buffer Rule Remove buffers from LC when not in use General Procedure for Troubleshooting e Examine the system front to back Repair all leaks e Verify that all settings eluants reagents valves etc are according to specifications e Have there been any changes in the system e Compare against reference conditions standard sample column parameter log as appropriate e Gather information observations manuals books technical assistance e Test your conclusions about the nature of the problem e Start working 4 12 When Priming the Reagent Pump is Difficult PCX5200 User s Manual Chapter 4 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 pump Ordinary primin
9. 4 6 Cleaning the post column system 4 15 Column installation 3 7 Column oven eeeeee rere erneut 2 6 Column protection na sene sen 1 2 Communicating with a computer 2 21 Corrosive eluants eene 3 5 D Depressurized ena eee venes 4 2 Design of an HPLC 1 4 Designinga post column system 1 5 TCR ECL OM MET 3 1 Detector connections cirie 3 6 Downloading firmware 2 21 E Electrical precaution 4 2 Electrical requirement 3 2 ENABLE key eem 2 10 Index F Filter pre column aaa aaa nave nave na oeuss 4 9 Filter reagent nacidas 4 10 P P 3 3 Flow diagrams eene 2 18 Flow conditioner 1 6 2 3 Flow conditioner panel 2 1 Front panels cccccccceecceseceseceneceecees 2 1 lu 4 12 PUSO HP 2 4 G Gas pressure system eere 2 9 Gas regulator s ena ee nese ee eee enese 2 9 ll E 1 2 il MONTH 3 7 H Heated reactor eu 4 11 HPLC system requirements 3 1 I Pica 3 2 3 3 Injector connections 3 6 Installing new check valves
10. The connection is labelled From Pump Using 1 16 inch x 0 020 inch ID capillary tubing connect the pressure sensor to the inlet of the injector or autosampler Use the fitting labelled To Injector 2 Alternate method for amino acid analysis with mobile phases with pH lt 3 to minimize corrosion of the sensor or some integrated HPLC systems to minimize tubing runs Use two pieces of 1 16 inch x 0 020 inch ID capillary tubing to connect a tee fitting between the outlet of the HPLC pump and the inlet of the injector Use a third piece of 1 16 inch x 0 020 inch ID capillary tubing to connect the third port of the tee to the lower inlet of the pressure sensor on the post column pressure panel labelled From Pump Install a high pressure plug in the upper outlet of the pressure sensor labelled To Injector Note on usage The post column interlock monitors the eluant pump pressure and turns OFF electrical power to the post column reactor reagent pump and column oven when the eluant pressure drops below 500 psig This pressure decrease may occur due to eluant pump malfunction empty reservoirs or a programmed shut down after the last sample The interlock turns off the reagent pumps to prevent backflow of reagents into the column which can ruin the column The interlock also defaults to OFF when a power loss occurs The PCX5200 instrument does not automatically turn on as the eluant pressure rises above 500 psi P
11. s Manual Chapter 4 10 11 13 l 1 Replace the pump 1 tighten the captive screw 2 reconnect the electrical connection 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 PCX5200 and prime the pump Follow steps 1 4 from the Cleaning Check valves section 2 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 washer faces upwards Remove plastic cover 2 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 Reassemble the pump head and wash system as shown in Figure 4 1 Hold the pump assembly firmly in place and reinstall it on the pump in line with the axis of the piston do not tilt the pump head
12. the LCD displays the current temperature of the heated reactor Reactor C lt Press and hold the REACTOR TEMP key the LCD displays the set temperature of the heated reactor Reactor set C By default the power to the heated reactor is controlled by the ENABLE function and cannot be overridden 3d Optional Press the coLUMN PRES key the LCD displays the current pressure of the HPLC column Column bar lt The lt sign indicates that the HPLC column pressure is below the minimum pressure for the PCX5200 to be Enabled This function was described earlier as the pressure interlock system to protect the column from reagent back flow The minimum and maximum pressures for the pressure interlock system were preset at Pickering Laboratories and we do not recommend any changes Press and hold the coLUMN PRES key While holding the coLUMN PRES key press the PROG key the LCD displays the maximum pressure Pres Max 260 bar the PROG LED turns orange To change the maximum pressure press and hold the PROG key While holding the PROG key press the A or V key to change the pressure we do not recommend any changes Press the V key the LCD displays the minimum pressure Pres Min 32 bar To change the minimum pressure press and hold the PROG key While holding the PROG key press the A or V key to change the pressure we do not recommend any changes Press the V key the LCD displays Set Autoz
13. 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 330 1988 631 633 Glyphosate e J E Cowell Analytical Residue Method for N Phosphono methylglycine and Aminomethylphosponic acid in Environmental Water Monsanto Method Number 86 63 1 1987 e 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 LCeGC 6 11 1988 994 997 e M V Pickering Modifying HPLC equipment to tolerate corrosive solutions LCeGC 6 9 1988 800 809 e J W Dolan and L R Snyder Troubleshooting LC Systems Humana Press Clifton NJ 1989 Reprints available from Pickering Laboratories Ref 1 PCX 5200 User s Manual References Amino Acid Analysis e D H Spackman W H Stein and S Moore Anal Chem 30 1958 1190 e M V Pi
14. C Hun Hetention time Peak width Average W Part 2 Post column reactors connected to column and detector Post column pumps off normal reactor temperature Hun Hetention time Peak width Average W Ratio VV 10 ODEratori vin Dale App A 3 PCX 5200 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 f_ 0 1 f fo f where f is eluant 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 Spare Parts Part Number Description 3102 9042 Replacement frit 0 5 um for pre column filter 3102 9040 Replacement frit 2 um for reagent filters 1100 2927 OPA Reactor 0 011 ID TFE tubing 1100 0281 0 5mL Coil Assembly only no heater 1100 2660 Heated Reactor 0 5mL 120 V other volumes on request 1100 2661 Heated Reactor 0 5mL 240 V 1100 0200 Restrictor for OPA NaOCI amp NaOH reagent with nuts amp ferrules 1100 0141 Restrictor Trione Ninhydrin with nuts amp ferrules 3106 1330 Seal 1 for reagent pump 3106 1310 Seal Kit for reagent pump includes 2 seals and seal installation tool 3106 1314 Inlet Check Valve for reagent pump 3106 1316 Outlet Check Valve for reagent pump 3106 1332 Piston sapphire for reagent pump with piston washing system 3106 1322 Pisto
15. Choose an eluant that elutes contaminants from the column for example methanol for a reversed phase column and regenerant for an ion exchange column 3 Set the HPLC pump to lt 0 1 mL min 4 Turn off the detector lamp 5 You may also program a slowdown method to accomplish all the above steps Step Time min Eluant Flow mL min 0 0 100 0 02 1 5 100 0 02 2 5 1 100 Normal flow 3 25 100 Normal flow 4 25 1 100 0 02 Normal flow rates are different for various columns Follow the instructions that came with your column Medium term up to 6 days I PCX5200 User s Manual Chapter 3 1 8 Turn off the PCX5200 either manually by pressing the ENABLE key or via the Slowdown program Set the HPLC pump at the normal flow rate to flush the system for 30 minutes Choose an eluant that elutes contaminants from the column Replace both reagents with water and draw 10 mL through each prime purge valve Replace the water in the reagent reservoir with water methanol approximately 1 1 Turn off the fluorescence detector and HPLC pump Loosen the fitting at the inlet of the 100 psi external back pressure regulator relieving pressure on the post column system Place paper towels under the back pressure regulator to absorb any escaping liquid Relieve the pressure in the reagent gauges by briefly opening the bypass valves E S Caution The long term shutdown should be performed pr
16. analytical column 2 Delivers the reagent 3 Heats the reaction It also has various features to make the analysis more convenient or reliable and features to protect the instrument itself from accidental damage The pressurized reservolr serves two purposes 1 It protects air sensitive reagents from oxidation 2 It helps the metering pump fill consistently by preventing cavitation The pump is a constant speed variable stroke piston pump The cam cycle is two seconds which makes for easy pulse damping The reagent pressure gauge does two things 1 It shows the pump pressure for diagnostic purposes 2 It absorbs pulses by the spring action of the Bourdon tube There is a packed bed restrictor just downstream to provide a load for the pressure gauge so that it will effectively absorb pulsations Referring to Fig 1 1 the anti siphon valve prevents the gas pressure in the reservoir from forcing liquid through the system with the power off The post column pressure gauge is primarily there to monitor the condition of the reactor There is a 500 psi relief valve in case there is a blockage in the reactor or detector The standard reactor is a PTFE capillary tube 0 011 I D wrapped on a heated mandrel The narrow diameter reduces band spreading and the PTFE is corrosion resistant There is a 100 psi 5 bar back pressure regulator on the exit line from the detector it suppresses boiling inside the hot reactor and prevents bubbl
