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MRC Spectro-97 Series Operation Manual

Fluorescent spectrophotometers
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Fluorescent Spectrophotometers
SPECTRO-97 SERIES
Operation Manual
PLEASE READ THIS MANUAL CAREFULLY BEFORE OPERATION
Hagavish st. Israel 58817 Tel: 972 3 5595252, Fax: 972 3 5594529
mrc@mrclab.com
MRC.4.18

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  • Page 1 Fluorescent Spectrophotometers SPECTRO-97 SERIES Operation Manual PLEASE READ THIS MANUAL CAREFULLY BEFORE OPERATION Hagavish st. Israel 58817 Tel: 972 3 5595252, Fax: 972 3 5594529 mrc@mrclab.com MRC.4.18...

  • Page 2: Preface

    Preface Thank you for purchasing the Fluorospectro- photometer of MRC Ltd. Please read this manual carefully before installation first time SPECTRO-97 using series Fluorospectrophotometer. Fluorometry is a high sensitive, high selective and modern analysis method. Fluorometry provides excitation spectrum, emission spectrum, including...

  • Page 3: Special Statement

    Special Statement Please read this manual carefully before installation or operation. The company will not take responsible for any trouble or damage due to unproper use. The company has the final interpretation of this manual. Modifications of the manual due to improvements of the instrument will not be announced.

  • Page 4: Safety Signs

    Safety Signs This sign indicates important part of the instrument. Please follow instructions. Please read and operate according to the instructions following. This sign indicates possible electric harm. Required by a professionally qualified personnel according to the appropriate procedures. (This label is attached on the power switch and trigger.) This sign indicates heat on surface.

  • Page 5: Precautions

    Precautions 1. The instrument is suitable for analysis in laboratory. If the instrument is needed outside the lab, please make the field work environment meets the environmental requirements of the laboratory. 2. Please use our original packaging when moving the instrument.
  • Page 6 machine or instrument error, shut down the instrument immediately. When the software is not operating properly, Start Task Manager to end the "NeoLG.exe" process, then restart the software and the instrument. 10. DO NOT loose the screws in the monochromator. Keep the environment clean.

  • Page 7: Table Of Contents

    Menu Preface ............................... 1 Special Statement ..........................2 Safety Signs ............................3 Precautions ............................4 P A R T I ：U s e r ’ s G u i d e ................ 9 1 Appearance & Performance ......................10 1.1 Appearance ........................10 1.1.1 Body ........................
  • Page 8 4.2.1 Maintenance ......................24 4.2.2 Light Source Replacement ..................24 Part II：Software Manual ....................... 25 5 Software Installation ........................26 5.1 Requirements ........................26 5.1.1 Hardware Requirements ..................26 5.1.2 System Requirements ..................... 26 5.2 Install SPECTRO-97 Software ..................26 6 How To Use The Software ......................
  • Page 9 Appendix VII： Derivative Operation on Spectrum............110 Appendix VIII： Smoothing ....................112 F8.1 Savitzky–Golay ..................... 112 F8.2 Mean ........................112 F8.3 Median ........................112 Appendix IX： Phosphorescence ..................113 F9.1 Theory ........................113 F9.2 Phosphorescence Wavelength Scan ..............113 F9.3 Phosphorescence Time Scan ................. 114 F9.4 Applications ......................

  • Page 10: Part I ：U S E R ' S G U I D E

    P A R T I ：U s e r ’s G u i d e...

  • Page 11: Appearance & Performance

    1 Appearance & Performance Appearance 1.1.1 Body Top Air-vent Panel Left Air-vent Pilot Light Power Switch Sample Compartment Cover Fig.1-1 Body Top Air-vent： Vent above the xenom light for air circulating. Please don’t touch. Left Air-vent：Vent beside the xenom light for air circulating. Power Switch：Please turn to chap.

  • Page 12: Interface

    1.1.2 Interface Fig.1-2 Side View USB Port RS232 Serial Port Fig.1-3 Interface USB Port：Link to PC with a USB cable. RS232 Serial Port：For debugging.

  • Page 13: Power & Switches

    1.1.3 Power & Switches Fig.1-4 Side View Main Power Power Switch Plug-in Main Power Xenom Fuse Light Xenom Light Fig.1-5 Power & Switches Switch Power Plug-in：For connecting power cable. Xenom Light Fuse：For xenom light fuse. Xenom Light Switch：Turn on/off the xenom light. Main Power Fuse：For main power fuse.

  • Page 14: Sample Compartment

    1.1.4 Sample Compartment Compartment Cover Sample Hold 图 1-4F97XP 荧光分光光度计主机侧图 Fig.1-6 Sample Compartment Sample Compartment：Sample hold inside. Sample Hold：For fixing sample pool. Fig.1-7 Quartz Fluorescence Sample Pool...

  • Page 15: Mode Of Operation

    1.2 Mode of Operation 1.2.1 Signal Processing & Control System Excitation Opitcal Monitor Gate Excitation Sample Xenon Light monochromator Photomultiplier Emission Controlled Xenon Light monochromator Processor Wavelengt Power Negative Pre-amplifier High Voltage Fig.1-8 Signal Processing & Control System...

