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horiba Fluorolog-3 - User Manual

horiba Fluorolog-3
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Table of Contents

Questions and Answers

  • K
    Karen WilliamsAug 18, 2025
    Why is the signal intensity low on my Horiba Measuring Instruments?
    • C
      Christopher ParkerAug 18, 2025
      If the signal intensity is low on your Horiba Measuring Instruments, several factors could be at play. First, ensure the lamp is correctly aligned and focused, and that the slits are properly adjusted. Confirm that the shutters are fully open and the polarizer is out of the light path. Also, verify that the high voltage is correctly set to the default value of 950 V. If the lamp is old, consider replacing it, especially if it has been in use for 1500–2000 hours. Note that if the lamp power supply is set to the wrong current rating, you should contact the Spex[®] Fluorescence Service Department, as the 450-W Xe lamp current should be 25 A.
  • J
    jamesrachelAug 21, 2025
    How to fix light not reaching the sample in horiba Fluorolog-3 Measuring Instruments?
    • J
      jeffrey67Aug 21, 2025
      If the light isn't reaching the sample in your Horiba Measuring Instruments, it could be due to several reasons. First, check if the excitation shutter is closed and open it using the software. Ensure that the slits are adjusted to the proper width. Verify that the lamp is turned on by pressing the lamp rocker switch and the Start button on the xenon-lamp power-supply front panel. Also, confirm that the excitation monochromator is correctly calibrated and recalibrate if necessary. Finally, make sure the sample turret is in the correct position by setting it via the software and verifying its position.
  • R
    Richard FrancoAug 23, 2025
    What to do if there is stray light in emission scan in horiba Fluorolog-3 Measuring Instruments?
    • P
      patriciapetersonAug 23, 2025
      If you observe stray light in the emission scan of your Horiba Measuring Instruments, it could be due to scattered light off the excitation wavelength. To resolve this, place bandpass filters in the excitation light path. Additionally, ensure that the emission-monochromator slit widths are not too wide; decrease them if necessary.
  • D
    Danielle OrozcoAug 26, 2025
    What to do if horiba Fluorolog-3 Measuring Instruments shows no signal?
    • D
      Dennis ButlerAug 26, 2025
      If you are getting no signal from your Horiba Measuring Instruments, there are a few things to check. First, ensure the lamp is on; if it's not, replace the xenon lamp. Also, verify that the high voltage is turned on through the software. Lastly, if the detectors are saturated, reduce the slit settings.
  • J
    joshua44Aug 27, 2025
    Why is there a noisy spectrum with magnetic stirrer on Horiba Measuring Instruments?
    • J
      James ThompsonAug 27, 2025
      If you notice a noisy spectrum with the magnetic stirrer in your Horiba Measuring Instruments, it's likely that the stirring speed is too fast. Try using a slower stirring speed.
  • J
    Jason RussellAug 30, 2025
    Why is the signal erratic on horiba Fluorolog-3 Measuring Instruments?
    • P
      Pamela ArellanoAug 30, 2025
      If you are experiencing erratic signals with your Horiba Measuring Instruments, several factors could be responsible. First, allow the lamp to warm up for 20 minutes before use, as the lamp may be unstable. Check the dark value to determine if there are any light leaks. Additionally, if a Raman band is superimposed on the fluorescence scan, change the excitation wavelength to move the Raman band away from the fluorescence peak, or run a blank scan of the solvent and subtract it from the fluorescence spectrum.
  • M
    Michael TaylorSep 1, 2025
    How to fix sample turret not operating on Horiba Measuring Instruments?
    • H
      Heather AtkinsonSep 1, 2025
      If the sample turret is not operating on your Horiba Measuring Instruments, first check the software status to ensure it is enabled. Also, verify that all cable connections are properly connected.
  • A
    Aaron DiazSep 3, 2025
    Why is there a large off-scale peak at twice the excitation wavelength on horiba Fluorolog-3 Measuring Instruments?
    • W
      William VelasquezSep 3, 2025
      If you observe a large off-scale peak at twice the excitation wavelength on your Horiba Measuring Instruments, it is likely due to Raman bands in aqueous solutions and solvents or 2[nd]-order effects from the spectrometer. To resolve this, use cut-on filters to eliminate the 2[nd]-order peak.
  • T
    Tina KelleySep 6, 2025
    What to do if see “Data file does not exist” error on horiba Fluorolog-3 Measuring Instruments?
    • P
      Paul DavidSep 6, 2025
      If you see a “Data file does not exist” or “file read error” message appear with your Horiba Measuring Instruments, ensure that you are logged into Windows[®] XP as an administrator or power user. Then, restart FluorEssence™.
  • J
    James ZunigaSep 8, 2025
    Why is there no change in signal intensity on Horiba Measuring Instruments?
    • T
      Tiffany MorrisSep 8, 2025
      If you're getting no change in signal intensity on your Horiba Measuring Instruments, it could be due to several reasons. Ensure that the monochromators are set to the correct wavelength based on the excitation and emission of the sample. If the detectors are saturated, adjust the slits, noting that the signal detector is linear to 2 × 10[6] cps in photon-counting mode and the reference detector saturates at 12 µA. Also, consider the possibility of optical density effects and self-absorption; if these are present, dilute the sample by a factor of 10 or 100 and retry the experiment.