17. left to right they are referred to as 1 Post column panel 2 Reagent 1 flow conditioner panel 3 Reagent 2 flow conditioner panel if applicable e On the post column panel the post column pressure gauge measures the liquid pressure at the first mixing tee This is effectively the pressure on the heated reactor ambient reactor and dectector This gauge indicates pressure when liquid is flowing through the system e The liquid connections to the pressure sensor are labelled From LC Pump and To Injector The pressure sensor is part of the safety interlock system The sensor requires 500 psi 35 bar before the module can be enabled e The liquid connection From Injector is also the pre column filter The filter element is a 0 5 um frit Cat No 3102 9042 e Note Only PEEK ferrules should be used to connect the tubing from the injector This is because the filter is made if a soft PEEK material and a stainless steel ferrule will damage it e To Detector bulkhead fitting should be connected to the detector with 0 010 inch 0 25 mm ID tubing e Over Pressure Relief is a safety relief valve that opens in case the post column pressure reaches gt 500 psi 35 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 piece of tubing from this fitting to a clean dry beaker Any evidence of liquid in this tubing ind
18. to be analyzed for carbamate insecticide residues the injector should be able to inject at least 200 uL and preferably 400 uL Important If the system will be used for amino acids glufosinate glyphosate polyamine or diquat amp paraquat analysis be aware that the 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 gt 9 a Tefzel or PEEK rotor seal must be installed e HPLC absorbance or fluorescence detector To prevent boiling in the heated reactor Pickering provide an external back pressure regulator that should be connected to the detector waste line The pressure rating of the detector flowcell must be 110 psi 7 5 bar If your detector flowcell is rated 3 1 Space Requirements Electrical Requirement Inert Gas Miscellaneous Supplies Chemistry Installation of the PCX5200 Layout of the HPLC and PCX5200 PCX5200 User s Manual Chapter 3 lower consult Pickering Laboratories e Integrator or data system e Because the Trione ninhydrin and OPA reactions are extremely sensitive the HPLC system must be thoroughly clean before using it with the PCX5200 Pay special attention to the cleanliness of eluant reservoirs and delivery tubings Space requirements for the entire HPLC system are determined by
19. your local 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 The main power switch is located in the power connector assembly 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 120 V systems use a fast acting 3 A 250 V 5 x 20 mm fuse type GMA3 Cat No 3543 0045 For 240 V systems use two fast acting 1 6 A 250 V 5 x 20 mm fuses meeting IEC127 specifications Cat No 3543 0044 The serial computer interface is the DB9F connector See the section later in this chapter about the RS232 serial port 2 4 PCX5200 User s Manual Chapter 2 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
20. 0 will resume normal operation 2 21 PCX5200 User s Manual Chapter 2 2 22 Unpacking Site Requirements HPLC System Requirements Chapter 3 Installation The PCX5200 instrument is shipped in one carton Application Kits may be shipped in one or more cartons each Report any carton damage to the carrier Unpack all 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 650 694 6700 or by fax at 650 968 0749 Store any standards in the freezer refridgerator immediately upon arrival Complete and mail the warranty registration included in this manual to receive instrument method updates and this User s Manual on Disk in Adobe Acrobat format Read all installation instructions and material safety data sheets MSDS s before operating your post column derivatization instrument and HPLC system The HPLC pumping system the injector or autosampler the detector and the integrator or data system must be supplied by the user e Binary or ternary gradient HPLC pump For carbamate or glyphosate applications the HPLC pump must be capable of binary gradient elution For amino acids or ALKION column applications the HPLC pump must be capable of ternary gradient elution e HPLC manual injector or autosampler The injector should be able to inject a 10 uL sample preferably by full loop injection If drinking water is
21. 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 Fur 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 e The Gas Inlet fitting is where inert gas enters the external gas regulator for pressurizing the reagent reservoirs Figure 2 8 The gas regulator requires an input Close 5 y 3 u Valve Open Sn EY P 1 1L Coated Reservoir P N 3107 0137 2 Cap Assembly P N Ke 0147 3 Gas Valve 4 Reagent shut off Valve Pin hole in 5 Gas connection tubing 1 8 SARAN P N 2103 0463 SEET 6 Plastic Nut and Lu P N 3101 0005 amp 3101 0006 ch 7 Reagent connection tubing Re 1 8 SARAN for OPA Trione agent Teflon for hydrolysis
22. ESET key each time will change to the next preset program Of course by pressing the A key instead of the V key you could go back to the previous preset program These 3a d steps are optional but it will help you to familiarize with other functions 3a Press the column TEMP key the LCD displays the current temperature of the column oven Column C lt The lt sign indicates that the temperature of the column oven is below that of the set point When the temperature of the column oven reaches the set point the sign will change to an sign If it is above the set point it becomes a gt sign The flashing sign indicates that the column oven is heating up Note the column oven warms up automatically in the Glyphosate and Amino Acids methods without enabling the PCX5200 The column oven does not warm up automatically in the Carbamates application By default the power to the column oven is controlled by the ENABLE function However the power cutoff to the column oven can be overridden so that the column oven remains on independent of the ENABLE function We will learn how to change this function in a later example PCX5200 User s Manual Chapter 2 3b Optional Press and hold the coLumn TEMP key the LCD displays the set temperature of the column oven Column set C In this example it is 55 C 3c Optional The REACTOR TEMP key operates the same way as the coLUMN TEMP key Press the REACTOR TEMP key
23. Interlocked NO NO Back YES Save Changes NO NO YES SET Save Preset 1 S name ij 1N name 1 Preset Name 2 S name 2 gt 2N name 2 Erase Preset 3 S name 3 3N name 3 Back YES 4 S name 4 4N name 4 5 S name 5 5N name 5 1 N name 1 2 N name 2 1 E name 1 3 N name 3 2 E name 2 4 N name 4 3 E name 3 5 N name 5 4 E name 4 Back YES 5 E name 5 Back YES Specifications Wetted Materials PCX5200 PCX5200 PEEK Ratings PCX5200 User s Manual 316 Stainless steel 17 4 PH Stainless steel Teflon FEP Saran PVDC Hastelloy C Kel F CTFE PEEK Viton Borosilicate glass Sapphire UHMW polyethylene 316 Stainless steel 17 4 PH Stainless steel Viton Teflon FEP Saran PVDC Kel F CTFE PEEK Tefzel ETFE Borosilicate glass Sapphire UHMW polyethylene Dimensions 37 cm x 38 cm x 38 cm h x w x d Weight 13 to 15 kg depending on configuration Electrical Power 100 120 V 50 60 Hz 1 7 A 200 W grounded supply or 200 240 V 50 60 Hz 0 8 A 200 W grounded supply Installation overvoltage category II Pollution degree 2 Environmental Indoor use only Altitude up to 2 000 m 6500 ft Ambient temperature 5 40 C 40 104 F Chapter 2 Relative humidity 80 31 C derated to 50 R H 40 C 2 20 RS232 Serial Port Downloading firmware Radio Frequency PCX5200 User s Manual Chapter 2 The RS232 serial port on the PCX5200 has three main
24. Operation Manual PCX 5200 Post column Derivatization Instrument PICKERING LABORATORIES Pickering Laboratories Inc 1280 Space Park Way Mountain View CA 94043 800 654 3330 International 650 694 6700 Fax 650 968 0749 Website http www pickeringlabs com E mail supportOpickeringlabs com Cat No 0101 0001 Version 6 January 2002 Pickering Laboratories Inc PCX 5200 User s Manual Contents PCX 5200 User s Manual Contents Contents Please CAG ecu BYS 1 A A PE BYS 1 Chapter 1 Introduction Why Post column Derivatization AE 1 1 MHOWCDOEBSAHL ee 1 1 What Are ine Requirements urlante 1 1 Pickering PFOXS200 A O e ty eae 1 2 Reagent DEIVI M 1 2 Colum Protech seu v ters vaut tm nnd ee 1 2 Supplement The Elements of an HPLC with Post column System 1 4 Design Of an ARE T i ot Pd Pet a Pd P 1 4 A simpler View or an EE ssa 1 5 Designing a Post column System ee 1 5 Chapter 2 PCX5200 System Components POX5200 Post colummn Instrument sea nn 2 1 Getting to KNOW YOUR PGKS20 gj r ci tnde erbe o E Di Hua Gas 2 1 e A 2 1 ematica 2 3 Bic cR NC TID EINE ea 2 4 eei ECT Mtr 2 6 ReagentiReseivo Sisa 2 8 External Reagent Pressurization System c coooonccnnccconnnnnccocooncnnonnnononnnononnnnnnnonncnnnnnnnannnncnnnannnns 2 9 Keypad and Liquid Crystal Display User Interface i 2 10 Wiele JE le
25. S ga kas tande een 2 13 Loading RE 2 13 SAVNI A FIOS G iana Emm 2 15 FION RE ele EEN 2 18 PRESET elle 2 18 A A 2 18 SIE 2 20 Wetted Materials POXS200 adas 2 20 POCXS200 PEEK snoot dodo an 2 20 Re Te EE 2 20 E Serial PON sen ne dei EE v ur tdv da f Ee cem M DP SUD ia 2 21 Downloading firmware nennen nennen nennen nnn nsn n nnn nennen nnne innen nnn nnns 2 21 Rado NEGU NCY siri e ni r e E 2 21 PCX 5200 User s Manual Contents Chapter 3 Installation Ue ala 3 1 SERGE ar ese e D eese lente md cu IE SEL UD EE 3 1 HPLG System e Han EE 3 1 Space EE enee een 3 2 Seite e E En EE 3 2 eros clicca 3 2 Eltere ansia 3 2 Installation or tne PGCX5200 2200er anne 3 2 Layout ot tne MPEG and POX5200 2 ee A 3 2 Note ABODUERBIOS EE 3 3 Tune 3 3 R ageni Reservoir GonneclloriS s E 3 3 Pressure interlock Connections aaa eee kena ee ee eee eee e eee nennen rere eee eee eee t ete reve eee te eee eee eee 3 4 InjeCIOr Gonneclions une een ili 3 6 Detector tee EE 3 6 Guard Analytical Column Installation aa ennn eee ee eee nete peer eee eee eee enen ee eee eee eee 3 7 A RU A A 3 8 Riston waslh SyS Misano 3 8 PRIMING TAS Roedgen Pimp sia ads 3 9 System TESTING e Veni lola ee 3 9 IER ee ES 3 10 NOMS a ii 3 10 Medi MSM o 3 11 ION 3 12 Chapter 4 Routine Maintenance amp Troubleshooting HITE SY STOMA TOSHNO eee 4 1 Test ONOMA O ANI EE 4 1 Paramaler POO EE 4 1 Precautions amp Problem Preventi
