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Agilent 1200 Series Fluorescence Detector

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1. c Barai DE12345678 Figure 52 Observing for Leaks 118 1200 Series FLD User M anual 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 a ur WN rPe Replace the front cover Maintenance 8 Leak funnel Leak funnel holder Leak tubing So QD G1321A Sarai DE123456 i Figure 53 Replacing Leak Handling System Parts 1200 Series FLD User M anual Leak sensor 119 8 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 and BCD
2. local user interface CAN Bus cable Analog detector signal 1 or 2 outputs per detector LAN to LC ChemStation location depends on detector Figure 22 1200 Series FLD User M anual Oooo000 Recommended Stack Configuration Rear View Installing the Detector AC power 3 47 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 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 120 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 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 48 1200 Series FLD User Manual Interface board LAN or BCD EXT RS 232C Installing the Detector 3 6 If an Agilent ChemStation is the controller connect either the LAN co
3. APG remote D lt serial number DE manufactured in Germany 6 2006 01 week of last 00130 product number serial number 28 major change number of unit 61321A FLD Serial No te Agilent Technologies Agilent Techostogis 76337 Weldbrosn Made in Germany Figure 17 CAN Configuration switch G1321A FLD Serial No CE safety standards amp proce 4 CES 66 0 oe configuration switch settings voltage range power consumption frequency 108 120 228 268 V 180 VA 58 08 Hr Electrical Connections 1200 Series FLD User Manual Introduction to the Fluorescence Detector 1 Instrument Layout The industrial design of the detector incorporates several innovative features It uses Agilent s E 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 th
4. 1200 Series FLD User M anual 75 4 First Steps with the Detector The upper trace was received with traditional wavelength switching 76 1 excitation WL at 260 nm Taena ave en 4 emission WL at 350 420 EaDAP Mens rysana 440 and 500 nm 3 Fluorene 10 Benzo b fluoranthene 4 Phenanthrene 11 Benzolk fluoranthene 5 Anthracene 12 Benz a pyrene 6 Fluoranthene 13 Dibenzo a hjanthracene LU 7 Pyrene 14 Benzolg h i perylene 5 15 Indeno 1 2 3 cd pyrene 180 Ex 275 Em 350 TT Reference 10 160 chromatogram with switching events 140 120 100 80 60 40 20 0 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 confirm 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
5. Select a Excitation wavelength in the low UV 230 260 nm THE gt gt 3 will cover nearly all a BT fai nm fluorescence in your P Cr TRO rm sample E jno nm DO NOT select additional emission ja wavelengths B C 300 to 500 D Doing so will B increase the scan Threshold POD Lu time and will lower the performance gt 0 2 min 4 s standarc 7 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 User M anual 79 4 _ First Steps with the Detector FLD1 A Ex 260 Em 350 FLD_PADT FLD_PAD1 D Lu 3 643 7211 5 300 T T T T T T o 2 4 6 8 10 12 min 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 80 1200 Series FLD User Manual DO NOT select additional excitation wavelengths B C D Doing so will increase the scan time and will lower the performance 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 op
6. 38 flow cell 38 GLP features 39 monochromators 37 performance 37 pulse frequency 37 safety and maintenance 39 wavelength accuracy 37 spectra wavelength shift 58 stack configuration 45 47 front view 45 rearview 47 T test functions 100 tests lamp intensity history 124 PMT gain test 57 troubleshooting error messages 100 status indicators 100 101 U unpacking 42 using EMF 30 UV degradation 16 124 W wavelength recalibration 100 wavelength shift of spectra 58 weight and dimensions 36 X xenon flash lamp 15 16 1200 Series FLD User 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 e maintenance e parts identification e safety and related information Agilent Technologies 2006 Printed in Germany 02 06 CHF 80010 G1321 90010 EE Agilent Technologies
7. spectra 220 nm 350 nm 420 nm 500M Emission 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 User M anual 71 4 72 First Steps with the Detector Procedure III 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 Spectral differences are caused by specific detector characteristics such as spectral resolution or light sources 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
8. D FLD1 A Ex 248 Em 317 FLD_RT RT_00003 D FLD1 A Ex 246 Em 317 FLD_RTIRT_00004 D Lu os 4 064 044 a e 8 sec eet 4 sec highly asm E 1sec Figure 47 Finding Best Response Time 94 1200 Series FLD User Manual 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 User M anual 95 5 How to optimize the Detector Reducing Stray Light 96 Cut off filters are used to remove stray light and 2 4 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 2 4 order light is the limiting fact
9. FLD User Manual 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 C 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 User M anual 103 6 Troubleshooting and Test Functions Agilent LC Diagnostic Software 104 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 maintenance positions e Agilent 1200 Series binary pump SL
10. and the spectra from a peak A match factor of 1 000 means identical spectra 1200 Series FLD User Manual 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 based on the emission spectra from a PNA reference sample Table 13 Peak confirmation using a fluorescence spectral library Meas Library CalTbl Signal Amount Purity Match Libary Name RetTime min min min ng Factor 4 859 4 800 5 178 1 1 47986e 1 1 993 Naphthalene em 6 764 7 000 7 162 1 2 16156e 1 1 998 Acenaphthene em 7 137 7 100 7 544 1 1 14864e 1 1 995 Fluorene em 8 005 8 000 8 453 1 2 56635e 1 1 969 Phenanthrene em 8 841 8 800 9 328 1 1 76064e 1 1 9983 Anthracene em 9 838 10 000 10 353 1 2 15360e 1 1 997 Fluoranthene em 10 439 10 400 10 988 1 8 00754e 2 1 1000 Pyrene em 12 826 12 800 13 469 1 1 40764e 1 1 998 Benz a
11. anthracene em 13 340 13 300 14 022 1 1 14082e 1 1 999 Chrysene em 15 274 15 200 16 052 1 6 90434e 1 1 999 Benzo b fluoranthene em 16 187 16 200 17 052 1 5 61791e 1 1 998 Benzo k fluoranthene em 16 865 16 900 17 804 1 5 58070e 1 1 99 Benz a pyrene em 18 586 18 600 19 645 1 5 17430e 1 1 999 Dibenz a h anthracene em 19 200 19 100 20 329 1 6 03334e 1 1 995 Benzo g h i perylene em 20 106 20 000 21 291 1 9 13648e 2 1 991 Indeno 1 2 3 cd pyrene em 1200 Series FLD User M anual 77 4 First Steps with the Detector Example Optimization for M ultiple Compounds 78 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 detector settings are shown in Figure 36 on page 79 Table 14 Chromatographic Conditions Mobile 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 1 yl 30 C PMT 15 4 seconds 1200 Series FLD User Manual First Steps with the Detector 4