Summary

Introduction to the Fluorolog-3

Chapter Overview

Summarizes the content of each chapter, providing a roadmap for the manual's structure.

Disclaimer and User Responsibilities

Outlines terms of use, user responsibilities, and limitations of liability for the product.

Safety Summary and Precautions

Presents general safety precautions and symbols used for operating the instrument safely.

Risks of Ultraviolet Exposure

Details hazards associated with ultraviolet light exposure and necessary protective measures.

Additional Risks of Xenon Lamps

Discusses hazards related to xenon lamps, including visible and infrared radiation.

Requirements and Installation

Surface Requirements

Specifies the necessary table stability and size for proper system installation.

Environmental Requirements

Outlines recommended temperature and humidity levels for optimal instrument operation.

Electrical Requirements

Details voltage, current, and circuit requirements for powering the instrument.

Initial Installation Procedure

Provides guidance for scheduling initial installation and subsequent assembly by authorized personnel.

System Description

Overview of System Operation

Explains the general operation of the Fluorolog®-3 system and its main components.

Basic System Components

Describes fundamental components: monochromators, sample compartment, detector, and accessories.

System Configurations

Details standard system configurations (FL3-11, FL3-12, FL3-21, FL3-22) and their specifications.

Standard and Custom Options

Lists available optional components for customizing system configurations and layouts.

System Operation

Turning On the System

Provides step-by-step instructions for powering on the lamp, accessories, and host computer.

Checking System Performance

Explains how to check system calibration, repeatability, and throughput for routine maintenance.

Excitation and Emission Calibration

Details procedures for verifying wavelength calibration of excitation and emission monochromators.

Water Raman Signal-to-Noise Ratio Calculation

Explains calculation of water-Raman S/N ratio as a measure of instrument sensitivity.

Useful Materials for System Characterization

Lists recommended substances for system sensitivity checks and lifetime measurements.

Data Acquisition

FluorEssence Toolbar Buttons Overview

Introduces special FluorEssence™ buttons for recording and presenting data.

Experiment Menu Button Usage

Explains how to use the Experiment Menu to select and set up various experiment types.

Previous Experiment Setup and Auto Run

Details using buttons to reset experiments or rerun previous setups without modifications.

Data File Creation and Processing

Guides on creating overlay files, 3D scans, batch experiments, and using calibration curves.

Gemini Alpha for Lifetime Data Modeling

Describes using Gemini Alpha for modeling TCSPC lifetime data and predicting lifetime components.

Running an Unknown Sample

Guides on finding preliminary emission maxima for unknown samples.

Using Corrected Signals in FluorEssence

Explains how to incorporate dark offset, blank subtraction, and correction factors for accurate signals.

Optimizing Data

Cuvette Preparation Procedures

Provides detailed steps for cleaning cuvettes using nitric acid and de-ionized water.

Sample Preparation Techniques

Discusses preparing solutions, small-volume samples, and solid samples for analysis.

Scan Techniques and Precautions

Covers precautions with solid sample holders and using filters in the optical path.

Data Collection Methods and S;N Improvement

Discusses collection methods, optimal wavelengths, G factor, and improving signal-to-noise ratio.