  • Page 16: Light Path

    1.2.2 Light Path Xenon Light Aspherical Mirror Photomultiplier Concav Slit Mirror3 Mirror4 Slit Slit Lens Photocell Gratin Transflective Mirror Mirror1 Lens Sampl Lens Slit Mirror2 Fig.1-9 Light Path...

  • Page 17: Modes For Measurement

    AD module detection, signal gain detection, motion parts detection etc. Test result will be displayed on the screen and easy for users to find problems. 1.4Performance Tab.1-1 Performance of SPECTRO-97 series Item Content 150W xenon lamp（Hamamatsu） Excitation Source...
  • Page 18 SPECTRO-97XP/SPECTRO-97Pro：2nm、5nm、10nm、20nm Emission Slit SPECTRO-97：10nm SPECTRO-97XP：±0.4nm Wavelength Accuracy SPECTRO-97/Pro SPECTRO-97：±1.0nm SPECTRO-97XP：≤0.2nm Wavelength Repeatability SPECTRO-97/SPECTRO-97Pro：≤0.5nm SPECTRO-97XP:Raman peak of water(P-P):S/N≥200(10nm Slit） Signal-to-Noise Ratio SPECTRO-97/97Pro:Raman peak of water(P-P):S/N≥150(10nm Slit） SPECTRO-97XP：≤5×10 g/ml（Quinine Sulfate Solution） Limit SPECTRO-97/97Pro：≤1×10 g/ml（Quinine Sulfate Solution） Linearity γ≥0.995 Peak Repeatability ≤1.5% Zero Drift：±0.3 Stability(10min) Value Limit：±1.5% Wavelength Scan...

  • Page 19: Booting & Shutting Down

    2 Booting & Shutting Down 2.1 Booting Status 2.1.1 Booting Put the instrument on steady platform. Make sure the main power and light power switch are off(switch on 0). Plug in the power line. There is a certain probability that the high voltage trigger of the Xenon light power will affect other electrical equipment around.

  • Page 20: Multi-Instruments Booting

    2.1.3 Multi-instruments Booting When using multiple instruments, please turn on all the xenon lights first, then turn on all the main power to reduce the influence of the high trigger voltage. 2.1.4 Initialization Initialization status will be displayed on computer software as Fig.1.3.2-1. 2.2 Power Off When connecting to PC, close the software first;...

  • Page 21: Installation

    3 Installation 3.1Environment The instrument is suitable for analysis in laboratory environment. For its work with computers, so need to meet the following working condition. Laboratory Environment 3.1.1 Temperature 10～30 ℃, humidity under 85%. Avoid corrosive gas and the organic and inorganic gases which are absorptive within the range of ultraviolet.
  • Page 22 Make sure the instrument and all accessories are correct according to the package list. Please contact us if there is any mistake. Table.3-1 Fluorescence spectrophotometer package list SPECTRO-97 series instrument One piece Fluorescence spectrophotometer One set software Power Cable...

  • Page 23: Installation

    3.3 Installation 3.3.1 Cleaning Remove the tape and clean the surface. 3.3.2 Check the Power Source Make sure the instrument power supply voltage and area voltage are correct. 3.3.3 Plug In Put the instrument on a stable work table about 10 cm away from wall. Plug in the power cable to the lab power.

  • Page 24: Wavelength Test

    ！ Attention： ①The energy of Raman is weak, so the measurement is easy to be interfered. If the result looks bad, please test again. ②In order to protect the PMT tube, when the gain is high(above 6), please don’t put high energy light source into the sample cell. ③Setting the gain will affect the fluorescent zero.

  • Page 25: Maintenance

    4 Maintenance 4.1Routine maintenance Always check whether it meets the requirements of the work environment in daily use. Keep the vents functioning while the instrument is on. ATTENTION: High temperature vents. Keep distance. Keep the instrument clean. Add a dust cover when not in use. Use water to clean the instrument appearance.

  • Page 26: Part Ii：software Manual

    Part II：Software Manual...

  • Page 27: Software Installation

    5 Software Installation Before reading this section, please read Part I carefully. Windows XP system is recommended. In order to run the software properly in Windows 7, please run the ． ．． ． ． ． ． ． ．． ． ．． ． ．． ． ． ． ． ． ． ．． ． ． ． ． ． ． ． ． ． ． ． ． ． ． ． ．． ．． ． ． ． ． ．． ． ．． ． ． software in administrator account.
  • Page 28 If the installation does not start, open X:\SETUP.EXE(X is the CD drive) as Fig.5-1.This manual is based on SPECTRO-97 Pro. Fig.5-1 Installation Click “Next” in Fig.5-1, input user name and company name. Fig.5-2 Information Click “Next” in Fig.5-2 to set install path. Default path is as in Fig.5-3: C:\Program...
  • Page 29 File\MRC\SPECTRO-97Pro Fluorescence spectrophotometer. Click “Change” to change the path. Click “Back” to go back, click “Cancel” to cancel. Fig.5-3 Install path Click “Next” to set account limit and shortcut path in fig.5-4. Fig.5-4 Shortcut path & account limit...
  • Page 30 Fig.5-5 Installation information Click “Next” if all the information is correct to start installation. Fig.5-6 Installing...
  • Page 31 Wait for the installation. When the installation is complete, a notice will pop out as fig.5-7. Then it is time to connect the instrument. Fig.5-7 Installation complete ATTENTION: CD serial number should be the same as the serial number of the instrument. Otherwise, the software does not work properly.