12. crystallization of buffer solutions This will lead into a blockage damage of the flow cell If the flow 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 2CHCl13 Og 2COClg 2ZHCI1 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 User M anual 145 A Appendix e Chromatographic grade ethers which can contain peroxides for example THF dioxane di isopropylether such ethers should be filtered through dry aluminium oxid
13. 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 Relative Intensity Aa UR rir iw VA a w w 500 w w w w Wavelenght nm Figure7 Lamp Energy Distribution vendor data 16 1200 Series FLD User 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 lt Grating EX inside Mirror Figure8 Mirror Assembly 1200 Series FLD User M anual 17 1 18 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 reflected 15t order dispersed light 800nm Figure9 Dispersion of Light by a Grating The grating is turned us
14. 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 User Manual 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 background Step 2 Optimize limits of detection and selectivity Excitation spectrum with emission at 440 nm emission spectrum with excitation at 250 nm of 1 ug ml quinidine Detector settings step size 5 nm PMT 12 Response time 4s To achieve optimum limits of detection and selectivity analysts must find out about the fluorescent properties of the compounds of interest Excitation and emis
15. 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 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 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 FL
16. 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 X Note Perform a wavelength verification to check the correct positioning of the flow cell as described in chapter Wavelength Verification and Calibration on page 125 1200 Series FLD User M anual 115 8 Maintenance How to use the Cuvette 116 The cuvette is used for off line measurements no flow system required and is basically a standard flow cell with a few changes e wide bore capillary connections for easier injections with a syringe e 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 cuvette Use the syringe see Cuvette Kit on page 133 to inject the compound Setup the parameters for the Fluorescence Scan under Special Setpoints aur WN FB Select Take Fluorescence Scan on the user interface to start the off line measurement 1200 Series FLD User Manual Maintenance 8 Flow Cell Flushing When required If flow cell is contaminated Tools requi
17. 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 User Manual 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 68704 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
18. takes about 5 minutes 3 You are now ready to change the settings of your detector 1200 Series FLD User M anual 59 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 Mobile 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 User Manual In this example additional excitation wavelengths B C D are used This will increase the scan time and may lower the performance First Steps with the Detector 2 Set the FLD setpoints according to Figure 25 on the local Control Module G1323B this information is split across separate screens r Signal Time r Multiple Wavelengths and Spectra Excitation A Emission Stoptime aod Lime 4 min Cc TID Multi Em ce 246 mo e jd nm g Use additional Excitation Posttime Off min Zeto Order C Zero Order Biv 230 nm Ch 250 nm D N 230 nm r
19. 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 User M anual 91 5 How to optimize the Detector Changing the Xenon Flash Lamp Frequency Modes The lamp flash frequency can be changed into the following modes Table 18 Flash Lamp Modes Positioning 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 Best sensitivity can be expected with no economy see Figure 46 FLD1 A Ex 246 Em 317 FLD_FLR FLR_0001 D FLD1 A Ex 246 Em 317 FLD_FLR FLR_0002 D Standard 296 Hz Economy 74 Hz Annn Figure 46 Xenon Flash Lamp Frequency 92 1200 Series FLD User 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 User M anual 93 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 248 Em 317 FLD_RTIRT_00002
20. 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 Intensity Test History 7 Figure 56 Lamp Intensity History 124 1200 Series FLD User Manual Maintenance 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 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 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 User M anual 12
21. 01 1446 5062 8592 5041 8388 5041 8389 5041 8387 5062 2463 5062 2462 5181 1516 5181 1519 G1369 60001 5023 0203 5023 0202 01046 60105 G1351 68701 132 1200 Series FLD User Manual Cuvette Kit Table 22 Cuvette Kit Item Description Parts and Materials for Maintenance Part Number 9 FLD Cuvette Kit 8 ul 20 bar includes Tubing flexible 1 meter SST Fitting QTY 1 SST front ferrule QTY 1 SST back ferrule QTY 1 PEEK fitting QTY 1 Needle Syringe Glass Syringe G1321 60007 79814 22406 0100 0043 0100 0044 0100 1516 9301 0407 9301 1446 1200 Series FLD User M anual 133 9 Parts and Materials for Maintenance Spare Parts 134 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 23 Spare Parts Description Part Number Cutoff filter k Cutoff filter k Cutoff filter k Cutoff filter 3 Photo multip Photo multip it 389 nm 408 nm 450 nm 500 nm 550 nm it 380 nm 399 nm 418 nm 470 nm 520 nm it 280 nm 295 nm 305 nm 335 nm 345 nm 70 nm ier Tube PMT R928HA 185 to 900 nm ier Tube PMT R3788HA 185 to 750 nm 5061 3327 5061 3328 5061 3329 1000 0822 contact Hamamatsu dealers 1200 Se
22. 200 Series Fluorescence Detector 10 1200 Series FLD User Manual Introduction to the Fluorescence Detector 1 How the Detector Operates Luminescence Detection Luminescence the emission of light occurs when molecules change from an 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 absorption energy level 2 hy N IY luminescence energy level 2 e hy NS ae ae ae V energy level 1 Figure2 Absorption of Light Versus Emission of Light 1200 Series FLD User M anual 11 1 12 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 rotationa
23. 32C LAN APG Remote ready start stop and shut down signals 1200 Series FLD User Manual Site Requirements and Specifications 2 Table3 Performance Specifications Agilent 1200 Series Fluorescence Detector Type Specification Comments Safety and maintenance GLP features Housing Environment Dimensions Weight Extensive diagnostics error 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 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 water All materials recyclable 0 to 40 C constant temperature at lt 95 humidity non condensing 140 mm x 345 mm x 435 mm 5 5 x 13 5 x 17 inches height x width x depth 11 5 kg 25 5 Ibs Reference conditions standard cell 8 ul response time 4 s flow 0 4 ml min LC grade Methanol 2 1 x 100 mm ODS column 1200 Series FLD User M anual 39 2 Site Requirements and Specifications 40 1200 Series FLD User Manual Agilent 1200 Series Fluorescence Detector User Manual 3 Installing the Detector Unpacking the Detector 42 Optimizing the Stack Configuration 45
