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FluoroMax-3 v. 3.1 (3 Mar 2006)

Data Acquisition

4-1

4: Data Acquisition

This chapter presents an introduction to the six special buttons used in FluorEssence™
to record and present data with the FluoroMax

-3. These buttons, located in FluorEs-

sence™’s main window, are:

Experiment
Menu

Previous Experiment
Setup

3D Scan to 3D Profile

Auto Run Previ-
ous

Experiment

Make Overlay

File

Get Peak Informa-
tion

For a detailed description of these six FluorEssence™ routines, see the

FluorEssence™

User’s Guide

and on-line help.

In addition, two methods for determining best excitation and emission wavelengths are
presented, in case these wavelengths are unknown for the sample.

Summary of Contents for FluoroMax-3

Page 1: ...n com www jobinyvon com France HORIBA Jobin Yvon S A S 16 18 rue du Canal 91165 Longjumeau Cedex Tel 33 0 1 64 54 13 00 Fax 33 0 1 69 09 93 19 www jobinyvon fr Japan HORIBA Ltd JY Optical Sales Dept H...

Page 2: ...itten permission from HORIBA Jobin Yvon Inc Requests for permission should be requested in writing Origin is a registered trademark of OriginLab Corporation Alconox is a registered trademark of Alcono...

Page 3: ...ration 3 1 Introduction 3 1 Controls and indicators 3 1 Turning on the system 3 2 Checking system performance 3 3 4 Data Acquisition 4 1 Experiment Menu button 4 2 Previous Experiment Setup button 4 4...

Page 4: ...ion Transmission Accessory 12 3 FL 1013 Liquid Nitrogen Dewar Assembly 12 6 Model 1908MOD Scatter Block Assembly 12 7 Model 1908 Standard Lamp Assembly 12 7 Sample cells 12 8 F 3000 Fiber Optic Mount...

Page 5: ...FluoroMax 3 v 3 1 3 Mar 2006 v 15 Declaration of Conformity 15 1 16 Index 16 1...

Page 6: ...FluoroMax 3 v 3 1 3 Mar 2006 vi...

Page 7: ...uoroMax P spectrofluorometer systems are State of the art optical components A personal computer FluorEssence for Windows the driving software The difference between the FluoroMax 3 and FluoroMax P is...

Page 8: ...Routine maintenance procedures such as replacing the lamp 7 Troubleshooting Potential sources of problems their most probable causes and possible solutions 8 Producing Correction Factors How to correc...

Page 9: ...ing from your use or misuse of any such products including without limitation to persons injured directly or indirectly in connection with your use or operation of the products The foregoing indemnifi...

Page 10: ...of use loss of data or loss of profits arising out of or in connection with our products or the use or possession thereof HORIBA Jobin Yvon is also in no event liable for damages on any theory of lia...

Page 11: ...re fully understood and met HORIBA Jobin Yvon Inc is not responsible for damage arising out of improper use of the equipment A CAUTION notice denotes a hazard It calls attention to an operating proced...

Page 12: ...o cause electric shock Danger to fingers This symbol warns the user that the equipment is heavy and can crush or injure the hand if precautions are not taken This symbol cautions the user that excessi...

Page 13: ...v 3 1 3 Mar 2006 Introduction 0 7 Wear appropriate safety goggles to protect the eyes Wear an appropriate face shield to protect the face General information is given concerning opera tion of the equ...

Page 14: ...ur lifetime The long term risks for large cumulative exposure include premature aging of the skin wrinkles and most seriously skin cancer and cataract Damage to vision is likely following exposure to...

Page 15: ...rk NORMAL EYEGLASSES OR CONTACTS OFFER VERY LIMITED PROTECTION Training For the use of UV sources new users must be trained by another member of the labora tory who in the opinion of the member of sta...

Page 16: ...ingle prolonged ex posure Excessive exposure to visible light can result in skin and eye damage Visible light sources that are not bright enough to cause retinal burns are not necessar ily safe to vie...

Page 17: ...ns In addition the heat deposited in the cornea may be conducted to the lens of the eye This heating of the lens is believed to be the cause of so called glass blowers cataracts because the heat trans...

Page 18: ...FluoroMax 3 v 3 1 3 Mar 2006 Introduction 0 12...

Page 19: ...roper training in cludes but is not limited to Understanding the risks of exposure to ultraviolet visible and infrared light and how to avoid unsafe exposures to these types of radiation Handling xeno...

Page 20: ...llation 1 2 Surface requirements A sturdy table or bench top Surface must hold 90 kg 200 lbs Surface should be about 27 72 69 cm 183 cm to hold spectrofluorometer computer and accessories comfortably...

Page 21: ...age the optics Warning For adequate cooling do not cover block or obstruct the vents on the left side and underside of the instrument Environmental requirements Temperature 59 86 F 15 30 C Maximum tem...

Page 22: ...quipment mal function or external faults For all instruments ground continuity is required for safe operation Any discontinuity in the ground line can make the in strument unsafe for use Do not operat...

Page 23: ...single packing carton If a host computer PC is ordered as a part of the system the PC is delivered in a few clearly labeled boxes All accessories cables software and manuals ordered with the system ar...

Page 24: ...rest it on the side of the laboratory bench where the system will stay d Place the instrument in its permanent location e Level the spectrofluorometer Adjust the four leveling feet on the bottom of t...

