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Agilent 1200 Series Fluorescence Detector

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1. Analog of Oo connector nuts its A lt gt _ CH NG de etezto ll J SS 4 L sy GERS 9 Remote SS ts Oe a a as a a Z S T 57 5 Bi GPIB a pele ee es fe ae ae 9 A e s SS Sas SS A EBA ER CN a 5 l gt K J gt lt b C ERGA Ge i T AEN IE GE Ha E a iy SS E n S a L eee Zo C J om 2 a ae J S Board recesses gt Figure 77 Unscrew Connectors from Board 4 Remove the front cover top cover and top foam section see Removing the Covers on page 198 5 Use a 5 mm and 7 mm wrench to unscrew the REMOTE and the GPIB connector and a 15 mm wrench to unscrew the nuts of the ANALOG connectors 212 1200 Series FLD Service M anual 6 Disconnect all connectors from the processor board When removing connectors counter hold with one hand on connector J 12 J 1 GPIB J 2 RS 232C J 3 REMOTE J 4 5 Analog output J6 7 CAN J11 J12 J14 J17 J18 J 20 J 21 J 22 Power supply EM Monochromator EX M onochromator NOT USED FLF board EM Monochromator Interface board Fan B Battery S1 Configuration switch eee mE T dL flo ECO J1 6e 7 2 J3 B J14 J12 J11 J17 J 18 il J20 m J21 Repairs 11 Figure 78 Location of Connectors on FLM Board 7 Remove the processor board Place the board on the ESD kit 8 On the new board check the switch setting of address switch S1 see
2. For correct assembling of the top and sides see Assembling the M ain Cover on page 256 Figure 85 Plastic Parts 1200 Series FLD Service M anual 273 13 Parts for Repair Foam Parts Table 38 Foam Parts Item Description Part Number 1 2 EPP foam kit includes bottom and top rear G1321 68702 3 Foam Pads located underneath the optical set of two G1321 68700 4 Guides for interface board 5041 8395 Do not order the individual part numbers imprinted on the foam Figure 86 Foam Parts 274 1200 Series FLD Service M anual Power and Status Light Pipes Table 39 Power and Status Light Pipes Item Description Power supply assembly Screw M4 x 0 7 8 mm lg to fix power supply at rear panel Washer 1 Power light pipe Status light pipe Power switch button gt _ gt WwW N Coupler for power supply actuator Parts for Repair 13 Part Number 0950 2528 0515 0910 2190 0409 5041 8382 5041 8384 5041 8381 5041 8383 Figure 87 Power and Status Light Pipe 1200 Series FLD Service M anual 275 13 Parts for Repair Leak Parts Table 40 Leak Parts Item 1 O Aa SP U N Description Leak sensor assembly Leak pan Leak funnel Leak funnel holder Corrugated tubing 120 mm lg re order 5 m Corrugated tubing 1200 mm lg re order 5 m Part Number 5061 3356 G1321 40511 5041 8388 5041 8389 5062 2463 5062 2463 Figure 88 Leak Parts 276 1200 Series FLD Service M anual
3. Agilent 1200 Series Fluorescence Detector G1321A Service M anual ot Agilent Technologies Notices Agilent Technologies Inc 2006 No part of this manual may be reproduced in any form or by any means including elec tronic storage and retrieval or translation into a foreign language without prior agree ment and written consent from Agilent Technologies Inc as governed by United States and international copyright laws M anual Part Number G1321 90110 Edition 02 06 Printed in Germany Agilent Technologies Hewlett Packard Strasse 8 76337 Waldbronn Germany M anual Structure The Service M anual G1321 90110 English contains the complete information about the Agilent 1200 Series Fluorescence Detector It is available as Adobe Reader file PDF only The User M anual G1321 90010 English and its localized versions contain a subset of the Service M anual and is shipped with the detector in printed matter Warranty The material contained in this docu ment is provided as is and is sub ject to being changed without notice in future editions Further to the max imum extent permitted by applicable law Agilent disclaims all warranties either express or implied with regard to this manual and any information contained herein including but not limited to the implied warranties of merchantability and fitness for a par ticular purpose Agilent shall not be liable for errors or for i
4. 10 Load the method WLEMTEST The FLD will change into the multi excitation mode and scan in the range of the expected maximum of 350 nm 20 nm 11 Repeat steps step 6 to step 8 ees ORE ONES OMe 0 00 MRE Ready Emission Figure 66 12 You can now select a stored spectra and evaluate print them The maximum should be found at 350 nm 3 nm The spectra on the control module is a real raw data spectrum Therefore it looks not that Smooth as on the Agilent ChemStation uses the spline algorithm If the limits are not met check for Interpretation of the Results on page 147 or perform Wavelength Calibration Procedure on page 164 1200 Series FLD Service M anual 163 8 Test Functions Wavelength Calibration Procedure When required If application requires or see Table 25 on page 153 Tools required Laboratory balance Parts required Glycogen Calibration Sample Syringe needle sample filter and PEEK fitting from the Accessory Kit see Accessory Kit on page 263 Steps 1 Preparation of the Glycogen Calibration Sample 2 Preparation of the Flow Cell 3 Wavelength Calibration 4 Verification using Check of Raman Band of Water on page 156 Preparation of the Glycogen Calibration Sample 1 To prepare 10 ml of the calibration solution you have to use 10 mg of the Glycogen sample a tolerance of 20 is not critical 2 Fill the prepared amount into a suitable bottle vial 3 Fill 10 ml of distilled water
5. 2 Select System Tests FLD 3 Select the function Enable test chromatogram and press Execute The logbook shows Simulated chromatogram enabled 4 Enter the Plot function and select Signal A and or Analog x set a time range of 10 minutes and the FLD signal from 1 to 50 LU 1200 Series FLD Service M anual 149 8 Test Functions If an Agilent ChemStation is connected the next step will start the Agilent ChemStation simultaneously and will overwrite the previous used Data File 5 Start the run After the run has finished the plot can be printed to a connected printer by pressing m and selecting Print Plot The test chromatogram is switched off automatically at the end of the run 150 1200 Series FLD Service M anual Test Functions 8 Using the Built in DAC Test This function is available from the Control Module G1323B ONLY The DAC test provides a test pattern as an analog output The output voltages analog 1 and analog 2 should show a constant value corresponding to the ZERO OFFSET value In addition to the constant voltage is a switched voltage with a duration of 12 seconds and a height of 10 uV see Figure 58 LISti ZERO Ses 31 614 LIST ATT 2 5 lt 4 CHT SF 5 i PLOT Limits Noise pp lt 5 pV STOP Figure58 DAC Test Example 1200 Series FLD Service M anual 151 8 Test Functions 1 Enable the function System Tests FLD Enable DAC Test Pattern 2 Start the plot mode
6. On board Battery An on board lithium battery buffers the electronic memory when the module is turned OFF For safety information on lithium batteries see Lithium Batteries Information on page 331 Analog Outputs There are two independent analog outputs 0 1 V full scale Digital data from the ASIC is converted into a pulse width modulated signal PWM The PWM signal is then fed to an amplitude modulator which precisely turns the reference signal on and off This signal then passes to a low pass filter section Interfaces For detailed information on interfaces see Interfaces on page 315 1200 Series FLD Service M anual 297 15 Hardware Information Detector Frontend Board FLF 298 Data Acquisition The FLF board provides circuitry to measure three different physical effects e Fluorescence mode peak value after flash e Phosphorescence mode integrated value after flash e Chemoluminescence mode amplified signal without flash operation In parallel to the fluorescence signal the reference signal is acquired to get a compensation of lamp drift and noise effects Both signals are amplified and buffered and routed to the ADC input multiplexer The 16 bit A D converter performs a conversion of the selected signal and additionally of the corresponding reference signal and several other signals e monochromator positions e board temperature e leak sensor status Then the digital data are polled by the
7. Service M anual 00 ee 4 First Steps with the Detector r Before You Start 62 Optimization Overview 63 Getting Started and Checkout 65 Method Development 69 Example Optimization for Multiple Compounds 84 This chapter guides you how to start the work with the detector ee Agilent Technologies 61 4 First Steps with the Detector Before You Start 62 Your normal LC grade solvents usually give good results most of the time But experience shows that baseline noise can be higher lower signal to noise ratio when impurities are in the solvents Flush your solvent delivery system for at least 15 minutes before checking sensitivity If your pump has multiple channels you should also flush the channels not in use 1200 Series FLD Service M anual First Steps with the Detector 4 Optimization Overview 1 Setting the right PMT value For most applications a setting of 10 is adequate The G1321A A D converter exhibits a large linear range making PMT switching unnecessary for most applications For example if at high concentrations a peak is cut off decrease the PMT setting Remember that low PMT settings decrease the signal to noise ratio The built in PMT gain test uses the parameters in the detector When using the PMT gain test the wavelength setting and lamp energy mode depending on Multiwavelength Mode and Lamp Economy will affect the pmt gain calculation If you have changed one or
8. Setting the 8 bit Configuration Switch on page 320 An incorrect switch setting for example TEST BOOT may cause the module to revert to a basic mode yellow or red flashing status light In this case turn OFF the module reset the address switches and turn ON the module again 1200 Series FLD Service M anual 213 11 Repairs 9 Install the new processor board and reconnect the connectors Assure that the board is fitted correctly into the board recess in the rear panel 10 Refit the screws at the REMOTE and GPIB connectors and the nuts of the ANALOG connectors 11 Reinstall the top foam section top cover and front cover see Replacing the Foams and Covers on page 254 12 Replace detector into the stack and reconnect the cables If anew FLM board is installed update the serial number information of the detector in the user interface see procedure below 13 Check the firmware revision of the module If it is older than the current firmware revision update the firmware using the standard firmware update procedure see Replacing the Detector s Firmware on page 185 214 1200 Series FLD Service M anual Repairs 11 Changing the Type and Serial Number When required If detector main board has been replaced Tools required User interface Parts required none Preparations Turn the detector on e Start the user interface When the main board has to be replaced the new board does not have serial numbe
9. 10 Maintenance Tests amp Calibrations The following tests are required after maintenance of lamps and flow cells e Lamp Intensity Test on page 187 e Wavelength Verification and Calibration on page 189 186 1200 Series FLD Service M anual Maintenance 10 Lamp Intensity Test When required If the flow cell or lamp has been replaced Tools required None Pre requisites clean flow cell flushed The intensity test scans an intensity spectrum via the reference diode 200 1200 nm in 1 nm steps and stores it in a diagnosis buffer The scan is displayed in a graphic window There is no further evaluation of the test Results of this test are stored as lamp history date code intensity Instrument Gl32l1A Serial Number DES2001563 Operator Wolfgang Date 09 01 2006 Time 11 26 30 File C CHEM32 2 DIAGNOSE FLD INT DGR Intensity counts 14000 12000 10000 8000 6000 1000 Wavelength nm Figure 73 Lamp Intensity Test Report 1200 Series FLD Service M anual 187 10 Maintenance The profile can vary from instrument to instrument It is dependig on the age of the lamp and the content of the flow cell use fresh water UV degradation especially below 250 nm is significantly higher compared to visible wavelength range Generally the LAMP ON during run setting or using economy mode will increase lamp life by a magnitude Lamp Intensity History Results of the lamp intens
10. Stoptime m a St min a Eu Multi Em Signal Time In this example 246 nm ie jid nm 2 Use additional Excitation additional excitation PGi Sates 5 Seton eee a aol J m B 230 nm wavelengths B C C 250 nm D are used This will D f 230 am increase the scan time and may lower Acquire Excitation Spectra the performance All 7 Range 230 to 400 nm Step Eo nm Thrashad 100 tii Figure 25 FLD Parameters 3 Start the run 4 The resulting chromatograms are shown in Figure 26 FLD1 B Ex 230 Em 317 FLD_ISO1FLD_ISO2 D FLD1 A Ex 246 Em 317 FLD_ISO4 FLD_ISO2 D tine 3 aia Gesah FLD_ISO1FLD_IS02 D Ex 250 nm A FLD_ISO1FLD_IS02 D T Ex 246 nm 200 i l 175 Biphenyl peak 150 Ex 230 nm ly pneny p 125 I 100 l ae 75 i i Je A Li Ex 290nm s 23B O ua oR E R a T Ot ae i Ea E OT a aa a 9 a E a a eee E ee E ee ae EEI E EET S 0 0 0 5 4 15 2 25 3 3 5 4 m Figure 26 Biphenyl peak with different excitation wavelengths 5 The excitation maxima is around 250 nm 1200 Series FLD Service M anual 67 4 First Steps with the Detector Observe the maxima via the isoabsorbance plot 1 Load the data file Apy 246 nm Agy 317 nm and open the isoabsorbance plot 2 The maximum Ay will be found around 250 nm Figure 27 Isoabsorbance Plot 68 1200 Series FLD Service M anual First Steps with the Detector 4 M ethod Development Fluorescence detectors are
11. The spectra acquisition mode allows automatic storage of spectra during a run The mode can be changed during the run by time programming The modes in detail are Table 43 Spectra Acquisition M odes M ode none apex all in peak all all without signals 1200 Series FLD Service M anual Description no spectra is stored if apex of a peak is detected one spectra is measured and stored In addition the next baseline spectra is stored The peak detectors will start and stop spectrum acquisition at begin and end of a detected peak In addition the next baseline spectra is stored periodically all spectra are stored The period depends on the spectra range and step and the number of selected chromatographical outputs Signals for screening all flashes are used for spectra acquisition no concentration connection is done as chromatographical signal the mean of each spectra is output stored 311 15 Hardware Information Optional Interface Boards 312 The Agilent 1200 Series modules have one optional board slot that allows addition of an interface board to the modules Table 44 Optional Interface Boards Description Part Number BCD Board G1351 68701 Fuse 250 mA four are on the board 2110 0004 LAN Communication Interface Board G1369A or G1369 60001 BCD Board The BCD board provides a BCD output for the bottle number of the Agilent 1200 Series autosampler and four external contacts The external
12. Use a flat screw 2 Unscrew screws A and B and disconnect connectors C driver to release the wires 237 1200 Series FLD Service M anual 11 Repairs 4 Remove the FLF board carefully in an upwards motion and 5 If you need to replace either the board or the PMT place it on an ESD protected mat assembly remove the PMT assembly from its socket 6 Replace the PMT assembly or the FLF board PMT assembly 7 Re install the PMT assembly in the socket Safety actuator FLR board REF diode 8 Replace the FLF board and fix the screws A and B and 9 Check that the safety switch is positioned correctly Look connectors C and D through board and casting 238 1200 Series FLD Service M anual Repairs 11 10 Reconnect the wires to the connector Use a flat screw 11 Put the wires into the released contacts using the driver to release the wire socket tweezers Order of wires left to right Set ie Ter Next Steps Reinstall the optical unit as described in Installing the Optical Unit on page 252 If you replaced a G1321 66501 FLF board by a newer FLF board update the FLD firmware to revision to latest version to make it compatible 1200 Series FLD Service M anual 239 11 Repairs Replacing FLL board and Trigger Pack When required If defective Tools required Screwdriver POZI 1 PT3 Hexagonal key 4mm and 2 5 mm Parts required FL
13. for lamp housing 0515 2548 Screw M4x 8 mm for FLF board 0515 2549 1200 Series FLD Service M anual 269 13 Parts for Repair 18 Cable chassis 19 Screw chassis 1 FLL Board Trigger Pack 16 Carbon filter mat 2 Xenon Flash Lamp below pad a GI A 7 Emission M onochromator i 9 Slit EM PMT aL l 3 Condenser EX 9 Slit EM PMT k 4 Slit EX YU DIE 10 Photomuliplier 15 Cutoff Filter 5 Mirror f K 11 Condenser EM 6 Excitation M onochromator 12 FLF Board underneath 8 Flow Cell 13 Reference diode FLR 14 Diffuser plate Figure 82 Optical Unit Parts 270 1200 Series FLD Service M anual Parts for Repair 13 Lamp Cover Parts Table 35 Lamp Cover Parts Item Description Part Number 1 Plate G1321 08100 2 Screw 2 5 mm hex 0515 1052 3 Quartz Window 1000 0999 Figure 83 Lamp Cover Parts 1200 Series FLD Service M anual 271 13 Parts for Repair Sheet M etal Kit Table 36 Sheet M etal Kit Parts Item Description Part Number 1 Sheet metal kit includes case and top cover G1321 68701 2 Screws M 3 for cover and flow cell door 5022 2112 Figure 84 Sheet Metal Kit Parts 272 1200 Series FLD Service M anual Parts for Repair 13 Plastic Parts Table 37 Plastics Parts Item Description Part Number 1 Front cover 5065 9982 2 Plastics includes base sides and top 5065 9985 3 Name plate Agilent 1200 Series 5042 8901
14. 0503 302 1200 Series FLD Service M anual Hardware Information 15 Detector Lamp Supply Board FLL FLL Board Lamp Voltage Lamp Energy Trigger Xenon Flash Ground Capacitor Pac Lamp Trigger Voltage Lamp Ignition Trigger Control Control Figure91 Block Diagram FLL Board Lamp Energy Capacitors The lamp high voltage circuit of the FLF board is used to charge the 4 energy capacitors of the FLL board The energy per flash depends on the applied charging voltage 1 2 C U A power diode freewheeling circuit prevents the backflowing current from generating negative lamp discharge currents which may damage the flash lamp Lamp Ignition Control An ignition voltage is switched by a thyristor to the TriggerPac ignition transformer to built a small ionized channel refer Igniting the Flash Lamp on page 299 A freewheeling diode forces the thyristor to switch OFF again to prevent lamp damage 1200 Series FLD Service M anual 303 15 Hardware Information Firmware Description The firmware of the instrument consists of two independent sections e anon instrument specific section called resident system e an instrument specific section called main system Resident System This resident section of the firmware is identical for all Agilent 1200 Series modules Its properties are e the complete communication capabilities GPIB CAN LAN and RS 232C e memory management e ability to update the firmware o
15. 1 WoO Co N OA oO A U N e e e eA eae e Uo A Wi N eaea O Signal Name EXT 1 EXT 1 EXT 2 EXT 2 EXT 3 EXT 3 EXT 4 EXT 4 Not connected Not connected Not connected Not connected Not connected Not connected Not connected 1200 Series FLD Service M anual Cables 14 RS 232 Cable Kit This kit contains a 9 pin female to 9 pin female Null Modem printer cable and one adapter Use the cable and adapter to connect Aligent Technologies instruments with 9 pin male RS 232 connectors to most PCs or printers Agilent 1200 module to PC RS 232 Cable Kit 34398As Instrument PC RX 2 2 RX TX 3 4 3 TX DTR 4 4 DTR GND 5 y 5 GND DSR 6 6 DSR RTS 7 7 RTS CTS 8 me 8 CTS RI 9 9 RI DB9 DB9 DB9 DB9 Male Female Female Male 1200 Series FLD Service M anual 293 14 Cables LAN Cables 294 Recommended Cables Table 42 Description Cross over network cable shielded 3 m long for point to point connection Twisted pair network cable shielded 7 m long for hub connections Part number 5023 0203 5023 0202 1200 Series FLD Service M anual Agilent 1200 Series Fluorescence Detector Service M anual 15 Hardware Information Detector Main Board FLM 296 Detector Frontend Board FLF 298 Detector Lamp Supply Board FLL 303 Firmware Description 304 Raw Data Conversion to Fluorescence LU Units 306 Optional Interface Boards 312 Interfaces 315 Setting the 8 bit Con
16. 100 Track amp Hold Ignite kes aes ste Ne Se 0 l 2 3 Time usec Figure 12 Measurement of Fluorescence 28 1200 Series FLD Service M anual Introduction to the Fluorescence Detector 1 Phosphorescence Detection An appropriate parameter set will be specified as soon as you chose the phosphorescence detection mode special setpoints under FLD parameter settings Flash Intensity 100 Phosphorescence 0 Measurement Time usec Figure 13 Measurement of Phosphorescence 1200 Series FLD Service M anual 29 1 30 Introduction to the Fluorescence Detector Processing of Raw Data If the lamp flashes at single wavelength and high power then the fluorescence data rate is 296 Hz That means that your sample is illuminated 296 times per second and any luminescence generated by the components eluted from the column is measured 296 times per second If the economy or multi wavelength mode is set then the flash frequency is TA Hz flash LAMP fluorescence phosphorescence ie time Figure 14 LAMP Frequency of Flash Fluorescence and Phosphorescence You can improve the signal to noise characteristics by disabling the economy mode Disabling the economy mode will shorten the lifetime of the lamp significantly Consider lifetime saving by switching off the lamp after the run is completed The data resolution is 20 bit at a response time
17. 2 While in the Tests screen press m m m dot m 3 From the box now displayed select the Command and press Enter 4 Into the box labeled Nester instruction enter the command TYPE XXXXX Letters and numbers are created using the up and down arrows XXXXX is the 5 character product number of the module being changed There must be a space between the word TYPE and the product number Examples TYPE G1321A to configure as a FLD If you enter the wrong type your module might not be accessible anymore with the Agilent ChemS tation In such a case re enter the TYPE command correctly 5 Now press the Execute key Below the box a reply line should then say Reply RA 0000 TYPE XXXXX XXXXX is what you just entered Turn the detector off then on again Turn on should be normal In the Records screen the product column should indicate the module you just entered If an Agilent ChemStation is also connected re boot it now 1200 Series FLD Service M anual Repairs 11 Exchanging the Fan When required If the fan is defective or noisy Tools required Screwdriver POZI 1 PT3 Parts required Fan assembly 3160 1017 The fan must be installed in the correct orientation to ensure optimum cooling and CAUTION operation of the detector Preparations for this procedure 1 Disconnect the emission and the excitation cables and from the detector main board Turn OFF the detector Disconnect the power cable Remove the flow cell Remov
18. 250 mA total of 4 are on the board Fan assembly Optical unit exchange assembly for additional optical unit parts see page 268 Xenon flash lamp M irror Excitation M onochromator assembly complete Emission M onochromator assembly complete Photomuliplier tube assembly PMT see also page 262 for other types Condenser EM same as EX Standard flow cell 8 ul 20 bar see also page 268 for cell screws Leak sensor assembly and Leak handling parts see page 276 Cable FLM FLF board comes with optical unit Front cover and plastic parts housing see page 273 Sheet metal parts see page 272 Foam parts see page 274 1200 Series FLD Service M anual Parts for Repair Part Number G1321 66500 G1321 69500 0380 0643 1251 7788 2940 0256 2190 0699 5181 1516 0950 2528 G1351 68701 G1369 60001 2110 0004 3160 1017 G1321 69002 2140 0600 1000 1000 G1321 60003 G1321 60004 1970 0201 1000 1124 G1321 60005 5061 3356 G1321 61600 13 267 13 Parts for Repair Optical Unit Assembly Table 34 Optical Unit Assembly Item Description Part Number Optical unit new comes with foam part and CableFLM FLF G1321 66002 Optical unit exchange assembly comes with foam part and CableFLM FLF G1321 69002 1 Flash Lamp Board FLL includes trigger pack G1321 66512 2 Xenon flash lamp 2140 0600 3 Condenser Excitation 1000 1136 4 Slit EX 2x4 G1321 08103 5 Mirror 1000 1000 6 Excitation M onochromator assembly complete G132
19. 332 Sound Emission 333 UV Radiation UV lamps only 334 Solvent Information 335 1200 Series FLD Service M anual 1200 Series FLD Service M anual Agilent Technologies on Internet 337 13 14 1200 Series FLD Service M anual Agilent 1200 Series Fluorescence Detector Service M anual 1 introduction to the Fluorescence Detector Introduction to the Detector 16 How the Detector Operates 17 Raman Effect 20 Optical Unit 21 Analytical Information From Primary Data 28 Electrical Connections 33 Instrument Layout 35 Early M aintenance Feedback EMF 36 This chapter gives an introduction to the detector instrument overview and internal connectors ee Agilent Technologies 15 1 Introduction to the Fluorescence Detector Introduction to the Detector The detector is designed for highest optical performance GLP compliance and easy maintenance It includes the following features e flash lamp for highest intensity and lowest detection limit e multi wavelength mode for on line spectra e spectra acquisition and simultaneous multi signal detection e optional cuvette is available and can be used for off line measurements e easy front access to flow cell for fast replacement and e built in wavelength accuracy verification For specifications see Performance Specifications on page 43 Figurel The Agilent 1200 Series Fluorescence Detector 16 1200 Series FLD Service M anual How the Detector Operat
20. 4 Detector Checklist Description Quantity Detector 1 Power cable 1 CAN cable 1 Flow cell 1 built in Optional flow cell cuvette as ordered User Manual 1 Accessory kit see Table 5 on page 49 1 48 1200 Series FLD Service M anual Installing the Detector 3 Detector Accessory Kit Contents Table5 Accessory Kit Contents Part Number G1321 68705 Description Part Number Quantity Teflon Tubing flexible i d 0 8 mm flow cell to waste 5062 2462 2m re order 5m Corrugated tubing to waste re order 5 m 5062 2463 1 2 m Fitting male PEEK 0100 1516 2 Capillary column detector one side preinstalled G1315 87311 1 380 mm lg 0 17 mm i d includes Ferrule front SST 0100 0043 1 Ferrule back SST 0100 0044 1 Fitting SST 79814 22406 1 Hex key set 1 5mm 8710 0641 1 Screwdriver hexagonal 4mm 100 mm long 5965 0027 1 Screwdriver hexagonal 2 5 mm 100 mm long 5965 0028 1 Needle Syringe 9301 0407 Glass Syringe 9301 1446 Calibration Sample Glycogen 5063 6597 Sample filter diameter 3 mm pore size 0 45 um 5061 3367 5 pack of 100 Wrench open end 1 4 5 16 inch 8710 0510 1 1200 Series FLD Service M anual 49 3 Installing the Detector Fitting male PEEK Tubing Figure19 Waste Tubing Parts ay Ferrule front O a 7 L This side is Saas preinstalled Fitting male SST Capillary Figure 20 Inlet Capillary Column Detector Parts 50 1200 Series FLD Service M anual Installing the Detector 3 Optimizi
21. Agilent 1200 Series Fluorescence Detector Service M anual 14 Cables Cable Overview 278 Analog Cables 280 Remote Cables 283 BCD Cables 288 Auxiliary Cable 290 CAN Cable 291 External Contact Cable 292 RS 232 Cable Kit 293 LAN Cables 294 This chapter provides information on cables used with the 1200 series of HPLC modules ee Agilent Technologies 277 14 Cables Cable Overview 278 Never use cables other than the ones supplied by Agilent Technologies to ensure proper functionality and compliance with safety or EMC regulations Table 41 Cables Overview Type Analog cables Remote cables Description 3390 2 3 integrators 3394 6 integrators 35900A A D converter General purpose Spade lugs 3390 integrator 3392 3 integrators 3394 integrator 3396A Series integrator 3396 Series II 3395A integrator see page 285 3396 Series III 3395B integrator Agilent 1200 1100 1050 modules 1046A FLD 1046A FLD 35900A A D converter 1090 liquid chromatographs Signal distribution module Part Number 01040 60101 35900 60750 35900 60750 01046 60105 01046 60203 01046 60206 01046 60210 03394 60600 03396 61010 5061 3378 5061 3378 5061 3378 01046 60202 01046 60202 1200 Series FLD Service M anual Table 41 Cables Overview continued Type Description BCD 3396 integrator cables General purpose Spade Lugs Cables 14 Part Number 03396 60560 G1351 81600 Auxi
22. Condensation will damage the system electronics If your detector was shipped in cold weather leave it in its box and allow it to warm up slowly to room temperature to avoid condensation 1200 Series FLD Service M anual 41 2 Site Requirements and Specifications Physical Specifications Table 2 Type Weight Dimensions width x depth x height Line voltage Line frequency Power consumption Ambient operating temperature Ambient non operating temperature Humidity Operating altitude Non operating altitude Safety standards IEC CSA UL EN 42 Physical Specifications Specification 11 5 kg 26 Ibs 345 x 435 x 140 mm 13 5 x 17 x 5 5 inches 100 240 VAC 10 50 or 60 Hz 5 180 VA 70W 239 BTU 0 40 C 32 104 F 40 70 C 4 158 F lt 95 at 25 40 C 77 104 F Up to 2000 m 6500 ft Up to 4600 m 14950 ft Installation category Il pollution degree 2 For indoor use only Comments Wide ranging capability Maximum Non condensing For storing the detector 1200 Series FLD Service M anual Site Requirements and Specifications 2 Performance Specifications Table3 Performance Specifications Agilent 1200 Series Fluorescence Detector Type Detection type Performance Specifications Light source Pulse frequency Excitation M onochromator Emission M onochromator Reference System Specification Comments M ulti signal fluorescence detector with
23. Detector 173 Using the ESD Strap 174 This chapter provides general information on maintenance and repair of the detector ee Agilent Technologies 169 9 Maintenance and Repair Introduction into Repairing the Fluorescence Detector 170 Simple Repairs The detector is designed for easy repair The most frequent repairs such as flow cell change can be done from the front of the detector with the detector in place in the system stack These repairs are described in Maintenance on page 175 Exchanging Internal Parts Some repairs may require exchange of defective internal parts Exchange of these parts including flash lamp requires removing the detector from the stack removing the covers and disassembling the detector The security lever at the power input socket prevents the detector cover from being removed when line power is still connected These repairs are described in Repairs on page 195 1200 Series FLD Service M anual Maintenance and Repair 9 Warnings and Cautions WARNING To prevent personal injury the power cable must be removed from the instrument before opening the detector cover Do not connect the power cable to the detector while the covers are removed WARNING To prevent personal injury be careful when getting in contact with sharp metal areas When working with solvents please observe appropriate safety procedures for example goggles safety gloves and protective clothing as des
24. Figure 45 the PMTGAIN was gradually raised from 4 up to 11 the peak is from the Agilent Technologies isocratic sample which was diluted 1000 times With increasing PMTGAIN there was an improvement in signal to noise up to 10 Above 10 the noise increased proportionately to the signal with no improvement in signal to noise FLD1 A 2 246 Em 317 FLD_PMTSiPMT_O001 D FLD1 A Ex 246 Em 317 FLD_PMT3 PMT_0002 D FLD1 A Ex 246 Em 317 FLD_PMT3 PMT_0003 D FLD1 A Ex 246 Em 317 FLD_PMT3 PMT_0004 D FLD1 A Ex 246 Em 347 FLD_PMT3 PMT_0005 D FLD1 A Ex 246 Em 347 FLD_PMT3 PMT_0006 D 7 i PMT Li io E NE 11 75 ne ne ee eee remem neni sss tad Meaty 10 R ee ese A nese ieee aes eee 9 5 a a Weeeieeeteeste cates 8 i rr ee ae Fo 1 a re Se ere 6 4 0 0 4 2 3 4 5 m Figure 45 Finding Best PM TGAIN for Biphenyl 96 1200 Series FLD Service M anual How to optimize the Detector 5 The reason for this is the fact that quantification of baselines especially at low background levels is not sufficient for statistically working filter methods For the best gain check your solvent under flow conditions with the auto gain function Do not use higher values than proposed by the system if not necessary because of excessive high fluorescence signals Use the PMT test to automatically determine the setting 1200 Series FLD Service M anual 97 5 How to optimize the Detector Changing the Xenon Fla
