horiba Fluorolog-3 Operation Manual
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horiba Fluorolog-3 Operation Manual

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  • Page 1 Artisan Technology Group is your source for quality new and certified-used/pre-owned equipment SERVICE CENTER REPAIRS WE BUY USED EQUIPMENT • FAST SHIPPING AND DELIVERY Experienced engineers and technicians on staff Sell your excess, underutilized, and idle used equipment at our full-service, in-house repair center We also offer credit for buy-backs and trade-ins •...
  • Page 2 Fluorolog-3 v. 2.2 (10 Sep 2002) Fluorolog ® Operation Manual Rev. 2.2 July 2002 http://www.isainc.com In the USA: Jobin Yvon Inc. 3880 Park Avenue, Edison, NJ 08820 In France: Japan: (81) 3/5823.0140 Tel: 1-732-494-8660 16-18, rue du Canal China: (86) 10/6836.6542...
  • Page 3 Fluorolog-3 v. 2.2 (11 Jul 2002) Copyright © 2002 by Jobin Yvon Inc. All rights reserved. No part of this work may be reproduced, stored, in a retrieval system, or transmitted in any form by any means, including electronic mechanical,...

  • Page 4: Table Of Contents

    Fluorolog-3 v. 2.2 (11 Jul 2002) Table of Contents 0: Introduction ....................0-1 ® About the Fluorolog -3..........................0-1 Chapter overview ............................ 0-2 Symbols used in this manual ........................0-4 1: Requirements & Installation ................1-1 Surface requirements ..........................1-1 Environmental requirements........................
  • Page 5 Fluorolog-3 v. 2.2 (11 Jul 2002) FL-1012 dual-position thermostatted cell holder .................. 6-21 Model 1933 solid sample holder......................6-23 FL-1039 xenon lamp housing ....................... 6-25 FL-1040 dual lamp housing ........................6-25 F-3005/6 autotitration injector....................... 6-26 Models F-3001, F-3002, and F-3003 microscope interfaces ............... 6-27 Model 1907 450-W xenon lamp......................
  • Page 6 Fluorolog-3 v. 2.2 (11 Jul 2002) Hardware .............................. 14-2 Software..............................14-4 Correcting data with the TRIAX ......................14-35 TRIAX 320 Specifications ........................14-38 Troubleshooting ..........................14-39 15: Technical Specifications ................15-1 Spectrofluorometer system........................15-2 Minimum computer requirements ......................15-4 Software..............................15-4 16: Glossary ....................

  • Page 7: 0: Introduction

    Fluorolog-3 v. 2.2 (11 Jul 2002) Introduction 0: Introduction ® About the Fluorolog ® The main parts of the Fluorolog -3 spectrofluorometer system are: • State-of-the-art optical components • A personal computer • DataMax for Windows™, the driving software. ®...

  • Page 8: Chapter Overview

    Fluorolog-3 v. 2.2 (11 Jul 2002) Introduction Chapter overview 1: Requirements & Installation Power and environmental requirements; select the best spot for the instrument. ® 2: System Description Various Fluorolog -3 configurations; their features and benefits. 3: System Operation Operation of the spectrofluorometer system, and calibration instructions.
  • Page 9 Fluorolog-3 v. 2.2 (11 Jul 2002) Introduction 14: Using TRIAX with the Fluorolog Special instructions on using a TRIAX ® ® imaging spectrometer with the Fluorolog system, including with a CCD detector. 15: Technical Specifications Instrument specifications and computer requirements.

  • Page 10: Symbols Used In This Manual

    Fluorolog-3 v. 2.2 (11 Jul 2002) Introduction Symbols used in this manual Certain symbols are used throughout the text for special conditions when operating the instruments: A hazardous condition exists, or danger exists that could damage the equipment. Jobin Yvon Inc. is not responsible for damage Warning: arising out of improper use of the equipment.

  • Page 11: 1: Requirements & Installation

    Fluorolog-3 v. 2.2 (11 Jul 2002) Requirements & Installation 1: Requirements & Installation Surface requirements • A sturdy table- or bench-top. • Table size varies according to the system configuration; an average size Warning: Do not split the of 38" × 60" (96.5 cm × 152.4 cm) is system between two tables.

  • Page 12: Environmental Requirements

    Fluorolog-3 v. 2.2 (11 Jul 2002) Requirements & Installation Environmental requirements • Temperature 72 ± 5°F (22 ± 3°C) • Humidity level ~70% • No special ventilation. Note: The standard xenon lamp ® provided with the Fluorolog -3 is ozone-free. The lamp housing contains an electrically powered fan that removes the heat.

  • Page 13: Electrical Requirements

    Fluorolog-3 v. 2.2 (11 Jul 2002) Requirements & Installation Electrical requirements • 115 V, 20 A or 220 V, 20 A; factory-set. • As an extra measure of caution, plug the xenon lamp into a circuit separate from Note: For the computer, Jobin the other components.

  • Page 14: Installation

    Fluorolog-3 v. 2.2 (11 Jul 2002) Requirements & Installation Installation Warning: Customer installation is not recommended. Special tools and several critical alignment verification procedures are required. ® ® Schedule the initial installation of a Fluorolog -3 by calling the Spex Fluorescence Service Department at (732) 494-8660 ×...

  • Page 15: 2: System Description

    Fluorolog-3 v. 2.2 (11 Jul 2002) System Description 2: System Description Overview General operation ® All Fluorolog -3 spectrofluorometers have common features: A source of radiation produces photons. The beam of light is Controller filtered by an excitation spectrometer that allows a...
  • Page 16 Fluorolog-3 v. 2.2 (11 Jul 2002) System Description Basic components Spectrometers ® The Fluorolog -3 comes equipped with either a single- or double-grating spectrometer in the excitation and emission positions. Double-grating spectrometers offer a significant increase in sensitivity, resolution and stray-light rejection.

  • Page 17: Configurations

    Fluorolog-3 v. 2.2 (11 Jul 2002) System Description Configurations ® The different configurations and various accessories available for the Fluorolog -3 system allow you to customize a system specific for today’s needs, while the interchangeability of the components and the inherent design enable the system to grow and change as new applications arise.
  • Page 18 Fluorolog-3 v. 2.2 (11 Jul 2002) System Description ® Fluorolog -3 Model FL3-11 ® The Fluorolog -3 Model FL3-11 is an economical system designed for routine fluorescence measurements. Source Single-grating Excitation Spectrometer Single-grating T-box Sample Compartment Module Emission Spectrometer The standard model FL3-11 comes equipped with: •...
  • Page 19 Fluorolog-3 v. 2.2 (11 Jul 2002) System Description ® Fluorolog -3 Model FL3-12 ® The Fluorolog -3 model FL3-12 provides optimum performance for highly scattering samples such as proteins, membranes, and solid samples. Source Single-grating Excitation Spectrometer Double-grating T-box Sample...
  • Page 20 Fluorolog-3 v. 2.2 (11 Jul 2002) System Description ® Fluorolog -3 Model FL3-21 ® The Fluorolog -3 model FL3-21 includes a double-grating spectrometer at the excitation position. Source Double-grating Excitation Spectrometer Single-grating T-box Sample Emission Spectrometer Compartment Module Features of the Model FL3-21 are: •...
  • Page 21 Fluorolog-3 v. 2.2 (11 Jul 2002) System Description ® Fluorolog -3 Model FL3-22 ® Because of the double-grating excitation and emission spectrometers, the Fluorolog model FL3-22 offers unsurpassed performance in resolution, sensitivity, and stray-light rejection. This system is perfect for highly scattering samples like lipids and proteins, or solids like powders, semiconductors, or phosphors.
  • Page 22 Fluorolog-3 v. 2.2 (11 Jul 2002) System Description Standard options The previously described systems represent the standard configurations. Each system, however, can be customized by selecting different components. Available options are listed below. For additional information, or for a list of the most recently developed products, ®...
  • Page 23 Fluorolog-3 v. 2.2 (11 Jul 2002) System Description Custom configurations With custom configurations, you can change the layout of the system to a T-format or add the high-performance TRIAX 320M imaging spectrograph at the emission port of the system. Using single-grating, double-grating, or the TRIAX 320M imaging spectrometer, you can create a system for almost any application.
  • Page 24 Fluorolog-3 v. 2.2 (11 Jul 2002) System Description Fluorolog®-3 Model FL3-XXX Systems with the T-configuration design were developed for T-format polarization or anisotropy and dual-emission spectroscopy. Models FL3-XXX (where the Xs are the type of spectrometer positioned at the excitation, first-emission, and second-emission positions, respectively) are available in numerous configurations.
  • Page 25 Fluorolog-3 v. 2.2 (11 Jul 2002) System Description ® Fluorolog -3 Model FL3-12-320M The fully automated TRIAX 320M imaging spectrometer can be a part of a custom ® Fluorolog -3 configuration. The imaging spectrograph offers the latest advances in optical design and automation.
  • Page 26 Fluorolog-3 v. 2.2 (11 Jul 2002) System Description Custom options Each system can be customized further by selecting different options. Available options are ® listed below. For additional information, or for a list of the newest options, contact a Spex Fluorescence Product Specialist.

  • Page 27: 3: System Operation

    Fluorolog-3 v. 2.2 (11 Jul 2002) System Operation 3: System Operation Turning on the system Start the lamp. The lamp must be turned on Warning: When the lamp is ® prior Fluorolog turned on, a large voltage is put accessories,...
  • Page 28 Fluorolog-3 v. 2.2 (11 Jul 2002) System Operation Start the peripheral devices. Turn on all peripheral devices such as printers and plotter (i.e., all devices other than the computer). Start the computer and software. Turn on the computer. In Windows™, click on the DataMax icon to start Instrument Control Center.

