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Agilent Technologies 1200 Series User Manual

Fluorescence detector
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Fluorescence Detector
G1321A
User Manual
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  • Page 1 Agilent 1200 Series Fluorescence Detector G1321A User Manual...
  • Page 2 It calls attention to an dance with the terms of such license. the Agilent 1200 Series Fluorescence operating procedure, practice, or Detector. It is available as Adobe Reader file the like that, if not correctly per- (PDF) only.
  • Page 3 This chapter provides general information on maintenance and repair of the detector. Maintenance This chapter describes the maintenance of the detector and the required tests. Parts and Materials for Maintenance This chapter provides information on parts for maintenance. 1200 Series FLD User Manual...
  • Page 4 Appendix This chapter provides safetey and other general information. 1200 Series FLD User Manual...

  • Page 5: Table Of Contents

    Processing of Raw Data Electrical Connections Instrument Layout Early Maintenance Feedback (EMF) EMF Counters Using the EMF Counters Site Requirements and Specifications Site Requirements Physical Specifications Performance Specifications Installing the Detector Unpacking the Detector Detector Accessory Kit Contents 1200 Series FLD User Manual...
  • Page 6 Design Features Help Optimization Check Performance Before You Start Finding the Best Wavelengths A Real Example Finding the Best Signal Amplification Changing the Xenon Flash Lamp Frequency Lamp life savings Selecting the Best Response Time Reducing Stray Light 1200 Series FLD User Manual...
  • Page 7 How to use the Cuvette Flow Cell Flushing Correcting Leaks Replacing Leak Handling System Parts Replacing the Interface Board Replacing the Detector’s Firmware Tests & Calibrations Lamp Intensity Test Wavelength Verification and Calibration Wavelength Calibration Procedure 1200 Series FLD User Manual...
  • Page 8 Parts and Materials for Maintenance Overview of Maintenance Parts Cuvette Kit Spare Parts Accessory Kit Appendix General Safety Information Lithium Batteries Information Radio Interference Sound Emission UV-Radiation (UV-lamps only) Solvent Information Agilent Technologies on Internet Index 1200 Series FLD User Manual...
  • Page 9 Agilent 1200 Series Fluorescence Detector User Manual Introduction to the Fluorescence Detector Introduction to the Detector How the Detector Operates Raman Effect Optical Unit Analytical Information From Primary Data Electrical Connections Instrument Layout Early Maintenance Feedback (EMF) This chapter gives an introduction to the detector, instrument overview and internal connectors.

  • Page 10: Introduction To The Fluorescence Detector

    • optional cuvette is available and can be used for off-line measurements • easy front access to flow cell for fast replacement and • built-in wavelength accuracy verification. For specifications, see “Performance Specifications” on page 37 Figure 1 The Agilent 1200 Series Fluorescence Detector. 1200 Series FLD User Manual...

  • Page 11: How The Detector Operates

    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. Figure 2 Absorption of Light Versus Emission of Light 1200 Series FLD User Manual...
  • Page 12 If a molecule emits light 10 seconds after it was illuminated then the process was fluorescence. If a molecule emits light longer than 10 seconds after illumination then the process was phosphorescence. 1200 Series FLD User Manual...
  • Page 13 The light which is emitted during phosphorescence therefore has less energy and is at a longer wavelength than fluorescence. Formula: E = h x In this equation: E is energy h is Planck's constant is the wavelength 1200 Series FLD User Manual...

  • Page 14: Raman Effect

    The particular molecule and its environment will determine what Raman signals will be observed (if any). A plot of Raman intensity versus Raman shift is a Raman spectrum. 1200 Series FLD User Manual...

  • Page 15: Optical Unit

    Flash lamp board EM Grating Trigger Pack assembly Xenon Flash lamp Slit EM Condenser EX Cutoff filter Slit EX Photo Multiplier Tube Mirror Condenser EM EX Grating assembly REF Diode Flow Cell Diffuser Figure 6 Optical Unit 1200 Series FLD User Manual...
  • Page 16 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 Wavelenght [nm] Figure 7 Lamp Energy Distribution (vendor data) 1200 Series FLD User Manual...
  • Page 17 The surface contains many minute grooves, 1200 of them per millimeter. The grating carries a blaze to show improved performance in the visible range. Grating (EX) inside Mirror Figure 8 Mirror Assembly 1200 Series FLD User Manual...
  • Page 18 The excitation and emission gratings are similar in design, but have different blaze wavelengths. The excitation grating reflects most 1 order light in the ultra-violet range around 250 nm, whereas the emission grating reflects better in the visible range around 400 nm. 1200 Series FLD User Manual...
  • Page 19 Figure 9 on page 18. The selected wavelength of light is reflected onto the slit in the wall of the photo-multiplier compartment of the optical unit. The bandwidth of the emitted light is 20 nm. 1200 Series FLD User Manual...
  • Page 20 PMTs are designed for specific wavelength ranges. The standard PMT offers optimum sensitivity from 200 to 600 nm. In the higher wavelength range a red-sensitive PMT can improve performance. For additional PMT types refer to“Spare Parts” on page 134. 1200 Series FLD User Manual...