26. The status LED turns green when the set temperatures are ready and the post column pump is on The status LED turns red if there is fault with the PCX5200 The following table summarizes the LED status Operational state ENABLE LED PUMP LED STATUS LED Enabled amp Ready green Enabled amp Not Ready green green or off orange Not Enabled off or orange off off Alarm off PCX5200 User s Manual Chapter 2 Keypad Examples Here are two examples to use the Keypad and Liquid Crystal Display User Interface Loading a Preset A How to start a routine Preset Analysis In this example we will load a Preset program Program 2 named Glyphosate enable the PCX5200 and then turn on the post column pump 1 Turn on main power switch in the back of the PCX5200 The POWER LED light turns green The ENABLE LED is off The pump LED is off The status LED is off Press and hold the PRESET key the LCD shows Load preset While holding down the PRESET key press the V key the LCD shows L Name of program for example Program number 1 is Carbamate the LCD then shows 1 L Carbamate The letter L stands for Load Of course in this example we want to use Program 2 With the PRESET key still held down press the V key a second time the LCD will now shows 2 L Glyphosate Release the PRESET key H Note there are five preset programs Pressing the V key while holding the PR
27. ame is completed Na AA OPA A space between words is allowed Press the PROG key to accept the name the LCD shows Back YES PCX5200 User s Manual Chapter 2 Press the PROG key to accept the LCD shows Save Preset Press the V key three times the LCD changes to Preset Name Erase Preset and then Back YES Press the PROG key to accept the LCD shows 5 Na AA Program number 5 is now ready PCX5200 User s Manual Flow Diagrams PRESET Key SES H Preset 1 Name 2 Name 3 Name 4 Name 5 Name PROG Key L 0 Pump hrs IN YES Pres Min bar COLUMN TEMP PROG Lcd C PID C RTD Set Interlock L Interlock OFF Interlock ON Back YES NO Chapter 2 For all of the diagrams below press and hold the function key i e PRESET TEMP then press V or PROG For PROG functions release the keys when the PROG LED turns orange COLUMN PRES PROG Pres Max bar Set Autozero Autozero NO Autozero YES Span Back YES Back NO Cycle sec Prop band Int reset C Offset Deriv C Span Deriv App Back YES Back YES NO NO Save Changes NO YES PCX5200 User s Manual Chapter 2 REACTOR TEMP PROG R PID Cycle sec Prop band Int reset R RTD R Offset Deriv R Span Deriv App t_ Back YES Back YES
28. be removed by loosening the captive thumb screws The Post column panel left can be removed by loosening the two captive thumb screws e The reagent pump with a piston wash system is behind the two reagent flow conditioner panels 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 the flow rate by about 0 1 mL min Important Make sure the piston wash system is flushed with 80 20 water methanol before turning on the post column pump k Im E 2 3 PCX5200 User s Manual Chapter 2 e The reagent filter is located just downstream of the prime purge valve in the reagent Figure 2 4 Reagent filter amp flow conditioner Rear Panel e panel The filters and elements are similar in appearance to the pre column filter but they are different The reagent filter element is a 2 um frit Cat No 3102 9040 BL EH PB Pa N PI Oz 1000 2000 5500 2 2500 LI 3000 The power connector is a standard IEC 320 type connector Use the appropriate power cord for
29. bing from the outlet of the flowcell to the external 100psi backpressure regulator using a 1 4 28 nut with a 1 16 inch reversed ferrule Caution The 100psi back pressure regulator is directional Do not reverse flow Caution The back pressure regulator provides 100 psi 7 bar of back pressure to the detector 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 detector has a flowcell pressure rating of less than 100 psi 7 bar contact Pickering Laboratories The minimum necessary backpressure is 75 psi 5 bar 4 Connect a 0 020 inch ID PTFE tubing to the outlet of the external 100 psi back pressure regulator Place the other end in an appropriately labelled waste container 5 Set the wavelengths of the detector as specified in the applications 6 Set the time constant to 2 4 seconds 7 Connect the signal cable from the detector to the input terminal of your data station Ensure that the polarity is correct refer to your HPLC instrument manual 3 6 Guard amp Analytical Column Installation PCX5200 User s Manual Chapter 3 1 A set of PEEK tubing is provided in the Application Kit for installing your Pickering column Select the column that you wish to install and the corresponding tubing set Refer to Figure 3 3 for column placement and door operation Open the column oven door Be careful not to knock off the reag
30. captive screw flip the flow conditioner upside down and place it behind the louver in front of the column oven You do not need to disconnect any tubings yet Repeat the same procedures for the second flow conditioner panel 4 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 1 16 fitting to prevent the check valve from moving 4 3 PCX5200 User s Manual Chapter 4 Remove the pump from the chassis by 1 loosening the captive screw and 2 unplugging the electrical connector Place the pump in a more accessible place to continue Remove the pump head from the pump as follows Figure 4 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 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 retai
31. cause 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 in 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 If you have the piston seal wash feature and the pump pressure exceeds 2500 psi for more than a couple minutes the high pressure piston seal will deform and start to leak After you have corrected the high pressure situation replace the piston seal Pump Seal You will need to purchase a seal kit 3106 1310 including two seals two back up Replacement washers 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 1 Flush the Eldex pump with water then shut down the PCX5200 and let the reactor cool Turn off the gas valve and vent the reservoirs Turn off the integrated 2 way valve on the reservoir cap 2 Open the column oven 3 Remove the flow conditioner panels center and right by loosening the captive screws Remove one flow conditioner at a time After removing the
32. ckering LCeGC 7 1988 484 7 e J A Grunau and J M Swiader J Chromatogr 594 1992 165 1 e A A Boulton G B Baker and J D Wood Eds Neuromethods 3 Amino Acids Humana Press Clifton NJ 1985 Chapter 1 Ref 2 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 CRX400 CHX700 PCX5200 Cat Nos 1152 1xxx and 1152 2xxx Analytical Columns Pickering s Analytical Columns are warranted to be free of defects in materials and workmanship under normal installation use and maintenance for the warranted time beginning 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 Ion Exchange columns for Amino Acid Analy
33. constant flow we need to add a reservoir pulse damper and a dam column The flow downstream from the dam is constant smooth baseline Designing a What happens if we simply add a post column pump a mixing tee and a reactor What Post column happens to the baseline The periodic noise returns created by the post column pump System Figure 1 5 Injector Thermostatted Heated Column amp Guard Reactor Back pressure Regulator Waste Eluants Baseline Post column Pump Reagent Figure 1 5 Bottle 1 5 Figure 1 6 Post Column Pressure PCX5200 User s Manual Chapter 1 However we do not need to invent anything new we just need a pulse damper and a column at Pickering we call it a restrictor Figure 1 6 in Pre column njector A CH i Thermostatted Heated z Column amp Guard Reactor ae 7 ook Damper YUOL gt Back pressure 6 QG Regulator o mM mM LC Pump reni Waste Eluants Baseline Restrictor Reagent Pressure Gauge Post column Pump e Reagent Bottle A Pickering flow conditioner includes a pressure gauge and a restrictor The Bourdon tube inside the pressure gauge is the pulse damper and the restrictor packed with very inert material is the column With the flow conditioner in place the baseline is now acceptable Add a second or third post column reagent Just follow the same procedure Another post column pump pressure gauge restr
34. ct acting keys ENABLE and PUMP keys cause an immediate change of state without any other keys involved 2 Status keys The coLumn TEMP coLUMN PRES REACTOR TEMP STATUS PROG and PRESET keys all display the current status of that function when pressed If the status message is a scrollable list the first line ends in to cue the user To select a menu item just press that key To adjust a setpoint press and hold that function key and use the A or V key menu selection takes effect when the function key is released To reach the advanced options menus hold a function key and press the PROG key The following table summarizes the keypad behavior e POWER indicator light LED is useful as an indicator that the PCX5200 is connected to a power source and the on off switch on the back of the PCX5200 is turned on Hold amp press PROG key ENABLE Toggle enabled state PUMP Toggle pump power STATUS Display ready not ready Reset elapsed time for alarm status Scrollable pump list of conditions elapsed service time COLUMN Display current Display setpoint adjust Interlock option PID TEMP temperature with AorV key sensor calibrations coLUMN Display current pressure Sensor calibrations over PRES and under pressure REACTOR Display current Display setpoint adjust PID sensor calibrations TEMP temperature with AorV key PRESET Display and name of Load one of 5 presets Save current setti
