Varian ProStar 363 Operation Manual
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Varian ProStar 363 Operation Manual

Fluorescence detector
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Varian Analytical Instruments
2700 Mitchell Drive
Walnut Creek, CA 94598-1675/USA
ProStar 363
Fluorescence Detector
Operation Manual
©Varian, Inc. 2000
03-914875-00: 1

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  • Page 1 Varian Analytical Instruments 2700 Mitchell Drive Walnut Creek, CA 94598-1675/USA ProStar 363 Fluorescence Detector Operation Manual ©Varian, Inc. 2000 03-914875-00: 1...

  • Page 3: Table Of Contents

    To Place a Marker on the Recorder ................28 Time Program......................29 Setting Up the Utilities ....................32 Setting Up Spectrum Acquisition Parameters and Output ........35 Collecting the Spectra ....................37 Setting the Recorder and Spectral Output Parameters..........38 ProStar 363 Fluorescence Detector...
  • Page 4 Selecting the GLP Function..................42 Example of Time Program Operation................ 44 Shutting Down the Detector ..................48 Flushing the Flow Cell After Use................... 48 Maintenance and Troubleshooting ................49 Replacing the Xenon Lamp................... 49 To Cool Down the Xenon Lamp: ................49 Lamp Replacement ....................
  • Page 5 Figures Figure 1 ProStar 363 Fluorescence Detector..............1 Figure 2 Power Receptacle Polarity ..................5 Figure 3 Door and Door Cap Orientation ................7 Figure 4 Door and Door Cap Assembly................8 Figure 5 Door Installation ....................9 Figure 6 Rear Panel......................10 Figure 7 Functional Block Diagram ...................16 Figure 8 Keyboard ......................17...
  • Page 6 03-914875-00:1...

  • Page 7: Introduction

    Introduction General Figure 1 ProStar 363 Fluorescence Detector ProStar 363 Fluorescence Detector...
  • Page 8 The ProStar 363 Fluorescence detector can be operated as a stand-alone HPLC detector or can be integrated into a Liquid Chromatography System controlled remotely from a Star Workstation. In the stand-alone mode of operation, all user inputs are made on the front panel keypad. All operator inputs are displayed on a front panel display consisting of two lines of forty characters each.

  • Page 9: Installation

    Installation General The modular design of the ProStar 363 Detector enables you to locate it anywhere within the limitations imposed by the length of the power cord and signal cables. In order to keep liquid dead volume as low as possible, the detector should be positioned so that distance between the column outlet to the flow cell inlet is as short as is practical.

  • Page 10: Unpacking

    Carefully unpack the instrument and place it on the bench top. After unpacking the system, carefully check the contents of each box against the packing list. If any part is missing or damaged, contact the nearest Varian dealer. Laboratory Requirements...

  • Page 11: Ground Terminal

    Neutral Hot (Line) (Line) Ground Ground 220 Vac Nominal 15 Amps 50 Hz 120 Vac Nominal 60 Hz Ground (green/yellow or green) Neutral (light blue or white) Hot (brown or black) Figure 2 Power Receptacle Polarity ProStar 363 Fluorescence Detector...

  • Page 12: Location

    Location The ProStar 363 Fluorescence Detector is a sensitive instrument and should always be handled with the degree of care appropriate to laboratory instrumentation. After unpacking, place the detector conveniently near your HPLC system.

  • Page 13: Installation Of Door Cap

    If the ProStar 363 is the top module in the stack, the door cap should be installed prior to installing the door on the module. If the ProStar 363 is not at the top of the stack, do not install the cap and proceed to the next section describing door installation.

  • Page 14: Installation And Removal Of Door

    Figure 4 Door and Door Cap Assembly Installation and Removal of Door The module door may be attached to the front of the ProStar 363 to cover the tubing connections to the flow cell, see Figure 5.

  • Page 15: Figure 5 Door Installation

    The door should now pivot on the pins and close against the magnetic latch. To remove the door, gently push down on the door and slide the lower hinge pin out of the lower hinge. Now lift and slide the door out. ProStar 363 Fluorescence Detector...

  • Page 16: Power And Signal Cable Connections (Rear Panel)

    If the ProStar 363 will be controlled by the Star Workstation, connect the serial cable (03-935462-01) to the serial connector on the back of the ProStar 363 and to a serial port on the Star Workstation. This same cable can be connected to the ProStar AutoSamplers and/or the ProStar 210/215 or SD-1 pumps.