25. PAC concept for the packaging of electronics and mechanical assemblies This concept is based upon the use of expanded polypropylene EPP layers of foam plastic spacers in which the mechanical and electronic boards components of the detector are placed This pack is then housed in a metal inner cabinet which is enclosed by a plastic external cabinet The advantages of this packaging technology are e virtual elimination of fixing screws bolts or ties reducing the number of components and increasing the speed of assembly disassembly e the plastic layers have air channels molded into them so that cooling air can be guided exactly to the required locations e the plastic layers help cushion the electronic and mechanical parts from physical shock and e the metal inner cabinet shields the internal electronics from electromagnetic interference and also helps to reduce or eliminate radio frequency emissions from the instrument itself 1200 Series FLD Service M anual 35 1 Introduction to the Fluorescence Detector Early Maintenance Feedback EM F 36 Maintenance requires the exchange of components which are subject to wear or stress Ideally the frequency at which components are exchanged should be based on the intensity of usage of the detector and the analytical conditions and not on a predefined time interval The early maintenance feedback EMF feature monitors the usage of specific components in the instrument and provides fe
26. Procedure 164 This chapter describes the detector s built in test functions fhe Agilent Technologies 137 8 Test Functions Diagram of Light Path The light path is shown in Figure 51 Emission Grating Reference Diode Mirror Diffuser gt 4 SQ vaca TE Excitation Grating Flash Tube Figure 51 Schematic of the Light Path 138 1200 Series FLD Service M anual Test Functions 8 Lamp Intensity Test The intensity test scans an intensity spectrum via the reference diode 200 1200 nm in 1 nm steps and stores it in a diagnosis buffer The scan is displayed in a graphic window There is no further evaluation of the test Results of this test are stored as lamp history date code intensity Instrument Gl32l1A Serial Number DES2001563 Operator Wolfgang Date 09 01 2006 Time 11 26 30 File C CHEM32 2 DIAGNOSE FLD INT DGR Intensity counts 14000 12000 10000 4000 1000 Wavelength inmi Figure 52 Lamp Intensity Test Report The profile can vary from instrument to instrument It is dependig on the age of the lamp and the content of the flow cell use fresh water 1200 Series FLD Service M anual 139 8 Test Functions UV degradation especially below 250 nm is significantly higher compared to visible wavelength range Generally the LAMP ON during run setting or using economy mode will increase lamp life by a magnitude Lamp Intensity History Results of
27. Replace detector into the stack 13 Reconnect the power cable and turn on the detector 1200 Series FLD Service M anual Repairs 11 Replacing Status Light Pipe When required If part was broken or removed Tools required Screwdriver POZI 1 PT3 Parts required Status light pipe 5041 8384 Preparations for this procedure 1 The status light pipe is clipped into the top cover Remove the front cover and top cover see Removing the Covers on page 198 i ia mt O pa f 2 Replace the top cover see Replacing the Foams and Covers on page 254 Y Aes K 3 Replace the detector into the stack and reconnect the cables and capillaries 4 Turn ON the detector 1200 Series FLD Service M anual 251 11 Repairs Installing the Optical Unit When required When all repairs on the optical unit have been completed Tools required Screwdriver POZI 1 PT3 Hexagonal key 4mm WARNING For safe operation make sure that the ground cable is reconnected Preparations for this procedure 1 Place the optical unit in the instrument Reconnect the grounding screw All previous work has been completed Power supply has been installed Fan has been installed The optical unit has its foam cover attached 252 1200 Series FLD Service M anual Repairs 11 2 Reconnect the leak sensor cable J 3 using your left hand 3 Reconnect the optical unit c
28. Y Ensure the leak sensor is connected correctly VY Exchange the leak sensor 1200 Series FLD Service M anual 119 7 Error Information Leak Sensor Short The leak sensor in the detector has failed short circuit The current through the leak sensor is dependent on temperature A leak is detected when solvent cools the leak sensor causing the leak sensor current to change within defined limits If the current increases above the upper limit the error message is generated Probable Causes e Defective leak sensor Suggested Actions VY Exchange the leak sensor 120 1200 Series FLD Service M anual Error Information 7 Compensation Sensor Open The ambient compensation sensor NTC on the FLM board in the detector has failed open circuit The resistance across the temperature compensation sensor NTC on the FLM board is dependent on ambient temperature The change in resistance is used by the leak circuit to compensate for ambient temperature changes If the resistance across the sensor increases above the upper limit the error message is generated Probable Causes e Defective FLM board Suggested Actions VY Exchange the FLM board 1200 Series FLD Service M anual 121 7 Error Information Compensation Sensor Short The ambient compensation sensor NTC on the FLM board in the detector has failed short circuit The resistance across the temperature compensation sensor NTC on the FLM board is dependen
29. a 1 sec 0 0 5 4 1 5 2 25 3 3 5 4 45 m Figure 47 Finding Best Response Time 100 1200 Series FLD Service M anual How to optimize the Detector 5 LC fluorescence detectors typically work with response times of 2 or 4 seconds The default of the Agilent 1200 Series fluorescence detector is 4 seconds It is important to know that comparing sensitivity requires using the same response time A response time of 4 seconds default is equivalent to a time constant of 1 8 seconds and appropriate for standard chromatographic conditions Responsetime 2 sec Responsetime 8 sec Figure 48 Separation of Peaks using Responsetime 1200 Series FLD Service M anual 101 5 How to optimize the Detector Reducing Stray Light 102 Cut off filters are used to remove stray light and 2nd order or higher stray light by allowing complete transmission above the cut off and little or no transmission below the cut off point They are used between excitation and emission gratings to prevent any stray excitation light from reaching the photomultiplier tube when it is measuring emission When the emission and excitation wavelengths are close together the distortion due to scattering severely limits the sensitivity When the emission wavelength is twice the excitation wavelength the 2nd order light is the limiting factor To explain the effect of such higher order light assume the detector is on but no sample is eluting through the flow cell The lamp se
30. and BCD outputs LAN Communication Interface board G1369A or G1369 60001 see LAN Communication Interface Board on page 314 Tools required None To replace the interface board unscrew the two screws remove the board slide in the new interface board and fix it with the board s screws Interface board Figure 72 Location of the Interface Board 184 1200 Series FLD Service M anual Maintenance 10 Replacing the Detector s Firmware When required If new version solves problems of currently installed version or after exchange of the detector main board FLM the version on board Is older than previous installed one Tools required LAN RS 232 Firmware Update Tool or Instant Pilot G4208A or Control M odule G1323B Parts required Firmware tools and documentation from Agilent web site Preparations Read update documentation provided with the Firmware Update Tool The installation of older firmware might be necessary e to keep all systems on the same validated revision or e if third part control software requires a special version To upgrade downgrade the detector s firmware the following steps have to be performed 1 Download the module s firmware the LAN RS 232 FW Update Tool Version 2 10 or above and the documentation from the Agilent web http www chem agilent com scripts cag_firmware asp 2 Load the firmware into the detector as described in the documentation 1200 Series FLD Service M anual 185
31. based on the emission spectra from a PNA reference sample Table 13 Peak confirmation using a fluorescence spectral library Meas Library CalTbl RetTime min 4 859 6 764 7 137 8 005 8 841 9 838 10 439 12 826 13 340 15 274 16 187 16 865 18 586 19 200 20 106 min 4 800 7 000 7 100 8 000 8 800 10 000 10 400 12 800 13 300 15 200 16 200 16 900 18 600 19 100 20 000 Signal Amount Purity Match Libary Name min ng Factor 5 178 1 1 47986e 1 1 993 Naphthalene em 7 162 1 2 16156e 1 1 998 Acenaphthene em 7 544 1 1 14864e 1 1 995 Fluorene em 8 453 1 2 56635e 1 1 969 Phenanthrene em 9 328 1 1 76064e 1 1 993 Anthracene em 10 353 1 2 15360e 1 1 997 Fluoranthene em 10 988 1 8 00754e 2 1 1000 Pyrene em 13 469 1 1 40764e 1 1 998 Benz a anthracene em 14 022 1 1 14082e 1 1 999 Chrysene em 16 052 1 6 90434e 1 1 999 Benzo b fluoranthene em 17 052 1 5 61791e 1 1 998 Benzo k fluoranthene em 17 804 1 5 58070e 1 1 999 Benz a pyrene em 19 645 1 5 17430e 1 1 999 Dibenz a h anthracene em 20 329 1 6 03334e 1 1 995 Benzo g h i perylene em 21 291 1 9 13648e 2 1 991 Indeno 1 2 3 cd pyrene em 1200 Series FLD Service M anual 83 4 First Steps with the Detector Example Optimization for M ultiple Compounds 84 Using PNAs as a sample this example uses the described scanning functions Setting the Chromatographic Conditions 1 This example uses the following chromatographic conditions the d
32. cables external contact 292 cables LAN cables 294 foams and pads 274 leak panels 276 main assemblies 260 267 optical unit 268 overview 260 266 plastics 273 power and status 275 sheet metal kit 272 peak detector 310 performance specifications 43 phosphorescence detection 29 photoluminescence 17 1200 Series FLD Service M anual photo multiplier tube figure 26 location of PMT 21 optional PMT types 236 PMT 25 replacing 236 physical specifications 42 humidity 42 line voltage and frequency 42 Operation temperature 42 power consumption 42 Safety standards 42 weight and dimensions 42 PMT gain 25 gain steps 31 gain test 63 Optional PM T types 236 photo multiplier tube 25 range 31 power considerations 40 power consumption 42 power cords 40 power supply description 325 specifications 326 R Raman 20 Raman S N test 144 rear view of module 34 55 reasons for doing a verification or calibration 153 recalibration of wavelength 106 138 reference diode 27 reference system 27 removing covers 198 200 repair flushing of flow cell 181 Repairs 195 Index repairs assembling main cover 256 cleaning the instrument 173 correction leaks 182 definition of 170 exchanging a flow cell 177 exchanging a lamp 201 exchanging EX condenser 206 exchanging internal parts 196 exchanging leak sensor 221 exchanging main board FLM 212 exchanging power supply 248 exch
33. complete Agilent 1200 System is shut down to prevent system damage This block consists of a PTC for the leak detection and a NTC for the ambient temperature compensation A leak would cool down the PTC and this change in resistance would generate a leak signal Digital Interface The control and data lines from and to the Fluorescence Detector Main Board FLM are buffered by Read and Write ports The signals from the main board allow setting of the signal multiplexers reference gain stages lamp current and to start the initial self calibration of the A D converter The port to the main board drives the A D converter data and several control lines like cell detection overcurrent detection and lamp cover safety switch position The signals driving the lamp are derived from the FPGA on the Main Board and have separate connections This allows the lamp timing control without processor interaction and guarantees a realtime control of the lamp operation 1200 Series FLD Service M anual 301 15 Hardware Information How to retrieve the FLF board revision Agilent ChemStation The FLF board revision can be retrieved with the following ChemStation command PRINT SENDMODULES LFLD FLFR The reply will be RA 0000 FLFR AAAA for revsion A boards Control M odule G1323B 1 Select FLD TESTS 2 Press m m m dot m 3 Select Command 4 Type FLFR 5 Press Execute Firmware revisions below A 03 70 do not know this command RE
34. contact closure contacts are relay contacts The maximum settings are 30 V AC DC 250 mA fused Board identification b E Processor BCD register N 12 BCD lt gt interface P connector Line driver 250mA 2 A External contact External Oo gt contacts Z 4x connector Figure 96 Block Diagram BCD Board 1200 Series FLD Service M anual Hardware Information 15 There are general purpose cables available to connect the BCD output see BCD Cables on page 288 and the external outputs see External Contact Cable on page 292 to external devices Table 45 Detailed connector layout 1200 Pin Signal name BCD digit 1 BCD5 20 2 BCD 7 80 3 BCD 6 40 4 BCD 4 10 5 BCD 0 1 6 BCD 3 8 7 BCD 2 4 8 BCD 1 2 9 Digital ground 10 BCD 11 800 11 BCD 10 400 12 BCD 9 200 13 BCD 8 100 14 not connected 15 5V 1200 Series FLD Service M anual 313 15 Hardware Information LAN Communication Interface Board One board is required per Agilent 1200 stack It is recommended to add the LAN board to the detector with highest data rate The LAN board can only be used together with a main board version G13XX 66520 for G1315A G1365A G1314A G1310A G1311A G1312A and G1313A or newer and on all other modules a DOS ChemStation software revision A 06 01 or above The following cards can be
35. e Parts and Materials for Maintenance o o Overview of Maintenance Parts 260 Cuvette Kit 261 Spare Parts 262 Accessory Kit 263 This chapter provides information on parts for maintenance agg Agilent Technologies 259 12 Parts and Materials for Maintenance Overview of Maintenance Parts Table 29 Maintenance Parts Item Description Control M odule G1323B or Instant Pilot G4208A Standard Flow Cell 8 ul 20 bar inlet i d length 0 17 mm 80 mm outlet i d length 0 25 mm 80 mm Cuvette 8 ul 20 bar see Cuvette Kit on page 261 inlet i d length 0 5 mm 80 mm outlet i d length 0 5 mm 80 mm Needle Syringe Glass Syringe Parts for wavelength calibration see Accessory Kit on page 263 Front cover Leak funnel Leak funnel holder Clip Corrugated tubing 120 mm lg re order 5 m Teflon Tubing flexible i d 0 8 mm flow cell to waste Cable CAN to Agilent 1200 Series modules 0 5 m Cable CAN to Agilent 1200 Series modules 1 m LAN Communication Interface Board G1369A Cross over network cable shielded 3 m long for point to point connection Twisted pair network cable shielded 7 m long for hub connections Analog cable BNC to general purpose spade lugs Interface board BCD BCD external contacts Part Number 61323 67001 64208 67001 G1321 60005 61321 60007 9301 0407 9301 1446 5062 8592 5041 8388 5041 8389 5041 8387 5062 2463 5062 2462 5181 1516
36. e g Agilent 3396 with ATTN 4 ZERO 50 CS 5 3 Stop the plot and disable the TAC Test System Tests FLD Disable DAC Test Pattern 4 Evaluate the noise should be lt 5 uV 152 1200 Series FLD Service M anual Test Functions 8 Wavelength Verification and Calibration The wavelength calibration is based on a Glycogen solution which acts as a strong elastic light scatterer refer to ASTM Test Method E388 72 1993 Spectral Bandwidth and Wavelength Accuracy of Fluorescence Spectrometers The Glycogen solution is introduced into the flow cell and then the built in wavelength calibration functionality is used The algorithm is based on evaluating different grating orders and calculating the wavelength scales of both excitation and emission monochromator by applying the fundamental grating equation A complete wavelength calibration is not always required In most cases a quick wavelength accuracy verification is sufficient enough see the table below Table 25 Reasons for doing a Verification or Calibration Verification WL calibration interest X GLP compliance X cell change X X lamp change X monochromator change X main board change X X optical unit change X only required if deviation is too large Prior to a wavelength calibration a wavelength accuracy verification should be performed see Check of Raman Band of Water on page 156 If the deviation is more than 3 nm the wavelength calibrat
37. into the vial and shake 4 Wait 5 minutes and shake again After 10 minutes the solution is ready 164 1200 Series FLD Service M anual Test Functions 8 Preparation of the Flow Cell Flush the flow cell with water Remove the inlet capillary from the flow cell Take the syringe and fix the needle to the syringe adapter Suck about 1 0 ml of the calibration sample into the syringe Keep the syringe in a horizontal position Remove t he needle Add the filter to the syringe and fit the needle to filter EH OF FP W N P sample filter Figure 67 Syringe with Sample Filter 8 Lift the needle tip and carefully eject approximately 0 5 ml to remove air out of the syringe and to flush the needle 9 Add the PEEK fitting to the needle tip and fix both at the flow cell inlet Do not inject the calibration sample without the sample filter 10 Slowly inject about 0 2 ml and wait for about 10 seconds to inject another 0 1 ml This will assure that the cell is filled properly 1200 Series FLD Service M anual 165 8 Test Functions Wavelength Calibration 1 From the user interface start the FLD Wavelength Calibration see Figure 68 Agilent ChemStation Diagnosis Maintenance FLD Calibration Instant Pilot G4208A Maintenance FLD Calibration Control Module G1323B System Tests FLD Calibrate If the wavelength calibration process fails refer to Wavelength Calibration Failed on page 130 2 Ifa deviation is
38. light which is emitted during phosphorescence therefore has less energy and is at a longer wavelength than fluorescence Formula E h x H In this equation E is energy h is Planck s constant lis the wavelength 1200 Series FLD Service M anual 19 1 Introduction to the Fluorescence Detector Raman Effect 20 The Raman effect arises when the incident light excites molecules in the sample which subsequently scatter the light While most of this scattered light is at the same wavelength as the incident light some is scattered at a different wavelength This inelastically scattered light is called Raman scatter It results from the molecule changing it s molecular motions ae Raman Scatter length as incidentlight Tew Wavelength Scattered Light Incident Light Figure5 Raman The energy difference between the incident light E and the Raman scattered light is equal to the energy involved in changing the molecule s vibrational state i e getting the molecule to vibrate E This energy difference is called the Raman shift E E E Several different Raman shifted signals will often be observed each being associated with different vibrational or rotational motions of molecules in the sample The particular molecule and its environment will determine what Raman signals will be observed if any A plot of Raman intensity versus Raman shift is a Raman spectrum 1200 Series FLD Service M anual
39. matrices simultaneous multi wavelength detection offers more reliability than timetable controlled wavelength switching The Agilent 1200 Series FLD can in addition acquire fluorescence spectra while it records the detector signals for quantitative analysis Therefore qualitative data are available for peak confirmation and purity checks in routine analysis 80 1200 Series FLD Service M anual First Steps with the Detector 4 Multi wavelength detection Time programmed wavelength switching traditionally is used to achieve low limits of detection and high selectivity in routine quantitative analysis Such switching is difficult if compounds elute closely and require a change in excitation or emission wavelength Peaks can be distorted and quantitation made impossible if wavelength switching occurs during the elution of a compound Very often this happens with complex matrices influencing the retention of compounds In spectral mode the Agilent 1200 Series FLD can acquire up to four different signals simultaneously All of them can be used for quantitative analysis Apart from complex matrices this is advantageous when watching for impurities at additional wavelengths It is also advantageous for reaching low limits of detection or increasing selectivity through optimum wavelength settings at any time The number of data points acquired per signal is reduced and thus limits of detection may be higher depending on the detector settings c
40. not connected to the FLM board e defective FLF board e defective FLM board Suggested Actions VY Check the connection between the FLF board and the FLM board VY Exchange the FLF board VY Exchange the FLM board 1200 Series FLD Service M anual 125 7 Error Information ADC Not Calibrated The analog to digital converter located on the FLF board cannot calibrate Probable Causes e defective ADC or other FLF electronics Suggested Actions VY Exchange the FLF board 126 1200 Series FLD Service M anual Error Information 7 A D Overflow This message is not implemented in firmware revision A 03 66 and below It indicates an overload situation of the A D converter sample signal The user interface will show a not ready condition for the FLD and an info event is written into the logbook If the message comes up during a run it includes the time of occurrence and when it disappears 1200 FLD 1 A D overflow RT is 0 32 min 16 33 24 02 11 99 1200 FLD 1 A D overflow finished RT is 0 67 min 16 33 46 02 11 99 If this condition is present prior to a run the not ready will prevent the system to start the run sequence Probable Causes e PMT setting to high e wavelength setting wrong Suggested Actions v reduce PMT gain v change wavelength setting 1200 Series FLD Service M anual 127 7 Error Information Flash Lamp Current Overflow The lamp current of the xenon flash lamp is monitored constantly If th
41. of 4 seconds default which is equivalent to a time constant of 1 8 seconds and appropriate for standard chromatographical conditions Weak signals may cause errors in 1200 Series FLD Service M anual Introduction to the Fluorescence Detector 1 quantification because of insufficient resolution Check your proposed PMTGAIN If it is significantly distant from your setting change your method or check the purity of your solvent You can amplify the signal using PMTGAIN Depending on the PMTGAIN you have set a multiple of electrons is generated for every photon falling on the photomultiplier You can quantify large and small peaks in the same chromatogram by adding PMTGAIN changes during the run into a timetable DVO tat OT sos CT T Uor sconce we Figure 15 PMTGAIN Amplification of Signal Check proposed PMTGAIN Deviations of more than 2 PMT gains should be corrected in the method Each PMTGAIN step is increased approximately by a factor of 2 range 0 18 To optimize your amplification for the peak with the highest emission raise the PMTGAIN setting until the best signal to noise is achieved After the photons are converted and multiplied into an electronic signal the signal at present analog is tracked and held beyond the photo multiplier After being held the signal is converted by an A to D converter to give one raw data point digital Eleven of these data points are bunched together as the first step
42. on 4 Re do steps 2 to 5 of Using the Instant Pilot G4208A on page 216 and correct the type information Enter the product number without R 5 Turn the detector off 6 Change the 8 bit Configuration Switch back to default settings see Setting the 8 bit Configuration Switch on page 320 7 Turn the detector ON again The Maintenance screen should display the correct type for this module Using the Control M odule G1323B 1 Connect the control module to the detector Turn ON the detector 2 On the control module press System F5 then Records F4 Using the up down arrows make sure that the detector is highlighted 3 Press FW Update F5 then m This will display a box which says Update Enter Serial 4 Press Enter This will display the box labeled Serial 5 Letters and numbers are created using the up and down arrows Into the box labeled Serial enter the 10 character serial number for the detector When the 10 character serial number is entered press Enter to highlight the complete serial number Then press Done F6 6 Turn the detector OFF then ON again The Records screen should display the correct serial number for this module 7 Ifa Agilent ChemStation is also connected restart the Agilent ChemStation now as well 1200 Series FLD Service M anual 217 11 Repairs 218 To change the product number go to the System screen 1 Press Tests F3 and select the detector and press Enter
43. solvents For example a 1 solution of acetic acid in methanol will attack steel e Solutions containing strong complexing agents for example EDTA ethylene diamine tetra acetic acid e Mixtures of carbon tetrachloride with 2 propanol or THF 336 1200 Series FLD Service M anual Appendix A Agilent Technologies on Internet For the latest information on products and services visit our worldwide web site on the Internet at http www agilent com Select Products Chemical Analysis It will provide also the latest firmware of the Agilent 1200 Series modules for download 1200 Series FLD Service M anual 337 A Appendix 338 1200 Series FLD Service M anual A cuvette 16 A D overflow 127 how to use 180 accessory kit 49 D accessory kit parts 263 accuracy of wavelength 43 Agilent on internet 337 algea 181 algea information 335 analog output 297 analog signal output 316 APG remote interface 317 DAC test 151 dark current test 141 degradation UV 22 140 188 delivery checklist 48 dimensions and weight 42 dispersion of light 24 E B early maintenance feedback EM F 36 battery electrical connections description of 33 EM F early maintenance feedback 36 emission condenser 21 emission grating 21 BCD LAN board 312 emission monochromator 24 bench space 41 emission slit 21 boards environment 41 interface board BCD LAN 312 location of connectors 213 C cable connec
44. that if not correctly performed or adhered to could result in damage to the product or loss of important data Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met A WARNING notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly per formed or adhered to could result in personal injury or death Do not proceed beyond a WARNING notice until the indicated condi tions are fully understood and met 1200 Series FLD Service M anual In This Guide 1200 Series FLD Service M anual Introduction to the Fluorescence Detector This chapter gives an introduction to the detector instrument overview and internal connectors Site Requirements and Specifications This chapter gives information on environmental requirements physical and performance specifications Installing the Detector This chapter describes the installation of the detector First Steps with the Detector This chapter guides you how to start the work with the detector How to optimize the Detector This chapter provides information on how to optimize the detector Troubleshooting and Test Functions This chapter gives an overview about the troubleshooting and diagnostic features and the different user interfaces Error Information This chapter describes the meaning of detector error messages and provides information on probable causes and
45. the lamp intensity test if the last one is older than one week are stored as lamp history date code intensity of four different wavelengths 250 nm 350 nm 450 and 600 nm in a buffer The data plot can be retrieved via the diagnostics and provides intensity data over a length of time MH Intensity Test History Figure 53 Lamp Intensity History 140 1200 Series FLD Service M anual Test Functions 8 Dark Current Test The dark current test measures the PMT signal with maximum and minimum gain while the lamp is OFF It also reads the signal of the reference diode The resulting values two via reference diode and two from PMT are shown ina table and checked against reasonable limits see below Instrument G1l3214 Serial Number DES2001563 Operator Wolfgang Date 09 01 2006 Time 11 35 34 Dark Current Test Results Specification Measured Result PMT dark current lt z 10000 cts 1227 ets Passed Reference diode dark current lt 5000 cts 1023 cts Passed Figure 54 Dark Current Test Report Dark Current Test Evaluation Test Failed Probable Causes e Defective PMT e Defective reference diode or A D converter Suggested Actions VY Exchange the PMT VY Exchange the FLF board 1200 Series FLD Service M anual 141 8 Test Functions Excitation and Emission Grating Resistance History This test runs automatically when the instrument is turned on not accessible as an external test It provide
46. though the instrument has been disconnected from its source of supply Dangerous voltages capable of causing serious personal injury are present in this instrument Use extreme caution when handling testing and adjusting Safety Symbols Table 60 shows safety symbols used on the instrument and in the manuals Table 60 Safety Symbols Symbol Description The apparatus is marked with this symbol when the user should refer to the AN instruction manual in order to protect the apparatus against damage Z Indicates dangerous voltages 1200 Series FLD Service M anual 329 A Appendix Table 60 Safety Symbols continued Symbol Description D Indicates a protected ground terminal Ml Eye damage may result from directly viewing the light produced by the deuterium a lamp used in this product Always turn off the deuterium lamp before opening the metal lamp door on the side of the instrument WARNING A warning alerts you to situations that could cause physical injury or damage to the equipment Do not proceed beyond a warning until you have fully understood and met the indicated conditions CAUTION A caution alerts you to situations that could cause a possible loss of data Do not proceed beyond a caution until you have fully understood and met the indicated conditions 330 1200 Series FLD Service M anual Appendix A Lithium Batteries Information Danger of explosion if battery is incorrectly replaced Replace
47. to fix the screw Replace the cutoff filter i E i ii i Next Steps e Replace the covers as described in Replacing the Foams and Covers on page 254 e Re install the flow cell and the front panel Re install the detector in the stack e Turn the lamp ON e Perform a wavelength verification to check the correct positioning of the replaced assemblies as described in chapter Diagram of Light Path on page 138 226 1200 Series FLD Service M anual Repairs 11 Replacing the Mirror Assembly When required If reflection is too low contaminated Tools required Screwdriver POZI 1 PT3 A pair of tweezers Parts required Mirror assembly 1000 1000 DO NOT touch the surface of any optical component during this procedure This will reduce the reflection efficiency One side of the mirror is marked R representing the rear side Preparations for this procedure 1 Disconnect the emission and the excitation cables and from the detector main board Turn OFF the detector Disconnect the power cable Remove the flow cell Remove detector from stack and place it on the workbench Remove the covers as described in Removing the Covers on page 198 1200 Series FLD Service M anual 227 Repairs 11 3 Remove the UV protector WN O uy WM Z D D O E O yu Ww Cc O T q w ZC w gt O w Se 5 q aD
48. tools needed for the installation and repair calibration of the detector Table 32 Accessory Kit Parts Item Description Accessory kit includes Corrugated tubing 120 mm lg re order 5 m Teflon Tubing flexible i d 0 8 mm flow cell to waste re order 5m Fitting male PEEK Qty 2 Capillary column detector 380 mm lg 0 17 i d includes items 4 5 and 6 not assembled Ferrule front SST gty 1 Ferrule back SST qty 1 Fitting SST gty 1 Screwdriver hexagonal 4 mm 100 mm long Screwdriver hexagonal 2 5 mm 100 mm long Needle Syringe Glass Syringe Calibration Sample Glycogen Sample filter diameter 3 mm pore size 0 45 um QYT 5 Hex key set1 5mm Wrench open end 1 4 5 16 inch 1200 Series FLD Service M anual Part Number G1321 68705 5062 2463 5062 2462 0100 1516 61315 87311 0100 0043 0100 0044 79814 22406 5965 0027 5965 0028 9301 0407 9301 1446 5063 6597 5061 3367 pack of 100 8710 0641 8710 0510 263 12 Parts and Materials for Maintenance Figure 79 Waste Tubing Parts J CF This end is oni l w pre installed Figure 80 Inlet Capillary Column Detector Parts 264 1200 Series FLD Service M anual Agilent 1200 Series Fluorescence Detector Service M anual 2 ee 13 909 Parts for Repair j e g Overview of M ain Assemblies 266 Optical Unit Assembly 268 Lamp Cover Parts 271 Sheet M etal Kit 272 Plastic Parts 273 Foam Parts 274 Power and Status Lig