  • Page 21: Reference System

    (depending on the EX-wavelength), the measured data are normalized. A diffuser is located in front of the reference diode (see Figure 6 on page 15). This diffuser is made of quartz, reduces light and allows integral measurement of the light. 1200 Series FLD User Manual...

  • Page 22: Analytical Information From Primary Data

    Figure 12. The luminescence is short-lived, therefore the fluorescence detector need only measure over a short period of time after the lamp has flashed. Intensity Track&Hold 100% Ignite Time [µsec] Figure 12 Measurement of Fluorescence 1200 Series FLD User Manual...

  • Page 23: Phosphorescence Detection

    Phosphorescence Detection An appropriate parameter set will be specified as soon as you chose the phosphorescence detection mode (special setpoints under FLD parameter settings). Flash Intensity Phosphorescence 100% Measurement Time [µsec] Figure 13 Measurement of Phosphorescence 1200 Series FLD User Manual...

  • Page 24: Processing Of Raw Data

    The data resolution is 20 bit at a response time of 4 seconds (default, which is equivalent to a time constant of 1.8 seconds and appropriate for standard chromatographical conditions). Weak signals may cause errors in 1200 Series FLD User Manual...
  • Page 25 The data is smoothed, without being reduced, by taking the mean of a number of points. The mean of the same points minus the first plus the next, and so on, is calculated so that there are the same number of 1200 Series FLD User Manual...
  • Page 26 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. Figure 16 RESPONSETIME: Signal-to-Noise Ratio 1200 Series FLD User Manual...

  • Page 27: 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 Agilent 1200 Series module data transfer and synchronization.
  • Page 28 Power manufactured in Germany 2006 Configuration switch week of last major change 00130 number of unit product number serial number safety standards configuration switch settings voltage range power consumption / frequency Figure 17 Electrical Connections 1200 Series FLD User Manual...

  • Page 29: Instrument Layout

    • the plastic layers help cushion the electronic and mechanical parts from physical shock, and • the metal inner cabinet shields the internal electronics from electromagnetic interference and also helps to reduce or eliminate radio frequency emissions from the instrument itself. 1200 Series FLD User Manual...

  • Page 30: Early Maintenance Feedback (Emf)

    296 Hz (Standard), 560 V 63 mJoule (18.8 W) 74 Hz (Economy), 560 V 63 mJoule (4.7 W) Rotation (Multi Ex/Em) 74 Hz (Standard), 950 V 180 mJoule (13.3 W) 74 Hz (Economy), 560 V 63 mJoule (4.7 W) 1200 Series FLD User Manual...
  • Page 31 Introduction to the Fluorescence Detector Number of flashes Number of flashes to 75% of intitial radiometric light output 0.05 Input energy per flash [J] Figure 18 Lamp life 1200 Series FLD User Manual...

  • Page 32: Using The Emf Counters

    EMF limits, and then reset the EMF counters to zero. The next time the EMF counters exceed the new EMF limits, the EMF flag will be displayed, providing a reminder that maintenance needs to be scheduled. 1200 Series FLD User Manual...
  • Page 33 Agilent 1200 Series Fluorescence Detector User Manual Site Requirements and Specifications Site Requirements Physical Specifications Performance Specifications This chapter gives information on environmental requirements, physical and performance specifications. Agilent Technologies...

  • Page 34: Site Requirements And Specifications

    It plugs into the power-input socket at the rear of the detector. The male end of each power cord is different and designed to match the wall socket of a particular country or region. 1200 Series FLD User Manual...
  • Page 35 2.5 cm (1.0 inches) of space on either side and approximately 8 cm (3.1 inches) in the rear for air circulation and electric connections. If the bench should carry a Agilent 1200 Series system, make sure that the bench is designed to bear the weight of all modules.

  • Page 36: Physical Specifications

    Operating altitude Up to 2000 m (6500 ft) Non-operating altitude Up to 4600 m (14950 ft) For storing the detector Safety standards: IEC, CSA, UL, EN Installation category II, pollution degree 2. For indoor use only. 1200 Series FLD User Manual...

  • Page 37: Performance Specifications

    Site Requirements and Specifications Performance Specifications Table 3 Performance Specifications Agilent 1200 Series Fluorescence Detector Type Specification Comments Detection type Multi-signal fluorescence detector with rapid on-line scanning capabilities and spectral data analysis Performance 10 fg Anthracene, see note below this table...
  • Page 38 Site Requirements and Specifications Table 3 Performance Specifications Agilent 1200 Series Fluorescence Detector Type Specification Comments Timetable programing: up to 4 signal wavelengths, response time, PMT Gain, baseline behavior (append, free, zero), spectral parameters Spectrum acquisition: Excitation or Emission spectra Scan speed: 28 ms per datapoint (e.g.
  • Page 39 Site Requirements and Specifications Table 3 Performance Specifications Agilent 1200 Series Fluorescence Detector Type Specification Comments Safety and Extensive diagnostics, error maintenance detection and display (through Instant Pilot G4208A, Control Module G1323B and ChemStation), leak detection, safe leak handling, leak output signal for shutdown of pumping system.
  • Page 40 Site Requirements and Specifications 1200 Series FLD User Manual...