  • Page 17: Table 1 Input/Output Terminals

    Installation If the ProStar 363 is to send data to a Star Workstation, the Processor or Data System output must be connected either to a CIM box, CIM in a pump, or an ADC board. Connect the ProStar 363 to the ADC board with the Fluorescence detector interface cable (03-936156-01).

  • Page 18: Hydraulic Connections

    Detector Outlet Backpressure Terminator A backpressure terminator assembly is included in the Standard Accessory Kit shipped with your ProStar 363 Detector. The back pressure terminator (03-919393-00) comes completely assembled and should be connected into the flow cell. The terminator applies about 40 psi back pressure to the flow cell to prevent outgassing and bubble formation in the light path which would cause an unstable baseline.

  • Page 19: Installing And Adjusting The Xenon Lamp

    The final part of installation is to install and align the xenon lamp. This is the same procedure that you will have to do when you replace a xenon lamp. Refer to procedures in Replacing the Xenon Lamp beginning on page 49. ProStar 363 Fluorescence Detector...
  • Page 20 03-914875-00:1...

  • Page 21: System Description

    System Description General The following paragraphs describe the ProStar 363 Detector as a stand-alone Fluorescence time-programmable detector controlled entirely from the local front panel keypad. When the instrument is being controlled from the Star Workstation, an [L] is displayed in the upper right hand corner of the two-line display.

  • Page 22: Optics Hardware

    Optics Hardware Diffusion plate Sample (Flow cell) Xenon lamp Excitation monochromator Monitor detector (Photodiode) Xenon lamp starter Emission Photomultiplier monochromator (Fluorescence monochromator Xenon lamp ballast Photomultiplier high voltage circuit Monitor side Sample side amplifier amplifier Operational circuit Electrical connection Recorder Optional path or Integrator Figure 7 Functional Block Diagram...

  • Page 23: Keyboard And Display

    All data entry is made via the keyboard on the front panel of the unit (see Figure 8). The role of each key is indicated in Table 3. For additional information about a key, see the detailed discussions in the Operation Section. Figure 8 Keyboard ProStar 363 Fluorescence Detector...

  • Page 24: Table 3 Keyboard

    Table 3 Keyboard Function Function Wave Used to set the Excitation and Used for inputting a decimal point. Length Fluorescence Wavelengths. Start/ Starts (Stops) the time program. Clear Used to remove the present data input Stop on the display before the Enter key is Starts (Stops) analog output of pressed (e.g., to erase incorrect data).

  • Page 25: Rear Panel

    Also, a back pressure restrictor should be put on the end of the waste line to prevent bubble formation. ProStar 363 Fluorescence Detector...

  • Page 26: Xenon Lamp

    Xenon Lamp The 150W xenon lamp is located on the right side of the instrument behind the flow cell as shown in Figure 10. Figure 10 Xenon Lamp Location The xenon lamp is mounted in a removable holder and adjusted with three screws located below at the bottom right of the lamp area.

  • Page 27: Operation

    LCD, refer to the Maintenance and Troubleshooting section. Liquid Crystal Display (LCD) Varian ProStar 363 Fluorescence Detector Firmware P/N xxxxxxx-xx (Year) After a short period of time, the monitor display presents: WL DRIVE & LAMP INITIALIZING...
  • Page 28 'L' is indicated when it is in the key-locked status. The “L” status is displayed when the ProStar 363 is being controlled by the Star Workstation. After the initialization process is complete, the unit should be allowed to warm up for approximately 30 minutes to meet specifications.

  • Page 29: Normal Operation

    Unit ID <8E1 19200> The Unit ID represents the address of the ProStar 363 on the serial bus. The addresses range from 90 to 99 so a unit ID of “0” means an address of 90, and a unit ID of 4 means an address of “4”.

  • Page 30: Setting Up The Instrument Conditions

    To change the Unit ID, Press Enter. The screen below appears. Unit ID (0-9) At this point, you can enter any number from 0 through 9 with 0 being the default. The values 0-3 are recognized by the Star Workstation and translates to Device Identifiers 90-93. Turn off the power to the system and then turn the power back on.
  • Page 31 If the indicated setting is acceptable, simply press the M key to go to the time program screen, or press the L key to go to the photomultiplier voltage setup screen. Press the Escape key to return to the monitor screen without changing the current setting. ProStar 363 Fluorescence Detector...
  • Page 32 Indicate if the time program should be used. USE TIME PROGRAM (NO=0, YES=1) Select the desired value and press Enter. The lamp select screen will be presented. (If the indicated time constant is acceptable, simply press the M key or the Enter key to present the lamp select screen, or press the L key to go to the Em bandwidth setting screen.