  • Page 32: How To Use The Software

    6 How to Use the Software 6.1 Before Use 6.1.1 Connect to PC The instrument connects to the computer via USB cable, the computer will automatically install the driver the first time connected, and please run software after the driver is successfully installed. 6.1.2 Link Procedure 1.

  • Page 33: Functions

    6.2 Functions 6.2.1 Measurement Modes There are 5 measurement modes: Wavelength Scan: （1） Shows the excitation spectra of the sample. （2） Shows the emission spectra of the sample. （3） Phosphorescence wavelength scan is available. （4） Supports automatic repeat scan. （5） Supports data printout. Time Scan: （1）...

  • Page 34: Interface

    6.2.2 Interface 6.2.2.1 Interface Modules Menu Tool bar Docu ments Info. Status Spectrum Information Fig.6-2 Interface modules...
  • Page 35 6.2.2.2 Modules & Functions 1. Menu & Tools Fig.6-3 Menu & Upper Tool bar Fig.6-4 Right Tool bar Provides instrument controls and settings. Tool bar are shortcuts for common features. 2. Document Browser Document Brower shows files saved in Wavelength Scan, Time Scan and Quantitative Analysis mode.
  • Page 36 Way of sorted files can be changed. Fig.6-7 File menu 3. Information Window Information window shows current data and info of the instrument.Including current fluorescence value, excitation wavelength, emission wavelength, excitation and emission slit, gain, the response time and optical gate condition. Double click to modify instrument parameters.
  • Page 37 6.2.2.3 Tool Bar Icon Function New Measurement Open Spectrum Save Spectrum Print Spectrum Show/Hide Status Show/Hide Spectrum Information Activate 2D Window Activate 3D Window Activate Quantitative Analysis Window Back to original coordinate Auto coordinate Y-axis enlarge 2 times Y-axis reduce 2 times Get/Cancel Axis Data Zoom In/Out Show/Hide Peaks...
  • Page 38 Start/Stop Open/Close Optical Gate Set Wavelength Run/Cancel Zero Adj. Spectrum Properties Print Data Peak Threshold Setting Spectrum Smoothing Spectrum derivation Spectrums Calculation Spectrums Comparison Table.6-1 Tool Bar Icons...

  • Page 39: Software Operation

    6.3 Software Operation 6.3.1 Wavelength Scan Wavelength Scan Flow chart Power On Online Run Software New Measurement Choose “Wavelength Scan” Set Parameters Confirm Parameters Start Scan Input File Name Confirm Scan Scan Complete Print Data Fig.6-9 Wavelength Scan Flow chart...
  • Page 40 6.3.1.1 New Measurement Create a new measurement. Select “Files”->“Create Method” or click to enter Create Method Window. Measurement Summary: Fig.6-10 Measurement Summary 1） Measure Mode: Choose “Wavelength Scan”. 2） Operator: Input operator’s name. 3） Instrument: The model of the connected instrument is indicated. 4）...
  • Page 41 Instrument: Fig.6-11 Scan Settings 1） Scan Mode: Excitation & Emission. A. In excitation mode, X axis is excitation wavelength, Y axis is fluorescence value. The instrument will do an excitation wavelength scan under a fixed emission wavelength. B. In emission mode, X axis is emission wavelength, Y axis is fluorescence value.
  • Page 42 measurement, it is the time until the start of the first measurement. 9） Excitation Slit: Set excitation slit.(SPECTRO-97 is fixed to 10nm) 10） Emission Slit: Set emission slit.(SPECTRO-97 is fixed to 10nm) 11） Gain(PMT): Set gain level by changing the PMT negative high voltage. 12）...
  • Page 43 Processing： Fig.6-13 Spectrum Processing 1） Processing choices : A list of data processing (Savitsky-Golay smooth, Mean smooth, Median smooth, Derivative) is shown. Select a data processing item, and click the rightward pointing arrow key between the Processing choices and Processing steps display fields. Then, the selected method appears in the Processing steps field.
  • Page 44 ： Report Fig.6-14 Data Printout 1） Output: Print Report or Save as Microsoft（R）Excel file. 2） Output options : Choose the printout data. Check the content in “Properties” button on the left after the scan. 3） Add Data: When “Spectrum Data” is checked, you can choose data section to printout.
  • Page 45 6） Click “Open” to open a saved method. It’s a *.FMTD file. 7） Click “Save” to save current settings. 6.3.1.2 Run Wavelength Scan Wavelength scan procedure: Standby->Ready->Start Wavelength Scan ->Move to Excitation(Emission) Wavelength -> Standby. File Name Click button to start a measurement. Input a file name or use system time as file name.
  • Page 46 6.3.1.3 Spectrum The instrument will do wavelength scan with all the parameters. Spectrum will be displayed in the spectrum window. The spectrum file will be automatically saved in the file browser window. Click to see details of the spectrum. Icons Function Reset Original Coordinate.
  • Page 47 Spectrum Smoothing Spectrum derivation Spectrums Calculation Spectrums Comparison Table.6-2 Functions for Spectrums 6.3.1.4 Printout Icon Functions Spectrum Properties Print Data Table.6-3 Functions for Printout...