49. used in liquid chromatography when superior limits of detection and selectivity are required Thorough method development including spectra acquisition is fundamental to achieve good results This chapter describes three different steps that can be taken with the Agilent 1200 Series fluorescence detector Table 7 gives an overview of how to benefit from the operation modes during these steps Table 7 Steps for thorough method development Fluorescence scan Signal mode Spectral mode multi wavelength detection Step 1 Step 2 Step 3 Check system Optimize limits of detection Setup routine methods and selectivity Find impurities forexample Determine simultaneously in solvents and reagents the excitation and emission Spectra of a pure compound Perform wavelength Use for lowest limits of Switching detection Determine Ex Em spectra for Collect online spectra all separated compounds ina__ perform library search Single run determine peak purity Activate up to four Deactivate wavelength wavelength simultaneously switching 1200 Series FLD Service M anual 69 4 First Steps with the Detector Step 1 Check the LC system for impurities A pure water sample was put into the flow cell Spectra were recorded at 5 nm step sizes 70 A critical issue in trace level fluorescence detection is to have an LC system free of fluorescent contamination Most contaminants derive from impure solvents Taking a fluorescence sc
50. used with the Agilent 1200 Series modules Table 46 LAN Boards Type Vendor Supported networks G1369A Agilent Technologies Fast Ethernet Ethernet 802 3 RJ 45 10 100Base TX G1369 60001 recommended for re ordering J 4106A Hewlett Packard Ethernet 802 3 RJ 45 10Base T J 4105A Hewlett Packard Token Ring 802 5 DB9 RJ 45 10Base T J 4100A Hewlett Packard Fast Ethernet Ethernet 802 3 RJ 45 10 100Base TX BNC 10Base2 These cards may be longer orderable Minimum firmware of the J etDirect cards is A 05 05 Recommended Cables Cross over network cable shielded 3 m long 5023 0203 for point to point connection Twisted pair network cable shielded 7 m long 5023 0202 for hub connections 314 1200 Series FLD Service M anual Hardware Information 15 Interfaces The Agilent 1200 Series modules provide the following interfaces Table 47 Agilent 1200 Series Interfaces Interface Type Pumps Autosampler DA Detector VW Detector Thermostatted Vacuum MW Detector RI Detector Column Degasser FL Detector Compartment CAN Yes Yes Yes Yes Yes No GPIB Yes Yes Yes Yes Yes No RS 232C Yes Yes Yes Yes Yes No Remote Yes Yes Yes Yes Yes Yes Analog Yes No 2 x 1x No Yes Interface board Yes Yes Yes Yes No No The vacuum degasser will have a special connector for specific use For details see description of main board e CAN connectors as interface to other Agilent 1200 Series modul
51. waste tubing from the accessory Kit accessory kit One side is already factory assembled Pre assembled Ep 58 1200 Series FLD Service M anual Installing the Detector 3 Note The fluorescence detector should be the last module in the flow system An additional detector should be installed before the fluorescence detector to prevent any overpressure to the quartz cell maximum 20 bar When working with detector behind the FLD on own risk determine the backpressure of this detector first b removing the column and the last detect and measuring system pressure at the application flow rate connecting the last detector without column and FLD and measuring the system pressure with flow the difference in measured pressure is due to the back pressure generated by the last detector and is the pressure seen by the FLD 5 Insert the flow cell and install the capillaries to the flow 6 Connect the waste tubing to the bottom waste fitting cell top is outlet bottom is inlet yp 1200 Series FLD Service M anual 59 3 Installing the Detector 7 Establish flow and observe if leaks occur 8 Replace the front cover The installation of the detector is now complete The detector should be operated with the front cover in place to protect the flow cell area against strong drafts from the ouside 60 1200 Series FLD Service M anual Agilent 1200 Series Fluorescence Detector
52. 00 to 3392 3 Integrators Connector Pin Pin SignalName Active 01046 60206 3392 3 Agilent 1200 TTL 3 1 White Digital ground NC 2 Brown Prepare run Low 11 3 Gray Start Low NC 4 Blue Shut down Low NC 5 Pink Not connected NC 6 Yellow Power on High 9 7 Red Ready High 4 Key 1 8 Green Stop Low NC 9 Black Start request Low Agilent 1200 to 3394 Integrators Connector Pin Pin Signal Name Active 01046 60210 3394 Agilent 1200 TTL 9 1 White Digital ground ER NC 2 Brown Preparerun Low 30 D 3 3 Gray Start Low K NC 4 Blue Shut down Low zo NC 5 Pink Not connected 10 NC 6 Yellow Power on High E 3 5 14 7 Red Ready High 6 8 Green Stop Low 1 9 Black Start request Low 13 15 Not connected 284 1200 Series FLD Service M anual Cables 14 START and STOP are connected via diodes to pin 3 of the 3394 connector Agilent 1200 to 3396A Integrators Connector Pin Pin SignalName Active 03394 60600 3394 Agilent 1200 TTL 9 1 White Digital ground eS NC 2 Brown Prepare run Low 80 JA 3 3 Gray Start Low NC 4 Blue Shut down Low NC 5 Pink Not connected 10 NC 6 Yellow Power on High ay 5 14 7 Red Ready High 1 8 Green Stop Low NC 9 Black Start request Low 13 15 Not connected Agilent 1200 to 3396 Series II 3395A Integrators Use the cable 03394 60600 and cut pin 5 on the integrator side Otherwise the inte
53. 1 60003 7 Emission M onochromator assembly complete G1321 60004 8 Standard Flow Cell 8 ul 20 bar G1321 60005 inlet i d length 0 17 mm 80 mm outlet i d length 0 25 mm 80 mm Cuvette 8 wl 20 bar G1321 60007 inlet i d length 0 5 mm 80 mm outlet i d length 0 5 mm 80 mm Screw for flow cell cuvette G1321 22400 9 Slit EM 2x8 G1321 08102 10 Photomuliplier tube assembly PMT see also page 236 for additional information 1970 0201 Photo multiplier Tube PMT R928HA 185 to 900 nm contact Photo multiplier Tube PM T R3788HA 185 to 750 nm soli 11 Condenser Emission 1000 1124 12 Front end Board FLF Rev D underneath the optical new requires firmware revision A 04 06 G1321 66531 or above 12 Front end Board FLF Rev D underneath the optical exchange assembly requires firmware G1321 69531 revision A 04 06 or above 268 1200 Series FLD Service M anual Parts for Repair 13 Table 34 Optical Unit Assembly continued Item Description Part Number 13 Reference Diode Board FLR G1321 66533 14 Diffuser plate in front of the reference diode board FLR 1000 1121 15 Cutoff Filter 295 nm for other type see page 262 5062 8530 16 Carbon filter mat 01046 42701 17 Cable FLM FLF board comes with optical unit G1321 61600 Foam Optics G1321 40103 18 Cable chassis G1321 61602 19 Screw M5 x 25 mm for chassis cable 0515 2574 Screw 2 5 mm hex for monochromator lamp pad condenser 1 slits etc 0515 1052 Screw M4 x 25 mm hex
54. 113 Shutdown 114 Remote Time out 115 Synchronization Lost 116 Leak 117 Leak SensorOpen 119 Leak Sensor Short 120 Compensation Sensor Open 121 Compensation Sensor Short 122 Fan Failed 123 Detector Error Messages 124 Lamp Cover Open 124 FLF Board not found 125 ADC Not Calibrated 126 A D Overflow 127 Flash Lamp Current Overflow 128 Flash Trigger Lost 129 Wavelength Calibration Failed 130 Wavelength Calibration Lost 131 Flow Cell Removed 132 No Peaks 133 Motor Errors 134 8 Test Functions Diagram of Light Path 138 Lamp Intensity Test 139 Lamp Intensity History 140 Dark Current Test 141 Excitation and Emission Grating Resistance History 142 Raman ASTM Signal to Noise Test 144 Procedure using an Agilent ChemStation 146 Using the Built in Test Chromatogram 148 Procedure using the Agilent ChemStation 149 Procedure using the Control Module G1323B 149 Using the Built in DAC Test 151 Wavelength Verification and Calibration 153 Check of Raman Band of Water 156 Using the Agilent ChemStation OQ PV 156 Using the Agilent ChemStation Manually 158 Using the Control Module G1323B 161 Wavelength Calibration Procedure 164 Preparation of the Glycogen Calibration Sample 164 Preparation of the Flow Cell 165 Wavelength Calibration 166 Verification 167 9 Maintenance and Repair Introduction into Repairing the Fluorescence Detector 1 0 Warnings and Cautions 171 1200 Series FLD Service M anual 10 Maintenance 11 Repairs 1
55. 1200 Series FLD Service M anual Error Information 7 General Error Messages General error messages are generic to all Agilent 1200 series HPLC modules Time out The time out threshold was exceeded set in sequence parameter on the Agilent ChemStation or Configure LC system Probable Causes e The analysis was completed successfully and the time out function switched off the pump as requested e A not ready state was present during a sequence or multiple injection run for a period longer than the time out threshold Suggested Actions Y Check the logbook for the occurrence and source of a not ready condition Restart the analysis where required 1200 Series FLD Service M anual 113 7 Error Information Shutdown An external instrument has generated a shut down signal through CAN or REMOTE lines The detector continually monitors the remote input connectors for status signals A LOW signal input on pin 4 of the remote connector generates the error message Probable Causes e Leak detected in an external instrument with a remote connection to the system e Shut down in an external instrument with a remote connection to the system e The degasser failed to generate sufficient vacuum for solvent degassing Suggested Actions V Fix the leak in the external instrument before restarting the pump Y Check external instruments for a shut down condition VY Check the degasser for an error condition Refer to the Referen
56. 1200 Series FLD Service M anual Test Functions 8 Using the Control M odule G1323B 1 Create the methods WLEMTEST and WLEXTEST as listed Table 27 on page 158 2 Copy the methods to your PC card and re boot the control module 3 On the Analysis screen press Method and copy the methods into the module Semmes ORE ODEs ORnce 0 00 NRE Ready WLEMTEST 08 07 98 10 19 E WWLEXTEST 08 07 98 10 28 Figure 63 Copying Methods into the FLD 4 Load the method WLEXTEST The FLD will change into the multi emission mode and scan in the range of the expected maximum of 397 nm 20 nm 5 Turn the pump ON and flush with water for a few minutes to assure a clean flow cell Flow rate should be 0 5 to 1 ml min and the baseline stable You may remove the flow cell and check for air bubbles After re inserting the cell turn the lamp ON 6 From the Analysis screen select Settings FL Detector Then press Spectrum this will open the window below 1200 Series FLD Service M anual 161 8 Test Functions Figure 64 Online Emission Spectra 7 Press Store a few times to store a few spectra in a temporary buffer 8 Press Eval to change to the following screen Emission i 4 E ee o B99 O4 ye me 303 304 O96 402 285 Figure 65 Evaluating Online Emission Spectra 162 1200 Series FLD Service M anual Test Functions 8 9 You can now select a stored spectra and evaluate print them The maximum should be found at 397 nm 3 nm
57. 200 Series FLD Service M anual Cleaning the Detector 173 Using the ESD Strap 174 Overview of Maintenance 1 6 Exchanging a Flow Cell 177 How to use the Cuvette 180 Flow Cell Flushing 181 Correcting Leaks 182 Replacing Leak Handling System Parts 183 Replacing the Interface Board 184 Replacing the Detector s Firmware 185 Tests amp Calibrations 186 Lamp Intensity Test 187 Wavelength Verification and Calibration 189 Wavelength Calibration Procedure 191 Preparation of the Glycogen Calibration Sample 191 Preparation of the Flow Cell 192 Wavelength Calibration 193 Cautions and Warnings 196 Removing the Covers 198 Removing the Rear Foam 200 Exchanging the Lamp 201 Exchanging the EX condenser 206 Replacing the Lamp Cover Window 209 10 Exchanging the Main Board 212 Changing the Type and SerialNumber 215 Using the Agilent ChemStation 215 Using the Instant Pilot G4208A 216 Using the Control Module G1323B 217 Exchanging the Fan 219 Exchanging the Leak Sensor or Leak Panel 221 Replacing the EM Condenser or Cutoff Filter 224 Replacing the M irror Assembly 227 Replacing the Slits 231 Removing the Optical Unit 233 Replacing PMT and or FLF board 236 Replacing FLL board and Trigger Pack 240 Replacing the EM and EX Grating 244 Exchanging the Power Supply 248 Replacing Status Light Pipe 251 Installing the Optical Unit 252 Replacing the Foams and Covers 254 Assembling the Main Cover 256 Parts and Materials for M a
58. 233 Replacing PMT and or FLF board 236 Replacing FLL board and Trigger Pack 240 Replacing the EM and EX Grating 244 Exchanging the Power Supply 248 Replacing Status Light Pipe 251 Installing the Optical Unit 252 Replacing the Foams and Covers 254 Assembling the Main Cover 256 This chapter describes the repairs of the detector ee Agilent Technologies 195 11 Repairs Cautions and Warnings The following procedures require opening the main cover of the detector Always ensure the detector is disconnected from the line power when the main cover is removed The security lever at the power input socket prevents the detector cover from being removed when line power is still connected To disconnect the detector from line unplug the power cord The power supply still uses some power even if the switch on the front panel is turned off When working with solvents please observe appropriate safety procedures for example goggles safety gloves and protective clothing as described in the material handling and safety data sheet supplied by the solvent vendor especially when toxic or hazardous solvents are used Always operate the detector with the top covers in place There will be a risk of damaging hardware due to overheating when operating the instrument without covers Electronic boards and components are sensitive to electronic discharge ESD In order to prevent damage always use an ESD protection when handling elec
59. 4 10 e 5 5 BCDO 1 pe O ie 8 6 6 BCD 3 8 O 7 7 BCD 2 4 8 8 BCD 1 2 9 9 Digital ground lt O m UI 5V Low 1200 Series FLD Service M anual 289 14 Cables Auxiliary Cable 290 One end of this cable provides a modular plug to be connected to the Agilent 1200 Series vacuum degasser The other end is for general purpose Agilent 1200 Series Degasser to general purposes Connector Color G1322 61600 White Brown Green _ Yellow Grey Pink Pin Agilent 1200 1 O A Aa WUWU N Signal Name Ground Pressure signal DC 5VIN Vent 1200 Series FLD Service M anual Cables 14 CAN Cable Both ends of this cable provide a modular plug to be connected to Agilent 1200 Series module s CAN bus connectors Agilent 1200 module to module 0 5 m 5181 1516 Agilent 1200 module to module 1 m 5181 1519 Agilent 1200 module to Control M odule G1323B G1323 81600 1200 Series FLD Service M anual 291 14 Cables External Contact Cable 292 O o O D on O ee 2 o os O One end of this cable provides a 15 pin plug to be connected to Agilent 1200 Series module s interface board The other end is for general purpose Agilent 1200 Series Interface Board to general purposes Connector G1103 61611 Color White Brown Green Yellow Grey Pink Blue Red Black Violet Grey pink Red blue White green Brown green White yellow Pin Agilent 1200
60. 5181 1519 G1369 60001 5023 0203 5023 0202 01046 60105 G1351 68701 260 1200 Series FLD Service M anual Parts and Materials for Maintenance 12 Cuvette Kit Table 30 Cuvette Kit Item Description Part Number FLD Cuvette Kit 8 ul 20 bar G1321 60007 includes Tubing flexible 1 meter SST Fitting QTY 1 79814 22406 SST front ferrule QTY 1 0100 0043 SST back ferrule QTY 1 0100 0044 PEEK fitting QTY 1 0100 1516 Needle Syringe 9301 0407 Glass Syringe 9301 1446 1200 Series FLD Service M anual 261 12 Parts and Materials for Maintenance Spare Parts 262 The spare parts listed below allow changes to the standard hardware configuration to adapt to specific application needs as it was possible on the HP 1046A Fluorescence detector The installation of these parts may affect the performance of the detector and may not fulfill the instruments specifications Table 31 Spare Parts Description Part Number Cutoff filter kit 389 nm 408 nm 450 nm 500 nm 550 nm Cutoff filter kit 380 nm 399 nm 418 nm 470 nm 520 nm Cutoff filter kit 280 nm 295 nm 305 nm 335 nm 345 nm Cutoff filter 370 nm Photo multiplier Tube PM T R928HA 185 to 900 nm 5061 3327 5061 3328 5061 3329 1000 0822 contact Hamamatsu dealers Photo multiplier Tube PM T R3788HA 185 to 750 nm 1200 Series FLD Service M anual Accessory Kit Parts and Materials for Maintenance 12 This kit contains some accessories and
61. 704 Fittings Qty 2 Part number 0100 1516 Capillary 150 mm long 0 17 mm i d Part number 5021 1817 Starting Your Detector 1 Turn ON the detector 2 Turn ON the lamp When the lamp is turned on the first time the instrument performs some internal checks and a calibration check which takes about 5 minutes 3 You are now ready to change the settings of your detector 1200 Series FLD Service M anual 65 First Steps with the Detector Setting the Chromatographic Conditions 1 Set up the system with the following chromatographic conditions and wait until the baseline gets stable Table6 Chromatographic Conditions M obile phases Column Sample Flow rate Compressibility A water Compressibility B Acetonitrile Stroke A and B Stop time Injection volume Oven temperature 1200 FLD Excitations Emission Wavelength FLD PMT Gain FLD Response time A water 35 B Acetonitrile 65 OSD Hypersil column 125 mm x 4 mm i d with 5 um particles Isocratic standard sample 1 10 diluted in methanol 1 5 ml min 46 115 auto 4 minutes 5 ul 30 C EX 246 nm EM 317 nm PMT 10 4 seconds 1200 Series FLD Service M anual First Steps with the Detector 4 2 Set the FLD setpoints according to Figure 25 on the local Control Module G1323B this information is split across separate screens FLD Signals System 7 E x Multiple Wavelengths and Spectra Excitation A Emission
62. D Service M anual 199 11 Repairs Removing the Rear Foam When required For all repairs inside the detector Parts required Depends on the work inside and the following procedures Preparations for this procedure 1 Using a pliers disconnect the cables from the main board Start with Removing the Covers on page 198 2 Carefully remove the rear foam 200 1200 Series FLD Service M anual Repairs 11 Exchanging the Lamp When required Lamp if noise or intensity exceeds limits or lamp does not ignite Tools required Screwdriver POZI 1 PT3 Hexagonal screwdriver 2 5 mm 100 mm long Hexagonal screwdriver 4mm 100 mm long Parts required Flash lamp 2140 0600 Wait until the lamp has cooled down before touching the lamp and or triggerpack assembly The instrument should not be operated without Xenon flash lamp Otherwise the Triggerpack electronics will be damaged When loosening or removing screws take care that they do not fall into the instrument This may result in a complete removal of other assemblies 1200 Series FLD Service M anual 201 11 Repairs Preparations for this procedure 1 Disconnect the emission and the excitation cables and from the detector main board Turn OFF the detector Disconnect the power cable Remove the flow cell or disconnect the tubings from the flow cell Remove detector from stack and place it on the workbench Remove the covers as described in R
63. Eut ony Paste Table Graphic Aro ezi fio Test Figure 36 Detector Settings for Emission Scan 2 Wait until the baseline stabilizes Complete the run 3 Load the signal In this example just the time range of 13 minutes is displayed 1200 Series FLD Service M anual 4 85 4 First Steps with the Detector FLD1 A Ex 260 Em 350 FLD_PAO1 FLD_PAQ1 D LU 7 211 3 543 N o 7 629 n e Q 2 4 6 3 10 12 mi Figure 37 Chromatogram from Emissions Scan 4 Use the isoabsorbance plot and evaluate the optimal emission wavelengths shown in the table below Figure 38 Isoabsorbance Plot from Emission Scan 86 1200 Series FLD Service M anual First Steps with the Detector 4 Table 15 Peak Time Emission Wavelength 1 5 3 min 330 nm 2 7 2 min 330 nm 3 7 6 min 310 nm 4 8 6 min 360 nm 5 10 6 min 445 nm 6 11 23 min 385 nm 5 Using the settings and the timetable from previous page do a second run for the evaluation of the optimal excitation wavelength See Figure 39 FLD Signals System i f g X Signal Time Multiple Wavelengths and Spectra Excitation A E mission Stoptime ae 7 min C Di C Multi Em i 260 nm f 3230 nm Use additional Excitation DO N oT select Postime Of min r additional excitation C Zem Order Zero Order m nm wavelengths B C E i hm D Doing so will D M 290 rm increase the scan Mea i 2 Acquire Excitation Spectra time
64. FPGA on the FLM main board to be available for calculations Flash Lamp Power Supply The flash lamp is operated with discharge voltages of 500 V or 1250 V The electronics design provides the voltages using a regulating pulse width modulator to switch a 24 V rail with a chopping frequency of 40 kHz toa transformer The resulting high voltage is rectified and fed back to the error amplifiers which generate a control feedback signal for the regulator to increase or decrease the duty cycle of the output signal An additional softstart circuitry and a limitation of the maximum voltage ramp up reduces the resulting noise emission of the power supply The complete flash voltage supply is isolated by optocouplers to avoid crosstalk to other circuits by the large switching currents 1200 Series FLD Service M anual Hardware Information 15 Flash Trigger Voltage Supply The flash lamp trigger assembly includes an ignition transformer which needs a 160 V pulse to generate a short high voltage pulse 10 kV for a reliable lamp ignition A second lamp power supply transformer output is used to generate this trigger voltage at both operating conditions Igniting the Flash Lamp The flash lamp is a plasma discharge lamp with an energy controlled intensity operated at 74 Hz or 296 Hz The applied voltage charges a capacitor array and forms a controlled discharge voltage difference between lamp anode and cathode Then an additional ignition electrode is
65. GPIB address of your detector The switches are preset to a default address see GPIB Default Addresses on page 321 which is recognized once the power is switched on e The CAN bus is a serial bus with high speed data transfer The two connectors for the CAN bus are used for internal Agilent 1200 Series module data transfer and synchronization e Two independent analog outputs provide signals for integrators or data handling systems e The interface board slot is used for external contacts and BCD bottle number output or LAN connections e The REMOTE connector may be used in combination with other analytical instruments from Agilent Technologies if you want to use features such as start stop common shut down prepare and so forth e With the appropriate software the RS 232C connector may be used to control the detector from a computer through a RS 232C connection This connector is activated and can be configured with the configuration switch next to the GPIB connector see Communication Settings for RS 232C Communication on page 322 See your software documentation for further information Together with a Control Module G1323B the RS 232C may be used to print screens to a connected printer e The power input socket accepts a line voltage of 100 240 V AC 10 with a line frequency of 50 or 60 Hz Maximum power consumption is 220 VA There is no voltage selector on your detector because the power supply has a wide ran
66. Introduction to the Fluorescence Detector 1 Optical Unit All the elements of the optical system shown in Figure 6 on page 21 including Xenon flash lamp excitation condenser excitation slit mirror excitation grating flow cell emission condenser cut off filter emission slit emission grating and photo multiplier tube are housed in the metal casting inside the detector compartment The fluorescence detector has grating grating optics enabling the selection of both excitation and emission wavelengths The flow cell can be accessed from the front of the fluorescence detector ash lamp boare Trigger Pack EM Grating assembly Xenon Flash lamp Slit EM Condenser EX Cutoff filter Slit EX Photo M ultiplier Tube Mirror Condenser EM EX Grating assembly REF Diode Flow Cell Diffuser Figure6 Optical Unit 1200 Series FLD Service M anual 21 1 22 Introduction to the Fluorescence Detector The radiation source is a xenon flash lamp The 3 us flash produces a continuous spectrum of light from 200 nm to 900 nm The light output distribution can be expressed as a percentage in 100 nm intervals see Figure 7 The lamp can be used for some 1000 hours depending on the sensitivity requirements You can economize during automatic operation using keyboard setpoints so the lamp flashes during your analysis only The lamp can be used until it no longer ignites but the noise level may increase with usage UV degradation
67. L Board G1321 66512 includes trigger pack W hen working on the optical unit a clean workbench with ESD protection mat must be CAUTION l available Otherwise optical components or electronic boards may be damaged WARNING The instrument should not be operated without Xenon flash lamp Otherwise the Triggerpack electronics will be damaged Preparations for this procedure 1 Place the optical unit on the ESD protection mat Optical unit has been removed as described in Removing the Optical Unit on page 233 The foam must be in place Otherwise cutoff filter and diffuser will fall out 240 1200 Series FLD Service M anual 2 Using the 4 mm hexagonal key unscrew the five screws and lift the cover 4 Remove the lamp as described in Exchanging the Lamp on page 201 ah L Repairs 11 3 Using the 2 5 mm hexagonal key unscrew the two screws and remove the pad A ln Z 1200 Series FLD Service M anual 5 Disconnect the wires from the connector Use a flat screw driver to release the wires me e T o Ba T am mm i f 241 11 Repairs 6 Carefully pull off the ferret from the wires and keep it ina 7 Insert the new FLL board Fit the wires through the holes Safe place T
68. LM replace monochromator assembly defective FLF board Ex or FLM board Em replace monochromator assembly replace FLM board check for connection to FLF Ex and FLM Em or replace monochromator assembly defective FLF board Ex or FLM board Em 1200 Series FLD Service M anual Table 20 Motor Errors continued Error Information 7 Message M otor Friction Too High EX 6709 EM 6710 M otor Position Not Found EX 6711 EM 6712 M otor Position Lost EX 6713 EM 6714 M otor Speed Too Low EX 6715 EM 6716 M otor Speed Unstable EX 6717 EM 6718 M otor Encoder Index Wrong EX 6717 EM 6 718 Description During initialization of the detector the excitation and emission grating resistance test provides the resistance history of the excitation and the emission grating drives The number of revolutions after switching off the drives is a measure of friction The history may show an increasing friction of the drive s over a length of time W hen the wavelength is changed the monochromator should move to the new position The position could not be found A mechanical shock to the instrument during operation may cause a movement of the monochromator The position is lost and the lamp will turn off For proper operation the monochromator gratings must run at a certain constant revolution For proper operation the monochromator gratings must run at a certain constant revolution The act
69. PMT can improve performance For additional PMT types refer to Spare Parts on page 262 1200 Series FLD Service M anual Introduction to the Fluorescence Detector 1 Reference System A reference diode located behind the flow cell measures the excitation EX light transmitted by the flow cell and corrects flash lamp fluctuations and long term intensity drift Because of a non linear output of the diode depending on the EX wavelength the measured data are normalized A diffuser is located in front of the reference diode see Figure 6 on page 21 This diffuser is made of quartz reduces light and allows integral measurement of the light 1200 Series FLD Service M anual 27 1 Introduction to the Fluorescence Detector Analytical Information From Primary Data We now know how the primary data from your sample is acquired in the optical unit But how can the data be used as information in analytical chemistry Depending on the chemistry of your application the luminescence measured by the fluorescence detector will have different characteristics You must decide using your knowledge of the sample what mode of detection you will use Fluorescence Detection When the lamp flashes the fluorescing compounds in your sample will luminesce almost simultaneously see Figure 12 The luminescence is short lived therefore the fluorescence detector need only measure over a short period of time after the lamp has flashed Intensity
70. SD cable J 20 protection required Next Steps Depending on the replacement you want to perform proceed to the following procedures 1200 Series FLD Service M anual 235 11 Repairs Replacing PMT and or FLF board When required If defective or if application specific PMT is required Tools required Screwdriver POZI 1 PT3 Hexagonal key 4mm and 2 5 mm Tweezers Parts required Photo multiplier Tube PM T 1970 0201 Photo multiplier Tube PM T for other application ranges please contact Hamamatsu dealers Alternative PM Ts are either R928HA 185 to 900 nm or R3788HA 185 to 750 nm No other PM Ts are recommended FLF Board G1321 69531 requires firmware revision A 04 06 or above FLR Reference Diode board G1321 66533 W hen working on the optical unit a clean workbench with ESD protection mat must be CAUTION l l available Otherwise optical components or electronic boards may be damaged 236 1200 Series FLD Service M anual 11 Repairs O prar O aD O Q am N LLI aD pi m Cc O Cc O O aD a wn Q mD _ mo O yD Q O w E J WwW UO Q i Preparations for this procedure Optical unit has been removed as described in Removing the Optical Unit on page 233 The foam must be in place Otherwise cutoff filter and diffuser will fall out 3 Disconnect the wires from the connector
71. ST S N 144 resistance of monochromators 142 test chromatogram 148 theory of operation and electronics 295 1200 Series FLD Service M anual Index troubleshooting error messages 106 112 no peaks 133 Status indicators 106 107 U unpacking 48 using EMF 36 UV degradation 22 140 188 W wavelength recalibration 106 138 wavelength calibration 153 wavelength calibration procedure 153 164 wavelength calibration steps 155 wavelength shift of spectra 64 weight and dimensions 42 X xenon flash lamp 21 22 342 Index 1200 Series FLD Service M anual 343 Index 344 1200 Series FLD Service M anual Index 1200 Series FLD Service M anual 345 Index 346 1200 Series FLD Service M anual Index 1200 Series FLD Service M anual 347 Index 348 1200 Series FLD Service M anual Index 1200 Series FLD Service M anual 349 Index 350 1200 Series FLD Service M anual Index 1200 Series FLD Service M anual 351 Index 352 1200 Series FLD Service M anual www agilent com In This Book This manual contains technical reference information about the Agilent 1200 Series fluorescence detector The manual describes the following e introcduction and specifications e installation e using and optimizing e troubleshooting and diagnose e maintenance and repair e parts identification e hardware information e safety and related information Agilent Technologie
72. Spectral differences are caused by specific detector characteristics such as spectral resolution or light SOULCES In practice combining a diode array detector with a fluorescence detector in series gives the full data set needed to achieve the optimum fluorescence excitation and emission wavelengths for a series of compounds in a single run With the UV Visible excitation spectra available from the diode array detector the fluorescence detector is set to acquire emission spectra with a fixed excitation wavelength in the low UV range The example is taken from the quality control of carbamates Samples are analyzed for the impurities 2 3 diamino phenazine DAP and 2 amino 3 hydroxyphenazine AHP Reference samples of DAP and AHP were analyzed with diode array and fluorescence detection Figure 9 shows the spectra obtained from both detectors for DAP The excitation spectrum of DAP is very Similar to the UV absorption spectrum from the diode array detector Figure 34 on page 79 shows the successful application of the method to a carbamate sample and a pure mixture of DAP and AHP for reference The column was overloaded with the non fluorescent carbamate 2 benzimidazole carbamic acid methylester MBC to see the known impurities AHP and DAP 1200 Series FLD Service M anual This is an impurity of carbamates The excitation spectrum in a second run shows the equivalence of UV spectra and fluorescence excitation spectra An excitation wavel