35. e for a particular class of substances so analytes of that class are more easily seen against a complex background So post column derivatization is used to increase sensitivity and selectivity in HPLC analysis The post column reaction system mixes the stream of effluent flowing from the HPLC column with a stream of reagent solution The mixture usually flows through a reactor to allow enough time for the chemical reactions to complete If the reaction is slow the reactor may be heated to speed things up Some reactions need two or more reagents added in sequence Finally the mixed streams pass into the detector typically UV VIS absorbance or fluorescence Of course a practical system requires metering pumps pulse damper thermostats and safety systems to give reliable results e Stability of Reagent The minimum reagent stability sufficient for routine work is one day This means that the yield and signal to noise ratio for a given sample must remain constant for at least 8 hours e Speed of Reaction The analytical separation is complete when the reagent is mixed with the column effluent Therefore in order to minimize band spreading it is important to keep the time therefore volume small between the mixing tee and the detector If the reaction is slow in excess of one minute an elevated temperature can be used to decrease the reaction time e Reproducibility Because the reaction is occurring on the fly as the combined c
36. e safety requirement of coated bottles they must not be substituted e Page 3 1 for the HPLC System Requirements especially for glyphosate or amino acid analysis For most ion exchange applications the HPLC components must be compatible with high pH regenerant e Pages 2 3 and 3 8 about the piston wash system of the post column pump 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 el correctly done or adhered to could result in loss of information or damage to or ee destruction 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 5200 User s Manual Before You Start The following symbols appear on the PCX5200 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 Il 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 e
37. ent bottle s if they are placed on top of the PCX5200 LOOSELY Connect the outlet of the guard column to the inlet of the analytical column Loosely fit the inlet of the guard column to the eluant heat exchanger Carefully lay the analytical column into its slot in the heating block Turn on the HPLC pump at 0 2 ml min at 100 of the column storage solution Wait for liquid to drip from the heat exchanger Tighten the connection to the guard Wait for liquid to drip from the inlet of the column and then tighten the connections A loose end of tubing with Fingertight fitting in the column oven leads to the first mixing tee Attach the loose end to the outlet of the analytical column Close the column oven door O O gf Figure 3 3 Reagent Pump Piston wash System 3 3 PCX5200 User s Manual Chapter 3 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 a syringe and flush the piston wash system with 80 20 water methanol 4 mL each time at least twice a day Alternatively you may c
38. er 7 Repeat the process for the other valve if applicable If priming the reagent pump is difficult see Chapter 4 p 4 13 Read the Application Manual if applicable to become familiar with the use of your instrument In Chapter 4 of this manual 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 e Parameter log of pressures temperatures and flows under standard initial conditions Appendix A e Chromatogram of test mix using Pickering standard conditions if applicable 3 9 Shutdown Short term up to 3 days PCX5200 User s Manual Chapter 3 Important If the system will not be used immediately after the installation the system must be shut down properly Generic shutdown procedures are given below but we advise you to follow the specific shutdown procedures in the Application Manual Upon completion of the analyses use one of the following two procedures to shut down the PCX5200 system properly These procedures can prevent potential column damage reaction coil blockage high background fluorescence reagent precipitation or other problems 1 Turn off the PCX5200 either manually by pressing the ENABLE key or via the Slowdown program see below 2 Set the HPLC pump at the normal flow rate to flush the system for at least 20 minutes to allow the heated reactor to cool
39. er 4 These filters are located on the backside of the flow conditioner panels inside the PCX5200 cabinet It is easily accessible 1 2 Be sure that the PCX5200 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 Remove the flow conditioner panels center and right by loosening the captive screws Remove one flow conditioner at a time After removing the captive screw 1 remove the 1 16 fitting from the pressure gauge tee on the back of the flow conditioner panel 2 then remove the fingertight fitting on the outlet of the antisiphon valve Set the detached flow conditioner panel in a more accessible place to continue Repeat the same procedures for the second flow conditioner panel Disconnect the tubings from the filter Use a 7 16 inch wrench and a 9 16 inch wrench to disassemble the filter Replace the filter element The correct filter element is 3102 9040 a 2 um frit It is superficially similar to the pre column 0 5 um filter so take care not to confuse them Tighten the fitting firmly Reconnect the tubings to the filter The arrow on the side of the filter shows the direction of flow Reconnect the flow conditioners The left pump head is Reagent 1 Do not overtighten fittings finger tighten the two fittings first and then tighten only the 1716
40. ero To autozero the pressure sensor make sure that the HPLC and post column pumps are off press the PROG key the LCD displays Autozero NO Press the A key the LCD displays Autozero YES Press the PROG key note that the PROG key acts like an ENTER key when the LCD displays a choice of YES or NO The LCD re displays Set Autozero Press the V key the LCD displays Pres Span 1710 Press the V key the LCD displays Back YES Press the PROG key to exit the LCD displays Column bar lt Note Before the last exit step by pressing the A key instead of the V key you could go back to the previous step 4 With the HPLC on at 0 4 mL min wait for the enable LED to turn orange then press Saving a Preset PCX5200 User s Manual Chapter 2 the ENABLE key The power LED remains green The ENABLE LED turns green The pump LED is off The status LED turns orange Once the temperatures of the heated reactor and column oven reach their set points press the PUMP key The power LED remains green The ENABLE LED remains green The pump LED turns green The status LED turns green Congratulations The system is ready to go of course it should be equilibrated for at least 15 min before your first injection B How to save new conditions as a Preset program In this example we will change the conditions of an un named Preset program Program 5 to a hyp
41. es from forming in the detector flowcell Usually the post column reagent will immediately damage the analytical column To prevent this there is a pressure switch connected between the HPLC pump and the injector that must sense 500 psi 35 bar before allowing the reagent pump and reactor to receive power If at any time pressure is lost because of fault or programmed shutdown the PCX5200 will gracefully shut itself off until a human turns it back on 1 2 PCX5200 User s Manual Chapter 1 Injector Pre column Gi Filter 0 5um 4 Thermostatted Column and Guard 500 psi Pressure Switch LC Pump Over pressure me relief valve O 525 psi Anti siphon Valve 40 psi LC Eluants Restrictor Bypass Valve The PCX5200 does three main things 1 Thermostats the column 2 Delivers the reagent u Waste 3 Heats the reaction Injector Pre column Gi Filter 0 5um 4 A Thermostatted X Column and Guard 500 psi Pressure Switch 3 Heated Reactor Back pressure Ja Regulator E Post column 100 psi Pressure Waste Reagent Filter 2um Pressure Post column Pump i Pressurized Reagent Bottle a 3 Heated Ambient Reactor Reactor EE Post column Back pressure a LC Pump C amp Over pressure Pressure Regulator H ES relief valve 100 psi B 525 psi Anti siphon Valves Waste 40 psi LC Eluants Restrictors Reagent Filters 2um Bypass Valves Reagent Pressure ES Was
42. g 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 1 This can be performed with the pump on or off it makes little difference 2 Connect the 20 mL priming syringe to the bypass Open the Bypass valve Draw liquid until no bubbles come through Empty the syringe and reconnect it to the bypass 3 Close the liquid shut off valve on top of the post column reagent reservoir 4 Pull a vacuum with the syringe Hold the vacuum until no more bubbles come out This causes the trapped bubbles to expand 5 While still holding a vacuum open the liquid shut off valve This sweeps the expanded bubbles out 6 Wait until about 5 mL of liquid has collected then 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 7 If the pump is not on turn it on 8 The pressure should come up within a fe
43. gno 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 este 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 5200 User s Manual Before You Start Power On En marche An Escendido Acceso Power Off teint Aus Apagado Spento Fuse Specification 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 Why Post column Derivatization How Does It Work What Are the Requirements Chapter 1 Introduction Post column derivatization also known as post column reaction renders visible certain compounds that are normally invisible This trick is accomplished after the separation by performing a chemical reaction on the substances that gives them an easily detectable physical property Typically you use a reaction that produces a strong color or makes a fluorescent product You can increase the sensitivity of detection by several orders of magnitude in favorable cases Most reagents are selectiv