  • Page 41: Installing The Detector

    Agilent 1200 Series Fluorescence Detector User Manual Installing the Detector Unpacking the Detector Optimizing the Stack Configuration Installing the Detector Flow Connections to the Detector This chapter describes the installation of the detector. Agilent Technologies...

  • Page 42: Unpacking The Detector

    Damaged Packaging If the delivery packaging shows signs of external damage, please call your Agilent Technologies sales and service office immediately. Inform your service representative that the detector may have been damaged during shipment. If there are signs of damage, please do not attempt to install the detector.

  • Page 43: Detector Accessory Kit Contents

    Screwdriver hexagonal 2.5 mm, 100 mm long 5965-0028 Needle-Syringe 9301-0407 Glass-Syringe 9301-1446 Calibration Sample, Glycogen 5063-6597 Sample filter, diameter=3 mm, pore size 0.45 µm 5061-3367 (pack of 100) Wrench open end 1/4 – 5/16 inch 8710-0510 1200 Series FLD User Manual...
  • Page 44 Installing the Detector Fitting male PEEK Tubing Figure 19 Waste Tubing Parts Ferrule front This side is Ferrule back preinstalled Fitting male SST Capillary Figure 20 Inlet Capillary (Column-Detector) Parts 1200 Series FLD User Manual...

  • Page 45: Optimizing The Stack Configuration

    Installing the Detector Optimizing the Stack Configuration If your detector is part of a complete Agilent 1200 Series system, you can ensure optimum performance by installing the following configuration. This configuration optimizes the system flow path, ensuring minimum delay volume.
  • Page 46 Installing the Detector Solvent cabinet Vacuum degasser Local User Pump Interface Autosampler Column compartment Detector Figure 21 Recommended Stack Configuration (Front View) 1200 Series FLD User Manual...
  • Page 47 CAN Bus cable to local user interface CAN Bus cable AC power Analog detector signal (1 or 2 outputs per detector) LAN to LC ChemStation (location depends on detector) Figure 22 Recommended Stack Configuration (Rear View) 1200 Series FLD User Manual...

  • Page 48: Installing The Detector

    Figure 23 Front View of Detector 4 Connect the power cable to the power connector at the rear of the detector. 5 Connect the CAN cable to other Agilent 1200 Series modules. 1200 Series FLD User Manual...
  • Page 49 The detector is turned ON when the line power switch is pressed and the green indicator N O T E lamp is illuminated. The detector is turned OFF when the line power switch is protruding and the green light is OFF. 1200 Series FLD User Manual...
  • Page 50 To disconnect the detector from line, unplug the power cord. The power supply still WA R N I N G uses some power, even if the power switch at the front panel is turned OFF. The detector was shipped with default configuration settings. N O T E 1200 Series FLD User Manual...

  • Page 51: Flow Connections To The Detector

    The flow cell is shipped with a filling of isopropanol (also recommended when the N O T E instrument and/or flow cell is shipped to another location). This is to avoid breakage due to subambient conditions. 1200 Series FLD User Manual...
  • Page 52 2 Locate the flow cell. gain access to the flow cell area. 3 Assemble the column detector capillary from the 4 Assemble the waste tubing from the accessory kit. accessory kit. One side is already factory-assembled. Pre-assembled 1200 Series FLD User Manual...
  • Page 53 - the difference in measured pressure is due to the back pressure generated by the last detector and is the pressure seen by the FLD. 5 Insert the flow cell and install the capillaries to the flow 6 Connect the waste tubing to the bottom waste fitting. cell (top is outlet, bottom is inlet). 1200 Series FLD User Manual...
  • Page 54 The installation of the detector is now complete. The detector should be operated with the front cover in place to protect the flow cell area N O T E against strong drafts from the ouside. 1200 Series FLD User Manual...
  • Page 55 Agilent 1200 Series Fluorescence Detector User Manual First Steps with the Detector Before You Start Optimization Overview Getting Started and Checkout Method Development Example: Optimization for Multiple Compounds This chapter guides you how to start the work with the detector.

  • Page 56: First Steps With The Detector

    Flush your solvent delivery system for at least 15 minutes before checking sensitivity. If your pump has multiple channels, you should also flush the channels not in use. 1200 Series FLD User Manual...

  • Page 57: Optimization Overview

    Most fluorescent active molecules absorb at 230 nm. Set the excitation wavelength to 230 nm and on-line scan the emission spectra (multi-emission mode). Then set the determined emission wavelength and perform a multi-excitation scan (multi-excitation mode) to find the best excitation wavelength. 1200 Series FLD User Manual...
  • Page 58 N O T E based absorbance spectra, you should consider large differences in the used optical bandwidth (FLD=20 nm) which cause a systematic wavelength maximum shift depending on the absorbance spectrum of the compound under evaluation. 1200 Series FLD User Manual...

  • Page 59: Getting Started And Checkout

    First Steps with the Detector Getting Started and Checkout This chapter describes the check out of the Agilent 1200 Series fluorescence detector using the Agilent isocratic checkout sample. When required If you want to checkout the detector Hardware required LC system with G1321A FLD...