  • Page 29: Checking System Performance

    Fluorolog-3 v. 2.2 (11 Jul 2002) System Operation Checking system performance Introduction Upon installation and as part of routine maintenance checks, examine the performance of ® the Fluorolog -3. Jobin Yvon Inc. recommends checking the system calibration before each day of use with the system. Scans of the xenon-lamp output and the Raman-scatter band of water are sufficient to verify system calibration, repeatability, and throughput.
  • Page 30 Fluorolog-3 v. 2.2 (11 Jul 2002) System Operation Excitation calibration check This calibration check verifies the wavelength calibration of your excitation monochroma- tor, using the reference photodiode located before the sample compartment. It is an excita- tion scan of the xenon lamp’s output, and should be the first check performed.
  • Page 31 Fluorolog-3 v. 2.2 (11 Jul 2002) System Operation converts to Excitation Acquistion. (For calibration and calibration verification, always adjust the excitation spectrometer first.) Set the scan parameters for the xenon lamp scan: In the Excitation Acquisition dialog box: Name the experimental parameters lamp.exp, Use a filename lamp.spc,...
  • Page 32 Fluorolog-3 v. 2.2 (11 Jul 2002) System Operation Click on Signals... This opens Signals dialog box. Enter R (reference), then click OK to close the box. Click on Slits... This opens the Slits dialog box. excitation entrance and exit slits to 0.5 mm.
  • Page 33 Fluorolog-3 v. 2.2 (11 Jul 2002) System Operation Lamp scan for single-monochromator systems: structure ~ 450 nm calibration peak at 467 nm broadband Xenon-lamp scan for an FL3-11 spectrofluorometer. Lamp scan for double-monochromator systems: On a double-monochromator system, the 467-nm line is no longer the most intense peak within the spectrum.
  • Page 34 Fluorolog-3 v. 2.2 (11 Jul 2002) System Operation Find the calibration peak for the xenon-lamp spectrum. The 467-nm peak is used for excitation calibration. The intensity of this spectrum should be noted for reference, although it is not used for instrument specifications.
  • Page 35 Fluorolog-3 v. 2.2 (11 Jul 2002) System Operation Excitation monochromator recalibration ® Re-calibration of the Fluorolog -3 is performed by moving to the position of the observed peak, going into Visual Instrument Setup, and telling the software the correct position at which this peak should be.
  • Page 36 Fluorolog-3 v. 2.2 (11 Jul 2002) System Operation Open the Visual Instrument Setup dialog box: Note: The Visual Instrument Setup will vary, depend- ing on the in- strument configu- ration. Click on the grating for the excitation monochromator. The Grating/Turret dialog box appears.
  • Page 37 Fluorolog-3 v. 2.2 (11 Jul 2002) System Operation Confirm that the excitation monochromator is calibrated by running another lamp scan in Run Experiment. This time the peak should occur at 467 ± 0.5 nm. 3-11 Artisan Technology Group - Quality Instrumentation ... Guaranteed | (888) 88-SOURCE | www.artisantg.com...
  • Page 38 Fluorolog-3 v. 2.2 (11 Jul 2002) System Operation Emission calibration check Note: The emission calibration of the instrument is directly affected by the calibration of the excitation monochromator. This calibration check verifies the wavelength calibration of the emission monochromator with the emission photomultiplier tube. It is an emission scan of the Raman-scatter band of water performed in right-angle mode.
  • Page 39 Fluorolog-3 v. 2.2 (11 Jul 2002) System Operation In the Emission Acquisition dialog box: Name the experimental setup water.exp, Use a filename water.spc, Start the scan at 365 nm, End the scan at 450 nm, Step the wavelength by 0.5 nm, Use an Integration Time of 0.5 s.
  • Page 40 Fluorolog-3 v. 2.2 (11 Jul 2002) System Operation Click on the Slits... button to open the Slits dialog box: Set the slits to 5 nm. Click OK to close the box. If your slits’ units bandpass units (nm), then open Visual Instrument Setup, select Options, then Units, and set the slits’...
  • Page 41 Fluorolog-3 v. 2.2 (11 Jul 2002) System Operation The Rayleigh scatter band or excitation band, which is Note: Observed throughput (and hence peak about 10 times the intensity intensity) is affected by lamp age and align- of the Raman band, occurs ment, slit settings, and sample purity.
  • Page 42 Fluorolog-3 v. 2.2 (11 Jul 2002) System Operation Emission recalibration ® Calibration for the Fluorolog -3 is performed by: • Moving to the position of the observed peak, • Going to the Visual Instrument Setup application, and • Telling the software the correct position for this peak.
  • Page 43 Fluorolog-3 v. 2.2 (11 Jul 2002) System Operation Open the Visual Instrument Setup dialog box: Note: The Visual Instrument Setup will vary, de- pending on the instrument con- figuration. Click on the grating for emission mono- chromator. The Grating/Turret dialog...
  • Page 44 Fluorolog-3 v. 2.2 (11 Jul 2002) System Operation Close Visual Instrument Setup. Confirm that the emission monochromator is calibrated, by running another water Raman scan in Run Experiment. This time the peak should occur at 397 ± 0.5 nm. 3-18...

  • Page 45: Notes On Excitation And Emission Recalibration

    Fluorolog-3 v. 2.2 (11 Jul 2002) System Operation Notes on excitation and emission calibration • Two experiments, lamp.exp and water.exp, have been defined and saved. They can be run, after the system is switched on each day, to check the calibration ®...

  • Page 46: Useful Materials For Characterizing System And Samples

    Fluorolog-3 v. 2.2 (11 Jul 2002) System Operation Useful materials for characterizing system and samples The following are materials that Jobin Yvon Inc. has found useful in determining system sensitivity or as standards for lifetime measurements. Substance CAS Number Purpose...

  • Page 47: 4: Optimizing Data Acquisition

    Fluorolog-3 v. 2.2 (10 Sep 2002) Optimizing Data Acquisition 4: Optimizing Data Acquisition Spectra can be enhanced by optimization of data acquisition. This chapter lists some meth- ods of optimizing sample preparation, spectrofluorometer setup, and data correction to get higher-quality data.

  • Page 48: Sample Preparation

    Fluorolog-3 v. 2.2 (10 Sep 2002) Optimizing Data Acquisition Sample preparation The typical fluorescence or phosphorescence sample is a solution analyzed in a standard cu- vette. The cuvette itself may contain materials that fluoresce. To prevent interference, Jobin Yvon Inc. recommends using non-fluorescing fused-silica cuvettes that have been cleaned as described above.

  • Page 49: Data Collection Techniques

    Fluorolog-3 v. 2.2 (10 Sep 2002) Optimizing Data Acquisition Data collection techniques Selecting the collection method The two basic collection methods are right-angle and front-face. In right-angle detection, the fluorescence is collected at 90° to the incident exciting beam. Right-angle detection is used primarily for clear solutions.
  • Page 50 Fluorolog-3 v. 2.2 (10 Sep 2002) Optimizing Data Acquisition Preliminary emission maximum Verify that all system components are on. Note: If you have not checked instrument performance, Jobin Yvon Inc. recommends acquiring a lamp spectrum and a wa- ter Raman spectrum, as outlined in the previous chapter, be- fore proceeding.
  • Page 51 Fluorolog-3 v. 2.2 (10 Sep 2002) Optimizing Data Acquisition On the Run Experiment toolbar, choose the Experiment button: This opens the Emission Acquisition dialog box: Click on Exp Type…. This opens the Select Experiment Type dialog box: Choose Emission Acquisition.
  • Page 52 Fluorolog-3 v. 2.2 (10 Sep 2002) Optimizing Data Acquisition Specify the data acquisition parameters. If unsure, try 300 nm, Add 15 nm to the exci- Try 550 nm. where many organic tation wavelength. samples absorb light. Try 2 nm for a rapid scan.
  • Page 53 Fluorolog-3 v. 2.2 (10 Sep 2002) Optimizing Data Acquisition Enter the data file name and experiment file name, and any comments desired to distinguish this experiment from others. Click Run to execute the experiment. With the preliminary emission spectrum on the screen, note the greatest intensity.
  • Page 54 Fluorolog-3 v. 2.2 (10 Sep 2002) Optimizing Data Acquisition Excitation scan The next step is to use the recently discovered emission maximum to determine the optimal excitation wavelength for the sample. The procedure is very similar to that outlined above.
  • Page 55 Fluorolog-3 v. 2.2 (10 Sep 2002) Optimizing Data Acquisition Enter S/R (Signal/Reference). Using produces spectrum corrected variations in lamp intensity with respect to time. Click OK to close the Signals dialog box. Enter the remaining parameters and, if desired, the data file...
  • Page 56 Fluorolog-3 v. 2.2 (10 Sep 2002) Optimizing Data Acquisition Measuring the G factor Note: Measure the G fac- The grating factor, or G factor, ought to be included tor before a polarization anytime polarization measurements are taken. The experiment. G factor corrects for variations in polarization wavelength-response for the emission optics and detectors.
  • Page 57 Fluorolog-3 v. 2.2 (10 Sep 2002) Optimizing Data Acquisition Either enter the G factor or have it meas- ured automatically during the scan. Note: For weak signals, enter the G factor, rather than measure it auto- matically. This may improve the S/N.
  • Page 58 Fluorolog-3 v. 2.2 (10 Sep 2002) Optimizing Data Acquisition Faster scans with polarizers can be taken with Polar Scan…. Here, the G factor is entered in the Quick Polarization window: Note: For detailed information on the G factor, see the Polarizers Operation Manual.
  • Page 59 Fluorolog-3 v. 2.2 (10 Sep 2002) Optimizing Data Acquisition Improving the signal-to-noise ratio Because of various hardware or software conditions, occasionally it is necessary to optimize the results of an experiment. The quality of acquired data is determined largely by the signal-to-noise (S/N) ratio. This is true especially for weakly fluorescing samples with low quantum yields.
  • Page 60 Fluorolog-3 v. 2.2 (10 Sep 2002) Optimizing Data Acquisition Determining the optimum integration time The length of time during which photons are counted and averaged for each data point is re- ferred to as the integration time. An unwanted portion of this signal comes from noise and dark counts (distortion inherent in the signal detector and its electronics when high voltage is applied).
  • Page 61 Fluorolog-3 v. 2.2 (10 Sep 2002) Optimizing Data Acquisition Scanning a sample multiple times Scanning a sample more than once, and averaging the scans together enhances the S/N ratio. , where n is the number of scans. In general, the S/N ratio improves by n To scan a sample multiple times, in an experiment dialog box, specify the number of scans in the Number of Scans field in Run Experiment.
  • Page 62 Fluorolog-3 v. 2.2 (10 Sep 2002) Optimizing Data Acquisition Selecting the appropriate bandpass The bandpass (wavelength spread) affects the resolution of your spectra. If the bandpass is too broad, narrow peaks separated by a small change in wavelength may be unresolved. For example, for two 2-nm peaks 5 nm apart, and a bandpass of 10 nm, one broad peak, instead of two well-defined ones, will be visible.
  • Page 63 Fluorolog-3 v. 2.2 (10 Sep 2002) Optimizing Data Acquisition Smoothing data Smoothing the data improves the appearance of your spectrum. Smoothing, as are most ® post-processing features, is handled by GRAMS/32 . By selecting Arithmetic from the main menu of Run Experiment, you can choose FFT (fast-Fourier transform), Binomial, or Savitsky-Golay smoothing.