Data Correction Procedures

Explains how to remove artifacts and apply correction factors during and after acquisition.

System Maintenance

External Case Cleaning

Provides instructions for cleaning the instrument's external case and exhaust filters.

Xenon Lamp Replacement and Adjustment

Discusses xenon lamp replacement, required tools, hazards, and adjustment for optimal sensitivity.

Lamp Housing Cleaning

Provides instructions for cleaning the lamp housing window and replacing the baffle.

Installing a New Photomultiplier Tube

Guides the user through replacing the photomultiplier tube, including safety precautions.

Grating and Mirror Information

Discusses standard gratings, their specifications, and mirror alignment procedures.

Troubleshooting

Troubleshooting Chart for Common Issues

A table listing problems, causes, and remedies for common issues encountered with the system.

Using Diagnostic Spectra for Analysis

Explains how to interpret diagnostic spectra, including lamp and water Raman scans.

Guidance for Further Assistance

Offers advice on steps before contacting service, including website resources and problem duplication.

Producing Correction Factors

Introduction to Radiometric Correction

Explains the need for radiometric correction factors to compensate for wavelength-dependent characteristics.

Correction Factor Kits and Accessories

Describes the 1908 Accessory and the F-3026 Correction Factor Kit.

Generating Emission Correction Factors

Details procedures for generating emission correction factors using the 1908 and F-3026 accessories.

Calculating Excitation Correction Factors

Details the process of calculating excitation correction factors using a rhodamine-B solution.

Using Correction-Factor Files in FluorEssence

Explains how to use acquired correction files within the Experiment Setup.

Automated Polarizers

Introduction to Polarization Measurements

Introduces polarized emission measurements for studying molecular rotations and changes.

Polarizer Installation and Alignment

Provides guidance on installing, removing, and checking polarizer alignment.

Re-alignment and Manual Alignment Procedures

Details automatic and manual alignment routines for polarizers, including safety cautions.

Using Automated Polarizers with Software

Discusses software choices for polarization measurements and applications.

Measuring the G Factor

Explains how to measure or input the G factor for polarization wavelength-response correction.

Polarizer Maintenance and Troubleshooting

Provides guidance on handling, cleaning, and troubleshooting polarizers.

Phosphorimeter Operation

Introduction to Phosphorimetry

Introduces the FL-1040 phosphorimeter for measuring long-lived luminescence and phosphorescence.

Phosphorimeter Theory and Data Acquisition

Explains principles of phosphorescence measurement and the data acquisition sequence.

Phosphorimeter Parameters and Applications

Explains key parameters and demonstrates applications like decay curves and mixture analysis.

Processing Phosphorimeter Data

Details the steps for processing phosphorimeter data, including fitting analytical curves.

Phosphorimeter Lamp Replacement

Provides instructions and warnings for replacing the xenon flash lamp in the phosphorimeter.

Phosphorimeter Troubleshooting

Lists common problems, causes, and remedies for the phosphorimeter accessory.

Applications

System Calibration and Data Correction

Highlights system calibration, accuracy, and use of data-correction techniques for reproducible spectra.

Detecting Sub-Picomolar Concentrations

Demonstrates the system's sensitivity in detecting trace concentrations of fluorescein.

Fluorescence Detection Techniques

Covers detection of highly scattering samples and quantum-yield calculations.

Characterizing Mixtures and IR Region Operation

Discusses synchronous scanning for mixtures and operation in the infrared region.

Time-Resolved and Low-Temperature Measurements

Explains time-resolved data acquisition and low-temperature scans for enhanced fluorescence.

Kinetic Monitoring and Front-Face Detection

Covers monitoring kinetic reactions and front-face detection for solid or absorbent samples.

Polarization for Biological Probe Detection

Explains fluorescence polarization for detecting trace biological probes and immunoassay techniques.

Xenon Lamp Information and Record of Use

Xenon Lamp Lifespan and Usage Record

Provides information on xenon lamp lifespan and a form for recording usage hours.

TRIAX Operation with the Fluorolog-3

Introduction to TRIAX Spectrometers

Introduces the TRIAX imaging spectrometer and its differences from standard monochromators.

TRIAX Hardware and Configuration

Details electrical power requirements, internal workings, and turret installation.