73. Status indicator green yellow red Line power switch with green light Figure 23 Front View of Detector 4 Connect the power cable to the power connector at the rear of the detector 5 Connect the CAN cable to other Agilent 1200 Series modules 54 1200 Series FLD Service M anual Installing the Detector 6 If an Agilent ChemStation is the controller connect either the LAN connection to the LAN interface board in the detector If an Agilent 1200 DAD MWD FLD is in the system the LAN should be connected to the DAD MWD FLD due to higher data load 7 Connect the analog cable s optional 8 Connect the APG remote cable optional for non Agilent Series instruments 9 Turn ON power by pushing the button at the lower left hand side of the detector The status LED should be green Security lever Interface board LAN or BCD EXT ie i an i J ee NY c o O QO C 2 of O yL N l 9 s Analog signal a X I J gt 3 gt 3 C pgi gt hs ee oe ae APG remote c fag S ae a I Pad L Dine RS 232C A Sa C JC Jg J gt lt J CAN GPIB 1200 Series FLD Service M anual Configuration switch Figure 24 Rear View of Detector The detector is turned ON when the line power switch is pressed and the green indicator lamp is illuminated The detector is turned OFF when the line power switch is protruding and the g
74. T QO N l D T 5 Use a pair of tweezers to lift off the top of the mirror holder 4 Locate the mirror assembly A ATA X gt f af Al LE ip f L Ji f oe a 1200 Series FLD Service M anual 228 Repairs 11 6 Remove the mirror assembly 7 Insert the new mirror into the holder The mirror surface faces towards the light The back side is marked as shown 1 0S EN ts 7 Mirror surface faces to this side Note If the mirror is inserted in the opposite direction the detector cannot be aligned correctly 8 Replace the mirror assembly and lock the top over the pin 9 Replace the UV protector 1200 Series FLD Service M anual 229 11 Repairs 10 Replace the front foam Next Steps e Replace the covers as described in Replacing the Foams and Covers on page 254 Re install the flow cell and the front panel Re install the detector in the stack Turn the lamp ON Perform a wavelength verification to check the correct positioning of the replaced assemblies as described in chapter Diagram of Light Path on page 138 230 1200 Series FLD Service M anual Repairs 11 Replacing the Slits When required If requ
75. able 5 Excitation position scan high resolution 260 s variable 6 n Emission rotation scan full circle 61s variable of scans depends on the required PMT gain 1 minute per scan 6 n Em rotation scan full circle instrument profile 9s 6 n Em rotation scan full circle instrument profile 9s 6 n Em rotation scan full circle instrument profile 9s 6 n Em rotation scan full circle instrument profile 9s 7 Emission rotation scan high resolution part 44s 8 Emission rotation scan high resolution part II 44s 9 Emission position scan low resolution 50 s variable 10 Emission position scan high resolution 250 s variable Variable times means that they could be a little bit longer When the lamp is of the calibration process will stop within the first two steps with Wavelength Calibration Failed see Wavelength Calibration Failed on page 130 1200 Series FLD Service M anual 155 8 Test Functions Check of Raman Band of Water This test is a verification of the wavelength calibration described in section Diagram of Light Path on page 138 using the Raman band of water This test requires the Agilent ChemStation Spectra module or the Control M odule G1323B and a running HPLC system If no OQ PV protocol is available use the procedure Using the Agilent ChemStation M anually on page 158 or Using the Control M odule G1323B on page 161 Using the Agilent ChemStation 0Q PV Set up th
76. able J 18 and the EM motor cable J 20 4 Insert the rear foam and reconnect cables EM monochromator and EX monochromator to the detector main board FLM Next Steps Replace the covers as described in Replacing the Foams and Covers on page 254 Re install the flow cell and the front panel Re install the detector in the stack Turn the lamp ON Perform a wavelength verification to check the correct positioning of the replaced assemblies as described in chapter Diagram of Light Path on page 138 1200 Series FLD Service M anual 253 11 Repairs Replacing the Foams and Covers If a new optical unit has been installed or assemblies within the optical unit have been replaced then refer Next Steps on page 247 When required When all repairs have been completed Tools required Screwdriver POZI 1 PT3 Prerequisites The detector is open and other procedures have been carried out The front cover across the optical unit should only be removed when required during a procedure This will keep dust away from optical components 1 Carefully insert the rear foam 2 Route the cables towards the main board and reconnect to their connectors 254 1200 Series FLD Service M anual 11 Repairs 4 Replace the cover 3 Slide the top plate towards the rear and fix the top plate va Ensure
77. al time requirement 316 1200 Series FLD Service M anual Hardware Information 15 Remote Interface The APG remote connector may be used in combination with other analytical instruments from Agilent Technologies if you want to use features such as common shut down prepare and so on Remote control allows easy connection between single instruments or systems to ensure coordinated analysis with simple coupling requirements The subminiature D connector is used The module provides one remote connector which is inputs outputs wired or technique To provide maximum safety within a distributed analysis system one line is dedicated to SHUT DOWN the system s critical parts in case any module detects a serious problem To detect whether all participating modules are switched on or properly powered one line is defined to summarize the POWER ON state of all connected modules Control of analysis is maintained by signal readiness READY for next analysis followed by START of run and optional STOP of run triggered on the respective lines In addition PREPARE and START REQUEST may be issued The signal level is defined as e standard TTL levels 0 V is logic true 5 V is false e fan out is 10 e input load is 2 2 kOhm against 5 V and e outputs are open collector type inputs outputs wired or technique Table 49 Remote Signal Distribution Pin Signal Description 1 DGND Digital ground 2 PREPARE L Request to prepare for anal
78. an is a convenient way to check the quality of the solvent in a few minutes This can be done for example by filling the FLD cuvette directly with the solvent for an offline measurement even before the start of a chromatographic run The result can be displayed as an isofluorescence plot or a three dimensional plot Different colors reflect different intensities Figure 28 shows a sample of slightly impure water which was planned for use as mobile phase The area where fluorescence of the contaminated water sample can be seen is between the stray light areas the first and second order Raleigh stray light and Raman stray light Impurity 1 order Raman 2 order Figure 28 Isofluorescence plot of a mobile phase Since excitation and emission wavelength are the same for Raleigh stray light the area of first order Raleigh stray light is visible in the left upper area of the diagram The Raman bands of water are seen below the first order Raleigh stray light Since the cut off filter cuts off light below 280 nm the second order Raleigh stray light starts above 560 nm 1200 Series FLD Service M anual First Steps with the Detector 4 Stray light acts in the same way as impurities in that it simulates background noise In both cases a higher noise level and therefore a higher limit of detection are obtained This indicates that high sensitivity measurements should be done away from wavelength settings that have a high stray light backgro
79. and will lower Line Time From To Step PMT Threshold Peakwidth B All T the performance Range 230 to 400 Hm Step 5 nm Threshold J100 LU Time Spectrum 945 me Peakwidth Responsetime p 0 2 min 4 s standart m Insert Append Eut Eopy Paste i PMT Gain 10 Test Figure 39 Detector Settings for Excitation Scan 1200 Series FLD Service M anual 87 4 First Steps with the Detector 6 Wait until the baseline stabilizes Start the run 7 Load the signal FLD1 A Ex 260 Em 330 TT FLD_PAO1 FLD_PAOD2 D 0 2 4 6 S 10 12 mi Figure 40 Chromatogram excitation scan at reference wavelength 260 330 nm 8 Use the isoabsorbance plot and evaluate the optimal excitation wavelengths in this example just in the time range of 13 minutes Figure 41 Isoabsorbance Plot Excitation 88 1200 Series FLD Service M anual Table 16 Peak 1 OH UOT S WW VN First Steps with the Detector 4 The table below shows the complete information about emission from Figure 38 on page 86 and excitation maxima Time 5 3 min 7 3 min 7 7 min 8 5 min 10 7 min 11 3 min Emission Wavelength 330 nm 330 nm 310 nm 360 nm 445 nm 385 nm Excitation Wavelength 220 280 nm 225 285 nm 265 nm 245 nm 280 nm 270 330 nm 1200 Series FLD Service M anual Evaluating The System Background The example below uses water 1 Pump solvent through your system 2 Set the fluore
80. anging the fan 219 installing the optical unit 252 introduction 170 of the detector 169 175 195 removing covers 198 200 removing optical unit 233 removing rear foam 200 replacing cutoff filter 224 replacing EM and EX Grating 244 replacing EM condenser 224 replacing FLF board 236 replacing FLL board 240 replacing foams and covers 254 replacing leak handling system 183 replacing mirror assembly 227 replacing PMT 236 replacing slits 231 replacing status light pipe 251 replacing trigger pack 240 using the ESD strap 174 warnings and cautions 170 replacing interface board BCD LAN 184 resistance of monochromators 142 responsetime 32 RS 232C cable kit to PC 293 communication settings 322 interface 318 settings 322 341 S safety information on lithium batteries 331 Standards 42 serial number entered on control module 217 entered on instant pilot 216 site requirements 40 Spare parts cutoff filters 262 specifications analog outputs 44 communications 44 flow cell 44 GLP features 45 monochromators 43 performance 43 pulse frequency 43 Safety and maintenance 45 wavelength accuracy 43 spectra acquisition modes 311 spectra wavelength shift 64 stack configuration 51 53 front view 51 rearview 53 T test chromatogram 148 test functions 106 138 tests DAC 151 dark current 141 functions 138 lamp intensity 139 lamp intensity history 140 188 PMT gain test 63 Raman A
81. arameters to be optimized in fluorescence detection are the excitation and emission wavelengths Generally it is assumed that the best excitation wavelength can be taken from the excitation spectrum acquired on a spectrofluorimeter It is also assumed that once the optimal excitation wavelength has been found for one particular instrument type this wavelength can also be applied to other instruments Both assumptions are wrong The optimum wavelength for the excitation depends on the absorption of the compounds but also on the instrument characteristics for example the lamp type and the gratings As most organic molecules absorb best in the ultra violet range the Agilent 1200 Series fluorescence detector was designed to give an optimum signal to noise ratio in the 210 nm to 360 nm range of the spectrum To achieve greatest sensitivity the absorbance wavelength of your sample molecule should match the wavelength range for your instrument In other words an excitation wavelength in the ultra violet range Your Agilent 1200 Series fluorescence detector has a broad excitation wavelength range but for higher sensitivity you should choose a wavelength in the ultra violet range near 250 nm The design elements that contribute to lower efficiency in the lower ultra violet range are the xenon flash lamp and the gratings Flash type lamps shift the optimum wavelength to lower wavelength ranges with the Agilent 1200 Series fluorescence detector to a m
82. ater Housing All materials recyclable Environment 0 to 40 C constant temperature at lt 95 humidity non condensing Dimensions 140 mm x 345 mm x 435 mm 5 5 x 13 5 x 17 inches height x width x depth Weight 11 5 kg 25 5 Ibs Reference conditions standard cell 8 ul response time 4 s flow 0 4 ml min LC grade M ethanol 2 1 x 100 mm ODS column 1200 Series FLD Service M anual 45 2 Site Requirements and Specifications 46 1200 Series FLD Service M anual Agilent 1200 Series Fluorescence Detector Service M anual 0 ee 3 Installing the Detector amp I j z Unpacking the Detector 48 Optimizing the Stack Configuration 51 Installing the Detector 54 Flow Connections to the Detector 57 This chapter describes the installation of the detector ee Agilent Technologies 47 3 Installing the Detector Unpacking the Detector Damaged Packaging If the delivery packaging shows signs of external damage please call your Agilent Technologies sales and service office immediately Inform your service representative that the detector may have been damaged during shipment CAUTION If there are signs of damage please do not attempt to install the detector Delivery Checklist Ensure all parts and materials have been delivered with the detector The delivery checklist is shown below Please report missing or damaged parts to your local Agilent Technologies sales and service office Table
83. aximum of 250 nm The excitation grating is blazed for highest efficiency at 300 nm 1200 Series FLD Service M anual How to optimize the Detector 5 A Real Example Although an excitation wavelength of 340 nm is quoted in the literature the Agilent 1200 Series fluorescence detector scan of orthophthalaldehyde a derivative of the amino acid alanine Figure 44 on page 95 shows a maximum between 220 nm and 240 nm LC Z 1046A EX of AAZHS1GL D ALA LC Z 1046A EM of AR HS1IGL D D U G u v ep0 3200 400 500 Excitation Wavelength nm Emission Figure 44 Scan Orthophthalaldehyde Derivative of Alanine When you are looking for the wavelength by scanning scan over the whole range As this example shows a maximum may be found in a completely different wavelength range When comparing fluorescence excitation spectra directly with DAD spectra or literature based absorbance spectra you should consider large differences in the used optical bandwidth FLD 20 nm which cause a systematic wavelength maximum shift depending on the absorbance spectrum of the compound under evaluation 1200 Series FLD Service M anual 95 5 How to optimize the Detector Finding the Best Signal Amplification Increasing the PMTGAIN increases the signal and the noise Up to a certain factor the increase in signal is higher than the increase in noise The step from gain to gain is equal to a factor of 2 which is the same as on the HP 1046A FLD In
84. band and wrap the exposed adhesive side firmly around your wrist 2 Unroll the rest of the band and peel the liner from the copper foil at the opposite end 3 Attach the copper foil to a convenient and exposed electrical ground Figure 69 Using the ESD Strap 174 1200 Series FLD Service M anual Agilent 1200 Series Fluorescence Detector Service M anual ee 10 Yo Maintenance Overview of Maintenance 176 Exchanging a Flow Cell 177 How to use the Cuvette 180 Flow Cell Flushing 181 Correcting Leaks 182 Replacing Leak Handling System Parts 183 Replacing the Interface Board 184 Replacing the Detector s Firmware 185 Tests amp Calibrations 186 Lamp Intensity Test 187 Wavelength Verification and Calibration 189 Wavelength Calibration Procedure 191 This chapter describes the maintenance of the detector and the required tests fhe Agilent Technologies 175 10 Maintenance Overview of Maintenance On the following pages repairs are described that can be carried out without opening the main cover Table 28 Simple Repairs Procedure Typical Frequency Notes Flow cell exchange If application requires a different flow cell type or if Complete Assembly defective A wavelength calibration check should be performed after replacement If the flow cell is removed and inserted then a quick calibration check is performed If this fails you must do a wavelength recalibration see Wavelength Verification and Cali
85. bration on page 189 Flow cell flushing If flow cell is contaminated Leak sensor drying If leak has occurred Check for leaks Leak handling System If broken or corroded Check for leaks replacement 176 1200 Series FLD Service M anual Maintenance 10 Exchanging a Flow Cell When required If an application needs a different type of flow cell or the flow cell is defective leaky Tools required Two 1 4 inch wrenches for capillary connections Parts required Standard flow cell 8 ul 20 bar G1321 60005 Cuvette for off line measurements 8 ul 20 bar G1321 60007 refer to How to use the Cuvette on page 180 for more information on usage DO NOT install the inlet capillary to the outlet connection of the flow cell This will result in poor performance Preparations for this procedure 1 Press the release buttons and remove the front cover for access to the flow cell area Turn off the flow 1200 Series FLD Service M anual 177 10 Maintenance 2 Disconnect the capillaries from the flow cell Note The label attached to the flow cell provides information on part number cell volume and maximum pressure The cell type will be automatically detected There are no parts that can be replaced on the flow cell If defective leaky the flow cell has to be replaced completely 178 3 Unscrew the thumb screws and pull the flow cell out of the compartment a TTT TTT 4 Insert the flow cell and tighten
86. ce Manual for the Agilent 1200 Series vacuum degasser 114 1200 Series FLD Service M anual Error Information 7 Remote Time out A not ready condition is still present on the remote input When an analysis is started the system expects all not ready conditions e g a not ready condition during detector balance to switch to run conditions within one minute of starting the analysis If a not ready condition is still present on the remote line after one minute the error message is generated Probable Causes e Not ready condition in one of the instruments connected to the remote line e Defective remote cable e Defective components in the instrument showing the not ready condition Suggested Actions Y Ensure the instrument showing the not ready condition is installed correctly and is set up correctly for analysis VY Exchange the remote cable VY Check the instrument for defects refer to the instrument s reference documentation 1200 Series FLD Service M anual 115 7 116 Error Information Synchronization Lost During an analysis the internal synchronization or communication between one or more of the modules in the system has failed The system processors continually monitor the system configuration If one or more of the modules is no longer recognized as being connected to the system the error message is generated Probable Causes e CAN cable disconnected e Defective CAN cable e Defective ma
87. cell is transported while temperatures are below 5 degree C it must be assured that the cell is filled with alcohol Aqueous solvents in the flow cell can built up algae Therefore do not leave aqueous solvents sitting in the flow cell Add small of organic solvents e g Acetonitrile or Methanol 5 Solvents Brown glass ware can avoid growth of algae Always filter solvents small particles can permanently block the capillaries Avoid the use of the following steel corrosive solvents e Solutions of alkali halides and their respective acids for example lithium iodide potassium chloride and so on e High concentrations of inorganic acids like nitric acid sulfuric acid especially at higher temperatures replace if your chromatography method allows by phosphoric acid or phosphate buffer which are less corrosive against stainless steel e Halogenated solvents or mixtures which form radicals and or acids for example 2CHCl O gt COCs 2HCI This reaction in which stainless steel probably acts as a catalyst occurs quickly with dried chloroform if the drying process removes the stabilizing alcohol 1200 Series FLD Service M anual 335 A Appendix e Chromatographic grade ethers which can contain peroxides for example THF dioxane di isopropylether such ethers should be filtered through dry aluminium oxide which adsorbs the peroxides e Solutions of organic acids acetic acid formic acid and so on in organic
88. components or electronic boards may be damaged The grating assembly is shipped with a special transport packaging and includes a protection ring DO NOT touch the grating surface and DO NOT bend the protection ring 244 1200 Series FLD Service M anual Preparations for this procedure Optical unit has been removed as described in Removing the Optical Unit on page 233 The cables from the position encoders at the rear of the EM or EX grating assembly should be disconnected Disconnect the encoder cable of the EX monochromator from FLF board 2 Use a 2 5 mm hex key to unscrew the 3 screws on the assembly you want to remove 1200 Series FLD Service M anual Repairs 11 1 Place the optical on the ESD protection mat and remove the front foam A Locate the EM and EX grating assembly As l SD oN 3 Carefully remove the grating assembly and the protection ring from its location Couple Spring Protection ring 245 11 Repairs 4 Insert the new protection ring into its position Do not bend the protection ring 6 Bring the top of the assembly into the position shown below and fix the 3 screws 246 5 For reinstallation route the cable first through the hole Carefully insert the grating into its position while pulling the cable slightly from below The couple spring has to be clipped into the protection ring TOP mark 7 Assure the UV protector cover is instal
89. correct fit with Z plane 6 Replace the waste funnel assmbly and the front panel 5 If required insert the interface board and fix the screws Next Steps D QO O q aD Bem p T J WN WM oc O om UO J WN aD a J O lt O J UO w WM oO WO ZC J WM UO Q aD oc s O WH O D WH D 5 oD WH a lt WH 5 am co ro O _ D 2 co S WH WH O D am O D oc O Q aD lt c See 5 H 255 1200 Series FLD Service M anual 11 Repairs Assembling the M ain Cover When required If cover is broken Tools required None Parts required Plastics kit 5062 8582 includes base top left and right The plastics kit contains all parts but it is not assembled WARNING If you mistakenly insert the left or right side in the opposite position you may not be able to remove that side from the top part 1 Place the top part on the bench and insert the left and right 2 Replace the cover sides into the top part 256 1200 Series FLD Service M anual Repairs 11 Next steps Replace the detector in the stack and reconnect the cables and capillaries Turn ON the detector 1200 Series FLD Service M anual 257 11 258 Repairs 1200 Series FLD Service M anual j Agilent 1200 Series Fluorescence Detector Service M anual 20 ee 12 7
90. cribed in the material handling and safety data sheet supplied by the solvent vendor especially when toxic or hazardous solvents are used Electronic boards and components are sensitive to electronic discharge ESD In order CAUTION l to prevent damage always use an ESD protection when handling electronic boards and components see Using the ESD Strap on page 174 CAUTION There iS a risk of damaging hardware due to overheating when operating the instrument without covers 1200 Series FLD Service M anual 171 9 Maintenance and Repair WARNING Eye damage may result from directly viewing the light produced by the Xenon flash lamp used in this product Always turn the xenon flash lamp off before removing It 172 1200 Series FLD Service M anual Maintenance and Repair 9 Cleaning the Detector The detector case should be kept clean Cleaning should be done with a soft cloth slightly dampened with water or a solution of water and mild detergent Do not use an excessively damp cloth allowing liquid to drip into the detector Do not let liquid drip into the detector It could cause shock hazard and it could damage the detector 1200 Series FLD Service M anual 173 9 Maintenance and Repair Using the ESD Strap Electronic boards are sensitive to electronic discharge ESD In order to prevent damage always use an ESD strap when handling electronic boards and components 1 Unwrap the first two folds of the
91. dious procedure it is applicable only when there is a limited number of compounds of interest The Agilent 1200 Series LC offers three different ways to obtain complete information on a compound s fluorescence Procedure I Take a fluorescence scan offline for a single compound as described above for the mobile phase This is done preferably with a manual FLD cuvette when pure compounds are available Procedure II Use two LC runs with the Agilent 1200 Series FLD to separate the compound mix under known conditions and acquire emission and excitation spectra separately Procedure III Use an Agilent 1200 Series FLD DAD combination and acquire UV Visible spectra equivalent to excitation spectra with the DAD and emission spectra with the FLD both in a single run Procedure Take a fluorescence scan Because fluorescence spectra traditionally have not been easily available with previous LC fluorescence detectors standard fluorescence spectrophotometers have been used in the past to acquire spectral information for unknown compounds Unfortunately this approach limits optimization as there are differences expected in optical design between an LC detector and a dedicated fluorescence spectrophotometer or even between detectors These differences can lead to variations for the optimum excitation and emission wavelengths The Agilent 1200 Series fluorescence detector offers a fluorescence scan that delivers all spectral information prev
92. displayed press Adjust and OK The history table will be updated To look at the history table ChemStation start a wavelength calibration and abort immediately No changes are made to the calibration at this time Rinse the flow cell with pure water at a minimum of 1 5 ml min to get rid of the Glycogen from the cell and the capillaries When organic solvent is sequentially applied without rinsing a blockage of capillaries may occur 166 1200 Series FLD Service M anual Test Functions 8 FLD Wavelength Calibration System 7 j X Hi S Calibration history Deviation Excitation Emission Time Date 0 0 nm 0 0 nm 14544 04 04 2001 0 6 nm 1 0 nm 10 00 53 06 10 2004 5 pP a a Fa p Excitation OOU O6nm O8nm 15 44 26 09 01 2006 J2nm 362 0nm 12 hm Emission UU Tznm 3620nm 12 rnm Excitation deviati 0 27 nm Emizsion deviatio 0 1 nm Abort OK Cancel Calibration settings not equal to measured ones To calibrate click Adjust Figure 68 Wavelength Calibration Verification 1 Refit the capillary to the flow cell Follow the procedure Check of Raman Band of Water on page 156 1200 Series FLD Service M anual 167 8 Test Functions 168 1200 Series FLD Service M anual Agilent 1200 Series Fluorescence Detector Service M anual 2 ee 9 Maintenance and Repair a j Introduction into Repairing the Fluorescence Detector 170 Warnings and Cautions 171 Cleaning the
93. ditonal Excitation E r 250 nm E r 250 nm DO p 7250 nm as Pump a Excitation 4 Emission stoptime no Limit f 350 am 297 nm Posttime Off 2 Zero Order Zero Order st Inert Append Eyt Tabe Graphic Timetable Acquire Excitation Spectra Cancel Help Line Time Em Ex Ex B E C Ex D Ex Spectra From 3 p fan wro signals Range 340 to 360 nim Step Zz nm Threshold Time Spectrum 283 ms Peakwidth Responsetime inset Append cw f Copy _ Paste Gein 10 Table Graphic een Short lt lt Special Setpoints Figure 62 Settings for EM EX Scan 2 Load the method WLEXTEST The FLD will change into the multi emission mode and scan in the range of the expected maximum of 397 nm 20 nm 1200 Series FLD Service M anual 159 8 Test Functions 3 Turn the pump ON and flush with water for a few minutes to assure a clean flow cell Flow rate should be 0 5 to 1 ml min and the baseline stable You may remove the flow cell and check for air bubbles After re inserting the cell turn the lamp ON 4 Open the Online Spectra plot and observe the maximum as shown in Figure 60 on page 157 left 5 Load the method WLEMTEST The FLD will change into the multi excitation mode and scan in the range of the expected maximum of 350 nm 20 nm 6 Open the Online Spectra plot and observe the maximum as shown in Figure 60 on page 157 right 160
94. driven with a high ignition voltage pulse to build up a small ionized channel from the ignition electrode to the cathode If this ionization is existent the voltage difference between anode and cathode leads to a flash discharge within a very short time 1 us After the flash the system pauses the regulator to force the ionized path to break down rapidly before charging the capacitors for the next cycle Flash Lamp Overcurrent Protection A special firmware controlled diagnostic circuitry shuts down the regulator when the charge current exceeds a critical value This may happen when the output voltage lines are shorted When an overcurrent condition is found the circuit stops the voltage regulator within the time between two flashes and the firmware reports an error signal when a restart of the regulator fails again Flash Voltage Safety Switch A safety switch stops the voltage regulator when the lamp cover is removed to avoid high voltages on board when electronic parts are accessible 1200 Series FLD Service M anual 299 15 Hardware Information Photomultiplier Tube Voltage Supply The photomultiplier tube needs an adjustable working voltage between 240 V and 820 V for correct operation The PMT gains are realized by variable operating voltage of the device A regulated pulse width modulator is used to generate the high voltage by switching the 24 V rail to a transformer The feedback loop has a D A converter controlled base voltage
95. e Use the syringe see Cuvette Kit on page 261 to inject the compound Setup the parameters for the Fluorescence Scan under Special Setpoints Select Take Fluorescence Scan on the user interface to start the off line measurement 1200 Series FLD Service M anual Maintenance 10 Flow Cell Flushing When required If flow cell is contaminated Tools required Glass syringe adapter Parts required Bidistilled water nitric acid 65 tubings to waste Aqueous solvents in the flow cell can built up algae Algae do fluoresce Therefore do not leave aqueous solvents in the flow cell for longer periods Add a small percentage of organic solvents e g Acetonitrile or M ethanol 5 In case the cell is contaminated follow the procedure below Flushing Procedure 1 Flush with bidistilled water 2 Flush with nitric acid 65 using a glass syringe 3 Leave this solution in the cell for about one hour 4 Flush with bidistilled water This concentration of nitric acid is dangerous and proper attention to safety should be given Also the nitric acid flushing procedure is not an infallible remedy for a dirty cell It is to be used as a last attempt to salvage the cell before cell replacement Note that the cell is a consumable item Do not exceed the pressure limit of 20 bar 0 2 M Pa 1200 Series FLD Service M anual 181 10 Maintenance Correcting Leaks When required If a leakage has occurred in the flow cell area or at
96. e current gets too high an error is generated and the lamp is turned OFF Probable Causes e shortage of flash lamp assembly e shortage of trigger pack assembly e defective FLL board Suggested Actions VY Exchange the flash lamp assembly VY Exchange the FLL board includes the trigger pack 128 1200 Series FLD Service M anual Error Information 7 Flash Trigger Lost This message is displayed when the flash trigger is no longer generated Probable Causes e firmware problem e defective encoder Suggested Actions VY Reboot the detector power cycle v Multi Mode Off exchange the firmware or replace the main board VY Multi Mode Ex replace EX monochromator assembly v Multi Mode Em replace EM monochromator assembly 1200 Series FLD Service M anual 129 7 130 Error Information Wavelength Calibration Failed This message may show up during a wavelength calibration If the expected deviation is larger than the specified wavelength accuracy the message Wavelength Calibration Failed is displayed and the instrument stays in a Not Ready condition Probable Causes e Flash lamp not ignited or position not correct e Cell position not correct e Solvent in the cell not clean or air bubble in the cell e monochromator assembly position not correct after replacement Suggested Actions VY Check the flash lamp image and position See also No Peaks on page 133 VY Check the cell position Y Fl
