UV/VIS 8500 DOUBLE-BEAM SPECTROPHOTOMETER OPERATION MANUAL TECHCOMP

Transcription

1 UV/VIS 8500 DOUBLE-BEAM SPECTROPHOTOMETER OPERATION MANUAL TECHCOMP

2 Copy Right Techcomp Limited Version: 1.2 All rights reserved worldwide. Printed in HONG KONG. Every effort has been made to avoid errors in text and diagrams, however, TECHCOMP Limited assumes no responsibility for any errors which may appear in this publication. It is the policy of TECHCOMP Limited to improve products as new techniques and components become available. TECHCOMP Limited therefore reserves the right to change specifications at any time. We would appreciate any comments on this publication. TECHCOMP Limited PO Box 72919, Kowloon Central Post Office, Kowloon, Hong Kong. Tel: (852) Fax: (852) Tlx: TECOP HX URL:

3 Chapter 1 System Installation 1.1 Introduction 1.2 Unpacking and Inspection 1.3 Power Requirements 1.4 Environmental Requirements 1.5 Electrical Connection 1.6 Initial System Calibration 1.7 Printer Setup Table of Contents Chapter 2 Functional Description 2.1 Introduction 2.2 Main Functions Fixed Points Measurement Wavelength Scanning Time Scanning Spectrum Processing Function Automatic System Initialization Function 2.3 Instrument Specifications Chapter 3 Routine Operation 3.1 Introduction 3.2 Key-Panel 3.3 Dynamic Keyboard 3.4 Sample Compartment 3.5 Main Menu Chapter 4 Fixed Points Measurement 4.1 Introduction 4.2 Selecting the Fixed Points Function 4.3 Selecting Data Acquisition Mode 4.4 Single Wavelength Measurement 4.5 Multiwavelength Measurement 4.6 Regression Curve Setup 4.7 Sample Test 4.8 Save Files 4.9 Load Files Chapter 5 Wavelength Scanning 5.1 Introduction 5.2 Selecting Wavelength Scan Mode 5.3 Step by Step Operation Selecting Data Acquisition Mode Entering Wavelength Scan Setup Variables Setting the Lamp Switching Wavelength Position 5.4 Collecting a Spectrum in Wavelength Scan

4 5.5 Spectrum Processing Introduction Rescale Zoom Peaks & Valleys Threshold Trace Derivative Adding Spectra Spectra Subtraction Save a Spectrum Load a Spectrum Unload a Spectrum Chapter 6 Time Scanning 6.1 Introduction 6.2 Selecting Time Scan Mode 6.3 Step by Step Operation Selecting Data Acquisition Mode Entering Time Scan Setup Variables Collecting a Spectrum in Time Scan 6.4 Spectrum Processing Introduction Rescale Peaks & Valleys Threshold Trace Derivative Save a Spectrum Load a Spectrum Unload a Spectrum Chapter 7 Maintenance 7.1 Introduction 7.2 Internal Diagnostics and Automatic Adjustments Verifying Wavelength Accuracy and Reproducibility Confirming the Spectrum Bandwidth Confirming Baseline Flatness Chapter 8 Replacement Parts 8.1 Introduction 8.2 Spare Parts 8.3 Lamp Replacement Replacing the Tungsten (WI) Lamp Assembly Replacing the Deuterium (D2) Lamp Assembly 8.4 Main Fuse Replacement

5 Chapter 1 System Installation 1.1 Introduction This chapter contains the necessary information for installing the instrument. The installation procedures involve unpacking, power requirements, environmental requirements and interfacing. Please note this instrument belongs to installation category 2 and pollution degree Unpacking and Inspection Use the following steps to unpack your UV8500 spectrophotometer. 1. Carefully inspect the shipping container for any signs of external damage. If any external damage is noted, contact your agent immediately and do not discard the box. 2. Place the carton in an upright position and open it. 3. Lift the instrument out of the carton and place it on a flat surface, free from dust, vibration, and away from direct sunlight. 4. Open the cover and visually inspect the instrument for loose, bent or broken parts. Report any damage immediately. 5. Check the instrument accessories against the delivery(shipping) note. 6. Please save all packing materials, as it maybe required for later transportation. 1.3 Power Requirements Check the power reference which is attached on the rear panel of the instrument. 8500D is used with power supply 110V/50~60 Hz, 8500E is used with 220V/50~60Hz. Make sure the instrument is grounded in your laboratory. The maximum tolerance of voltage variation is ±10%. Note: Power Rating: 110 or 220VAC/ 50-60Hz, 6A Max. Main Fuse Rating: 250VAC/6A, Quick Blow(F).

6 1.4 Environmental Requirements Place the instrument on a flat, non-vibrating work bench. Avoid direct sunlight and preferable in an air-conditioning environment. Make sure that the back and right side of the instrument have at least 15 cm of clearance from the wall. Insufficient ventilation will cause overheat of the instrument. Note: Working Temperature: 5 C ~ 30 C Relative Humidity: < 85% Altitude: Up to 2000m 1.5 Electrical Connection Use the following steps to complete electrical connection of UV Locate the module power cord in the set of accessories that came with your instrument. Plug its female end into the receptacle of the power entry product, which is located on the back panel of UV8500. The power entry product combines the power socket, main switch, main fuses and power rating display as a single unit. The following diagram is sketch of the back panel of UV MOUSE TECHCOMP LTD. HONGKONG VOLTS FRE.: ***V 50/60Hz SERIAL NO.: H****** PRINTER 2. Plug the male end of the module power cord into an appropriate, grounded wall outlet or power strip. Warning: Do not use a plug adapter to ground the modular cord into a 2- prong(ungrounded) electrical outlet. You may expose yourself to dangerous electrical shock in the event of certain types of instrument failure. 3. Install a Windows 95 compatible mouse on the serial port of UV Install a Windows 95 compatible printer on the parallel port of UV8500.

