Spectrophotometer. Operational Manual

Transcription

1 Spectrophotometer Operational Manual V1.0 Revision 1 for Alpha-1860/1900 Laxco, Inc. 1

2 Contents General information... 3 Safety... 3 Electrical... 3 Warning... 3 Performance... 4 Radio Interference... 4 Introduction... 4 Working Principle... 4 Unpacking Instructions:... 5 Specifications... 6 Installation:... 6 Operation:... 7 Description of keys... 7 Turn on spectrophotometer... 8 Basic operation... 9 Set wavelength (Set wavelength in Basic mode ) Load, delete data or curve (in Wavelength Scan ) Save data or curve (Save curve in Wavelength Scan ) Analyze Sample Before measurement Basic Mode Quantitative Mode Wavelength Scan Kinetics Mode DNA/Protein Mode Multi Wavelength Mode System Utility Wavelength Reset Printer Setup Lamp Service Clock Setup Refresh Dark Current Connect to PC Beeper on/off Selection Language Refresh System Baseline Delete entire saved files Restore default settings Appendix A Appendix B

3 General information The apparatus described in this manual is designed to be used by properly trained personnel in a suitable equipped laboratory. For the correct and safe use of this apparatus it is essential that laboratory personnel follow generally accepted safe procedures in addition to the safety precautions called for in this manual. The covers on this instrument may be removed for servicing. However, the inside of the power supply unit is a hazardous area and its cover should not be removed under any circumstances. There are no serviceable components inside this power supply unit. For all instruments, avoid touching the high voltage power supply at all times. Some of the chemicals used in spectrophotometry are corrosive and/or inflammable and samples may be radioactive, toxic, or potentially pathogens. Care should be taken to follow the normal laboratory procedures for handling chemicals and samples. Safety Read the following before installing and using the instrument and its accessories. The instruments mentioned in this manual should be operated by appropriate laboratory technicians. Electrical The power cord shall be inserted in a socket provided with a protective earth contact. The protective action must not be negated by the use of an extension cord without a protective conductor. Warning Any interruption of the protective conductor inside or outside the apparatus or disconnection of the protective earth terminal is likely to make the apparatus dangerous. Intentional interruption is prohibited. Whenever it is likely that the protection has been impaired, the apparatus shall be made inoperative and be secured against any unintended operation. NEVER touch or handle the power supply on the instruments due to the high voltage. The protection is likely to be impaired if, for example, the apparatus Shows visible damage Fails to perform the intended measurements 3

4 Has been subjected to prolonged storage under unfavorable conditions. Has been subjected to severe transport stresses Performance To ensure that the instrument is working within its specification, please verify proper operation before making important measurements. The performance checks should be at the particular reference to wavelength and absorbance accuracy. Radio Interference For compliance with the EMC standards referred to in the EC Declaration of Conformity, it is necessary that only shielded cables supplied by the factory are used when connecting the instrument to computers and accessories. Introduction The Alpha-1860, Fig. 1 and the Alpha-1900, Fig. 2, are general purpose instrument designed to meet the needs of the conventional laboratory, They are ideal for various applications, such as: Chemistry, Biochemistry, Petro chemistry, Environmental Protection, Food and Beverage Labs, Water and Waste Water Labs and other fields of quality control and research. The Alpha-1860 and 1900 incorporates a dot matrix LCD display for photometric results, easy operation and wavelength range of 190nm to 1100nm. The instruments are ideal for measurements in the visible and ultraviolet wavelength region of the electromagnetic spectrum. Alpha-1860 Fig 1 Alpha-1900 Fig 2 Working Principle The spectrophotometer consists of five parts: 1) Halogen and deuterium lamp to supply the light; 2) A Monochromator to isolate the wavelength of interest and eliminate the unwanted 4