Page 25: ...ecommended location for the PC is just to the right of the spectro fluorometer but other positions are possible b Follow the instructions for the host PC to set up the computer system including the CP...

Page 26: ...Jobin Yvon Inc the software installa tion is complete If the computer is not from HORIBA Jobin Yvon Inc perform the installation Contact a HORIBA Jobin Yvon Inc Sales Representative for recommended sp...

Page 27: ...Software emulation Emulating the FluorEssence software means letting the computer act as though the FluoroMax is properly connected even if it isn t 1 Disconnect the communications cable from the host...

Page 28: ...Initialization Process window opens Under the Status column warning symbols appear for the hardware devices noting that they were Not Found Thus FluorEssence chooses the Emulate radio button as the de...

Page 29: ...ator which selects a band of wavelengths This monochromatic excitation light is directed onto a sample which emits luminescence The luminescence is directed into a second emission mono chromator which...

Page 30: ...r not on diagram Illuminator xenon arc lamp 1 The continuous light source is a 150 W ozone free xenon arc lamp Light from the lamp is collected by a diamond turned elliptical mirror and then focused o...

Page 31: ...rtical grooves A spectrum is obtained by rotating the gratings and recording the intensity values at each wavelength The gratings in the Fluoro Max 3 contain 1200 grooves mm 1 and are blazed at 330 nm...

Page 32: ...nm 0 50 2 125 2 1 175 4 994 5 2 00 8 500 8 5 Shutters An excitation shutter standard on the FluoroMax 3 is located just after the excitation monochromator s exit slit The shutter protects samples from...

Page 33: ...detector This detector is an R928P photomultiplier tube which sends the signal to a photon counting module The detector s response ranges from 180 850 nm with dark counts 1000 counts per second cps T...

Page 34: ...tails Electronics and controllers 1a and 7 The right rear and bottom of the FluoroMax 3 houses the electronics for running the lamp instrument scans and measurements Older generation of slave controll...

Page 35: ...to the host computer via a null modem serial link The controller houses the CTI counter timer integrator card for all instrument con trol and data acquisition Monochromator 180F boards and optional a...

Page 36: ...FluoroMax 3 v 3 22 Feb 2005 System Description 2 8...

Page 37: ...the monochromators While doing these procedures how to define a scan run a scan and optimize system settings to obtain the best results is explained Controls and indicators Power switch The power swit...

Page 38: ...efore take care in case tiny lamp shards exit the ventilation fans Turning on the system 1 Turn on the FluoroMax 3 Turn the power switch to the ON 1 position When the xenon lamp is lit the Spex logo i...

Page 39: ...ation at a particular wavelength in this step Repeatability is the ability of the system to produce consistent spectra Throughput is the amount of signal passing through and detected by the system The...

Page 40: ...ated before the sample compartment It is an excitation scan of the xenon lamp s output and should be the first check performed 1 Close the lid of the sample compartment 2 On the main FluorEssence tool...

Page 41: ...Monochromator 1200 grooves mm Initial wave length Final wave length Increment Slits bandpass Excitation 200 nm 600 nm 1 nm 1 nm Emission 350 nm 1 nm Default detector parameters for the xenon lamp scan...

Page 42: ...0 nm 7 Calibrate the excitation monochromator if re quired a Expand the plot by clicking the Expand button b Click the cursor button to start the Cursor function c Click on the graph near the peak to...

Page 43: ...m Operation 3 7 This example shows the peak actually at 477 nm which is 10 nm too high Therefore we must recalibrate the monochromator f Click the Previous Experiment button The Experiment Setup windo...

Page 44: ...1 3 Mar 2006 System Operation 3 8 g Click the RTC button on the lower right The Real Time Control window opens h Click the Monos icon to view the monochromators index card then click the excitation mo...

Page 45: ...tation 1 button The Calibrate window opens k In Peak Of Interest enter the actual or expected position of the peak it ought to be 467 nm l Click OK m At the bottom right of the Real Time Control windo...

Page 46: ...e should be research quality triple distilled or deionized water HPLC grade 18 M spec or equivalent water is suggested for the Raman scan Impure sam ples of water will cause elevated background levels...

Page 47: ...FluoroMax 3 v 3 1 3 Mar 2006 System Operation 3 11 4 Choose Spectra The Experiment Type window appears 5 Choose Emission The water Raman experiment automati cally loads...

Page 48: ...th Final wave length Incre ment Slits band pass Excitation 350 nm 5 nm Emission 365 nm 450 nm 1 nm 5 nm Detector parameters for the water Raman scan Detector Signal Integration time Units Signal S1 10...

Page 49: ...t settings and sample pu rity As the xenon lamp ages the throughput of the system will decline slowly Therefore low water Raman peak intensity may indicate a need to replace the xenon lamp 8 If the wa...

Page 50: ...BA Jobin Yvon we define the S N ratio as the difference of peak and back ground signal divided by the square root of the background signal S N S S S peak background background Our method The peak sign...

Page 51: ...00 4790 The other method similarly gives a water Raman S N of S N S S N peak background rms background 501500 10500 44 6 11000 HORIBA Jobin Yvon feels that the first method is correct although it give...

Page 52: ...e system is switched on each day to check the calibration and performance of the FluoroMax 3 HORIBA Jobin Yvon Inc recommends monitoring the number of hours of xenon lamp use via the hour meter Additi...