7 1.6 Initial System Calibration Power on the UV8500 unit after electrical connection, the system will perform an initial calibration automatically. The calibration procedure includes: entering into Windows95 environment, electronic system check, deuterium lamp check, tungsten lamp check, wavelength calibration, and system baseline measurement. UV8500 software will be started automatically after the unit entered into Windows 95 environment.

8 1.7 Printer Setup Canon Bubble-Jet BJ-10e is set as default printer in UV8500. If you want to use another type of printer, first you should install it by using the Add Printer wizard of Windows95, then in UV8500 software, set the newly installed printer as defaulted. The following procedure shows you how to set a newly installed printer as defaulted in Steps: 1. On the File menu, click Print Setup. 2. Click the arrow next to Name, and select the printer type. 3. Click OK.

9 Chapter 2 Functional Description 2.1 Introduction The UV8500 spectrophotometer is designed to perform qualitative and quantitative photometric analyses in the ultraviolet and visible regions. A complete functional description of this instrument is introduced in this chapter. 2.2 Main Functions This section introduces the main functions in UV Fixed Points Measurement Single Wavelength Photometric Measurement UV8500 lets you read the photometric value(%transmittance or Absorbance) at a single wavelength quickly and conveniently. The wavelength slew speed reaches 6,000nm/min. Multiwavelength Photometric Measurement Up to 20 wavelength points can be set up in the multi-wavelength photometric measurement mode. Results will be grouped into a table format automatically. Result can be saved in built-in memory or floppies for later use. Concentration Measurement The UV8500 provides the following capabilities in concentration measurement. You can use up to 3 wavelength measurement to generate the regression curve. You can use up to 20 standards to set up the regression curve. UV8500 will calculate the working curve using a linear equation that fits the data. Abnormal reading for standard and samples can be deleted and modified after measurement. You can enter factor values to generate regression curves. Analytical results can be print out on a printer. Method and results can be saved in built-in memory or floppy for later use.

10 2.2.2 Wavelength Scanning Features You can acquire wavelength spectrum accurately and conveniently with selection of steps of 0.1, 0.2, 0.5, 1.0 and 4.0nm which corresponsing to speed ranges from 10 nm/min to 3,200nm/min. Light source switching position can be user defined from 320nm to 360nm. Light sources and filters are automatically changed during scanning. Peaks and valleys will be automatically detected after scanning. User can define the peak threshold. Powerful spectrum processing functions are provided. ( see section 5.5 ) Spectra can be printed out on a printer. Analytical results can also be saved in the built-in memory or floppy diskettes for later use Time Scanning The UV8500 allows you to record the absorbance or transmittance value of a sample as a function of time at a specified wavelength. On-line display of absorbance or transmittance with time graduations on the abscissa. Spectra can be printed out on a printer. Analytical results can also be saved in built-in memory or floppy diskettes for later use. Photometric values at specified intervals can be printed in table format. Data can also be exported to EXCEL software for statistical calculations.

11 2.2.4 Spectrum Processing Function The UV8500 spectrophotometer can save measured spectra in built-in memory or floppy diskettes for later processing. Spectrum processing functions include: Trace a Spectrum A cursor can be moved to a desired point with mouse in the spectrum displayed on the LCD screen and the photomatric data at this point is displayed. Automatic Peak Detection After a scanning is completed, peaks and valleys can be automatically detected and listed out in a table format. They will also be labelled on the spectrum. Scale Expansion Simultaneous expansion of X and Y axes are provided with Zoom function. Display range can also be changed though Display Setup function. Differentiation You can calculate and display the first through fourth derivative spectra for a given spectrum. Derivative spectra are useful for enhancing spectral data that are not readily apparent in absorbance spectrum. Calculations Between Spectra You can calculate addition and subtraction between two spectra with the resulting data displayed on the LCD Automatic System Initialization Function The UV8500 performs the following calibration and self-diagnosis functions automatically. Memory Check Lamp Ignition Check Wavelength Drive Mechanism Check Automatic Wavelength Calibration

12 2.3 Instrument Specifications Item Description Monochromator Double Beam CT Arrangement, Holographic diffraction grating Spectral Bandwidth < 2.0 nm Wavelength Range 190 to 1100nm Wavelength Setability 0.1nm increment Wavelength Accuracy ±0.5nm (ASTM method #E275-83) Wavelength Reproducibility 0.3nm (ASTM method #E275-83) Photometric Mode Absorbance & % Transmittance Photometric Range to 3.000A; 0 to 300%T Photometric Accuracy ±0.003A; ±0.5%T(0.000 to 1.000A) Photometric 0.002A; 0.3%T(0.000 to 1.000A) Reproducibility Photometric Drift ±0.001A/hr Instrument Noise < 0.2%T Baseline Flatness ±0.005A Stray Light <0.05%T (ASTM method #E387-72) Wavelength Slew Speed 6,000nm/min (full wavelength range) Wavelength Scan Speed 60 to 3,200nm/min Kinetic Sampling Interval 0.5s, 1s, 6s, 30s and 60s Light Source Deuterium Lamp, Tungsten-Halogen Lamp Light Source Switching Automatic light source switching, User selectable within a range from 320 to 360nm Detector Dual Silicon Photodiode Detectors Display UV8500 : 8.4 backlight LCD display (optional color LCD display) UV8500PC: Computer Monitor Sample Compartment 100 mm beam spacing 12.5cm (W) x 21.5cm (L) x 13.0cm (H) Dimensions 61.0cm (W) x 52.0cm (L) x 24.0cm (H) Power Source 110 or 220VAC, 50/60Hz Note: Please note that the instrument can be used at altitude up to 2000 meter. This instrument belongs to installation category 2 and pollution degree 1.