  • Page 48: Time Scan

    6.3.2 Time Scan Time Scan Flow Chart Power On Initialization Open software Create a measurement Select “Time Scan” Set Parameters Confirm Parameters Start Scanning Input file name Confirm Scan Scan Complete Printout Fig.6-18 Time Scan Flow Chart...
  • Page 49 6.3.2.1 Create a Measurement Click “Files”->“Create Measurement” or click to create a new measurement. General: Fig.6-19 General Measurement: Choose “Time Scan”. Operator: Input operator’s name. Instrument : The model of the connected instrument is indicated. Comment: Enter a description or notes on measuring conditions.
  • Page 50 Instrument Fig.6-20 Scan Settings Scan Mode: Fluorescence and phosphorescence mode available* A. The instrument will do Fluorescence scan in Fluorescence mode. B. The instrument will do phosphorescence scan in phosphorescence mode. A phosphorescence excitation time input window will be activate before the scan.* Phosphorescence excitation time: Set the excitation time in phosphorescence mode.* Emission Wavelength: Input emission wavelength (200nm-900nm).
  • Page 51 Monitor: Fig.6-21 Data Display 1、 Y Axis: Input the min point and max point of Y axis. 2、 Auto Adjust Y Axis: Y axis will automatically set by spectrum data. Processing： Fig.6-22 Spectrum Processing 2） Processing choices : A list of data processing (Savitsky-Golay smooth, Mean smooth, Median smooth, Derivative) is shown.
  • Page 52 appears in the Processing steps field. 3） Processing steps : The processing sequence is displayed. To delete a processing method, first select the method, and then click the leftward-pointing arrow key between the Processing choices and processing steps display fields. Then, the selected method disappears from the Processing steps field.
  • Page 53 6.3.2.2 Run Time Scan Time scan procedure: Standby->Ready->Start Time Scan -> Standby. Save File: Click to start scan.Input file name in the pop out window or use system time as file name. Stop Scan: Click to stop the scan. 6.3.2.3 Spectrum Processing The instrument will do Time Scan with all the parameters.
  • Page 54 Start/Stop Open/Close Optical Gate Set Wavelength Run/Cancel Zero Adj. Spectrum Smoothing Spectrum derivation Spectrums Calculation Spectrums Comparison Table.6-4 Functions for Spectrum 6.3.2.4 Printout Data Icon Functions Spectrum Properties Print Data Table.6-5 Functions for Printout...

  • Page 55: Quantitative Analysis

    6.3.3 Quantitative Analysis Quantitative Analysis flow chart Power On Initialization Open Software Create a measurement Choose Quanti-Analysis Set Parameters Confirm Parameters Get fluorescence Input file name values of standard sample Build regression curve Get unknown sample value Printout Data Complete Fig.6-24 Quantitative-Analysis Flow Chart...
  • Page 56 6.3.3.1 Create a Measurement Click “Files”->“Create Measurement” or click to create a new measurement. General: Fig.6-25 General Measurement: Choose “Time Scan”. Operator: Input operator’s name. Instrument : The model of the connected instrument is indicated. Comment: Enter a description or notes on measuring conditions.
  • Page 57 Quantitative analysis Fig.6-26 Quantitative analysis 1、 QA Options : A. Type : Select the method of creating a calibration curve. Wavelength only. B. Number of wavelengths: the number of wavelengths used in Quantitative analysis. Available from 1 to 3.(Details in appendix 4) C.
  • Page 58 Instrument Fig.6-27 Instrument Data Mode: Fluorescence mode. Wavelength Mode: “Fixed Excitation Wavelength” and “Fixed Emission Wavelength”. The Wavelength 1, 2, 3 are related to the number of wavelengths in tab “Quantitative Parameters”. A. When the number of wavelengths is 1, it would be the same whether in “Fixed Excitation Wavelength”...
  • Page 59 2(200-900nm). Wavelength 3: Input Excitation Wavelength 3 and Emission Wavelength 3(200-900nm). Pre-Excitation Time: The instrument allows light to illuminate the sample pre-excitation time before measurement. During this time the instrument will not do fluorescence measurement. The time of this part is to stabilize the excitation light and the sample.
  • Page 60 Standards Fig.6-28 Standards Sample table: Sample table gives a list of standards for sample measurement or calibration curve preparation. This table contains the items listed below. Lines: Number of Standard Samples. 1) “Update”: By clicking this button, sample numbers are set by the entered number of samples.
  • Page 61 Report Fig.6-29 Report tab Output: Print Data or Save as Microsoft（R）Excel file. Output options : Choose the printout data. Check the content in “Properties” button on the left after the scan. Clear Data: Clear current data section. Click “Defaults” to reset the settings to default. Click “Open”...
  • Page 62 6.3.3.2 Quantitative-Analysis Interface Click ”OK” in the last step to enter Quantitative-Analysis interface. Standard Menu & Toolbar Sample File Browser Curve Unknown Sample Property Information Status Fig.6-30 Quantitative-Analysis Interface Menu, Toolbar, File Browser, Information and Status are the same as Chap.6.2.2.2.
  • Page 63 6.3.3.3 Conducting Measurement In the Standards window, you can modify sample name, description and concentration; add or delete sample; check the fluorescence value of samples. Fig.6-32 Standards Window Modify sample name, description and concentration: Click “Edit”, then double click in the table to modify the content you want. Then click “OK” to confirm. Measure a fluorescence value: Click to select a sample in the table, then click “Start”...
  • Page 64 Fig.6-33 Regression Curve 6.3.3.4 Measurement of the sample When the regression curve is created, you can start measuring the sample. Operate the test sample in samples window as Fig.6-34. There are functions in the sample window: Measure, Modify, Delete and Clear. Fig.6-34 Sample Window 1、...
  • Page 65 3、 Delete sample: Click “Edit” button, then click the line you want to delete and click “delete” button to delete the sample. Click “OK” to confirm the modification and back to test sample window. 4、 Clear sample list: Click “Edit” button, then click “Clear” button. Click “OK” to confirm the modification and back to test sample window.