  • Page 60: Setting The Chromatographic Conditions

    Stop time 4 minutes Injection volume 5 µl Oven temperature (1200) 30°C FLD Excitations/Emission Wavelength EX = 246 nm, EM = 317 nm FLD PMT Gain PMT = 10 FLD Response time 4 seconds 1200 Series FLD User Manual...
  • Page 61 3 Start the run. 4 The resulting chromatograms are shown in Figure Ex=250 nm Ex=246 nm Biphenyl peak Ex=230 nm Ex=290 nm Figure 26 Biphenyl peak with different excitation wavelengths 5 The excitation maxima is around 250 nm. 1200 Series FLD User Manual...

  • Page 62: Observe The Maxima Via The Isoabsorbance Plot

    Observe the maxima via the isoabsorbance plot =246 nm, λ 1 Load the data file (λ =317 nm) and open the isoabsorbance plot. 2 The maximum λ will be found around 250 nm. Figure 27 Isoabsorbance Plot 1200 Series FLD User Manual...

  • Page 63: Method Development

    Thorough method development, including spectra acquisition, is fundamental to achieve good results. This chapter describes three different steps that can be taken with the Agilent 1200 Series fluorescence detector. Table 7 gives an overview of how to benefit from the operation modes during these steps.

  • Page 64: Step 1: Check The Lc System For Impurities

    The Raman bands of water are seen below the first-order Raleigh stray light. Since the cut-off filter cuts off light below 280 nm, the second-order Raleigh stray light starts above 560 nm. 1200 Series FLD User Manual...

  • Page 65: Step 2: Optimize Limits Of Detection And Selectivity

    440 nm, emission spectrum with excitation at 250 nm of 1 ug/ml quinidine. Detector settings: step size 5 nm, PMT 12, Response time 4 s. Figure 29 Excitation and emission spectra of quinidine 1200 Series FLD User Manual...
  • Page 66 This is done preferably with a manual FLD cuvette when pure compounds are available. Procedure II - Use two LC runs with the Agilent 1200 Series FLD to separate the compound mix under known conditions and acquire emission and excitation spectra separately.
  • Page 67 68: All excitation and emission spectra of Quinidine (1 µg/ml) are shown in graphic. Fluorescence intensity is plotted vs excitation and emission wavelengths. Detector settings: step size 5 nm, PMT 12, Response time 4 s 1200 Series FLD User Manual...
  • Page 68 First Steps with the Detector straylight 350 nm Ex 315 nm Ex 250 nm Ex 1. order Ex axis Em axis Figure 30 Characterization of a pure compound from a fluorescence scan 1200 Series FLD User Manual...
  • Page 69 The area of high intensity (red) is caused by stray light when emission spectra overlap with the excitation wavelength. This can be avoided by fitting the spectral range automatically. Excitation at 260 nm is most appropriate for all PNAs. 1200 Series FLD User Manual...
  • Page 70 4 s, step size 5 nm This shows the isofluorescence plot of emission spectra for 15 PNAs (5 µg/ml) with a fixed excitation wavelengths (260 nm). Figure 31 Optimization of the time-program for the emission wavelength 1200 Series FLD User Manual...
  • Page 71 Table 10 Timetable for the analysis of 15 polynuclear aromatic hydrocarbons Time [min] Exitation Wavelength [nm] Emission Wavelength [nm] 19.0 This timetable gives the conditions for optimum detection based on the results of two chromatographic runs. 1200 Series FLD User Manual...
  • Page 72 First Steps with the Detector Procedure III - Make a single run with the Agilent 1200 Series DAD/FLD combination For most organic compounds, UV-spectra from diode array detectors are nearly identical to fluorescence excitation spectra. Spectral differences are caused by specific detector characteristics such as spectral resolution or light sources.
  • Page 73 UV-spectrum and fluorescence spectra for 2,3-diaminophenazine (DAP) The two upper traces are obtained using two different excitation wavelengths. The lower trace is a pure standard of the known impurities. Figure 34 Qualitive analysis of MBC (2-benzimidazole carbamic acid methylester) and impurities 1200 Series FLD User Manual...

  • Page 74: Step 3: Set Up Routine Methods

    LC methods must be rugged enough. With difficult matrices, simultaneous multi-wavelength detection offers more reliability than timetable-controlled wavelength switching. The Agilent 1200 Series FLD can, in addition, acquire fluorescence spectra while it records the detector signals for quantitative analysis. Therefore qualitative data are available for peak confirmation and purity checks in routine analysis.
  • Page 75 Very often this happens with complex matrices, influencing the retention of compounds. In spectral mode, the Agilent 1200 Series FLD can acquire up to four different signals simultaneously. All of them can be used for quantitative analysis.
  • Page 76 PNA analysis. The match factor given in the report for each peak indicates the degree of similarity between the reference spectrum and the spectra from a peak. A match factor of 1,000 means identical spectra. 1200 Series FLD User Manual...
  • Page 77 13.340 13.300 14.022 1.14082e-1 Chrysene@em 15.274 15.200 16.052 6.90434e-1 Benzo(b)fluoranthene@em 16.187 16.200 17.052 5.61791e-1 Benzo(k)fluoranthene@em 16.865 16.900 17.804 5.58070e-1 Benz(a)pyrene@em 18.586 18.600 19.645 5.17430e-1 Dibenz(a,h)anthracene@em 19.200 19.100 20.329 6.03334e-1 Benzo(g,h,i)perylene@em 20.106 20.000 21.291 9.13648e-2 Indeno(1,2,3-cd)pyrene@em 1200 Series FLD User Manual...