Page 53: ...FluorEs sence s main window are Experiment Menu Previous Experiment Setup 3D Scan to 3D Profile Auto Run Previ ous Experiment Make Overlay File Get Peak Informa tion For a detailed description of the...

Page 54: ...connected accessories such as a temperature bath MicroMax etc Only those scans that can be run using the available hardware configuration are active scans that cannot be taken are grayed out Calibrat...

Page 55: ...n 7 Insert the sample into the sample compartment and close the sample compartment s cover 8 Click Run The collected spectrum is displayed on the Intermediate Display screen After all data are recorde...

Page 56: ...n The Previous Experiment Setup button resets the experiment to the previous experi ment used with minor modifications to the hardware possible 1 After an experiment is loaded click the Previous Exper...

Page 57: ...been loaded and run Auto Run Previous Experiment button The Auto Run Previous Experiment button reruns the last experiment loaded without modifications 1 Click the Auto Run Previous Experiment button...

Page 58: ...ly with an active graph Make Overlay File button The Make Overlay File button creates an SPC file for use as an overlay file 2 Click the Auto Run Previous Experiment button in the toolbar The Save As...

Page 59: ...n extracts emission profiles from an excitation emission matrix 1 Open excitation emission matrix data 2 Click the 3D Scan to 3D Profile button in the toolbar The Profiles window appears 3 Grab and mo...

Page 60: ...n 4 8 4 Click the Arbitrary Line button to choose an arbi trary profile Grab an end of the profile line and move to the desired location on the matrix The profiles are updated 5 To return to perpendic...

Page 61: ...her parameters for that peak 1 Open a data set 2 Click the Get Peak Information button in the toolbar A shaded area with two tabs appears over the peak and a red line calculates a baseline for the pea...

Page 62: ...running an emission scan to find the peak emission value Then an excitation scan is run using the determined peak emission value 1 Find the preliminary emission maximum The object of this step is to a...

Page 63: ...viously saved file h Verify that experimental parameters are correct Be sure to check all parameters under all icons in the left hand column i Set the scan parameters Most of these parameters are a tr...

Page 64: ...j Insert the sample into the sample compartment and close the sample compartment s cover k Click Run The scan starts l With the spectrum on the screen note the greatest intensity If the signal exceed...

Page 65: ...FluoroMax 3 v 3 1 3 Mar 2006 Data Acquisition 4 13 b Choose Spectra The Experiment Type menu appears c Choose Excitation d Click Next The Experiment Setup window appears...

Page 66: ...s maximum excitation wavelength If the raw S channel signal 20 A reduce slits and rescan i Note the excitation peak This is the optimum excitation peak position 3 Find the optimal emission peak a Use...

Page 67: ...ults in light scattering from the cuvettes 5 Optimizing Data Spectra can be enhanced by optimization of data acquisition This chapter lists some methods of optimizing sample preparation spectrofluorom...

Page 68: ...mL cell 5 mm 5 mm cross section is also available Solid samples Solid samples usually are mounted in the Model 1933 Solid Sample Holder with the fluorescence collected from the front surface of the s...

Page 69: ...ground fluorescence persists recrystallize the sample to eliminate organic impurities and then dissolve it in an appropriate solvent for analysis Biological samples For reproducible results some sampl...

Page 70: ...e emission slits The photograph at right illustrates how a 60 angle to the excitation keeps the incoming excitation light away from the emission monochromator s entrance Use filters in the optical pat...

Page 71: ...sed to the front surface of the sample and fluorescence emission is collected from this region at an angle that minimizes reflected and scattered light With front face detection set the front face of...

Page 72: ...erimental parameters are constant In other cases the system can measure the G factor automatically before an experimental run G factors are incorporated into the Anisot ropy scan type 1 In the FluorEs...

Page 73: ...olarizers Operation Manual 1 In the Experiment Setup window click the De tectors icon This shows the parameters related to detectors including the G factor in the Polarization area 2 Click the G Facto...

Page 74: ...he signal to noise ratio S N This is true especially for weakly fluorescing samples with low quantum yields The signal to noise ratio can be improved by Using the appropriate integration time Scanning...

Page 75: ...creases the S N by over 40 as shown below For an integration time of 1 second S N t 1 2 1 2 1 1 For an integration time of 2 seconds S N t 1 2 1 2 2 1414 or approximately 42 Because S N determines the...

Page 76: ...nces the S N In general the S N improves by n1 2 where n is the number of scans To scan a sample multiple times 1 Open the Experiment Setup window 2 Choose the Detectors icon 3 Specify the number of s...

Page 77: ...l spectrum examine the results If two adjacent peaks are not resolved i e separated satisfactorily reduce the increment If the spectrum is described by an excessive number of data points increase the...

Page 78: ...ses through the excitation spectrometer to the sample The emission spectrometer slits control the amount of fluorescence recorded by the signal detector Signal level is proportional to the square of t...

Page 79: ...eraging or Savitsky Golay smoothing See the on line Origin help for additional information regarding smoothing data In general start with a 9 or 11 point smooth for a time base measurement To select t...

Page 80: ...with the instrument are sets of excitation and emission correction factors to eliminate response characteristics These files1 xcorrect and mcorrect are included with the software Items that may be con...

Page 81: ...ch particular instrument and cannot be swapped 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...

Page 82: ...button and browse for the desired correction file Detector Correction factor file name S mcorrect R xcorrect T if used tcorrect 2 Choose correction when setting up the experi ment a In the Experiment...