24. 5 8 Maintenance FLD Wavelength Calibration System 2 Figure 57 Wavelength Calibration NOTE When the lamp is of the calibration process will stop within the first two steps with Wavelength Calibration Failed 126 1200 Series FLD User Manual Maintenance 8 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 135 Steps 1 Preparation of the Glycogen Calibration Sample 2 Preparation of the Flow Cell 3 Wavelength Calibration 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 Wait 5 minutes and shake again After 10 minutes the solution is ready 1200 Series FLD User M anual 127 8 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
25. Agilent 1200 Series Fluorescence Detector G1321A User Manual Eee 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 Manual Part Number 61321 90010 Edition 02 06 Printed in Germany Agilent Technologies Hewlett Packard Strasse 8 76337 Waldbronn Germany Manual Structure The User Manual G1321 90010 English and its localized versions contain a subset of the Service M anual and is shipped with the detector in printed matter The Service M anual G1321 90110 English contains the complete information about the Agilent 1200 Series Fluorescence Detector Itis available as Adobe Reader file PDF only Latest versions of the manuals can be obtained from the Agilent webWarranty 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 ticu
26. Appendix A Lithium Batteries Information Danger of explosion if battery is incorrectly replaced Replace 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 leverandgren 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 User M anual 141 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 M easurement 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 142 1200 Series FLD U
27. D User M anual 25 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 Foye Nts i 0000000000 bunched data COJO OC OO00O points O O D GIOCOO000 OO O D O O 0000 DO QIOCOO boxcar fitr OO DIDXD O lO O O CODD OODGDIOO C0000 0 O O DG O ooge D O filtered data 000080008 points Mv S N ratio JK Ps Figure 16 RESPONSETIME Signal to Noise Ratio 1200 Series FLD User Manual 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 GPIB address of your detector The switches are preset to a default address 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 contac
28. 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 User Manual Agilent 1200 Series Fluorescence Detector User Manual 7 Maintenance and Repair Introduction into Repairing the Fluorescence Detector 106 Warnings and Cautions 107 Cleaning the Detector 109 Using the ESD Strap 110 This chapter provides general information on maintenance and repair of the detector ae Agilent Technologies 105 7 Maintenance and Repair Introduction into Repairing the Fluorescence Detector 106 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 111 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
29. Iimetable Figure 25 FLD Parameters 3 Start the run Acquire Excitation Spectra All Me Range 230 to 400 nm Step j5 nm Threehald NOO 111 4 The resulting chromatograms are shown in Figure 26 x FLD1 B Ex 230 Em 317 FLD_ISO1 FLD_ISO2 D FLD1 A Ex 246 Em 317 FLD_ISO1 FLD_IS02 D FLD1 C Ex 260 Em 317 FLD_ISO1 FLD_ISO2 D a FLD1 D Ex 200 Em 317 FLD_ISO1 FLD_IS02 D Ex 246 nm Ex 250 nm 60 Ex 230 nm Biphenyl peak 75 so H Ex 290 nm z E a E E A E E E A Figure 26 Biphenyl peak with different excitation wavelengths 5 The excitation maxima is around 250 nm 1200 Series FLD User M anual 61 4 4 First Steps with the Detector Observe the maxima via the isoabsorbance plot 1 Load the data file Agpx 246 nm Agy 317 nm and open the isoabsorbance plot 2 The maximum Ag will be found around 250 nm Figure 27 Isoabsorbance Plot 62 1200 Series FLD User Manual First Steps with the Detector 4 Method Development Fluorescence detectors are 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 Tab
30. Installing the Detector 48 Flow Connections to the Detector 51 This chapter describes the installation of the detector ott Agilent Technologies 41 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 Table4 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 43 1 42 1200 Series FLD User Manual 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 12m Fitting male PEEK 0100 1516 2 Capillary column detector one side preinstalled G1315 87311 1 380 mm lg 0 17 mm i d includes Ferr
31. 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 73 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 User Manual 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 wavelength 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 First Steps with the Detector 4 Norm 35 30 25 20 15 10 5 Excitation Emission 0 200 250 300 350 400 450 500 550 Wavelength nm Figure 33 UV spectrum and fluorescence spectra for 2 3 diaminophenazine DAP 2 amino 3 QH phenazine LU Un
32. 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 in the Service Manual 1200 Series FLD User Manual Maintenance and Repair 7 Warnings and Cautions 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 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 described 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 to prevent damage always use an ESD protection when handling electronic boards and components see Using the ESD Strap on page 110 There is a risk of damaging hardware due to overheating when operating the instrument without covers 1200 Series FLD User M anual 107 7 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 108 1200 Series FLD User Manual M
33. after maintenance of lamps and flow cells e Lamp Intensity Test on page 123 e Wavelength Verification and Calibration on page 125 122 1200 Series FLD User Manual Maintenance 8 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 G1i321A Serial Number DE92001563 Operator Wolfgang Date 09 01 2006 Time 11 26 30 File C CHEM32 2 DIAGNOSE FLD_INT DGR Intensity Plot Intensity counts 14000 12000 10000 8000 6000 4000 2000 ot T T T T T T T T T T T T T T T T 200 400 600 800 1000 Wavelength nm Figure 55 Lamp Intensity Test Report 1200 Series FLD User M anual 123 8 Maintenance NOTE 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
34. aintenance and Repair 7 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 WARNING Do not let liquid drip into the detector It could cause shock hazard and it could damage the detector 1200 Series FLD User M anual 109 7 Maintenance and Repair Using the ESD Strap 110 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 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 Figure51 Using the ESD Strap 1200 Series FLD User Manual Agilent 1200 Series Fluorescence Detector User Manual 8 Maintenance Overview of Maintenance 112 Exchanging a Flow Cell 113 How to use the Cuvette 116 Flow Cell Flushing 117 Correcting Leaks 118 Replacing Leak Handling System Parts 119 Replacing the Interface Board 120 Replacing the Detector s Firmware 121 Tests amp Calibrations 122 Lamp Intensity Test 123 Wavelength Verification and Calibration 125 Wavelength Calibration Procedure 127 This ch
35. ange 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 maximum of 250 nm The excitation grating is blazed for highest efficiency at 300 nm 1200 Series FLD User Manual 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 89 shows a maximum between 220 nm and 240 nm LC Z 1846A EX of AAYHSIGL D ALA LC Z 1046A EM of AR ZHSIGL D Scaled 20 508 Excitation 400 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 s