97. e 30 Characterization of a pure compound from a fluorescence scan First Steps with the Detector 4 Procedure Il Take two LC runs with the FLD The conditions for the separation of organic compounds such as polyaromatic nuclear hydrocarbons PNAs are well described in various standard methods including commonly used EPA and DIN methods Achieving the best detection levels requires checking for the optimum excitation and emission wavelengths for all compounds Yet taking fluorescence scans individually makes this a tedious process A better approach is to acquire spectra online for all compounds during a run This speeds up method development tremendously Two runs are sufficient for optimization During the first run one wavelength is chosen in the low UV range for the excitation wavelength and one emission wavelength in the spectral range for the emission wavelength Most fluorophores show strong absorption at these wavelengths and the quantum yield is high Excitation is sufficient for collecting emission spectra Figure 31 on page 76 contains all emission spectra obtained in a single run from a mix of 15 PNAs This set of spectra is used to set up a timetable for optimum emission wavelengths for all compounds The individual compound spectra in the isofluorescence plot show that at least three emission wavelengths are needed to detect all 15 PNAs properly Table8 Timetable for PNA analysis 0 min 350 nm for naphthalene to phena
98. e HPLC system and the ChemStation Flush the flow cell with clean bi distilled water Turn ON the FLD lamp Select Verification OQ PV Create a New Instrument Verification Select the Excitation Wavelength Accuracy and the Emission Wavelength Accuracy checks Raman Band of Water Set the limits to 3 nm on both tests ul FPF WN fF fF OS Run the verification The FLD will change into the multi excitation mode with emission wavelength at 397 nm and scan in the range of the expected maximum of 350 nm 20 nm As result the maxima should be found at 350 nm 3 nm see Figure 60 on page 157 The FLD will change into the multi emission mode with excitation wavelength at 350 nm and scan in the range of the expected maximum of 397 nm 20 nm 156 1200 Series FLD Service M anual Test Functions 8 As result the maxima should be found at 397 nm 3 nm see Figure 60 If the limits are not met check for Interpretation of the Results on page 147 or perform Wavelength Calibration Procedure on page 164 FLD 0 765 0 7 LU Ex Em 397 of WLEsS03 0 FLO 0 697 29 4 LU E m E5350 of WLE M sos gt EM 397 nm EX 350 nm fixed fixed Figure 60 Excitation and Emission Spectrum 1200 Series FLD Service M anual 157 8 158 Test Functions Using the Agilent ChemStation Manually 1 Create the methods WLEMTEST and WLEXTEST as listed Table 27 Table 27 Method Settings Setting Pea
99. e detector from stack and place it on the workbench Remove the covers as described in Removing the Covers on page 198 1200 Series FLD Service M anual 219 11 Repairs 2 Carefully remove the rear foam 3 Lift the main board slightly Bring the fan cable up and disconnect the fan cable Pull out the fan sim ee 4 When the fan is inserted assure that the air flow is from 5 Carefully insert the rear foam Reconnect the the rear towards the front of the detector monochromator cables to the main board Left is excitation right is emission Insert the fan Lift the main board slightly Reconnect the fan cable and route it underneath the board Next Steps e Replace the covers as described in Replacing the Foams and Covers on page 254 Re install the detector in the stack 220 1200 Series FLD Service M anual Repairs 11 Exchanging the Leak Sensor or Leak Panel When required If defective Tools required Screwdriver POZI 1 PT3 Flat screwdriver Parts required Leak sensor assembly 5061 3356 Leak Pan G1321 40511 Preparations for this procedure 1 Locate the leak sensor cable Turn OFF the detector Disconnect the power cable Remove the flow cell Remove detector from stack and place it on the workbench Remove the covers as described in Removing the C
100. e is needed because the main power supply is safe against any short circuits or overload conditions on the output lines When overload conditions occur the power supply turns off all output voltages Turning the line power off and on again resets the power supply to normal operation if the cause of the overload condition has been removed An over temperature sensor in the main power supply is used to turn off output voltages if the temperature exceeds the acceptable limit for example if the cooling fan of the instrument fails To reset the main power supply to normal operating conditions turn the instrument off wait until it is approximately at ambient temperature and turn the instrument on again The following table gives the specifications of the main power supply Table 59 Main Power Supply Specifications Maximum power 130W Continuous output Line Input 100 240 volts AC Wide ranging 10 line frequency of 50 60 Hz Output 1 24V 4 5 A maximum total power consumption of 24 V and 36 V must not exceed 107 W Output 2 36V 2 5 A maximum Output 3 5V 3A Output 4 15V 0 3A Output 5 15V 0 3A 1200 Series FLD Service M anual This chapter provides safetey and other general information Agilent 1200 Series Fluorescence Detector Service M anual A Appendix General Safety Information 328 Lithium Batteries Information 331 Radio Interference 332 SoundEmission 333 UV Radiation UV lamps only 334 Solvent Informati
101. e used to check the signal path from the detector to the ChemStation and the data analysis or via the analog output to the integrator or data system An example is shown in Figure 57 The chromatogram is continuously repeated until a stop is executed either by means of a stop time or manually FLD1 A Test Chromatogram DIAGNOSE TESTCRO1 D 2 774 2 193 p 1 423 b 4 222 0 0 5 1 1 5 2 2 5 3 3 5 4 4 5 mi Figure 57 Built in Test Chromatogram default settings 148 The peak height is always the same but the area and the retention time depend on the set peakwidth see examples below 1200 Series FLD Service M anual Test Functions 8 Table 24 Peakwidth gt 0 05 min response time 1 s fast Stop time 1 2 minutes Peakwidth gt 0 20 min response time 4 s STD Stop time 4 8 minutes Procedure using the Agilent ChemStation 1 Load the FLD default parameter peakwidth to gt 0 2 minutes standard set stop time to 4 8 minutes 2 Complete the Sample Information no injection 3 Type into the command line or as pre run command PRINT SENDMODULES LFLD SIMU DFLT 4 The logbook shows Preparing for a simulation 5 Start the run The results are processed like a regular chromatogram The test chromatogram is switched off automatically at the end of the run Procedure using the Control M odule G1323B 1 Load the FLD default parameter peakwidth to gt 0 2 minutes standard set stop time to 4 8 minutes
102. ed without Xenon flash lamp Otherwise the Triggerpack electronics will be damaged When loosening or removing screws take care that they do not fall into the instrument This may result in a complete removal of other assemblies 206 1200 Series FLD Service M anual Repairs 11 Preparations for this procedure 1 Disconnect the emission and the excitation cables and from the detector main board Turn OFF the detector Disconnect the power cable Remove the flow cell or disconnect the tubings from the flow cell Remove detector from stack and place it on the workbench Remove the covers as described in Removing the Covers on page 198 2 Carefully remove the rear foam 3 Using the 4 mm hexagonal key unscrew the five screws remove the cover and place it on the bench without turning it around DO NOT unscrew the grounding screw marked with X HES 1200 Series FLD Service M anual 207 11 Repairs 4 Using a 2 5 mm hexagonal key loosen the screw and 5 Insert the new or cleaned condenser shift it completely to remove the condenser There is a mechanical stop for the its mechanical stop and fit the screw condenser 6 Replace the lamp housing cover and fix the screws Carefully insert the rear foam Reconnect the monochromator cables to the main board Left is excitation right is emission Next Steps e Replace the covers as described in Replacing
103. edback when the user selectable limits have been exceeded The visual feedback in the user interface provides an indication that maintenance procedures should be scheduled EM F Counters The detector provides three EMF counters for the lamp The counters increment with lamp use and can be assigned a maximum limit which provides visual feedback in the user interface when the limit is exceeded The counters can be reset to zero after the lamp is exchanged The detector provides the following EMF counters e number of flashes low power mode multiples of 1000 flashes e number of flashes high power mode multiples of 1000 flashes e Flash Lamp Life Time value of 0 100 as expected lifetime factor calculated from combined expected lifetime low power and high power flashes Figure 18 on page 37 shows the lamp life based on number of flashes vs the input energy The lamp flash frequency energy can be changed into the following modes Table1 Flash Lamp M odes Position 296 Hz Standard 560 V 63 mj oule 18 8 W 74 Hz Economy 560 V 63 mJ oule 4 7 W Rotation M ulti Ex Em 74 Hz Standard 950 V 180 mj oule 13 3 W 74 Hz Economy 560 V 63 mJ oule 4 7 W 1200 Series FLD Service M anual Number of flashes Figure 18 Introduction to the Fluorescence Detector Number of flashes to 75 of intitial radiometric light i output 03 Input energy per flash 0 05 02 Lamp life 1200 Series FLD Serv
104. emoving the Covers on page 198 2 Carefully remove the rear foam 3 Using the 4 mm hexagonal key unscrew the five screws remove the cover and place it on the bench without turning it around DO NOT unscrew the grounding screw marked with X 202 1200 Series FLD Service M anual Repairs 11 4 Using the 2 5 mm hexagonal key unscrew the two screws 5 Loosen the screws of the trigger pack holder a few turns and remove the brass pad together with its holder 6 Tip the holder to release the lamp and trigger pack Slide WARNING the cables of the trigger pack through the holder ore 2 While pulling off the lamp be careful that you do not scrape your skin on the metal plates Do not remove the bottom pad Otherwise the lamp 5 i l will loose its correct position during reinstallation AN ps 1200 Series FLD Service M anual 203 11 Repairs 7 Remove the lamp carefully from the trigger pack 9 Place the lamp on the bottom pad 8 Carfully insert the new lamp completely into the trigger pack and slide the cables through the holder 10 Refit the trigger pack holder by moving the lamp towards 11 Place the pad with the holder on the stand offs The TOP the condenser Leave a distance of about 0 5 mm betwe
105. en Sign should be directed towards the trigger pack Refit the each other Refit the screws Screws Note During the next step no horizontal movement should be made to the pad 204 1200 Series FLD Service M anual Repairs 11 12 While pressing the top pad down onto the stand offs 13 Replace the lamp housing cover and fix the screws tighten the screws Turn the trigger pack such as its Carefully insert the rear foam Reconnect the marker stays in line with the TOP sign of the brass monochromator cables to the main board Left is holder excitation right is emission Next Steps Replace the covers as described in Replacing the Foam and the Top Cover on page 149 Re install the detector in the stack Enter the replacement of the lamp in the maintenance logbook this will reset the lamp counter Turn the lamp ON Perform a wavelength verification to check the correct positioning of the replaced assemblies as described in chapter Diagram of Light Path on page 138 1200 Series FLD Service M anual 205 11 Repairs Exchanging the EX condenser When required Condenser if contaminated Tools required Screwdriver POZI 1 PT3 Hexagonal screwdriver 2 5 mm 100 mm long Hexagonal screwdriver 4 mm 100 mm long Parts required EX condenser Assembly 1000 1136 Wait until the lamp has cooled down before touching the lamp and or triggerpack assembly The instrument should not be operat
106. ength at 265 nm was used for taking the emission spectrum and an emission wavelength at 540 nm was used for taking the excitation Spectrum The two upper traces are obtained using two different excitation wavelengths The lower trace is a pure standard of the known impurities Norm 35 30 First Steps with the Detector 4 265 nm 430 nm UV Excitation DAU spectra Emission ina 200 250 300 350 400 450 500 550 Wavelength nm Figure 33 UV spectrum and fluorescence spectra for 2 3 diaminophenazine DAP 2 amino 3 QH phenazine Unknown 3 diaminaphenazine MBC 265 540 nm 430 540 nm Standard 0 2 A 6 8 10 12 Time min Figure 34 Qualitive analysis of MBC 2 benzimidazole carbamic acid methylester and impurities 1200 Series FLD Service M anual 79 4 First Steps with the Detector Table 11 Conditions for Figure 33 and Figure 34 on page 79 Column Mobile phase Gradient Flow rate Column temperature Injection volume FLD settings Step 3 Set up routine methods Zorbax SB 2 x 50 mm PNA 5 um A water B acetonitrile 0 minutes 5 10 minutes 15 0 4 ml min 35 C 5 ul PMT 12 response time 4s step size 5 nm Ex 265 nm and 430 nm Em 540 nm In routine analysis sample matrices can have a significant influence on retention times For reliable results sample preparation must be thorough to avoid interferences or LC methods must be rugged enough With difficult
107. ens and passes through a second slit Before the luminescence reaches the emission monochromator a cut off filter removes light below a certain wavelength to reduce noise from 1 order scatter and 2 order stray light see Figure 9 on page 24 The selected wavelength of light is reflected onto the slit in the wall of the photo multiplier compartment of the optical unit The bandwidth of the emitted light is 20 nm 1200 Series FLD Service M anual 25 1 26 Introduction to the Fluorescence Detector On the photocathode Figure 11 incident photons generate electrons These electrons are accelerated by an electrical field between several arc shaped dynodes Depending on the voltage difference between any pair of dynodes an incident electron may spark off further electrons which accelerate onto the next dynode An avalanche effect results finally so many electrons are generated that a current can be measured The amplification is a function of the voltage at the dynodes and is microprocessor controlled You can set the amplification using the PMTGAIN function incident light Figure 11 Photo multiplier Tube This type of so called side on photo multiplier is compact ensuring fast response conserving the advantages of the short optical path shown in Figure 6 on page 21 PMTs are designed for specific wavelength ranges The standard PMT offers optimum sensitivity from 200 to 600 nm In the higher wavelength range a red sensitive
108. er than the ones supplied by Agilent Technologies to ensure proper functionality and compliance with safety or EMC regulations Bench Space The detector dimensions and weight see Table 2 on page 42 allows you to place the detector on almost any desk or laboratory bench It needs an additional 2 5 cm 1 0 inches of space on either side and approximately 8 cm 3 1 inches in the rear for air circulation and electric connections If the bench should carry a Agilent 1200 Series system make sure that the bench is designed to bear the weight of all modules The detector should be operated in a horizontal position Environment Your detector will work within the specifications at ambient temperatures and relative humidity described in Table 2 on page 42 ASTM drift tests require a temperature change below 2 C hour 3 6 F hour over one hour period Our published drift specification refer also to Performance Specifications on page 43 is based on these conditions Larger ambient temperature changes will result in larger drift Better drift performance depends on better control of the temperature fluctuations To realize the highest performance minimize the frequency and the amplitude of the temperature changes to below 1 C hour 1 8 F hour Turbulences around one minute or less can be ignored Do not store ship or use your detector under conditions where temperature fluctuations could cause condensation within the detector
109. error condition always interrupts the analysis 1200 Series FLD Service M anual User Interfaces Troubleshooting and Test Functions 6 Depending on the user interface the available tests vary All test descriptions are based on the Agilent ChemStation as user interface Some descriptions are only available in the Service Manual Table 19 Test Functions avaible vs User Interface Test ChemStation Instant Pilot Control M odule G4208A G1323B D A Converter No No Yes Test Chromatogram Yes C No Yes Wavelength Calibration Yes Yes M Yes Lamp Intensity Yes No Yes Dark Current Yes No No via command M section M aintenance D section Diagnose The Agilent Control M odule G1323B does not do any calculations So there will be no reports generated with passed failed information 1200 Series FLD Service M anual 109 6 Troubleshooting and Test Functions Agilent LC Diagnostic Software 110 The Agilent LC diagnostic software is an application independent tool that provides troubleshooting capabilities for the Agilent 1200 Series modules It provides for all 1200 Series LC the possibility of a first guided diagnostic for typical HPLC symptoms and a status report stored as Adobe Acrobat pdf or as a printable file to assist users evaluating the instrument state At the introduction following modules will be fully supported by the software including module tests and calibrations as well as injector steps and mai
110. es Luminescence Detection Luminescence the emission of light occurs when molecules change from an Introduction to the Fluorescence Detector 1 excited state to their ground state Molecules can be excited by different forms of energy each with its own excitation process For example when the excitation energy is light the process is called photoluminescence In basic cases the emission of light is the reverse of absorption see Figure 2 With sodium vapor for example the absorption and emission spectra are a single line at the same wavelength The absorption and emission spectra of organic molecules in solution produce bands instead of lines ny Lt tl er Ga energy level 2 energy lever 4 energy level 2 energy level 1 luminescence Pt ee Se Figure2 Absorption of Light Versus Emission of Light 1200 Series FLD Service M anual 17 1 18 Introduction to the Fluorescence Detector When a more complex molecule transforms from its ground energy state into an excited state the absorbed energy is distributed into various vibrational and rotational sub levels When this same molecule returns to the ground state this vibrational and rotational energy is first lost by relaxation without any radiation Then the molecule transforms from this energy level to one of the vibrational and rotational sub levels of its ground state emitting light see Figure 3 The characteristic maxima of abs
111. es e GPIB connector as interface to the Agilent ChemStation e RS 232C as interface to a computer e REMOTE connector as interface to other Agilent products e Analog Output connector s for signal output and e Interface slot for specific interfacing external contacts BCD LAN and so on For identification and location of the connectors Figure 24 on page 55 WARNING Never use cables other than the ones supplied by Agilent Technologies to ensure proper functionality and compliance with safety or EMC regulations see Cable Overview on page 278 1200 Series FLD Service M anual 315 15 Hardware Information Analog Signal Output The analog signal output can be distributed to a recording device For details refer to the description of the module s main board GPIB Interface The GPIB connector is used to connect the module with a computer The address and control switches next to the GPIB connector determine the GPIB address of your module The switches are preset to a default address and recognized by the operating software from Agilent Technologies Table 48 Default Addresses Autosampler 28 Autosampler 28 Pump 22 RID 29 FLD 23 VW D 24 Autosampler HP 1050 18 Agilent 8453A 25 Pump HP 1050 16 DAD MWD 26 VW D HP 1050 10 Column Compartment 27 DAD HP 1050 17 CAN Interface The CAN is an intermodule communication interface It is a 2 wire serial bus system supporting high speed data communication and re
112. especially below 250 nm is significantly higher compared to Visible wavelength range Generally the LAMP ON during run setting or using economy mode will increase lamp life by a magnitude Relative Intensity Sal a Pa bed l 300 400 00 600 100 800 900 Wavelenght nm Figure7 Lamp Energy Distribution vendor data 1200 Series FLD Service M anual Introduction to the Fluorescence Detector 1 The radiation emitted by the lamp is dispersed and reflected by the excitation monochromator grating onto the cell entrance slit The holographic concave grating is the main part of the monochromator dispersing and reflecting the incident light The surface contains many minute grooves 1200 of them per millimeter The grating carries a blaze to show improved performance in the visible range Grating EX inside Mirror Figure8 Mirror Assembly 1200 Series FLD Service M anual 23 1 24 Introduction to the Fluorescence Detector The geometry of the grooves is optimized to reflect almost all of the incident light in the 1 order and disperse it with about 70 efficiency in the ultra violet range Most of the remaining 30 of the light is reflected at zero order with no dispersion Figure 9 illustrates the light path at the surface of the grating grating 50 v a reflected 15t order dispersed light se ow 200nm 800nm Figure9 Dispers
113. etector settings are shown in Figure 36 on page 85 Table 14 Chromatographic Conditions M obile phases Column Sample Flow rate Compressibility A water Compressibility B Acetonitrile Stroke A and B Time Table Stop time Post time Injection volume Oven temperature 1200 FLD PMT Gain FLD Response time A water 50 B Acetonitrile 50 Vydac C18 PNA 250 mmx 2 1 mm i d with 5 um particles PAH 0 5 ng 0 4 ml min 46 115 auto at 0 minutes B 50 at 3 minutes B 60 at 14 5 minutes B 90 at 22 5 minutes B 95 26 minutes 8 minutes l yl 30 C PMT 15 4 seconds 1200 Series FLD Service M anual sone Saute Signal Time Multiple Wavelengths and Spectr wavelength in the low UV Excitatiors E mission A Skoptime e min C DE C Multi Es E munien 230 260 nm ThS p z260 nm ff 550 nm 4 Use additional Emission i FPosttime Off min will cover nearly all Zero Order Zero Order F ET 410 rir fluorescence in your E eme sample D AD om Timetable Acquire Emission Spectra DO NOT select Line Time Ex EmA Em B EmC Em D Em Spectra Fr All additional emission 200 500 wavelengths B C ange 300 to 500 nm Step 5 nm D Doing so will increase the scan time and will lower the performance First Steps with the Detector Threshold fi D0 LU TimerSpectrum 1107 ms Feakwidth Responsetime gt 0 2 min 4 3 standart 4l nert Append
114. f the main system Main System Its properties are e the complete communication capabilities GPIB CAN LAN and RS 232C e memory management e ability to update the firmware of the resident system In addition the main system comprises the instrument functions that are divided into common functions like e run synchronization through APG remote e error handling e diagnostic functions e or module specific functions like internal events such as lamp control filter movements raw data collection and conversion to absorbance see Raw Data Conversion to Fluorescence LU Units on page 306 304 1200 Series FLD Service M anual Hardware Information 15 Firmware Updates Firmware updates can be done using your user interface e instant pilot G4208A with files from a USB memory stick or e handheld Control Module G1323B with files from a PC card or e aPC Firmware Update Tool with files from hard disk or CD ROM The file naming conventions are 13821A_A602_zz dlb where XxXxxx is the product number e g 1321A for the G1321A FLD and vvvv is the revision number for example A602 is revision A 06 02 and ZZ is the build number of the firmware For instructions refer to your user interface Update of main system can be done in the resident system only Update of the resident system can be done in the main system only main FW update Resident System Main System resident FW update Figure 92 Firmware U
115. figuration Switch 320 The Main Power Supply Assembly 325 This chapter describes the detector in more detail on hardware and electronics ofits Agilent Technologies 295 15 Hardware Information Detector M ain Board FLM This board controls all information and activities of all assemblies within the detector Through interfaces CAN GPIB RS 232C or LAN connected to the user interface the operator enters parameters changes modes and controls the detector Main Power Supply Interfaces MIO Analog CAN GPIB RS232 LAN Outputs Agilent 1200 FLM Core Processor ASIC Soa e i Supply Timing Control M otor Power 6 Flash Lamp A 4 Analog Photomultiplier p gt Reference Diode B Figure 89 Detector Electronic Overview 296 1200 Series FLD Service M anual Hardware Information 15 Excitation Emission M onochromator M otor Control A chopped motor driver IC provides the signal to run the excitation emission monochromator motor in positioning or rotation mode Encoder Signal Acquisition Each monochromator includes an optical encoder to get the actual position of the device The two encoder signals are amplified and prepared for A D conversion Fan Drive The operation of the fan is controlled by the main processor and runs with constant revolution The fan produces a sense signal which is derived from the revolution This sense signal is used for diagnostics
116. firm peak identity as assigned by retention time Now fluorescence detectors provide an additional tool for automated peak confirmation and purity control No additional run is necessary after the quantitative analysis During method development fluorescence excitation and emission spectra are collected from reference standards and entered into a library at the choice of the method developer All spectral data from unknown samples can then be compared automatically with library data Table 3 illustrates this principle using a PNA analysis The match factor given in the report for each peak indicates the degree of similarity between the reference spectrum and the spectra from a peak A match factor of 1 000 means identical spectra 1200 Series FLD Service M anual First Steps with the Detector 4 In addition the purity of a peak can be investigated by comparing spectra obtained within a single peak When a peak is calculated to be within the user defined purity limits the purity factor is the mean purity value of all spectra that are within the purity limits The reliability of the purity and the match factor depends on the quality of spectra recorded Because of the lower number of data points available with the fluorescence detector in general the match factors and purity data obtained show stronger deviations compared to data from the diode array detector even if the compounds are identical Table 13 shows an automated library search
117. g it around DO NOT unscrew the grounding screw marked with X HHA f 4 Using the 2 5 mm hexagonal Key unscrew one screws and loosen the other one Slide the window holder out of the window area TTH A et ng Ow XE The quartz window may be cleaned with alcohol e g ethanol y W Note 210 1200 Series FLD Service M anual Repairs 11 7 Slide the window holder across the window and fix the screws LH L ie 6 Insert the cleaned or new window Next Steps e Replace the covers as described in Replacing the Foam and the Top Cover on page149 Re install the detector in the stack e Turn the lamp ON 8 Replace the lamp housing cover and fix the screws Carefully insert the rear foam Reconnect the monochromator cables to the main board Left is excitation right is emission 1200 Series FLD Service M anual 211 11 Repairs Exchanging the Main Board When required If detector main board is defective or for repair on other assemblies Tools required Screwdriver POZI 1 PT3 Flat screw driver Hexagonal wrenches 5 mm 7 mm and 15 mm Parts required Detector main board FLM G1321 69500 exchange assembly 1 Turn OFF the lamp 2 Switch OFF the module and disconnect the cables 3 Remove module from stack and place it on the workbench
118. g the Optical Unit When required For following repairs power supply FLL board Trigger Pack FLF board and PMT Tools required Screwdriver POZI 1 PT3 Hexagonal key 4mm Parts required Optical unit G1315 69002 exchange assembly comes with foam or individual parts depending on the following procedures W hen working on the optical unit a clean workbench with ESD protection mat must be CAUTION l available Otherwise optical components or electronic boards may be damaged DO NOT remove the foam from the optical unit unless it is required during a procedure Otherwise parts can fall out 1200 Series FLD Service M anual 233 11 Repairs Preparations for this procedure 1 Disconnect cables EM monochromator and EX monochromator from the detector main board FLM Turn OFF the detector Disconnect the power cable Disconnect capillaries Remove detector from stack and place it on the workbench Remove the covers as described in Removing the Covers on page 198 Remove the flow cell 2 Carefully remove the rear foam A Leave the other foam 3 Disconnect the leak sensor cable J 3 and unscrew the on the assembly grounding screw 234 1200 Series FLD Service M anual Repairs 11 4 Disconnect the optical unit cable J 18 and the EM motor 5 Remove the optical unit and place it on the bench E
119. ging capability There are no externally accessible fuses because automatic electronic fuses are implemented in the power supply The security lever at the power input socket prevents removal of the detector cover when line power is still connected 1200 Series FLD Service M anual 33 1 Introduction to the Fluorescence Detector Never use cables other than the ones supplied by Agilent Technologies to ensure proper functionality and compliance with safety or EMC regulations Security lever Interface board Analog signal APG remote a a ue p 2 J gt lt J serial number CAN DE manufactured in Germany ov rower 6 2006 01 week of last major change Configuration switch 00130 number of unit product number G1321A FD G1321A FLD serial number Serial No Serial No ye Agilent Technologies CEs bd safety standards it i c im Wiii LRSJPAD QOP ICES MAAN 001 m e configuration switch Con 1 settings refer to Setting ase T T ra the 8 bit Configuration a ee eRe PE C1 fools foal Agilent Teckostages JEII Waldbioen Ne N voltage range Made in Germany im vA Stet power consumption I Figure 17 34 frequency Electrical Connections 1200 Series FLD Service M anual Introduction to the Fluorescence Detector 1 Instrument Layout The industrial design of the detector incorporates several innovative features It uses Agilent s E
120. grator prints START not ready 1200 Series FLD Service M anual 285 14 Cables Agilent 1200 to 3396 Series III 3395B Integrators Connector Pin Pin SignalName Active 03396 61010 33XX Agilent 1200 TTL 9 1 White Digital ground En NC 2 Brown Preparerun Low 30 JA 3 3 Gray Start Low NC 4 Blue Shut down Low a NC 5 Pink Not connected io NC 6 Yellow Power on High OF 14 7 Red Ready High 4 8 Green Stop Low NC 9 Black Startrequest Low 13 15 Not connected Agilent 1200 to HP 1050 HP 1046A or Agilent 35900 A D Converters Connector Pin Pin Signal Name Active 5061 3378 HP 1050 Agilent 1200 TTL 1 White 1 White Digital ground 2 Brown 2 Brown Prepare run Low O 3 Gray 3 Gray Start Low oe a 4 Blue 4 Blue Shut down Low m o 5 Pink 5 Pink Not connected es 6 Yellow 6 Yellow Power on High 7 Red 7 Red Ready High 8 Green 8 Green Stop Low 9 Black 9 Black Start request Low 286 1200 Series FLD Service M anual Agilent 1200 to HP 1090 LC or Signal Distribution M odule Cables 14 Connector Pin Pin SignalName Active 01046 60202 HP 1090 Agilent 1200 TTL 1 1 White Digital ground NC 2 Brown Prepare run Low 8 4 3 Gray Start Low aa 7 4 Bl
121. gs in Table 23 Table 22 Settings for Single Wavelength Specifications Time EX EM PMT Baseline 0 350 397 12 Free 20 30 350 450 12 Free Table 23 Settings for Dual Wavelength Specifications M ulti EM Scan Time EX EM A EM_B Spectra From To Step PMT Baseline FitSpectra 00 00 350 397 450 None 280 450 10 12 Free OFF 20 30 350 450 450 None 280 450 10 12 Free OFF 1200 Series FLD Service M anual Test Functions 8 Formula for the Raman ASTM S N value see Figure 56 for details RamanASTM H ight RamanCurrent Height DarkCurrent ASTMNoise Td oe og PT rane Oe es A ee 8 iu ASTMNoise p i time 5 to 20 minutes Height Raman Current if average between 5 and 10 minutes Height Dark Current s average between 21 and 22 minutes 6 Li E ra ry aa is i il J Figure 56 Raman ASTM signal noise calculation 1200 Series FLD Service M anual 145 8 146 Test Functions Procedure using an Agilent ChemStation 1 1 Set up the HPLC system and the ChemStation Flush the flow cell with clean bi distilled water 2 Turn ON the FLD lamp 3 Select Verification OQ PV Assure that the FLD signal is signal 1 in case you use an additional detector in the system Otherwise wrong calculations may be done due to wrong signals Single Wavelength Verification 4 5 6 7 8 Create a New Instrument Verification Select the Signal to Noise Temp test Set the limits to 500 Run the verificatio