  • Page 33: Wavelength Range Setup Screen

    Press, Numeric key for the desired excitation wavelength, the Enter key → Monitor setup screen If the indicated fluorescence wavelength is acceptable, simply press the M key to go to the monitor screen, or press the L key ProStar 363 Fluorescence Detector...

  • Page 34: To Adjust The Auto Zero

    to return to the excitation wavelength screen. As an alternative, press the Escape key to return to the monitor screen without changing the current setting. To Adjust the Auto Zero Auto Zero Press the Auto Zero key to adjust the FL intensity to zero. This will affect the reading on the monitor screen and the output signal level.

  • Page 35: Time Program

    Enter the number for the program to be created or modified. The relevant execution program number is changed at the same time. To return to the monitor screen without changing the value, press the Escape key. ProStar 363 Fluorescence Detector...
  • Page 36 When a program is selected, it is necessary to specify whether it should be modified or a new program should be created. MODIFY=0, NEW=1 Numeric key → Enter key Set up the time program The first line of the time program is indicated below. TIME BASE (A/Z=1, HOLD=2) 0.0 250 350...
  • Page 37 To remove a stored numeric value, use the Delete key. To go to the previous or next step, use the  or  keys. • After the final step is set up, press the Escape key to return to the monitor screen. ProStar 363 Fluorescence Detector...

  • Page 38: Setting Up The Utilities

    Executing the Time Program Start/ Stop Start the time program by pressing the Start/Stop key. The status indicator “R” appears and indicates the program is running. To stop execution of the time program, press the Start/Stop key. Setting Up the Utilities Utility/ Setup To access the Utility/Setup setup screen, press the Utility/...
  • Page 39 (see Figure 11a). In such a case, the offset function is used to adjust the shifted baseline to the allowable input signal range of the data processor (see Figure 11b). ProStar 363 Fluorescence Detector...

  • Page 40: Figure 11 Example Of Offsetting

    Figure 11 Example of Offsetting Set the monitor display mode. DISPLAY MODE (FL=1, Ex<M>=2, Em<M>=3) Numeric key → Enter key The currently specified value is displayed; the default value is 1. Input Value Meaning FL mode Enter 1 for fluorescence intensity measurement. (Normal status screen) Ex<M>...

  • Page 41: Setting Up Spectrum Acquisition Parameters And Output

    Enter 1 to define the excitation scan range. Enter 2 to define the fluorescence scan range. NOTE: The system will present either the Ex SCANNING WL RANGE screen or the Em SCANNING WL RANGE when Enter is selected. ProStar 363 Fluorescence Detector...
  • Page 42 Specify an excitation scan range. Indicate the starting wavelength (minimum value). (The information in this section is not relevant in Em Scan Mode.) Ex SCANNING WL RANGE (200-800) nm 200 - 550 1 nm step Enter the desired wavelength with the numeric keypad and press the Enter key to change the value.

  • Page 43: Collecting The Spectra

    When collecting a spectrum, there should be a sample in the cell and the flow rate should be zero (0). Specify a storage file. SPECTRUM MEMORY NO. (1-8) MODE: Em Numeric key → Enter key ProStar 363 Fluorescence Detector...

  • Page 44: Setting The Recorder And Spectral Output Parameters

    The scanning starts with the display below. Up to eight spectra can be stored. The currently selected scan mode is displayed by the MODE field. The spectral memory numbers (input values) are used for identification of stored spectra. When a spectral memory number is specified, the spectrum in that memory location is deleted and the new spectrum data is stored.
  • Page 45 RCD SPEED (40 nm/min=1, 60 nm/min=2) Indicate the output speed for the recorder. Numeric key → ENTER key The currently specified recorder speed is displayed; the default Recorder Speed value is 2. ProStar 363 Fluorescence Detector...
  • Page 46 Input Value Meaning 40 nm/min=1 60 nm/min=2 RECORD SPECTRUM NO. (1-8) Assign a spectral number to output data Numeric key → Enter key The currently specified value is displayed; the default value is 1. A value ranging from 1 to 8 can be entered using the Numeric keys.
  • Page 47 Workstation by pressing the Start key on the ProStar 363 module in System Control. For more information about scanning form the Star Workstation, see the ProStar 363 control information in the Star Workstation manual. NOTE 1:...