44. icates a fault condition 2 1 PCX5200 User s Manual Chapter 2 PCX 5200 TON ENABLE i Ka Ze COLUMN REACTOR REAGENT 1 REAGENT 2 Ze gt PICKERING C Ji J J Model PCX 5200 Post Column Derivatizer j j j STATUS l PRES I J x J 1500 e Ga 1500 7NNN 400 1000 2000 1000 2000 200 80 800 2500 800 2 2500 1000 3000 3000 POST COLUMN REAGENT 1 REAGENT 2 PRESSURE PRESSURE PRESSURE TO INJECTOR OVER us FROM PRESSURE BYPASS 1 BYPASS 2 Figure 2 1 Front view pt INJECTOR RELIEF F ER TZ of the two reagent N 7 P es TO PCX5200 system FROM DETECTOR PUMP Ell Po Po POWER PCX 5200 ON p au PICKERING PUMP rn mr TEMP i I Er LABORATORIES A Model PCX 5200 Post Column Derivatizer ENABLE KA D 2 COLUMN REACTOR REAGENT 1 REAGENT 2 j j j STATUS PRES PROG POST COLUMN Ll PRESSURE Figure 2 2 jute OVER FROM PRESSURE Exposed VIEW of E INJECTOR m RELIEF the two reagent N A PCX5200 system and PON DETECTOR post column pumps 2 2
45. icates the reactor is heating e Pressing and holding the REACTOR TEMP key shows the set temperature of the reactor To change the setpoint press and hold the REACTOR TEMP key and press the A key to increase or press the V key to decrease e The recommended maximum temperature for the column heater is 75 C A thermal safety switch limits the heater at ca 80 C e 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 limits the heater at ca 150 C Figure 2 8 Keypad and LCD interface 2 11 PCX5200 User s Manual Chapter 2 e There are four LED status lights 1 POWER LED is green when the power cable is connected and the power switch is on 2 ENABLE LED The ENABLE LED is orange when two conditions are fulfilled 1 The LC column pressure is at least 500 psi 34 bar The required LC column pressure to enable the PCX5200 is adjustable however we recommend that this set pressure remains at 500 psi 11 The ENABLE key is not pressed It is green when two conditions are fulfilled 1 The LC column pressure is at least 500 psi 34 bar 11 The ENABLE key has been pressed 3 PUMP LED is green when two conditions are fulfilled 1 The PCX5200 is enabled 11 The PUMP key has been pressed 4 status LED turns orange when the PCX5200 is enabled but the set temperatures are not ready or the post column pump is off
46. ictor mixing tee and reactor We add a post column pressure gauge Fig 1 1 to determine if there is any blockages in the reactors Note that the pressure in the post column gauge is stable because it is down stream from the analytical column and the restrictors i e standing down stream from the reservoir and dam in our earlier river example Now you are ready for Chapter 2 1 6 PCX5200 Post column Instrument Getting to Know Your PCX5200 Front Panels Chapter 2 PCX5200 System Components The PCX5200 is available for one reagent or two reagent 120V or 240V operation and is shipped completely assembled calibrated and tested The one reagent PCX5200 consists of a simplex reagent pump heated reactor column heater backflow and over pressure safety devices filters and flow conditioner reagent reservoir Saran gas tubing and other accessories Note that the one reagent PCX5200 can be easily upgraded to the two reagent PCX5200 The two reagent PCX5200 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 If this system was purchased to analyze for glufosinate amino acids carbamates or glyphosate you should have an appropriate column kit and chemistry manual for your application Terminologies There are three removable front panels Figure 2 1 From
47. in Vorhaben die bei unkorrektem Befolgen der Vorschriften zu einer Verletzung oder einer lebensgefahrlichen Situation fuhren konnen 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 piu affinch le condizioni e instruzioni sono completamente chiare BYS 2 PCX 5200 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 a 70 C Pour votre s curit priere de porter des gants isolants Dieses Warnsymbol kennzeichnet eine heiBe Oberflache oder eine Gefahr durch hohe Temperaturen Es macht aufmerksam auf den Heizblock des Saulenofens der heiBer als 70 C sein kann Zu Ihrer Sicherheit sollten Sie isolierende Handschuhe tragen wenn der Saulenofen warm ist Este si
48. ior to any work on the HPLC or PCX5200 Failure to do so could defeat the safety systems 3 11 Long term 7 days or longer PCX5200 User s Manual Chapter 3 1 Replace the reagent s with water and draw 10 mL through the prime purge valve 2 Set the HPLC pump at the normal flow rate Choose an eluant that elutes contaminants from the column Turn on the PCX5200 to flush the system for 15 minutes Set the reactor temperature to lt 60 C 3 Turn the reagent pump on and flush the entire system for 10 minutes 4 Depressurize the system by disconnecting the To Detector fitting Disconnect the outlet of the column and remove the column and guard Plug them tightly 5 Replace the column and guard with a restrictor or small ID tubing and unions so there are no open lines 6 Remove any buffers from the HPLC and replace them with water 7 Increase the flow rate of the HPLC to give 500 psi on the post column system 8 Flush the entire system for 30 minutes then replace the water on the HPLC with 80 20 water methanol 9 Replace the water in the reagent reservoir with water methanol 80 20 10 Start the HPLC and reagents pumps and flush the system for another 10 minutes 11 Relieve the pressure in the reagent gauge s by briefly opening the bypass valve s 12 Let the system drain for 1 2 minutes 13 Loosen the fitting at the inlet of the 100 psi external back pressure regulator relieving pressure on the post c
49. led From Injector on the front post column gauge panel of the PCX5200 1 Be sure that the PCX5200 is shut down and depressurized disconnect the to detector fitting before changing the filter 2 Disconnect the capillary tubing from the fitting Use a 7 16 wrench to remove the filter element 3 The correct filter element is 3102 9042 with 0 5 um porosity It is superficially similar to the 2 um reagent filter so take care not to confuse them 4 Replace the filter element Tighten the filter element firmly 5 Reconnect the capillary tubing PCX 5200 po ENABLE v x COLUMN REACTOR REAGENT 1 REAGENT 2 gt gt PUMP a TEMP TEMP PRE O SET Been ei Za gt gt gt STATUS gt ka i PROG I I I I PICKERING LABORATORIES Model PCX 5200 Post Column Derivatizer gt fy S a 1600 7 400 600 1000 2000 200 800 500 2500 1000 1 3000 7 LL ER POST COLUMN REAGENT 1 PRESSURE PRESSURE ro Inlet Filter INJECTOR OVER Se FROM PRESSURE BYPASS 1 PAR INJECTOR RELIEF IN pl dich E Bes TO FROM DETECTOR PUMP nz A pert Figure 4 5 0 5 um Inlet filter 1000 2000 500 Z 2500 3000 REAGENT 2 PRESSURE BYPASS 2 FAN NUS pa Reagent Filter PCX5200 User s Manual Chapt
50. n oc KO Eldex 25 Hooked End Blunt End 58 N a Piston Seal Figure 4 3 Piston seal Seal Back up Washer replacement in the wash Installation Tool Retainer Assy H system with installation tool se 9 yn mc JO Eldex D Hooked End Blunt End 58 N a UJ Piston Seal 4 5 PCX5200 User s Manual Chapter 4 Check valves Always work with check valves in a clean area to prevent dust and dirt from entering Cleaning Check valves the pump The check valves are the hexagonal shaped components on the pump head Figure 4 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 1 2 open end wrench 5 32 hex wrench Open end wrenches are not included Cleaning check valves is easy and very effective and should be your first consideration in the event of failure 1 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 Turn off the integrated 2 way valve on the reservoir cap Remove the flow conditioner panels center and right by loosening the captive screws Remove one flow conditioner at a time After removing the captive screw flip the flow conditioner upside down and place it behind the louver in front of the column oven You do not need to disconnect any tubings Repeat the same procedures for the
51. n Guide Retainer 3106 1324 Liquid End Assembly 3107 0137 Reagent bottle coated 1 liter borosilicate with cap for storage 3107 0300 Reagent bottle assembly cap with integrated 2 way valve 2103 0463 Tubing Saran 1 8 OD x 0 063 ID per 3 ft 90 cm 3104 0081 Seal Kit for bypass valve 3101 0060 Nut Fingertight for 1 16 plastic tubing 3102 1202 Nut male Upchurch type 10 32 1 16 3102 2102 Ferrule Upchurch type 1 16 3102 1402 Nut male Valco type 10 32 1 16 3102 2402 Ferrule Valco type 1 16 3103 1030 Tubing stainless steel 1 16 OD x 0 010 ID x 30 cm 2101 0212 Tubing TFE 1 16 OD x 0 011 ID per 3 ft 90 cm 2101 0225 Tubing TFE 1 16 OD x 0 025 ID per 3 ft 90 cm waste line 3101 0007 Nut 1 4 28 x 1 16 3101 0008 Ferrule for 1 4 28 x 1 16 3101 0005 Nut 1 4 28 x 1 8 3101 0006 Ferrule for 1 4 28 x 1 8 3102 1518 Nut Lite Touch for 10 32 1 16 3102 2507 Ferrule Lite Touch for 10 32 1 16 3543 0045 Fuse for PCX5200 120 V 3543 0044 Fuse for PCX5200 240 V App B 1 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
52. nation 2 Connect the reagent line s from the rear of the PCX5200 to the reservoir s Connect the Trione ninhydrin or OPA reagent bottle to the Saran tubing Connect the other 3 3 Pressure interlock Connections 1 Standard 2 Alternate for Amino Acids amp others PCX5200 User s Manual Chapter 3 reagent to the Teflon tubing Each of these tubings has a 1 4 28 nut and ferrule already in place There are three fittings on each bottle cap the reagent tubing goes to the fitting on the side of the bottle cap facing the on off valve Note Ensure that the reagent outlet and gas lines connected to an OPA or Trione reservoir are Saran amber color Saran tubing is necessary because of its low permeability to oxygen 3 Optionally place a small plastic container in case of spills on top of the PCX5200 and put the reservoir s in it Caution The reagent bottles 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 PCX5200 system Replacement 1 L 2 L or 5 L reagent bottles may be ordered directly from Pickering Laboratories You may use either of two ways to connect the pressure sensor to the HPLC system 1 Standard method for most applications and HPLC systems Using 1 16 inch x 0 020 inch ID capillary tubing connect the outlet of the HPLC pump directly to the pressure sensor on the post column panel