13 Chapter 3 Basic Operation 3.1 Introduction This chapter provides information for basic operation of UV8500/8500PC spectrophotometer. 3.2 Key-Panel (UV8500 only) The illustration below shows the operation panel of UV8500. Tab A 7 B C D 8 E 9 F Y Z SAVE SHUTS DOWN Caps G 4 H I 5 J K 6 L GOTOλ AU / ZE PRINT Shift M 1 N O P 2 Q 3 R CLEAR EXIT ENTER Ctrl S T U V W 0 X S/P STOP Description of Key Functions The description below gives the key functions that are available during operation. KEY DESCRIPTION Tab Use this key to select items Caps Input Character font Shift Input Characters of the upper position Ctrl Input characters of the lower position 0 ~ 9 Input Values: ( input numeric values ) - Minus?iDecimal Space GO TO λ Use for single wavelength photometric measurement CLEAR Clear error inputs, unwanted data, delete files S/P START/PAUSE the measurement(wavelength scan or time scan) SAVE Save current working files into memory AUTO ZERO Absorbance autozero calibration EXIT Return from sub-menu to main menu STOP Stop the measurement(wavelength scan or time scan) SHUTS DOWN Exit the UV8500 software

14 PRINT connected ENTER Left Arrow Right Arrow Up Arrow Down Arrow Print out current working files(data or spectra) on the printer Confirm entered value or selections Moves the highlight or the cursor left Moves the highlight or the cursor right Moves the highlight or the cursor up Moves the highlight or the cursor down 3.3 Dynamic Keyboard UV8500 provides a dynamic keyboard in the software. Every time the software asks you to input characters, a button representing the dynamic keyboard appears. Clicking this button, the dynamic keyboard will be shown on the LCD as the following. 3.4 Sample Compartment The following figure shows you the structure of sample compartment. REFERENCE LENS DETECTORS SAMPLE

15 3.5 Main Menu After entering the UV8500 software, the Main Menu appears on the display as shown in the illustration below. Three main functions are listed on the dialog box. They are Wave Scan, Fixed Points and Time Scan. The Fixed Points function includes multi-wavelength photometric measurement and concentration measurement. Status Bar Menu bar and Toolbar are both provided in UV8500 software, so you have two ways to select a desired function. On the menu bar, use your keypad or mouse to select the desired function. Almost all the functions listed in the menu bar can be reached by clicking a corresponding button in the toolbar.

16 Many quick-click buttons are provided in the Toolbar. In the following table, we give a brief introduction of these buttons. Button Description Button Description Start a Measurement Pause a Measurement Stop a Measurement Display and Print Setting Load a Spectrum Save a File Remove a Spectrum Print B Wavelength Scan Measurement Time Scan Measurement Background Calibration Fixed Points Measurement Go To λ Zero Calibration Method Setup Set Absorbance as Photometric Mode Activate the ZOOM Function Activate the Trace Cursor Set %Transmittance as Photometric Mode Display Range Setup Restore Zoomed(expanded) Axes to Their Original Scales Add Two Spectra Subtract one Spectrum from Another List out Peaks of a Spectrum Get a Derivative from a Spectrum List out Valleys of a Spectrum Define the Peak/Valley Threshold

17

18 Chapter 4 Fixed Points Measurement 4.1 Introduction This chapter describes three functions in the Fixed Points Measurement. Single Wavelength Photometric Measurement Multi-wavelength Photometric Measurement (up to 20 wavelength points) Concentration Measurement, which include regression curve setup and reading unknown samples. 4.2 Fixed Points Mode Method 1 On the File menu, click New, the following dialog box will be popped out. Either: (a) Use mouse to click Fixed Points from the pop out menu, then click OK, or (b) Use the arrow key on your keypad to select this item and press ENTER. Method 2 Click button on the toolbar. 4.3 Data Acquisition Mode Use the following steps to select the data Acquisition mode(%t, ABS) that you wish to use in the Fixed Points measurement. Step: Click button in the toolbar to select the %transmittance mode or click button to select the absorbance mode. The status bar will be changed accordingly. Abs.: *.**** %T: ***.*

19 4.4 Single Wavelength Measurement The UV8500 provides you a convenient method to measure photometric value at a fixed wavelength. Step: 1. Press key GO TO λ in the keypad or click button in the toolbar. 2. Key in the desired wavelength position and click OK. The wavelength setability is 0.1nm. Please note the wavelength should be within the range from 190nm to 1100nm. 3. Place a reference and a sample in the sample compartment. The wavelength position and photometric value will be displayed in the status bar. For example, if the wavelength position is set to be 555.0nm and Abs. value at this point is 0.226, the status bar will be shown as Ready Pos.: 555.0nm Abs.: Note: The single wavelength photomatric measurement function is also available in other sub-menus.

20 4.5 Multiwavelength Photometric Measurement Mode This section shows you the procedure in the multi-wavelength photometric measurement. Steps: 1. Click the button in the toolbar. The method dialog box will be popped out. 2. Type the number of wavelength points in the Number of points box, or click the up/down arrow next to the box to set the wavelength points. Leave the two boxes of Calculate Concentration and Use Standard Sample blank. 3. Keyin the wavelength positions in the wavelength box. For example, we set six wavelength points, which are 500nm, 510nm, 520nm, 780nm, 770nm and 760nm.

21 4. Click the Sample tab, the LCD will be shown as the following. The control menu contains six buttons, which are Start, Delete, Modify, Recalculate, Title/Format and Print Data. 5. Place a sample and a reference in the sample compartment. 6. Click Start to run a new measurement. The display will be changed as the following. 7. UV8500 will read the photometric value of sample 1 at the fixed wavelength automatically. Keyin the sample name in the Name box with keypad or dynamic keyboard. 8. Click OK after UV8500 complete the measurement, the photometric data for sample 1 will be listed in the sample table. 9. Repeat step 3 to step 8 to test all samples.