  • Page 48: Selecting The Glp Function

    Selecting the GLP Function Access the GLP function setup screen. These functions are used to enter information about a variety of security functions and performance related issues. Select the GLP function item to be edited. CONFIDENCE: KEY LOCK=1, CHECK=2, <1-4> 1 LOGBOOK=3, LAMP CHANGE=4 Press the desired Numeric key and press the Enter key.
  • Page 49 As an example, if the lamp was changed on 3-11-00 and it is being changed on 11-27-00, make certain that 00 is reentered before Enter is selected. ProStar 363 Fluorescence Detector...

  • Page 50: Example Of Time Program Operation

    Example of Time Program Operation This selection describes how a time-based program operates. The events indicated in the figure below will be used in the discussion. Setting up a Time Program Auto zero Hold Start Stop Time (minutes) 20.0 10.0 Ex (nm) Em (nm) Hold...
  • Page 51 Initial Value Input Range TIME 0.0 to 600.0 200 to 850, 0 250 to 900, 0 BASE 1, 2 START STOP TIME PROGRAM NO. (1-9) 1 key → Enter key MODIFY=0, NEW=1 1 key → Enter key ProStar 363 Fluorescence Detector...
  • Page 52 ( 0 key →) Enter key → ( 2 5 0 key →) Enter key → ( 3 5 0 key →) Enter key → ( 1 key →) Enter key → TIME Ex BASE (A/Z=1, HOLD=2) 1 0 key → Enter key → 2 7 0 key →...
  • Page 53 _ 10.0 270 To access the next line, press the  key. TIME Ex Em BASE(A/Z=1, HOLD=2) _ 20.0 270 To return to the first line of the program press the Escape key. TIME PROG LAMP [I] (90) ProStar 363 Fluorescence Detector...

  • Page 54: Shutting Down The Detector

    To execute the Time Program press the Start/Stop key. TIME PROG LAMP [I] (90) Program number Measured value Measurement wavelength Elapsed time On completion of the time program, measurement is automatically terminated and the display returns to the status screen. At this point, the wavelength settings are those that were on the status screen before the time program was executed.

  • Page 55: Maintenance And Troubleshooting

    Leave the power to the instrument on and wait for approximately 30 minutes. The xenon lamp must be removed from the detector prior C A U TIO N to shipping the unit. Failure to follow this procedure will result in a broken lamp. ProStar 363 Fluorescence Detector...
  • Page 56 • The xenon lamp remains hot for a while WARN ING : even after power-off and can severely B U R N H A ZA R D burn you if touched. • Although the xenon lamp will be automatically extinguished by a safety mechanism when the light source cover is opened, before replacement of the lamp, turn power off and wait for about 2...
  • Page 57 Cause Eye Damage! • The xenon lamp radiates intense ultraviolet light when it is on. • Do not look at the xenon lamp directly when it is lit. • Wear tinted safety glasses to prevent possible eye damage ProStar 363 Fluorescence Detector...

  • Page 58: Lamp Replacement

    Lamp Replacement After the xenon lamp is cooled down, turn off the Power switch. Then, remove the lamp holder (with the xenon lamp mounted) by reversing steps (1) to (6) beginning on page 53. Remove the xenon lamp by reversing steps (3) and (4) beginning on page 53.

  • Page 59: Installing The Xenon Lamp

    30 minutes. Then, open the light source cover. 1. Make sure the Power switch is turned off before installing the xenon lamp. To remove the light source cover, first loosen the four mounting screws indicated in Figure 12. ProStar 363 Fluorescence Detector...

  • Page 60: Figure 12 Removal Of Light Source Cover

    Mounting Screws Mounting Screws Light source cover Figure 12 Removal of Light Source Cover • Explosion of the xenon lamp could WA R N ING : cause serious injury. E XP LO SIO N H A ZA R D High-pressure gas at about 1 MPa is filled in the lamp at room temperature.
  • Page 61 If a lamp with its cathode consumed excessively is used continuously, the pressure inside the lamp bulb may become too high, causing explosion. To prevent this, replace the lamp with a new one immediately if its cathode has been consumed significantly ProStar 363 Fluorescence Detector...

  • Page 62: Figure 13 Xenon Lamp And Its Holder

    Metal fixture Ceramic plate Anode nut Anode cord Lamp holder Anode terminal l (marked '+') Protruding part of the bulb Lamp bulb Cathode terminal Cathode nut Cathode lead plate Cathode cord Figure 13 Xenon Lamp and Its Holder Mount the lamp so that the protruding part of the bulb comes to the instrument front panel side (see Figure 14).

