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Agilent 1260 Infinity Fluorescence Detector

1. KT x r Signal Time m Multiple Wavelengths and Spectra Excitation Emission A Stoptime anme 4 nin C Off C MutiEx Multi Em c 3505 nm ce aio nm Use additional Emission Posttime Off i Zero Order C Zero Order z 3 Jai B FAO rm C17 410 nm D 10 nm r Timetable E Line Tiao Ex EmA EnB ERC EMD Em Spaan Fi lo All w o signals Range 377 to 417 nm Step fi nm r Signal Time r Multiple Wavelenaths and Spectra Excitation Emission Stoptime ar tm 4 min C O Muli Ex JO Muli Em e 50 nm 397 nm Use addtional Excitation osttime Of s o ij C ZecGrde C Zeto Order R Jmn B F 250 rm Insert Append CF 250 nm DT 250 nm Table Graphic Timetable Cancel Line Time Em Ex A Ex 8 Ex C Ex D Ex Spectra From oe Range 340 to 360 nm Step fi nm Threshold 010 Ll Time Spectrum 283 ms FE Peakgiah esponsetne e al gt p 0 2 min 4 s standar iser Append E Copy Paste Tobe Grohic PMT Gain M0 Test pen TE shat lt lt Special Setpoint Figure 69 Settings for EM EX Scan Agilent 1260 FLD User Manual 175 176 Test Functions 2 Load the method WLEXTEST The FLD will change into the multi emission mode and scan in the range of the expected maximum of 397 nm 20 nm 3 Start the pump and flush the cell with water for a few minutes to assure a clean flow cell Flow rate should be 0 5 to 1 ml min and the baseline s
2. KA 7 O cy z D 2 over cies eccccecce data points p 1 fin High S N rato I AFA if IY A ay Vv RESPONSETIME Signal to Noise Ratio Agilent 1260 FLD User Manual Introduction to the Fluorescence Detector System Overview Leak and Waste Handling The 1200 Infinity Series has been designed for safe leak and waste handling It is important that all security concepts are understood and instructions are carefully followed Agilent 1260 FLD User Manual 1 29 1 30 Introduction to the Fluorescence Detector System Overview Figure 17 Leak and waste handling concept overview typical stack configuration as an example Agilent 1260 FLD User Manual Introduction to the Fluorescence Detector 1 The solvent cabinet 1 is designed to store a maximum volume of 6 L solvent The maximum volume for an individual bottle stored in the solvent cabinet should not exceed 2 5 L For details see the usage guideline for the Agilent 1200 Infinity Series Solvent Cabinets a printed copy of the guideline has been shipped with the solvent cabinet electronic copies are available on the Internet The leak pan 2 individually designed in each module guides solvents to the front of the module The concept covers also leakages on internal parts e g the detector s flow cell The leak sensor in the leak pan stops the running system as soon as the leak dete
3. Notices Agilent Technologies Inc 2010 2012 2013 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 90014 Rev B Edition 11 2013 Printed in Germany Agilent Technologies Hewlett Packard Strasse 8 76337 Waldbronn This product may be used as a com ponent of an in vitro diagnostic sys tem if the system is registered with the appropriate authorities and com plies with the relevant regulations Otherwise it is intended only for gen eral laboratory use Warranty The material contained in this docu ment is provided as is and is sub ject to being changed without notice in future editions Further to the max imum extent permitted by applicable law Agilent disclaims all warranties either express or implied with regard to this manual and any information contained herein including but not limited to the implied warranties of merchantability and fitness for a par ticular purpose Agilent shall not be liable for errors or for incidental or consequential damages in connection with the furnishing use or perfor mance of this document or of any information contained herein Should Agilent and the user have a separate written agr
4. p n 5061 3378 Pin 35900 Pin Agilent Signal Name Active A D module TTL 1 White 1 White Digital ground 2 Brown 2 Brown Prepare run Low 3 Gray 3 Gray Start Low 4 Blue 4 Blue Shut down Low 5 Pink 5 Pink Not connected 6 Yellow 6 Yellow Power on High 7 Red 7 Red Ready High 8 Green 8 Green Stop Low 9 Black 9 Black Start request Low Agilent 1260 FLD User Manual 213 11 Identifying Cables Agilent Module to General Purpose p n 01046 60201 Wire Color Pin Agilent Signal Name Active module TTL 7 White 1 Digital ground a Ol ou Brown 2 Prepare run Low 00 pes DE KEY Gray 3 Start Low oo o0 S iE Blue 4 Shut down Low og Pink 5 Not oa connected 5 5 15 Q Yellow 6 Power on High Red 7 Ready High Green 8 Stop Low Black 9 Start request Low 214 Agilent 1260 FLD User Manual BCD Cables Identifying Cables 11 One end of these cables provides a 15 pin BCD connector to be connected to the Agilent modules The other end depends on the instrument to be connected to Agilent Module to General Purpose p n G1351 81600 Wire Color Pin Agilent Signal Name BCD Digit module Green 1 BCD 5 20 pea Violet 2 BCD 7 80 AA Blue 3 BCD 6 40 a ee Yellow 4 BCD 4 10 Black 5 BCD 0 1 Ras Orange 6 BCD 3 8 Red 7 BCD 2 4 Brown 8 BCD 1 2 Gray 9 Digital ground Gray Gray pink 10 BCD 11 800 Red blue 11 BCD 10 400
5. EM position scan high resolution Description This procedure performs a Wavelength Verification and Recalibration Result Name Value Ex 1 300 nm Em 3 400 nm e Wavelength Calibration E x Do you want to calibrate the detector using the wavelength verification results Figure 63 Wavelength Calibration Agilent Lab Advisor Agilent 1260 FLD User Manual 169 Test Functions Table 29 Wavelength Calibration Steps Step Description Duration 1 Preparation max 30s Excitation rotation scan full circle 60s 3 Excitation rotation scan high resolution 44s 4 Excitation position scan low resolution 55 s variable 5 Excitation position scan high resolution 260 s variable 6 n Emission rotation scan full circle of scans depends on the required PMT gain 1 61 s variable minute per scan 6 n Em rotation scan full circle instrument profile 9s 6 n Em rotation scan full circle instrument profile 9s 6 n Em rotation scan full circle instrument profile 9s 6 n Em rotation scan full circle instrument profile 9s 7 Emission rotation scan high resolution part 44s 8 Emission rotation scan high resolution part II 44s 9 Emission position scan low resolution 50 s variable 10 Emission position scan high resolution 250 s variable Variable times means that they could be a little bit longer When the lamp is off the calibration process will stop within the first two steps w
6. Mode Select sw1 sw2 SW3 sw4 SW5 SW6 SW7 sws TEST BOOT 1 1 0 0 1 0 0 0 Forced Cold Start A forced cold start can be used to bring the module into a defined mode with default parameter settings CAUTION Loss of data Forced cold start erases all methods and data stored in the non volatile memory Exceptions are calibration settings diagnosis and repair log books which will not be erased gt Save your methods and data before executing a forced cold start If you use the following switch settings and power the instrument up again a forced cold start has been completed Table 43 Forced Cold Start Settings without on board LAN Mode Select sw1 sw2 SW3 sw4 SW5 SW6 SW7 sws TEST BOOT 1 1 0 0 0 0 0 1 242 Agilent 1260 FLD User Manual Hardware Information 12 Early Maintenance Feedback 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 module 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 EMF counters increment with use and can be assigned a m
7. 1 These cards may be no longer orderable Minimum firmware of these Hewlett Packard JetDirect cards is A 05 05 Recommended LAN Cables p n Description 5023 0203 Cross over network cable shielded 3 m for point to point connection 5023 0202 Twisted pair network cable shielded 7 m for point to point connection Agilent 1260 FLD User Manual Hardware Information 12 Electrical Connections The CAN bus is a serial bus with high speed data transfer The two connectors for the CAN bus are used for internal module data transfer and synchronization e Two independent analog outputs provide signals for integrators or data handling The interface board slot is used for external contacts and BCD bottle number output or LAN connections e The REMOTE connector may be used in combination with other analytical instruments from Agilent Technologies if you want to use features such as start stop common shut down prepare and so on e With the appropriate software the RS 232C connector may be used to control the module from a computer through a RS 232C connection This connector is activated and can be configured with the configuration switch e The power input socket accepts a line voltage of 100 240 VAC 10 with a line frequency of 50 or 60 Hz Maximum power consumption varies by module There is no voltage selector on your module because the power supply has wide ranging capability There are no externally accessible fu
8. 172 Wavelength Accuracy Test Description The test uses the Raman band of water to determine the excitation and G1321B DEABC00159 1260 FLD ii i Passed 2 21 2012 2 26 39 PM 2 21 2012 2 29 38 PM n M Test Procedure Name Value Sree WL Accuracy EX Deviation 070mm Y 2 Wavelength Accuracy Test Step 1 EM san Y 3 Wavelength Accuracy Test Step 2 EX scan WL Accuracy EM Deviation W 4 Evaluate Data Figure 65 Wavelength Accuracy Test with Lab Advisor If the test fails observe the maxima of the EX or EM side under the Signals tab Intensity LU Interpolated Excitation Peak 350 nm 350 Wavelength nm Figure 66 Example of good EX maxima If the plots do not have a maximum around EX 397 nm and EX 350 nm 3 nm the test fails see figure below Refer to Interpretation of the Results on page 173 Agilent 1260 FLD User Manual Test Functions 8 Wavelength Accuracy Test Interpolated Excitation Peak 350 nm 350 Wavelength nm Interpolated Emission Peak 397 nm Intensity LU Figure 67 Example of bad EX EM maxima no maximum found Interpretation of the Results If the test fails check for Y correctly positioned flow cell Y clean flow cell flush with isopropanol and clean bi distilled water V no air bubble s check via fluorescence scan or visual check of cell cuvette V solvent inlet filter may create air bubbles in flow cell V check optical path
9. VVV is the revision number for example 102 is revision 1 02 XXX is the build number of the firmware For instructions on firmware updates refer to section Replacing Firmware in chapter Maintenance or use the documentation provided with the Firmware Update Tools Update of main system can be done in the resident system only Update of the resident system can be done in the main system only Main and resident firmware must be from the same set Main FW update Resident System Main System Resident FW Update Figure 79 Firmware Update Mechanism Agilent 1260 FLD User Manual 223 12 Hardware Information 224 Some modules are limited in downgrading due to their main board version or their initial firmware revision For example a G1315C DAD SL cannot be downgraded below firmware revision B 01 02 or to a A xx xx Some modules can be re branded e g G1314C to G1314B to allow operation in specific control software environments In this case the feature set of the target type are use and the feature set of the original are lost After re branding e g from G1314B to G1314C the original feature set is available again All these specific informations are described in the documentation provided with the firmware update tools The firmware update tools firmware and documentation are available from the Agilent web http www chem agilent com _layouts agilent downloadFirmware aspx whid 69761 Agilent 1260 FLD User Manua
10. r Reference diode Diffuser Mirror Flow cell or cuvette re Excitation grating Flash tube Figure 56 Schematic of the Light Path Agilent 1260 FLD User Manual 159 8 Test Functions Lamp Intensity Test Lamp Intensity Test The intensity test scans an intensity spectrum via the reference diode 200 1200 nm in 1 nm steps and stores it in a diagnosis buffer The scan is displayed in a graphic window There is no further evaluation of the test Results of this test are stored as lamp history date code intensity General Signals Test Name Intensity Test Description The test scans the Intensity spectrum generated by the Xenon Module G1321A DE92991563 E Status Passed Start Time 5 19 2010 2 15 50 PM Stop Time 5719 2010 2 17 38 PM Intensity Counts 11422 10000 1 1 1 1 T 600 700 800 1000 1100 Wavelength nm Figure 57 Lamp Intensity Test Agilent Lab Advisor 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 160 Agilent 1260 FLD User Manual Lamp Intensity History Test Functions 8 Lamp Intensity Test Results of the lamp intensity test if the last one is older than on
11. Optimization for Multiple Compounds 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 NOTE 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 The wavelength range and step number defines the duration Using the maximum range the scan would take approximately 10 minutes FLD Special Setpoints System 2 Figure 46 FLD Special Settings Agilent 1260 FLD User Manual 107 4 Using the Fluorescence Detector 4 Define a data file name and take a fluorescence scan After the scan is completed the isoabsorbance scan results appear see Figure 47 on page 108 A low background will improve the signal to noise see also Reducing Stray Light on page 135 440 p Raleigh scattering Note This white area 360 is normally dark blue N Raman scattering of water 2nd order of cutoff 340 320 300 280 260 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 bay Figure 47 Fluorescence Scan of Water 108 Agilent 1260 FLD User Manual Using the Fluorescence Detector 4 How to collect spectra with modes SPECTRA ALL IN PEAK and APEX SPECTRA ONLY This section describes how to overcome a malfunction in the current imp
12. RS 232C LAN APG Remote ready start stop and shut down signals Extensive support for troubleshooting and maintenance is provided by the Instant Pilot Agilent Lab Advisor and the Chromatography Data System Safety related features are leak detection safe leak handling leak output signal for shutdown of pumping system and low voltages in major maintenance areas Early maintenance feedback EMF 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 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 recommended range see FLD Scaling Range and Operating Conditions Agilent 1260 FLD User Manual Site Requirements and Specifications 2 Table5 Performance Specifications Agilent 1260 Infinity Fluorescence Detector G1321C Type Specification Comments Detection type One signal wavelength excitation and Programmable single Performance specifications Light source Pulse frequency Maximum data rate Excitation monochromator Emission monochromator Reference system Timetable programing emission Single wavelength operation RAMAN H30 gt 500 noise reference me
13. Selecting the Best Response Time 133 Reducing Stray Light 135 ee Agilent Technologies 119 5 Optimizing the Detector Optimization Overview 120 Some features e g spectrum acquisition multi wavelength detection described in this chapter are not available on the G1321C 1260 Infinity Fluorescence Detector PMT Gain Test The PMT Gain test was provided in the classic Agilent ChemStation within the settings of the FLD It is not available in the Agilent OpenLAB CDS and G4208A Instant Pilot The PMT Gain test has been incorporated into in the Agilent Lab Advisor B 02 04 093 The PMT Gain Test can be found under Instrument Control gt Special Commands 1 Setting the right PMT value For most applications a setting of 10 is adequate see Finding the Best Signal Amplification on page 125 The FLD 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 th
14. The leak pan outlet of the upper module must be vertically positioned above the leak tray of the lower module see Figure 22 on page 58 2 Connect data and power cables to the modules see section Installing the Module below 3 Connect capillaries and tubes to the modules see section Flow Connections to the module below or the relevant system manual WARNING Toxic flammable and hazardous solvents samples and reagents gt Keep solvent path free from blockages gt Keep the flow path closed in case the pump in the system is equipped with a passive inlet valve solvent may leak out due to hydrostatic pressure even if your instrument is off gt Avoid loops gt Tubes must not sag gt Do not bend tubes gt Do not immerse tube end in waste liquid gt Do not intubate tubes in other tubes gt For correct tubing follow instructions on label attached to the module Agilent 1260 FLD User Manual 59 3 Installing the Module sa i tallatinn Infn Inst lle iON INnTori Warning Attention Warnung Attenzione Advertencia Disconnect power cord before moving D branchez le cordon secteur avant tout d placement Entfernen Sie das Netzkabel bevor Sie das Gerat bewegen Scollegare il cavo di alimentazione prima di spostare lo strumento Desconectar el aparato de la red antes de moverlo L 2J I Figure 23 Warning label illustration for correct waste tubing 60 Agilent 1260 FLD User Manua
15. 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 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 Agilent 1260 FLD User Manual 83 4 Using the Fluorescence Detector Step 2 Optimize Limits of Detection and Selectivity Excitation spectrum with emission at 440 nm emission spectrum with excitation at 250 nm of 1 pg ml quinidine Detector settings Step size 5 nm PMT 12 Response time 4s 84 To achieve optimum limits of detection and selectivity analysts must find out about the fluorescent properties of the compounds of interest Excitation and emission wavelengths can be selected for optimum limits of detection and best selectivity In general fluorescence spectra obtained with different instruments may show significant differences depending on the hardware and software used The traditional approach is to extract an appropriate excitation wavelength from the UV spectrum that is similar to the fluorescence excitation spectrum see Figure 33 on page 84 and to record the emission spectrum Then with an optim
16. 1 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 54 on page 136 Agilent 1260 FLD User Manual 135 5 Optimizing the Detector LU Exitation 300nm Second order light 600nm Stray light Fluorescence no filter es E 200M Wavelength nm Figure 54 Reducing Stray Light 136 Agilent 1260 FLD User Manual Agilent 1260 FLD User Manual 6 Troubleshooting and Diagnostics Overview of the Module s Indicators and Test Functions 138 Status Indicators 139 Power Supply Indicator 139 Module Status Indicator 140 User Interfaces 141 Agilent Lab Advisor Software 142 This chapter gives an overview about the troubleshooting and diagnostic features and the different user interfaces Apg Agilent Technologies 137 6 Troubleshooting and Diagnostics Overview of the Module s Indicators and Test Functions Status Indicators The module is provided with two status indicators which indicate the operational state prerun run and error states of the module The status indicators provide a quick visual check of the operation of the module Error Messages In the event of an electronic mechanical or hydraulic failure the module generates an error message in the user interface For each message a short descripti
17. 2002 96 EC adopted by EU Commission on 13 February 2003 is introducing producer responsibility on all Electric and Electronic appliances from 13 August 2005 z This product complies with the WEEE Directive 2002 96 EC marking requirements The affixed label indicates that you must not discard this electrical electronic product in domestic household waste Product Category With reference to the equipment types in the WEEE Directive Annex l this product is classed as a Monitoring and Control instrumentation product Do not dispose off in domestic household waste To return unwanted products contact your local Agilent office or see www agilent com for more information Agilent 1260 FLD User Manual 249 13 Appendix Lithium Batteries Information Lithium batteries may not be disposed off into the domestic waste Transportation of discharged Lithium batteries through carriers regulated by IATA ICAO ADR RID IMDG is not allowed Danger of explosion if battery is incorrectly replaced gt Discharged Lithium batteries shall be disposed off locally according to national waste disposal regulations for batteries gt Replace only with the same or equivalent type recommended by the equipment manufacturer Lithiumbatteri Eksplosionsfare ved fejlagtig handtering Udskiftning ma kun ske med batteri af samme fabrikat og type gt Lever det brugte batteri tilbage til leverand ren Lithiumbatteri Eksplosionsf
18. 4 min C Off MultiEx Multi Em 250 nm ff Jao nm I Use additional Excitation C Zeto Order Zero Order ae at 3 ji B M 250 mm cr 50 rn O B 250 Am metab ann G fs a Acquire Excitation Spectra Line Time PMT Thse hold Peakwidth Baveline nang 230 ta E m Step 5 hint hhreshald fi 00 LU ime spectrum 63y Peakwidth Responsetime gt 0 2 min 4 s standard T T D Insert Append Cut Cop pA a Table C Graphic PMT Gain 70 Test Cancel Help Short lt lt Special Setpoints When you change PKWD you should change PDPW also Enter in the Timetable at 0 0 min a PDPW PKWD 2 e g PKWD 0 2 min PDPW 0 05 min In case of a longer chromatogram and peak broadening later on you can increase the PDPW value by 1 step with an additional entry in the Timetable THRS and PDPW influence the peak triggered spectra acquisition You can change THRS in the FLD s setup screen PDPW can only be changed with the Peakwidth field in the Timetable Agilent 1260 FLD User Manual 111 4 112 Using the Fluorescence Detector Notes The peak detection algorithm works best when a peak is reduced to 8 16 data points The FLD collects the data points with an internal data rate of 74 08 Hz 13 50 ms 1 signal only The data reduction is only influenced by the PDPWparameter When PDPW is too low the peak detector does not find a peak instead it assum
19. 4 seconds It is important to know that comparing sensitivity requires using the same response time A response time of 4 s default is equivalent to a time constant of 1 8 s and appropriate for standard chromatographic conditions Nu Nt Response time 2 sec Response time 8 sec Figure 53 Separation of Peaks using Responsetime Agilent 1260 FLD User Manual 133 5 134 Optimizing the Detector Peakwidth Settings Do not use peak width shorter than necessary Peakwidth enables you to select the peak width response time for your analysis The peak width is defined as the width of a peak in minutes at half the peak height Set the peak width to the narrowest expected peak in your chromatogram The peak width sets the optimum response time for your detector The peak detector ignores any peaks that are considerably narrower or wider than the peak width setting The response time is the time between 10 and 90 of the output signal in response to an input step function Limits When you set the peak width in minutes the corresponding response time is set automatically and the appropriate data rate for signal and spectra acquisition is selected as shown in the table below Table 20 Peakwidth Setting Peak Width Data Rate At half Response Hz ms height min sec gt 0 0016 0 016 144 93 6 9 G1321B K1321B with firmware A 06 54 and above lt 0 003 0 03 74 07 13 5 G1321B C K1321B gt 0 003 0 06 37 04 27 0 gt
20. Approx Time 20 min Status Running PITTI Test Procedure Result Name Value W 1 Check Prerequisites Ee 1 300 nm 2 Wavelength Verification Preparation Em 3 400 nm 4 3 WL Verification Step 1 EX rotation scan full circle Y 4 WL Verification Step 2 EX rotation scan high resolution Y 5 WL Verification Step 3 EX position scan low resolution Y 6 WL Verification Step 4 EX position scan high resolution 7 WL Verification Step 5 EM rotation scans full circle Y 8 WL Verification Step 6 EM rotation scan high resolution part Y 9 WL Verification Step 7 EM rotation scan high resolution part In Y 10 WL Verification Step 8 EM position scan low resolution 7A 11 WL Verification Step 9 EM position scan high resolution tt il 12 Calibrate Detector e Wavelength Calibration Es Do you want to calibrate the detector using the wavelength verification results Figure 71 Wavelength Calibration Agilent Lab Advisor Agilent 1260 FLD User Manual 179 8 Test Functions Wavelength Calibration Procedure WL Calibration History nvon os maz E E 19 AEEA a 2 0772172009 11 31 08725 2006 1205 02 01709 2006 16 02 0170972006 1530 06 i E Figure 72 Calibration History Agilent Lab Advisor under Module Info FLD Wavelength Calibration System 2 Figure 73 Wavelength Calibration Agilent ChemStation NOTE To look at the history table ChemS
21. Communication Interface Board Hardware Information 12 The Agilent modules have one optional board slot that allows to add an interface board to the modules Some modules do not have this interface slot Refer to Interfaces on page 232 for details p n Description G1369B or Interface board LAN G1369 60002 OR G1369C or Interface board LAN G1369 60012 One board is required per Agilent 1260 Infinity instrument It is recommended to add the LAN board to the detector with highest data rate For the configuration of the G1369 LAN Communication Interface card refer to its documentation The following cards can be used with the Agilent Table 33 LAN Boards Type Vendor 1260 Infinity modules Supported networks Interface board LAN G1369B Agilent Technologies or G1369 60002 or Interface board LAN G1369C or G1369 60012 LAN Communication Interface Agilent Technologies board G1369A or G1369 60001 J4106A Hewlett Packard Fast Ethernet Ethernet 802 3 RJ 45 10 100Base TX recommended for re ordering Fast Ethernet Ethernet 802 3 RJ 45 10 100Base TX obsolete Ethernet 802 3 RJ 45 10Base T Agilent 1260 FLD User Manual 227 12 Hardware Information 228 Table 33 LAN Boards Type Vendor Supported networks J4105A Hewlett Packard Token Ring 802 5 DB9 RJ 45 10Base T J4100A Hewlett Packard Fast Ethernet Ethernet 802 3 RJ 45 10 100Base TX BNC 10Base2