5 second order radiation; 3) A sample compartment to accommodate the sample solution; 4) A detector to receive the transmitted light and convert it to an electrical signal; and 5) A digital display to indicate absorbance or transmittance. The block diagram below illustrates the relationship between these parts. Block diagram for the Spectrophotometer In your spectrophotometer, light from the lamp is focused on the entrance slit of the monochromator where the collimating mirror directs the beam onto the grating. The grating disperses the light beam to produce the spectrum, a portion of which is focused on the exit slit of the monochromator by a collimating mirror. From here the beam is passed to a sample compartment through filters, which helps to eliminate unwanted second order radiation from the diffraction grating. Upon leaving the sample compartment, the beam is passed to the silicon photodiode detector and causes the detector to produce an electrical signal that is displayed on the digital display. The Alpha-1860 /1900 incorporate an USB1.0 for connecting to a PC for the Application Software, and include a parallel port for the optional built-in thermal printer for Alpha Alpha can drive a USB 2.0 printer with an optional driving modular. Unpacking Instructions: Carefully unpack the contents and check the materials against the following packing list to ensure that you have received everything in good condition. Description Packing List Quantity Spectrophotometer...1 Mains Lead.1 Cuvettes.. Set of 4, glass Set of 2, quartz Dust Cover..1 Manual. 1 5

6 Specifications Alpha-1860 Alpha-1900 Wavelength Range nm Spectral Band pass 1.8 nm 2 nm Wavelength Accuracy ±0.5 nm Wavelength Repeatability ±0.2 nm Stray Radiant Energy <0.05%@220 nm & 340 nm Photometric Range 0-200%T, A Baseline flatness <0.002A( nm) <0.001A( nm) Drift 500 nm 500 nm Power Requirements Vac Hz Dimensions 570W 460L 230H 660W 600L 280H Light Source Tungsten Halogen/Deuterium Weight: 16kg 28.5kg Installation: After carefully unpacking the contents, check the materials with the packing list (page 5) to ensure that you have received everything in good condition. Place the instrument in a suitable location away from direct sunlight. In order to have the best performance from your instrument, keep it as far as possible from any strong magnetic or electrical fields or any electrical device that may generate high-frequency fields. Set the unit up in an area that is free of dust, corrosive gases and strong vibrations. Remove any obstructions or materials that could hinder the flow of air under and around the instrument. Use the appropriate power cord and plug into a grounded outlet. Turn on the spectrophotometer. Allow it to warm up for at least 15 minutes before taking any readings. We suggest you then do the Calibrate System with the Search 656.1nm to set the wavelength to the deuterium lamp emission line. NOTE: This symbol means Caution, Risk of Danger. Refer to this Manual. 6

7 Operation: Description of keys User can perform all operations by pressing the keys and all the results and operation information are displayed on the LCD, Fig 4. Fig 4 LOAD Load data or curve saved before; SAVE Save data or curve; SETλ Set wavelength; 0Abs/100%T Blank or scan the base line; PRINT Print test results or screen START Start testing or scanning sample; ESC/STOP Exit to previous screen or cancel the operation; ENTER Confirm the inputted data or selected item; Go into next setup or screen; F1 - F4 Function based on the information on the screen; 0-9 Input number or letter, consecutively press a numeric key to select a character; +/-/. Input +,- or dot; CLEAR Clear all characters when you are inputting or clear curve displays on the screen; <, > Change x scale; Search point after scan; < clear a character;, Change y scale; Search peak after scan; Scroll items for selecting; Change capital/small letter last typed in; Browse the items for selection; CELL Set cell position. 7

8 Turn on spectrophotometer Turn on spectrophotometer by pressing the Power Switch (IO) (on the back of instrument ). The instrument starts to initiate and follows the steps as listed below: a. The instrument will implement initiation first. Figure 5 shows you all items need to be checked. If all checks are passed, instrument will warm up for 15 minutes, you may press ESC to skip, the screen display as Fig 6. Select No to skip to main menu (Fig 8) or select Yes (recommended) to calibrate system (Fig7).The calibrating process includes get dark current, searching 656.1nm and check energy. After finishing the calibration steps, instrument will go to the main menu, (Fig 8). b. If the data in memory has been lost, the instrument will directly calibrate system without any other choice for the user. c. If no auto-cell changer is installed, cell #1 will disappear as in Fig 8 d. Each checked item is displayed as one line. Note: For Alpha-1900, when System Calibration appears on the screen, the user has no choice to select Yes or No, but you can press ESC/STOP to skip the system calibration, but it is strongly recommended for you not. Ten (10) seconds later, the instrument will automatically implement the System Calibration. Fig 5 Fig 6 8

9 Fig 7 Fig 8 Basic operation Blank Push the blank cuvette with reference solution into the light path. Press 0Abs/100%T to blank,(fig 9) Note: If the reference solution is too thick, Energy Low will appear following the Blanking on the screen. If Energy too Low appears following the Blanking, the test will be paused and Warning will appear on the screen. 9