  • Page 64: Correcting Data

    Fluorolog-3 v. 2.2 (10 Sep 2002) Optimizing Data Acquisition Correcting data Introduction Collecting accurate information about the fluorescent or phosphorescent properties of a sample depends upon several factors: • Equipment specifications • Sample characteristics • Timing considerations. To ensure that the spectra collected indicate the actual properties of the sample and not ex- ternal conditions, data often must be corrected.
  • Page 65 Fluorolog-3 v. 2.2 (10 Sep 2002) Optimizing Data Acquisition During acquisition Data can be acquired either as raw data or as corrected data. A spectrum composed of raw data exhibits the effects of system parameters, while a corrected spectrum displays only the properties related to the sample.
  • Page 66 Fluorolog-3 v. 2.2 (10 Sep 2002) Optimizing Data Acquisition After acquisition To apply the correction factors after the data have been acquired, multiply the data file by the appropriate correction factor file (mcorrect or xcorrect). Make sure the trace to be corrected is active in the Run Experiment window.
  • Page 67 Fluorolog-3 v. 2.2 (10 Sep 2002) Optimizing Data Acquisition dialog box. The name of the file appears in the Term File area. Click Apply. The trace that appears on the screen is a result of the mathematical operation, giving a cor- rected spectrum.

  • Page 68: 5: System Maintenance

    Fluorolog-3 v. 2.2 (11 Jul 2002) System Maintenance 5: System Maintenance ® The Fluorolog -3 spectrofluorometer requires very little maintenance. The outside panels may be wiped with a damp cloth to remove dust and fingerprints. The lamp is the only com- ponent that has to be replaced routinely.
  • Page 69 Fluorolog-3 v. 2.2 (11 Jul 2002) System Maintenance Replacement The replacement xenon lamp is packed in the manufacturer’s box and must be installed in the lamp housing. Read all the Warning: Do not remove the pro- packing material including instructions and...
  • Page 70 Fluorolog-3 v. 2.2 (11 Jul 2002) System Maintenance To open the lamp housing: Remove the 10 Phillips-head screws on top of the lamp housing. Lift off the cover. This exposes the lamp assembly: Upper bracket Lamp Lower bracket Mirror Top view of the lamp housing with cover removed.
  • Page 71 Fluorolog-3 v. 2.2 (11 Jul 2002) System Maintenance To remove an existing lamp, Remove the 5/32" cap screws from the upper and lower brackets, freeing the positive and negative power leads. upper bracket 7/64" cap screw 5/32" cap screws Remove the 7/64" cap screw from the upper bracket.
  • Page 72 Fluorolog-3 v. 2.2 (11 Jul 2002) System Maintenance To insert a xenon lamp Be sure the cover of the lamp housing is removed. Note: Jobin Yvon Inc. provides new bulbs with leads and connectors of the proper length and size. Other manufac- turers’...
  • Page 73 Fluorolog-3 v. 2.2 (11 Jul 2002) System Maintenance The positive end is marked with a “+”, and should be pointing upward. Insert and tighten the 7/64" cap screw on the upper bracket. Secure the upper lamp lead and upper power...
  • Page 74 Fluorolog-3 v. 2.2 (11 Jul 2002) System Maintenance Plug the power cord from the lamp power supply into an outlet with the proper line voltage. Note: After installation, the lamp Warning: DO NOT operate should burn in for 24 hours. After this system from an un- the burn-in, the lamp’s position...
  • Page 75 Fluorolog-3 v. 2.2 (11 Jul 2002) System Maintenance Adjustment Once the lamp is installed (and after a 24-hour burn-in period), it may need an adjustment to ® maximize the sensitivity of the Fluorolog -3. To do this, Jobin Yvon Inc. recommends run-...
  • Page 76 Fluorolog-3 v. 2.2 (11 Jul 2002) System Maintenance Start Calibrate the emis- Is the water Ra- sion monochroma- man peak at 397 tor. Go to System ± 0.5 nm? Operation. Lamp is Continue Is the peak inten- properly with step sity as listed in the adjusted.
  • Page 77 Fluorolog-3 v. 2.2 (11 Jul 2002) System Maintenance Insert a 5/64" Allen wrench inside the middle port on the top of the lamp housing. Focus Horizontal adjustment Vertical adjustment Note: To reach the set screw, you may have to fish around...
  • Page 78 Fluorolog-3 v. 2.2 (11 Jul 2002) System Maintenance Record the date that the new lamp was installed, as well as its maximum intensity. Save the protective cover of the new bulb, for when the bulb must be replaced in the future.

  • Page 79: Lamp

    Fluorolog-3 v. 2.2 (11 Jul 2002) System Maintenance Lamp housing Clean the window in front of the lamp housing once a year, or more frequently if needed. Note: Dust reduces the excitation light transmission, resulting in lower system sensitivity. Remove 10...
  • Page 80 Fluorolog-3 v. 2.2 (11 Jul 2002) System Maintenance If the cap screws were removed, replace them. Replace the baffle, and re-install the 2 screws holding it in place. Re-install the cover, and secure it with the 10 Phillips-head screws. 5-13...

  • Page 81: Emission Signal Detector

    Fluorolog-3 v. 2.2 (11 Jul 2002) System Maintenance Emission signal detector There is a wide range of photomultipliers available to optimize different wavelength re- gions. When the R928P photomultiplier is replaced with a different detector, the emission correction factors must be updated (see Chapter 11: Producing Correction Factors).

  • Page 82: Reference Signal Detector

    Fluorolog-3 v. 2.2 (11 Jul 2002) System Maintenance Reference signal detector The reference signal detector is a state-of-the art silicon diode that requires no routine main- tenance. 5-15 Artisan Technology Group - Quality Instrumentation ... Guaranteed | (888) 88-SOURCE | www.artisantg.com...

  • Page 83: Gratings

    Fluorolog-3 v. 2.2 (11 Jul 2002) System Maintenance Gratings The excitation and emission spectrometer gratings are 1200 grooves/mm, and are blazed at 330 nm and 500 nm, respectively. If an application requires that the system be optimized for a particular region, the gratings can be changed by following a simple procedure.
  • Page 84 Fluorolog-3 v. 2.2 (11 Jul 2002) System Maintenance Remove the existing grating Close the slits in the emission spectrometer. Turn off all instrument power. Remove the Phillips-head screws holding the lid on the monochromator. Warning: A circuit board and critical...
  • Page 85 Fluorolog-3 v. 2.2 (11 Jul 2002) System Maintenance Release the grating from the stand. Loosen the thumbwheel screw securing the grating to the grating stand. Back view of grating assembly. Pull the grating away from the stand. 5-18 Artisan Technology Group - Quality Instrumentation ... Guaranteed | (888) 88-SOURCE | www.artisantg.com...
  • Page 86 Fluorolog-3 v. 2.2 (11 Jul 2002) System Maintenance To insert a new grating, Place the 3 mounting pins on the back of the grating into the matching mounting slots on the grating stand. Tighten the thumbscrew on the back of the grating stand.

  • Page 87: Mirrors

    Fluorolog-3 v. 2.2 (11 Jul 2002) System Maintenance Mirrors Mirrors are aligned at the factory and usually do not need realignment. Only the Model 1692M Selection Mirror (an option) can be adjusted easily to optimize the fluorescence sig- nal from the sample. This mirror should be adjusted periodically if low signal is caused by poor alignment at the slit position.