22. Figure 2 Absorption of Light Versus Emission of Light 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 Agilent 1260 FLD User Manual Introduction to the Fluorescence Detector 1 energy level to one of the vibrational and rotational sub levels of its ground state emitting light see Figure 3 on page 13 The characteristic maxima of absorption for a substance is its Apy and for emission its Apy absorption emission S radiationless transition Figure 3 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 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 cal
23. Hz Standard 560 V 74 Hz Standard 950 V 63 mJ 18 8 W 63 mJ 4 7 W 180 mJ 13 3 W 63 mJ 4 7 W Best sensitivity can be expected with no economy see Figure 51 on page 131 FLD1 A Ex 246 Em 317 FLD_FLRFLR_0001 D Lu 084 065 044 024 FLD1 A Ex 246 Em 317 FLD_FLR FLR_0002 D Standard 296 Hz T T o5 4 Re Economy 74 Hz l u tas z _ Figure 51 Agilent 1260 FLD User Manual Xenon Flash Lamp Frequency T T 35 4 131 5 5 132 Optimizing 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 a combination of the above Agilent 1260 FLD User Manual Optimizing the Detector 5 Selecting the Best Response Time Selecting the Best Response Time Data reduction using the RESPONSETIME function will increase your signal to noise ratio For example see Figure 52 on page 133 FLD1 A E245 Em 317 FLD_RTIRT_00002 D FLD1 A Ex 248 Em 317 FLD_RT RT_00003 0 FLD1 A Ex 248 Em 317 FLD_RT RT_00004 0 w sieht 8 sec 4 sec ad adany lr ArWNe paper Jasons AM ery eny ofey 1 sec ooeen Figure 52 Finding Best Response Time LC fluorescence detectors typically work with response times of 2 or 4 s The default of the module is
24. Replacing Leak Handling System Parts When If the parts are corroded or broken Parts required p n Description 1 5041 8388 Leak funnel 1 5041 8389 Leak funnel holder 1 5042 9974 Leak tubing 1 5 m 120 mm required 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 ow A WwW N Replace the front cover Leak funnel Leak funnel holder Leak tubing it Figure 75 Replacing Leak Handling System Parts 196 Agilent 1260 FLD User Manual Maintenance 9 Replacing the Interface Board When For all repairs inside the detector or for installation of the board Parts required p n Description 1 G1351 68701 Interface board BCD with external contacts and BCD outputs 1 G1369B or Interface board LAN G1369 60002 OR 1 G1369C or Interface board LAN G1369 60012 1 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 76 Location of the Interface Board Agilent 1260 FLD User Manual 197 9 Maintenance Replacing Module Firmware When Tools required OR OR Parts required Preparations 198 The installation of newer firmware might be necessary if a newer version solves problems of olde
25. Vespel which is also used by Agilent Polyimide is stable in a pH range between 1 and 10 and in most organic solvents It is incompatible with concentrated mineral acids e g sulphuric acid glacial acetic acid DMSO and THF It is also degraded by nucleophilic substances like ammonia e g ammonium salts in basic conditions or acetates Polyethylene PE Agilent uses UHMW ultra high molecular weight PE PTFE blends for yellow piston and wash seals which are used in 1290 Infinity pumps and for normal phase applications in 1260 Infinity pumps Polyethylene has a good stability for most common inorganic solvents including acids and bases in a pH range of 1 to 12 5 It is compatible to many organic solvents used in chromatographic systems like methanol acetonitrile and isopropanol It has limited stability with aliphatic aromatic and halogenated hydrocarbons THF phenol and derivatives concentrated acids and bases For normal phase applications the maximum pressure should be limited to 200 bar Agilent 1260 FLD User Manual Using the Fluorescence Detector 4 Tantalum Ta Tantalum is inert to most common HPLC solvents and almost all acids except fluoric acid and acids with free sulfur trioxide It can be corroded by strong bases e g hydroxide solutions gt 10 diethylamine It is not recommended for the use with fluoric acid and fluorides Stainless Steel ST Stainless steel is inert against many common solvents It
26. White green 12 BCD 9 200 Brown green 13 BCD 8 100 notconnected 14 notconnected 15 5V Low Agilent 1260 FLD User Manual 215 11 Identifying Cables Agilent Module to 3396 Integrators p n 03396 60560 Pin 3396 Pin Agilent Signal Name BCD Digit module an 1 1 BCD 5 20 2 2 BCD7 80 Se 15 oO Z 3 3 BCD 6 40 oO So 4 4 BCD 4 10 s je 2 5 5 BCDO 1 fie e9 6 6 BCD 3 8 7 7 BCD 2 4 8 8 BCD 1 2 9 9 Digital ground NC 15 5V Low 216 Agilent 1260 FLD User Manual CAN LAN Cables Identifying Cables 11 Both ends of this cable provide a modular plug to be connected to Agilent modules CAN or LAN connectors CAN Cables p n 5181 1516 5181 1519 LAN Cables p n 5023 0203 5023 0202 Agilent 1260 FLD User Manual Description CAN cable Agilent module to module 0 5 m CAN cable Agilent module to module 1 m Description Cross over network cable shielded 3 m for point to point connection Twisted pair network cable shielded 7 m for point to point connection 217 11 Identifying Cables External Contact Cable c50 o o ol 10 6 15 41 One end of this cable provides a 15 pin plug to be connected to Agilent modules interface board The other end is for general purpose Agilent Module Interface Board to general purposes p n G1103 61611 Color Pin Agilent Signal Name module White 1 EXT 1 Brown 2 EXT 1 Green 3 EXT 2 Yellow
27. a function of the voltage at the dynodes and is microprocessor controlled You can set the amplification using the PMTGAIN function Opaque photocathode Anode Incident light Arc shaped dynodes 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 17 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 Agilent 1260 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 by the flow cell and corrects flash lamp fluctuations and long term intensity drift Because of a non linear output of the diode depending on the EX wavelength the measured data are normalized A diffuser is located in front of the reference diode see Figure 6 on page 17 This diffuser is made of quartz reduces light and allows integral measurement of the light Agilent 1260 FLD User Manual 23 1 Introduction to the Fluorescence Detector Analytical Information From Primary Data 24 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 Dependi
28. be UP plus required mode For normal operation use the default best settings Switch settings provide configuration parameters for serial communication protocol and instrument specific initialization procedures With the introduction of the Agilent 1260 Infinity all GPIB interfaces have been removed The preferred communication is LAN Agilent 1260 FLD User Manual 239 12 Hardware Information 240 The following tables represent the configuration switch settings for the modules without on board LAN only Table 37 8 bit Configuration Switch without on board LAN Mode Select 1 2 3 4 5 6 7 8 RS 232C 0 1 Baudrate Data Parity Bits Reserved 1 0 Reserved TEST BOOT 1 1 RSVD SYS RSVD RSVD FC The LAN settings are done on the LAN Interface Card G1369B C Refer to the documentation provided with the card Communication Settings for RS 232C The communication protocol used in the column compartment supports only hardware handshake CTS RTR Switches 1 in down and 2 in up position define that the RS 232C parameters will be changed Once the change has been completed the column instrument must be powered up again in order to store the values in the non volatile memory Table 38 Communication Settings for RS 232C Communication without on board LAN Mode Select 1 2 3 4 5 6 7 8 Rs 232c 0 1 Baudrate Data Bits Parity Use the following tables for selecting the setting which you want to us
29. by means of a stop time or manually The peak height is always the same but the area and the retention time depend on the set peakwidth see example below Procedure Using the Agilent Lab Advisor This procedure works for all Agilent 1200 Infinity detectors DAD MWD VWD FLD and RID The example figure is from the RID detector 1 Assure that the default LC method is loaded via the control software 2 Start the Agilent Lab Advisor software B 01 03 SP4 or later and open the detector s Tools selection 3 Open the test chromatogram screen Tools Test Chromatogram Current Status Disabled Switch Test Chromatogram on Switch Test Chromatogram off 4 Turn the Test Chromatogram on 5 Change to the detector s Module Service Center and add the detector signal to the Signal Plot window Agilent 1260 FLD User Manual Test Functions 8 Using the Built in Test Chromatogram 6 To start a test chromatogram enter in the command line STRT Set heater temperature 35 Send Generic command line stor 7 Send A Signal configuration Available signals Signal A mA G13 x Add Select signal Signal A nRIU G13 gt Remove IV Autoscroll Generic plot RA 0000 STAT G13624 DE91600336 5 4 2010 3 19 20 PM RA 0000 STOP G13624 DE91600336 5 4 2010 3 24 11 PM Time min Figure 62 Test Chromatogram with Agilent Lab Advisor 7 To stop the test chromatogram enter in the command line STOP NOTE The test chromatogra
30. 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 on page 28 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 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 Agilent 1260 FLD User Manual 27 1 28 Introduction to the Fluorescence Detector Figure 16 Small S N ratio Nf My aia gaseeecees O0 ODIO 00000 O points RESPONSETIME 125 O O 0 D D O O O OO gives 3 points Boxcar S per boxcar filter
31. fitting using your fingers Second Step Installation to Connector In the second step Second Step Installation to Hard Connectors on page 257 or Second Step Installation to Soft Connectors on page 258 a wrench is used to rotate the fitting relative to the finger tight position by a defined angle For each of the cases mentioned above there is a recommended range in which the fitting is tight Staying below this range could create a leak either a visible one or a micro leak potentially biasing measurement results Exceeding the recommended range could damage the capillary Alternatively a torque wrench may be used The target torque for all connections is about 0 7 Nm When using a torque wrench read instructions for that tool carefully as wrong handling may easily miss the correct torque Second Step Installation to Hard Connectors Use this procedure for hard connectors made from metal titanium or ceramics In the system these are connections to and from the analytical head of the autosampler connections to injection valve and needle and to a metal column Agilent 1260 FLD User Manual 257 13 Appendix First installation of a capillary to a hard connector 1 When tightening a fitting for the first time start from the finger tight position which is not necessarily a vertical wrench position and rotate the wrench by 135 180 Staying below 135 grey arrow will be insufficiently tight more tha
32. i o i 5 o 05 4 15 EN 25 3 35 4 min Figure 30 Biphenyl Peak With Different Excitation Wavelengths The excitation maxima are around 250 nm Agilent 1260 FLD User Manual Using the Fluorescence Detector 4 Observe the Maxima via the Isoabsorbance Plot 1 Load the data file Agx 246 nm Agy 317 nm and open the isoabsorbance plot 2 The maximum Agy will be found around 250 nm Figure 31 Isoabsorbance Plot Agilent 1260 FLD User Manual 81 4 Using the Fluorescence Detector 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 fluorescence detector Table 9 on page 82 gives an overview of how to benefit from the operation modes during these steps Table9 Steps for thorough method development Step 1 Check system Step 2 Optimize limits of detection and selectivity Step 3 Set up routine methods 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 switching Determine Ex Em spectra for all separated compounds in a single run Activate up to four
33. li Condenser lens JAN SA REF Diode Viana as a Ij j Diffuser Figure 6 Optical Unit The radiation source is a xenon flash lamp The 3 us flash produces a continuous spectrum of light from 200 nm to 900 nm The light output distribution can be expressed as a percentage in 100 nm intervals see Figure 7 on page 18 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 Agilent 1260 FLD User Manual 17 1 Introduction to the Fluorescence Detector 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 Relative Intensity a ial Ly Vi lh i C po 300 400 500 600 700 800 900 Wavelenght nm Figure 7 Lamp Energy Distribution vendor data 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 r
34. light is OFF NOTE The detector was shipped with default configuration settings NOTE The GPIB interface has been removed with the introduction of the 1260 Infinity modules Agilent 1260 FLD User Manual 63 3 Installing the Module Flow Connections to the Module BIO inert Tools required Parts required Preparations For bio inert modules use bio inert parts only Description Wrench 1 4 5 16 inch for capillary connections p n Description G1321 68755 Accessory kit Detector is installed in the LC system Toxic flammable and hazardous solvents samples and reagents The handling of solvents samples and reagents can hold health and safety risks gt When working with these substances observe appropriate safety procedures for example by wearing goggles safety gloves and protective clothing as described in the material handling and safety data sheet supplied by the vendor and follow good laboratory practice gt The volume of substances should be reduced to the minimum required for the analysis gt Do not operate the instrument in an explosive atmosphere 64 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 Agilent 1260 FLD User Manual Installing the Module 3 Flow Connections to the Module 1 Press the release buttons and remove
35. line spectra G1321B SPECTRA e spectra acquisition and simultaneous multi signal detection G1321B SPECTRA optional cuvette is available and can be used for off line measurements e easy front access to flow cell for fast replacement and e built in wavelength accuracy verification For specifications see Performance Specifications on page 40 Agilent 1260 FLD User Manual Introduction to the Fluorescence Detector Introduction to the Detector Figure 1 Agilent 1260 FLD User Manual The Agilent 1260 Infinity Fluorescence Detector 1 Introduction to the Fluorescence Detector How the Detector Operates 12 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 on page 12 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 e _ energy level 2 hv ww RT NS Ne _ energy level 1 luminescence energy level 2 hv IVINS VP energy level 1
36. mL syringe 8 pL volume Bio inert 8 uL volume and 20 bar 2 MPa pressure maximum pH 1 12 Micro 4 uL volume and 20 bar 2 MPa pressure maximum Agilent ChemStation for LC Agilent Instant Pilot G4208A with limited spectral data analysis and printing of spectra 100 LU is the recommended range see FLD Scaling Range and Operating Conditions Recorder integrator 100 mV or 1 V output range gt 100 LU two outputs Controller area network CAN RS 232C LAN APG Remote ready start stop and shut down signals Extensive support for troubleshooting and maintenance is provided by the Instant Pilot Agilent Lab Advisor and the Chromatography Data System Safety related features are leak detection safe leak handling leak output signal for shutdown of pumping system and low voltages in major maintenance areas 44 Agilent 1260 FLD User Manual Site Requirements and Specifications 2 Table5 Performance Specifications Agilent 1260 Infinity Fluorescence Detector G1321C Type Specification Comments GLP features Early maintenance feedback EMF 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 Housing All materials recyclable Environment 0 40 C constant temperature at lt 95 humidity non condensing D
37. of external damage please call your Agilent Technologies sales and service office immediately Inform your service representative that the instrument may have been damaged during shipment CAUTION Defective on arrival problems If there are signs of damage please do not attempt to install the module Inspection by Agilent is required to evaluate if the instrument is in good condition or damaged gt Notify your Agilent sales and service office about the damage gt An Agilent service representative will inspect the instrument at your site and initiate appropriate actions 50 Agilent 1260 FLD User Manual Delivery Checklist Installing the Module 3 Ensure all parts and materials have been delivered with your module The delivery checklist is shown below For parts identification please check the illustrated parts breakdown in Parts for Maintenance on page 201 Please report any missing or damaged parts to your local Agilent Technologies sales and service office Table 7 Detector Checklist Description Quantity Detector 1 Power cable 1 CAN cable 1 Flow cell as ordered Optional flow cell cuvette as ordered User Manual on Documentation CD part of the 1 per order shipment not module specific Accessory kit see Standard Accessory Kit on page 204 1 Agilent 1260 FLD User Manual 51 3 Installing the Module Optimizing the Stack Configuration If your module is part of a comple
38. overload the 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 To check for leaks establish a flow and observe the flow cell outside of the cell compartment and all capillary connections Agilent 1260 FLD User Manual 191 9 Maintenance 5 Replace the front cover Perform a wavelength verification to check the correct positioning of the flow cell as described in chapter Wavelength Verification and Calibration on page 168 192 Agilent 1260 FLD User Manual Maintenance 9 How to use the Cuvette 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 1 Install the cuvette instead of the standard flow cell 2 Connect the waste tubing to the outlet of the cuvette 3 Use the syringe see Cuvette Kit on page 203 to inject the compound 4 Setup the parameters for the Fluorescence Scan under Special Setpoints 5 Select Take Fluorescence Scan on the user interface to start the off line measurement Agilent 1260 FLD User Manual 193 9 Maintenance Flow Cell Flushing When Tools required Parts required If flow cell is contaminated Description Glass syringe Adap
39. parts in a bio inert system DO NOT install the inlet capillary to the outlet connection of the flow cell This will result in poor performance In case the flow cell is not used for some time stored then flush the flow cell with iso propanol and close the cell with Plug Screw Fitting 0100 1259 Agilent 1260 FLD User Manual 189 9 Maintenance Exchanging a Flow Cell 1 Press the release buttons and remove the front cover for 2 Disconnect the capillaries from the flow cell access to the flow cell area 190 Agilent 1260 FLD User Manual Maintenance 9 3 Unscrew the thumb screws and pull the flow cell out of the compartment 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 install the inlet capillary to the outlet connection of the flow cell This will result in poor performance or damage If an additional detector is added to the system the fluorescence detector should be the last detector in the flow path except for evaporative detectors like LC MSD Otherwise the back pressure generated by the other detector may
40. the not ready will prevent the system to start the run sequence With firmware revision A 06 11 and above the A D overflow leads into a flat peak in the chromatogram For details see Visualization of ADC Limits on page 129 Probable cause Suggested actions 1 PMT setting to high Reduce PMT gain 2 Wavelength setting wrong Change wavelength setting Agilent 1260 FLD User Manual Error Information 7 Flash Lamp Current Overflow Error ID 6704 The lamp current of the xenon flash lamp is monitored constantly If the current gets too high an error is generated and the lamp is turned OFF Probable cause Suggested actions 1 Short circuit of trigger pack assembly or Please contact your Agilent service defective FLL board representative 2 Short circuit of flash lamp assembly Please contact your Agilent service representative No light at reference diode despite lamp is on Error ID 6721 e Revision A B C Front End Board FLF There is no feedback mechanism that checks whether the lamp is ON If no peaks are shown in the chromatogram the user interface shows the module still in Ready Perform a Lamp Intensity Test see Lamp Intensity Test on page 160 first If flat use below steps e Revision D Front End Board FLF The flashing of the xenon flash lamp is monitored constantly If the Lamp has not flashed for more than 100 times in series an error is generated and the lamp is turned OFF Probable cause Sugges
41. this does not help a main board replacement is required Agilent 1260 FLD User Manual Troubleshooting and Diagnostics 6 User Interfaces 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 21 Test Functions avaible vs User Interface Test ChemStation Instant Pilot G4208A Lab Advisor D A Converter No No Yes Test Chromatogram Yes C No Yes Wavelength Yes Yes M Yes Calibration Lamp Intensity Yes No Yes Dark Current Yes No Yes via command M section Maintenance D section Diagnose Agilent 1260 FLD User Manual 141 Troubleshooting and Diagnostics Agilent Lab Advisor Software 142 The Agilent Lab Advisor software is a standalone product that can be used with or without data system Agilent Lab Advisor software helps to manage the lab for high quality chromatographic results and can monitor in real time a single Agilent LC or all the Agilent GCs and LCs configured on the lab intranet Agilent Lab Advisor software provides diagnostic capabilities for all Agilent 1200 Infinity Series modules This includes diagnostic capabilities calibration procedures and maintenance routines for all the maintenance routines The Agilent Lab Advisor software also allows users to monitor the status of their LC instruments The Early Maintenance Feedback EMF feature helps to ca
42. to the following TLVs Threshold Limit Values according to the American Conference of Governmental Industrial Hygienists Table 45 UV Radiation Limits Exposure day Effective Irradiance 8 hours 0 1 pW cm2 10 minutes 5 0 pW cm2 Typically the radiation values are much smaller than these limits Table 46 UV Radiation Typical Values Position Effective Irradiance Lamp installed 50 cm distance average 0 016 pW cm2 Lamp installed 50 cm distance maximum 0 14 pW cm2 Agilent 1260 FLD User Manual 253 13 Appendix Solvent Information Flow Cell To protect optimal functionality of your 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 e If the flow cell is transported while temperatures are below 5 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 a small of organic solvents e g acetonitrile or methanol 5 Use of Solvents Observe the following recommendations on the use of solvents Brown glass ware can avoid growth of algae e Small particles can permanently block capillaries and valves Therefore always filter solvents through 0 4 um filters e Avoid the use of the following steel corrosive solvents Solutions of alkali halides and their respective acids for example lithiu
43. to the main Please contact your Agilent service board representative 2 Defective leak sensor Please contact your Agilent service representative 3 Leak sensor incorrectly routed being Please contact your Agilent service pinched by a metal component representative Agilent 1260 FLD User Manual Error Information 7 Leak Sensor Short Error ID 0082 The leak sensor in the module has failed short circuit The current through the leak sensor is dependent on temperature A leak is detected when solvent cools the leak sensor causing the leak sensor current to change within defined limits If the current increases above the upper limit the error message is generated Probable cause Suggested actions 1 Defective leak sensor Please contact your Agilent service representative Compensation Sensor Open Error ID 0081 The ambient compensation sensor NTC on the main board in the module has failed open circuit The resistance across the temperature compensation sensor NTC on the main board is dependent on ambient temperature The change in resistance is used by the leak circuit to compensate for ambient temperature changes If the resistance across the sensor increases above the upper limit the error message is generated Probable cause Suggested actions 1 Defective main board Please contact your Agilent service representative Agilent 1260 FLD User Manual 149 7 150 Error Information Compensation Sensor Shor
44. 0 005 0 12 37 04 27 0 gt 0 01 0 25 37 04 27 0 gt 0 025 0 5 18 52 54 0 G1321A B C K1321B gt 0 05 1 0 9 26 108 0 gt 0 1 2 0 4 63 216 0 gt 0 2 4 0 2 31 432 0 gt 04 8 0 1 16 864 0 Agilent 1260 FLD User Manual Optimizing the Detector 5 Reducing Stray Light 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 factor To explain the effect of such higher order light assume the detector is on but no sample is eluting through the flow cell The lamp 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
45. 0 counts max range of the ADC converter lt A filter smoothes the peak making it not clearly visible that the max intensity is reached Also peak area and peak height are distorted which leads to poor linearity performance Note that max LU is not a fix number but depends on the intensity of the reference gt channel LU New implementation with firmware A 06 11 or above i i i While any sample value within the filter width is i in state ADC overflow the max possible LU is displayed in chromatogram Note that max LU is slightly dependent on lamp drift and lamp noise but strongly dependent on the excitation wavelength ix Milter width __ gt bo ae 4 t As a result the ADC overflow is visible as a real flat peak in the chromatogram showing the user that the setting of the detector parameter PMT gain or the concentration of the solution is set to high The transfer of methods 1 1 from one FLD to another may result into the above ADC overflow problem For details see FLD Scaling Range and Operating Conditions on page 126 FLD Scaling Range and Operating Conditions 130 Agilent 1260 FLD User Manual Changing the Xenon Flash Lamp Frequency Modes Optimizing the Detector The lamp flash frequency can be changed into the following modes Table 19 Flash Lamp Modes Positioning Rotation Multi Ex Em 74 Hz Economy 560 V 74 Hz Economy 560 V 296