TRIAX Software Setup for CCD Acquisition

Explains setting up the Experiment Setup window for CCD acquisition with a TRIAX.

Real Time Control for TRIAX Parameters

Details adjusting TRIAX parameters in Real Time Control, including grating and mirror settings.

System Configuration and Detector Temperature

Guides on using System Configuration to set the cooled detector's temperature.

TRIAX Data Correction Procedures

Explains correction-factor file naming conventions and data correction for TRIAX systems.

TRIAX 320 Specifications

Lists the technical specifications for the TRIAX 320 spectrograph.

TRIAX Troubleshooting Guide

Provides a troubleshooting chart for common problems encountered with the TRIAX spectrograph.

Introduction to Lifetime Measurements

Lifetime Measurement Methods

Outlines two methods for upgrading to lifetime capability: frequency domain and TCSPC.

Fluorolog-Tau-3 Lifetime System

Describes the Fluorolog®-Tau-3 system for frequency domain lifetime measurements.

Lifetime Measurement Techniques

Explains frequency and time domain lifetime measurement techniques and their parameters.

Types of Lifetime Scans

Details the four available scan types: Lifetime, Lifetime-resolved, Anisotropy-decay, and Time-resolved.

Technical Specifications

Spectrofluorometer System Overview

Lists the main components of the Fluorolog®-3 system and its control by a PC.

Detailed Spectrofluorometer System Specifications

Details specifications for excitation source, monochromators, sample compartment, and detectors.

Minimum Computer Requirements

Lists the minimum hardware and software requirements for the host computer.

Components & Accessories

Introduction to System Configuration

Introduces how the Fluorolog®-3 can be configured with various components and accessories.

Liquid Nitrogen Dewar Assembly (FL-1013)

Describes the FL-1013 Dewar assembly for low-temperature measurements.

Sample Cells (HPLC, Micro, Quartz, Reduced Volume)

Lists and describes various sample cells for different applications.

Correction Factor Kits (F-3026, 1908 Accessory)

Details correction factor kits for calibrating spectral response.

Cryostats and Detectors

Describes cryostats, CCD detectors, and signal detectors for spectral acquisition.

Fiber Optic Mounts and Filters

Details fiber optic mounts for remote sensing and cut-on UV-Visible filters.

Thermostatted Cell Holders

Describes cell holders for maintaining samples at constant temperatures with magnetic stirrers.

Solid Sample Holder (1933)

Details the 1933 holder designed for samples like thin films, powders, and microscope slides.

Xenon Lamp Housings and Autotitration Injector

Describes xenon lamp housings and the autotitration injector accessory.

MicroMax Plate Reader and Polarizers

Details the MicroMax plate reader and L/T-format polarizers for automated measurements.

Quantum-Yield Accessory and Stopped-Flow Accessory

Describes accessories for studying fluorescence and monitoring fast reactions.

TCSPC Upgrades and Temperature Bath

Explains TCSPC upgrades for lifetime capability and the F-1000/1 Temperature Bath.

Reassembly Instructions

Host Computer and Spectrofluorometer Assembly

Guides on connecting the host computer and assembling the spectrofluorometer modules.

Cable Connections and Powering Up

Illustrates connectors, SpectrAcq rear panel, and guides on connecting power cables.

Glossary

Glossary Terms A-C

Defines terms starting with A, B, and C, related to spectroscopy and instrumentation.

Glossary Terms C-F

Defines terms starting with C, D, E, and F, related to spectroscopy and instrumentation.

Glossary Terms F-M

Defines terms starting with F, G, H, I, L, M, related to spectroscopy and instrumentation.

Glossary Terms R-T

Defines terms starting with R, S, T, related to spectroscopy and instrumentation.

Glossary Terms T-X

Defines terms starting with T, X, related to spectroscopy and instrumentation.

Bibliography

Index

horiba Fluorolog-3 Specifications

General IconGeneral
Excitation Source450 W xenon arc lamp
DetectorPhotomultiplier tube (PMT)
SoftwareFluorEssence
Emission MonochromatorDouble grating
Excitation MonochromatorDouble grating
Sample CompartmentStandard and optional accessories available
Wavelength Range200 to 850 nm (standard), up to 1700 nm (optional)

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