Page 83: ...v 3 1 3 Mar 2006 Optimizing Data 5 17 c Click the Add button to add the corrected signal to the Formulas ta ble The corrected signal appears in the Formulas table d Run the experiment with the correc...

Page 84: ...main FluorEssence window 2 Choose Analysis A drop down menu appears 3 Select Simple Math This opens the Math Functions dialog box The chosen trace should appear in the Data field If not browse for it...

Page 85: ...ce the lamp Obtaining good spectral results depends on the xenon lamp Keep track of lamp usage with the hour meter After 1200 1500 h of use the lamp output decreases significantly indicating that the...

Page 86: ...a nently damage eyes Do not touch the focusing lens back scatter mirror or the surface of the lamp Fin gerprints will be burned onto these surfaces when the lamp is ignited Changing the lamp 1 Switch...

Page 87: ...from the lamp housing cover b Gently rotate the lamp housing cover with its cooling fans at tached c Rotate the cover backwards and set it behind the instrument so that electrical connections are not...

Page 88: ...node side a While holding the metal anode top portion of the lamp loosen the height adjustment above the lamp with a 1 8 Allen key until the lamp is removable b Press down against the spring action No...

Page 89: ...h the cathode connection to the new lamp b Secure the connection with the new lamp s thumbscrew c Attach the anode connection to the new lamp d Secure the connection with the new lamp s thumbscrew e R...

Page 90: ...rse lamp adjustments immediately After a 6 h burn in set the fine ad justments For lamp replacement the major adjustment is to optimize the height screw that was loosened to remove the old xenon lamp...

Page 91: ...a Move the excitation monochromator to 350 nm and the emission mono chromator to 397 nm b Set the slits to 5 nm bandpass c Open the excitation shutter and emission shutter d Set the integration time...

Page 92: ...improve the water Raman signal No Replace the lamp housing cover Replace the FluoroMax 3 cover STOP The FluoroMax 3 is ready for operation After repeated ad justments is the signal still low No Yes Go...

Page 93: ...pply has a capacity of 150 300 W and is found in the center back of the instrument In order to suppress the emf spike emitted from the lamp s power supply during arcing and start up of the lamp severa...

Page 94: ...bottom cover of the instrument Remove all screws on the bottom cover except those that attach the dust screens for the exhaust fans The leveling feet do not need to be removed before detaching the bo...

Page 95: ...ore attempting repairs or testing with meters DO NOT ATTEMPT ANY REPAIRS OR INSTRUMENT EVALUATION WITHOUT THE EXPRESS PERMISSION OF SPEX FLUORESCENCE SERVICE The emission photomultiplier tube is coupl...

Page 96: ...FluoroMax 3 v 3 1 3 Mar 2006 Maintenance 6 12...

Page 97: ...rating Call the Spex Fluorescence Service Depart ment Lamp is too old Replace lamp 150 W lamp has lamp lifetime 1200 1500 h Signal intensity is low Shutter s closed Open all shutters Signal intensity...

Page 98: ...try scan Stirring speed is too fast Use slower stirring speed Noisy spectrum with magnetic stirrer Stirring bar is too large light beam is striking it Use a smaller stirring bar available from BelArt...

Page 99: ...an SVGA monitor to the VIDEO connector and a keyboard to the KBD connector c Switch on the FluoroMax A number of messages scroll down the monitor The last message should be something similar to CMOS...

Page 100: ...C SCSI Boot Up Floppy Disk Enabled Boot Up NumLock Status On Section CHIPSET FEATURES SETUP Settings to change VGA Shared Memory Size 0 5 MB Section POWER MANAGEMENT SETUP Settings to change Power Man...

Page 101: ...ing the hardware or software parameters that should be adjusted The following spectra occur with explanations about problems leading to their appearance Lamp scan Running a lamp scan verifies system i...

Page 102: ...or lamp scan of 150 W Xe lamp Note low resolution in the area near the 467 nm peak This lack of spectral resolution appears because the slit widths are set too wide To re solve this problem narrow the...

Page 103: ...5 W avelength nm Intensity 10 5 counts s Contaminated water or dirty cuvette note high background Clean water Water Raman spectra Contaminated water Running a water Raman scan helps identify abnormali...

Page 104: ...problem goes away then the problem was due to the cuvette surface Clean or use a different cuvette Or 1 Clean the cuvette 2 Fill with fresh double distilled deionized water If the problem goes away th...

Page 105: ...tice the high level of stray light below 380 nm in the water Raman spectrum To correct this problem 1 Inspect the cuvette surface for fingerprints and scratches 2 Clean the cuvette or use a new one 3...

Page 106: ...back on 2 Make sure all accessories are properly configured and turned on as needed 3 Following the instructions in Chapter 3 System Operation run a lamp scan and a water Raman scan to make sure the s...

Page 107: ...der Help choose About FluorEssence This opens the About FluorEs sence window The version of the software both FluorEs sence and Origin is listed here 9 Determine the SpectrAcq firmware version a Open...

Page 108: ...ting 7 12 c Move the mouse over the detectors table in the Select area The SpectrAcq firmware version appears in a small pop up window If the problem persists or is unlisted call the Spex Fluorescence...

Page 109: ...hose required for quantum yield determinations spectrometer response characteris tics must be eliminated Corrections are made for each of these potential problems by using radiometric correction facto...

Page 110: ...lamp as a function of wavelength are known dividing the irradiance values by the lamp spectrum results in a set of relative correction values These values can then be applied to the raw fluo rescence...