46. 1321C 1260 500 3000 FLF board revision D and above G1321B 1260 500 3000 300 FLF board revision D and above G1321A 1200 500 300 FLF board revision D and above G1321A 1100 400 FLF board revision B and above G1321A 1100 200 FLF board revision A Conditions Standard flow cell G1321 60005 G5615 60005 flow of 0 25 mL min of water The Dark and Dual WL values are just additional specifications Only the Raman value is used for the standard instrument checkout 162 Agilent 1260 FLD User Manual Test Functions 8 The specification single wavelength at signal can be measured with the Agilent Lab Advisor All others not used for standard checkout have to be set up manually with the information from Table 26 on page 163 and Table 27 on page 164 Table 24 Raman Signal to Noise Test Conditions Duration approximately 23 minutes Standard Flow Cell G1321 60005 G5615 60005 Solvent LC grade water degassed Flow rate 0 25 mL min Specification single wavelength at signal gt 500 according to settings in Table 25 on page 163 Specification single wavelength at gt 3000 according to settings in Table 26 on background page 163 Specification dual wavelength gt 300 according to settings in Table 27 on page 164 Table 25 Settings for Single Wavelength Specifications at signal Time EX EM PMT Baseline 0 350 397 12 Free 20 30 350 450 12 Free Table 26 Settings for Single Wavelength Specifications at backgro
47. 4 EXT 2 Grey 5 EXT 3 Pink 6 EXT 3 Blue 7 EXT 4 Red 8 EXT 4 Black 9 Not connected Violet 10 Not connected Grey pink 11 Not connected Red blue 12 Not connected White green 13 Not connected Brown green 14 Not connected White yellow 15 Not connected 218 Agilent 1260 FLD User Manual Identifying Cables 11 Agilent Module to PC p n Description G1530 60600 RS 232 cable 2 m RS232 61601 RS 232 cable 2 5 m Instrument to PC 9 to 9 pin female This cable has special pin out and is not compatible with connecting printers and plotters It s also called Null Modem Cable with full handshaking where the wiring is made between pins 1 1 2 3 3 2 4 6 5 5 6 4 7 8 8 7 9 9 5181 1561 RS 232 cable 8 m Agilent 1260 FLD User Manual 219 11 Identifying Cables Agilent Module to PC 220 Agilent 1260 FLD User Manual Agilent 1260 FLD User Manual 12 Hardware Information Firmware Description 222 Optional Interface Boards 225 Electrical Connections 229 Rear view of the module 230 Serial Number Information 231 Interfaces 232 Overview Interfaces 235 Setting the 8 bit Configuration Switch without On board LAN 239 Communication Settings for RS 232C 240 Special Settings 242 Early Maintenance Feedback 243 Instrument Layout 244 This chapter describes the detector in more detail on hardware and electronics te Agilent Technologies 221 12 Hardware Information Firmware Description 222 The firmware of the instrument consist
48. 7 266 Agilent 1260 FLD User Manual Index Agilent 1260 FLD User Manual 267 www agilent com In This Book This manual contains technical reference information about the Agilent 1260 Infinity Fluorescence Detector G1321B SPECTRA G1321C and Agilent 1100 1200 Series Fluorescence Detector G1321A obsolete introduction and specifications e installation e using and optimizing troubleshooting and diagnose e maintenance e parts identification safety and related information Agilent Technologies 2010 2012 2013 Printed in Germany 11 2013 G1321 90014 Rev B EE Agilent Technologies
49. 78 Agilent 1260 FLD User Manual Using the Fluorescence Detector 4 Setting the Chromatographic Conditions 1 Set up the system with the following chromatographic conditions and wait until the baseline gets stable Table 8 Chromatographic Conditions Mobile phases A water 35 B Acetonitrile 65 Column OSD Hypersil column 125 mm x 4 mm i d with 5 um particles Sample Isocratic standard sample 1 10 diluted in methanol Flow rate 1 5 ml min Compressibility A water 46 Compressibility B Acetonitrile 115 Stroke A and B auto Stop time 4 min Injection volume 5 ul Oven temperature 1200 30 C FLD Excitations Emission Wavelength EX 246 nm EM 317 nm FLD PMT Gain PMT 10 FLD Response time 4s 2 Set the FLD setpoints according to Figure 29 on page 80 Agilent 1260 FLD User Manual 79 4 Using the Fluorescence Detector Getting Started and Checkout In this example FLD Signals System 2 additional excitation wavelenghts B C D are used This will sunl increase the scan time and may lower the performance Figure 29 FLD Parameters 3 Start the run The resulting chromatograms are shown below FLD1 B Ex 230 Em 317 FLD_ISO1 FLD_ISO2 D FLD1 A Ex 246 Em 317 FLD_ISO1 FLD_ISO2 D PLDI D Exe260 En 917 FLD ISONFLD 1802 0 Ex 246 nm Ex 250 nm Lu m l 175 i Biphenyl peak i Ex 230 nm E pane 125 E 100 E 75 i E s E Ex 290 nm lf 7
50. 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 c d pyrene em 98 Agilent 1260 FLD User Manual Using the Fluorescence Detector 4 Example Optimization for Multiple Compounds Example Optimization for Multiple Compounds Using PNAs as a sample this example uses the described scanning functions Agilent 1260 FLD User Manual 99 Using the Fluorescence Detector Setting the Chromatographic Conditions This example uses the following chromatographic conditions the detector settings are shown in Figure 40 on page 101 Table 16 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 mm x 2 1 mm i d with 5 ym particles PAH 0 5 ng 0 4 ml min 46 115 auto at 0 min B 50 at 3 min B 60 at 14 5 min B 90 at 22 5 min B 95 26 min 8 min 1 pl 30 C PMT 15 4s Agilent 1260 FLD User Manual Using the Fluorescence Detector 4 Example Optimization for Multiple Compounds Select a Excitation FLD Signals System 2 wavelength in the low UV 230 260 nm This will cover nea
51. Flow Cell Flushing 194 Correcting Leaks 195 Replacing Leak Handling System Parts 196 Replacing the Interface Board 197 Replacing Module Firmware 198 Tests and Calibrations 199 10 Parts for Maintenance 201 Overview of Maintenance Parts 202 Cuvette Kit 203 Accessory Kit 204 11 Identifying Cables 207 Cable Overview 208 Analog Cables 210 Remote Cables 212 BCD Cables 215 CAN LAN Cables 217 External Contact Cable 218 Agilent Module to PC 219 12 Hardware Information 221 Firmware Description 222 Optional Interface Boards 225 Electrical Connections 229 Interfaces 232 Setting the 8 bit Configuration Switch without On board LAN 239 Early Maintenance Feedback 243 Instrument Layout 244 Agilent 1260 FLD User Manual Contents 13 Appendix 245 General Safety Information 246 The Waste Electrical and Electronic Equipment WEEE Directive 2002 96 EC 249 Lithium Batteries Information 250 Radio Interference 251 Sound Emission 252 UV Radiation UV lamps only 253 Solvent Information 254 Installation of Stainless Steel Cladded PEEK Capillaries 256 Agilent Technologies on Internet 262 8 Agilent 1260 FLD User Manual Agilent 1260 FLD User Manual 1 Introduction to the Fluorescence Detector Introduction to the Detector 10 How the Detector Operates 12 Raman Effect 15 Optical Unit 16 Reference System 23 Analytical Information From Primary Data 24 Fluorescence Detection 24 Phosphorescence Detection 25 Proc
52. Fluorescence Detector Type Specification Comments Safety and maintenance GLP features Housing Environment Dimensions Weight Extensive diagnostics error detection and display through Instant Pilot G4208A and ChemStation leak detection safe leak handling leak output signal for shutdown of pumping system Low voltages in major maintenance areas Early maintenance feedback EMF 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 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 Agilent 1260 FLD User Manual Agilent 1260 FLD User Manual 3 Installing the Module Unpacking the Module 50 Optimizing the Stack Configuration 52 One Stack Configuration 53 Two Stack Configuration 55 Installation Information on Leak and Waste Handling 57 Installing the Module 61 Flow Connections to the Module 64 Installing Capillaries 68 This chapter gives information about the preferred stack setup for your system and the installation of the module pe Agilent Technologies 49 3 Installing the Module Unpacking the Module Damaged Packaging If the delivery packaging shows signs
53. Instruction menu DMUL X XX This setting is resident to the instrument even for firmware updates and is independent of the software environment The level of LU is no measure of instrument sensitivity At the lowest concentration limit limit of detection the signal to noise e g by Raman S N Test is the only measure that can accurately be used to compare chromatograms and results and to confirm the performance of the instrument For low background and highest sensitivity keep the flow cell clean and use always fresh water to prevent biological background from native fluorescence by algae and bacteria Agilent 1260 FLD User Manual Optimizing the Detector 5 Visualization of ADC Limits A new firmware A 06 11 for the Fluorescence Detector G1321A B has been released that includes a new feature the Visualization of ADC Limits Up to firmware A 06 10 an ADC overflow was not visible in the chromatogram under certain method conditions Overflow could be concealed by smoothing of a filter and thus not visible for the user In the Agilent ChemStation the ADC overflow event was only shown in the logbook This problem did only occur if the Peakwidth Responsetime parameter has been set similar or larger than the real width of the chromatographic peak Agilent 1260 FLD User Manual 129 5 Optimizing the Detector gt no 5 Raw ADC counts 2 ee aan ae s gy ee The measured light intensity is limited by the a 6553
54. Keep the syringe in a horizontal position Remove the needle Agilent 1260 FLD User Manual 177 8 Test Functions g Add the filter to the syringe and fit the needle to filter Sample filter Figure 70 Syringe with Sample Filter h Lift the needle tip and carefully eject approximately 0 5 ml to remove air out of the syringe and to flush the needle i 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 j 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 3 Wavelength Calibration a From the user interface start the FLD wavelength calibration see Figure 73 on page 180 Agilent Lab Advisor Calibrations Agilent ChemStation Diagnosis gt Maintenance gt FLD Calibration Instant Pilot G4208A Maintenance gt FLD gt Calibration If the wavelength calibration process fails refer to Wavelength Calibration Failed on page 154 178 Agilent 1260 FLD User Manual Test Functions 8 Wavelength Calibration Procedure b If a deviation is displayed press Yes Lab Advisor to adjust to new values or Adjust and OK ChemStation see next page The history table will be updated General Limits Test Name Wavelength Calibration Description This procedure performs a Wavelength Verification and Recalibration Module G13214 DE92991563
55. LD User Manual Maintenance 9 Warnings and Cautions WARNING Toxic flammable and hazardous solvents samples and reagents The handling of solvents samples and reagents can hold health and safety risks gt When working with these substances observe appropriate safety procedures for example by wearing goggles safety gloves and protective clothing as described in the material handling and safety data sheet supplied by the vendor and follow good laboratory practice gt The volume of substances should be reduced to the minimum required for the analysis gt Do not operate the instrument in an explosive atmosphere WARNING Eye damage by detector light 0 Eye damage may result from directly viewing the UV light produced by the lamp of the optical system used in this product gt Always turn the lamp of the optical system off before removing it WARNING Electrical shock Repair work at the module can lead to personal injuries e g shock hazard when the cover is opened gt Do not remove the cover of the module gt Only certified persons are authorized to carry out repairs inside the module Agilent 1260 FLD User Manual 185 9 Maintenance Personal injury or damage to the product Agilent is not responsible for any damages caused in whole or in part by improper use of the products unauthorized alterations adjustments or modifications to the products failure to comply with procedu
56. N cable Agilent module to module 1 m LAN cables p n Description 5023 0203 Cross over network cable shielded 3 m for point to point connection 5023 0202 Twisted pair network cable shielded 7 m for point to point connection RS 232 cables p n Description G1530 60600 RS 232 cable 2 m RS232 61601 RS 232 cable 2 5 m Instrument to PC 9 to 9 pin female This cable has special pin out and is not compatible with connecting printers and plotters It s also called Null Modem Cable with full handshaking where the wiring is made between pins 1 1 2 3 3 2 4 6 5 5 6 4 7 8 8 7 9 9 5181 1561 RS 232 cable 8 m Agilent 1260 FLD User Manual 209 11 Identifying Cables Analog Cables One end of these cables provides a BNC connector to be connected to Agilent modules The other end depends on the instrument to which connection is being made Agilent Module to 3394 6 Integrators p n 35900 60750 Pin 3394 6 Pin Agilent Signal Name module 1 Not connected 2 Shield Analog 3 Center Analog Agilent Module to BNC Connector p n 8120 1840 Pin BNC Pin Agilent Signal Name module Shield Shield Analog an Center Center Analog 210 Agilent 1260 FLD User Manual Agilent Module to General Purpose Identifying Cables 11 p n 01046 60105 Pin Agilent Signal Name module Not connected Black Analog ES Red Analog a Agilent 1260 FLD User Manual 211 11 Ide
57. Performance Specifications Agilent 1260 Infinity Fluorescence Detector G1321B Type Specification Comments Detection type Performance specifications Light source Pulse frequency Maximum data rate Excitation monochromator Multi signal fluorescence detector with rapid on line scanning capabilities and spectral data analysis Single wavelength operation e RAMAN H30 gt 500 noise reference measured at signal Ex 350 nm Em 397 nm dark value 450 nm standard flow cell RAMAN H30 gt 3000 noise reference measured at dark value Ex 350 nm Em 397 nm dark value 450 nm standard flow cell Dual wavelength operation RAMAN H20 gt 300 Ex 350 nm Em 397 nm and Ex 350 nm Em 450 nm standard flow cell Xenon Flash Lamp normal mode 20 W economy mode 5 W lifetime 4000 h 296 Hz for single signal mode 74 Hz for economy mode 74 Hz 145 Hz Range settable 200 nm 1200 nm and zero order Bandwidth 20 nm fixed Monochromator concave holographic grating F 1 6 blaze 300 nm see note below this table see Service Manual for details 145 Hz with firmware A 06 54 and above 40 Agilent 1260 FLD User Manual Table 4 G1321B Site Requirements and Specifications 2 Performance Specifications Agilent 1260 Infinity Fluorescence Detector Type Specification Comments Emission monochromator Reference system Timetable programing Spectrum acquisition Waveleng
58. Test Functions 8 Wavelength Verification and Calibration The duration of the wavelength calibration is about 15 minutes plus setup time for the NOTE ae es calibration sample and system Depending on the maximum intensity found during this scan the PMT gain will be changed automatically and requires an additional 1 minute per scan Table 29 on page 170 shows the steps performed during the wavelength calibration The excitation grating and the emission grating are calibrated using Rayleigh stray light from the flow cell or cuvette measured with the photomultiplier tube General Limits Test Name Wavelength Calibration Module G13214 DE92991563 Approx Time 20 min Status Running Test Procedure 2 3 4 WL Verification 5 WL Verification 6 WL Verification 7 8 9 WL Verification WL Verification Mss WL Verification In 10 WL Verification TAL VTALA LSS 11 WL Verification 3 5 1 Check Prerequisites WL Verification Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 Step 9 Calibrate Detector Wavelength Verification Preparation EX rotation scan full circle EX rotation scan high resolution EX position scan low resolution EX position scan high resolution EM rotation scans full circle EM rotation scan high resolution part EM rotation scan high resolution part EM position scan low resolution
59. able C Graphic gt 0 1 min 2s gt 0 2 min 4 s standard gt 0 4 min 8 s slow Cancel Help Short lt lt Agilent 1260 FLD User Manual Using the Fluorescence Detector 4 Solvent Information Observe the following recommendations on the use of solvents e Follow recommendations for avoiding the growth of algae see pump manuals e Small particles can permanently block capillaries and valves Therefore always filter solvents through 0 4 um filters e Avoid or minimize the use of solvents that may corrode parts in the flow path Consider specifications for the pH range given for different materials like flow cells valve materials etc and recommendations in subsequent sections Material Information Materials in the flow path are carefully selected based on Agilent s experiences in developing highest quality instruments for HPLC analysis over several decades These materials exhibit excellent robustness under typical HPLC conditions For any special conditions please consult the material information section or contact Agilent Disclaimer Subsequent data were collected from external resources and are meant as a reference Agilent cannot guarantee the correctness and completeness of such information Data is based on compatibility libraries which are not specific for estimating the long term life time under specific but highly variable conditions of UHPLC systems solvents solvent mixt
60. age in the range described in Table 3 on page 39 Consequently there is no voltage selector in the rear of the module There are also no externally accessible fuses because automatic electronic fuses are implemented in the power supply Hazard of electrical shock or damage of your instrumentation can result if the devices are connected to a line voltage higher than specified gt Connect your instrument to the specified line voltage only The module is partially energized when switched off as long as the power cord is plugged in Repair work at the module can lead to personal injuries e g electrical shock when the cover is opened and the module is connected to power gt Always unplug the power cable before opening the cover gt Do not connect the power cable to the instrument while the covers are removed CAUTION Inaccessible power plug In case of emergency it must be possible to disconnect the instrument from the power line at any time gt Make sure the power connector of the instrument can be easily reached and unplugged gt Provide sufficient space behind the power socket of the instrument to unplug the cable 36 Agilent 1260 FLD User Manual Site Requirements and Specifications 2 Power Cords Different power cords are offered as options with the module The female end of all power cords is identical It plugs into the power input socket at the rear The male end of each power cord is differ
61. age of the equipment The protection provided by the equipment may be impaired gt The operator of this instrument is advised to use the equipment in a manner as specified in this manual Safety Standards This is a Safety Class I instrument provided with terminal for protective earthing and has been manufactured and tested according to international safety standards Operation Before applying power comply with the installation section Additionally the following must be observed Do not remove instrument covers when operating Before the instrument is switched on all protective earth terminals extension cords auto transformers and devices connected to it must be connected to a protective earth via a ground socket Any interruption of the protective earth grounding will cause a potential shock hazard that could result in serious personal injury Whenever it is likely that the protection has been impaired 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 Agilent 1260 FLD User Manual 247 13 Appendix 248 replacement The use of repaired fuses and the short circuiting of fuse holders must be avoided Some adjustments described in the manual are made with power supplied to the instrument and protective covers removed Energy available at many poin
62. ange 18 Agilent 1260 FLD User Manual Introduction to the Fluorescence Detector 1 Grating EX inside Figure 8 Mirror Assembly 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 on page 20 illustrates the light path at the surface of the grating Agilent 1260 FLD User Manual 19 1 20 Introduction to the Fluorescence Detector G o A 5 amp amp E a 7 i S 2 aw Em reflected 1 st orderdispersed light 200 nm 800 nm Figure 9 Dispersion of Light by a Grating The grating is turned using a 3 phase brushless DC motor the position of the grating determining the wavelength or wavelength range of the light falling onto the flow cell The grating can be programmed to change its position and therefore the wavelength during a run For spectra acquisition and multi wavelength detection the grating rotates at 4000 rpm The excitation and emission gratings are similar in design but have different blaze wavelengths The excitation grating reflects most 1 order light in the ultra violet range around 250 nm whereas the emission grating reflects better in the visible range around 400 nm The flow cell is a solid fused silica body with a maximum ba
63. ar of the detector 5 Connect the CAN cable to other modules 6 If an Agilent ChemStation is the controller connect the LAN connection to the LAN interface board in the detector The detector DAD MWD FLD VWD RID is the preferred access point for control via LAN due to higher data load 7 Connect the analog cable s optional 8 Connect the APG remote cable optional for non Agilent Series instruments 62 Agilent 1260 FLD User Manual Installing the Module 3 Installing the Module 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 Configuration switch co O 90 BSS Ss Interface board a ny e LAN or BCD EXT O z az le TA Analog signal RS 232C oo EE E e o FIFAB A00 s ii THRA APG remote an IUEI cewe ERREN GPIB only 1100 1200 Power Figure 25 Rear View of Detector NOTE 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
64. are Ved udskiftning benyttes kun batteri som anbefalt av apparatfabrikanten gt Brukt batteri returneres appararleverandoren 250 Bij dit apparaat zijn batterijen geleverd Wanneer deze leeg zijn moet u ze niet weggooien maar inleveren als KCA Agilent 1260 FLD User Manual Appendix 13 Radio Interference Cables supplied by Agilent Technologies are screened to provide optimized protection against radio interference All cables are in compliance with safety or EMC regulations Test and Measurement If test and measurement equipment is operated with unscreened cables 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 Agilent 1260 FLD User Manual 251 13 Appendix 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 252 Agilent 1260 FLD User Manual Appendix 13 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
65. asured at signal Ex 350 nm Em 397 nm dark value 450 nm standard flow cell RAMAN H30 gt 3000 noise reference measured at dark value Ex 350 nm Em 397 nm dark value 450 nm standard flow cell Xenon Flash Lamp normal mode 20 W economy mode 5 W lifetime 4000 h 296 Hz for single signal mode 74 Hz for economy mode 74 Hz Range settable 200 nm 1200 nm and zero order Bandwidth 20 nm fixed Monochromator concave holographic grating F 1 6 blaze 300 nm Range settable 200 nm 1200 nm and zero order Bandwidth 20 nm fixed Monochromator concave holographic grating F 1 6 blaze 400 nm in line excitation measurement up to 4 signal wavelengths response time PMT Gain baseline behavior append free zero spectral parameters wavelength excitation and emission fluorescence detector see note below this table see Service Manual for details Agilent 1260 FLD User Manual 43 2 Site Requirements and Specifications Table 5 G1321C Performance Specifications Agilent 1260 Infinity Fluorescence Detector Type Specification Comments Wavelength characteristic Flow cells Control and data evaluation Analog outputs Communications Safety and maintenance Repeatability 0 2 nm Accuracy 3 nm setting Standard 8 pL volume and 20 bar 2 MPa pressure maximum fused silica block Optional e Fluorescence cuvette for offline spectroscopic measurements with 1
66. at Are Error Messages Error messages are displayed in the user interface when an electronic mechanical or hydraulic flow path failure occurs which requires attention before the analysis can be continued for example repair or exchange of consumables is necessary In the event of such a failure the red status indicator at the front of the module is switched on and an entry is written into the module logbook If an error occurs outside a method run other modules will not be informed about this error If it occurs within a method run all connected modules will get a notification all LEDs get red and the run will be stopped Depending on the module type this stop is implemented differently For example for a pump the flow will be stopped for safety reasons For a detector the lamp will stay on in order to avoid equilibration time Depending on the error type the next run can only be started if the error has been resolved for example liquid from a leak has been dried Errors for presumably single time events can be recovered by switching on the system in the user interface Special handling is done in case of a leak As a leak is a potential safety issue and may have occurred at a different module from where it has been observed a leak always causes a shutdown of all modules even outside a method run In all cases error propagation is done via the CAN bus or via an APG remote cable see documentation for the APG interface Agi