22 10. Click Print Data to print out the table displayed. The following table shows the format of the printout. Fixed Points Measurement By Techcomp UV8500 Sample name 500.0nm 510.0nm 520.0nm 780.0nm 770.0nm 760.0nm Sample-1 Sample-2 Sample-3 *Abs unit is used. Note: The Title/Format button enables you to definite your report title and the print format. Click the Title/Format button in the control menu, the following dialog box will appear. You can change the Main Title, Second Title and Foot Note in this dialog box.

23 4.6 Linear Regression Analysis Method Set Up There are two methods to set up the linear regression curve. You can use standards to set up the regression curve or just key in the parameters manually. Use the following steps to select the method that you want to use. Steps 1. Click the Method Tab. 2. Enter the number of wavelength points in the Number of points box, or click the up/down arrow next to the box to set the wavelength points. 3. Key in the wavelength positions in Wavelength boxes. 4. Tick the Calculate Concentration check box to activate concentration calculation. If you leave this check box blank, you will only get results in absorbance or in %transmittance. 5. Tick the Use Standard Samples check box if you want to set up the regression curve with prepared standards. Leave this check box blank if you want to use the existing standard curve parameters. Wavelength Points One to three wavelength points can be used in the concentration measurement. The calculation functions are different according to the number of wavelength points. 1. One Wavelength A=A 2. Two Wavelengths A= A2-A1 3. Three Wavelengths A=A2-(mA1+nA3)/(λ3-λ1) where m=λ3-λ2, n=λ2-λ Key in the curve parameters to set up the regression curve The regression curve is represented by the following function. C = C0 + C1 *Abs C0 represents the intercept and C1 is the slope. Keyin the value of C0 and C1 in their boxes.

24 4.6.3 Using Standards for Calibration Curve Setup This section shows you the procedure of using standard to set up the calibration curve. In the following example, we use two wavelength points, which are 260.0nm and 280.0nm to set up the regression curve. steps 1. Click the Standard tab. The following dialog box will be popped out. 2. Click the B button for background correction. 3. If a blank is prepared, place cuvettes which contain blank solution in both the reference and sample holders. Click for zero correction. 4. Place standard 1 in the sample compartment.

25 5. Click Start to run a measurement. 6. Key in the concentration value of standard 1 in the box of Conc. %. 7. You can define a new name for the standard in the Name box, otherwise, it will be defaulted as Standard After UV8500 completes the measurement of standard 1, click OK. The photometric data, A and concentration will be shown in the standard table. Repeat step 4 to step 7 to measure all the standards prepared. 9. Incorrect results can be deleted. To do this, first click the standard name in the sample column, then click Delete. The following dialog box will be shown on the display. Click Yes to delete the standard reading.

26 10. Click the Fitting tab to display the regression curve. 11. Display range can be modified by clicking the Display Setting tab. After you changed the display parameters in the above dialog box, click Apply, the fitting display will be changed according to your settings. Note: The unit of concentration you set in the above dialog box should be the same as the one you have set for your standards.

27 4.7 Sample Test The following procedure shows you the step to read samples. 1. Click the Sample tab. The interface will display as the following. 2. Place sample 1 in the sample cuvette holder. 3. Click START to run a measurement. 4. UV8500 will read out the photometric value of sample 1 at the fixed wavelength positions automatically. Type the sample name in the Name box, otherwise, it will be defaulted as Sample Click OK, the photomatric readout for Sample-1 will be listed in the sample table. DeltaAbs and concentration value of Sample-1 will also be displayed in column 3 and column 4.

28 6. Repeat the step 2 to 5 to test all the unknown samples prepared. 7. Click Print Data to print out the table displayed. The following table shows the format of the printout. Fixed Points Measurement By UV8500 Sample name 260.0nm 280.0nm DeltaAbs % Sample-1 Sample-2 Sample-3 *Abs unit is used. Note: The Title/Format button enables you to definite your report title and the print format. Click the Title/Format button in the control menu, the following dialog box will be popped out. You can change the Main Title, Second Title and Foot Note in this dialog box.

29 4.8 Save Files Step: 1. On the File menu, click Save, or click in the toolbar. The interface will be changed as the following. 2. Select a folder and key in a file name in the File name box. The file type for fixed points measurement is defaulted as *.fix. There are two ways to key in a filename. One is to use the membrane keypad. The other is to use the dynamic keyboard, which will be popped out by clicking the button located beside the File name box. 3. Click Save.

30 4.9 Load Files Steps : 1. On the File menu, click Open, or click in toolbar. The interface will be changed as the following. 2. Select a folder and a filename. 3. Click OK to open the file selected.

31 Chapter 5 Wavelength Scanning 5.1 Introduction This chapter describes how to collect a spectrum while using Wavelength Scan function. 5.2 Selecting Wavelength Scan Mode Method 1 On the File menu, click New, the following dialog box will be popped out. Either: (a) Use mouse to click Wave Scan from the pop out menu, then click OK, or (b) Use the arrow key on your keypad to select this item and press ENTER. Method 2 Click button on the toolbar. The LCD will display the wavelength scan interface as below.

32 5.3 Setting the Lamp Switching Wavelength Position During system initialization, UV8500 will automatically compare the energy of D2 lamp and tungsten lamp within the range from 320nm to 360nm to set the lamp switching point. You can set a new switching point to replace the current one through the following steps. Step: 1. On the UV8500 menu, click D2/W Switch Point, the interface will be changed as the following. 2. Key in the lamp switching wavelength position in the New point box. It should be within the range from 320nm to 360nm. 3. Click Setup button or press ENTER on the keypad. The interface will return to the wavelength scan sub-menu.