36. apter describes the maintenance of the detector and the required tests Apg Agilent Technologies 111 8 Maintenance Overview of Maintenance On the following pages repairs are described that can be carried out without opening the main cover Table 20 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 Calibration on page 125 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 112 1200 Series FLD User Manual Maintenance 8 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 116 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 Prep
37. arations 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 User M anual 113 8 Maintenance 2 Disconnect the capillaries from the flow cell 3 Unscrew the thumb screws and pull the flow cell out of the compartment W4 i TTT S AN 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 4 Insert the flow cell and tighten the thumb screws Reconnect the capillaries to the flow cell DO NOT insta the inlet capillary to the outlet connection of the flow cell This will result in poor performance or damage S FTTH a all gt C N L7 5 114 1200 Series FLD User Manual Maintenance 8 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
38. cription of 27 EMF early maintenance feedback 30 emission condenser 15 emission grating 15 emission monochromator 18 emission slit 15 environment 35 ESD electrostatic discharge strap 110 excitation condenser 15 excitation grating 15 excitation monochromator 17 excitation slit 15 F eatures safety and maintenance 39 flash frequency 24 flow cell 15 19 fluorescence and phosphorescence 12 fluorescence detection 22 fluorescence spectral libraries for peak confirmation 76 flushing of flow cell 117 front view of module 48 G GLP features 39 glycogen 127 H How the Detector Operates 11 how to use the cuvette 116 humidity 36 information on lithium batteries 141 installation accessory kit 43 bench space 35 delivery checklist 42 environment 35 flow connections 51 of flow cell and capillaries 51 of the detector 48 physical specifications 36 power considerations 34 power cords 34 site requirements 34 unpacking 42 instrument layout 29 internet 147 Introduction to the Detector 10 L lamp intensity history 124 leaks correcting 118 line voltage and frequency 36 luminescence 11 M Maintenance 105 111 method development 63 1 check the LC system for impurities 64 2 optimize limits of detection and selectivity 65 3 set up routine methods 74 fluorescence spectral libraries for peak confirmation 76 multi wavelength detection 75 take a fluoresc
39. e 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 User M anual 29 1 Introduction to the Fluorescence Detector Early Maintenance Feedback EMF 30 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 feedback when the user selectable limits have been exceeded The visual feedback in the user interface provides an indication that maintenance procedures should be scheduled EMF 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 mu
40. e which adsorbs the peroxides e Solutions of organic acids acetic acid formic acid and so on in organic 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 146 1200 Series FLD User Manual 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 User M anual 147 A Appendix 148 1200 Series FLD User Manual Index A accessory kit 43 accessory kit parts 135 accuracy of wavelength 37 Agilent on internet 147 algea 117 algea information 145 battery safety information 141 bench space 35 C cable connecting APG remote 47 connecting CAN 47 connecting GPIB 47 connecting LAN 47 connecting the ChemStation 47 connecting the power 47 calibration sample 127 cut off filter 15 cutoff filter other type 134 cuvette 10 how to use 116 D degradation UV 16 124 delivery checklist 42 dimensions and weight 36 dispersion of light 18 early maintenance feedback EM F 30 electrical connections 1200 Series FLD User M anual des
41. ence scan 66 mirror 15 monochromator EM 15 18 EX 15 17 multi wavelength detection 75 149 Index 0 off line measurements 10 operation of the detector 11 operation temperature 36 Optical unit overview 15 optimization example 78 P Parts 131 parts identification 131 accessory kit 135 main assemblies 132 overview 132 performance specifications 37 phosphorescence detection 23 photoluminescence 11 photo multiplier tube figure 20 location of PMT 15 PMT 19 physical specifications 36 humidity 36 line voltage and frequency 36 operation temperature 36 power consumption 36 safety standards 36 weight and dimensions 36 PMT gain 19 gain steps 25 gain test 57 photo multiplier tube 19 range 25 power considerations 34 power consumption 36 power cords 34 R Raman 14 rear view of module 28 49 recalibration of wavelength 100 reference diode 21 reference system 21 repair flushing of flow cell 117 150 repairs cleaning the instrument 109 correction leaks 118 definition of 106 exchanging a flow cell 113 introduction 106 of the detector 105 111 replacing leak handling system 119 using the ESD strap 110 warnings and cautions 106 replacing interface board BCD LAN 120 responsetime 26 S safety information on lithium batteries 141 standards 36 site requirements 34 spare parts cutoff filters 134 specifications analog outputs 38 communications
42. ention to an operat ing procedure practice or the like 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 User Manual In This Guide 1200 Series FLD User 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 Maintenance and Repair This chapter provides general information on maintenance a
43. es FLD User Manual Agilent 1200 Series Fluorescence Detector User Manual Pa ee 9 e Parts and Materials for Maintenance J Overview of Maintenance Parts 132 Cuvette Kit 133 Spare Parts 134 Accessory Kit 135 This chapter provides information on parts for maintenance ott Agilent Technologies 131 9 Parts and Materials for Maintenance Overview of Maintenance Parts Table 21 Maintenance Parts Item Description Part Number 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 133 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 135 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 G1323 67001 64208 67001 G1321 60005 G1321 60007 9301 0407 93
44. fit the needle to filter ny GO UP WN sample filter Figure 58 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 128 1200 Series FLD User Manual Maintenance 8 Wavelength Calibration 1 From the user interface start the FLD Wavelength Calibration 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 in the Service Manual 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 User M anual 129 8 Maintenance 130 1200 Seri
45. formation 141 Radio Interference 142 SoundEmission 143 UV Radiation UV lamps only 144 Solvent Information 145 Agilent Technologies on Internet 147 ee Agilent Technologies 137 A Appendix General Safety Information 138 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