Page 111: ...tems like the FluoroMax 3 spectrofluoro meter however data usually are collected in units of photons s 1 cm 2 nm To convert the units multiply each irradiance value by the wavelength at which it is va...

Page 112: ...Simple Math Tool window under the Analysis menu divide the mcorrect file by this minimum signal intensity a constant c Save this new file as mcorrect That is overwrite the existing mcorrect file This...

Page 113: ...on from 240 600 nm when a ratio acquisition mode is selected i e S R More accurate measure ments require that compensation be applied for the difference in optical path between the detector and the sa...

Page 114: ...FluoroMax 3 v 3 1 7 Mar 2006 Producing Correction Factors 8 6 b Open the Real Time Control c Set the excitation and emission monochromators to 467 nm and 630 nm respectively...

Page 115: ...ote the slit width the slit width discovered in this step will be used to run the scan g Close the Real Time Control and revert to the Experiment Setup dialog box h Close the dialog box and open the E...

Page 116: ...he Formulas table d Remove both S1 and R1 from the Formulas table e In the Signal column click S1 In the Operations column click the division sign In the Signal column click R1 In the Formula box S1 R...

Page 117: ...ducing Correction Factors 8 9 As with the corrected emission factors find the minimum data point and divide the file by that value 8 Save the normalized file as xcorrect This overwrites the existing e...

Page 118: ...en the Correction check box is activated in the Experiment Setup window 1 In the FluorEssence toolbar choose Collect A drop down menu appears 2 Choose Advanced Setup A sub menu appears 3 Click System...

Page 119: ...e the Instrument Correction Files icon The Instrument Correction Files area appears 6 If there are no active fields in the Instrument Correction Files area click Insert 7 In sequence a Choose the Dete...

Page 120: ...n the File column browse for the appropriate correction factor file 8 When all necessary detectors have an associated correction factor file click OK The new correction factor files are now ready to b...

Page 121: ...ction The FluoroMax P includes a phosphorimeter that is a programmable pulsed source and selectable signal gating from the reference detector Switching between the pulsed lamp and continuous lamp is c...

Page 122: ...400 1 ms 4000 10 ms 40 000 100 ms 400 000 1 s 4 000 000 With for example 100 flashes of integrated over 10 ms each the maximum detectable signal is 100 flashes 40 000 counts 4 000 000 counts total A t...

Page 123: ...r FluorEssence parameters govern the sequence in a phosphorimeter experiment These parameters automatically appear on the phosphorimeter experiment acquisition dialog box Initial Delay Sets the time i...

Page 124: ...and then increase it until acceptable results are ob served If the Sample Window is too long the detector will record spuri ous background signal If the Sample Window is too short com ponents of the l...

Page 125: ...With the Phos experiment type in the Fluorescence Experiment Menu create a phospho rescence decay curve as in the screenshot from FluorEssence shown above This is an example using data from TbCl3 aq...

Page 126: ...delay Notice the wave length shift in the 593 nm peak marked with a gray line from initially mostly europium fluorescence short lifetime to a mixture of terbium longer lifetime and europium fluoresce...

Page 127: ...ontaining a mixture of components can be analyzed through fitting its phosphorescence decay curve Here is a phosphorescence decay of an aqueous mixture of EuCl3 and TbCl3 whose different lifetimes hav...

Page 128: ...FluorEssence features Experiment Setup window The Experiment Setup window under the Detectors icon includes a Phosphorimeter area indicating that the phosphorimeter is available Experiment Type window...

Page 129: ...of parameters for the emission of EuCl3 Use an excitation monochromator set to 393 nm the emission monochromator should start at 570 nm end at 750 nm with an increment of 1 nm Decay by Delay Decay by...

Page 130: ...Open the graph to be processed 2 Click on the data points to be processed 3 In the toolbar choose Analysis A drop down menu appears 4 Choose Fit 5 From the sub menu choose the type of analytical curv...

Page 131: ...g the lamp cover unplug the FluoroMax P s power cord and wait at least 15 min while the internal capacitors discharge Never operate the lamp with the cover removed Warning Xenon lamps are an explosion...

Page 132: ...ok directly at the xenon arc or its reflection Intense radiation can perma nently damage eyes Do not touch the focusing lens back scatter mirror or the surface of the lamp Fingerprints will be burned...

Page 133: ...ent by grasping opposite corners of the cover and raising upward If the cover sticks gently work each side upward until the cover slides smoothly off 5 Remove the lamp housing cover at the rear of the...

Page 134: ...failure Handle the flash lamp with tissues cloth gloves or soft cloths only c Discard the old flash lamp in a safe and appropriate manner 7 Insert the new flash lamp a Align the pins with the lamp soc...

Page 135: ...ed emission components allows calcula tion of the type and extent of rotational motions of the molecule The third step is measurement of emission The polarized components of fluorescence emission are...

Page 136: ...emission spectrometer The spectral bandpass of the emission also affects G Thus a pre calculated G factor can be applied to experiments in which instrumental factors emission wavelength and emission...

Page 137: ...arization units mP for very small changes in the polarization Polarization geometries Polarization measurements are taken in two basic geometries L format uses two polarizers which are both rotated be...

Page 138: ...ome polarization bias Specifically when the rotational correlation time of a fluorophore is similar to the fluorescence life time the effect can be significant To record spectra that are free of rotat...