53. ner 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 The back up washer is usually 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 10 This step is only necessary if there are signs of corrosion on the stainless steel piston 11 holder in step 7 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 i Outlet Check valve DURAN Compression Ring CID Check valve Figure 4 1 Pump assembly with wash system 1 Back up Washer 2 Piston Seal 3 Wash Cylinder 4 Inlet Port of the Wash System 5 Outlet Port of the Wash System 6 O ring 4 4 PCX5200 User s Manual Chapter 4 12 Insert the installation tool blunt end into the retainer assembly and then int
54. ngs as a current preset program with AorV key preset edit name erase preset Display scrollable list of s n date of manufacture version PCX5200 User s Manual Chapter 2 e ENABLE key enables the power to the heated reactor and column oven The interlock system requires the pressure switch to sense over 35 bar 500 psi before the PCX5200 can be enabled This means that the LC pump must be running before pressing ENABLE can start the PCX5200 When enabled the ENABLE LED turns green e PUMP key controls power to the reagent pump To power the pump the ENABLE LED must be green The pump LED turns green when the pump is on e Pressing the column TEMP key shows the process temperature of the column oven to the nearest degree and one of the three signs lt and gt indicating that the process temperature is less than equal to or greater than the set temperature respectively A flashing indicates the column is heating e Pressing and holding the COLUMN TEMP key shows the set temperature of the column oven To change the setpoint press and hold the coLumn TEMP key and press the A key to increase or press the V key to decrease e Pressing the REACTOR TEMP key shows the process temperature of the reactor to the nearest degree and one of the three signs lt and gt indicating that the process temperature is less than equal to or greater than the set temperature respectively A flashing ind
55. 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 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 O O O O O mm O U aa a a ES ES ES ES O ES Lele SES a a GE sy WW 6 L_ _ O O O O WS 2 m es gt LI N La N 2 O Figure 2 5 One fuse for 120V systems two fuses for 240V systems 2 5 PCX5200 User s Manual Chapter 2 Column Oven The column oven is located in the front of the instrument Simply lift the hinged lid to gain access to it There is space for one analytical and guard column e The heater block is slotted to receive the analytical column 5 10 15 and 25 cm long 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 0 17 mm ID to minimize loss of efficiency Figure 2 6 Column di Ris gt DEA H E Lu Guard column E O O gf
56. o 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 4 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 Optional The piston seal Figure 4 3 in the wash system also requires periodic replacement However it needs not be changed as often as the primary seal Follow steps 8 and 12 if you desire to replace the seal in the wash system 13 Reassemble the pump head and wash system as shown in Figure 4 1 Hold the pump assembly firmly in place and reinstall it on the pump in line with the axis of the piston do not tilt the pump head sideways 14 If the post column pump is a duplex pump repeat steps 6 13 for reagent 2 side 15 Replace the pump tighten the captive screw and reconnect the electrical connection 16 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 17 Open the intergrated 2 way valve on the reservoir and turn on the gas Start the PCX5200 and prime the pump Figure 4 2 Piston seal Seal Back up Washer replacement with Installation Tool Retainer Assy installation tool 9 y
57. olumn and reagent stream flows toward the detector the reproducibility is linked to the flowrate precision of the pumps and to the temperature Accordingly even an incomplete reaction will be as repeatable as the retention time for any given species The completeness of the reaction then is not strictly necessary for reproducibility but it is important for maximum sensitivity e Minimal Detector Response of Reagents The color or background fluorescence of the reagent or its by products represents a continuous noise source Because the reagent is present in excess relative to the analyte the analyte s signal could be obliterated by the reagent s strong background signal The baseline noise is proportional to the background signal 1 1 Pickering PCX5200 Reagent Delivery Column Protection PCX5200 User s Manual Chapter 1 e Solubility All species must remain in solution including the combined components of the eluants and the reagent s as well as the newly formed derivative s Precipitates can block capillary tubes burst reactors and foul detector flowcells e Uniformity of flow The baseline noise is a function of the flow noise in the eluant and reagent pumps Non uniform flow causes non uniform mixing leading to modulation of the background signal which appears as noise Refractive index noise can be even more objectionable than absorbance noise The PCX5200 does three main things Figure 1 1 1 Thermostats the
58. olumn system Place paper towels under the back pressure regulator to absorb any escaping liquid 14 Turn off the gas at the toggle valve of the gas manifold assembly and vent the reservolrs 15 Turn off the main power of the PCX5200 16 Turn off the HPLC system 17 Turn off the inert gas source 3 12 Initial System Testing Test Chromatogram Paramater Log Chapter 4 Routine Maintenance amp Troubleshooting Your Pickering PCX5200 will require some routine maintenance to stay in top condition Ordinarily little maintenance is needed beyond good operating procedures For example keeping the back side of the piston wash system wet with a mixture of water methanol significantly extends seal life 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 PCX5200 as required in the applications Collect two chromatograms to be sure that the system is stable and repeatable 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 paramete
59. on a 4 2 Moble ue EE 4 2 BACK 1IOW PIEVENIION ces lalla 4 2 Reaccion gite iUo PNE a T LE 4 2 Electriical Precaulioni ee tate i dne hatuin dauerte eil 4 2 AGUHN EM AIMT MAM CO es ai TT TEES 4 3 ReagentrRU Nara Aaa 4 3 Pump Seal Replacement i 4 3 Sii VIVES c TH la 4 6 Cleaning Check valves nennen nnne nnnni nnn teke eee eee teen eee tee 4 6 Removing Check valves nassen id 4 7 Installing New Check valves ii 4 7 Reblilding GheekVvalVves ssrne ni ail 4 8 Pre column Filter 02000 Bnnnn anne anne anne nnnnn anne nnnnnnnnne nenne nenne nenne nnnnnnnnnnnnnnennnnnnnnne 4 9 Reagent e ME 4 10 Ambient Reactor not applicable to single reagent PCX5200 ena nenen eee eee nete 4 11 PCX 5200 User s Manual Contents mice blei te WEE 4 11 US e A 4 12 Troubleshooting GUISO naar 4 12 BONS ee RR AA 4 12 When Priming the Reagent Pump is Difficult i 4 13 interpretation of EE 4 14 IWOTeagen RER 52 00blog 4 14 Te EA 4 14 Cleaning the Post column System i 4 15 FIROS eR 4 15 ad TEE 4 15 Rendere 4 15 is Ee EE 4 15 Appendix A POXS200 Parameter COO srl ira A 1 An Eder Ve Leite det A 2 Post column Band spreading Test ee nene pe serie nese rere re pene teve nennen A 3 Appendix B PRAN Siro Pe Ue A e IA B 1 References Limited Warranty Index Before You Start Please Read Please Read Page 2 8 for th
60. onnect it in series with the piston seal wash system on your HPLC so that you can flush them at the same time 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 the piston and the scratched piston will in turn cause the primary seal to fail Je gt l f Waste Syringe and Luer adapter gt Syringe and Luer adapter Figure 3 4 3 8 Priming the Reagent Pump System Testing amp Verification PCX5200 User s Manual Chapter 3 1 Ensure that the reagent and gas supply tubings are connected to their proper fittings on the reservoirs 2 At the start it is best to fill the reservoirs with methanol or a mixture of methanol water 3 Connect a 20 mL disposable syringe to the Luer fitting in the center of one of the prime purge valves 4 Open the prime purge valve 1 2 to 1 full turn CCW and let the flow exit into the syringe 5 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 6 Close the valve remove the syringe and wash the Luer fitting with a little wat
61. or goggles laboratory coat gloves and other appropriate safety clothing Please read and understand the instructions in the MSDS s shipped with the chemicals If the MSDS s are missing please contact Pickering Laboratories and we can instantly fax you a copy Thoroughly clean any leaks from fittings with water and dry with paper towels especially if the solution is a buffer or hydroxide Standing salt and hydroxide solution are corrosive Soak up spills with rags paper towels or sponge Clean spill area with a wet towel and thoroughly dry Do not spray water directly into the instrument Vacuum clean inside the post column system once a year 1 Open the column oven door 2 The reagent flow conditioners can be removed by removing two captive screws Set the flow conditioners upside down in the front of the column oven You do not need to disconnect any tubings 3 The post column panel can be removed by removing two captive screws Set this panel sideway in front of the PCX5200 4 Vacuum clean the inside 5 Reinstall the panels Follow the directions listed above to get inside the system 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 thoroughly dry Do not spray water directly into the electrical part of the post column pump PCX5200 User s Manual Chapter 4 Appendix A PCX 5200 Parameter Log Reagent Pump Calibration Pump 1