46. g 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 J i HT H wT Y PO HOT OROI Ss lt QD SS 1200 Series FLD User M anual 53 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 54 1200 Series FLD User Manual Agilent 1200 Series Fluorescence Detector User Manual 6 ee 4 e First Steps with the Detector e Before You Start 56 Optimization Overview 57 Getting Started and Checkout 59 Method Development 63 Example Optimization for Multiple Compounds 78 This chapter guides you how to start the wor
47. gh 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 compared 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 76 Table 12 Conditions for Figure 35 on page 76 Column Vydac 2 1 x 250 mm PNA 5 um Mobile 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 C Injection volume 2 ul FLD settings PMT 12 response time 4s
48. h 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 CGnulti 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 User M anual 57 4 58 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 lifetime 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
49. he 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 User M anual 13 1 Introduction to the Fluorescence Detector Raman Effect 14 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 Raleigh Scatter Raman Scatter same wavelength length as incident light ar ule wave Scattered Light Incident Light Sample Figure5 Raman The energy difference between the incident light E and the Raman scattered light E 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 Ky E Es 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 spectru
50. hock hazard or damage of your instrumentation can result if the devices are connected to a line voltage higher than specified Make sure to have easy access to the power cable of the instrument in order to disconnect the instrument from line 34 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 User Manual 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 other 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 36 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 posi
51. ing 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 1 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 User Manual 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 a VLCC LLL LL LLL ukxx kcknteccrrrceit ce KKK INN 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 lens 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 18 The selected wavelength of light is
52. ity Maximum Ambient operating temperature Ambient non operating temperature Humidity Operating altitude Non operating altitude Safety standards IEC CSA UL EN 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 Non condensing For storing the detector 36 1200 Series FLD User Manual Site Requirements and Specifications 2 Performance Specifications Table3 Performance Specifications Agilent 1200 Series Fluorescence Detector Type Specification Comments Detection type Multi signal fluorescence detector with rapid on line scanning capabilities and spectral data analysis Performance 10 fg Anthracene see note below this table Specifications Light source Pulse frequency Excitation Monochromator Emission Monochromator Reference System 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 H20 gt 300 with Ex 350 nm Em 397 nm dark value 450 nm standard flow cell time constant 4 seconds 8 seconds responsetime see Service M anual for details see Service M anual for details Xenon Flash Lamp normal mode 20 W economy mode 5 W 296 Hz for single signal mode 74 Hz for spec
53. k with the detector a Agilent Technologies 55 4 First Steps with the Detector Before You Start 56 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 User Manual 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 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 hig
54. king 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 User M anual 51 3 Installing the Detector 1 Press the release buttons and remove the front cover to gain access to the flow cell area 2 Locate the flow cell OD 3 Assemble the column detector capillary from the accessory kit One side is already factory assembled Pre assembled 4 Assemble the waste tubing from the accessory kit 52 1200 Series FLD User 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 by removing the column and the last detect and measurin
55. known 2 3 diaminophenazine 0 8 0 6 265 540 nm 0 4 430 540 nm 0 2 0 Standard 0 2 4 6 8 10 12 Time min Figure 34 Qualitive analysis of MBC 2 benzimidazole carbamic acid methylester and impurities 1200 Series FLD User M anual 73 4 First Steps with the Detector Table 11 Conditions for Figure 33 and Figure 34 on page 73 Column Mobile phase Gradient Flow rate Column temperature Injection volume FLD settings 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 5nm Ex 265 nm and 430 nm Em 540 nm Step 3 Set up routine methods 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 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 74 1200 Series FLD User Manual First Steps with the Detector 4 Multi wavelength detection Time programmed wavelength switching traditionally is used to achieve low limits of detection and hi
56. l sub levels of its ground state emitting light see Figure 3 The characteristic maxima of absorption for a substance is its Agy and for emission its Apy absorption emission A m1 radiationless transition t ENANA 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 User Manual 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 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 T
57. lar purpose Agilent shall not be liable for errors or for incidental 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 Ifa 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 att
58. le7 Steps for thorough method development Step 1 Step 2 Step 3 Check system Optimize limits of detection Set up routine methods and selectivity Fluorescence scan Signal mode Spectral mode multi wavelength detection Find impurities for example 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 Emspectrafor 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 User M anual 63 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 64 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 scan 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
59. ltiples 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 31 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 Modes 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 User M anual Number of flashes Figure 18 Introduction to the Fluorescence Detector Number of flashes to 75 of intitial radiometric light output 0 Input energy per flash Lamp life 1200 Series FLD User M anual 1 31 1 32 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
60. m 1200 Series FLD User Manual Introduction to the Fluorescence Detector 1 Optical Unit All the elements of the optical system shown in Figure 6 on page 15 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 board 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 my REF Diode Flow Ce Diffuser Figure6 Optical Unit 1200 Series FLD User M anual 15 1 Introduction to the Fluorescence Detector The radiation source is a xenon flash lamp The 3 s 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