  • Page 66: Scan

    6.3.4 3D-Scan 3D fluorescence spectrum has a characteristic of the "fingerprint". We can get all kinds of information on the sample map through the analysis of three-dimensional fluorescence spectrum, Including the excitation wavelength of Rayleigh scattering and secondary scattering, Raman shift samples, the optimal excitation wavelength, the best fluorescence peak wavelength.
  • Page 67 Fig.6-35 3D Scan Procedure 6.3.4.1 Create a method First of all, create a method. Click “File”“New Method” or click to enter the window below in Fig.6-36 to create a method. General: Fig.6-36 General tab Measure Mode: Choose “3D Scan”. Operator: Input operator’s name. Instrument : The model of the connected instrument is indicated.
  • Page 68 Instrument: Fig.6-37 Instrument tab 1） Scan Mode: Fluorescence mode. 2） Excitation Start Wavelength: Input excitation wavelength (200nm-900nm). 3） Excitation End Wavelength: Input excitation wavelength (200nm-900nm). 4） Excitation Sampling Interval： In the 3-dimensional measurement mode, emission spectrum measurement is repeated while shifting the excitation wavelength.
  • Page 69 measurement for suppressing sample deterioration due to the energy of excitation beam and opened when measurement starts. When you put a check mark at the head, the shutter will close and open at start of measurement. The shutter will close again when measuring wavelength begins returning to the start wavelength after measurement in the wavelength scan mode.
  • Page 70 smooth", "smooth mean", "median Smooth" and "derivative" are available. 1) Select a method in “available” window, then press to move the method to “selected” window. 2) Select a method in “selected” window, then press to move the method to “available” window. 2、...
  • Page 71 6.3.4.2 Run Wavelength Scan The status will be: “Standby” “Ready to scan” “Moving excitation wavelength” “Moving emission wavelength” “Start wavelength scan” “Move emission wavelength” “Standby”. A. File Name Click the start measurement button , there will be a popout window to input a file name.
  • Page 72 6.3.4.3 3D Spectrum Processing A. Spectrum Display When the scan is running, spectrum data will be displayed in the spectrum window as 2D figure. The spectrum will be displayed in the spectrum window as 3D figure after scan.The spectrum file will be automatically saved. Tool bar 3D window Fig.6-41 3D Scan Interface...
  • Page 73 C. Spectrum Window 3D Spectrum Tab: 3-D Scan data can be displayed in 3D view. The spectrum is a composition of emission wavelength axis, the excitation wavelength axis fluorescence value axis, contour and color notification. Tabs Buttons Fig.6-42 3D Spectrum Tab Click these tabs to show 3D view, Contour view and 3D contour view.
  • Page 74 spectrum. Click to change the depth. Hold left key and move the mouse to adjust the depth of the spectrum. Hold right key to move the spectrum. Tab.6-8 Spectrum Controls Contour Tab: Shows the contour line in 2D view as Fig.6-43. Contour lines Emission spectrum Excitation spectrum...
  • Page 75 3D Contour Tab: Shows all the 2D spectrums in 3D view as Fig.6-44. Tabs Buttons Fig.6-44 3D Contour Tab 6.3.4.4 Printout Icon Function Shows the spectrum information. Select printout format and content. Click to printout data. Tab.6-9 Printout Functions...