  • Page 88: Automated 4-Position Turret

    Fluorolog-3 v. 2.2 (11 Jul 2002) System Maintenance Automated 4-position turret If a circulating bath is used to regulate the turret’s temperature, periodically replace the flu- ids in the bath. Note: Over time, bacteria can grow in the temperature bath, or the water can be- come hard.

  • Page 89: 6: Components & Accessories

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories 6: Components & Accessories ® The Fluorolog -3 can be configured to obtain optimum results for a variety of applica- tions. The basic system, regardless of configuration, consists of slits, detectors, a xenon lamp with power supply, and a sampling module.

  • Page 90: Itemized List

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories Itemized List Fluorolog -3 Accessories ® Item Model Page Accessory, Absorption/Transmission 1940 Adapter, Micro Cell (See Cell, Micro) 1923A Adapter, Micro Cell (See Cell, Micro) 1924A Assembly, Liquid Nitrogen Dewar FL-1013...

  • Page 91: Model 1940 Absorption/Transmission Accessory

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories Model 1940 Absorption/Transmission Accessory The Model 1940 Absorption/Transmission Accessory slightly displaces the sample from its normal position and directs the transmitted light into the collection optics with a mirror mounted at 45°.
  • Page 92 Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories Place the cuvette containing the blank in the sample holder. Set the emission spectrometer position to 0 nm. Select acquisition mode S/R. Run an excitation scan over the absorption range of the sample.
  • Page 93 Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories Adjust the high voltage HV2, as needed, so that the reference signal is ~ 1 µA (saturation of the reference detector is 10 µA). Acquire transmission spectra. Either Position the emission spectrometer at 0 nm, and use the S/R acquisition mode.

  • Page 94: Fl-1013 Liquid Nitrogen Dewar Assembly

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories FL-1013 Liquid Nitrogen Dewar Assembly For phosphorescence or delayed fluorescence measurements, samples are often frozen at liquid-nitrogen temperature (77 K) to preserve the fragile triplet state. The sample is placed in the quartz cell and slowly immersed in the liquid-nitrogen-filled Dewar flask.

  • Page 95: Model 1908Mod Scatter Block Assembly

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories Model 1908MOD Scatter Block Assembly The Scatter Block Assembly includes a white scatter block assembly and a clamp holder for use with a user- supplied standard lamp and regulated power supply.

  • Page 96: Sample Cells

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories Sample Cells Model 1955 HPLC Flow Cell With a sample capacity of 20 µL, this non-fluorescing fused silica cell is ideal for on- line monitoring of fluorescent samples. The cell maintains high sensitivity because it has a large aperture for collecting the excitation light to the sample and fluorescence emission from the sample.

  • Page 97: Model 1967 Photodiode Reference Detector

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories Model 1967 Photodiode Reference Detector The Photodiode Reference Assembly monitors the xenon lamp up to 1 µm. This acces- ® sory is standard in the Fluorolog -3 package, but a photomultiplier can be substituted instead (necessary for phosphorescence measurements).

  • Page 98: Ccd Detectors

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories CCD Detectors For multichannel spectral acquisition, many charge-coupled devices are available to suit the researcher’s needs. Both air-cooled and liquid-nitrogen-cooled CCDs can be in- ® serted into the Fluorolog -3. Available options include extended-UV and near-IR de- tection, various pixel sizes and arrays, and maximum-coverage options.

  • Page 99: Model 1911F Room Temperature Signal Detector

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories Model 1911F Room Temperature Signal Detector ® The Fluorolog -3 includes a room temperature R928P emission signal detector. This detector is mounted to the emission spectrometer and operated in the photon-counting mode.

  • Page 100: Model 1914F Thermoelectrically Cooled Signal Detector

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories Model 1914F Thermoelectrically Cooled Signal Detector As a rule, cooling a detector improves the S/N ratio by reducing the inherent dark count or noise. For the standard signal detector, Model 1911F (described above), cooling re- duces the dark count from 1000 cps to 20 cps;...

  • Page 101: Fl-1030 Thermoelectrically Cooled Near-Ir Photomultiplier Tube

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories FL-1030 Thermoelectrically Cooled Near- IR Photomultiplier Tube For spectral measurements extending into the near-infrared, the FL-1030 Thermoelec- trically Cooled PMT is perfect. Included in the FL-1030 is the thermoelectrically cooled housing. The InGaAs detector has a spectral range from 250 nm all the way to 1050 nm.

  • Page 102: F-3000 Fiber Optic Mount

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories F-3000 Fiber Optic Mount Now you can study marine environments, skin and hair, or other large samples in situ! For those users who want to examine samples unable to be inserted into the sample compartment, the F-3000 Fiber Optic Mount (plus fiber-optic bundles) allows remote sensing of fluorescence.

  • Page 103: Model 1938 Cut-On Filter

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories Model 1938 Cut-On Filter The Model 1938 Cut-On Filter Set consists of 5 filters with dimensions of 1" × 2". To properly position filter, Model FL-1010 Filter Holder is required. Cut-on filters are used to eliminate second-order effects of the gratings.

  • Page 104: Fl-1001 Front-Face Viewing Option

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories FL-1001 Front-Face Viewing Option Designed to examine fluorescence from the surface of solid samples, the FL-1001 Front-Face Viewing Option includes a swing-away mirror. This allows the researcher to change from front-face and right-angle data collection instantly. In the front-face col- lection mode, the viewing angle is 22.5°.

  • Page 105: Gratings

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories Gratings ® Many gratings are available to replace the standard model in the Fluorolog -3. Rulings with the following specifications are available: • 300 grooves/mm • 600 grooves/mm • 1200 grooves/mm In addition, a number of different blazes can be purchased: •...

  • Page 106: Fl-1010 Cut-On Filter Holder

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories FL-1010 Cut-On Filter Holder Cut-on filters are used to eliminate second-order effects of the gratings. The single- beam and T-box sampling compartments have three slots that can hold the FL-1010 Fil- ter Holder.

  • Page 107: Fl-1011 Four-Position Thermostatted Cell Holder

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories FL-1011 Four-Position Thermostatted Cell Holder The FL-1011 Four-Position Thermostatted Cell Holder keeps a sample at a constant temperature from –20°C to +80°C. The temperature is maintained by an ethylene- glycol–water mixture pumped through from an external circulating temperature bath (not included).
  • Page 108 Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories Place the sample in a 10 mm × 10 mm cuvette and insert a magnetic stirring bar. (The stirring bar is available from Bel-Art Products, Pequannock, NJ) Place a cuvette in each holder.

  • Page 109: Fl-1012 Dual-Position Thermostatted Cell Holder

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories FL-1012 Dual-Position Thermostatted Cell Holder The FL-1012 Dual-Position Thermostatted Cell Holder keeps a sample at a constant temperature from –20°C to +80°C. The temperature is maintained by an ethylene- glycol–water mixture pumped through from an external circulating temperature bath (not included).
  • Page 110 Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories Place your sample in a 10 mm × 10 mm cuvette and insert a magnetic stirring bar. (The stirring bar is available from Bel-Art Products, Pequannock, NJ) Place a cuvette in each holder.

  • Page 111: Model 1933 Solid Sample Holder

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories Model 1933 Solid Sample Holder The Model 1933 Solid Sample Holder is designed for samples such as thin films, pow- ders, pellets, microscope slides, and fibers. The holder consists of a base with graduated dial, upon which a bracket, a spring clip, and a sample block rest.
  • Page 112 Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories For samples such as thin films, microscope slides, fibers, or other materials: Place the material on the block on the side opposite that of the well. Insert the block between the bracket and spring clip.

  • Page 113: Fl-1039 Xenon Lamp Housing

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories FL-1039 Xenon Lamp Housing The FL-1039 is the standard lamp housing for the 450-W Xenon Lamp. The power supply is included internally. FL-1040 Dual Lamp Housing The FL-1040 Dual Lamp Housing contains both the standard continuous 450-W xenon lamp and a UV xenon flash tube.

  • Page 114: F-3005/6 Autotitration Injector

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories F-3005/6 Autotitration Injector For controlled, automatic injection of aliquots into the sample of your choice, the F- 3005/6 Autotitration Injector is just the thing, available in both 110-V (F-3005) and 220-V (F-3006) models. The F-3005/6 comes with dual syringes, for complete control over dispensing and aspirating volumes of liquids into and out of the sample cell.

  • Page 115: Models F-3001, F-3002, And F-3003 Microscope Interfaces

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories Models F-3001, F-3002, and F-3003 Microscope Fiber-Optic Interfaces ® The Microscope Fiber-Optic Interface eases the use of the Fluorolog -3 systems for fluorescence-microscopy measurements. Interfaces are available for Nikon, Olympus, and Zeiss microscopes.

  • Page 116: Model 1907 450-W Xenon Lamp

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories Model 1907 450-W Xenon Lamp The Model 1907 450-W xenon lamp delivers light from 240 nm to 850 nm for sample excitation. The lamp has an approximate life of 2000 hours, and is ozone-free. The lamp is designed to fit into the FL-1039 Xenon Lamp Housing and the FL-1040 Dual Lamp Housing.

  • Page 117: F-3004 Sample Heater/Cooler Peltier Thermocouple Drive

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories F-3004 Sample Heater/Cooler Peltier Thermocouple Drive ® For rapid control of the sample’s temperature in the Fluorolog -3’s sample compart- ment, choose the F-3004 Peltier Drive. Instead of messy fluids, the Peltier device heats and cools the sample thermoelectrically and fast! The temperature range is –10°C to...