61. n overview about the troubleshooting and diagnostic features and the different user interfaces Apg Agilent Technologies 99 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 101 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 in the Service Manual 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 in the Service Manual Test Functions A series of test functions are available for troubleshooting and operational verification after exchanging internal components see Test Functions in the Service Manual 100 1200 Series FLD User Manual Troubleshooting and Test Functions 6 Status Indicators Two status indicator
62. nd repair of the detector Maintenance This chapter describes the maintenance of the detector and the required tests Parts and Materials for Maintenance This chapter provides information on parts for maintenance A Appendix This chapter provides safetey and other general information 1200 Series FLD User Manual Contents 1 Introduction to the Fluorescence Detector Introduction to the Detector 10 How the Detector Operates 11 Raman Effect 14 Optical Unit 15 Reference System 21 Analytical Information From Primary Data 22 Fluorescence Detection 22 Phosphorescence Detection 23 Processing of Raw Data 24 Electrical Connections 27 Instrument Layout 29 Early Maintenance Feedback EMF 30 EMF Counters 30 Using the EMF Counters 32 2 Site Requirements and Specifications Site Requirements 34 Physical Specifications 36 Performance Specifications 37 3 installing the Detector Unpacking the Detector 42 Detector Accessory Kit Contents 43 1200 Series FLD User M anual Optimizing the Stack Configuration 45 Installing the Detector 48 Flow Connections to the Detector 51 4 First Steps with the Detector Before You Start 56 Optimization Overview 57 Getting Started and Checkout 59 Starting Your Detector 59 Setting the Chromatographic Conditions 60 Observe the maxima via the isoabsorbance plot 62 Method Development 63 Step 1 Check the LC system for impurities 64 Step 2 Optimize limits of detection and selectivi
63. ng Stray Light on page 96 440 ae Raleigh scattering 360 340 300 280 260 200 220 240 260 280 300 320 En Raman scattering of water 340 360 380 400 420 440 Note This white area is normally dark blue Pa 2nd order of cutoff 460 480 500 520 540 560 580 Figure 43 Fluorescence Scan of Water 84 1200 Series FLD User Manual This chapter provides information on how to optimize the detector Agilent 1200 Series Fluorescence Detector User Manual 5 How to optimize the Detector Optimization Overview 86 Design Features Help Optimization 87 Finding the Best Wavelengths 88 Finding the Best Signal Amplification 90 Changing the Xenon Flash Lamp Frequency 92 Selecting the Best Response Time 94 Reducing Stray Light 96 ot Agilent Technologies 85 5 How to optimize the Detector Optimization Overview Refer to Optimization Overview on page 57 for details 86 1200 Series FLD User Manual 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 gi
64. ngths 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 70 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 phenanthrene 8 2 min 420 nm for anthracene to benzo g h 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 E
65. nnection to the LAN interface board in the detector If an Agilent 1200 DAD M W D 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 WAP OW yO Analog signal 3 APG remote UUUPULULY J i b AN 1 J lt j a J CAN GPIB Power Configuration switch Figure24 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 green light is OFF 1200 Series FLD User M anual 49 3 Installing the Detector WARNING To disconnect the detector from line unplug the power cord The power 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 50 1200 Series FLD User Manual 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 43 Two wrenches 1 4 5 16 inch for capillary connections WARNING When wor
66. or 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 sends 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 at 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 97 1200 Series FLD User Manual How to optimize the Detector 5 Exitation 300nm Second order light 600nm Stray light Fluorescence no filter filter 280 nm a E S T A Wavelength nm Figure 49 Reducing Stray Light 1200 Series FLD User M anual 97 5 How to optimize the Detector 98 1200 Series FLD User Manual Agilent 1200 Series Fluorescence Detector User Manual 6 Troubleshooting and Test Functions Overview of the Detector s Indicators and Test Functions 100 Status Indicators 101 User Interfaces 103 Agilent LC Diagnostic Software 104 This chapter gives a
67. osphorescence detection mode special setpoints under FLD parameter settings Flash Intensity Phosph 100 osphorescence E Measurement Time usec Figure 13 Measurement of Phosphorescence 1200 Series FLD User M anual 23 1 24 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 74 Hz flash fluorescence vA SS mal i phosphorescence 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 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 User Manual Introduction to the Fluorescence Detector 1 quantification because of insufficient resolution Check your proposed PMTGAIN If
68. outputs LAN Communication Interface board G1369A or G1369 60001 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 54 Location of the Interface Board 120 1200 Series FLD User Manual Maintenance 8 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 Module 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 00 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 User M anual 121 8 Maintenance Tests amp Calibrations The following tests are required
69. 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 User Manual Agilent 1200 Series Fluorescence Detector User Manual 2 Site Requirements and Specifications Site Requirements 34 Physical Specifications 36 Performance Specifications 37 This chapter gives information on environmental requirements physical and performance specifications ee Agilent Technologies 33 2 Site Requirements and Specifications Site Requirements 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 36 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 S
70. r M aintenance A Appendix Index Overview of Maintenance Parts 132 Cuvette Kit 133 Spare Parts 134 Accessory Kit 135 General Safety Information 138 Lithium Batteries Information 141 Radio Interference 142 Sound Emission 143 UV Radiation UV lamps only 144 Solvent Information 145 Agilent Technologies on Intemet 147 1200 Series FLD User M anual Agilent 1200 Series Fluorescence Detector User Manual 1 Introduction to the Fluorescence Detector Introduction to the Detector 10 How the Detector Operates 11 Raman Effect 14 Optical Unit 15 Analytical Information From Primary Data 22 Electrical Connections 27 Instrument Layout 29 Early Maintenance Feedback EMF 30 This chapter gives an introduction to the detector instrument overview and internal connectors a Agilent Technologies 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 flash lamp for highest intensity and lowest detection limit multi wavelength mode for on line spectra spectra acquisition and simultaneous multi signal detection optional cuvette is available and can be used for off line measurements easy front access to flow cell for fast replacement and built in wavelength accuracy verification For specifications see Performance Specifications on page 37 Figurel The Agilent 1