67. at 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 Agilent 1260 FLD User Manual Optimizing the Detector 5 Finding the Best Wavelengths 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 module was designed to give an optimum signal to noise ratio in the 210 nm to 360 nm range of the spectrum To achieve greatest sensitivity the absorbance wavelength of your sample molecule should match the wavelength range for your instrument In other words an excitation wavelength in the ultra violet range Your module has a broad excitation wavelength range but for higher sensitivity you should choose a wav
68. atus Passed Start Time 4 16 2013 2 41 16 PM Stop Time 4 16 2013 3 06 24 PM Test Procedure Result Name Value Cheik Pereas Raman ASTM 1337 94 SNR Measurement Part 1 Preparation Minimum Raman ASTM Limit 400 SNR Measurement Part 2 Raman and Noise Drift 10 346 LU h Measurement Part 3 Noise Measurement Part 4 set EM grating to 450nm Measurement Part 5 Dark Current Measurement Part 6 set EM grating to 350nm and PMT 10 Measurement Part 7 Rayleigh WPRVVCVCVgs Evaluate Data Figure 61 Raman ASTM Signal to Noise Test Agilent Lab Advisor In case of failing this test as shown above see Interpretation of the Results on page 165 Interpretation of the Results If the test shows low Raman values check for vY correctly positioned flow cell Y clean flow cell flush with clean bi distilled water Y no air bubble s check via fluorescence scan or visual check of cell cuvette V solvent inlet filter may create air bubbles in flow cell Agilent 1260 FLD User Manual 165 Test Functions Using the Built in Test Chromatogram 166 This function is available from the Agilent ChemStation Lab Advisor and Instant Pilot The built in Test Chromatogram can be used to check the signal path from the detector to the data system and the data analysis or via the analog output to the integrator or data system The chromatogram is continuously repeated until a stop is executed either
69. aximum limit which provides visual feedback in the user interface when the limit is exceeded Some counters can be reset to zero after the required maintenance procedure Using the EMF Counters The user settable EMF limits for the EMF Counters enable the early maintenance feedback to be adapted to specific user requirements The useful maintenance cycle is dependent on the requirements for use Therefore the definition of the maximum limits need to be determined based on the specific operating conditions of the instrument Setting the EMF Limits The setting of the EMF limits must be optimized over one or two maintenance cycles Initially the default EMF limits should be set When instrument performance indicates maintenance is necessary take note of the values displayed by the EMF 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 Agilent 1260 FLD User Manual 243 12 Hardware Information Instrument Layout The industrial design of the module 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 el
70. bols Symbol Description The apparatus is marked with this symbol when the user should refer to the instruction manual in order to protect risk of harm to the operator and to protect the apparatus against damage Indicates dangerous voltages Indicates a protected ground terminal Indicates eye damage may result from directly viewing the light produced by the deuterium lamp used in this product The apparatus is marked with this symbol when hot surfaces are available A and the user should not touch it when heated up WARNING es alerts you to situations that could cause physical injury or death gt 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 loss of data or damage of equipment gt Do not proceed beyond a caution until you have fully understood and met the indicated conditions 246 Agilent 1260 FLD User Manual Appendix 13 General Safety Information 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 WARNING Ensure the proper us
71. cates one of six possible module conditions 140 When the status indicator is OFF and power switch light is on the module is in a prerun condition and is ready to begin an analysis A green status indicator indicates the module is performing an analysis run mode A yellow indicator indicates a not ready condition The module is in a not ready state when it is waiting for a specific condition to be reached or completed for example immediately after changing a set point or while a self test procedure is running An error condition is indicated when the status indicator is red An error condition indicates the module has detected an internal problem which affects correct operation of the module Usually an error condition requires attention e g leak defective internal components An error condition always interrupts the analysis If the error occurs during analysis it is propagated within the LC system i e a red LED may indicate a problem of a different module Use the status display of your user interface for finding the root cause module of the error A blinking indicator indicates that the module is in resident mode e g during update of main firmware A fast blinking indicator indicates that the module is in a low level error mode In such a case try to re boot the module or try a cold start see Special Settings on page 242 Then try a firmware update see Replacing Module Firmware on page 198 If
72. ce Overview of Maintenance Parts 202 Cuvette Kit 203 Accessory Kit 204 This chapter provides information on parts for maintenance ot Agilent Technologies 201 10 Parts for Maintenance Overview of Maintenance Parts p n Description G1321 60005 OR G1321 60015 OR G5615 60005 G5615 68755 G1321 60007 9301 0407 9301 1446 5067 4691 5041 8388 5041 8389 5041 8387 5062 2463 5062 2462 5181 1516 5181 1519 G1369B or G1369 60002 5023 0203 5023 0202 01046 60105 G1351 68701 Flow cell 8 uL 20 bar pH 1 9 5 Flow cell 4 uL 20 bar pH 1 9 5 requires a 0 12 mm i d capillary e g p n G1316 87318 300 mm long part of Capillary kit for 0 12 mm id p n G1316 68716 Bio inert flow cell 8 uL 20 bar pH 1 12 includes Capillary Kit Flow Cells BIO p n G5615 68755 Capillary Kit Flow Cells BIO includes Capillary PK 0 18 mm x 1 5 m and PEEK Fittings 10 PK p n 5063 6591 FLD Cuvette Kit 8 pL 20 bar Needle Syringe Front Panel DAD VWD FLD 1260 1290 Leak funnel Leak funnel Tube clip Corrugated tubing PP 6 5 mm id 5 m Tube PTFE 0 8 mm x 2 m re order 5 m CAN cable Agilent module to module 0 5 m CAN cable Agilent module to module 1 m Interface board LAN Cross over network cable shielded 3 m for point to point connection Twisted pair network cable shielded 7 m for point to point connection Agilent module to general purpose Analog Interface board BCD with external contact
73. ck pressure of 20 bar Excessive back pressure will result in destruction of the cell Agilent 1260 FLD User Manual Introduction to the Fluorescence Detector 1 Operating the detector close to waste with low back pressure is recommended A slit is integrated to the body NS SK PILILIDILIDLLLLLOLLLLLLLOR 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 20 The selected wavelength of light is reflected onto the slit in the wall of the photo multiplier compartment of the optical unit The bandwidth of the emitted light is 20 nm Agilent 1260 FLD User Manual 21 1 22 Introduction to the Fluorescence Detector On the photocathode Figure 11 on page 22 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
74. connected to the system the error message is generated Probable cause Suggested actions 1 CAN cable disconnected Ensure all the CAN cables are connected correctly Ensure all CAN cables are installed correctly 2 Defective CAN cable Exchange the CAN cable 3 Defective main board in another module Switch off the system Restart the system and determine which module or modules are not recognized by the system Agilent 1260 FLD User Manual 147 7 148 Error Information Leak Error ID 0064 A leak was detected in the module The signals from the two temperature sensors leak sensor and board mounted temperature compensation sensor are used by the leak algorithm to determine whether a leak is present When a leak occurs the leak sensor is cooled by the solvent This changes the resistance of the leak sensor which is sensed by the leak sensor circuit on the main board Probable cause Suggested actions 1 Loose fittings Ensure all fittings are tight 2 Broken capillary Exchange defective capillaries Leak Sensor Open Error ID 0083 The leak sensor in the module has failed open circuit The current through the leak sensor is dependent on temperature A leak is detected when solvent cools the leak sensor causing the leak sensor current to change within defined limits If the current falls outside the lower limit the error message is generated Probable cause Suggested actions 1 Leak sensor not connected
75. ction level is reached The leak pan s outlet port 8 A guides excessive overfill from one module to the next as the solvent flows into the next module s leak funnel 8 B and the connected corrugated waste tube 3 C The corrugated waste tube guides the solvent to the next lower positioned module s leak tray and sensor The waste tube of the sampler s needle wash port 4 guides solvents to waste The condense drain outlet of the autosampler cooler 5 guides condensate to waste The waste tube of the purge valve 6 guides solvents to waste The waste tube connected to the leak pan outlet on each of the bottom instruments 7 guides the solvent to a suitable waste container Agilent 1260 FLD User Manual 31 1 Introduction to the Fluorescence Detector Bio inert Materials For the Agilent 1260 Infinity Bio inert LC system Agilent Technologies uses highest quality materials in the flow path also referred to as wetted parts which are widely accepted by life scientists as they are known for optimum inertness to biological samples and ensure best compatibility with common samples and solvents over a wide pH range Explicitly the complete flow path is free of stainless steel and free of other alloys containing metals such as iron nickel cobalt chromium molybdenum or copper which can interfere with biological samples The flow downstream of the sample introduction contains no metals whatsoever Table 2 Bio inert mate
76. ction may not be tight CAUTION Potential leak or damage of the Bio inert ZDV Union gt To avoid leaks or a damage to the Bio inert ZDV union follow the procedure below in the prescribed sequence 1 Install the capillary at the end marked with a 2 Install the second capillary at the other end ring indentation Agilent 1260 FLD User Manual 73 3 Installing the Module Installing Capillaries 74 Agilent 1260 FLD User Manual Agilent 1260 FLD User Manual 4 Using the Fluorescence Detector Leak and Waste Handling 76 Before You Start 77 Getting Started and Checkout 78 Starting Your Detector 78 Setting the Chromatographic Conditions 79 Observe the Maxima via the Isoabsorbance Plot 81 Method Development 82 Step 1 Check the LC System for Impurities 83 Step 2 Optimize Limits of Detection and Selectivity 84 Step 3 Set up Routine Methods 95 Example Optimization for Multiple Compounds 99 How to collect spectra with modes SPECTRA ALL IN PEAK and APEX SPECTRA ONLY 109 Solvent Information 113 This chapter guides you how to start the work with the detector oh Agilent Technologies 75 4 Using the Fluorescence Detector Leak and Waste Handling WARNING Toxic flammable and hazardous solvents samples and reagents The handling of solvents samples and reagents can hold health and safety risks gt When working with these substances observe appropriate safety procedures for example by wearing go
77. d Like Carbon DLC Diamond Like Carbon is inert to almost all common acids bases and solvents There are no documented incompatibilities for HPLC applications Fused silica and Quartz Si04 Fused silica is used in 1290 Infinity Flow Cells and capillaries Quartz is used for classical flow cell windows It is inert against all common solvents and acids except hydrofluoric acid and acidic solvents containing fluorides It is corroded by strong bases and should not be used above pH 12 at room temperature The corrosion of flow cell windows can negatively affect measurement results For a pH greater than 12 the use of flow cells with sapphire windows is recommended Gold Gold is inert to all common HPLC solvents acids and bases within the specified pH range It can be corroded by complexing cyanides and concentrated acids like aqua regia Zirconium Oxide Zr0 Zirconium Oxide is inert to almost all common acids bases and solvents There are no documented incompatibilities for HPLC applications Platinum Iridium Platinum Iridium is inert to almost all common acids bases and solvents There are no documented incompatibilities for HPLC applications Fluorinated polymers PTFE PFA FEP FFKM Fluorinated polymers like PTFE polytetrafluorethylene PFA perfluoroalkoxy and FEP fluorinated ethylene propylene are inert to almost all common acids bases and solvents FFKM is perfluorinated rubber which is also resistant to most che
78. d to the system it is recommended to form two stacks Some users prefer the lower height of this arrangement even without the autosampler thermostat A slightly longer capillary is required between the pump and autosampler See Figure 20 on page 55 and Figure 21 on page 56 Instant Pilot Detector Column compartment Solvent cabinet Degasser optional Autosampler ALS Fraction collector Thermostat for the ALS Fraction collector optional Figure 20 Recommended Two Stack Configuration for 1260 Infinity Front View Agilent 1260 FLD User Manual 55 3 Installing the Module Optimizing the Stack Configuration LAN to control software RAN Bus cable to Instant Pilot utosampler Fraction Cu lector C Il fu BELLU Remote cable Figure 21 Recommended Two Stack Configuration for 1260 Infinity Rear View 56 Agilent 1260 FLD User Manual Installing the Module 3 Installation Information on Leak and Waste Handling The Agilent 1200 Infinity Series has been designed for safe leak and waste handling It is important that all security concepts are understood and instructions are carefully followed WARNING Toxic flammable and hazardous solvents samples and reagents The handling of solvents samples and reagents can hold health and safety risks gt When working with these substances observe appropriate safety procedures for example by weari
79. dule provides one remote connector which is inputs outputs wired or technique To provide maximum safety within a distributed analysis system one line is dedicated to SHUTDOWN the system s critical parts in case any module detects a serious problem To detect whether all participating modules are switched on or properly powered one line is defined to summarize the POWER ON state of all connected modules Control of analysis is maintained by signal readiness READY for next analysis followed by START of run and optional STOP of run triggered on the respective lines In addition PREPARE and START REQUEST may be issued The signal levels are defined as e standard TTL levels 0 V is logic true 5 0 V is false fan out is 10 e input load is 2 2 kOhm against 5 0 V and e output are open collector type inputs outputs wired or technique All common TTL circuits operate with a 5 V power supply A TTL signal is defined as low or L when between 0 V and 0 8 V and high or H when between 2 0 V and 5 0 V with respect to the ground terminal Agilent 1260 FLD User Manual 237 12 Hardware Information Table 36 Remote Signal Distribution Pin Signal Description 1 DGND Digital ground 2 PREPARE L Request to prepare for analysis for example calibration detector lamp on Receiver is any module performing pre analysis activities 3 START L Request to start run timetable Receiver is any module performing run time contr
80. e do a second run for the evaluation of the optimal excitation wavelength See Figure 43 on page 104 FLD Signals System 2 DO NOT select additional excitation wavelengths B C D Doing so will increase the scan time and will lower the performance Figure 43 Detector Settings for Excitation Scan 5 Wait until the baseline stabilizes Start the run 104 Agilent 1260 FLD User Manual Using the Fluorescence Detector 4 6 Load the signal FLD1 A Ex 260 Em 330 TT FLD_PADT FLD_PAD2 D Lu 904 804 704 604 504 ao 4 32 204 t T T T T T T o 2 4 6 8 10 12 mi Figure 44 Chromatogram Excitation Scan at Reference Wavelength 260 330 nm 7 Use the isoabsorbance plot and evaluate the optimal excitation wavelengths in this example just in the time range of 13 minutes 380 360 340 320 300 280 260 240 220 200 Figure 45 Isoabsorbance Plot Excitation The table below shows the complete information about emission from Figure 42 on page 103 and excitation maxima Agilent 1260 FLD User Manual 105 4 Using the Fluorescence Detector Table 18 Peak Time Emission Wavelength Excitation Wavelength 1 5 3 min 330 nm 220 280 nm 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 106 Agilent 1260 FLD User Manual Using the Fluorescence Detector 4 Example
81. e 10 Benzo b fluoranthene Wave engt 4 Phenanthrene 11 Benzo k fluoranthene switching 5 Anthracene 12 Benz a pyrene 6 Fluoranthene 13 Dibenzo a h anthracene 7 Pyrene 14 Benzo g h i perylene 180 Ex 275 Em 350 TT 5 15 Indeno 1 2 3 cd pyrene JReterenc 10 160 chromatogram with switching events 140 120 0 5 10 15 20 25 Time min Figure 39 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 15 on page 98 illustrates this principle using a PNA analysis The match factor given in the report for each peak indicates the degree of similarity between the reference spectrum and the spectra from a peak A match factor of 1 000 means identical spectra Agilent 1260 FLD User Manual 97 4 Using the Fluorescence Detector In addition the purity of a peak can be investigated by comparing spectra obtained within a single peak Wh
82. e 64 Installing Capillaries 68 4 Using the Fluorescence Detector 75 Leak and Waste Handling 76 Before You Start 77 Getting Started and Checkout 78 Method Development 82 Example Optimization for Multiple Compounds 99 How to collect spectra with modes SPECTRA ALL IN PEAK and APEX SPECTRA ONLY 109 Solvent Information 113 Agilent 1260 FLD User Manual Contents Optimizing the Detector 119 Optimization Overview 120 Design Features Help Optimization 122 Finding the Best Wavelengths 123 Finding the Best Signal Amplification 125 Changing the Xenon Flash Lamp Frequency 131 Selecting the Best Response Time 133 Reducing Stray Light 135 Troubleshooting and Diagnostics 137 Overview of the Module s Indicators and Test Functions 138 Status Indicators 139 User Interfaces 141 Agilent Lab Advisor Software 142 Error Information 143 What Are Error Messages 145 General Error Messages 146 Detector Error Messages 151 Test Functions 157 Introduction 158 Diagram of Light Path 159 Lamp Intensity Test 160 Raman ASTM Signal to Noise Test 162 Using the Built in Test Chromatogram 166 Wavelength Verification and Calibration 168 Wavelength Accuracy Test 171 Wavelength Calibration Procedure 177 Agilent 1260 FLD User Manual Contents 9 Maintenance 183 Introduction to Maintenance 184 Warnings and Cautions 185 Overview of Maintenance 187 Cleaning the Module 188 Exchanging a Flow Cell 189 How to use the Cuvette 193
83. e PMT gain test 2 Using an appropriate response time For most applications a setting of 4 seconds is adequate see Selecting the Best Response Time on page 133 Only for high speed analyses short columns at high flow rates a lower setting is recommended Bear in mind that even if the response time is too high fast peaks will appear a little smaller and broader but retention time and peak areas are still correct and reproducible Agilent 1260 FLD User Manual Optimizing the Detector 5 3 Finding the optimum wavelength Most fluorescent active molecules absorb at 230 nm see Finding the Best Wavelengths on page 123 Set the excitation wavelength to 230 nm and on line scan the emission spectra multi emission mode Then set the determined emission wavelength and perform a multi excitation scan multi excitation mode to find the best excitation wavelength 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
84. e are summarized in Table 12 on page 91 Table 12 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 Agilent 1260 FLD User Manual 91 4 92 Using the Fluorescence Detector Procedure III Make a single run with a 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 The example is taken from the quality control of carbamates Samples are analyzed for the impurities 2 3 diaminophenazine DAP and 2 amino 3 hydroxyphenazine AHP Reference samples of DAP and AHP were analyzed with diode array and fluorescence detection Table on page 93 shows the spectra obtained from both detectors fo
85. e for RS 232C communication The number 0 means that the switch is down and 1 means that the switch is up Agilent 1260 FLD User Manual Hardware Information 12 Table 39 Baudrate Settings without on board LAN Switches Baud Rate Switches Baud Rate 3 4 5 3 4 5 0 0 0 9600 1 0 0 9600 0 0 1 1200 1 0 1 14400 0 1 0 2400 1 1 0 19200 0 1 1 4800 1 1 1 38400 Table 40 Data Bit Settings without on board LAN Switch 6 Data Word Size 0 7 Bit Communication 1 8 Bit Communication Table 41 Parity Settings without on board LAN Switches Parity 7 8 0 0 No Parity 0 1 Odd Parity 1 1 Even Parity One start bit and one stop bit are always used not selectable Per default the module will turn into 19200 baud 8 data bit with no parity Agilent 1260 FLD User Manual 241 12 Hardware Information Special Settings The special settings are required for specific actions normally in a service case Boot Resident Firmware update procedures may require this mode in case of firmware loading errors main firmware part If you use the following switch settings and power the instrument up again the instrument firmware stays in the resident mode It is not operable as a module It only uses basic functions of the operating system for example for communication In this mode the main firmware can be loaded using update utilities Table 42 Boot Resident Settings without on board LAN
86. e high pressure flow path after sample introduction loop needle seat capillary through the thermostatted column compartment heat exchangers to the column Such capillaries need to be installed carefully in order to keep them tight without damaging them by over tightening Handling of stainless steel cladded PEEK capillaries Be careful when installing stainless steel cladded PEEK capillaries The correct torque must be applied to avoid leaks potentially causing measurement problems or damage to the capillary gt Follow the procedure below for a correct installation 256 Installation procedure The amount of force torque needing to be applied to install the capillary depends on the female connector to which the capillary is installed and whether the material of that connector is soft or hard Compared with hard connectors a greater tightening angle is required for soft connectors to achieve same torque e whether the capillary is installed for the first time or subsequent times For the first time a greater tightening angle needs to be applied The installation consists of two steps In the first step the fitting is installed finger tight without using tools Finger tight means that the fitting will grip and hold the capillary This brings the fitting to the appropriate start position marked as 0 below for the second step Agilent 1260 FLD User Manual Appendix 13 First Step Finger tight Fitting 1 Tighten the
87. e setting Agilent 1260 FLD User Manual 125 5 Optimizing the Detector FLD Scaling Range and Operating Conditions When using different FLD e The signal height of individual G1321 FLD modules may exceed the recommended signal range 0 100 LU Under certain circumstances this could lead to clipped peaks Different G1321 FLD modules show different signal heights with identical methods This is not a problem in general but could be inconvenient when operating more than one G1321 FLD in the lab Both scaling issues can be resolved Refer to Optimize the PMT Gain Level on page 126 Optimize the PMT Gain Level Start the PMT Gain Test with your operating conditions used method parameter EX EM wavelength solvent flow rate The resulting PMT value will give you the best signal to noise performance with the maximum usable signal range for this method and this specific instrument For another FLD this PMT level may vary based on the individual PMT Gain Test The figure below demonstrates the impact of changing the PMT Gain 126 Agilent 1260 FLD User Manual Optimizing the Detector 5 PMT Behaviour of FLD Noise LU Signal LU SIN 1000 00 1000 100 00 100 10 00 S useable PMT range gt 1 00 E 10 0 10 0 01 EE EES OW 25734 5 46 T S 9 10 112 AS 44S AG Aly 18 PMT Gain Signal here is the Peak Height of the highe
88. e 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 Available tables Lamp Intensity History Reference Diode Counts at 250nm Reference Diode Counts at 350nm Reference Diode Counts at 450nm Reference Diode Counts at 600nm 01 28 2013 14 15 2143 2994 7166 3150 12 17 2012 13 55 10 9 9 9 12 17 2012 13 55 9 9 1 10 12 17 2012 13 49 10 n 10 10 10 29 2012 16 48 388 2120 5776 2766 12 08 2011 10 39 88 1004 1227 935 12 06 2011 11 31 576 2155 5532 2678 Figure 58 Lamp Intensity History Agilent Lab Advisor under Module Info Agilent 1260 FLD User Manual 161 8 Test Functions Raman ASTM Signal to Noise Test This test verifies the Raman ASTM signal to noise for the G1321 FLD detectors Intensity LU ka r T T r F j R H z Zz Signal Part 1 Preparation d Part2 Raman and Noise Part 3 Noise EM d Part 4 se grating E Part 5 Dark Current Time min Figure 59 Raman ASTM Signal to Noise Test Lab Advisor Depending on the version of the detector the specification has changed Table 23 Raman ASTM Signal to Noise Specification Instrument SNR Specification SNR Specification Comment Raman Dark Dual WL G