Page 139: ...and complete polarizer orders are shipped with pre aligned polarizers marked for excitation X or emission M and are locked in their collars Store the polarizer crystals in a dust free environment in...

Page 140: ...rement of the polarization or anisotropy within the software using the Anisotropy scan type or use of the Remeasure Anisotropy Only utility click Advanced and the Polarizer Alignment window opens The...

Page 141: ...1 5 million cps on S 11 Record the four permutations of the polarizers VV HV VH and HH 12 Subtract the dark counts from each permutation For example VV VV dark counts 13 Calculate the polarization ra...

Page 142: ...ements and locates the optimal positions for each autopolarizer After completion the anisot ropy for the scattering solution is measured and displayed for user approval of the alignment If approved th...

Page 143: ...ct Dark recommended Reset to Mechanical Zero only if the polarizers are definitely miscalibrated This deletes the previous calibration 6 Place the LUDOX or glycogen in the sample holder 7 Click the Co...

Page 144: ...n channel S or T 8 Approve or retry the measurement based on satisfaction with the result 9 To quit hit the Cancel button at any time during the procedure Alignment 1 Turn off power to the polarizers...

Page 145: ...Sheets MSDS before using colloidal silica or glycogen 6 Insert the LUDOX or glycogen sample into the sample holder 7 Start the software if not yet running and go to Real Time Control 8 Set all monoch...

Page 146: ...1050 V for R1527 12 Set slits to 5 nm bandpass for all monochroma tors 13 Set scatterer concentration to give 1 1 5 106 cps on S 14 Rotate the excitation polarizer to a rough maxi mum 15 Set the pola...

Page 147: ...ps on S channel 18 Measure polarization ratio Equation 9 If the polarization ratio 100 then the alignment is acceptable Otherwise repeat steps 15 18 19 Secure the polarizers in their collars 20 Verify...

Page 148: ...f emission anisotropy or polarization at fixed wavelengths This is used for binding assays kinetics of molecular size or shape change temperature ef fects on rotational motion of fluorophores e g phas...

Page 149: ...utopolarizers load an instrument configuration with autopolarizers Real Time Control Real Time Control manipulates the polarizers and other instrument settings to observe and optimize the spectrofluor...

Page 150: ...Setup runs all scanning options for the autopolarizers First choose the type of scan using polar izers in the Fluorescence Main Ex periment Menu The Experiment Setup window appears Adjust polarizer p...

Page 151: ...stant wavelength analysis experiment that is to take polariza tion acquisitions at fixed excita tion emission wavelength pairs choose Anisotropy from the Fluo rescence Main Experiment Menu The Experim...

Page 152: ...Polarizers 10 18 Make sure to use the appropriate Signal in the Signal Algebra area Add it to the Formulas table Click the checkbox to measure G factor s during the scan or specify G factor s beforeh...

Page 153: ...ntain cali bration in a drawer or cabinet Wrap the polarizers in lens tissue to keep them dust free and for protection and then place them in a plastic bag The automated accesso ries should also be st...

Page 154: ...and the result Refer to the appropriate section of this man ual and the software manuals if necessary 3 If the problem persists or is not listed Call the Fluorescence Service Department by phone at 7...

Page 155: ...ly set slits Set slits for 1 106 cps in VV Signals much less than this give excessive contribu tion from dark noise while signals 2 106 cps are in non linear region Low polarization ratio System misal...

Page 156: ...FluoroMax 3 v 3 1 6 Mar 2006 Automated Polarizers 10 22...

Page 157: ...tor An emission monochromator An emission detector A FluoroMax P system adds A motorized mirror to change between light sources A pulsed xenon lamp A phosphorimeter control module Each system is contr...

Page 158: ...00 nm s 1 Accuracy 0 5 nm Step Size 0 0625 100 nm Range 0 950 nm physical Gratings Excitation 330 nm blaze 220 600 nm optical range Emission 500 nm blaze 290 850 nm optical range Sample Module The sam...

Page 159: ...ve humidity 75 Power 5 A 120 V 60 Hz or 2 5 A 240 V 50 Hz single phase AC Phosphorimeter FluoroMax P only The following components and specifications also apply to the FluoroMax P Source UV xenon flas...

Page 160: ...disk drive Hard disk At least 80 GB of free storage CD ROM drive Required Memory 128 MB RAM 256 MB Recommended Video display Video resolution of at least 1024 768 Keyboard A 104 key keyboard plus USB...

Page 161: ...s all the accessories and components in al phabetical order available for the FluoroMax 3 spectrofluorometers A brief descrip tion of each is included in the following sections Like the list presented...

Page 162: ...ilter cut on 2 2 1939 12 10 Holder filter FL 1010 12 11 Holder four position variable temp control w magnetic stirrer FL 1011 12 12 Holder dual position variable temp control w magnetic stirrer FL 101...

Page 163: ...40 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 Installation 1 Remo...

Page 164: ...he signal without saturating the detector 7 Similarly locate the maximum reference signal of the lamp spectrum 8 Set the excitation monochromator to that position 9 Adjust the high voltage HV2 as need...

Page 165: ...utral density filter a Acquire a synchronous scan with an offset of 0 nm over the absorption range of the blank b Acquire a synchronous scan with an offset of 0 nm over the absorption range of the sam...

Page 166: ...Dewar flask The white Teflon cone in the bottom of the Dewar flask keeps the quartz sam ple tube centered in the Dewar flask The Teflon cover on the top of the Dewar flask holds any ex cess liquid nit...