  • Page 76: Synchronous Scan

    6.3.5 Synchronous Scan Synchronous Scan flow chart Boot Initialization Run software Create a measurement Choose Synchronous scan Set parameters Confirm parameters Start scanning Input file name Confirm file name Scan complete Printout Fig.6-45 Synchronous Scan flow chart...
  • Page 77 6.3.5.1 Create a measurement Click “File”“Create measurement” or click icon to create a measurement. General Fig.6-46 General 1、 Measurement: Choose “Synchronous scan” 2、 Operator: Input operator name. 3、 Instrument: The model of the connected instrument. 4、 Comments: Enter a description or notes on measuring conditions.
  • Page 78 Instrument Fig.6-47 Instrument Tab 1、 Scan mode: Constant Wavelength Synchronous Fluorescence (CWSF) & Constant Energy Synchronous Fluorescence (CESF). Constant Wavelength Synchronous Fluorescence: Excitation and emission sides scan at the same time in a fixed wavelength difference. Constant Energy Synchronous Fluorescence: Excitation and emission sides scan at the same time in a fixed energy difference.
  • Page 79 6、 Scan Interval: Shows data sampling interval according to the scan speed. 7、 Delay: After pressing the Measure button, measurement is started following the delay time set here. It is used for temperature stabilization, etc. 8、 Excitation Slit ： Select a slit width for the excitation side. （SPECTRO-97 is fixed to 10nm）...
  • Page 80 Monitor Fig.6-48 Monitor Tab 1、 Y Axis: Enter the start point and end point of Y axis. 2、 Auto Y Axis: Automatically adjust Y axis when a check mark on. Processing Fig.6-49 Processing Tab 1、 Processing choices: Used for obtaining an average spectrum in repeat measurement.
  • Page 81 2) The processing sequence is displayed. To delete a processing method, first select the method, and then click the leftward-pointing arrow key between the Processing choices and Processing steps display fields. Then, the selected method disappears from the Processing steps field. 2、...
  • Page 82 6.3.5.2 Run Synchronous Scan There is a changing process running synchronous scan: “Standby”“Ready”“Start wavelength scan”“Moving excitation and emission wavelength”“Standby” 一、 File Name Click to enter a file name. Click OK to start the scan. Keep it blank to use the current system time as file name.
  • Page 83 Start/Stop Open/Close Optical Gate Set Wavelength Run/Cancel Zero Adj. Spectrum Smoothing Spectrum derivation Spectrums Calculation Spectrums Comparison Tab.6-10 Toolbar 6.3.5.4 Printout Data Icon Functions Spectrum Properties Print Data Tab.6-11 Toolbar...

  • Page 84: More Convenient Operating Methods

    6.3.6 MORE CONVENIENT OPERATING METHODS 6.3.6.1 Arithmetic Operation between Spectra Arithmetic Operation Procedures Boot Initialization Run software Scan to get a spectrum Enter calculation interface Choose spectrum type Choose target Choose calculate method Calculate Save & printout Fig.6-51 Flowchart...
  • Page 85 Arithmetic Operation includes addition, subtraction, multiplication and division among the same type of spectrum. Click on the right to enter the spectrum compare window as Fig.6-52. Target spectrum 2 Target spectrum Function Result Spectrum files 1 Spectrum type Spectrum files 2 Spectrum type Fig.6-52Arithmetic Operation Arithmetic Operation Interface:...
  • Page 86 6.3.6.2 Overlaid Display of Spectra Overlaid Display of Spectra Boot Initialization Run software Scan to get spectrum Enter spectrum interface Choose spectrum type Choose target spectrum Enter overlaid interface View results Save & Printout Fig.6-53 Flowchart...
  • Page 87 This function is convenient for comparison of two or more spectra of the same type. Click to enter the overlaid interface as in Fig.6-54. Type Files Fig.6-54Overlaid Display Interface Interface: Type: Choose the spectrum type. Excitation spectrum, emission spectrum, time scan spectrum and synchronization scan spectrum are available. Files: Shows the spectrum files of the type above.
  • Page 88 6.3.6.3 Detection of Spectral Peaks Detection of Spectral Peaks Procedure Boot Initialization Run software Scan to get spectrum Enter Peak finding Set threshold Show/Hide peak Coordinates Save & Printout Fig.6-56 Flowchart The spectral peaks and valleys are detectable automatically. Select the Find Peaks command from the Data menu or click button, and a window as in Fig.
  • Page 89 6.3.6.4 Smoothing of Spectrum Smoothing of Spectrum Procedure Boot Initialization Run software Scan to get spectrum Enter Smoothing Set function type & parameters View results Save & printout Fig.6-58 Flowchart Smoothing is to reduce the noise of spectrum. Click to set smoothing parameters in Fig.6-59. Select function type, Smoothing order, Number of points and Number of times then click “OK”...
  • Page 90 Type Smoothing order Number of points Number of times Savitsky-Go The highest power Set the number of points number of the polynomial used smoothing operations. calculation.(odd number) Mean --------------- Set the number of points number to be used in calculation. smoothing operations.
  • Page 91 6.3.6.5 Derivative Operation on Spectrum Derivative Operation Procedure Boot Initialization Run software Scan to get spectrum Derivative Operation Set order View result Save & printout Fig.6-60 Flowchart Derivative Operation on Spectrum is to enhance the resolution of peaks. Derivation can distinguish various disturbances affecting the shape of the spectrum peaks.