  • Page 63: Figure 14 Protrusion Of Lamp Bulb

    Mount the xenon lamp so that the protruding part of the bulb comes to the instrument front panel side (see Figure 14). A mistake in the positioning may cause an increased light loss and preclude obtaining the desired performance from the instrument. ProStar 363 Fluorescence Detector...

  • Page 64: Figure 15 Xenon Lamp Mounted In Holder

    Metal fixture Anode cord Anode nut Ceramic plate Lamp bulb Protruding part Lamp holder Cathode lead plate Cathode cord Cathode nut Figure 15 Xenon Lamp Mounted in Holder Be sure to secure the anode and cathode nuts WA R N ING : when attaching the xenon lamp.

  • Page 65: Adjusting The Position Of The Xenon Lamp

    This adjustment will ensure that the optimum radiation from the lamp is focused into the optical system and will maximize the sensitivity. To adjust the lamp position, use the three adjust screws located at the right side of the instrument. ProStar 363 Fluorescence Detector...

  • Page 66: Figure 18 Adjusting The Lamp Position

    HORIZONTAL ADJ For adjustment in the horizontal direction. VERTICAL ADJ For adjustment in the vertical direction. FOCUS For adjustment of the focal point of light source. Adjustment of the lamp position involves a coarse adjustment (in which the position of the light image is monitored) and a fine adjustment (in which the signal detected by the monitor photomultiplier is observed).
  • Page 67 (rainbow color) becomes brightest. You may need to readjust all screws three or four times before obtaining the most intense light amount. NOTE: Allow the lamp to stabilize for approximately 15 minutes after it has been turned on, before optimizing the position. ProStar 363 Fluorescence Detector...

  • Page 68: Fine Adjustment

    Observe excitation beam on paper Figure 19 Observation of Excitation Beam Fine Adjustment 1. Remount the flow cell unit. The flow cell should be flushed with MeOH and blown dry with clean air. 2. Change display mode: Utility/Setup → 3 (Display Mode) → Enter → 2 (Ex) → Enter Now the status display monitors Excitation energy.
  • Page 69 When a maximum value is reached, fine adjustment of the lamp position is completed. 5. After lamp alignment is complete change display mode back to Fluorescence. Utility/Setup → 3 (Display Mode) → Enter 1 (FL) → Enter ProStar 363 Fluorescence Detector...

  • Page 70: Flow Cell Unit Maintenance

    Flow Cell Unit Maintenance Checking and Cleaning the Flow Cell If the inside of the flow cell is contaminated, it must be cleaned. The following items are required for cleaning the flow cell. • A glass syringe with capacity of approximately 10 mL •...

  • Page 71: Figure 20 Flow Cell Cleaning

    From pump If pressure is too high, the flow cell may be damaged or C A U TIO N broken. When removing air bubbles, set the pump pressure limit to 150 psi (10 atm) or less. ProStar 363 Fluorescence Detector...

  • Page 72: Disassembly Of The Flow Cell

    Disassembly of the Flow Cell If the contamination in the flow cell cannot be removed by the aforementioned cleaning procedure, disassemble the cell and clean its parts as instructed below. Shielding plate Setscrews A Figure 21 Flow Cell Unit 1. Loosen setscrews A and detach the shielding plate. 2.

  • Page 73: Cleaning Of Flow Cell

    Using a glass container may scratch the cell or prevent obtaining the desired performance from the instrument. Retaining Flow cell Cell window Cell clamp screw 1 Retaining screw 2 Figure 22 Disassembly of Flow Cell Unit ProStar 363 Fluorescence Detector...

  • Page 74: Assembly Of Flow Cell

    Assembly of Flow Cell 1. Assemble the flow cell into the cell holder in the orientation shown in Figure 23 and fix it with the cell clamp. 2. Tighten the upper and lower retaining screws. Enlarged view of flow cell (Top View) Transmitted Excitation...

  • Page 75: Leakage Of The Flow Cell

    If liquid comes out of the drain port located at the lower right of the instrument during measurement, stop the pump immediately and turn off the Power switch. Then, follow steps (1) to (5) above. ProStar 363 Fluorescence Detector...

  • Page 76: Flow Cell Storage

    Flow Cell Storage If the flow cell will be left unused for less than a few days, clean the flow cell thoroughly by pumping distilled water or alcohol through it and then storing it in a safe place. If the period is more than a few days, the inside of the cell may be filled completely with a solvent like ethanol or acetonitrile and then stored.