62. othetical Na amino acid program with sodium hypochlorite and OPA derivatizations First let us review the parameters 1 2 3 4 m Column oven 55 C Column oven remains on at all time i e not slaved to the pressure interlock Heated reactor 36 C Save as Preset program 5 and title it Na AA OPA To adjust the set temperature of the column oven press and hold the coLumn TEMP key and then press the A or V key to obtain a readout of Column set 55 C Next we want to set up the column oven to warm up automatically By default the power to the column oven is controlled by the ENABLE function Press and hold the coLumn TEMP key then press the PROG key the LCD displays C PID Press the V key twice the LCD displays C RTD first then would display Set interlock Press the PROG key to Enter the Set interlock function Note this is also another example of the PROG key acting like an Enter key The LCD displays Interlock ON Press the V key the LCD displays Interlock OFF Press the PROG key to Exit the Set interlock function PCX5200 User s Manual Chapter 2 Press the V key the LCD displays Back YES Press the PROG key to Exit the Column oven programming functions the LCD displays Save Changes NO Press the A key the LCD displays Save Changes YES Press the PROG key to Accept the changes The LCD now displays Col
63. oubleshooting section in the Application Manual for some easy fixes Generally chemical clogging problems can be solved with chemistry Changing the heated or ambient reactor is your last resort after attempts to clean the reactor have failed 1 Shutdown the post column system and let the reactor cool for at least 30 min 2 Remove the post column panel by loosening the two captive thumb screws 3 Trace the two tubings from the heated reactor to the post column panel and disconnect their fingertight fittings 4 Loosen the two captive thumb screws that hold the right side of the reactor to the mounting bracket 5 Disconnect press and pull the electrical connector to the heated reactor 6 Replace the heated reactor 7 Reconnect the electrical connector 8 Reconnect the captive thumb screws on the reactor bracket 9 Reconnect the two fingertight fittings Do not overtighten The inlet and outlet tubings of the heated reactor are interchangeable 10 Start the LC pump and inspect for leaks 11 Reinstall the post column panel 4 11 Fuse Troubleshooting Guide Advice PCX5200 User s Manual Chapter 4 The line fuse is on the back panel in the power inlet module between the cord connector and the power switch 1 Remove the cord from the power inlet 2 Use a small flat screwdriver to pry up the fuse holder then pull the fuse out 3 Only use the correct type of fuse for the 120 V instrument one each GMA3 type 3 A
64. oxidizing reagent P N 2102 0463 Figure 2 7 E Reagent reservoir A Gas Inlet Reagent Pump 2 8 PCX5200 User s Manual Chapter 2 External Reagent pressure of 45 75 psi 3 5 bar to function properly Pressurization System e Gas is controlled by the toggle valve Lever ON pressurizes the manifold e The gas 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 allows the gas to vent from the relief valve rapidly depleting a gas cylinder e Gas tubing for all reservoirs are 1 8 OD Saran 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 e Reagent tubing is pre connected to the pump at the factory The protuding tubing from the rear of the PCX5200 should be connected to the reagent bottle cap directly facing the shut off valve Connect the Trione ninhydrin or OPA reagent to the Saran tubing and connect the Hydrolysis or Oxidizing reagent to the Teflon tubing Nuts and reversed ferrules 1 4 28 are provided e The reser
65. purposes The first is to allow your data system to communicate with the PCX5200 The second is to download presets and operating parameters to the EEPROM The third is to download new system firmware into the flash memory Pickering Laboratories does not provide software for your chromatography data system you may write your own or use third pary software For a complete description on the RS232 Port please contact Pickering Laboratories Inc Pickering Laboratories may announce free firmware bug fixes or paid firmware enhancements You may use a standard terminal emulation software to download the firmware file to the PCX5200 Instructions for how to do this will accompany the firmware package We need your comments to support the firmware If you discover a bug report it in writing to our Customer Service Department We will also consider your written suggestions for new features or other enhancements This device complies with Part 15 Class A of the FCC Rules Operation is subject to the following two conditions 1 This device may not cause harmful interference and 2 this device must accept any interference received including interference that may cause undesired operation External radio frequency interference may cause improper operation of the PCX5200 Such interference causes incorrect pressure measurements that may result in shutdown conditions Remove the PCX5200 from the source of interference and reset the instrument the PCX520
66. r amp Valco reversed for low pressure 1 4 28 1 Using the 3m tan colored 1 8 inch Saran tubing PN 2103 0463 connect the Gas In port on the external gas regulator to a supply of inert gas at 45 75 psi 3 5 bar When 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 to the ON position to start gas flow Let the gas system purge for about one minute Switch the toggle valve down to OFF 2 Using the 0 5m Saran tubing PN 2103 0463 connect the reservoir to the gas outlet manifold There are three fittings on each bottle cap the gas tubing goes to the fitting on top of the bottle cap Each of these tubings has a 1 4 28 nut and ferrule already in place Plug any unused outlets of the manifold Note Do not use polyolefin or fluorocarbon tubing The Saran tubing is impermeable to oxygen and is used to prevent degradation of oxygen sensitive reagents Fluorocarbon tubing is 1 000 times more oxygen permeable than Saran tubing 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 lint free cellulose tissue Avoid touching the tubings or the interior of the reservoir with your skin and do not leave caps and lines dangling without a reservoir because this will cause contami
67. r 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 4 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 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 4 1 Precautions amp Problem Prevention Mobile Phase Back flow Prevention Reactor Precaution
68. ress the ENABLE button to enable the instrument 3 4 PCX5200 User s Manual dd PCX 5200 gt Lv COLUMN REACTOR REAGENT 1 REAGENT 2 pus r PICKERING TEMP I LABORATORIES leet Model PCX 5200 SOUPE co Post Column Derivatizer STATUS PRES i i i i i i PROG o oy Lo Injector JI IR 500 M 1500 Se 1500 ZN 400 600 1000 2000 1000 2000 200 800 500 2500 500 2500 1000 3000 3000 x J Il POST COLUMN REAGENT 1 REAGENT 2 PRESSURE PRESSURE PRESSURE OVER FADM PRESSURE BYPASS 1 BYPASS 2 INJERTOR RELIEF BS X 9 TO DETECTOR Standard method to connect the Pressure interlock Lid PCX 5200 gt ENABLE D COLUMN REACTOR REAGENT 1 REAGENT 2 775 E PICKERING PUMP TEMP Ser LABORATORIES sud Model PCX 5200 M E MES E E Post Column Derivatizer STATUS PRES I i PROG ea Lo Pac D d Injector 500 1500 ER 1500 ZN 400 600 1000 2000 1000 2000 200 800 500 2500 500 2500 1000 3000 3000 d N A ff POST COLUMN REAGENT 1 REAGENT 2 PRESSURE PRESSURE PRESSURE OVER PRESSURE BYPASS 1 BYPASS 2 RELIEF fc D NS TO DETECTOR Alternate method to connect the Pressure interlock ideal for corrosive eluants Chapter 3 3 5 Injector Connections Detector Connections PCX5200 User s Manual Chapter 3 5 baaHo
69. s Electrical Precaution PCX5200 User s Manual Chapter 4 e Use Pickering Laboratories reagents and eluants The quality of the chemicals is excellent and the cost is low relative to the worth of your analytical results The one year warranty does not cover damage caused by poor quality reagents and eluants not purchased from Pickering Laboratories e Use the proper start up and shutdown procedures consistently see Chapter 1 e 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 e 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 e 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 The PCX5200 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 35 bar before the reagent pump can be engaged The 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
70. second flow conditioner panel 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 1 16 fitting to prevent the check valve from moving Remove the pump from the chassis by loosening the captive screw and unplugging the electrical connector Place the pump in a more accessible place to continue Remove the pump head from the pump as follows Figure 4 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 Reassemble the pump head and wash system as shown in Figure 4 1 Hold the pump assembly firmly in place and reinstall it on the pump in line with the axis of the piston do not tilt the pump head sideways If the post column pump is a duplex pump repeat steps 6 8 for reagent 2 side 4 6 Removing Check valves Installing New Check valves PCX5200 User
71. sideways 4 7 Rebuilding Check valves PCX5200 User s Manual Chapter 4 1 Remove the check valve cartridge from its housing Figure 4 4 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 hammer on the dowel pin Do not allow the valve parts to fall out onto a hard surface 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 4 Press a new Kel F valve seal into the valve housing 5 Slide the check valve cartridge into the valve housing making certain the cartridge is oriented correctly 6 Pressa second valve seal into the valve housing This valve seal will extend approximately 0 02 0 03 from the valve housing Outlet cartridge detail Note orientation Housing outlet valve a Outlet valve insert ie N Outlet valve cartridge IES Outlet A Valve seal check valve Inlet check valve gt Valve seal Inlet valve cartridge Inlet valve insert LI Inlet cartridge detail E Housing inlet valve Note orientation Figure 4 4 Check valve assemblies 4 8 PCX5200 User s Manual Pre column Filter The filter is located inside the fitting label