  • Page 78: Example: Optimization For Multiple Compounds

    14.5 minutes %B=90 at 22.5 minutes %B=95 Stop time 26 minutes Post time 8 minutes Injection volume 1 µl Oven temperature (1200) 30°C FLD PMT Gain PMT = 15 FLD Response time 4 seconds 1200 Series FLD User Manual...
  • Page 79 Figure 36 Detector Settings for Emission Scan 2 Wait until the baseline stabilizes. Complete the run. 3 Load the signal. (In this example just the time range of 13 minutes is displayed). 1200 Series FLD User Manual...
  • Page 80 First Steps with the Detector Figure 37 Chromatogram from Emissions Scan 4 Use the isoabsorbance plot and evaluate the optimal emission wavelengths, shown in the table below. Figure 38 Isoabsorbance Plot from Emission Scan 1200 Series FLD User Manual...
  • Page 81 See Figure DO NOT select additional excitation wavelengths (B, C, D). Doing so will increase the scan time and will lower the performance Figure 39 Detector Settings for Excitation Scan 1200 Series FLD User Manual...
  • Page 82 Chromatogram - excitation scan at reference wavelength 260/330 nm 8 Use the isoabsorbance plot and evaluate the optimal excitation wavelengths (in this example just in the time range of 13 minutes). Figure 41 Isoabsorbance Plot - Excitation 1200 Series FLD User Manual...
  • Page 83 2 Set the fluorescence scan range under FLD special setpoints according to your needs. The scan time will increase when the range is enlarged. With the default values, the scan N O T E takes about 2 minutes. 3 Set PMT gain to 16. 1200 Series FLD User Manual...
  • Page 84 A low background will improve the signal-to-noise, see also “Reducing Stray Light” N O T E page 96. Raleigh scattering Note: This white area is normally dark blue. Raman scattering of water 2nd order of cutoff Figure 43 Fluorescence Scan of Water 1200 Series FLD User Manual...
  • Page 85 Agilent 1200 Series Fluorescence Detector User Manual How to optimize the Detector Optimization Overview Design Features Help Optimization Finding the Best Wavelengths Finding the Best Signal Amplification Changing the Xenon Flash Lamp Frequency Selecting the Best Response Time Reducing Stray Light This chapter provides information on how to optimize the detector.

  • Page 86: How To Optimize The Detector

    How to optimize the Detector Optimization Overview Refer to “Optimization Overview” on page 57 for details. 1200 Series FLD User Manual...

  • Page 87: Design Features Help Optimization

    How to optimize the Detector Design Features Help Optimization The Agilent 1200 Series fluorescence detector has several features you can use to optimize detection: Table 17 PMTGAIN Amplification factor LAMP Flash frequency RESPONSETIME Data reduction interval Check Performance Before You Start Before you start you should check that your detector is performing according to the specifications published by Agilent Technologies.

  • Page 88: Finding The Best Wavelengths

    Flash-type lamps shift the optimum wavelength to lower wavelength ranges with the Agilent 1200 Series fluorescence detector to a maximum of 250 nm. The excitation grating is blazed for highest efficiency at 300 nm.

  • Page 89: A Real Example

    How to optimize the Detector A Real Example Although an excitation wavelength of 340 nm is quoted in the literature the Agilent 1200 Series fluorescence detector scan of orthophthalaldehyde, a derivative of the amino acid alanine, (Figure 44 on page 89) shows a maximum between 220 nm and 240 nm.

  • Page 90: Finding The Best Signal Amplification

    Figure 45 the PMTGAIN was gradually raised from 4 up to 11 (the peak is from the Agilent Technologies isocratic sample which was diluted 1000 times). With increasing PMTGAIN there was an improvement in signal-to-noise up to 10. Above 10 the noise increased proportionately to the signal with no improvement in signal-to-noise.
  • Page 91 For the best gain, check your solvent under flow conditions with the auto-gain function. Do not use higher values than proposed by the system, if not necessary, because of excessive high fluorescence signals. Use the PMT test to automatically determine the setting. 1200 Series FLD User Manual...

  • Page 92: Changing The Xenon Flash Lamp Frequency

    74 Hz (Standard), 950 V 180 mJoule (13.3 W) 74 Hz (Economy), 560 V 63 mJoule (4.7 W) Best sensitivity can be expected with “no economy”, see Figure Standard=296 Hz Economy=74 Hz Figure 46 Xenon Flash Lamp Frequency 1200 Series FLD User Manual...

  • Page 93: Lamp Life Savings

    There are three ways to save lamp life: • switch to “lamp on during run” without loss of sensitivity. • switch to “economy” mode with a certain loss of sensitivity. • a combination of the above. 1200 Series FLD User Manual...