89. ectronic boards components of the module are placed This pack is then housed in a metal inner cabinet which is enclosed by a plastic external cabinet The advantages of this packaging technology are e virtual elimination of fixing screws bolts or ties reducing the number of components and increasing the speed of assembly disassembly e the plastic layers have air channels molded into them so that cooling air can be guided exactly to the required locations 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 244 Agilent 1260 FLD User Manual Agilent 1260 FLD User Manual 13 Appendix General Safety Information 246 The Waste Electrical and Electronic Equipment WEEE Directive 2002 96 EC 249 Lithium Batteries Information 250 Radio Interference 251 Sound Emission 252 UV Radiation UV lamps only 253 Solvent Information 254 Installation of Stainless Steel Cladded PEEK Capillaries 256 First Step Finger tight Fitting 257 Second Step Installation to Connector 257 Removing Capillaries 261 Agilent Technologies on Internet 262 This chapter provides safetey and other general information ate Agilent Technologies 245 13 Appendix General Safety Information Safety Symbols Table 44 Safety Sym
90. eement 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 If software is for use in the performance of a U S Government prime contract or subcon tract Software is delivered and licensed as Commercial computer software as defined in DFAR 252 227 7014 June 1995 or as a commercial item as defined in FAR 2 101 a or as Restricted computer soft ware as defined in FAR 52 227 19 June 1987 or any equivalent agency regulation or contract clause Use duplication or dis closure of Software is subject to Agilent Technologies standard commercial license terms and non DOD Departments and Agencies of the U S Government will receive no greater than Restricted Rights as defined in FAR 52 227 19 c 1 2 June 1987 U S Government users will receive no greater than Limited Rights as defined in FAR 52 227 14 June 1987 or DFAR 252 227 7015 b 2 November 1995 as applicable in any technical data Safety Notices CAUTION A CAUTION 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 damage to t
91. ehavior append free zero spectral parameters 46 Agilent 1260 FLD User Manual Table 6 G1321A Site Requirements and Specifications 2 Performance Specifications Agilent 1200 Series Fluorescence Detector Type Specification Comments Spectrum acquisition Wavelength characteristic Flow cells Control and data evaluation Analog outputs 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 pL volume and 20 bar 2 MPa pressure maximum fused silica block Optional Fluorescence cuvette for offline spectroscopic measurements with 1 mL syringe 8 pL volume Bio inert 8 uL volume and 20 bar 2 MPa pressure maximum pH 1 12 e Micro 4 uL volume and 20 bar 2 MPa pressure maximum Agilent ChemStation for LC Agilent Instant Pilot G4208A with limited spectral data analysis and printing of spectra 100 LU is the recommended range see FLD Scaling Recorder integrator 100 mV or 1 V output range gt 100 LU two outputs Range and Operating Conditions Communications Controller area network CAN RS 232C LAN APG Remote ready start stop and shut down signals 47 Agilent 1260 FLD User Manual 2 48 Site Requirements and Specifications Table 6 G1321A Performance Specifications Agilent 1200 Series
92. elength 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 module to a maximum of 250 nm The excitation grating is blazed for highest efficiency at 300 nm Agilent 1260 FLD User Manual 123 5 124 Optimizing the Detector A Real Example Although an excitation wavelength of 340 nm is quoted in the literature the module scan of orthophthalaldehyde a derivative of the amino acid alanine Figure 48 on page 124 shows a maximum between 220 nm and 240 nm LC Z 1046A EX of AAYHS1IGL D ALA Lc Z 1046A EM of AAZHSIGL D Scaled 500 Emission 200 400 Excitation Wavelength nm Figure 48 Scan Orthophthalaldehyde Derivative of Alanine When you are looking for the wavelength by scanning scan over the whole range As this example shows a maximum may be found in a completely different wavelength range When comparing fluorescence excitation spectra directly with DAD spectra or literature based absorbance spectra you should consider large differences in the used optical bandwidth FLD 20 nm which cause a systematic wavelength maximum shift depending on the absorbance spectrum of the compound under evaluation Agilent 1260 FLD User Manual Optimizing the Detector 5 Finding the Best Signal Amplificat
93. en 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 15 on page 98 shows an automated library search based on the emission spectra from a PNA reference sample Table 15 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 1 993 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 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 9
94. ent and designed to match the wall socket of a particular country or region Absence of ground connection or use of unspecified power cord The absence of ground connection or the use of unspecified power cord can lead to electric shock or short circuit gt Never operate your instrumentation from a power outlet that has no ground connection gt Never use a power cord other than the Agilent Technologies power cord designed for your region Use of unsupplied cables Using cables not supplied by Agilent Technologies can lead to damage of the electronic components or personal injury gt Never use cables other than the ones supplied by Agilent Technologies to ensure proper functionality and compliance with safety or EMC regulations Unintended use of supplied power cords Using power cords for unintended purposes can lead to personal injury or damage of electronic equipment gt Never use the power cords that Agilent Technologies supplies with this instrument for any other equipment Agilent 1260 FLD User Manual 37 2 Site Requirements and Specifications Bench Space The module dimensions and weight see Table 3 on page 39 allow you to place the module 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 8 1 inches in the rear for air circulation and electric connections If the bench shall carry a complete HPLC system make sure that
95. ent labels provide the following information CCYWWSSSSS Format CC country of manufacturing DE Germany e JP Japan e CN China YWW year and week of last major manufacturing change e g 820 could be week 20 of 1998 or 2008 SSSSS real serial number Agilent 1260 FLD User Manual 231 12 Hardware Information Interfaces The Agilent 1200 Infinity Series modules provide the following interfaces Table 34 Agilent 1200 Infinity Series Interfaces Module CAN LAN BCD LAN optional on board RS 232 Analog APG Remote Special Pumps G1310B Iso Pump 2 Yes No G1311B Quat Pump G1311C Quat Pump VL G1312B Bin Pump K1312B Bin Pump Clinical Ed G1312C Bin Pump VL 1376A Cap Pump G2226A Nano Pump G5611A Bio inert Quat Pump G4220A B Bin Pump 2 No Yes G4204A Quat Pump G1361A Prep Pump 2 Yes No Samplers G1329B ALS 2 Yes No G2260A Prep ALS G1364B FC PS 2 Yes No G1364C FC AS G1364D FC uS G1367E HiP ALS K1367E HiP ALS Clinical Ed G1377A HiP micro ALS G2258A DL ALS G5664A Bio inert FC AS G5667A Bio inert Autosampler G4226A ALS 2 Yes No Yes Yes Yes Yes Yes Yes No No No No No Yes Yes Yes Yes Yes Yes CAN DC OUT for CAN slaves CAN DC OUT for CAN slaves THERMOSTAT for G1330B K1330B THERMOSTAT for G1330B K1330B CAN DC OUT for CAN slaves 232 Agilent 1260 FLD User Manual Hardware Information 12 Table 34 Agilent 1200 Infini
96. es a rising falling baseline at begin end of the peak In case PDPW is too big the peak detector assumes that the peak is noise The peak detector works online on the current chromatogram This means that begin apex end of a peak is recognized with delay Additionally the points of spectra are sequentially acquired This means that the acquisition of wide range spectra lasts much longer than the acquisition of a short range spectrum When you have a fast chromatography it is nearly impossible to collect a clean APEX spectra the first last points of the spectra are acquired before after you have the highest concentration in the detector s cell How long the acquisition of single spectra lasts is shown in the FLD s setup screen FLD Signals FLD_1563 xl Signal Time r Multiple Wavelengths and Spectra Excitation A Emission Stoptime no Limit 4 min C Off MultiEx Multi Em 250 mm 1410 nm Use additional Excitation Posttime Off 4 min E a Zero Order C Zero Order E Sipin CT 4250 nm D 7 250 rn Timetable 3 a Acquire Excitation Spectra Line Time Em Ex A Ex B Ex C Ex D Ex Spectra From Range 230 to 380 nm 5 nm Time Spectrum 837 ms m Peakwidth Responsetine gt 0 2 min 4 s standard z r lt 0 005 min lt 0 12 s gt 0 005 min 0 12 s gt 0 010 min 0 25 s gt 0 025 min 0 5 s gt 0 05 min 1 s fast Step st Insert Append ut T
97. es and the different user interfaces Agilent 1260 FLD User Manual 3 In This Guide 7 Error Information This chapter describes the meaning of error messages and provides information on probable causes and suggested actions how to recover from error conditions 8 Test Functions This chapter describes the detector s built in test functions 9 Maintenance This chapter provides general information on maintenance of the detector 10 Parts for Maintenance This chapter provides information on parts for maintenance 11 Identifying Cables This chapter provides information on cables used with the Agilent 1200 Infinity Series modules 12 Hardware Information This chapter describes the detector in more detail on hardware and electronics 13 Appendix This chapter provides safetey and other general information Agilent 1260 FLD User Manual Contents Contents 1 Introduction to the Fluorescence Detector 9 Introduction to the Detector 10 How the Detector Operates 12 Raman Effect 15 Optical Unit 16 Analytical Information From Primary Data 24 System Overview 29 Bio inert Materials 32 2 Site Requirements and Specifications 35 Site Requirements 36 Physical Specifications 39 Performance Specifications 40 3 Installing the Module 49 Unpacking the Module 50 Optimizing the Stack Configuration 52 Installation Information on Leak and Waste Handling 57 Installing the Module 61 Flow Connections to the Modul
98. ese wavelengths and the quantum yield is high Excitation is sufficient for collecting emission spectra Table on page 90 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 Table 10 Timetable for PNA analysis 0 min 350 nm for naphthalene to phenanthrene 8 2 min 420 nm for anthracene to benzo g h i perylene 19 0 min 500 nm for indeno 1 2 3 c d 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 36 on page 91 The area of high intensity red is caused by stray light when emission spectra overlap with the excitation wavelength This can be avoided by fitting the spectral range automatically Excitation at 260 nm is most appropriate for all PNAs Agilent 1260 FLD User Manual Using the Fluorescence Detector 4 Table 11 Conditions for Optimization of PNA analysis according to figures below Column Mobile phase Gradient Flow rate Column temperature Injection volume FLD settings Vydac 2 1 x 200 mm PNA 5 pm A water B acetonitrile 50 50 3 minutes 60 14 minutes 90 22 minutes 100 0 4 ml min 18 C 5 ul PMT 12 respo
99. essing of Raw Data 25 System Overview 29 Leak and Waste Handling 29 Bio inert Materials 32 This chapter gives an introduction to the detector and instrument overview Apg Agilent Technologies 1 Introduction to the Fluorescence Detector Introduction to the Detector 10 Detector Versions Table 1 Detector versions Version Description G1321C G1321B SPECTRA G1321A Introduced as 1260 Infinity FLD without spectra and multi signal capabilities in June 2013 Maximum data rate is 74 Hz Instrument firmware is A 06 54 Controlled by Instant Pilot with firmware B 02 16 Driver A 02 08 Agilent OpenLAB CDS ChemStation Edition C 01 05 OpenLAB EZChromEdition EE A 04 05 ICF A 02 01 and Lab Advisor B 02 04 The G1321C cannot be converted to G1321A B Introduced as 1260 Infinity FLD with spectra and multi signal capabilities in June 2010 Maximum data rate is 74 Hz The G1321B can be converted to G1321A emulation mode With the introduction of the G1321C the data rate was increased to maximum 144 9 Hz instrument firmware A 06 54 Introduced as 1100 Series FLD with spectra and multi signal capabilities in August 1998 Maximum data rate is 18 Hz Obsoleted with introduction of the G1321B FLD The detector is designed for highest optical performance GLP compliance and easy maintenance It includes the following features e flash lamp for highest intensity and lowest detection limit e multi wavelength mode for on
100. ested actions 1 Lamp cover removed Please contact your Agilent service representative FLF Board not found Error ID 6620 6730 The FLF board could not be found by the main board FLM This message comes together with some other message generated on the FLF board e g Leak Probable cause Suggested actions 1 FLF board not connected to the FLM board Please contact your Agilent service representative 2 Defective FLF board Please contact your Agilent service representative 3 Defective FLM board Please contact your Agilent service representative Agilent 1260 FLD User Manual 151 7 7 152 Error Information ADC Not Calibrated Error ID 6621 6732 The analog to digital converter located on the FLF board cannot calibrate Probable cause Suggested actions 1 Defective ADC or other FLF electronics Please contact your Agilent service representative A D Overflow Error ID 6618 6619 This message is not implemented in firmware revision A 03 66 and below It indicates an overload situation of the A D converter sample signal The user interface will show a not ready condition for the FLD and an info event is written into the logbook If the message comes up during a run it includes the time of occurrence and when it disappears 1200 FLD 1 A D overflow RT is 0 32 min 16 33 24 02 11 99 1200 FLD 1 A D overflow finished RT is 0 67 min 16 33 46 02 11 99 If this condition is present prior to a run
101. face to other modules e LAN connector as interface to the control software e RS 232C as interface to a computer e REMOTE connector as interface to other Agilent products e Analog output connector s for signal output 234 Agilent 1260 FLD User Manual Hardware Information 12 Overview Interfaces CAN The CAN is inter module communication interface It is a 2 wire serial bus system supporting high speed data communication and real time requirement LAN The modules have either an interface slot for an LAN card e g Agilent G1369B C LAN Interface or they have an on board LAN interface e g detectors G1315C D DAD and G1365C D MWD This interface allows the control of the module system via a PC with the appropriate control software Some modules have neither on board LAN nor an interface slot for a LAN card e g G1170A Valve Drive or G4227A Flex Cube These are hosted modules and require a Host module with firmware B 06 40 or later or with additional G1369C LAN Card If an Agilent detector DAD MWD FLD VWD RID is in the system the LAN should be connected to the DAD MWD FLD VWD RID due to higher data load If no Agilent detector is part of the system the LAN interface should be installed in the pump or autosampler RS 232C Serial The RS 232C connector is used to control the module from a computer through RS 232C connection using the appropriate software This connector can be configured with the configuration switch mod
102. for Figure 34 on page 87 All excitation and emission spectra of Quinidine 1 ug 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 86 Agilent 1260 FLD User Manual straylight 1 order 350 nm Ex 315 nm Ex C x 440 00 nmfE y 250 00 nm z 1 87 LU X Cube o 02 D4 DE os 1 t2 14 16 13 2 28 24 25 Ox Oy z 250 nm Ex Expand z Data view Projection Print oe Ex axis Em axis Figure 34 Characterization of a pure compound from a fluorescence scan 4 88 Using the Fluorescence Detector Procedure Il Take two LC runs with the FLD The conditions for the separation of organic compounds such as polyaromatic nuclear hydrocarbons PNAs are well described in various standard methods including commonly used EPA and DIN methods Achieving the best detection levels requires checking for the optimum excitation and emission wavelengths for all compounds Yet taking fluorescence scans individually makes this a tedious process A better approach is to acquire spectra online for all compounds during a run This speeds up method development tremendously Two runs are sufficient for optimization During the first run one wavelength is chosen in the low UV range for the excitation wavelength and one emission wavelength in the spectral range for the emission wavelength Most fluorophores show strong absorption at th
103. for contamination service Y check alignment of lamp trigger pack assembly service V perform a Wavelength Calibration Agilent 1260 FLD User Manual 173 Test Functions Using the Agilent ChemStation Manually 1 Create the methods WLEMTEST and WLEXTEST as listed Table 30 on page 174 Table 30 Method Settings Setting Check of EM WL 397 nm Check of EX WL 350 nm WLEMTEST WLEXTEST Peak Width gt 0 2 min 4 s standard gt 0 2 min 4 s standard Fit Spectral Range OFF OFF PMT Gain 12 12 Flash Lamp ON ON Spectrum Range Store Spectra EX Wavelength EM Wavelength Multi WL Settings EM 367 417 nm step 1 nm All w o signal 350 nm ON 397 nm OFF Multi EM EX 330 380 nm step 1 nm All w o signal 350 nm OFF 397 nm ON Multi EX FLD Special Setpoints System 2 m Phosphorescence Detection Mode Of On Delay Gate m Fluorescence Scan Range From To Step Excitation 220 feo Bo nm Emission fao 500 Bo nm Time Scan 137 s x Baseline Behavior Append C Free C Zero Signal Polarity Positive C Negative J Fit Spectral Range m Lamp I Only On During Run I Economy Mode now 74 Hz High Lamp Current J Enable analysis when lamp is off Lamp Energy Reference On Off Restore Defaults Cancel Help Agilent 1260 FLD User Manual Test Functions 8 Wavelength Accuracy Test Figure 68 Special Setpoints Settings
104. ggles safety gloves and protective clothing as described in the material handling and safety data sheet supplied by the vendor and follow good laboratory practice gt The volume of substances should be reduced to the minimum required for the analysis gt Do not operate the instrument in an explosive atmosphere gt Never exceed the maximal permissible volume of solvents 6 L in the solvent cabinet gt Do not use bottles that exceed the maximum permissible volume as specified in the usage guideline for the Agilent 1200 Infinity Series Solvent Cabinets gt Arrange the bottles as specified in the usage guideline for the solvent cabinet gt A printed copy of the guideline has been shipped with the solvent cabinet electronic copies are available on the Internet gt The residual free volume in the appropriate waste container must be large enough to collect the waste liquid gt Check the filling level of the waste container regularly gt To achieve maximal safety check the correct installation regularly 76 Recommendations for Solvent Cabinet For details see the usage guideline for the Agilent 1200 Infinity Series Solvent Cabinets For details on correct installation see Installation Information on Leak and Waste Handling on page 57 Agilent 1260 FLD User Manual Using the Fluorescence Detector 4 Before You Start Your normal LC grade solvents usually give good results most of the time But e
105. gth detection 95 take a fluorescence scan 85 mirror 16 monochromator EM 20 16 EX 18 16 multi wavelength detection 95 non operating altitude 39 non operating temperature 39 0 off line measurements 10 operating Altitude 39 operating temperature 39 operation of the detector 12 Agilent 1260 FLD User Manual optical unit overview 16 optimization example 99 stack configuration 52 P packaging damaged 50 parts identification accessory kit 204 cables 207 overview 202 parts damaged 51 missing 51 peakwidth settings 134 peakwidth selecting 133 40 43 46 phosphorescence detection 25 photoluminescence 12 photo multiplier tube location of PMT 16 PMT 21 physical specifications 39 PMT gain test 120 gain 125 21 photo multiplier tube 21 range 27 power consideration 36 performance specifications power consumption 39 power cords 37 power supply indicator 139 radio interference 251 Raman S N test 162 Raman 15 Agilent 1260 FLD User Manual recalibration of wavelength 138 159 reference diode 23 reference system 23 23 remote cable 212 repairs cautions and warnings 185 correction leaks 195 exchanging a flow cell 189 of the detector 183 replacing firmware 198 replacing leak handling system 196 replacing interface board BCD LAN 197 response time settings 134 response time selecting 133 responsetine 27 RS 232C cable 219 communication settings 240 S safety class 247 safety infor
106. gures above Column Zorbax SB 2 x 50 mm PNA 5 pm Mobile phase A water B acetonitrile Gradient 0 minutes 5 10 minutes 15 Flow rate 0 4 ml min Column temperature 35 C Injection volume 5 ul FLD settings PMT 12 response time 4 s step size 5 nm Ex 265 nm and 430 nm Em 540 nm 94 Agilent 1260 FLD User Manual Using the Fluorescence Detector 4 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 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 Multi wavelength detection Time programmed wavelength switching traditionally is used to achieve low limits of detection and high selectivity in routine quantitative analysis Such switching is difficult if compounds elute closely and require a change in excitation or emission wavelength Peaks can be distorted and quantitation made impossible if wavelength switching occurs during the elution of a compound Very often this happens with complex matrices influencing the retention of compou
107. he product or loss of important data Do not proceed beyond a CAUTION notice until the indicated condi tions 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 performed or adhered to could result in personal injury or death Do not proceed beyond a WARNING notice until the indi cated conditions are fully under stood and met Agilent 1260 FLD User Manual In This Guide In This Guide This manual covers e the Agilent 1260 Infinity Fluorescence Detector G1321B SPECTRA e the Agilent 1260 Infinity Fluorescence Detector G1321C and e the Agilent 1200 Series Fluorescence Detector G1321A obsolete 1 Introduction to the Fluorescence Detector This chapter gives an introduction to the detector and instrument overview 2 Site Requirements and Specifications This chapter provides information on environmental requirements physical and performance specifications 3 Installing the Module This chapter gives information about the preferred stack setup for your system and the installation of the module 4 Using the Fluorescence Detector This chapter guides you how to start the work with the detector 5 Optimizing the Detector This chapter provides information on how to optimize the detector 6 Troubleshooting and Diagnostics This chapter gives an overview about the troubleshooting and diagnostic featur
108. imensions 140 mm x 345 mm x 435 mm 5 5 x 13 5 x 17 inches height x width x depth Weight 11 5 kg 25 5 Ibs Agilent 1260 FLD User Manual 45 2 Site Requirements and Specifications Table6 Performance Specifications Agilent 1200 Series Fluorescence Detector G1321A Type Specification Comments Detection type Multi signal fluorescence detector with Performance specifications Light source Pulse frequency Maximum data rate Excitation monochromator Emission monochromator Reference system Timetable programing rapid on line scanning capabilities and spectral data analysis Single wavelength operation see note below this RAMAN H370 gt 500 noise reference table measured at signal see Service Manual for details Ex 350 nm Em 397 nm dark value 450 nm standard flow cell Dual wavelength operation RAMAN H20 gt 300 Ex 350 nm Em 397 nm and Ex 350 nm Em 450 nm standard flow cell Xenon Flash Lamp normal mode 20 W economy mode 5 W lifetime 4000 h 296 Hz for single signal mode 74 Hz for economy mode 37 Hz Range settable 200 nm 1200 nm and zero order Bandwidth 20 nm fixed Monochromator concave holographic grating F 1 6 blaze 300 nm Range settable 200 nm 1200 nm and zero order Bandwidth 20 nm fixed Monochromator concave holographic grating F 1 6 blaze 400 nm in line excitation measurement up to 4 signal wavelengths response time PMT Gain baseline b
109. increased by switching it on just for analysis In contrast to other LC detectors the fluorescence detector equilibrates within seconds after the lamp is switched ON For highest reproducibility and linearity change the lamp setting to always ON default is on only during run One hour of initial warm up of the instrument is recommended 6 Do not overpressurize the detector 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 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 Agilent 1260 FLD User Manual 121 5 Optimizing the Detector Design Features Help Optimization 122 The module has several features you can use to optimize detection PMTGAIN Amplification factor LAMP Flash frequency RESPONSETIME Data reduction interval Check Performance Before You Start Before you start you should check that your detector is performing according to the specifications published by Agilent Technologies Your normal LC grade solvents may give good results most of the time but our experience shows th