72. sis Glyphosate and ALKION columns are warranted for 90 days Silica based reversed phase columns are warranted for 30 days How to Obtain Warranty Service If there is a problem with your Instrument or Analytical Column within the Warranty period immediately notify Pickering at 800 654 3330 if calling from outside U S A use 650 694 6700 If the Instrument or Analytical Column was not purchased directly from Pickering please contact the vendor where it was purchased 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 1280 Space Park Way Mountain View CA 94043 U S A A Alkal EEN 3 1 Ambient reactor eene 4 11 Anti siphon valve aaa aaa nana ana ann nene nore nese 1 2 Application kits sa aaa aaa aaa ana e ene enen eee eenese 3 1 B Back flow prevention 4 2 Back pressure regulator 1 3 3 1 3 6 ER cali een 3 1 Bourdon tube cia 1 2 C Calibration column oven reactor 2 19 CUO EE 3 4 3 6 Check valves cce eee eene nne 4 6 Cleaning check valves
73. some time to leak down to zero Follow these procedures to avoid such accidents e Never disconnect any fittings between the HPLC pump and the column until the post column system has been shut down and depressurized loosen the to dectector fitting e Any leaky fittings between the HPLC pump and the column can permit backflow in the event of an unattended shutdown e When removing the column remove the outlet fitting first e Always follow the proper shutdown procedures See Chapter 1 e Do not operate the heated reactor above the boiling point of the eluant unless the external 100 psi back pressure regulator is connected to the waste line of the detector Boiling inside the reactor causes precipitates to form e Do not operate the reactor above 130 C This can weaken and deform the PTFE tubing e Do not operate with a post column pressure above 600 psi e Always use the correct fuse 4 2 PCX5200 User s Manual Chapter 4 Routine The PCX5200 uses a custom made Eldex reagent pump with piston wash The piston Maintenance seals require periodic replacement The length of service to be expected from the seal Reagent Pump depends on a wide variety of factors including whether proper shutdown procedures AAA were followed how often the system was turned on and off and whether the piston wash system was wetted It is critically important that the seal be replaced immediately upon failure or better yet before failure be
74. te Pressurized en Reagent Bottles Figure 1 1 gt Pickering post column systems one and two reagent 1 3 PCX5200 User s Manual Chapter 1 The Elements of This next section is a simplified view of a post column system and it is intended to help an HPLC with novice HPLC operators Post column Sytem Design of an HPLC In order to understand post column HPLC we need to understand the design of an HPLC If we connect an HPLC pump directly to a detector with nothing in between the baseline from the detector shows a periodic noise Figure 1 2 the time period is equivalent to the pump stroke Injector Detector LC Pump Baseline Waste Figure 1 2 Een Now add a commercial pulse damper The baseline is still not smooth the periodic noise is still there although less pronounced Figure 1 3 Pulse Detector SHA AJ Baseline LC Pump Waste Figure 1 3 cante 1 4 PCX5200 User s Manual Chapter 1 What we need is a column The column does more than separation It creates a back pressure It is the combination of the pulse damper and the column that creates a smooth baseline Figure 1 4 Pre column Filter Thermostatted Column amp Guard Baseline Waste Figure 1 4 Eluants A Simpler View Let us use a river as an example If it rains the river swells If it stops raining the level of an HPLC goes down As the level fluctuates it is equivalent to a periodic noise To obtain a
75. the brand of HPLC pump and detector in use Minimum benchtop space required for the Pickering system is approximately 17 inches 42 cm long by 17 inches 42 cm 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 reservoir s The PCX5200 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 Trione ninhydrin or OPA reagent use oxygen impermeable tubing for the entire gas supply line Saran or metal Unless the Installation and Training service has been purchased the user will need to provide adequate lengths of capillary tubing to connect HPLC pump and injector to pressure sensor 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 The user also needs to check the chemistry requirement for the specific application Placement of the LC system and detector can be on either side of the PCX5200 For most cases it is best to place the LC system on the left side of the PCX5200 The connections for reagent lines are from the rear The gas manifold and the reservoir s can be placed on top The column oven is in the front of the instrument the oven door swings up about 5 inches 12 cm
76. umn C lt The flashing symbol indicates that the column oven is heating up 3 To adjust the set temperature of the heated reactor press and hold the REACTOR TEMP key and then press the A or V key to obtain a readout of Reactor set 36 C 4 Press and hold the PRESET key and press the PROG key the LCD shows Save preset Press the PROG key to Enter the Save preset function the LCD shows 1 S Carbamate Since we do not want to write over this program press the V key until the LCD shows 5 S Note that program 5 may or may not have a name assigned to it yet If it is already in use the LCD would show 5 S name of program If that is the case press the A or V key until the LCD shows just S If all five preset programs are filled you need to write over one of them If you do not want to override any of the five preset programs press the V key until the LCD shows Back YES and press the PROG key to exit Press the PROG key to save the presently set conditions as a Preset Program Once again this step will override any previously set conditions for this particular Preset program The LCD shows 5 with a flashing cursor after it Press the A key to choose the letter N Press the 1 gt key to move the flashing marker to the next space Press the A key to choose the letter a Lower case characters come after the upper case characters Repeat until the desired n
77. voir 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 On the side of the cap away from the on off valve there is a 1 4 28 fitting you may optionally connect a tube here to carry vapors to an exhaust vent e When changing reagent first turn off the gas using the toggle valve on the manifold 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 The keypad and liquid crystal display LCD user interface instructions are in three complementary parts The first part is a general description of the keys and their Gas outlets S 3 I Figure 2 8 Gas Pressure System C C o o d d On off switch s Gas inlet 2 9 Keypad and Liquid Crystal Display User Interface PCX5200 User s Manual Chapter 2 functions the second part is comprised of examples the third part shows diagrams The keypad has eleven keys five LEDs and a liquid crystal display For routine operation of the instrument there are preset parameters to get the system up and running in minimum time The keys can be divided into two groups 1 Dire
78. w seconds If it does not begin pulsing within 30 sec repeat steps 3 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 Interpretation of Pressures 3 Two reagent PCX5200 One reagent PCX5200 PCX5200 User s Manual Chapter 4 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 Note These pressures are typical and may vary slightly depending on the system and the application It is very important to know the initial pressures specific to your system Condition Normal Pre column filter blocked Heated reactor obstructed Ambient reactor obstructed Reagent 1 not pumping Reagent 2 not pumping Restrictor 1 blocked Restrictor 2 blocked Condition Normal Pre column filter blocked Reactor obstructed Reagent not pumping Restrictor blocked Column 1200 T T Column 1200 T T Post Column Reagent 1 Reagent 2 250 1500 1500 te gt gt gt gt gt Post Column Reagent 250 1500 gt gt There is also detailed section on troubleshooting in the Application Manual 4 14 Cleaning the Post column System Fittings External Internal Internal Spill PCX5200 User s Manual Chapter 4 Always wear safety glasses
79. wever the default power off to the column oven can be overriden so that the column oven remains on independent of the post column interlock Enable function 1 Caution Operating the reagent pumps when the HPLC pump is not producing eluant 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 eluant flow can cause precipitation and complete blockage of the post column reactor Connect the outlet of the injector or autosampler to the bulkhead fitting 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 5 um filter element The bulkhead fitting is made of PEEK use the PEEK nut and ferrule that comes with the PCX5200 to avoid damaging the fitting 1 Install the HPLC detector referring to the manufacturer s manual supplied with the instrument 2 Connect the inlet of the detector flowcell to the bulkhead union labelled To Detector Use 1 16 inch x 0 010 inch ID capillary tubing Note to Hewlett Packard 1046A end users Replace the 0 12 mm 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 25 mm ID tubing HP Cat No 79881 67302 or Pickering Cat No 3110 6045 blue tubing to reduce the back pressure 8 Connect a 1 16 inch x 0 020 inch ID tu

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