  • Page 118: Phosphorimeter Accessory

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories Phosphorimeter Accessory The phosphorimeter adds a programmable, pulsed excitation source and selectable sig- nal gating from the signal photomultiplier tube. This provides time-discrimination ca- pability to sort out the lifetimes of simultaneous, competing luminescence emissions.

  • Page 119: Micromax Microwell Plate Reader

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories MicroMax Microwell Plate Reader The MicroMax Microwell Titer-Plate Reader allows multiple samples to be scanned in one experiment. The MicroMax is controlled through the DataMax software via a serial port to the host computer. The titer plate moves beneath a stationary optical beam, and fluorescence measurements are collected with top-reading geometry.

  • Page 120: Fl-1044 L-Format Polarizer & Fl-1045 T-Format Polarizer

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories FL-1044 L-Format Polarizer & FL-1045 T- Format Polarizer For L-format spectrofluorometers, the FL-1044 dual polarizer is ideal. The kit includes two polarizers, to be placed at the entrance and the exit of the T-box. The polarizers are fully automated, and are adjustable to within 1°...

  • Page 121: Fl-1015 Injector Port

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories FL-1015 Injector Port For the study of reaction kinetics, such as Ca measurements, the FL-1015 Injector Port is ideal. This accessory allows additions of small volumes via a syringe or pipette to the sample cell without removing the lid of the sample compartment.

  • Page 122: F-1000/1 Temperature Bath

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories F-1000/1 Temperature Bath For studies of samples whose properties are temperature-dependent, use the F-1000/1 Temperature Bath. The controller circulates fluids externally, with tubes leading to the sample chamber. The temperature range is from –25°C to +150°C. Sensor and all ca- bles are included with the F-1000/1.

  • Page 123: Model Trig-15/25 External Trigger Accessory

    Fluorolog-3 v. 2.2 (10 Sep 2002) Components & Accessories Model TRIG-15/25 External Trigger Accessory The TRIG-15/25 accessory permits the fluorescence system to be operated with almost any external trigger stimulus. Data acquisition can be synchronized with external events, either automatically following a voltage pulse (minimum 3 V above ground), or manually by pushing a button on a trigger-release cable.

  • Page 124: 7: Troubleshooting

    Fluorolog-3 v. 2.2 (31 Jul 2002) Troubleshooting 7: Troubleshooting ® The Fluorolog -3 spectrofluorometer system has been designed to operate reliably and predictably. Should a problem occur, examine the chart below, and try the steps listed on the following pages.
  • Page 125 Fluorolog-3 v. 2.2 (31 Jul 2002) Troubleshooting Communication prob- Boot disk corrupted. Use backup boot disk. If no backup boot disk lems between com- is available, call Fluorescence Service De- puter and instrument. partment. Cables are improperly con- Check communications cables’ connections.

  • Page 126: Using Diagnostic Spectra

    0.35 467 nm 0.25 0.15 0.05 Wavelength (nm) Scan of good quality 450-W xenon lamp in Fluorolog-3 with single excitation monochromator. Artisan Technology Group - Quality Instrumentation ... Guaranteed | (888) 88-SOURCE | www.artisantg.com...
  • Page 127 Fluorolog-3 v. 2.2 (31 Jul 2002) Troubleshooting The following lamp scan spectrum shows poor resolution in the area around the peak. 0.4473 Xenon-lamp peaks are unresolved 0.3355 0.2236 0.1118 Wavelength (nm) Lamp scan of 150-W Xe lamp. Note poor resolution in the area near the 467-nm peak.
  • Page 128 Fluorolog-3 v. 2.2 (31 Jul 2002) Troubleshooting Water Raman spectra Contaminated water Running a water Raman scan helps identify abnormalities as a result of accessory prob- lems or miscalibration. The following spectrum is normal: 397 nm Wavelength (nm) Clean water Raman scan.
  • Page 129 Fluorolog-3 v. 2.2 (31 Jul 2002) Troubleshooting If the problem goes away, then the problem was due to the cuvette surface. Clean or use a different cuvette. Clean the cuvette. Fill with fresh, double-distilled, deionized water. If the problem goes away, then the problem was due to contaminated water.
  • Page 130 Fluorolog-3 v. 2.2 (31 Jul 2002) Troubleshooting Stray light In the following diagram, notice the high level of stray-light below 380 nm in the water Raman spectrum. Wavelength (nm) High stray light in a water Raman scan. To correct this problem, Inspect the cuvette surface for fingerprints and scratches.

  • Page 131: Further Assistance

    Fluorolog-3 v. 2.2 (31 Jul 2002) Troubleshooting Further assistance... ® Read all software and accessory manuals before contacting the Spex Fluorescence Service Department. Often the manuals show the problem’s cause and a method of so- lution. Technical support is available for both hardware and software troubleshooting.
  • Page 132 Fluorolog-3 v. 2.2 (31 Jul 2002) Troubleshooting In Run Experiment toolbar, open the About DataMax window. Make a note of the software’s and instrument’s serial numbers, and instrument configuration, including all accessories. ® If the problem persists or is unlisted, call the Spex Fluorescence Service Department at (732) 494-8660 ×...

  • Page 133: 8: Introduction To Lifetime Measurements

    Fluorolog-3 v.2.2 (10 Sep 2002) Introduction to Lifetime Measurements 8: Introduction to Lifetime Measurements Introduction ® Although the Fluorolog -3 spectroscopy system is fully capable of performing lifetime measurements, it was designed to excel in the area of fluorescence measurements. Op- erating within a lifetime range of 10 ps to 10 µs and a frequency range of 0.2 to 310...

  • Page 134: Lifetime Measurements

    Fluorolog-3 v.2.2 (10 Sep 2002) Introduction to Lifetime Measurements Lifetime measurements The lifetime software is based on acquisition of frequency-domain lifetime measure- ments. In this technique, the excitation light is sinusoidally modulated. The emission from the sample is a forced response to the excitation, and therefore is modulated at the same frequency as the excitation light.
  • Page 135 Fluorolog-3 v.2.2 (10 Sep 2002) Introduction to Lifetime Measurements φ ωτ τ ω − φ − ω τ τ ω − − Artisan Technology Group - Quality Instrumentation ... Guaranteed | (888) 88-SOURCE | www.artisantg.com...

  • Page 136: Types Of Lifetime Scans

    Fluorolog-3 v.2.2 (10 Sep 2002) Introduction to Lifetime Measurements Types of lifetime scans The type of scan defines which measurement will be acquired. In lifetime operation, four scan types are available: • Lifetime • Lifetime-resolved • Dynamic depolarization • Time-resolved.

  • Page 137: 9: Xenon Lamp Information & Record Of Use Form

    Fluorolog-3 v. 2.2 (10 Sep 2002) Xenon Lamp Information 9: Xenon Lamp Information & Record of Use Form Xenon lamps typically are used in fluorescence instruments because they provide a ® continuous output from 240 nm to 600 nm. In the Fluorolog -3 spectrofluorometers, the standard xenon lamp is ozone-free.

  • Page 138: Xenon Lamp Record Of Use

    Fluorolog-3 v. 2.2 (10 Sep 2002) Xenon Lamp Information Xenon Lamp Record of Use Page _____ of _______ In Service Current Time Time Total Time Date Operator Date (Hours/Min.) Total Hours Artisan Technology Group - Quality Instrumentation ... Guaranteed | (888) 88-SOURCE | www.artisantg.com...

  • Page 139: 10: Applications

    Fluorolog-3 v. 2.2 (11 Jul 2002) Applications 10: Applications Introduction ® The Fluorolog series of spectrofluorometers have earned the reputation for being the ® most sensitive in the world. Because the Fluorolog -3 provides superior optical and electronic components combined with a comprehensive selection of accessories, it pro- vides the highest sensitivity and selectivity for all types of samples.
  • Page 140 Fluorolog-3 v. 2.2 (11 Jul 2002) Applications 0.225 0.150 0.075 Wavelength (nm) 0.41 0.308 0.205 0.103 Wavelength (nm) Spectra of azulene. Upper plot: corrected (solid line) and uncorrected (broken line) excitation spectra. Lower plot: an absorption spectrum. The broken line in the upper plot traces the uncorrected excitation spectrum of an az- ®...

  • Page 141: Detecting Sub-Picomolar Concentrations Of Fluorescein

    Fluorolog-3 v. 2.2 (11 Jul 2002) Applications Detecting sub-picomolar concentrations of fluorescein Instrument sensitivity is often expressed in terms of the limit of detection of a standard ® substance. The superior sensitivity of Fluorolog -3 systems is demonstrated by their ability to detect sub-picomolar concentrations of standard substances such as fluo- rescein.

  • Page 142: Fluorescence Detection Of Highly Scattering Samples

    Fluorolog-3 v. 2.2 (11 Jul 2002) Applications Fluorescence detection of highly scattering samples Qualitative and quantitative determinations normally are difficult to ascertain from highly scattering samples. Typically, fluorescence signals are dwarfed by stray or scat- ® tered light from the sample. The flexibility of the Fluorolog...

  • Page 143: Operating In The Ir Region

    Fluorolog-3 v. 2.2 (11 Jul 2002) Applications Perylene 436 nm Anthracene 382 nm 2,3-Benzofluorene Fluorene 342 nm 303 nm Acenaphthene 323 nm Wavelength (nm) Emission (solid line) and synchronous (broken line) scans of a mixture of polynuclear aromatic hydrocarbons. ®...