33 5.4 Step by Step Operation This section describes how to operate the UV8500 spectrophotometer in the Wavelength Scan Mode Selecting Data Acquisition Mode Use the following steps to select the data Acquisition mode(%t, ABS) that you wish to use in the wavelength scanning measurement. Click button in the toolbar to select the %transmittance mode or click button to select the absorbance mode Entering Wavelength Scan Setup Variables You can use the following steps to set up the variables for wavelength scan. 1. Click button in the toolbar. The interface will be changed as the following. 2. Key in the lower limit of scan range in the From box. Acceptable entries range from 180 to Key in the upper limit of scan range in the To box. Acceptable entries range from 180 to Click the arrow next to Step, and select a scan interval. Six scan intervals can be selected from 0.1nm, 0.2nm, 0.5nm, 1.0nm, 2.0nm and 4.0nm. 5. Click OK, the interface will return to the wavelength scan sub-menu.

34 5.5 Collecting a Spectrum in Wavelength Scan Once you have set up the operation conditions for wavelength scan, you are ready to collect a spectrum. The following procedure shows you how to collect a spectrum. 1. Place a sample in the sample cuvette holder. Close the cover of the sample compartment. 2. Click button in the toolbar. The instrument will start scanning automatically. 3. During scanning, if you want to stop it, click in the toolbar. The scan procedure can also be paused by clicking the button. Click this button again, the instrument will continue scanning. 4. The real time spectrum will be displayed on the LCD during scanning.

35 Baseline During scanning, the unit will use the system baseline which was checked during system initilization. For particular experiments where the background matrix is a strong absorber, we suggest you run the baseline again with the following steps. 1. Place sample cuvettes which contain a reference or blank solution in both the reference and sample cuvette holders. 2. On the UV8500 menu, click Autozero, or click button in the toolbar. The unit will begin the collection of baseline automatically. The new baseline will be stored in the baseline memory and will be used in all subsequent experiments until you run a new baseline to replace the current one.

36 5.5 Spectrum Processing Introduction Once you have acquired and displayed a spectrum, the following options are available. 1. RESCALE 2. ZOOM 3. PEAKS & VALLEYS 4. TRACE 5. ARITHMETIC PROCESS 6. FILES The following table shows you the general functions of spectrum processing. Option Variable Range Function RESCALE X-Axis 190 to 1100nm Reset the display scale of a spectrum Y-Axis -1 to 3 ZOOM X-Axis 190 to 1100nm Expands either or both axes for more detailed viewing. Y-Axis -1 to 3 PEAKS & VALLEYS TABLE List out the peaks and valleys of a spectrum TRACE ARITHMETIC PROCESS FILES THRESHOLD ABS=0.001 to 1.000, in increments, %T=0.1 to in 0.1 increments A + B A - B DERIV SAVE LOAD UNLOAD List the Y-axis values over which the instrument detects a peak or valley Permits reading of values from the on-screen spectrum using the cursor Sums together two spectra Subtracts one spectrum from another Displays a 1st through 4th order derivative spectrum save the current processing spectrum load a spectrum saved before Remove a spectrum from display

37

38 Select a Current Spectrum As UV8500 can displayed several spectra overlaid on the screen, you should specify the spectrum before you want to process it. Step: Down Arrow 1. Click the down arrow. All spectra displayed will be listed in the pull-down menu. 2. Click the spectrum you want to select. Its name will be listed in the Name Box and we call it as Current Spectrum.

39 5.5.2 RESCALE The following steps shows you how to change the display range of a spectrum. 1. On the Settings menu, click Display Range, or click the button in the toolbar. The interface will be changed as follows. 2. Key in the display range variables for x-axis and y-axis. The maximum range for X- axis is from 180 to 1100 and the maximum range for Y-axis is from -1 to You can also set a display intervals on both X and Y axes. To do this, first tick the box of Manual Settings and then key in the intervals. 4. Click OK. Note: The button state. in the toolbar lets you restore the display settings to the defaulted

40 5.5.3 ZOOM By using a mouse, you can zoom a part of the spectrum more conveniently. Steps. 1. Click the button in the toolbar. 2. Position the cursor in the upper-left corner of the area you want to select. 3. Hold the left mouse button to drag the cursor to outline the spectrum area you want to enlarge. 4. Release the mouse button. The spectrum which displayed within the outlined area will be enlarged. The following is an example of this function. Zoomed Area Note: The button state. in the top tools bar lets you restore the display settings to the defaulted

41 5.5.4 PEAKS & VALLEYS List the Peaks and Valleys of a spectrum Once a spectrum is displayed on the screen and it is appointed as Current Spectrum. (refer to section 5.5.1) On the View menu, click Peaks, Or clicking button in the toolbar. All peaks detected will be listed in a table format beside the spectrum. On the View menu, click Valleys, Or clicking button in the toolbar. Valleys of the spectrum displayed will be listed. Following is an example of this function. Peaks are listed above the spectrum, while valleys are listed below it. In the table, peaks are noted with,while valleys are noted with.

42 5.5.5 THRESHOLD The threshold value measures the absorbance from a valley to peak. If the value is greater than the one you choose for Threshold, the instrument will detect it and report it as a peak. Therefore, smaller Threshold values will detect smaller peaks and even background noise, if you lower the value far enough. UV8500 let you change the threshold value which will be used in peak and valley detection. On the Settings menu, click Peak Height, or click the button, the following dialog box will be popped out. Type the threshold value in the box and click OK. UV8500 will then use the new threshold to detect peaks and valleys.

43 5.5.6 TRACE Step: 1. Once a spectrum is displayed on the screen and it is appointed as Current Spectrum. (refer to section ) on the View menu, click Search, or click the button in the toolbar. The interface will be changed as the following. 2. A crosshair cursor will appear with x and y axes values displayed. 3. Move the crosshair cursor left or right on the spectrum. 4. The data in the cursor window indicate the X-axis and Y-axis values for the current cursor location. 5. Double click the left mouse button to release the crosshair cursor.