Page 167: ...s an NIST traceable calibrated 200 W quartz tungsten halogen lamp with irradiance values Regulated 6 5 A power supply with lamp holder 1908MOD Scatter Block Assembly see above Emission correction fact...

Page 168: ...mm cell holder Model 1924 Micro Cell with Model 1924A Adapter This non fluorescing fused silica cylindrical cell holds 250 L A magnetic stirrer cannot be used with this cell The 1924A Adapter is requi...

Page 169: ...m the ex citation spectrometer onto the fiber optic bundle and then directed to the sample Fluo rescence emission from the sample is directed back through the bundle and into the front face collection...

Page 170: ...ectrum install the filter holder and the appropriate cut on filter in the excitation light path Model 1939 Cut On Filter The Model 1939 Cut On Filter set consists of five 2 2 filters with cut on wave...

Page 171: ...iminate second order effects of the gratings The sample compartment has three slots that can hold the FL 1010 Fil ter Holder Refer to either Model 1939 Cut On Filter or Model 1938 Cut On Filter for a...

Page 172: ...mostatted 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 temperatu...

Page 173: ...and insert a magnetic stirring bar The stirring bar is available from Bel Art Products Pequannock NJ 2 Place a cuvette in each holder 3 Allow the samples to reach the desired temperature 4 Turn on th...

Page 174: ...tted 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 ba...

Page 175: ...only one sample is mixed at a time Use 1 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 2 Place a cuvet...

Page 176: ...ng angle of rotation upon which a bracket a spring clip and a sample block rest Model 1933 Solid Sample Holder with sample block nearby Installation 1 Remove the present holder 2 Position the base on...

Page 177: ...minimizes stray and reflected light off the sur face of the sample For samples such as thin films microscope slides fibers or other materials 1 Place the material on the block on the side opposite tha...

Page 178: ...e 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 vol...

Page 179: ...xcitation light to the micro scope s stage and emission light to the emission monochromator plus a sam ple compartment adapter to direct light in and out of the FluoroMax Microscope Interface Fiber op...

Page 180: ...150 W xenon lamp delivers light from 240 nm to 850 nm for sample excitation The lamp has an approximate life of 1500 hours and is ozone free Caution This lamp emits intense light and heat and contains...

Page 181: ...004 Peltier Drive Instead of messy fluids the Peltier device heats and cools the sample thermoelectrically and fast The temperature range is 10 C to 120 C To prevent condensation of moisture on chille...

Page 182: ...enon lamp installed in the FluoroMax 3 housing No manual adjustments are necessary The FM 2005 provides FluorEssence controlled flashlamp delays number of flashes per data point signal gating and acqu...

Page 183: ...a stationary optical beam and fluorescence measurements are collected with top reading geometry Thus any titer plates even disposable ones may be used Up to 384 well plates may be inserted into the Mi...

Page 184: ...uorometers such as the FluoroMax 3 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...

Page 185: ...dditions of small volumes via a syringe or pipette to the sample cell without removing the lid of the sample compartment With the injec tor in place a lock tight seal is achieved prevented both light...

Page 186: ...sphere has an internal diameter of 4 10 cm Of special interest is the measurement of photoluminescence quantum yields of such materials especially for thin solid films Measurement of quantum yields o...

Page 187: ...sample economy The SFA series stopped flow accessory permits observation of the reaction rate of two reactants forced through a mixing chamber and into an observation cell The reactant solutions are...

Page 188: ...strips away the noise rather than adding noise to your signal the way an analog system does The pulsed source used in the TCSPC upgrade is our NanoLED solid state pulsed di ode which can be ordered f...

Page 189: ...n cause severe burns 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 tube...

Page 190: ...r manually pushing a button on a trigger release cable Multiple trigger events are recorded and stored with the as sociated data file A TTL trigger output also is provided for activating external de v...

Page 191: ...ers mount the FM 2007 Windows on the inside of the sample compart ment Go directly to step 2a FM 2007 Windows for the FluoroMax 3 sample compartment Installing the windows 1 Prepare the instrument a R...

Page 192: ...tering the sample compartment 3 Install purge port a Remove plastic original plug using a 3 16 Allen key b Screw in manually the new metal purge port c Tighten with a 7 16 wrench d Remove the plastic...

Page 193: ...the total light intensity The measurement of anisotropy can provide insight into molecular size and shape as well as the environment that sur rounds it Autopolarizers An automated device to hold and p...

Page 194: ...outine Then an as say may be completed with the measurements based on concentra tion Corrected emission scan An emission scan that has been corrected for the wavelength re sponse of the emission monoc...

Page 195: ...rder to run the Dark Offset correction Datafile A file used to store spectral data constant wavelength analysis data or other recorded data In FluorEssence the most common datafile is the spectral fil...

Page 196: ...lli mating mirror the reflection grating blazed at 330 nm and a focus ing mirror with slit apertures at the entrance and exit An excitation shutter is located directly after the excitation exit slit t...

Page 197: ...hs and their fluorescence lifetimes Fluorophores are used to provide information on concentration size shape and binding in a particular medium Good fluorophores are stable over wide pH and temperatur...

Page 198: ...ion from the excited singlet state to the excited triplet state before returning to the ground state This transition in volves a change of spin that is quantum mechanically forbidden giv ing a much lo...