71. red 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 User M anual 117 8 Maintenance Correcting Leaks When required If a leakage has occurred in the flow cell area or at 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
72. ree 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 User Manual 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 68 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 User M anual 67 straylight 1 order 350nm Ex 315 nm Ex 250 nm Ex M L JO x x 440 00 nmlE y 250 00 nm 2 187 LU X Cube o 02 D4 Of og 1 12 14 15 13 2 2B 24 26 28 3 32 Jt 35 38 4 PrE KAES ere E E a E S tore tii ith fre S er Sere rer ere ee i vhs Ox Oy z Expand z Dita view Projection Print cose Ex axis Em axis Figure 30 Characterization of a pure compound from a fluorescence scan First Steps with the Detector 4 Procedure II 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 wavele
73. 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 User M anual 19 1 20 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 15 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 PMT can improve performance For additional PMT types refer to Spare Parts on page 134 1200 Series FLD User Manual Introduction to the Fluorescence Detector 1 Reference System A reference diode located behind the flow cell measures the excitation EX light transmitted b
74. ries FLD User Manual Parts and Materials for Maintenance 9 Accessory Kit This kit contains some accessories and tools needed for the installation and repair calibration of the detector Table 24 Accessory Kit Parts Item Description Part Number Accessory kit G1321 68705 includes Corrugated tubing 120 mm lg re order 5 m 5062 2463 1 Teflon Tubing flexible i d 0 8 mm flow cell to waste 5062 2462 re order 5 m 2 Fitting male PEEK Qty 2 0100 1516 3 Capillary column detector 380 mm lg 0 17 i d G1315 87311 includes items 4 5 and 6 not assembled 4 Ferrule front SST qty 1 0100 0043 5 Ferrule back SST gty 1 0100 0044 6 Fitting SST qty 1 79814 22406 Screwdriver hexagonal 4 mm 100 mm long 5965 0027 Screwdriver hexagonal 2 5 mm 100 mm long 5965 0028 Needle Syringe 9301 0407 Glass Syringe 9301 1446 Calibration Sample Glycogen 5063 6597 Sample filter diameter 3 mm pore size 0 45 um QYT 5 5061 3367 pack of 100 Hex key set 1 5mm 8710 0641 Wrench open end 1 4 5 16 inch 8710 0510 1200 Series FLD User M anual 135 9 Parts and Materials for Maintenance AR me Figure 59 Waste Tubing Parts O Co This end is OO pre installed Figure 60 Inlet Capillary Column Detector Parts 136 1200 Series FLD User Manual This chapter provides safetey and other general information Agilent 1200 Series Fluorescence Detector User Manual A Appendix General Safety Information 138 Lithium Batteries In
75. rocedure 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 I 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 previously obtained with a standard fluorescence spectrophotometer independent of the LC fluorescence detector Figure 30 on page 68 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 th
76. s are located on the front of the detector The lower left indicates the power supply status the upper right indicates the detector status Status indicator _ green yellow red r 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 User M anual 101 6 102 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 error condition always interrupts the analysis 1200 Series
77. ser Manual Appendix A Sound Emission Manufacturer s Declaration This statement is provided to comply 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 User M anual 143 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 26 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 27 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 144 1200 Series FLD User Manual 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
78. sion 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 Norm Excitation Emission 250 300 350 400 450 500 550 600 Wavelength nm Figure 29 Excitation and emission spectra of quinidine 1200 Series FLD User M anual 65 4 66 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 64 for each compound Since this is a tedious 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 P
79. that could result in serious personal injury Whenever it is likely that the protection has been impaired 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 User Manual 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
80. timal excitation wavelength See Figure 39 FLD Signals System 2 x rm Signal Excitation A Emission 260 nm 1330 nm Zero Order C Zero Order Time r Multiple Wavelengths and Spectra Stoptime F 21 min Cor Cuties Multi Em Use additional Excitation Posttime Off 4 min om ao e 1200 Series FLD User M anual Kil Insert Append Cut Copy Paste Figure 39 Detector Settings for Excitation Scan C F 250 nm D 7 290 nm Acquire Excitation Spectra All X Range 230 to 400 nm Step 5 nm Threshold 1 00 LU Time Spectrum 945 ms r Peakwidth Responsetime cl gt l f gt 0 2 min 4 s standart x PMT Gain 10 Test 81 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_PADT FLD_PAD2 D 204 104 T T T T T T o 2 4 6 8 10 12 min Figure 40 Chromatogram excitation scan at reference wavelength 260 330 nm 8 Use the isoabsorbance plot and evaluate the optimal excitation wavelengths Cin this example just in the time range of 13 minutes Figure 41 Isoabsorbance Plot Excitation 82 1200 Series FLD User Manual First Steps with the Detector 4 The table below shows the complete information about emission from Figure 38 on page 80 and excitation maxima Table 16 Peak Time Emission Wavelength Excitation Wa
81. tion Environment Your detector will work within the specifications at ambient temperatures and relative humidity described in Table 2 on page 36 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 37 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 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 User M anual 35 2 Site Requirements and Specifications Physical Specifications Table 2 Physical Specifications Type Weight Dimensions width x depth x height Specification 11 5 kg 26 Ibs 345 x 435 x 140 mm 13 5 x 17 x 5 5 inches Comments Line voltage Line frequency Power consumption 100 240 VAC 10 50 or 60Hz 5 180 VA 70 W 239 BTU Wide ranging capabil
82. tral 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 User M anual 37 2 Site Requirements and Specifications Table 3 Type Performance Specifications Agilent 1200 Series Fluorescence Detector Specification Comments Timetable programing Spectrum acquisition Wavelength characteristic Flow cells Control and data evaluation Analog outputs Communications 38 up to 4 signal wavelengths response time PMT Gain baseline behavior append free zero spectral parameters Excitation or Emission 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 10 luminescence units two outputs Controller area network CAN GPIB RS 2