10 Fig 9 NOTE: (1) Blanking is automatic after a wavelength change (2) Make sure no obstacle in the light path and don t open sample compartment lid during blanking. (3) The dark current will not be taken after power on, if you bypass the calibrating system. It is recommended that you take the dark current after warm up. See page 44. Set wavelength (Set wavelength in Basic mode ) Press SET λ (Fig. 10) Fig 10 Use numeric keypad to input wavelength 700nm (Fig 11). 10

11 Fig 11 Press ENTER to change the wavelength from 500.0nm to 700.0nm (Fig 12), and then blank automatically; after blanking, the screen displays as (Fig 13). Fig 12 Fig 13 Load, delete data or curve (in Wavelength Scan ) Press 3 or move the reverse display stripe to line 3 in Fig. 8. Go into Wavelength Scan. After LOAD being pressed, the first file (ABC.wav) in memory will appear on the bottom line of screen. Shown as Fig 14, press or to browse the files stored in memory. Then if: a. ENTER be pressed, the file selected will be loaded and displays on the screen.(fig 15). Note: (1) The file selected must match WL scan test s type. If not, the file type error will appear on the right of top line. (2) Different tests have different file types. Refer to table 1 on page 12. b. CLEAR be pressed the file selected will be deleted by selecting Yes. 11

12 Fig 14 Test Quantitative Curve Quantitative Test Result WL Scan Kinetics DNA/Protein Multi WL Fig 15 Table 1 File Type ***.fit ***.qua ***.wav ***.kin ***.dna ***.mul Save data or curve (Save curve in Wavelength Scan ) Press SAVE in Fig. 16 to save curve. Name the curve by pressing the numeric keypad (Fig 17), press ENTER to confirm. Note (1). Press the numeric key repeatedly to scroll characters and pressing, to alter capitalization. Table 2 shows all characters available. (2). If the name already exists in memory, the warning duplicated name, are you sure? will appear. Yes for overwrite and No for Exit. (3).The length of filename is 4 characters or less. 12

13 Fig 16 Fig 17 Table 2 key representing key representing key representing 0 0,+,-,*,/ 1 1,#,?,:,I 2 2,A,B,C,= 3 3,D,E,F,% 4 4,G,H,I,{ 5 5,J,K,L,} 6 6,M,N,O,~ 7 7,P,Q,R,S, 8 8,T,U,V, 9 9,W,X,Y,Z +/-/. -,., Print test report. Show Print the report in Basic mode, Fig18. Press PRINT to print the report about curve or data you have loaded or tested, (Fig 19). 13

14 Fig 18 Fig 19 Analyze Sample Before measurement Make a blank reference solution by filling a clean cuvette (or test tube) half full with distilled or de-ionized water or other specified solvent. Wipe the cuvette with tissue to remove the fingerprints and droplets of liquid. Fit the blank cuvette into the 4-cell linear changer and place the cuvette in the slot nearest you. For the Alpha Series, push the changer so that the cuvette is in the light path (Push the rod in). Close the lid. For different user requirements, we have provided different test methods. Basic Mode Push the blank cuvette into the light path. In main menu (Fig8), press 1 to enter Basic mode test. After automatically blanking, it will display as (Fig 20). Press ESC/STOP to exit. Fig20 choice. There are three modes (T%, Abs, conc/factor) for you to select by pressing F2 to make 14

15 Fig 21 1: Abs mode Push the blank cuvette into the light path. Press F2 to select Abs mode, Press 0Abs/100%T for Blanking, and then Push the sample into light path to take reading(fig 21) 2: T% mode The operation is the same as Abs test mode but pressing F2 to select T% mode. 3: Conc/Factor mode Press F1 to select a concentration unit. If no unit is suitable for your test, please select the item Other (Fig 22), press enter and input a new unit by pressing the numeric keypad. (Fig 23) Fig 22 Fig Push the blank cuvette into the light path and then press 0Abs/100%T to blank. There are now two choices for you to take: 15

16 3.1.1 Press F3 to input known Factor, Fig 24. Then push the sample into light path to take reading of concentration, Fig 25. Fig 24 Fig Push sample of known concentration into the light path and press F4 to input known Standard Concentration (Fig. 26). Then push the sample into light path to take a reading of the concentration. (Fig. 27) Note: (1) You can select wavelength at any time by pressing SET λ. After your selection, instrument always blanks automatically. (2) If Factor value is more than 9999, the out of range will display on screen. (3) All results can be printed, if the optional built in printer is installed. 16