44 5.5.7 DERIVATIVE Steps: 1. Once a spectrum is displayed on the screen and it is appointed as Current Spectrum, (refer to section 5.5.1) on the Compute menu, click Derivative, or click the button in the toolbar. The following dialogue box will be popped out. 2. Key in the class of derivative and type a name for the result spectrum, then click OK. The result spectrum will be displayed overlaid with the original one.

45 5.5.8 Adding Spectra Adding spectra can assist in the development of artificial structure of spectra in multicomponent mixtures. UV8500 will only adding two spectra that are already displayed on the screen, so before arithmetic processing, load two spectra from the memory. In the following example, we have two spectra saved in directory D:\UV8500\Samples, which are 1.wav and 2.wav. Step 1. On the Compute menu, click Add, or click the button in the toolbar. The following dialogue box will be popped out. 2. Click the down arrow next to Source 1 to select a spectrum and define it as source 1. Select a spectrum for Source 2 in the same way. UV8500 will not allow you to select a spectrum not displayed on the screen or select a same spectrum twice. 3. Key in a name for the Result spectrum and click OK. The UV8500 will start processing with the result displayed on the screen.

46 5.5.9 Spectra Subtraction Subtracting one spectrum from another has been a classical technique to offset spectral interference from the spectrum of interest. UV8500 will only process subtraction of two spectra that are already displayed on the screen, so before arithmetic processing, load two spectra from the memory. In the following example, we have two spectra saved in directory D:\UV8500\Samples, they are 1.wav and 2.wav. Steps 1. On the Compute menu, click Sub, or click the button in the toolbar. The following dialogue box will be popped out. 2. Click the down arrow next to Source 1 to select a spectrum and define it as source 1. Select a spectrum for Source 2 in the same way. UV8500 will not allow you to select a spectrum not displayed on the screen or select a same spectrum twice. 3. Key in a name for the Result spectrum and click OK. The UV8500 will start processing with the result displayed on the screen.

47 SAVE A SPECTRUM Step: 1. On the File menu, click Save, or click button in the toolbar. The interface will be changed as the following. 2. Select a folder and key in a file name in the File name box. The file type for wavelength scanning is defaulted as *.wav. There are two ways to key in a filename. One is to use the membrane keypad. The other is to use the dynamic keyboard, which will be popped out by clicking the icon located beside the File name box. The following dialog box will be popped out after you clicking this icon. 3. Click Save.

48 LOAD A SPECTRUM Step: 1. Click the button in the toolbar. The interface will be changed as the following. 2. Select the directory and select the file name. 3. Click Open.

49 Unload a spectrum You can unload a spectrum if you do not want it displayed on the screen. For example, we suppose that three spectra are displaying on the LCD. They are demo2.wav, 1.wav and 2.wav and we want to remove 2.wav from the screen. Step: 1. Click the down arrow. All spectra displayed will be listed in the pull-down menu. 2. Click 2.wav and appoint it as Current Spectrum(refer to section 5.5.1). 3. Click the button in the toolbar, 2.wav will be removed from the display. This will not removed the spectrum from the memory, 2.wav is still saved in directory D:\UV8500\samples\.

50

51 Chapter 6 Time Scanning (Kinetic Analysis) 6.1 Introduction This chapter tells you how to obtain the absorbance or transmittance value for a sample as a function of time at a given wavelength. 6.2 Selecting Time Scan Mode Method 1 On the File menu, click New, the following dialog box will appear. Either: (a) Use mouse to click Time Scan from the pop out menu, then click OK, or (b) Use the arrow key on your keypad to select this item and press ENTER. Method 2 Click icon following. in the toolbar. The LCD will display the time scan interface as the

52 6.3 Step by Step Operation This section describe how to operate the UV8500 spectrophotometer in the Time Scan Mode Selecting Data Acquisition Mode Click button in the toolbar to select the %transmittance mode or click button to select the absorbance mode Entering Time Scan Setup Variables You can use the following steps to set up the variables for time scan. Step : 1. Click button in the toolbar. A dialogue box will be popped out as follows. 2. Key in the wavelength, total time(in seconds) and scan step in the above dialog box. The wavelength range should be within 190 to 1100nm. The upper limit for total time is 10 5 second, around 27 hours and 42 minutes. Four scan interval can be selected from 0.5s, 1s, 60s, and 120s. 3. Click OK, the interface will be returned to the Time Scan sub-menu.

53 6.3.3 Collecting a Spectrum in Time Scan Once you have set up the operation conditions, you are ready to collect a spectrum. To collect a spectrum in time scan, follow this procedure: 1. Close the cover of the sample compartment, on the UV8500 menu, click Background, B or click button on the toolbar. UV8500 will run a background correction at this wavelength point automatically. Then the status bar will show OK. 2. Place sample cuvettes which contain a reference or blank solution in both the reference and sample cuvette holders. On the UV8500 menu, click Autozero, or click button in the toolbar. 3. Take out the reference or blank in the sample holder, place a sample in it and close the cover. 4. Click button in the toolbar. The instrument will start scanning automatically. 5. You can stop scanning by clicking the button. The scan procedure can also be paused by clicking the button. Click this button again, the instrument will continue scanning. 6. The spectrum will be displayed on the LCD during scanning. For rate measurement, please refer to section 6.4.6, use the Derivative Function to calculate slope.