  • Page 92: Instrument Settings

    6.3.6.6 Instrument Settings This is to change the save path, file name and spectrum type. Click “Settings” “Options” to open settings window in Fig.6-62. A. General tab： Fig.6-62 General tab Default Method: Instrument use default parameters. Specified method: Instrument uses specified parameters. Default Directory: Instrument uses default path.
  • Page 93 Add Subhead when repeated scan: Automatically add a number suffix to file names. From: Set the start number. Places: Set number digits. Reset after rename: Auto reset number when using another name. C. Spectrum Tab Fig.6-64 Spectrum Tab (Curve & Coordinates) Fig.6-65 Spectrum Tab (Background Setup) Curve &...
  • Page 94 width in “Spectrum information” window. Grid color: Sets the table color in “Spectrum information” window. Grid width: Sets the curve width in “Spectrum information” window. Grid style: Sets table style as solid, dash, dot, dashdot, dashdotdot and clear in “Spectrum” tab. Panel color: Sets the background color in “Spectrum information”...
  • Page 95 6.3.6.7 Rename & Delete Spectrum Files Right click on a file in file browser. You can delete or rename in the pop-out menu in Fig.6-66. Fig.6-66 Edit File MAKE SURE YOU WANT TO DELETE THE FILE BEFORE YOU ！ DELETE IT. 6.3.6.7 Reorder Files Sort by menu: There are 4 ways to sort files: “Name in ASC order”, “Name in DESC order”, “Time in ASC order”...

  • Page 96: Part Iii：appendix

    PART III：APPENDIX...

  • Page 97: Appendix I: Fluorescence & Phosphorescence

    Appendix I: Fluorescence & Phosphorescence F1.1 Theory Schematic diagram of luminous Lowest energ excited state Stable stage Fluorescence Absorb Nonradiative transition Excitation Stable stage Excited stage Phosphorescence Stable stage Triplet F1-1 Schematic diagram of luminous F1-2 Typical Organic Molecular Energy Level 1- Vibrational relaxation；...
  • Page 98 excited state. A part of the excitation (absorbed) energy is lost on vibration relaxation, i.e., radiationless transition to the lowest vibrational level takes place in the excited state. And, eventually the molecule returns to the ground state while emitting fluorescent radiation. Also, if radiationless transition to the triplet state takes place, then phosphorescence is emitted during triplet-to-singlet transition (from the excited triplet state to the ground singlet state).
  • Page 99 absorption of radiation) to a higher energy level, either an excited singlet state or an excited triplet state will form. A singlet state is a molecular electronic state such that all electron spins are paired. That is, the spin of the excited electron is still paired with the ground state electron (a pair of electrons in the same energy level must have opposite spins, per the Pauli exclusion principle).

  • Page 100: F1.2 Fluorescence Analysis

    F1.2 Fluorescence analysis The ratio of the number of photons emitted during fluorescence to the number of photons absorbed is called the quantum efficiency of fluorescence. The larger the quantum efficiency a substance has, the more fluorescence it emits. Also, the intensity of florescence emitted from a substance is proportional to the quantity of light absorbed by it.

  • Page 101: Appendix Ii： Measurement Of Instrumental Response

    Appendix II： MEASUREMENT OF INSTRUMENTAL RESPONSE F2.1 Theory Spectrum correction is performed to enable measuring a true spectrum by eliminating instrumental response such as wavelength characteristics of the monochromator or detectors. The measurement of instrumental response is needed to perform spectrum correction. “Instrumental Response” is the function to measure and save the instrumental response.
  • Page 102 Excitation light Rhodamine B Excitation light Emission Rhodamine B Emission F2-1 Direction 1 Excitation light Excitation light Rhodamine B Rhodamine B Emission Emission F2-2 Direction 2 Pour Rhodamine B into a triangular cell in the procedure illustrated in F2-1 or F2-2.

  • Page 103: F2.3 Measurement Of Instrumental Response On Emission Side

    F2.3 Measurement of Instrumental Response on Emission Side This is the function to obtain the instrumental response on the emission side such as wavelength characteristics of the emission monochromator and detector (photomultiplier). The instrumental response on the emission side is determined by measuring a combination of instrumental response on both the excitation and emission sides by synchronous wavelength scan, and dividing it by the instrumental response on the excitation side preliminarily measured.

  • Page 104: Appendix Iii： Raman Scattering Of Water & Detection Limit Of Quinine Sulfate

    Appendix III： Detection Raman Scattering of Water & Limit of Quinine Sulfate F3.1 Raman Scattering of Water In fluorescence measurement, spectra having different natures from that of fluorescence may be observed. These are called Rayleigh scattering spectrum and Raman scattering spectra; the former appearing at the same wavelength position as the excitation spectrum, and the latter appearing at the longer-wavelength side near Rayleigh scattering.