  • Page 144: Low-Temperature Scans

    Fluorolog-3 v. 2.2 (11 Jul 2002) Applications Because triplet transitions are “forbidden” quantum-mechanically, the average phos- phorescence decay times are generally longer, ranging from a few microseconds to sev- eral seconds. Thus, phosphorescence offers a longer observation period for monitoring reactions, looking at environmental effects on a sample, or following changes in the hydrodynamic characteristics of macromolecular systems.

  • Page 145: Polarization To Detect Trace Quantities Of Biological Probes

    Fluorolog-3 v. 2.2 (11 Jul 2002) Applications microscope slides. A swing-away mirror is positioned to allow collection of sample luminescence at 90° to the excitation beam, or front-face at 22.5°. In front-face view- ing, the fluorescence is collected from the sample’s surface.

  • Page 146: 11: Producing Correction Factors

    Fluorolog-3 v. 2.2 (31 Jul 2002) Producing Correction Factors 11: Producing Correction Factors Introduction Collecting accurate information about the fluorescent or phosphorescent properties of a sample depends upon several factors: equipment, sample, and timing. To ensure that the spectra are indicative of the actual sample properties and not of external conditions, data often must be corrected.

  • Page 147: Generating Emission Correction Factors

    Fluorolog-3 v. 2.2 (31 Jul 2002) Producing Correction Factors Generating emission correction factors Required kits Emission correction factors should be updated periodically or whenever different grat- ings or signal detectors are installed. The correction factors can be updated either at the ®...
  • Page 148 Fluorolog-3 v. 2.2 (31 Jul 2002) Producing Correction Factors Generation Open the Real Time Display. Turn off the high voltage to the emission photomultiplier tube. Close the slits. Place the 1908MOD Scatter Block Assembly in the sample chamber, so that light is directed toward the right angle.
  • Page 149 Fluorolog-3 v. 2.2 (31 Jul 2002) Producing Correction Factors Attach the 2 wire ends of the standard lamp to the lamp holder posts. Warning: Do not touch the lamp. Use cotton gloves or The positive (+) lead goes to lens paper.
  • Page 150 Fluorolog-3 v. 2.2 (31 Jul 2002) Producing Correction Factors Connect the 2 wire leads from the constant- current power supply to the lamp holder. Attach the red wire to the red clip and the black wire to the black clip, located on the sides of the lamp holder.
  • Page 151 Fluorolog-3 v. 2.2 (31 Jul 2002) Producing Correction Factors Determine the dark counts Place the sample lid over the mask to block light to the detector. In Run Experiment, select the Experiment button. This opens the Emission Acquisition dialog box.
  • Page 152 Fluorolog-3 v. 2.2 (31 Jul 2002) Producing Correction Factors stdlamp blank Wavelength (nm) Blank and lamp spectra. Your spectrum should appear similar to the one pictured above. Its actual ® appearance, however, depends on the configuration of your Fluorolog system. The lamp scan was acquired with gratings in the emission spectrometer blazed in the visible region and an R928P red-sensitive photomultiplier as the detector.

  • Page 153: Calculating Emission Correction Factors

    Fluorolog-3 v. 2.2 (31 Jul 2002) Producing Correction Factors Calculating emission correction factors Introduction Irradiance values for a standard lamp, packaged with the lamp, are usually expressed in –6 –2 ® W·cm ·nm. With photon-counting systems like the Fluorolog -3 spectrofluorome- –1...
  • Page 154 Fluorolog-3 v. 2.2 (31 Jul 2002) Producing Correction Factors Choose Emission Acquisition. Click OK to close the Select Experiment Type dialog box. Enter the following parameters: Start the scan at 300 nm. End the scan at 850 nm. Increment the...
  • Page 155 Fluorolog-3 v. 2.2 (31 Jul 2002) Producing Correction Factors Click Signals... to Click HV to open the Signals open the High Voltage dialog dialog box: box: Turn off the volt- age to the detec- tors. Select S (signal) detector. Click OK to close this By turning off the high voltage, a file which box.
  • Page 156 Fluorolog-3 v. 2.2 (31 Jul 2002) Producing Correction Factors Because this file was created with the high voltage off, each intensity value is zero. The irradiance values calculated in a previous step will be entered into the intensity locations for each wavelength.
  • Page 157 Fluorolog-3 v. 2.2 (31 Jul 2002) Producing Correction Factors 4000 3000 2000 1000 Wavelength (nm) IRR file. Now you have the two files: IRR and stdlamp2. These files are required to ® calculate the emission correction factors for the Fluorolog -3 system.
  • Page 158 Fluorolog-3 v. 2.2 (31 Jul 2002) Producing Correction Factors Calculate the correction factors Using the Arithmetic menu in Run Experiment, divide IRR by stdlamp2, and name the resulting file mcorrect. Note: Naming the file mcorrect will overwrite the mcorrect file supplied with the software.
  • Page 159 Fluorolog-3 v. 2.2 (31 Jul 2002) Producing Correction Factors ® The correction factors shown above were acquired for a Fluorolog -3 system with 500-nm blazed gratings in the emission spectrometer and a red-sensitive R928P photomultiplier detector. Once the emission correction factors have been found, determination of the excitation correction factors may be necessary.

  • Page 160: Calculating Excitation Correction Factors

    Fluorolog-3 v. 2.2 (31 Jul 2002) Producing Correction Factors Calculating excitation correction factors The photodiode reference detector handles the bulk of excitation correction from 240– 600 nm when a ratio acquisition mode is selected (e.g., S/R for single-beam and T-box sampling modules).
  • Page 161 Fluorolog-3 v. 2.2 (31 Jul 2002) Producing Correction Factors The slit width discovered in this step will be used to run the Note: Obtain a signal intensity of scan. no greater than 1.5 × 10 cps. With the Real Time Display still running, open Run Experiment.
  • Page 162 Fluorolog-3 v. 2.2 (31 Jul 2002) Producing Correction Factors Click Signals..This opens the Signals dialog box: Enter S/R as the Selected Signal. Click OK to close the Signals box. Click Slits..This opens the Slits dialog box: Use the XFER...
  • Page 163 Fluorolog-3 v. 2.2 (31 Jul 2002) Producing Correction Factors Start the scan at 240 nm. End the scan at 600 nm. Increment by 5 nm after each data point. Set the Inte- gration Time to 5 s. Perform one scan.
  • Page 164 Fluorolog-3 v. 2.2 (31 Jul 2002) Producing Correction Factors Find the minimum data point and divide the file by that value. Save the normalized file as xcorrect. This overwrites the existing excitation correction-factor file. To acquire corrected data for an experiment, enter the name of this file in the COR- RECTION factor file field in the Data Acquisition Parameters dialog box.

  • Page 165: 12: Determining The Plateau Voltage

    Fluorolog-3 v. 2.2 (11 Jul 2002) Determining the Plateau Voltage 12: Determining the Plateau Voltage Introduction The plateau voltage is the voltage at which the signal intensity is maximized, while the dark-count intensity is minimized. The emission signal detector requires a voltage input to operate.
  • Page 166 Fluorolog-3 v. 2.2 (11 Jul 2002) Determining the Plateau Voltage High Voltage Signal Dark Counts (Volts) (cps) (cps) 1000 1050 1100 1150 1200 12-2 Artisan Technology Group - Quality Instrumentation ... Guaranteed | (888) 88-SOURCE | www.artisantg.com...

  • Page 167: Procedure

    Fluorolog-3 v. 2.2 (11 Jul 2002) Determining the Plateau Voltage Procedure Set up the instrument ® Turn on the xenon lamp, Fluorolog -3 system, peripherals, and computer. Enter the DataMax software. Run a xenon-lamp spectrum and the water Raman scans.
  • Page 168 Fluorolog-3 v. 2.2 (11 Jul 2002) Determining the Plateau Voltage Open the programmable excitation shutter by clicking on the shutter icon. Enter 650 V in the S voltage text box. Click on the right arrow on the Continuous area. This records data in Prompt-Step mode (see the DataMax manual for details).
  • Page 169 Fluorolog-3 v. 2.2 (11 Jul 2002) Determining the Plateau Voltage High Voltage Signal Dark Counts (Volts) (cps) (cps) 8.7 × 10 1.28 × 10 1.52 × 10 1.68 × 10 1.74 × 10 1.80 × 10 1.81 × 10 1000 1.96 ×...
  • Page 170 Fluorolog-3 v. 2.2 (11 Jul 2002) Determining the Plateau Voltage From these graphs, at ~1000 V there is a plateau both in the signal and dark counts. Therefore, the point at which there is maximum voltage with minimum dark counts 1000 V—the...

  • Page 171: 13: Reassemby Instructions

    Fluorolog-3 v. 2.2 (11 Jul 2002) Reassembly Instructions 13: Reassembly Instructions ® The Fluorolog -3 system consists of four main components: • Personal computer Warning: Jobin Yvon Inc. does • Color monitor not recommend reassembly by • Expanded keyboard ®...

  • Page 172: Spectrofluorometer Assembly

    Fluorolog-3 v. 2.2 (11 Jul 2002) Reassembly Instructions Spectrofluorometer assembly ® Once the computer has been assembled, the external components of the Fluorolog ® spectrofluorometer must be connected. The modules of the Fluorolog -3 fit together in a seamless configuration. Each module and the lamp housing has alignment studs,...