54 6.4 Spectrum Processing Introduction After you have acquired and displayed a spectrum, the following options are available: 1. RESCALE 2. PEAKS & VALLEYS 3. TRACE 4. ARITHMETIC PROCESS 5. FILES The following table shows you the general functions of spectrum processing. Option Variable Range Function RESCALE X-Axis 0 to (second) Expands either or both axes for more detailed viewing Y-Axis -1 to 3 PEAKS & VALLEYS TABLE List out the peaks and valleys of a spectrum TRACE THRESHOLD ABS=0.001 to 1.000, in increments, %T=0.1 to in 0.1 increments List the Y-axis values over which the instrument detects a peak or valley Permits reading of values from the on-screen spectrum using the cursor PROCESS DERIV Displays a 1st through 4th order derivative spectrum FILES SAVE LOAD UNLOAD save the current processing spectrum load a spectrum saved before Remove a spectrum from display

55 Appoint a Current Spectrum As UV8500 can displayed several spectra overlaid on the screen, you should specify the spectrum before you want to process it. Step: Down Arrow 1. Click the down arrow. All spectra displayed will be listed in the pull-down menu. 2. Click the spectrum you want to select. Its name will be listed in the Name Box and we call it as Current Spectrum.

56 6.4.2 RESCALE The following steps shows you how to change the display range of a spectrum. 1. On the Settings menu, click Display Range, or click the button in the toolbar. The interface will be changed as the following. 2. Key in the display range variables. The maximum range for X-axis is from 0 to and the maximum range for Y-axis is from -1 to 3 if the photometric mode is absorbance. 3. Click OK. Note: The button state. in the top tools bar lets you restore the display settings to the defaulted

57 6.4.3 PEAKS & VALLEYS List the Peaks and Valleys of a spectrum Once a spectrum is displayed on the screen and it is appointed as Current Spectrum,(refer to section 6.4.1) on the View menu, click Peaks, or click button in the toolbar. All peaks detected will be listed in a table format beside the spectrum. On the View menu, click Valleys, Or clicking button in the toolbar. Valleys of the spectrum displayed will be listed. Following is an example of this function.

58 6.4.4 THRESHOLD The threshold value measures the absorbance from a valley to peak. If the value is greater than the one you choose for Threshold, the instrument will detect it and report it as a peak. Therefore, smaller Threshold values will detect lower peaks and even background noise, if you lower the value far enough. UV8500 let you change the threshold value which will be used in peak and valley detection. Click the button on the toolbar, the following dialog box will be popped out. Type the threshold value in the box and click OK. UV8500 will then use the new threshold to detect peaks and valleys.

59 6.4.5 TRACE Step: 1. Once a spectrum is displayed on the screen and it is appointed as Current Spectrum, (refer to section 6.4.1) click the button in the top tools bar. The interface will be changed as the following. 2. A crosshair cursor will appear. Move the crosshair cursor left or right on the spectrum. 3. The data in the cursor window indicate the X-axis and Y-axis values for the current cursor location. 4. Double click the left mouse button to will release the crosshair cursor.

60 6.4.6 DERIVATIVE Steps: 1. Once a spectrum is displayed on the screen and it is appointed as Current Spectrum, (refer to section ) on the Compute menu, click Derivative, or click the button on the toolbar. The following dialogue box will be popped out. 2. Key in the class of derivative and select a name for the result spectrum, then click OK. The result spectrum will be displayed overlaid with the original one.

61 6.4.7 SAVE A SPECTRUM Step: 1. On the File menu, click Save, or click button on the toolbar. The interface will be changed as the following. 2. Select a folder and key in a file name in the File name box. The file type for time scan measurement is defaulted as *.tim. There are two ways to key in a filename. One is to use the membrane keypad. The other is to use the dynamic keyboard, which will be popped out by clicking the button next to the File name box. The following diagram is a draft of the dynamic keyboard. 3. Click Save.

62 6.4.8 LOAD A SPECTRUM Step: 1. Click the button on the toolbar. The interface will be changed as the following. 2. Select the file directory and select the file name. 3. Click Open.

63 6.4.9 Unload a spectrum You can unload a spectrum if you do not want it to be displayed on the screen. For example, we suppose there are two spectra displayed on the LCD. They are inktest.tim and test1.tim. Now we want to remove test1.tim from the screen. Step: 1. Click the down arrow. All spectra displayed will be listed in the pull-down menu. 2. Click test1.tim and appoint it as Current Spectrum(refer to section 6.4.1). 3. Click the button in the toolbar, test1.tim will be removed from the display. This will not removed the spectrum from the memory, test1.tim is still saved in directory D:\UV8500\samples\.

64

65 Chapter 7 Maintenance 7.1 Introduction The UV8500 spectrophotometer is designed to require minimal maintenance. This section describes the routine maintenance tasks you should perform and the diagnostic functions that are used to characterize the instrument. 7.2 Internal Diagnostics and Automatic Adjustments The UV8500 spectrophotometer executes a self-diagnostic each time it is power on. If you want to conduct certain performance verifications manually, follow the procedures listed below Verifying Wavelength Accuracy and Reproducibility Definition Wavelength Accuracy: the deviation of the average wavelength reading at an absorption band or emission band from the known wavelength of the band. Wavelength Reproducibility: a measure of the ability of a spectrophotometer to return to the same spectral position as measured by an absorption band or emission band of known wavelength when the instrument is reset or read at a given wavelength. Standard Most spectrophotometric methods employ compounds or unknown mixtures for the purpose of calibrating instruments photometrically at specified analytical wavelengths. We can provide you with a calibration standard ( Cat no. CS-100, holmium oxide glass ) to verify your spectrophotometer performance. The absorption spectrum of CS100 is obtained by scanning it in the whole wavelength range. Absorption peak wavelength values of holmium oxide glass are listed below. Band No. Wav. (nm) Band No. Wav. (nm) Band No. Wav. (nm) Absorption peaks of holmium oxide glass Method Use Wave Scan function to scan the CS-100 standard from 250 to 650nm. We suggest you to use 0.1nm as scanning step. After you have acquired and displayed the scanned spectrum, list out the peaks.