110. ing Agilent 1260 FLD User Manual 69 3 Installing the Module Installing Capillaries 2 Insert the fitting to the receiving port and push the capillary to the bottom of the port 3 Finger tighten the nut into the port until snug 70 Agilent 1260 FLD User Manual Installing the Module 3 Installing Capillaries 4 Use Fitting mounting tool 5043 0915 or a 5 mm hex wrench for fixing the fitting maximum torque 0 8 Nm CAUTION Potential damage of capillaries gt Do not remove fittings from used capillaries Agilent 1260 FLD User Manual 71 3 72 Installing the Module 5 When using UHP FF fittings with bioinert capillaries do not try to remove fittings from these capillaries Bio inert capillaries are using a PEEK front end which may expand under pressure especially when being in contact with some organic solvents If a fitting is moved across an expanded PEEK end there is a risk of damaging the capillary by ripping off its end Before re installing such capillaries push the ferrule towards the rear site for a small distance Rear Front Figure 28 Capillary fitting Agilent 1260 FLD User Manual Installing the Module 3 Installing Capillaries Installation of the Bio inert Zero Dead Volume ZDV Union The Bio inert ZDV p n 5067 4741 union has two different connectors where capillaries need to be installed in the correct sequence Otherwise an inset of the union may be damaged and the conne
111. ion 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 49 on page 125 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_PMT3 PMT_0001 0 FLD1 A Ex 246 Em 317 FLD_PMT3 PMT_0002 D FLD1 A Ex 248 Em 317 FLD_PMT3 PMT_0003 0 FLD1 A Ex 246 Em 317 FLD_PMT3 PMT_0004 D FLD1 A Ex 248 Em 317 FLD_PMT3 PMT_0005 0 FLD1 A Ex 246 Em 317 FLD_PMT3 PMT_0006 D i PMT ji 104 im 1 z eS PER ee pes 10 So E N E A E I AE E 9 A e Mtl act a las 8 28 seer ea 6 4 Figure 49 Finding Best PMTGAIN for Biphenyl The reason for this is the fact that quantification of baselines especially at low background levels is not sufficient for statistically working filter methods For the best gain check your solvent under flow conditions with the auto gain function Do not use higher values than proposed by the system if not necessary because of excessive high fluorescence signals Use the PMT test to automatically determine th
112. 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 Agilent 1260 FLD User Manual 67 3 Installing the Module Installing Capillaries Figure 26 In May 20138 Agilent has introduced new UHP FF fittings which are designed for improved robustness and ease of use Previous fittings require careful handling Therefore it is important to know which fittings are used in the system The figure below illustrates the differences between new and previous capillaries 1 4 inch fitting New bio inert capillary and UHP FF fitting Figure 27 Previous bio inert capillary and fitting with nose For handling instructions of capillaries and fittings used in modules before delivery of the new UHP FF fittings introduced in May 2013 refer to Installation of Stainless Steel Cladded PEEK Capillaries on page 256 To work on bio inert capillaries produced before May 2013 you will need a1 4 inch wrench instead of the 5 mm mounting tool Agilent 1260 FLD User Manual Installing the Module 3 Installing Capillaries Installing UHP FF Fittings Tools required p n Description 5043 0915 Fitting mounting tool for bio inert capillaries Parts required p n Description Capillaries and For details refer to the part section of the manual Fittings 1 Slide the fitting on the capillary Let the capillary jut out 5 mm Capillary Fitt
113. is stable in the presence of acids and bases in a pH range of 1 to 12 5 It can be corroded by acids below pH 2 3 It can also corrode in following solvents Solutions of alkali halides their respective acids for example lithium iodide potassium chloride and so on and aqueous solutions of halogens e High concentrations of inorganic acids like nitric acid sulfuric acid and organic solvents 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 2 CHCl Oo 2 COCl 2 HCl This reaction in which stainless steel probably acts as a catalyst occurs quickly with dried chloroform if the drying process removes the stabilizing alcohol e Chromatographic grade ethers which can contain peroxides for example THF dioxane di isopropylether Such ethers should be filtered through dry aluminium oxide which adsorbs the peroxides e Solutions of organic acids acetic acid formic acid and so on in organic 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 Mixtures of carbon tetrachloride with 2 propanol or THF Agilent 1260 FLD User Manual 115 4 116 Using the Fluorescence Detector Diamon
114. ith Wavelength Calibration Failed see Wavelength Calibration Failed on page 154 If you encounter calibration problems 1 Check for air bubbles in the flow cell 2 Flush the flow cell with isopropanol 3 Change the water 170 Agilent 1260 FLD User Manual Test Functions 8 Wavelength Accuracy Test Using the Agilent Lab Advisor Set up the HPLC system and the Agilent Lab Advisor Flush the flow cell with clean bi distilled water Turn on the FLD lamp Run the Wavelength Accuracy Test oF Ww N The FLD will change into the multi excitation mode with emission wavelength at 397 nm and scan in the range of the expected maximum of 350 nm 20 nm As result the maxima should be found at 350 nm 3 nm see Figure 64 on page 171 The FLD will change into the multi emission mode with excitation wavelength at 350 nm and scan in the range of the expected maximum of 397 nm 20 nm As result the maxima should be found at 397 nm 3 nm see Figure 64 on page 171 FLO1 0 765 0 7 LU Ex Em 397 of WLEXSO3 D FLD1 0 697 29 4 LU Em Ex 350 of WLEMSO3 0 tu EX 350 nm fixed ace 3 nm NJ 0 370 380 390 40 410 nm Figure 64 Excitation and Emission Spectrum expected results Agilent 1260 FLD User Manual 171 Test Functions Wavelength Accuracy Test NOTE If the plots do not have a maximum around EM 397 nm and EX 350 nm 3 nm the test fails Refer to Interpretation of the Results on page 173
115. l Hardware Information 12 Optional Interface Boards BCD External Contact Board The Agilent 1200 Infinity Series modules have one optional board slot that allows to add an interface board to the modules Some modules do not have this interface slot Refer to Interfaces on page 232 for details Optional Interface Boards p n Description G1351 68701 Interface board BCD with external contacts and BCD outputs 2110 0004 Fuse for BCD board 250 mA The BCD board provides a BCD output for the bottle number of the Agilent 1200 Series autosampler and four external contacts The external contact closure contacts are relay contacts The maximum settings are 30 V AC DC 250 mA fused Board identification 12 Processor BCD BCD interface register connector Line driver RFI filter 250mA External 4x External contacts lt contact connector DRF filter Agilent 1260 FLD User Manual 225 12 Hardware Information There are general purpose cables available to connect the BCD output see BCD Cables on page 215 and the external outputs see External Contact Cable on page 218 to external devices Table 32 Detailed connector layout 1200 Pin Signal name BCD digit 1 BCD 5 20 2 BCD 7 80 3 BCD 6 40 4 BCD 4 10 5 BCD 0 1 6 BCD 3 8 7 BCD 2 4 8 BCD 1 2 9 Digital ground 10 BCD 11 800 11 BCD 10 400 12 BCD 9 200 13 BCD 8 100 15 5V Low 226 Agilent 1260 FLD User Manual LAN
116. l Installing the Module 3 Installing the Module Parts required Software required Preparations Description Power cord For other cables see Cable Overview on page 208 Agilent Data System and or Instant Pilot G4208A Locate bench space Provide power connections Unpack the detector Module is partially energized when switched off as long as the power cord is plugged in Repair work at the module can lead to personal injuries e g shock hazard when the cover is opened and the module is connected to power gt Make sure that it is always possible to access the power plug gt Remove the power cable from the instrument before opening the cover gt Do not connect the power cable to the Instrument while the covers are removed 1 Install the LAN interface board in the detector if required see Replacing the Interface Board on page 197 2 Place the detector in the stack or on the bench in a horizontal position Agilent 1260 FLD User Manual 61 3 Installing the Module 3 Ensure the line power switch at the front of the detector is OFF Status indicator i green yellow red S g GA i eae OE DaKSa TE Line power switch with green light Figure 24 Front View of Detector 4 Connect the power cable to the power connector at the re
117. led triplet state T see Figure 4 on page 14 Agilent 1260 FLD User Manual 13 1 Introduction to the Fluorescence Detector spin change Ne Phosphorescence Figure 4 Phosphorescence Energy Transitions The molecule must change its spin back again before it can return to its ground state Since the chance of colliding with another molecule with the necessary spin for change is slight the molecule remains in its triplet state for some time During the second spin change the molecule loses more energy by relaxing without radiation The light which is emitted during phosphorescence therefore has less energy and is at a longer wavelength than fluorescence Formula where E Energy h Planck s constant A Wavelength c speed of light 14 Agilent 1260 FLD User Manual Introduction to the Fluorescence Detector 1 Raman Effect 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 Raman Scatter Raleigh Scatter new wavelength same wavelength as incident light Scattered Light Incident Light Figure 5 Raman The energy difference between the incident light E and the Raman scattered light E is eq
118. lementation of the Agilent ChemStation with the Fluorescence Detector G1321A B In these modes spectra intermittently are not collected into the data file The peak triggered spectra acquisition in the FLD is controlled by 2 parameters THRS Threshold and PDPW PeakDetector PeakWidth In addition the parameter PKWD Detector PeakWidth only influences the filtering of the chromatogram 1 Set the parameters THRS PDPW and PKWD accordingly to the current chromatogram Best results for collecting peak triggered spectra are gathered when PDPW is 2 steps lower than PKWD see Peakwidth Settings on page 134 Agilent 1260 FLD User Manual 109 4 Using the Fluorescence Detector How to collect spectra with modes SPECTRA ALL IN PEAK and APEX SPECTRA ONLY 2 In the FLD s setup screen there are 2 fields to enter the PKWD Peakwidth Responsetime and the THRS Threshold visible when Multi EX or Multi EM is selected Defaults are PKWD 6 0 2 min THRS 5 000 LU FLD Signals FLD_1563 a am E An a nit Shot lt lt eee gt 0 4 min 8s slow 110 Agilent 1260 FLD User Manual Using the Fluorescence Detector 4 The selected values are fixed during the run Changes of PDPW are only possible using the Peakwidth field in the Timetable visible when Multi EX or Multi EM is selected FLD Signals FLD_1563 x r Signal m Time m Multiple Wavelengths and Spectra Excitation A Emission Stoptime no Limit
119. lent 1260 FLD User Manual 145 7 Error Information General Error Messages 146 General error messages are generic to all Agilent series HPLC modules and may show up on other modules as well Error ID 0062 The timeout threshold was exceeded Probable cause 1 The analysis was completed successfully and the timeout function switched off the module as requested 2 Anot ready condition was present during a sequence or multiple injection run for a period longer than the timeout threshold Shutdown Error ID 0063 Suggested actions Check the logbook for the occurrence and source of a not ready condition Restart the analysis where required Check the logbook for the occurrence and source of a not ready condition Restart the analysis where required An external instrument has generated a shutdown signal on the remote line The module continually monitors the remote input connectors for status signals A LOW signal input on pin 4 of the remote connector generates the error message Probable cause 1 Leak detected in another module with a CAN connection to the system 2 Leak detected in an external instrument with a remote connection to the system 3 Shut down in an external instrument with a remote connection to the system 4 The degasser failed to generate sufficient vacuum for solvent degassing Suggested actions Fix the leak in the external instrument before restarting the module Fix
120. luorescence Detector Flash Lamp a Fluorescense Phosphorescence 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 s default which is equivalent to a time constant of 1 8 s and appropriate for standard chromatographical conditions Weak signals may cause errors in quantification because of insufficient resolution Check your proposed PMTGAIN If it is significantly distant from your setting change your method or check the purity of your solvent See also Finding the Best Signal Amplification on page 125 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 Agilent 1260 FLD User Manual Introduction to the Fluorescence Detector 1 Phosphorescence Fluorescense Figure 15 PMITGAIN 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
121. m iodide potassium chloride and so on High concentrations of inorganic acids like sulfuric acid and nitric acid especially at higher temperatures if your chromatography method allows replace by phosphoric acid or phosphate buffer which are less corrosive against stainless steel Halogenated solvents or mixtures which form radicals and or acids for example 2CHCls O gt 2COCI 2HC1 This reaction in which stainless steel probably acts as a catalyst occurs quickly with dried chloroform if the drying process removes the stabilizing alcohol 254 Agilent 1260 FLD User Manual Appendix 13 Chromatographic grade ethers which can contain peroxides for example THF dioxane di isopropylether such ethers should be filtered through dry aluminium oxide which adsorbs the peroxides Solvents containing strong complexing agents e g EDTA Mixtures of carbon tetrachloride with 2 propanol or THF Agilent 1260 FLD User Manual 255 13 Appendix Installation of Stainless Steel Cladded PEEK Capillaries CAUTION This installation procedure applies for capillaries and corresponding fittings used in modules delivered before January 2013 For current capillaries and fittings see Installing UHP FF Fittings on page 69 The 1260 Infinity Bio inert LC system uses PEEK capillaries that are cladded with stainless steel These capillaries combine the high pressure stability of steel with the inertness of PEEK They are used in th
122. m is switched off automatically at the end of a run Agilent 1260 FLD User Manual 167 Test Functions Wavelength Verification and Calibration 168 The wavelength calibration is based on a Glycogen solution which acts as a strong elastic light scatterer refer to ASTM Test Method E388 72 1993 Spectral Bandwidth and Wavelength Accuracy of Fluorescence Spectrometers The Glycogen solution is introduced into the flow cell and then the built in wavelength calibration functionality is used The algorithm is based on evaluating different grating orders and calculating the wavelength scales of both excitation and emission monochromator by applying the fundamental grating equation A complete wavelength calibration is not always required In most cases a quick wavelength accuracy verification is sufficient enough see Table 28 on page 168 Table 28 Reasons for doing a Verification or Calibration Verification WL calibration interest X GLP compliance X cell change X X lamp change X X monochromator change X main board change X optical unit change X X only required if deviation is too large Prior to a wavelength calibration a wavelength accuracy verification should be performed see Wavelength Accuracy Test on page 171 If the deviation is more than 3 nm the wavelength calibration should be done as described in Wavelength Calibration Procedure on page 177 Agilent 1260 FLD User Manual
123. mation lithium batteries 250 safety general information 247 standards 39 symbols 246 selecting peakwidth 133 response time 133 serial number information 231 231 settings peakwidth 134 response time 134 shutdown 146 Index site requirements 35 power cords 37 solvent information 254 solvents 254 sound emission 252 special interfaces 238 special settings boot resident 242 forced cold start 242 specification physical 39 specifications analog outputs 42 44 47 communications 42 44 47 flow cell 41 44 47 GLP features 42 45 48 monochromators 40 43 46 performance 40 43 46 pulse frequency 40 43 46 safety and maintenance 42 44 48 wavelength accuracy 40 spectra wavelength shift 121 stack configuration front view 55 rearview 56 status indicator 140 stray light 135 system setup and installation optimizing stack configuration 52 T temperature sensor 148 test chromatogram 166 test functions 138 159 tests functions 159 lamp intensity history 161 lamp intensity 160 PMT gaintest 120 265 Index Raman AST S N 162 test chromatogram 166 timeout 146 troubleshooting error messages 138 145 status indicators 139 138 U unpacking 50 UV degradation 18 160 V voltage range 39 W warnings and cautions 185 wavelength calibration procedure 168 177 wavelength calibration 168 wavelength shift of spectra 121 wavelength recalibration 138 159 weight 39 X xenon flash lamp 16 1
124. micals As an elastomer it may swell in some organic solvents like halogenated hydrocarbons TFE PDD copolymer tubings which are used in all Agilent degassers except G1322A are not compatible with fluorinated solvents like Freon Fluorinert or Vertrel They have limited life time in the presence of Agilent 1260 FLD User Manual Using the Fluorescence Detector 4 Hexafluoroisopropanol HFIP To ensure the longest possible life with HFIP it is best to dedicate a particular chamber to this solvent not to switch solvents and not to let dry out the chamber For optimizing the life of the pressure sensor do not leave HFIP in the chamber when the unit is off Sapphire Ruby and Al 03 based ceramics Sapphire ruby and ceramics based on aluminum oxide Al Og are inert to almost all common acids bases and solvents There are no documented incompatibilities for HPLC applications Agilent 1260 FLD User Manual 117 4 Using the Fluorescence Detector Solvent Information 118 Agilent 1260 FLD User Manual This chapter provides information on how to optimize the detector Agilent 1260 FLD User Manual 5 Optimizing the Detector Optimization Overview 120 Design Features Help Optimization 122 Check Performance Before You Start 122 Finding the Best Wavelengths 123 A Real Example 124 Finding the Best Signal Amplification 125 FLD Scaling Range and Operating Conditions 126 Changing the Xenon Flash Lamp Frequency 131 Lamp Life Savings 132
125. n 180 red arrow could damage the capillary possibly leaky D 180 recommended range Second and subsequent installations of a capillary to a hard connector 1 When tightening the fitting for the second and subsequent times again start from the finger tight position which is not necessarily a vertical wrench position and rotate the wrench by 90 135 Staying below 90 grey arrow could be insufficiently tight more than 135 red arrow could damage the capillary o ossibly eaky 90 recommended range o Second Step Installation to Soft Connectors Use this procedure for soft connectors which are typically made from PEEK These are the following connections e to and from all bio inert valves injection valve in the autosampler and valves in the thermostatted column compartment and 1290 Infinity Valve Drive e bio inert ZDV unions detector flow cells multi draw upgrade kit capillary to capillary connections for example for heat exchangers 258 Agilent 1260 FLD User Manual Appendix 13 to the autosampler needle and e to PEEK columns like many bio inert columns For the installation of bio inert ZDV unions see Installation of the Bio inert Zero Dead Volume ZDV Union on page 73 First installation of a capillary to a soft connector 1 When tightening a fitting for the first time start from the finger tight position which does not necessarily need t
126. n Test Chromatogram 166 Procedure Using the Agilent Lab Advisor 166 Wavelength Verification and Calibration 168 Wavelength Accuracy Test 171 Using the Agilent Lab Advisor 171 Interpretation of the Results 173 Using the Agilent ChemStation Manually 174 Wavelength Calibration Procedure 177 This chapter describes the detector s built in test functions ate Agilent Technologies 157 8 Test Functions Introduction All tests are described based on the Agilent Lab Advisor Software B 02 03 Other user interfaces may not provide any test or just a few Table 22 Interfaces and available test functions Interface Comment Available Function Agilent Instrument Utilities Maintenance tests are e Intensity available WL Calibration Agilent Lab Advisor All tests are available e Intensity Agilent ChemStation Some tests may be available E Adding of temperature Agilent Instant Pilot Some tests are available ASTM Drift and Noise Dark Current D A Converter WL Accuracy WL Calibration Test Chromatogram Tools Spectra Scan Tools Module Infos Tools Diagnostic Tools Some of Lab Advisor tests Intensity WL Calibration Spectra Scan Tools Module Infos Tools Diagnostic For details on the use of the interface refer to the documentation interface 158 Agilent 1260 FLD User Manual Test Functions 8 Diagram of Light Path The light path is shown in Figure 56 on page 159 Emission grating
127. nds In spectral mode the 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 Table on page 97 Agilent 1260 FLD User Manual 95 4 96 Using the Fluorescence Detector Table 14 Conditions for simultanoeus multi wavelength detection for PNA analysis see figure below Column Vydac 2 1 x 250 mm PNA 5 um Mobile phase A water B acetonitrile 50 50 Gradient 3 min 60 14 5 min 90 22 5 min 95 Flow rate 0 4 mL min Column temperature 22 C Injection volume 2 ul FLD settings PMT 12 response time 4s Agilent 1260 FLD User Manual Using the Fluorescence Detector 4 The upper trace was received with 1 excitation WL at 260 nm 1 Naphthalene 8 Benz a anthracene traditional 4 emission WL at 350 420 2 Acenaphthene 9 Chrysene h 440 and 500 nm 3 Fluoren
128. ng goggles safety gloves and protective clothing as described in the material handling and safety data sheet supplied by the vendor and follow good laboratory practice gt The volume of substances should be reduced to the minimum required for the analysis gt Never exceed the maximal permissible volume of solvents 6 L in the solvent cabinet gt Do not use bottles that exceed the maximum permissible volume as specified in the usage guideline for the Agilent 1200 Infinity Series Solvent Cabinets gt Arrange the bottles as specified in the usage guideline for the solvent cabinet gt A printed copy of the guideline has been shipped with the solvent cabinet electronic copies are available on the Internet Recommendations for Solvent Cabinet For details see the usage guideline for the Agilent 1200 Infinity Series Solvent Cabinets Agilent 1260 FLD User Manual 57 3 Installing the Module Installation Information on Leak and Waste Handling Figure 22 Leak and waste handling overview typical stack configuration as an example 58 Agilent 1260 FLD User Manual Installing the Module 3 1 Solvent cabinet Leak pan 3 Leak pan s outlet port A leak funnel B and corrugated waste tube C 4 Waste tube of the sampler s needle wash 5 Condense drain outlet of the autosampler cooler 6 Waste tube of the purge valve 7 Waste tube 1 Stack the modules according to the adequate stack configuration
129. ng 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 on page 24 The luminescence is short lived therefore the fluorescence detector need only measure over a short period of time after the lamp has flashed Intensity Track and Hold Ignite Time psec Figure 12 Measurement of Fluorescence Agilent 1260 FLD User Manual Introduction to the Fluorescence Detector 1 Phosphorescence Detection An appropriate parameter set will be specified as soon as you chose the phosphorescence detection mode special setpoints under FLD parameter settings Flash Phosphorescence i Measurement 30 100 150 200 Time psec Figure 13 Measurement of Phosphorescence 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 Agilent 1260 FLD User Manual 25 1 26 Introduction to the F
130. nnecting the power 56 external contact 218 LAN 217 remote 212 RS 232 219 cables analog 208 BCD 208 CAN 209 LAN 209 overview 208 remote 208 RS 232 209 calibration sample 1 77 CAN cable 217 cautions and warnings 185 cleaning 188 Communication settings RS 232C 240 compensation sensor open 149 compensation sensor short 150 condensation 38 configuration Index one stack 53 two stack 55 cut off filter 16 cuvette howtouse 193 D defect on arrival 50 degradation UV 18 160 delivery checklist 51 dimensions 39 E electrical connections descriptions of 229 EMF early maintenance feedback 243 emission condenser lens 16 emission grating 16 emission monochromator 20 emission slit 16 error messages A D Overflow 152 ADC Not Calibrated 152 compensation sensor open 149 compensation sensor short 150 fan failed 150 Flash Lamp Current Overflow 153 Flash Trigger Lost 154 FLF Board not found 151 Flow Cell Removed 155 Lamp Cover Open 151 leak sensor open 148 leak sensor short 149 263 Index leak 148 lost CAN partner 147 motor errors 156 remote timeout 147 shutdown 146 timeout 146 Wavelength Calibration Failed 154 Wavelength Calibration Lost 155 excitation condenser lens 16 excitation grating 16 excitation monochromator 18 excitation slit 16 external contact cable 218 external contacts BCD board 225 F fan failed 150 features safety and maintenance 42 44 48 firmware description 222 main system 222 re