  • Page 173: Cable Connections

    Fluorolog-3 v. 2.2 (11 Jul 2002) Reassembly Instructions Cable connections These connectors connect spectrofluorometer components with BNC Connector System Controller (SAC) D-shell Connector computer system. MHV Connector SHV Connector 4-Pin Circular Connector The jacks on the rear of the SAC are shown to...
  • Page 174 Fluorolog-3 v. 2.2 (11 Jul 2002) Reassembly Instructions The cable diagram below shows the path of each cable. During installation, refer to the schematic to ensure proper system interconnections. mains mains mains Lamp System housing controller (SAC) Mono drive Excitation...
  • Page 175 Fluorolog-3 v. 2.2 (11 Jul 2002) Reassembly Instructions Connecting the reference detector to the SAC ® The Fluorolog -3 spectrofluorometer system comes standard with a silicon-photodiode reference detector inside the sample-compartment module. The connections to the ref- erence detector are on the outside front of the sample compartment module. The sample compartment module (reference detector) is joined with the SAC via a split cable;...
  • Page 176 Fluorolog-3 v. 2.2 (11 Jul 2002) Reassembly Instructions Take the end of the #33977 cable that has two connectors. Plug the end with the 4-pin circular connector into the POWER jack of the DM302, and the BNC plug into the OUT jack of the DM302.

  • Page 177: Connecting Power Cables

    Fluorolog-3 v. 2.2 (11 Jul 2002) Reassembly Instructions Connecting power cables Several components in the system have AC power cables. These items include: • SAC • Computer (and peripherals) • Lamp ® • System Accessories (such as MicroMax , temperature bath, etc.)

  • Page 178: Introduction

    ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog 14: TRIAX operation with the Fluorolog ® Introduction Some users choose the TRIAX series of imaging spectrometers as a building block in a ® Fluorolog -3 spectrofluorometer. Using a TRIAX imaging spectrometer on the emis- sion side of the sample mount offers the option of detection with a CCD, to create an image of the dispersed fluorescence for subsequent analysis.

  • Page 179: Hardware

    ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Hardware Warning: Some users may wish to remove the mono- Electrical chromator and replace it with a TRIAX. Always switch off the power to the monochromator and TRIAX before...
  • Page 180 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Automated slits The TRIAX has automated adjustable slits at the entrance (2 mm maximum) and exit (7 mm maximum) ports. The slit width may be adjusted in steps of 0.0125 mm. For a 0.6- mm slit on the TRIAX 320, dispersion is 1.58 mm.
  • Page 181 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Communication between TRIAX and SpectrAcq A cable runs between the TRIAX and SpectrAcq for proper communication. To the right is a photograph of the cable attachment to the TRIAX.

  • Page 182: Software

    ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Software Visual Instrument Setup The TRIAX appears as a standard monochromator icon within Visual Instrument Setup’s instrument layout. Moving the flip mirrors For those TRIAX accessories with dual...
  • Page 183 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Rotating the turret to a different grating Click the grating icon. The Grating/Turret window opens. Click the desired grating’s radio button. Allow several seconds for the turret to rotate to the new position.
  • Page 184 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog The New Value dialog box closes. Run a scan with the grating, to find a known peak. Jobin Yvon Inc. suggests running a xenon-lamp scan to find the 467-nm peak, or a water-Raman scan to find the 397-nm peak.
  • Page 185 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Adjusting slit widths Click on an icon of a slit. Note: A CCD is mounted on the side (lateral) exit, while a PMT is mounted on the front exit.
  • Page 186 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog CCD Detector The CCD detector has a special symbol in the Visual Instrument Setup: Click on the CCD icon. The Acquisition Channel window opens. Choose Data Units. Choose Gain Level.
  • Page 187 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Run Experiment Within the DataMax main window, CCD experiments using a TRIAX may be per- formed. To run a CCD acquisition, Choose Collect. A drop-down menu appears. Choose Experiment….
  • Page 188 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog In this dialog box—as with other Experiment Acquisition dialog boxes—the user defines the CCD experiment fully. Several functions specific to the CCD- acquisition type are available, such as:...
  • Page 189 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog CCD Areas A CCD detector can be divided into several data-collection areas, each acting inde- pendently. CCD Areas controls how to divide up the CCD chip into multiple detectors.
  • Page 190 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Visible only with two or more active areas, Normalize lets the user choose an area as the normalization standard. DataMax performs a Dark Subtract on all data, then divides all areas by the normalization standard.
  • Page 191 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog CCD Regions In Scan mode, a CCD detector can take “snapshots” of various spectral regions, Warning: Based on the charac- which can be attached together to create a teristics of multichannel detec- single complete spectrum.
  • Page 192 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Two methods specify how to acquire data: the Position index card, setting the central wavelength, and the Range index card, setting the region on the CCD detector. Position method Click on the Position index-card tab.
  • Page 193 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Range method Click on the Range index-card tab. Enter the starting wavelength in From. Enter the ending wavelength in To. Enter the integration time in Intg. time. For piecing together several “snapshots” with automatic baseline matching, click Auto Adjust.
  • Page 194 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Image and Scan These radio buttons choose between two detection formats: Using the CCD chip as a two-dimensional detector (Image), or columns rows Binning columns of pixels together to create a one-dimensional detector (Scan) for re- cording standard spectra.
  • Page 195 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Integration Time This is the amount of time that the CCD chip is exposed to the sample’s luminescence. Enter the Integration Time, in s, in the data-entry field.
  • Page 196 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Accumulations An “accumulation” is one exposure to the luminescence for a particular active CCD area. Accumulations are added together if more than one accumulation is specified. Enter the desired number of accumulations in the data-entry field.
  • Page 197 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Mirrors… At present, this button is inactive in DataMax. 14-20 Artisan Technology Group - Quality Instrumentation ... Guaranteed | (888) 88-SOURCE | www.artisantg.com...
  • Page 198 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Correction The Correction button is useful only when working with a PMT. Warning: Correction factors pertain only to the photomultiplier tube or other single-point detector, and NOT to a 2-dimensional CCD detector.
  • Page 199 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Fast Kinetics Mode Fast Kinetics Mode is a special high-speed data-acquisition mode that uses only a por- tion of the CCD (top or bottom) as the active area. The Fast Kinetics Mode checkbox contains hidden data-entry fields.
  • Page 200 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog In Continuous mode, Choose the number of Accumulations. Set the Integration Time. Choose the Time Increment from the start of one exposure to the start of the next exposure.
  • Page 201 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Flush The Flush option can remove all residual charges from the CCD chip before data ac- quisition. The drop-down menu contains three choices: • Flush Before Each Acquisition Clear out all charges before an accumulation.
  • Page 202 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Linearization Use this function to re-align the TRIAX if the peaks’ wavelengths are correctly as- signed near the center of the CCD, but are incorrect near the edges.
  • Page 203 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Do so. Click OK. If you choose to have DataMax calculate them, Click OK. The Linearization Parameters window closes, and the Linearization Procedure dialog box appears. Choose the...
  • Page 204 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Click CCD Regions. The Acquisition Setup window opens. Choose the Position index-card tab. Five different spectra are required. The fol- lowing example uses a CCD that is 2000 pixels wide ×...
  • Page 205 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Enter the integration time. Enter other required parameters, e.g., slit widths. Click Run. One Scan/Multi Peaks, for one source with at least five known lines within the CCD’s area.
  • Page 206 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Choose the Position in- dex-card tab. The spectrum must contain five known peaks on different ar- eas of the CCD chip. Set Nb Region to 1. Click OK.
  • Page 207 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Autoscaling the display AutoScale is always on, and cannot be switched off. 14-30 Artisan Technology Group - Quality Instrumentation ... Guaranteed | (888) 88-SOURCE | www.artisantg.com...
  • Page 208 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Real Time Display Unlike typical systems, Real Time Display with a TRIAX and CCD must be used with the Run Experiment window open simultaneously. The Real Time Display is used to optimize the CCD detector and TRIAX settings for the best experimental results.
  • Page 209 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Note: The Counts window does not appear in Real Time Display with a CCD detector, because the system is collecting data over many pix- els, not reporting a single number.
  • Page 210 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog CCD button The CCD button provides information about the CCD chip. Click CCD. The Area List window opens. Chip Height and Chip Width This shows the size of the...
  • Page 211 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog matically (Areas Definitions) and graphically (Areas Views). Comments: Unlimited amount of information may be entered, but only the first 80 charac- ters are displayed. Linearization Normally, data are taken as...

  • Page 212: Correcting Data With The Triax

    ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Correcting data with the TRIAX Introduction General comments about data correction are found in Chapter 4: Optimizing Data Ac- quisition. This section deals with the procedures that are different when using the TRIAX.
  • Page 213 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Here are some examples of deciphering the emission correction-factor file: Mcorrect.spc The default emission correction-factor file for an instru- ment, with no TRIAX, with only one emission grating and detector.
  • Page 214 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog After acquisition To apply the correction factors after the data have been acquired, multiply the data file by the appropriate correction factor file (mc… or xc…). Make sure the trace to be corrected is active in the Run Experiment window.

  • Page 215: Triax 320 Specifications

    ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog TRIAX 320 Specifications Focal length 0.32 m Entrance-aperture ratio f/4.1 Grating size 68 mm × 68 mm Image magnification at exit 1.00 Scanning range 0–1500 nm (with 1200-grooves/mm grating) Multichannel coverage 79.2 nm over 30-mm array width (with 1200-...