17 Fig 26 Fig 27 Quantitative Mode Press 2 in Main Menu for Quantitative Test, Fig 28. Press ESC/STOP to exit. Fig Press F1 to select unit of concentration, Fig 29. Fig Press SETλ to select correction methods and enter the wavelength. There are three correction methods (single, Iso absorbance and 3 point, Fig

18 Note: Please refer to the Appendix B for the correction method. Fig 30 Press F2 in Fig 28 for more items to select. (Fig. 31) Fig Press F1 in Fig 31 to select fitting method. There are 4 methods for you to choose: Linear fit, linear fit through zero, square fit and cubic fit. Press, to select and ENTER confirm. Let us select Linear fit as an example. Fig 31A Fig31A 3.1 Press F2 in Fig 31 to input a known standard curve.(fig 32 & Fig 33) 18

19 Fig 32 Fig 33 Note: The constants to be entered are depending on which fitting method is selected. Table 3 lists the Equations. Table 3 Fitting Method Fitting Equation constants linear fit through zero C=K1 A K1, r* Linear fit C=K0+K1 A K0,K1,r* square fit C=K0+K1 A+K2 A 2 K0,K1,K2 cubic fit C=K0+K1 A+K2 A 2 +K3 A 3 K0,K1,K2,K3 * r : regression co-efficient, default=1 3.2 Press F3 in Fig 31 to establish a standard curve by measuring a group of standard samples, ( Fig 34). Enter standard concentrations of samples by pressing the numeric keypad followed by ENTER. Press or to modify the inputted data. (Fig. 35) 19

20 Fig 34 Fig 35 Press ESC/STOP to end input at any number of the standard sample, maximum input standard sample is 10. Before measuring the standard, blank is necessary. (Fig. 36) Fig 36 Push the blank cuvette into the light path, press 0Abs/%100T, the instrument will step to the wavelength and blank. See Fig 37. Fig 37 Pull the first sample cuvette of known concentration into the light path, Press START to get the absorbance of standard sample. After finishing the measurement of 3 standard sample, the standard curve equation has come out. (Fig. 38) 20

21 Fig 38 Press F4 to draw the curve. You can get a different curve by pressing F1 to select a different fitting method. See Fig. 39 to Fig. 42. For linear fits, r represent fitting coefficient of linear regression. r=1 is best fit usually r is very close to 1. Note: If there are few standard samples, it is not suitable for selecting square fitting, especially cubic fitting, otherwise invalid fitting results will be obtained. Fig 39 linear through zero fit Fig 40 linear fit 21

22 Fig 41 Square fit Fig 42 Cubic fit 4. Press SAVE to save calibration if necessary, press ESC/STOP to exit 5. Quantitative Test Before test, the standard curve must be obtained. There are three ways for you to obtain. 5.1 The standard curve established and saved in the instrument s memory. In Fig. 38, press Load and then press or to select the file with type ***fit. Press ENTER TO confirm. 5.2 Known standard curve, which is not saved in the instrument. Refer to 3.1 on page 18. Input a known standard curve directly. 5.3 Use the standard samples for the test. At first, the standard curve must be established. Please refer to 3.2 on page 19. Note: All sample results must be taken in screen Fig 28. Follow the steps below to measure: 5.4 Push the blank cuvette with reference solution into the light path and press 0Abs/100%T. 5.5 Pull the sample cuvette into the light path, press START & take the readings on the screen. (Fig. 43) 22

23 Fig If there is more than one sample, repeat step 5.5 for the next sample 5.7 Press (SAVE) to save the results and fitting parameters. 5.8 Print Test Report Press PRINT to print the test report (Fig 44). Fig 44 Wavelength Scan Press 3 in main menu(fig 8) for WL Scan test (Fig 45). ESC/STOP to exit. To load a previous curve, press LOAD and select a previously stored curve (.wav). Fig 45 1 Scan sample 23

24 1.1 Press F1 to setup scan parameters, input the start wavelength, and end wavelength by pressing the numeric keypad (Fig 46). The Alpha-series spectrophotometer always scans from high to low wavelength. Browse and select the items of scan step and scan speed by pressing or. Fig 46 Scan step allows the selection of 0.1nm, 0.2nm, 0.5nm,1nm,2nm and 5nm. Scan speed allows the selection of HI, MEDIUM and LOW. For survey scan we suggest 5nm, HI. For detailed scan we suggest 0.5nm, HI 1.2 Press F2 to select the test mode, Abs, %T or E (Fig 47). Fig Push the blank cuvette into the light path, press 0Abs/100%T to scan the base line (Fig 48). Press ESC/STOP to stop scanning; 24