  • Page 105: F3.3 Detection Limit Of Quinine Sulfate

    S/N： （Formula F3-3） Detection Limit of F3.3 Quinine Sulfate Set the excitation wavelength at 350nm and the emission wavelength at 450nm. Alternately measure 1 10 quinine sulfate and 0.05mol/L sulfuric acid blank solution fluorescence values. – F i0 （Formula F3-4） i1 : Fluorescence of standard quinine sulfate solution i0 : Fluorescent of Standard blank solution Average fluorescent: ∑...

  • Page 106: Appendix Iv： Quantitative Analysis Wavelength Method

    Appendix IV ： Quantitative analysis wavelength method F4.1 Single Wavelength λ F4-1 Single wavelength Fluorescence F is the value on the curve at λ F4.2 Double Wavelengths λ λ F4-2 Double wavelengths λ λ In F4-2, are the Fluorescence at...

  • Page 107: F4.3 Triple Wavelengths

    F4.3 Triple Wavelengths λ λ λ F4-3 Triple wavelengths λ λ λ In F4-3, are the Fluorescence at λ λ λ λ F – λ λ...

  • Page 108: Appendix V： Details On Quantitative

    Appendix V： DETAILS ON QUANTITATIVE SPECTRO-97 series provide 3 calibration types: Linear working curve, Quadratic working curve and Cubic working curve. All of them are not forced through the 0 coordinates. F5.1 Linear Working Curve (1st order) The calculation formula is as follows : （Formula F5-1）...

  • Page 109: F5.3 The Correlation Coefficient

    （Formula F5-5） （Formula F5-5） ∑ ∑ ∑ （Formula F5-7） ∑ ∑ S FF （Formula F5-8） ∑ ∙ ∑ ∑ S FC （Formula F5-9） ∑ ∙ ∑ ∑ S FF （Formula F5-10） ∑ ∙ ∑ ∑ S F C （Formula F5-11） ∑...

  • Page 110: Appendix Vi： Synchronous Scan

    Appendix VI： Synchronous Scan The difference between Synchronous scan and other usual scan is the excitation side and emission side scan at the same time. The fluorescent value and wavelength spectrum is synchronous fluorescent spectrum. F6.1 Constant Wavelength Difference Constant wavelength difference is to keep a fixed wavelength difference between excitation wavelength and emission wavelength in the scanning process(Δλ= λex- λem = constant).

  • Page 111: Appendix Vii： Derivative Operation On Spectrum

    Appendix VII： Derivative Operation on Spectrum The derivative of a function of a real variable measures the sensitivity to change of a quantity (a function or dependent variable) which is determined by another quantity (the independent variable). F7-1 Derivative of the function There are many ways of derivative operation on spectrum.
  • Page 112 curves are seriously overlapping, but in n=2/4 the peaks are clearer. Higher order derivative can eliminate the low order background curves. The spectrum shape is complicated after derivative, but it raises the resolution and sensitivity.

  • Page 113: Appendix Viii： Smoothing

    With modern analytical instruments increasing speed and automation, multiple accumulate and smoothing technology has become a common method of noise reduction. SPECTRO-97 series provide 3 smoothing methods: Savitsky-Golay, Mean and Median. Savitzky–Golay F8.1 A Savitzky–Golay filter is a digital filter that can be applied to a set of digital data...

  • Page 114: Appendix Ix： Phosphorescence

    Since phosphorescence usually last longer than fluorescence, it is possible to use SPECTRO-97 series for phosphorescent analysis. There is a shutter to do chopper operation. When the sample is excited excitation light, use the shutter to chop the light path, the sample is no longer irradiated by excitation light.

  • Page 115: F9.3 Phosphorescence Time Scan

    too short, the excitation may not be chopped, then the phosphorescence may contain interference of excitation light. We recommend to set the delay time no less than 18ms. Phosphorescence Time Scan F9.3 Phosphorescence wavelength scan is to do a time scan of the phosphorescence of samples to get information of phosphorescence life or phosphorescence decay rate.

  • Page 116: Appendix X： Chemiluminescence

    Appendix X： Chemiluminescence Chemiluminescence is the emission of light, as the result of a chemical reaction. There may also be limited emission of heat. Given reactants A and B, with an excited intermediate [◊], [A] + [B] → [◊] → [Products] + light The decay of this excited state[◊] to a lower energy level causes light emission.

  • Page 117: Appendix Xi： Multiple Excitation Scattering

    Appendix XI： Multiple Excitation Scattering Due to the grating monochromator factors, during the wavelength scan (including 2D & 3D), the photomultiplier tube will receive multiple scattered light from excitation light. Scattering occurs on the wavelengths which is 2 or 3 times of excitation wavelength.

  • Page 118: Appendix Xii： Accessories

    Appendix XII： Accessories Type Name Description 10mm 4-way quartz cell Quartz fluorescence cell 10mm 10mm 4-way glass cell Glass fluorescence cell 10mm Optional Fuse Fuse（2A/5A） spare USB cable USB cable parts Power cable Power cable Eliminate the scattering of excitation Cut-off filter Computer for the instrument For measuring membrane kind sample...

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