  • Page 216: Troubleshooting

    ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Troubleshooting Should there be a problem with the TRIAX spectrograph, check the following chart for ® possible problems. Try the remedies listed on these pages before contacting Spex Fluorescence Service.
  • Page 217 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Light leak. Follow directions above for detecting light leaks. Improper grounding. Exit DataMax. Turn off spectrometer. Rearrange power connections so that spec- trograph, source, and detector are connected...
  • Page 218 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog CCD Areas..14-11, 14-12, 14-26, 14-28 CCD chip 14-4, 14-12, 14-13, 14-16, 14-17, 14-18, 14-33, 14-34 CCD controller .....14-4, 14-9, 14-40 .arl ....14-13, 14-15, 14-16, 14-34 CCD detector..14-9, 14-12, 14-14, 14-15, 14-31 CCD icon ..........
  • Page 219 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog Experiment….........14-10 laser ............14-40 Last Read Out Time ......14-23 lateral ......... 14-1, 14-3, 14-32 Lateral............ 14-5 layout ..........14-5, 14-32 ..........faraday cage 14-40 Linearization 14-12, 14-25, 14-26, 14-28, Fast Kinetics Mode ....
  • Page 220 ® Fluorolog-3 v. 2.2 (9 Aug 2002) TRIAX operation with the Fluorolog power supply..........14-2 specifications, TRIAX......14-38 SpectrAcq ..........14-4 ® Spex Fluorescence Service....14-39 R928P........14-35, 14-36 Raman ............14-7 Term File ..........14-37 Range........14-15, 14-16 Term File*K .......... 14-37 Real Time Display ......

  • Page 221: 15: Technical Specifications

    Fluorolog-3 v. 2.2 (12 Jul 2002) Technical Specifications 15: Technical Specifications ® Each Fluorolog -3 system consists of: • An excitation source • An excitation spectrometer • A sampling module with reference detector • At least one emission spectrometer • At least one emission detector.

  • Page 222: Spectrofluorometer System

    Fluorolog-3 v. 2.2 (12 Jul 2002) Technical Specifications Spectrofluorometer system ® The Fluorolog -3 spectrofluorometer consists of modules and components controlled by the specialized software. Although the system can be configured in various ways for ® a variety of applications, the basic (standard) Fluorolog...
  • Page 223 Fluorolog-3 v. 2.2 (12 Jul 2002) Technical Specifications Lifetime Option Lifetime range: 10 picoseconds to 10 microseconds Frequency range: 0.2 to 310 MHz. Double-distilled deionized ICP-grade water Raman scan 4000:1 Sensitivity S/N at 397 nm, 5-nm bandpass, 1 s integration time, background noise first standard deviation at 450 nm.

  • Page 224: Minimum Computer Requirements

    Fluorolog-3 v. 2.2 (12 Jul 2002) Technical Specifications Minimum computer requirements • Pentium III 90 MHz • 64 megabytes memory (more is recommended) • Minimum 4-gigabyte hard drive • SVGA display card and SVGA monitor to match display card • At least one 3½" high-density floppy drive •...

  • Page 225: 16: Glossary

    Fluorolog-3 v. 2.2 (12 Jul 2002) Glossary 16: Glossary 3D excitation/emission This maps a specified emission-scan wavelength range using various display excitation wavelengths. 3D synchronous/offset A scan that maps a specified synchronous scan using various offset scan wavelengths between the spectrometers.
  • Page 226 Fluorolog-3 v. 2.2 (12 Jul 2002) Glossary tector signal is ratioed to the reference signal, which provides 90% of the corrected excitation scan. To obtain a completely correct scan, the excitation scan acquired in the manner described above is multi- plied by correction factors.
  • Page 227 Fluorolog-3 v. 2.2 (12 Jul 2002) Glossary Front-face detection A mode of detection in which fluorescence is collected off the front surface of the sample. Front-face detection usually is selected for samples such as powders, thin films, pellets, cells on a cover-slip, and solids.
  • Page 228 Fluorolog-3 v. 2.2 (12 Jul 2002) Glossary –1 dent radiation is constant in energy units (cm Rayleigh scattering Light scattering from particles whose dimensions are much smaller than the wavelength of incident light. The scattered light is of the same energy as the incident light. Rayleigh scatter shows scatter ra-...
  • Page 229 Fluorolog-3 v. 2.2 (12 Jul 2002) Glossary other experiments. Triplet state (T The spin-paired ground or excited state formed from the excited singlet state when paired electrons become unpaired. The triplet state emits phosphorescence. Tyndall scattering Scatter that occurs from small particles in colloidal suspensions.

  • Page 230: 17: Bibliography

    Fluorolog-3 v. 2.2 (12 Jul 2002) Bibliography 17: Bibliography P.M. Bayley and R.E. Dale, Spectroscopy and the Dynamics of Molecular Biological Systems, Academic Press, London, 1985. R. Becker, Theory and Interpretation of Fluorescence and Phosphorescence, Wiley-Interscience, 1969. B. Berlman, Handbook of Fluorescence Spectra in Aromatic Molecules, Vols. I & II, Academic Press, New York, 1965 &...
  • Page 231 Fluorolog-3 v. 2.2 (12 Jul 2002) Bibliography Joseph R. Lakowicz, Principles of Fluorescence Spectroscopy, 2 ed., Plenum Press, New York, 1999. _______, Ed., Topics in Fluorescence Spectroscopy, Vols. 1–5, Plenum Press, New York, 1991–1998. _______, Badri P. Melinal, Enrico Gratton, “Recent Developments in Frequency- Domain Fluorometry,”...
  • Page 232 Fluorolog-3 v. 2.2 (12 Jul 2002) Bibliography E.L. Wehry, Ed., Modern Fluorescence Spectroscopy, Vol. 1–4, Plenum Press, New York, 1981. C.E. White and R.J. Argauer, Fluorescence Analysis: A Practical Approach, Marcel Dekker, 1970. J.D. Winefordner, et al., Luminescence Spectrometry in Analytical Chemistry, Wiley-Interscience, New York, 1972.

  • Page 233: 18: Index

    Fluorolog-3 v. 2.2 (11 Jul 2002) Requirements & Installation 18: Index Key to the entries: front-face viewing option......6-16 grating............6-17 Times New Roman font..subject or HPLC flow cell.......... 6-8 keyword injector port..........6-33 lamp housing, dual........6-25 Arial font........command, lamp housing, universal......
  • Page 234 Fluorolog-3 v. 2.2 (11 Jul 2002) Index Area List ....... 14-12–13, 14-34 CCD shutter ........14-4, 14-23 Areas Views ....... 14-12, 14-33–34 CE marking..........15-1 Arithmetic4-17, 4-20, 11-6, 11-11, 11-13, Center ........14-15, 14-27 11-18, 14-37 center wavelength CCD ............14-36 .arl ......14-13, 14-15–16, 14-34 Change ...........
  • Page 235 Fluorolog-3 v. 2.2 (11 Jul 2002) Index Data Units..........14-9 F-3004 Sample Heater/Cooler Peltier DataMax0-1, 4-18, 7-8, 8-1, 10-1, 12-3, 14-1, Thermocouple Drive ......6-29 14-5, 14-10, 14-13, 14-20, 14-25–26, 14- F-3005/6 Autotitration Injector ....6-26 factors, correction......4-18, 11-1 29, 14-31, 14-34, 14-39–40, 15-4 D-connector ..........13-2...
  • Page 236 Fluorolog-3 v. 2.2 (11 Jul 2002) Index grating4-16, 4-18, 5-8, 5-16–19, 6-7, 6-15, 6- 17–18, 10-1, 14-1–2, 14-6–7, 14-15–16, 14-28, 14-35–36, 14-38 lamp grating factor....... See G factor adjustment...........5-8 exposing ............5-3 grating icon ..........14-6 grating turret..........14-1 inserting............5-5 Grating/Turret......3-10, 3-17, 14-6 removing .............5-4...
  • Page 237 Fluorolog-3 v. 2.2 (11 Jul 2002) Index MicroMax .........3-1, 6-2, 6-31 New Polarization Sample......4-10 New Value ........... 14-6–7 Microscope Interface .......6-27 mirror icon ..........14-5 Nikon ..........6-2, 6-27 Mirrors… ........14-11, 14-20 nitric acid........... 4-1 mm............. 3-6, 6-8 nm ............3-14 Model 1630 Field Lens Adapter ....6-12...
  • Page 238 Fluorolog-3 v. 2.2 (11 Jul 2002) Index Polarization..........4-11 rhodamine-B ..........11-15 polarizer ....4-10–12, 6-32, 7-1, 10-7 ribbon cables.......... 14-39 Polarizer Settings ........4-10 right-angle data collection ......4-3 rose Bengal ..........3-20 poor resolution .......... 7-4 POPOP ............ 3-20 RTD-CCD..........14-31 Position ......14-15, 14-27, 14-29...
  • Page 239 Fluorolog-3 v. 2.2 (11 Jul 2002) Index slits2-4–7, 3-6, 3-14, 4-4, 4-16, 5-8, 5-17, 6- troubleshooting, TRIAX ....... 14-39 1, 6-4–5, 7-1–2, 7-4, 7-6–7, 8-1, 11-3, 11- turret ........14-2, 14-6, 14-39 5, 11-15, 14-3, 14-8, 14-28–29, 14-32, 14- Slits ...3-6, 3-14, 4-6, 11-17, 14-8, 14-32 Slits...
  • Page 240 Artisan Technology Group is your source for quality new and certified-used/pre-owned equipment SERVICE CENTER REPAIRS WE BUY USED EQUIPMENT • FAST SHIPPING AND DELIVERY Experienced engineers and technicians on staff Sell your excess, underutilized, and idle used equipment at our full-service, in-house repair center We also offer credit for buy-backs and trade-ins •...

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