  • Page 77: Troubleshooting

    The xenon lamp radiates intense ultraviolet light when it is on. • Do not look at the xenon lamp directly when it is lit. Be sure to wear tinted safety glasses to prevent possible eye damage. ProStar 363 Fluorescence Detector...

  • Page 78: Table 4 Troubleshooting

    Table 4 Troubleshooting Symptom Cause Check Remedy . Self-diagnosis The power cord plug is Visual check Plug in the power (initialization) is not not securely plugged in. cord securely. performed when the Power switch is turned The fuse is blown. Check the fuse for Replace the fuse continuity.
  • Page 79 Visual check Clean the flow cell. Xenon lamp off-position Check lamp energy Adjust lamp level position. Error in flow cell Visual check Correctly mount flow mounting cell. Flow cell surface Visual check Replace flow cell. scratched ProStar 363 Fluorescence Detector...

  • Page 80: Error Messages

    DRIVE MECHANISM properly set during the self- nearest Varian service center. ERROR diagnosis test (initialization). ROM ERROR Error in ROM Notify the nearest Varian service center. C-RAM ERROR Error in RAM Notify the nearest Varian service center. CHECK TIME The allowable maximum number...
  • Page 81 If this is the case, enter the relevant value again. In the event of PARAMETER ERROR, the wavelength calibration value is deleted. In this case, it is necessary to perform the wavelength calibration procedure. ProStar 363 Fluorescence Detector...
  • Page 82 03-914875-00:1...

  • Page 83: Appendix

    Changeable in 5 steps, corresponding to time constants of 0.1, 0.5, 2.0, 4.0, 8.0 seconds. Auto zero range 0 to 1000 Offset 0 to 1000 (settable in steps of 1) Spectrum memory Up to 8 excitation or emission spectra can be stored. ProStar 363 Fluorescence Detector...
  • Page 84 Marker Marker output of approx. 0.6 mV at recorder terminal (corresponding to about 6 graduations on chart) Instrument parameter setting Following parameters are settable and stored in battery-backed-up memory when power is turned off. Full scale range Spectrum measuring range Photomultiplier applied voltage Offset value Time program...

  • Page 85: Accessories

    (e.g., a year) will be less than that of a newly acquired lamp. It is suggested that the user maintain a small supply of spare lamps and reorder lamps as required, rather than purchasing a large number of lamps at one time. ProStar 363 Fluorescence Detector...

  • Page 86: Raman Scattering

    Raman Scattering When fluorescence is measured, two additional peaks may appear in the spectrum. The Rayleigh peak appears at the excitation wavelength and is due to scattered light, while the Raman peak appears at longer wavelength than the excitation. The position of the Raman band is dependent on the excitation wavelength, while the position of the fluorescence is independent of the excitation wavelength.

  • Page 87: Figure 25 Raman Spectrum Of Water

    (nm) Excitation wavelength Figure 25 Raman Spectrum of Water Table 5 Raman Spectral Peak Position at Each Excitation Wavelength (Excitation Water Ethanol Cyclohexane Carbon Tetrachloride Chloroform Wavelength) Excitation wavelength, — — and Raman peak position (nm) ProStar 363 Fluorescence Detector...

  • Page 88: Samples Containing High Concentration Of The Compound Of Interest

    Samples Containing High Concentration of the Compound of Interest When the concentration of the compound of interest is relatively high, the concentration reported by fluorescence detection may be lower than the actual concentration. This phenomenon is due to the “Inner Filter effect” which is depicted in Figure 26. In this example, the concentration of the compound of interest is sufficiently high that essentially all of the excitation radiation is absorbed by molecules that are close to the entrance slit.

  • Page 89: Figure 27 Re-Absorption Of Fluorescence

    480 and 720 nm, respectively. To eliminate second and third order radiation, it is necessary to place a short-wavelength cutoff filter in the path of fluorescing radiation (before the fluorescence monochromator). ProStar 363 Fluorescence Detector...

  • Page 90: Figure 28 Measurement Example Of Fluorescence Spectrum

    An Example of a Fluorescence Spectrum Figure 28 provides an example of a fluorescence spectrum which contains the various types of peaks indicated above. In most cases, the fluorescence of the compound of interest lies closer to the excitation than shown in Figure 28, and overlapping of peak of interest and the second order radiation is rarely a problem.

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