122. hen pull out the FLL board and the Ferret C FLL Board ie FLL Board A F ain Bis Bile Ferret Ges x iis VS 7 Y VY 8 Put the wires into the released contacts using the 9 Reinstall the lamp as described in Exchanging the tweezers Lamp on page 201 Order of wires left to right 242 1200 Series FLD Service M anual Repairs 11 10 Replace the lamp housing cover Insert the optical unit Next Steps into its location and carefully insert the rear foam A Themeconnece Wecanics Replace the covers as described in Replacing the Foams and Covers on page 254 Re install the detector in the stack Reset the lamp counter as described in the user interface documentation Turn the lamp ON Perform a wavelength verification to check the correct positioning of the replaced assemblies as described in chapter Diagram of Light Path on page 138 1200 Series FLD Service M anual 243 11 Repairs Replacing the EM and EX Grating When required If defective Tools required Screwdriver POZI 1 PT3 Hexagonal key 4mm and 2 5 mm Parts required EM Grating assembly G1321 60004 includes protection ring EX Grating assembly G1321 60003 includes protection ring W hen working on the optical unit a clean workbench with ESD protection mat must be CAUTION i available Otherwise optical
123. ht Pipes 275 Leak Parts 276 This chapter provides information on parts for repair agg Agilent Technologies 265 13 Parts for Repair Overview of Main Assemblies Figure 81 shows the main assemblies and their locations geq 3 Interface board Pt H not shown O J Tee BS Pope 2 Power Supply 14 Cable FLM FLF 1 FLM Board 5 Optical unit E a l A II JHH HHH JLL HHHI HHH nil Ls nig igi eae immense eee eee el eS E ee E ee e 6 Flash lamp N ll For other parts T i f within the optical 7 f H F 9 Emission unit see page 268 F A fe 7 M onochromator immi ia 7 Mirror 10 Photomuliplier 11 condenser EM 8 Excitation M onochromator is a a SS BS i _ 12 Flow cell 13 Leak Sensor Figure 81 Main Assemblies 266 1200 Series FLD Service M anual Table 33 Main Assemblies Item 1 1 oO O N DM 1 amp 11 12 13 14 Description Detector main board FLM new Detector main board FLM exchange assembly Hexagonal nut for GPIB connector Hexagonal nut for RS 232C connector Nut for analog connector Washer for analog connector Cable CAN to Agilent 1200 Series modules 0 5 m Power supply for power and status light parts see page 275 Interface board BCD BCD external contacts optional optional see page 312 LAN Communication Interface Board G1369A LAN optional see page 314 Fuse for BCD board
124. ice M anual 1 37 1 38 Introduction to the Fluorescence Detector Using the EMF Counters The user selectable EMF limits for the EMF counters enable the early maintenance feedback to be adapted to specific user requirements The useful lamp burn time is dependent on the requirements for the analysis high or low sensitivity analysis wavelength etc therefore the definition of the maximum limits need to be determined based on the specific operating conditions of the instrument Setting the EM F Limits The setting of the EMF limits must be optimized over one or two maintenance cycles Initially no EMF limit should be set When instrument performance indicates maintenance is necessary take note of the values displayed by lamp counters Enter these values or values slightly less than the displayed values as EMF limits and then reset the EMF counters to zero The next time the EMF counters exceed the new EMF limits the EMF flag will be displayed providing a reminder that maintenance needs to be scheduled 1200 Series FLD Service M anual Agilent 1200 Series Fluorescence Detector Service M anual ee 2 Site Requirements and Specifications B 6 j e z Site Requirements 40 Physical Specifications 42 Performance Specifications 43 This chapter gives information on environmental requirements physical and performance specifications ee Agilent Technologies 39 2 Site Requirements and Specification
125. ifetime can significantly be increased by switching it on just for analysis In contrast to other LC detectors the G1321A fluorescence detector equilibrates within seconds after the lamp is switched ON For highest reproducibility and linearity change the lamp setting to always ON default is on only during run One hour of initial warm up of the instrument is recommended 6 Do not overpressurize the detector quartz flow cell Be aware to not exceed a 20 bar pressure drop after the flow cell when hooking up additional devices like other detectors or a fraction collector It s better to place a UV detector before the G1321A fluorescence detector When comparing fluorescence excitation spectra directly with DAD spectra or literature based absorbance spectra you should consider large differences in the used optical bandwidth FLD 20 nm which cause a systematic wavelength maximum shift depending on the absorbance spectrum of the compound under evaluation 1200 Series FLD Service M anual First Steps with the Detector 4 Getting Started and Checkout This chapter describes the check out of the Agilent 1200 Series fluorescence detector using the Agilent isocratic checkout sample When required If you want to checkout the detector Hardware required LC system with G1321A FLD Parts required Start up Kit 5063 6528 includes LC cartridge Hypersil ODS 5um 125x4mm with CIS cartridge holder Agilent isocratic checkout sample Part number 01080 68
126. ilent ChemStation Diagnosis Maintenance FLD Calibration Instant Pilot G4208A Maintenance FLD Calibration Control Module G1323B System Tests FLD Calibrate If the wavelength calibration process fails refer to Wavelength Calibration Failed on page 130 2 Ifa deviation is displayed press Adjust and OK The history table will be updated To look at the history table ChemStation start a wavelength calibration and abort immediately No changes are made to the calibration at this time Rinse the flow cell with pure water at a minimum of 1 5 ml min to get rid of the Glycogen from the cell and the capillaries When organic solvent is sequentially applied without rinsing a blockage of capillaries may occur 1200 Series FLD Service M anual 193 10 Maintenance 194 1200 Series FLD Service M anual Agilent 1200 Series Fluorescence Detector Service M anual 11 Repairs Cautions and Warnings 196 Removing the Covers 198 Removing the RearFoam 200 Exchanging the Lamp 201 Exchanging the EX condenser 206 Replacing the Lamp Cover Window 209 Exchanging the Main Board 212 Changing the Type and Serial Number 215 Using the Agilent ChemStation 215 Using the Instant Pilot G4208A 216 Using the Control Module G1323B 217 Exchanging the Fan 219 Exchanging the Leak Sensor or Leak Panel 221 Replacing the EM Condenser or Cutoff Filter 224 Replacing the MirrorAssembly 227 Replacing the Slits 231 Removing the Optical Unit
127. in board in another module Suggested Actions VY Ensure all the CAN cables are connected correctly VY Switch off the system Restart the system and determine which module or modules are not recognized by the system Y Ensure all CAN cables are installed correctly 1200 Series FLD Service M anual Error Information 7 Leak A leak was detected in the detector The signals from the two temperature sensors leak sensor and board mounted temperature compensation sensor are used by the leak algorithm to determine whether a leak is present When a leak occurs the leak sensor is cooled by the solvent This changes the resistance of the leak sensor which is sensed by the leak sensor circuit on the FLM board Probable Causes e Loose fittings e Broken capillary e Leaking flow cell Suggested Actions VY Ensure all fittings are tight Y Exchange defective capillaries Y Exchange flow cell formation Detector leak error may be caused by the Agilent 1200 Series sampler In each sampler injection sequence step 2 ejects the mobile phase stored in the metering head during the previous injection This mobile phase is ejected through the short plastic tube connected to port 4 of the sampler switching valve The output of port 4 is integrated into the sampler s contingency leak drain system which eventually terminates in the leak pan of the bottom module of the stack the detector With normal injection volumes and run times
128. indicates the detector status Status indicator green yellow red ae Si a foo S A AS Ir f AN f f ff A i o if li We 7N _ CON YY n K Wi Nn ST lt SS Line power switch with green light Figure 50 Location of Status Indicators Power Supply Indicator The power supply indicator is integrated into the main power switch When the indicator is illuminated green the power is ON 1200 Series FLD Service M anual 107 6 108 Troubleshooting and Test Functions Detector Status Indicator The detector status indicator indicates one of four possible detector conditions e When the status indicator is OFF and power switch light is on the detector is in a prerun condition and is ready to begin an analysis A green status indicator indicates the detector is performing an analysis run mode A yellow indicator indicates a not ready condition The detector is ina not ready state when it is waiting for a specific condition to be reached or completed for example immediately after changing a setpoint or while a self test procedure is running An error condition is indicated when the status indicator is red An error condition indicates the detector has detected an internal problem which affects correct operation of the detector Usually an error condition requires attention e g leak defective internal components An
129. ing Check of Raman Band of Water on page 156 Preparation of the Glycogen Calibration Sample 1 To prepare 10 ml of the calibration solution you have to use 10 mg of the Glycogen sample a tolerance of 20 is not critical 2 Fill the prepared amount into a suitable bottle vial 3 Fill 10 ml of distilled water into the vial and shake 4 Wait 5 minutes and shake again After 10 minutes the solution is ready 1200 Series FLD Service M anual 191 10 Maintenance Preparation of the Flow Cell Flush the flow cell with water Remove the inlet capillary from the flow cell Take the syringe and fix the needle to the syringe adapter Suck about 1 0 ml of the calibration sample into the syringe Keep the syringe in a horizontal position Remove t he needle Add the filter to the syringe and fit the needle to filter EH OF FP W N P sample filter Figure 76 Syringe with Sample Filter 8 Lift the needle tip and carefully eject approximately 0 5 ml to remove air out of the syringe and to flush the needle 9 Add the PEEK fitting to the needle tip and fix both at the flow cell inlet Do not inject the calibration sample without the sample filter 10 Slowly inject about 0 2 ml and wait for about 10 seconds to inject another 0 1 ml This will assure that the cell is filled properly 192 1200 Series FLD Service M anual Maintenance 10 Wavelength Calibration 1 From the user interface start the FLD Wavelength Calibration Ag
130. intenance Overview of Maintenance Parts 260 Cuvette Kit 261 Spare Parts 262 Accessory Kit 263 1200 Series FLD Service M anual 13 Parts for Repair Overview of Main Assemblies 266 Optical Unit Assembly 268 Lamp Cover Parts 2 1 Sheet Metal Kit 272 Plastic Parts 273 Foam Parts 2 4 Power and Status Light Pipes 275 Leak Parts 27 6 14 Cables Cable Overview 278 Analog Cables 280 Remote Cables 283 BCD Cables 288 Auxiliary Cable 290 CAN Cable 291 External Contact Cable 292 RS 232 Cable Kit 293 LAN Cables 294 15 Hardware Information Detector Main Board FLM 296 Detector Frontend Board FLF 298 How to retrieve the FLF board revision 302 Detector Lamp Supply Board FLL 303 1200 Series FLD Service M anual 11 12 A Appendix Firmware Description 304 Firmware Updates 305 Raw Data Conversion to Fluorescence LU Units 306 Data flow for chromatographic output 307 Data flow for spectral output 308 Spectra Acquisition Modes 311 Optional Interface Boards 312 BCD Board 312 LAN Communication Interface Board 314 Interfaces 315 Analog Signal Output 316 GPIB Interface 316 CAN Interface 316 Remote Interface 317 RS 232C 318 Setting the 8 bit Configuration Switch 320 GPIB Default Addresses 321 Communication Settings for RS 232C Communication 322 Forced Cold Start Settings 323 Stay Resident Settings 324 The Main Power Supply Assembly 325 General Safety Information 328 Lithium Batteries Information 331 Radio Interference
131. ion of Light by a Grating The grating is turned using a 3 phase brushless DC motor the position of the grating determining the wavelength or wavelength range of the light falling onto the flow cell The grating can be programmed to change its position and therefore the wavelength during a run For spectra acquisition and multi wavelength detection the grating rotates at 4000 rpm The excitation and emission gratings are similar in design but have different blaze wavelengths The excitation grating reflects most 15t order light in the ultra violet range around 250 nm whereas the emission grating reflects better in the visible range around 400 nm 1200 Series FLD Service M anual Introduction to the Fluorescence Detector 1 The flow cell is a solid quartz body with a maximum back pressure of 20 bar Excessive back pressure will result in destruction of the cell Operating the detector close to waste with low back pressure is recommended A slit is integrated to the quartz body SSSSSSSSSSSSSSSSSSSSSS5SS5 UL LLL ILLL LLL LLL LALLA LLLA LL Ss Z SSM LLL h kh hhh Mk he LLL LLL LL SESS SESE ADR AN E l SG Co S Ntt Z Z Z ay AG NA A ANS Z Z Z A Z LLki ki kh hh kh hh kl hh hl hk hh lk lh lk hee EEE SEE EEE ELIE SIE Figure 10 Cross Section of Flow Cell The luminescence from the sample in the flow cell is collected at right angles to the incident light by a second l
132. ion should be done as described in Wavelength Calibration Procedure on page 164 1200 Series FLD Service M anual 153 8 Test Functions The duration of the wavelength calibration is about 15 minutes plus setup time for the calibration sample and system Depending on the maximum intensity found during this scan the PMT gain will be changed automatically and requires an additional 1 minute per scan Table 26 on page 155 shows the steps performed during the wavelength calibration The excitation grating and the emission grating are calibrated using Rayleigh stray light from the flow cell or cuvette measured with the photomultiplier tube FLO Wavelength Calibration System 2 q X oo O S Excitation VUUUPU PPT J2nm 362 0nm 12 rnm Emission Cee eye J2nm 362 0 nm 12 hm Calibration history Deviation Excitation Emission Time Date 0 0 nm O0nm 1454 41 04 04 2001 0 6 nm TV 0nm 10 00 53 06 70 2004 0 6 nm Onm 1544 26 05 01 2006 Excitation deviati 0 2 nm Emizsion deviatio 0 1 nm Abort OF Cancel Calibration settings not equal to measured ones To calibrate click Adjust Figure 59 Wavelength Calibration 154 1200 Series FLD Service M anual Test Functions 8 Table 26 Wavelength Calibration Steps Step Description Duration 1 Preparation max 30s 2 Excitation rotation scan full circle 60s 3 Excitation rotation scan high resolution 44s 4 Excitation position scan low resolution 55 s vari
133. iously obtained with a standard fluorescence spectrophotometer independent of the LC fluorescence detector Figure 30 on page 74 shows the complete information for quinidine as obtained with the Agilent 1200 Series FLD and a manual cuvette in a single offline measurement The optima for excitation and emission wavelengths can be extracted as coordinates of the maxima in the three dimensional plot One of the three maxima in the center of the plot can be chosen to define the excitation wavelength The selection depends on the additional compounds 1200 Series FLD Service M anual First Steps with the Detector 4 that are going to be analyzed in the chromatographic run and the background noise that may be different upon excitation at 250 nm 315 nm or 350 nm The maximum of emission is observed at 440 nm Details for Figure 30 on page 74 All excitation and emission spectra of Quinidine 1 g ml are shown in graphic Fluorescence intensity is plotted vs excitation and emission wavelengths Detector settings step size 5 nm PMT 12 Response time 4 s 1200 Series FLD Service M anual 73 Straylight 1 order 350 nm Ex 315 nm Ex 250 nm Ex ENE a a a E x 440 00 nm E y 250 00 nmi z 1 87 LU OK Cube o o2 D t O 0s 1 12 1 4 16 15 2 22 z 26 25 al 42 Jd 16 48 L l l l l l l l l l l l l l l l l l l l l C x O y ei z pit tit tii tid tod pid tod pat tit A I IIi i i LI OI LiL it ti tot LL tut LiL I Ex axis Em axis Figur
134. ired Tools required Screwdriver POZI 1 PT3 A pair of tweezers Parts required Slit EX G1321 08103 Slit EM G1321 08102 also used for PMT DO NOT touch the surface of any optical component during this procedure This will deteriorate the reflection process Preparations for this procedure 1 Carefully remove the front foam holding the foam as shown Turn OFF the detector Disconnect the power cable Remove the flow cell Remove detector from stack and place it on the workbench Remove the covers as described in Removing the Covers on page 198 1200 Series FLD Service M anual 231 11 Repairs 2 Remove the UV protector 3 Locate the three slits from left to right PMT EM EX 4 Use a pair of tweezers to replace the slit Next Steps Replace the UV protector and the front foam Replace the covers as described in Replacing the Foams and Covers on page 254 Re install the flow cell and the front panel Re install the detector in the stack Turn the lamp ON Perform a wavelength verification to check the correct positioning of the replaced assemblies as described in chapter Diagram of Light Path on page 138 232 1200 Series FLD Service M anual Repairs 11 Removin
135. ity test if the last one is older than one week are stored as lamp history date code intensity of four different wavelengths 250 nm 350 nm 450 and 600 nm in a buffer The data plot can be retrieved via the diagnostics and provides intensity data over a length of time M Intensity Test History OF x 250nm counts 7000 6000 5000 4000 vo N XN N N N N 14 Time Days 350nm counts 7000 6000 s000 4000 4 3 2 14 Time Days 450nm counts 10000 a 8000 Pi 6000 p 14 Time Days 600nm counts 4000 3500 2000 2500 14 Time Days Figure 74 Lamp Intensity History 188 1200 Series FLD Service M anual Maintenance 10 Wavelength Verification and Calibration The wavelength calibration is based on a Glycogen solution which acts as a strong elastic light scatterer refer to ASTM Test Method E388 72 1993 Spectral Bandwidth and Wavelength Accuracy of Fluorescence Spectrometers The Glycogen solution is introduced into the flow cell and then the built in wavelength calibration functionality is used The algorithm is based on evaluating different grating orders and calculating the wavelength scales of both excitation and emission monochromator by applying the fundamental grating equation The duration of the wavelength calibration is about 15 minutes plus setup time for the calibration sample and system Depending on the maxi
136. k Width Fit Spectral Range PMT Gain Flash Lamp Spectrum Range Check of EM WL 397 nm WLEM TEST gt 0 2 min 4s standard OFF 10 ON EM 377 417 nm step 1 nm Store Spectra All w 0 signal EX Wavelength 350 nm ON EM Wavelength 397 nm OFF Multi WL Settings M ulti EM x Prgaphorescence Detection Mode Puseecence Scan Morgs fe Det i On Fim Ta Gip ETEN Er s MET 20 f mm Davee Sear TAF amp Lamp Bacelne Behavior fe fijara C Fia 7 Feo tf Positive Meggive M iei Da Duar Aan Eciam Mod near 74 Hr High Lamp Cungri Erabi anajen whan lamp m ot Lamp Energy Aolenenes w Om Of Festers eime Cancel Hee Figure 61 Special Setpoints Settings Check of EX WL 350 nm WLEXTEST gt 0 2 min 4s standard OFF 10 ON EX 330 370 nm step 1 nm All w 0 signal 350 nm OFF 397 nm ON M ulti EX 1200 Series FLD Service M anual Test Functions 8 FLD Signals System X Signal Time Multiple Wavelengths and Spectra Stoptime PSPAD a pi Cor Muti EC Muti Em Use additional Emission Posttime Ot 4 i E no 28 Cp JAT mm D aio Fini Excitation Emission E m 0 m C Zer Order Zero Order Timetable Line Time Ex Em Em Em C EmD Em Spectra Fri Acquire Emission Spectra Jal wo signals Range a77 to AF nim Shep z Ai Ca i eT FLD Signals System E x Signal Time Multiple Wavelengths and Spectra CE Mut Ey Multi Em Wee ad
137. l Re install the detector in the stack 1200 Series FLD Service M anual 223 11 Repairs Replacing the EM Condenser or Cutoff Filter When required If light throughput is too low Tools required Screwdriver POZI 1 PT3 Hexagonal key 2 5 mm Parts required Condenser assembly EM 1000 1124 Cutoff filter 295 nm 5062 8530 Preparations for this procedure 1 Disconnect the emission and the excitation cables and from the detector main board Turn OFF the detector Disconnect the power cable Remove the flow cell Remove detector from stack and place it on the workbench e Remove the covers as described in Removing the Covers on page 198 e Remove the flow cell 224 1200 Series FLD Service M anual Repairs 11 2 Carefully remove the front foam holding the foam as Note shown Locate the condenser assembly and the cutoff filter 3 The cutoff filter can be replaced just by sliding it out and ack in 1200 Series FLD Service M anual 225 11 Repairs 5 Slide out the condenser assembly and place it in a safe 6 Slide in the condenser assembly completely with the location larger lens diameter towards the cutoff filter ek Or IS a Ag Y E ENA JR 7 Use a 2 5 mm hexagonal key
138. lashes that are measured to get spectral information are collected to get one spectra 1200 Series FLD Service M anual 309 15 Hardware Information 310 Concentration Correction For every measured spectrum a certain time is required During this time the concentration within the flow cell may be changed This can effect the spectra To avoid this distortion every spectra point is corrected with the quasi simultaneous measured concentration of the chromatographic channel A As point of reference the mid point of each spectra is used Concentration correction is not done e If the cuvette is used e g fluorescence scan e If all spectra without signal is used as spectra acquisition mode Peak Detector The peak detector PD always uses signal A as the pilot signal The PD is adjusted by means of two setpoints peakwidth and threshold Both setpoints are time programmable While the PD threshold is a method parameter of its own the PD peakwidth parameter is derived from the general peakwidth PKWD parameter At the start of the run the value for the PD peakwidth is set equal to the PKWD setpoint The peak detector recognizes changes in the slope of this signal upslope apex downslope and baseline and stores spectra according to the operator s instructions During the run the PD peakwidth parameter can be changed by time programming 1200 Series FLD Service M anual Hardware Information 15 Spectra Acquisition M odes
139. led Replace the foam on the optical unit 1200 Series FLD Service M anual Repairs 11 Next Steps Reconnect the encoder cable of the EX monochromator to FLF board Reinstall the optical unit as described in Installing the Optical Unit on page 252 Enter the replacement in the maintenance logbook Reset the monochromator settings to default using the diagnose functions In this case Wavelength Calibration Lost is displayed and the instrument stays in Not Ready condition until you perform a wavelength calibration Perform a wavelength verification to check the correct positioning of the replaced assemblies as described in chapter Diagram of Light Path on page 138 1200 Series FLD Service M anual 247 11 Repairs Exchanging the Power Supply When required If defective Tools required Screwdriver POZI 1 PT3 Wrench 1 4 inch Wrench 5 mm Wrench 7 mm Parts required Power supply 0950 2528 The repair of the power supply assembly comprises exchanging the complete assembly No serviceable parts are inside Preparations for this procedure Turn OFF the lamp Switch OFF the detector and disconnect the cables Remove the detector from the stack and place it on the workbench Remove the front cover and top cover see Removing the Covers on page 198 Remove the processor board see Exchanging the M ain Board on page 212 Remove the fan assembly see Exchanging the Fan on page 219 Re
140. lent 1200 Series Fluorescence Detector Service M anual 5 How to optimize the Detector Optimization Overview 92 Design Features Help Optimization 93 Finding the Best Wavelengths 94 Finding the Best Signal Amplification 96 Changing the Xenon Flash Lamp Frequency 98 Selecting the Best Response Time 100 Reducing Stray Light 102 ee Agilent Technologies 91 5 How to optimize the Detector Optimization Overview Refer to Optimization Overview on page 63 for details 92 1200 Series FLD Service M anual How to optimize the Detector 5 Design Features Help Optimization The Agilent 1200 Series fluorescence detector has several features you can use to optimize detection Table 17 PMTGAIN Amplification factor LAMP Flash frequency RESPONSETIME Data reduction interval Check Performance Before You Start Before you start you should check that your detector is performing according to the specifications published by Agilent Technologies Your normal LC grade solvents may give good results most of the time but our experience shows that baseline noise can be higher with LC grade solvents than with fluorescence grade solvents Flush your solvent delivery system for at least 15 minutes before checking sensitivity If your pump has multiple channels you should also flush the channels not in use 1200 Series FLD Service M anual 93 5 How to optimize the Detector Finding the Best Wavelengths 94 The most important p
141. liary Agilent 1200 Series vacuum degasser 61322 61600 CAN Agilent 1200 module to module 0 5 m cables Agilent 1200 module to module 1 m Agilent 1200 module to Control M odule G1323B 9181 1516 9181 1519 61323 81600 External Agilent 1200 Series interface board to general purpose G1103 61611 contacts GPIB Agilent 1200 module to Agilent ChemStation 1 m 10833A cable Agilent 1200 module to Agilent ChemStation 2 m 10833B RS 232 Agilent 1200 module to a computer 34398A cable This kit contains a 9 pin female to 9 pin female N ull M odem printer cable and one adapter LAN cable Cross over network cable shielded 3 m long 5023 0203 for point to point connection Twisted pair network cable shielded 7 m long 5023 0202 for hub connections 1200 Series FLD Service M anual 279 14 Cables R Analog Cables O One end of these cables provides a BNC connector to be connected to Agilent 1200 Series modules The other end depends on the instrument to which connection is being made Agilent 1200 to 3390 2 3 Integrators Connector Pin Pin Signal Name 01040 60101 3390 2 3 Agilent 1200 1 Shield Ground 2 Not connected 8 3 Center Signal 4 Connected to pin 6 5 Shield Analog Ta 6 Connected to pin 4 1 Key 8 Not connected 280 1200 Series FLD Service M anual Cable
142. more parameter s you have to press OK to write down the new settings into the FLD Then re enter FLD Signals and start the PMT gain test 2 Using an appropriate response time For most applications a setting of 4 seconds is adequate Only for high speed analyses short columns at high flow rates a lower setting is recommended Bear in mind that even if the response time is too high fast peaks will appear a little smaller and broader but retention time and peak areas are still correct and reproducible 3 Finding the optimum wavelength Most fluorescent active molecules absorb at 230 nm Set the excitation wavelength to 230 nm and on line scan the emission spectra multi emission mode Then set the determined emission wavelength and perform a multi excitation scan multi excitation mode to find the best excitation wavelength 1200 Series FLD Service M anual 63 4 64 First Steps with the Detector 4 Evaluating fluorescence spectra In contrast to diode array based UV detectors where UV spectra are evaluated by taking a spectrum at the peak maximum and selecting a reference spectrum at the baseline correct fluorescence spectra are obtained by selecting a peak maximum spectrum and a reference around the inflection points Selecting reference spectra at the baseline is not useful because the spectrum on the baseline is very noisy no light 5 Switching lamp ON only for analysis Unless maximum sensitivity is needed the lamp l
143. move the optical unit see Removing the Optical Unit on page 233 248 1200 Series FLD Service M anual Repairs 11 1 Carefully remove the bottom foam piece by sliding it out 2 Unscrew the power supply at the rear of the module towards the rear 3 Press down the power switch light pipe to remove it from 4 Remove the power supply completely the coupler Re use the coupler on the new power supply LIME III IIIS Power switch light The repair of the power supply assembly is comprised of exchanging the complete assembly No serviceable parts are inside 1200 Series FLD Service M anual 249 11 Repairs 5 Insert the power supply into its location and fix it with the screws at the rear panel 7 Reinstall bottom foam piece Slide it in underneath the leak drain 250 6 Press down and clip in the power switch light pipe into the power supply LUMI UII LUVANA RIL Power switch light pipe 8 Reinstall the processor board see Exchanging the M ain Board on page 212 9 Reinstall the fan assembly see Exchanging the Fan on page 219 10 Reinstall the optical unit see Installing the Optical Unit on page 252 11 Reinstall the front cover top cover see Replacing the Foams and Covers on page 254 12
144. mum intensity found during this scan the PMT gain will be changed automatically and requires an additional 1 minute per scan The excitation grating and the emission grating are calibrated using Rayleigh stray light from the flow cell or cuvette measured with the photomultiplier tube 1200 Series FLD Service M anual 189 10 Maintenance 190 FLD Wavelength Calibration System 2 Calibration history apl Deviation Excitation Emission Time Date o N COnm O lt F0nm 14 54 41 04 04 2001 Excitation UUU O 6 Aili 1 0 Film 10 00 53 06 10 2004 12am 3620nm 12nm 0 6 nm Qnm 1544 26 09 01 2006 Emission PUUPUU J2nm 362 0nm 12 nm Excitation deviati 0 2 nm Emizsion deviatio 0 1 nm Abort OK Cancel Calibration settings not equal to measured ones To calibrate click Adjust Figure 75 Wavelength Calibration When the lamp is of the calibration process will stop within the first two steps with Wavelength Calibration Failed 1200 Series FLD Service M anual Maintenance 10 Wavelength Calibration Procedure When required If application requires or after replacement of flow cell or lamp Tools required Laboratory balance Parts required Glycogen Calibration Sample Syringe needle sample filter and PEEK fitting from the Accessory Kit see Accessory Kit on page 263 Steps 1 Preparation of the Glycogen Calibration Sample 2 Preparation of the Flow Cell 3 Wavelength Calibration 4 Verification us
145. n When the run is completed a report is displayed and the status line shows the Raman signal noise ratio value should be gt 500 Dual Wavelength Verification aq ur W N P 7 8 9 Open M ethod and Run Control Open method OQFLDSNT M Modify the time table according to Table 23 on page 144 Save the method as OQFLDSNT2 M Open sequence OQFLDSNT S Modify the sequence to use a different store location and and to call up method OQFLDSNT2 M Save the sequence as OQFLDSNT2 S Create a New Instrument Verification Select the Signal to Noise Temp test 10 Select customize sequence and select OQFLDSNT2 S 11 Set the limits to 300 1200 Series FLD Service M anual Test Functions 8 12 Run the verification 13 When the run is completed a report is displayed and the status line shows the Raman signal noise ratio value should be gt 300 Interpretation of the Results If the test shows low Raman values check for Y correctly positioned flow cell v clean flow cell flush with clean bi distilled water Y no air bubble s check via fluorescence scan or visual check of cell cuvette Y solvent inlet filter may create air bubbles in flow cell 1200 Series FLD Service M anual 147 8 Test Functions Using the Built in Test Chromatogram LU 35 30 25 20 0 This function is available from the Agilent ChemStation and the Control Module G1323B The built in Test Chromatogram can b