83. ts 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 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 ranging 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 WARNING Never use cables other than the ones supplied by Agilent Technologies to ensure proper functionality and compliance with safety or EMC regulations 1200 Series FLD User M anual 27 1 Introduction to the Fluorescence Detector Interface board Security lever ia Analog signal RS 232C
84. ty 65 Step 3 Set up routine methods 74 Example Optimization for Multiple Compounds 78 5 How to optimize the Detector Optimization Overview 86 Design Features Help Optimization 87 Check Performance Before You Start 87 Finding the Best Wavelengths 88 A Real Example 89 Finding the Best Signal Amplification 90 Changing the Xenon Flash Lamp Frequency 92 Lamp life savings 93 Selecting the Best Response Time 94 Reducing Stray Light 96 1200 Series FLD User M anual 6 Troubleshooting and Test Functions Overview of the Detector s Indicators and Test Functions 100 Status Indicators 101 Power Supply Indicator 101 Detector Status Indicator 102 User Interfaces 103 Agilent LC Diagnostic Software 104 7 Maintenance and Repair 8 Maintenance 1200 Series FLD User M anual Introduction into Repairing the Fluorescence Detector 106 Warnings and Cautions 107 Cleaning the Detector 109 Using the ESD Strap 110 Overview of Maintenance 112 Exchanging a Flow Cell 113 How to use the Cuvette 116 Flow Cell Flushing 117 Correcting Leaks 118 Replacing Leak Handling System Parts 119 Replacing the Interface Board 120 Replacing the Detector s Firmware 121 Tests amp Calibrations 122 Lamp Intensity Test 123 Wavelength Verification and Calibration 125 Wavelength Calibration Procedure 127 Preparation of the Glycogen Calibration Sample 127 Preparation of the Flow Cell 128 Wavelength Calibration 129 9 Parts and Materials fo
85. ule 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 4 mm 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 16inch 8710 0510 1 1200 Series FLD User M anual 43 3 Installing the Detector Fitting male PEEK Tubing Figure 19 Waste Tubing Parts ee y Ferrule front O x gt a l Co This side is Ferrule back preinstalled Fitting male SST x Capillary Figure 20 Inlet Capillary Column Detector Parts 44 1200 Series FLD User Manual Installing the Detector 3 Optimizing 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 User M anual 45 3 Installing the Detector Solvent cabinet Vacuum degasser Local User Interface Pump Autosampler Column compartment Detector Figure 21 Recommended Stack Configuration Front View 46 1200 Series FLD User Manual Remote cable CAN Bus cable to
86. unauthorized modification to the instrument Capacitors inside the instrument may still be charged even 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 25 shows safety symbols used on the instrument and in the manuals Table 25 Safety Symbols Symbol Description The apparatus is marked with this symbol when the user should refer to the A instruction manual in order to protect the apparatus against damage p Indicates dangerous voltages 1200 Series FLD User M anual 139 A Appendix Table 25 Safety Symbols continued Symbol Description Indicates a protected ground terminal A Eye damage may result from directly viewing the light produced by the deuterium n d 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 140 1200 Series FLD User M anual
87. ve 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 User M anual 87 5 How to optimize the Detector Finding the Best Wavelengths 88 The most important parameters 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 r
88. velength 1 5 3 min 330 nm 220 280 nm 2 7 3 min 330 nm 225 285 nm 3 7 7 min 310 nm 265 nm 4 8 5 min 360 nm 245 nm 5 10 7 min 445 nm 280 nm 6 11 3 min 385 nm 270 330 nm 1200 Series FLD User M anual Evaluating The System Background The example below uses water 1 Pump solvent through your system 2 Set the fluorescence 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 83 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 gt Phasphorescence Detection Mode r Fluorescence Scan Range Of On From To Step Delay foo is Excitation 220 a00 5 nm Gate 000 ps Emission 200 500 fs nm Time Scan 143 s x Baseline Behavior Append Free C Zero Signal Polarity Positive Negative IV Fit Spectral Range m Lamp V Only On During Run J Economy Mode now 74 Hz High Lamp Current J Enable analysis when lamp is off Lamp Energy Reference On Off 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 84 A low background will improve the signal to noise see also Reduci
89. xcitation at 260 nm is most appropriate for all PNAs 1200 Series FLD User M anual 69 4 First Steps with the Detector This shows the isofluorescence plot of emission spectra for 15 PNAs 5 pg ml witha fixed excitation wavelengths 260 nm 70 Table9 Conditions for Figure 31 and Figure 32 on page 71 Column Vydac 2 1 x 200 mm PNA 5 um Mobile 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 5nm 1 Naphthalene 8 Benzlajanthracene 2 Acenaphthene 9 Chrysene 3 Fluorene 10 Benzo b fluoranthene 4 Phenanthrene 11 Benzo k fluoranthene 5 Anthracene 12 Benzla pyrene 6 Fluoranthene 13 Dibenzolahjanthracene 7 Pyrene 14 Benzolg h i perylene ii LU 60 15 Indeno 1 2 3 cd pyrene 50 40 30 20 10 300 nm Figure 31 LS ON 1253 ES 17 5 20 22 5 Time min Ein spectra fixed Ex Optimization of the time program for the emission wavelength 1200 Series FLD User Manual First Steps with the Detector 4 1 Naphthalene 8 Benzlajanthracene 2 Acenaphthene 9 Chrysene 3 Fluorene 10 Benzo b fluoranthene 4 Phenanthrene 11 Benzo k fluoranthene 5 Anthracene 12 Benz a pyrene 6 Fluoranthene 13 Dibenzo ah anthracene 7 Pyrene 14 Benzolg h i perylene 15 Indeno 1 2 3 cd pyrene 11 0 25 5 7 5 10 125 15 175 20 22 5 Time min 400 nm TET Excitation
90. y 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 15 This diffuser is made of quartz reduces light and allows integral measurement of the light 1200 Series FLD User M anual 21 1 Introduction to the Fluorescence Detector Analytical Information From Primary Data 22 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 100 Track amp Hold Ignite ane 0 1 2 3 Time usec Figure 12 Measurement of Fluorescence 1200 Series FLD User M anual Introduction to the Fluorescence Detector 1 Phosphorescence Detection An appropriate parameter set will be specified as soon as you chose the ph
91. ystematic wavelength maximum shift depending on the absorbance spectrum of the compound under evaluation 1200 Series FLD User M anual 89 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 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 Ex 248 Em 317 FLD_PMTS PMT_0001 D FLD1 A Ex 246 Em 317 FLD_PMT3 PMT_0002 D FLD1 A Ex 248 Em 317 FLD_PMT3 PMT_0003 D FLD1 A Ex 248 Em 317 FLD_PMT3 PMT_0004 D FLD1 A Ex 248 Em 317 FLD_PMT3 PMT_0005 D FLD1 A Ex 248 Em 317 FLD_PMT3 PMT_0008 D Figure 45 90 Finding Best PMTGAIN for Biphenyl PMT 11 10 A O0 COO 1200 Series FLD User 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

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