  • Page 94: Selecting The Best Response Time

    How to optimize the Detector Selecting the Best Response Time Data reduction using the RESPONSETIME function will increase your signal-to-noise ratio. For example, see Figure 8 sec 4 sec 1 sec Figure 47 Finding Best Response Time 1200 Series FLD User Manual...
  • Page 95 How to optimize the Detector LC fluorescence detectors typically work with response times of 2 or 4 seconds. The default of the Agilent 1200 Series fluorescence detector is 4 seconds. It is important to know that comparing sensitivity requires using the same response time.

  • Page 96: Reducing Stray Light

    To remove this stray light, you need a cut-off filter around 280 nm. Because of a known set of applications a 295 nm cut-off filter is built-in for undisturbed application up to 560 nm without compromises (see Figure 49 page 97). 1200 Series FLD User Manual...
  • Page 97 How to optimize the Detector Exitation 300nm Second order light 600nm Stray light no filter filter 280 nm Wavelength [nm] Figure 49 Reducing Stray Light 1200 Series FLD User Manual...
  • Page 98 How to optimize the Detector 1200 Series FLD User Manual...
  • Page 99 Agilent 1200 Series Fluorescence Detector User Manual Troubleshooting and Test Functions Overview of the Detector’s Indicators and Test Functions Status Indicators User Interfaces Agilent LC Diagnostic Software This chapter gives an overview about the troubleshooting and diagnostic features and the different user interfaces.

  • Page 100: Troubleshooting And Test Functions

    “Wavelength Verification and Calibration” in the Service Manual. Test Functions A series of test functions are available for troubleshooting and operational verification after exchanging internal components, see “Test Functions” in the Service Manual. 1200 Series FLD User Manual...

  • Page 101: Status Indicators

    Line power switch with green light Figure 50 Location of Status Indicators Power Supply Indicator The power supply indicator is integrated into the main power switch. When the indicator is illuminated (green) the power is ON. 1200 Series FLD User Manual...

  • Page 102: Detector Status Indicator

    • An error condition is indicated when the status indicator is red. An error condition indicates the detector has detected an internal problem which affects correct operation of the detector. Usually, an error condition requires attention (e.g. leak, defective internal components). An error condition always interrupts the analysis. 1200 Series FLD User Manual...

  • Page 103: User Interfaces

    Yes (M) Lamp Intensity Dark Current via command section Maintenance section Diagnose The Agilent Control Module (G1323B) does not do any calculations. So there will be no N O T E reports generated with passed/failed information. 1200 Series FLD User Manual...

  • Page 104: Agilent Lc Diagnostic Software

    The Agilent LC diagnostic software is an application independent tool that provides troubleshooting capabilities for the Agilent 1200 Series modules. It provides for all 1200 Series LC the possibility of a first guided diagnostic for typical HPLC symptoms and a status report stored as Adobe Acrobat pdf or as a printable file to assist users evaluating the instrument state.

  • Page 105: Maintenance And Repair

    Agilent 1200 Series Fluorescence Detector User Manual Maintenance and Repair Introduction into Repairing the Fluorescence Detector Warnings and Cautions Cleaning the Detector Using the ESD Strap This chapter provides general information on maintenance and repair of the detector. Agilent Technologies...

  • Page 106: Introduction Into Repairing The Fluorescence Detector

    The security lever at the power input socket prevents the detector cover from being removed when line power is still connected. These repairs are described in “Repairs” in the Service Manual. 1200 Series FLD User Manual...

  • Page 107: Warnings And Cautions

    ESD protection when handling electronic boards and components (see “Using the ESD Strap” on page 110). There is a risk of damaging hardware due to overheating when operating the C A U T I O N instrument without covers. 1200 Series FLD User Manual...
  • Page 108 Eye damage may result from directly viewing the light produced by the Xenon flash WA R N I N G lamp used in this product. Always turn the xenon flash lamp off before removing it. 1200 Series FLD User Manual...

  • Page 109: Cleaning The Detector

    Do not use an excessively damp cloth allowing liquid to drip into the detector. Do not let liquid drip into the detector. It could cause shock hazard and it could WA R N I N G damage the detector. 1200 Series FLD User Manual...

  • Page 110: Using The Esd Strap

    2 Unroll the rest of the band and peel the liner from the copper foil at the opposite end. 3 Attach the copper foil to a convenient and exposed electrical ground. Figure 51 Using the ESD Strap 1200 Series FLD User Manual...

  • Page 111: Maintenance

    Agilent 1200 Series Fluorescence Detector User Manual Maintenance Overview of Maintenance Exchanging a Flow Cell How to use the Cuvette Flow Cell Flushing Correcting Leaks Replacing Leak Handling System Parts Replacing the Interface Board Replacing the Detector’s Firmware Tests & Calibrations...

  • Page 112: Overview Of Maintenance

    “Wavelength Verification and Calibration” on page 125. Flow cell flushing If flow cell is contaminated Leak sensor drying If leak has occurred. Check for leaks. Leak handling System If broken or corroded. Check for leaks. replacement 1200 Series FLD User Manual...