146. n during the restart of the Agilent ChemStation After restart the serial number type you have just entered can be seen under the Instrument menu of the main user interface screen Using the Instant Pilot G4208A 216 1 2 Connect the Instant Pilot to the detector Turn ON the detector On the Instant Pilot s Welcome screen press More then select Maintenance Using the up down arrows select the detector where you have to change the product number or serial number Press PN SN This will display a screen where you can enter the product number and or serial number Make your changes using the information from the product label of your detector If you enter the wrong type your module might not be accessible anymore with the Agilent ChemStation and the Instant Pilot unsupported module In such a case follow the Recover Instructions on page 217 Press OK to highlight the complete command Press Done to transfer the information into the main board s memory Press Cancel quit the process Turn the detector OFF then ON again The Maintenance screen should display the correct serial number for this module 1200 Series FLD Service M anual Repairs 11 8 If an Agilent ChemStation is also connected restart the Agilent ChemStation now as well Recover Instructions 1 Turn off the detector 2 Change the 8 bit Configuration Switch to Resident see Stay Resident Settings on page 324 3 Turn the detector
147. n spectra Scan speed 28 ms per datapoint e g 0 6 s spectrum 200 400 nm 10 nm step Step size 1 20 nm Spectra storage All Repeatability 0 2 nm Accuracy 3 nm setting Standard 8 ul volume and 20 bar 2 MPa pressure maximum quartz Optional Fluorescence cuvette for offline Spectroscopic measurements with 1 ml syringe 8 ul volume quartz Agilent ChemStation for LC Agilent Instant Pilot G4208A or Agilent Control M odule G1323B with limited spectral data analysis and printing of spectra Recorder integrator 100 mV or 1 V output range gt 102 luminescence units two outputs Controller area network CAN GPIB RS 232C LAN APG Remote ready start stop and shut down signals 1200 Series FLD Service M anual Site Requirements and Specifications 2 Table3 Performance Specifications Agilent 1200 Series Fluorescence Detector Type Specification Comments Safety and Extensive diagnostics error maintenance detection and display through Instant Pilot G4208A Control M odule G1323B and ChemStation leak detection safe leak handling leak output Signal for shutdown of pumping system Low voltages in major maintenance areas GLP features Early maintenance feedback EM F for continuous tracking of instrument usage in terms of lamp burn time with user settable limits and feedback messages Electronic records of maintenance and errors Verification of wavelength accuracy using the Raman band of w
148. nce diode collect data for complete spectra concentration correction Control M odule ChemStation Online M onitor Spline algorithm raw smoothed Spectra output LU Data Analysis Figure 95 Data flow for spectral output 308 1200 Series FLD Service M anual Hardware Information 15 Raw Signal Acquisition PMT and Reference Diode Each raw data value of the PMT and the reference diode signal is measured synchronized to the flashes of the xenon flash lamp with a 16 bit analog to digital converter Dark Subtraction The dark value of the PMT and the reference signal are measured during any power cycle of the detector without flash lamp on These values are stored and subtracted from any PMT and reference diode value Normalize Reference Diode Signal The reference diode located behind the flow cell measures the excitation EX light within the flow cell and corrects flash lamp fluctuations Because of a non linear output of the diode depending on the EX wavelength the measured data are now normalized to get better real light level values within the flow cell Divide PMT Reference Diode With this calculation the output of the detector will suppress lamp effects like lamp flash fluctuations or lamp lifetime Filtering FIR filters finite impulse response are used to filter the given raw data and output it to a the digital data bus and the analog outputs Spectra Collect data to complete spectra All f
149. ncidental or consequential damages in connec tion with the furnishing use or per formance of this document or of any information contained herein Should Agilent and the user have a separate written agreement with warranty terms covering the material in this document that conflict with these terms the warranty terms in the sep arate agreement shall control Technology Licenses The hardware and or software described in this document are furnished under a license and may be used or copied only in accor dance with the terms of such license Restricted Rights Legend Software and technical data rights granted to federal government customers include only those rights customarily provided to end user Customers of Software Agilent provides this customary commercial license in Software and technical data pursuant to FAR 12 211 Technical Data and FAR 12 212 Computer Software and for Department of Defense purchases DFARS 252 227 7015 Technical Data Commercial Items and DFARS 227 7202 3 Rights in Commercial Computer Software or Computer Software Documentation If a federal government or other public sector Customer has a need for rights not conveyed under these terms it must negotiate with Agilent to establish acceptable terms in a written agreement executed by all relevant parties Safety Notices CAUTION A CAUTION notice denotes a haz ard It calls attention to an operat ing procedure practice or the like
150. nds 1 million photons into the flow cell at for example 280 nm Scattering on the surface of the flow cell and scattering from the molecules of solvent allow 0 1 of this light to leave the cell through the window at right angles to the incident light Without a cut off filter these remaining 1000 photons will reach the emission grating 90 will be reflected totally without dispersion onto the photomultiplier The other 10 disperses at 280 nm 1t order and at 560 nm 2 order To remove this stray light you need a cut off filter around 280 nm Because of a known set of applications a 295 nm cut off filter is built in for undisturbed application up to 560 nm without compromises see Figure 49 on page 103 1200 Series FLD Service M anual How to optimize the Detector 5 LU Exitation 300nm Second order light 600nm Stray light Fluorescence no filter filter 280 nm Wavelength nm Figure 49 Reducing Stray Light 1200 Series FLD Service M anual 103 5 How to optimize the Detector 104 1200 Series FLD Service M anual Agilent 1200 Series Fluorescence Detector Service M anual c00 ee 6 Troubleshooting and Test Functions amp a Overview of the Detector s Indicators and Test Functions 106 Status Indicators 107 User Interfaces 109 Agilent LC Diagnostic Software 110 This chapter gives an overview about the troubleshooting and diagnostic features and the different user inte
151. ng the Stack Configuration If your detector is part of a complete Agilent 1200 Series system you can ensure optimum performance by installing the following configuration This configuration optimizes the system flow path ensuring minimum delay volume 1200 Series FLD Service M anual 51 3 Installing the Detector Solvent cabinet Vacuum degasser Local User Pump Interface Autosampler Column compartment Detector Figure 21 Recommended Stack Configuration Front View 52 1200 Series FLD Service M anual Installing the Detector 3 Remote cable CAN Bus cable to local user interface o o o o Oo o Oo CAN Bus cable Analog detector AC power Signal 1 or 2 outputs per detector LAN to LC ChemStation location depends on detector Figure 22 Recommended Stack Configuration Rear View 1200 Series FLD Service M anual 53 3 Installing the Detector Installing the Detector Preparations Locate bench space Provide power connections Unpack the detector Parts required Detector Power cord for other cables see below and Cable Overview on page 278 Agilent ChemStation and or Instant Pilot G4208A or Control Module G1323B 1 Install the LAN interface board in the detector if required see Replacing the Interface Board on page 184 2 Place the detector in the stack or on the bench in a horizontal position 3 Ensure the line power switch at the front of the detector is OFF
152. ns other than being both up will allow for normal operation If you use the following switch settings and power the instrument up again the instrument firmware stays in the resident part that is it is not operable as a detector It only uses basic functions of the operating system for example for communication Table 58 Stay Resident Settings M ode Select TEST BOOT ESN EC CC To return to normal operation set switches back to your GPIB or RS 232C configuration settings 324 1200 Series FLD Service M anual Hardware Information 15 The Main Power Supply Assembly The main power supply comprises a closed assembly no onsite repair possibility The power supply provides all DC voltages used in the module except for the voltages supplied by the lamp power supply to the deuterium and tungsten lamps in the detectors The line voltage can vary in a range from 100 240 volts AC 10 and needs no manual setting MPS 436V a 36V not used supply line i a voltage circuits a 5V switching 5V 3A regulator linear voltage gt TNR regulators 5 3 Figure 99 Main Power Supply M PS Blockdiagram To disconnect the instrument from line unplug the power cord The power supply still uses some power even if the power switch on the front panel is turned OFF 1200 Series FLD Service M anual 325 15 Hardware Information 326 No accessible hardware fus
153. ntenance positions e Agilent 1200 Series binary pump SL G1312B e Agilent 1200 Series high performance autosampler SL G1367B e Agilent 1200 Series thermostatted column compartment SL G1316B e Agilent 1200 Series diode array detector SL G1315C With further releases of the diagnostic software all Agilent 1200 Series HPLC modules will be fully supported This diagnostic software provides tests and diagnostic features that may differ from the descriptions in this manual For details refer to the help files provided with the diagnostic software 1200 Series FLD Service M anual Agilent 1200 Series Fluorescence Detector Service M anual ee 7 Tbe Error Information amp j z What Are Error Messages 112 e General ErrorMessages 113 Detector ErrorMessages 124 This chapter describes the meaning of detector error messages and provides information on probable causes and suggested actions how to recover from error conditions gt Agilent Technologies 111 7 Error Information What Are Error Messages Error messages are displayed in the user interface when an electronic mechanical or hydraulic flow path failure occurs which requires attention before the analysis can be continued for example repair or exchange of consumables is necessary In the event of such a failure the red status indicator at the front of the detector is switched ON and an entry is written into the detector logbook 112
154. nthrene 8 2 min 420 nm for anthracene to benzo g h l perylene 19 0 min 500 nm for indeno 1 2 3 cd pyrene In the second run three setpoints for emission wavelengths are entered into the time program and excitation spectra are recorded as shown in figure 8 The area of high intensity red is caused by stray light when emission spectra overlap with the excitation wavelength This can be avoided by fitting the spectral range automatically Excitation at 260 nm is most appropriate for all PNAs 1200 Series FLD Service M anual 75 4 First Steps with the Detector This shows the isofluorescence plot of emission spectra for 15 PNAs 5 pg mi witha fixed excitation wavelengths 260 nm 76 Table9 Conditions for Figure 31 and Figure 32 on page 77 Column Vydac 2 1 x 200 mm PNA 5 um M obile phase A water B acetonitrile 50 50 Gradient 3 minutes 60 14 minutes 90 22 minutes 100 Flow rate 0 4 ml min Column temperature 18 C Injection volume 5 Ul FLD settings PMT 12 response time 4s step size 5 nm 1 Naphthalene 8 Benzlaljanthracene 2 Acenaphthene 9 3 Fluorene 10 Benzolb fluoranthene 4 Phenanthrene 11 Benzo kifluoranthene 5 Anthracene 12 Benzlajpyrene 6 Fluoranthene 13 Dibenzolahjanthracene 7 Pyrene 14 Benzolyg h iperylene i LU 60 15 Indenol1 2 3 cd pyrene 12 50 40 8 10 7 0 2 5 5 75 12 5 15 17 5 20 22 5 Time min 600 nm 300 nm Em spectra fixed Ex Figure 31 Optimization of the time p
155. of data processing Bunching improves your signal to noise ratio The bunched data shown as larger black dots in Figure 16 is then filtered using a boxcar filter The data is smoothed without being reduced by taking the mean of a number of points The mean of the same points minus the first plus the next and so on is calculated so that there are the same number of 1200 Series FLD Service M anual 31 1 Introduction to the Fluorescence Detector bunched and filtered points as the original bunched points You can define the length of the boxcar element using the RESPONSETIME function the longer the RESPONSETIME the greater the number of data points averaged A four fold increase in RESPONSETIME for example 1 sec to 4 sec doubles the signal to noise ratio small S N ratio uf Lal PEI bunched data GAA OO OOO points ID I SG DAD DCO OO OO D PODQDIOCOO boxcar fitr OOODODDOCOO OOO O O O O YIO O ODD DD Depo 0o00 0 d d ODOO tiltered data 00 000000 points high S N ratio Figure 16 RESPONSETIME Signal to Noise Ratio 32 1200 Series FLD Service M anual Introduction to the Fluorescence Detector 1 Electrical Connections e The GPIB connector is used to connect the detector with a computer The address and control switch module next to the GPIB connector determines the
156. ompared to the signal mode PNA analysis for example can be performed with simultaneous multi wavelength detection instead of wavelength switching With four different wavelengths for emission all 15 PNAs can be monitored Figure 35 on page 82 Table 12 Conditions for Figure 35 on page 82 Column Vydac 2 1 x 250 mm PNA 5 um M obile phase A water B acetonitrile 50 50 Gradient 3 minutes 60 14 5 minutes 90 22 5 minutes 95 Flow rate 0 4 ml min Column temperature 22 Injection volume 2 ul FLD settings PMT 12 response time 4s 1200 Series FLD Service M anual 81 4 First Steps with the Detector The upper trace was received with traditional wavelength switching 82 1 Naphthalene 2 Acenaphthene 3 Fluorene 4 Phenanthrene 5 Anthracene 6 Fluoranthene 8 Benzlajanthracene 9 Chrysene 10 Benzo b fluoranthene 11 Benzolkifluoranthene 12 Benzla pyrene 13 Dibenzola hlanthracene 1 excitation WL at 760 nm 4emission WL at350 420 440 and 500 nm LU 7 Pyrene 14 Benzolg h ijperylene 5 15 Indenol1 2 3 cd pyrene 180 afere Em 350 TT Reference 160 chromatogram with switching events 140 120 2 4 f 15 Ma 3 W Th 13 14 80 j 60 4 q 40 vE AH 0 Ex 260 Em 42 Mi I 0 5 10 15 20 25 Time min Figure 35 Simultaneous multi wavelength detection for PNA analysis Previously only diode array detectors and mass spectrometric detectors could deliver spectral information on line to con
157. on 335 Agilent Technologies on Internet 337 gt Agilent Technologies 327 A Appendix General Safety Information 328 The following general safety precautions must be observed during all phases of operation service and repair of this instrument Failure to comply with these precautions or with specific warnings elsewhere in this manual violates safety standards of design manufacture and intended use of the instrument Agilent Technologies assumes no liability for the customer s failure to comply with these requirements General This is a Safety Class I instrument provided with terminal for protective earthing and has been manufactured and tested according to international safety standards This instrument is designed and certified as a general purpose laboratory instrument for research and routine application only It is not certified for in vitro or medical applications Operation Before applying power comply with the installation section Additionally the following must be observed Do not remove instrument covers when operating Before the instrument is switched on all protective earth terminals extension cords auto transformers and devices connected to it must be connected to a protective earth via a ground socket Any interruption of the protective earth grounding will cause a potential shock hazard that could result in serious personal injury Whenever it is likely that the protection has been impaired
158. only with the same or equivalent type recommended by the equipment manufacturer Lithium batteries may not be disposed off into the domestic waste Transportation of discharged Lithium batteries through carriers regulated by IATA ICAO ADR RID IM DG is not allowed Discharged Lithium batteries shall be disposed off locally according to national waste disposal regulations for batteries Lithiumbatteri Eksplosionsfare ved fejlagtig handtering Udskiftning ma kun ske med batteri af samme fabrikat og type Lever det brugte batteri tilbage til leverand ren Lithiumbatteri Eksplosionsfare Ved udskiftning benyttes kun batteri som anbefalt av apparatfabrikanten Brukt batteri returneres appararleverandoren Bij dit apparaat zijn batterijen geleverd Wanneer deze leeg zijn moet u ze niet weggooien maar inleveren als KCA 1200 Series FLD Service M anual 331 A Appendix Radio Interference Never use cables other than the ones supplied by Agilent Technologies to ensure proper functionality and compliance with safety or EMC regulations Test and Measurement If test and measurement equipment is operated with equipment unscreened cables and or used for measurements on open set ups the user has to assure that under operating conditions the radio interference limits are still met within the premises 332 1200 Series FLD Service M anual Appendix A Sound Emission M anufacturer s Declaration This statement is provided to com
159. ormance Specifications 43 3 Installing the Detector Unpacking the Detector 48 Detector Accessory Kit Contents 49 1200 Series FLD Service M anual 5 Optimizing the Stack Configuration 51 Installing the Detector 54 Flow Connections to the Detector 57 4 First Steps with the Detector Before You Start 62 Optimization Overview 63 Getting Started and Checkout 65 Starting Your Detector 65 Setting the Chromatographic Conditions 66 Observe the maxima via the isoabsorbance plot 68 Method Development 69 Step 1 Check the LC system for impurities 70 Step 2 Optimize limits of detection and selectivity 71 Step 3 Set up routine methods 80 Example Optimization for Multiple Compounds 84 5 How to optimize the Detector Optimization Overview 92 Design Features Help Optimization 93 Check Performance Before You Start 93 Finding the Best Wavelengths 94 A Real Example 95 Finding the Best Signal Amplification 96 Changing the Xenon Flash Lamp Frequency 98 Lamp life savings 99 Selecting the Best Response Time 100 Reducing Stray Light 102 1200 Series FLD Service M anual 6 Troubleshooting and Test Functions Overview of the Detector s Indicators and Test Functions 106 Status Indicators 107 Power Supply Indicator 107 Detector Status Indicator 108 User Interfaces 109 Agilent LC Diagnostic Software 110 7 Error Information 1200 Series FLD Service M anual What Are Error Messages 112 General ErrorMessages 113 Time out
160. orption for a substance is its Apy and for emission its Ary absorption emission A i i radiationless transition 1 Figure3 Relationship of Excitation and Emission Wavelengths Photoluminescence is the collective name for two phenomena fluorescence and phosphorescence which differ from each other in one characteristic way the delay of emission after excitation If a molecule emits light 10 to 10 seconds after it was illuminated then the process was fluorescence If a molecule emits light longer than 10 seconds after illumination then the process was phosphorescence 1200 Series FLD Service M anual Introduction to the Fluorescence Detector 1 Phosphorescence is a longer process because one of the electrons involved in the excitation changes its spin during a collision with a molecule of solvent for example The excited molecule is now in a so called triplet state T see Figure 4 Ss spin change A wo lt lt a Phosphorescence gt Figure4 Phosphorescence Energy Transitions The molecule must change its spin back again before it can return to its ground state Since the chance of colliding with another molecule with the necessary spin for change is slight the molecule remains in its triplet state for some time During the second spin change the molecule loses more energy by relaxing without radiation The
161. overflow 127 ADC not calibrated 126 compensation sensor open 121 compensation sensor short 122 encoder index not found 134 fan failed 123 flash lamp current overflow 128 flash trigger lost 129 FLF board not found 125 flow cell removed 132 lamp cover open 124 leak 117 leak sensor open 119 leak sensor short 120 motor encoder index wrong 135 motor friction too high 135 motor messages 134 motor or encoder not found 134 motor position not found or lost 135 motor speed too low or unstable 135 remote timeout 115 shutdown 114 synchronization lost 116 time out 113 wavelength calibration failed 130 wavelength calibration lost 131 method development 69 1 check the LC system for impurities 70 2 optimize limits of detection and selectivity 71 3 set up routine methods 80 fluorescence spectral libraries for peak confirmation 82 multi wavelength detection 81 take a fluorescence scan 72 mirror 21 monochromator EM 21 24 EX 21 23 multi wavelength detection 81 340 N no peaks 133 0 off line measurements 16 operation of the detector 17 Operation temperature 42 operation theory of electronics 295 Optical unit overview 21 optimization example 84 p Parts 259 265 parts identification 259 265 accessory kit 263 cable overview 278 cables 277 cables analog 280 cables APG remote 283 cables auxiliary 290 cables BCD 288 cables CAN 291
162. overs on page 198 Remove the flow cell 1200 Series FLD Service M anual 221 11 Repairs 2 Disconnect the leak sensor cable using your left hand 3 Pull out the leak sensor Note 4 Using flat screw driver release the leak base two clasps If you want to replace just the leak sensor continue with step 8 a 222 1200 Series FLD Service M anual Repairs 11 5 Use a flat screw driver to lift the leak base from the base 6 Remove the leak base completely oo Ty mi mit i a 8 Replace the leak sensor using your left hand and 7 Replace the leak pan into the cabinet bottom Press the reconnect the connector leak pan completely down into its recess S Next Steps Replace the covers as described in Replacing the Foams and Covers on page 254 e Re install the flow cell and the front pane
163. pdate M echanism 1200 Series FLD Service M anual 305 15 Hardware Information Raw Data Conversion to Fluorescence LU Units LU light or luminescence units is used as new unit and will indicate the light intensity not comparable with the physical unit Lumen These units are calculated from the PMT photomuliplier tube in the emission path and a reference diode located behind the flow cell see figure below Xenon Emission flash lamp monochromator Lens Lens Photomultiplier Mirror Excitation Sample Photodiode monochromator Figure 93 Optical Diagram of the FLD 306 1200 Series FLD Service M anual Hardware Information 15 Data flow for chromatographic output Influences Influences PMT Raw Signal PMT gain eee position Dark subtraction subtraction Normalization of reference signal EX wavelength Divide PMT Reference Diode Peakwidth will be executed Filtering Channel A B C D at each flash Responsetime assigned for chromatographic output Peak Detector Output LU Figure 94 Data flow for chromatographic output For multi channel output any single flash data point will be processed in a separate independent filter 1200 Series FLD Service M anual 307 15 Hardware Information Data flow for spectral output PMT raw signal reference acquisition raw signal position acquisition positio dark subtraction subtraction normalization of reference signal divide PMT refere
164. ply with the requirements of the German Sound Emission Directive of 18 January 1991 This product has a sound pressure emission at the operator position lt 70 dB e Sound Pressure Lp lt 70 dB A e At Operator Position e Normal Operation e According to ISO 7779 1988 EN 27779 1991 Type Test 1200 Series FLD Service M anual 333 A Appendix UV Radiation UV lamps only Emissions of ultraviolet radiation 200 315 nm from this product is limited such that radiant exposure incident upon the unprotected skin or eye of operator or service personnel is limited to the following TLVs Threshold Limit Values according to the American Conference of Governmental Industrial Hygienists Table 61 UV Radiation Limits Exposure day Effective Irradiance 8 hours 0 1 uW cm 10 minutes 5 0 uW cm Typically the radiation values are much smaller than these limits Table 62 UV Radiation Typical Values Position Effective Irradiance Lamp installed 50 cm distance average 0 016 uW cm Lamp installed 50 cm distance maximum 0 14 uW cm 334 1200 Series FLD Service M anual Appendix A Solvent Information Observe the following recommendations on the use of solvents Flow Cell Avoid the use of alkaline solutions pH gt 9 5 which can attack quartz and thus impair the optical properties of the flow cell Prevent any crystallization of buffer solutions This will lead into a blockage damage of the flow cell If the flow
165. r For some modules e g pumps or auto samplers the type has to be changed multiple usage boards Use the information from the serial number plate of your module The changes become active after the reboot of the module Using the Agilent ChemStation Module serial numbers are entered by typing specific commands on the command line at the bottom of the main user interface screen 1 To enter a module serial number type the following command into the command line print sendmoduleS lfld Ser YYYYYYYYYY Where YYYYYYYYYY is the 10 character serial number of the module in question The first two characters are letters which should be capitalized The reply line will respond with RA 0000 SER followed by the module serial number you just entered To change the type of the module use the following command print sendmodules lfld TYPE XXXXX 1200 Series FLD Service M anual 215 11 Repairs Where XXXXX is the 5 character product number of the module e g G1314A If you enter the wrong type your module will not be accessible anymore In sucha case see Using the Instant Pilot G4208A on page 216 or Using the Control Module G1323B on page 217 for recovering Turn OFF the detector then ON again Then restart the Agilent ChemStation If the serial number you have just entered is different than the original module serial number you will be given the opportunity to edit the configure 1200 access scree
166. rapid on line Scanning capabilities and spectral data analysis 10 fg Anthracene see note below this table Ex 250 nm Em 400 nm RAMAN single wavelength H20 gt 500 with Ex 350 nm Em 397 nm dark value 450 nm Standard flow cell time constant 4 seconds 8 seconds responsetime RAMAN dual wavelength H 0 see Raman ASTM Signal to Noise gt 300 with Ex 350 nm Test on page 144 Em 397 nm dark value 450 nm Standard flow cell time constant 4 seconds 8 seconds responsetime see Raman ASTM Signal to Noise Test on page 144 Xenon Flash Lamp normal mode 20 W economy mode 5 W 296 Hz for single signal mode 74 Hz for spectral mode Range 200 nm 700 nm and zero order Bandwidth 20 nm fixed M onochromator concave holographic grating F 1 6 blaze 300 nm Range 280 nm 900 nm and zero order Bandwidth 20 nm fixed M onochromator concave holographic grating F 1 6 blaze 400 nm in line excitation measurement 1200 Series FLD Service M anual 43 2 Site Requirements and Specifications Table 3 Performance Specifications Agilent 1200 Series Fluorescence Detector Type Timetable programing Spectrum acquisition Wavelength characteristic Flow cells Control and data evaluation Analog outputs Communications Aa Specification Comments up to 4 signal wavelengths response time PMT Gain baseline behavior append free zero spectral parameters Excitation or Emissio
167. rdware Information 15 Table 50 RS 232C Connection Table Pin Direction Function 1 In DCD 2 In RxD 3 Out TxD 4 Out DTR 5 Ground 6 In DSR 7 Out RTS 8 In CTS 9 In RI Instrument PC DCD 1 1 DCD RX 2 2 RX TX 3 3 TX DTR 4 4 DTR GND 5 Y 5 GND DSR 6 6 DSR RTS D a 7 RTS CTS 8 on 8 CTS RI 9 9 RI DB9 DB9 DB9 DB9 Male Female Female Male Figure 97 RS 232 Cable 1200 Series FLD Service M anual 319 15 Hardware Information Setting the 8 bit Configuration Switch 320 The 8 bit configuration switch is located next to the GPIB connector Switch settings provide configuration parameters for GPIB address serial communication protocol and instrument specific initialization procedures Factory setting is shown for the fluorescence detector Figure 98 8 bit Configuration Switch Table 51 8 bit Configuration Switch RS 232C 1 Baud rate Data Parity Bits a fe Reserved o 1200 Series FLD Service M anual Hardware Information 15 Switches 1 and 2 define which set of parameters for example for GPIB RS 232C and so on will be changed Once the change has been completed the instrument must be powered up again in order to store the values in the non volatile memory In the non volatile memory the parameters are kept regardless of whether you turn the instrument off and on again They will be kept until the same set of parameters is changed and the power is reset All other previously
168. reen light is OFF 55 3 3 Installing the Detector 56 To disconnect the detector from line unplug the power cord The pow er supply still uses some power even if the power switch at the front panel is turned OFF The detector was shipped with default configuration settings To change these settings see Setting the 8 bit Configuration Switch on page 320 1200 Series FLD Service M anual Installing the Detector 3 Flow Connections to the Detector Preparations Detector is installed in the LC system Parts required Other modules Parts from accessory kit see Detector Accessory Kit Contents on page 49 Two wrenches 1 4 5 16 inch for capillary connections WARNING When working with solvents please observe appropriate safety procedures for example goggles safety gloves and protective clothing as described in the material handling and safety data sheet supplied by the solvent vendor especially when toxic or hazardous solvents are used The flow cell is shipped with a filling of isopropanol also recommended when the instrument and or flow cell is shipped to another location This is to avoid breakage due to Subambient conditions 1200 Series FLD Service M anual 57 3 Installing the Detector 1 Press the release buttons and remove the front cover to 2 Locate the flow cell gain access to the flow cell area 3 Assemble the column detector capillary from the 4 Assemble the
169. rfaces agg Agilent Technologies 105 6 Troubleshooting and Test Functions Overview of the Detector s Indicators and Test Functions Status Indicators The detector is provided with two status indicators which indicate the operational state prerun run and error states of the detector The status indicators provide a quick visual check of the operation of the detector see page 107 Error Messages In the event of an electronic mechanical or hydraulic failure the detector generates an error message in the user interface For each message a short description of the failure a list of probable causes of the problem and a list of suggested actions to fix the problem are provided see Error Information on page 111 Wavelength Recalibration Wavelength recalibration is recommended after repair of internal components to ensure correct operation of the detector The detector uses specific properties of the excitation and emission light characteristics see Wavelength Verification and Calibration on page 189 Test Functions A series of test functions are available for troubleshooting and operational verification after exchanging internal components see Test Functions on page 137 106 1200 Series FLD Service M anual Troubleshooting and Test Functions 6 Status Indicators Two status indicators are located on the front of the detector The lower left indicates the power supply status the upper right
170. rogram for the emission wavelength 1200 Series FLD Service M anual 1 Naphthalene 2 Acenaphthene 3 Fluorene 4 Phenanthrene Anthracene 6 Fluoranthene 7 Pyrene LU 60 50 40 30 20 1 10 0 25 5 400 nm 220 nm 350 nm 11 12 13 14 15 na 9 420 nm 500 nm Emission First Steps with the Detector 4 Benzlalanthracene Chrysene Benzolb fluoranthene Benzolk fluoranthene Benzlajpyrene Dibenzolahjanthracene Benzolg h ijperylene Indenol1 2 3 cd pyrene 11 12 10 1314 7 LIW ANS 10 12 5 15 17 5 20 225 Time min Excitation spectra switching Figure 32 Optimization of the time program for the excitation wavelength The obtained data are combined to setup the time table for the excitation wavelength for best limit of detection and selectivity The optimized switching events for this example are summarized in Table 10 Table 10 Timetable for the analysis of 15 polynuclear aromatic hydrocarbons Time min Exitation Wavelength nm Emission Wavelength nm 0 260 350 8 2 260 420 19 0 260 500 This timetable gives the conditions for optimum detection based on the results of two chromatographic runs 1200 Series FLD Service M anual 77 4 78 First Steps with the Detector Procedure Ill Make a single run with the Agilent 1200 Series DAD FLD combination For most organic compounds UV spectra from diode array detectors are nearly identical to fluorescence excitation spectra