Page 199: ...n SPC extension Multifiles may be used in their entirety in FluorEssence as 3D files or they may be split up into individual two dimensional spectra using multi file utilities Multigroup scan This exp...

Page 200: ...tion P A measurement of the fluorescence polarization of a sample defined as the linear polarizer s component s intensity divided by the natural light intensity The measurement of polarization provide...

Page 201: ...e compartment Use this accessory to run up to four samples at one time for a small assay or to run blanks with the samples simultaneously Automated sample changers are thermostatted and possess magnet...

Page 202: ...tware The water Raman band is scanned with 350 nm excitation and the 397 nm peak is assigned as such in the soft ware for the emission monochromators Spectral correction The removal of the wavelength...

Page 203: ...unt Throughput The amount of light that passes through the spectrofluorometer for a particular measurement The throughput usually is measured as the counts per second measured on the water Raman band...

Page 204: ...ve a large change in energy and typically is a non radiative transition In larger electronic transitions such as fluorescence a molecule drops from the lowest vibrational level of the excited state to...

Page 205: ...R F Chen et al Biochemical Fluorescence Concepts Vol I II 1964 1970 J N Demas Excited State Lifetime Measurements Academic Press New York 1983 Enrico Gratton David M Jameson and Robert D Hall Multifre...

Page 206: ...ts and Their Usage in Spectrochemical Analysis VI Molecular Luminescence Spectroscopy Pure App Chem 53 1953 1981 J N Miller ed Standardization Fluorescence Spectrometry Techniques in Visible and Ultra...

Page 207: ...L Wehry ed Modern Fluorescence Spectroscopy Vol 1 4 Plenum Press New York 1975 1981 C E White and R J Argauer Fluorescence Analysis A Practical Approach Marcel Dekker New York 1970 J D Winefordner S...

Page 208: ...FluoroMax 3 v 3 1 6 Mar 2006 Bibliography 14 4...

Page 209: ...0 4 2 Performance Criteria A EN 61000 4 4 2kV Performance Criteria A Supplementary Information The product herewith complies with the requirements of the Low Voltage Directive 73 23 EEC as amended by...

Page 210: ...FluoroMax 3 v 3 1 6 Mar 2006 Declaration of Conformity 15 2...

Page 211: ...4 9 4 400144 1 5 5 53057 1 5 8 81038 1 5 81106 1 5 9 98015 1 5 98020 1 5 A About FluorEssence window 7 11 About FluorEssence 7 11 Absorption Transmission accessory 12 3 Accessories icon 10 4 10 7 8 1...

Page 212: ...nced Setup 5 15 8 10 Advanced button 10 6 10 8 Alconox 5 1 alignment 10 5 8 10 10 10 13 10 21 Allen wrenches 1 5 6 1 6 4 6 5 6 7 12 32 Analysis 5 13 5 18 9 10 menu 8 4 9 2 anisotropy 10 1 4 10 8 10 10...

Page 213: ...12 4 14 5 6 5 9 5 12 5 14 6 9 11 7 1 8 1 2 8 5 8 11 12 9 2 9 4 11 1 2 12 4 12 7 Detector 5 16 column 8 11 Detectors icon 5 7 5 10 5 16 7 11 8 8 8 12 9 8 Dewar flask 12 6 dimensions 11 2 disclaimer 0 3...

Page 214: ...22 23 fluorophore 10 1 10 4 10 14 FM 2003 12 12 12 14 FM 2005 12 22 FM 2007 12 31 FM 2013 12 28 focus xenon lamp 6 7 Formula box 8 8 Formulas table 5 17 8 8 10 18 Four Position Thermostatted Cell Hold...

Page 215: ...ifetime 10 1 liquid nitrogen 12 6 Liquid Nitrogen Dewar Assembly 12 6 Lorentzian distribution 5 11 Ludox 10 6 10 8 9 10 11 10 21 M M 10 13 mA 3 5 3 12 macromolecules 10 1 magic angle 10 4 10 14 magnet...

Page 216: ...2 4 5 11 5 12 mode 2 4 photodiode 2 2 2 4 5 3 4 6 11 8 4 5 11 2 photomultiplier tube 2 2 2 5 6 11 8 4 9 2 10 7 photon counting module 2 5 photon pulse pile up 2 5 photoselection 10 1 10 4 pinned colla...

Page 217: ...ard 1 5 training 1 1 Sample Cell 12 8 sample changer 3 3 3 10 6 10 7 1 7 2 sample compartment 2 2 2 4 3 4 3 10 4 3 4 12 6 10 8 5 10 6 10 8 11 3 12 9 11 12 19 12 21 12 25 26 12 28 29 lid 3 4 4 3 window...

Page 218: ...0 System Configuration window 5 15 8 10 system controller 2 1 System Initialization Process window 1 10 T T 10 3 10 10 10 21 TbCl3 aq 9 5 9 7 T box 12 9 12 24 tcorrect 5 16 TCSPC upgrade 12 28 technic...

Page 219: ...FluoroMax 3 sample compartment 12 31 wrenches Allen 1 5 X X 10 13 xcorrect 5 14 5 16 5 18 8 1 8 8 10 xenon lamp 0 10 11 1 1 2 2 2 5 2 7 3 1 4 3 10 3 16 5 14 6 1 3 6 5 7 6 9 7 1 9 12 11 1 2 12 20 12 22...

Page 220: ...FluoroMax 3 v 3 1 25 Apr 2006 Index 16 10...

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