25 Fig Pull the sample cuvette into the light path, press START to scan the sample (Fig. 49) ESC/STOP to stop scanning. When scan has finished, the beeper beeps 3 times (Fig. 50). Fig 49 Fig If you want to change the scale, press < or > to change x scale (Fig 51), input upper limit and lower limit by pressing the numeric keypad. To change y scale press or (Fig 52). After these inputs the instrument will redraw the curve (Fig 53). 25

26 Fig. 51 change X Scale Fig. 52 Change Y Scale Fig. 53 After X Scale, Y Scale changed 1.6 Press F3 to go to setup page Fig54A, press F1 in Fig54A to set Peak height Fig 54B of searching peak of Abs/%T value. There are two ways for you to search (Fig 54B). 26

27 Fig 54A Fig 54B a. Peak to peak, press F1 to set peak height and input value by pressing the numeric keypad (Fig 55). Press to search the peak from left to right and press to search from right to left. The value of every peak found will be displayed on the screen one at a time (Fig 56). Fig 55 27

28 Fig 56 b. Point to point, Press > to search the point from left to right and press <) to search from right to left. The search step interval is the same as the scan step. The value of every point searched will be displayed on the screen. 1.7 Save Curve Press SAVE to save the curve. Note: Load/Save requires the first scan display page (Fig.50). If in Search Page, press ESC to return to the required page 1.8 Print Test Report Press PRINT to print the curve you have loaded or scanned (Fig 57). Note: The report always is printed as in Fig

29 Fig

30 Kinetics Mode Please press 4 in main menu (Fig8) for Kinetics. (Fig. 58) Please press ESC/STOP to exit. To load a previous kinetics result, press (LOAD) and select a previously stored result (.kin) Fig Press F1 to set Total Time, Delay Time, Time interval, and input the value by pressing the numeric keypad, Fig 59, Fig 60, Fig 61. Fig 59 Set total time Fig 60 Set Delay time 30

31 Fig Select the test mode ( Abs or %T ) by pressing F2, Fig. 62. Fig Set wavelength by pressing SETλ.Place the blank cuvette into the light path, press 0Abs/100%T for blanking. 4. Place the sample cuvette into the light path, press START to scan the sample. After the delay time, the beeper beeps 3 times and time -scan starts. Fig 63,At the end of the time-scan, the beeper also beeps 3 times, Fig 64. Fig. 63 Time-scanning 31

32 Fig. 64 Time scan finish 5. Press F3 to process the data with the Calculation of Enzyme kinetics reaction rate, and enter Begin Time, End Time and Factor (Fig 65, Fig 66, Fig 67) and the value in I.U. will be calculated and displayed (Fig 68). The average straight line between the Begin Time and End Time will be calculated. The gradient of this line gives the rate of change of ΔA/min. Note: I.U.=Factor ΔA/min Fig. 65 Set Begin Time Fig. 66 Set End Time 32

33 Fig. 67 Set Factor Fig. 68 Calculate Reaction Rate 6. If you want to change the scale, please refer to page Press F4 to search the Abs/%T value in relation to the time axis. Search point to point by pressing < or >. Please refer to 1.5 of WL scan. 8. Save Curve Press SAVE to save curve. Note: Load/Save requires the first kinetics display page Fig 64. Press ESC if in Search to return to the required page. 9. Print Test Report Press PRINT to print the curve you have loaded or scanned, Fig

34 Fig 69 DNA/Protein Mode Press 5 in main menu for DNA/Protein, (Fig. 70) ESC/STOP to exit. Note: The algorithm of the test refers to Appendix A. Fig 70 To load previous DNA results, press (LOAD) and select a previously stored result (.dna) 34

35 1. To use a simpler or different algorithm, you can enter your own values for f1-f4. Press F1 to set f1-f4. Input the value by pressing the numeric keypad (Fig 71). Fig Press F2 to select test mode. Absorbance difference 1 is for testing at the wavelength 260 nm, 280 nm and 320 nm (optional), and the Absorbance difference 2 is for testing at the wavelength 260 nm, 280 nm and 320 nm (optional), Fig 72. Then select with/without reference. If selected with reference (NO), the A ref. will be 0 (Fig 73). Fig 72 Fig Press F3 to select the unit of concentration (Fig 74). 35