131. nse time 4 s step size 5 nm Agilent 1260 FLD User Manual 89 4 Using the Fluorescence Detector Method Development This shows the isofluorescence plot of emission spectra for 15 PNAs 5 pg ml with a fixed excitation wavelength 260 nm 90 1 Naphthalene 2 Acenaphthene 3 Fluorene 4 Phenanthrene 5 Anthracene 6 Fluoranthene 7 Pyrene LU 8 Benz a anthracene 9 Chrysene 10 Benzo b fluoranthene 11 Benzo k fluoranthene 12 Benz a pyrene 13 Dibenzo ah anthracene 14 Benzo g h i perylene 15 Indeno 1 2 3 cd pyrene 600 nm 300 nm LS 10 1255 515 S 20 22 5 Time min Em pectra fixed Ex Figure 35 Optimization of the time program for the emission wavelength Agilent 1260 FLD User Manual Using the Fluorescence Detector 4 1 Naphthalene 8 Benz a anthracene 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 Benzo g h i perylene 15 Indeno 1 2 3 cd pyrene 11 LU 60 50 40 30 20 10 0 2 5 5 75 10 12 5 15 17 5 20 22 5 Time min 400 nm Exitation spectra 220 nm 350 nm 420 nm 500 nm Emission switching Figure 36 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 exampl
132. nt 1260 FLD User Manual 205 10 10 Parts for Maintenance Accessory Kit 206 Agilent 1260 FLD User Manual Agilent 1260 FLD User Manual 11 Identifying Cables Cable Overview 208 210 Remote Cables 212 BCD Cables 215 CAN LAN Cables 217 External Contact Cable 218 Agilent Module to PC 219 Analog Cables This chapter provides information on cables used with the Agilent 1200 Infinity Series modules Ee Agilent Technologies 207 11 Identifying Cables Cable Overview 208 Never use cables other than the ones supplied by Agilent Technologies to ensure proper functionality and compliance with safety or EMC regulations Analog cables p n 35900 60750 35900 60750 01046 60105 Remote cables p n 03394 60600 03396 61010 5061 3378 01046 60201 BCD cables p n 03396 60560 G1351 81600 Description Agilent module to 3394 6 integrators Agilent 35900A A D converter Analog cable BNC to general purpose spade lugs Description Agilent module to 3396A Series integrators 3396 Series Il 3395A integrator see details in section Remote Cables on page 212 Agilent module to 3396 Series III 3395B integrators Remote Cable Agilent module to general purpose Description Agilent module to 3396 integrators Agilent module to general purpose Agilent 1260 FLD User Manual Identifying Cables 11 CAN cables p n Description 5181 1516 CAN cable Agilent module to module 0 5 m 5181 1519 CA
133. ntifying Cables Remote Cables 212 One end of these cables provides a Agilent Technologies APG Analytical Products Group remote connector to be connected to Agilent modules The other end depends on the instrument to be connected to Agilent Module to 3396A Integrators p n 03394 60600 Pin 3396A Pin Agilent Signal Name Active module TTL 9 1 White Digital ground NC 2 Brown Prepare run Low 80 A 3 3 Gray Start Low z NC 4 Blue Shut down Low e oa NC 5 Pink Not Ls connected NC 6 Yellow Power on High 5 14 7 Red Ready High 1 8 Green Stop Low NC 9 Black Start request Low 13 15 Not connected Agilent Module to 3396 Series II 3395A Integrators Use the cable Agilent module to 3396A Series I integrators 03394 60600 and cut pin 5 on the integrator side Otherwise the integrator prints START not ready Agilent 1260 FLD User Manual Identifying Cables 11 Agilent Module to 3396 Series III 3395B Integrators p n 03396 61010 Pin 33XX Pin Agilent Signal Name Active module TTL 9 1 White Digital ground ae NC 2 Brown Prepare run Low o i 3 3 Gray Start Low z NC 4 Blue Shut down Low a NC 5 Pink Not Ls connected Se NC 6 Yellow Power on High 14 7 Red Ready High 4 8 Green Stop Low NC 9 Black Start request Low 13 15 Not connected Agilent Module to Agilent 35900 A D Converters
134. o be a vertical wrench position and rotate the wrench by 180 210 Staying below 180 grey arrow will not be sufficiently tight more than 210 red arrow could damage the capillary 0 possibly leaky 210 recommended180 range Second and subsequent installations of a capillary to a soft connector 1 When tightening the fitting for the second and subsequent times again start from the finger tight position which is not necessarily a vertical wrench position and rotate the wrench by 135 180 Staying below 135 grey arrow could be insufficiently tight enough more than 180 red arrow could damage the capillary 0 ssibl eaky ror 135 180 recommended range Agilent 1260 FLD User Manual 259 13 Appendix 10 Summary for Second Step Table 47 Summary for second step 2 dStep First installation Subsequent installations Hard connectors 0 ossibly eaky D 135 180 recommended range Soft connectors ossibly eaky 210 recommended 180 range possibly leaky 90 ecommended range e possibly leaky 135 180 recommended range 260 Agilent 1260 FLD User Manual Appendix 13 Removing Capillaries CAUTION Potential damage of capillaries gt Do not remove fittings from used capillaries To keep the flow path free of stainless steel the front end of the capillary is made of PEEK Under high p
135. olled activities 4 SHUT DOWN L System has serious problem for example leak stops pump Receiver is any module capable to reduce safety risk 5 Not used 6 POWER ON H All modules connected to system are switched on Receiver is any module relying on operation of others 7 READY H System is ready for next analysis Receiver is any sequence controller 8 STOP L Request to reach system ready state as soon as possible for example stop run abort or finish and stop injection Receiver is any module performing run time controlled activities 9 START REQUEST L Request to start injection cycle for example by start key on any module Receiver is the autosampler Special Interfaces There is no special interface for this module 238 Agilent 1260 FLD User Manual Hardware Information 12 Setting the 8 bit Configuration Switch without On board LAN The 8 bit configuration switch is located at the rear of the module This module does not have its own on board LAN interface It can be controlled through the LAN interface of another module and a CAN connection to that module i SS Y Figure 82 Configuration switch settings depend on configured mode All modules without on board LAN default should be ALL DIPS DOWN best settings Bootp mode for LAN and 19200 baud 8 data bit 1 stop bit with no parity for RS 232 DIP 1 DOWN and DIP 2 UP allows special RS 232 settings e for boot test modes DIPS 1 2 must
136. on of the failure a list of probable causes of the problem and a list of suggested actions to fix the problem are provided see chapter Error Information Test Functions A series of test functions are available for troubleshooting and operational verification after exchanging internal components see Tests and Calibrations 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 Calibration Procedure on page 177 138 Agilent 1260 FLD User Manual Troubleshooting and Diagnostics 6 Status Indicators Two status indicators are located on the front of the module The lower left indicates the power supply status the upper right indicates the module status A a Status indicator green yellow red E s Gaia I a oy 50s EA Line power switch with green light Figure 55 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 Agilent 1260 FLD User Manual 139 6 Troubleshooting and Diagnostics Module Status Indicator The module status indicator indi
137. r DAP The excitation spectrum of DAP is very similar to the UV absorption spectrum from the diode array detector Table on page 94 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 Agilent 1260 FLD User Manual Using the Fluorescence Detector 4 This is an impurity of carbamates The Norm 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 A for taking the ie es excitation spectrum Emission 200 250 300 350 400 450 500 550 Wavelength nm Figure 37 UV spectrum and fluorescence spectra for 2 3 diaminophenazine DAP Agilent 1260 FLD User Manual 93 4 Using the Fluorescence Detector The two upper traces are obtained using 2 amino 3 OH phenazine two different LU Unknown excitation wavelengths The lower trace is a 0 8 pure standard of the known impurities 2 3 diaminophenazine 0 6 265 540 nm 0 4 430 540 nm 0 2 Standard Time min Figure 38 Qualitive analysis of MBC 2 benzimidazole carbamic acid methylester and impurities Table 13 Conditions for analysis of DAP and MBC according to fi
138. r versions or to keep all systems on the same validated revision The installation of older firmware might be necessary to keep all systems on the same validated revision or if anew module with newer firmware is added to a system or if third party control software requires a special version Description LAN RS 232 Firmware Update Tool Agilent Lab Advisor software Instant Pilot G4208A only if supported by module Description 1 Firmware tools and documentation from Agilent web site Read update documentation provided with the Firmware Update Tool To upgrade downgrade the module s firmware carry out the following steps 1 Download the required module firmware the latest LAN RS 232 FW Update Tool and the documentation from the Agilent web http www chem agilent com _layouts agilent downloadFirmware aspx whid 69761 2 For loading the firmware into the module follow the instructions in the documentation Module Specific Information There is no specific information for this module Agilent 1260 FLD User Manual Maintenance 9 Tests and Calibrations The following tests are required after maintenance of lamps and flow cells e Lamp Intensity Test on page 160 e Wavelength Verification and Calibration on page 168 Agilent 1260 FLD User Manual 199 9 Maintenance Tests and Calibrations 200 Agilent 1260 FLD User Manual Agilent 1260 FLD User Manual e e 10 r Parts for Maintenan
139. res in Agilent product user guides or use of the products in violation of applicable laws rules or regulations gt Use your Agilent products only in the manner described in the Agilent product user guides CAUTION Safety standards for external equipment gt If you connect external equipment to the instrument make sure that you only use accessory units tested and approved according to the safety standards appropriate for the type of external equipment 186 Agilent 1260 FLD User Manual Overview of Maintenance Maintenance 9 The following pages describe maintenance simple repairs of the detector that can be carried out without opening the main cover Table 31 Simple Repairs Procedure Typical Frequency Notes Flow cell exchange If application requires a different flow cell type or if defective Flow cell flushing If flow cell is contaminated Leak sensor drying If leak has occurred Leak handling System If broken or corroded replacement Complete Assembly 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 168 Check for leaks Check for leaks Agilent 1260 FLD User Manual 187 9 Maintenance Cleaning the Module To keep the module case clean use a soft cloth slightl
140. ressure or when in contact with some solvents PEEK can expand to the shape of the connector where the capillary is installed If the capillary is removed this may become visible as a small step In such cases do not try to pull the fitting from the capillary as this can destroy the front part of the capillary Instead carefully pull it to the rear During installation of the capillary the fitting will end up in the correct position ON ee Rear Front Figure 83 Capillary fitting Agilent 1260 FLD User Manual 261 13 Appendix 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 262 Agilent 1260 FLD User Manual Index 8 bit configuration switch without On Board LAN 239 A accessory kit parts 204 accuracy of wavelength 40 Agilent Lab Advisor software 142 Agilent Lab Advisor 142 Agilent oninternet 262 algae 254 254 algea 194 ambient non operating temperature 39 ambient operating temperature 39 analog signal 236 analog cable 210 apgremote 237 battery safety information 250 BCD board external contacts 225 BCD cable 215 bench space 38 bio inert 189 materials 32 board HP JetDirect card 227 boards Agilent 1260 FLD User Manual LAN card 227 C cable analog 210 BCD 215 CAN 217 connecting APG remote 56 connecting CAN 56 connecting LAN 56 connecting the ChemStation 56 co
141. rials used in Agilent 1260 Infinity Systems Module Materials Agilent 1260 Infinity Bio inert Quaternary Pump G5611A Agilent 1260 Infinity Bio inert High Performance Autosampler G5667A Agilent 1260 Infinity Bio inert Manual Injector G5628A Agilent 1260 Infinity Bio inert Analytical Fraction Collector G5664A Bio inert Flow Cells Standard flow cell bio inert 10 mm 13 pL 120 bar 12 MPa for MWD DAD includes Capillary Kit Flow Cells BIO p n G5615 68755 G5615 60022 for Agilent 1260 Infinity Diode Array Detectors DAD G1315C D Max Light Cartridge Cell Bio inert 10 mm V s 1 0 pL G5615 60018 and Max Light Cartridge Cell Bio inert 60 mm V s 4 0 uL G5615 60017 for Agilent 1200 Infinity Series Diode Array Detectors DAD G4212A B Bio inert flow cell 8 uL 20 bar pH 1 12 includes Capillary Kit Flow Cells BIO p n G5615 68755 G5615 60005 for Agilent 1260 Infinity Fluorescence Detector FLD G1321B Titanium gold platinum iridium ceramic ruby PTFE PEEK Upstream of sample introduction Titanium gold PTFE PEEK ceramic Downstream of sample introduction PEEK ceramic PEEK ceramic PEEK ceramic PTFE PEEK ceramic sapphire PTFE PEEK fused silica PEEK fused silica PTFE 32 Agilent 1260 FLD User Manual Introduction to the Fluorescence Detector 1 Table 2 Bio inert materials used in Agilent 1260 Infinity Systems Module Materials Bio inert heat e
142. rly all fluorescence in your x T i sample r A rm jan DO NOT select 200 to 500 additional emission 5 wavelengths B C Threshold M00 Lu D Doing so will increase the scan Free time and will lower the performance Figure 40 Detector Settings for Emission Scan 1 Wait until the baseline stabilizes Complete the run Agilent 1260 FLD User Manual 101 4 102 Using the Fluorescence Detector Example Optimization for Multiple Compounds 2 Load the signal In this example just the time range of 13 min is displayed FLD A Ex 260 Em 350 FLD_PADT FLD_PAD1 D Lu 9 543 254 7211 204 7 629 5 300 t T T T T T T o 2 4 6 8 10 12 Figure 41 Chromatogram from Emissions Scan Agilent 1260 FLD User Manual Using the Fluorescence Detector 4 Example Optimization for Multiple Compounds 3 Use the isoabsorbance plot and evaluate the optimal emission wavelengths shown in the table below 480 460 440 420 400 380 340 320 300 min Figure 42 Isoabsorbance Plot from Emission Scan Table 17 Peak Time Emission Wavelength 1 5 3 min 330 nm 2 12 min 330 nm i so 76min 3100m l 4 7 86 min 360 nm 5 10 6 min 445 nm 6 11 23 min 385 nm g Agilent 1260 FLD User Manual 103 4 Using the Fluorescence Detector Example Optimization for Multiple Compounds 4 Using the settings and the timetable from previous pag
143. rry out preventive maintenance In addition users can generate a status report for each individual LC instrument The tests and diagnostic features as provided by the Agilent Lab Advisor software may differ from the descriptions in this manual For details refer to the Agilent Lab Advisor software help files The Instrument Utilities is a basic version of the Lab Advisor with limited functionality required for installation use and maintenance No advanced repair troubleshooting and monitoring functionality is included Agilent 1260 FLD User Manual Agilent 1260 FLD User Manual 7 Error Information What Are Error Messages 145 General Error Messages 146 Timeout 146 Shutdown 146 Remote Timeout 147 Lost CAN Partner 147 Leak 148 Leak Sensor Open 148 Leak Sensor Short 149 Compensation Sensor Open 149 Compensation Sensor Short 150 Fan Failed 150 Detector Error Messages 151 Lamp Cover Open 151 FLF Board notfound 151 ADC Not Calibrated 152 A D Overflow 152 Flash Lamp Current Overflow 153 No light at reference diode despite lamp ison 153 Flash Trigger Lost 154 Wavelength Calibration Failed 154 Wavelength Calibration Lost 155 Flow Cell Removed 155 Motor Errors 156 ote Agilent Technologies 143 7 Error Information This chapter describes the meaning of error messages and provides information on probable causes and suggested actions how to recover from error conditions 144 Agilent 1260 FLD User Manual Error Information 7 Wh
144. s and BCD outputs Parts for wavelength calibration see Standard Accessory Kit on page 204 202 Agilent 1260 FLD User Manual Cuvette Kit p n G1321 60007 5062 2462 79814 22406 0100 0043 0100 0044 0100 1516 9301 0407 9301 1446 Agilent 1260 FLD User Manual Parts for Maintenance 10 Description FLD Cuvette Kit 8 uL 20 bar includes Tube PTFE 0 8 mm x 2 m re order 5 m ST Fitting ST front ferrule ST back ferrule Fitting male PEEK 2 pk Needle Syringe 203 10 Parts for Maintenance Accessory Kit Standard Accessory Kit Accessory kit G1321 68755 contains some accessories and tools needed for the installation and repair calibration of the detector Item p n Description 1 5062 2462 Tube PTFE 0 8 mm x 2 m re order 5 m 2 0100 1516 Fitting male PEEK 2 pk 3 G1315 87311 Capillary ST 0 17 mm x 380 mm S S Column to detector includes ST ferrule front ST ferrule back and ST fitting 4 0100 0043 ST front ferrule 5 0100 0044 ST back ferrule 6 79814 22406 ST Fitting 2 Figure 77 Waste Tubing Parts 204 Agilent 1260 FLD User Manual Parts for Maintenance 3 This end is pre installed Figure 78 Inlet Capillary Column Detector Parts Capillary Kit Flow Cells BIO Capillary Kit Flow Cells BIO includes Capillary PK 0 18 mm x 1 5 m and PEEK Fittings 10 PK p n 5063 6591 G5615 68755 includes p n Description 0890 1763 Capillary PK 0 18 mm x 1 5 m 5063 6591 PEEK Fittings 10 PK Agile
145. s of two independent sections a non instrument specific section called resident system e an instrument specific section called main system Resident System This resident section of the firmware is identical for all Agilent 1100 1200 1220 1260 1290 series modules Its properties are the complete communication capabilities CAN LAN and RS 232C memory management e ability to update the firmware of the main system Main System Its properties are the complete communication capabilities CAN LAN and RS 232C memory management e ability to update the firmware of the resident system In addition the main system comprises the instrument functions that are divided into common functions like e run synchronization through APG remote e error handling diagnostic functions or module specific functions like internal events such as lamp control filter movements raw data collection and conversion to absorbance Firmware Updates Firmware updates can be done using your user interface e PC and Firmware Update Tool with local files on the hard disk Agilent 1260 FLD User Manual Hardware Information 12 Instant Pilot G4208A with files from a USB Flash Disk e Agilent Lab Advisor software B 01 03 and above The file naming conventions are PPPP_RVVV_XXX dlb where PPPP is the product number for example 1315AB for the G1315A B DAD R the firmware revision for example A for G1315B or B for the G1315C DAD
146. ses because automatic electronic fuses are implemented in the power supply Never use cables other than the ones supplied by Agilent Technologies to ensure proper functionality and compliance with safety or EMC regulations Agilent 1260 FLD User Manual 229 12 Hardware Information Rear view of the module i gt 3 o den l wR aa es m QOQ tlm E DOOU D a a P er A Sma OMT OBE A 22222 e E Z gE e l Sz2222 E mm ma oF O SZZ e el E 1 Aai i I4 ma dt yt Me J Figure 80 Rear View of Detector Electrical Connections and Label The GPIB interface has been removed with the introduction of the 1260 Infinity modules 230 Agilent 1260 FLD User Manual Hardware Information 12 Serial Number Information Serial Number Information 1260 Infinity The serial number information on the instrument labels provide the following information CCXZZ00000 Format CC Country of manufacturing DE Germany JP Japan e CN China X Alphabetic character A Z used by manufacturing ZZ Alpha numeric code 0 9 A Z where each combination unambiguously denotes a module there can be more than one code for the same module 00000 Serial number Serial Number Information 1200 Series and 1290 Infinity The serial number information on the instrum
147. sident system 222 update tool 223 updates 222 198 upgrade downgrade 198 16 20 254 solvent information 254 flow cell fluorescence and phosphorescence 13 fluorescence detection 24 frequency range 39 front view of module 61 G general error messages 146 GLP features 42 45 48 glycogen 177 264 How the Detector Operates 12 how to use the cuvette 193 HP JetDirect card 227 humidity 39 installation bench space 38 flow connections 64 of flow cell and capillaries 64 of the detector 61 site requirements 35 instrument layout 244 interfaces 232 internet 262 Introduction to the Detector 10 L lamp intensity history 161 lamp intensity test 160 LAN cable 217 communication interface board 227 eak sensor open 148 eak sensor short 149 eak 148 eaks correcting 195 ine frequency 39 ine voltage 39 ithium batteries 250 ost CAN partner 147 uminescence 12 M maintenance definition of 184 feedback 243 overview 187 replacing firmware 198 materials bio inert 32 message A D Overflow 152 ADC Not Calibrated 152 Flash Lamp Current Overflow 153 Flash Trigger Lost 154 FLF Board not found 151 Flow Cell Removed 155 Lamp Cover Open 151 motor messages 106 remote timeout 147 Wavelength Calibration Failed 154 Wavelength Calibration Lost 155 method development 1 check the LC system for impurities 83 2 optimize limits of detection and selectivity 84 3 setup routine methods 95 multi wavelen
148. st concentration to measure Noise ist the peak to peak baseline noise The highest LU level in this example is about 220 LU gt in this case the limit of the detector may vary gt Highest PMT Level to not exceed highest concentration level here is 9 But between PMT Level 5 and 9 the signal and noise increases with the same factor gt So S N is constant Below PMT Level 5 noise of electronic and other purpose get dominant gt PMT Level 5 is the optimal PMT Level because a S N cannot be increased and b maximum headroom for higher concentrations IMPORTANT the LU numbers and the PMT border levels here 5 and 9 may vary by application ex em wavelength solvent and by instrument Figure 50 PMT Gain Behavior In this example the maximum output is around 220 LU and further increase of the PMT above 9 results in a signal overload clipping and drop of signal to noise value 1 Set the PMT Gain Level Now check with your highest concentration amount that your highest peak does not clip or overflow If this check is ok you finished the PMT Gain Level optimization Continue with Set your Luminescence Units in LU If the check shows that the highest concentration doesn t fit to the selected range e g by clipping you may decrease the sensitivity of your FLD by gradually decreasing the PMT Level by 1 to get roughly half the signal height by each step Be aware that by that step you will lose sensitivity a
149. t Error ID 0080 The ambient compensation sensor NTC on the main board in the module has failed open circuit The resistance across the temperature compensation sensor NTC on the main board is dependent on ambient temperature The change in resistance is used by the leak circuit to compensate for ambient temperature changes If the resistance across the sensor falls below the lower limit the error message is generated Probable cause Suggested actions 1 Defective main board Please contact your Agilent service representative Fan Failed Error ID 0068 The cooling fan in the module has failed The hall sensor on the fan shaft is used by the main board to monitor the fan speed If the fan speed falls below a certain limit for a certain length of time the error message is generated Depending on the module assemblies e g the lamp in the detector are turned off to assure that the module does not overheat inside Probable cause Suggested actions 1 Fan cable disconnected Please contact your Agilent service representative 2 Defective fan Please contact your Agilent service representative 3 Defective main board Please contact your Agilent service representative Agilent 1260 FLD User Manual Error Information Detector Error Messages Lamp Cover Open Error ID 6622 6731 The lamp cover in the optical compartment has been removed The lamp cannot be turned on while this message is on Probable cause Sugg
150. t low signal levels LOD Agilent 1260 FLD User Manual 127 5 128 Optimizing the Detector 2 Set your Luminescence Units in LU If you are not satisfied with the LU output level of the detector or if you want to align the output of multiple instruments with different output levels you can scale each instrument output The recommended setting of the G1321 FLD is around 100 LU for the highest peak height to get optimum signal to noise and signal range Lower LU values normally do not influence the performance of the instrument if PMT Gain Test was executed fine For analog out less than 100 LU is optimum to get best analog signal performance with the default attenuation of 100 LU 1 V Adapt your LU setting such that your maximum signal level under default attenuation is between 50 to 80 LU analog output equivalent to 500 mV to 800 mV After correct PMT Setting you can scale any instrument to your favorable LU level We recommend not exceeding around 100 LU The parameter of choice is called Scale factor and is applicable by the local controller the Instant Pilot B 02 07 or later In case older revisions are used the Scale factor can be entered using the command line of Agilent ChemStation PRINT SENDMODULES LFLD DMUL x xx Instant Pilot Service Mode FLD then type DMUL x xx and press SEND e LAN RS 232 Firmware Update Tool via Send Instruction menu DMUL x xx e Agilent Lab Advisor Software via