  • Page 113: Exchanging A Flow Cell

    DO NOT install the inlet capillary to the outlet connection of the flow cell. This will result in N O T E poor performance. Preparations for this procedure: 1 Press the release buttons and remove the front cover for access to the flow cell area. • Turn off the flow. 1200 Series FLD User Manual...
  • Page 114 This will result in poor performance or damage. 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. 1200 Series FLD User Manual...
  • Page 115 (outside of the cell compartment) and all capillary connections. Note: Perform a wavelength verification to check the correct positioning of the flow cell, as described in chapter “Wavelength Verification and Calibration” on page 125. 1200 Series FLD User Manual...

  • Page 116: How To Use The Cuvette

    3 Use the syringe (see “Cuvette Kit” on page 133) 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. 1200 Series FLD User Manual...

  • Page 117: Flow Cell Flushing

    It is to be used as a last attempt to salvage the cell before cell replacement. Note that the cell is a consumable item. Do not exceed the pressure limit of 20 bar (0.2 MPa). N O T E 1200 Series FLD User Manual...

  • Page 118: Correcting Leaks

    2 Use tissue to dry the leak sensor area and the leak pan. 3 Observe the capillary connections and the flow cell area for leaks and correct, if required. 4 Replace the front cover. Figure 52 Observing for Leaks 1200 Series FLD User Manual...

  • Page 119: Replacing Leak Handling System Parts

    4 Insert the leak funnel with the tubing in its position. 5 Insert the leak funnel into the leak funnel holder. 6 Replace the front cover. Leak funnel Leak funnel holder Leak tubing Leak sensor Figure 53 Replacing Leak Handling System Parts 1200 Series FLD User Manual...

  • Page 120: Replacing The Interface Board

    None To replace the interface board unscrew the two screws, remove the board, slide in the new interface board and fix it with the board’s screws. Interface board Figure 54 Location of the Interface Board 1200 Series FLD User Manual...

  • Page 121: Replacing The Detector's Firmware

    1 Download the module’s firmware, the LAN/RS-232 FW Update Tool Version 2.00 or above and the documentation from the Agilent web http://www.chem.agilent.com/scripts/cag_firmware.asp. 2 Load the firmware into the detector as described in the documentation. 1200 Series FLD User Manual...

  • Page 122: Tests & Calibrations

    Maintenance Tests & Calibrations The following tests are required after maintenance of lamps and flow cells: • “Lamp Intensity Test” on page 123. • “Wavelength Verification and Calibration” on page 125. 1200 Series FLD User Manual...

  • Page 123: Lamp Intensity Test

    (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). Figure 55 Lamp Intensity Test (Report) 1200 Series FLD User Manual...
  • Page 124 (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. Figure 56 Lamp Intensity History 1200 Series FLD User Manual...

  • Page 125: Wavelength Verification And Calibration

    PMT gain will be changed automatically and requires an additional 1 minute per scan. The excitation grating and the emission grating are calibrated using Rayleigh stray light from the flow cell or cuvette measured with the photomultiplier tube. 1200 Series FLD User Manual...
  • Page 126 Maintenance Figure 57 Wavelength Calibration When the lamp is of, the calibration process will stop within the first two steps with N O T E “Wavelength Calibration Failed”. 1200 Series FLD User Manual...

  • Page 127: Wavelength Calibration Procedure

    2 Fill the prepared amount into a suitable bottle/vial. 3 Fill 10 ml of distilled water into the vial and shake. 4 Wait 5 minutes and shake again. After 10 minutes the solution is ready. 1200 Series FLD User Manual...

  • Page 128: Preparation Of The Flow Cell

    Do not inject the calibration sample without the sample filter. N O T E 10 Slowly inject about 0.2 ml and wait for about 10 seconds to inject another 0.1 ml. This will assure that the cell is filled properly. 1200 Series FLD User Manual...

  • Page 129: Wavelength Calibration

    Rinse the flow cell with pure water at a minimum of 1.5 ml/min to get rid of the Glycogen N O T E from the cell and the capillaries. When organic solvent is sequentially applied (without rinsing), a blockage of capillaries may occur. 1200 Series FLD User Manual...
  • Page 130 Maintenance 1200 Series FLD User Manual...

  • Page 131: 9 Parts And Materials For Maintenance

    Agilent 1200 Series Fluorescence Detector User Manual Parts and Materials for Maintenance Overview of Maintenance Parts Cuvette Kit Spare Parts Accessory Kit This chapter provides information on parts for maintenance. Agilent Technologies...

  • Page 132: Overview Of Maintenance Parts

    Corrugated tubing, 120 mm lg, re-order 5 m 5062-2463 Teflon Tubing flexible i.d. 0.8 mm (flow cell to waste) 5062-2462 Cable CAN to Agilent 1200 Series modules (0.5 m) 5181-1516 Cable CAN to Agilent 1200 Series modules (1 m) 5181-1519...

  • Page 133: Cuvette Kit

    FLD Cuvette Kit, 8 µl, 20 bar G1321-60007 includes Tubing, flexible, 1 meter SST Fitting, QTY=1 79814-22406 SST front ferrule, QTY=1 0100-0043 SST back ferrule, QTY=1 0100-0044 PEEK fitting, QTY=1 0100-1516 Needle-Syringe 9301-0407 Glass-Syringe 9301-1446 1200 Series FLD User Manual...