36 Fig Push the blank cuvette into the light path, then press 0Abs/100%T, Fig 75. Fig Pull the sample cuvette into the light path, press START to test the sample. The test result will be displayed on the screen, Fig 76. Fig If there is more than one sample, repeat step 5 for the next sample. 7. After multi samples have measured, press <) or > for searching. Input the sample number, the result will be displayed on the screen. Press or to browse the test results one by one. 36

37 8. Recall the default Press F4 to recall the default of the f1-f4. 9. Save Data Press SAVE to save test results. Input the filename and press ENTER to confirm, Fig 77. Fig Print Test Report Press PRINT to print the test result (Fig 78). Multi Wavelength Mode Fig 78 Press 6 in main menu for Multi WL, (Fig 79). ESC/STOP to exit. Fig 79 37

38 To load previous Multi Wavelength results, press (LOAD) and select previously stored results (.mul). 1. Press F1 to setup a group of wavelengths for testing by pressing the numeric keypad followed by ENTER. or to modify the inputted data Fig Press ESC/STOP to finish setup and exit. Note: It is recommended to enter the wavelengths from higher to lower. Fig 80 After press F1 Fig. 81 Input 800 nm Fig. 82 Input 750 nm 38

39 Fig. 83 Ten Wavelengths Finish In Fig 83, press ESC/STOP to go to Fig. 84. Fig Push the blank cuvette into the light path, then press 0Abs/100%T to Blank. 3. Pull the sample cuvette into the light path, press START to test. The test results will be displayed on the screen, Fig 85. Fig If there is more than one sample, repeat step 2-3 for the next sample. Note: When the test has finished, the wavelength will go to the first one. 5. Press <) or > for searching. Input the sample number, the result will be displayed 39

40 on the screen. Press or to browse the test results one by one. 6. Save Data Press SAVE to save data. 7. Print Test Report Press PRINT to print the test results (Fig 86). Fig 86 System Utility Press 7 in Main menu for System Service Routines, (Fig. 87) ESC/STOP to exit.. Fig 87 Wavelength Reset Press 1 in Fig 87 to reset wavelength. The instrument will implement re-check Dark Current, Search 656.1nm to calibrate the wavelength. If you are worried about the wavelength accuracy after a long time test cycle, you can select this item. Printer Setup Press 2 in Fig 87 to setup printer (Fig 88). ESC/STOP to Exit. 40

41 1. Press 1 in Fig 88 to Reset Printer. Fig Press 2 in Fig 88 to select print port, (LPT or Comm.), Fig. 89. Fig Press 3 in Fig 88 to select printer, Fig90. Fig Press 4 in Fig 88 to select print mode. If you select Print screen mode, a little icon will be displayed on the top line of the screen, (Fig 91), if you select Print report mode, the little icon will disappear. 41

42 Fig91 Lamp Service Press 3 in Fig. 87 to set lamp, Fig. 92. ESC/STOP to exit. Item 6 is only for Alpha There is NO item 6 display for Alpha Fig Press 1 in Fig 92 to switch on/off deuterium lamp and press 3 to switch Halogen lamp. When deuterium lamp and/or Halogen lamp are turned off, the D2 and W icon will change to no fill in Fig. 93. Fig Press 2 or 4 in Fig 92 to reset usage time of deuterium lamp or Halogen lamp (Fig 94). Press or to select Yes or No, and then press ENTER. Usually you need reset the lamp s usage time after the new lamp install. 42

43 Fig Press 5 in Fig. 92 to set the switch change point of D2 and W lamp (Fig. 95). Fig Press 6 in Fig. 92 to locate the light source. The instrument always locates the light source at 546.0nm.This item is only for Alpha Clock Setup Press 4 In Fig 87 to set the display mode and modify the clock (Fig 96). ESC/STOP to exit. Fig Press 1 or 2 in Fig 96 to modify time or date by pressing the numeric keypad and 43

44 press PRINT (Fig. 97). Fig Press 3 in Fig. 96 to set the date display on the top right corner of the screen. 3. Press 4 in Fig. 96 to set the time display on the top right corner of the screen. Refresh Dark Current Press 5 In Fig. 87 to get dark current, Fig. 98. Fig. 98 The dark solutions are more likely to be affected by drifting dark current. It is recommended to refresh the dark current before you take measurements of very dark solutions. Connect to PC Press 6 in Fig. 87 to connect to a computer, the screen displays as in Fig. 99. Press ESC/STOP to exit. 44