66 Calculation Calculate the difference of each observed wavelength from the average and sum the differences. The wavelength accuracy is the difference between the true wavelength and the averaged observed wavelength. Wavelength accuracy can be calculated as follows: n λ = 1/n λi - λr i=1 where: λi = observed wavelength value, λr = true or reference wavelength value, n = number of measurements The average value of the differences is the wavelength reproducibility which can be expressed as: n δλ = max λi - 1/n λi i=1 where: λi = observed wavelength value, n = number of measurements

67 7.2.2 Confirming the Spectrum Bandwidth Definition The bandwidth, in nanometers of the segment of the spectrum that will be isolated by the monochromator. It is the range of wavelength between the points at which the transmittance is equal to one-half the peak. Steps 1. Enter the interface of Wave Scan. 2. On the UV8500 menu, click D2 Spectral Slitwidth. The instrument will automatically scanned the deuterium lamp energy around its emission peak at 656.1nm. 3. Print out the scanned spectrum. Calculations Confirm the bandwidth as instructed and illustrated below: 1. Measure the peak width in nm at half-height. 2. If the value is below 2nm, then the bandwidth is normal. x 5 A x 1/2A Bandwidth Bandwidth T t

68 7.2.3 Confirming Baseline Flatness The baseline flatness is defined by the amount of baseline drift, excluding electronic noise. Be sure to warm up the instrument sufficiently (approx. 2 hours), and then proceed as instructed below: 1. From the main menu, select Wave Scan function. 2. Set up the wavelength scan variables as shown in the following table. DATA MODE ABS START WAVE nm STOP WAVE nm UP SCALE LOW SCALE SCAN STEP 0.2nm LAMP SWITCH 340nm 3. Click OK to acquire the spectrum. The baseline is normal if it is within ±0.005 Abs in the whole wavelength range.

69 Chapter 8 Replacement Parts 8.1 Introduction The UV8500 contains only a few parts that are considered to be spare parts. It is good preventive maintenance to keep these spare parts on hand to minimize instrument downtime in case of need. 8.2 Spare Parts The following parts are recommended to be kept on hand as spares: 1. Tungsten (WI) Lamp Assembly 500 hours expected service life (P/N: A) 2. Deuterium (D2) Lamp Assembly 300 hours expected service life (P/N: A) 3. Main Power Fuse 250VAC/6A, 50~60Hz, quick blow(f) Ver.: 1.2

70 8.3 Lamp Replacement Please observe the following safety precaution when using the lamp installation procedures to install a new lamp in your UV8500 spectrophotometer. WARNING: Do not attempt to replace either lamp with the power switch on. Both lamps operate at high temperatures that can cause skin burns on contact. Also, potentially injurious voltages will remain in the lamp connections for a short period of time after the instrument is turned off. For these reasons, turn the instrument off and allow a minimum of 20 minutes to allow lamp cool down before making a replacement. Removing the lamp compartment cover will expedite the lamp cooling process. Caution: Do not touch the reflective surface of the focusing reflector and the bulb. Any finger prints on these surfaces must be removed with ethanol Ver.: 1.2

71 8.3.1 Replacing the Tungsten (WI) Lamp Assembly Follow these steps and precautions to replace a tungsten (WI) lamp: 1. Turn off the instrument and disconnect its power cord from the power source. Allow a minimum of 20 minutes to pass so that the lamp cools down and the lighting circuitry completely discharges before proceeding to step Remove the slide door which is located on the top of the lamp compartment. 3. Unscrew the two locking nuts which fasten the lamp compartment cover. Then lift off the cover and set it aside. The illustration below shows the location of the main components inside the lamp compartment. Focussing Reflector Connection Terminal D2 L WI Lamp 4. Unscrew the two locking screws in the connection terminal for WI lamp. The connection terminal is illustrated in the following diagram. The two blue wires are connected to the WI lamp. D2 WI 5. Unscrew the two screws which fasten the WI lamp assembly on the chassis. 6. Take out the lamp holder and replace it with a new one. Note: You need not to take the lamp off the lamp holder as these two parts should only be replaced as a whole assembly. 7. Lock the new lamp assembly on the chassis with two fastening screws. 8. Fasten the two blue wires in the connection terminal for WI Lamp. 9. Close the lamp compartment cover and re-tighten the lamp compartment screws. 10. Install the slide door on the instrument cover Replacing the Deuterium (D2) Lamp Assembly Blue Wires Follow these steps and precautions to replace a tungsten (WI) lamp: Ver.: 1.2

72 1. Turn off the instrument and disconnect its power cord from the power source. Allow a minimum of 20 minutes to pass so that the lamp cools down and the lighting circuitry completely discharges before proceeding to step Remove the slide door which is located on the top of the lamp compartment. 3. Unscrew the two locking nuts which fasten the lamp compartment cover. Then lift off the cover and set it aside. The illustration below shows the location of the main components inside the lamp compartment. Focussing Reflector Connection Terminal D2 L WI Lamp 4. To replace the D2 lamp, you should unscrew the three locking screws in the connection terminal for deuterium lamp first. The connection terminal is illustrated in the following diagram. The color of the high voltage one is different from the low voltage ones. Please note that the high voltage should be connected to a specified position while the positions of the low voltage ones are interchangeable. D2 W High Voltage 5. Unscrew the two screws which fasten the D2 lamp holder on the chassis. Low Voltage 6. Take out the lamp holder and replace it with a new one. Note: You need not to take the lamp off the lamp holder as these two parts should only be replaced as a whole assembly. 7. Lock the new lamp assembly on the chassis with two fastening screws. 8. Fasten the three color wires in the connection terminal for D2 Lamp. 9. Close the lamp compartment cover and re-tighten the lamp compartment screws Ver.: 1.2