171. s Site Requirements 40 A suitable environment is important to ensure optimal performance of the detector Power Consideration The detector power supply has wide ranging capabilities and accepts any line voltage in the range mentioned in Table 2 on page 42 Consequently there is no voltage selector in the rear of the detector There are also no externally accessible fuses because automatic electronic fuses are implemented in the power supply To disconnect the detector from line unplug the power cord The power supply still uses some power even if the power switch on the front panel is turned off Shock hazard or damage of your instrumentation can result if the devices are connected to a line voltage higher than specified M ake sure to have easy access to the power cable of the instrument in order to disconnect the instrument from line Power Cords Different power cords are offered as options with the detector The female end of all power cords is identical It plugs into the power input socket at the rear of the detector The male end of each power cord is different and designed to match the wall socket of a particular country or region 1200 Series FLD Service M anual Site Requirements and Specifications 2 Never operate your instrumentation from a power outlet that has no ground connection Never use a power cord other than the Agilent Technologies power cord designed for your region Never use cables oth
172. s 14 Agilent 1200 to 3394 6 Integrators Connector Pin Pin Signal Name 35900 60750 3394 6 Agilent 1200 1 Not connected 2 Shield Analog 3 Center Analog nS T o Agilent 1200 to BNC Connector Connector Pin Pin Signal Name 8120 1840 BNC Agilent 1200 Shield Shield Analog Center Center Analog 1200 Series FLD Service M anual 281 14 Cables Agilent 1200 to General Purpose Connector 01046 60105 P Pin 3394 6 1 2 3 Pin Agilent 1200 Black Red Signal Name Not connected Analog Analog 282 1200 Series FLD Service M anual Remote Cables Cables 14 O opao One end of these cables provides a Agilent Technologies APG Analytical Products Group remote connector to be connected to Agilent 1200 Series modules The other end depends on the instrument to be connected to Agilent 1200 to 3390 Integrators Connector Pin 01046 60203 3390 2 NC 7 NC D NC NC NC NC NC Pin Signal Name Agilent 1200 1 White Digital ground 2 Brown Prepare run 3 Gray Start 4 Blue Shut down 5 Pink Not connected 6 Yellow Power on 7 Red Ready 8 Green Stop 9 Black Start request Active TTL Low Low Low High High Low Low 1200 Series FLD Service M anual 283 14 Cables Agilent 12
173. s 2006 Printed in Germany 02 06 GL34T BOTTO 61321 90110 ee Agilent Technologies
174. s the resistance history of the excitation and the emission grating drives The number of revolutions after switching off the drives is a measure of friction The history may show an increasing friction of the drive s over a length of time The history data contains the data time information and the number of turns The data plot can be retrieved via the diagnostics Bao be fal Corto Hesna e Aer B x l0 ig Me Taree 5 a a r m0 Sa 8 h slut tee s Hij 14 mW 5 fi ia Pe ai EG Pri 180 H 0 Tini Figure 55 Resistance History 142 1200 Series FLD Service M anual Test Functions 8 Friction Evaluation M otor Error Probable Causes e Friction too high lt 12 turns e Defective monochromator assembly Suggested Actions VY Exchange the monochromator assembly 1200 Series FLD Service M anual 143 8 Test Functions Raman ASTM Signal to Noise Test 144 These tests verify the Raman ASTM signal to noise for e single wavelength EX 350 nm EM 397 nm or e dual wavelength EX 350 nm EM 397 nm EMp 450 nm Table 21 Raman Signal to Noise Test Conditions Duration approximately 23 minutes Report Style Agilent ChemStation Performance N oise Noise Determination 5 to 20 minutes Solvent LC grade water degassed Flow rate 0 5 1 ml min Specification single wavelength gt 500 according to settings in Table 22 Specification dual wavelength gt 300 according to settin
175. scence scan range under FLD special setpoints according to your needs The scan time will increase when the range is enlarged With the default values the scan takes about 2 minutes 3 Set PMT gain to 16 89 4 First Steps with the Detector The wavelength range and step number defines the duration Using the maximum range the scan would take approximately 10 minutes FLD Special Setpoints System 2 q X Phosphorescence Detection Mode Fluorescence Scan Range f pif C On Delay jao Le From To Step Excitation 220 400 E nm Emission 200 500 E nm Time Scan 143 3 Gate 200 0 es Lamp Baseline Behavior Append Free Zero M Only On During Run D Economy Mode Signal Polarity now 4 Hz High Lamp Current Enable analysis when lamp is off Positive Negative Lamp Energy Reference On f Off W Fit Spectral Range Figure 42 FLD special settings 4 Define a data file name and take a fluorescence scan After the scan is completed the isoabsorbance scan results appear see Figure 43 on page 90 A low background will improve the signal to noise see also Reducing Stray Light on page 102 Raleigh scattering Note This white area is normally dark blue Raman scattering of water 2nd order of cutoff Figure 43 Fluorescence Scan of Water 90 1200 Series FLD Service M anual This chapter provides information on how to optimize the detector Agi
176. sh Lamp Frequency 98 M odes The lamp flash frequency can be changed into the following modes Table 18 Flash Lamp M odes Positioning 296 Hz Standard 560 V 74 Hz Economy 560 V Rotation M ulti Ex Em 74 Hz Standard 950 V 74 Hz Economy 560 V 63 mj oule 18 8 W 63 mJ oule 4 7 W 180 mJ oule 13 3 W 63 mJ oule 4 7 W Best sensitivity can be expected with no economy see Figure 46 FLD1 A Ex 246 Em 317 FLD_FLRIFLR_0001 D FLD1 A Ex 246 Em 317 FLD_FLRiFLR_0002 D Standard 296 Hz 0 2 gf nae ee a a weal we Tai 0 0 5 1 1 5 2 Figure 46 Xenon Flash Lamp Frequency Economy 74 Hz i 1200 Series FLD Service M anual How to optimize the Detector Lamp life savings There are three ways to save lamp life e switch to lamp on during run without loss of sensitivity e switch to economy mode with a certain loss of sensitivity e a combination of the above 1200 Series FLD Service M anual 99 5 How to optimize the Detector Selecting the Best Response Time Data reduction using the RESPONSETIME function will increase your signal to noise ratio For example see Figure 47 FLD1 A Ex 246 Em 317 FLD_RT RT_OOOO2 D FLD1 A Ex 246 Em 317 FLD_RT RT_OOO003 D FLD1 A Ex 246 Em 317 FLD_RT RT_OO004 D LU 4 08 a 04 ee re LE E A EE A a 8 Sec 0 2 aaar gt l nent Aeran 4 sec
177. st on page 139 If no profile available very low counts replace FLL board Trigger pack VY replace Xenon flash lamp Y perform a Check of Raman Band of Water on page 156 to check the wavelength calibration Y replace FLF board 1200 Series FLD Service M anual 133 7 Error Information M otor Errors M onochromator motor errors may show up during the initialization or during operation of the detector There are individual messages for either the excitation or the emission side If an error occurs do a lamp ignition This will clear the error and a re initialization of the motors is performed Table 20 Motor Errors Message M otor Or Encoder Not Found EX 6705 EM 6706 Encoder Index Not Found EX 6707 EM 6708 134 Description During initialization of the detector the excitation and emission monochromator are activated During initialization of the detector the excitation and emission monochromator are activated and the encoder should generate an index Probable Causes encoder cables mixed on FLM board monochromator assembly not connected monochromator or encoder defective monochromator motor power driver defective encoder defective encoder electric defective monochromator defective or missing one phase monochromator motor power driver defective Suggested Actions check encoder connections to FLM check motor connections to FLF Ex and FLM Em and encoders to F
178. stored configuration settings will still remain in the non volatile memory In this way you can store more than one set of parameters using the same 8 bit configuration switch twice for example for both GPIB and RS 2382C GPIB Default Addresses If you just want to change the GPIB address and need a detailed procedure refer to the Installing Your Agilent ChemStation System handbook Default GPIB address is set to the following addresses Table 52 Default Addresses for Agilent 1200 Series M odules Module Address Binary Address Pump 22 00010110 FLD 23 00010111 VWD 24 00011000 Agilent 8453A 25 00011101 DAD MWD 26 00011010 Column compartment 27 00011011 Autosampler 28 00011100 RID 29 00011101 where 0 means that the switch is down and 1 means that the switch is up 1200 Series FLD Service M anual 321 15 Hardware Information Communication Settings for RS 232C Communication The communication protocol used in this instrument supports only hardware handshake CTS RTS Switches 1 in down and 2 in up position define that the RS 232C parameters will be changed Once the change has been completed the instrument must be powered up again in order to store the values in the non volatile memory Table 53 Communication Settings for RS 232C Communication Modeselect ja f2 fa Je b fje p fje Bits Use the following tables for selecting the setting which you want to use for RS 232C communication The number 0 means that the s
179. suggested actions how to recover from error conditions Test Functions This chapter describes the detector s built in test functions Maintenance and Repair This chapter provides general information on maintenance and repair of the detector 10 11 12 13 14 15 Maintenance This chapter describes the maintenance of the detector and the required tests Repairs This chapter describes the repairs of the detector Parts and Materials for Maintenance This chapter provides information on parts for maintenance Parts for Repair This chapter provides information on parts for repair Cables This chapter provides information on cables used with the 1200 series of HPLC modules Hardware Information This chapter describes the detector in more detail on hardware and electronics Appendix This chapter provides safetey and other general information 1200 Series FLD Service M anual Contents 1 Introduction to the Fluorescence Detector Introduction to the Detector 16 How the Detector Operates 1 7 Raman Effect 20 Optical Unit 21 Reference System 2 7 Analytical Information From Primary Data 28 Fluorescence Detection 28 Phosphorescence Detection 29 Processing of Raw Data 30 Electrical Connections 33 Instrument Layout 35 Early Maintenance Feedback EMF 36 EMF Counters 36 Using the EMF Counters 38 2 Site Requirements and Specifications Site Requirements 40 Physical Specifications 42 Perf
180. t on ambient temperature The change in resistance is used by the leak circuit to compensate for ambient temperature changes If the resistance across the sensor falls below the lower limit the error message is generated Probable Causes e Defective FLF board Suggested Actions VY Exchange the FLF board 122 1200 Series FLD Service M anual Error Information 7 Fan Failed The cooling fan in the detector has failed The hall sensor on the fan shaft is used by the FLM board to monitor the fan speed If the fan speed falls below two revolutions second for more than five seconds the error message is generated Probable Causes e Fan cable disconnected e Defective fan e Defective FLM board Suggested Actions VY Ensure the fan is connected correctly VY Exchange fan VY Exchange the FLM board 1200 Series FLD Service M anual 123 7 Error Information Detector Error Messages These errors are detector specific Lamp Cover Open The lamp cover in the optical compartment has been removed The lamp cannot be turned on while this message is on Probable Causes e lamp cover removed Suggested Actions VY close lamp cover and tighten the screws 124 1200 Series FLD Service M anual Error Information 7 FLF Board not found The FLF board could not be found by the main board FLM This message comes together with some other message generated on the FLF board e g Leak Probable Causes e FLF board
181. the Foam and the Top Cover on page 149 Re install the detector in the stack Enter the replacement of the lamp in the maintenance logbook this will reset the lamp counter Turn the lamp ON Perform a wavelength verification to check the correct positioning of the replaced assemblies as described in chapter Diagram of Light Path on page 138 208 1200 Series FLD Service M anual Repairs 11 Replacing the Lamp Cover Window When required If window Is blocking the light throughput due to contamination Tools required Screwdriver POZI 1 PT3 Hexagonal screwdriver 2 5 mm 100 mm long Hexagonal screwdriver 4 mm 100 mm long Parts required Quartz Window 1000 0999 When loosening or removing screws take care that they do not fall into the instrument This may result in a complete removal of other assemblies Preparations for this procedure 1 Disconnect the emission and the excitation cables and from the detector main board Turn OFF the detector Disconnect the power cable Remove the flow cell or disconnect the tubings from the flow cell Remove detector from stack and place it on the workbench Remove the covers as described in Removing the Covers on page 198 1200 Series FLD Service M anual 209 11 Repairs 2 Carefully remove the rear foam 3 Using the 4 mm hexagonal key unscrew the five screws remove the cover and place it on the bench without turnin
182. the capillary connections Tools required Tissue Two 1 4 inch wrenches for capillary connections Parts required None 1 Remove the front cover 2 Use tissue to dry the leak sensor area and the leak pan 3 Observe the capillary connections and the flow cell area for leaks and correct if required 4 Replace the front cover mM Sariai DE12345678 Figure 70 Observing for Leaks 182 1200 Series FLD Service M anual Maintenance 10 Replacing Leak Handling System Parts When required If the parts are corroded or broken Tools required None Parts required Leak funnel 5061 3356 Leak funnel holder 5041 8389 Leak tubing 120 mm 0890 1711 Remove the front cover Pull the leak funnel out of the leak funnel holder Pull out the leak funnel with the tubing Insert the leak funnel with the tubing in its position Insert the leak funnel into the leak funnel holder aq ur W N P Replace the front cover Leak funnel Leak funnel holder Leak tubing G1321A Sariai 0E123456 Leak sensor Figure 71 Replacing Leak Handling System Parts 1200 Series FLD Service M anual 183 10 Maintenance Replacing the Interface Board When required For all repairs inside the detector or for installation of the board Part required Interface board BCD G1351 68701 with external contacts
183. the instrument must be made inoperative and be secured against any intended operation Make sure that only fuses with the required rated current and of the specified type normal blow time delay and so on are used for replacement The use of repaired fuses and the short circuiting of fuseholders must be avoided 1200 Series FLD Service M anual Appendix A The operator of this instrument is advised that if the equipment is used in a manner not specified in this manual the protection provided by the equipment may be impaired Some adjustments described in the manual are made with power supplied to the instrument and protective covers removed Energy available at many points may if contacted result in personal injury Any adjustment maintenance and repair of the opened instrument under voltage should be avoided as much as possible When inevitable this should be carried out by a skilled person who is aware of the hazard involved Do not attempt internal service or adjustment unless another person capable of rendering first aid and resuscitation is present Do not replace components with power cable connected Do not operate the instrument in the presence of flammable gases or fumes Operation of any electrical instrument in such an environment constitutes a definite safety hazard Do not install substitute parts or make any unauthorized modification to the instrument Capacitors inside the instrument may still be charged even
184. the output of port 4 is small and evaporates right in the sampler leak pan However the output of port 4 is significant and a substantial volume of ejected mobile phase reaches the detector leak pan 1200 Series FLD Service M anual 117 7 Error Information There are two possible fixes Select the one which is most convenient 1 The waste drain plumbing orientation shown in step 6 on page 59 eliminates the possibility of any leak drainage from above reaching the detector leak pan The leak drain for the detector can be connected to the detector s leak drain fitting and taken to waste separately 2 If itis desired that the system has only one leak drain tube then it s possible in increase the length of the small plastic tube which is connected to port 4 of the sampler switching valve This tube can then be taken to waste separately The tube which normally serves as the detector cell outlet tube can be used for this purpose 118 1200 Series FLD Service M anual Error Information 7 Leak Sensor Open The leak sensor in the detector has failed open circuit The current through the leak sensor is dependent on temperature A leak is detected when solvent cools the leak sensor causing the leak sensor current to change within defined limits If the current falls outside the lower limit the error message is generated Probable Causes e Leak sensor not connected to the FLM board e Defective leak sensor Suggested Actions
185. the thumb screws Reconnect the capillaries to the flow cell DO NOT install the inlet capillary to the outlet connection of the flow cell This will result in poor performance or damage 1200 Series FLD Service M anual Maintenance 10 Note If an additional detector is added to the system the fluorescence detector should be the last detector in the flow path except for evaporative detectors like LC M SD Otherwise the back pressure generated by the other detector may overload the quartz flow cell and will lead to a defective cell maximum pressure is 20 bar 2 MPa Always use the outlet capillary set supplied with the accessory kit Note 5 Replace the front cover To check for leaks establish a flow and observe the flow cell outside of the cell compartment and all capillary connections Note Perform a wavelength verification to check the correct positioning of the flow cell as described in chapter Wavelength Verification and Calibration on page 189 1200 Series FLD Service M anual 179 10 Maintenance How to use the Cuvette 180 The cuvette is used for off line measurements no flow system required and is basically a standard flow cell with a few changes ur W N P wide bore capillary connections for easier injections with a syringe identification lever for cell auto recognition system Install the cuvette instead of the standard flow cell Connect the waste tubing to the outlet of the cuvett
186. ting APG remote 53 connecting CAN 53 connecting GPIB 53 connecting LAN 53 connecting the ChemStation 53 connecting the power 53 overview and identification 278 calibration sample 164 191 CAN interface 316 check of Raman band of water 156 configuration switch default settings 320 description and factory settings 320 cut off filter 21 cutoff filter other type 262 description 297 location on FLM board 213 safety information 331 BCD board 312 1200 Series FLD Service M anual Index error messages 112 A D overflow 127 ADC not calibrated 126 compensation sensor open 121 compensation sensor short 122 encoder index not found 134 fan failed 123 flash lamp current overflow 128 flash trigger lost 129 FLF board not found 125 flow cell removed 132 lamp cover open 124 leak 117 leak sensor open 119 leak sensor short 120 motor encoder index wrong 135 motor errors 134 motor friction too high 135 motor or encoder not found 134 motor position not found or 135 motor speed too low or unstable 135 remote timeout 115 shutdown 114 synchronization lost 116 time out 113 wavelength calibration failed 130 wavelength calibration lost 131 ESD electrostatic discharge strap 174 excitation condenser 21 excitation grating 21 excitation monochromator 23 excitation slit 21 F features safety and maintenance 45 firmware description 304 main system 304 raw data conversion 306 resident s
187. to set the desired output voltage The PMT output delivers a current signal which is proportional to the detected light and to the actual working voltage FLM Board Board Temp Reference Sensor Diode PMT voltage 4 Supply Signal conditioning amp gain FLL Flash Lamp Board Pre Amplifier amp signal conditioning EX M otor EX Mono e aromato Driver Lamp Cover A D Converter Safety Switch 16 bit Cell Read Write Leak Sensor Leak Detection Port circuit Sensor FLM Board Figure 90 Block diagram FLF 300 1200 Series FLD Service M anual Hardware Information 15 Reference System A reference photodiode is located behind the cell and monitors the incoming radiation through a diffusor window The data are used to compensate the fluorescence data for drift effects The reference path provides multiple gain stages which are set automatically according to the actual intensity Cell Detection The system detects if a cell is inserted and switches off lamp operation PMT high voltage and monochromator rotation if no cell is present to prevent injury by UV radiation high voltage or rotating parts to the user Emission M onochromator M otor Control A chopped motor driver IC provides the signal to run the excitation monochromator motor in positioning or rotation mode Leak Sensor A leak sensor is mounted in the instrument s front chassis to detect leakage of the cell assy In case of a leakage the
188. tronic boards and components see Using the ESD Strap on page 174 1200 Series FLD Service M anual Repairs 11 WARNING Eye damage may result from directly viewing the light produced by the xenon flash lamp used in this product 1200 Series FLD Service M anual 197 11 Repairs Removing the Covers When required For all repairs inside the detector Tools required Screwdriver POZI 1 PT3 Parts required Depends on the work inside and the following procedures CAUTION There will be a risk of damaging hardware due to overheating when operating the instrument without covers Preparations for this procedure 1 Press the release buttons and remove the front cover Unclip and remove the waste funnel assemly Turn OFF the detector Disconnect the power cable Remove the flow cell Remove detector from stack and place it on the workbench 198 1200 Series FLD Service M anual 2 Install the ESD strap M ove the power lock across the power inlet and lift the clips on the rear of the cover 4 Lift the cover up and slide it towards the rear Warning Repairs 11 3 If installed unscrew and remove the interface board Place the board on the ESD kit 5 Unscrew the screws at the rear of the top plate Slide the plate towards the front and remove It Do not connect a power plug to the detector module after removing the top covers 1200 Series FL
189. ual encoder pattern is checked against a known pattern 1200 Series FLD Service M anual Probable Causes friction too high defective monochromator assembly defective monochromator assembly Short mechanical shock message appears intermittently without mechanical shock revolution too low defective monochromator assembly encoder was replaced and has a different pattern or no reset of pattern was made encoder lost position completely Suggested Actions replace monochromator assembly replace monochromator assembly reset monochromator settings replace monochromator assembly re ignite the lamp replace monochromator assembly replace monochromator assembly re ignite the lamp replace monochromator assembly reset pattern via user interface and recalibrate replace monochromator assembly 135 7 Error Information 136 1200 Series FLD Service M anual Agilent 1200 Series Fluorescence Detector Service M anual ee 8 Yo Test Functions Diagram of Light Path 138 Lamp Intensity Test 139 Dark Current Test 141 Excitation and Emission Grating Resistance History 142 Raman ASTM Signal to Noise Test 144 Using the Built in Test Chromatogram 148 Using the Built in DAC Test 151 Wavelength Verification and Calibration 153 Check of Raman Band of Water 156 Using the Agilent ChemStation OQ PV 156 Using the Agilent ChemStation Manually 158 Using the Control Module G1323B 161 Wavelength Calibration
190. ue Shut down Low 1a als 8 5 Pink Not connected NC 6 Yellow Power on High 3 7 Red Ready High 5 Key 6 8 Green Stop Low NC 9 Black Startrequest Low Agilent 1200 to General Purpose Connector Pin Pin SignalName Active 01046 60201 Universal Agilent 1200 TTL 1 White Digital ground 5 2 Brown Prepare run Low A 1 3 Gray Start Low gal KEY m1 4 Blue Shut down Low a 5 Pink Not connected 6 Yellow Power on High Joy 7 Red Ready High 8 Green Stop Low 9 Black Start request Low 1200 Series FLD Service M anual 287 14 Cables BCD Cables One end of these cables provides a 15 pin BCD connector to be connected to the Agilent 1200 Series modules The other end depends on the instrument to be connected to Agilent 1200 to General Purpose Connector Wire Color Pin SignalName BCD Digit G1351 81600 Agilent 1200 Green 1 BCD 5 20 Violet 2 BCD 7 80 Blue 3 BCD 6 40 Ze Yellow 4 BCD 4 10 Black 5 BCD 0 1 NSS Orange 6 BCD 3 8 Red 7 BCD 2 4 Brown 8 BCD 1 2 Gray 9 Digital ground Gray Gray pink 10 BCD 11 800 Red blue 11 BCD 10 400 White green 12 BCD 9 200 Brown green 13 BCD 8 100 notconnected 14 notconnected 15 5 V Low 288 1200 Series FLD Service M anual Cables 14 Agilent 1200 to 3396 Integrators Connector Pin Pin SignalName BCD Digit 03396 60560 3392 3 Agilent 1200 1 1 BCD5 20 O 2 2 BCD 7 80 ae J 3 3 BCD 6 40 ies ae 4 4 BCD
191. und Step 2 Optimize limits of detection and selectivity To achieve optimum limits of detection and selectivity analysts must find out about the fluorescent properties of the compounds of interest Excitation and emission wavelengths can be selected for optimum limits of detection and best selectivity In general fluorescence spectra obtained with different instruments may show significant differences depending on the hardware and software used The traditional approach is to extract an appropriate excitation wavelength from the UV spectrum that is similar to the fluorescence excitation spectrum see Figure 29 and to record the emission spectrum Then with an optimum emission wavelength determined the excitation spectrum is acquired Excitation spectrum Norm with emission at 40 440 nm emission 35 spectrum with excitation at 250 nm 30 iini niii of 1 ug ml quinidine 25 Detector settings 20 step size 5 nm PMT 15 12 Response time 4 s 10 5 0 250 300 350 400 450 500 550 600 Wavelength nm Figure 29 Excitation and emission spectra of quinidine 1200 Series FLD Service M anual 71 4 72 First Steps with the Detector These tasks have to be repeated for each compound using either a fluorescence spectrophotometer or stop flow conditions in LC Usually each compound requires a separate run As a result a set of excitation and emission spectrum is obtained Figure 28 on page 70 for each compound Since this is a te
192. ush the flow cell VY Reset monochromator settings and re run the wavelength calibration 1200 Series FLD Service M anual Error Information 7 Wavelength Calibration Lost After exchanging the monochromator assemblies the calibration factors should be reset to defaults values a new FLM board comes with default values In this case Wavelength Calibration Lost is displayed and the instrument stays in a Not Ready condition Probable Causes e Reset of monochromator settings after exchange e Replacement of FLM board Suggested Actions V Perform a wavelength calibration 1200 Series FLD Service M anual 131 7 Error Information Flow Cell Removed The detector has an automatic cell recognition system When the flow cell is removed the lamp is turned off and a NOT READY condition exists If the flow cell is removed during an analysis a SHUT DOWN is generated Probable Causes e Flow cell has been removed during analysis Suggested Actions VY Insert flow cell and turn on the lamp 132 1200 Series FLD Service M anual Error Information 7 No Peaks If no peaks are shown in the chromatogram the user interface shows the module still in Ready There is no feedback mechanism that checks whether the lamp is ON Probable Causes e defective FLL board Trigger pack e defective Xenon flash lamp e wrong position of monochromator e FLF board defective Suggested Actions V Perform a Lamp Intensity Te
193. witch is down and 1 means that the switch is up Table 54 Baud Rate Settings Switches Baud Rate Switches Baud Rate Table 55 Data Bit Settings Switch 6 Data Word Size OO 7 Bit Communication 8 Bit Communication 322 1200 Series FLD Service M anual Hardware Information 15 Table 56 Parity Settings ms my E C One start bit and one stop bit are always used not selectable Per default the module will turn into 19200 baud 8 data bit with no parity Forced Cold Start Settings Switches 1 and 2 do not force storage of this set of parameters in non volatile memory Returning switches 1 and 2 to other positions other than being both up will allow for normal operation Forced cold start erases all methods and data stored in the non volatile memory Exceptions are diagnose and repair log books which will not be erased If you use the following switch settings and power the instrument up again a forced cold start has been completed Table 57 Forced Cold Start Settings M ode Select TEST BOOT To return to normal operation set switches back to your GPIB or RS 232 configuration settings 1200 Series FLD Service M anual 323 15 Hardware Information Stay Resident Settings Firmware update procedures may require this mode in case of firmware loading errors Switches 1 and 2 do not force storage of this set of parameters in non volatile memory Returning switches 1 and 2 to other positio
194. ysis for example calibration detector lamp on Receiver is any module performing pre analysis activities 3 START L Request to start run timetable Receiver is any module performing run time controlled activities 4 SHUT DOWN L System has serious problem for example leak stops pump Receiver is any module capable to reduce safety risk 5 Not used 1200 Series FLD Service M anual 317 15 Hardware Information 318 Table 49 Remote Signal Distribution continued Pin Signal Description 6 POWER ON H All modules connected to system are switched on Receiver is any module relying on operation of others 7 READY H System is ready for next analysis Receiver is any sequence controller 8 STOP L Request to reach system ready state as soon as possible for example stop run abort or finish and stop injection Receiver is any module performing run time controlled activities 9 START REQUEST L Request to start injection cycle for example by start key on any module Receiver is the autosampler RS 232C The RS 232C connector is used to control the instrument from a computer through RS 232C connection using the appropriate software This connector can be activated by the configuration switch module next to the GPIB connector The RS 232C is designed as DCE Data Communication Equipment with a 9 pin male SUB D type connector The pins are defined as follows 1200 Series FLD Service M anual Ha
195. ystem 304 updates 305 firmware updates 305 flash frequency 30 339 flat baseline no peaks 133 FLM board analog output 297 battery 297 encoder signal acquisition 297 EX EM motor control 297 fan drive 297 interfaces 297 flow cell 21 25 fluorescence and phosphorescence 18 fluorescence detection 28 fluorescence spectral libraries for peak confirmation 82 flushing of flow cell 181 front view of module 54 fuses BCD board 312 power supply 326 G GLP features 45 glycogen 164 191 GPIB default addresses 316 321 interface 316 H How the Detector Operates 17 how to use the cuvette 180 humidity 42 information on lithium batteries 331 1200 Series FLD Service M anual installation accessory kit 49 bench space 41 delivery checklist 48 environment 41 flow connections 57 of flow cell and capillaries 57 of the detector 54 physical specifications 42 power considerations 40 power cords 40 site requirements 40 unpacking 48 instrument layout 35 interface board BCD LAN 312 interfaces analog signal output 316 APG remote 317 CAN 316 GPIB 316 overview 315 RS 232C 318 internet 337 Introduction to the Detector 16 L lamp intensity history 140 188 lamp intensity test 139 LAN cables 294 interface board 312 LAN interface board 314 leaks correcting 182 line voltage and frequency 42 luminescence 17 M Maintenance 169 175 Index message A D

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