151. table You may remove the flow cell and check for air bubbles After re inserting the cell turn the lamp ON 4 Open the Online Spectra plot and observe the maximum as shown in Figure 64 on page 171 Cleft 5 Load the method WLEMTEST The FLD will change into the multi excitation mode and scan in the range of the expected maximum of 350 nm 20 nm 6 Open the Online Spectra plot and observe the maximum as shown in Figure 64 on page 171 right Agilent 1260 FLD User Manual Test Functions 8 Wavelength Calibration Procedure When If application requires or see Table 29 on page 170 Tools required Description Laboratory balance Parts required p n Description 5063 6597 Calibration Sample Glycogen 9301 1446 Syringe 9301 0407 Needle 5190 5111 Syringe filter 0 45 pm 100 pk 0100 1516 Fitting male PEEK 2 pk 1 Preparation of the Glycogen Calibration Sample 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 Fill the prepared amount into a suitable bottle vial c Fill 10 ml of distilled water into the vial and shake Wait 5 minutes and shake again After 10 minutes the solution is ready 2 Preparation of the Flow Cell a b o a 0 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
152. tation start a wavelength calibration and abort immediately No changes are made to the calibration at this time 180 Agilent 1260 FLD User Manual Test Functions 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 4 Verification using Wavelength Accuracy Test on page 171 a Refit the capillary to the flow cell b Follow the procedure Wavelength Accuracy Test on page 171 Agilent 1260 FLD User Manual 181 8 Test Functions Wavelength Calibration Procedure 182 Agilent 1260 FLD User Manual This chapter provides general information on maintenance of the detector Agilent 1260 FLD User Manual 9 Maintenance Introduction to Maintenance 184 Warnings and Cautions 185 Overview of Maintenance 187 Cleaning the Module 188 Exchanging a Flow Cell 189 How to use the Cuvette 193 Flow Cell Flushing 194 Correcting Leaks 195 Replacing Leak Handling System Parts 196 Replacing the Interface Board 197 Replacing Module Firmware 198 Tests and Calibrations 199 Apg Agilent Technologies 183 9 Maintenance Introduction to Maintenance The module is designed for easy maintenance Maintenance can be done from the front with module in place in the system stack There are no serviceable parts inside Do not open the module 184 Agilent 1260 F
153. te Agilent 1260 Infinity Liquid Chromatograph you can ensure optimum performance by installing the following configurations These configurations optimize the system flow path ensuring minimum delay volume 52 Agilent 1260 FLD User Manual One Stack Configuration Installing the Module 3 Optimizing the Stack Configuration Ensure optimum performance by installing the modules of the Agilent 1260 Infinity LC System in the following configuration See Figure 18 on page 53 and Figure 19 on page 54 This configuration optimizes the flow path for minimum delay volume and minimizes the bench space required Solvent cabinet Vacuum degasser Instant Pilot Autosampler Column compartment Detector Figure 18 Recommended Stack Configuration for 1260 Infinity Front View Agilent 1260 FLD User Manual 53 3 Installing the Module Optimizing the Stack Configuration TTT TTT Remote cable CAN Bus cable to Instant Pilot AC power CAN Bus cable Analog detector signal 1 or 2 outputs per detector LAN to LC ChemStation location depends on detector Figure 19 Recommended Stack Configuration for 1260 Infinity Rear View 54 Agilent 1260 FLD User Manual Installing the Module 3 Optimizing the Stack Configuration Two Stack Configuration To avoid excessive height of the stack when the autosampler thermostat is adde
154. ted actions 1 Defective Hardware Please contact your Agilent service representative Agilent 1260 FLD User Manual 153 7 154 Error Information Flash Trigger Lost Error ID 6722 This message is displayed when the flash trigger is no longer generated Probable cause 1 Firmware problem 2 Multi Mode Off 3 Defective encoder Wavelength Calibration Failed Error ID 6703 Suggested actions Reboot the detector power cycle Please contact your Agilent service representative Please contact your Agilent service representative This message may show up during a wavelength calibration If the expected deviation is larger than the specified wavelength accuracy the message Wavelength Calibration Failed is displayed and the instrument stays in a Not Ready condition Probable cause 1 Flash lamp not ignited or position not correct 2 Cell position not correct 3 Solvent in the cell not clean or air bubble in the cell 4 monochromator assembly position not correct after replacement Suggested actions Please contact your Agilent service representative Check the cell position Flush the flow cell Please contact your Agilent service representative Agilent 1260 FLD User Manual Error Information 7 Wavelength Calibration Lost Error ID 6691 After exchanging the monochromator assemblies the calibration factors should be reset to defaults values a new FLM board comes
155. ter Description 1 Bidistilled water nitric acid 65 tubings to waste Dangerous concentration of nitric acid 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 gt Give proper attention to safety 194 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 Methanol 5 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 Do not exceed the pressure limit of 20 bar 0 2 MPa Agilent 1260 FLD User Manual Maintenance 9 Correcting Leaks When If a leakage has occurred in the flow cell area or at the capillary connections Tools required p n Description Tissue Wrench 1 4 inch for capillary connections 5043 0915 Fitting mounting tool for bio inert capillaries 1 Remove the front cover Use tissue to dry the leak sensor area and the leak pan Observe the capillary connections and the flow cell area for leaks and correct if required 4 Replace the front cover a a Figure 74 Observing for Leaks Agilent 1260 FLD User Manual 195 9 Maintenance
156. th characteristic Flow cells Control and data evaluation Range settable 200 nm 1200 nm and zero order Bandwidth 20 nm fixed Monochromator concave holographic grating F 1 6 blaze 400 nm in line excitation measurement 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 pL volume and 20 bar 2 MPa pressure maximum fused silica block Optional Fluorescence cuvette for offline spectroscopic measurements with 1 mL syringe 8 pL volume Bio inert 8 uL volume and 20 bar 2 MPa pressure maximum pH 1 12 Micro 4 pL volume and 20 bar 2 MPa pressure maximum Agilent ChemStation for LC Agilent Instant Pilot G4208A with limited spectral data analysis and printing of spectra Agilent 1260 FLD User Manual 41 2 42 Site Requirements and Specifications Table4 Performance Specifications Agilent 1260 Infinity Fluorescence Detector G1321B Type Specification Comments Analog outputs Recorder integrator 100 mV or 1 V 100 LU is the Communications Safety and maintenance GLP features Housing Environment Dimensions Weight output range gt 100 LU two outputs Controller area network CAN
157. the bench is designed to bear the weight of all modules The module should be operated in a horizontal position Condensation CAUTION Condensation within the module Condensation will damage the system electronics gt Do not store ship or use your module under conditions where temperature fluctuations could cause condensation within the module gt If your module was shipped in cold weather leave it in its box and allow it to warm slowly to room temperature to avoid condensation 38 Agilent 1260 FLD User Manual Physical Specifications Site Requirements and Specifications 2 Table3 Physical Specifications Type Specification Comments Weight 11 5 kg 26 Ibs Dimensions 140 x 345 x 435 mm height x width x depth 7 x 13 5 x 17 inches Line voltage 100 240 VAC 10 Wide ranging capability Line frequency 50 or 60 Hz 5 Power consumption 180 VA 70 W 239 BTU Maximum Ambient operating temperature Ambient non operating temperature Humidity Operating altitude Non operating altitude Safety standards IEC CSA UL 0 40 C 32 104 F 40 70 C 40 158 F lt 95 rh at 40 C 104 F Up to 2000 m 6562 ft Up to 4600 m 15091 ft Installation category II Pollution degree 2 Non condensing For storing the module For indoor use only Agilent 1260 FLD User Manual 39 2 Site Requirements and Specifications Performance Specifications Table4
158. the front cover to 2 Locate the flow cell gain access to the flow cell area Agilent 1260 FLD User Manual 65 3 Installing the Module 3 Assemble the column detector capillary from the Pre assembled accessory kit One side is already factory assembled 4 Assemble the waste tubing from the accessory kit 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 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 measuring system pressure at the application flow rate connecting the last detector without column and FLD and measuring the system pressure with flow the difference in measured pressure is due to the back pressure generated by the last detector and is the pressure seen by the FLD 66 Agilent 1260 FLD User Manual Installing the Module 3 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 a 7 MOR Ag ile EN A i N Olan Ta 7 Establish flow and observe if leaks occur 8 Replace the front cover The installation of the detector
159. the leak in the external instrument before restarting the module Check external instruments for a shut down condition Check the vacuum degasser for an error condition Refer to the Service Manual for the degasser or the 1260 pump that has the degasser built in Agilent 1260 FLD User Manual Error Information 7 Remote Timeout Error ID 0070 A not ready condition is still present on the remote input When an analysis is started the system expects all not ready conditions for example a not ready condition during detector balance to switch to run conditions within one minute of starting the analysis If a not ready condition is still present on the remote line after one minute the error message is generated Probable cause Suggested actions 1 Not ready condition in one of the instruments Ensure the instrument showing the not ready connected to the remote line condition is installed correctly and is set up correctly for analysis 2 Defective remote cable Exchange the remote cable 3 Defective components in the instrument Check the instrument for defects refer to the showing the not ready condition instrument s documentation Lost CAN Partner Error ID 0071 During an analysis the internal synchronization or communication between one or more of the modules in the system has failed The system processors continually monitor the system configuration If one or more of the modules is no longer recognized as being
160. tra 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 1260 Infinity 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 34 on page 87 shows the complete information for quinidine as obtained with the Agilent 1260 Infinity Fluorescence Detector and a manual cuvette in a single offline measurement The optima for excitation and emission wavelengths can be extracted as coordinates of the maxima in the three dimensional plot One of the three maxima in the center of the plot can be chosen to define the excitation wavelength The selection depends on the additional compounds that are going to be Agilent 1260 FLD User Manual 85 4 Using the Fluorescence Detector 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
161. ts may if contacted result in personal injury Any adjustment maintenance and repair of the opened instrument under voltage should be avoided whenever possible When inevitable this has to be carried out by a skilled person who is aware of the hazard involved Do not attempt internal service or adjustment unless another person capable of rendering first aid and resuscitation is present Do not replace components with power cable connected Do not operate the instrument in the presence of flammable gases or fumes Operation of any electrical instrument in such an environment constitutes a definite safety hazard Do not install substitute parts or make any unauthorized modification to the instrument Capacitors inside the instrument may still be charged even 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 When working with solvents observe appropriate safety procedures for example goggles safety gloves and protective clothing as described in the material handling and safety data sheet by the solvent vendor especially when toxic or hazardous solvents are used Agilent 1260 FLD User Manual Appendix 13 The Waste Electrical and Electronic Equipment WEEE Directive 2002 96 EC Abstract The Waste Electrical and Electronic Equipment WEEE Directive
162. ty Series Interfaces Module CAN LAN BCD LAN RS 232 Analog APG Special optional on board Remote Detectors G1314B VWD VL 2 Yes No Yes 1 Yes G1314C VWD VL G1314E F VWD 2 No Yes Yes 1 Yes K1314F Clinical Ed G4212A B DAD 2 No Yes Yes 1 Yes K4212B DAD Clinical Ed G1315C DAD VL 2 No Yes Yes 2 Yes G1365C MWD G1315D DAD VL G1365D MWD VL G1321B FLD 2 Yes No Yes 2 Yes K1321B FLD Clinical Ed G1321C FLD G1362A RID 2 Yes No Yes 1 Yes G4280A ELSD No No No Yes Yes Yes EXT Contact AUTOZERO Others G1170A Valve Drive 2 No No No No No 1 G1316A C TCC 2 No No Yes No Yes K1316C TCC Clinical Ed G1322A DEG No No No No No Yes AUX K1322A DEG Clinical Ed G1379B DEG No No No Yes No Yes G4225A DEG No No No Yes No Yes K4225A DEG Clinical Ed Agilent 1260 FLD User Manual 233 12 Hardware Information Table 34 Agilent 1200 Infinity Series Interfaces Module CAN LAN BCD LAN RS 232 Analog APG Special optional on board Remote G4227A Flex Cube 2 No No No No No CAN DC OUT for CAN slaves 1 G4240A CHIP CUBE 2 Yes No Yes No Yes CAN DC OUT for CAN slaves THERMOSTAT for G1330A B NOT USED K1330B 1 Requires a HOST module with on board LAN e g G4212A or G4220A with minimum firmware B 06 40 or C 06 40 or with ad ditional G1369C LAN Card The detector DAD MWD FLD VWD RID is the preferred access point for control via LAN The inter module communication is done via CAN e CAN connectors as inter
163. ual 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 Ey Ej 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 spectrum Agilent 1260 FLD User Manual 15 1 Introduction to the Fluorescence Detector Optical Unit 16 All the elements of the optical system shown in Figure 6 on page 17 including Xenon flash lamp excitation condenser lens excitation slit mirror excitation grating flow cell emission condenser lens 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 Agilent 1260 FLD User Manual Flash lamp board Trigger Pack Xenon Flash lamp Condenser lens EX Slit EX Mirror EX Grating assembly Flow Cell Introduction to the Fluorescence Detector 1 Optical Unit EM Grating assembly Slit EM Cutoff filter Photo Multiplier Tube aw N
164. ule at the rear of the module Refer to Communication Settings for RS 232C There is no configuration possible on main boards with on board LAN These are pre configured for 19200 baud e 8 data bit with no parity and one start bit and one stop bit are always used not selectable The RS 232C is designed as DCE data communication equipment with a 9 pin male SUB D type connector The pins are defined as Agilent 1260 FLD User Manual 235 12 Hardware Information 236 Table 35 RS 232C Connection Table Pin Direction Function 1 In DCD 2 In RxD 3 Out TxD 4 Out DTR 5 Ground 6 In DSR 7 Out RTS 8 In CTS 9 In RI Instrument PC DCD 1 1 DCD RX 2 n 2 RX TX 3 X 3 TX DTR 4 4 DTR GND 5 5 GND DSR 6 6 DSR RTS 7 V 7 RTS CTS 8 M 8 CTS RI 9 9 RI DB9 DB9 DB9 DB9 Male Female Female Male Figure 81 RS 232 Cable Analog Signal Output The analog signal output can be distributed to a recording device For details refer to the description of the module s main board Agilent 1260 FLD User Manual Hardware Information 12 APG Remote The APG Remote connector may be used in combination with other analytical instruments from Agilent Technologies if you want to use features as common shut down prepare and so on Remote control allows easy connection between single instruments or systems to ensure coordinated analysis with simple coupling requirements The subminiature D connector is used The mo
165. um emission wavelength determined the excitation spectrum is acquired Norm Excitation Emission 250 300 350 400 450 500 550 600 Wavelength nm Figure 33 Excitation and emission spectra of quinidine Agilent 1260 FLD User Manual Using the Fluorescence Detector 4 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 32 on page 83 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 Infinity Series LC offers three different ways to obtain complete information on a compound s fluorescence Procedure I Take a fluorescence scan offline for a single compound as described above for the mobile phase This is done preferably with a manual FLD cuvette when pure compounds are available Procedure II Use two LC runs with the Agilent 1260 Infinity Fluorescence Detector to separate the compound mix under known conditions and acquire emission and excitation spectra separately Procedure III Use an Agilent 1200 Infinty Series FLD DAD combination and acquire UV Visible spectra equivalent to excitation spectra with the DAD and emission spectra with the FLD both in a single run Procedure Take a fluorescence scan Because fluorescence spec
166. und Time EX EM PMT Baseline 0 350 450 14 Free 20 30 350 397 14 Free Agilent 1260 FLD User Manual 163 8 Test Functions Table 27 Settings for Dual Wavelength Specifications Multi EM Scan Time EX EM_ EM_ Spectr From To Ste PM Baseline Fit A B a p T Spectra 00 0 350 397 450 None 280 450 10 12 Free OFF 0 20 3 350 450 450 None 280 450 10 12 Free OFF 0 Formulas for the Raman ASTM S N value see Figure 60 on page 164 for details SNR_ Raman mean_raman ex 350 em 397 mean_background ex 350 em 450 noise_raman ex 350 em 397 SNR_Dark mean_raman ex 350 em 397 mean_background ex 350 em 450 noise_background ex 350 em 450 FLD1 A Ex 350 Em 397 TT OOPV OOFLDSNT2 13 01 2006 13 05 11 F LDSN2000001 D Lu E i A ASTMNoise_ a time 5 to 20 minutes Mean Raman Current average between 5 5 and 10 minutes Mean Dark Current average between 21and 22 minutes l ms as r r TES E o 25 5 75 10 125 15 Figure 60 Raman ASTM signal noise calculation 164 Agilent 1260 FLD User Manual Test Functions 8 Using the Agilent Lab Advisor 1 Set up the HPLC system and the Lab Advisor 2 Flush the flow cell with clean bi distilled water 3 Start the test in the Lab Advisor Test Name Raman ASTM Signal Noise Test Description The test determines the detector noise and drift over a period of 15 Module G1321A DE81700118 FLD LL A E St
167. ures and samples Information can also not be generalized due to catalytic effects of impurities like metal ions complexing agents oxygen etc Apart from pure chemical corrosion other effects like electro corrosion electrostatic charging especially for non conductive organic solvents swelling of polymer parts etc need to be considered Most data available refers to room temperature typically 20 25 C 68 77 F If corrosion is possible it usually accelerates at higher temperatures If in doubt please consult technical literature on chemical compatibility of materials Agilent 1260 FLD User Manual 113 4 114 Using the Fluorescence Detector PEEK PEEK Polyether Ether Ketones combines excellent properties regarding biocompatibility chemical resistance mechanical and thermal stability PEEK is therefore the material of choice for UHPLC and biochemical instrumentation It is stable in a wide pH range and inert to many common solvents There is still a number of known incompatibilities with chemicals such as chloroform methylene chloride THF DMSO strong acids nitric acid gt 10 sulphuric acid gt 10 sulfonic acids trichloroacetic acid halogenes or aequous halogene solutions phenol and derivatives cresols salicylic acid etc Polyimide Agilent uses semi crystalline polyimide for rotor seals in valves and needle seats in autosamplers One supplier of polyimide is DuPont which brands polyimide as
168. wavelengths simultaneously Use for lowest limits of detection Collect online spectra perform library search determine peak purity Deactivate wavelength switching 82 Agilent 1260 FLD User Manual Using the Fluorescence Detector 4 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 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 32 on page 83 shows a sample of slightly impure water which was planned for use as mobile phase The area where fluorescence of the contaminated water sample can be seen is between the stray light areas the first and second order Raleigh stray light and Raman stray light Impurity 1 order Raman 2 order Figure 32 lsofluorescence 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
169. with default values In this case Wavelength Calibration Lost is displayed and the instrument stays in a Not Ready condition Probable cause Suggested actions 1 Reset of monochromator settings after Perform a wavelength calibration exchange 2 Replacement of FLM board Perform a wavelength calibration Flow Cell Removed Error ID 6616 6702 6760 The detector has an automatic cell recognition system When the flow cell is removed the lamp is turned off and a NOTREADY condition exists If the flow cell is removed during an analysis a SHUT DOWN is generated Probable cause Suggested actions 1 Flow cell has been removed during analysis Insert flow cell and turn on the lamp Agilent 1260 FLD User Manual 155 7 Error Information Motor Errors Monochromator motor errors may show up during the initialization or during operation of the detector There are individual messages for either the excitation or the emission side If an error occurs do a lamp ignition This will clear the error and a re initialization of the motors is performed If motor errors are displayed please contact your Agilent service representative 156 Agilent 1260 FLD User Manual Agilent 1260 FLD User Manual 8 Test Functions Introduction 158 Diagram of Light Path 159 Lamp Intensity Test 160 Lamp Intensity History 161 Raman ASTM Signal to Noise Test 162 Using the Agilent Lab Advisor 165 Interpretation of the Results 165 Using the Built i
170. xchanger G5616 60050 PEEK steel cladded for Agilent 1290 Infinity Thermostatted Column Compartment G1316C Bio inert Valve heads G4235A G5631A G5639A PEEK ceramic Alz03 based Bio inert Connection capillaries Upstream of sample introduction Titanium Downstream of sample introduction e Agilent uses stainless steel cladded PEEK capillaries which keep the flow path free of steel and provide pressure stability to more than 600 bar To ensure optimum bio compatibility of your Agilent 1260 Infinity Bio inert LC system do not include non inert standard modules or parts to the flow path Do not use any parts that are not labeled as Agilent Bio inert For solvent compatibility of these materials see Material Information on page 113 Agilent 1260 FLD User Manual 33 1 34 Introduction to the Fluorescence Detector Bio inert Materials Agilent 1260 FLD User Manual Agilent 1260 FLD User Manual e00 ee 2 r Site Requirements and Specifications Site Requirements 36 Physical Specifications 39 Performance Specifications 40 This chapter provides information on environmental requirements physical and performance specifications Agg Agilent Technologies 35 2 Site Requirements and Specifications Site Requirements A suitable environment is important to ensure optimal performance of the instrument Power Considerations The module power supply has wide ranging capability It accepts any line volt
171. xperience 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 For optimal results refer to Optimizing the Detector on page 119 Some features e g spectrum acquisition multi wavelength detection described in this chapter are not available on the G1321C FLD Agilent 1260 FLD User Manual 77 4 Using the Fluorescence Detector Getting Started and Checkout This chapter describes the check out of the Agilent 1260 Infinity Fluorescence Detector using the Agilent isocratic checkout sample Starting Your Detector When If you want to checkout the detector Parts required p n Description 1 5063 6528 Start up Kit includes 1 LC Column and parts listed below 1 01080 68704 Agilent isocratic checkout sample This 0 5 mL ampoule contains 0 15 wt dimethylphthalate 0 15 wt diethylphthalate 0 01 wt biphenyl 0 03 wt o terphenyl in methanol 1 0100 1516 Fitting male PEEK 2 pk 1 5021 1817 Capillary ST 0 17 mm x 150 mm Hardware required LC system with FLD Turn ON the detector 2 Turn ON the lamp When the lamp is turned on the first time the instrument performs some internal checks and a calibration check which takes about 5 minutes 3 You are now ready to change the settings of your detector
172. y dampened with water or a solution of water and mild detergent WARNING Liquid dripping into the electronic compartment of your module can cause shock hazard and damage the module gt Do not use an excessively damp cloth during cleaning gt Drain all solvent lines before opening any connections in the flow path 188 Agilent 1260 FLD User Manual Maintenance 9 Exchanging a Flow Cell Linert When Tools required OR Parts required Preparations CAUTION For bio inert modules use bio inert parts only If an application needs a different type of flow cell or the flow cell is defective leaky p n Description Wrench 1 4 inch for capillary connections 5043 0915 Fitting mounting tool for bio inert capillaries p n Description 1 G1321 60005 Flow cell 8 uL 20 bar pH 1 9 5 1 G1321 60015 Flow cell 4 uL 20 bar pH 1 9 5 1 G5615 60005 Bio inert flow cell 8 uL 20 bar pH 1 12 includes Capillary Kit Flow Cells BIO p n G5615 68755 1 G1321 60007 FLD Cuvette Kit 8 uL 20 bar Turn off the flow Sample degradation and contamination of the instrument Metal parts in the flow path can interact with the bio molecules in the sample leading to sample degradation and contamination gt For bio inert applications always use dedicated bio inert parts which can be identified by the bio inert symbol or other markers described in this manual gt Do not mix bio inert and non inert modules or

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