  • Page 134: Spare Parts

    Cutoff filter kit: 280 nm, 295 nm, 305 nm, 335 nm, 345 nm 5061-3329 Cutoff filter 370 nm 1000-0822 Photo-multiplier Tube (PMT) R928HA (185 to 900 nm) contact Hamamatsu dealers Photo-multiplier Tube (PMT) R3788HA (185 to 750 nm) 1200 Series FLD User Manual...

  • Page 135: Accessory Kit

    Glass-Syringe 9301-1446 Calibration Sample, Glycogen 5063-6597 Sample filter, diameter=3 mm, pore size 0.45 µm, QYT=5 5061-3367 (pack of 100) Hex key set 1 – 5 mm 8710-0641 Wrench open end 1/4 – 5/16 inch 8710-0510 1200 Series FLD User Manual...
  • Page 136 Parts and Materials for Maintenance Figure 59 Waste Tubing Parts This end is pre-installed Figure 60 Inlet Capillary (Column-Detector) Parts 1200 Series FLD User Manual...
  • Page 137 Agilent 1200 Series Fluorescence Detector User Manual Appendix General Safety Information Lithium Batteries Information Radio Interference Sound Emission UV-Radiation (UV-lamps only) Solvent Information Agilent Technologies on Internet This chapter provides safetey and other general information. Agilent Technologies...

  • Page 138: A Appendix

    Make sure that only fuses with the required rated current and of the specified type (normal blow, time delay, and so on) are used for replacement. The use of repaired fuses and the short-circuiting of fuseholders must be avoided. 1200 Series FLD User Manual...
  • Page 139 Table 25 Safety Symbols Symbol Description The apparatus is marked with this symbol when the user should refer to the instruction manual in order to protect the apparatus against damage. Indicates dangerous voltages. 1200 Series FLD User Manual...
  • Page 140 A caution alerts you to situations that could cause a possible loss of data. Do not C A U T I O N proceed beyond a caution until you have fully understood and met the indicated conditions. 1200 Series FLD User Manual...

  • Page 141: Lithium Batteries Information

    WA R N I N G av apparatfabrikanten. Brukt batteri returneres appararleverandoren. Bij dit apparaat zijn batterijen geleverd. Wanneer deze leeg zijn, moet u ze niet weggooien N O T E maar inleveren als KCA. 1200 Series FLD User Manual...

  • Page 142: Radio Interference

    Appendix Radio Interference Never use cables other than the ones supplied by Agilent Technologies to ensure proper functionality and compliance with safety or EMC regulations. Test and Measurement If test and measurement equipment is operated with equipment unscreened cables and/or used for measurements on open set-ups, the user has to assure that under operating conditions the radio interference limits are still met within the premises.

  • Page 143: Sound Emission

    This product has a sound pressure emission (at the operator position) < 70 dB. • Sound Pressure Lp < 70 dB (A) • At Operator Position • Normal Operation • According to ISO 7779:1988/EN 27779/1991 (Type Test) 1200 Series FLD User Manual...

  • Page 144: Uv-Radiation (Uv-Lamps Only)

    10 minutes 5.0 µW/cm Typically the radiation values are much smaller than these limits: Table 27 UV-Radiation Typical Values Position Effective Irradiance Lamp installed, 50-cm distance average 0.016 µW/cm Lamp installed, 50-cm distance maximum 0.14 µW/cm 1200 Series FLD User Manual...

  • Page 145: Solvent Information

    • Halogenated solvents or mixtures which form radicals and/or acids, for example: → 2CHCl 2COCl + 2HCl This reaction, in which stainless steel probably acts as a catalyst, occurs quickly with dried chloroform if the drying process removes the stabilizing alcohol. 1200 Series FLD User Manual...
  • Page 146 For example, a 1-% solution of acetic acid in methanol will attack steel. • Solutions containing strong complexing agents (for example, EDTA, ethylene diamine tetra-acetic acid). • Mixtures of carbon tetrachloride with 2-propanol or THF. 1200 Series FLD User Manual...

  • Page 147: Agilent Technologies On Internet

    For the latest information on products and services visit our worldwide web site on the Internet at: http://www.agilent.com Select “Products” - “Chemical Analysis” It will provide also the latest firmware of the Agilent 1200 Series modules for download. 1200 Series FLD User Manual...
  • Page 148 Appendix 1200 Series FLD User Manual...
  • Page 149 EM, 15, early maintenance feedback (EMF), on lithium batteries, EX, 15, electrical connections installation multi wavelength detection, 1200 Series FLD User Manual...
  • Page 150 PMT gain test, Raman, troubleshooting rear view of module, 28, error messages, recalibration of wavelength, status indicators, 100, reference diode, reference system, repair flushing of flow cell, unpacking, 1200 Series FLD User Manual...
  • Page 152 In This Book This manual contains technical reference information about the Agilent 1200 Series fluorescence detector. The manual describes the following: • introcduction and specifications, • installation, • using and optimizing, • troubleshooting, • maintenance, • parts identification, • safety and related information.

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