45 Fig. 99 If the instrument does handshake with the computer successfully, the screen will change to Fig Fig. 100 Beeper on/off Press 7 in Fig. 87 to enable/disable the beeps of the keypad. Selection Language Press 8 in Fig. 87 to select display language. Refresh System Baseline Press 9 in Fig 87. to refresh the system baseline. The energy over the full range of wavelengths can be balanced by selecting this option. Delete entire saved files Move the reverse display stripe to line 10 and then press ENTER to Delete entire saved files. Please take care when using this action. Double confirmation is required for this operation, Fig 101, Fig

46 Fig. 101 First Confirm Fig. 101 Second Confirm Restore default settings Move the reverse display stripe to line 11 and then press ENTER to restore default settings if you select this option, instrument will restore the defaults of all test modes and methods. The defaults are: Basic Mode: Abs The change wavelength of UV zone and visible zone: 339 nm; Concentration factor: 1; Quantitative Mode: Method: Single WL WL: 546 nm Unit: mg/l Sample Time: 1 Curve: Ratio Coeff.: 1 Edit Standard: 0 Wavelength Scan: Scan wavelength range: 1100 nm-190 nm Scan step: 1.0 nm Scan speed: Hi Scan mode: Abs Display range: 0-3 A 46

47 DNA/Protein Mode: measure wavelength: 280 nm, 260 nm reference wavelength: 320 nm calculate factor: f1=62.90, f2=36, f3=1552, f4=757.3 concentration unit: ug/ml, Kinetics Analysis: Scan time: 180 s interval time: 1.0 s delay time: 3 s test mode: Absorbance display range: 0-3 A Multi-Wavelength Mode: The number: 1(546 nm) Mode: Abs 47

48 Appendix A DNA/Protein Test Algorithm Test Name Method Wavelength (s) Calculations Parameters Displayed Units DNA MEASUREMENT DNA/Protein Concentratio n and DNA purity Absorbance difference (260,280) Absorbance difference (260,230) Absorbance ratio A1=A260n m A2=A280n m A ref=a320n m (optional) A1=A260n m A2=A230n m A ref=a320n m (optional) A1=A260n m A2=A280n m or A2=A230n m A ref=a320n m (optional) DNA concentration: (A1-Aref)f1- (A2-Aref)f2 Protein concentration: (A2-Aref)f3- (A1-Aref)f4 DNA concentration: (A1-Aref)f1- (A2-Aref)f2 Protein concentration (A2-Aref)f3- (A1-Aref)f4 Ratio= A1-Aref A2-Aref f1=62.9 f2=36.0 f3=1552 f4=757.3 f1=49.1 f2=3.48 f3=183 f4=75.8 None DNA: μg/ml Protein: μ g/ml No units(ratio) 48

49 Appendix B A number of correction techniques can be used to eliminate or reduce interference errors. In general, if the source of the error is known and is consistent from sample to sample, the error can be eliminated. On the other hand, if the source is unknown and varies from sample to sample, the error can be reduced but cannot be eliminated. Correction techniques always require data from at least two wavelengths. The more sophisticated correction techniques require multiple wave length or spectral data. A.1 Iso absorbance When a known interfering component with a known spectrum is present, the error introduced by this component at the analytical wavelength for the target analyst can be eliminated by selecting a reference wavelength at which the interfering compound exhibits the same absorbance as it does at the analytical wavelength. The absorbance at this reference wavelength is subtracted from the absorbance at the analytical wavelength, as shown in Figure A1.The residual absorbance is the true absorbance of the target analyst. This technique is less reliable when the spectra of the analyst and interfere are highly similar. Moreover, it can be corrected for only one interference. Fig A1 Iso absorbance correction A.2 Three-point correction The three-point, or Morton-Stubbs, correction uses two reference wavelengths, usually those on either side of the analytical wavelength. The background interfering absorbance at the analytical wavelength is then estimated using linear interpolation, (see Figure A2).This method represents an improvement over the singlewavelength reference technique because it corrects for any background absorbance that exhibits a linear relationship to the wavelength. In many cases, if the wavelength range is narrow, it will be a reasonable correction for nonlinear background absorbance such as that resulting from 49

50 scattering of from a complex matrix. Fig A2 50